key: cord- -pxryt wn authors: leroy, eric; gonzalez, jean paul title: filovirus research in gabon and equatorial africa: the experience of a research center in the heart of africa date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: pxryt wn health research programs targeting the population of gabon and equatorial africa at the international center for medical research in franceville (cirmf), gabon, have evolved during the years since its inception in in accordance with emerging diseases. since the reemergence of ebola virus in central africa, the cirmf “emerging viral disease unit” developed diagnostic tools and epidemiologic strategies and transfers of such technology to support the response of the national public health system and the world health organization to epidemics of ebola virus disease. the unit carries out a unique investigation program on the natural history of the filoviruses, emergence of epidemics, and ebola virus pathogenesis. in addition, academic training is provided at all levels to regional and international students covering emerging conditions (host factors, molecular biology, genetics) that favor the spread of viral diseases. the international centre for medical researches of franceville (cirmf) was founded in by his excellency el hadj omar bongo ondimba, president of the gabonese republic, and mr. pierre guillaumat, the chairman of the petroleum company, total gabon. the centre was inaugurated on december th, with the participation of numerous internationally eminent scientists (figure ) . in the s, viral hemorrhagic fevers became a focus of attention in equatorial. the decision to develop a high security laboratory for the study of ebola virus disease came after a ebola virus disease outbreak in mayibout area, gabon. the main objective was to develop the potential for rapid and specific diagnosis on viral hemorrhagic fevers and to have a backup for field investigation of severe viral hemorrhagic fever epidemics. because the unique expertise and interaction of the cirmf team along with the international world health organization (who) teams for ebola virus disease, the gabonese government agreed to such a project. the first bsl + (including negative pressure and glove box) laboratory was built in , mostly financed by the foreign ministry of france ( figure ). this laboratory was built in a period of quasi "emergence" of successive ebola virus disease outbreaks in gabon, and the plans to upgrade the bsl + laboratory were not efficient. a specific research unit was founded in to study emerging infectious diseases: the emerging viral disease unit (umve). thanks to work done between and , both in the field and in research, cirmf became a national reference laboratory and a who collaborating center in the equatorial african region. a second high security laboratory for risk group / agents, mostly funded by the total gabon oil company and the gabonese government, was built between and on cirmf campus ( figure ). this bsl- laboratory was commissioned by a combined team of experts from the pasteur institute of paris, national institute of health and medical research, france, and jean mérieux p laboratory of lyon. on hectares, the cirmf campus has a working space of , square meters composed of a main building, laboratories, service buildings, and living accommodations (figure ). the present high containment and high security laboratory, operated by the emerging viral diseases unit (umve), is one of laboratories in africa that can manipulate risk group / agents (i.e., ebola, marburg, and crimean-congo hemorrhagic fever viruses). research, including isolation and characterization of these highly pathogenic viruses is performed in accordance with international rules defined by who on the handling risk group / agents updated equipment includes a double door autoclave, thermo regulated cabinet, a high security centrifuge system, a conventional photonic microscope, a virus isolation unit, and two independent rooms the can be shut down alternatively after decontamination when necessary. an uninterruptable controlled electrical supply for refrigeration, computer systems, and other systems is ensured by two back-up power plants. the telecommunication network consists of mobile phones and the internet through a dedicated satellite antenna. other service units consist of a primate center, the gorilla and chimpanzee study station in lopé national park, and the dienga health observatory. investigators from this observatory conduct field studies on the prevalence of viral and parasitic diseases and their implications for public health [ ] dedicated to medical research, the primate center houses more than primates belonging to ten different african species (e.g., chimpanzees (pan troglodytes), gorillas (gorilla sp), mandrills (mandrillus sphinx), guenons (cercopithecus sp.), collared mangabeys (cercocebus torquatus), greater spot-nosed monkeys (cercopithecus nictitans), vervet monkeys (chlorocebus pygerythrus) and an asian macaque (macaca sp.) colony. one of the largest primate centers in africa, the primate center is equipped with level a and a animal facilities for scientific research protocols. the great apes are housed in large open-air facilities. semi-free living colony of twelve forested hectares harbor about half of the primates at the primate center. at the gorillas and chimpanzee study station, researchers study ecological approaches to the emergence of zoonotic diseases, inter-species transmission of pathogens, and disease outbreaks in humans and wild animals [ ] . running costs are funded by the ministry of economy, gabon, the national petroleum company of total-gabon, and the ministry of foreign and european affairs, france. several international agencies participate in a variety of financial supports including scientists' salaries, equipment, research projects, and academic grants (e.g., ird, who, united states agency for international development, national center for scientific research, france). technical laboratory training support of gabonese teams and other african countries has been one of the major aims of cirmf. the umve actively participates in the academic training at the regional graduate school and the different state universities of equatorial africa. a special relationship with the "health sciences university" of libreville and the "sciences and technology university" of masuku in franceville encourages collaborative research projects with teachers and supports students of the faculties of medicine and sciences in the preparation of doctoral theses. cirmf receives doctoral and post-doctoral scientists from other universities of developed countries (e.g., bonn, marseille, montpellier, and tübingen universities). continuing medical education in the form of post-doctoral workshops are held at the cirmf for discussion and demonstration of modern techniques. as a national reference laboratory, cirmf has the following roles: diagnosis of suspected cases during outbreaks of viral hemorrhagic fevers or severe clinical infectious syndromes; development of new methods for diagnosing such infections; surveillance of animal fatalities in reservoir or intermediate hosts; and intervention during outbreaks of unknown etiology. cirmf diagnosed infections of more than pathogens that could not be identified in other biology laboratories throughout the country. cirmf maintains close ties to several components of the national healthcare system, such as amissa bongo regional hospital in franceville or the sino-gabonese friendship hospital. in order to facilitate national and international scientific exchanges including scientists, equipment, biological specimens, the capital of gabon, libreville, is part of cirmf operational system. hosted by the university of health sciences, libreville, one laboratory is now operational. tight connections with other scientific teams in libreville are under development (i.e.,: military hospital, libreville; general hospital, libreville; a. schweitzer lambarene foundation). the emerging viral diseases unit, cirmf, proposes forming a research partnership to study infectious diseases transmitted by animals of the tropical rain forests regions of equatorial africa. the proposed partnership builds on such existing collaborations of several years between the major research centers of other french speaking equatorial african countries, namely the national public health laboratory in brazzaville, republic of the congo, and the institute for biomedical research in kinshasa, democratic republic of congo. an international partnership with the ird, marseille, france, and the institute of virology, bonn, germany, will assist in the development of this regional partnership. with the who regional office, brazzaville, republic of the congo, the umve-cirmf field team participates along with other international partners (e.g., centers for disease control and prevention, usa; laboratory centre for disease control and national microbiology laboratory, winnipeg, canada; p laboratory of the national institute for communicable diseases, south africa) to respond to all ebola virus disease epidemics in africa. cirmf aims to use laboratory and field expertise be a regional focal resource in conjunction with local health authorities to organize epidemic responses. cirmf expertise is also offered from by entering into laboratory-to-laboratory agreements. also, an informal international laboratory network for the diagnosis and surveillance of severe infectious clinical syndromes includes: the institut national de recherche biomédicale, democratic republic of the congo; laboratoire national de santé publique de brazzaville, republic of the congo; metabiota/laboratoire des maladies emergentes, yaoundé, cameroon; pasteur institute, bangui, central african republic; institute of virology, bonn university, germany; and p jean mérieux lyon, france. exchange of materials, equipment, and personnel is facilitated through memorandums of understanding. cirmf holds more than , samples of various origins in a biological repository, which is accessible to the international scientific community. the uvme assists the national public health system in consolidating and formalizing microbiological monitoring of the equatorial african sub-region. ultimately, cirmf will be positioned as a center of excellence for microbiological surveillance and research in a global network. developing diagnostic tools and strategies is the main driver to improve surveillance and research of emerging viral diseases. a strategic choice was made to link syndromes to an etiological agent, including hemorrhagic syndromes. to isolate and diagnose highly pathogenic viruses, a progressive and diversified methodology was applied. the first approach used real-time virus-specific pcr (qrt-pcr). if the first approach was not successful, conventional rt-pcr was implemented using degenerate consensus primers targeting conserved regions of the genome. ultimately, random amplification of nucleotide sequences was directly applied to the original biological material (dna chip re-sequencing, meta-genomic pyrosequencing ( life sciences, branford, ct)). umve studied the factors implicated in the three steps that led to ebola virus and marburg virus diseases emergence in humans. these steps include: the identification of reservoir species, the circulation within the natural host, the crossing to intermediary animal species, and finally the direct transmission to humans from great apes and fruit bats. antibodies and nucleotide sequences specific for ebola virus were detected in the liver and spleen of fruit bat belonging to three species (hypsignathus monstrosus, epomops franqueti, myonycteris torquata) in gabon and republic of the congo (figure ). antibodies and nucleotide sequences specific for marburg virus were found in the egyptian fruit bat (rousettus aegyptiacus) in gabon, suggesting that bats might be reservoirs for filoviruses [ ] [ ] [ ] . we showed that ebola virus caused extensive epizootics among gorillas and chimpanzees, killing thousands of animals during the last decade in parts of gabon and republic of the congo [ ] . we characterized the viral variants associated with all ebola virus disease outbreaks that occurred between and and developed new epidemiological models of ebola virus disease epidemics, based on the identification of several independent epidemic chains. the identification of multiple variants during the gabon/republic of congo outbreak and two phylogenetically divergent lineages suggest independent introductions into great ape and human populations following multiple viral spillovers from a reservoir host [ ] [ ] . in this "multi-emergence" hypothesis, ebola virus disease outbreaks would occur episodically during certain ecological conditions caused by habitat disturbances or climatic phenomena. this hypothesis also implicitly assumes that ebola virus was present in equatorial africa long before the first documented disease outbreak in , as supported by various serological surveys. furthermore, we recently showed that the luebo outbreak in the democratic republic of the congo was linked to massive fruit bat migration, strongly suggesting that humans could be infected directly by bats or by consumption of bats [ ] . in the study of immunological mechanisms of ebola virus disease humans, we showed that fatal infection is associated with aberrant innate immunity and global suppression of adaptive immunity [, [ ] [ ] [ ] [ ] . the innate immune reaction is characterized by a 'cytokine storm', with a hyper secretion of numerous pro-inflammatory cytokines, chemokines and growth factors, and by the noteworthy absence of antiviral interferon (ifn)-α [ , , ] . immunosuppression of adaptive immunity is characterized by very low levels of circulating cytokines produced by t lymphocytes and by massive loss of peripheral cd and cd lymphocytes, probably through fas/fasl-mediated apoptosis. finally, we hypothesized that a viral protein with super-antigen activity might be involved in the massive t cell apoptosis [ ] . in striking contrast with fatal outcome, effective control of ebola virus infection is associated with balanced immune responses in survivors. asymptomatic ebola virus infection was demonstrated in humans during the - disease outbreak in gabon [ ] . asymptomatic infection was associated with an early strong inflammatory response that may be involved in the early inhibition of viral replication [ , ] . consistent with this discovery, we showed a decade later that a large fraction of the human population living in forested areas of gabon has both humoral and cellular immunity to ebola virus [ ] . in the absence of identified risk factors, the high prevalence of 'immune' individuals suggests a common source of human exposure such as fruits contaminated by bat saliva. initially focused on ebola virus disease, umve science policy was redirected since by expanding the main research themes to other emerging viral diseases that could threaten public health in the congo basin (table ) [ ] [ ] [ ] [ ] . cirmf is geographically isolated from the capital of gabon, libreville. the libreville office is essential to the franceville headquarters as it coordinates visits from staff on field missions, and forwards imported equipment to headquarters. the capital is accessible by a -hour ride in a four-wheel drive vehicle or in a train (three times/week schedule) covering km. due to tropical weather, four weekly domestic plane rotations often fly on an inconsistent schedule. ultimately cirmf needs to be largely autonomous in term of electrical power (i.e.,: unexpected fuel supply disruption), cold chain with the necessity to maintain in situ a unit of liquid nitrogen production (repository), and purified water supply. cirmf is uniquely suited to study infectious diseases of the congolese tropical rain forest, the second world's largest rain forest. as a central point of a north-south transect of the rain forest, the center is able to study the biodiversity of africa including animal species, microbes, and parasites. cirmf is dedicated to conduct medical research of the highest standard, and is the only facility of its type in equatorial africa. with unrivalled infrastructure, multiple sites, and multidisciplinary teams, the center promotes a modern healthcare system in gabon. cirmf teams are engaged in trans-disciplinary projects bringing together specialists from the health sciences, biological sciences, veterinary medicine, conservation, the humanities, and environmental sciences. the center welcomes partnerships from around the world to work on global human health issues. coronaviridae coronavirus/hcov nl /gabon - human + coronavirus/hcov hku /gabon - human + coronavirus/hcov oc /gabon - human + available online fruit bats as reservoirs of ebola virus spatial and temporal patterns of zaire ebolavirus antibody prevalence in the possible reservoir bat species marburg virus infection detected in a common african bat multiple ebola virus transmission events and rapid decline of central african wildlife isolates of zaire ebolavirus from wild apes reveal genetic lineage and recombinants human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in ebola virus-infected patients apoptosis in fatal ebola infection. does the virus toll the bell for the immune system human fatal zaire ebola virus infection is associated with an aberrant innate immunity and massive lymphocyte apoptosis association of kir ds and kir ds with fatal outcome in ebola virus infection lansoud-soukate, j. inflammatory responses in ebola virus-infected patients the acute phase of chikungunya virus infection in humans is associated with strong innate immunity and t cd cell activation evidence for ebola virus superantigen activity human asymptomatic ebola infection and strong inflammatory response early immune responses accompanying human asymptomatic ebola infections high prevalence of both humoral and cellular immunity to zaire ebolavirus among rural populations in gabon recent introduction and rapid dissemination of chikungunya virus and dengue virus serotype associated with human and mosquito co-infections in re-emergence of crimean-congo hemorrhagic fever virus in central africa type wild poliovirus and putative enterovirus in an outbreak of acute flaccid paralysis in congo concurrent chikungunya and dengue virus infections during simultaneous outbreaks this article is an open access article distributed under the terms and conditions of the creative commons attribution license we would like to thank all the umve-cirmf and the health ecology research unit of cirmf work teams. we acknowledge the support and funding of the gabonese government and the oil company total-gabon. we acknowledge foreign agencies that largely contributed to the success of the "cirmf filovirus program": the french ministry of foreign and european affairs through the french embassy in gabon; metabiota and global viral (alias gvfi) for technical and equipment support; and the institute for research development, for funding research teams and providing high performance equipment. pneumovirus/hrsv /gabon - human +/- the authors declare no conflict of interest. key: cord- - t d vvo authors: li, yongfeng; li, lian-feng; yu, shaoxiong; wang, xiao; zhang, lingkai; yu, jiahui; xie, libao; li, weike; ali, razim; qiu, hua-ji title: applications of replicating-competent reporter-expressing viruses in diagnostic and molecular virology date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: t d vvo commonly used tests based on wild-type viruses, such as immunostaining, cannot meet the demands for rapid detection of viral replication, high-throughput screening for antivirals, as well as for tracking viral proteins or virus transport in real time. notably, the development of replicating-competent reporter-expressing viruses (rcrevs) has provided an excellent option to detect directly viral replication without the use of secondary labeling, which represents a significant advance in virology. this article reviews the applications of rcrevs in diagnostic and molecular virology, including rapid neutralization tests, high-throughput screening systems, identification of viral receptors and virus-host interactions, dynamics of viral infections in vitro and in vivo, vaccination approaches and others. however, there remain various challenges associated with rcrevs, including pathogenicity alterations due to the insertion of a reporter gene, instability or loss of the reporter gene expression, or attenuation of reporter signals in vivo. despite all these limitations, rcrevs have become powerful tools for both basic and applied virology with the development of new technologies for generating rcrevs, the inventions of novel reporters and the better understanding of regulation of viral replication. the commonly used tests based on wild-type viruses, such as immunostaining, are often time-consuming and labor-intensive. furthermore, these methods cannot meet the demands for high-throughput screening (hts) of antivirals, rapid, sensitive and quantitative detection of neutralizing antibodies (nabs), visual tracking of viral proteins or viruses in vitro and in vivo and other fields of virology. replicating-competent reporter-expressing viruses (rcrevs) are one type of artificially modified viruses that not only retain the viral genetic characteristics but also possess the new properties of the reporter genes, which represent a useful tool for quantitative analysis of viral replication and tracking viral protein transport in both living cells and animals. to date, advances in technologies enable the generation of rcrevs, which have been successfully applied in diagnostic and molecular virology. currently, reverse genetics systems for many viruses have been well-established [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , providing powerful tools for generating rcrevs. since some viruses possess a large genome, they usually permit a large extrinsic genetic insertion without impairing viral replication. for example, vaccinia virus (vacv) contains a kb genome, capable of accepting up to kb insertion [ ] . however, for most rna and some dna viruses containing a small-sized genome, a recurring difficulty in generating rcrevs is the genetic instability, especially for a larger reporter gene. for some viruses with a segmented rna genome, the insertion of a large reporter gene into the genome is difficult or even impossible to achieve. commonly used reporters in rcrevs include fluorescent proteins, such as enhanced green fluorescent protein (egfp) (green), gfp mutants (enhanced cyan fluorescent protein (ecfp) (blue), mcherry (red) and venus (yellow)), far-red fluorescent reporters (red fluorescent protein (rfp), katushka , dtomato and dsred)), near-infrared fluorescent proteins (irfps) and tetracysteine (tc); bioluminescent reporters, such as firefly luciferase (fluc), renilla luciferase (rluc) and gaussia luciferase (gluc); in addition to other reporters, such as neomycin-resistance gene (neo r ) and cre recombinase. these reporters are mainly used to rapidly quantify viral replication and track viral proteins or viruses by living imaging in vitro and in vivo. however, different reporters may have different influences on the biological properties of various viruses, and the loss of the reporter gene expression is a significant concern for some rcrevs. therefore, choosing a suitable reporter is a critical decision on designing rcrevs. for the planned applications, the reporters with a smaller size may be a promising option due to their minimum effects on the viral biology. for example, the rluc gene ( bp) is better than the fluc gene ( bp) and has a minimal influence on the growth of the engineered classical swine fever virus (csfv) expressing the reporters [ , ] . various strategies associated with the reporter gene expression have been developed. an extensively used expression strategy is to fuse the reporters to one of the viral proteins. for instance, the rluc activities from engineered csfv carrying the rluc fused to the viral n pro protein were detected [ ] . a nonessential viral gene can be replaced with a reporter gene to generate a reporter virus. in addition, the cre-loxp recombination is widely used to control reporter gene expression in cell cultures or animal models. notably, reporters can be expressed from an additional transcriptional unit (atu), in which a reporter gene is generally flanked by highly conserved gene start-and-stop signals. for instance, gfp was expressed as a separate protein from the atu in the recombinant peste des petits ruminants virus (pprv) [ ] . furthermore, the reporters can be expressed separately by introduction of an internal ribosome entry site (ires) or foot-and-mouth disease virus (fmdv) a self-cleaving peptide ( a) (llnfdllklagdvesnpgÓp), which is able to undergo self-cleavage allowing simultaneous expression. for example, recombinant alphaviruses expressing a separate fluc by a-mediated cleavage were successfully used to screen viral receptors [ ] . since the properties of rcrevs and the stability of reporter genes may vary among different strategies, the selection of expression strategy is another principal consideration on designing rcrevs for specific applications. notably, the same strategy might lead to different effects on the growth of the same virus due to the distinct insertion site. for example, a recombinant respiratory syncytial virus (rsv) expressing a reporter protein from an atu upstream of ns displayed negligible attenuation in cell cultures [ ] , whereas the rsv expressing a reporter from an atu inserted between f and g genes was significantly attenuated in cell cultures [ ] . additionally, the use of a peptide to achieve expression of a separate reporter might constitute a promising approach as a peptide is small and can readily be self-cleaving while minimizing the possibility of the loss of functions of the viral proteins. the neutralization immunofluorescence test (nift) is currently a gold standard for the detection of nabs against many noncytopathogenic viruses. however, nift is labor-intensive and time-consuming due to the necessary incubation and staining procedures. it would be convenient to use rcrevs to detect nabs directly without immunostaining. there are many successful applications of rcrevs harboring egfp, rluc or fluc in the rapid neutralization tests [ , [ ] [ ] [ ] [ ] [ ] . for viruses causing slight or no cytopathic effects (cpes) in cultured cells, the egfp reporter can be chosen to generate rcrevs for quantifying nabs with higher specificity through direct observation of egfp fluorescence. due to the structural characteristics of egfp, the fluorescence of egfp fused to a viral protein may be attenuated or quenched. therefore, egfp should be separated from the viral protein by introduction of an atu, ires or a sequence when constructing the rcrevs. owing to the simple assaying for gluc activity compared with the fluc, rluc and other bioluminescent reporters, it is advantageous to determine the neutralizing antibody titers based on gluc-expressing viruses. notably, attenuated rcrevs can also be applied for rapid neutralization tests due to high sensitivity and operational simplicity for detection of the reporters. antiviral compounds, interferon-stimulated genes (isgs) or small interfering rnas (sirnas) have potential applications in the treatment of many diseases. the traditional screening methods of them are developed by a cell-based hts, in which the treated cells were observed under a microscope for the inhibitory activity of the compounds for cpes [ ], enzyme-linked immunosorbent assay (elisa) [ ] or fluoresces-linked immunosorbent assay [ ] . using these approaches, the scientists have screened and identified a series of small antiviral molecules or inhibitors [ , ] . however, the traditional methods based on wild-type viruses are inefficient for antiviral screening. to overcome this problem, rcrevs have been applied for the purpose of antiviral screening, because rcrevs can target the complete virus life cycle and offer a higher throughput of antiviral screening than traditional assays. rcrevs represent powerful screening tools for identifying antiviral compounds against various highly pathogenic viruses [ - ]. for example, a high-throughput assay for zaire ebov has been established using the recombinant ebov expressing the egfp reporter gene [ ] . interestingly, reporter viruses in combination with other approaches, such as rnai, have been applied to screen anti-csfv isgs [ ] , which is time-and cost-effective. importantly, rcrevs with slightly reduced growth ability compared with the wild-type viruses can also be applied for screening antiviral isgs. in addition, rcrevs can be used for sirnas hts with high efficiency. for instance, a reporter csfv expressing the fluc gene has been used to screen antiviral sirnas efficiently [ ] . recently, a recombinant ebov carrying a luciferase reporter was used to screen sirnas with higher screening efficiency than the wild-type virus [ ] . however, there are some problems associated with rcrevs in hts applications. first, the interference of compound fluorescence may occur when screening antivirals using fluorescent reporter-expressing viruses. second, the antiviral effects of screened out antivirals by rcrevs need to be verified with the parental viruses. furthermore, the antiviral effects may be different between rcrevs and the wild-type viruses due to the occasionally inclusive fluorescence signals for the wild-type viruses in indirect immunofluorescent assay (ifa) and higher sensitivity for rcrevs in fluc/rluc activity assay. third, rcrevs are not ideal tools for screening of antivirals targeting specific step(s) of viral infection, since rcrevs can undergo a complete virus life cycle. for example, currently, a set of isgs against hepatitis c virus (hcv), yellow fever virus (yfv), west nile virus (wnv), chikungunya virus (chikv), venezuelan equine encephalitis virus (veev) and human immunodeficiency virus (hiv- ) have been documented, but their exact antiviral step(s) remain(s) unknown [ ] [ ] [ ] [ ] . a practical challenge lies in the explanations of their antiviral mechanisms for antiviral isgs screened by rcrevs. despite these limitations, the following strategies may address some of the above issues. fluc, rluc and other bioluminescent reporters provide a viable alternative to fluorescent reporters in hts assays for drug discovery [ ] . this facilitates the development of highly sensitive, cell-based reporter assays [ ] , eliminates the problem of compound fluorescence [ ] , and possesses several advantages such as high reliability, convenience and adaptability to hts assays. remarkably, primary hts followed by validation using traditional assays based on the parental viruses will greatly aid the discovery of novel antivirals against infectious diseases. finally, the use of replicons or pseudoparticles would help to identify the step(s) of the viral life cycle as the potential targets of antivirals. identification of cellular receptors facilitates understanding of the mechanisms of virus entry into host cells [ , ] . moreover, the receptors are regarded as promising targets for development of novel antivirals [ ] [ ] [ ] [ ] . while reporter-expressing pseudoparticles are widely used to screen viral receptors [ , ] , rcrevs carrying fluc [ , ] , gfp [ ] or neo r [ ] as new useful tools have been applied for screening of viral receptors (table ). since rcrevs can infect the cells with multiple life cycles in contrast to pseudoparticles, more false-positive receptors may be screened. in spite of these few limitations, rcrevs are still powerful tools to screen viral receptors in combination with unsusceptible cells and cdna library derived from susceptible cells [ , ] or a set of sirnas against a number of genes encoding cell membrane proteins [ , ] (table ) . with the development of reverse genetics systems, rcrevs provide an ideal tool for monitoring the dynamics of viral infection progression in vitro and in vivo due to eliminating the need for secondary labeling, which represents a significant advance in the study of the biology of viruses (table ) . rcrevs carrying a gfp reporter gene have been successfully used for tracking viral protein(s) or viral infection in vitro and in vivo [ ] [ ] [ ] [ ] , which indicates that the gfp reporter gene is suitable for generating rcrevs to track viral proteins either in cell cultures or animal models. furthermore, gfps in rcrevs can be expressed efficiently in rodent brain for a long time [ ] and show lower autofluorescence in the tissue [ ] . therefore, gfp may be a promising option when rcrevs are used to study the infection of viruses replicating in the brain. additionally, an engineered virus expressing the split-green fluorescent protein (split-gfp) in the presence of cell lines expressing the complementing gfp can facilitate the tracking of viral infection in living cells [ ] . compared with the most commonly used egfp tag, the tc tag enables the fusion protein to fluoresce more quickly, with a minimum risk of disrupting the overall structure and function of the targeted protein [ ] . the tc-labeling technology has led to successful tracking of the nonstructural or structural proteins of diverse viruses [ ] [ ] [ ] [ ] [ ] [ ] . however, since the tc-tag technology contains a biarsenical labeling process [ , ] , the engineered replication-competent tc-tagged viruses are not suitable for tracking viral protein in vivo. in addition, recombinant canine distemper virus (cdv) expressing dtomato was used to investigate the routes of virus spread in vivo [ ] . a fully functional recombinant pneumonia virus of mice (pvm) with katushka has been developed to track infection of target cells in vivo [ ] . compared with far-red gfp-like proteins, irfp has a substantially higher signal-to-background ratio in a mouse model due to its infrared-shifted spectra [ , ] . interestingly, the cre recombinase as a reporter is used to generate rcrevs for visualizing virus infection in engineered cell lines or transgenic animals harboring a loxp-flanked fluorescent marker upstream of another otherwise silenced fluorescent reporter [ ] . table . applications of representative rcrevs in virus tracking and live imaging in vitro and in vivo. green fluorescent protein (gfp) herpes simplex virus (hsv) compartmentalization of protein by autofluorescent particles [ ] borna disease virus (bdv) in rodent brains [ ] canine distemper virus (cdv) routes of virus spread in vivo [ ] vesicular stomatitis virus (vsv) intracellular transport [ ] tetracysteine (tc) influenza a virus visualization of ns protein nuclear import in virus-infected cells in real time [ ] classical swine fever virus (csfv) nucleus import and export [ ] hepatitis c virus (hcv) virus particle assembly [ ] human immunodeficiency virus (hiv) viral component complexes [ ] de novo hiv production [ recently, several influenza viruses expressing fluorescent proteins of different colors ("color-flu" viruses expressing ecfp, egfp, venus or mcherry) or a toolbox of influenza a and b reporter viruses were generated to facilitate the study of viral infection in in vivo models. whole-mount images of transparent lung tissues were obtained using a fluorescent stereomicroscope [ ] [ ] [ ] [ ] [ ] . in addition, bioluminescent and fluorescent dual-reporter marek's disease viruses are engineered to track viral replication in cell cultures or animal models [ ] . in the future, "color" or dual-reporter viruses will be powerful tools to analyze viral infection at the cellular level in vivo to better understand the pathogenesis of various viruses. notably, reporters fused with viral proteins are very suitable for investigating the localization and distribution of the proteins in infected living cells. rcrevs will help advance virus-related live-imaging studies in vitro and in vivo, which allow localization of the infection and tracking of changes in the distribution of viruses in animals in real time. elucidating various aspects of pathogen-host interactions is essential for the comprehensive understanding of pathogenesis. compared with the most frequently used techniques for mapping of virus-host interactions, the approaches based on rcrevs can recapitulate the virus life cycle [ ] . split-gluc (gluc and gluc ) has been applied for identification of virus-host interactions. for example, a recombinant influenza virus carrying a gluc -tagged polymerase subunit is used to infect the cultured cells expressing a pool of gluc -tagged cellular proteins involved in nucleocytoplasmic-transporting pathways for confirming virus-host interactions [ ] . in addition, split-gfp reporter has huge potential in this application. however, the reporter activity based on the interactions of rcrevs with the cellular proteins may not be detected due to the interference of the space structure. the rcrevs are also useful in modified live vaccines containing genetic markers, which have been developed for many viruses by inserting egfp as a positive marker [ ] [ ] [ ] . for example, a genetically marked recombinant rinderpest vaccine expressing gfp has been developed [ ] . in addition, a recombinant gfp-tagged prrsv containing a deletion of an immunogenic epitope, in accompany with the diagnostic tests (gfp-and epitope-based elisas), enables serological differentiation between the marker virus-infected animals and those infected with the wild-type virus [ ] . a recombinant viral hemorrhagic septicemia virus (vhsv) harboring rfp gene was utilized to evaluate vhsv-based viral-vectored vaccines [ ] . more recently, the marker vaccine vsmegfp-hclv 'utr in the context of the csfv shimen strain was generated by inserting egfp to create a positive marker [ ] . for those viruses causing cpes, rcrevs can be used as an intermediate to generate and purify expected variants. for example, a novel ge-deleted pseudorabies virus (prv) was obtained by ge/gi-deleted virus expressing egfp [ ] . in addition, katushka as a reporter was used to evaluate a novel reverse genetic system of rsv [ ] . interestingly, oncolytic recombinant viruses harboring reporter genes have been developed and applied for the disease progression tracking and accurate visualization of tumor burden [ ] . since oncolytic viruses selectively infect as well as replicate within cancer cells, the recombinant oncolytic viruses expressing reporter genes, particularly for far-red fluorescent proteins, will be a promising option for real-time monitoring of viral infection in cancer tissues [ ] . while rcrevs harboring a reporter fused to a viral protein are the most suitable for studying the localization of the protein in infected cells, rcrevs carrying separate reporters are useful for other basic research purposes. for example, the preferential translation of viral rnas over host rnas during vsv infection has been demonstrated by the egfp reporter expressed from the recombinant vsv [ ] . recently, the contribution of ebov proteins in modulating dendritic cells (dc) maturation was investigated using the recombinant virus carrying egfp [ ] . furthermore, unique profiles of rfp expression acquired from thousands of co-infected cells with viable and defective viruses showed how the interference of defective viruses acts at multiple steps of infection [ ] . firstly, a practical challenge for some viruses lies in not allowing the insertion of reporter genes. as we stated above, it is difficult to insert a reporter gene into the genome of influenza viruses. despite the challenge, reporter-expressing influenza viruses have been developed and applied in basic science [ , , [ ] [ ] [ ] [ ] [ ] [ ] . to address the question, there are three necessary considerations, including the reporter protein itself, expression strategy, and structure of the viral protein. for example, the loop/linker regions are usually chosen to insert the tc tag based on the structure of ns of influenza viruses [ ] . although rcrevs have been developed and applied in vitro and in vivo, one question arises regarding the expression stability of the reporter gene in rcrevs during the viral replication in vitro and in vivo [ , ] . one potential consequence of rcrevs' attenuation is the purging of the inserted reporter from the viral genome. in this regard, we need to better understand the mechanism of regulation of viral genome replication and gene expression [ , ] , the association between structure and function of viral proteins, as well as the application of novel reporters such as nanoluc due to its small size [ ] . one of the biggest challenges is that rcrevs are possibly attenuated and may not accurately reflect natural infections [ , ] , which partially limits the applications of the rcrevs, especially in vivo. replacement of currently used expression strategies may be a promising approach to overcoming this problem. as an example, ires or a peptide-encoding sequence has been used to express separately the reporter from viral protein [ , ] . importantly, the use of split-gfp or split-luciferase may not compromise viral replication competency due to their smaller sizes [ , ] . however, whether these reporter viruses will be attenuated in vivo needs further investigation in the future. more recently, it has been reported that after mouse adaptation, influenza virus h n expressing the venus reporter gene became more pathogenic to mice and the venus gene was more highly and stably expressed [ ] , which may be another promising avenue that maintains the pathogenicity of the reporter viruses. luciferase imaging uses the luciferases to catalyze reactions that produce visible light in vivo at body temperature, which is used to determine the sites of virus replication, monitor viral dissemination in real time [ ] . however, there are many caveats in the process of obtaining accurate luciferase imaging [ ] . for example, the reporter signal from rcrevs is attenuated when in vivo imaging in tissues. despite these limitations, luciferases will still become major reporters for in vivo imaging in real time in the future as they have a number of advantages compared with the fluorescent reporters, such as no intrinsic autoluminescence. in addition, irfps are in high demand for in vivo imaging, which exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies with multicolor imaging. finally, in view of the advantages and disadvantages of different reporters, there seems no universal reporter for various applications. fortunately, ever-increasing novel reporters, including gfp mutants, "red-shifted" analogs of luciferase, variants of luciferase and novel luciferase nanoluc, can be chosen to design rcrevs for specific purposes. moreover, the dual-reporter rcrevs may be widely used to address the scientific questions. although reporter-based assays require costly automated imaging equipment, the detection of the reporter gene expression could be also performed with inexpensive, small and simple-to-use equipment, such as a pcr device based on the development of the technologies discussed in this article. rcrevs have proved themselves to be powerful tools for applied and basic sciences. despite their limitations, rcrevs have many more far-reaching benefits in virus research: a genome-wide rnai screening for host factors required for 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the use of transcription terminators to generate transgenic lines of chinese hamster ovary cells (cho) with stable and high level of reporter gene expression importance of codon usage for the temporal regulation of viral gene expression engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate rescue of influenza virus expressing gfp from the ns reading frame characterization of a neuraminidase-deficient influenza a virus as a potential gene delivery vector and a live vaccine multiple proteins from a self-processing polyprotein molecular determinants of virulence and stability of a reporter-expressing h n influenza a virus applications of bioluminescence imaging to antiviral research and therapy: multiple luciferase enzymes and quantitation imaging luciferase-expressing viruses acknowledgments: this work was supported by natural science foundation of china (no. and no. ) and the natural science foundation of heilongjiang province of china (no. qc ). we thank lintao liu at lerner research institute, united states of america for improving the language of the manuscript. key: cord- - zisomq authors: wang, xue; tan, jiying; biswas, santanu; zhao, jiangqin; devadas, krishnakumar; ye, zhiping; hewlett, indira title: pandemic influenza a (h n ) virus infection increases apoptosis and hiv- replication in hiv- infected jurkat cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: zisomq influenza virus infection has a significant impact on public health, since it is a major cause of morbidity and mortality. it is not well-known whether influenza virus infection affects cell death and human immunodeficiency virus (hiv)- replication in hiv- -infected patients. using a lymphoma cell line, jurkat, we examined the in vitro effects of pandemic influenza a (h n ) virus (ph n ) infection on cell death and hiv- rna production in infected cells. we found that ph n infection increased apoptotic cell death through fas and bax-mediated pathways in hiv- -infected jurkat cells. infection with ph n virus could promote hiv- rna production by activating host transcription factors including nuclear factor kappa-light-chain-enhancer of activated b cells (nf-ĸb), nuclear factor of activated t-cells (nfat) and activator protein (ap- ) through mitogen-activated protein kinases (mapk) pathways and t-cell antigen receptor (tcr)-related pathways. the replication of hiv- latent infection could be reactivated by ph n infection through tcr and apoptotic pathways. these data indicate that hiv- replication can be activated by ph n virus in hiv- -infected cells resulting in induction of cell death through apoptotic pathways. it is estimated by the world health organization (who) that approximately . ( . ~ . ) million persons worldwide are infected with human immunodeficiency virus (hiv), which is the etiologic agent of acquired immunodeficiency syndrome (aids), including . million in the united states [ ] . influenza a virus can cause acute respiratory infection in humans and animals throughout the world, and has continued to be a significant public health threat, leading to substantial global morbidity and mortality and an average of approximately , deaths annually in the united states alone [ ] . the impact of influenza virus on hiv infection has not been well investigated. little is known about influenza virus infection in hiv-positive individual. hiv infection has been shown to be related to worse prognosis of influenza. hiv-infected patients in canada who also had pandemic influenza a (h n ) virus (ph n ) infection had more severe illness than those who did not have the co-infection [ ] , and fatality was higher than for patients who were not co-infected in california (usa) [ ] . recent reports indicate that adults with aids experience substantially elevated influenza-associated mortality [ , ] . influenza virus infection has been associated with viremia in human and animal models. viral rna has been detected in blood in severe human ph n infection [ ] [ ] [ ] . encapsidated ph n rna is stable in blood derived matrices [ ] and influenza viruses can be transmitted by blood transfusion in ferrets [ ] . normally, influenza a virus infection is confined to the airways where the virus replicates in respiratory epithelial cells. cumulated reports indicate that influenza viruses can infect and replicate in blood cells, such as dendritic cells [ ] , primary monocytes/macrophages, and t cells [ , ] . infection with hiv- can result in apoptotic cell death through activation of both death receptor-mediated and bax/mitochondrial-mediated apoptotic pathways [ ] , which cause a progressive depletion of a select group of immune cells namely the cd + t helper cells leading to immunodeficiency. while hiv directly and selectively infects cd + t cells, the low levels of infected cells in patients is discordant with the rate of cd + t cell decline and argues against the role of direct infection in cd loss [ ] . a viral protein, neuraminidase (na), derived from the human influenza virus was reported to enhance the level of hiv- -mediated syncytium formation and hiv- replication [ , ] . however, it is not known whether influenza a virus in blood affects hiv- replication, or reactivates hiv- replication in hiv- -infected cells. here, we showed that pandemic influenza a (h n ) virus infection increased apoptotic cell death and hiv- replication in hiv- infected jurkat cells. rabbit polyclonal antibodies against bax, bcl-x l (b-cell lymphoma-extra large), caspase- , caspase- , cavoelin- , cd , cd , cytochrome c, fadd (fas-associated protein with death domain), fas, flip ((fadd-like il- β-converting enzyme)-inhibitory protein), p , zap- (zeta-chain-associated protein kinase ), and gapdh (glyceraldehyde -phosphate dehydrogenase were from santa cruz biotechnology (santa cruz, ca, usa). ap- , erk (extracellular-signal-regulated kinases), jnk (c-jun n-terminal kinases), p , nfat, and nf-kb p were bought from cell signaling technology, inc. (danvers, ma, usa). all other chemicals were from sigma (st. louis, mo, usa). the human jurkat t cell line (clone je . ) and j . , a latently hiv infected cell line cloned by limiting dilution from hiv-infected jurkat cells were obtained from national institutes of health (nih) aids reagent program (germantown, md, usa) and cultured at ˝c in % co in rpmi medium containing % fetal calf serum, mm glutamine, µg/ml penicillin, and µg/ml streptomycin. for hiv- infection, jurkat cells (clone je . ) were seeded at ˆ cells/ml for h, and infected with known amounts of hiv- (hiv- mn strain, copies per cells) for h, washed twice with pbs, and cultured for periods of time indicated. quantitative real-time reverse-transcriptase (rt) pcr was used for quantitation of viral rna. viral rna was isolated from µl of culture supernatant by using the qiaamp viral rna mini kit (qiagen inc., valencia, ca, usa) according to the manufacturer's protocol. the primers and taqman probe were designed in the gag capsid (p ) region, which is the variable region among most of the hiv- subtype b isolate sequences according to genbank database. the forward primer was -gacatcaagcagccatgcaa- , corresponding to nucleotides - , and the reverse primer was -ctatcccattctgcagcttcct- , corresponding to nucleotides - . the taqman probe was oligonucleotide -attgatggt ctcttttaaca- , corresponding to nucleotides - , coupled with a reporter dye ( -carboxy fluorescein) (fam) at the end and a non-fluorescent quencher and a minor groove binder (mgb), which is a tm enhancer, at the end. the nucleic acids were amplified and detected in an automated taqman analyzer by using quantitect™ probe rt-pcr kit (qiagen inc.). the -µl pcr mixture consisted of nm primers and nm probe. following three thermal steps at ˝c for min, at ˝c for min, and at ˝c for min. cycles of two-step pcr at ˝c for s and at ˝c for min were performed. the data are expressed as copy numbers/ml. known concentrations of hiv- (mn) viral rna (serially diluted: to copies) were used as templates and quantitative rt-pcr performed to generate a standard curve. each value represents the average concentration of six reactions in triple isolated repeats based on the standard curve. enzchek ® caspase- assay kit # , z-devd-amc substrate from invitrogen (grand island, ny, usa) was used to test caspase- activity, which was performed according to the manufacturer's instruction. cell viability was determined by trypan blue exclusion analysis (life technologies, waltham, ma, usa). to generate membrane and cytoplasmic lysates, subcellular protein fractionation kit for cultured cells was used (thermo scientific, rockford, il, usa). lysis of cells generated membrane and cytoplasmic protein extracts. the protocol was performed according to the manufacturer's instructions. normalized portions of each extract ( µg) were analyzed by western blotting using specific antibodies against proteins from various cellular compartments, including cytoplasmic (cytochrome c) and plasma membrane (caveolin- ) ( figure s ). proteins were isolated from the culture of jurkat cells with ripa buffer ( ˆpbs, % (v/v) np- , . % (w/v) sodium deoxycholate, . % (w/v) sds, . mg/ml pmsf, µl/ml aprotinin, mm sodium orthovanadate). equal amounts of protein were boiled in the loading buffer ( mm tris-hcl, mm dtt, % sds, . % bromphenol blue, % glycerol) and separated on sds-page and blotted onto polyvinylidene difluoride membranes. for immunoprecipitation (ip), µg of antibody was added to µg of total protein in µl, rotated for h at ˝c, then incubated with µl of protein a-sucrose beads (santa cruz biotechnology) for another h, spun down at ˆg, and washed three times with ripa buffer. the data represented are from three independent experiments. the unpaired student's t test was used for data analyses as indicated, and a value of p < . was considered very significant (**). it was reported that influenza virus infection could cause severe complications in hiv- -infected individuals leading to an increased risk of complications and death compared with uninfected individuals [ , ] . we found that ph n could infect and replicate in jurkat cells and primary cd + t cells ( figure s ). to examine whether ph n infection increased cell death due to hiv- infection, jurkat cells were infected with hiv- for three days, followed by incubation in the presence of ph n for another three days. as shown in figure a , ph n infection caused increased cell death in hiv- infected cells compared with uninfected cells (control). we also tested caspase- activities ( figure b ,c) and found that more caspase- was activated by ph n in hiv- infected cells than uninfected cells (control). we also obtained the similar results with using primary cd t cells incubated with hiv- for seven days and then infected with ph n for another three days ( figure s a ,b). the total cell lysates were subjected to test caspase- activities with enzchek®caspase- assay kit # -z-devd-amc substrate from invitrogen (grand island, ny, usa), which was performed according to the manufacturer's instruction; (c) the total cell lysates with ripa buffer were subjected to western blot analysis to detect caspase- .the unpaired student's t test was used for data analyses and a value of p < . was considered very significant (**) relative to control. these data indicated that ph n infection caused cell death through apoptotic signaling pathways in t cells. recently, we found that ph n is able to induce apoptotic cell death in a cells [ ] . to determine whether ph n can promote death through fas-mediated apoptotic pathway in jurkat cells, total cell lysates from cells infected with hiv- followed by infection with ph n for an additional three days were used for ip with anti-fas antibody and western blotting with anti-caspase- antibody to evaluate death-inducing signaling complex (disc) formation. as shown in figure a , more disc formation was found with the ph n /hiv- infection relative to hiv- infection or ph n alone. figure b showed that high level of expression of p and bax proteins was detected in jurkat cells infected with ph n infection and hiv- -infected cells, compared with ph n or hiv- alone. these data indicate that pandemic influenza a (h n ) infection can induce both fas-mediated and bax-mediated apoptotic pathways. a higher level of activation of both apoptotic signaling pathways was found in ph n infected jurkat cells after hiv- infection. recent reports indicate that ph n infection was associated with increase in mortality in patients with late and advanced hiv disease [ , ] and increased cell death was found in ph n -infected jurkat cells after hiv- infection ( figure a) . we determined whether ph n infection could influence hiv- replication. as shown in figure a , jurkat cells infected with hiv- and incubated with ph n for another three days, had higher levels of hiv- rna relative to cell with hiv- infection alone, suggesting that ph n infection increases hiv- replication in co-infected cells in jurkat cells and in cd + t cells ( figure s c ). it has been well-known that some host transcription factors are required for hiv replication, including nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb), nfat, ap- , which are up-regulated when t-cells become activated [ ] [ ] [ ] . they stimulate viral gene expression through transcription with the long terminal repeat (ltr) of hiv- [ ] . cells treated with ph n had higher level of nf-kb phosphorylation and increased protein expression of nfat and ap- ( figure b ) relative to hiv- infection alone, suggesting ph n infection can activate host transcription factors required for hiv- replication in jurkat cells. the mitogen-activated protein kinases (mapks) are central components of signal transduction pathways activated by diverse extracellular stimuli. several studies have shown that the mapk signal pathway can positively regulate replication of hiv- , and pathway inhibitors can block hiv- replication [ , ] . our results indicate that ph n infection promotes mapk pathway molecules, such as p , erk, and jnk phosphorylation ( figure c) . these data approve the conclusion that pandemic influenza a (h n ) infection increases hiv- replication in infected cells through activation of nf-kb, nfat, ap- , and mapk pathways that are necessary for hiv- replication. it is well known that cd is the receptor of hiv- [ , ] . hiv- gp binds to the cd molecule on the surface of host cells [ ] . since hiv- entry takes place in lipid rafts of the plasma membrane [ ] , we examined whether ph n infection affects cd expression on the plasma membrane in jurkat cells infected with hiv- and ph n . as shown in figure a the t cell activation and proliferation control hiv- replication and gene expression [ ] ; and activation of t cells through involvement of tcr, cd /cd , or cd , can trigger and activate downstream molecules, such as zap- and pkc , and which activate transcription factors, ap- nfat and nf-κb [ , ] , and increase hiv- gene expression and replication directed by the hiv- ltr [ ] . activation events of t cells require association with lipid rafts of the plasma membrane [ , ] . our results showed that ph n infection increased more zap- and pkc expression on plasma membranes relative to hiv- infection alone ( figure b,c) . these data indicate that pandemic influenza a (h n ) infection can increase accumulation of cd protein and induce t cell signaling and activate host transcription factors required for hiv- replication. hiv- infection is presently readily controlled with combination antiretroviral therapy. the persistence of hiv- in the face of potent antiretroviral drugs appears to be largely due to the ability of the virus to establish a state of latent infection in a long-lived population of resting memory cd + t cells [ ] . this latent reservoir is widely considered to be the major barrier to curing infection and is currently the subject of intense research [ ] . to determine whether ph n infection can reactivate hiv- latent infection, j . cells were infected with ph n for three or seven days days. as shown in figure a , measure with real-time pcr analysis showed that significant high level of hiv- rna production was detected in ph n infected cell relative to non-ph n infected controls. cell fractionation showed that ph n infection caused increased accumulation of cd and zap- proteins in plasma membrane in j . cells ( figure b ). western blotting analysis showed that ph n infection increased nfat expression and activated nf-kb signaling ( figure c ). ph n infection could activate both apoptotic pathways with an increased expression of pre-apoptotic proteins (such as fas, disc formation, fadd, and bax) and a decreased expression of anti-apoptotic proteins (such as flip and bcl- ) (figure ), suggesting that apoptotic pathways may be also involved in ph n -induced reactivation of hiv- replication. these data indicate that pandemic influenza a (h n ) infection is able to reactivate latent hiv- infection in vitro. during the h n influenza a virus pandemic, it was reported that some patients developed severe pneumonia leading to acute respiratory distress syndrome (ards) and multiple organ dysfunction, associated with high ( %- %) mortality [ , ] . however, there is little information available on the direct interaction between influenza virus and the host immune system that pertains to severe disease outcomes. it has been reported that pathogenesis due to influenza infections was associated with alterations in the lymphohematopoietic system, leading to the destruction of lymphocytes and histopathological necrosis of lymphoid tissues [ , ] . there is little information available on whether and how influenza infection could damage the immune system directly and alter its ability to control other infectious pathogens that ultimately are detrimental to the host. our study showed that pandemic influenza a (h n ) virus infection is able to enhance cell death in hiv- -infected cells (figure ) . hiv- infection may be associated with a worse prognosis of influenza and adults with aids experience substantially elevated influenza-associated mortality, which has declined with widespread introduction of haart (highly-active anti-retroviral therapy), but not completely eliminated [ ] . in hiv patients, well controlled on haart, pandemic influenza a (h n ) virus infection has resulted in a similar clinical outcome and prognosis to that of non-hiv patients [ ] and oseltamivir treatment has been known to shorten the duration of influenza viral shedding in children [ ] and adults [ , ] . cd cell count and plasma hiv- rna did not differ before and - weeks after influenza a h n diagnosis in the presence of oseltamivir treatment in the hiv-positive group [ ] . however, hiv- -infected subjects that had progressed to immune deficiency presented an increased mortality by ph n [ ] . this information suggests that hiv- activation, induced by influenza virus infection, can be controlled by oseltamivir in "normal" hiv-positive persons on haart treatment, and may not be controlled in hiv-infected patients under illness condition [ ] . influenza a virus induces apoptotic death in infected epithelial, lymphocyte, and phagocytic cells [ , , , ] , and apoptosis is a source of tissue damage during infection [ ] . distal lung epithelial cell apoptosis and apoptosis-resulting in necrosis were described as a classical feature of h n -or pandemic h n -induced ards [ ] . epithelial cell apoptosis is generally seen as a major underlying cause of diffuse alveolar damage in many forms of ards [ ] . so far, it has been clear that influenza infection can affect both intrinsic and extrinsic apoptotic pathways [ ] . it was also reported that after influenza virus infection, some cd + cells in the spleen and thymus may express viral proteins [ ] , and a population of t cells is susceptible to influenza a virus in infected mice [ , ] . influenza virus infection has been shown to activate tcr-signaling pathways in jurkat cells [ ] and pandemic influenza a (h n ) virus infection causes apoptotic death in hiv- -infected jurkat cells (figures and b,c) . previously, we reported that pre-apoptotic molecules from apoptotic pathways increase hiv- replication, while anti-apoptotic proteins inhibit viral rna yield [ ] . consistent with our previous observation, pandemic influenza a (h n ) virus infection can increase hiv- replication with up-regulation of pro-apoptotic molecules, down-regulation of anti-apoptotic proteins, and with an increased activation of t cells through tcr-related signaling pathways ( figures c, and ) required for t-cell activation and hiv- replication; however the detailed mechanisms by which pandemic influenza a (h n ) virus infection induces hiv- replication needs further study. in conclusion, our results demonstrate that pandemic influenza a (h n ) virus 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eradication and cure pneumonia and respiratory failure from swine-origin influenza a (h n ) in mexico critically ill patients with influenza a(h n ) in mexico apoptosis: a mechanism of cell killing by influenza a and b viruses depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza a (h n ) virus isolated from humans clinical presentation and prognosis of the h n influenza a infection in hiv- -infected patients: a spanish multicenter study pandemic influenza a (h n ) outbreak among school-aged hiv- -infected children acute respiratory distress syndrome due to influenza virus a/h n v in a patient with hiv/hcv co-infection influenza a h n in hiv-infected adults aryl hydrocarbon receptor activation during influenza virus infection unveils a novel pathway of ifn-gamma production by phagocytic cells early upregulation of acute respiratory distress syndrome-associated cytokines promotes lethal disease in an aged-mouse model of severe acute respiratory syndrome coronavirus infection influenza h n and h n virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation role of itk signalling in the interaction between influenza a virus and t-cells antigen signal strength during priming determines effector cd t cell function and antigen sensitivity during influenza virus challenge the authors wish to acknowledge of mohan haleyurgirisetty and viswanath ragupathy for critical review of this manuscript, and acknowledge of the nih aids reagent repository for the reagents. this work was supported by an internal cber modernizing science grant. the findings and conclusions in this article have not been formally disseminated by the food and drug administration and should not be construed to represent any agency determination or policy.author contributions: x.w. and i.h. conceived and designed the experiments; x.w., j.t., s.b., j.z., and k.d. performed the experiments; and x.w. and z.y. analyzed the data, and x.w. and i.h. wrote the paper. all authors have read and approved the final manuscript. the authors declare no conflict of interest. key: cord- -f v hhr authors: hung, ting-chun; jassey, alagie; lin, chien-ju; liu, ching-hsuan; lin, chun-ching; yen, ming-hong; lin, liang-tzung title: methanolic extract of rhizoma coptidis inhibits the early viral entry steps of hepatitis c virus infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: f v hhr hepatitis c virus (hcv) remains an important public health threat with approximately million carriers worldwide who are at risk of developing hepatitis c-associated end-stage liver diseases. despite improvement of hcv treatment using the novel direct-acting antivirals (daas) targeting viral replication, there is a lack of prophylactic measures for protection against hcv infection. identifying novel antivirals such as those that target viral entry could help broaden the therapeutic arsenal against hcv. herein, we investigated the anti-hcv activity of the methanolic extract from rhizoma coptidis (rc), a widely used traditional chinese medicine documented by the who and experimentally reported to possess several pharmacological functions including antiviral effects. using the cell culture-derived hcv system, we demonstrated that rc dose-dependently inhibited hcv infection of huh- . cells at non-cytotoxic concentrations. in particular, rc blocked hcv attachment and entry/fusion into the host cells without exerting any significant effect on the cell-free viral particles or modulating key host cell entry factors to hcv. moreover, rc robustly suppressed hcv pseudoparticles infection of huh- . cells and impeded infection by several hcv genotypes. collectively, our results identified rc as a potent antagonist to hcv entry with potential pan-genotypic properties, which deserves further evaluation for use as an anti-hcv agent. hepatitis c virus (hcv) is an important liver pathogen belonging to the flaviviridae family with an enveloped positive single-stranded rna genome. hcv has seven genotypes (genotype ~ ) and a genome size of about . kb which encodes a polyprotein that is approximately amino acids long. the polyprotein upon translation is processed by viral and host proteases to yield matured protein including structural proteins, core, e , e , and p ion channel, as well as non-structural proteins ns , ns , ns a, ns b, ns a, and ns b [ ] . hcv entry into the host hepatocytes is mediated by interaction basal media for all viral infection analyses consisted of dulbecco's modified eagle's medium (gibco-invitrogen, carlsbad, ca, usa) containing % fetal bovine serum. rhizoma coptidis roots from coptis chinensis franch (id#kew- from the plant list [ ] ) were obtained from local pharmacy store (kaohsiung, taiwan) and authenticated by dr. ming-hong yen using anatomical methods as well as by hplc analysis through comparison to known molecular standards as previously described [ , ] . a voucher specimen was deposited at the kaohsiung medical university herbarium (ctm-rcc ). for methanol extraction [ ] , the roots were washed, dried, and homogenized before extraction with % methanol, followed by concentration in vacuo. the methanolic rc stock was dissolved in dimethyl sulfoxide (dmso; sigma, st. louis, mo, usa) prior to use. huh- . cells seeded at × cells/well in -well plates overnight were treated with increasing concentrations of rc for days. the cells were then washed twice with phosphate buffered saline (pbs) before xtt cell viability analysis as previously described [ ] . for examining antiviral activity, huh- . cells ( × cells/well in -well plates) were concurrently treated with the virus (moi = . ) and the test drug at various concentrations before incubation at • c for days. the anti-hcv activity was determined by measuring the luciferase reporter signals using the biolux™ gaussia luciferase assay kit (new england biolabs; pickering, on, canada) and a luminometer (promega; madison, wi, usa) as previously reported [ ] . data were expressed as percent (%) hcv infectivity from test treatments relative to medium control (virus only). ifn-α (sigma) served as positive control. the time-of-drug-addition assay which provides information on the target of test agents in the viral life cycle consisted of pre-treatment, co-addition treatment, and post-infection treatment, and was performed as previously described [ ] . for all analyses, huh- . cells were seeded in -well plates at a density of × cells/well overnight and infection with hcvcc was carried out at moi = . . luciferase activity for all conditions was determined as described earlier following h of incubation at • c. the synchronized infection analysis to determine the effect of test agents on early viral entry was performed as previously described [ ] . to examine viral inactivation, the test drug was incubated with the cell-free virus particles prior to diluting the virus-drug mixture to a subtherapeutic concentration and infecting the host cells (final hcvcc moi = . ). to investigate the influence on viral attachment, pre-chilled huh- . cells were challenged with the hcvcc (moi = . ) in the presence/absence of the test agent at • c, which allows binding of the viral particles to the host cells while precluding entry. to test for impact on viral entry/fusion, huh- . cells were pre-bound with hcvcc (moi = . ) at • c and then shifted to • c incubation in the presence/absence of the test drug. for all the above analyses, huh- . cells were seeded in -well plates ( × cells/well) and luciferase activity for all conditions was determined as described earlier following h of incubation at • c. the enzyme-linked immunosorbent assay (elisa)-based binding assay was performed as previously described [ ] . briefly, pre-chilled huh- . cells were challenged with hcvcc in the presence/absence of test drug at • c for h before washing with pbs and fixing the cells with % paraformaldehyde. cell-bound virus was detected using primary anti-hcv e antibody ( : ; austral biological, san ramon, ca, usa) and secondary goat anti-mouse igg conjugated with horseradish peroxidase antibody ( : , invitrogen), followed by assessment with tmb ( , ', , '-tetramethylbenzidine) two-component microwell peroxidase substrate kit (kpl; gaithersburg, md, usa) and absorbance reading at nm with a elx microplate reader (instrument, inc. ; winooski, vt, usa). retroviral pseudoparticles bearing hcv glycoproteins e /e were produced following a previously described method [ ] with some modifications. a pcdna . plasmid vector (invitrogen) containing complete hcv core and e /e glycoproteins of the hc-j ch strain (genotype a; nc_ ) was co-transfected in conjunction with the pnl.luc.env − r + construct (env-defective retroviral backbone encoding firefly luciferase; kindly provided by dr. Éric a. cohen) into t cells using omnifect™ (transomic technologies inc.; huntsville, al, usa). supernatant containing the viral pseudoparticles was harvested and filtered ( . µm) before being concentrated using % (v/v) polyethylene glycol and stored at − • c before use. for the infectivity experiment, the hcv pseudoparticles (hcvpp) were used to inoculate huh- . cell monolayers in -well plates in the presence or absence of the test agent for h at • c. the cells are then washed with pbs and further incubated in basal media for h before being harvested and assessed for reporter luciferase activity using the firefly luciferase assay kit (promega) and a luminometer. viral infectivity was calculated as percent (%) relative light units (rlu) compared to control (virus only). antiviral activity against recombinant hcv carrying glycoproteins from genotype b (j /jfh ), a (s /jfh ), and a (qc /jfh ) viruses (kindly provided by dr. jens bukh) [ , ] was performed using a previously reported method [ ] with some modifications. huh- . cells ( × cells/well in -well plates) were challenged with the viruses at moi = . in the presence/absence of the test agent. after days of further incubation, detection of viral infectivity was performed by immunofluorescence staining of hcv foci using primary mouse anti-core clone b antibody ( : ; anogen; mississauga, on, canada), which cross-reacts different hcv core antigenic determinants, and a secondary alexa fluor goat anti-mouse igg (h+l) ( : ; invitrogen) [ ] . hcv-positive foci were quantitated and results were plotted against the dmso control [ ] . statistical analysis was conducted using one-way analysis of variance (anova) followed by tukey's multiple comparison test or unpaired t test. a p value of less than . (p < . ) was considered to be statistically significant. all data are expressed as means ± standard error of the means (sem) from three independent experiments. given the numerous pharmacological properties of rc, we hypothesized that the medicinal herb could potentially possess antiviral activities against hcv. to explore this possibility, huh- . cells were infected with gaussia luciferase reporter-tagged hcvcc in the presence of different concentrations of the methanolic extract of rc and the luciferase activity was subsequently assessed to determine viral infectivity. a cytotoxicity assay was simultaneously performed on the cells with the same drug concentrations. our results showed that rc could inhibit hcv infection dose-dependently and up to µg/ml without inducing significant cytotoxicity ( figure a ). the % cytotoxic concentration (cc ), the % effective concentration (ec ), and the selective index (si = cc /ec ) were determined to be . ± . µg/ml, . ± . µg/ml, and . , respectively ( figure b ). since the antiviral efficacy of rc appeared to be comparable between and µg/ml, a concentration of µg/ml was chosen for the remainder of our experiments. given the numerous pharmacological properties of rc, we hypothesized that the medicinal herb could potentially possess antiviral activities against hcv. to explore this possibility, huh- . cells were infected with gaussia luciferase reporter-tagged hcvcc in the presence of different concentrations of the methanolic extract of rc and the luciferase activity was subsequently assessed to determine viral infectivity. a cytotoxicity assay was simultaneously performed on the cells with the same drug concentrations. our results showed that rc could inhibit hcv infection dosedependently and up to μg/ml without inducing significant cytotoxicity ( figure a ). the % cytotoxic concentration (cc ), the % effective concentration (ec ), and the selective index (si = cc /ec ) were determined to be .  . μg/ml, . ± . μg/ml, and . , respectively ( figure b ). since the antiviral efficacy of rc appeared to be comparable between and μg/ml, a concentration of μg/ml was chosen for the remainder of our experiments. in order to narrow down the window of antiviral activity from rc, we performed a time-ofdrug-addition assay wherein the drug was either added to cells h before hcvcc infection ("pretreatment"), concurrently added at the time of viral infection ("co-addition"), or added after infection ("post-infection") and incubated for days before measuring the luciferase reporter activity. results indicated no significant difference in hcv infectivity in the presence or absence of rc during pretreatment (figure ) , suggesting that the drug does not appear to modulate the host cells before hcv infection. in contrast, rc significantly inhibited hcv infectivity when simultaneously added during the viral challenge as indicated by the substantial drop in the luciferase signal (figure ) . only a moderate decrease in viral infectivity was observed when the drug was added after the establishment of viral infection. in contrast, ifn-α, which served as positive control, effectively inhibited the hcvcc infection in all types of treatment. thus, rc's anti-hcv activity appeared in order to narrow down the window of antiviral activity from rc, we performed a time-of-drug-addition assay wherein the drug was either added to cells h before hcvcc infection ("pre-treatment"), concurrently added at the time of viral infection ("co-addition"), or added after infection ("post-infection") and incubated for days before measuring the luciferase reporter activity. results indicated no significant difference in hcv infectivity in the presence or absence of rc during pretreatment (figure ) , suggesting that the drug does not appear to modulate the host cells before hcv infection. in contrast, rc significantly inhibited hcv infectivity when simultaneously added during the viral challenge as indicated by the substantial drop in the luciferase signal (figure ) . only a moderate decrease in viral infectivity was observed when the drug was added after the establishment of viral infection. in contrast, ifn-α, which served as positive control, effectively inhibited the hcvcc infection in all types of treatment. thus, rc's anti-hcv activity appeared strongest when concurrently present on the host cell with the viral particles, suggesting that its inhibitory effect mainly targeted the early phase of the hcv infection, including viral entry. viruses , , x for peer review of strongest when concurrently present on the host cell with the viral particles, suggesting that its inhibitory effect mainly targeted the early phase of the hcv infection, including viral entry. to further characterize the mechanism(s) underlying rc's anti-hcv effect, which was strongest when rc was simultaneously present with the virus on the host cell surface, we performed a synchronized infection assay on early viral entry. to test whether rc could inactivate the cell-free viral particles, the drug was pre-incubated with the hcvcc for h before dilution and infecting huh- . cells. the drug-virus mixture was then diluted x to yield a non-effective concentration which was subsequently added to the cells and incubated for days. luciferase readings following days of incubation showed no significant difference between samples treated with or without rc, suggesting that the drug does not impact the free viral particles ( figure a ). to determine the effect of rc on viral attachment, we specifically added rc during hcv cell binding at °c, which allows for virus binding but precludes viral internalization, and then tested the reporter readout at the end of the incubation. results demonstrated a significant decrease in the luciferase reporter activity, which indicated that rc interfered with the attachment of the virus to the host cells ( figure a ). to ascertain whether rc influenced the post-attachment viral entry/fusion step, huh- . cells were prebound with hcv at °c before shifting the temperature to °c in the presence of rc. similar to the effect observed on viral attachment, our data revealed a significant decrease in hcv infectivity at the end of the incubation, suggesting that rc equally blocked hcv entry/fusion ( figure a ). to further characterize the mechanism(s) underlying rc's anti-hcv effect, which was strongest when rc was simultaneously present with the virus on the host cell surface, we performed a synchronized infection assay on early viral entry. to test whether rc could inactivate the cell-free viral particles, the drug was pre-incubated with the hcvcc for h before dilution and infecting huh- . cells. the drug-virus mixture was then diluted x to yield a non-effective concentration which was subsequently added to the cells and incubated for days. luciferase readings following days of incubation showed no significant difference between samples treated with or without rc, suggesting that the drug does not impact the free viral particles ( figure a ). to determine the effect of rc on viral attachment, we specifically added rc during hcv cell binding at • c, which allows for virus binding but precludes viral internalization, and then tested the reporter readout at the end of the incubation. results demonstrated a significant decrease in the luciferase reporter activity, which indicated that rc interfered with the attachment of the virus to the host cells ( figure a ). to ascertain whether rc influenced the post-attachment viral entry/fusion step, huh- . cells were pre-bound with hcv at • c before shifting the temperature to • c in the presence of rc. similar to the effect observed on viral attachment, our data revealed a significant decrease in hcv infectivity at the end of the incubation, suggesting that rc equally blocked hcv entry/fusion ( figure a) . to directly validate our finding that rc inhibits hcv attachment to the host cells, we employed an elisa-based binding assay. huh- . cells were inoculated with hcvcc in the presence of rc at • c for h, after which the cells were washed, fixed, and subjected to colorimetric analysis by detecting cell surface-bound hcv particles using anti-hcv e antibody. as depicted in figure b , rc treatment dose-dependently decreased viral attachment as demonstrated by the sharp decline in the absorbance signal, which is in agreement with the above results. hcv entry steps are mediated by viral glycoproteins [ ] . based on the inhibitory effects observed from rc against hcv entry steps, and to validate its antiviral potency against hcv entry, we further examined the impact of rc treatment on infection by retroviral pseudoparticles bearing hcv glycoproteins e /e (hcvpp). specifically, luciferase-tagged hcvpp were used to infect huh- . cells in the presence or absence of rc, before further incubating the cells and assessing luciferase reporter activity as a readout of viral infectivity. as demonstrated in figure c , rc robustly suppressed hcvpp infection of the huh- . hepatoma cells compared to the dmso control. this result therefore confirms and validates the antiviral capacity of rc against the viral entry stage of hcv. hcv infection of hepatocytes is a well-orchestrated process involving the engagement of several host factors including cd , sr-bi, cldn- , and ocln. in addition, apoe has also been reported to play a role in mediating hcv entry [ ] . given that rc treatment blocked hcv infection mainly by targeting viral entry, we next asked whether the drug could modulate the expression of key host cells entry factors. to this end, huh- . cells were pre-treated with rc for h before harvesting the cell lysates for western blot analysis. as depicted in figure d , pre-treatment of huh- . cells with rc did not alter the expression of cd , sr-bi, cldn- , and ocln. similarly, the expression of apoe was not affected by rc treatment ( figure d) . apob, which participates in hcv virion release was included for comparison and was neither affected by the drug treatment. these results therefore suggested that rc did not inhibit hcv infection via influencing the host cells and is consistent with our data from the pre-treatment analysis (figure ). since rc appeared to primarily target hcv entry, we finally sought to examine whether the medicinal herb also possesses a pan-genotypic activity against hcv. for this purpose, huh- . cells were seeded and infected with recombinant hcvcc expressing glycoproteins from genotypes b (j /jfh ), a (s /jfh ), and a (qc /jfh ) in the presence of the rc before further incubation and analysis of hcv infectivity by immunofluorescence staining. results showed a significant decrease in viral infection across all the tested hcv genotypes when rc was present, suggesting that it may possess a pan-genotypic activity against hcv (figure ) . continuous identification of novel antivirals with various modes of action is important given that development of drug resistance is commonplace especially with viruses that exhibit genetic variability, including hcv. in this study, we demonstrated for the first time that the methanolic extract of rc robustly inhibited hcv infection. specifically, rc mainly targeted the hcv early viral entry steps such as attachment and entry/fusion to the host cells. interestingly, rc also inhibited hcv infection of several other genotypes. our results therefore identified rc as a promising antagonist with pan-genotypic function against hcv entry, which could be useful for developing hcv prophylaxis. currently no approved therapeutic treatment exists for targeting hcv entry, and patients with chronic hepatitis c are at risk for end-stage liver diseases such as cirrhosis and liver cancer, which necessitate liver transplantation. importantly, donor livers inadvertently become re-infected almost immediately after transplantation in hepatitis c patients [ ] . given that the daas in current use cannot prevent liver graft re-infection and have the propensity to select for drug-resistant mutants, combining entry inhibitors with the daas would be expected to broaden the treatment strategies against hepatitis c especially in the liver transplant setting. interestingly, previous studies have demonstrated that combining daas and virus entry inhibitors can produce a synergistic effect to improve drug efficacy [ ] . our discovery that rc can antagonize hcv entry makes it an ideal candidate for use in liver transplant scenarios and to test in combination with the daas for better therapeutic efficacy. natural resources such as plant materials serve as excellent starting point for antiviral discovery [ ] . rc's anti-hcv bioactivity identifies the medicinal herb as an important antiviral source for the treatment of hepatitis c. various medicinal plant extracts have been shown to possess anti-hcv properties and thereafter served as source for further identifying small molecule inhibitors. examples of those that specifically target hcv entry include silibinin and silymarin from silybum marianum [ , ] , gallic acid found in limonium sinense [ ] , loliolide and the butenolide ( r, s)- dihydromenisdaurilide derived from phyllanthus urinaria [ , ] , ladanein isolated from marrubium peregrinum l. (lamiaceae) [ ] , and bupleurum kaoi and its terpenoid saikosaponin b [ ] . while the molecular constituents in rc contributing to its anti-hcv activity remain to be identified, the alkaloids are potential candidate bioactives which are known to be the major components of rc and are typically present in the alcoholic extracts of the herb [ ] . examples include berberine, coptisine, palmatine, epiberberine, columbamine, jatrorrhizine, and groenlandicine, among other major compounds [ ] . whether a single compound or a combination of them contributes to rc's antiviral actions against hcv remains to be explored. further in-depth analysis would be required to fully elucidate the bioactive ingredients responsible for rc's anti-hcv effect. our results indicated that rc specifically blocked hcv attachment and entry/fusion into the host cells without significantly influencing the cell-free viral particles or modulating key host cell continuous identification of novel antivirals with various modes of action is important given that development of drug resistance is commonplace especially with viruses that exhibit genetic variability, including hcv. in this study, we demonstrated for the first time that the methanolic extract of rc robustly inhibited hcv infection. specifically, rc mainly targeted the hcv early viral entry steps such as attachment and entry/fusion to the host cells. interestingly, rc also inhibited hcv infection of several other genotypes. our results therefore identified rc as a promising antagonist with pan-genotypic function against hcv entry, which could be useful for developing hcv prophylaxis. currently no approved therapeutic treatment exists for targeting hcv entry, and patients with chronic hepatitis c are at risk for end-stage liver diseases such as cirrhosis and liver cancer, which necessitate liver transplantation. importantly, donor livers inadvertently become re-infected almost immediately after transplantation in hepatitis c patients [ ] . given that the daas in current use cannot prevent liver graft re-infection and have the propensity to select for drug-resistant mutants, combining entry inhibitors with the daas would be expected to broaden the treatment strategies against hepatitis c especially in the liver transplant setting. interestingly, previous studies have demonstrated that combining daas and virus entry inhibitors can produce a synergistic effect to improve drug efficacy [ ] . our discovery that rc can antagonize hcv entry makes it an ideal candidate for use in liver transplant scenarios and to test in combination with the daas for better therapeutic efficacy. natural resources such as plant materials serve as excellent starting point for antiviral discovery [ ] . rc's anti-hcv bioactivity identifies the medicinal herb as an important antiviral source for the treatment of hepatitis c. various medicinal plant extracts have been shown to possess anti-hcv properties and thereafter served as source for further identifying small molecule inhibitors. examples of those that specifically target hcv entry include silibinin and silymarin from silybum marianum [ , ] , gallic acid found in limonium sinense [ ] , loliolide and the butenolide ( r, s)- -dihydromenisdaurilide derived from phyllanthus urinaria [ , ] , ladanein isolated from marrubium peregrinum l. (lamiaceae) [ ] , and bupleurum kaoi and its terpenoid saikosaponin b [ ] . while the molecular constituents in rc contributing to its anti-hcv activity remain to be identified, the alkaloids are potential candidate bioactives which are known to be the major components of rc and are typically present in the alcoholic extracts of the herb [ ] . examples include berberine, coptisine, palmatine, epiberberine, columbamine, jatrorrhizine, and groenlandicine, among other major compounds [ ] . whether a single compound or a combination of them contributes to rc's antiviral actions against hcv remains to be explored. further in-depth analysis would be required to fully elucidate the bioactive ingredients responsible for rc's anti-hcv effect. our results indicated that rc specifically blocked hcv attachment and entry/fusion into the host cells without significantly influencing the cell-free viral particles or modulating key host cell entry factors to hcv. the ability of rc to inhibit hcvpp infection suggests that its mechanistic target(s) may involve the hcv glycoproteins-mediated interactions with the host cell. a potential mechanism is the transient interaction(s) with the hcv glycoproteins that are inadequate to inactivate free viral particles but sufficiently effective to hinder contact with cell surface receptors/co-receptors. additional mechanisms of action could also include concentration-dependent reversible conformational alterations in receptors/co-receptors or viral particles' structure to block viral entry. we also noted a moderate effect from rc in the post-infection stage (figure ) , albeit less pronounced compared to its impact in inhibiting hcv entry steps. this effect was not entirely due to inhibition of entry in subsequent de novo infection cycles, since treatment of subgenomic hcv replicon cells with rc also showed a moderate decrease in viral rna in a preliminary experiment ( figure s ). it is possible that rc may possess multiple mechanisms against hcv infection, which would necessitate further studies for clarification. nonetheless, our results clearly demonstrated that rc most potently inhibited hcv entry. therefore, we suggest that rc could be further explored for prophylactic/therapeutic management of hepatitis c. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / / s , figure s : effect of rc treatment on hcv subgenomic replicon cells. replication of hepatitis c virus the mechanism of hcv entry into host cells strategies to preclude hepatitis c virus entry roles of lipoproteins and apolipoproteins in particle formation of hepatitis c virus neglected but important role of apolipoprotein e exchange in hepatitis c virus infection antiviral treatment of hepatitis c drug interactions with new hepatitis c oral drugs ledipasvir/sofosbuvir (harvoni): improving options for hepatitis c virus infection hepatitis c virus treatment in the real world: optimising treatment and access to therapies coptidis rhizoma and its main bioactive components: recent advances in chemical investigation, quality evaluation and pharmacological activity english version; ministry of health and welfare anti-inflammatory and antimicrobial effects of heat-clearing chinese herbs: a current review anti-herpes simplex virus effects of berberine from coptidis rhizoma, a major component of a chinese herbal medicine in vitro inhibition of coronavirus replications by the traditionally used medicinal herbal extracts, cimicifuga rhizoma, meliae cortex, coptidis rhizoma, and phellodendron cortex coptidis rhizoma extract inhibits replication of respiratory syncytial virus in vitro and in vivo by inducing antiviral state broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry the plant list-version . characterization of protoberberine alkaloids in coptidis rhizoma (huanglian) by hplc with esi-ms/ms application of analytical and preparative high-speed counter-current chromatography for separation of alkaloids from coptis chinensis franch hydrolyzable tannins (chebulagic acid and punicalagin) target viral glycoprotein-glycosaminoglycan interactions to inhibit herpes simplex virus entry and cell-to-cell spread limonium sinense and gallic acid suppress hepatitis c virus infection by blocking early viral entry saikosaponin b is a naturally occurring terpenoid that efficiently inhibits hepatitis c virus entry studying hcv cell entry with hcv pseudoparticles (hcvpp) robust hepatitis c genotype a cell culture releasing adapted intergenotypic a/ a (s /jfh ) viruses development and characterization of hepatitis c virus genotype - cell culture systems: role of cd and scavenger receptor class b type i and effect of antiviral drugs highly bioavailable silibinin nanoparticles inhibit hcv infection activity-based and fraction-guided analysis of phyllanthus urinaria identifies loliolide as a potent inhibitor of hepatitis c virus entry hepatitis c and liver transplantation: enhancing outcomes and should patients be retransplanted synergy of entry inhibitors with direct-acting antivirals uncovers novel combinations for prevention and treatment of hepatitis c antiviral natural products and herbal medicines multiple effects of silymarin on the hepatitis c virus lifecycle silibinin inhibits hepatitis c virus entry into hepatocytes by hindering clathrin-dependent trafficking a plant-derived flavonoid inhibits entry of all hcv genotypes into human hepatocytes the authors would like to thank charles m. rice, jens bukh, and Éric a. cohen for reagents, christopher d. richardson for experimental support for the antiviral assay against the various hcv genotypes, and shun-pang chang and chueh-yao chung for technical assistance. the authors declare no conflicts of interest. key: cord- - o viv authors: rahe, michael c.; murtaugh, michael p. title: mechanisms of adaptive immunity to porcine reproductive and respiratory syndrome virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: o viv the adaptive immune response is necessary for the development of protective immunity against infectious diseases. porcine reproductive and respiratory syndrome virus (prrsv), a genetically heterogeneous and rapidly evolving rna virus, is the most burdensome pathogen of swine health and wellbeing worldwide. viral infection induces antigen-specific immunity that ultimately clears the infection. however, the resulting immune memory, induced by virulent or attenuated vaccine viruses, is inconsistently protective against diverse viral strains. the immunological mechanisms by which primary and memory protection are generated and used are not well understood. here, we summarize current knowledge regarding cellular and humoral components of the adaptive immune response to prrsv infection that mediate primary and memory immune protection against viruses. porcine reproductive and respiratory syndrome virus (prrsv) is the most severe enemy of porcine health and wellbeing. the highly mutable, enveloped, rna virus was discovered nearly years ago but, while extensive research has been carried out and many vaccines have been developed, there is still no reproducible immunological intervention that develops a broadly protective immune response against virulent prrsv. prrs disease was first described on farms in north carolina in the usa at the end of the s. outbreaks were marked by reproductive losses, post-weaning pneumonia, and increased mortality in growing pigs. initial efforts to identify an etiological agent responsible for the new disease syndrome were unsuccessful, leading to the disease being temporarily designated mystery swine disease (msd) in north america. koch's postulates for msd were fulfilled in with a previously unidentified rna virus discovered in europe, named lelystad virus [ , ] . the discovery was quickly followed by isolation of the virus, initially referred to as swine infertility and respiratory syndrome virus or sirs virus, in north america [ ] . the name prrsv was introduced in and encompasses prrsv- (genotypes first isolated in europe) and prrsv- (genotypes first isolated in north america) [ , ] . today, both virus types are globally distributed, with prrsv- viruses predominantly in europe and prrsv- viruses largely in north america, asia and south america [ ] . recent discovery of multiple arteriviral nucleotide sequences in nonhuman primates has led to a reclassification of prrsv as two distinct viruses, prrsv- and prrsv- [ ] . here, we use the generic prrsv to refer broadly to both viruses when evidence indicates that are equivalent, and the specific prrsv- and prrsv- is used when a distinction is desired. the reasoning is based on the many similarities of the two viruses in fine details of genome structure and organization, transcriptional strategy, host preference, clinical signs of disease, and prrsv infects cells of the macrophage/monocyte lineage, including dendritic cells [ ] [ ] [ ] [ ] . permissive cells express cluster of differentiation (cd) , a hemoglobin-haptoglobin scavenger, which is the necessary receptor for prrsv infection and replication [ ] [ ] [ ] . macrophages and dendritic cells are common members of the mononuclear phagocyte system that plays a varied, and important, role in many aspects of tissue remodeling, development, immunity and immunopathology [ ] . classically designated as part of the innate immune system, these leukocytes are critical for the development of a productive adaptive immune response. macrophages and, particularly, dendritic cells take up and present antigen to t cells and b cells, thus initiating an adaptive immune response against the presented antigen [ , ]. if a pathogen is able to infect and destroy, manipulate, or maintain itself within macrophages or dendritic cells, it then has the potential to modulate the immune response into a favorable situation for its own replication and survival. therefore, many pathogens employ strategies for macrophage infection as a way to make the host more amenable to infection. recent research into mycobacterium tuberculosis (mtb) has shown that, after phagocytosis, the bacterium arrests phagosome maturation and intra-phagosome lipolysis resulting in mtb survival and an increased supply of nutrients for growth [ , ] . human immunodeficiency virus (hiv) infects macrophages to establish reservoirs within the host for the chronic stage of the disease when cd + t cells are largely depleted and neutralizing antibodies may be present [ ] [ ] [ ] . leishmania major is a protozoan which infects phagocytes to subvert the immune system. the parasite expresses glycoprotein (gp) , a multifaceted surface-expressed pathogenicity factor that is responsible for preventing antigen presentation and killing by natural killer (nk) cells [ ] [ ] [ ] . indeed, there are many more examples of burdensome pathogens which target phagocytic cells, especially macrophages and dendritic cells, in an attempt to gain a foothold within the immune system and allow for unchecked survival and replication [ ] [ ] [ ] . prrsv is one of these pathogens. the ability of prrsv to subvert the immune system has not been investigated as extensively as more prominent pathogens of humans, such as hiv. prrsv has been shown to inhibit the production, or the downstream effects, of type interferons, particularly interferon (ifn)-α, on intracellular signaling [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . interestingly, multiple prrsv proteins (nonstructural protein (nsp) , nsp , nsp , nsp , nsp and nucleocapsid) have been reported to possess interferon inhibiting abilities. in addition, a number of in vivo experiments have reproduced earlier in vitro findings showing that interferon-α is inhibited during the early stages of prrsv infection [ , , ] . while the impact of type interferon suppression is likely to create a favorable environment for the virus to replicate and survive in phagocytic cells, it is still unclear what effect, if any, suppression of type interferon activity has on the adaptive immune response to infection [ ] . future investigations could clarify the relative contributions of viral proteins on modulation of interferon production and their impacts on viral growth, survival, and the subsequent development of the adaptive immune response. apart from interfering with interferon expression, prrsv has also displayed the in vitro ability to subvert the immune system by spreading from cell to cell. recent work has uncovered the ability of the virus to spread infectious viral rna, several replicases, and certain structural proteins between cells via intercellular nanotubules [ , ] . while this activity theoretically allows for prrsv to avoid neutralizing antibodies, the presence and significance of this mechanism in prrsv pathogenesis has yet to be fully elucidated. future studies are needed to determine if this process operates in naturally permissive macrophages and dendritic cells, if it can be interrupted, for example by intracellular antibodies, and what effect it might have on viral propagation [ , ] . vaccines depend upon innate immune stimulation to promote effective adaptive immune response to antigen, resulting in production of antibodies and cytotoxic t cell responses. the ability of a pathogen to successfully infect and replicate within innate immune cells makes the development of a protective immune response more difficult. as a result, the generation of effective vaccines against pathogens that target immune cells is fraught with challenges. extensive variation in viral genetics, primary immune responses, and cross-protection indicates that much remains to be learned about cellular pathogenesis in order to arrive at better immunological solutions. immunosuppression refers to suppression of the immune system and its ability to fight infection. hiv and infectious bursal disease virus are examples of viral infections that destroy entire lymphoid cell populations that ablate or disable adaptive immune responses. lymphoproliferative cancers block cellular differentiation and deprive the body of mature, effector lymphocytes, thus causing immunosuppression in a different manner. prrsv does neither; infection does not lead to severe lymphoid depletion or ablation, and it does not interfere profoundly with lymphocyte differentiation or maturation. leukocyte perturbations in lymphoid tissues are associated with prrsv infection, suggesting that adaptive immunity might be weakened, though not destroyed [ ] [ ] [ ] [ ] [ ] [ ] . the immune system also maintains peripheral tolerance to self and commensal bacteria through immunosuppressive mechanisms that include regulatory t cells (tregs), characterized as cd + cd + forkhead box p (foxp ) + t lymphocytes [ ] . treg suppressive properties were discovered when thymectomized or treg-depleted mice succumbed to autoimmune reactions [ , ] . tregs suppress effector and effector memory t cell proliferation by cytokine deprivation leading to polyclonal apoptosis, and by suppression of antigen presenting cells by cytotoxic t lymphocyte-associated antigen- (ctla- ) and other mechanisms [ ] . studies in prrsv infections give an ambiguous picture about the role of tregs. prrsv- strains are reported to induce a strong treg response which included transforming growth factor (tgf)β- secretion in vitro as well as in vivo [ , ] . other studies did not show treg responses to infection with either prrsv- or prrsv- [ , ] . interleukin- (il- ), an immunosuppressive cytokine expressed by various cell types including tregs, was induced by prrsv- vaccination in weaned pigs in one study, but was not induced in weaned or adult pigs in another study [ ] . additional in vitro and in vivo studies reported il- mrna transcription and cytokine production after prrsv infection [ ] [ ] [ ] . however, kinetic analysis in serum of viremic pigs of various ages showed that elevated il- levels were primarily a function of age and were not associated with infection status [ ] . the only exception was in weaned pigs infected with a virulent virus, in which a transient increase was associated with viral pathogenesis [ ] . on balance, the immunological evidence for prrsv inducing a state of immunosuppression does not appear to be compelling. secondary infections following prrs disease outbreak in swine herds, suggesting a reduced ability to fight infection, is an alternative indicator of immunosuppression. an early study showed concurrent pulmonary bacterial infections in % of prrs cases [ ] . however, the study did not determine if bacterial infections were present before the prrs outbreaks. the immunosuppression question also was addressed in more controlled settings using dual infection models with prrsv and various bacterial species. a summary of published literature in showed no predisposition to bacterial disease in of coinfection models, three ambiguous outcomes, and four cases in which severity of disease was increased [ ] . more recent studies found a positive association between prrsv infection and replication of porcine circovirus (pcv ) or swine influenza virus [ , ] . it is possible that bacterial infections in swine herds increase following prrs outbreaks due an increased burden of viral infection on host resilience to pathogen burden. subclinical viral and bacterial infections are common, with pcv , salmonella enterica, haemophilus parasuis, various mycoplasma species, leptospira, and escherichia coli being examples. control of infection is maintained by a combination of immune resistance to microbial replication and tissue tolerance to damage. in a coinfection model of influenza virus and legionella pneumophila, it was clearly demonstrated that l. pneumophila infection was subclinical in healthy mice, but was lethal in the presence of influenza virus [ ] . overwhelming disease was due to loss of tissue resilience, since the bacterial load was unchanged [ ] . this model might account for mortalities observed in experimental swine following prrsv exposure [ ] . given the variable results of prrsv coinfection models in swine and an alternative mechanism for increased disease in prrsv-infected herds, generalized immunosuppression does not appear to be a key feature of prrsv pathogenesis. prrsv, like many viruses, has developed countermeasures to host immune responses that enable it to survive and replicate for extended periods of time before the infection is resolved. prrsv modulation of intracellular antiviral defense mechanisms has been reviewed extensively [ ] . the effects of prrsv infection on adaptive immune response, i.e., antigen-specific t cell, b cell, and antibody responses, are less well characterized. the antiviral response of t cells to prrsv, examined primarily by the ifnγ enzyme-linked immunospot (elispot), appears to develop slowly over a period of weeks, and is not associated with changes in viral loads in blood or in infected lung and lymphoid tissues [ , ] . peripheral blood mononuclear cells (pbmc) from young, weaned pigs show limited ifnγ responses even when stimulated by phytohemagluttinin, which might account for the low anti-prrsv responsiveness after re-stimulation in vitro [ ] . however, pbmc from growing pigs and mature sows, which showed higher levels of ifnγ sensitivity, still showed limited responsiveness [ ] . these findings indicate that prrsv may interfere with specific cell-mediated immunity, but more direct evidence is needed for a fuller understanding. by contrast, the interaction of prrsv with pigs does not appear to retard or attenuate the development of humoral immunity or b cell differentiation. induction of antibody responses to prrsv proteins, both structural and non-structural, occurred in the same time frame as antibody responses to keyhole limpet hemocyanin (klh), an irrelevant protein antigen [ ] . the antibody response to klh was also the same in the presence or absence of prrsv infection [ ] . similarly, prrsv infection did not inhibit cellular or humoral immune protection in response to pseudorabies virus vaccination [ ] . thus, the adaptive b cell response is not delayed or suppressed by prrsv. an extended viremia and prolonged survival in lymphoid tissues is characteristic of prrsv infection. these features show that prrsv has mechanisms of immune avoidance that are not present in viruses such as influenza virus and foot and mouth disease virus, in which sterilizing immunity is achieved within - days. it appears from the findings of field observations and experimental investigations that some type of prrsv-specific t cell interference is present, whereas specific b cell inhibition or a generalized state of immunosuppression are not immunological hallmarks of prrsv infection. the antibody response to prrsv typically dominates discussions of prrsv immunity, as neutralizing antibodies are the crucial component of immune-mediated protection against most viral infections [ , ]. as a result, shortly after the identification of prrsv as the causative agent of mystery swine disease, there was a strong push to identify the presence and dynamic response of neutralizing antibodies against prrsv and then to characterize their specificity for prrsv variants. early work suggested that neutralizing antibodies against homologous prrsv could be found as early as - days after inoculation [ ]. however, this was likely the non-affinity matured immunoglobulin (ig)m response, as anti-swine igm ablated the previously observed neutralizing activity. subsequent research showed that the high affinity neutralizing igg response, detected at around - days post-inoculation, is specific for the inoculating virus with partial neutralizing activity against heterologous viruses [ ] [ ] [ ] [ ] [ ] . following the identification of prrsv neutralizing antibodies, the effectiveness of immunoglobulins in protecting against infection was evaluated with passive transfer studies. these experiments displayed the effectiveness of neutralizing antibodies at preventing clinical infection and disease against homologous challenge [ , ] . however, these studies also showed that immune protection can be quite limited, especially between prrsv- and prrsv- [ ]. within prrsv- or prrsv- , protection against homologous inoculation is consistently solid, whereas protection against heterologous challenge is variable for unclear reasons [ - ]. however, genetic similarity, based primarily on orf sequence comparisons, shows no relationship with degree of protection [ ]. these results appeared to explain the potential field problem, in which vaccinated or live virus inoculated animals become infected with a variant prrsv genetically different enough from the inoculating strain to evade the immune system, propagate, and then cause disease. hence, ever since the mutability, antigenic variability, and resultant immunological elusiveness of prrsv were first appreciated, a broadly neutralizing antibody response to prrsv has been coveted by immunologists and practitioners [ ] . recent research shows that there are animals capable of developing a broadly neutralizing antibody response to genetically disparate viruses [ , ] . however, this immune capability has only been found in a proportion of animals in groups of similar genetics age, sex, and exposure history [ ] . the seemingly random ability of some animals to develop broadly neutralizing antibodies suggests that the inherent variation of the adaptive immune response may play a role in conferring broadly neutralizing capabilities to certain animals. investigations into this ability are needed at the lymphocyte level and while the obvious target is the b cell, t cells cannot be overlooked, as the induction of a humoral immune response requires antigen-specific t cell driven help [ , ] . therefore, animals able to develop a strong neutralizing antibody response would require both b cells and t cells that are capable of recognizing neutralizing epitopes. the conditions needed to achieve cross-neutralizing antibody production are not known, but may involve multiple exposures to the same or different virus isolates. sows with high titered, broadly neutralizing antibodies were found in herds with multiple exposures to virulent field viruses [ ] . in an experimental study, cross-neutralization was reported in animals exposed first to a prrsv vaccine strain followed by homologous or heterologous virus challenge [ ] . however, the majority of data analyzed were below the neutralization assay cutoff. duration of viremia, up to days, was linked with increased breadth of neutralizing antibodies following a single viral infection [ ] . however, since cross-neutralization activity and titer data were not presented, it was not possible to further interpret the results. the animals were not subsequently challenged, so it is not known if the cross-neutralizing activity in serum was predictive of protection. other studies showed that significant neutralizing antibody responses are not commonly observed during viremic infection of young pigs, as well as in adult sows [ , [ ] [ ] [ ] . recently, vaccinology research in hiv has shown that sequential immunizations, tailored for specific stages of the immune response, may be useful for inducing broadly neutralizing antibodies [ ] [ ] [ ] . the approach is based on the finding that early immune responses to hiv resulted in neutralizing antibodies against the circulating virus which quickly led to immune escape of the virus and the ineffectiveness of generated antibodies. the antibody-resistant virus then stimulated a secondary antibody response which again selected for antibody resistant virus. this virus-antibody hide and seek continued, eventually resulting in the selection of several neutralization targets of the virus as well as the generation of broadly neutralizing antibodies [ ] [ ] [ ] . cloning of the antibodies showed that somatic mutations are generally necessary for antibody neutralizing capabilities against hiv- [ , ] . these findings have shown that the b cell response of the host adapts in the germinal center as the virus evolves, suggesting that tailored sequential immunization could lead to the development of a broadly neutralizing antibody response [ ] . the consistent generation of a broadly neutralizing antibody response to prrsv on the herd level has evaded the swine health industry since the emergence of prrsv. there are multiple proposed mechanisms by which prrsv may evade or inhibit the development, or the effectiveness, of a neutralizing antibody response, such as glycan shielding of envelope glycoprotein (gp) or gp [ , ] , the existence of decoy epitopes in gp [ ] , lymphocyte dysregulation [ ] , and inhibition of the innate immune response [ ] . comprehension of defense mechanisms employed by prrsv makes the development of a broadly neutralizing immune response appear to be a daunting task. however, as previously shown, some animals are capable of developing such a response. simply, the key to adapting the immune phenomenon of some animals to a vaccine capable of inducing broadly protective immunity in many animals lies in identifying conserved epitopes on surface proteins which are necessary for infection. while the purported targets of neutralization have been extensively discussed in recent reviews, it is worth noting that several epitopes on the membrane (m) protein, gp , gp , gp , and gp , have been shown, or implicated, to harbor neutralizing activity [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, knocking out only cd in the pig is sufficient to render animals non-susceptible to prrsv infection and replication [ , , ] . it is proposed that following endocytosis, cd associates with the virus within the endosome, resulting in uncoating of the virus and the release of the viral genome into the cellular cytoplasm [ ] . since cd is necessary for viral infection and replication, the logical next step is to identify the conserved regions of viral surface proteins, most likely the minor glycoproteins (gp , gp , and gp ), that interact with cd [ , ] . traditionally, the non-neutralizing antibody response to prrsv has been considered useful only for its ability to identify if an animal had been exposed and seroconverted to virus. indeed, there are many structural and non-structural proteins of prrsv which make this possible through their ability to induce a robust humoral immune response [ , , ] . however, recent research on other pathogens has shown that non-neutralizing antibodies may play a much larger role in immunity than was previously appreciated [ ] [ ] [ ] [ ] . alternative antibody functions, such as antibody dependent cell-mediated cytotoxicity (adcc), antibody-dependent complement-mediated cytotoxicity (cdc), and antibody-dependent complement-mediated virolysis may be important in the clearance of virus and virally infected cells from an animal. to our knowledge, there are only two published papers investigating non-neutralizing antibody functions in the context of prrsv infection [ , ] . both of these in vitro studies utilized a prrsv- virus and failed to find an effect of adcc and cdc on infected cells. however, experiments focused on prrsv- viruses with extended time points beyond h are warranted. a more extensive review of non-neutralizing antibody functions can be found in the cited review [ ] . if antibodies are the most important effectors of the immune system against viral infection, then b cells that make the antibodies are the most important cells. previous research on the interaction between prrsv and the porcine b cell is contradictory. it has recently been suggested that prrsv infection results in lymphocyte apoptosis and immune impairment [ ] . several sources have shown that prrsv largely or exclusively induces a specific humoral response to infection [ , ] . other studies report that prrsv infection results primarily in polyclonal b cell activation leading to hypergammaglobulinemia and the development of immune complexes [ ] [ ] [ ] [ ] . the majority of work describing infection leading to polyclonal activation and hypergammaglobulinemia was performed in germ-free isolator piglets. this model is very effective for comparing b cell and antibody repertoire development in the fetus, as the germ-free status of the pigs removes many of the variables present when experiments are performed on conventionally reared animals [ ] . however, these animals are deprived of the microflora and maternal antibodies to which conventional animals are exposed. as a result, the translation of immunological outcomes observed in isolator pigs to conventional pigs must be performed with caution. studies in mice show that the immune systems of specific-pathogen free laboratory mice are similar to neonatal human immune systems, whereas feral mice displayed immune systems more comparable to adult humans. effectively, the immune systems of germ-free animals may not display "normal" immune system phenotypes due to the lack of exposure to microflora [ , ] . the development of protective humoral immunity, after vaccination or exposure to a pathogen, is dependent upon two lines of defense. the first immune defense is secreted antibodies, first from short-lived and then from long-lived, plasma cells residing somewhere in the body (figure ). the second line of defense is memory b cells (figure ). memory cells are sentinels against reinfection which are activated upon antigen recognition to proliferate and differentiate into antibody secreting plasma cells, thus rapidly boosting circulating antibody titers with high affinity class switched antibodies [ ] . antibody repertoire development in the fetus, as the germ-free status of the pigs removes many of the variables present when experiments are performed on conventionally reared animals [ ] . however, these animals are deprived of the microflora and maternal antibodies to which conventional animals are exposed. as a result, the translation of immunological outcomes observed in isolator pigs to conventional pigs must be performed with caution. studies in mice show that the immune systems of specific-pathogen free laboratory mice are similar to neonatal human immune systems, whereas feral mice displayed immune systems more comparable to adult humans. effectively, the immune systems of germ-free animals may not display "normal" immune system phenotypes due to the lack of exposure to microflora [ , ] . the development of protective humoral immunity, after vaccination or exposure to a pathogen, is dependent upon two lines of defense. the first immune defense is secreted antibodies, first from short-lived and then from long-lived, plasma cells residing somewhere in the body (figure ). the second line of defense is memory b cells (figure ). memory cells are sentinels against reinfection which are activated upon antigen recognition to proliferate and differentiate into antibody secreting plasma cells, thus rapidly boosting circulating antibody titers with high affinity class switched antibodies [ ] . currently, there is scant research on the memory b cell response to prrsv. strong memory responses have been shown against nsp , nsp , n, and the end of gp [ , ] . the specific memory b cells are abundant in tonsil, lymph nodes draining the lungs and reproductive tract, and spleen. unfortunately, there are many questions about the porcine memory response to prrsv which have yet to be answered, including if memory cell kinetics closely mimic antibody kinetics, the response of prrsv-specific memory pools upon homologous or heterologous viral challenge, and the importance of these cells in conferring protection against challenge. the development of sensitive and specific reagents, such as b cell tetramers, is a first step in being able to answer these critical questions. additionally, it is possible that the key to understanding the broadly neutralizing response to prrsv lies within circulating or lymphoid organ resident memory b cells. the potential to investigate these cells for identification of heavy and light chain antibody sequences is reviewed in rahe and murtaugh [ ] . plasma cells are terminally differentiated b cells responsible for making antibodies. apart from the immature plasmablast, two types of plasma cells have been defined in the mouse and human [ , ] . short-lived plasma cells quickly boost antibody titers while long-lived plasma cells maintain circulating antibody titers in the face of continual antibody degradation. mulupuri et al. identified prrsv-specific plasma cells in several secondary lymphoid organs, such as the spleen, tonsil, sternal lymph node, and inguinal lymph node [ ] . interestingly, no prrsv-specific or klh-specific plasma cells were found in the bone marrow of immune pigs [ ] . this was surprising, as the bone marrow has been long considered as the reservoir for long-lived plasma cells in both mice and humans [ ] [ ] [ ] . it then begs the question, do pigs have long-lived plasma cells and, if so, where do they reside? mulupuri et al. found prrsv and klh specific plasma cells in secondary lymphoid organs days after inoculation [ ] . however, these cells may not be "long lived" as the prolonged viremia of prrsv may result in a somewhat continuous stimulation of memory b cells resulting in the appearance of this plasma cell population in secondary lymphoid organs. it seems unlikely that pigs do not have long lived plasma cells, as the half-life of porcine antibodies in serum is, on average, approximately nine days [ , ] . therefore, without long lived plasma cells, pigs would quickly lose humoral protection as antibody titers waned. the identification of the anatomic location as well as the understanding of mechanisms for inducing a strong long lived plasma cell response may be important for future vaccine design as well as comprehending host-pathogen interactions. interestingly, even though neutralizing antibodies have historically garnered the majority of attention in prrsv immunology, it is well-known that pigs readily control infection in the absence of neutralizing antibodies. furthermore, viremia is reported in the presence of neutralizing antibodies [ , ] . therefore, there must be other facets of the immune system which effectively function to control infection and eliminate prrsv from the host. while some of this activity may be attributed to non-neutralizing functions of antibodies, the t cell response to infection demands further investigation. a recent prrs immunity review summarized previous research on functional t cell subsets, and prrsv epitope targets, as well as gaps in t cell immunity [ ] . here, we provide context for the understanding of novel results that have not been comprehensively reviewed. early research on the t cell response to prrsv identified a large, transient decrease in the cd + /cd + t cell ratio early, usually within the first week, in the course of infection [ ] . the change in this ratio could have been due to a temporary loss of cd + cells through apoptosis or to an increase in cd + cells due to antigen-specific proliferation [ ] . the importance of these findings for clearance of prrsv or protection from infection were not known at the time, and other explanations, such as fluxes in cell populations between spleen, other lymphoid tissues, and blood could not be discounted. experiments to address the helper t cell type /helper t cell type (th /th ) paradigm in the pig showed that prrsv induced a strong th response, as expected, identified in vivo by an increased expression of th -specification factor tbx (t-bet) in cd + cells [ ] . however, the finding is at odds with previously reports indicating that prrsv infection results in the production of il- , a cytokine classically associated with a th phenotype. similarly, monocyte-derived dendritic cells (mo-dcs) infected with prrsv down regulate swine leukocyte antigen (sla)-i, sla-ii, cd and cd as well as promote il- secretion over il- secretion [ ] . delineation of the th /th response to prrsv, elucidation of th /th -specific cytokine markers in swine, as well as identifying associated cytokine responses of dendritic cells within secondary lymphoid organs where t cell proliferation and differentiation is most likely to occur, would help to resolve these outstanding questions [ ] . the th cell has classically been identified, in mouse and human, as playing an important role in extracellular bacterial immunity through the production of the pro-inflammatory cytokines, il- a, il- f, and il- [ , ] . il- producing th cells are known to exist in the pig [ ] . the importance of this t cell subset in the context of prrsv infection has recently been investigated. a strain of chinese highly pathogenic prrsv (hp-prrsv) appeared to suppress th cells in the peripheral blood and lungs of pigs, resulting in an increased susceptibility to secondary bacterial infections [ ] . remarkably, the effect was prrsv strain-specific, as a non-hp prrsv strain failed to elicit the same response. future research into the t cell response to prrsv, especially with t cell tetramers and functional elispots, will be essential for the characterization of both cd + and cd + antigen specific t cells. understanding how antigen-specific t cells interact with both infected and uninfected antigen presenting macrophages and dendritic cells will be helpful for advancing the field of prrsv immunity. the natural killer cell is an innate lymphoid cell which can have a profound impact on adaptive immunity, but is also able to induce an early and rapid innate response against pathogens through a variety of mechanisms. nk cells produce cytokines, such as ifnγ, show cytotoxic activity against infected cells not expressing mhci, can induce dendritic cell maturation, and effect the destruction of infected cells in adcc [ ] . however, nk cells may deploy even more extensive and important functions in porcine immunity than are currently realized. an early clue that nk cells were involved in innate responses to prrsv was a sharp peak in serum ifnγ shortly after infection [ ] . the acute response was attributed to nk cells, as the result was deemed too early for a t cell response, and suggested that decreased viral burdens in the lung prior to humoral or t cell responses could be due to the function of nk cells. however, it is known that porcine macrophages are also capable of producing ifnγ in the presence of prrsv infection [ , ] . furthermore, prrsv appears to suppress the nk cell response without significantly affecting nk cell numbers [ ] [ ] [ ] [ ] . the cause of this suppression has yet to be determined, although viral proteins, rather than soluble factors from cells, may be responsible [ ] . potential roles of additional nk cell functions, such as adcc, in prrsv immunity are poorly understood [ ] . prrsv has tormented the health and wellbeing of swine worldwide since its discovery in the late s. unfortunately, after almost years of research into the porcine immune response to prrsv, there is still no effective means for inducing a broadly protective immune response at the herd level. the reasons for this failure are not completely known, but presumably include mechanisms by which the virus subverts the immune system. the ability of the virus to rapidly mutate while not losing fitness challenges the host immune system to keep pace. at the same time, infection of macrophages, a key player in immunoregulation, challenges both innate and adaptive immune cell mobilization as well as induction of a coordinated response that is needed for effective control and elimination of the virus. fortunately, foundational advances in the understanding of viral pathogenesis and immunity are enabling more informative investigations. the identification of cd as the necessary and sufficient receptor for infection supports the implications of broadly neutralizing antibodies that a conserved target is present on all prrsv. understanding how prrsv surface glycoproteins interact with cd should lead to the identification of conserved epitopes which are necessary for infection. if, as appears to be the case, there is only one conserved way into the cell, then there must be a conserved viral sequence, or structure, which enables viral entry. furthermore, the knowledge that pigs eventually develop sterilizing immunity, if given enough time, supports the concept that conserved epitopes exist on the virus. therefore, the study of mature animals, which have cleared the virus, may provide the key to understanding how the immune system eventually gets the upper hand on the virus and cures infection. even with seminal advances in several aspects of the study of prrsv, there remains much to be understood and clarified. currently, the published literature presents conflicting views on many aspects of prrsv adaptive immunity, especially related to t and b cell responses and the production, or inhibition, of cytokines in the face of infection. the continued development of antigen-specific reagents, of high sensitivity and specificity, is needed for understanding how the host responds to prrsv infection. furthermore, it is important that future prrsv studies focus on the relevant host animal, the conventional pig. while the study of this outbred animal species is perhaps challenging at times, it affords the ability to study the host-pathogen interaction in the only species in which the virus naturally interacts. additionally, knowledge gained about the immunology of conventional pigs will accelerate immunological elucidation of other pig-pathogen interactions. in conclusion, prrsv continues to be the most burdensome pathogen of pigs worldwide, 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expression of antimicrobial peptides influenza a inhibits th -mediated host defense against bacterial pneumonia in mice cd engagement strongly induces cd expression on porcine dendritic cells and polarizes the t cell immune response toward th natural killer cells in host defense against veterinary pathogens infection with porcine reproductive and respiratory syndrome virus stimulates an early gamma interferon response in the serum of pigs expression of interferon-gamma and tumour necrosis factor-alpha in pigs experimentally infected with porcine reproductive and respiratory syndrome virus immunohistochemical staining of ifn-γ positive cells in porcine reproductive and respiratory syndrome virus-infected lungs porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions porcine reproductive and respiratory syndrome virus induces pronounced immune modulatory responses at mucosal tissues in the parental vaccine strain vr infected pigs the authors declare no conflicts of interest. key: cord- - hlj r authors: brauburger, kristina; hume, adam j.; mühlberger, elke; olejnik, judith title: forty-five years of marburg virus research date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: hlj r in , the first reported filovirus hemorrhagic fever outbreak took place in germany and the former yugoslavia. the causative agent that was identified during this outbreak, marburg virus, is one of the most deadly human pathogens. this article provides a comprehensive overview of our current knowledge about marburg virus disease ranging from ecology to pathogenesis and molecular biology. marburg virus (marv) first appeared in august , when laboratory workers in marburg and frankfurt, germany and belgrade, yugoslavia (now serbia) were infected with a previously unknown infectious agent. the patients ( primary, six secondary infections) developed severe disease that progressed to a fatal outcome in seven of the cases. an additional case showing symptoms of disease was diagnosed retrospectively (reviewed in [ ] ). the source of infection was traced back to african green monkeys (chlorocebus aethiops) that had been imported from uganda and were shipped to all three locations. the primary infections ironically occurred when the monkeys were necropsied for the purpose of obtaining kidney cells to culture poliomyelitis vaccine strains. in the remarkable period of less than three months the etiologic agent was isolated, characterized, and identified by the joint effort open access of scientists in marburg and hamburg [ ] and was later confirmed by kunz and colleagues [ ] and kissling and colleagues [ ] . the pathogen was named marburg virus after the city with the most cases and represented the first isolation of a filovirus. erroneously, a study published in 'the lancet' claiming that the mysterious disease was caused by rickettsia or chlamydia has frequently been cited as the first report on the causative agent of marburg virus disease (mvd) [ ] . it was not until that the now better-known member of the family, ebola virus (ebov), first emerged in africa [ , ] . shortly thereafter marburgviruses and ebolaviruses were classified together in a newly established family termed filoviridae, so-named after their distinctive thread-like structure (filum being latin for thread). marv had not been heard of for eight years, when a young australian who had traveled throughout zimbabwe was admitted to a hospital in johannesburg, south africa with symptoms reminiscent of those observed during the outbreak in europe [ ] . when he died and the infection spread to his travel companion and later also to a nurse, lassa fever was initially suspected resulting in strict barrier nursing techniques and isolation of the patients and their primary contacts. this lead to a quick containment of the outbreak, and while the secondary cases recovered, marv was identified as the causative agent of the disease. in the following years from through , only sporadic outbreaks that affected small numbers of individuals were caused by marv on the african continent (table , figure a ). as the case fatality rates associated with mvd were also lower than those seen in the devastating outbreaks associated with ebov disease that reached up to %, marv was long thought to be less threatening (table ) . however, this view had to be revised as marv reemerged in two large outbreaks occurring in the democratic republic of the congo (drc) in - [ ] and then, for the first time also in western africa, in angola, in - [ ] . the total number of cases and the high fatality rates ( % in drc and % in angola) revealed that marv was as big of a threat for public health as ebov [ , ] . the variation observed in disease severity and case fatality rates between these outbreaks versus the initial one in may depend on many complicating/mitigating factors. these include quality and availability of medical care, infectious dose and route of infection, differences in host population susceptibility (depending on immune and nutritional status) and genetics, inherent differences in viral variant virulence, and the prevalence of co-infections (particularly malaria and aids in patients from sub-saharan africa) [ ] . the assumption that marv angola might be inherently more virulent than other marv variants has been proposed mainly based on infection studies with nonhuman primates (nhp) [ ] [ ] [ ] but is a matter of debate [ ] . the genomes of the angolan isolates differ about % at nucleotide level from the majority of the east african marv isolates, including the ones from [ ] . there is no evidence so far that the observed genetic differences result in higher virulence in humans. the drc outbreak was unique, as there were at least nine different virus variants circulating in the tested patients indicative of several different spillover events from the natural reservoir to the human population [ ] . in contrast, sequence data from the angolan outbreak suggested a single introduction of marv to an unidentified index patient and subsequent spread via person-to-person contact. the viral genomes showed a remarkably high genetic stability within this outbreak. identical marv genomes were isolated from patients even after two to three human to human transmissions [ ] . mvd is considered a zoonotic disease that is thought to persist in a healthy reservoir host in the endemic areas in africa. humans and nhps are spillover hosts and show a high rate of fatal disease outcomes. several large-scale attempts to identify the natural host of filovirus infection throughout sub-saharan africa had been undertaken in the years since filoviruses first emerged with frustratingly little success [ ] [ ] [ ] [ ] . consistent with ecologic niche modeling of outbreaks and epidemiological patterns, isolated cases have suggested that ebov is endemic in the rain forests of central and western africa while marv is more prevalent in open, dry areas of eastern, south-central africa [ , ] . almost all of the primary infections of natural mvd outbreaks so far have been linked to human entry into caves inhabited by bats (e.g., cave visitors, mine workers) (table ) . thus, bats have long been suspected to play an important role in the transmission cycle of the disease [ , , ] . in , evidence was detected for marv infection of the common egyptian fruit bat (rousettus aegyptiacus) [ , ] (figure ), and marv was isolated from healthy infected r. aegyptiacus bats caught in the same year in uganda [ ] . the bats were collected in kitaka cave around the same time as human infections occurred that had been linked to the cave (table and see above, . epidemiology). genomic analysis of the few isolates of marv acquired from bats showed that the sequences matched closely to the marv genomes isolated from patient samples (figure b) . this was also the case for partial marv sequences isolated from bats inhabiting the goroumbwa mine in the drc that was suspected to be the major location for several independent spillover events to gold miners between and . the bat marv sequences were closely related to the distinct isolates that had been reported during these outbreaks in humans [ ] . a study analyzing marv prevalence in bat populations in gabon found marv-specific nucleic acids in r. aegyptiacus bats in several local caves [ ] . together with previous data showing a high prevalence of marv-specific antibodies in gabonese bat populations [ ] and with an observed relation of the isolated sequences with previously reported gabonese bat isolates [ ] this study viruses , suggests that marv is enzootic in gabon and raises the concern of further spread of marv into other countries. therefore, close ecological as well as serological surveillance of the bat populations in sub-saharan africa could help to predict and prevent further mvd outbreaks especially in areas where bats are still used as a food source. it is not currently clear whether r. aegyptiacus bats are the exclusive reservoir for marv or if other bat species reported to be positive for viral antibodies and rna are also natural reservoirs or merely intermediate hosts [ ] . the genus marburgvirus includes a single species, marburg marburgvirus (formerly referred to as lake victoria marburgvirus) [ , ] . phylogenetic analysis based on genomic sequence data suggests that the known members of this species can be assigned to at least five different lineages of which four are very closely related (nucleotide sequences differ up to %) while the fifth is divergent (a nucleotide difference of %) ( figure b) [ , , ] . as the genomic divergence between all isolates is below %-the cutoff for the classification of the five different ebolaviruses into five different species-the five marburgvirus lineages were recently reclassified as two viruses. ravn virus (ravv) is represented by the ravn isolates from , one isolate from the drc outbreak in - , and one human and several bat isolates from infections that took place in uganda in . marburg virus (marv) is represented by all other sequenced isolates ( figure b , table ) [ ] . for the sake of simplicity, in this review the abbreviation "marv" is used for all marburgviruses and the abbreviation "ebov" for all ebolaviruses. initial mvd patients are believed to contract the virus via exposure to an infected animal: either a reservoir host (several bat species) or a spill-over host such as nhps as described in the first mvd outbreak (see above, . epidemiology) [ , ] . following transmission to humans, spread of the virus between individuals is the result of direct contact with blood or other body fluids (saliva, sweat, stool, urine, tears, and breast milk) from infected patients. typical risks of exposure include administration of medical care to infected individuals as well as handling of corpses without use of proper protection [ ] . of particular note, virus has been found in tears, semen, and in a liver biopsy weeks to months following the onset of symptoms highlighting the importance of monitoring convalescent patients [ , [ ] [ ] [ ] . much of what we know about typical mvd symptoms comes primarily from clinical data obtained during the three largest recorded mvd outbreaks: the outbreak in germany and yugoslavia, the - outbreak in the drc, and the - outbreak in angola. although the case fatality rates were significantly higher in the latter outbreaks, most of the clinical symptoms observed were similar. based on the most reliable documented cases of exposure and subsequent illness, mvd has an incubation period ranging from to days (typically to days), which is likely modulated by factors such as infectious dose and possibly by route of infection. the course of mvd has conventionally been broken down into three phases [ ] : an initial generalization phase, an early organ phase, and either a late organ phase or convalescence phase depending upon disease outcome [ ] . a summary of mvd symptoms is reviewed below [ , [ ] [ ] [ ] [ ] . the onset of illness begins with generic flu-like symptoms; a characteristic high fever (typically - o c), severe headache, chills, myalgia, prostration, and malaise. for many patients ( - %) this is followed by rapid debilitation characterized by gastrointestinal symptoms including anorexia, abdominal pain, severe nausea, vomiting, and watery diarrhea. starting on day four to five patients commonly develop enanthem, dysphasia, and pharyngitis. additionally, a characteristic maculopapular rash is typically the first distinctive feature indicating a filovirus infection versus influenza or malaria. other common symptoms include lymphadenopathy, leukopenia, and thrombocytopenia. many of the initial symptoms may persist in the early organ phase, and patients may sustain a high fever. they may additionally display neurological symptoms including encephalitis, confusion, delirium, irritability, and aggression. patients can also develop dyspnea and abnormal vascular permeability, particularly conjunctival injection and edema. during the latter part of this phase more than % of patients present with some form of clear hemorrhagic manifestation such as petechiae, mucosal bleeding, melena, bloody diarrhea, hematemesis, and ecchymoses. due to the unusualness of hemorrhagic symptoms, diseases caused by filoviruses have sometimes been referred to as hemorrhagic fevers (marburg hemorrhagic fever (mhf) and ebola hemorrhagic fever (ehf)), although these terms are currently disfavored since not all patients display hemorrhagic symptoms. at this stage, multiple organs are affected including the pancreas, kidney, and liver. elevated serum activity of a number of liver enzymes including sgot and sgpt have been observed in most patients sampled. the late stages of mvd result in one of two potential outcomes: patients either succumb to the disease or enter a prolonged phase of recuperation. typical preagonal symptoms include restlessness, obtundation, confusion, dementia, convulsions, reduced circulation due to severe dehydration, metabolic disturbances, severe diffuse coagulopathy, multiorgan failure, shock, and coma. fatalities typically occur - days following the onset of symptoms, with death usually the resulting of shock and multiorgan failure. non-fatal cases are typified by an extensive convalescent period during which myalgia, exhaustion, sweating, peeling of the skin at the sites of rash, partial amnesia, and secondary infections are all common. prevention of newly emerging marv infections and effective containment during ongoing outbreaks is both essential and challenging, as there is currently no licensed vaccine or treatment available for general use. following the outbreak of mvd in europe and cases of infection with ebolavirus reston in imported crab-eating macaques (macaca fascicularis) in the usa in / and as well as in italy (reviewed in [ ] ), strict quarantine procedures have been put in place that have so far prevented infections acquired by imported nhps into non-endemic countries [ , ] . to avoid the spread of filoviruses by tourists, python cave was closed to the public following the diagnosis of the dutch patient in . the prevention and control of outbreaks and infections in endemic countries is much more challenging. in the past, joint efforts of teams from the who, doctors without borders, the red cross, the cdc and others in collaboration with the local ministries of health have been undertaken to cease the spread of mvd. the main focus of outbreak control is the prevention of secondary transmission and further primary infections. the first measures in response to a mvd outbreak include setting up isolation wards in hospitals to assure rapid isolation of marv-infected patients and prevent person-to-person transmission (figure b ). proper and fast laboratory diagnosis of suspected cases is key to eliminate further spread. nosocomial infections were commonly seen in earlier outbreaks [ , , ] . however, reinforcement of barrier nursing techniques and education of health care workers have limited these infections in recent outbreaks ( figure a ). epidemiological surveillance has been crucial in the identification of index cases as well as the predominant modes of transmission. in endemic areas, secondary infections mainly occurred while taking care of ill patients and family members or during traditional burial practices involving close contact to corpses [ ] . therefore, the execution of safe burial and disinfection techniques and information campaigns to educate the local population are essential in order to contain the spread of infections in endemic areas ( figure a ) [ , , ] . biosafety and epidemiological efforts alone were not sufficient for efficient outbreak control during large outbreaks, emphasizing the need for additional psychosocial support of the affected communities [ ] . the fast progression and high lethality rates associated with mvd even-and especially-after hospital admission resulted in a high level of fear and suspicion by the resident population. the fact that health care workers wearing recommended personal protective equipment (ppe) were fully masked and not identifiable further increased anxiety (figure b ). this resulted in the hiding of infected family members and verbal and in some cases physical aggression towards members of aid organizations [ ] . communicating necessary protective measures while respectfully considering the affected families' and communities' traditions and culture during ongoing outbreaks is therefore essential for successful outbreak management. the recent identification of bats as the potential reservoirs for marv as well as ebov [ , , , , ] will help to increase not only the public awareness, but also the effectiveness of the preventive measures taken in endemic areas to minimize contact with infected animals (i.e. closing of bat inhabited caves for the public, serosurveillance of bat populations) [ , ] . this is a challenging task, emphasized by the fact that during the last cluster of marv infections linked to a gold mine in uganda, the miner hired to enforce the restricted access to the mine got infected. the mine had been closed in response to the ongoing outbreak and even though he was aware of the risk, he had entered the mine without the suggested ppe [ ] . later, the bat population of this mine was eliminated by the owner by means of fumigation [ ] . as bats of most species are endangered, this does not seem a viable option and educational campaigns aimed at villagers living close to bat-inhabited caves as well as tourist groups and tour operators might prove more sustainable in the future. in , during the first reported filovirus disease outbreak in europe, the identification of the previously unknown causative agent of the deadly disease was performed by electron microscopy (em) (figure ). the unusual filamentous structure of the particles led to some confusion and it was even suggested that the causative agent of the disease might be related to the spiral-shaped leptospira, a genus of the spirochaetes bacteria [ ] . others concluded that the observed particles were viruses morphologically related to rhabdoviruses and named the newly discovered pathogen marburg virus [ , ] . marburg virions are pleomorphic particles, which appear as rod-or ring-like, crook-or sixshaped, or branched structures. cryo-em analysis of purified virions showed that about % of viral particles released from infected vero cells were filamentous, % were six-shaped, and % were round [ ] . the same study revealed a mean particle length of nm and a mean diameter of nm. previous conventional em studies showed that the marv particles were uniformly nm in diameter, whereas the length varied widely with virions measuring up to , nm. the average particle length was nm [ ] [ ] [ ] . the reported differences in particle size might be due to experimental differences between cryo-em and conventional em [ ] . notably, marv particles are considerably shorter than ebov virions, although marv genomes are slightly longer than ebov genomes [ , ] . marv particles are surrounded by a host-derived membrane that is coated with spikes of - nm in length, which are formed by trimers of the viral glycoprotein (gp) ( figure ) [ ] [ ] [ ] [ ] [ ] [ ] . the central core of the viral particles is the ribonucleoprotein complex (nucleocapsid) formed by the viral rna genome and tightly associated nucleocapsid proteins ( figure ). the nucleocapsids are highly organized tubular structures with an outer diameter of - nm and an electron-dense central axis of - nm. the central axis is surrounded by a helical capsid with cross-striations at a nm interval [ ] [ ] [ ] . a recently published detailed cryo-electron tomography analysis of marv virions has shed some light on the structural organization of the nucleocapsids. reconstructions of virion-associated nucleocapsids using subtomogram-averaging analysis revealed that the marv nucleocapsids form a left-handed helix with a pitch of . nm and a flexible average symmetry of . protrusions per turn with two inner lobes of density per protrusion. the inner lobes represent the nucleoprotein (np), suggesting that the marv nucleocapsid contains an average number of . np molecules per turn with each np molecule packaging six rna bases [ ] . marv nucleocapsids show directionality, having a pointed and a barbed tip [ ] . the nonsegmented negative-sense (nns) rna genomes of the various marv isolates range in size from , to , nts and contain seven monocistronic genes in a linear order ( figure ) [ , ] . each gene is composed of a highly conserved transcription start and stop signal, an unusually long ' and ' untranslated region, and the open reading frame (orf). the genes are either separated by short intergenic regions that range from to nts, or the transcription stop signal of the upstream gene and the transcription start signal of the downstream gene overlap, sharing five highly conserved nts ( figure ). the structure of this gene overlap is found among all filoviruses and is unique among members of the order mononegavirales (for review see [ ] ). the ' and ' genome ends are extracistronic regulatory regions that contain cis-acting signals essential for transcription and replication, including transcription and replication promoters. there are generally two types of genomic replication promoters for nns rna viruses: a bipartite promoter found in members of the paramyxovirus subfamily paramyxovirinae and one continuous more compact replication promoter for rhabdo-and pneumoviruses [ ] . the bipartite promoter structure of the paramyxovirinae subfamily is associated with the "rule of six", i.e., the total genome length must be a multiple of six [ ] . given that filoviruses do not obey the rule of six, it was surprising that mapping of the marv genomic replication promoter revealed a bipartite structure. the ' genome end, the leader, comprises nts and contains the first promoter element of the bipartite genomic replication promoter. the second promoter element consists of a (unnnnn) motif with three conserved uridine residues separated from each other by five non-conserved nucleotides. the un ( ) hexamers are located within the ' untranslated region of the first marv gene, the np gene, and are separated from the first promoter region by the nts long transcription start signal. substitutions in the np transcription start signal do not affect replication activity but do interfere with transcription initiation [ ] . the ' extracistronic region, the trailer, spans the last nts of the marv genome and contains the complement of the antigenomic replication promoter (see below, . . transcription and replication). the structure of the marv antigenomic promoter has not yet been determined. however, due to the presence of un ( ) hexamers it is likely that it is of bipartite nature, similar to the genomic promoter. a common feature of the leader and trailer regions of all nns rna viruses is a high degree of complementarity of the - most terminal ' and ' nucleotides [ ] . although filoviruses share this feature, both the leader and the trailer also have the capability to form an internal secondary structure, which is not the case for the leaders and trailers of other nns rna viruses [ , [ ] [ ] [ ] . the marv genome encodes seven structural proteins listed in table . marv has a single surface protein, gp, which is encoded by the fourth gene and mediates attachment to target cells and virus entry [ ] . gp is a type i transmembrane protein which is inserted into the viral envelope in the form of homotrimeric spikes [ ] . in contrast to ebolaviruses, which use transcriptional editing to express the membrane-bound gp and at least two nonstructural glycoproteins [ ] [ ] [ ] [ ] , the marv gp gene contains a single open reading frame (orf) encoding the full-length gp. during its transport from the endoplasmic reticulum (er) to the plasma membrane via the secretory pathway, the precursor gp is the target of various posttranslational modifications including glycosylation [ , ] , acylation [ ] , and phosphorylation [ ] . gp is heavily glycosylated by complex and high mannose-type n-linked glycans as well as by mucin-type o-linked glycans, with the carbohydrates contributing about % of the apparent molecular weight of the protein [ , , ] . similar to ebov gp, the o-linked glycans and many of the n-linked oligosaccharides are clustered in a mucin-like domain [ ] . after synthesis in the er, the precursor gp is cleaved at amino acid by furin or a furin-like protease in the trans golgi network, resulting in two disulfide-linked subunits, gp ( kd) and gp ( kd) [ ] . while the ectodomain, which is mainly formed by gp , mediates binding to entry factors and receptors [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , the transmembrane subunit gp contains the fusion peptide and is presumed to mediate fusion of the viral and the cellular membrane based on similarity to ebov gp both at the amino acid and structural level [ , ] . the amino acid long transmembrane domain of gp is required for the incorporation of gp into virions [ ] . in addition, the cytoplasmic tail of gp is involved in enhancing the efficiency of viral entry by maintaining the structure of the ectodomain [ ] . the receptor binding domain of marv gp was mapped to the aminoterminal region of gp spanning amino acids to [ ] , whereas the highly glycosylated mucin-like domain is not essential for virus entry [ ] . an important step in marv entry is the proteolytic activation of gp by endosomal proteases, facilitating binding of the receptor binding region to the endosomal entry factor niemann-pick c protein (see below, . . entry) [ , ] . besides its function in entry and budding, gp may also play a role in immune evasion. the ifn-inducible antiviral protein tetherin was shown to block the release of vp -induced virus-like marv and ebov particles, suggesting that tetherin might act as a restriction factor for filovirus release [ , ] . however, co-expression of gp was sufficient to counteract the antiviral activity of tetherin by a yet unknown mechanism [ , ] . it is possible that gp not only subverts innate immune responses but also suppresses the adaptive immune response. filoviral gp subunits, including marv gp , contain a domain resembling an immunosuppressive motif found in retroviral envelope proteins [ ] . a -mer peptide corresponding to the putative immunosuppressive domain of marv gp was shown to induce lymphocyte death and suppression of cytokine responses [ ] . it is not yet known if this motif plays a role in the induction of lymphocyte apoptosis observed in marv infection. finally, it has been suggested that shedding of the ectodomain of membrane-bound ebov gp by tumor necrosis factor α-converting enzyme (tace) may play a role in blocking the activity of neutralizing antibodies during infection [ ] . it has been reported for marv that considerable amounts of gp shed from infected cells, although it is not clear if marv gp is a target for tace cleavage [ , ] . the matrix protein vp is encoded by the third marv gene and is the counterpart of the m proteins of other nns rna viruses. vp plays a major role in the formation of virions by redistributing nucleocapsids from the perinuclear region to the plasma membrane, recruiting gp to the sites of budding, and mediating particle release [ ] [ ] [ ] . overexpression of vp led to reduced reporter gene expression of marv minigenomes, suggesting a regulatory role of vp in transcription and/or replication [ ] . as a peripheral membrane protein, vp coats the inner side of the virion's membrane ( figure ) [ ] . cryo-em tomography studies suggest that vp associates with the nucleocapsid through flexible interactions [ ] . it can be easily removed from the nucleocapsid by salt dissociation, indicating that it is only loosely connected to the nucleocapsid [ ] . after synthesis in the cytoplasm of the infected cell, vp associates rapidly with cellular membranes and accumulates in membranous structures of the late endosomal compartment, the multivesicular bodies. a minor portion of vp is also found in association with viral nucleocapsids and in inclusions. additionally, vp appears in patches beneath the plasma membrane where it is transported via the retrograde late endosomal pathway [ , , ] . similar to ebov vp , marv vp is the major factor in particle formation and budding. expression of vp in the absence of other viral proteins leads to the formation and release of filamentous virus-like particles (vlps) resembling authentic virions. this process is enhanced in the presence of gp [ , [ ] [ ] [ ] . the role of vp during budding is described in more detail below (see . . budding). compared to ebov vp , little is known about the structure of marv vp . the n-terminal domain of marv vp folds into ring-like structures, which have the tendency to polymerize into rod-like structures. while ebov vp has been shown to form hexamers and octamers, the stoichiometry of marv vp oligomers is not known [ ] . marv vp is phosphorylated at several tyrosine residues located in the n-terminal region of the protein. a non-phosphorylatable mutant of vp is impaired in its ability to recruit nucleocapsids to the sites of budding, but is still able to efficiently induce particle release [ ] . vp also possesses a pppy late domain motif in its amino terminus which is important for its interaction with components of the endosomal sorting complex required for transport (escrt) machinery in order to mediate budding, including tumor susceptibility gene (tsg ) and the membrane-bound e ubiquitin ligase nedd . [ , , ] . besides the pppy motif, other motifs and single amino acids have been found to be important for particle release [ , ] . besides its role as a classical matrix protein, marv vp also acts as a virulence factor by counteracting the innate immune response and determining the host tropism for marv [ , ] . marv vp blocks the phosphorylation of janus kinases, which play an important role in multiple signaling pathways by phosphorylating and activating stat proteins ( figure ). when marv-infected cells were treated with various stimuli, including ifnα, ifnγ, and il , it was shown that the stat proteins were neither phosphorylated nor translocated into the nucleus [ , ] . it was then shown that in marv-infected cells treated with exogenous stimuli, janus kinases were also not phosphorylated and vp was identified as the viral protein inhibiting ifn signaling. it is believed that jak is the target for vp , however, the mechanism of vp -induced inhibition is not completely understood [ ] . intriguingly, ebov is also able to block ifn signaling by employing a completely different mechanism. ebov vp blocks the nuclear translocation of phosphorylated stat proteins by binding to stat and importins involved in the nuclear transport of specific stat proteins ( figure ) ( [ ] , reviewed in [ ] ). when marv was adapted to non-or less permissive animals, such as mouse and guinea pig, the adapted viruses showed mutations in vp . two of the amino acid changes in the mouse-adapted marv vp have been shown to be essential for the inhibition of ifn signaling in mouse cells, underlining the importance of ifn suppression for the virulence and host specificity of marv [ , , ] . the protein product of the sixth gene, vp , is unique to the filovirus family. vp is generally addressed as a second, minor matrix protein. however, cryo-electron tomography analysis of viral particles showed that vp is located in close proximity to the nucleocapsid proteins, suggesting that it might be part of the nucleocapsid complex [ ] . vp can easily be released from virion-associated nucleocapsids by treatment with increasing salt concentrations, indicating that it is only loosely connected to the nucleocapsid [ ] . intracellular localization studies of vp showed that a minor part of the protein (approx. %) is weakly bound to cellular membranes, including filopodia enriched with vp . vp is also distributed diffusely in the cytoplasm, relocalizes to nucleocapsid-containing inclusions, and is found in association with free nucleocapsids. co-expression of np and vp is sufficient to direct vp to np inclusions in the cytoplasm [ ] . functional studies on marv vp suggest that the protein is important for the release of viral particles in the context of infection, although it influences neither the morphology of vp -derived vlps nor the efficiency of vlp release. in addition, rnai-mediated knockdown of vp in marv-infected cells had no impact on viral genome replication, indicating that vp is involved in a step after replication and before budding [ ] . according to the model that has been proposed based on these data, vp is involved in the maturation of transport-competent nucleocapsids and/or mediates the interaction between nucleocapsids and budding sites at the plasma membrane [ ] . there is also evidence that marv vp affects transcription and replication in a transcription and replication competent vlp system [ ] . structural information about marv vp is very limited. it has been shown that it forms oligomers, preferentially tetramers [ ] . structure prediction studies have proposed an ancestral link between vp and the armadillo repeat family [ ] . the marv nucleocapsid complex consists of the genomic rna and four tightly associated proteins, np, vp , vp , and l ( figure ). encapsidation of the viral rna by the nucleocapsid proteins protects it from both rnase degradation and detection by cellular pattern recognition receptors. similar to the genomic rna, the antigenomic rna, a replicative intermediate, is also encapsidated by the nucleocapsid proteins (see below, . . transcription and replication). in contrast, the viral mrnas are not encapsidated [ ] . the nucleocapsid rather than naked rna serves as the template for viral transcription and replication. in a marv minigenome system, np, vp , and l are essential for transcription and replication [ , ] . the role of vp in marv transcription and replication is not well understood and the steps in genome amplification that require, or do not require, vp are not yet defined. the nucleoprotein np enwraps the genomic and antigenomic rnas. replication and transcription activity in a marv minigenome system depends on the presence of np [ ] . when np is expressed in the absence of other nucleocapsid proteins, it self-assembles into highly organized helical tubular structures that resemble the nucleocapsids in infected cells, indicating that it is the driving force for nucleocapsid formation [ , , ] . recently, it has been shown that the conserved n-terminal residues of marv np are sufficient to form the helical structure of the nucleocapsid core [ ] . indeed, np serves as a viral hub protein. it forms interactions with most of the other viral proteins, leading to the subcellular redistribution of these proteins. the strong binding of np to the nucleocapsid proteins vp and vp redirects both proteins into np-derived inclusions [ ] . a bipartite coiled-coil motif in the central part of np has been shown to play an important role for self-assembly and np-vp interaction [ ] . as mentioned above, there is also a weak interaction between np and vp , leading to the partial relocalization of vp into np-containing inclusions [ , ] . in addition, np interacts with vp , which is important for the transport of newly synthesized nucleocapsids to the plasma membrane [ , , , ] . interestingly, np contains a c-terminal late domain motif, psap, which has been shown to be required for budding. np recruits tsg , a component of the escrt i complex, through its late domain motif, leading to enhanced vp -induced budding [ ] . np is heavily phosphorylated at serine and threonine residues clustered in seven regions in the c-terminal part of the protein. only the phosphorylated form of np is incorporated into virions [ , ] . recent studies suggest that the phosphorylation level in region ii modulates transcription and/or replication activity [ ] . vp is a polymerase cofactor and essential for transcription and replication. together with the catalytic subunit l, vp forms the rna-dependent rna polymerase complex [ , ] . vp is tightly associated with np and serves as a bridging protein between the nucleocapsid complex and l. without vp , l is not associated with the nucleocapsids which serve as the templates for viral transcription and replication [ , ] . vp forms homo-oligomers mediated by a coiled-coil motif located in the n-terminal part of the protein. homo-oligomerization of vp is essential for its interaction with l but not needed for redistribution of vp into np-derived inclusions [ ] . vp shares many features with the phospho (p) proteins of other nns rna viruses, including its position as the second gene in the viral genome and its role in transcription and replication. however, in contrast to the p proteins, vp is either not or only very weakly phosphorylated [ ] . besides its function in transcription and replication, marv vp acts as an ifn antagonist. while the impact of ebov vp on the host's antiviral response has been intensively investigated (reviewed in [ ] ), much less information is available about similar functions of marv vp . when we tested marv vp for its ability to block ifn induction in a reporter gene assay, it blocked reporter gene expression as efficiently as ebov vp (unpublished data). in addition, bosio and colleagues [ ] reported that expression of marv vp in the absence of other viral proteins was sufficient to completely block the induction of ifnα in stimulated human dendritic cells. besides its ability to inhibit the induction of type i ifn, ebov vp has been shown to block the activation of the antiviral protein pkr and to interfere with rna silencing pathways. importantly, ebov vp is a dsrna binding protein. the c-terminus of ebov vp contains a domain with patches of basic amino acids which is important for dsrna binding and the protein's inhibitory functions (for review see [ ] ). this c-terminal region, the so-called ifn inhibitory domain, is conserved in marv vp [ ] , suggesting that marv vp possesses similar inhibitory functions. marv and ebov vp proteins show many structural similarities. both marv and ebov vp proteins are tightly associated with the nucleocapsid via their binding to np ( figure ) [ , ] . both are highly phosphorylated at n-terminally located serine and threonine residues, and phosphorylation is crucial for their interaction with np [ , ] . both contain an unusual c h zn binding domain, which is essential for the function of ebov vp as transcription initiation factor, but whose functional relevance for marv vp is not known [ ] . it has also been shown that ebov vp forms hexamers [ , ] , binds single-stranded rna [ ] , and interacts with l [ ] . however, to date, similar data for marv vp are not available. the role of marv vp in viral transcription and replication is not well understood. in contrast to ebov vp , which plays an important role in regulating transcription initiation [ , , [ ] [ ] [ ] , marv vp is not essential for transcription or replication activity in a marv minigenome system [ , ] . nevertheless, it seems to play an important role in viral amplification, since rescue of a full-length marv clone is only successful in the presence of vp [ ] . in addition, down-regulation of vp by rna interference in marv-infected cells led to the reduction of both viral protein synthesis and virion production [ ] . among the nns rna viruses, only the members of the subfamily pneumovirinae possess a protein similar to vp , m - , which functions as a transcription processivity factor [ ] . the major component of the marv polymerase complex, l, has an estimated molecular weight of kd [ ] . it is essential for transcription and replication and together with vp forms the rna-dependent rna polymerase complex (see above, . . viral proteins, vp ). l contains the enzymatic functions of the polymerase. the binding site for vp has been mapped to the n-terminal amino acid residues of l [ , ] . the l proteins of the nns rna viruses are highly conserved multifunctional proteins, which are organized in functional domains [ ] . based on this conservation with other nns rna polymerases, marv l is believed to carry out rna synthesis, capping, and polyadenylation of viral mrnas although these functions have not been shown experimentally. to date most of the studies characterizing the marv replication cycle have utilized recombinant systems, allowing for these experiments to be performed in a biosafety level (bsl- ) context, unfettered by the restrictions of a bsl- setting. surrogate systems mimicking specific steps in the marv replication cycle include marv gp-pseudotyped retroviruses or recombinant vesiculoviruses expressing gp to study entry, vlps to study budding, and minigenome systems to study replication and transcription. while such experiments allow for the more facile examination of the marv replication cycle, the findings must be recapitulated with infectious marv since all surrogate systems lack elements of the infectious virus such as the distinct morphological features and virion protein composition of marv. marburg virus entry consists of three distinct phases: cellular attachment, endocytosis, and fusion ( figure ) . based on the sequence similarity between ebov and marv gps many investigators have presumed identical functions and characteristics between the filovirus glycoproteins. this is presumptuous given the differences in glycosylation and sialic acid linkages [ ] and dependence upon endosomal proteases (see below, . . . endocytosis). despite the existence of a number of detailed studies and structural analyses of ebov gp [ ] [ ] [ ] [ ] , relatively few mechanistic studies of marv gp have been performed [ ] , although one recent post-fusion structure of marv gp has been reported [ ] . the structure of marv gp is nearly identical to that of ebov gp , indicating that the mechanisms of fusion between the two viruses is likely conserved [ ] . ( ), gp is cleaved by endosomal proteases ( ) facilitating binding to npc , the entry receptor ( ). fusion is mediated in a ph-dependent manner by gp . following release of viral nucleocapsid into the cytosol ( ), transcription of the viral genome takes place ( ) . mrna is subsequently translated by the host cell machinery ( ) . synthesis of gp takes place at the er and undergoes multiple posttranslational modifications on its way through the classical secretory pathway ( ) . positive sense antigenomes are synthesized from the incoming viral genomes ( ) . these intermediate products then serve as templates to replicate new negative sense genomes ( ) . after cleavage in the golgi, gp is transported to multivesicular bodies (mvb) and to the cell membrane where budding takes place ( ) . nucleocapsids and vp are also recruited to sites of viral budding ( ) , which is driven by vp ( ). marv gp mediates both cell attachment and fusion of the virus. there is convincing evidence that initial virus attachment at the cell surface can occur via the binding of gp carbohydrates to various cellular c-type lectins, including the hepatocyte-specific asgp-r [ ] , dc-sign and dc-signr (also known as l-sign) [ , , ] , hmgl [ , ] , and lsectin [ , ] . other cell surface proteins have also been implicated in facilitating marv entry including the tam receptor protein kinases ax , dtk, and mer [ ] , and tim- [ ] . however, although these proteins may play a role in attachment or entry of certain cell types, the ability of marv to infect cells lacking these receptors [ , , ] indicates that there might be redundancy in cellular molecules required for marv attachment to cells. a number of key residues of ebov gp that are involved in virion incorporation and gp-mediated entry have been identified [ ] and found to play a similar role in marv gp [ ] , indicating that the viruses might utilize similar mechanisms to enter the cell. following attachment, marburg virions undergo endocytosis mediated through a mechanism that currently remains undetermined. (figure ) [ , ] initial studies investigating caveolin-mediated endocytosis showed that depletion of host cell cholesterol reduced viral infectivity but presented no direct evidence of caveolae involvement [ ] . in addition, studies examining the role of caveolae in ebov endocytosis are conflicting [ , ] . a major role for clathrin in marv entry has also been proposed based upon the ability of chlorpromazine (an inhibitor of both clathrin-mediated endocytosis and macropinocytosis) as well as rnai-knockdown of clathrin heavy chain to inhibit marv gp-pseudotyped hiv- entry [ ] . a caveat to these analyses of marv endocytosis is that they were performed only in the context of marv gp-pseudotyped retroviruses which lack the characteristic filamentous morphology and size of marburg virions. while other reports have verified that cholesterol is important for live marv particle uptake [ ] , canonical caveolae-and clathrin-mediated mechanisms are unlikely to be the primary mechanism of marv entry due to steric issues. the typical marv particle size (average nm) is much larger than canonical caveolae ( - nm) or clathrin-coated pits (up to nm) whereas pseudotyped murine leukemia virus (mlv) ( × nm) and vesicular stomatitis virus (vsv) ( × nm) are not [ ] . these findings indicate that involvement of the caveolae-and clathrin-mediated endocytic pathways for virus entry may therefore be the result of the artificial nature of the pseudotyped virions and highlights the need to confirm such experiments with live marv. macropinocytosis has been identified as a major entry pathway of ebov by research using the morphologically more relevant vlps and live ebov [ ] [ ] [ ] [ ] . although none of these analyses examined the role of this pathway during marv entry, it remains an intriguing possibility given the cholesterol-dependence and large size of macropinocytotic vesicles (up to - µm) [ ] . another important process in marv entry is believed to occur while virions are being trafficked within endocytic vesicles; the proteolytic cleavage of gp . endosomal cleavage of gp has been shown to be critical for the efficient entry of marv [ , ] . the current model for marv entry involves the cleavage of gp by host endosomal cysteine proteases. this removal of a large portion of gp (including the mucin-like domain) is believed to expose the putative receptor-binding domain based on studies conducted with ebov gp [ , ] . studies examining the roles of endosomal proteases on the entry of marv and ebov have produced mixed results. experiments analyzing recombinant vsv expressing ebov gp indicate a primary role for cathepsin b (catb) and minor role for cathepsin l (catl) [ ] . entry of recombinant vsv particles containing marv gp was inhibited when cells were treated with an inhibitor of both catb and catl [ ] . these reports are confounded by a report conducted with infectious marburg and ebola viruses in which catb and catl inhibitors greatly reduced ebov infection but showed mixed results with marv [ ] . yet two other, more recent analyses determined that catb was not required for marv entry (although over-expression did enhance infectivity) and that catl was required for entry into mouse embryonic fibroblasts but not vero cells, t cells, or human macrophages [ , ] . these data as well as the ability of other proteases to greatly diminish marv infectivity [ , ] , indicate that although catb and catl likely play a role in cleavage and activation of gp in certain cell types, other endosomal proteases may also be able to facilitate gp activation via cleavage. recently, two independent studies elegantly showed the requirement of the endosomal cholesterol transporter niemann-pick c (npc ) for the entry of both marv gp-pseudotyped viruses (vsv and mlv) as well as infectious marv [ , ] . it was also shown that npc catalytic activity is not required for ebov infection indicating that specific binding to npc rather than its role in cholesterol transport is required, although this was not tested for marv [ ] . in one of the studies identifying npc as the marv entry receptor, it was also determined that members of the homotypic fusion and vacuole protein-sorting (hops) complex were important for ebov entry, although they appeared to play a less important role in marv entry [ ] . the current model of ebov and marv fusion is that gp cleavage by endosomal proteases removes heavily glycosylated domains, exposing the receptor binding domain on gp and enabling binding to npc [ ] . the membrane-bound fusogenic gp undergoes a low ph-dependent rearrangement to an extended conformation resulting in the fusion of virion and endo-lysosomal membranes [ ] . in support of the ph-dependence of gp-mediated fusion, pre-treatment of cells with ammonium chloride prevented entry of a marv gp-pseudotyped virus [ ] . a recent report with live marv showed that ammonium chloride inhibited entry and replication, but that bafilomycin a , which specifically inhibits vacuolar-type h(+) atpase and prevents re-acidification of vesicles of the central vacuolar system, surprisingly had no effect [ ] . following viral fusion with the endosomal membrane, the nucleocapsid is released into the cytoplasm (figure ). after the nucleocapsid is released into the cytoplasm of the infected cell, transcription and replication of the viral rna genome takes place (figure ). the first morphological sign of viral replication observed by em analysis is the appearance of granular material containing rna and viral proteins in the cytoplasm of the infected cells at h post infection. later on, tubular structures can be detected in the granular material representing the newly synthesized nucleocapsids embedded in the viral inclusions [ ] . while experimental data on the sites of marv replication and transcription are not available, recent studies on ebov have shown that viral replication takes place in the inclusions, while transcription was observed prior to inclusion formation [ ] . the encapsidated negative-sense rna genome is transcribed resulting in seven monocistronic mrnas by the viral polymerase. they are co-transcriptionally capped and polyadenylated and subsequently translated by the cellular machinery (figure ) . the genomic rna also serves as the template for the production of positive-sense antigenomes, which are complementary copies of the genomes. the antigenomes are encapsidated by the nucleocapsid proteins and are in turn used as templates for genome synthesis (figure ) (for review see [ ] ). as mentioned above, np, vp , l, and probably vp are needed for viral transcription and replication. analogous to ebov, it is conceivable that vp and vp inhibit transcription and replication [ , , ] . it is hypothesized that negative regulators of replication convert the active polymerase complex into an inactive state, resulting in mature and transport-competent nucleocapsids. following assembly, newly synthesized nucleocapsids are recruited to the sites of virus budding (figure ) . release of viral particles is mainly mediated by vp via recruitment of nucleocapsids from the inclusions to the plasma membrane, recruiting gp to the sites of budding, and inducing the formation and release of filamentous vlps. vp -induced budding is enhanced by np, gp, and vp [ , , ] . as is the case with many other viruses, marv exploits the vesicular transport machinery of the infected cell for viral egress, including the copii vesicular transport system and the escrt machinery. the copii vesicular transport system is used by vp for its intracellular trafficking to the multivesicular bodies, where marv budding takes place [ , ] . cellular proteins that promote particle release and are linked to the escrt machinery include tsg , vps a/b, and nedd . [ , , , ] . marv budding not only takes place at internal membranes but also at the plasma membrane [ , , ] . in cell culture, marv particles are preferentially released at filopodia, filamentous cellular protrusions [ , , ] . filopodia are used by cells to explore the extracellular environment, which includes neighboring cells, and it is believed that viral particles can bud directly into adjacent cells via filopodia-mediated cell-to-cell contact [ , ] . budding at filopodia depends on actin and is not sensitive to the depolymerization of microtubules [ ] . marv budding was observed at the basolateral membrane of polarized epithelial cells and hepatocytes [ , ] , whereas viral particles were predominantly released from the apical membrane of infected endothelial cells [ ] , suggesting that cell-type specific components determine the sites of virus release. electron tomography studies of marv-infected cells led to the following model for marv particle release: the budding process is initiated when intracellular nucleocapsids associate laterally with the plasma membrane. starting from one end, the nucleocapsids are then subsequently wrapped by the plasma membrane until the viral particles protrude vertically from the cell surface. the release of infectious filamentous marv from cultured cells peaked at - days post infection, when the cells were still intact. at d post infection, when most of the cells were vesiculated, the released virions were round or bent and infectivity was decreased [ ] . determination of the nucleocapsid orientation at the sites of budding by -d reconstructions revealed that the pointed tip of the budding nucleocapsids is oriented towards the membrane, indicating that marv budding is directional [ ] . marv infections usually occur by direct contact with infected body fluids or direct personal contact with infected animals or humans. the viruses enter the body through small skin lesions or mucosal membranes (reviewed in [ ] ). cells of the mononuclear phagocyte system, including monocytes, macrophages and dendritic cells, are early target cells of marv, as shown in different experimental animal models [ , [ ] [ ] [ ] [ ] [ ] [ ] . marv replication was observed as early as hours post infection in macrophages of infected guinea pigs [ ] , and infected monocytes have been found in cynomolgus macaques at days post infection [ ] . monocytes and macrophages were also identified as early target cells in human patients [ ] . this has been confirmed by cell culture experiments showing that primary human monocytes and macrophages are highly susceptible to marv infection and produce infectious virus [ ] [ ] [ ] . in addition, primary human monocyte-derived dendritic cells (mdcs) and endothelial cells support marv replication [ , , ] . early sites of virus replication are the lymph nodes, liver, and spleen where the most severe necrotic lesions are observed [ , , , , , , ] . these organs contain high numbers of monocytes and macrophages. migration of infected monocytes and macrophages into surrounding tissues or transport of free virus via the lymph-or bloodstream is believed to facilitate the dissemination to multiple organs, resulting in a systemic infection [ , ] . cell-free virus has been observed in the tissue and organs of infected animals, and high levels of virus have been detected in the blood [ ] [ ] [ ] , , , , ] . besides monocytes, macrophages, and dendritic cells, a wide range of cell types including hepatocytes, adrenal cortical and medullary cells and fibroblasts are permissive to marv infection [ , , [ ] [ ] [ ] [ ] , , ] . endothelial cells are late target cells during marv infection in multiple tissues. whether or not replication of marv in endothelial cells is associated with the observed vascular impairment during mvd is a matter of debate [ , ] . only low numbers of infected endothelial cells are observed in nhp infection and therefore changes in the endothelium are likely caused by paracrine effects of cytokines [ ] . in late stages of infection marv particles can be isolated from nearly every organ [ , , , , ] . despite high viral load and necrotic lesions, only minor inflammation is observed in infected tissues and organs, indicating a dysregulated immune response [ , , , ] . strong liver pathology is observed, including increased serum activity of liver enzymes. this might influence synthesis of clotting factors and contribute to the observed coagulation defects in mvd [ , , , ] . these factors together with systemic virus replication and associated pathology probably trigger the multiorgan failure associated with fatal cases. although lymphocytes are not susceptible to marv infection [ , , , ] , massive bystander lymphocyte apoptosis is a hallmark of mvd [ , , , , , ] . however, the molecular mechanisms for lymphocyte depletion and the role it may play in the pathogenesis of mvd are far from being understood. cytokine secretion may play a role in the induction of lymphocyte apoptosis, since marv-infected cells secrete cytokines known to induce apoptosis, including tnfα [ , , , ] . increased levels of tnfα have been observed in infected rhesus macaques [ ] and mice [ ] , although no increase was observed in infected cynomolgus macaques [ ] . elevated tnfα levels may also play a role in the formation of endothelial gaps in the context of marv infection [ , ] . in addition, increased survival of marv-infected guinea pigs treated with anti-tnfα antibodies suggests that tnfα indeed plays an important role in mvd pathogenesis [ ] . increased serum levels of additional proinflammatory cytokines and chemokines have been observed in infected nhps and in mice, but the reported data are not completely consistent [ , , , , ] . cytokine and chemokine secretion has also been observed in infected primary human monocytes and macrophages [ , ] . however, data about the cytokine levels in the serum of marv-infected patients are not available, but high levels of cytokines have been observed in ebov-infected patients [ ] [ ] [ ] [ ] . upregulation of the proinflammatory cytokines il (mediator of fever and acute inflammatory response) and il (chemoattraction of neutrophils and macrophages) is consistently found in infected nhps, with macrophages and plasmacytoid dendritic cells (pdcs) serving as the main sources of il secretion in the spleen [ , , ] . primary human monocytes and macrophages produce both il and il after infection [ ] . elevated levels of il have also been detected in marv-infected mice [ ] . increased levels of il β mrna and secreted protein were detected in primary human cells [ , ] , but contradictory data have been reported for the nhp model. one study reported elevated il β levels in final disease stages [ ] , whereas no change was observed in another study [ ] . ifnα levels were elevated in infected nhps and mice [ , , ] . however, no change in ifnα levels was detected in another study of infected nhps [ ] . it is unclear whether or not the observed differences are due to different marv variants being used for the studies. serum levels of several chemokines were also found to be elevated during marv infection of nhps and mice, including macrophage inflammatory proteins (mip) and monocyte chemotactic protein (mcp- ) [ , , ] . the involvement of multiple cell types along with the possible role of non-infected cells in the secretion of cytokines further complicates the analysis of existing data. primary human monocytes and macrophages are activated by marv infection inducing the secretion of cytokines. induction of cytokines has also been described using uv-inactivated marv, suggesting that viral replication might not be needed for the observed cytokine increase [ ] . in contrast, marv-infected mdcs show no upregulation of activation markers, do not secrete cytokines, and fail to stimulate t cells [ ] . mdcs treated with vlps containing marv vp and gp show functional mdc responses including cytokine secretion indicating that marv replication is required to inhibit mdc activation [ ] . however, infection of mdcs with marv did not prevent lps-induced tnfα production whereas dsrna-dependent ifnα secretion was inhibited [ ] , suggesting differential regulation of cytokines by marv. interestingly, pdcs in the spleen were identified as the major source of secreted ifnα in marv infected nhps, but secretion most likely occurs from non-infected cells [ ] . it has been shown for ebov that pdcs are not productively infected due to impairment of viral entry [ ] . these results suggest that secretion of cytokines by non-infected bystander cells might play an important role during marv pathogenesis. taken together, marv infection induces both an increase in the production of proinflammatory cytokines and high levels of chemokines, but the molecular mechanisms causing these changes are not well understood. to date four different animal models have been established for marv infection: nhps, mice, guinea pigs, and hamsters. the nhp model best reflects the symptoms and pathology observed in human cases (described in . clinical manifestations and reviewed in [ , ] ) with uniform lethality in cynomolgus and rhesus macaques as well as african green monkeys [ , , , , , , [ ] [ ] [ ] . the disease symptoms are generally the same for all types of nhps. the animals develop febrile illness with high fever, anorexia, weight loss and unresponsiveness. death is observed after - days and thrombocytopenia, lymphopenia, blood coagulation abnormalities and hemorrhages are observed. squirrel monkeys have also been successfully infected with marv, showing typical disease symptoms [ ] . recently, a small nhp model using marmosets has been developed recapitulating the features of human infections except for the typical maculopapular rash development that is observed in other nhps and humans infected with marv [ ] . rodents with an intact immune system do not develop disease after infection with marv. marv variants musoke and ci and ravv variant ravn were adapted to severe-combined immunodeficiency (scid) mice by serial passaging, reducing the time to death from - days to - days for all tested virus variants [ ] . further passaging of the scid mouse-adapted marburgviruses in immunocompetent mice was used to establish mouse models for ravv ravn and marv ci [ , ] . successful adaptation by serial passaging was also used to generate lethal infection models for both guinea pig [ , ] and hamster [ , ] . coagulation abnormalities, typical rash development, and hemorrhagic manifestations (especially in mice) are not as pronounced as in the nhp model [ ] ; (reviewed in [ ] ). neuropathogenicity, recapitulating the cns involvement described during the first human mvd outbreak in germany [ , , ] , has only been observed in the hamster model [ ] . it is not clear if cns pathology is developed in other animal models as no brain pathology has been observed in mice [ ] and cynomolgus macaques [ ] . nevertheless, virus has been isolated from brain from marv-infected marmosets, showing micro-hemorrhages [ ] . sequence comparison of the rodent-adapted viruses to the human marv isolates revealed several mutations. sixty-one nucleotide changes in the mouse-adapted ravv ravn variant were detected in the orfs of np, vp , vp , and vp ( amino acid changes in total) or untranslated regions (vp , vp , gp, vp ) [ ] . in a second study analyzing genome changes during mouse adaptation, nucleotide changes during adaptation of ravv ravn and changes for marv ci were described, with most amino acid changes occurring in vp [ ] . during guinea pig adaptation, only nucleotide changes were observed, resulting in four amino acid changes. one of these changes was located in vp and the other mutations were detected in the viral polymerase l [ ] . the only amino acid exchange detected in both the mouse-and the guinea pig-adapted marv is amino acid in vp (asp to asn). this was also the first mutation detected during mouse adaptation for both ravv ravn and marv ci , as analyzed by sequencing of serial passages [ ] . this is of particular interest because vp has been shown to function as an inhibitor of ifn signaling (see above, . . viral proteins, vp ) [ , ] . both ifn receptor-and stat -deficient mice develop disease using non-adapted marv, highlighting the importance of the ifn pathway for the control of mvd [ ] [ ] [ ] . virological, serological, and molecular diagnostic methods for the detection for marv are available, including virus isolation, elisa, rt-pcr, em, and immunohistochemistry (summarized in [ , ] ). during outbreak settings, mobile laboratories commonly use pcr and/or elisa analysis for rapid screening. sensitive elisa assays have been developed for detection of viral antigen or virus-specific antibodies using overexpressed marv np or gp [ ] [ ] [ ] [ ] . detection of filoviruses by pcr is the only assay currently available to distinguish between different virus variants for a variety of tissue and fluid specimens. the use of a combination of virus-specific primer sets for conventional rt-pcr makes the detection of all know filoviruses in a single assay possible [ ] . for more sensitive and quantitative detection, real-time rt-pcr-based assays have been developed for the detection of marv [ , [ ] [ ] [ ] [ ] [ ] . feasibility of real-time rt-pcr in the field was successfully proven during the marv outbreak in uíge, angola using an improved field laboratory-adapted rna isolation protocol [ ] . a network of european bsl- facilities in collaboration with a company (qiagen) developed the first commercial prototype of a real-time-rt-pcr assay for the detection of filoviruses [ ] . a recently developed assay, rt loop-mediated isothermal amplification (lamp), has the potential to significantly improve field diagnosis of marv infections, by eliminating the need of pcr machines [ ] . initial approaches using inactivated virus to develop a vaccine against marv were unsuccessful or had contradictory results [ ] . in addition, successful protection of rodents did not always translate into protection of nhps. for example, inactivated marv protects guinea pigs from lethal marv challenge but only % of challenged nhps survived [ , [ ] [ ] [ ] . a panel of different approaches has been used in order to develop successful vaccines for marv (reviewed in [ , ] ). recombinant gp expressed from insect cells or a dna vaccine based on gp only partially protected guinea pigs, but use of a combination of both vaccines resulted in % survival of guinea pigs [ ] . in another study, complete protection of guinea pigs using a different gp dna vaccine was reported, but only four of six vaccinated nhps survived the challenge with marv, showing incomplete protection [ ] . increased doses of a codon-optimized dna vaccine resulted in % survival of nhps, although some animals developed symptoms before recovering. in comparison to other vaccine candidates, a poor induction of virus-specific antibodies was observed using a dna vaccine [ ] . a codon-optimized dna vaccine elicited a strong antibody response and resulted in complete protection of mice with no clinical symptoms observed [ ] . vaccine candidates based on the venezuelan equine encephalitis virus (veev) replicon system expressing either marv gp along with np or gp alone completely protected guinea pigs and nhps [ ] . a vaccine based on vlps represents an additional candidate for protection against marv [ ] . complete protection of guinea pigs has been demonstrated with a vlp-based vaccine containing marv gp, with induction of virus-specific antibodies. protection with this vaccine relied on a functional cd + t cell response, whereas depletion of cd + t cells did not ablate the protective response [ ] . vlps containing marv musoke gp provided cross-protection in animals challenged with marv ci or ravv ravn in both guinea pigs and nhps [ , ] . another approach to marv vaccines is the use of viral vectors expressing marv gp. to date, two different systems have been established based on replication-defective adenoviral vectors or recombinant vsv expressing marv gp. the adenovirus-based vaccine successfully protects guinea pigs and nhps, and provides cross-protection. high levels of cross-reactive marv-specific igg and t cell responses are induced, indicating an induction of an immune response [ , ] . preexisting immunity against the adenovirus strain ad might pose a problem for its successful use in humans (reviewed in [ ] ). the vsv-based vaccine completely protects nhps and additionally has proven successful in post-exposure treatment (reviewed in [ ] ). a single immunization with recombinant vsv expressing marv musoke gp resulted in % protection of cynomolgus macaques challenged by intramuscular injection or aerosol exposure and protected against ravv ravn and marv angola [ , , ] . although marv-specific igg were produced, only low levels of neutralizing antibodies were detected [ , ] . surprisingly, t cell-mediated responses were not observed in nhps vaccinated with recombinant vsv expressing marv gp [ , ] . safety is a concern for this vaccine, especially for immunocompromised individuals, as it is a replication-competent vsv vector. however, in all vsv-based filovirus vaccine studies vsv viremia was observed only shortly after immunization. additionally, the vsv-based filovirus gp vaccine was well tolerated and protective in immunocompromised mice and nhps and lacked neurovirulence in nhps [ ] [ ] [ ] (reviewed in [ ] ). cross-protection has not been observed in animals vaccinated with marv-based vaccines and subsequently challenged with ebov, while combined marv and ebov vaccines have been successful in protection against both viruses [ , , ] . to date no approved treatment is available for marv infection. supportive care (fluids, anti-microbials, blood transfusions) has been the primary treatment of patients during mvd outbreaks. in the guinea pig model various treatments had some success as reflected by prolonged survival or increased survival rates. applied treatments included cytokine inhibition, ifn treatment, or antibody transfer. the tested treatments were unsuccessful in the nhp model (reviewed in [ , ] ). a third of ebov-infected nhps survived, however, following treatment with recombinant nematode coagulant protein , while only one of six marv-infected animals survived [ , ] . treatment using antisense technology to block viral protein expression using phosphorodiamidate morpholino oligomers (pmo) beginning to minutes after marv infection completely protected nhps [ ] . additionally, a small molecule inhibitor showed complete protection of marv-infected mice when administered h after infection but has not been tested in nhps [ ] . the vsv-based vaccine expressing marv gp has also been demonstrated to be effective as a post-exposure treatment. a hundred percent survival of nhps was 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of the l gene and ' trailer region of ebola virus the ebola virus genomic replication promoter is bipartite and follows the rule of six marburg virus gene encodes the virion membrane protein, a type i transmembrane glycoprotein gp mrna of ebola virus is edited by the ebola virus polymerase and by t and vaccinia virus polymerases the virion glycoproteins of ebola viruses are encoded in two reading frames and are expressed through transcriptional editing the nonstructural small glycoprotein sgp of ebola virus is secreted as an antiparallel-orientated homodimer a new ebola virus nonstructural glycoprotein expressed through rna editing intracellular transport and processing of the marburg virus surface protein in vertebrate and insect cells acylation of the marburg virus glycoprotein the marburg virus surface protein gp is phosphorylated at its ectodomain carbohydrate structure of marburg virus glycoprotein characterization of filoviruses based on differences in structure and antigenicity of the virion glycoprotein proteolytic processing of marburg virus glycoprotein the asialoglycoprotein receptor is a potential liver-specific receptor for marburg virus folate receptor-alpha is a cofactor for cellular entry by marburg and ebola viruses dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus tyro family-mediated cell entry of ebola and marburg viruses human macrophage c-type lectin specific for galactose and n-acetylgalactosamine promotes filovirus entry different potential of c-type lectinmediated entry between marburg virus strains t-cell immunoglobulin and mucin domain (tim- ) is a receptor for zaire ebolavirus and lake victoria marburgvirus ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection crystal structure of the ebola virus membrane fusion subunit, gp , from the envelope glycoprotein ectodomain crystal structure of the marburg virus gp core domain in its post-fusion conformation role of the transmembrane domain of marburg virus surface protein gp in assembly of the viral envelope the cytoplasmic domain of marburg virus gp modulates early steps of viral infection conserved receptor-binding domains of lake victoria marburgvirus and zaire ebolavirus bind a common receptor characterization of marburg virus glycoprotein in viral entry an interferon-alpha-induced tethering mechanism inhibits hiv- and ebola virus particle release but is counteracted by the hiv- vpu protein. cell host microbe broad-spectrum inhibition of retroviral and filoviral particle release by tetherin tetherin-mediated restriction of filovirus budding is antagonized by the ebola glycoprotein the ebola virus glycoprotein and hiv- vpu employ different strategies to counteract the antiviral factor tetherin the gp-protein of marburg virus contains the region similar to the 'immunosuppressive domain' of oncogenic retrovirus p e proteins implication of a retrovirus-like glycoprotein peptide in the immunopathogenesis of ebola and marburg viruses ectodomain shedding of the glycoprotein gp of ebola virus sorting of marburg virus surface protein and virus release take place at opposite surfaces of infected polarized epithelial cells interactions with the host cell tsg is recruited by a late domain of the nucleocapsid protein to support budding of marburg virus-like particles phosphorylation of marburg virus matrix protein vp triggers assembly of nucleocapsids with the viral envelope at the plasma membrane establishment and application of an infectious virus-like particle system for marburg virus vp , the matrix protein of marburg virus, is associated with membranes of the late endosomal compartment interactions of marburg virus nucleocapsid proteins the matrix protein of marburg virus is transported to the plasma membrane along cellular membranes: exploiting the retrograde late endosomal pathway generation of marburg virus-like particles by co-expression of glycoprotein and matrix protein multivesicular bodies as a platform for formation of the marburg virus envelope vacuolar protein sorting pathway contributes to the release of marburg virus oligomerization and polymerization of the filovirus matrix protein vp interaction of tsg with marburg virus vp depends on the pppy motif, but not the pt/sap motif as in viruses , the case of ebola virus, and tsg plays a critical role in the budding of marburg virus-like particles induced by vp , np, and gp regulation of marburg virus (marv) budding by nedd . : a different ww domain of nedd . is critical for binding to marv and ebola virus vp conserved motifs within ebola and marburg virus vp proteins are important for stability, localization, and subsequent budding of virus-like particles identification of amino acids in marburg virus vp that are important for virus-like particle budding marburg virus vp antagonizes interferon signaling in a species-specific manner marburg virus evades interferon responses by a mechanism distinct from ebola virus global suppression of the host antiviral response by ebolaand marburgviruses: increased antagonism of the type i interferon response is associated with enhanced virulence the ebola virus interferon antagonist vp directly binds stat and has a novel, pyramidal fold filoviral immune evasion mechanisms development and characterization of a mouse model for marburg hemorrhagic fever key genomic changes necessary for an in vivo lethal mouse marburgvirus variant selection process vp of marburg virus influences formation of infectious particles fold prediction of vp protein of ebola and marburg viruses using de novo fragment assembly three of the four nucleocapsid proteins of marburg virus, np, vp , and l, are sufficient to mediate replication and transcription of marburg virus-specific monocistronic minigenomes ultrastructural organization of recombinant marburg virus nucleoprotein: comparison with marburg virus inclusions morphology of marburg virus np-rna nucleocapsid formation and rna synthesis of marburg virus is dependent on two coiled coil motifs in the nucleoprotein characterization of filovirus protein-protein interactions in mammalian cells using bimolecular complementation genus-specific recruitment of filovirus ribonucleoprotein complexes into budding particles the nucleoprotein of marburg virus is phosphorylated the nucleoprotein of marburg virus is target for multiple cellular kinases phosphorylation of marburg virus np region ii modulates viral rna synthesis comparison of the transcription and replication strategies of marburg virus and ebola virus by using artificial replication systems homo-oligomerization of marburgvirus vp is essential for its function in replication and transcription co-and posttranslational modifications and functions of marburg virus proteins ebola and marburg viruses replicate in monocyte-derived dendritic cells without inducing the production of cytokines and full maturation a c-terminal basic amino acid motif of zaire ebolavirus vp is essential for type i interferon antagonism and displays high identity with the rna-binding domain of another interferon antagonist, the ns protein of influenza a virus phosphorylation of vp impairs ebola virus transcription phosphorylation of marburg virus vp at serines and is critical for its interaction with np inclusions ebola virus transcription activator vp is a zinc-binding protein oligomerization of ebola virus vp is essential for viral transcription and can be inhibited by a synthetic peptide crystal structure of the c-terminal domain of ebola virus vp reveals a role in transcription and nucleocapsid association the ebola virus vp is an rna binding protein the ebola virus ribonucleoprotein complex: a novel vp -l interaction identified ebola virus vp -mediated transcription is regulated by rna secondary structure formation role of ebola virus vp in transcription reinitiation role of vp phosphorylation in the ebola virus replication cycle rescue of recombinant marburg virus from cdna is dependent on nucleocapsid protein vp inhibition of marburg virus protein expression and viral release by rna interference transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand rna virus sequence comparison of five polymerases (l proteins) of unsegmented negative-strand rna viruses: theoretical assignment of functional domains characterization of the receptor-binding domain of ebola glycoprotein in viral entry comprehensive analysis of ebola virus gp in viral entry structural basis for membrane fusion by enveloped viruses covalent modifications of the ebola virus glycoprotein lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus the dc-sign-related lectin lsectin mediates antigen capture and pathogen binding by human myeloid cells lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha association of the caveola vesicular system with cellular entry by filoviruses folate receptor alpha and caveolae are not required for ebola virus glycoprotein-mediated viral infection differential requirements for clathrin endocytic pathway components in cellular entry by ebola and marburg glycoprotein pseudovirions analysis of filovirus entry into vero e cells, using inhibitors of endocytosis, endosomal acidification, structural integrity, and cathepsin (b and l) activity ebola virus enters host cells by macropinocytosis and clathrinmediated endocytosis the tyro receptor kinase axl enhances macropinocytosis of zaire ebolavirus the ebola virus glycoprotein mediates entry via a non-classical dynamin-dependent macropinocytic pathway ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and aate endosomes virus entry by macropinocytosis filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences cathepsins b and l activate ebola but not marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of tmprss expression endosomal proteolysis of the ebola virus glycoprotein is necessary for infection role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein distinct mechanisms of entry by envelope glycoproteins of marburg and ebola (zaire) viruses inclusion bodies are a site of ebola virus replication both matrix proteins of ebola virus contribute to the regulation of viral genome replication and transcription ebola virus (ebov) vp inhibits transcription and replication of the ebov genome ebola virus matrix protein vp uses the copii transport system for its intracellular transport budding of marburgvirus is associated with filopodia electron tomography reveals the steps in filovirus budding recombinant marburg virus expressing egfp allows rapid screening of virus growth and real-time visualization of virus spread basolateral budding of marburg virus: vp retargets viral glycoprotein gp to the basolateral surface marburg virus and mononuclear phagocytes: study of interactions) cellular immune response to marburg virus infection in cynomolgus macaques pathogenesis of marburg hemorrhagic fever in cynomolgus macaques development of a model for marburgvirus based on severe-combined immunodeficiency mice ebola virus infection in guinea pigs: presumable role of granulomatous inflammation in pathogenesis marburg hemorrhagic fever: report of a case studied by immunohistochemistry and electron microscopy filovirusinduced endothelial leakage triggered by infected monocytes/macrophages breakdown of paraendothelial barrier function during marburg virus infection is associated with early tyrosine phosphorylation of platelet endothelial cell adhesion molecule- infection and activation of monocytes by marburg and ebola viruses apoptosis induced in vitro and in vivo during infection by ebola and marburg viruses the morbid anatomy of marburg virus disease marburg and ebola hemorrhagic fevers: does the primary course of infection depend on the accessibility of organ-specific macrophages? viral hemorrhagic fever-a vascular disease? postexposure treatment of marburg virus infection marburg agent disease: in monkeys respiratory marburg virus infection in guinea pigs marburg agent disease: pathology vestn ross akad med nauk inactivated marburg virus elicits a nonprotective immune response in rhesus monkeys induction of immune mediators in human cultured mononuclear cells by marburg virus markedly elevated levels of interferon (ifn)-gamma, ifn-alpha, interleukin (il)- , il- , and tumor necrosis factor-alpha associated with fatal ebola virus infection human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis monocyte-derived human macrophages and peripheral blood mononuclear cells infected with ebola virus secrete mip- alpha and tnf-alpha and inhibit poly-ic-induced ifn-alpha in vitro serology and cytokine profiles in patients infected with the newly discovered bundibugyo ebolavirus ebola and marburg virus-like particles activate human myeloid dendritic cells ebola virus failure to stimulate plasmacytoid dendritic cell interferon responses correlates with impaired cellular entry vervet monkey disease. experiment infection of guinea pigs and monkeys with the causative agent certain pathogenetic characteristics of a disease in monkeys in infected with the marburg virus by an airborne route the sensitivity of different experimental animals to the marburg virus a small nonhuman primate model for filovirus-induced disease genomic differences between guinea pig lethal and nonlethal marburg 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filoviruses virus-like particles exhibit potential as a pan-filovirus vaccine for both ebola and marburg viral infections monovalent virus-like particle vaccine protects guinea pigs and nonhuman primates against infection with multiple marburg viruses de novo syntheses of marburg virus antigens from adenovirus vectors induce potent humoral and cellular immune responses recombinant vesicular stomatitis virus-based vaccines against ebola and marburg virus infections vesicular stomatitis virusbased vaccines protect nonhuman primates against aerosol challenge with ebola and marburg viruses live attenuated recombinant vaccine protects nonhuman primates against ebola and marburg viruses assessment of a vesicular stomatitis virus-based vaccine by use of the mouse model of ebola virus hemorrhagic fever vesicular stomatitis virus-based ebola vaccine is well-tolerated and protects immunocompromised nonhuman primates recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates single-injection vaccine protects nonhuman primates against infection with marburg virus and three species of ebola virus vaccine to confer to nonhuman primates complete protection against multistrain ebola and marburg virus infections treatment of ebola virus infection with a recombinant inhibitor of factor viia/tissue factor: a study in rhesus monkeys advanced antisense therapies for postexposure protection against lethal filovirus infections antiviral activity of a small-molecule inhibitor of filovirus infection postexposure protection against marburg haemorrhagic fever with recombinant vesicular stomatitis virus vectors in non-human primates: an efficacy assessment this article is an open access article distributed under the terms and conditions of the creative commons attribution license the authors thank s. carroll and j. towner, cdc, atlanta, ga for providing the phylogenetic tree analysis shown in figure b , bobbie rae erickson, cdc, atlanta, ga for providing the photograph shown in figure , and w. slenczka, university of marburg, germany for providing the electron micrograph shown in figure . the picture in figure a was taken from [ ] the photographs shown in figure b were taken from [ ] . this work was supported by national institutes of health (nih) grants u -ai and ai (new england regional center of excellence-kasper, subaward - ). the authors declare no conflict of interest. key: cord- -wjf c a authors: friis-nielsen, jens; kjartansdóttir, kristín rós; mollerup, sarah; asplund, maria; mourier, tobias; jensen, randi holm; hansen, thomas arn; rey-iglesia, alba; richter, stine raith; nielsen, ida broman; alquezar-planas, david e.; olsen, pernille v. s.; vinner, lasse; fridholm, helena; nielsen, lars peter; willerslev, eske; sicheritz-pontén, thomas; lund, ole; hansen, anders johannes; izarzugaza, jose m. g.; brunak, søren title: identification of known and novel recurrent viral sequences in data from multiple patients and multiple cancers date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: wjf c a virus discovery from high throughput sequencing data often follows a bottom-up approach where taxonomic annotation takes place prior to association to disease. albeit effective in some cases, the approach fails to detect novel pathogens and remote variants not present in reference databases. we have developed a species independent pipeline that utilises sequence clustering for the identification of nucleotide sequences that co-occur across multiple sequencing data instances. we applied the workflow to sequencing libraries from cancer samples of different cancer and tissue types, non-template controls, and test samples. recurrent sequences were statistically associated to biological, methodological or technical features with the aim to identify novel pathogens or plausible contaminants that may associate to a particular kit or method. we provide examples of identified inhabitants of the healthy tissue flora as well as experimental contaminants. unmapped sequences that co-occur with high statistical significance potentially represent the unknown sequence space where novel pathogens can be identified. the international agency for research on cancer (iarc) lists several biological species with carcinogenic potential in humans [ ] . this list comprises a bacterium (species helicobacter pylori), three parasitic flukes (clonorchis sinensis, opisthorchis viverrini and schistosoma haematobium), and seven viruses: human papillomaviruses (hpv), human immunodeficiency virus- (hiv- ), epstein-barr virus (ebv), hepatitis b and c virus (hbv and hcv), kaposi's sarcoma-associated herpesvirus (kshv), and human t-cell lymphotropic virus type (htlv- ). with the advent and spread of low-cost sequencing technologies, many viruses were discovered in the last decade [ ] [ ] [ ] [ ] [ ] [ ] [ ] . one interesting discovery that fuelled the search for oncoviruses was merkel cell polyomavirus (mcpyv) found to be clonally integrated in merkel cell carcinomas [ , ] . the computational biology community has promptly responded to the growing need for specialised algorithms and pipelines to analyse the wealth of data [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . table s summarises the main features of some of the common approaches. in spite of particularities in the implementation, these methodologies share key conceptual similarities: first, sequencing reads or assembled contigs that originate from the host are identified and discarded, a process termed computational subtraction [ , ] . when the genomes or the concentrations of foreign species are small compared to host genomes, this step eliminates a substantial fraction of the total sequencing reads. second, the remaining non-host sequences are compared to a library of known reference sequences for taxonomic characterisation. the aforementioned methods identify species present across multiple samples, and the recurrence of a given viral entity may indicate an association to disease [ , ] . albeit conceptually valid, this bottom-up approach is inherently limited to the pre-existence of the organism in the reference databases, whereas novel oncoviruses showing partial or no similarity to known sequences will be missed. current efforts aiming at estimating and characterising metagenomic diversity are far from a complete mapping of the (viral) sequence-space [ ] . in fact, it is common to observe that a small but significant amount of unknown sequences, the so-called dark matter [ ] , goes through the current analysis pipelines without proper characterisation and is discarded from further analyses [ , , ] . here, we propose a method capable of identifying the recurrence of sequences across related samples independently of their existence in reference databases. our top-down approach compares samples and establishes recurrence prior to the taxonomic characterisation of the sequences. thus, enabling the identification of both known and novel biological entities. our method has conceptual similarities to the work of andreatta et al. [ ] where clustering of genes is used to find families that are predominantly found in pathogenic bacteria. attending to koch's postulates as modified by fredericks and relman [ ] , sequences from biological entities with a causative or facilitator role would be present in diseased samples and absent in healthy controls. in addition, recent studies documented the presence of contaminating and/or artefactual sequences that source from the laboratory kits and reagents used for sample processing and library preparation [ , [ ] [ ] [ ] [ ] [ ] . if not properly addressed, these confounding observations may lead to erroneous conclusions [ , ] . our method ascertains the statistical associations between recurrent sequences and a collection of features that describe the samples with respect to tissue, disease type, laboratory method, etc. additionally, the presence of other known technical problems, such as cluster invasion on the sequencing flow cells [ ] , might be detected. the study was conducted in accordance with the declaration of helsinki. two ethical boards reviewed the protocol of this study: the regional committee on health research ethics (case no. h- - -fsp ) and the national committee on health research ethics (case no. ). because the study used only samples that were anonymised at collection both boards waived the need for informed consent in compliance with the national legislation in denmark. two hundred and fifty-two cancer samples of different types were collected from various locations in denmark and hungary. cancer samples of malignant melanoma, acute myeloid leukaemia (aml), b-cell chronic lymphocytic leukaemia (b-cll), chronic myelogenous leukaemia (cml), and t-lineage acute lymphoblastic leukaemia (t-all; n = ) were obtained from aarhus university hospital, denmark. b-cell precursor acute lymphoblastic leukaemia (bcp-all), oropharyngeal head and neck cancer, testicular cancer, and t-all (n = ) were obtained from rigshospitalet, denmark (copenhagen university hospital). basal cell carcinoma, and mycosis fungoides (cutaneous t-cell lymphoma) were obtained from bispebjerg hospital (copenhagen university hospital). samples of bladder cancer, breast cancer, colon cancer, as well as ascites fluid of breast cancer, colon cancer, ovarian cancer, and pancreatic cancer were obtained from the danish cancer biobank, herlev hospital, denmark. b-cell lymphoma cell lines were obtained from aalborg university hospital, denmark. vulva cancer samples were obtained from the national institute of oncology, budapest, hungary. libraries were prepared at the center for geogenetics (cgg), university of copenhagen, denmark based on seven different methods for sample processing comprising five different enrichment methods and shotgun sequencing targeting total dna or rna (table s ). the enrichment methods used in the current work were circular genome amplification, sequence capture with retrovirus probes, virion enrichment (dna and rna), and mrna enrichment. further details on sample processing and library preparation have been published elsewhere [ , , ] , except for mrna enrichment which was performed using dynabeads mrna direct extraction kit (thermo fisher scientific, waltham, ma, usa) followed by scriptseq v rna-seq library preparation kit as for total rna analysis [ ] . ultimately, the data set consisted of dna and rna libraries, for which ˆ bp paired end sequencing was performed using the illumina hiseq platform at bgi-europe, copenhagen, denmark. the sequencing libraries thus originated from different cancer samples, non-template controls, and exogenous controls. the distribution of methods, libraries and controls for each sample type is provided in table s . samples were preferably analysed with multiple methods, thus out of samples were analysed with more than one laboratory method (table s ). the datasets went through a sequential pipeline with modules (in order) of preprocessing, computational subtraction of host sequences, low-complexity sequence removal, sequence assembly, clustering, association to metadata features, and taxonomical annotation. figure provides a schematic representation of the pipeline used to identify recurrent sequences across related samples. demultiplexing was performed using a local python script to partition the reads based on exact matches in the fastq header lines to the multiplexed indices provided. preprocessing of reads was performed for all datasets in parallel using adapterremoval [ ] with the following parameters {-trimns, -trimqualities, -minquality , -minlength , -collapse, -outputcollapsed, -outputcollapsedtruncated, -singleton}. read ends were trimmed for low quality base calls. reads were discarded if the length after trimming fell below bp. in these cases, the other read in a pair was kept as a singleton. overlapping paired reads from short inserts were collapsed into a single read if the overlap was longer than bp, according to the default behaviour of adapterremoval. preprocessed reads were filtered if they showed homology to the human reference genome, which included decoys and alternative sequences from version gca_ . (grch ) of the genome reference consortium (downloaded august , ) . mapping to the human genome was done using bwa [ ] version . . -r with the mem alignment algorithm and default parameters. all mapped reads without sequence alignment/map (sam) [ ] flag were discarded. single-unmapped reads from read pairs were kept. human depleted reads were filtered for low-complexity regions using the ncbi-blast associated module dustmasker [ ] and default parameters. reads containing low-complexity stretches of bp or longer were discarded. assembly of the remaining (non-human, high complexity) reads was performed with idba-ud [ ] and parameters {-precorrection}. contigs shorter than bp were discarded. a total of , , contigs, originating from the data sets, went through the entire pipeline. contigs ranged from bp to , bp with an overall n of bp. contigs from all data sets were pooled and clustered based on pairwise sequence homology using cd-hit [ ] , in fast mode. we chose parameters for clustering that maximised grouping of similar sequences while minimising inclusion of unrelated sequences. we table s from where we chose the final settings {-c . -as . -g } the datasets were described with a panel of different binary metadata features, for example tissue or disease characteristics (table s ) . features logically assessed whether they related to a particular dataset or not. features describing less than five datasets were removed. additionally, features that correlated perfectly in terms of matthew's correlation coefficient (mcc =˘ ) were merged. these filters resulted in unique features (table s ) . biological features (n = ) defined sample type, for instance tissue or disease category. methodological features (n = ) described specifics for sample preparation such as extraction kits, enrichment methods, polymerases, primers, buffer, filters used, or the laboratory where the work was performed, etc. technical features (n = ) defined the flow cell lane identifiers and whether resequencing was performed. the distributions of datasets and samples across the features are provided in table s . associations in the clustered contigs and metadata features were evaluated with fisher's exact test using a one-tailed alternative hypothesis (greater) and calculated in r using the function fisher.test [ ] . annotation of taxonomy was performed in two rounds. first aligning contigs with blastn [ ] with parameters {-evalue . } using default {-task megablast} to a frozen version of the ncbi nucleotide database nt (downloaded february ). secondly, using blastx with parameters {-evalue . } of all unmapped contig stretches to a frozen version of the ncbi non-redundant protein database nr (downloaded february ). the best hit by highest bit-score was kept for each contig. the taxonomy database (downloaded february ) was used to translate all genbank identifiers from hits to taxonomy identifiers. the taxonomy identifiers were then used to obtain the complete taxonomical lineage and extract scientific names of species. the abundances of all species in each cluster were used to calculate the species evenness index as defined by mulder et al. [ ] . clusters were annotated as the most abundant species in each cluster. the software to use after the assembly step has been uploaded at https://github.com/ jensfriisnielsen/sequence_recurrence. sequence clusters that have been described in detail throughout the manuscript have been included as supplementary files. clustering performance depends on the adequate selection of parameters. we experimented with a variety of configurations described by c xay ygz where x,y,y,z varied. the variables denote minimum percentage of sequence identity x (c x), minimum percentage of alignment length y (ay y) based on mode y of shortest (as) or longest (al) contig in alignment, as well as using local (g ) or global (g ) alignment mode z (gz). for example, a configuration encoded c as g would represent a clustering that requires global alignments with a % minimum sequence identity over % of the length of the shortest contig. the full list of investigated parameter combinations can be found in table s . we chose the parameters based on the performance of the clustering of expected contaminant sequences from avian leukosis virus (accession id ay ) [ ] and other related avian retroviruses (ars) such as avian myeloblastosis virus [ ] . ars are used in the manufacture of the reverse transcriptase failsafe pcr enzyme (epicentre, madison, wi, usa) included in the utilized scriptseq v rna-seq library preparation kit (illumina, san diego, ca, usa). this kit is commonly used for preparation of rna libraries [ ] . we identified clusters containing contigs that aligned to species of the alpharetrovirus genus (ncbi taxa-id: ) according to blastn and blastx hereafter referred to as ar clusters. all contigs in ar clusters were resolved with blastn and blastx and two metrics were considered for ar clusters. as the first performance metric, we computed the odds ratios (ors) of the associations between the presence of ar in the clusters and the use of the scriptseq kit. we used a ˆ contingency table defining the sets of libraries: ar positive and scriptseq positive (arpssp); ar positive and scriptseq negative (arpssn); ar negative and scriptseq positive (arnssp); ar negative and scriptseq negative (arnssn). or is then defined as the ratio arpsspˆarnssn / (arpssnˆarnssp) and describes the strength of the association between clusters and features. ors above indicate association between the presence of the ar virus and the use of the scriptseq kit. ors for all ar clusters were inspected in different parameter settings ( figure s ). the ors varied mostly block-wise with the parameters. the largest differences observed were between usages of the shortest or longest sequence in alignments with the alignment length filter. associations from the shortest mode tended to have higher dispersion in the range of ors. furthermore, one block of clustering results using global alignment mode, alignment length based on the shortest contig, and a minimum sequence identity of % (c ˆasyg ), had an overall high range of ors as well as the highest minimum values. this suggested that the clustering was able to reproduce the association between ar clusters and the scriptseq kit. in contrast, the clustering with parameter settings c as g had a very broad range of ors corresponding to a skewed clustering where some clusters had incorporated most sequences and left other clusters with only a few contigs. as a second performance metric we computed the species evenness [ ] indices of the ar clusters represented in figure s . the species evenness index is a score that derives from the shannon's diversity index [ ] and compares the abundance of each species within a cluster. an index of zero is assigned to clusters that are constituted uniquely by contigs mapping to a single species. contrarily, scores closer to would indicate that the cluster points to several species and that these are equally abundant. in our experiment, we favoured lower evenness indices as they indicate that clusters were able to single out species correctly. for example, parameter settings c al g generally had a high level of species evenness (median . ) in clusters, suggesting an incorrect separation of species. in stark contrast, a block of parameters using global alignment mode, alignment length based on shortest sequence, % minimal sequence identity, and a minimum alignment length of / / / / % of the shortest sequence (c asyg ) all had a median species evenness of . this group of parameter settings also showed desirable performance in terms of or, as mentioned before. generally it seemed that global mode (g ) had better ors than local mode (g ) when keeping other parameters fixed. additionally keeping % minimal sequence identity (c ) and varying minimal length of alignment in shortest mode (as) seemed stable in both ors and species evenness indices indicating that these close parameter settings were generally good. we chose to proceed with a clustering based on global alignments with a % minimum sequence identity over % of the length of the shortest contig (c as g ). this configuration resulted in a total of , clusters. of these, , clusters contained contigs from at least five different data sets and represented , different contigs. the full distribution of cluster sizes can be found in table s . the associations between the clusters and the binary metadata features were assessed using a fisher's one tailed exact test. there were , significant associations having p-value < . e- , corresponding to a . significance level when using bonferroni's correction for multiple testing [ ] . the significant associations were arranged in unique clusters and with unique features. the distribution of the significant associations showed that recurrent sequences originated from diverse sources and that individual clusters often associated to more than one feature (figure ). furthermore it is evident that the clusters tend to group in their associations. likely, these groupings represent one or more organisms. we investigated the nature of the clusters accounting only for the associated feature with the smallest p-value; hereafter described as the strongest associations. there were unique features involved in all the strongest associations. the distribution of p-values for each feature is represented in figure . the strongest associations were distributed according to biological, methodological and technical associations. these unique features were arranged in biological, methodological and technical features. most p-values were above e- and associations with lower p-values were to a few methodological features annotated as extraction kits: qiaamp dna mini kit (f ) (qiagen, hilden, germany), dnase/rnase: promega dnase (f ) (promega, madison, wi, usa), and dnase/rnase: promega dnase stop solution (f ), purification kit: rneasy minelute, qiagen (f ), library build: nebnext, new england biolabs (f ) (new england biolabs, ipswitch, ma, usa), and scriptseq v rna-seq, illumina (f ); the latter with a minimum p-value of . e- . the boxes span the first and third quartiles. the dark band inside each box represents the median. the whiskers of the boxes extend to the lowest and highest values within a distance of . times the interquartile range. as can be seen, most p-values were above e- , but a few methodological features have associated clusters with very low p-values, such as f , f , f , f , f , and f . the library preparation kit scriptseq v rna-seq, illumina (f ) displays strongly associated clusters with p-values as low as . e- that mapped as species avian myeloblastosis-associated virus. clusters that were annotated as ncbi species parvovirus nih/cqv were associated to laboratory-kit rneasy minelute, qiagen (f ) with minimal p-value . e- . finally, a cluster annotated as acanthocystis turfacea chlorella virus mn . (atcv) was associated to dnase/rnase: promega dnase stop solution (f ) with p-value = . e- . using blast and the ncbi taxonomy database a taxonomic characterisation was attempted for the , contigs in the clusters. this resulted in a taxonomical annotation of clusters using blastn and an additional clusters when using blastx. for clusters, neither blastn nor blastx found significant species in the database. these clusters remained uncharacterised (table ) . we found that almost all clusters significantly associated to biological features could be annotated ( of ) in contrast to non-biologically associated clusters ( of ). a total of unique species were annotated corresponding to clusters. the human microbiome project (hmp) [ ] defines a collection of reference genomes built from metagenomic samples and associates these to specific sites and tissues across human body sites. we used this data set of associations as a confirmation that our pipeline was able to correctly detect and taxonomically characterise recurrent biologically relevant sequences. hmp provides a list of commensal organisms commonly found in the three sites that relate to our samples: the gastrointestinal tract, oral cavity and urogenital tract. we observed the strongest, significant associations between the expected organisms and biopsies from colon cancer, oral cavity cancer, and vulva cancer. the taxonomical characterisation of these clusters is described in table . seven clusters significantly associated to colon cancer biopsies describing four different organisms that inhabit the gastrointestinal tract according to hmp, and clusters significantly associated to oral cavity cancer describing different organisms present in the oral cavity in hmp. finally, we also discovered a cluster significantly associated to vulva cancer annotated as species campylobacter ureolyticus (p-value = . e- ), an inhabitant of the urogenital tract as described by hmp. table . taxonomical characterisation of certain biologically associated clusters. the clusters are significantly associated with lowest p-values to biological features and the species annotations are described by hmp. in cases where several clusters shared the annotated species, the lowest p-value of the associations is given #sig: number of significant clusters. cluster in the methodological associations, we correctly detected the strong known association (p-value: . e- ) of avian myeloblastosis-associated virus (accession l . ) used in the manufacture of the scriptseq v rna-seq library preparation kit (f ). as the clustering parameters were evaluated with this known contaminant, this is expected. furthermore, we annotated clusters as ncbi taxonomy species parvovirus nih-cqv (accession kc . ; ncbi taxa-id ), an established contaminant [ , ] . the associated feature with lowest p-value to the parvovirus clusters suggested a contamination from the rneasy minelute purification kit (f ) manufactured by qiagen (p-value: . e- ). in addition, a single cluster annotated as ncbi taxonomy species acanthocystis turfacea chlorella virus mn . (accession jx . , taxa-id ) with lowest associated p-value (p-value = . e- ) to laboratory kit dnase/rnase: promega dnase stop solution (f ). atcv- was previously reported as a contaminant [ ] . in addition to the sequences that were characterised in the previous step, we found examples of uncharacterised clusters. the contigs in these clusters varied substantially in length ranging from a minimum of bp to a maximum of . kb (n = bp). our approach provides the capability to discover these recurrent novel sequences, but also permits the investigation of their plausible origin. most associations were methodological (table ), probably sourcing from nucleotide sequences contained in various laboratory kits (figure ) . for instance, out of the methodologically associated clusters, there were associated clusters to the laboratory reagent dnase/rnase: promega dnase stop solution (minimum p-value: . e- ). additionally, recurring sequences were attributed to technical issues of the flow cell lanes (minimum p-value: . e- in feature ). in total, unmapped clusters were associated to a biological feature, namely oral cavity cancer, with the longest contig of each cluster at , , , and bp and with respective p-values of . e- , . e- , . e- , and . e- . to further clarify the unresolved biologically associated sequences, we manually investigated the cluster representatives using the newest databases (december ) at the ncbi web-interfaces for blastn, blastx and ccd v. . (conserved domains) [ ] with default parameters and an e-value < . (table ) . all cluster representatives could be explained as commensal bacteria related to the oral cavity as described by hmp. in order of increasing length, the cluster representatives were identified as: prevotella veroralis, prevotella veroralis, prevotella fusca jcm , and peptostreptococcus anaerobius as the best hits with percent sequence identity: %, %, %, and %, respectively. cluster representatives and contained both bacterial and phage-like conserved domains. the super family duf is of unknown function but related to bacteria and the nd super family is the nicotine adenine dinucleotide (nadh) dehydrogenase subunit involved in electron transport. conversely, phage_base_v is related to the tail of phages and rve is an integrase domain that could also be explained as part of a transposon. likely these sequences derived from less well-described parts of the microbiome. usually, virus discovery in shotgun sequencing studies involves processing millions of reads in a viroinformatics pipeline. existing tools typically offer a comprehensive taxonomical description of a single sample that is compared to the taxonomy of other samples to determine their relevance. a downside of this bottom-up methodology is that novel sequences that cannot be sufficiently well characterised in the first round are often discarded in the process. another disadvantage is that potential contaminants will have to be controlled for in the post-processing of the data, an effort that is often omitted [ ] . in the present study, we have presented a methodology to categorise recurring sequences according to experimental origin and metadata features. additionally, using this methodology we could replicate both biological and methodological sequence associations known from the literature as well as pinpoint new unannotated recurring sequences. in this study, we had no datasets and features of healthy biological controls. we included a comparison to published reference genomes from hmp to validate that biologically co-occurring sequences can be found with the presented methodology. in this case, we are most likely observing normal biological inhabitants of the tissue samples, something our metadata scheme does not account for. the disease association of many of these organisms is obviously not fully known, and some of them could be related to disease features outside the cancer domain, features that we did not include in the present study. optimising clustering parameters for one virus family might not result in the optimal separation of other families. here, we optimised clustering parameters to rediscover the association of sequences to a known laboratory kit. using these clustering parameters may result in a non-optimal separation of clusters that biologically belonged together, or the reverse problem-merged clusters that reflected different biological units. optimal separation is likely problem-specific. different taxonomic units would require the use of different clustering parameters to separate. however, choosing taxonomy-specific parameters requires a working hypothesis of the most likely findings. here, we focused on the general problem of associating sequences to features using a known association to guide the choice of clustering parameters. a combination of several features may be the true foundation of particular sequences but this was not explored in this work. there may also be situations where a combination of clusters is the correct association to a particular feature. for instance, a virus that is present with a low titre may be sequenced sporadically resulting in less than full coverage and several non-coherent contigs from different viral genome regions. each cluster may include an incomplete amount of data sets and thereby artificially show a weaker association. merged and viewed as one, the incomplete clusters will have the correct strength of association. a grouping based on taxonomy, or a more data-driven approach that cluster sequence groups based on the associated datasets as seen in figure , could be included as another iteration to properly strengthen the statistical associations. furthermore, forming clusters only by internal sequence identity may also miss pathogenic scenarios such as an oncovirus and any necessary helper viruses that do not share homology to the oncovirus. in the present study, we used a majority vote to assign taxonomy. there could be other ways to assign taxonomy, for instance, using a lowest common ancestor (lca) strategy. a majority vote will likely introduce some false assignments if there are distant taxa involved in the sequence group present in nearly equal fractions. a lca strategy can handle this but may reduce the taxonomic resolution to a level where there is no real gain of information. after determining what the significantly co-occurring sequence groups are, more effort might resolve interesting unmapped contigs. for instance, use of more sensitive alignment algorithms, profile hidden markov models (hmms), gene predictors, artificial neural networks trained on specific signals such as viral capsid sequences [ ] , or pcr extraction followed by sanger sequencing might provide the relevant clues. however, that was not within the scope of this study. the major advantage of the top-down approach is that it works without prior knowledge of the sequences. it is not dependent on reference sequence databases to single out the promising candidates for further analysis. the top-down method can determine the relevance of unknown sequences upfront while also systematically controlling for contamination by design. most of the annotated sequences found in this study were sequenced from cancer specimens. however, it is apparent from the association analysis that several viral sequences detected are possibly contaminants or technical artefacts. furthermore, the unmapped clusters are retained and easily arranged by relevance according to the nature of their associated features. having this information helps precipitate a prioritised list of sequence candidates the quality of the associations will depend on the experimental design, sampling, available metadata, as well as the rigorousness and standardisation of both working routines and annotations. we stress the point that care must still be taken when formulating hypotheses and in the interpretation of associations. virus discovery using high-throughput sequencing and especially characterising clinical samples is a challenge. many viral discovery pipelines rely on similarity to reference databases as the most compelling argument for identifying putative sequences of medical or biological importance. although a necessary step in the analysis, it has the downside of not considering novel sequences not included in reference sets as well as not considering the origins of the discoveries. there are many examples of contamination and technical artefacts; therefore, potential 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grierson, sylvia; cheney, tanya; steinbach, falko; choudhury, bhudipa; williamson, susanna title: uk pigs at the time of slaughter: investigation into the correlation of infection with prrsv and hev date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: xq iq ai hepatitis e virus (hev) and porcine reproductive and respiratory syndrome virus (prrsv) and are both globally prevalent in the pig population. while hev does not cause clinical disease in pigs, its zoonotic potential has raised concerns in the food safety sector. prrs has become endemic in the united kingdom (uk) since its introduction in , and continues to cause considerable economic losses to the swine industry. a better understanding of the current prevalence and diversity of prrsv and hev in the uk, and their potential association, is needed to assess risks and target control measures appropriately. this study used plasma, tonsil, and cecal content samples previously collected from pigs in abattoirs in england and northern ireland to study the prevalence of several pathogens including prrsv and hev. the diversity of prrsv strains detected in these samples was analyzed by sequencing open reading frame (orf ), revealing no substantial difference in prrsv strains from these clinically unaffected pigs relative to those from clinical cases of disease in the uk. despite the potential immuno-modulatory effect of prrsv infection, previously demonstrated to affect salmonella and hev shedding profiles, no significant association was found between positive prrsv status and positive hev status. hepatitis e virus (hev) is the cause of hepatitis e in humans, typically a self-limiting hepatitis but more serious in those with pre-existing liver conditions and in the immunocompromised [ ] . in pigs, hev infection alone does not cause clinical disease. hev genotypes hev- and hev- are the cause of sporadic cases of hepatitis e in developed countries, and are ubiquitous in the pig population worldwide [ ] . hepatitis e is a foodborne zoonosis, for which pork or pork products from infected pigs is one of the risks identified in europe [ , ] and consumption of processed pork products in the united kingdom (uk) has been shown to be associated with an increased risk of acquiring hev [ ] . hence, there is a need to better understand factors influencing hev entering the food chain. porcine reproductive and respiratory syndrome virus (prrsv) was first confirmed in the uk in and is now considered endemic [ ] . the economic and welfare impacts of the disease are considerable, as both the breeder and grower segments of the pig industry are affected [ ] . all prrsv infections in the uk characterized to date have been identified as being caused by genotype virus, but the genetic diversity of the virus is continually increasing [ ] . the phylogenetic analyses of uk prrsv sequences have previously been based on data from samples submitted for diagnostic purposes, originating from clinical cases of prrs, thereby possibly introducing a bias in our coverage of circulating strains. it is therefore possible that prrsv strains circulating in apparently healthy pigs in the uk may represent a different subset from those causing disease. prrsv infection has been suggested to modulate pig immune responses, thereby rendering pigs more susceptible to other infections [ ] [ ] [ ] . for example, previous studies have shown significant associations between prrsv presence and salmonella shedding [ ] . salines et al. [ ] reported that experimental co-infection of hev and prrsv affected the dynamics of hev infection. however, a direct immune-regulation in infected pigs could not be confirmed for genotype prrsv [ ] , rather suggesting a role for co-infection viruses influencing each other more directly. moreover, less pathogenic strains of genotype prrsv seem to cause a more persistent infection than highly pathogenic ones which are better resolved by the immune response [ ] . notably, viruses closely related to the modified-live vaccine used in the uk have previously been found circulating on farms with clinical prrs [ ] . in , an abattoir-based study was undertaken to estimate the prevalence of various pathogens including hev and prrsv in uk-reared pigs at slaughter and seroprevalence for prrsv was . % ( / ) [ ] , while hev seroprevalence was . % ( / ). approximately . % of pigs were hev viremic ( / ) and around one in five pigs had evidence of an active hev infection ( / ), with hev rna detected in serum or cecal contents [ ] . to follow up these studies, we report here on ( ) the investigation of prrsv active infection (rna in tonsil) using the same abattoir survey sample-set and ( ) an analysis of the correlation of prrsv and hev infection in these pigs, which could be of significance for the control of both diseases and in informing farming practices for reducing hev in the food chain. the overall study design for the abattoir survey has been described previously [ ] . briefly, qualifying pigs were sampled at abattoirs in england and northern ireland, between january and may . these pigs originated from farms, with between and pigs from each. the majority of pigs were from farms in england ( . %), followed by northern ireland ( . %), scotland ( . %), and wales ( . %), which is representative of the uk pig population [ ] . of the samples collected from each selected carcass along the processing line, those relevant for the investigation of hev and prrsv were a blood sample (whole blood with ethylenediaminetetraacetic acid (edta)), tonsil, and the whole cecum. antibody to prrsv had been detected in of plasma samples, with additional samples being inconclusive [ ] . only of these plasma samples were also analyzed for the presence of antibodies to hev, with of these being sero-positive for prrsv. tonsil samples from all seropositive or inconclusive pigs were then tested by a real-time reverse transcription polymerase chain reaction (rt-pcr) to detect prrsv nucleic acid. the tonsils of four other pigs for which plasma samples-and hence enzyme-linked immunosorbent assay (elisa) results-were missing were also tested by pcr, while for five seropositive animals the tonsil samples were not available, so a total of tonsil samples were analyzed; only of these animals also had matching hev pcr data. rna was extracted from the tonsil samples using the magna pure lc dna isolation kit ii (tissue) following the manufacturer's instructions (roche diagnostics, burgess hill, uk), setting the sample volume to µl and the elution volume to µl. a real-time rt-pcr assay targeting the nucleocapsid (orf ) gene of prrsv and differentiating genotypes and was used [ ] with a stratagene mx p qpcr system (agilent genomics, wokingham, uk). sequencing of the prrsv open reading frame (orf ) gene was subsequently performed on nucleic acids from tonsil samples testing positive in the diagnostic prrsv pcr to characterize the viruses present [ ] . the pcr amplicons were purified using the beckman ampure solid phase reversible immobilisation (spri) technique (beckman coulter ltd., high wycombe, uk). cycle sequencing was performed using forward and reverse primers and abi bigdye chemistry (applied biosystems ltd., warrington, uk) at the end of which the dye terminators were removed using beckman cleanseq spri (beckman coulter ltd., high wycombe, uk). samples were sequenced on an abi capillary electrophoresis dna analyser (applied biosystems ltd., warrington, uk) and the raw data analysed by abi seqscape software (applied biosystems ltd., warrington, uk). the resulting sequences were aligned with reference sequences from genbank, and with other uk prrsv orf sequences from samples submitted to the animal and plant health agency (apha) for prrs diagnostic testing between and , using the clustalw algorithm [ ] in mega [ ] . the phylogenetic tree was generated in mega using the neighbour-joining method [ ] , with the evolutionary distances being computed using the maximum composite likelihood method [ ] . the sequences obtained were deposited in genbank with accession numbers mf to mf . existing data for prrsv seropositivity [ ] and hev seropositivity and active infection [ ] were collated with data obtained in this study for prrsv active infection. associations between the two viruses, or antibodies to them, in the same carcasses were investigated using χ tests, with stata v. (statacorp, college station, tx, usa). collated data for prrsv seropositivity and hev seropositivity and active infection (rna in plasma and/ or cecal contents) were available for pigs. collated data for prrsv active infection (rna in tonsil) and seropositivity and active infection for hev were available for pigs. prrsv rna was detected in of tonsil samples. this corresponds to . % of the prrsv seropositive finisher pigs showing active prrsv infection at slaughter. importantly, all pcr-positive samples were of genotype (european), which is endemic in the uk, and no genotype virus, which is exotic to the uk, was detected. while the elisa-positive pigs tested by pcr originated from farms in counties, pcr-positive pigs were only identified from farms in eight of those counties. almost two-thirds of the pcr-positive pigs were from farms in east anglia or east riding and north lincolnshire. while seropositivity had been found to vary significantly between age groups (p = . ) with the highest level found in pigs aged less than six months ( . %) and lowest in those aged > months ( . %) [ ] , the prevalence of prrsv rna-positive tonsils was similar across the age groups (table ) . sequencing of the orf prrsv gene was undertaken on of the tonsil samples from which prrsv rna was detected. two pcr-positive tonsils were not suitable for sequencing as there was insufficient viral nucleic acid in the samples. six samples did not yield useable sequence data. all of the sequences confirm that the viruses belong to prrsv genotype . only three of the sequences may be considered to possibly originate from the currently licensed attenuated vaccine, with greater than % similarity between the sample and vaccine strain orf sequences ( . %, . %, and %). the phylogenetic trees (figure ) illustrate the genetic diversity of the orf genes from the samples in this study in comparison to the vaccine virus licensed in the uk at the time and published reference sequences representing the different genotypes and subtypes ( figure a ) and in more detail, in the context of previously sequenced viruses specifically from uk pigs between and (unpublished data) ( figure b ). in the within-uk analysis, there is no clear association between geographic origin and the clade in which the prrsv strains belong. all of the sequences are found in clades where other uk strains were already identified, and no distinct clustering is observed. analyses were performed to identify potential associations between prrsv serology or pcr status (prrsv rna detected in tonsil sample) and hev serology or pcr status (hev rna detected in serum or cecal contents). these are summarized in tables and . of the six animals that were pcr positive for both hev and prrsv, one was less than six months old, three were six months of age, and two were greater than six months of age. there was no evidence from this study that prrsv infection, as detected by serology or pcr, increased the likelihood of hev infection in pigs at the time of slaughter. the only associations identified suggested that for the pigs in this study, prrsv seropositive animals were less likely ( . % vs. . %, p = . ) to be hev pcr positive in plasma or cecum (table ) ; prrsv seropositive animals were less likely ( . % vs. . %, p = . ) to be hev pcr positive in plasma (table ) ; prrsv pcr positive animals were less likely ( . % vs. . %, p = . ) to be hev seropositive (table ) . it has been suggested that infection with prrsv renders pigs more susceptible to secondary bacterial [ , , , ] or viral [ , ] infections. in this study, we further investigated the active prrsv infection at slaughter age and the association between prrsv infection and an active hev infection in pigs entering the food chain in the uk. since the majority of uk pig farms that vaccinate against prrsv use a live vaccine [ ] it was considered that prrsv in both the vaccinated and naturally infected pigs may modulate hev infection. the abattoir survey had found a prevalence of antibodies to prrsv in slaughter-age pigs of . % [ ] . antibody to vaccine and field prrsv cannot be distinguished but vaccination of rearing pigs is less common than that of breeding pigs in the uk and is generally performed when there is an expectation of field prrsv challenge during the rearing period. therefore, seropositivity in finishers is considered a reasonable indicator of the presence of prrsv infection on the respective rearing units. from the prrsv seropositive pigs in the study, the prevalence of prrs viral rna in tonsils, where prrsv may persist up to days post infection [ ] , was . %. as tonsils from most seronegative pigs were not tested by pcr, it is possible that detection of a few prrsv-positive pigs in the early stages of infection were missed, although prrsv rna was not detected in any of the tonsils from seronegative pigs in a pilot study (data not shown). as the pigs were not showing obvious clinical signs, this is a significant finding, highlighting that a proportion of healthy pigs from prrsv-infected units may be infectious at slaughter, and if still shedding virus, may be able to transmit the virus on to other farms, for example through contaminated vehicles [ ] , underlining the need for good biosecurity during and after transport to slaughter. these findings triggered the genetic characterization of the viruses in order to further evaluate the potential risks associated with their presence. overall, the sequences from the abattoir samples did not cluster separately from those from clinical cases submitted for diagnostic testing, and they appear to be representative of the overall diversity of prrsv strains circulating in the uk. none of them was from an eastern european subtype of genotype prrsv. of the successfully-sequenced viruses, three showed greater than . % similarity to the modified-live vaccine used in the uk at the time. these 'vaccine-like' viruses may derive from vaccine virus, and have been identified in previous years, although at a lower rate [ ] , possibly because most samples previously sequenced were from disease outbreaks, whereas these were detected in clinically normal pigs. the other viruses showed between . % and . % similarity to the vaccine sequence. although the degree of genetic difference of a field prrs virus from the vaccine strain cannot alone predict the degree of protection that would be afforded by the vaccine to infection by the field virus, nor allow for a determination of the strain's pathogenicity, these results further illustrate the diverse nature of field prrsv in the uk. interestingly, some of the viruses from this study are sufficiently similar to one another to be potentially linked epidemiologically, even when they originate from pigs from different geographic regions. conversely, for two pigs from the same farm, the viruses detected in them did not show a great degree of similarity. there was no relation between four prrsv sequences from animals co-infected with hev (no sequence data was available for the other two), as they each grouped into different clusters in the phylogenetic analysis, one being homologous with the porcilis prrs vaccine strain sequence. the sequences from the abattoir samples did not cluster separately from those from clinical cases submitted for diagnostic testing, and they appear to fit within the overall diversity of prrsv strains previously found to be circulating in the uk. the existence of infected slaughter-age pigs with the potential, if shedding, to transmit the virus to susceptible pigs is thereby confirmed. we found no evidence in this study that exposure to, or infection with prrsv enhanced hev infection rates in pigs entering the food chain. indeed, pigs exposed to prrsv were less likely to be viremic (p = . ). the age-range of the six co-infected pigs identified reflected that of the overall population sampled. the calculated virus load in plasma for hev showed no association with either prrsv serology or pcr status (data not shown). several other studies have specifically investigated prrsv and hev co-infection [ , , ] , some by experimental infection. one report of an association of co-infection with disease was restricted to investigation of a single pig [ ] . martelli et al. [ ] found no association between these pathogens in an investigation of diagnostic submissions. the abattoir survey had investigated the presence of a number of other pathogens in clinically healthy pigs, but no evidence was found that the prrsv-seropositive pigs were more likely to carry salmonella or yersinia or have antibodies to toxoplasma [ ] . interestingly, salines et al. [ ] reported that experimental prrsv and hev co-infection did affect the hev infection dynamics in five-week old pigs with no maternal antibody for these two endemic viruses. the simultaneous co-infection resulted in delays to both the latent period ( . vs. . days with hev alone) and the humoral response ( . vs. . days) to hev, as well as increasing the infectious period ( . vs. . days), in association with an increased hev viral load. in contrast, several other studies have failed to demonstrate any association between prrsv infection and the dynamics of other viral infections [ , , ] . while the present study failed to show any positive association overall between prrsv and hev infections, this may be an age-dependent effect, and variation in the timing of the respective infections may also affect their outcomes. future investigations of natural prrsv and hev co-infections should perhaps be directed towards younger pigs, since these were under-represented in this study, and may provide different outcomes. a field study showed that the majority of pigs were infected by weeks of age [ ] . experimental infections to further characterize prrsv and hev co-infections must consider non-simultaneous infections, as they may be more relevant to the situation in the field. other viral co-infections such as porcine circovirus type (pcv- ) and prrsv or hev also remain to be investigated, with at least one report of fatal disease associated with hev and pcv- [ ] . an active hev infection in pigs entering the food chain is a potential risk to public health and there is a need to better understand factors that may influence this. the uk abattoir survey had found that one in five pigs had an active hev infection as they entered the food chain [ ] . this same finding of active hev infections in slaughter-age pigs is found worldwide [ ] [ ] [ ] [ ] . there was no evidence from the current study that prrsv infection adversely affected the proportion of hev infected pigs entering the food chain. further studies are needed to investigate factors influencing the dynamics of hev infection in the pig and within farms and that may then be used to inform means of reducing infection in slaughter-age pigs. no association was found between prrsv and hev infections in the slaughter age pigs sampled. in addition, there was no difference in strain diversity of prrsv sampled from clinically unaffected pigs in this study relative to those identified from clinical cases of disease in the uk. hepatitis e: an emerging infection in developed countries zoonotic origin of hepatitis e hepatitis e virus in england and wales: indigenous infection is associated with the consumption of processed pork products the cost of endemic disease in pig production porcine reproductive and respiratory syndrome virus: genetic diversity of recent british isolates in utero infection with prrs virus modulates cellular functions of blood monocytes and alveolar lung macrophages in piglets pathogenesis 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virus in swine and effluent samples from slaughterhouses in brazil genetic characterization and serological prevalence of swine hepatitis e virus in shandong province the authors declare no conflict of interest. the funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. key: cord- -hjx d rq authors: márquez-jurado, silvia; nogales, aitor; Ávila-pérez, ginés; iborra, francisco j.; martínez-sobrido, luis; almazán, fernando title: an alanine-to-valine substitution in the residue of zika virus ns a protein affects viral rna synthesis and attenuates the virus in vivo date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: hjx d rq the recent outbreaks of zika virus (zikv), its association with guillain–barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat zikv disease. in this respect, infectious clones constitute excellent tools to accomplish these goals. however, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. to bypass this problem, several alternative approaches have been applied for the generation of zikv clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. here, we report a simple and novel dna-launched approach based on the use of a bacterial artificial chromosome (bac) to generate a cdna clone of rio grande do norte natal zikv strain. the sequence was identified from the brain tissue of an aborted fetus with microcephaly. the bac clone was fully stable in bacteria and the infectious virus was efficiently recovered in vero cells through direct delivery of the cdna clone. the rescued virus yielded high titers in vero cells and was pathogenic in a validated mouse model (a mice) of zikv infection. furthermore, using this infectious clone we have generated a mutant zikv containing a single amino acid substitution (a v) in the ns a protein that presented reduced viral rna synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with zikv wild-type. this bac approach provides a stable and reliable reverse genetic system for zikv that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies. zika virus (zikv) is a recently emerged mosquito-borne member of the family flaviviridae, which was declared by the word health organization (who) as a global public health emergency on february [ , ] . like other flaviviruses, the viral particle is constituted by an inner nucleocapsid composed of the capsid (c) protein associated with the viral genomic rna (grna), surrounded by a lipid bilayer that contains the structural membrane (m) and envelope (e) proteins, which are arranged nucleus from the cmv promoter with a second amplification step in the cytoplasm driven by the viral polymerase. the recombinant virus rescued from the bac clone was fully infectious in vitro and in vivo. the zikv-rgn infectious clone was further used to evaluate the effect of a single amino acid change (alanine to valine) at residue of the ns a protein on viral rna synthesis and pathogenesis in vivo. we found that this unique single amino acid substitution impairs viral rna synthesis in cell culture and results in viral attenuation in a mice. remarkably, a single dose of the mutant virus was sufficient to induce protection against challenge with the parental wild-type (wt) zikv. these results demonstrate the reliability and potential of our bac approach to study zikv biology and to facilitate the development of vaccine and antiviral strategies. vero (a kidney epithelial cell line from an african green monkey) and a (an human adenocarcinomic alveolar epithelial cell line) cells were purchased from the american type culture collection (atcc, ccl- ) and were grown and maintained at • c and % co in growth medium, consisting in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) (hyclone, thermofisher scientific, madrid, spain), mm l-glutamine (sigma-aldrich, madrid, spain), % nonessential amino acids (sigma-aldrich), u/ml penicillin (sigma-aldrich) and µg/ml streptomycin (sigma-aldrich). the recombinant zikv-rgn wt (rzikv-rgn) or ns a mutant (rzikv-rgn-mns a) viruses were propagated in vero cells with virus growth medium (dmem supplemented with % fbs, mm l-glutamine, % nonessential amino acids, u/ml penicillin and µg/ml streptomycin) at • c and % co . for virus stocks preparation, to % confluent monolayers of vero cells were infected with a multiplicity of infection (moi) of . plaque forming units (pfu) per cell in virus growth medium and incubated at • c under % co . after - days of infection, the tissue culture supernatants were collected, clarified by centrifugation at × g for min, and stored in small aliquots at − • c. the bac plasmid pbelobac [ ] , kindly provided by h. shizuya (california institute of technology, pasadena, ca, usa), was used to assemble the zikv-rgn infectious cdna clone. this plasmid is a synthetic low-copy-number plasmid (one copy per cell) based on the escherichia coli (e. coli) f-factor [ ] that minimize the toxicity problems in the bacteria of exogenous sequences. e. coli dh b cells (invitrogen, thermofisher scientific) were used to amplify the bac plasmids. electrocompetent dh b cells (invitrogen, thermofisher scientific) were transformed by electroporation using a micropulser unit (bio-rad, madrid, spain), according to the manufacturer's instructions. bac-based plasmids were isolated and purified using the large-construct kit (qiagen, hilden, germany), following the manufacturer's specifications. we have assembled a zikv infectious cdna clone in the bac plasmid pbelobac , based on the data of the full-length sequence of the zikv clinical strain rgn [ ] deposited in the genbank (accession number ku ). this strain was selected because the full-length sequence was obtained directly from the virus-infected brain tissue of an aborted fetus with microcephaly in brazil in and therefore represents a good candidate to study zikv pathogenesis. the first step for the assembly of the full-length cdna clone was the selection of the restriction sites pmli, afei, and bstbi (genomic positions , and , respectively), which are unique in the viral genome ( figure a ). after that, four overlapping dna fragments covering the entire viral genome (zikv to zikv ) and flanked by the appropriate restriction sites, were generated by chemical synthesis (bio basic, inc., toronto, canada) ( figure b ). zikv fragment contained the cmv promoter precisely fused to the first nucleotides of the viral genome flanked at the '-end by apali and asci (absent in the viral genome) sites and at the '-end by a multiple-cloning site containing the selected restriction sites (pmli, afei and bstbi) followed by mlui (absent in the viral genome) and bamhi. fragments zikv (flanked by pmli and afei) and zikv (flanked by afei and bstbi) covered the genomic regions - and - , respectively. zikv fragment contained the restriction site bstbi, the last nucleotides of the viral genome, the hepatitis delta virus (hdv) ribozyme, the bovine growth hormone (bgh) termination and polyadenylation sequences, and the mlui restriction site. the infectious clone was assembled into pbelobac by sequential cloning of these four overlapping dna fragments. briefly, fragment zikv was digested with apali and bamhi and cloned into pbelobac −afei (a pbelobac without the afei restriction site) digested with the same enzymes, to generate the intermediate plasmid pbac-zikv . then, this plasmid was used as the backbone for the sequential cloning of the remaining overlapping dna fragments (zikv to zikv ) into the multicloning site of the intermediate plasmid (contains the restriction sites selected, pmli, afei, bstbi and mlui) to generate the full-length cdna clone pbac-zikv-rgn ( figure b) . the genetic integrity of the cdna clone was verified throughout the assembly process by extensive restriction analysis and sequencing. in all cases, the bacterial strain dh b (invitrogen, thermofisher scientific) was used as the e. coli host for all the cloning steps and the propagation of the bac cdna clone. to recover the infectious virus, vero cells on -well plates were grown to % confluence in growth medium without antibiotics, and transfected with µg of the bac cdna clone using µl of lipofectamine (invitrogen, thermofisher scientific), following the manufacturer's specifications. after h of incubation at • c, the transfection medium was replaced with fresh growth medium and the cells incubated at • c. aliquots of the culture supernatants were collected at h intervals for virus titer determination by plaque assay on vero cells. after five to seven days of transfection, when the cytopathic effect (cpe) was clear, cell culture supernatants were harvested and the recovered virus was cloned by three rounds of plaque purification. to determine the complete genome sequence of the rescued viruses, virions from supernatant of infected vero cells (moi of . pfu/cell) were purified through a % (w/v) sucrose cushion. viral rna was isolate from the purified virus with the qiaamp viral rna minikit (qiagen) following the manufacturer's instructions and deep-sequenced at the university of rochester genomics research center using illumina miseq (illumina, san diego, ca, usa). briefly, . µg of total viral rna was fragmented by controlled sonication and a dna library was generated using the nebnext mrna library prep master mix set for illumina (new england biolabs, ipswich, ma, usa), according to the manufacturer's instructions. after analyzing the library for size and quality (bio-analyzer; agilent technologies, inc., santa clara, ca, usa), deep-sequencing was performed using miseq (illumina) and the raw sequencing reads analyzed using swarm custom software. the genomic 'and '-terminal sequences were determined by the rapid amplification of cdna ends (race) using the '/ ' race second generation kit (roche, basilea, switzerland) with a polya-tail added to the cdna prior to the ' race reaction using polya polymerase (new england biolabs), following the manufacturer's instructions. to analyze the genetic stability of the recombinant zikv harboring the point mutation a v in the coding region of the ns a protein (rzikv-rgn-mns a), total rna was purified from vero cells infected with viruses from passage (p ) to passage (p ) using the rneasy minikit (qiagen), according to the manufacturer's specifications. purified rna ( ng) was reverse transcribed (rt) with random hexamer primers using the high-capacity cdna transcription kit (life technologies, thermofisher scientific), and the cdna was amplified by pcr with the forward primer zikv- vs viruses , , of ( '-gaggaatggtgctgcagg- '), spanning nucleotides to of the viral genome, and the reverse primer zikv- rs ( '-gcttgacatctccccag- ') , complementary to nucleotides to of the viral genome. finally, the amplicons generated covering the region encoding ns a and ns b proteins (genomic region - ) were sequenced by sanger sequencing (macrogen europe, amsterdam, netherlands) using specific oligonucleotides. vero cells seeded into -well plates at - % of confluence were infected with µl of serial -fold dilutions of the virus in virus growth medium without fbs for h at • c. after viral absorption, the viral inoculum was removed and the cells overlaid with ml of virus growth medium containing % deae-dextran (sigma-aldrich) and . % agar noble (difco, thermofisher scientific). after - days of incubation at • c under % co , the cells were fixed with % formaldehyde for h at room temperature, the overlaid removed, and the viral plaques visualized by staining with . % crystal violet in % methanol or by immunostaining with µg/ml of the pan-flavivirus e protein monoclonal antibody (mab) g (bei resources; nr- ) using the vectastain abc kit (vector laboratories inc., burlingame, ca, usa). visible plaques were counted and virus titers were calculated as pfu/ml. vero and a cells seeded into -well plates at % of confluence were infected with the indicated viruses diluted in virus growth medium without fbs at the specified mois. after h of absorption at • c in % co , the virus inoculum was removed, the cell monolayers washed twice with pbs, and . ml of fresh virus growth medium was added to each well. cells were incubated at • c under % co and at selected time points, aliquots of tissue culture supernatants were collected and virus titers determined by plaque assay in vero cells as described above. viral rna synthesis was evaluated by quantitative rt-pcr (rt-qpcr). total intracellular rna from uninfected or infected vero cells was purified using the rneasy minikit (qiagen) and total cdna was synthetized from ng of purified rna using random hexamer primers and the high-capacity cdna transcription kit (life technologies, thermofisher scientific), following the manufacturer's specifications. using this cdna, the level of viral rna was further quantified by qpcr using a custom taqman assay specific for zikv-rgn rna. this taqman assay is constituted by the forward primer '-gaagagcatccagccagagaa- ' (spanning nucleotides to of the viral genome), the reverse primer '-ctgggagccatgaactgaca- ' (complementary to nucleotides to of the viral genome), and the probe '-fam-tggagtaccggataatg- iabkfq- ' (covering nucleotides to of the viral genome). to normalize for differences in rna sampling, the expression of the histone h b (reference housekeeping gene) was analyzed using a specific taqman gene expression assay (rh _s ; life technologies, thermofisher scientific). data were acquired with a real-time pcr system (life technologies, thermofisher scientific) and analyzed with abi prism software v . . . the relative quantifications were performed using the cycle threshold ( −∆∆ct ) method [ ] . all experiments and data analysis were miqe (minimum information for publication of quantitative real-time pcr experiments) compliant [ ] . the expression of zikv e protein was analyzed by indirect immunofluorescence assay (ifa). vero cells grown on coverslips in -well plates at - % of confluence were infected with the rescued rzikvs at the indicated mois. at selected time points post-infection, cells were fixed with % paraformaldehyde in mm hepes ph . during min at room temperature and then permeabilized with . % triton x- in pbs for min. after that, cells were treated for h at room temperature with blocking solution ( % fbs in pbs) and incubated with µg/ml of the pan-flavivirus e protein mab g (bei resources; nr- ) in blocking solution for h at room temperature. after three washed with pbs, cells were incubated at room temperature for h with donkey anti-mouse antibody conjugated to alexa fluor (invitrogen, thermofisher scientific) diluted : in blocking solution, extensively washed with pbs, and incubated for min with dapi ( ', '-diamidino- -phenylindole) (sigma-aldrich) diluted : in pbs for nuclear staining. finally, coverslips were mounted in prolong gold antifade reagent (invitrogen, thermofisher scientific) and analyzed on a leica sp confocal microscope. immunofluorescence acquired images were processed and analyzed with imagej . b software [ ] . for the evaluation of the virus-specific antibodies levels present in the sera of vaccinated mice, enzyme-linked immunosorbent assays (elisas) were performed as previously described [ ] . briefly, -well plates were coated with cell lysates from mock-or zikv-infected vero cells and incubated overnight at • c. the coated wells were washed with pbs, blocked with % bsa in pbs, and then incubated with two-fold dilutions (starting dilution of : ) of mice sera for h at • c. after that, plates were washed with water and incubated with hrp-conjugated goat anti-mouse igg ( : ; southern biotech, birmingham, al, usa) for h at • c. reactions were developed with tetramethylbenzidine (tmb) substrate (biolegend, san diego, ca, usa) for min at room temperature, quenched with n h so , and read at nm in a vmax kinetic microplate reader (molecular devices, san jose, ca, usa). the in vivo studies were performed in type-i interferon (ifn) receptor deficient (ifnr-/-) a mice (the jackson laboratory, bar harbor, me, usa) maintained in the animal care facility at the university of rochester under specific pathogen-free conditions. in this animal model, subcutaneous (s.c.) or intraperitoneal (i.p.) infection with zikv induces neurological disease and the animals succumb to viral infection, with high viral load in blood, brain, spin cord, and testes, consistent with manifestations of zikv infection in humans. although deficient in innate ifn responses, a mice retain their adaptive immunity and have been successfully used as a suitable model for testing antivirals and vaccines [ ] [ ] [ ] . to evaluate virus pathogenicity, female -to- -week-old a mice (n = ) were first anesthetized i.p. with a mixture of ketamine ( µg per gram of body weight) and xylazine ( µg per gram of body weight), and then mock-infected (pbs) or infected s.c. in the footpad with the indicated doses of rzikv-rgn or rzikv-rgn-mns a diluted in pbs in a final volume of µl. after viral infection, animals were monitored daily for morbidity (body weight loss and disease signs, including hunching, ruffling and hind limb paralysis) and mortality (survival) over days. mice showing more than % of body weight loss or severe paralysis were considered to have reached the experimental endpoint and were humanely euthanized. to correlate development of clinical symptoms and death with virus replication, -to- -week-old mice (n = ) were infected as described above and the viral titers in serum were determined at days (n = ) and (n = ) by plaque assay and immunostaining using the pan-flavivirus e protein mab g as indicated before. to evaluate the protection efficacy of the rzikv-rgn-mns a, female -to- -week-old a mice (n = ) were first anesthetized i.p. as indicated above, and then mock-immunized (pbs) or immunized s.c. in the footpad with pfu of rzikv-rgn-mns a diluted in pbs in a final volume of µl. at days post-immunization, mouse sera were collected by submandibular bleeding and the presence of total antibodies against zikv-rgn was evaluated by elisa. twenty-four hours after bleeding, mice were challenged s.c. in the footpad with pfu of rzikv-rgn and their morbidity and mortality monitored over days as previously described. to determine viral replication, challenged -to- -week-old a mice (n = ) were bleeding at days (n = ) and (n = ) post-challenge and viruses , , of zikv viremia was determined by plaque assay and immunostaining using the pan-flavivirus e protein mab g as previously described. for quantitative analyses, a two-tailed, unpaired student t test was used to analyze differences in mean values between groups. all results were expressed as mean ± standard deviations of the means. p values of < . were considered significant. for mice experiments, the meier log-rank test was used to compare survival data and the reed and muench method to determine the mouse lethal dose (mld ). graphpad prism v . software was used for all statistical analysis. all animal protocols were approved by the university of rochester committee of animal resources (protocol number: ucar- - / ; approval date: / / ) and complied with the recommendations in the guide for the care and use of laboratory animals of the national research council [ ] . to overcome the toxicity problems associated to several flavivirus sequences during its propagation in bacteria, we used the bac plasmid pbelobac (a single-copy plasmid derived from the e. coli f-factor) [ ] to assemble a zikv infectious cdna clone, based on the genome sequence of the rgn strain of zikv (genbank accession number ku ) [ ] (figure ). this zikv-rgn strain was selected because it has no laboratory passage history and the full-length genome sequence was obtained from a zikv-infected fetus with microcephaly in [ ] , constituting a good candidate to further study zikv pathogenesis. after appropriate selection of unique restriction sites in the zikv-rgn genome ( figure a ), four overlapping dna fragments (zikv to zikv ), spanning the full-length viral genome and flanked for the selected restriction sites, were chemically synthesized, and sequentially cloned into pbelobac to generate the infectious cdna clone pbac-zikv-rgn ( figure b ). fragment zikv contained the cmv immediate-early promoter to allow the expression of the viral rna in the nucleus by the cellular rna polymerase ii [ ] and fragment zikv was flanked at the '-end by the hdv ribozyme followed by the bgh termination and polyadenylation sequences to produce synthetic rnas bearing authentic '-ends of the viral genome. this dna-lunched system ensures capping of the viral rna and allows the recovery of infectious virus from the transfected cdna clone without the need of an in vitro transcription step. once assembled, the full-length sequence of the zikv-rgn bac clone was determined and no changes were detected to that reported for the zikv-rgn strain (genbank accession number ku ). finally, to confirm the stability of this synthetic infectious cdna clone in bacteria, the bac clone was passaged in e. coli dh b cells for more than two hundred generations and the genetic integrity of the passaged infectious clone analyzed by restriction endonuclease analysis and sequencing. no differences were detected, demonstrating that the zikv-rgn bac clone was fully stable in bacteria and that the bac approach is a reliable and simple method to generate zikv infectious cdna clones. to recover the infectious virus ( figure ), vero cells were transiently transfected with the bac cdna clone using lipofectamine and virus production analyzed during seven days. in contrast to mock-transfected cells, increasing amounts of infectious virus were detected in the tissue culture supernatant of cells transfected with the infectious clone, with peak titers around pfu/ml on day five ( figure a ). to further confirm the identity of the rescued virus, vero cells were infected with an moi of . pfu/cell of the rescue virus and monitored for cpe induction and viral e protein expression by ifa using the pan-flavivirus e protein mab g ( figure b ). the rescued virus induced a clear cpe, characterized by the presence of rounded and birefringent cells, and high levels of e protein expression were detected in the perinuclear region of infected cells. these results demonstrated that the zikv-rgn infectious bac cdna clone produces high titers of rzikv-rgn directly after transfection of susceptible vero cells. to recover the infectious virus ( figure ), vero cells were transiently transfected with the bac cdna clone using lipofectamine and virus production analyzed during seven days. in contrast to mock-transfected cells, increasing amounts of infectious virus were detected in the tissue culture supernatant of cells transfected with the infectious clone, with peak titers around pfu/ml on day five (figure a ). to further confirm the identity of the rescued virus, vero cells were infected with an moi of . pfu/cell of the rescue virus and monitored for cpe induction and viral e protein expression by ifa using the pan-flavivirus e protein mab g ( figure b ). the rescued virus induced a clear cpe, characterized by the presence of rounded and birefringent cells, and high levels of e protein expression were detected in the perinuclear region of infected cells. these results demonstrated that the zikv-rgn infectious bac cdna clone produces high titers of rzikv-rgn directly after transfection of susceptible vero cells. once the identity of the rescued virus was confirmed, it was cloned by three round of plaque purification, and its phenotypic and genotypic properties were determined. analysis of the growth kinetics revealed that the rzikv-rgn replicated efficiently in both vero and a cells, reaching peak titers of approximately and pfu/ml at hpi, respectively ( figure a ). in addition, the rescued virus generated homogeneous plaques of about mm in size after four days of infection in vero cells ( figure b ). finally, the genetic identity of the virus was analyzed by deep-sequencing of two independent clones. full-genome sequencing of both viral clones revealed that both clones presented the same sequence that the cdna clone. overall, these results demonstrate the feasibility of generating infectious rzikvs using a bac-based approach. once the identity of the rescued virus was confirmed, it was cloned by three round of plaque purification, and its phenotypic and genotypic properties were determined. analysis of the growth kinetics revealed that the rzikv-rgn replicated efficiently in both vero and a cells, reaching peak titers of approximately and pfu/ml at hpi, respectively ( figure a ). in addition, the rescued virus generated homogeneous plaques of about mm in size after four days of infection in vero cells ( figure b ). finally, the genetic identity of the virus was analyzed by deep-sequencing of two independent clones. full-genome sequencing of both viral clones revealed that both clones presented the same sequence that the cdna clone. overall, these results demonstrate the feasibility of generating infectious rzikvs using a bac-based approach. once the identity of the rescued virus was confirmed, it was cloned by three round of plaque purification, and its phenotypic and genotypic properties were determined. analysis of the growth kinetics revealed that the rzikv-rgn replicated efficiently in both vero and a cells, reaching peak titers of approximately and pfu/ml at hpi, respectively ( figure a ). in addition, the rescued virus generated homogeneous plaques of about mm in size after four days of infection in vero cells ( figure b ). finally, the genetic identity of the virus was analyzed by deep-sequencing of two independent clones. full-genome sequencing of both viral clones revealed that both clones presented the same sequence that the cdna clone. overall, these results demonstrate the feasibility of generating infectious rzikvs using a bac-based approach. to determine whether the rescued rzikv-rgn was pathogenic in vivo (figure ) , groups of five female -to- -week-old a mice (ifnr-/-) were inoculated s.c. in the footpad with pbs (as negative control) or with different doses of rzikv-rgn ( , and pfu per animal) and the morbidity (body weight loss and disease signs) and survival were monitored daily over days ( figure a ). as expected, weight loss and survival correlated with the inoculated dose. mice infected with pfu did not show disease symptoms, only a slight reduction in body weight was detected on days to , and all of them survived. in the case of mice infected with pfu, they presented some symptoms of disease (hunching and reduced mobility) and weight loss from days seven to nine (with a maximum of % on day nine), but all of them recovered the initial body weight and survived. in contrast, animals infected with pfu showed hind limb paralysis, rapidly lost weight, and all of them succumbed to viral infection between days seven and eight post-infection ( figure a ). using the reed & muench method we determined that the mld of rzikv-rgn was approximately × pfu. to further analyze whether the virulence observed correlated with viral replication, viral titers in mouse sera were analyzed at days two and four post-infection ( figure b ). as expected, the viremia in the infected animals was dose dependent, reaching the highest titers at day two after inoculation. at day four post-inoculation a significant reduction of the viral titers was observed. in the case of animals infected with or pfu, viremia was not detected or only detected in one of the three infected mice, respectively ( figure b ). overall, these results indicated that the rzikv-rgn recovered from the infectious clone is virulent in mice but only at high ( pfu) dose. to determine whether the rescued rzikv-rgn was pathogenic in vivo (figure ) , groups of five female -to- -week-old a mice (ifnr-/-) were inoculated s.c. in the footpad with pbs (as negative control) or with different doses of rzikv-rgn ( , and pfu per animal) and the morbidity (body weight loss and disease signs) and survival were monitored daily over days ( figure a ). as expected, weight loss and survival correlated with the inoculated dose. mice infected with pfu did not show disease symptoms, only a slight reduction in body weight was detected on days to , and all of them survived. in the case of mice infected with pfu, they presented some symptoms of disease (hunching and reduced mobility) and weight loss from days seven to nine (with a maximum of % on day nine), but all of them recovered the initial body weight and survived. in contrast, animals infected with pfu showed hind limb paralysis, rapidly lost weight, and all of them succumbed to viral infection between days seven and eight post-infection ( figure a ). using the reed & muench method we determined that the mld of rzikv-rgn was approximately × pfu. to further analyze whether the virulence observed correlated with viral replication, viral titers in mouse sera were analyzed at days two and four post-infection ( figure b ). as expected, the viremia in the infected animals was dose dependent, reaching the highest titers at day two after inoculation. at day four post-inoculation a significant reduction of the viral titers was observed. in the case of animals infected with or pfu, viremia was not detected or only detected in one of the three infected mice, respectively ( figure b ). overall, these results indicated that the rzikv-rgn recovered from the infectious clone is virulent in mice but only at high ( pfu) dose. week-old a mice (five mice per group) were mock-infected (pbs) or infected s.c. in the footpad with the indicated pfu of rzikv-rgn, and body weight loss (expressed as the percentage of starting weight, left panel) and survival (right panel) were monitored daily during days. mice that lost more than % of their initial body weight or presented hind limb paralysis were humanely euthanized. error bars represent standard deviations of the mean for each group of mice. asterisks indicate that the differences between viral doses of and are statistically significant when data are compared using the unpaired t test (*, p < . ; **, p < . ). (b) viral titers in mice sera. female -to- -week-old a mice (six mice per group) were infected with the indicated pfu of rzikv-rgn as described above, and viral titers in sera were determined at days two and four after infection (three animals per time point) by plaque assay and immunostaining using the pan-flavivirus e protein mab g . symbols represent data from individual mice and bars the geometric means of viral titers. asterisks indicate that the differences in viral titers between experimental samples are statistically significant when data are compared using the unpaired t test (**, p < . ; ***, p < . ). &: virus not detected in two mice; nd: virus not detected. the detection limit of the assay ( pfu/ml) is indicate as a dashed line. during the assembly of the pbac-zikv-rgn infectious clone, we detected the presence of a point mutation in the ns a protein, which was introduced during the chemical synthesis of fragment zikv . this mutation consists in a cytosine-to-thymidine substitution at genomic position , resulting in an alanine-to-valine change in the residue of the ns a protein (a v). because this mutation consists of a conservative amino acid change, we decided to explore the possibility of using this mutation as a genetic marker. to this end, the infectious clone pbac-zikv-rgn-mns a was generated by replacing the zikv wt fragment for that containing the ns a a v mutation. this infectious clone was fully stable in bacteria and no additional mutations were observed after sequencing the full-length clone. after that, vero cells were transfected with the mutant infectious clone and the recovery efficiency of the rzikv-rgn-mns a mutant virus was compared to that of the parental rzikv-rgn virus ( figure ). although the infectious virus was recovered in both cases, virus production was one logarithm lower in the case of the mutant rzikv-rgn-mns a, reaching maximum titers of pfu/ml at seven days post-transfection ( figure a ). when the plaque phenotype was analyzed, we found that the plaque size of the mutant rzikv-rgn-mns a was smaller (more than a -fold reduction) than that of the parental rzikv-rgn ( figure b ), indicating that the a v mutation, despite of being a conservative substitution, caused reduction in plaque size and virus production. in addition, an in silico analysis was performed to evaluate the frequency of amino acid residues of the ns a protein in more than zikv strains sequences deposited in the database [ ] (https://www.viprbrc.org/brc/home.spg?decorator=flavi). this analysis indicated that amino acid a is highly conserved, since the % of the analyzed zikv sequences contained an alanine residue at this position. to further confirm the effect of the ns a a v mutation on virus production, the growth kinetics at high ( pfu/cell) and low ( . pfu/cell) moi of the mutant virus were compared to those of the parental virus ( figure c ). again, a reduction of about one logarithmic unit in virus production was detected in vero cells infected with the mutant virus both at high and low moi ( figure c ). taken into consideration that flavivirus ns a protein is involved in regulation of rna replication and virus assembly [ ] , we further analyzed whether the reduction in plaque size and virus production of the mutant virus was associated with reduced viral rna synthesis. to this end, the production of viral rna in vero cells infected with either the parental or mutant viruses at an moi of . pfu/cell was analyzed at and hpi by rt-qpcr using a custom taqman assay specific for zikv-rgn genome ( figure d ). at both times, a -fold reduction in the levels of viral rna was observed in cells infected with the mutant virus ( figure d ), confirming that ns a a v mutation at least impairs viral rna synthesis. in agreement with these data, a reduction in the expression levels of zikv e protein was observed by ifa in vero cells infected with the mutant virus in comparison to cells infected with the parental virus ( figure e ). finally, to discard the presence of other undesired mutations, the full-length sequence of the mutant virus was determined by deep-sequencing, and no mutations other than ns a a v were detected. collectively, these results indicated that ns a a v mutation alone affected zikv growth in vero cells at least by impairing viral rna synthesis. to investigate whether the reduced rna synthesis of rzikv-rgn-mns a in vero cells could result in viral attenuation in vivo, the ability of the mutant virus to induce pathogenesis was analyzed in a mice and compared with that of the parental rzikv-rgn ( figure ). to that end, groups of five female -to- -week-old a mice were inoculated s.c. in the footpad with pfu of either rzikv-rgn or rzikv-rgn-mns a, or with pbs as a negative control, and the body weight loss and survival were monitored daily over days. in contrast to mice infected with rzikv-rgn that quickly lost weight and all of them died at day eight after infection, mice infected with the mutant rzikv-rgn-mns a did not presented any clinical signs of infection or weight loss and all of them survived to viral infection ( figure a ). to further analyze the correlation of the attenuation of the mutant virus with viral replication, presence of the virus in mice sera was analyzed at days two and four post-infection. in agreement with the pathogenicity data, mice infected with the mutant virus presented lower viremia than mice infected with the parental virus ( figure b ). the mutant virus was only detected at day two after infection and at lower titers (approximately . logarithms lower) than the parental virus. as an internal control of the experiment, the plaque phenotype of the viruses recovered from the blood of infected mice were analyzed. as expected, rzikv-rgn formed big plaques while the mutant rzikv-rgn-mns a formed small plaques ( figure c ). these results indicated that rzikv-rgn-mns a was highly attenuated in mice, as compared to rzikv-rgn, and that this attenuation may be due to a lower replication of the rzikv-rgn-mns a mutant virus. after elucidating that rzikv-rgn-mns a was attenuated in vivo, its ability to induce protection against a challenge with the parental rzikv-rgn was analyzed (figure ) . to that end, groups of five female -to- -week-old a mice were vaccinated s.c. in the footpad with pfu of rzikv-rgn-mns a or mock-vaccinated with pbs. twenty days after vaccination, blood samples were collected to evaluate the humoral response. one day later, mice were challenged with a lethal dose ( pfu) of rzikv-rgn and the body weight loss and survival were analyzed daily over figure . pathogenesis of rzikv-rgn-mns a in a mice. (a) weight loss and mortality. female -to- -week-old a mice (five mice per group) were mock-infected (pbs) or infected s.c. in the footpad with pfu of rzikv-rgn or rzikv-rgn-mns a, and body weight loss (expressed as the percentage of starting weight, left panel) and survival (right panel) were monitored daily during days. mice that lost more than % of their initial body weight or presented hind limb paralysis were humanely euthanized. error bars represent standard deviations of the mean for each group of mice. (b) viral titers in mice sera. female -to- -week-old a mice (six mice per group) were infected with pfu of rzikv-rgn (wt) or rzikv-rgn-mns a (mut) as described above, and viral titers in sera were determined at days two and four after infection (three animals per time point) by plaque assay and immunostaining using the pan-flavivirus e protein mab g . symbols represent data from individual mice and bars the geometric means of viral titers. asterisks indicate that the differences between rzikv-rgn and rzikv-rgn-mns a are statistically significant when data are compared using the unpaired t test (***, p < . ). nd: virus not detected. the detection limit of the assay ( pfu/ml) is indicate as a dashed line. (c) plaque phenotype. vero cells at % confluence ( -well plate format) were infected with pfu of rzikv-rgn (left) or rzikv-rgn-mns a (right) recovered from infected mice at day two post-infection and the plaque size evaluated by plaque assay and immunostaining using the pan-flavivirus e protein mab g . after elucidating that rzikv-rgn-mns a was attenuated in vivo, its ability to induce protection against a challenge with the parental rzikv-rgn was analyzed (figure ) . to that end, groups of five female -to- -week-old a mice were vaccinated s.c. in the footpad with pfu of rzikv-rgn-mns a or mock-vaccinated with pbs. twenty days after vaccination, blood samples were collected to evaluate the humoral response. one day later, mice were challenged with a lethal dose ( pfu) of rzikv-rgn and the body weight loss and survival were analyzed daily over days. as expected, mice vaccinated with pbs lost weight rapidly, showed clear symptoms of disease, and all of them succumbed to challenge with rzikv-rgn. in contrast, mice vaccinated with rzikv-rgn-mns a did not lose weight and all of them survived the challenge with rzikv-rgn ( figure a ), indicating that a single immunization dose with rzikv-rgn-mns a is enough to induce full protection against zikv-rgn. in agreement with these data, a strong humoral response against zikv-rgn was observed in mice vaccinated with rzikv-rgn-mns a ( figure b ) and no viremia was detected in sera samples of vaccinated mice at days two and four after challenge ( figure c ). figure a ), indicating that a single immunization dose with rzikv-rgn-mns a is enough to induce full protection against zikv-rgn. in agreement with these data, a strong humoral response against zikv-rgn was observed in mice vaccinated with rzikv-rgn-mns a ( figure b ) and no viremia was detected in sera samples of vaccinated mice at days two and four after challenge ( figure c ). once confirmed that rzikv-rgn-mns a was attenuated in vivo and induces protection against zikv-rgn in mice, the genetic stability of the mutant virus was analyzed in vero cells, in order to test the possible use of this mutant virus as a base for the development of a live-attenuated zikv vaccine (figure ). to this end, both mutant (rzikv-rgn-mns a) and parental (rzikv-rgn) viruses were passaged five times in vero cells (p to p ) and the virus plaque phenotype, growth kinetics and the sequence of ns a were analyzed for each passage. analysis of the plaque phenotype showed that the parental virus presented the expected plaque size throughout all passaging. in contrast, for the mutant virus a reversion to parental plaque phenotype was observed. this reversion started at p ( % of big plaques), clearly increased at p ( % of big plaques) and was complete at p ( figure a ). after that, we analyzed whether this plaque size reversion of the mutant rzikv-rgn-mns a correlated with an increase in virus replication to levels of that of the parental virus. to that end, the growth kinetics (moi of pfu/cell) of the mutant virus from p and p were compared to those of the parental virus. growth curve analysis showed that in contrast to the mutant virus from p , the virus from p replicated to the same levels as the parental virus ( figure b ), suggesting that the mutant virus reverted to the wt sequence during its propagation in vero cells. to confirm these observations, the ns a coding region of mutant viruses from p to p was amplified by rt-pcr and sequenced. sequence analysis confirmed the reversion of the a v mutation to the wt sequence. although the instability and reversion of the mutant virus to the wt sequence during its propagation in vero cells limits the use of this mutant for vaccine development, these data further support the importance of this ns a residue for virus replication. at hpi, cell culture supernatants were collected and used to infect fresh vero cells. this process was repeated four more times and virus stocks of passages to (p to p ) were generated. (a) plaque size. vero cells were infected with the different passages (p to p ) of rzikv-rgn or rzikv-rgn-mns a, and at four days post-infection the viral plaques were visualized by immunostaining using the pan-flavivirus e protein mab g . (b) growth kinetics. vero cells at % confluence ( -well plate format; triplicates) were infected (moi of pfu/cell) with p and p of rzikv-rgn or rzikv-rgn-mns a and at the indicated hpi, virus titers were determined by plaque assay. error bars represent standard deviations of the mean from three experiments. asterisks indicate that the differences between rzikv-rgn-mns a p and the experimental samples, rzikv-rgn p , rzikv-rgn p and rzikv-rgn-mns a p , are statistically significant when data are compared using the unpaired t test (***, p < . ). analysis of the plaque phenotype showed that the parental virus presented the expected plaque size throughout all passaging. in contrast, for the mutant virus a reversion to parental plaque phenotype was observed. this reversion started at p ( % of big plaques), clearly increased at p ( % of big plaques) and was complete at p ( figure a ). after that, we analyzed whether this plaque size reversion of the mutant rzikv-rgn-mns a correlated with an increase in virus replication to levels of that of the parental virus. to that end, the growth kinetics (moi of pfu/cell) of the mutant virus from p and p were compared to those of the parental virus. growth curve analysis showed that in contrast to the mutant virus from p , the virus from p replicated to the same levels as the parental virus ( figure b ), suggesting that the mutant virus reverted to the wt sequence during its propagation in vero cells. to confirm these observations, the ns a coding region of mutant viruses from p to p was amplified by rt-pcr and sequenced. sequence analysis confirmed the reversion of the a v mutation to the wt sequence. although the instability and reversion of the mutant virus to the wt sequence during its propagation in vero cells limits the use of this mutant for vaccine development, these data further support the importance of this ns a residue for virus replication. moreover, these data also suggest that zikv ns a protein represents a good target for the development of antivirals against zikv infection. the significance of zikv to public health due its association with guillain-barré syndrome and fetal abnormalities [ ] [ ] [ ] [ ] [ ] [ ] , together with the lack of approved antiviral agents or vaccines, have triggered a global effort to study this flavivirus in order to develop effective strategies to prevent and control zikv infection in humans. in this respect, the development and implementation of reverse genetic approaches for zikv provide investigators with a novel and powerful experimental tool to study both the biology and pathogenesis of zikv as well as the development of attenuated forms of zikv for their implementation as live-attenuated vaccines. however, as described for other flaviviruses, the generation of zikv infectious clones using traditional approaches are very difficult due to the toxicity and instability of some viral sequences when they were propagated as cloned cdna in bacteria [ ] [ ] [ ] . in the past two years, several approaches that overcomes this toxicity problem have been applied for the successfully generation of zikv infectious clones. these include the use of low-copy plasmids [ , ] , insertion of introns to disrupt toxic sequences [ ] [ ] [ ] , mutational silencing of cbps present in the viral genome [ ] , in vitro ligation of cdna fragments [ , , ] , the isa method [ , ] , and the cper approach [ ] . although very useful, some of these approaches are laborious, time consuming and present several disadvantages. for instance, most of them need in vitro ligation and transcription steps that complicate the assembly and reduce the recovery efficiency. moreover, these low recovery efficiencies increase the presence of undesired mutations that could result in in vitro and/or in vivo attenuation, limiting the use of these infectious clones for certain studies. others, such as the mutational silencing of cbps, in which a high number of silent mutations have to be introduced, could affect viral fitness. finally, the use of low-copy plasmids has been shown to be effective for several zikv strains but not for others, probably due the different degrees of toxicity of rna sequences of different strains [ , , , ] . here, we describe a powerful approach for the generation of an infectious cdna clone of the zikv-rgn strain in a single plasmid, based on the use of a combination of synthetic biology and bacs. the full-length cdna copy of the zikv-rgn strain was generated from four synthetic dna fragments and cloned in the bac plasmid pbelobac [ ] under the control of the cmv promoter, which allows the expression of the viral rna in the nucleus [ ] , and flanked at the '-end by the hdv ribozyme and the bgh polyadenylation and termination sequences to produce synthetic rnas bearing authentic '-ends of the viral genome. the bac cdna clone was fully stable during its propagation in bacteria and the functional infectious virus was rescued after direct transfection of susceptible vero cells that was pathogenic in a mice. a zikv-rgn infectious clone generated using the cper approach has been recently reported [ ] . however, in contrast to our results, the rescued virus was asymptomatic and nonlethal in female -to- -week-old a mice infected with doses of to ccid ( % cell culture infective doses) via the s.c. route. whether the differences in pathogenicity among this rzikv-rgn and ours are related to the experimental approach (cper versus bac), the age of the mice ( -to- -week-old versus -to- -week-old) or the moi used to infect the mice ( - ccid versus pfu) remain to be evaluated. although other zikv reverse genetic systems have been reported (discussed above), the bac approach constitutes an useful alternative that presents important advantages: (i) the bac plasmids present a strictly controlled replication leading to only one plasmid per cell and therefore minimize the toxicity associated with several flavivirus sequences when amplified in bacteria [ ] . this allows the easy and direct manipulation of the viral genome for molecular studies; (ii) similarly to other approaches using polii-driven promoters, the bac approach results in intracellular expression of the viral rna [ , , , , ] , allowing the capping of the viral rna and the recovery of infectious virus without the need of an in vitro transcription step. although some splicing events could occur during the nuclear expression of the viral genome, mainly due to the presence of donor and acceptor putative sequences in the viral genome, the efficiency of this phenomenon is very low and does not affect the recovery of infectious viruses [ ] ; (iii) like other systems based on transfection of dna constructs [ , , , , ] , bac cdna clones present a higher efficiency of transfection than rna transcripts in mammalian cells. this allows higher efficiencies of virus recovery, reducing the passages in cell culture to get a viral stock and therefore, the possibility of introducing undesired mutations by cell culture adaptation; (iv) the manipulation of bac cdna clones is relatively easy and similar to that of conventional plasmids with slight modifications due to the presence of only one plasmid copy per cell. in addition to standard protocols, the bac cdna clones could also be efficiently modified into e. coli by homologous recombination using a two-step approach that combine the red recombination system and counterselection with the homing endonuclease i-scei [ ] [ ] [ ] [ ] ; and (v) the bac approach has been successfully used to engineer infectious clones of other flaviviruses, including denv [ ] , and several coronaviruses that contain the largest viral rna genome known and similar toxicity problems to those described for flaviviruses [ , [ ] [ ] [ ] [ ] . these data highlight the potential of the bac approach for the rapid and reliable construction of stable infectious clones of emerging flavivirus and other similar rna viruses with unstable viral genomes when amplified as cdnas in bacteria. the zikv reverse genetic system described in this article was further used to study the effect of a single amino acid substitution (a v) in the viral ns a protein on virus growth in cultured cells and pathogenesis in vivo. our results suggested that this single amino acid change impaired viral rna synthesis and virus production in cell culture and highly attenuated the virus in mice. however, we cannot discard that this mutation in the ns a protein could affect other steps in the replication cycle of the virus. flavivirus ns a protein is a -kda hydrophobic protein associated with the endoplasmic reticulum that contains eight transmembrane domains. it is a multifunctional protein that has been involved in viral rna synthesis [ , ] , virus assembly [ , ] , membrane rearrangement [ ] , and immunomodulation of innate immune response [ ] [ ] [ ] . by homology with the denv ns a topology, the zikv ns a a v mutation maps in the last transmembrane domain, for which no specific function has been reported. therefore, our data constitute the first evidence of a role of this ns a domain in viral rna synthesis. on the other hand, it is important to note that the ns a a v mutation promoted a -fold reduction in viral rna synthesis and more than -fold reduction in virus production. this reduced virus production could be a consequence of the rna synthesis impairment. however, since the reduction in virus production is higher than that observed in rna synthesis and that flavivirus ns a protein is also involved in virus assembly, we cannot discard an additional effect of a v mutation in zikv assembly. in addition, we have found that the mutant virus was attenuated in a mice. this attenuation could be explained as a consequence of the lower rna synthesis of the mutant virus. however, an additional effect of the a v mutation on the putative immunomodulatory role of ns a protein [ ] [ ] [ ] , leading to virulence attenuation, cannot be discarded. future studies will be required to determine whether this mutation affects only viral rna synthesis or also virus assembly and immunomodulation of the host defenses. importantly, we have shown that immunization with a single dose of pfu of the mutant rzikv-rgn-mns a induced protection against a lethal challenge with the parental rzikv-rgn, suggesting the potential implementation of this ns a mutant as the base of a live-attenuated vaccine. unfortunately, the mutant rzikv-rgn-mns a was instable and reverted to the wt sequence during its propagation in vero cells, limiting the use of this mutation alone for vaccine development. however, this instability and the high conservation of the amino acid a of the ns a protein among zikv strains highlights the importance of this ns a residue for virus replication, and therefore the potential use of ns a protein as a good target for antiviral development against zikv infection. in summary, we have developed a powerful zikv reverse genetic system based on the use of bacs that has allowed us to identify a single point mutation in the ns a protein that attenuates the virus in vitro and in vivo. this infectious clone system provides a valuable tool to the research community to explore zikv molecular biology, viral determinants of zikv pathogenesis, virus-host interactions, and vaccine and antivirals developments. an update on zika virus infection who calls off global zika emergency the . å resolution cryo-em structure of zika virus probing molecular insights into zika virus(-)host interactions zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria simultaneous outbreaks of dengue, chikungunya and zika virus infections: diagnosis challenge in a returning traveller with nonspecific febrile illness the global threat of zika virus to pregnancy: epidemiology, clinical perspectives, mechanisms, and impact zika virus outbreak on yap island, federated states of micronesia zika virus, french polynesia, south pacific zika virus in the americas: early epidemiological and genetic findings four in florida are infected with zika from local mosquitoes zika virus was transmitted by sexual contact in texas, health officials report defining the syndrome associated with congenital zika virus infection the brazilian zika virus strain causes birth defects in experimental models guillain-barre syndrome after zika virus infection in brazil zika virus disrupts neural progenitor development and leads to microcephaly in mice zika virus infection during pregnancy in mice causes placental damage and fetal demise zika virus associated with microcephaly potential of selected senegalese aedes spp. mosquitoes (diptera: culicidae) to transmit zika virus viral determinants and vector competence of zika virus transmission transmission of zika virus through breast milk and other breastfeeding-related bodily-fluids: a systematic review the expanding spectrum of modes of transmission of zika virus: a global concern flavivirus reverse genetic systems, construction techniques and applications: a historical perspective successful propagation of flavivirus infectious cdnas by a novel method to reduce the cryptic bacterial promoter activity of virus genomes functional cdna clones of the flaviviridae: strategies and applications recovery of the zika virus through an in vitro ligation approach a reverse genetics platform that spans the zika virus family tree zika virus encoding nonglycosylated envelope protein is attenuated and defective in neuroinvasion an infectious cdna clone of zika virus to study viral virulence, mosquito transmission, and antiviral inhibitors characterization of cis-acting rna elements of zika virus by using a self-splicing ribozyme-dependent infectious clone rescue of the zika virus prototype strain with a cytomegalovirus promoter-driven cdna clone a full-length infectious cdna clone of zika virus from the epidemic in brazil as a genetic platform for studies of virus-host interactions and vaccine development development and characterization of recombinant virus generated from a new world zika virus infectious clone simple reverse genetics systems for asian and african zika viruses a robust method for the rapid generation of recombinant zika virus expressing the gfp reporter gene a reverse genetics system for zika virus based on a simple molecular cloning strategy de novo generation and characterization of new zika virus isolate using sequence data from a microcephaly case development of a novel dna-launched dengue virus type infectious clone assembled in a bacterial artificial chromosome complete nucleotide sequence of two generations of a bacterial artificial chromosome cloning vector cloning and stable maintenance of -kilobase-pair fragments of human dna in escherichia coli using an f-factor-based vector analysis of relative gene expression data using real-time quantitative pcr and the (−∆∆ c(t)) method. methods the miqe guidelines: minimum information for publication of quantitative real-time pcr experiments an open-source platform for biological-image analysis canine influenza viruses with modified ns proteins for the development of live-attenuated vaccines a susceptible mouse model for zika virus infection a mouse model of zika virus pathogenesis characterization of a novel murine model to study zika virus committee for the update of the guide for the care and use of laboratory animals sindbis virus dna-based expression vectors: utility for in vitro and in vivo gene transfer virus pathogen resource (vipr), faviviridae. available online engineering the largest rna virus genome as an infectious bacterial artificial chromosome targeted modification of a human β-globin locus bac clone using get recombination and an i-scei counterselection cassette a highly efficient escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of bac dna two-step red-mediated recombination for versatile high-efficiency markerless dna manipulation in escherichia coli a new logic for dna engineering using recombination in escherichia coli construction of a severe acute respiratory syndrome coronavirus infectious cdna clone and a replicon to study coronavirus rna synthesis engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate molecular characterization of feline infectious peritonitis virus strain df- and studies of the role of orf abc in viral cell tropism recovery of a neurovirulent human coronavirus oc from an infectious cdna clone subcellular localization and some biochemical properties of the flavivirus kunjin nonstructural proteins ns a and ns a mutations in west nile virus nonstructural proteins that facilitate replicon persistence in vitro attenuate virus replication in vitro and in vivo mutations in the yellow fever virus nonstructural protein ns a selectively block production of infectious particles role of nonstructural protein ns a in flavivirus assembly analysis of adaptive mutations in kunjin virus replicon rna reveals a novel role for the flavivirus nonstructural protein ns a in inhibition of β interferon promoter-driven transcription a single amino acid substitution in the west nile virus nonstructural protein ns a disables its ability to inhibit α/β interferon induction and attenuates virus virulence in mice subversion of interferon by dengue virus we are grateful to carla gómez and snezhana dimitrova for technical assistance in the bac clone generation and mice experiments, respectively. we also thank sylvia gutiérrez and ana oña at the cnb advanced microscopy facility for their valuable support in immunofluorescence microscopy analysis. the authors declare no conflict of interest. the funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, and in the decision to publish the results. key: cord- -synm cyw authors: hou, jiun-nan; chen, tien-huang; chiang, yi-hsuan; peng, jing-yun; yang, tsong-han; cheng, chih-chieh; sofiyatun, eny; chiu, cheng-hsun; chiang-ni, chuan; chen, wei-june title: perk signal-modulated protein translation promotes the survivability of dengue virus-infected mosquito cells and extends viral replication date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: synm cyw survival of mosquitoes from dengue virus (denv) infection is a prerequisite of viral transmission to the host. this study aimed to see how mosquito cells can survive the infection during prosperous replication of the virus. in c / cells, global protein translation was shut down after infection by denv type (denv ). however, it returned to a normal level when infected cells were treated with an inhibitor of the protein kinase rna (pkr)-like er kinase (perk) signaling pathway. based on a -methylguanosine ′-triphosphate (m gtp) pull-down assay, the eukaryotic translation initiation factor f (eif f) complex was also identified in denv -infected cells. this suggests that most mosquito proteins are synthesized via canonical cap-dependent translation. when the perk signal pathway was inhibited, both accumulation of reactive oxygen species and changes in the mitochondrial membrane potential increased. this suggested that er stress response was alleviated through the perk-mediated shutdown of global proteins in denv -infected c / cells. in the meantime, the activities of caspases- and - and the apoptosis-related cell death rate increased in c / cells with perk inhibition. this reflected that the perk-signaling pathway is involved in determining cell survival, presumably by reducing denv -induced er stress. looking at the perk downstream target, α-subunit of eukaryotic initiation factor (eif α), an increased phosphorylation status was only shown in infected c / cells. this indicated that recruitment of ribosome binding to the mrna ′-cap structure could have been impaired in cap-dependent translation. it turned out that shutdown of cellular protein translation resulted in a pro-survival effect on mosquito cells in response to denv infection. as synthesis of viral proteins was not affected by the perk signal pathway, an alternate mode other than cap-dependent translation may be utilized. this finding provides insights into elucidating how the perk signal pathway modulates dynamic translation of proteins and helps mosquito cells survive continuous replication of the denv . it was ecologically important for virus amplification in mosquitoes and transmission to humans. dengue fever is caused by infection of dengue viruses (denvs), leading to a broad spectrum of clinical symptoms including asymptomatic or undifferentiated febrile illness and sometimes severe forms of symptoms such as dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) [ ] . the virus is naturally transmitted between humans by aedes mosquitoes, primarily aedes aegypti [ ] , meaning that it is able to grow in both arthropod and human/mammalian cells. denvs and other flaviviruses are dependent on the host endoplasm reticulum (er) for translation, replication and packaging of their genome [ ] . it results in inducing the unfolded protein response (upr) because of the accumulation of misfolded or unfolded proteins in the er [ ] . the upr is composed of three different classes of er stress transducers, i.e., inositol-requiring protein- (ire ), activating transcription factor- (atf ) and protein kinase rna (pkr)-like er kinase (perk) signaling pathways [ ] . all of these consist of a luminal domain and a cytosolic domain; the luminal domain recognizes unfolded/misfolded proteins inside the er, while the cytosolic domain relays signals to turn on downstream genes [ ] . denv infection in mammalian cells mostly leads to death, necrosis and/or apoptosis, due to the persistence of the upr [ , ] ; which has also been observed in mosquito cells [ ] . virus infection in eukaryotic cells frequently shuts down cellular protein translation due to the need to recruit ribosomes to translate viral proteins required for replication [ ] . such translational control by the virus may help cells subvert er stress [ ] . the perk signal pathway may be an important factor as it is usually activated and subsequently phosphorylates the downstream effector, α-subunit of eukaryotic initiation factor (eif α), which functions to suppress general protein translation [ , ] . modulation of the upr in denv-infected cells was found to override the shutting down of translation, delay cell death and prolong the viral life cycle [ ] . the upr was also identified in mosquito cells with denv infection [ ] . however, it usually does not impair the growth of those cells [ ] , frequently leading to persistent infection in mosquito cells [ , ] . regulation of the upr via virus-controlled translation of cellular proteins could be critical for a cell's fate during infection in both mammalian and mosquito cells. protein translation initiation of messenger rna (mrna) in cells is generally implemented by the eukaryotic translation initiation factor f (eif f) complex, which is composed of the cap-binding protein, eif e, the rna helicase, eif a, the adaptor protein, eif g and other essential proteins [ ] . the complex functions to link -methylgpppg caps at the -end and poly-a tails at the -end [ ] . this machinery requires the binding of eif e to the cap structure during initiation of mrna translation. therefore, this process is called cap-dependent translation and is implemented by most proteins translated for physiological purposes [ ] . cap-dependent translation is initiated by activation of the mammalian target of rapamycin (tor; mtor) that phosphorylates eif e-bp in response to extracellular stimuli in such viral infections [ ] . for instance, inhibiting cap-dependent translation may thus shut down protein translation of cells infected by the encephalomyocarditis virus (emcv) or vesicular stomatitis virus (vsv) [ , ] . based on our previous reports about mosquito cell responses to denv infection [ , ] , we herein aimed to demonstrate and discuss how denv regulates protein translation and subsequent cell survival via the perk signaling pathway in mosquito cells. hypothetically, the result could be applied to elucidate how the mosquito vector can healthily transmit the denv and maybe other arboviruses to the host. denv (new guinea c strain) was propagated in c / cells derived from ae. albopictus. cells were cultured with minimal essential medium (mem; gibco tm , invitrogen, carlsbad, ca, usa), which was supplemented with % fetal bovine serum (fbs) (life technologies, grand island, ny, usa), % non-essential amino acids, g/ml hepes (sigma, st. louis, mo, usa), . g/ml sodium bicarbonate (nahco ) and . % antibiotic-antimycotic (gibco tm , invitrogen) at • c in a closed system without a supply of co . the propagated virus was titrated with baby hamster kidney (bhk)- cells (kindly provided by chwan-chuen king, national taiwan university, taipei, taiwan), which were maintained in mem containing % fbs, % non-essential amino acids, . g/ml nahco and . % of an antimycotic (gibco tm , invitrogen) at • c in an incubator with a % co atmosphere [ ] . to inactivate the denv , a virus suspension was treated with a low-intensity ultraviolet (uv) source ( nm; mj/cm ) for min on ice in the spectrolinker xl- uv crosslinker (spectronics, westbury, ny, usa). the extraction of total rna from mock-or denv -infected c / cells using isol-rna lysis reagent ( prime gaithersburg, md, usa) followed the protocol provided by the manufacture. the extracted rna was subsequently reversely transcribed to complementary dna (cdna) using the m-mlv reverse transcriptase (invitrogen, carlsbad, ca, usa). briefly, the components including µg of extracted rna, µl mm random hexamer primer (fermentas, glen burnie, md, usa) and µl mm dntp mix (fermentas) were added into a . -ml microcentrifuge tube, then filled with distilled water up to µl of the total volume. the mixture was heated at • c for min, then µl of × first-stand buffer, µl . m dtt, µl rnase outtm ribonucleases inhibitor (invitrogen) and µl of m-mlv reverse transcriptase were added. the final mixture was incubated at • c for min, followed by at • c for min. the formed cdna was stored at − • c for further experiments. a part of extracted cdna was measured by a q-pcr to quantify the expression level of specific genes. reagents prepared for q-pcr included forward and reverse primers ( . µm), µl sybr green supermix (kapa biosystems, wilmington, ma, usa), µl cdna template and . µl ddh o, making a final volume of µl. the thermal cycles included min of activation with amplitaq at • c and cycles of amplification consisting of s for denaturation at • c and s for amplification at • c, with an applied biosystems real-time pcr system (applied biosystems, carlsbad, ca, usa). primer pairs used in this experiment are listed as follows: s rrna forward, -attgacggaagggcaccaccag; s rrna reverse, -agaacggccatgcaccactacc (for the internal control); d v forward, -tggaccgacaaagacagattctt; d v reverse, -cgyccytgcagcattccaa (for denv ). c / cells were treated with tunicamycin (tm; emd millipore, billerica, ma, usa), an er stress inducer, to induce a strong er stress response in mosquito cells. briefly, . × c / cells were seeded in a -cm dish and incubated at • c for h. cells were subsequently treated with dimethyl sulfoxide (dmso) or tunicamycin at a concentration of , , and µg/ml for h at • c. treated cells were then harvested to measure the efficiency of protein synthesis using the surface sensing of translation (sunset) method followed by western blot analysis. one group of those cells further added with µm perk inhibitor was subjected to detection of eif , also by western blotting. c / cells were seeded in -well plates with cells per well and incubated at • c for h. except for those used as the control, cells were then infected with denv at a multiplicity of infection (moi) of . at various intervals of time in hours post-infection (hpi), cells were treated with µm puromycin (sigma) and incubated at • c for min, thus labeling nascent polypeptide chains. cells collected from each well were then subjected to a western blot analysis. a cap pull-down assay using immobilized γ-aminiphenyl-m gtp c -spacer beads (ac- s, jena bioscience, jena, germany) was carried out to precipitate components of the intracellularly formed eif complex. in brief, c / cells were lysed in np lysis buffer ( mm tris-hcl, mm nacl, mm edta and % np- ) with m naf, mm na vo , × protease inhibitor cocktail (bioshop, burlingtom, on, canada) and × serine/threonine phosphatase inhibitor cocktail (bionovas, toronto, on, canada). about - µg of cell lysates were incubated with - µl of beads for h at • c with gentle rotation. after incubation, beads were pelletized, and the supernatant was used as the run-off lysate. pelleted beads were washed twice with np- lysis buffer. washed beads were then boiled in µl of × sodium dodecyl sulfate (sds) protein loading dye for min. after centrifugation at , rpm for min, the supernatants were stored at − • c for a further western blot analysis. in the present study, protein profiles from c / cells were analyzed by western blotting using the corresponding antibodies listed as follows: anti-actin (clone c ) mouse monoclonal antibody (mab; diluted : ; emd millipore), anti-puromycin ( d ) mouse mab (diluted : ; emd millipore), anti-eif α rabbit polyclonal antibody (pab; diluted : ; # , cell signaling technology, danvers, ma, usa,), anti-phospho-eif α (ser ) rabbit pab (diluted : ; # , cell signaling technology), p s kinase (c- ) rabbit pab (diluted : ; santa cruz biotechnology, sc- , dallas, tx, usa), phosphorylated (phospho)-drosophila p s kinase (thr ) rabbit pab (diluted : ; # , cell signaling technology), eif e-bp rabbit pab (diluted : ; produced by our lab), phospho- e-bp (thr / ) rabbit mab (diluted : ; # , cell signaling technology), eif e rabbit pab (diluted : ; # , cell signaling technology), eif a (f ) rabbit pab (diluted : ; # , cell signaling technology), pabp rabbit pab (diluted : ; # , cell signaling technology), anti-flavivirus ns (diluted : ; #yh , yao-hong biotechnology, taipei, taiwan) and anti-dengue capsid mouse mab (diluted : ; kind gift from oscar g, c perng, national cheng kung university, tainan, taiwan). goat anti-mouse immunoglobulin g (igg) conjugated with a horseradish peroxidase (hrp) antibody (diluted : ; emd millipore) and goat anti-rabbit igg conjugated with an hrp antibody (diluted : ; sigma) were used as secondary antibodies. briefly, c / cells were harvested at various intervals of time post-infection by centrifugation of rpm at • c for min. pellets were re-suspended in µl of ripa lysis buffer consisting of mm tris-hcl, mm nacl, mm edta, . % deoxycholic acid, % np- , . % triton-x , m naf, mm na vo , × protease inhibitor cocktail (bioshop) and × serine/threonine phosphatase inhibitor cocktail (bionovas). subsequently, whole-cell extracts were run on % or % sds-polyacrylamide gels, and separated proteins were electrically transferred onto polyvinylidene difluoride (pvdf) membranes with a . -µm pore size (emd millipore). after being blocked with % non-fat milk in tbs- . % tween at room temperature for h, the membrane was washed with tbs- . % tween and then incubated with one of the mentioned primary antibodies diluted in tbs- % bsa at • c overnight. after another wash, the membrane was incubated with a selected secondary antibody diluted in tbs- % non-fat milk at room temperature for h. after the membrane had been washed again, images were developed using chemiluminescence reagent (perkinelmer tm viruses , , of life science, waltham, ma, usa) and visualized with an x-ray film processor (kodak x-omat , kodak, rochester, ny, usa). activation of perk can be inhibited by treatment with an inhibitor (perki; gsk ) (emd millipore). in practice, . × c / cells were seeded in a -cm dish and incubated at • c for h. cells infected with denv at an moi of were maintained in culture medium containing dmso or µm perki at indicated time points. mock-infected cells were used as a control in the present study. cells from each treatment were harvested at indicated time points for further experiments. the mitochondrial membrane potential (mmp) was analyzed using a mitocapture mitochondrial apoptosis detection fluorometric kit (biovision, milpitas, ca, usa). in brief, cells were collected from each treatment and centrifuged at rpm for min, and then, the supernatant was discarded. pelletized cells were gently suspended by adding ml of mitocapture solution and incubated at • c for min in the dark. after another centrifugation at rpm for min, pelleted cells were re-suspended with ml of incubation buffer and analyzed with a facscan flow cytometer (bd biosciences, san jose, ca, usa). reactive oxygen species (ros) representing oxidative stress were measured using dihydroethidium (dhe) staining (sigma) and , -dichlorodihydrofluorescein diacetate (h dcfda) staining (invitrogen) to respectively estimate the accumulation of superoxide anions and h o , following instructions provided by the manufacturers. briefly, c / cells from each treatment were collected and centrifuged at rpm for min and then re-suspended in ml of phosphate-buffered saline (pbs) containing µm dhe or µm h dcfda. these cells were incubated at • c for min in the dark, subsequently centrifuged at rpm for min and then re-suspended in ml of pbs. collected cells were subjected to analysis with a facscan flow cytometer (bd biosciences) using red fluorescence (channel fl ) to detect superoxide anions and green fluorescence (channel fl ) for h o . apoptotic cells were detected using an annexin v-fitc/pi apoptosis detection kit (biovision), following the manufacturer's instructions. in brief, c / cells from each treatment were collected by centrifugation at rpm for min. after being gently suspended in µl of binding buffer, cells were incubated with a mixture containing µl annexin v-fitc and µl propidium iodide (pi) at • c for min in the dark. the annexin v-fitc/pi-bound cells were ultimately analyzed with a facscan flow cytometer (bd biosciences). activities of caspases- and - were detected using a caspglow tm fluorescein caspase- staining kit (biovision) and a caspglow tm fluorescein caspase- staining kit (biovision), following the protocol from the manufacturer. briefly, harvested c / cells were centrifuged at rpm for min and then re-suspended in µl pbs; in which µl of fitc-deve-fmk for caspase- or µl of fitc-lehd-fmk for caspase- was added and incubated at • c for h in the dark. after centrifuging at rpm for min and discarding the supernatant, cells washed with and re-suspended in wash buffer were subjected to analysis with a facscan flow cytometer (bd biosciences). c / cells were collected by centrifugation at rpm and • c for min. after the supernatant was removed, the cell pellet was fixed in ice-cold % ethanol in a − • c freezer for h. the fixation solution was removed by centrifugation at rpm and • c for min, and then, the cell pellet was washed with pbs. these cells were treated with . % triton x- and . % rnase a (sigma) in pbs for h at • c. after a final centrifugation, pelletized cells were stained with µg/ml of pi (sigma) in pbs at • c for min in the dark. the cellular dna content was measured using a facscan flow cytometer (bd biosciences, san jose, ca, usa). the double luciferase reporter assay was used in this study to evaluate the efficiency of cap-dependent protein translation by measuring the reactions of firefly and renilla luciferases in c / cells. briefly, . × c / cells were seeded in a -cm dish for h, followed by co-transfection with plasmids containing firefly luciferase or renilla luciferase reporter genes. the transfected cells were distributed evenly in each well of a -well culture plate ( × cells per well) for h. cells were then infected with the denv (at an moi of ), followed by h of adsorption before the addition of fresh culture medium. cells collected at , and hpi were washed with pbs and then incubated µl passive lysis buffer (promega, madison, wi, usa) for min at room temperature. after the cell lysate was centrifuged at , rpm for s, a mixture of µl of supernatant and µl of luciferase measurement for assay reagent ii (promega) was intermediately analyzed, after µl of stop & glo ® reagent (promega) were added to stop the reaction of firefly luciferase, with a glomax ® / luminometer (promega). each comparison between two means was analyzed by student's t-test at a significance level of ≤ . , or %. for those more than two means, a one-way analysis of variance (anova) was used for the statistical test at the same level of significance. sunset is a nonradioactive fluorescence-activated cell sorting-based assay that allows the monitoring and quantification of global protein synthesis in cells [ ] . herein, the total protein amount in c / cells with denv infection was shown to be reduced at hpi, and this persisted to hpi ( figure a ); changes between infected and uninfected cells at both time points significantly differed (student t-test; ** p < . ) ( figure b) . the viral ns protein can only be detected in cells inoculated with infectious denv , but not the virus inactivated by treatment with uv light ( figure c ). furthermore, protein shutdown was not shown in cells inoculated with uv-inactivated denv for h, while it was observed in untreated denv ( figure d ). in the meantime, total proteins were obviously reduced in cells treated with different concentrations of tunicamycin (tm), which is known as a strong er stress inducer ( figure e ). this reveals that shutdown of global protein translation triggered by denv entering c / cells was compatible to that treated with the er stress inducer. sunset also showed that total protein synthesis decreased in cells infected with untreated denv , but there was no obvious change in cells inoculated with a uv-inactivated virus; (e) in c / cells treated with tunicamycin (tm), a strong endoplasmic reticular (er) stress inducer, for h, the expression of total proteins decreased, even when the concentration of the drug was as low as μg/ml. cellular proteins were generally synthesized via cap-dependent protein translation, which was evaluated in denv -infected c / cells by transfection with a dual luciferase detection system. the system utilizes a plasmid containing firefly and renilla luciferases, the promoter of which triggers the translation of both reporter proteins in a cap-dependent manner. results showed that both firefly and renilla luciferases had significantly reduced activities in c / cells with denv infection at , and hpi compared to the relative luciferase activities of mock-infected cells (student's t-test; * p < . , ** p < . , *** p < . ) (figure a ). with the m gtp pull-down assay followed by sds-page and coomassie blue staining, only a few proteins were shown, representing specific proteins that had been pulled down ( figure b ). by detection with western blot analysis using specific antibodies, components of the eif f complex, including eif e, eif a and pabp, were indifferently identified in both mock-and denv -infected cells after pull-down with m gtp, especially in cells with denv infection for h ( figure c ). this indicates that an eif f complex serving as the capdependent protein translation machinery had been formed; however, denv infection may have interfered with subsequent protein translation. protein synthesis rate was analyzed by western blot with an antibody against puromycin. western blot analysis of actin and coomassie blue staining of total cellular protein were included to prove equal protein loading. results showed that the total protein amount had been reduced by hpi, and this persisted to hpi; (b) histograms represent the quantification of the intensities of puromycin-incorporated bands from three independent experiments. changes between virus-and mock-infected cells significantly differed at both time points (student's t-test; ** p < . ); (c) the viral ns protein was only detected in cells infected by infectious denv , not in those inoculated with a uv-inactivated virus according to the results of the immunofluorescent assay; (d) the method sunset also showed that total protein synthesis decreased in cells infected with untreated denv , but there was no obvious change in cells inoculated with a uv-inactivated virus; (e) in c / cells treated with tunicamycin (tm), a strong endoplasmic reticular (er) stress inducer, for h, the expression of total proteins decreased, even when the concentration of the drug was as low as µg/ml. cellular proteins were generally synthesized via cap-dependent protein translation, which was evaluated in denv -infected c / cells by transfection with a dual luciferase detection system. the system utilizes a plasmid containing firefly and renilla luciferases, the promoter of which triggers the translation of both reporter proteins in a cap-dependent manner. results showed that both firefly and renilla luciferases had significantly reduced activities in c / cells with denv infection at , and hpi compared to the relative luciferase activities of mock-infected cells (student's t-test; * p < . , ** p < . , *** p < . ) (figure a ). with the m gtp pull-down assay followed by sds-page and coomassie blue staining, only a few proteins were shown, representing specific proteins that had been pulled down ( figure b ). by detection with western blot analysis using specific antibodies, components of the eif f complex, including eif e, eif a and pabp, were indifferently identified in both mock-and denv -infected cells after pull-down with m gtp, especially in cells with denv infection for h ( figure c ). this indicates that an eif f complex serving as the cap-dependent protein translation machinery had been formed; however, denv infection may have interfered with subsequent protein translation. with an m gtp pull-down assay followed by sds-page, only specific proteins were left and are shown on the gel; (c) detection by western blotting with specific antibodies, proteins, including eif e, eif a and poly-a binding protein (pabp), were identified without significant difference in both mock-and denv -infected cells after pull-down by m gtp in addition to that found in run-off cell lysate both at and hpi. this indicated that an eif f complex that served as the cap-dependent protein translation machinery had been formed. since regulation of cap-dependent translation has been related to tor signaling [ ] , to access the activation of the tor signaling pathway, its downstream target gene eif e-bp was detected due to the absence of an appropriate antibody for recognizing tor activation in mosquito cells. the result showed that eif e-bp was phosphorylated at a very early stage of infection by denv (< hpi); however, it was at a lower level in a later stage of infection (> hpi). in spite of this, such changes may depend on the expressed protein according to the ratios between the phosphorylated form and the amount of eif e-bp ( figure ) . another of tor's downstream genes s k was also shown to be phosphorylated, as shown in mammalian cells infected by west nile virus [ ] , particularly in a later stage of infection, e.g., hpi in c / cells, although its protein expression level remained at a constant level throughout the same period of infection ( figure ). results revealed that the tor signaling pathway may have retained its activity involved in controlling cap-dependent protein translation in c / cells infected by the denv . this suggests that most cellular proteins are synthesized principally via cap-dependent translation in c / cells. since ns viral proteins were persistently expressed throughout the period of infection for h in denv -infected c / cells (figure ), viral proteins may be translated by an alternate mode of translation disregarding the tor signaling pathway [ ] . viruses , , of since regulation of cap-dependent translation has been related to tor signaling [ ] , to access the activation of the tor signaling pathway, its downstream target gene eif e-bp was detected due to the absence of an appropriate antibody for recognizing tor activation in mosquito cells. the result showed that eif e-bp was phosphorylated at a very early stage of infection by denv (< hpi); however, it was at a lower level in a later stage of infection (> hpi). in spite of this, such changes may depend on the expressed protein according to the ratios between the phosphorylated form and the amount of eif e-bp ( figure ) . another of tor's downstream genes s k was also shown to be phosphorylated, as shown in mammalian cells infected by west nile virus [ ] , particularly in a later stage of infection, e.g., hpi in c / cells, although its protein expression level remained at a constant level throughout the same period of infection ( figure ). results revealed that the tor signaling pathway may have retained its activity involved in controlling cap-dependent protein translation in c / cells infected by the denv . this suggests that most cellular proteins are synthesized principally via cap-dependent translation in c / cells. since ns viral proteins were persistently expressed throughout the period of infection for h in denv -infected c / cells (figure ), viral proteins may be translated by an alternate mode of translation disregarding the tor signaling pathway [ ] . -infection (hpi) ) and was at a lower level at a later stage of infection (> hpi), compatible with a reduction in eif e-bp expression. according to the ratio of phosphorylation/total eif e-bp, its change of phosphorylated level may be dependent on the expression of eif e-bp. one of tor's downstream genes, s kinase (s k), was usually phosphorylated particularly at hpi in c / cells, even though its protein level remained at a constant level throughout the same period of infection. in the meantime, ns viral proteins were persistently expressed throughout the -h infection period. the level of actin was included to prove equal loading of protein in each line. this reveals that the tor signaling pathway may have been activated and involved in controlling cap-dependent translation of cellular proteins. in order to check the role of the perk signaling pathway in cellular protein synthesis, a perk inhibitor (gsk ) at a concentration of μm was used to treat denv -infected c / cells. though off-target-effects have ever been of concern, this drug seems to be specific enough for observation on its functional trend of perk inhibition [ , ] . the result showed that inhibition of total protein translation was recovered ( figure a) . statistically, it returned to a level close to normal, figure . activation of the target of rapamycin (tor) signaling pathway by the dengue virus (denv ) in c / cells. according to the western blotting analysis carried out in denv -infected c / cells, eif e-bp was shown to be phosphorylated at a very early stage of infection by the denv (< h post-infection (hpi)) and was at a lower level at a later stage of infection (> hpi), compatible with a reduction in eif e-bp expression. according to the ratio of phosphorylation/total eif e-bp, its change of phosphorylated level may be dependent on the expression of eif e-bp. one of tor's downstream genes, s kinase (s k), was usually phosphorylated particularly at hpi in c / cells, even though its protein level remained at a constant level throughout the same period of infection. in the meantime, ns viral proteins were persistently expressed throughout the -h infection period. the level of actin was included to prove equal loading of protein in each line. this reveals that the tor signaling pathway may have been activated and involved in controlling cap-dependent translation of cellular proteins. in order to check the role of the perk signaling pathway in cellular protein synthesis, a perk inhibitor (gsk ) at a concentration of µm was used to treat denv -infected c / cells. though off-target-effects have ever been of concern, this drug seems to be specific enough for observation on its functional trend of perk inhibition [ , ] . the result showed that inhibition viruses , , of of total protein translation was recovered ( figure a ). statistically, it returned to a level close to normal, showing an insignificant difference between perki-treated cells with and without infected by denv (student's t-test; p > . ) ( figure b) . dengue viral proteins, capsid (c) and ns , remained to be synthesized in perk inhibitor-treated c / cells even those that have been infected by denv for h ( figure c ). no significant difference of protein amounts was shown between cells with and without perk inhibitor treatment ( figure d ). meanwhile, no significant difference of viral rna level was shown between cells with and without perk inhibitor treatment ( figure e ). in addition, no obvious morphological defects were observed in cells treated with the inhibitor for up to h ( figure f ), indicating that at µm, the perk inhibitor did not have adverse effects on cells. moreover, reduced protein translation in cells treated with tunicamycin (tm) was recovered by adding the perk inhibitor at µm ( figure g ). in the meantime, phosphorylated (p-) eif α was shown to increase after treatment with tm, but returned to be a lower level of amount when the perk inhibitor was added ( figure g ). the eif α is known to be a key downstream target of the perk signaling pathway and involved in protein translation in eukaryotic cells [ ] . this suggested that the perk signaling pathway is involved in regulating cellular protein translation when er stress is induced by denv in mosquito cells. viruses , , of showing an insignificant difference between perki-treated cells with and without infected by denv (student's t-test; p > . ) ( figure b ). dengue viral proteins, capsid (c) and ns , remained to be synthesized in perk inhibitor-treated c / cells even those that have been infected by denv for h ( figure c ). no significant difference of protein amounts was shown between cells with and without perk inhibitor treatment ( figure d ). meanwhile, no significant difference of viral rna level was shown between cells with and without perk inhibitor treatment ( figure e ). in addition, no obvious morphological defects were observed in cells treated with the inhibitor for up to h ( figure f ), indicating that at μm, the perk inhibitor did not have adverse effects on cells. moreover, reduced protein translation in cells treated with tunicamycin (tm) was recovered by adding the perk inhibitor at μm ( figure g ). in the meantime, phosphorylated (p-) eif α was shown to increase after treatment with tm, but returned to be a lower level of amount when the perk inhibitor was added ( figure g ). the eif α is known to be a key downstream target of the perk signaling pathway and involved in protein translation in eukaryotic cells [ ] . this suggested that the perk signaling pathway is involved in regulating cellular protein translation when er stress is induced by denv in mosquito cells. the mmp generally changes due to a transition in mitochondrial permeability, which has been demonstrated to change in c / cells, as well as in mammalian bhk- cells infected by denv for h [ ] . to evaluate the mmp in this study, facscan flow cytometry was used to measure the fluorescein isothiocyanate (fitc) channel for green mitocapture monomers in mock-and denv -infected c / cells with and without treatment with the perk inhibitor. results revealed a difference in the induction of the mmp between cells in the two groups, particularly at hpi. quantitatively, the fluorescent intensity of denv -infected c / cells treated with the perk inhibitor at hpi ( . -fold) significantly increased compared to the two control groups (by . -fold compared to untreated cells and . -fold compared to dmso-treated cells) (student's t-test; *** p < . ), while no statistical difference was shown at hpi ( figure ). this indicates that a change in the mmp is one of the essential steps possibly involved in the perk signaling pathway. in fact, it is an important step in the process of inducing apoptosis in cells under er stress [ ] . adding the perk inhibitor ( μm). the phosphorylated eif α elicited by treatment with tm turned out to be a lower level of amount according to the western blotting analysis. it reflected that the perk signaling pathway regulates cellular protein translation after endoplasmic reticular stress was induced in c / cells. the mmp generally changes due to a transition in mitochondrial permeability, which has been demonstrated to change in c / cells, as well as in mammalian bhk- cells infected by denv for h [ ] . to evaluate the mmp in this study, facscan flow cytometry was used to measure the fluorescein isothiocyanate (fitc) channel for green mitocapture monomers in mock-and denv infected c / cells with and without treatment with the perk inhibitor. results revealed a difference in the induction of the mmp between cells in the two groups, particularly at hpi. quantitatively, the fluorescent intensity of denv -infected c / cells treated with the perk inhibitor at hpi ( . -fold) significantly increased compared to the two control groups (by . -fold compared to untreated cells and . -fold compared to dmso-treated cells) (student's t-test; *** p < . ), while no statistical difference was shown at hpi ( figure ). this indicates that a change in the mmp is one of the essential steps possibly involved in the perk signaling pathway. in fact, it is an important step in the process of inducing apoptosis in cells under er stress [ ] . superoxide anions (o -) are one of the main ros and are usually converted to hydrogen peroxide (h o ); both of these are free radicals that may accumulate and induce the er stress in the cell [ ] . using facscan flow cytometry, the relative fluorescent intensity of superoxide anions detected in c / cells with denv infection for h did not significantly change ( . -fold) even when a perk superoxide anions (o -) are one of the main ros and are usually converted to hydrogen peroxide (h o ); both of these are free radicals that may accumulate and induce the er stress in the cell [ ] . using facscan flow cytometry, the relative fluorescent intensity of superoxide anions detected in c / cells with denv infection for h did not significantly change ( . -fold) even when a perk inhibitor was applied ( figure a ). nevertheless, it had significantly increased by hpi (a . -fold increase) compared to cells in the control groups ( . -and . -fold for untreated and dmso-treated cells, respectively) (student's t-test; *** p < . ). a similar changing trend of hydrogen peroxide was shown in the same cell groups, leading to a significant increase at hpi ( . -fold; student's t-test; * p < . , *** p< . ), but not at hpi ( . -fold) ( figure b ). this reflects that the perk signaling pathway may be involved in changing the accumulation of ros in c / cells with denv infection. increase) compared to cells in the control groups ( . -and . -fold for untreated and dmso-treated cells, respectively) (student's t-test; *** p < . ). a similar changing trend of hydrogen peroxide was shown in the same cell groups, leading to a significant increase at hpi ( . -fold; student's t-test; * p < . , *** p< . ), but not at hpi ( . -fold) ( figure b ). this reflects that the perk signaling pathway may be involved in changing the accumulation of ros in c / cells with denv infection. the sub-g phase corresponds to cells with fragmented dna genome, thus representing the cells undergoing apoptosis during the cell cycle. in this study, the cell cycle progression was detected by pi staining in denv -infected c / cells treated with a perk inhibitor, revealing that there was a higher rate ( . % at hpi) of cells in the sub-g phase ( figure a ). when denv -infected cells the sub-g phase corresponds to cells with fragmented dna genome, thus representing the cells undergoing apoptosis during the cell cycle. in this study, the cell cycle progression was detected by pi staining in denv -infected c / cells treated with a perk inhibitor, revealing that there was a higher rate ( . % at hpi) of cells in the sub-g phase ( figure a ). when denv -infected cells were stained with annexin v-fitc/pi, slight apoptosis-related cell death was found to have occurred at hpi ( figure b ), but it had significantly increased by hpi ( figure c ). there was a statistically-significant difference between cells treated with the perk inhibitor followed by denv infection compared to the other two control groups with infection (student's t-test; *** p < . ) ( figure d ). activation of caspases- (the initiator) and - (the effector), which leads to apoptosis, was previously demonstrated in c / cells with denv infection [ ] . in this study, we further assessed its occurrence in denv -infected c / cells with denv infection. results showed that no evident change in caspase- had occurred in cells by hpi, even when they had been treated with the perk inhibitor, while it had obviously increased at pi (a . -fold increase) compared to the two control groups ( . -fold for untreated and . -fold for dmso-treated cells) (student's t-test; *** p < . ) ( figure a) . a similar changing trend of caspase- , remaining unchanged at hpi, but significantly increased at hpi (a . -fold increase), was shown compared to the control groups ( . -fold for untreated and . -fold for dmso-treated cells) (student's t-test; *** p < . ) ( figure b ). this suggests that the denv -induced perk signaling pathway plays an important role in c / cells, allowing them to avoid the induction of apoptosis in response to infection. were stained with annexin v-fitc/pi, slight apoptosis-related cell death was found to have occurred at hpi ( figure b ), but it had significantly increased by hpi ( figure c ). there was a statistically-significant difference between cells treated with the perk inhibitor followed by denv infection compared to the other two control groups with infection (student's t-test; *** p < . ) ( figure d ). activation of caspases- (the initiator) and - (the effector), which leads to apoptosis, was previously demonstrated in c / cells with denv infection [ ] . in this study, we further assessed its occurrence in denv -infected c / cells with denv infection. results showed that no evident change in caspase- had occurred in cells by hpi, even when they had been treated with the perk inhibitor, while it had obviously increased at pi (a . -fold increase) compared to the two control groups ( . -fold for untreated and . -fold for dmso-treated cells) (student's t-test; *** p < . ) ( figure a) . a similar changing trend of caspase- , remaining unchanged at hpi, but significantly increased at hpi (a . -fold increase), was shown compared to the control groups ( . -fold for untreated and . -fold for dmso-treated cells) (student's t-test; *** p < . ) ( figure b ). this suggests that the denv -induced perk signaling pathway plays an important role in c / cells, allowing them to avoid the induction of apoptosis in response to infection. as mentioned above, eif α is involved in protein translation in eukaryotic cells via the perk signaling pathway. since that are no available tools for directly detecting perk activation of insect cells in hand, measuring phosphorylation of eif α was used as the alternative way showing perk activation in this study. when observing its activity, an increased degree of phosphorylation in response to viral infection was detected, while its protein amount remained stable. importantly, phosphorylated eif α was significantly reduced when the perk inhibitor was applied to infected cells ( figure a ). this indicates that phosphorylation of eif α triggered by denv infection is deeply involved in the translation of cellular proteins through the perk signaling pathway in c / cells. quantitatively, the level of phosphorylated eif α significantly increased (student's t-test; ** p < , even when they were treated with a perk inhibitor ( µm). however, it had increased by hpi ( . -fold increase) compared to the two control groups ( . -and . -fold for untreated and dmso-treated cells, respectively; student's t-test; *** p < . ); (b) a similar changing trend of caspase- was seen in the same cell group, having remained unchanged at hpi, but having significantly increased at hpi ( . -fold increase) compared to the control groups ( . -fold for the untreated and . -fold for the dmso-treated group; student's t-test; *** p < . ). very likely, the denv -induced perk signaling pathway was important for c / cells avoiding induction of apoptosis during infection. as mentioned above, eif α is involved in protein translation in eukaryotic cells via the perk signaling pathway. since that are no available tools for directly detecting perk activation of insect cells in hand, measuring phosphorylation of eif α was used as the alternative way showing perk activation in this study. when observing its activity, an increased degree of phosphorylation in response to viral infection was detected, while its protein amount remained stable. importantly, phosphorylated eif α was significantly reduced when the perk inhibitor was applied to infected cells ( figure a ). this indicates that phosphorylation of eif α triggered by denv infection is deeply involved in the translation of cellular proteins through the perk signaling pathway in c / cells. quantitatively, the level of phosphorylated eif α significantly increased (student's t-test; ** p < . ) in denv -infected cells without perk inhibitor treatment and in those treated with dmso only. in contrast, no evident change was shown in virus-infected cells treated with a perk inhibitor (student's t-test; p > . ) ( figure b ). in the meantime, synthesis of the ns viral protein did not show evident change even in the cells that were infected for h. this suggested that translation of viral proteins may not utilize a canonical cap-dependent translation mechanism in c / cells. viruses , , of . ) in denv -infected cells without perk inhibitor treatment and in those treated with dmso only. in contrast, no evident change was shown in virus-infected cells treated with a perk inhibitor (student's t-test; p > . ) ( figure b ). in the meantime, synthesis of the ns viral protein did not show evident change even in the cells that were infected for h. this suggested that translation of viral proteins may not utilize a canonical cap-dependent translation mechanism in c / cells. translation of global cellular proteins in this study was shown to decrease in response to denv infection. a similar reaction was also observed in mammalian cells infected with lytic rna and dna viruses [ , ] . this suggests that arboviral infections in host cells, either mammal or mosquito figure . activity of eif α in the perk signaling pathway during protein synthesis in dengue virus (denv )-infected c / cells. (a) the c / cells were infected with denv at moi of one, then treated with µm perk inhibitor or dmso. the activity of eif α was shown to increase the degree of phosphorylation in response to viral infection, while its protein level mostly remained at a stable level. on the other hand, the amount of phosphorylated eif α was significantly reduced when the perk inhibitor was applied to infected cells. as synthesis of the ns viral protein did not show an evident change even when cells had been infected for h, an alternative mechanism for translation of viral proteins in c / cells may exist; (b) statistically, levels of phosphorylated eif α significantly increased (student's t-test; ** p < . ) in cells without perk inhibitor treatment and in those cells treated with dmso only. in contrast, there was no significant difference between denv -infected cells and the control groups (student's t-test; p > . ). it revealed that the perk signaling pathway may have been important during protein synthesis in denv -infected mosquito cells. translation of global cellular proteins in this study was shown to decrease in response to denv infection. a similar reaction was also observed in mammalian cells infected with lytic rna and dna viruses [ , ] . this suggests that arboviral infections in host cells, either mammal or mosquito derived, change the translation of cellular proteins [ ] . within cells, proteins are generally synthesized using mrna as a template; this has been highly conserved across species of eukaryotes throughout evolution [ ] . it usually follows a cap-dependent translation mode [ ] ; which is generally regulated by activated mtor, which contributes to the phosphorylation and activation of eif e-bp and the ribosomal protein, s kinase (s k) [ ] . a tor-mediated pathway may have been involved in such cap-dependent translation, which may be inhibited when dephosphorylated eif e-bp binds to eif e and thus prevents the formation of the eif f complex [ ] . this is compatible with our observations in denv -infected c / cells, indicating that global cellular proteins are translated by a cap-dependent translation mode [ ] . normally, they are processed by the s ribosomal subunit that binds in the vicinity of the cap structure at the -end of mrna and scans until an aug codon in the mrna is encountered [ , ] . components of the eif f complex, including eif e, eif a and pabp, were identified according to the m gtp pull-down assay performed in this study. the results firmly revealed that canonical cap-dependent protein translation occurred in mosquito cells even when they were infected by the denv , as the formation of the eif f complex was obviously observed in c / cells. the dual luciferase reporter assay in this study also supported that denv infection negatively regulates cap-dependent translation in c / cells. eif α being obviously phosphorylated in c / cells with denv infection firmly demonstrates that met-trnai binding to the s ribosome may have been impaired, even though the eif f complex eventually formed [ , ] . the upr as mentioned above is caused by the accumulation of unfolded or misfolded proteins in the er; which actually resulted in changes in x-box binding protein (xbp ) and splicing activity [ ] . the upr generally leads to activation of three signaling pathways including perk, ire and atf [ , ] . apparently, the perk signaling pathway is highly involved in modulating the dynamic translation of cellular proteins and subsequently regulating er stress induced by denv infection. interestingly, decreased translation of global cellular proteins in denv -infected c / cells significantly recovered once a perk inhibitor (gsk ) was applied to cells. this indicates that the perk signaling pathway was activated in response to denv infection, leading to suppressed translation of mosquito proteins. in the meantime, perk inhibition in this study was further shown to increase mmp changes and ros accumulation, indicating that denv -induced er stress may be reduced by activating the perk signal pathway since such cellular responses were not evidently shown in uninfected perki-treated cells (figures s -s ) . it is known that gsk is a small molecule that inhibits perk phosphorylation [ ] . thus, it may reduce eif α phosphorylation [ ] , resulting in enhanced efficiency of protein translation and the increase of er stress levels in denv -infected c / cells. in addition to er stress, initiator and effector caspases and the apoptosis rate also obviously increased in denv -infected c / cells after the perk signal pathway was inhibited. this revealed that denv -induced er stress in mosquito cells can be alleviated via the shutdown of cellular proteins by eif α, which was phosphorylated by the perk signal pathway. undoubtedly, this pro-survival "byproduct" of mosquito cells was also advantageous for viral replication. indeed, a similar feature was also observed infected by denv [ ] . flaviviruses are required to compete for the machinery of a host cell during replication due to the lack of an intrinsic translational apparatus [ ] . hypothetically, viral rnas retain their translation efficiency by utilizing ribosomes released from host mrnas [ ] . this feature ensures maximal viral replication and the possibility of evading a host's defenses [ , ] . however, it raises an interesting question of how the virus translates its proteins for continuous replication. although denvs possess an m gpppn cap structure at the -end of genomic rna, they lack a poly a-tail at the -end [ ] . as a result, denvs may utilize a unique mode for protein translation, likely a cap-independent process [ , ] . cyclization between conserved complementary sequences in both terminal regions of rna was reported to be the way that proteins are translated in denvs [ ] . a panel of host genes involved in antioxidant defense and/or anti-apoptotic effects was demonstrated to be upregulated in mosquito cells with denv infection [ , ] . as global proteins of infected cells are usually shut down after infection by denv , pro-survival genes may be selectively upregulated. this suggests that there might be second machinery in addition to the mode of cap-dependent translation. many genes involved in antioxidant defense actually possess internal ribosome entry site (ires)-containing mrnas, which may be utilized for an alternative mode of protein translation [ , ] . the er-stress marker, bip/grp , is known to be the first cellular mrna with a mode of translation by an ires element in its -utr [ ] . bip/grp is significantly upregulated in response to denv infection in c / cells [ ] ; this is eventually beneficial for cell survival under stress induced by virus infection [ ] . as no change in translation of this gene was seen in cells treated with the perk inhibitor [ ] , we presumed that this gene may shift its translation from a cap-dependent to a cap-independent mode. apparently, translation of only mrnas with the structure for canonical cap-dependent protein translation was reduced in mosquito cells with denv infection. denv virus infection in mosquito cells causes a regulatory cascade of protein translation ( figure ) . specifically, infection causes the upr that activates the perk signal pathway. global cellular proteins are thus shut down via impairment of the recruitment of ribosomes that bind to the mrna -cap structure. in the meantime, the accumulation of er stress is reduced, helping infected cells survive continuous amplification of the virus by activating antioxidant defenses and anti-apoptotic effects. this novel finding provides insights into elucidating the perk-mediated modulating web involved in dynamic protein synthesis, cell survival and virus replication in cells derived from the mosquito vector. this phenomenon is relatively important for elucidating how the mosquito vector can healthily serve as a transmitter of denv and may-be other arboviruses, as well. selectively upregulated. this suggests that there might be second machinery in addition to the mode of cap-dependent translation. many genes involved in antioxidant defense actually possess internal ribosome entry site (ires)-containing mrnas, which may be utilized for an alternative mode of protein translation [ , ] . the er-stress marker, bip/grp , is known to be the first cellular mrna with a mode of translation by an ires element in its ′-utr [ ] . bip/grp is significantly upregulated in response to denv infection in c / cells [ ] ; this is eventually beneficial for cell survival under stress induced by virus infection [ ] . as no change in translation of this gene was seen in cells treated with the perk inhibitor [ ] , we presumed that this gene may shift its translation from a cap-dependent to a cap-independent mode. apparently, translation of only mrnas with the structure for canonical cap-dependent protein translation was reduced in mosquito cells with denv infection. denv virus infection in mosquito cells 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controls the switch between cap-dependent and internal ribosomal entry site-mediated translation regulation and function of ribosomal protein s kinase (s k) within mtor signalling networks an integrated stress response regulates amino acid metabolism and resistance to oxidative stress protein kinase r-like er kinase and its role in endoplasmic reticulum stress-decided cell fate xbp -mediated bip/grp upregulation copes with oxidative stress in mosquito cells during dengue virus infection er stress-induced cell death mechanisms dengue-induced autophagy, virus replication and protection from cell death require er stress (perk) pathway activation translational control of viral gene expression in eukaryotes. microbiol viral mechanisms of immune evasion molecular biology of flaviviruses dengue virus-induced regulation of the host cell translational machinery. braz essential role of cyclization sequences in flavivirus rna replication selective mrna translation during eif phosphorylation induces expression of ibtkα translation of glucose-regulated protein /immunoglobulin heavy-chain binding protein mrna is increased in poliovirus-infected cells at a time when cap-dependent translation of cellular mrna is inhibited cellular ires-mediated translation: the war of itafs in pathophysiological states no change in translation of bip/grp in c / cells treated with the perk inhibitor the following are available online at www.mdpi.com/ - / / / /s . the authors would like to thank der-jen tang, hsi-chien su and chao-fu yang for their technical assistance. this study was financially supported by a grant from the ministry of science and technology, taiwan (most - -b- - -my ), and in part by chang gung memorial hospital (cmrpd f ~ ).author contributions: wei-june chen conceived of the project; jiun-nan hou and wei-june chen designed the experiments; jiun-nan hou, tien-huang chen, yi-hsuan chiang, jing-yun peng, tsong-han yang, chih-chieh cheng and eny sofiyatun performed most of the experiments; yi-hsuan chiang and jing-yun peng prepared the viruses; cheng-hsun chiu and chuan chiang-ni contributed materials and participated in the discussion; jiun-nan hou and wei-june chen wrote the manuscript; wei-june chen supervised the work and edited the final version of the manuscript, which was read and approved by all authors. the authors declare no conflict of interest. key: cord- - aonqyub authors: royle, jamie; dobson, samuel john; müller, marietta; macdonald, andrew title: emerging roles of viroporins encoded by dna viruses: novel targets for antivirals? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: aonqyub studies have highlighted the essential nature of a group of small, highly hydrophobic, membrane embedded, channel-forming proteins in the life cycles of a growing number of rna viruses. these viroporins mediate the flow of ions and a range of solutes across cellular membranes and are necessary for manipulating a myriad of host processes. as such they contribute to all stages of the virus life cycle. recent discoveries have identified proteins encoded by the small dna tumor viruses that display a number of viroporin like properties. this review article summarizes the recent developments in our understanding of these novel viroporins; describes their roles in the virus life cycles and in pathogenesis and speculates on their potential as targets for anti-viral therapeutic intervention. as obligate intracellular parasites, viruses have evolved a myriad of strategies to manipulate the host cell environment to one that is conducive for virus replication. research over recent decades has identified a group of virus-encoded proteins able to mediate the passage of ions and solutes across cellular membranes, termed viroporins [ , ] . the majority of viroporins described are small (less than amino acids) and contain one or two transmembrane domains (tmd), although a small number of larger viroporins have been shown to encode up to three putative tmd [ ] . whilst high-resolution structural information is currently only available for a limited number of viroporins [ ] [ ] [ ] [ ] [ ] , a bio-informatic approach has often been successfully employed to identify key features in viroporins that lack any structural information [ , ] . their small size necessitates that viroporins must oligomerize in membranes to form an active channel complex. formation of these high-order complexes is often observed in mild detergents such as , -diheptanoyl-sn-glycero- -phosphocholine (dhpc) [ , ] and is likely to be mediated by hydrophobic interactions between the tmd of each monomer; although in some viroporins basic residues adjacent to the tmd may facilitate membrane binding and insertion [ , ] . known viroporins have been placed into distinctive classes based on the number of tmd and the orientation of their carboxyl termini relative to the endoplasmic reticulum (er) membrane [ ] . undoubtedly, this classification system is useful for cataloguing the expanding number of viroporins, however, it will need to adapt in order to accommodate those few viroporins encoding three tmd and it will also need to take into account the dynamic nature of membrane proteins in lipids, which are capable of altering the orientation of their termini depending on the lipid environment [ , ] . modulation of ionic homeostasis within specific cellular compartments allows for viroporins to manipulate a wide range of cellular processes from autophagy [ ] [ ] [ ] , trafficking [ , ] , inflammation [ , ] , transformation [ ] to cell survival [ ] . due to these broad perturbations to host cell physiology, it is not surprising that viroporin function has been shown to assist in all stages of the virus life cycle including entry, membrane penetration, genome replication and virus egress [ , ] . the existence of virus encoded pore-forming proteins was initially postulated nearly four decades ago [ ] . however, it was observations that the m protein of influenza a virus (iav) was able to form a tetramer [ ] and raise intracellular ph [ , ] that provided the first clues to its role as an ion channel. pioneering studies in xenopus oocytes demonstrated m -dependent currents that could be blocked by addition of the anti-viral compound amantadine [ ] . following this, viroporins were swiftly identified from a number of rna virus families. whilst iav m remains the paradigm, viroporins have now been described in a number of virus families including the flaviviridae, picornaviridae, retroviridae, coronaviridae, reoviridae and paramyxoviridae [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . to date, the overwhelming majority of viroporins have been identified in rna viruses. however, given the myriad of cellular processes that they are able to modulate, it is logical to assume that all viruses might benefit from encoding such a protein. recent findings have now identified proteins that exhibit a number of viroporin characteristics encoded by members of the polyomaviridae and papillomaviridae. this review will summarize our understanding of these putative viroporins, describe their known functions and attempt to highlight how possible ion channel activity may aid the life cycles of these small dna tumor viruses. the polyomaviridae are small, non-enveloped, double-stranded dna viruses that infect a wide range of species [ ] . the family was named after the founding member, polyomavirus, which caused "many tumors" in mice [ ] , followed by the prototypic primate polyomavirus, simian vacuolating agent (sv ), from the rhesus monkey [ ] . the first two human polyomaviruses discovered in , jc and bk, were named after the index cases, and cause serious disease in the immunocompromised [ ] . the last decade has seen the discovery of several novel human polyomaviruses, including merkel cell polyomavirus, which causes an aggressive skin cancer [ , ] . these discoveries have led to resurgence in interest in polyomavirus biology and to the roles of virus encoded proteins in pathogenesis. in this regard, two members of the family have recently been shown to encode proteins with viroporin characteristics. in , progressive multifocal leukoencephalopathy (pml), a potentially fatal demyelinating disease of the brain, was discovered and later attributed to a novel polyomavirus termed jc [ ] . jc is widespread amongst the adult population, with studies suggesting infection rates are upwards of %, and possibly as high as % [ ] . despite the high prevalence, pml incidence is extremely low due to the tendency of jc to result in an asymptomatic latent infection of the kidneys, lymphatic system and bone marrow in immunocompetent individuals [ ] . activation of the virus occurs almost exclusively in immunocompromised patients and is characterised by a lytic infection of oligodendrocytes resulting in demyelination and development of pml, although it is not clear when the virus infects the central nervous system. before the introduction of antiviral therapies to combat the progression to aids, pml was a prominent feature of the hiv phenotype. since then, there has been a resurgence in pml cases as a wide range of immunosuppressant therapies are being applied to combat autoimmune diseases such as multiple sclerosis (ms) and aid in the acceptance of transplanted tissues [ ] . links between jc and a range of human cancers have also been reported, although these are disputed [ ] . similar to most polyomaviruses, jc encodes for early proteins, which constitute the small and large t antigens and their many splice variants, which engage with many host processes to ensure a cellular milieu is available that is conducive to virus replication. in addition to this, three late structural proteins are expressed. these include the major capsid protein, vp , and the vp /vp minor capsid proteins [ ] . jc encodes for an additional late protein termed agnoprotein [ ] . agnoprotein is only expressed by a limited number of polyomaviruses including the related bk and sv . the amino acid agnoprotein is highly basic and contains a central hydrophobic region capable of forming an amphipathic helix [ , ] . in concert with residues in the amino terminal region, this amphipathic helix is required for localization to the er and membrane insertion [ ] . biochemical analysis shows that residues - within the amphipathic helix are necessary and sufficient for dimer and oligomer formation [ ] . agnoprotein oligomers are stable in sds and do not depend on disulphide bridge formation [ ] . recent nuclear magnetic resonance (nmr) data confirmed the formation of an amphipathic helix between leu and phe [ ] . the basic nature of the protein may provide flexibility within the amino and carboxyl termini increasing the range of interactions with host partners. for a list of known jc agnoprotein binding partners see table . viroporin activity is associated with increased plasma membrane permeability, resulting in elevated cytosolic calcium levels [ ] . similar to several known viroporins, agnoprotein expression manipulates host trafficking pathways to allow transport of agnoprotein to the cell surface to mediate plasma membrane permeabilization [ ] . to achieve this, agnoprotein interacts with the δ sub-unit of adaptor protein complex (ap- ), inhibiting its function and as a consequence preventing trafficking of agnoprotein to the lysosome for destruction. substitution of two basic amino acids in the amino terminal region of agnoprotein (arg /lys ) prevented the interaction with ap- , perturbed sub-cellular localization and abrogated the plasma membrane permeabilization seen with the wild type protein. moreover, jc viruses containing either an agnoprotein deletion or alanine substitution of the basic residues resulted in a comparable defect in virion release from infected cells [ , ] . the conclusion of these studies is that the basic residues are a critical requirement for agnoprotein function, potentially by contributing towards viroporin activity. mutations of a similar basic motif in the hcv p protein also impaired viroporin activity, as assessed using a carboxylfluorescein dye release assay, however, further investigation in fact revealed that the mutant protein was no longer able to integrate correctly into membranes [ ] . it is plausible that by mutating the basic loop in agnoprotein, membrane integration has been perturbed which would be expected to have broader impacts on agnoprotein function beyond inhibiting viroporin function. agnoprotein has multiple roles within the jc life cycle, and some of these are known to require residues within the amphipathic helix, and as such may depend upon viroporin function. binding of the large t antigen to the viral origin of replication is enhanced in the presence of agnoprotein [ ] . agnoproteins containing mutations that would be expected to impair amphipathic helix formation display decreased large t binding and significantly reduced virus genome replication [ ] . some mutations within the amphipathic helix impact protein stability, resulting in reduced agnoprotein expression [ ] . as such the impact on virus replication might arise as a result of less agnoprotein rather than loss of putative viroporin activity. nevertheless, reduced jc replication is a feature observed in some agnoprotein deletion viruses, indicating that modulation of replication is a bona fide function of agnoprotein [ ] . as polyomavirus virions assemble in the nucleus, they are first required to exit this organelle into the cytoplasm prior to the egress from the infected cell [ ] . since complete lysis of the nuclear membrane would abolish the integrity of the infected cell and impair virus replication, jc has evolved to instead alter the nuclear envelope (ne) to mediate virus egress. jc infected cells show protrusions and invaginations in the ne, mediated through binding of agnoprotein to heterochromatin protein- (hp- ) [ ] . hp- normally interacts with the lamin-b receptor (lbr) to assist with the reassembly of the ne after cell division [ ] . however, the amino terminal domain of agnoprotein is capable of binding to hp- inducing disassociation from lbr and resulting in morphological changes to the ne, allowing escape of progeny virions [ ] . whether viroporin activity is required for this function is not clear, although binding to hp- requires the amino terminal residues, which are outside the putative tmd. agnoprotein has been shown to be subject to extensive post-translation modification and this is likely a key regulator of function. protein kinase c (pkc) can phosphorylate jc agnoprotein on three identified residues; ser , ser and thr [ ] . jc viruses containing alanine substitutions at these sites were less able to maintain an active infection, although they displayed enhanced agnoprotein expression levels at early time points during infection. subsequent studies identified an antagonistic role for protein phosphatase a (pp a) in agnoprotein phosphorylation [ ] . agnoprotein dephosphorylated by pp a was shown to inhibit jc replication to levels comparable with the alanine substitution mutants. mechanistically, jc small t antigen has been shown to bind to both pp a and agnoprotein, and is thought to reduce the ability of pp a to dephosphorylate agnoprotein [ ] . interestingly, depletion of pp a from jc infected cells using sirna also resulted in reduced virus replication [ ] . this raises the intriguing possibility that both phosphorylated and de-phosphorylated forms of agnoprotein have defined functions, and interplay between the two contributes to the successful replication and propagation of jc. these functions, however, have yet to be delineated. phosphorylation has been shown to be important in regulating the sub-cellular localization of proteins, as such it is plausible that the phosphorylation state of agnoprotein may determine its sub-cellular location and hence the role it plays in replication. whether it is also required to regulate viroporin function has not been shown. however, given the findings of sawa and colleagues that viroporin function is needed at the cell surface, it is a possibility that phosphorylation of agnoprotein may also contribute to localizing the channel to where it is required [ ] . regulation of viroporins by post-translational modifications would add another tier of control to these important proteins. jc agnoprotein shares significant identity with the agnoproteins encoded by the related viruses bk and sv ( % across the whole protein, rising to % in the amino terminal region) [ ] (figure ). as expected from such a high homology, agnoproteins of bk and sv appear to perform similar functions during the virus life cycle, although with some subtle differences. though less well studied, loss of agnoprotein appears to correlate with defects in the late stages of the virus life cycle and to perturb egress. in addition, agnoprotein may also play a role in packaging the sv genome [ ] . bk egress has recently been shown to be sensitive to the dids compound [ ] . given that dids is a broad-spectrum inhibitor of ion channels, it is possible that the target of its actions is an agnoprotein viroporin. it would be interesting to assess the impact of dids on virus egress from an agnoprotein knockout virus. the wide-ranging roles of agnoproteins identified from both jc and bk coupled with the deleterious effects seen in the agnoprotein deficient viruses warrants a serious analysis of the potential for agnoprotein as a target of direct acting antiviral therapeutics. groups have previously generated sirna to target jc agnoprotein and have had success of inhibiting viral replication in mice infected brains, justifying the premise of targeting this protein for inhibition [ ] . such studies would be expedited by a direct analysis of channel activity using recombinant agnoprotein, similar to the pioneering work on the hcv p protein [ ] . not only would this affirm channel function but would also provide a platform from which to screen the large libraries of compounds displaying viroporin inhibitory properties available. three late proteins have been shown to aid in sv entry and release by virtue of their viroporin-like properties. vp and vp are generated from successive met residues within the vp messenger rna so they share a common carboxyl-terminus. they are classed as minor constituents of the virus particle, being present at a stoichiometry of one copy of a vp or vp protein per vp pentamer [ ] . given that sv is a non-enveloped virus, it has evolved strategies to allow transport of the incoming virion through the membranous environment of the cytoplasm to the nucleus to initiate genome replication. a number of studies have shown that both vp and vp form pores in cellular membranes and may aid in delivery of the sv virion into the nucleus [ , ] . mutation of residues within putative tmd prevented membrane targeting and when engineered into sv genomes resulted in reduced infectivity [ ] . sv also encodes a unique very late protein termed vp , encoded by the same transcript as vp /vp . unlike other late proteins, vp is thought not to be incorporated into sv capsids. vp the wide-ranging roles of agnoproteins identified from both jc and bk coupled with the deleterious effects seen in the agnoprotein deficient viruses warrants a serious analysis of the potential for agnoprotein as a target of direct acting antiviral therapeutics. groups have previously generated sirna to target jc agnoprotein and have had success of inhibiting viral replication in mice infected brains, justifying the premise of targeting this protein for inhibition [ ] . such studies would be expedited by a direct analysis of channel activity using recombinant agnoprotein, similar to the pioneering work on the hcv p protein [ ] . not only would this affirm channel function but would also provide a platform from which to screen the large libraries of compounds displaying viroporin inhibitory properties available. three late proteins have been shown to aid in sv entry and release by virtue of their viroporin-like properties. vp and vp are generated from successive met residues within the vp messenger rna so they share a common carboxyl-terminus. they are classed as minor constituents of the virus particle, being present at a stoichiometry of one copy of a vp or vp protein per vp pentamer [ ] . given that sv is a non-enveloped virus, it has evolved strategies to allow transport of the incoming virion through the membranous environment of the cytoplasm to the nucleus to initiate genome replication. a number of studies have shown that both vp and vp form pores in cellular membranes and may aid in delivery of the sv virion into the nucleus [ , ] . mutation of residues within putative tmd prevented membrane targeting and when engineered into sv genomes resulted in reduced infectivity [ ] . sv also encodes a unique very late protein termed vp , encoded by the same transcript as vp /vp . unlike other late proteins, vp is thought not to be incorporated into sv capsids. vp encodes a single tmd and associates with membranes, where it generates a channel of defined pore size and increases membrane permeability. vp is expressed at least h later than other late proteins, indicating a potential role in virus release [ ] . vp channel activity is regulated by the lipid environment and shows a greater activity in liposomes mimicking the composition of the plasma membrane, and sv viruses lacking vp exhibit a significant defect in virus spread. together, these data indicate that vp is a viroporin that functions specifically during the latter stages of the sv life cycle [ ] [ ] [ ] [ ] . whether other mammalian polyomaviruses encode a vp protein is not clear. it is currently uncertain why sv might require vp when other polyomaviruses do not. jc agnoprotein has been shown to permeabilize the plasma membrane [ ] , and may therefore fulfil this role. however, given the sequence similarity it is likely that sv agnoprotein may also serve as a viroporin. it is possible that there is either redundancy or co-operation between agnoprotein and vp . these questions will remain unanswered until viroporin function is confirmed in sv agnoprotein. the papillomaviridae contains an extensive array of different papillomavirus (pv) types capable of infecting a variety of animal species, of which at least have currently been isolated from humans [ ] . like polyomavirus, they are small, non-enveloped double stranded dna viruses around nm in diameter [ ] . most pv encode six early (e , e , e , e , e and e ) and two late structural genes (l and l ). approximately hpv types, termed high-risk, are the causative agents of several ano-genital and oral malignancies [ ] . of these, hpv and hpv are the most important and are responsible for approximately % of the cervical cancer cases, and for the deaths of approximately , women in alone [ ] . low risk hpv are usually cleared by the body and vaccines are available for prophylactic treatment of high risk hpv, but there is as of yet no therapeutics for existing cases of hpv infection. three gene products e , e and e mediate the transforming potential of high-risk hpv. e and e are the major drivers of keratinocyte proliferation [ ] . they are necessary to maintain the cell cycle of differentiating keratinocytes and they achieve this by binding to and inactivating the retinoblastoma (prb) and p tumor suppressor proteins [ ] . repression of e and e in various cell lines has been shown to induce cellular senescence and halt differentiation. the e protein is the least understood of the three oncoproteins [ ] . hpv e is a highly hydrophobic, amino acid membrane protein that resides in the lumen of cytoplasmic membranes and interacts with a growing number of cellular partners [ , [ ] [ ] [ ] [ ] [ ] (figure a) . the triple membrane spanning topology of hpv e was determined by partial membrane permeabilization fluorescence studies in cells and these support the idea that the e amino terminus resides in the lumen of the er with a carboxyl terminus exposed to the cytosol [ ] . lack of specific antibodies preclude localization studies from virus infected cells, however, epitope tagged e has been shown by over-expression studies to predominantly localize to er and golgi membranes [ ] . cell surface localization has also been noted [ ] , although this has been disputed by others [ ] . sequence analysis demonstrates that a recognizable e gene is missing from several hpv types (beta, gamma and mu). further, viruses that do encode an e open reading frame show significant sequence divergence, with the resulting protein product ranging in size from approximately - amino acids. such significant divergence might indicate distinctive roles for e within the hpv life cycle, dependent on the specific virus type. importantly, all high-risk cancer causing hpv types encode an e protein similar to hpv , of approximately acids termed e alpha [ ] . hpv e oligomerizes in vitro and in cells, with oligomer formation driven not by the presence of disulphide linkages between cysteine residues, rather by hydrophobic interactions between individual monomers [ , ] . in our laboratory demonstrated that the e oligomer could mediate the controlled release of the small molecule carboxyflourescein using a liposome dye release assay [ ] . whilst some questions have been raised as to the validity of this indirect measure of channel activity [ ] , it has been widely used with multiple channels (e.g., hcv p , classical swine fever virus (csfv) p , respiratory syncytial virus (rsv) sh), and has provided important insights into their activity [ , , ] . the stoichiometry of hpv e was predicted to be hexameric using in silico modelling and subsequently confirmed by native page electrophoresis and transmission electron microscopy [ ] (figure b ). these complexes displayed channel-forming activity with a defined pore size, and activity was increased in acidic ph. sensitivity of e to the adamantane derivative rimantadine [ ] and the alkyl imino-sugar nn-dnj (our unpublished data) was demonstrated in vitro, as well as to a novel small molecule inhibitor generated using in silico modelling of the e channel and subsequent docking analysis [ ] . journal , volume, page-page fever virus (csfv) p , respiratory syncytial virus (rsv) sh), and has provided important insights into their activity [ , , ] . the stoichiometry of hpv e was predicted to be hexameric using in silico modelling and subsequently confirmed by native page electrophoresis and transmission electron microscopy [ ] (figure b ). these complexes displayed channel-forming activity with a defined pore size, and activity was increased in acidic ph. sensitivity of e to the adamantane derivative rimantadine [ ] and the alkyl imino-sugar nn-dnj (our unpublished data) was demonstrated in vitro, as well as to a novel small molecule inhibitor generated using in silico modelling of the e channel and subsequent docking analysis [ ] . e is predicted to have three transmembrane domains (tmds; boxes) based on the hydrophobic nature of its amino acids. membrane permeabilization assays demonstrate that the carboxyl-terminus extends into the cytosol while the amino-terminus is directed towards the endoplasmic reticulum (er) lumen. the first tmd gives rise to the subcellular localization of e and mediates binding to mhc class i molecules and calnexin. the second tmd facilitates the recently identified interaction with the transmembrane protein yipf as well as the k subunit of the h + -atpase. further functions of e such as the increase of koilocytosis, activation of egfr signaling and induction of endosome alkalinization are exerted via the third tmd; (b) the sequence of hpv e was obtained from uniprot and the secondary structure predicted using psipred and memsat- and energy minimized. the model for an e monomer contained three tmd and had the lowest energy and was used to build a hexameric model using the protocol described previously [ ] . each monomer in the model was minimized individually to restore the symmetry and was refined using prime (schrodinger inc). the oligomeric state of hpv e was confirmed by native page and transmission electron microscopy [ ] . e expression induces anchorage-independent growth in murine nih t cells and mitogenic effects in primary human foreskin epithelial cells [ , ] . transformation has been recapitulated in vivo using transgenic mouse models where e is expressed in epithelial cells under the control of the keratin- promoter. in these mice, e expression was associated with hyperplasia and tumor formation [ , ] . a number of studies have demonstrated the importance of the epidermal growth figure . hpv e is a tmd viroporin. (a) hpv e is predicted to have three transmembrane domains (tmds; boxes) based on the hydrophobic nature of its amino acids. membrane permeabilization assays demonstrate that the carboxyl-terminus extends into the cytosol while the amino-terminus is directed towards the endoplasmic reticulum (er) lumen. the first tmd gives rise to the subcellular localization of e and mediates binding to mhc class i molecules and calnexin. the second tmd facilitates the recently identified interaction with the transmembrane protein yipf as well as the k subunit of the h`-atpase. further functions of e such as the increase of koilocytosis, activation of egfr signaling and induction of endosome alkalinization are exerted via the third tmd; (b) the sequence of hpv e was obtained from uniprot and the secondary structure predicted using psipred and memsat- and energy minimized. the model for an e monomer contained three tmd and had the lowest energy and was used to build a hexameric model using the protocol described previously [ ] . each monomer in the model was minimized individually to restore the symmetry and was refined using prime (schrodinger inc). the oligomeric state of hpv e was confirmed by native page and transmission electron microscopy [ ] . e expression induces anchorage-independent growth in murine nih t cells and mitogenic effects in primary human foreskin epithelial cells [ , ] . transformation has been recapitulated in vivo using transgenic mouse models where e is expressed in epithelial cells under the control of the keratin- promoter. in these mice, e expression was associated with hyperplasia and tumor formation [ , ] . a number of studies have demonstrated the importance of the epidermal growth factor (egf) receptor (egfr) for e -induced transformation [ , ] , and these have since been reinforced by genetic studies which showed that transgenic mice expressing e failed to produce tumors when crossed with mice encoding for an inactive egfr [ ] . thus the contribution of e to host cell transformation appears dependent on manipulation of the egfr. studies aimed at understanding the role of e in the productive hpv life cycle show that e is required to maintain the proliferative status of infected cells as they undergo terminal differentiation, in order to allow virus genome replication. moreover, e causes a delay to the early stages of keratinocyte differentiation to achieve this. using small molecule inhibitors targeting egfr we have been able to show that these processes are also dependent on active egfr signalling (our unpublished data). the precise mechanism by which e manipulates the egfr is unclear. the current consensus model is that e expression is associated with a deacidification of endosomes [ ] . this prevents the normal degradative trafficking pathways, which would culminate in deposition of active egfr in the lysosome and termination of egfr signalling. in e expressing cells active egfr appears to be re-routed into recycling endosomes and returned to the plasma membrane, where it maintains mitogenic signalling [ ] . deacidification of endosomes might result from specific interactions with host binding partners [ ] , or, similar to iav m and hepatitis c virus p , might indicate a requirement for direct proton channel activity [ , ] . in support of this idea, enhanced egfr signalling observed in e expressing cells was reduced by the small molecule inhibitors that prevented carboxyfluorescein release in the in vitro dye release assays [ ] . these data suggest that the oncogenic activity of e may be directly linked to viroporin function. if this proves to be the case then e would represent the first example of an oncogenic viroporin. the development of specific mutant e proteins that lack channel activity will need to be tested in these assays in order to validate the small molecule inhibitor studies, which can be fraught with off-target effects. moreover, it will be of great interest to generate viruses containing these mutants in order to study at what stage of the hpv life cycle any putative viroporin function is necessary. the viroporin function was described for e from hpv [ ] . given that e proteins from the other high-risk hpv types are predicted to adopt a similar three tmd topology, it will be of interest to determine whether viroporin function is conserved amongst this group of viruses. whilst the presence of a defined pore size of < nm was confirmed in these studies [ ] , future work should focus on determining the ion selectivity of the e channel to conclusively determine whether there is a preference for protons or whether ion selectivity fluctuates dependent on the particular membrane environment, as has been shown for other viroporins [ ] . finally, although e is not thought to be expressed in cervical cancer cells due to the integrated nature of the genome [ ] , it is postulated to play a role in the early stages of cancer development. given the limited treatment options for those already infected with hpv, ongoing research into the development of e inhibitors with greater potency suited to drug development programs appears feasible given our findings [ ] . thus, there is potential for drug development targeting e , yet whether this will ultimately prove relevant in the post-vaccination era remains to be seen. viroporins are multi-faceted viral proteins shown to play key roles in the life cycles of a number of important human pathogens. the relatively recent identification of such proteins encoded by small dna tumor viruses offers the opportunity to understand their contribution towards the productive life cycle of these viruses and ultimately their pathogenesis. analysis of viroporin deletion mutants in these viruses will undoubtedly help to delineate their functions and may provide an important insight into how they manipulate critical host functions. the large economic and health burden associated with virus infection, alongside the rapid increase in 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cord- - j yic authors: donato, celeste; vijaykrishna, dhanasekaran title: the broad host range and genetic diversity of mammalian and avian astroviruses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: j yic astroviruses are a diverse family of viruses that infect a wide range of mammalian and avian hosts. here we describe the phylogenetic diversity and current classification methodology of astroviruses based on the orf b and orf genes, highlighting the propensity of astroviruses to undergo interspecies transmission and genetic recombination which greatly increase diversity and complicate attempts at a unified and comprehensive classification strategy. astroviruses (astvs) were first described in as small viruses that are - nm in diameter with icosahedral morphology. astroviruses are named due to the distinct five-pointed or six-pointed star-like appearance of some virions when visualized under an electron microscope (em); astrovirus is derived from the greek word astron meaning star [ ] [ ] [ ] . astroviruses were first described from human infants with diarrhea and were subsequently identified in the young of numerous mammalian and avian species [ , ] . human astroviurses (hastvs) have been recognized as one of the major causes of acute gastroenteritis in children, associated with - % of infections [ ] . transmission of hastv occurs via the fecal-oral route, person-to-person contact, or contaminated food or water. following an incubation period of - days, symptoms including diarrhea, vomiting, abdominal pain, and fever are often reported [ , ] . whilst primarily associated with asymptomatic or diarrheal disease in humans, there are several reports of central nervous system (cns) complications such as acute flaccid paralysis [ ] , meningitis, and encephalitis [ , ] . animal astroviurses, have been isolated from numerous mammalian and avian species. in animals, astrovirus infection may be asymptomatic or associated with enteric disease and a range of other symptoms indicative of the involvement of other organ systems including hepatitis and nephritis in avian species [ , ] , and neurological symptoms in cattle [ ] [ ] [ ] and mink [ ] . astroviruses are classified within the unassigned astroviridae family and are non-enveloped viruses characterized by a positive sense, single-stranded rna (ssrna) genome . - . kb long comprised of a -untranslated region (utr), three open reading frames (orfs)-orf a, orf b, and orf , a -utr, and a poly a tail [ ] . the orf a region encodes a non-structural polyprotein (serine protease), orf b encodes a polyprotein including the rna-dependent rna polymerase (rdrp), and orf encodes the viral capsid protein [ ] . a further orf, termed orfx, has been observed in classic hastvs and some mammalian astroviurses, overlapping the end of orf which may be translated through a leaking scanning mechanism [ ] . astroviruses exhibit several distinctive features in addition to a distinctive morphology. the viruses lack a rna-helicase domain encoded within the genome and utilize a ribosomal frameshifting mechanism to translate the rdrp, which distinguishes the astroviridae family from other non-enveloped ssrna virus families such as picornaviridae and caliciviridae [ , ] . the greatest diversity in the genome is within the orf region, which is characterized by a highly-conserved n-terminal domain (amino acids (aa) - ), a hypervariable domain (aa - ) which is believed to form the capsid spike and contribute to receptor binding, and a highly acidic c-terminal domain [ , ] . the wide host range of astroviruses and the high degree of genetic diversity present within the astroviridae family have complicated attempts at a unified classification method. astroviruses are classified within the unassigned astroviridae family, which was initially comprised of a single genus (astrovirus) based on virion morphology [ ] . the classification of the astrovirus genus within the family astroviridae was recognized by the international committee for taxonomy of viruses (ictv) in and the classification scheme has been modified numerous times over the intervening years [ ] . in , two genera were recognized; mamastrovirus (mastv) and avastrovirus (aastv), that were known to infect mammalian and avian species, respectively, and viruses were classified solely on the species of origin [ ] . however, the advent of sequence based characterization rendered this approach inadequate, revealing that viruses isolated from different species can be genetically similar (reflecting prevalent interspecies transmission of viruses) and also revealing a large range of diversity of viruses within a single host species. with this in mind, the classification system proposed by the ictv astroviridae study group in recommended classification based on the amino acid sequence of the orf genome region, recommending that different strains of the same astrovirus species should share > % identity in the capsid proteins [ ] . additionally, there are proposals to define distinct variants within a recognized astrovirus species, with a variant defined as sharing < - % nucleotide similarity to the reference or prototype strain of each species or if phylogenetic analysis is used, a distance of > . based on analysis of the capsid protein [ , ] . astroviruses within the mamastrovirus genus are derived from numerous mammalian species in addition to humans (hastv), including farmed species such as pigs (pastv), sheep (oastv), cattle (boastv), domesticated animals including cats (fastv), and dogs (caastv), rodents and small mammals including mink (miastv), bats (bastv), rats (rastv), mice, rabbit (rabastv), fox, marmot (hhmastv), porcupine, shrew, vole, and larger species including deer (ccastv), monkeys, water buffalo (bufastv), yak, camel (dcastv), and cheetah (chastv) (figure a,b) . viruses from the mamastrovirus genus have also been characterized from marine mammals including stellar sea lion (sslastv) and california sea lions (cslastv), minke whale, orca whale, and bottlenose dolphins (bdastv) (figure a ,b) [ ] . the current ictv classification reveals recognized species of mamastrovirus (mastv- - ) within two genogroups gi and gii; mamastrovirus (gi.a-human); mamastrovirus (gi.b-feline); mamastrovirus (gi.c-porcine); mamastrovirus (gi.d-california sea lion); mamastrovirus (gi.e-canine); mamastrovirus (gi.f-human); mamastrovirus (gi.g-bottlenose dolphin); mamastrovirus (gii.a-human); mamastrovirus (gii.b-human); mamastrovirus (gii.c-mink); mamastrovirus (gii.d-california sea lion), mamastrovirus (gii.e-bat); mamastrovirus (gii.f-ovine); and mamastroviruses - (gii.g to gii.l-bat species), and numerous other strains awaiting classification, some of which are considered as tentative new species (designated by a ∧ symbol in the phylogenetic trees) (figure a ) [ , ] . viruses from the avastrovirus genus have been characterized from numerous farmed avian species including turkeys (tastv), ducks (dastv), chicken (castv), guineafowl (gfastv), pigeon (piastv), goose, as well as wild aquatic and terrestrial birds including heron, doves, penguins, and many other species (figure a) . the three species originally recognized within the genus were avastrovirus gi.a comprised of turkey astrovirus (tastv- ), avastrovirus gi.b comprised of avian nephritis virus (anv- ), avian nephritis virus (anv- ), and avastrovirus gii.a comprised of turkey astrovirus (tastv- ) and duck astrovirus dastv/c-ngb [ ] . avastrovirus gi.a, avastrovirus gi.b, and avastrovirus gii.a were renamed avastrovirus (aastv- ), avastrovirus (aastv- ), and avastrovirus (aastv- ), respectively [ ] . viruses from the avastrovirus genus have been characterized from numerous farmed avian species including turkeys (tastv), ducks (dastv), chicken (castv), guineafowl (gfastv), pigeon (piastv), goose, as well as wild aquatic and terrestrial birds including heron, doves, penguins, and many other species (figure a) . the three species originally recognized within the genus were avastrovirus gi.a comprised of turkey astrovirus (tastv- ), avastrovirus gi.b comprised of avian nephritis virus (anv- ), avian nephritis virus (anv- ), and avastrovirus gii.a comprised of turkey astrovirus (tastv- ) and duck astrovirus dastv/c-ngb [ ] . avastrovirus gi.a, avastrovirus gi.b, and avastrovirus gii.a were renamed avastrovirus (aastv- ), avastrovirus (aastv- ), and avastrovirus (aastv- ), respectively [ ] . currently, classification into species is based on the phylogenetic analysis of the amino acid sequence of the full length orf region of the genome that encodes the capsid. however, the limited number of capsid sequences available compared to rdrp sequences makes consistent classification difficult, especially with some novel viruses incompletely sequenced. there are numerous unclassified astroviruses, particularly isolated from aquatic and terrestrial wild birds which, according to the ictv, are "related viruses which may be members of the avastrovirus genus but have not been approved as species" [ ] . astrovirus infection in humans has been primarily associated with diarrhea and vomiting, accounting for up to % of sporadic gastroenteritis cases in some regions [ ] . cns complications associated with astrovirus infection have been reported in recent years, including acute flaccid paralysis, with some fatalities reported in children with underlying immune disorders [ , ] . historically human astroviruses (hastv) were classified into five serotypes in [ ] . subsequent molecular characterization based on viral reactivity to polyclonal antibodies and nucleotide sequence analysis led to the recognition of eight serotypes (hastv- - ), now termed "classic" hastv [ , , ] . the relatively recent advent of next generation sequencing (ngs) and metagenomic analysis has led to the identification of numerous novel strains considered "non-classic" hastv [ ] . currently, hastvs are classified within the species mastv- (hastv- - ), mastv- (mlb - ), mastv- (va /hmo-a, va , va , bf ), and mastv- (va /hmo-c, va /hmo-b) [ ] (figure a ). the mastv- species is comprised of hastv- - , and surveillance has revealed that hastv- is the most commonly detected type in children, followed by hastv- - , whereas hastv- - have been rarely detected [ ] . hastv- and hastv- have been associated with infection of older children and longer duration of diarrhea (> days) [ , ] . a hastv- strain was also isolated from an infant with fatal meningoencephalitis [ ] . based upon the phylogenetic analysis of the orf region, different lineages within each hastv type have been proposed; hastv- (hastv- a-d) and hastv- (hastv- a-d) have been divided into four lineages, whereas hastv- (hastv- a-b) and hastv- (hastv- a-c) have been classified into two and three lineages, respectively [ ] . the first "non-classic" hastv strain characterized was mlb , the virus was detected in a stool sample from a year old australian child with acute diarrhea in ; the child had previously received a liver transplant [ ] . the majority of mlb strains characterized to date have been detected in india, kenya, and japan with limited detected in the usa, china, bhutan, egypt, brazil, and italy and prevalence has been reported in the range of . % to % [ ] . however, a seroepidemiologic study in the usa revealed that primary exposure to mlb occurs in childhood and that seropositivity reached % by adulthood suggesting the widespread circulation of the virus in the human population [ ] . mlb viruses were first identified in vellore, india [ ] with the majority of strains subsequently identified in japan, the gambia, and switzerland with limited detection in turkey, usa, kenya, china, and thailand and prevalence reported in the range of . % to . % [ ] . mlb has been associated with meningitis and other cns complications and has been detected in immunocompromised children [ ] . mlb viruses were first detected in india in [ ] , with subsequent detection in kenya and the gambia and the prevalence in stools ranges from . % to . % [ , ] . there is a dual naming system for some hastv species due to the simultaneous characterization of these viruses by different researchers; these viruses are termed va/hmo named for va-virginia and hmo-human-mink-ovine-like viruses, due to their genetic relatedness to previously characterized mink and sheep viruses [ ] . in , va /hmo-a strains were detected in children with non-polio acute flaccid paralysis in nigeria, pakistan [ ] , and india [ ] . the prevalence of va /hmo-a viruses in stools ranges from . % to . %, with strains also detected in egypt, japan, usa, kenya, and china [ ] . va has only been detected in nepal [ ] and the bf strain has only detected in burkina faso [ ] . the va /hmo-b viruses were first identified in vellore, india [ ] with sporadic detection in nigeria, pakistan, and nepal with prevalence ranging from . % to . % [ ] . va /hmo-c viruses were first detected in during an outbreak of diarrhea in children in virginia, usa [ ] . va /hmo-c viruses have been detected and associated with encephalitis in immunocompromized children and adults [ , , ] and acute respiratory disease [ ] . the prevalence of va /hmo-c viruses in stools ranges from . % to . % in diarrheic and non-diarrheic subjects worldwide, with limited detection in nepal, japan, tanzania, the gambia, france, and the u.k. [ , , ] . seroprevalence of va /hmo-c has been reported at % in adults [ ] . the divergent human astrovirus-like virus tentatively named bastrovirus was isolated from patients in the netherlands. the capsid region is homologous to the capsid of hastv whilst the rdrp region is more closely related to members of the hepeviridae family. this virus remains to be classified by the icvt, however the capsid regions clusters closest to the mlb - viruses suggesting an evolutionary relationship to these divergent human astroviruses [ ] (figure a ). revealing the geographic and host diversity of this virus, divergent bastrovirus strains have also been isolated from bat, pig, and rat species in vietnam, forming a distinct cluster of strains that also await classification (figure a ). mamastroviruses are capable of infecting a wide range of mammalian species including companion animals (cats and dogs), intensively farmed species (pigs and cattle), as well as terrestrial and aquatic wild mammalian species. unexpectedly there is no clear clustering of viruses separated by hosts of terrestrial or aquatic origins (figure a,b) . in addition to the classified mastv species, there are numerous divergent viruses isolated from diverse species which likely represent new species awaiting formal classification (figure a ) [ ] . not surprisingly, domesticated animals such as cats and dogs harbor astrovirus strains more closely related to hastv than the viruses harboured by many other animal species. feline astrovirus was first identified in , and feline strains form a small discrete cluster defined as the species mastv- , closely related to the human strains comprising the species mastv- [ ] (figure a ). based on evolutionary analysis, an interspecies transmission pathway has been hypothesised whereby porcine strains may have been transmitted to cats and subsequently to humans, possibly involving other intermediary species suggesting sustained interspecies transmission events [ ] . characterization of an outbreak of diarrhea in a group of captive cheetahs in a breeding facility identified an astrovirus strain most closely related to feline strains (figure a ), however it is not clear if this is a recent transmission from domesticated cats or if the virus is circulating independently in cheetahs [ ] . despite dogs also being a companion animal, canine astrovirus, first described in the s, are not as closely related to human strains as feline strains appear to be, based on phylogenetic analysis (figure a,b) . canine strains form a small, discrete cluster comprising species mastv- , within a lineage comprised of dolphin and sea lion clades representing species mastv- and - [ , ] , and feline, porcine, and human strains are more distantly related within the same lineage (figure a) . a large diverse lineage, largely comprised of unclassified viruses or tentatively classified viruses awaiting approval encompasses divergent marmot and rabbit strains (mastv- ), small, discrete clusters of porcine/ovine/bovine strains (mastv- ), divergent rat strains (mastv- ), and unclassified mouse and mink strains forming a small discrete cluster (figure a) . numerous porcine strains also cluster within this lineage; pastv comprise mamastrovirus and were first detected by em in pigs in the u.k. and the usa [ ] , and are now recognized to have worldwide distribution [ ] . there is a high prevalence of astrovirus detection in pigs, up to % in some studies, suggesting that pigs may be persistently infected with various strains of pastv [ ] . there are five recognized lineages (pastv - ) reflecting the diversity of strains, suggesting swine are highly permissive to astrovirus infection often without symptoms of disease (figure a,b) . this diversity likely reflects the varying origins of these viruses, in particular highlighting frequent interspecies transmission and recombination events [ ] [ ] [ ] . a study in the usa revealed the high level of co-infections in pigs ( . %), revealing the frequent opportunity for recombination, especially between viruses of different lineages [ ] . the close clustering between some porcine and humans strains, in particular mastv- and mastv- viruses, reflects the close contact between these species with the close sharing of environments and co-housing documented in some countries facilitating frequent interspecies transmission [ ] . wild boars showing no symptoms of enteric disease, housed in a captive breeding park with no contact with domesticated pigs, were found to harbor astrovirus strains with a high degree of genetic similarity to porcine strains comprising mastv- and suggests that the virus may be derived from commonly circulating porcine strains [ ] (figure a,b) . porcupine strains isolated in china also cluster with unclassified pastv- strains suggesting further interspecies transmission of these viruses (figure a ) [ ] . unexpectedly, limited astrovirus strains have been isolated from sheep. ovine astrovirus was first identified by em in [ ] , and oastv- clusters with bovine strains comprising mastv- and a second strain from a healthy sheep characterized in , oastv- , clusters with porcine and bovine strains comprising mastv- [ ] (figure a) . similarly, astroviruses are not associated with a significant burden of diarrheal disease in bovine species. the first bovine astrovirus was detected in england in [ ] and bovine astrovirus strains have been detection in association with neurological disease, including encephalitis (boastv-ch /neuros ) [ ] [ ] [ ] [ ] [ ] [ ] and diarrheal disease in calves in south korea [ ] and cattle and buffalo calves in china [ ] . two serotypes were previously recognized, boastv- and boastv- [ ] , however based on phylogenetic analysis there are multiple lineages of boastv strains circulating in farmed bovine populations, and the close clustering of bovine, porcine, and ovine strains in multiple lineages reflects the common interspecies transmission events that occur between farmed animals (figure a,b) . phylogenetic analysis also reveals that bovine-like astrovirus strains have been isolated from numerous wild species including water buffalo, yak, and european roe deer (ccastv- and ccastv- ) suffering from gastroenteritis [ ] . unclassified astroviruses from dromedary camels (dcastv) [ ] also cluster in a lineage comprised of porcine and bovine strains, further suggesting multiple interspecies transmission events (figure a) . substantial astrovirus strain diversity has been observed in small mammals, primarily rodents and bats, forming both species-specific clusters reflecting endemic transmission and co-clustering of strains with other host species reflecting widespread interspecies transmission (figure a,b) . novel murine astrovirus strains have been isolated from laboratory mice in the usa and japan [ , ] . divergent viruses have also been detected, such as those detected in rats (mastv- ), highlighting the need for more detailed detection and characterization of these viruses to better understand the role these animals play in astrovirus transmission between varied species. one of the mammalian species with the highest burden of symptomatic astrovirus infection is mink; infection is associated with pre-weaning diarrhea syndrome and the neurological condition "shaking mink syndrome" [ , ] . mink viruses cluster within multiple lineages suggesting that the species is permissive to infection with multiple, diverse lineages of viruses (figure a) . bat astroviruses were first detected in in hong kong [ ] and subsequently detected in bats in china [ , ] , north america [ ] , germany [ ] , hungary [ ] , and the czech republic [ ] from numerous bat species, with detection rates ranging from % to % in miniopterus magnater bats and from % to % of miniopterus pusillus bats sampled in hong kong [ ] . a diverse population of viruses appears to be highly prevalent in bats without causing disease [ ] (figures and ) . the majority of bat astroviruses are divergent from other characterized mammalian astroviruses, and display a high degree of genetic diversity forming numerous recognized and proposed species (figure a) . some bat sequences clustered with strains from other species including fox, cattle, and mice, suggesting that bats are highly permissive to infection with diverse astrovirus strains from multiple hosts and play a key role in astrovirus diversity and interspecies transmission (figure a,b) . astroviruses have been detected in aquatic mammalian species including californian sea lions, steller sea lion, bottlenose dolphin, killer whale, and minke whale [ ] . the strains from these aquatic species do not cluster together, instead forming multiple discrete clusters, suggesting several transmission events from terrestrial mammals to aquatic mammals (figure a) . minke whale and bdastv- strains are divergent to characterized astrovirus strains, with bdastv- strains comprising mastv- and a diverse group of sea lion viruses comprising the mastv- cluster with porcine and canine strains (figure a ). there are numerous divergent strains that are yet to be classified that may reflect interspecies transmission events. a single divergent feline strain along with a fox strain cluster within a diverse lineage comprised of human hmo strains (mastv- and - ), sea lion (mastv- ), unclassified sea lion, mink (mastv- ), and bat (mastv- ) strains. a himalayan marmot strain clusters with mastv- strains (figure a,b ). there appears to be a large diversity of mastv strains not captured by the currently available sequences, highlighting the need for more detailed sampling and characterization of animal strains. the isolation of astroviruses from avian species predates their isolation in humans, with disease in ducklings described in , however the virus was not formally recognized as an astrovirus until [ , ] . avian astroviruses have been documented to cause infection in poultry leading to economic losses in farms and affecting food production worldwide [ ] . avian astroviruses have been associated with a spectrum of disease ranging from subclinical infection in seemingly healthy adult birds to heavy flock losses. pleomorphic symptoms include enteritis in turkeys, chickens, and guineafowl, mild growth depression and nephritis in chickens, and hepatitis in ducklings [ ] . astrovirus infection has been implicated in pre-hatching mortality in ducklings and goslings [ ] . in addition to aastv- comprised of tastv- , aastv- comprised of anv and anv , and aastv- comprised of tastv- and duck astrovirus (dastv- ), there are numerous yet-to-be classified viruses, including turkey astrovirus (tastv- ), chicken astrovirus a (castv-a), chicken astrovirus b (castv-b) [ , ] , gfastv, and multiple viruses isolated from ducks including duck hepatitis virus (dhv- /dastv- ) [ ] , dhv- /dastv- -like viruses, dastv-cph (dastv- ) [ ] , dastv- , dastv-yp/dastv- -like, and diverse viruses isolated from wild birds [ ] . avian astrovirus have been detected in outbreaks of enteric disease in turkey poults, and avian astrovirus were first reported as an agent of gastroenteritis and mortality in young turkeys in , associated with a condition known as poult enteritis mortality syndrome (pems) [ , ] . subsequently there have been sporadic reports of astrovirus outbreaks in turkeys, associated with enteritis and growth depression [ ] . based on serological and genetic analysis, two types of tastv have been recognized (tastv- and tastv- ). tastv- comprises the avastrovirus species and was first described in the u.k. [ ] . tastv- has limited detection in other avian species with sporadic detection in chicken and ducks and forms a discrete cluster in both the capsid and rdrp phylogenetic analysis (figure a,b) . tastv- was identified in [ ] , and is likely to be classified within the species avastrovirus and is primarily associated with pems [ ] . tastv- is the predominant tastv lineage, with a wider global circulation and greater genetic diversity compared to tastv- , reflected by the co-circulation of multiple sub-lineages (figure a,b) . based on phylogenetic analysis of the rdrp gene there appears to be limited interspecies transmission of tastv- -like viruses detected in chicken and duck (figure b) . astroviruses infecting guineafowl are closely related to tastv- strains based on analysis of the rdrp region [ ] , however the capsid region is distinct to tastv- strains, forming a discrete cluster in a lineage of unassigned viruses suggesting they are closely related to castv-b strains (figure a) . these gfastvs were possibly derived from recombination and interspecies events followed by sustained transmission in the guineafowl population. this highlights the limitation of classifying viruses based on a single region of the genome. chicken astrovirus has been associated with runting-stunting syndrome (rss) in chickens characterized by poor weight gain, lower feed conversion, and mortality resulting in economic losses [ ] , and "white chicks" disease associated with increased mortality of embryos and chicks, weakness, and white plumage [ ] . currently two serotypes of castv have been described [ ] , and both serotypes form discrete clusters in the phylogenetic analysis of the capsid region within a lineage of unclassified viruses (likely to be classified within aastv- ). the castv-a viruses form a smaller, discrete lineage, clustering closest to dastv- strains (figure a) . castv-b strains form a large lineage clustering closest to gfastv strains. based on phylogenetic analysis of the small region of the rdrp gene commonly sequenced, it does not allow castv-a and -b strains to form discrete clusters as seen in the capsid analysis, possibly reflecting multiple recombination events in castv strains (figure b ). castv strains have limited detection in other avian species with sporadic detection in pigeon and duck (figure a,b) . avian nephritis virus was identified in association with intestinal nephritis in chickens and growth retardation. the first serotype of anv (anv- ) was isolated from a healthy broiler chick in [ ] and was initially regarded as a picornavirus, subsequently reclassified within the astroviridae family in [ ] . based on capsid phylogenetic analysis, anv- strains cluster within the aastv- lineage forming a small, discrete, relatively conserved cluster (figure a) . the second serotype (anv- ) was later described from chicks with stunted growth [ ] . based on capsid phylogenetic analysis, anv- strains cluster within the aastv- lineage, forming a larger, more diverse sub-lineage compared to anv- . anv- and anv- viruses have also been sporadically detected in ducks and turkeys (figure a) . a third serotype (anv- ) detected in chickens and turkeys with rss and locomotion impairment has been proposed based on sequencing of the orf a region, however complete genome sequences are unavailable for adequate comparisons to the recognized serotypes [ ] . the virus designated pigeon anv (p-anv) was detected during an outbreak of gastrointestinal illness in young pigeons in china [ ] . p-anv represents an interspecies transmission event from chickens to pigeon; based on phylogenetic analysis, p-anv strains share a high degree of genetic similarity to anv- strains and cluster closely with strains also circulating in china suggesting a localized transmission event, and these viruses should be considered as anv- viruses and do not require a distinct designation (figure a ) [ ] . based on phylogenetic analysis of the small region of the rdrp gene commonly sequenced, it does not allow anv and strains to form discrete clusters as characterized in the capsid analysis (figure b ). whilst this may reflect common recombination events between anv strains, the analysis of a small region does not adequately allow for distinct clustering. astrovirus infection in ducks has been associated with a highly contagious and fatal hepatitis, historically known as duck hepatitis virus type (dhv- ), which was described in the u.k. and subsequently serotype dhv- was isolated in the usa [ , , , ] . the th ictv report classified dhv- and dhv- as dastv- and dastv- , respectively [ ] . dastv- strains form a small, discrete cluster within the aastv- lineage based on phylogenetic analysis of the capsid region, clustering closest to tastv- strains (figure a) . dastv- viruses, currently unclassified, form a discrete, highly conserved cluster closest to castv-a strains (figure a) . based on phylogenetic analysis of the rdrp region, dastv- is closely related to other unclassified duck strains whilst dastv- strains are more closely related to tastv- strains (figure b ). further unclassified serotypes have been described; dastv- -cph, dastv/c-ngb, and dastv- [ ] . duck astrovirus dastv- /cph has been suggested to transmit horizontally and vertically [ ] and dastv- /cph viruses have also been detected in goslings [ ] . phylogenetic analysis of the rdrp region highlights the diversity of aastv strains circulating in duck species (figure b) . strains endemic to ducks have limited detection in other avian species including geese (figure a,b) . astroviruses have been detected in numerous wild aquatic species including teals, pintails, and shovelers (belonging to the order anseriformes), sanderlings (order charadriiformes), and herons and spoonbills (order pelecaniformes) [ , ] . fewer land dwelling wild birds have been found to harbor avian astroviruses including doves and pigeons (order colombiformes), european roller (order coraciiformes), and black-naped monarch (order passeriformes) [ , , , ] . these viruses are highly divergent and largely unclassified, suggesting these viruses are endemic to the wild bird population (figure a,b) . the migratory behavior of some of these species provides ideal conditions for virus dissemination and diversification as during migration there is a high degree of co-mingling and increased density of birds of different species that may originate from varied geographic regions [ ] . phylogenies indicate that the genetic diversity of mamastroviruses has been shaped by extensive interspecies transmission events that have occurred in the past between wild and domestic species and humans. however, the inference of astrovirus interspecies transmission events is hampered due to (a) sequencing of only a small portion of the genome; (b) inadequate sampling; or (c) that the event occurred early during the divergence of astrovirus lineages. a few exceptions where host-jumps are apparent have been in livestock where a greater level of sampling has occurred. this includes hosts with a greater level of interaction (e.g., mastv- in ovine and bovine) or host genetic similarly (e.g., mastv- in wild boars and domestic pigs). with the exception of hastv- strains associated with fatal meningoencephalitis, it is interesting to note that viruses from multiple species, all recognized to cause neurological symptoms, are closely related including human va /hmo-c viruses and mink and bovine viruses also associated with neurological symptoms, suggesting that these related viruses may have a distinct phenotype compared to other mastv strains (figure a,b) . astrovirus strains identified from fecal samples of multiple non-human primate species from wild, captive, and peri-urban environments in bangladesh and cambodia reveal multiple interspecies transmission events, with viruses closely related to the va/hmo lineage of human viruses, and non-human mammalian and avian astroviruses (figure a,b) [ ] . similarly, there appears to be evidence for a high degree of cross species transmission of avian astroviruses between farmed poultry species as described in the above sections. there also appears to be transmission between avian and mammalian species. the highly divergent strains isolated from european roller (er/szal /hun/ and er/bmtk /hun/ ) exhibited low identity to avian and mammalian astroviruses, cluster within the mastv lineage, and were likely derived from multiple recombination and interspecies transmission events [ ] . the carnivorous diet of this avian species may have facilitated the interspecies transmission event between a rodent or small mammal species. this is the only report of a mamastrovirus strain in an avian species; in contrast, there have been more reports of avastrovirus strains detected in mammalian species which may reflect greater sampling density of the mammalian population. a highly divergent group of mink strains detected in china represent a novel clade of astroviruses that were distantly related to previously described mink astrovirus and were closely related to chicken and turkey astroviruses (figure a,b) [ ] . these viruses are recombinant strains with the capsid region clustering with castv-b strains and the rdrp region clustering with human mastv strains and castv strains (figure a,b) . there have been two avastrovirus strains detected in humans; a strain clustering close to anv- strains detected in turkey and chicken was isolated from a child in the gambia, and a strain clustering with anv- strains detected in chicken was isolated from a child in kenya [ ] . serological studies have been used to screen human sera from poultry workers for antibodies to tastv- , with up to % of participants positive with the highest detection in abattoir workers and turkey growers [ ] , suggesting avian strains may be readily transmitted to humans under prolonged close contact. the important role that ecotones play in astrovirus cross-species transmission has been proposed [ ] . ecotones are ecological transition areas such as small and medium sized farms which rear multiple species. the co-rearing of poultry such as domestic ducks, chickens, turkey, and guineafowl can facilitate transmission between these species but also transmission to wild birds [ ] . farms and abattoirs have also been recognized as environments facilitating transmission between livestock species and to farm and abattoir workers [ ] . many other species have contact with livestock in a farming environment; in addition to wild species, companion animals such as cats and dogs and other peri-domestic animals have contact with livestock and their biological waste providing substantial opportunities for cross-species transmission. astroviruses also persist in bodies of water making the aquatic environment ideal for the transmission of viruses infecting avian species, aquatic mammalian species, and possible transmission between terrestrial and aquatic species. untreated or inadequately treated sewage and waste water from domestic and farmed areas can reach fresh and marine bodies of water transmitting human and animal viruses. astroviruses have been detected in the environment and the durability of the virus in this environment may greatly contribute to cross-species transmission within and between terrestrial and aquatic species, generating significant diversity [ ] . in addition to interspecies transmission which generates significant diversity in astrovirus species, both intra-species and inter-species recombination can rapidly generate novel, divergent viruses. full-and partial-genome sequence analysis has identified multiple strains that have undergone recombination events, which are predominately located within the orf b/orf junction region of the genome, which is a region with an rna secondary structure predicted to contain a stable hairpin structure [ , , [ ] [ ] [ ] [ ] . a virus with a recombination event within the orf a region has been identified [ ] . some recombinants appear to be highly stable and show widespread detection [ ] , whilst others are detected sporadically as single strains. numerous human recombinant strains have been reported between hastv strains, including strains with hastv- (orf b) and hastv- (orf ) parental viruses, hastv- (orf b) and hastv- (orf ) parental viruses, hastv- (orf b) and hastv- (orf ), and va (orf b) and mlb (orf ) parental viruses [ , [ ] [ ] [ ] . divergent species often represent recombination events between strains of different species. in , the study conducted by rivera et al. suggested the possibility of a recombination event between human and california sea lion astrovirus strains [ ] . a recombinant strain derived from porcine astrovirus and human hastv- strains was reported from piglets and children from various regions of colombia [ ] . anv strains that appear to be derived from recombination events between anv- (orf b) and anv- (orf ) have been described in the usa [ ] . the increased sampling density of numerous host species combined with the more prevalent use of ngs technologies and viral metagenomic studies will increase the detection of novel strains, further driving the need for a unified, complex, and encompassing classification system. the previously common practice of sequencing a relatively conserved rdrp amplicon of - bp renders many sequences available in genbank of little use in detailed phylogenetic analysis, as does the frequent missing metadata regarding species of isolation, country, and date of collection. sequencing small regions is not adequate to determine if a virus strain is a novel, divergent strain or a recombinant virus. recommendations should be made to encourage full genome sequencing where possible and the deposition of associated host and demographic information. although analysis of amino acid sequences of the capsid region is required for classification, it leads to confusion regarding appropriate phylogenetic analysis, with highly inconsistent publication of nucleotide and amino acid trees further complicating attempts to clarify diversity and classification. the topologies of amino acid trees differ to those of nucleotide trees, particularly for the analysis of mastv, and whilst amino acid trees are required for classification, nucleotide trees may be more appropriate for describing within species diversity (figures s and s ) . incorporating a standardized nomenclature to aid in classification has proven invaluable in the classification of numerous viruses, including rotavirus and influenza [ ] . adopting a nomenclature that records the appropriate metadata associated with sample collection including host, location, date of collection, and determined species and serotype as proposed by martella and colleagues would vastly improve the usability of strains for more complex analyses [ ] . the following are available online at www.mdpi.com/ - / / / /s , figure s : maximum-likelihood phylogenetic tree of mastv (a) capsid nucleotide and (b) capsid amino acid sequences, figure s : maximum-likelihood phylogenetic tree of mastv rdrp, figure s : maximum-likelihood phylogenetic tree of aastv (a) capsid nucleotide and (b) capsid amino acid sequences, figure s : maximum-likelihood phylogenetic tree of aastv rdrp. the authors declare no conflict of interest. nm particles in faeces in infantile gastroenteritis ultrastructure of human astrovirus serotype letter: viruses and gastroenteritis in infants detection and transmission of nm virus particles (astroviruses) in faeces of lambs with diarrhoea duck hepatitis: vaccination against two serological types epidemiology of astrovirus infection in children the changing epidemiology of astrovirus-associated gastroenteritis: a review molecular epidemiology of childhood astrovirus infection in child care centers multiple novel astrovirus species in human stool astrovirus mlb , a new gastroenteric virus 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astroviruses and caliciviruses from acute enteritis of calves exploring the virome of cattle with non-suppurative encephalitis of unknown etiology by metagenomics bouzalas, i. identification of a second encephalitis-associated astrovirus in cattle detection of astrovirus in historical cases of european sporadic bovine encephalitis phylogenetic analysis of bovine astrovirus in korean cattle molecular epidemiology and phylogenetic analysis of diverse bovine astroviruses associated with diarrhea in cattle and water buffalo calves in china serotypes of bovine astrovirus identification and characterization of deer astroviruses identification of an astrovirus commonly infecting laboratory mice in the us and japan adaptive immunity restricts replication of novel murine astroviruses astrovirus epidemiologically linked to pre-weaning diarrhoea in mink novel astroviruses in insectivorous bats detection of diverse astroviruses from bats in china isolation and phylogenetic characterization of bat astroviruses in southern china bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses amplification of emerging viruses in a bat colony novel european lineages of bat astroviruses identified in hungary detection of diverse novel bat astrovirus sequences in the czech republic. vector borne zoonotic dis astrovirus-like particles associated with hepatitis in ducklings molecular characterization of avian astroviruses a novel group of avian astroviruses in wild aquatic birds astroviruses associated with stunting and pre-hatching mortality in duck and goose embryos a seroprevalence investigation of chicken astrovirus infections determination of the full length sequence of a chicken astrovirus suggests a different replication mechanism complete sequence of a novel duck astrovirus detection of astroviruses in turkey faeces by direct electron microscopy induction of functional defects in macrophages by a poult enteritis and mortality 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and sewage treatment plants, evaluated by a competitive reverse transcription-pcr high evolutionary rate of human astrovirus nationwide surveillance study of human astrovirus infections in an italian paediatric population detection of all serotypes of human astrovirus by the polymerase chain reaction evidence of a recombinant wild-type human astrovirus strain from a kenyan child with gastroenteritis lineage diversification and recombination in type- human astroviruses novel astroviruses in children uniformity of rotavirus strain nomenclature proposed by the rotavirus classification working group (rcwg) key: cord- -ion n b authors: de silva senapathi, upasama; abdul-cader, mohamed sarjoon; amarasinghe, aruna; van marle, guido; czub, markus; gomis, susantha; abdul-careem, mohamed faizal title: the in ovo delivery of cpg oligonucleotides protects against infectious bronchitis with the recruitment of immune cells into the respiratory tract of chickens date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ion n b the in ovo delivery of cytosine-guanosine (cpg) oligodeoxynucleotides (odns) protects chickens against many bacterial and viral infections, by activating the toll-like receptor (tlr) signaling pathway. although the delivery of cpg odns in ovo at embryo day (ed) has been shown to reduce infectious bronchitis virus (ibv) loads in embryonic chicken lungs pre-hatch, whether in ovo delivered cpg odns are capable of protecting chickens against a post-hatch challenge is unknown. thus, our objectives were to determine the protective effect of the in ovo delivery of cpg odns at ed against ibv infection encountered post-hatch and, then, to investigate the mechanisms of protection. we found significantly higher survival rates and reduced ibv infection in the chickens following the pre-treatment of the ed eggs with cpg odns. at days post infection (dpi), we found an increased recruitment of macrophages, cluster of differentiation (cd) α+ and cd + t lymphocytes, and an up-regulation of interferon (ifn)-γ mrna in the respiratory tract of the chickens. overall, it may be inferred that cpg odns, when delivered in ovo, provide protection against ibv infection induced morbidity and mortality with an enhanced immune response. infectious bronchitis (ib) is mainly an acute and severe disease of the respiratory system of chickens [ ] . the causative agent, infectious bronchitis virus (ibv), belongs to the family coronaviridae [ ] . there is increasing evidence of ibv infection being reported in birds other than chickens [ , ] . although ibv induced changes are observed primarily in the mucosal surfaces of the respiratory tract, the virus is also known to cause pathology in the female reproductive tract and kidneys, with a varying degree of severity dependent upon the type of strain that infects and replicates in the aforementioned tissues [ ] [ ] [ ] . ever since the first record of ib in the early s [ ] , periodic ib outbreaks associated with the isolation of heterogeneous strains of ibv have been reported globally [ , ] . major losses to the broiler meat industry are due to carcass condemnation at processing, a poor feed conversion ratio resulting in poor weight gain, and mortality. ibv is considered a highly infectious agent with near % morbidity, and with mortality reaching - % [ , ] . in breeder and layer flocks, the major losses are due to reduced egg production during and after infection with ibv. the egg drop during ibv infection has been estimated to be between - % [ ] . furthermore, the downgrading of eggs because of a poor internal egg quality and egg shell quality also account for considerable production losses [ ] . the standard preventive measures, such as strict quarantine and biosecurity measures [ ] , do not seem to sufficiently control the disease. thus far, the most efficient method for controlling ibv is by vaccination [ ] . the immunization of chickens against ibv is mainly by live attenuated and killed vaccines [ ] . however, the emergence of variant ibv strains/serotypes arising from vaccinated flocks, among other factors, has led to vaccine failure and ib outbreaks. thus, the development of novel approaches as an alternative or adjunct in order to control the current measures against ibv is becoming increasingly important. toll-like receptor (tlr)s are a family of germ line encoded pattern recognition receptors (prrs) expressed on the surface or within the endosomal compartments of cells [ ] . these receptors are crucial for recognizing whole or segments of microbial pathogens, and they initiate key host immune defenses against inciting agents. among the tlrs, tlr (in mammals)/tlr (in birds) are the only receptors capable of distinguishing bacterial, parasitic, and viral dna containing cytosine-guanosine (cpg) motifs [ ] . several studies have demonstrated the immunostimulatory and therapeutic success of cpg oligodeoxynucleotides (odns) application in various host-pathogen interaction models [ ] [ ] [ ] . the protection provided by cpg odns against lethal challenges of extracellular bacteria, such as escherichia coli [ ] and salmonella typhimurium [ ] , and viruses, such as low pathogenic avian influenza virus [ ] and infectious laryngotracheitis virus (iltv) [ , ] , in chickens have been well documented. cpg odns are known to induce an array of cytokines; chemokines; and effecter molecules, such as interferon (ifn) α, β and γ, interleukin (il)- β, il- , il- , il- , tumor necrosis factor (tnf)-α, and nitric oxide (no) [ ] [ ] [ ] . these effecter molecules are believed to play a pivotal role in protecting the host against intra and extra cellular pathogens. whilst activating a variety of immune cells, it plays an integral role in bridging the innate immune system with the adaptive immune system directing immune responses toward t helper (th) response [ ] . a study that pre-treated chicken embryos with class b cpg odns at embryo day (ed) in ovo, and then challenged them with ibv ark strain the day after (ed ) , showed an increased up-regulation of ifn-γ, il- β, il- , il- , and oligoadenylate synthetase (oas) a in the embryonic spleen [ ] . additionally, the authors saw a significant reduction in the ibv nuclear (n) gene mrna expression in various embryonic tissues pre-treated with cpg odns, compared with the control, highlighting the value of cpg odn treatment in ibv control. however, they did not demonstrate whether the in ovo cpg odn delivery is effective against the ibv challenge encountered post-hatch. in this study, we determined whether the cpg odns delivered in ovo could provide protection against a post-hatch ibv challenge. furthermore, we looked into several cytokines and immune cells that may be activated with such protection. we found that in ovo delivery was protective against ibv challenge post hatch, suggesting a potential lasting protective effect of cpg odns towards ibv infection, which could be exploited for developing control measures. the specific pathogen free (spf) eggs from white leghorn layer hens were obtained from the canadian food inspection agency (cfia), ottawa, and were incubated according to the manufacturer's instructions in digital egg incubators (kingsuromax and rcom maru deluxe max, autoelex co., ltd., gimhae, gyeongnam, korea), located at the health research innovation centre (hric) , university of calgary. all of the animal care protocols as well as the use of live chickens, embryos, and spf eggs in our experiments, have been reviewed and approved by the health science animal care committee (hsacc, ac - , november ). at ed , the incubated eggs were candled in order to select viable eggs for further incubation, and the hatched birds were transported and housed in high containment poultry isolators at the prion/virology animal facility, hric, university of calgary, with access to ad libitum food, water, and necessary veterinary care. the ibv massachusetts (m) strain was purchased from the american type culture collection (atcc, manassas, va, usa) and was used in all of the experiments. nine day old spf viable eggs were used to propagate m strain of ibv, and the allantoic fluid was harvested at dpi by careful aspiration. the end point dilution assay was employed to assess the viral titers using ed spf eggs, and was expressed as a % embryo infectious dose (eid ) [ ] . the synthetic cpg odns, class b cpg motifs recognized by chicken tlr . the control odns were diluted in pbs to the same concentration ( µg in µl per egg), and were delivered via the same route (n = ). the in ovo tlr ligand delivery was carried out as described previously [ ] . on day post-hatch, the birds in both groups were infected with ibv m strain intra-trachealy, at a dose rate of . × eid per bird, and were monitored for days post-infection (dpi) for disease progression and outcome. the humane end point of the birds was determined based on the clinical score of each bird (ruffled feathers and huddling together = , droopy wings = , depression = , mild increase in respiratory rate = , increased respiratory rate with constant beak opening = , severe increased respiratory rate marked by gasping = , and body weight loss = ). the clinical score of was considered the humane endpoint. the eggs were then incubated for days until hatching. on the day of hatching, a subset of cpg odn-treated chickens (n = ) were infected with ibv m strain intra-trachealy at a dose rate of . × eid per bird, while maintaining the rest of the birds in that treatment group as uninfected controls (n = ). similarly, a subset of the control odn-treated chickens was infected with ibv m strain with the same dose (n = ), with the remaining birds being the control odn treated birds (n = ), and all the pbs treated birds (n = ) were kept as controls. the birds were weighed, wing tagged and after infection, were placed in separate isolators until the subsets of the animals were euthanized at (n = - per group) and (n = - per group) dpi. the clinical signs were observed and recorded daily as described, and the oro-pharyngeal and cloacal swab samples obtained using puritan ® unitranz-rt ® media transport systems (vwr, edmonton, ab, canada) at and dpi, and the ibv genome load were quantified following the rna extraction. simultaneously, the lung tissue was collected at and dpi in rna save ® (biological industries, froggabio, toronto on, canada), in order to determine the viral genome loads in lungs. to evaluate the ibv n antigen in the tracheal mucosal epithelium, the tracheal tissues from the dpi birds were collected and preserved in an optimum cutting temperature (oct) compound (tissue-tek ® , sakura finetek usa inc, torrance, ca, usa), and were snap frozen in dry ice until use in immunofluorescent assay. to observe the histopathology, the tracheal tissues of the dpi birds were fixed in % neutral buffered formalin (vwr international, west chester, pa, usa) and sent to the histopathology diagnostic services unit at the university of calgary, faculty of veterinary medicine, for hematoxylin and eosin (h and e) staining. additionally, the trachea and lung tissues were collected in an oct compound (tissue-tek ® , sakura finetek usa inc, torrance, ca, usa), snap frozen, and subjected to immunofluorescent assay so as to quantify the key innate and adaptive immune cells. another portion of the tissues were collected iusing rna save ® (biological industries, froggabio, toronto on, canada) for the cytokine mrna expression analysis. the animal numbers represent the total number of animals in two independent experiments. the total rna from the lungs collected at and dpi was extracted using a trizol reagent (invitrogen, canada inc., burlington, on, canada), according to the manufacturer's guidelines. for the rna extraction of oro-pharyngeal and cloacal swabs, the e.z.n.a. ® viral rna kit (omega bio-tek inc., norcross, ga, usa) protocol was adopted as per manufacturer's guidelines. the concentration of extracted rna was measured using nanodrop spectrophotometer (thermoscientific, wilmington, de, usa), with the absorbance at a / nm wavelength. two µg of total rna from the tissue samples and ng of total rna from the swab samples were used to synthesize the cdna with the use of the high capacity cdna reverse transcription kit (invitrogen life technologies, carlsbad, ca, usa), as per manufacturer's guidelines. a rt-pcr assay was carried out using fast sybr ® green master mix (invitrogen, burlington, on, canada) in order to quantify the ibv n gene and cytokine mrna expressions. rt-pcr assays were conducted in a well un-skirted, low profile pcr plate (vwr, edmonton, ab, canada), where the final reaction volume of the qpcr was maintained at µl. each qpcr run consisted of samples of interest, a positive control/s (gene specific plasmid), negative reverse transcriptase (nrt) control (cdna construct without the multiscribe reverse transcriptase enzyme), and negative template (ntc) control. all of the cdna samples originating from the tissues, along with the plasmid dilution series used to generate the standard curves, were run in triplicate. the target genes were quantified in relation to the β actin housekeeping gene. the target gene and the housekeeping gene for each sample was run on the same plate. five picomolar (pm) of different gene specific primers (forward and reverse primers) were used in each reaction (supplementary table s ). the change in the mrna expression of the cytokines was assessed using the pfaffl method [ ] . the optimum parameters used in the thermal cycler (cfx -c ) (bio-rad laboratories, mississauga, on, canada) were • c for seconds (s) of pre-incubation, • c for s, and • c for s for amplification cycles. a melting curve analysis was performed between • c and • c, with a . • c raise in temperature every s. the acquisition of fluorescent signals was performed at • c for s. for the cluster of differentiation (cd) α+ cell and macrophage (kul +) of the lung and trachea, µm thick sections were cut from the oct preserved tissues and were fixed using cold acetone for minutes (min). the tissues were then blocked by adding % goat serum diluted in a trizma buffered saline (tbs) buffer (trizma base: . g; nacl: g in l of distilled water; ph . ) at room temperature for min. after tipping off the excess blocking buffer, as the primary antibodies, the mouse monoclonal antibody specific for chicken macrophages/monocytes, kul + (southern biotech, birmingham, alabama, usa), cd α (ct- , southern biotech, birmingham, alabama, usa), was used in a : dilution in a % goat serum for min. the secondary antibody, goat anti-mouse igg (h+l) conjugated with dylight ® (red fluorescence) (bethyl laboratories inc., montgomery, tx, usa) was then used in a : in % goat serum for hour (h), followed by adding vectashield ® mounting medium with , -diamidine- -phenylindole dihydrochloride (dapi, vector laboratories inc., burlingame, ca, usa) (blue fluorescence), placing cover slips and edges sealed with lacquer as the final step. for the cd + t cell staining, before blocking the tissues with % goat serum, sections were blocked with avidin followed by biotin (vector laboratories, inc., burlingame, ca, usa), each with min incubation periods, in between washing with tbs-t for min twice and with pbs for min once. after blocking with % goat serum for min, a primary antibody, cd (ct- , southern biotech, birmingham, alabama, usa) was added in a : dilution in % goat serum for min. next, biotinylated goat anti-mouse igg (h+l) (southern biotech, birmingham, alabama, usa) was used as a secondary antibody in a : dilution in a % blocking buffer, and was incubated for min. then, dylight ® (green fluorescence) streptavidin in a : dilution was added for min, followed by a final step of mounting the slides with a vectashield ® mounting medium with dapi (vector laboratories inc., burlingame, ca, usa). all of the incubations were performed in a humidifying chamber at room temperature. each incubation with an antibody was followed by washing the slides in a tbs-t buffer for min twice and in pbs for min once. for the quantification of the tissue kul +, cd + cells, and cd α+ cells, five areas with maximum positive fluorescent signals of kul +, cd + cells, and cd α+ cells per tissue section were captured at x magnification, along with the corresponding nuclear stained (dapi) areas. the images were then subjected to fluorescent intensity quantification using image j software (national institute of health, bethesda, md, usa). the fluorescent intensities for the dylight ® (kul +, cd α+ cells) and dylight ® (cd + cells) positive signals were expressed relative to the total area (as estimated by nuclear staining with dapi), and were given as a percentage. a log-rank test was used to identify the differences in survival percentage. the kruskal-wallis test followed by the mann-whitney u test were used to identify the group differences in the clinical score data for each time point. the differences among the two groups were identified using othe student's t test. one-way analysis of variance (anova) followed by the students-newman-keuls test were used to identify the group differences in all of the other experiments. the grubbs' outlier test was performed in order to identify the outliers before the data was analyzed. the data in the graphs are shown in the original scale of the measurements. however, because of the non-normality and inability to satisfy the model assumptions of data belonging to the cell counts and cytokine mrna expression, a natural log transformation was applied to these data sets prior to analysis. model statistics were performed using graphpad prism software , la jolla, ca, usa. a normality test, generation of histograms, box plots, and q-q plots were performed in r statistical software, r studio version . . , boston, ma, usa. * = significant at p ≤ . , ** = significant at p ≤ . *** = significant at p ≤ . . we observed a significant increase in the survival rate of the cpg odn-treated chickens (p < . ) when compared with the control odn-treated chickens, as seen in figure a . also, the clinical signs in the cpg odn-treated ibv-infected group were significantly milder compared with the control odn-treated and ibv-infected group at dpi (p < . , figure b) . , and the rest were kept as in ovo cpg pre-treated uninfected controls (n = ). similarly, a subset of birds in the in ovo control odn-treated birds was infected with ibv (n = ), and the remaining birds were kept as in ovo control odn-treated uninfected controls (n = ). the in ovo pbs treated birds were kept as uninfected controls (n = ). a subset of birds from each group was sacrificed at dpi (n = - per group), and the remaining birds were sacrificed at dpi (n = - ) in order to obtain lung tissue. (c) ibv genome loads in oro-pharyngeal swabs at and dpi, (d) ibv genome loads in cloacal swabs at and dpi, and (e) ibv genome loads in and dpi lung. (f-g) the quantitative data and representative figures from the immunofluorescent assay of the trachea for ibv n antigen is presented. scale bar = µm (h) representative images of histological observations of trachea are given. control odns -ibv: severe epithelial metaplasia with severe cellular infiltration, germinal center formation is seen (a), superficial epithelial layer has become squamous with complete loss of cilia (b) and mucus glands not detected. cpg odns-ibv: pseudostratified simple columnar epithelium and intact ciliated epithelia (arrow) is evident where some have become rounded, and a few mucus secreting glands have been distorted and elongated (arrow head). cpg odns-control, control odns-control, and pbs-control: no lesions, normal pseudostratified ciliated columnar epithelium (c) with mucus secreting glands. log-rank test was used to identify the differences in the survival rate, and the kruskal-wallis test followed by the mann-whitney u test were used to identify the differences in the clinical scores at selected time points. the student's t test was performed to identify group differences in the oropharyngeal and cloacal genome loads, and one-way analysis of variance (anova) followed by the students-newman-keuls post hoc test was used to identify the differences in the lung ibv genome loads and ibv n antigen amount in the trachea. the differences were considered significant at * = significant at p ≤ . , ** = significant at p ≤ . *** = significant at p ≤ . . c-h: the animal numbers and results represent the pooled data of the two independent experiments. in order to assess the ibv genome loads in the lungs, and to determine the degree of virus shedding through the feco-oral route, the ibv n gene was quantified at and dpi in the birds that were pre-treated with in ovo cpg odns, control odns, and pbs. we observed a significant reduction in the viral genome loads in the oro-pharyngeal swabs collected and dpi (p < . ; figure c ), but did not observe a difference in the ibv genome loads in the cloacal swabs ( and dpi) between the treatment groups (p > . ; figure d ). however, significantly lower levels of lung viral genome load in the in ovo cpg odn pre-treated ibv infected group compared to in ovo control odn pre-treated ibv-infected group were observed at dpi (p < . ; figure e ). at dpi, although the control odn pretreated ibv infected lung had a significantly higher ibv genome load when compared with the uninfected controls (p < . ; figure e) , the difference of the ibv genome load in the lungs between two ibv infected groups was not significant (p > . ; figure e ). we observed a significant reduction of the ibv-n antigen in the tracheal mucosal epithelium of the in ovo cpg odn pre-treated-ibv infected birds compared with the in ovo control odn pre-treated-ibv infected birds (p < . ; figure f-g) . this was also seen in the histology of the trachea for the degree of tracheal damage following ibv infection. the mucosal epithelium of the in ovo control odn treated-ibv infected group showed severe metaplasia with severe mononuclear cell infiltration, where the superficial epithelial layer had been replaced by squamous cells. a complete erosion/loss of the entire mucosae was evident in several areas of the trachea. also, mucus secreting glands were absent from the remaining mucosae. in contrast, in the in ovo cpg odn pre-treated-ibv infected birds, the epithelium was a pseudostratified simple columnar epithelium mostly with intact cilia on the surface. mucus glands were present with some distortion and elongation (figure h ). in general, the in ovo cpg odn pre-treated ibv infected group recorded higher macrophage and cd + and cd α+ t numbers in the trachea compared with the uninfected and control odn pre-treated groups, although not all of the specific comparisons reached statistical significance (figure a-c) . similarly, the in ovo cpg odn pre-treated ibv infected group recorded higher macrophage and cd + and cd α+ t numbers in lungs compared to uninfected and control odn pre-treated groups, although not all of the specific comparisons reached statistical significance (figure d-e) . in the trachea and lungs, the macrophage and cd α+ t recruitment patterns, respectively, indicated that the in ovo delivered cpg odns are capable of increasing the recruitment of these cells in both the ibv infected and uninfected chickens (figure a the quantitative data following immunofluorescent assays done for the trachea (a) macrophages, (b) cluster of differentiation (cd) + t cells, and (c) cd α+ t cells are given. the quantitative data following the immunofluorescent assays done for lung (d) macrophages, (e) cd + t cells, and (f) cd α+ t cells are given. one-way anova followed by the students-newman-keuls post hoc test were used to identify the group differences. the differences were considered significant at * = significant at p ≤ . , ** = significant at p ≤ . *** = significant at p ≤ . . the results represent the pooled data of two independent experiments. considering that we observed a significant reduction in the ibv induced morbidity and mortality of in ovo cpg odn pre-treated birds correlating with varying degrees of increased macrophages, cd +, and cd α+ t cells in the tracheal and lung tissues, we needed to further elucidate the mechanisms by which these immune cells were efficiently recruited. several cytokine mrna expression levels in the lungs were analyzed at dpi, and our data showed a significant increase in the up-regulation of only the ifn-γ mrna expression in the in ovo cpg odn pre-treated lungs compared with the in ovo control odn pre-treated lungs, in both the ibv infected (p < . ) and uninfected (p < . ) groups (figure a-c) . in ovo delivery of poultry vaccines has been performed routinely for decades by the poultry industry [ ] . in ovo delivery targets the deposition of cpg odns in the amniotic cavity. subsequently, the ingestion of cpg odns containing amniotic fluid by the developing embryo distributes cpg odns in the respiratory and gastrointestinal tracts, leading to immune cell recruitment in these two body systems [ ] . we have shown in this study that cpg odns when delivered in ovo are capable of protecting young chickens against a post-hatch ibv infection induced ib. in ovo cpg odn-treated birds displayed reduced ibv viral loads in the lungs and a decreased ibv replication and pathology in the trachea, which is associated with high survival rates and low morbidity. we found that the macrophages in the trachea and cd + and cd α+ t cells in the lungs play important roles in this process, as increases in these cells were observed in the in ovo cpg odns treated group. lastly, we saw an up-regulation of ifn-γ mrna in the in ovo cpg odn pre-treated lungs, suggesting the critical role of this cytokine in the in ovo cpg odn-induced clearance of ibv infection. the host survival after day post-hatch ibv infection was seen as significant in the presence of cpg odn administration in ovo compared with the controls, and a similar protective effect of in ovo delivered cpg odns has been recorded against the post-hatch iltv infection [ , ] , and e. coli and salmonella thypimurium septicemia [ , ] . in the current study, the protection-mediated by the in ovo administered cpg odns was associated with significantly lower ibv replication in the trachea and ibv genome loads in the lungs at and dpi. consequently, the ibv genome loads in the oro-pharyngeal swabs were significantly reduced at and dpi. however, we did not observe a significant reduction in the ibv genome loads in the cloacal swabs at and dpi, because of the high variability of the ibv genome loads within the control odn pre-treated group. it is difficult to explain why we observed a discrepancy in the ibv genome loads between the oro-pharyngeal and cloacal swabs, as the in ovo delivered cpg odns have been shown to recruite immune cells into the gastrointestinal mucosa [ ] . our data confirm that, when delivered in ovo, the cpg odns are able to recruit macrophages into the trachea at dpi (four days of age), when compared to the in ovo delivered control odns in both the ibv infected and uninfected groups. previously, we saw that the in ovo delivered cpg odns increased the macrophages in the trachea at one day of age [ , ] . this macrophage recruitment to the trachea is associated with a lower ibv replication in the tissue, and it is possible that the cpg odn-mediated increase of the macrophages seen in the trachea in this study, played a central role in limiting the viral replication by three possible mechanisms. first, these cells may have efficiently and rapidly phagocytized the virus-infected cells and aided in virus elimination. second, they may have alerted the adaptive immune system to the invasion via active antigen presentation to the t cells. third, it may have contributed to the t and b cell activation and proliferation through the release of cytokines [ , ] . our observation of the increased recruitment of cd + and cd α+ t cells in the lungs following in ovo cpg odns delivery indicated that cpg odns could act as a mitogen, as has been shown previously [ ] . this cpg odn-mediated increased cd + and cd α+ t cell recruitment also could be due to the increased survival of these t cells in the lungs [ ] . interestingly, we saw an expansion of the cd α+ t cell population, but not the cd + t cell population in the in ovo cpg odn pre-treated-ibv infected lungs. although a portion of this increase of cd α+ cell recruitment could be potentially attributable to the ibv specific cd + t cells, we did not determine whether these cd α+ t cells are in deed ibv specific. we are at a loss as to why we did not see a similar cd + and cd α+ t cell response in the trachea, but it is possible that the in ovo delivered cpg odn-mediated cd α+ t cell recruitment is tissue specific [ ] . it is also important to note that our data is limited to dpi, and we do not know whether the in ovo delivered cpg odn-mediated cd + and cd α+ t cell recruitments in the trachea are occurring in other time points. of the examined immune mediators, ifn-γ, a dominant product of the t helper (th) type cells, was upregulated in the cpg odn pre-treated ibv infected and uninfected groups, when compared with the control odn pre-treated ibv infected and uninfected groups. the source of the lung ifn-γ mrna could be the cd + t cell lymphocytes and cd α+ cytotoxic lymphocytes [ , ] , and we observed an increased recruitment of the cd + and cd α+ t cells in the lungs in our experiment. two other immune mediators that were induced by the cpg odns and originated from the innate immune cells, such as the macrophages in the lungs, are the chemoattractant, il- β, and the no production inducer, inos [ ] . in the current study, we did not observe that the cpg odns or ibv induced the mrna expression of il- β or inos. this discrepancy in the cpg odn-mediated lack of il- β and inos expression can be explained by the difference in the time points observed. thapa et al. [ ] observed an increase il- β mrna expression in the lungs pre-hatch, and we observed a lack of il- β mrna expression post-hatch following in ovo cpg odns delivery. the significance of the observations described in our study are two-fold. first, we found that the in ovo administration of cpg odns is capable of limiting ibv replication in the lungs and trachea, leading to an increased survival and reduced morbidity in the early post-hatch birds. second, in our study, the early recruitment and maintenance of key immune cells, such as cd α+ and cd + t cells and macrophages, and the up-regulated ifn-γ mrna, exhibited not only an initiation of the early innate response, but also an effective and early adaptive host response mediated by the cpg odns, which would facilitate protection against the ibv infections encountered in birds in their immediate post-hatch life. further experiments elucidating the mechanisms of the cpg odn-mediated adoptive response, such as cell-and antibody-mediated immune responses in chickens and the duration of protection provided by this ligand against ibv, would be greatly beneficial in order to better understand the protective effects of cpg odns, and may aid in the development of more effective ibv control measures. to conclude, we show that the cpg odn-mediated protective response against post-hatch encountered ibv infection is associated with the up-regulation of ifn-γ mrna expression (in the lungs) and the enhanced recruitment of macrophages (in trachea) and cd + and cd α+ t cells (in the lungs). our findings, although preliminary, may provide a basis for developing novel control strategies in the long term against ibv infection in chickens. infectious bronchitis virus types: incidence in the united states induction of innate immune response following infectious bronchitis corona virus infection in the respiratory tract of chickens pathogenicity of australian strains of avian infectious bronchitis virus isolation of avian infectious 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oligodeoxynucleotide and double-stranded rna synergize to enhance nitric oxide production and mrna expression of inducible nitric oxide synthase, pro-inflammatory cytokines and chemokines in chicken monocytes a novel toll-like receptor that recognizes bacterial dna protection of chickens against escherichia coli infections by dna containing cpg motifs protection of neonatal broiler chicks against salmonella typhimurium septicemia by dna containing cpg motifs toll-like receptor (tlr) signalling-mediated antiviral response against avian influenza virus infection correlates with macrophage recruitment and nitric oxide production in ovo delivery of cpg dna reduces avian infectious laryngotracheitis virus induced mortality and morbidity in ovo cpg dna delivery increases innate and adaptive immune cells in respiratory, gastrointestinal and immune systems post-hatch correlating with lower infectious laryngotracheitis virus infection cpg oligodeoxynucleotides activate innate immune response that suppresses infectious bronchitis virus replication in chicken embryos cpg-odns induced changes in cytokine/chemokines genes expression associated with suppression of infectious bronchitis virus replication in chicken lungs bacterial dna-induced nk cell ifn-γ production is dependent on macrophage secretion of il- interactions between bacterial cpg-dna and tlr bridge innate and adaptive immunity a simple method of estimating fifty per cent endpoints the use of real-time quantitative pcr for the analysis of cytokine mrna levels hatchery vaccination against poultry viral diseases: potential mechanisms and limitations immunostimulatory cpg-modified plasmid dna enhances il- , tnf-α, and no production by bovine macrophages interleukin- -induced promotion of t-cell differentiation in mice immunized with killed listeria monocytogenes creating space: an antigen-independent, cpg-induced peripheral expansion of naive and memory t lymphocytes in a full t-cell compartment distinct effects of t-bet in th lineage commitment and ifn-γ production in cd and cd t cells il- up-regulates il- receptor expression on t cells, th cells, and b cells: synergism with il- for ifn-γ production this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to acknowledge the staff of the prion/virology animal facility at foothill campus, university of calgary, for the experimental animal management. the authors declare no conflicts of interest. key: cord- - zjrunzc authors: faye, martin; faye, oumar; diagne, moussa moise; fall, gamou; weidmann, manfred; sembene, mbacke; sall, amadou alpha; faye, ousmane title: full-genome characterization and genetic evolution of west african isolates of bagaza virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: zjrunzc bagaza virus is a mosquito-borne flavivirus, first isolated in in central african republic. it has currently been identified in mosquito pools collected in the field in west and central africa. emergence in wild birds in europe and serological evidence in encephalitis patients in india raise questions on its genetic evolution and the diversity of isolates circulating in africa. to better understand genetic diversity and evolution of bagaza virus, we describe the full-genome characterization of west african isolates, sampled from to . parameters such as genetic distances, n-glycosylation patterns, recombination events, selective pressures, and its codon adaptation to human genes are assessed. our study is noteworthy for the observation of n-glycosylation and recombination in bagaza virus and provides insight into its indian origin from the th century. interestingly, evidence of bagaza virus codon adaptation to human house-keeping genes is also observed to be higher than those of other flaviviruses well known in human infections. genetic variations on genome of west african bagaza virus could play an important role in generating diversity and may promote bagaza virus adaptation to other vertebrates and become an important threat in human health. bagaza virus (bagv) belongs to the flaviridae family, flavivirus genus and ntaya serological group. bagv is a mosquito-transmitted virus, which was first isolated in the bagaza district of the central african republic (car), in , from a pool of mixed-species female culex spp. mosquitoes during entomological investigations [ ] . as is characteristic of flaviviruses, bagv possesses a linear single-stranded, positive-sense rna genome [ ] . the bagv genome is , nucleotides in length, encoding a single polyprotein ( amino acids) from which viral proteins are derived, and flanked by and untranslated regions (utrs) of and nt, respectively [ ] . bagv has been isolated repeatedly with a high titer from different species of mosquitoes in central and west african countries [ ] [ ] [ ] , and in india, where serological investigations suggested subclinical infections in humans [ , ] . despite this widespread circulation of bagv, outbreaks involving humans or animals have not yet been reported from these countries. subsequently, in september , bagv was associated with a high mortality among game birds (partridges and pheasants) in southern spain, the first detection of bagv in europe and the first isolation from a vertebrate host [ , ] . however, it is not surprising that bagv infects birds since it has been shown to be synonymous with israel turkey all virus strains analyzed in this study were derived from the who collaborating center (http://apps.who.int/whocc/detail.aspx?cc_ref=sen- &cc_code=sen) for arboviruses and viral hemorrhagic viruses in senegal at institut pasteur de dakar (dakar, senegal) ( table ). viral stocks were prepared by inoculating viral strains into aedes albopictus (c / ) cell line in leibovitz (l- ) growth medium viruses , , of (gibcobrl, grand island, ny, usa) supplemented with % fetal bovine serum (fbs) (gibcobrl, grand island, ny, usa), % tryptose phosphate and antibiotics (sigma, gmbh, germany). bagv infection was confirmed after days of propagation by immunofluorescence assay (ifa) using specific hyper-immune mouse ascitic fluid, as previously described [ ] . cultures supernatants were collected for virus rna isolation. extraction of viral rna from supernatants was performed with the qiaamp viral rna mini kit (qiagen, heiden, germany) according to manufacturer's instructions. extracted rna was frozen at − • c prior to downstream applications. real-time rt-pcr (reverse transcriptase-polymerase chain reaction) was performed using the quantitect ® probe rt-pcr kit (qiagen, heiden, germany) in a final volume of µl following previously established protocols and primers [ ] . reverse-transcription was performed using the amv kit (promega, madison, wi, usa) following manufacturer's instructions and cdna were stored at − • c. the polymerase chain reaction with each primer set was carried out in a final volume of µl using the gotaq ® dna polymerase kit (promega, madison, wi, usa) according to manufacturer's instructions. briefly, µl (around µg) of cdna was added to µl of a rt-pcr mix containing mm mgcl , mm of dntp, x reaction buffer, u gotaq polymerase, . µl of nuclease-free water and pmols of each primer (sense and antisense). pcr was carried using the following conditions: an initial incubation at • c for min, followed by cycles of • c for min, min at melting temperature of primers, and • c according to the length of pcr product and • c during min. subsequently, µl of each pcr product was analyzed by gel electrophoresis on % agarose gels stained with ethidium bromide to check the size of amplified fragments by comparison to a dna molecular weight marker (hyperladder™ kb, bioline, taunton, ma, usa). the dna bands from the pcr amplification were purified (qiaquick gel extraction kit, qiagen, heiden, germany) and sequenced from both ends for each positive sample (beckmann coulter, high wycombe, uk). sequencing of the and termini of the viral genome was performed using a race kit (invitrogen, carlsbad, ca, usa) and a race kit (roche, basel, switzerland) following the manufacturer's protocols. additional sequences representing strains from central african republic, india, strain related to spanish wild bird's outbreak in , the itv and the ntaya virus were obtained from genbank, with the following accession numbers, respectively: ay , eu , hq , kc and nc_ . full-length genome sequences bagv isolates were obtained by assembling overlapping nucleotide sequences using the unipro ugene software (http://ugene.net/download.html) [ ] . multiple alignments of full-genome sequences were carried out by using muscle algorithm (http://www.drive .com/muscle/) [ ] within unipro ugene software. based on these alignments, we investigated the genetic properties of these different isolates circulating in west africa, such as genome length and location of main conserved amino acid motifs previously described in mosquito-borne flaviviruses (mbfvs) with sometimes mutations which include no physicochemical properties changes [ ] . comparatively, conservation of these motifs was also assessed in culex flavivirus (cxfv) and aedes flavivirus (aefv) (insect-specific flaviviruses; (isfs)) and in modoc virus (modv) and rio bravo virus (rbv) (vertebrate-specific flaviviruses, also known as no known vector flaviviruses (nkvfs)). we also searched for evidence of informative amino acid sites among bagv sequences using the divein web server (https://indra.mullins.microbiol.washington.edu/divein/) [ ] . the genetic divergence between previously available bagv complete genomes and new characterized sequences was also assessed at the nucleotide and protein levels. prediction of n-glycosylated sites on the genome of bagv were performed by submitting complete polyproteins on online version of netnglyc . server (http://www.cbs.dtu.dk/services/ netnglyc/). n-linked glycosylation is a post-translational event whereby carbohydrates are added to asparagines, which occur in the consensus sequence asn-xaa-ser/thr, where xaa is any amino acid except proline. "potential" scores of predicted n-glycosylated sites across the protein chain from n-to c-terminal were illustrated using the default threshold of . and the "jury agreement" indicates how many of the nine networks support the prediction [ ] . the rnaz method [ ] implemented in the vienna rna websuite (http://rna.tbi.univie.ac.at/) [ ] was used to detect thermodynamically stable and evolutionarily conserved structural rna domains on complete non-coding regions of the west african bagv isolates characterized in this study and the isolates from spain and car, because complete non-coding sequences are not currently available for the isolate from india. the rnaz method use an algorithm which testing a large set of well-known conserved structural rna domains and reports a "rna classification probability" or p-value as a measure of thermodynamic stability. structural rna domains with p > . are classified as stable [ ] . furthermore, the optimal secondary structures were predicted with a minimum free energy using the rnaalifold method [ ] implemented also in the vienna rna websuite that use a dynamic programming algorithm with rna parameters as previously described [ ] . furthermore, previously described organization of conserved sequences (cs) [ ] was analyzed on predicted secondary structures of the utr, considering possible repetitions of these cs. thus, a conserved sequence was considered as imperfect when it presented three or more differences with corresponding consensus sequence previously described [ ] , marked by a deletion, an insertion, or a substitution. a bayesian phylogenetic analysis for estimation of data quality and selection of the best-fit nucleotide substitution model were performed using mega . (https://www.megasoftware.net/) with a discrete gamma distribution (+g) with rate categories. thus, a total of different nucleotide substitution models were tested and model with the lowest bic score (bayesian information criterion) was considered to describe the best substitution pattern. further parameters as aicc value (akaike information criterion, corrected) and maximum likelihood value (lnl) are also estimated [ ] . a maximum likelihood tree was then constructed with complete polyprotein sequences from insect-specific flaviviruses, no known vector flaviviruses, tick-born flaviviruses, mosquito-borne flaviviruses, the orfs from new characterized west african bagv isolates and bagv sequences previously available from spain (hq - , kr - ), india (eu ) and car (ay ). tree was inferred using fasttree v . . (http://www.microbesonline.org/fasttree/) [ ] , where nucleotide substitution was modeled using general time-reversible with a proportion of invariant sites (gtr+i). nodes were labeled with local support values, which were computed with the shimodaira-hasegawa test (sh-like) for replications. topology was visualized by figtree v. . . (http://tree.bio.ed.ac.uk/software/figtree/). to prevent potential biases during phylogenetic inference due to recombination, all polyprotein sequences were analyzed using seven methods (rdp, geneconv, maxchi, bootscan, chimaera, siscan, and seq) implemented in the recombination detection program (rdp beta . ) to uncover evidence for recombination events [ ] . the disentangle recombination signals option was "on" and the linear sequence setting was used. the remaining settings were kept at their default values. only events with p-values < × − that were detected by four or more methods were considered to represent strong evidence for recombination using permutations and the bonferroni correction [ ] implemented in the rdp program to prevent false positive results. a chi-square test was used to determine if the sequence identity between a recombinant isolate and a given parent was significantly different both inside and outside the recombinant region. in addition, a bootscan analysis including the recombinant and the parental strains determined above was also performed to confirm these putative recombination events. the occurrence of recombination in bagv genomes was also investigated with a method called genetic algorithms for recombination detection (gard) implemented in datamonkey web server (http://datamonkey.org) [ ] , that estimates breakpoints based on a genetic algorithm. the statistical significance of putative breakpoints was evaluated through kishino-hasegawa (hk) tests; breakpoints were considered significant if their p value was < . . separate neighbor-joining (nj) trees were constructed for identified putative recombinant region and non-recombinant alignment partitions dictated by the breakpoint locations. phylogenetic trees were inferred using the percentage of bootstrap replications under the appropriate model of nucleotide substitution. recombination can mislead inference of positive natural selection if it is not properly accounted for. if recombination was identified, these potential recombinant sequences were excluded from further analyses to avoid inferential biases [ ] . the non-synonymous/synonymous rate ratio (dn/ds) is a widely used method to detect positive selection. the statistical test dn/ds permitted to distinguish diversifying or positive selection (dn/ds > ) from negative or purifying selection (dn/ds < ). positive selection is inferred when the rate of non-synonymous (dn) substitutions is higher than that of synonymous (ds) substitutions (dn > ds). episodes of positive selection in each gene of bagv were analyzed using methods of estimation among individual sites and internal sites on branches of the phylogenetic tree. for this, a total of alignment partitions were performed corresponding to c, prm, e, ns , ns a, ns b, ns , ns a, ns b and ns proteins. as site model, we used the single-likelihood ancestor counting (slac) that estimated the difference between non-synonymous (dn) and synonymous (ds) rates per codon site at . significance level. the fast unconstrained bayesian approximation (fubar) method which evaluated episodic positive selection at each site in the alignment at posterior probability ≥ . was also used [ ] . the mixed effects model of evolution (meme) was also conducted at a . significance level for estimation selective pressure changes among codon sites. finally, branch-site random effects likelihood (branch-site rel) analysis was used to evaluate evidence of diversifying selection on specific branches in the phylogenetic tree at a proportion of sites, considering p-values less than . as significant. all four methods were conducted with hyphy package implemented in datamonkey web server [ ] . an episode of positive diversifying selection in concern of a region was considered if it was detected by at least two different methods. the evolutionary analysis was performed using a strict clock gmrf bayesian skyride coalescent tree prior [ , ] . the gtr substitution model was used with gamma rate categories. the bayesian markov chain monte carlo (mcmc) algorithms using beast v . . (http://beast.community/) [ ] were employed to estimate the rate of bagv evolution from first isolation to . mcmc analyses were run for million generations, sampling every thousand to ensure convergence of estimates. population size (ess) above was assessed using the analysis program tracer v . (http://beast.bio.ed.ac.uk/tracer). the posterior distribution of trees obtained from the beast analysis was also used to obtain the bayesian maximum clade credibility (mcc) tree for these sequences generated by treeannotator v. . . (http://beast.community/treeannotator) (from million) after removing % of the runs burn-in and visualized by figtree v. . . . the codon adaptation index (cai) is a measure of the synonymous codon usage bias making comparisons of codon usage preferences in different organisms and assessing the adaptation of viral genes to their hosts [ , ] . cai was applied in many recent studies involving humans and rna viruses [ ] [ ] [ ] . to know if there is evidence of bagv adaptation for codon usage in humans, the cai was calculated for each isolate. to calculate normalized cai, full-length polyprotein sequences of west african bagv isolates and previously available bagv sequences from spain were compared to that of human using caical v . program (http://genomes.urv.es/caical/) [ ] . first, we obtained a "raw" cai (rcai) and then, the cai was normalized by the "expected neutral cai" (ecai) value based on random viral sequences using similar length, codon composition, gc-content and human amino acid usage. indeed, a table for human codon usage containing the entirety of human coding genes is publicly available [ ] . based on this table, we created a new table where only the identified human housekeeping genes were considered [ ] . normalized cai threshold was obtained by calculating rcai/ecai values and a value above ' ' is higher than neutral and considered as evidence of codon adaptation to the reference set of codon preferences [ ] . cai values obtained for bagv were then compared to those of others mbfvs well known to infect humans such as dengue virus (denv), usutu virus (usuv), wnv, zika virus (zikv) and yellow fever virus (yfv), nkv flaviviruses (modv and rbv) and isfs (cxfv and aefv), using the non-parametric wilcoxon test with r program. a p-value less than . was considered as significant. sequences of tobacco mosaic virus (tmv) were compared to human codons and used as negative control to provide an example that results for codon adaptation to human house-keeping genes are robust and not false positives or anomalies. as there are no known cases of human infection, or evidence of human adaptation for tmv, we expected all sequences to have a lower cai threshold than the calculated cai. in this study, a total of full-genome sequences ( , bp) of west african bagv isolates were obtained by sequencing overlapping pcr amplifications covering the genome and by using race (rapid amplification of cdna ends) techniques for the terminal ends and deposited in genbank (www.ncbi.nlm.nih.gov/genbank/) (accession numbers: mf - ) ( table ). analysis of new characterized bagv complete open reading frames (orfs) was performed at nucleotide and amino acid levels including previously available sequences from car (isolate dakarb _car_ , accession no. ay ) and spain (isolate spain_h_ , accession no. hq ) into multiple sequence alignments. the polyprotein length of the newly sequenced west african bagv isolates was determined with respect to gene sizes (table ) . although the utr was similar in length, the utr of these west african isolates was either nt or nt longer than those of sequences from car and spain, respectively. in the utr, positions , and had nucleotide changes that were distinguishable for west african isolates. nucleotide changes a to c at position and t to c at position were seen in west african sequences, and a t to c change at position was observed only in sequences of the isolates ard _dakar-bango_sen_ and ara _dezidougou_ci_ . interestingly, the utr can be divided into three sections; a proximal highly variable section constituted by the first nucleotides following the stop codon, a second highly conservative section located between nucleotide positions and and a moderately variable distal region comprising the last nucleotides. in this distal section, utr sequences of west african isolates presented insertions of nt and nt, compared to the isolates from car and spanish (kr - ), respectively. pairwise genetic distances of coding sequences were evaluated at nucleotide and amino acid levels between isolates characterized in this study and in comparison with previously available bagv sequences ( figure ). nucleotide sequences of bagv isolated from senegal showed a mean distance of . % ± . % ( . - . %). this lowest genetic distance was also apparent at amino acid level with a mean distance of . % ± . % ( . - . %). here, we described location of main conserved amino acid motifs on bagv proteins using in silico analysis of complete genome sequences of the west african bagv isolates characterized in this study and sequences from india, car and spain. most of highly conserved amino acid motifs localized across e, ns , ns and ns proteins of mvfs were identified in the bagv genomes, sometimes with presence of conservative amino acid mutations (positions highlighted in black) or non-conservative amino acid mutations (positions highlighted in red) ( table ) in protein ns , all analyzed motifs were conserved, but nc t p and t a were observed in sequences of isolates ard _barkedji_sen_ and eu _ _india_ , respectively. the bagv isolate ard _dakar-bango_sen_ also contains nc p t. in ns , the conserved motif identified at positions - contains nc a p and p l for bagv isolates ard _diawara_sen_ and ard _diawara_ sen_ , whereas nc l s in comparison to sequence of the isolate ara _dezidougou_ci_ from côte d'ivoire, senegalese bagv isolates showed a higher mean distance of . % ± . % ( . - . %) at nucleotide level. however, this highest genetic distance was less apparent at amino acid level with a mean distance of . % ± . % ( . - . %). furthermore, mean distances of . % ( . - . %), . % ( . - . %), . % ( . - . %) were recorded at nucleotide level between senegalese bagv isolates and the isolate from car, spain, and eu __ _india_ , respectively while respective mean distances were . % ( . - . %), . % ( . - . %), . % ( . - . %) at amino acid level. a differentiation coefficient value of . was also observed between these west african bagv isolates and previously available sequences. here, we described location of main conserved amino acid motifs on bagv proteins using in silico analysis of complete genome sequences of the west african bagv isolates characterized in this study and sequences from india, car and spain. most of highly conserved amino acid motifs localized across e, ns , ns and ns proteins of mvfs were identified in the bagv genomes, sometimes with presence of conservative amino acid mutations (positions highlighted in black) or non-conservative amino acid mutations (positions highlighted in red) ( table ) in protein ns , all analyzed motifs were conserved, but nc t p and t a were observed in sequences of isolates ard _barkedji_sen_ and eu _ _india_ , respectively. the bagv isolate ard _dakar-bango_sen_ also contains nc p t. in ns , the conserved motif identified at positions - contains nc a p and p l for bagv isolates ard _ diawara_sen_ and ard _diawara_ sen_ , whereas nc l s is present in ard _diawara_sen_ , and ard _barkedji_sen_ . a non-conserved motif at positions - contained nc f l in all bagv sequences analyzed and isolate ard _diawara_sen_ contained additional nc t p and d y. a non-conserved motif in ns at positions - contained nc t n in all bagv sequences. bagv isolate ard _dakar-bango_sen_ has two supplementary mutations s w and s p. in addition, these mbfvs amino acid motifs were also mostly conserved in bagv, nkvfs and cxfv (isfs) than in aefv (isfs). non-conservative amino acid mutations on the bagv polyprotein might be associated to phenotypic differences of bagv isolates. in addition, the presence of phylogenetically informative sites was assessed on the divein web server. the identified site lap is harbored by the conserved motif laptrvv previously identified in ns protein of flaviviruses [ ] and presents nc mutations in the genome of three bagv isolates. in addition, phylogenetically informative sites iega and griwna identified in ns b and ns , showed combined variations in the genome of the car isolate (dkgq and rtdmec, respectively) and the senegalese bagv isolates ard _diawara_sen_ (rraa and griwna, respectively) and ard _barkedji_sen_ (rrss and rtdmec, respectively) ( figure ). non-conservative amino acid mutations on the bagv polyprotein might be associated to phenotypic differences of bagv isolates. in addition, the presence of phylogenetically informative sites was assessed on the divein web server. the identified site lap is harbored by the conserved motif laptrvv previously identified in ns protein of flaviviruses [ ] and presents nc mutations in the genome of three bagv isolates. in addition, phylogenetically informative sites iega and griwna identified in ns b and ns , showed combined variations in the genome of the car isolate (dkgq and rtdmec, respectively) and the senegalese bagv isolates ard _diawara_sen_ (rraa and griwna, respectively) and ard _barkedji_sen_ (rrss and rtdmec, respectively) ( figure ). prediction of n-glycosylation sites was performed using complete genome sequences of the west african bagv isolates characterized in this study and sequences from india, car and spain on the divein web server. the "potential" score represents the averaged output of nine neural networks and the "jury agreement" indicates how many of the nine networks support the prediction. in total, eight n-glycosylated motifs were identified in the bagv genome (potential > . ) including two highly probable sites (potential > . and jury agreement of / ). despite high potential ( . ) and jury agreement ( / ), the motif (asn-x-thr) nptd identified at position was not considered to be glycosylated because it contained a proline known to preclude the n-glycosylation by rendering inaccessible the asparagine in the majority of cases (figure ). this motif was in the domain iii region prediction of n-glycosylation sites was performed using complete genome sequences of the west african bagv isolates characterized in this study and sequences from india, car and spain on the divein web server. the "potential" score represents the averaged output of nine neural networks and the "jury agreement" indicates how many of the nine networks support the prediction. in total, eight n-glycosylated motifs were identified in the bagv genome (potential > . ) including two highly probable sites (potential > . and jury agreement of / ). despite high potential ( . ) and jury agreement ( / ), the motif (asn-x-thr) nptd identified at position was not considered to be glycosylated because it contained a proline known to preclude the n-glycosylation by rendering inaccessible the asparagine in the majority of cases (figure ). this motif was in the domain iii region of the e protein of all bagv isolates. however, a second (asn-x-ser) motif nfsl was highly predicted (score . ( / )) and suggested an n-linked glycosylation site at the residue asn- in the ns b protein. interestingly, we also found six others probable n-glycosylation at different positions on the bagv polyprotein including one site (nysi) harboring, the nys motif at the th position ( th position of the e protein), previously described as a virulence factor for wnv and denv. of the e protein of all bagv isolates. however, a second (asn-x-ser) motif nfsl was highly predicted (score . ( / )) and suggested an n-linked glycosylation site at the residue asn- in the ns b protein. interestingly, we also found six others probable n-glycosylation at different positions on the bagv polyprotein including one site (nysi) harboring, the nys motif at the th position ( th position of the e protein), previously described as a virulence factor for wnv and denv. the "potential" score is the averaged output of nine neural networks and the "jury agreement" indicates how many of the nine networks support the prediction. the n-glyc result column shows one of the following outputs for predictions. nglycosylated sites highly predicted by the nine networks (potential > . and jury agreement of / ) are highlighted in red and the site previously reported as virulence factor on e protein of flaviviruses is colored in blue. assessment of thermodynamically stable and evolutionarily conserved structural rna domains was performed using complete non-coding sequences of the west african bagv isolates characterized in this study and the isolate from spain. the rnaz method implemented in the vienna figure . prediction of n-glycosylation on bagaza virus genome. predictions were performed using the netnglyc . server. a position with a potential (green vertical lines) crossing the threshold (horizontal red line at . ) is predicted glycosylated. the "potential" score is the averaged output of nine neural networks and the "jury agreement" indicates how many of the nine networks support the prediction. the n-glyc result column shows one of the following outputs for predictions. n-glycosylated sites highly predicted by the nine networks (potential > . and jury agreement of / ) are highlighted in red and the site previously reported as virulence factor on e protein of flaviviruses is colored in blue. assessment of thermodynamically stable and evolutionarily conserved structural rna domains was performed using complete non-coding sequences of the west african bagv isolates characterized in this study and the isolate from spain. the rnaz method implemented in the vienna rna websuite was used to identify conserved structural rna domains in the utrs of bagv characterized by a p > . . using the rnaz method, highly conserved structural rna domains was not identified in the utr of bagv genome while a total of four highly conserved structural rna domains were determined in the region with respective classification probabilities of . , . , . and . ( figure s ). however, the rnaalifold method implemented in the vienna rna websuite server predicted that, as in the genome of other members of the genus flavivirus, bagv has a shorter utr (≈ nt), consisting of a pair of conserved stem-loops (sl-a and sl-b) (figure ) . sl-a serves as promoter of viral polymerase activity followed by a shorter loop which contains a cyclisation sequence upstream of the aug (sl-b) . the secondary structure of bagv's utr could be divided in three parts; a highly variable domain following the stop codon and consisting in an au-rich stem-loop (sl-i), a second domain with highly conserved sequence and two stem-loops (sl-ii and sl-iii) and dumbbell structures (db and db ), and the moderately conserved distal domain which contains the complementary cyclisation elements. in the intermediate domain, the sl-ii presented a pseudoknot pk preceding a short conserved loop (rcs ). this structural motif was repeated in a stem-loop sl-iii with pk and cs . these stem-loops were followed by dumbbell structures db and db that presented conserved loop rcs connected with a pseudoknot pk and its repetition cs , respectively [ ] . thus, organization of conserved sequences on consensus secondary structure of bagv's utr was structured rcs -cs -rcs -cs -imcs . indeed, cs was imperfect (imcs ) only on sequences of west african bagv isolates with a total of nine substitutions compared to the corresponding consensus sequence previously described [ ] . viruses , , x of rna websuite was used to identify conserved structural rna domains in the utrs of bagv characterized by a p > . . using the rnaz method, highly conserved structural rna domains was not identified in the ′ utr of bagv genome while a total of four highly conserved structural rna domains were determined in the ′ region with respective classification probabilities of . , . , . and . ( figure s ). however, the rnaalifold method implemented in the vienna rna websuite server predicted that, as in the genome of other members of the genus flavivirus, bagv has a shorter ′ utr (≈ nt), consisting of a pair of conserved stem-loops (sl-a and sl-b) (figure ) . sl-a serves as promoter of viral polymerase activity followed by a shorter loop which contains a cyclisation sequence upstream of the ′ aug (sl-b) . the secondary structure of bagv's ′ utr could be divided in three parts; a highly variable domain following the stop codon and consisting in an au-rich stem-loop (sl-i), a second domain with highly conserved sequence and two stem-loops (sl-ii and sl-iii) and dumbbell structures (db and db ), and the moderately conserved distal domain which contains the complementary cyclisation elements. in the intermediate domain, the sl-ii presented a pseudoknot pk preceding a short conserved loop (rcs ). this structural motif was repeated in a stem-loop sl-iii with pk and cs . these stem-loops were followed by dumbbell structures db and db that presented conserved loop rcs connected with a pseudoknot pk and its repetition cs , respectively [ ] . thus, organization of conserved sequences on consensus secondary structure of bagv's ′ utr was structured rcs -cs -rcs -cs -imcs . indeed, cs was imperfect (imcs ) only on sequences of west african bagv isolates with a total of nine substitutions compared to the corresponding consensus sequence previously described [ ] . the bayesian phylogenetic analysis for estimation of data quality and selection of the best-fit nucleotide substitution model were performed using mega . with a discrete gamma distribution (+g) with rate categories. the general time-reversible with a discrete gamma distribution and a proportion of invariant sites (gtr+i) was the best nucleotide substitution model for our sequences data presenting score values of the bayesian phylogenetic analysis for estimation of data quality and selection of the best-fit nucleotide substitution model were performed using mega . with a discrete gamma distribution (+g) with rate categories. the general time-reversible with a discrete gamma distribution and a proportion of invariant sites (gtr+i) was the best nucleotide substitution model for our sequences data presenting score values of , . , , . and − , . for bic, aicc and lnl criteria, respectively. the maximum likelihood (ml) tree was inferred using fasttree v . . [ ] on our total data set including the complete polyprotein sequences of west african bagv isolates circulating in senegal and côte d'ivoire from to , the bagv sequences from spain, the bagv sequences from india and car and complete polyproteins from different flaviviruses, with , bp alignment length ( figure ). a gtr+i model was used, as selected by bayesian criteria. nodes were labeled with local support values computed with bootstrap replications using the shimodaira-hasegawa (sh) test. the phylogeny of complete bagv genome sequences presented evidence of a single bagv phylogenetic group. furthermore, we observed also that israel meningo-encephalitis turkey virus (itv) was closed to bagv in genetic relatedness [ ] . viruses , , x of respectively. the maximum likelihood (ml) tree was inferred using fasttree v . . [ ] on our total data set including the complete polyprotein sequences of west african bagv isolates circulating in senegal and côte d'ivoire from to , the bagv sequences from spain, the bagv sequences from india and car and complete polyproteins from different flaviviruses, with , bp alignment length ( figure ). a gtr+i model was used, as selected by bayesian criteria. nodes were labeled with local support values computed with bootstrap replications using the shimodaira-hasegawa (sh) test. the phylogeny of complete bagv genome sequences presented evidence of a single bagv phylogenetic group. furthermore, we observed also that israel meningoencephalitis turkey virus (itv) was closed to bagv in genetic relatedness [ ] . given the major implications of recombination events for evolution, pathogenicity, or diagnosis of non-segmented positive rna viruses like flaviviruses [ ] , it is clearly important to determine their occurrence in the bagv genome. the rdp beta . program used for assessment of recombination events on complete polyprotein sequences [ ] revealed evidence of only one highly credible recombination event from the e protein to ns b, with estimated breakpoints at positions and of bagv genome. this recombination event involved the isolate ard _dakar-bango_sen_ originating from saint-louis, in the north of senegal ( figure ). considering the isolates ard _ barkedji_sen_ and ard _barkedji_sen_ as respective minor and major parents of the isolate ard _dakar-bango_sen_ (similarity of . % and %, respectively), this recombination event was found by rdp, geneconv, bootscan, maxchi, chimaera, sisscan and seq methods and supported by significant p-values of . × - , . × − , . × − , . × − , . × − , . × − and . × − , respectively. the bootscan and gard analyzes identified one significant recombination breakpoint at nucleotide position corresponding to the e protein, supported by a lhs p-value of . and a rhs p-value of . . this breakpoint divides the bagv genome into two regions: one that encodes the structural proteins and another that encodes the non-structural proteins. phylogenetic trees were constructed using bootstrap replications and midpoint rooted for clarity only (figure ). this recombination event led to a mismatch between nj phylogenetic trees constructed using comparison of nucleotides sequences of recombinant (positions - ) and non-recombinant genomic regions (positions - and - , ). the tree is midpoint-rooted, nodes are labeled with local support values computed using the shimodaira-hasegawa (sh) test for bootstrap replications, species names are color-coded as follows: new characterized bagv isolatesdark blue with dots; previous sequences of bagv-dark blue; mosquito-borne flaviviruses (mbfvs)-green; dual-host affiliated isfs (disfs)-red; no known vector (nkv) flavivirusesyellow; tick-born flaviviruses (tbfvs)-light blue; classical isfs (cisfs)-orange. given the major implications of recombination events for evolution, pathogenicity, or diagnosis of non-segmented positive rna viruses like flaviviruses [ ] , it is clearly important to determine their occurrence in the bagv genome. the rdp beta . program used for assessment of recombination events on complete polyprotein sequences [ ] revealed evidence of only one highly credible recombination event from the e protein to ns b, with estimated breakpoints at positions and of bagv genome. this recombination event involved the isolate ard _dakar-bango_sen_ originating from saint-louis, in the north of senegal (figure ). considering the isolates ard _barkedji_sen_ and ard _barkedji_sen_ as respective minor and major parents of the isolate ard _dakar-bango_sen_ (similarity of . % and %, respectively), this recombination event was found by rdp, geneconv, bootscan, maxchi, chimaera, sisscan and seq methods and supported by significant p-values of . × - , . × − , . × − , . × − , . × − , . × − and . × − , respectively. the bootscan and gard analyzes identified one significant recombination breakpoint at nucleotide position corresponding to the e protein, supported by a lhs p-value of . and a rhs p-value of . . this breakpoint divides the bagv genome into two regions: one that encodes the structural proteins and another that encodes the non-structural proteins. phylogenetic trees were constructed using bootstrap replications and midpoint rooted for clarity only (figure ). this recombination event led to a mismatch between nj phylogenetic trees constructed using comparison of nucleotides sequences of recombinant (positions - ) and non-recombinant genomic regions (positions - and - , ). the structural and non-structural coding regions were analyzed separately for estimation of sites and branches under positive diversifying selection, applying four different methods to ensure consistency of these events along of bagv sequences. using this approach, we found several sites under strong negative selection and most of them were in the e, ns and ns proteins (table ) . however, the significant evidence (p < . ) of episodic positive selection was obtained for all the coding genes, except for the prm, ns b and ns a regions. all positively selected sites estimated by the fubar model (posterior probability ≥ . ) were also identified by the meme method (p < . ). thus, an important number of positively selected sites were detected; interestingly, the majority of such sites were in the e, ns and ns proteins. branch-site analysis showed also a total of branches evaluating under positive selection (p < . ) and the highest proportion was in the e and ns proteins. the structural and non-structural coding regions were analyzed separately for estimation of sites and branches under positive diversifying selection, applying four different methods to ensure consistency of these events along of bagv sequences. using this approach, we found several sites under strong negative selection and most of them were in the e, ns and ns proteins (table ) . however, the significant evidence (p < . ) of episodic positive selection was obtained for all the coding genes, except for the prm, ns b and ns a regions. all positively selected sites estimated by the fubar model (posterior probability ≥ . ) were also identified by the meme method (p < . ). thus, an important number of positively selected sites were detected; interestingly, the majority of such sites were in the e, ns and ns proteins. branch-site analysis showed also a total of branches evaluating under positive selection (p < . ) and the highest proportion was in the e and ns proteins. pervasive diversifying selection at posterior probability ≥ . with fubar model; episodic diversifying selection at . significance level with slac and meme models; episodic diversifying selection at p-value p ≤ . with branch-sites rel model. mcmc convergence was obtained for three independent runs with million generations, which were sufficient to obtain a proper sample for the posterior at mcmc stationarity assessed by effective sample sizes (ess) above for each gene. furthermore, the evolutionary rates (µ) and the highest posterior densities (hpd with % of confidence interval) were . × evidence of bagv adaptation to human house-keeping genes was analyzed by calculating cai indices using complete coding polyprotein sequences of west african bagv isolates and bagv sequences available from spain, in comparison to other mbfvs such as denv, usuv, wnv, zikv and yfv, nkv flaviviruses (modv and rbv) and isfs (cxfv and aefv). cai values > were obtained for polyprotein sequences of all bagv isolates. thus, there is evidence that bagv could have adaptation to the human genes ( figure ). modv(mean cai: . and median cai: . ), rbv (mean cai: . and median cai: . ) and yfv (mean cai: . and median cai: . ) showed the highest cai values for human housekeeping genes and were significantly different to spanish and west african bagv isolates (wilcoxon rank sum test, p-values ranging from . to . × − ). compared to those of spanish isolates, sequences of west african bagv isolates presented significantly higher cai values (mean cai: . and median cai: . , wilcoxon rank sum test, p-value < . ). in addition, they were also higher than denv serotype (denv- ) (wilcoxon rank sum test, p-value < . × − ). however, cai values of west african bagv isolates were lower than those of denv- (wilcoxon rank sum test, w = , p-value = . × − ) and comparable to cai values given by denv- and denv- (wilcoxon rank sum test, w = , p-value = . and w = , p-value = . , respectively). interestingly, cai values of west african isolates were also significantly higher than those obtained for other mbfvs well known to infect human such as usuv (wilcoxon rank sum test, p-value < . × − ), wnv (wilcoxon rank sum test, p-value < . × − ), zikv (wilcoxon rank sum test, p-value < . × − ) and isfs (means cai: . and . and median cai: . and . for cxfv and aefv, respectively) which showed low evidence evidence of bagv adaptation to human house-keeping genes was analyzed by calculating cai indices using complete coding polyprotein sequences of west african bagv isolates and bagv sequences available from spain, in comparison to other mbfvs such as denv, usuv, wnv, zikv and yfv, nkv flaviviruses (modv and rbv) and isfs (cxfv and aefv). cai values > were obtained for polyprotein sequences of all bagv isolates. thus, there is evidence that bagv could have adaptation to the human genes ( figure ). modv(mean cai: . and median cai: . ), rbv (mean cai: . and median cai: . ) and yfv (mean cai: . and median cai: . ) showed the highest cai values for human housekeeping genes and were significantly different to spanish and west african bagv isolates (wilcoxon rank sum test, p-values ranging from . to . × − ). compared to those of spanish isolates, sequences of west african bagv isolates presented significantly higher cai values (mean cai: . and median cai: . , wilcoxon rank sum test, p-value < . ). in addition, they were also higher than denv serotype (denv- ) (wilcoxon rank sum test, p-value < . × − ). however, cai values of west african bagv isolates were lower than those of denv- (wilcoxon rank sum test, w = , p-value = . × − ) and comparable to cai values given by denv- and denv- (wilcoxon rank sum test, w = , p-value = . and w = , p-value = . , respectively). interestingly, cai values of west african isolates were also significantly higher than those obtained for other mbfvs well known to infect human such as usuv (wilcoxon rank sum test, p-value < . × − ), wnv (wilcoxon rank sum test, p-value < . × − ), zikv (wilcoxon rank sum test, p-value < . × − ) and isfs (means cai: . and . and median cai: . and . for cxfv and aefv, respectively) which showed low evidence for codon adaptation towards human housekeeping genes (wilcoxon rank sum test, p-value < . × − ). although cai results for isfs were significantly lower to human housekeeping genes, we did not find any significant difference between cxfv and aefv codon adaptation. compared to codon usage of human genes, sequences of tobacco mosaic virus (tmv) showed mean and median cai values of . and . , respectively. with an increasing number of emergent and re-emergent pathogens involved in human encephalitis, it is important to try to better understand which viruses have a potential to emerge causing human infection in the future. since its first isolation, bagv was only detected in mosquito pools collected in the field during entomological investigations in west and central africa and in india [ ] . however, in , bagv was identified as the cause of an encephalitis outbreak in wild birds circulating in southern spain [ ] . in a possible host-switching event [ ] , bagv could acquire future adaptation to other vertebrates such as humans [ ] . in this study, genetic properties of bagv isolates circulating in west africa, the evolutionary phylogeny of bagv and evidence of bagv adaptation to human house-keeping genes were evaluated in comparison with different flavivirus groups. genomes of west african bagv strains isolated from mosquito pools collected in the field from to showed similarities in terms of gene lengths when compared with polyprotein sequences of previously available isolates from car and spain. low amino acid distances observed between west african isolates (< %) in comparison with previously non-west african sequences (< %) combined with the weak coefficient of differentiation (< . ) revealed evidence of a low genetic diversity of bagv sequences analyzed in this study as previously described [ ] . in addition, the west african bagv isolates were more closely related to the car isolate. genome sequences originating from bagv isolates from other geographic locations would be helpful to understand if this low diversity is secluded to west-africa. although the ′ utr was conserved between isolates, the ′ utr of west african isolates varied in terms of length and structure. as in other mosquito-borne flavivirus genomes, bagv genome harbored structural rna domains both in ′ and ′ utrs which play a major role in flaviviral replication and interactions with host proteins and regulate cellular response to infection [ , ] . with an increasing number of emergent and re-emergent pathogens involved in human encephalitis, it is important to try to better understand which viruses have a potential to emerge causing human infection in the future. since its first isolation, bagv was only detected in mosquito pools collected in the field during entomological investigations in west and central africa and in india [ ] . however, in , bagv was identified as the cause of an encephalitis outbreak in wild birds circulating in southern spain [ ] . in a possible host-switching event [ ] , bagv could acquire future adaptation to other vertebrates such as humans [ ] . in this study, genetic properties of bagv isolates circulating in west africa, the evolutionary phylogeny of bagv and evidence of bagv adaptation to human house-keeping genes were evaluated in comparison with different flavivirus groups. genomes of west african bagv strains isolated from mosquito pools collected in the field from to showed similarities in terms of gene lengths when compared with polyprotein sequences of previously available isolates from car and spain. low amino acid distances observed between west african isolates (< %) in comparison with previously non-west african sequences (< %) combined with the weak coefficient of differentiation (< . ) revealed evidence of a low genetic diversity of bagv sequences analyzed in this study as previously described [ ] . in addition, the west african bagv isolates were more closely related to the car isolate. genome sequences originating from bagv isolates from other geographic locations would be helpful to understand if this low diversity is secluded to west-africa. although the utr was conserved between isolates, the utr of west african isolates varied in terms of length and structure. as in other mosquito-borne flavivirus genomes, bagv genome harbored structural rna domains both in and utrs which play a major role in flaviviral replication and interactions with host proteins and regulate cellular response to infection [ , ] . however, differences in determination of structural rna domains in utr between the rnaz and the rnaalifold methods used in this study could be attributed to variations in algorithm of analysis used by each method [ ] [ ] [ ] [ ] . the small subgenomic rna (sfrna) identified in the utr of bagv is generated through incomplete degradation of the viral genome by cellular - exonuclease xrn [ , ] and plays an important role in viral pathogenicity [ ] and modulation of host responses [ , ] . in addition, the stable ' terminus region of the sfrna following the dumbbell structures (db and db ) and complementary to the terminus of the utr, was shown to be necessary in genomic rna cyclisation for viral replication and translation [ ] . the sfrna can be in competition with the utr of genomic rna in binding to proteins of viral replication complexes (rc) [ ] and/or cellular machinery [ ] . thus, it slows down the replication or translation and assembly of particles [ ] . the utr region is important for translation and replication of the rna genome through interactions with viral and host proteins, genome stabilization, and rna packaging [ ] . a better understanding of the potential impact of utr variations in replication of bagv could be important in the study of mechanisms implicated in their pathogenicity [ , ] . most motifs linked to virulence previously described in these proteins of other mbfvs were conserved among bagv isolates. however, some non-conservative mutations were identified in e, ns , ns and ns . in general, non-conservative amino acid mutations (nc) are spontaneous, rare, and hazardous, and then represent the main causes of genetic diversity. thus, non-conservative mutations observed on bagv genome could modulate viral phenotypes of particular isolates in mechanisms such as virus cell entry, replication, production of viral particles, and assembly, and cause modifications in post-translational regulation as previously demonstrated for other flaviviruses such as denv [ ] [ ] [ ] . the e protein is involved in cell receptor recognition, attachment, cell fusion, tropism, and virulence [ ] . ns is the most conserved non-structural protein of flaviviruses. associated with the other non-structural proteins, the ns protein plays an important function in viral replication and assembly and viral escape to host innate immune response [ ] . the ns protein is the main component of the replication machinery and ensures multiple functions in viral evasion to host antiviral response and in production and assembly of infectious viral particles [ ] . the ns protein is the largest viral protein that serves as the rna-dependent rna polymerase (rdrp) and performs multiple functions essential for viral replication, including processing the viral polyprotein, replicating the viral rna. sharing these motifs of virulence mostly with mbfvs, nkvfs and cxfv than with aefv showed that bagv could be more closely related to mbfvs transmitted by culex mosquitoes and could explain frequent bagv isolations mainly from mosquitoes of culex genus and its capacity to infect vertebrates such as wild birds [ , , , , ] . in addition, the west african bagv isolates characterized in our study were mainly isolated from culex poicilipes and culex neavei mosquitoes which have been reported as potential vectors for flaviviruses such as wnv [ ] . culex neavei was also found as a competent vector able to transmit flaviviruses such as usuv and wnv [ , ] . despite no available data on culex poicilipes competency to transmit flaviviruses, these two mosquito species belonging to culex genus could play an important role in natural transmission of bagv to vertebrates such as wild birds since another member of the culex genus, culex tritaeniorhynchus, has been found competent to transmit bagv to mice [ ] . the phylogenetically informative sites identified on the bagv genome located mainly in ns , ns b and ns proteins, respectively, could have a considerable impact in viral fitness on host for corresponding west african isolates. in addition, the prediction of the n-glycosylated sites at different positions on bagv genome such as asn in ns b and the nysi motif at th position of the e protein showed that post-translational modifications may influence acquisition or loss of capacity in mechanisms such as pathogenicity, evasion of innate immune pathways. indeed, flaviviruses ns b plays an important role in replication of viral rna facilitating the formation of replication complexes and modulating host innate immune response such as interferons, micrornas and rna interference, formation of stress granules and the unfolded protein responses [ ] [ ] [ ] . a previous study had shown that n-glycosylation of ns b of denv does not affect the protein stability but causes a considerable reduction in efficiency of viral production [ ] . presence of a glycosylation site and an informative site in the viral ns b protein could influence the efficiency of viral replication and the outer shape of the virion. the presence of the n-linked glycosylation motif nys had been previously reported at / th and th on the e protein of denv and wnv (lineage strains and some neuroinvasive lineage strains), respectively, involving in receptor binding, viral morphogenesis, viral infectivity, and tropism [ ] [ ] [ ] [ ] [ ] . since glycosylation is a means of evasion to immune recognition within the host by masking particular antigenic sites from recognition by neutralizing antibodies, it could increase the diversity of the glycosylation on viral proteins [ , ] . nevertheless, it could be important in future studies to determine whether the predicted glycosylation sites are really used (asparagine-linked) using specific enzymatic digestion by endo h and peptide n-glycosidase (pngase f) [ ] . our data suggest the ability of bagv to develop phenotypically important variations and potentially adaptation to new vertebrate hosts such as humans. however, to understand better the impact of variation on these predicted n-glycosylation sites and the identified phylogenetically important variations would require in vitro studies with reverse genetically engineered infectious clones on mosquito or mammalian cell lines and in vivo experiments in mosquitoes or in animal models like mouse [ , ] . however, antibodies against bagv proteins or infectious clone are currently not available for bagv. the identification of natural recombination events between virus isolates is important for our understanding of virus evolution. in our study, we identified a recombination event in the e protein bagv. recombination was documented in other members of the mosquito-borne flavivirus group [ , ] , but had not yet been demonstrated to occur in bagv. identification of recombination breakpoints and the graphical detection of conflicting phylogenetic signals gave confirmation of this recombination event in e protein of the senegalese isolate ard _dakar-bango_sen_ as previously described for zikv [ , ] . nevertheless, the precise molecular mechanisms of the template switches are unknown. the e protein is highly important because it encodes the most important antigen with regards to virus biology and humoral immunity. therefore, large-scale genetic changes in this region, as might be brought about by recombination, could have significant impact on virus phenotype [ ] . the estimation of the selection pressures acting on each protein of bagv demonstrated episodes of strong negative selection in functionally important proteins. these results suggested frequent purging of deleterious polymorphisms in the bagv genome that could be associated with accumulation of synonymous mutations during bagv transmission [ ] . however, location of more significant episodes of positive selection in the e, ns and ns proteins indicated that they could represent preferential selection targets during bagv evolution [ ] . indeed, the e protein of flaviviruses plays a crucial role in early steps of host cell binding and viral entry and represents a main target for immune responses influencing antigenic response and positive selection on the e protein is a hallmark of the emergence of flaviviruses [ , ] . positive selection episodes have been also previously reported for the denv- capsid, however, the impact needs to be further investigated [ ] . likewise, non-structural proteins could also be targets of positive selection. the ns protein is essential for viral rna replication, is involved in immune system evasion, and represents the major positive selection target during speciation of arthropod-born flaviviruses such as denv and zikv [ ] . ns a and ns b proteins have been shown to antagonize the interferon response during denv infection [ ] and changes in these regions would be evolutionary advantageous selecting for bagv strains with strong innate immunity suppression mechanisms. mutations in the ns b protein were also seen to modulate several phenotype mechanisms of flaviviruses, such as pathogenesis [ ] , viral adaptation [ ] , replication [ ] , neurovirulence [ ] and host preferences [ ] . thus, presence of positively selected sites in ns b of bagv isolates could have major impact in its natural evolution. ns and ns proteins are crucial for viral replication, since non-conservative changes in these regions could modify process of protease and atpase/helicase functions of ns protein [ ] and rna polymerase activity of ns protein [ ] . these several polymorphic amino acid coding sites in the bagv genome suggest that these proteins may be experiencing relatively adaptive changes in the natural evolution and they should be prioritized in future experimental studies. despite the evidence of a single phylogenetic group for bagv sequences analyzed in our study, the evolutionary rates are expected in accordance to proteins functions; the ns representing the polymerase and the most conserved protein [ ] . the inferred bayesian mcc trees indicated a single introduction of bagv into europe and africa from india, contrary to other african flaviviruses as wnv [ ] and zikv [ ] , suggesting an indian origin of bagv. estimated times from the mrca suggested a distant origin of west african bagv sequences analyzed in this study from the th century. thus, further phylodynamic analyzes based on more complete sequences could be interesting for determining geographical pathways and potential evolution patterns in correlation with bagv spread from india to african and european continents. the codon adaptation index (cai) represents a reliable bio-informatics approach to measure the synonymous codon usage bias and to assess the adaptation of viral genes to their hosts [ ] . flaviviruses can infect and replicate in hosts of different phyla. therefore, their versatility in gene expression and protein synthesis and changes in the viral rna genome could affect the fitness of the virus in a specific host relating to dinucleotide frequencies, codon preferences, and codon pair biases [ ] [ ] [ ] . nevertheless, ecology, different virus-host relationships, biogeographical migrations of flavivirus species and genetic differences may explain observed differences in flaviviral codon usage preference to human housekeeping genes [ , , ] . in particular, nkv flaviviruses were only isolated from vertebrates and are maintained in nature by horizontal transmission between vertebrate hosts [ , ] . although isfs were thought to sustain their populations in their respective insect vectors in the absence of mammal reservoirs, so lower translational efficiency in vertebrates could be expected [ , ] . in addition, the highest cais of yfv and denv could be related to their long histories of infection in humans [ ] . indeed, yfv and denv are maintained in endemic and sylvatic cycles, which conducted to repeated epidemics for more than one hundred years. however, yfv showed generally a higher virulence in human infections, particularly when it is compared to denv infections reported in africa [ ] . this could explain the higher cai values of yfv towards codon usage of the human housekeeping genes. with evidence of adaptation to human house-keeping genes, bagv could be potential cause of infection in vertebrates, such as humans. considering the highest cai values of west african bagv isolates when compared to isolates responsible of the spanish wild bird's outbreak in [ ] , bagv adaptation to vertebrate species such as birds could have led to an extension of adaptation to other species as shown in a previous virus study [ ] . interestingly, west african bagv isolates showed a higher evidence of codon adaptation than mbfvs well known to infect humans, such as wnv which is a major cause of human encephalitis in usa and responsible of recent outbreaks in europe [ ] and zikv associated with microcephaly in fetuses and newborns during the outbreak in brazil in [ ] . thus, further comparison of codon adaptation indexes of other bagv genomic regions, such as the utr, among isolates that differ in biological, ecological, and genetic characteristics could help to characterize the evolutionary adaptation of bagv genomes to vertebrate hosts [ , ] . nevertheless, to ensure the potential of bagv to be involved in human encephalitis cases, it would be important to evaluate its pathogenicity on human induced pluripotent stem cell lines (ipsc) capable of differentiating into brain microvascular endothelial cells (bmecs) and constituting a robust model of the human blood-brain barrier [ ] . otherwise, the ipsc cells can also generate primitive neural stem cells (nscs), which can differentiate into neurons, astrocytes, or 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review of their discovery, host range, mode of transmission, superinfection exclusion potential and genomic organization yellow fever virus: genetic and phenotypic diversity and implications for detection, prevention and therapy identification of host genes leading to west nile virus encephalitis in mice brain using rna-seq analysis zika virus: an update on epidemiology, pathology, molecular biology, and animal model genome-wide analysis reveals class and gene specific codon usage adaptation in avian paramyxoviruses an isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes and neurons derived from human induced pluripotent stem cells efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells this research received no specific grant from any funding agency in the public, commercial, key: cord- -p buyrcj authors: batista, mariana n.; braga, ana cláudia s.; campos, guilherme rodrigues fernandes; souza, marcos michel; de matos, renata prandini adum; lopes, tairine zara; candido, natalia maria; lima, maria leticia duarte; machado, francielly cristina; de andrade, stephane tereza queiroz; bittar, cíntia; nogueira, maurício l.; carneiro, bruno m.; mariutti, ricardo b.; arni, raghuvir krishnaswamy; calmon, marilia freitas; rahal, paula title: natural products isolated from oriental medicinal herbs inactivate zika virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: p buyrcj zika virus (zikv) has been associated with serious health conditions, and an intense search to discover different ways to prevent and treat zikv infection is underway. berberine and emodin possess several pharmacological properties and have been shown to be particularly effective against the entry and replication of several viruses. we show that emodin and berberine trigger a virucidal effect on zikv. when the virus was exposed to µm of berberine, a reduction of . % in the infectivity was observed; when emodin was used ( µm), this reduction was approximately . %. dynamic light scattering data showed that both compounds significantly reduce the hydrodynamic radius of virus particle in solution. we report here that berberine and emodin, two natural compounds, have strong virucidal effect in zika virus. zika virus (zikv), a member of the flaviviridae family and the flavivirus genus, is named after the forest where it was first identified in [ ] . for approximately years only a few minor epidemics have been identified, none of which had any major consequences. however, when this virus started to circulate in brazil in , a correlation was observed between zikv infection in pregnant women and poor fetal development, leading to severe conditions such as microcephaly and other neurological diseases [ ] . although the exact mechanism by which zikv triggers these diseases is still unclear, some relevant information is now available. for example, it is known that the virus can penetrate the placenta [ ] , infect progenitor neural cells, and disrupt development [ ] . in addition to fetal disease, zikv has also been implicated in the development of the guillain-barré syndrome in adults [ ] . strategies to combat zikv infection include the development of vaccines [ ] and the screening of molecules that inhibit the different phases of the viral lifecycle [ , ] . berberine is an isoquinoline alkaloid belonging to the structural class of protoberberines and is encountered in many plants including berberis vulgaris [ ] . it exhibits several pharmacological properties and is particularly effective against entry and replication of many viruses, including the human cytomegalovirus (hcmv) [ ] , herpes simplex virus (hsv) [ ] , influenza virus [ ] , chikungunya virus (chikv), and other alphaviruses [ ] . emodin, an anthraquinone derivative, is also a naturally occurring compound derived from the chinese herbs rheum palmatum [ ] , polygonum multiflorum [ ] , aloe vera [ ] , and cassia obtusifolia [ ] . emodin possesses a wide spectrum of pharmacological effects, including antiviral activity, against coxsakie b virus (cvb ), human respiratory syncytial virus (hrsv) [ ] , influenza a virus [ ] , epstein-barr virus (ebv) [ ] , herpes simplex virus (hsv) [ ] , hepatitis b virus (hbv) [ ] , and japanese encephalitis virus (jev), which is another flavivirus [ ] . in this study, we tested the activity of the natural compounds berberine and emodin for their ability to inhibit zikv infection. stock solutions of emodin and berberine were diluted in . % dimethyl sulfoxide (dmso, sigma-aldrich, st. louis, mo, usa). the working solutions were obtained by dilution, in different concentrations, of both drugs in dulbecco's modified eagle's medium (dmem) (sigma-aldrich, st. louis, mo, usa). vero e cells (atcc crl- tm ), donated by dr. maurício lacerda nogueira, were cultured in dmem (supplemented with % fetal bovine serum (cutlab, campinas, brazil), %, u/ml of penicillin, and µg/ml of streptomycin (invitrogen, new york, ny, usa) and maintained in a humidified % co incubator at • c. to test the inhibitory potential of berberine and emodin, a brazilian zika virus strain isolated from a febrile patient in northeast brazil [ ] was used. an aliquot of this virus was inoculated onto aedes albopictus mosquito cells, clone c / (atcc ® crl- ™), and incubated in leibovitz's l- medium (cutlab, campinas, brazil) supplemented with % fetal bovine serum, %, u/ml of penicillin, and µg/ml of streptomycin for - days until the first cytopathic effects were observed. the same viral passage was used in all experiments. the cytotoxicity of berberine and emodin was evaluated in order to select the optimal non-toxic concentration of each compound in vero e cells. for this assay, × vero e cells were seeded to each well of a -well plate and incubated for h at • c. media was removed and replaced with dmem containing different concentrations of berberine ( - µm) or emodin ( - µm). the effects of the compounds on the cells were analyzed at h, h and h after treatment. the supernatants were removed, and a solution of -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt; mg/ml) (sigma-aldrich, st. louis, mo, usa) was added to each well. the plates were then incubated for min at • c. subsequent to incubation, formazan crystals were solubilized with µl dmso (sigma-aldrich, st. louis, mo, usa), and the absorbance was measured at nm. the ability of berberine and emodin to inhibit zikv in a pre-entry step, acting directly on the viral particle, was tested by a virucidal assay. briefly, × cells per well were seeded in a -well plate. berberine or emodin were incubated at different concentrations (berberine: - µm and emodin: . - µm) with zikv ( plaque-forming unit/ ml (pfu/ml)) for h at • c. a control, zikv incubated with the drug diluent (dmem + dmso . %), was also performed. subsequently, the drug-containing supernatant was titrated in vero e cells and incubated for h. cells were fixed with % formaldehyde for min before staining with a crystal violet ethanolic solution. the titer of the virus treated with different concentration of each compound was determined and compared to the control (diluent only). we also tested the influence of berberine and emodin on cell receptors involved in virus entry. for these experiment, one day prior to the assay, × cells/well were seeded onto a -well. on the following day, the culture growth medium was replaced with dmem containing µm of berberine or µm of emodin and incubation proceeded for h at • c. after one hour, the medium containing the compounds was removed, and the cell monolayer was washed thrice with pbs. the control was incubated only with dmem + . % dmso (drug diluent). the treated cells were then infected with zikv and incubated at • c for h. the virus was then removed, culture media was added and cells were incubated for h at • c with % co . after fixation with % formaldehyde and staining with crystal violet, the number of foci was determined and compared to the non-treated control. the half maximal inhibitory concentration (ic ) and half maximal cytotoxic concentration (cc ) of berberine and emodin were calculated using non-linear regression analysis. the ic and cc values, were used to calculate the selectivity index (si) of each compound (si = cc /ic ). the si suggests potential effectiveness, where the higher the value, the more promising the drug. dynamic light scattering (dls) measurements of virions were carried out using freshly purified samples of zikv in polystyrene curettes with optical path lengths of cm and at a concentration of . × pfu/ml in filtered pbs buffer (ph . ) after incubation for h at • c, both in the absence and presence of µm emodin or µm berberine. the results presented are the average values of scans of s, each obtained with a zetasizer nano s (malvern, uk) equipped with a he-ne laser ( . nm). the scattered light was detected at • and the experimental data were analyzed using the zetasizer software. the results of viral inhibition were calculated as a percentage of the negative control (medium plus drug diluent). all statistical analyses were performed by one-way anova with tukey's post-test using the graphpad prism . software (graphpad software, san diego, ca, usa). we initially screened berberine and emodin for cellular cytotoxicity. the higher non-toxic concentrations were µm for berberine and µm for emodin (more than % cell viability) in all experimental times (figure ). by non-linear regression analysis, the cc values were calculated as µm for berberine and . µm for emodin. after determining the highest non-toxic concentration for each compound, a virucidal assay was performed to test the ability of these compounds to impair the virus particle infectivity. the supernatant containing infectious virus particles was incubated for h at different concentrations of each compound (berberine: - µm and emodin: . - µm) and subsequently used to infect vero e cells. after h, a considerable reduction of the foci numbers was observed for both compounds in a dose-dependent manner. when the viral particles were incubated with µm of berberine, plaques reduced from . × pfu/ml (control) to . × pfu/ml, a reduction of . % in zikv infectivity. when the particles were exposed to µm of emodin, this reduction was approximately . % (from . × pfu/ml to . × pfu/ml) ( figure ). the ic values for berberine and emodin were . µm and . µm respectively, leading to a si of . for berberine and . for emodin. although at reduced concentrations, berberine had a lower ability to inhibit the virus, all tested concentrations of berberine presented a significant reduction of foci number compared to the control (p < . ) (figure a) . emodin inhibited the virus with a significant reduction of foci number for all tested concentrations (p < . ), except for . µm ( figure b ). after determining the highest non-toxic concentration for each compound, a virucidal assay was performed to test the ability of these compounds to impair the virus particle infectivity. the supernatant containing infectious virus particles was incubated for h at different concentrations of each compound (berberine: - µm and emodin: . - µm) and subsequently used to infect vero e cells. after h, a considerable reduction of the foci numbers was observed for both compounds in a dose-dependent manner. when the viral particles were incubated with µm of berberine, plaques reduced from . × pfu/ml (control) to . × pfu/ml, a reduction of . % in zikv infectivity. when the particles were exposed to µm of emodin, this reduction was approximately . % (from . × pfu/ml to . × pfu/ml) (figure ). the ic values for berberine and emodin were . µm and . µm respectively, leading to a si of . for berberine and . for emodin. although at reduced concentrations, berberine had a lower ability to inhibit the virus, all tested concentrations of berberine presented a significant reduction of foci number compared to the control (p < . ) (figure a) . emodin inhibited the virus with a significant reduction of foci number for all tested concentrations (p < . ), except for . µm ( figure b ). finally, we tested the compounds as a pre-treatment, exposing the cells to each compound prior to infection. vero e cells were incubated with berberine or emodin for h at the highest non-toxic concentrations prior to zikv infection. pre-treatment with berberine had no effect on virus entry. on the other hand, emodin reduced entry by . % (plaques reduced from × pfu/ml (control) to . × pfu/ml) (figure ) . the hydrodynamic radius of zikv was determined both before and after incubation with berberine or emodin. the purified zikv particles are monodisperse (red line, figure ) with a correlation coefficient close to unity (supplementary figure s ) and the addition of µm of emodin finally, we tested the compounds as a pre-treatment, exposing the cells to each compound prior to infection. vero e cells were incubated with berberine or emodin for h at the highest non-toxic concentrations prior to zikv infection. pre-treatment with berberine had no effect on virus entry. on the other hand, emodin reduced entry by . % (plaques reduced from × pfu/ml (control) to . × pfu/ml) (figure ). finally, we tested the compounds as a pre-treatment, exposing the cells to each compound prior to infection. vero e cells were incubated with berberine or emodin for h at the highest non-toxic concentrations prior to zikv infection. pre-treatment with berberine had no effect on virus entry. on the other hand, emodin reduced entry by . % (plaques reduced from × pfu/ml (control) to . × pfu/ml) (figure ) . the hydrodynamic radius of zikv was determined both before and after incubation with berberine or emodin. the purified zikv particles are monodisperse (red line, figure ) with a correlation coefficient close to unity (supplementary figure s ) and the addition of µm of emodin the hydrodynamic radius of zikv was determined both before and after incubation with berberine or emodin. the purified zikv particles are monodisperse (red line, figure ) with a correlation coefficient close to unity (supplementary figure s ) and the addition of µm of emodin (green line, figure ) or µm berberine (blue, figure ) led to a significant reduction of the hydrodynamic radius of the samples. zikv incubated with emodin presented a sharper profile than when incubated with berberine. since the drugs were dissolved in dmso, dls measurements were also conducted with dmso at the same concentration as the diluent, and did not indicate any significant difference with the native virus in a pbs buffer. viruses , , x for peer review of (green line, figure ) or µm berberine (blue, figure ) led to a significant reduction of the hydrodynamic radius of the samples. zikv incubated with emodin presented a sharper profile than when incubated with berberine. since the drugs were dissolved in dmso, dls measurements were also conducted with dmso at the same concentration as the diluent, and did not indicate any significant difference with the native virus in a pbs buffer. berberine and emodin, when evaluated in vitro, have virucidal effects on zika virus. berberine reduced virus infectivity by almost % in vero e cells. this compound is present in two herbs widely used in natural therapies, coptis sp. and berberis sp. [ ] , and has been shown to be effective in controlling glycemia and lipid metabolism [ ] . from an antimicrobial point of view, berberine inhibited many viruses, in particular influenza virus [ ] , chikv [ ] , and hsvs [ ] . in these studies, berberine was effective at later stages of the viral cycle, such as during uncoating and replication. however, it should be noted that the viruses mentioned are not flavivirus. emodin is an anthraquinone derivative present in several types of herbs and used primarily in eastern medicine for the treatment of various health conditions [ ] . this compound has also been shown to inhibit several viruses using different mechanisms; for example, emodin can block the coronavirus channel protein and inhibit the process of viral particle release from cells [ ] . this compound also blocked the entry step of coxsackieviruses [ ] and hsv [ ] . we also observed a modest ( . %) effect of emodin as a pre-treatment, therefore interfering in virus entry. however, the mechanism of action is not clear. in previous studies, the effect of emodin on virus entry was berberine and emodin, when evaluated in vitro, have virucidal effects on zika virus. berberine reduced virus infectivity by almost % in vero e cells. this compound is present in two herbs widely used in natural therapies, coptis sp. and berberis sp. [ ] , and has been shown to be effective in controlling glycemia and lipid metabolism [ ] . from an antimicrobial point of view, berberine inhibited many viruses, in particular influenza virus [ ] , chikv [ ] , and hsvs [ ] . in these studies, berberine was effective at later stages of the viral cycle, such as during uncoating and replication. however, it should be noted that the viruses mentioned are not flavivirus. emodin is an anthraquinone derivative present in several types of herbs and used primarily in eastern medicine for the treatment of various health conditions [ ] . this compound has also been shown to inhibit several viruses using different mechanisms; for example, emodin can block the coronavirus channel protein and inhibit the process of viral particle release from cells [ ] . this compound also blocked the entry step of coxsackieviruses [ ] and hsv [ ] . we also observed a modest ( . %) effect of emodin as a pre-treatment, therefore interfering in virus entry. however, the mechanism of action is not clear. in previous studies, the effect of emodin on virus entry was determined through its high affinity for the envelope phospholipid bilayer, and this interaction can lead to the rupture and destruction of the hsv- and vhsv viral particles [ , ] . a wide spectrum of biological activities has been described for emodin through different mechanisms of action [ ] [ ] [ ] , ] and among these, some are directly related to induced changes of the viral envelopes, such as % disruption of the herpes simplex virus type (hsv- ) envelope [ ] . the interaction of emodin with phospholipids and its effects in the integrity of vesicles and lipid extracts from escherichia coli through the perturbation of the physical properties of the bilayer have been reported [ , ] , supporting the direct effect of this anthraquinone on biological membranes. berberine has also been described to act directly on biomembranes [ ] or by inducing the increase of membrane permeability [ ] . dls measurements indicated differences in the estimated hydrodynamic radii of the virion samples before and after incubation with emodin or berberine. the calculated hydrodynamic diameter is indicative of the apparent size of the dynamic hydrated/solvated particle including the hydration sphere which encapsulates the virus particle. thus, the observed change in the apparent hydrodynamic radius of the infectious viral particle upon addition of emodin and berberine could indicate disorder or disruption of the bilayer, or alterations in the solvation characteristics of the viral particles in solution. compounds that act inhibiting virus before the attachment to the host cell, such as berberine and emodin, can be used prophylactically at low concentrations since the number of infectious particles in a primary infection is relatively low. in addition, berberine can also spread to various organs of the body, such as the liver, kidneys, muscles, and brain, and remain there for a considerable period of time [ ] . this is a desired characteristic for an antiviral targeting zika virus when maintained at low concentrations, since many cell lines and tissues has been shown to be permissive to this virus [ ] [ ] [ ] . thus, infection via mosquito bites or through sexual contact could be controlled. another beneficial feature of berberine is that it has low toxicity and no serious adverse effects in humans [ ] [ ] [ ] . some applications of berberine and emodin have encountered a few obstacles in pharmaceutical development, such as poor aqueous solubility [ , ] and rapid metabolism [ , ] . recently, several nanoparticulate delivery systems for berberine and emodin have been reported, which have attempted to address the major pharmaceutical concerns associated with its systemic administration, with lipid-based nanocarriers being the most investigated [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . zika virus has drawn much attention within the scientific community since as a result of the outbreak in brazil. efforts have been made to identify effective drugs to treat this virus and to elucidate its fundamental characteristics. in this study, we showed that berberine and emodin, two natural compounds, have strong virucidal effect in vitro, directly impairing the zikv particles. after additional studies, given the high cellular viability in the active concentrations both compounds are good candidates for treatment of zika virus-infected patients and possibly for preventive treatment in low doses in endemic areas. zika virus. i. isolations and serological specificity zika virus infection during pregnancy and microcephaly occurrence: a review of literature and brazilian data zika virus infection during pregnancy in mice causes placental damage and fetal demise zika virus impairs growth in human neurospheres and brain organoids guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study a susceptible mouse model for zika 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phenylboronic acid-functionalized nanoparticles for emodin delivery key: cord- - np brx authors: campos, samuel k. title: subcellular trafficking of the papillomavirus genome during initial infection: the remarkable abilities of minor capsid protein l date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: np brx since , our understanding of human papillomavirus (hpv) subcellular trafficking has undergone a drastic paradigm shift. work from multiple laboratories has revealed that hpv has evolved a unique means to deliver its viral genome (vdna) to the cell nucleus, relying on myriad host cell proteins and processes. the major breakthrough finding from these recent endeavors has been the realization of l -dependent utilization of cellular sorting factors for the retrograde transport of vdna away from degradative endo/lysosomal compartments to the golgi, prior to mitosis-dependent nuclear accumulation of l /vdna. an overview of current models of hpv entry, subcellular trafficking, and the role of l during initial infection is provided below, highlighting unresolved questions and gaps in knowledge. hpvs infect and replicate in cutaneous and mucosal epithelium (skin and oral/genital mucosa). of the hundreds of hpv types [ ] [ ] [ ] , a set of about hpv "high-risk" types are associated with cervical, anogenital, and oropharyngeal cancers. an additional set of "low-risk" mucosal types cause benign anogenital warts. hpvs are currently the most common sexually transmitted infection, and collectively, these viruses account for % of cancers worldwide [ ] [ ] [ ] . as for most other dna viruses, a successful hpv infection requires that the viral genome (vdna) be transported from an extracellular encapsidated state (i.e., viral particles) to a free unencapsidated state within the host cell nucleus, to allow for viral gene expression and vdna replication. the non-enveloped hpv capsid, comprised of two proteins (l and l ), is the molecular machine that accomplishes this task. seventy-two pentamers of the major capsid protein l form the nm icosahedral particle, which together with l , encapsidate the vdna. the minor capsid protein l is present in variable, but low, amounts, with a maximal occupancy of molecules per virion [ ] . most studies report a range between and molecules of l per virion [ , ] , and my lab generally estimates - copies of l per particle as measured by coomassie staining of sds-page gels and densitometry. although cryoem reconstructions indicate that the bulk of l density resides beneath the capsid surface underneath the l pentamers [ ] , it is important to remember that regions of l are known to be exposed on the surface of the virion [ ] [ ] [ ] . likewise, it is important to consider that individual l molecules can likely assume different conformations or configurations within the virion, although this has yet to be proven. it should be noted that many of the findings summarized in this review were found using differentiation-independent pseudovirus (psv) or quasivirus (qsv) systems for generating infectious or reporter-containing hpv virions in tt cells [ ] . while more "native" systems for the generation of hpv virions like organotypic raft systems might be ideal, the tt psv/qsv system has been invaluable for basic research on hpv, as the system enables high titer production of highly purified infectious particles, as well as the means to generate virions that package l fusions, noninfectious l mutants, and -ethynyl- ′-deoxyuridine (edu)-labeled vdna for a variety of experimental systems. for a recent review on the differences between psv/qsv and raft-derived hpv, see [ ] . virion binding to extracellular heparin sulfate proteoglycans (hspgs) induces conformational changes in both the l and l capsid proteins of the viral particle, and subsequent transfer of the virion to a cell surface entry receptor complex [ ] [ ] [ ] . while bound, cell surface kallekrein- (klk ) and furin cleave the l and l capsids, respectively, an important "priming" event that ensures the proper subsequent subcellular trafficking of l /vdna [ ] [ ] [ ] . endocytosis of the virion occurs through an actin-dependent process with similarities to macropinocytosis [ ] . tetraspanin cd and its associated α β , α β and α β integrin partners, growth factor receptor tyrosine kinases, annexin a , and the cytoskeletal adaptor obscurin-like (obsl ) have been implicated in tetraspanin-enriched microdomain (tem)-dependent hpv entry [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (figure ). while endocytosis of individual cell surface-bound particles can be quite rapid, overall bulk populationlevel internalization is asynchronous and slow, occurring on the time scale of many hours [ ] . cd and the associated tems likely coordinate organization and assembly of entry receptor complexes; once assembled and bound to virion, these complexes facilitate rapid entry. similar scaffolding roles for other tetraspanins have been reported for other enveloped and nonenveloped viruses [ , ] . it should be noted that many of the findings summarized in this review were found using differentiation-independent pseudovirus (psv) or quasivirus (qsv) systems for generating infectious or reporter-containing hpv virions in tt cells [ ] . while more "native" systems for the generation of hpv virions like organotypic raft systems might be ideal, the tt psv/qsv system has been invaluable for basic research on hpv, as the system enables high titer production of highly purified infectious particles, as well as the means to generate virions that package l fusions, non-infectious l mutants, and -ethynyl- -deoxyuridine (edu)-labeled vdna for a variety of experimental systems. for a recent review on the differences between psv/qsv and raft-derived hpv, see [ ] . virion binding to extracellular heparin sulfate proteoglycans (hspgs) induces conformational changes in both the l and l capsid proteins of the viral particle, and subsequent transfer of the virion to a cell surface entry receptor complex [ ] [ ] [ ] . while bound, cell surface kallekrein- (klk ) and furin cleave the l and l capsids, respectively, an important "priming" event that ensures the proper subsequent subcellular trafficking of l /vdna [ ] [ ] [ ] . endocytosis of the virion occurs through an actin-dependent process with similarities to macropinocytosis [ ] . tetraspanin cd and its associated α β , α β and α β integrin partners, growth factor receptor tyrosine kinases, annexin a , and the cytoskeletal adaptor obscurin-like (obsl ) have been implicated in tetraspanin-enriched microdomain (tem)-dependent hpv entry [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (figure ). while endocytosis of individual cell surface-bound particles can be quite rapid, overall bulk population-level internalization is asynchronous and slow, occurring on the time scale of many hours [ ] . cd and the associated tems likely coordinate organization and assembly of entry receptor complexes; once assembled and bound to virion, these complexes facilitate rapid entry. similar scaffolding roles for other tetraspanins have been reported for other enveloped and nonenveloped viruses [ , ] . early subcellular trafficking and uncoating. internalized virions, primed by cleavage on the cell surface, enter the endolysosomal pathway and begin ph-dependent uncoating and l insertion/penetration. l recruitment of cytosolic sorting factors including sorting nexins (snxs) and retromer modulates the trafficking pathway. retromer binding is important for ee to le/mvb transport. retrograde transport of l /vdna from le/mvbs to the trans-golgi network (tgn) occurs in a furin-, cyclophilin-γ-sec-, and ph-dependent manner. cleavage of l by the host protease furin occurs on the surface of host cells and on the extracellular matrix (ecm) in response to binding of the virion to hspgs [ ] . this cleavage occurs c-terminal to the final arginine residue of a conserved consensus site (rtkr, residues - for hpv ), removing twelve n-terminal residues of hpv l [ ] (figure ). the molecular basis for the requirement of this cleavage remains unknown, but inhibition of cleavage through mutation of the cleavage site or by biochemical inhibition of furin results in aberrant trafficking of the l /vdna complex and potent abrogation of infection [ ] . cleavage appears to trigger a conformational change in capsid and/or l structure, as the conserved and neutralizing rg- epitope (residues - for hpv [ ] ) becomes accessible to antibody staining shortly after virion binding in a furin-dependent manner [ ] . cell surface cyclophilins (peptidyl-prolyl isomerases, ppis) also appear to modulate the conformation of l , as rg- epitope exposure is sensitive to cyclosporine a, a broad ppi inhibitor [ ] . rg- epitope exposure was initially believed to be a convenient marker for furin cleavage, and cyclophilins were believed to control l accessibility and susceptibility to furin, but recent work disfavors this idea, as furin cleavage still occurs despite ppi inhibition of rg- exposure [ ] . thus, while rg- staining is a convenient marker for an l conformational change that is both furin-and cyclophilin-dependent, it is not a direct readout for furin proteolysis of l , as cleavage can occur without rg- exposure. shortly after entry, early trafficking of hpv is modulated by the tetraspanin cd and its partners syntenin/alix [ ] . these molecules are necessary for sorting of hpv virions from early endosomes (ee) into acidic late endosome (le) and multivesicular bodies (mvbs), a prerequisite for capsid disassembly, uncoating, and segregation of l /vdna from l ( figure ). when mvb trafficking of hpv was interrupted by knockdown of either cd or syntenin, subcellular transport cleavage of l by the host protease furin occurs on the surface of host cells and on the extracellular matrix (ecm) in response to binding of the virion to hspgs [ ] . this cleavage occurs c-terminal to the final arginine residue of a conserved consensus site (rtkr, residues - for hpv ), removing twelve n-terminal residues of hpv l [ ] (figure ). the molecular basis for the requirement of this cleavage remains unknown, but inhibition of cleavage through mutation of the cleavage site or by biochemical inhibition of furin results in aberrant trafficking of the l /vdna complex and potent abrogation of infection [ ] . cleavage appears to trigger a conformational change in capsid and/or l structure, as the conserved and neutralizing rg- epitope (residues - for hpv [ ] ) becomes accessible to antibody staining shortly after virion binding in a furin-dependent manner [ ] . cell surface cyclophilins (peptidyl-prolyl isomerases, ppis) also appear to modulate the conformation of l , as rg- epitope exposure is sensitive to cyclosporine a, a broad ppi inhibitor [ ] . rg- epitope exposure was initially believed to be a convenient marker for furin cleavage, and cyclophilins were believed to control l accessibility and susceptibility to furin, but recent work disfavors this idea, as furin cleavage still occurs despite ppi inhibition of rg- exposure [ ] . thus, while rg- staining is a convenient marker for an l conformational change that is both furin-and cyclophilin-dependent, it is not a direct readout for furin proteolysis of l , as cleavage can occur without rg- exposure. shortly after entry, early trafficking of hpv is modulated by the tetraspanin cd and its partners syntenin/alix [ ] . these molecules are necessary for sorting of hpv virions from early endosomes (ee) into acidic late endosome (le) and multivesicular bodies (mvbs), a prerequisite for capsid disassembly, uncoating, and segregation of l /vdna from l ( figure ). when mvb trafficking of hpv was interrupted by knockdown of either cd or syntenin, subcellular transport of l /vdna viruses , , of was altered and infection was partially blocked, demonstrating a requirement for virion transport into mvbs [ ] . accordingly, components of the escrt machinery, a group of cytosolic multisubunit complexes that facilitate endosomal maturation and mvb biogenesis, are involved in efficient hpv infection [ , ] . endosomal acidification is a strict requirement for hpv infection [ , , ] , but it remains unclear if it is simply needed for proper endosomal maturation and mvb biogenesis, or if acidification itself also triggers conformational changes in the hpv capsid or host proteins that are required for downstream processes like capsid uncoating and vdna trafficking. acid-dependent cathepsin proteases further cleave and process the l capsid within the endolysosomal compartments. capsid proteolysis and disassembly can be visualized by immunofluorescence with the monoclonal antibody l - [ ] , specific for l residues - , located in central cavities underneath each of the l pentamers. this region is only available for binding to l - after capsid disassembly; around h post infection [ ] . while useful for marking capsid disassembly, l - reactivity does not reveal true infectious uncoating, as staining is blocked by cathepsin inhibitors with no effect on infectivity [ ] . within le/mvbs the l /vdna complex segregates away from the partially degraded l capsid in a cyclophilin-dependent manner [ ] . the complex then traffics to the trans-golgi network (tgn) in a retromer-dependent manner [ , , ] , where it resides until the onset of mitosis [ ] [ ] [ ] . recent work has shown that a fraction of the l capsid, in the form of conformationally intact pentamers, accompanies the l /vdna complex to the tgn and nucleus, but a functional role for these l pentamers remains unclear [ ] . colocalization of l /vdna with tgn markers is well established, but several groups have reported transport of vdna to more distal retrograde sites. partial colocalization of vdna with cis/medial-golgi markers like gm and giantin has been reported [ , ] . likewise, sensitive techniques like the proximity ligation assay [ ] have suggested that l /vdna retrograde traffics past the golgi to the er [ ] . whether these represent primary or alternative routes of infection, or even unproductive dead ends, is not clear. it is worth noting that the dynamic flux of proteins within the secretory compartments makes it difficult to precisely determine where colocalization is occurring by microscopy. many er proteins contain a c-terminal kdel sequence, and although they are maintained within the er at steady state, they are constantly trafficking into the golgi, where they must be recycled back through kdel cargo receptor [ , ] . sensitive techniques like the proximity ligation assay using such kdel-containing er proteins must therefore be interpreted with caution. since retrograde trafficking of l /vdna to more distal compartments has not been well established, this review will simply refer to the final retrograde destination of l /vdna as the "tgn". the retromer, a trimer of vps , vps , and vps , is a cytosolic sorting adaptor complex that binds to peptide motifs within the intracellular domains and cytosolic tails of membrane-bound receptors destined for the tgn. retromer works in concert with molecules like rab b, rab a, and members of the sorting nexin family, including snx , snx , and the bar-domain sorting nexins (snx-bar), to sort cargos from a variety of endosomal compartments to the tgn [ , ] . l contains conserved hydrophobic retromer-binding sites near the c-terminus (fyl at residues - and yyml at residues - for hpv , see figure ). mutation of these sites prevents association of l with retromer, and blocks the trafficking of l /vdna to the tgn, instead causing an accumulation within eea -positive endocytic compartments [ ] suggesting a retromer-dependent sorting event away from ee compartments ( figure ). likewise, sirna knockdown of retromer components also prevents l /vdna from reaching the tgn [ ] . in addition to retromer, l is capable of interaction with snx and snx to direct endosomal and retrograde trafficking of the vdna [ , ] . the interaction with snx through a conserved motif (npxy, residues - for hpv , see figure ) is believed to occur very early after entry. one recent study observed recruitment of snx to hpv positive endosomes by h post infection, a phenotype that was dependent on the conserved npxy motif within l [ ] . the snx -l interaction likely promotes retention/recycling viruses , , of of the l /vdna complex within the endosomal compartment, preventing the rapid trafficking and degradation of l /vdna within lysosomal compartments [ ] . perhaps the virion requires a relatively long retention time in moderately acidic ee and le/mvb environments for efficient uncoating, partitioning of the l /vdna subviral complex from l , and/or recruitment of the retromer? mutation of the npxy motif or knockdown of snx results in aberrant l /vdna trafficking and decreased infectivity [ ] . snx uses its ferm domain to bind to cargo harboring the npxy motif. snx is another ferm domain-containing snx involved in l /vdna trafficking but, unlike snx , snx does not interact with l through the conserved npxy motif. in addition to a ferm domain, snx also contains a pdz domain, which mediates interaction with l through a non-canonical pdz ligand located somewhere in between residues - of hpv l [ ] . notably, both snx and snx have been implicated in efficient retrograde trafficking through the retromer [ , ] , raising the possibility that cooperative interactions of l with these snxs may somehow promote retromer-dependent tgn localization. recent work has revealed the existence of an additional trimeric sorting complex called the retriever, which functions in concert with snx , the ccc, and wash complexes to sort cargo from degradative to recycling compartments. retriever consists of three subunits-dscr , c orf , and vps , a subunit in common with the retromer [ ] . hpv infection is decreased upon knockdown of retriever components dscr and c orf , as well as ccc components ccdc and ccdc , suggesting a role for this novel pathway in hpv infection [ ] . escrt proteins, tetraspanins, snxs, and retromer all have physiological roles in subcellular trafficking and protein transport, and thus many of these natural pathways and components are exploited and commandeered by different viruses for entry or assembly. perhaps the most mysterious host factor necessary for hpv infection is the multisubunit intramembrane protease γ-secretase (γ-sec), which appears to be a unique requirement of papillomaviruses. the γ-sec complex is a transmembrane protease comprised of four subunits: presenilin / (ps / ), nicastrin (nic), aph a/b, and pen . two isoforms exist for both ps and aph , so there is heterogeneity among cellular γ-sec complexes [ ] . γ-sec catalyzes the intramembrane cleavage of tmds from a wide variety of membrane proteins [ ] , and is perhaps best known as an important component of the notch signaling pathway and the biogenesis of aβ peptides from amyloid precursor protein (app) [ ] [ ] [ ] [ ] . biochemical inhibition of γ-sec or knockdown of any of the four subunits results in a potent block of hpv infection [ , ] . in a screen of a diverse panel of different mucosal and cutaneous hpv types, sensitivity to γ-sec inhibition was the most conserved feature among the alpha and beta hpv types tested, even higher than sensitivity to furin inhibition [ ] . the molecular basis for the γ-sec requirement is unknown, but inhibition of γ-sec activity results in a failure of l /vdna to reach the tgn, even though l appears to exit eea positive endosomal compartments [ ] (figure ). this is in contrast to retromer knockdown, which causes an accumulation of vdna within eea endosomes [ ] . this may suggest that, in the absence of γ-sec activity, l /vdna never exits the mvb/le compartments, and instead continues to lysosomes for degradation; or that γ-sec controls trafficking of l /vdna to a discrete intermediate compartment, between the mvb and the tgn. consistent with the observed effects on l /vdna trafficking, hpv is only sensitive to γ-sec inhibition during the first - h of infection [ ] , and γ-sec inhibition has no effect on post-tgn trafficking of vdna [ ] . failure of l /vdna to reach the tgn in the absence of γ-sec activity means that the retrograde trafficking pathway utilized by hpv involves more than just the canonical players like retromer and snxs, suggesting that new γ-sec-dependent retrograde pathways may exist and are being exploited by papillomaviruses. the catalytic ps / subunit of γ-sec are known to modulate protein trafficking, lysosomal maturation, and ca + homeostasis independently of γ-sec-activity, but direct connections to retrograde trafficking pathways are scant [ ] . retromer has, however, been implicated in retrograde-dependent trafficking and cleavage of gamma secretase substrates, including app [ , ] . how does l , the minor capsid protein from a non-enveloped virus, complexed with the vdna within the lumen of intracellular vesicular compartments, interact with a variety of cytosolic sorting molecules to direct its own transport to the tgn? evidence from multiple laboratories suggests that l can interact with and span across vesicular membranes, thereby allowing l to gain access to the cytosol, to recruit cytosolic factors necessary for retrograde trafficking (figure ). post-tgn transit, full translocation across the limiting membrane, and nuclear accumulation of the l /vdna complex requires mitosis and is summarized in greater detail in latter sections of this review. for the sake of consistency within the field, the following nomenclature is proposed for the -step process describing l 's remarkable ability to shift from being a soluble protein to a transmembrane protein and back again: ( ) insertion-within the lumen, part(s) of l insert(s) into the local membrane. how does the l protein initially interact with and insert into membranes? when naturally or ectopically expressed, l is a soluble nuclear protein, not a membrane protein [ , ] . yet, during infection, l must somehow interact with and cross membranes; how is this accomplished? in , a conserved "membrane destabilizing peptide" near the c-terminus of l (syymlrkrrkrlpy, residues - for hpv , see figure ) was identified as having a role in the endosomal escape of l /vdna via membrane disruption or destabilization [ ] . using synthetic c-terminal peptides from hpv l , containing the corresponding membrane destabilization moiety (syfilrrrrkrfpyfftdvrvaa, residues - ), in vitro cytotoxicity and propidium iodide uptake experiments showed a ph-dependent ability to disrupt cellular membranes. while it is possible that this c-terminal region aids in membrane insertion of l within the acidic endosomes, it is important to note that a direct role in l membrane insertion has yet to be demonstrated. regardless of how l initially inserts into membranes during infection, multiple groups have published indirect evidence that l interacts with cytosolic factors and thus must protrude into the cytosol across vesicular membranes. in , my laboratory identified a glycine-rich transmembrane-like domain (tmd) towards the n-terminus of l (ilqygsmgvffgglgigtgsgtg, residues - of hpv , see figure ) [ ] . taking advantage of tmd-flanking monoclonal antibody epitopes and using elegant immunofluorescence staining procedures, the sapp laboratory has since demonstrated that l utilizes this tmd to span intracellular vesicular membranes with residues c-terminal of the tmd being cytosolic, consistent with a type-i transmembrane topology [ ] . moreover, additional data from the sapp laboratory suggest the vdna remains lumenal within these intracellular vesicles [ ] . exactly how l is able to insert into the membrane and span across remains unknown, but endosomal acidification seems to be required to adopt this conformation [ ] , although it remains to be determined if this may simply reflect a requirement for l capsid disassembly or l /l partitioning, rather than low ph having a direct effect on l protein structure or conformation. thus, virion-associated l appears to be an inducible transmembrane protein, with the ability to insert into membranes and adopt a transmembrane configuration, to drive vdna subcellular trafficking by physically linking the lumenal vdna to host cytosolic sorting proteins (figure ). l 's function to facilitate vdna delivery across the limiting membrane is analogous to that of many bacterial toxins, which penetrate intracellular membranes and deliver toxin domains to the cytosol. many of these bacterial toxins including diphtheria toxin, anthrax toxin protective antigen, shiga toxin, and pseudomonas exotoxin a [ , ] also rely on proteolytic activation by furin and other proteases to trigger conformational changes and structural rearrangements that underlie toxin membrane insertion and penetration. given the requirement for furin in tgn localization of l /vdna, it is very likely that cleavage triggers a structural change that enables l to insert and protrude into the local membrane via the tmd to recruit cytosolic snxs and retromer. until structural data on l is obtained, the nature of any cleavage-induced conformational changes will remain elusive. although direct evidence is lacking, the tmd itself may play a role in the initial insertion of l into membranes. it is noteworthy that the l tmd is quite similar to the fusion peptides (fps) from many type-i fusogenic glycoproteins of enveloped viruses of the orthomyxoviridae, paramyxoviridae, and retroviridae families [ ] . these fusion peptides generally consist of~ apolar residues, and are typically enriched for glycine, a composition believed to impart conformational flexibility. the structurally dynamic nature of these fusion peptides is thought to be critical for their ability to partition into and destabilize local membranes [ ] [ ] [ ] [ ] . it is also noteworthy that, like l , these viral fusion proteins require "priming" by proteolytic cleavage and "activation" by environmental cues like low ph or receptor binding [ ] . it is conceptually challenging to envision how l could achieve a type-i transmembrane state upon insertion of its n-terminal tmd-l would have to essentially drag residues c-terminal to the tmd across the membrane. perhaps cooperative interactions between the tmd, the c-terminal membrane disruption peptide, and the membrane are required for insertion and protrusion of l . how does γ-sec facilitate l /vdna trafficking? no interaction between l and the γ-sec complex has been reported, although it is tempting to envision that l could interact with the complex via its tmd while protruding through the membrane. alternatively, γ-sec activity could somehow be required for l to initially insert into membranes to achieve membrane protrusion. this latter possibility would be consistent with the trafficking defects observed upon inhibition of gamma secretase [ ] , as failure to insert and protrude through the membrane would be expected to block tgn localization, and may instead cause trafficking away from eea positive early endosomes into a degradative lysosomal pathway. although no evidence exists supporting a role for γ-sec in hpv endocytosis, it has been found as part of a "tetraspanin interactome", associated with many of the same molecules believed to be part of the initial entry receptor complex, including tetraspanins cd and cd , integrins α and β , and annexin-a [ ] . the γ-sec complex may therefore be present locally during uncoating when l presumably adopts the protruding conformation ( figure ). in this scenario, one could also imagine l actually being a substrate for γ-sec cleavage, triggering a conformational change of some kind upon cleavage of the l tmd. while attractive, there is no published evidence supporting this notion. this begs the question-if l is not a substrate for γ-sec then how does inhibition of γ-sec catalysis affect l /vdna trafficking so drastically? perhaps γ-sec cleavage of a cellular protein somehow modulates trafficking, or l may actually be a "pseudosubstrate", interacting with γ-sec without cleavage. tmd substrates of γ-sec are believed to first dock into a substrate binding site prior to transfer to the catalytic active site for proteolysis [ ] . γ-sec inhibitors perturb the global structure of the γ-sec complex and may therefore prevent initial substrate docking [ , ] . as discussed above, many host proteins and pathways are expoited by hpv to facilitate virion trafficking and viral infection, but some proteins can restrict hpv infection suggesting an inherent anti-papillomaviral function. the endosomal protein stannin restricts hpv infection by rerouting virions away from the tgn to degradative compartments [ ] . similarly, the α-defensin hd alters l /vdna trafficking, accelerating the degradation of virions within le/lysosomal compartments to restrict hpv infection [ ] . stannin appears to work by blocking association of l with retromer, to prevent retrograde trafficking of l /vdna, causing an increased accumulation of l /vdna in lamp -positive lysosomal compartments [ ] . this is in contrast to retromer knockdown or mutation of retromer binding sites in l , which instead cause a trafficking block within eea -positive endosomal compartments [ ] . rather than directly inhibiting the l -retromer association, stannin likely blocks the insertion and protrusion of l within vesicular membranes to indirectly prevent binding and recruitment of retromer. similarly, hd may induce aberrant trafficking of l /vdna by directly binding the virion to interfere with l insertion and protrusion [ ] . interestingly, interferon gamma has recently been found to restrict hpv infection in an l -dependent and type-specific manner, decreasing the proteolytic degradation of l capsid and causing a block of l /vdna in le/mvb/lysosomal compartments [ ] . cathepsin proteases also appear to limit hpv, as infection is increased upon genetic knockout, sirna silencing, or biochemical inhibition [ , ] ; this is contrary to other non-enveloped viruses, like reoviruses and adeno-associated viruses, which depend on these endosomal proteases for uncoating [ , ] . vimentin is another recently reported inhibitory factor, found to limit infection at the level of viral entry [ ] . much will be gained from further mechanistic studies of these inhibitory host factors. prior to , the consensus view was that the l /vdna complex egressed from endosomal compartments into the cytosol, as is the case for many other non-enveloped viruses [ ] [ ] [ ] . thus, discovery of the tgn as an important stop in the retrograde route of incoming l /vdna was a major advance in the field [ , ] . initially, the l /vdna was believed to penetrate the tgn directly and wait in the cytosol prior to nuclear entry. cell cycle progression into mitosis is known to be important for hpv infection [ ] , and nuclear envelope breakdown was thought to facilitate l /vdna transfer from the cytosol into the nucleus like some retroviruses [ , ] . recent work supports a model where, at the onset of mitosis, when the golgi and tgn naturally begins to fragment and vesiculate, the vesicle-bound vdna egresses from what was the interphase tgn ( figure ). the sapp laboratory pioneered an edu/vdna staining technique based on selective membrane permeabilization and sequential edu labeling to demonstrate the lumenal state of the vdna [ ] . while the exact nature of these vesicles remains unknown, immunofluorescence microscopy reveals that these vdna-containing vesicles stain negative for classical tgn markers like tgn and p , and appear to migrate along microtubules, clustering around the centrioles during progression from g /m to prometaphase [ , ] . l likely remains in the protruding conformation, spanning across the limiting membrane to coordinate microtubule-dependent traffic of these vdna-containing vesicles along the mitotic spindle [ ] . these vesicles eventually make their way to the condensed chromosomes, and by metaphase, vdna can be seen to be associated with and presumably bound to the host chromosomes [ , , ] (figure ). from there, the chromosome-bound vdna is partitioned into daughter cells. in this manner, infection of both daughter cells is favored at an moi > . when does the l /vdna complex fully translocate across the limiting membrane? while useful for observing the trafficking of vdna during hpv infection, standard subcellular localization of edu-labeled vdna by microscopy is insufficient to reveal the actual translocation event. using their specialized immunofluorescence protocol for sequential fluorophore-azide conjugation of edu-labeled vdna in differentially permeabilized cells, the sapp laboratory concluded that vdna became cytosolic sometime during g , well after mitosis [ ] . in this model, l /vdna would reside within these unique mitotic transport vesicles, bound to chromosomes for an extended period of time (figure ) , until translocation occurred sometime in g after completion of mitosis. to better understand translocation, my laboratory has developed an alternative platform to detect and measure this elusive process. our system is based on the biotin-protein ligase bira, from escherichia coli. the bira enzyme will covalently attach a biotin molecule to a specific lysine residue of a short peptide substrate, termed the biotin acceptor peptide (bap). the bap is a specific substrate for bacterial bira, and is not recognized by mammalian biotin-protein ligases [ , ] . by generating hpv pseudoviruses that encapsidate a functional l -bira fusion and a hacat keratinocyte line that stably expresses a cytosolic gfp-bap fusion, we have set up a two-component compartmentalization assay to detect l -bira translocation. in this system, lumenal l -bira is separated from the cytosolic gfp-bap substrate by limiting membranes. only upon translocation of l will bira encounter the bap; thus, biotinylation of gfp-bap is a readout for translocation. however, it should be noted that, using this system, we found that l translocation required tgn localization of l /vdna and cell cycle progression past g /m. timecourse experiments with synchronized hacat-gfp-bap cells demonstrated that the earliest biotinylated gfp-bap signal was detected at or just prior to the onset of mitosis. although these experiments were performed with a bulk population, we believe it suggests that l translocation (or at least of the c-terminus of l ) begins during mitosis (figure ) , well before transition into g . moreover, this timing of l /vdna translocation would be consistent with the visual "jump" of vdna from a punctate pericentriolar distribution in prometaphase to being chromosome-bound by metaphase [ ] . the chromosome binding ability of l was first reported in [ ] , and since then, the schelhaas group has mapped a minimal chromatin binding region (cbr, residues - for hpv ) within l [ ] . interestingly, the ability of ectopically expressed l -gfp fusion to associate with mitotic chromatin was found to require cell cycle progression into prometaphase. this finding is suggestive that either the interaction between l and mitotic chromatin is indirect, requiring a prometaphase-specific factor, or that l is post-translationally modified during prometaphase to somehow activate its chromatin binding ability. substitution of specific residues (ival; - , r / , and rtr; - ) were found to completely abrogate the chromatin binding activity while mutation of rr / resulted in a partial-inhibition cbr function. when these same residues were mutated in reporter-expressing psv, packaged with either l or l -bira, the same phenotypes were upon entry into mitosis, l /vdna remains vesicle-bound but loses coincidence with tgn markers. these l /vdna-containing vesicles likely travel along astral microtubules in the minus-end direction towards the centrosome, where they accumulate during prometaphase. the vesicles likely switch polarity and travel along the spindle microtubules in the plus-end direction to reach the host chromosomes by metaphase. chromosome-bound l /vdna partitions with host chromosomes, eventually localizing to pml bodies of the daughter cells. chromosome binding of l /vdna is through the chromatin binding region (cbr) of l and mutation of this region causes a block in translocation, with vesicular l /vdna becoming reabsorbed back into the nascent golgi after mitosis. chromosome-bound l /vdna may be in a membrane-bound vesiclular state, or may have penetrated the limiting membrane upon chromosome binding, further work is needed to clarify this stage of the hpv life cycle. using this system, we found that l translocation required tgn localization of l /vdna and cell cycle progression past g /m. timecourse experiments with synchronized hacat-gfp-bap cells demonstrated that the earliest biotinylated gfp-bap signal was detected at or just prior to the onset of mitosis. although these experiments were performed with a bulk population, we believe it suggests that l translocation (or at least of the c-terminus of l ) begins during mitosis (figure ) , well before transition into g . moreover, this timing of l /vdna translocation would be consistent with the visual "jump" of vdna from a punctate pericentriolar distribution in prometaphase to being chromosome-bound by metaphase [ ] . the chromosome binding ability of l was first reported in [ ] , and since then, the schelhaas group has mapped a minimal chromatin binding region (cbr, residues - for hpv ) within l [ ] . interestingly, the ability of ectopically expressed l -gfp fusion to associate with mitotic chromatin was found to require cell cycle progression into prometaphase. this finding is suggestive that either the interaction between l and mitotic chromatin is indirect, requiring a prometaphase-specific factor, or that l is post-translationally modified during prometaphase to somehow activate its chromatin binding ability. substitution of specific residues (ival; - , r / , and rtr; - ) were found to completely abrogate the chromatin binding activity while mutation of rr / resulted in a partial-inhibition cbr function. when these same residues were mutated in reporter-expressing psv, packaged with either l or l -bira, the same phenotypes were observed-infectivity and translocation were completely blocked for ival; - , r / , and rtr; - and partially blocked for rr / [ , ] . the striking correlation between the ability of l to bind chromatin and to translocate during infection supports a model whereby chromatin binding is required for l translocation [ , ] . a mechanistic linkage between these processes favors a model where translocation of l /vdna out of the mitotic vesicles occurs while these compartments encounter mitotic chromosomes during prometaphase, and is supported by appearance of translocation signal in timecourse experiments [ ] . while further work is needed to validate one model over another, it should be noted that the two models do not have to be mutually exclusive. translocation studies based on l -bira may in fact only be revealing exposure of the l c-terminus, and full translocation of vdna could be occurring at a later time post-mitosis, as suggested by the sapp laboratory. alternatively, the nature of these vesicles is unknown, and if their lipid composition and detergent solubility changes during mitosis, it could affect sequential labeling efficiency or edu-labeled vdna availability to fluorophore-azides after differential detergent permeabilization. as mentioned above, immunofluorescence staining with mabs specific for l epitopes flanking the tmd suggest a type-i topology for l protrusion, with the n-terminal~ residues being lumenal, a~ residue tmd, and the c-terminal~ residues being cytosolic [ ] (figure ). this topology is in agreement with the placement of known snx and retromer binding motifs within l , as well as the newly defined cbr (figure ) . however, translocation studies with psv encapsidating the l -bira fusion are suggestive of a different topology for l . hpv infection in the presence of s-phase blockers like aphidicolin traps incoming l /vdna at the tgn, likely in the protruding conformation [ ] . this block is reversible, as removal of the drug releases cell cycle inhibition and enables synchronized egress and translocation of l /vdna out of the tgn upon entry into mitosis [ ] . this data, however, is not in agreement with a strict type-i membrane topology for l . the lack of translocation signal in the presence of aphidicolin implies that the c-terminus of l -bira is not cytosolic, as it would be in a type-i topology. rather, it suggests that bira is either lumenal or is somehow obstructed from engaging the gfp-bap substrate when l is protruding from endosomal and tgn compartments, only becoming accessible to the cytosol upon entry into mitosis. a double-pass topology of protruding l -bira would result in a lumenal c-terminal bira (figure ) . it should be noted that the c-terminal membrane destabilization peptide bears no resemblance to a conventional tmd, and no other membrane-spanning regions of l have been identified, so it is unclear how l could span the membrane twice to keep the c-terminal bira fusion lumenal. membrane-spanning bacterial toxins, including anthrax toxin, diphtheria toxin, botulinum toxin, tetanus toxin, and clostridium difficile toxins, form pores through which they can extrude themselves into the cytosol by a variety of protein translocation mechanisms [ , ] . oligomerization of individual l molecules, each with a single membrane-spanning tmd, could theoretically enable a double-pass topology by extrusion of the l c-termini back into the lumen through such a pore. this hypothetical configuration would place a central portion of l within the cytosol to recruit sorting factors and direct traffic of the associated vdna ( figure ). it should also be noted that such a double-pass topology would still be consistent with the immunofluorescence data supporting a type-i topology [ ] . clearly, much more work is needed to understand the protruding conformation of l during hpv infection. into mitosis [ ] . this data, however, is not in agreement with a strict type-i membrane topology for l . the lack of translocation signal in the presence of aphidicolin implies that the c-terminus of l -bira is not cytosolic, as it would be in a type-i topology. rather, it suggests that bira is either lumenal or is somehow obstructed from engaging the gfp-bap substrate when l is protruding from endosomal and tgn compartments, only becoming accessible to the cytosol upon entry into mitosis. a double-pass topology of protruding l -bira would result in a lumenal c-terminal bira (figure ). immunofluorescence and trypsin susceptibility data [ ] . (a) in the type-i model, the n-terminus remains lumenal with all ~ residues downstream of the tmd being cytosolic to recruit sorting factors. l -bira would be expected to biotinylate substrate in this model, contradicting the actual data [ ] . (b) in the double-pass model, both the n-and c-termini would be lumenal, with the bulk of l being cytosolic. l -bira would not be expected to biotinylate substrate as observed. however, the means by which l spans the membrane a second time is difficult to conceptualize, as the protein only has one tmd towards the n-terminus [ ] . in vitro data suggest both the n-and c-termini are capable of non-speciific dsdna binding through electrostatic interactions [ , ] . immunofluorescence and trypsin susceptibility data [ ] . (a) in the type-i model, the n-terminus remains lumenal with all~ residues downstream of the tmd being cytosolic to recruit sorting factors. l -bira would be expected to biotinylate substrate in this model, contradicting the actual data [ ] . (b) in the double-pass model, both the n-and c-termini would be lumenal, with the bulk of l being cytosolic. l -bira would not be expected to biotinylate substrate as observed. however, the means by which l spans the membrane a second time is difficult to conceptualize, as the protein only has one tmd towards the n-terminus [ ] . in vitro data suggest both the n-and c-termini are capable of non-speciific dsdna binding through electrostatic interactions [ , ] . regardless of the precise mechanisms of l translocation, the minor capsid protein eventually leaves vesicular compartments and is seen along with vdna within interphase nuclei of infected cells, localized to punctate nuclear foci called promyelocytic leukemia (pml) nuclear domains, also known as pml oncogenic domains (pods), or nd bodies [ ] . pml bodies are small nuclear structures, organized by the pml protein for which they are named. these dynamic domains are present in most cells, and are assembled and remodeled in response to a variety of cellular stresses, including infection, innate immune triggers/interferon (ifn), heat shock, dna damage pathways, and metabolic stress [ ] [ ] [ ] . pml bodies modulate a wide variety of cellular responses via recruitment, retention, and modification of numerous proteins including the transcriptional repressor daxx, tumor suppressor sp , transcriptional regulator atrx, dna helicase blm, kinase hipk , and a multitude of other host proteins. the pml protein, which has many different isoforms, is critical to the assembly of pml bodies and recruitment of host proteins to these foci [ ] . many pml-associated proteins are either directly conjugated to small ubiquitin-like modifier (sumo) proteins or contain short linear sumo-interaction motifs (sims), or both. in addition to pml oligomerization, sumoylation and sumo-sim networks are believed to be important to pml assembly and dynamics [ ] . given the role of pml bodies in innate antiviral responses, many viruses have been shown to target pml bodies or induce degradation or remodeling of specific pml components [ , ] . pml bodies have been shown to be important for efficient infection from reporter-expressing hpv pseudoviruses, as well as authentic bpv virions [ ] , suggesting that the vdna is actively targeted to these sites by l upon infection. ectopically expressed l can localize to pml bodies, remodeling them through recruitment of daxx and depletion of sp [ ] . in older studies, the ability of gfp-l fusions to localize and induce remodeling of pml bodies was mapped to a c-terminal region of l (residues - for hpv ) [ ] . l can itself be sumoylated at a conserved lysine residue (k for hpv ) when ectopically expressed, but recent work suggests this modification is not important for pml body localization [ , ] . rather, a moderately conserved sim (dival, residues - for hpv , see figure ) has been implicated in pml localization of ectopically expressed untagged full length, l [ ] . precise mechanisms of l -dependent pml body remodeling have yet to be worked out but ectopic overexpression studies must be interpreted with caution since the mode of l gene transfection/delivery has been shown to heavily influence nuclear/pml localization of l [ ] . recent work suggests that the pml component sp restricts hpv transcription and vdna replication [ ] , favoring a model whereby incoming l might promote a nuclear environment conducive for early hpv gene transcription and genome maintenance in basal cells. during cell division, pml bodies show increased dynamics and disperse into the cytosol during open mitosis. only after exit from mitosis and reformation of the nuclear envelope are pml bodies assembled de novo, and recruitment of daxx and sp is observed [ ] . whether l recruits pml to nucleate de novo assembly of pml bodies in the vicinity of the vdna after mitosis or whether the l /vdna complex is targeted to newly formed pml bodies in early g remains to be determined. likewise, much remains to be discovered regarding preferential remodeling of pml components like daxx and sp immediately after mitotic translocation of l /vdna, and the consequences of this remodeling for infection, immune evasion, and viral persistence. in addition to role(s) in vdna packaging, virion assembly, and particle stability [ ] , minor capsid protein l is tasked with ensuring nuclear delivery of the vdna during hpv infection. this feat is accomplished via some remarkable means for a viral capsid protein present in low copy numbers. l is able to partition vdna away from degradative endolysosomal compartments, instead diverting it to the tgn. l does this by possessing properties of an "inducible transmembrane" protein, with the ability to insert into and protrude across local vesicular membranes using a transmembrane-like domain. portions of l containing conserved sorting motifs are exposed to the cytosol, recruiting cellular sorting factors that dictate retrograde trafficking of l /vdna to the tgn. upon entry into mitosis, the vesicular l /vdna complex separates from the dispersed golgi towards the pericentriolar region and by metaphase the vdna can be seen to be associated with condensed chromosomes. whether the visual association of vdna with mitotic chromosomes represents full translocation of l /vdna across limiting membranes, or simply vdna-filled post-golgi vesicles bound to chromosomes, remains to be shown. together with recent work on the chromatin-binding abilities of l , translocation studies using a novel bira-based approach suggest that chromatin binding is necessary for translocation. timecourse experiments with synchronized cells suggest that translocation is concurrent with or slightly after the onset of mitosis. in contrast, sequential fluor-azide conjugation of edu-labeled vdna after differential detergent permeabilization suggests that translocation, as defined by the liberation of vdna from membrane-bound compartments, occurs post-mitosis in g . regardless of the specific mechanisms and timing of translocation, l /vdna localizes to pml bodies of the daughter cells and likely functions to promote efficient viral gene expression. additional efforts are needed to further define the mechanisms of l 's remarkable abilities. structural studies are needed to understand the nature of the l /vdna complex within viral particles, the molecular basis underlying the requirement for furin, the consequences of cleavage, and the mechanisms of l -membrane interaction. further work is needed to understand and define the nature of l protrusion through limiting membranes, specifically the topology of l within these membranes, and to identity any host proteins that may be necessary for l -membrane insertion and protrusion. more work is necessary to identify the nature of the post-golgi vesicles in which l resides upon entry into mitosis, and again to define the host proteins that may be interacting with l to enable subcellular transport of these compartments and eventual translocation. elucidation of the timing and mechanisms of actual l /vdna translocation will require new approaches. finally, the precise role(s) of virion-derived l in the remodeling of pml bodies, establishment of infection, initial viral gene expression, and potential immunoevasion all represent exciting avenues of future endeavors. deep sequencing extends the diversity of human papillomaviruses in human skin evolution of the papillomaviridae the papillomavirus episteme: a major update to the papillomavirus sequence database the biology and life-cycle of human papillomaviruses global burden of human papillomavirus and related diseases carcinogenic human papillomavirus infection arrangement of l within the papillomavirus capsid antibody competition reveals surface location of hpv l minor capsid protein residues - cross-neutralization of cutaneous and mucosal papillomavirus types with anti-sera to the amino terminus of l neutralization of hpv , , , and pseudovirions with antisera induced by immunizing rabbits with synthetic peptides representing segments of the hpv minor capsid protein l surface region l , the minor capsid protein of papillomavirus the l minor capsid protein of low-risk human papillomavirus type interacts with host nuclear import receptors and viral dna the positively charged termini of l minor capsid protein required for bovine papillomavirus infection function separately in nuclear import and dna binding nuclear import strategies of high-risk hpv l minor capsid protein interaction of human papillomavirus (hpv) type capsid proteins with hpv dna requires an intact l n-terminal sequence production of papillomavirus-based gene transfer vectors papillomavirus infectious pathways: a comparison of systems hpv entry into cells concepts of papillomavirus entry into host cells cruising the cellular highways: how human papillomavirus travels from the surface to the nucleus cleavage of the papillomavirus minor capsid protein, l , at a furin consensus site is necessary for infection kallikrein- proteolytically processes human papillomaviruses in the extracellular space to facilitate entry into host cells identification of a role for the trans-golgi network in human papillomavirus pseudovirus infection entry of human papillomavirus type by actin-dependent, clathrin-and lipid raft-independent endocytosis hpv infection of hacats is dependent on β integrin, and α integrin processing annexin a and s a regulate human papillomavirus type entry and intracellular trafficking in human keratinocytes tetraspanin cd mediates papillomavirus type endocytosis clathrin-and caveolin-independent entry of human papillomavirus type -involvement of tetraspanin-enriched microdomains (tems) human papillomavirus types , , and share similar endocytic requirements for entry essential roles for soluble virion-associated heparan sulfonated proteoglycans and growth factors in human papillomavirus infections the s a subunit of the annexin a heterotetramer facilitates l -mediated human papillomavirus infection the cytoskeletal adaptor obscurin-like interacts with the human papillomavirus (hpv ) capsid protein l and is required for hpv endocytosis the tetraspanin cd facilitates mers-coronavirus entry by scaffolding host cell receptors and proteases cd and hepatitis c virus (hcv) infection. viruses the role of furin in papillomavirus infection furin cleavage of l during papillomavirus infection: minimal dependence on cyclophilins a protective and broadly cross-neutralizing epitope of human papillomavirus l mechanisms of human papillomavirus type neutralization by l cross-neutralizing and l type-specific antibodies target cell cyclophilins facilitate human papillomavirus type infection the cd -syntenin- complex controls post-endocytic trafficking of oncogenic human papillomaviruses human papillomavirus infection requires the tsg component of the escrt machinery the vps component of the escrt machinery plays an essential role in hpv infectious entry and capsid disassembly inhibition by cellular vacuolar atpase impairs human papillomavirus uncoating and infection caveolin- -dependent infectious entry of human papillomavirus type in human keratinocytes proceeds to the endosomal pathway for ph-dependent uncoating analysis of type-restricted and cross-reactive epitopes on virus-like particles of human papillomavirus type and in infected tissues using monoclonal antibodies to the major capsid protein cyclophilins facilitate dissociation of the human papillomavirus type capsid protein l from the l /dna complex following virus entry genome-wide sirna screen identifies the retromer as a cellular entry factor for human papillomavirus direct binding of retromer to human papillomavirus type minor capsid protein l mediates endosome exit during viral infection a central region in the minor capsid protein of papillomaviruses facilitates viral genome tethering and membrane penetration for mitotic nuclear entry large scale rnai reveals the requirement of nuclear envelope breakdown for nuclear import of human papillomaviruses translocation of the papillomavirus l /vdna complex across the limiting membrane requires the onset of mitosis human papillomavirus major capsid protein l remains associated with the incoming viral genome throughout the entry process identification of trappc as a host factor required for human papillomavirus cell entry application of the proximity-dependent assay and fluorescence imaging approaches to study viral entry pathways vesicular trafficking of incoming human papillomavirus to the golgi apparatus and endoplasmic reticulum requires γ-secretase activity protein sorting at the er-golgi interface golgi enzymes do not cycle through the endoplasmic reticulum during protein secretion or mitosis retromer: a master conductor of endosome sorting human papillomavirus l facilitates viral escape from late endosomes via sorting nexin a novel pdz domain interaction mediates the binding between human papillomavirus l and sorting nexin and modulates virion trafficking characterizing the spatio-temporal role of sorting nexin in human papillomavirus trafficking snx regulates notch pathway and pancreas development through the retromer-dependent recycling of jag retriever is a multiprotein complex for retromer-independent endosomal cargo recycling presenilins and γ-secretase: structure, function, and role in alzheimer disease. cold spring harb substrate specificity of γ-secretase and other intramembrane proteases a greek tragedy: the growing complexity of alzheimer amyloid precursor protein proteolysis a presenilin- -dependent gamma-secretase-like protease mediates release of notch intracellular domain total inactivation of gamma-secretase activity in presenilin-deficient embryonic stem cells presenilins are required for γ-secretase cleavage of β-app and transmembrane cleavage of notch- papillomavirus infection requires gamma secretase impact of inhibitors and l antibodies upon the infectivity of diverse alpha and beta human papillomavirus types beyond γ-secretase activity: the multifunctional nature of presenilins in cell signalling pathways amyloid precursor protein (app) traffics from the cell surface via endosomes for amyloid β (aβ) production in the trans-golgi network sorting through the cell biology of alzheimer's disease: intracellular pathways to pathogenesis expression of the l and e genes of the human papillomavirus type in female genital dysplasias self-assembly of human papillomavirus type capsids by expression of the l protein alone or by coexpression of the l and l capsid proteins a membrane-destabilizing peptide in capsid protein l is required for egress of papillomavirus genomes from endosomes a transmembrane domain and gxxxg motifs within l are essential for papillomavirus infection topography of the human papillomavirus minor capsid protein l during vesicular trafficking of infectious entry incoming human papillomavirus type genome resides in a vesicular compartment throughout mitosis furin-induced cleavage and activation of shiga toxin proteolytic activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular proteases fusion peptides and the mechanism of viral fusion structure and function of membrane fusion peptides the complete influenza hemagglutinin fusion domain adopts a tight helical hairpin arrangement at the lipid:water interface de novo design of conformationally flexible transmembrane peptides driving membrane fusion structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme analysis of the γ-secretase interactome and validation of its association with tetraspanin-enriched microdomains structure, mechanism and inhibition of gamma-secretase and presenilin-like proteases differential effects of inhibitors on the γ-secretase complex. mechanistic implications structural biology of presenilin complexes the cellular endosomal protein stannin inhibits intracellular trafficking of human papillomavirus during virus entry α-defensin hd inhibits human papillomavirus infection via capsid stabilization and redirection to the lysosome interferon gamma prevents infectious entry of human papillomavirus via an l -dependent mechanism human papillomavirus type does not require cathepsin l or b for infection a two-hybrid screen identifies cathepsins b and l as uncoating factors for adeno-associated virus and cathepsin l and cathepsin b mediate reovirus disassembly in murine fibroblast cells vimentin modulates infectious internalization of human papillomavirus pseudovirions current understanding of the mechanism of hpv infection how viruses enter animal cells penetration of nonenveloped viruses into the cytoplasm establishment of human papillomavirus infection requires cell cycle progression passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a residue consensus peptide specifies biotinylation in escherichia coli mechanism of diphtheria toxin catalytic domain delivery to the eukaryotic cell cytosol and the cellular factors that directly participate in the process on the translocation of botulinum and tetanus neurotoxins across the membrane of acidic intracellular compartments establishment of papillomavirus infection is enhanced by promyelocytic leukemia protein (pml) expression pml nuclear bodies pml nuclear bodies: regulation, function and therapeutic perspectives emerging role of pml nuclear bodies in innate immune signaling dynamics of component exchange at pml nuclear bodies oxidative stress-induced assembly of pml nuclear bodies controls sumoylation of partner proteins dna viruses and viral proteins that interact with pml nuclear bodies pml and pml nuclear bodies: implications in antiviral defence reorganization of nuclear domain induced by papillomavirus capsid protein l dissection of human papillomavirus type l domains involved in nuclear domains (nd) homing and reorganization an l sumo interacting motif is important for pml localization and infection of human papillomavirus type modification of human papillomavirus minor capsid protein l by sumoylation factors influencing subcellular localization of the human papillomavirus l minor structural protein sp provides intrinsic immunity against human papillomavirus infection live cell dynamics of promyelocytic leukemia nuclear bodies upon entry into and exit from mitosis sincere apologies to all whose work was neither discussed nor cited due to space limitations. i thank koenraad van doorslaer, brittany forte, shuaizhi li, and matthew bronnimann for critical reading of the manuscript. samuel k. campos is supported by grant r ai from the national institute for allergy and infectious diseases. key: cord- -oz hras authors: nelson, elizabeth a.; barnes, alyson b.; wiehle, ronald d.; fontenot, gregory k.; hoenen, thomas; white, judith m. title: clomiphene and its isomers block ebola virus particle entry and infection with similar potency: potential therapeutic implications date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: oz hras the outbreak of ebola virus (ebov) in western africa highlighted the need for anti-ebov therapeutics. clomiphene is a u.s. food and drug administration (fda)-approved drug that blocks ebov entry and infection in cells and significantly protects ebov-challenged mice. as provided, clomiphene is, approximately, a : mixture of two stereoisomers, enclomiphene and zuclomiphene. the pharmacokinetic properties of the two isomers vary, but both accumulate in the eye and male reproductive tract, tissues in which ebov can persist. here we compared the ability of clomiphene and its isomers to inhibit ebov using viral-like particle (vlp) entry and transcription/replication-competent vlp (trvlp) assays. clomiphene and its isomers inhibited the entry and infection of vlps and trvlps with similar potencies. this was demonstrated with vlps bearing the glycoproteins from three filoviruses (ebov mayinga, ebov makona, and marburg virus) and in two cell lines ( t/ and vero e ). visual problems have been noted in ebov survivors, and viral rna has been isolated from semen up to nine months post-infection. since the clomiphene isomers accumulate in these affected tissues, clomiphene or one of its isomers warrants consideration as an anti-ebov agent, for example, to potentially help ameliorate symptoms in ebov survivors. the epidemic of ebola virus (ebov) that swept through western africa beginning in december , was the worst outbreak of this deadly hemorrhagic fever virus in recorded history. over , individuals were infected, over , died, and there are currently an estimated , survivors [ , ] . in addition to the tragic loss of life, many survivors are now suffering from sequelae that arose after they recovered from their ebov infections; these sequelae include visual problems, hearing loss, body aches, severe fatigue, and memory loss [ , ] . moreover, infectious ebov has been shown to persist in semen for six months, ebov rna has been found in semen as late as nine months post-infection, and at least one case of sexual transmission has been reported [ , ] . currently there are no approved drugs with which to treat ebov patients. consequently, during the recent outbreak several agents were administered to patients on a compassionate care transcription/replication-competent viral-like particles (trvlps) were prepared (under biosafety level (bsl ) conditions) as described in [ , ] . briefly, to prepare a p stock, % confluent hek t/ cells, seeded h prior in six well plates, were transfected with pcaggs-np, pcaggs-vp , pcaggs-vp , pcaggs-l, a tetracistronic minigenome plasmid, and pcaggs-t polymerase using transit-lt (mirus, madison, wi, usa). the minigenome plasmid encodes renilla luciferase, as well as the matrix proteins vp and vp and the gp envelope protein from ebov. h post transfection, the medium in each well was replaced with ml fresh growth medium containing % fbs. h after transfection, the medium (containing trvlps harboring the renilla luciferase-containing mini-genome) was harvested, pooled, and cleared of cellular debris by centrifugation for min at ˆg. p and p stocks were prepared by serial passaging of p stocks on hek t/ target cells freshly transfected with pcaggs-np, pcaggs-vp , pcaggs-vp , pcaggs-l, and pcaggs-tim . trvlps (p , p , and p ) were stored on ice and used within two weeks. entry reporter viral-like particles (vlps) bearing gps from mayinga and makona ebov, and from marburg virus (marv; angola isolate, gift of dr. christopher broder (uniformed services university of the health sciences, bethesda, md, usa) were prepared essentially as described in previous work [ , , , ] . in brief, % confluent hek t/ cells were transfected with cdnas encoding ebov or marv gp, vp , mcherry-vp , and β-lactamase-vp (βlam-vp ) (although not utilized in this study, mcherry-vp in the vlps can be used to monitor vlp binding to and trafficking within cells as described in [ , ] ). the cell medium was collected and h post-transfection and was cleared of cellular debris. vlps in the cleared medium were pelleted through a % sucrose cushion by centrifugation, resuspended in hm buffer ( mm hepes, mm mes, mm nacl, ph . ), and repelleted. the final vlp pellet was resuspended ( : starting volume of medium) in % sucrose-hm. the total protein concentration of the vlps was determined by bicinchoninic acid (bca) assay. all entry-reporter vlp preparations were assessed by western blot analyses (for the presence of the indicated glycoprotein as well as ebov vp ) and titered on vero e cells to confirm entry competency. entry-reporter vlps were frozen in single use aliquots at´ ˝c and were used within four months. infection of hek t/ cells by trvlps was assayed as described [ , ] with minor modifications. in brief, to prepare target cells, hek t/ cells were seeded in parallel opaque white -well plates (bd falcon, thermofisher scientific, waltham, ma, usa) for trvlp and cell viability assays and in clear-bottom -well plates to assess cell density and transfection efficacy. at - h post seeding, when the cells were approximately % confluent, the cells were transfected with (per well) . ng pcaggs-np, . ng pcaggs-vp , . ng pcaggs-vp , . ng pcaggs-l, and . ng pcaggs-tim using . µl transit-lt . these plasmids are necessary for the target cells to support entry and replication of incoming trvlps. to assess transfection efficacy and to provide a negative control, wells on each plate were transfected as above, but with a green fluorescent protein (gfp) expression plasmid in place of pcaggs-l. - h post transfection, the medium was removed and these target hek t/ cells were pre-treated with the indicated concentration(s) of the indicated drug(s) (dmso for mock) diluted in opti-mem (omem, gibco life technologies via university of virginia tissue culture facility, charlottesville, va, usa) for h at ˝c in a % co incubator. to assess trvlp infection, the pretreatment solution was removed and replaced with µl or µl trvlps diluted to µl in growth medium containing % scs and the indicated concentration(s) of the indicated drug(s) (dmso for mock). the cells were then incubated for h at ˝c in a % co incubator, after which the medium was replaced with µl of fresh growth medium containing % scs. µl of renillaglo substrate (promega, madison, wi, usa) was then added to each well and the plate was immediately analyzed on a glomax plate reader. to assess cell viability, the pretreatment solution was removed and replaced with µl fresh growth medium containing % scs and the indicated concentration(s) of the indicated drug(s) (dmso for mock). the cells were then incubated for h at ˝c in a % co incubator, after which the medium was replaced with µl of fresh growth medium containing % scs. µl of celltiter-glo . (promega) was then added to each well and the plate placed on a jitterbug orbital shaker (boekel scientific, thermofisher scientific, waltham, ma, usa) set at rpm for min at room temperature (rt). the plate was then incubated at rt for min, after which the luminescent signal was detected using a synergy ht (biotek, winooski, vt, usa) plate reader. vlp entry assays were performed as described [ , , ] with minor modifications. in brief, - , vero e or t/ cells were seeded per well of a clear -well plate. at - h post seeding, when the cells were~ %- % confluent, the cells were treated with the indicated drug(s) at the indicated concentration(s) (dmso for mock) diluted in omem for h at ˝c in a % co incubator. vlps diluted in omem in the presence of the indicated drug(s) at the indicated concentration(s) (dmso for mock) were bound to the cells by spinfection ( ˆg) for h at ˝c. the cells were then incubated for h in a ˝c, % co incubator. the βlam substrate ccf -am (life technologies, thermofisher scientific, waltham, ma, usa) was then loaded into the cells as previously described, but with mm instead of mm probenecid (mp biomedicals, thermofisher scientific, waltham, ma, usa) in the loading buffer. the cells were incubated overnight at rt and then fixed and analyzed by flow cytometry as described [ , , ] . to measure cell viability, - , vero e cells, seeded and grown as above but in -well opaque white plates were treated (as above) for vlp entry, but vlps were not added and ccf -am was not loaded. after the overnight incubation at rt (see above), the medium was removed from the cells and replaced with µl of fresh medium per well. microliters (per well) of celltiter-glo . was then added. the plate was placed on a jitterbug orbital shaker ( rpm) for min at rt. the plate was then incubated at rt for min, after which the luminescent signal was detected using a biotek synergy ht plate reader. for both trvlp infection and vlp entry, raw data were normalized to mock infection/entry signals, and percent inhibition of infection/entry was determined. graphpad prism . was used to perform non-linear regression analyses (log (agonist) vs. response; variable slope) of the percent inhibition values. inhibitory concentration, % (ic ) and % (ic ) values were interpolated based on the regression curve, and ec values were automatically generated. the graphpad program "ecanything" [ ] was used to determine ec values, which are based on the ec and hill slope values from the regression analysis. clomiphene is a~ : mixture of two stereoisomers: enclomiphene and zuclomiphene. their primary metabolites are -hydroxy-enclomiphene and, to a lesser extent, -hydroxy-zuclomiphene. we first compared the ability of clomiphene, enclomiphene, zuclomiphene, -hydroxy-enclomiphene, and -hydroxy-zuclomiphene to block ebov using the trvlp life cycle modeling system described by watt and hoenen [ , ] . as seen in figure a , both isomers of clomiphene (enclomiphene and zuclomiphene) as well as the -hydroxy metabolites of each ( -hydroxy-enclomiphene, and -hydroxy-zuclomiphene) displayed dose-dependent inhibition of trvlp infection similar to that seen with the parent mixture (clomiphene) when cells were pretreated for h with increasing concentrations of compound. at a dose of µm, all five forms of clomiphene showed approximately equal potency, reducing the trvlp (renilla luciferase) signal by approximately -fold. parallel sets of cells showed no evidence of cytotoxicity by any form of clomiphene ( figure b ). our previous findings indicated that clomiphene blocks ebov infection by blocking entry of viral particles into the cell cytoplasm [ , ] ; this effect appears to be at the level of fusion between the membrane of the viral particle and the limiting membrane of an niemann-pick disease, type c positive (npc + ) endolysosome, the site of ebov fusion [ ] [ ] [ ] . we, therefore, compared the effects of clomiphene, enclomiphene, zuclomiphene, -hydroxy-enclomiphene, and -hydroxyzuclomiphene on ebov entry using entry reporter vlps, as described previously [ , , ] . at a concentration of μm, clomiphene, both of its isomers (enclomiphene and zuclomiphene), as well as the -hydroxy metabolite of each isomer blocked vlp entry (figure a) , with no evidence of cytotoxicity ( figure. b ). in this system, -hydroxy-zuclomiphene appeared more potent than the our previous findings indicated that clomiphene blocks ebov infection by blocking entry of viral particles into the cell cytoplasm [ , ] ; this effect appears to be at the level of fusion between the membrane of the viral particle and the limiting membrane of an niemann-pick disease, type c positive (npc + ) endolysosome, the site of ebov fusion [ ] [ ] [ ] . we, therefore, compared the effects of clomiphene, enclomiphene, zuclomiphene, -hydroxy-enclomiphene, and -hydroxy-zuclomiphene on ebov entry using entry reporter vlps, as described previously [ , , ] . at a concentration of µm, clomiphene, both of its isomers (enclomiphene and zuclomiphene), as well as the -hydroxy metabolite of each isomer blocked vlp entry (figure a) , with no evidence of cytotoxicity ( figure b ). in this system, -hydroxy-zuclomiphene appeared more potent than the other forms of clomiphene ( figure a ), but this is likely an assay-dependent result, as -hydroxy-zuclomiphene was not more potent in the trvlp assay ( figure a ). other forms of clomiphene ( figure a ), but this is likely an assay-dependent result, as -hydroxyzuclomiphene was not more potent in the trvlp assay ( figure a ). as elaborated in sections and , enclomiphene and zuclomiphene exhibit different pharmacological properties. notably, zuclomiphene has a longer half-life [ , ] , and evidence suggests that the zuclomiphene component is responsible for more of the adverse side effects seen in mice treated with clomiphene [ ] . we, therefore, compared clomiphene, enclomiphene, and zuclomiphene in more detail for their effects on trvlp infection and vlp entry. we first compared clomiphene and enclomiphene in the trvlp infection and vlp entry systems using eight-point dose response curves. as seen in figure , clomiphene and enclomiphene showed similar dose-dependent inhibition of trvlp infection. in this experiment the calculated ic for clomiphene was . μm and that for enclomiphene was . μm. clomiphene and enclomiphene also showed similar dose-dependent inhibition in the vlp entry system; the ic for clomiphene was . μm and for enclomiphene was . μm (figure ). similar results were seen when comparing eight doses of clomiphene and zuclomiphene in trvlp infection ( figure a ) and vlp entry ( figure b ) as elaborated in sections and , enclomiphene and zuclomiphene exhibit different pharmacological properties. notably, zuclomiphene has a longer half-life [ , ] , and evidence suggests that the zuclomiphene component is responsible for more of the adverse side effects seen in mice treated with clomiphene [ ] . we, therefore, compared clomiphene, enclomiphene, and zuclomiphene in more detail for their effects on trvlp infection and vlp entry. we first compared clomiphene and enclomiphene in the trvlp infection and vlp entry systems using eight-point dose response curves. as seen in figure , clomiphene and enclomiphene showed similar dose-dependent inhibition of trvlp infection. in this experiment the calculated ic for clomiphene was . µm and that for enclomiphene was . µm. clomiphene and enclomiphene also showed similar dose-dependent inhibition in the vlp entry system; the ic for clomiphene was . µm and for enclomiphene was . µm (figure ). similar results were seen when comparing eight doses of clomiphene and zuclomiphene in trvlp infection ( figure a ) and vlp entry ( figure b ) assays. the ic and ic values from all of the eight-point dose response curves (figures - ) are given in table . table . previous work using authentic ebov in a bsl lab showed that clomiphene blocks infections in multiple cell types including vero e (kidney), hepg (liver), and huvec (endothelial) cells, and by all strains of filoviruses tested: three strains of ebola virus and two of marburg virus [ , ] . here we used assays that can be conducted in a bsl laboratory to begin to test whether enclomiphene shows similar breadth as a filovirus inhibitor. we first used the trvlp system to compare the efficacy of clomiphene and enclomiphene in vero e cells. the trvlp experiments presented thus far were conducted in hek t/ cells, which are optimal for this assay because they are highly transfected with the multiple plasmids needed to produce trvlps and to assay their lifecycle capacity [ , ] . vero e cells are also good targets for this assay. as seen in figure , clomiphene and enclomiphene blocked trvlp infection in vero e cells (with similar dose profiles). treated samples; and (b) hek t/ were seeded, treated with clomiphene or zuclomiphene, and vlps bearing ebov gp (mayinga strain) were bound to the cells as described in the legend to figure . cytoplasmic entry was assayed as described in the legend to figure . data ± sd are from triplicate samples normalized to the average % entry in mock treated cells ( . % and . % for the two sets of drugs). previous work using authentic ebov in a bsl lab showed that clomiphene blocks infections in multiple cell types including vero e (kidney), hepg (liver), and huvec (endothelial) cells, and by all strains of filoviruses tested: three strains of ebola virus and two of marburg virus [ , ] . here we used assays that can be conducted in a bsl laboratory to begin to test whether enclomiphene shows similar breadth as a filovirus inhibitor. we first used the trvlp system to compare the efficacy of clomiphene and enclomiphene in vero e cells. the trvlp experiments presented thus far were conducted in hek t/ cells, which are optimal for this assay because they are highly transfected with the multiple plasmids needed to produce trvlps and to assay their lifecycle capacity [ , ] . vero e cells are also good targets for this assay. as seen in figure , clomiphene and enclomiphene blocked trvlp infection in vero e cells (with similar dose profiles). the current trvlp system utilizes plasmids encoding proteins from the mayinga ( ) isolate of ebov [ , ] , an oft-used reference strain. we, therefore, utilized entry reporter vlps, which can accommodate glycoproteins from other viruses [ , ] , to ask whether the ability of enclomiphene to block ebov entry extends to other filoviruses. as seen in figure a , enclomiphene exerted a similar effect as clomiphene in blocking entry mediated by the gp from makona ebov, the isolate that caused the - outbreak in western africa, and, as seen in figure b , enclomiphene inhibited entry mediated by a marburg virus gp, representing a different genus within the filovirus family, similar to the effects of clomiphene. the current trvlp system utilizes plasmids encoding proteins from the mayinga ( ) isolate of ebov [ , ] , an oft-used reference strain. we, therefore, utilized entry reporter vlps, which can accommodate glycoproteins from other viruses [ , ] , to ask whether the ability of enclomiphene to block ebov entry extends to other filoviruses. as seen in figure a , enclomiphene exerted a similar effect as clomiphene in blocking entry mediated by the gp from makona ebov, the isolate that caused the - outbreak in western africa, and, as seen in figure b , enclomiphene inhibited entry mediated by a marburg virus gp, representing a different genus within the filovirus family, similar to the effects of clomiphene. clomiphene emerged from two independent screens of fda-approved drugs for anti-ebov activity in tissue culture cells [ ] [ ] [ ] , and was also found to provide %- % protection in female mice challenged with a lethal dose of ebov [ , ] . further analyses demonstrated that clomiphene blocks ebov entry into the host cell cytoplasm after virus particles are transported to endolysosomes [ , ] , the site of ebov fusion and cytoplasmic entry [ ] [ ] [ ] . hence, clomiphene interferes with the process of ebov fusion with the endolysosomal membrane. clomiphene, as used to treat female infertility, is a ~ : mixture of two stereoisomers, enclomiphene and zuclomiphene. since zuclomiphene is thought to impart more side effects [ ] , enclomiphene is being developed for the treatment of secondary hypogonadism (low testosterone) [ ] . the main objective of this study was to assess the abilities of enclomiphene and zuclomiphene to block ebov infection, as the individual isomers might offer (different) advantages in different clinical settings. we found that enclomiphene and zuclomiphene were highly similar to clomiphene in their ability to block trvlp infection and vlp entry, which are validated surrogate assays for ebov infection and ebov entry, respectively. clomiphene, enclomiphene, and zuclomiphene inhibited vlp entry and trvlp infection with similar ic values. where tested, they were equally effective to each other in two cell types and for entry mediated by three filoviral gps. -hydroxy-enclomiphene, and -hydroxy-zuclomiphene also blocked ebov trvlp infection and vlp entry under the clomiphene emerged from two independent screens of fda-approved drugs for anti-ebov activity in tissue culture cells [ ] [ ] [ ] , and was also found to provide %- % protection in female mice challenged with a lethal dose of ebov [ , ] . further analyses demonstrated that clomiphene blocks ebov entry into the host cell cytoplasm after virus particles are transported to endolysosomes [ , ] , the site of ebov fusion and cytoplasmic entry [ ] [ ] [ ] . hence, clomiphene interferes with the process of ebov fusion with the endolysosomal membrane. clomiphene, as used to treat female infertility, is a~ : mixture of two stereoisomers, enclomiphene and zuclomiphene. since zuclomiphene is thought to impart more side effects [ ] , enclomiphene is being developed for the treatment of secondary hypogonadism (low testosterone) [ ] . the main objective of this study was to assess the abilities of enclomiphene and zuclomiphene to block ebov infection, as the individual isomers might offer (different) advantages in different clinical settings. we found that enclomiphene and zuclomiphene were highly similar to clomiphene in their ability to block trvlp infection and vlp entry, which are validated surrogate assays for ebov infection and ebov entry, respectively. clomiphene, enclomiphene, and zuclomiphene inhibited vlp entry and trvlp infection with similar ic values. where tested, they were equally effective to each other in two cell types and for entry mediated by three filoviral gps. -hydroxy-enclomiphene, and -hydroxy-zuclomiphene also blocked ebov trvlp infection and vlp entry under the conditions tested. therefore, we propose that, like clomiphene, enclomiphene and zuclomiphene have potential as pan-filoviral inhibitors in multiple cell types. approximately , individuals died during the recent outbreak of makona ebov, and another~ , have survived their infection [ ] . tragically, many of these survivors are displaying post ebola virus disease syndromes, including visual problems, hearing loss, and excessive fatigue [ , ] . in addition, infectious ebov has been found in semen for up to six months [ ] , raising concerns of sexual transmission [ ] . hence, drugs that accumulate in tissues of the eye and male reproductive tract could be particularly helpful for certain ebola virus survivors. in this respect it is interesting that, in mice, both enclomiphene and zuclomiphene persist in the eye and male reproductive tract longer than in other tissues, with zuclomiphene maintained at higher levels than enclomiphene. zuclomiphene was also found to persist in the brain, whereas enclomiphene was not [ ] . clomiphene provided up to % protection in the lethal mouse model of ebov infection when administered to female mice at a dose of mg/kg on days , , , , , and post-infection, with no apparent ill effects on surviving mice. the maximum serum concentration (c max ) for a mg/kg dose (intra-peritoneal administration, ip) of clomiphene to female mice was . µm (lisa johansen, personal communication) and, hence, above the ic for blockade of ebov infection in tissue culture cells [ , ] and above the ic values reported here for clomiphene and its isomers in blocking ebov vlp entry and trvlp infection ( table ). the standard dose of clomiphene to treat female infertility is mg/day (per os). with this (oral) dose, reported c max values (summed for enclomiphene and zuclomiphene) range from . to . µm (table ) ,~ - -fold below the ic ( . µm in vero e cells) for anti-ebov activity [ , ] . therefore, for potential treatment of acute ebov infections, higher doses of clomiphene would certainly be needed, likely in combination with other anti-ebov drugs. the human equivalent dose (to the mouse dosing employed in [ ] ) for an adult weighing kg would be mg clomiphene every other day [ ] . in this respect it is noteworthy that higher doses of clomiphene (up to mg/day) have been administered to non-responding (anovulating) females [ , ] , and that considerably higher c max values were obtained when patients were administered the standard dose of clomiphene ( mg), but by intravenous administration (iv) instead of the oral route (table ) [ ] . hence, it is conceivable that higher doses of clomiphene could be administered (perhaps as part of a cocktail) to treat acute ebov infections. irrespective of the utility of clomiphene for acute ebov infections, given the persistence of the clomiphene isomers in the eye and male reproductive tract [ ] , clomiphene (alone or in a combination) may have utility in the management of post ebola virus disease symptoms in ebov survivors. for example, enclomiphene and zuclomiphene accumulate~ -to~ -fold, respectively, in the harderian gland (located in the orbit) and the uveal tract of the mouse eye. similar accumulation of zuclomiphene was seen in tissues of the male mouse reproductive tract [ ] . if such accrual occurs in humans, the standard dose ( mgs per day, per os) of clomiphene, which contains both isomers, could be sufficient to reach anti-viral levels in the eye and male reproductive tract; the levels would more likely be sufficient if clomiphene were given at a higher dose and/or in combination with another anti-ebov agent. interestingly, no sperm were seen in the testes and epididymes of male mice treated for days with mg/kg/day of zuclomiphene [ ] . since the two isomers of clomiphene and their hydroxylated metabolites all blocked ebov vlp entry and replication in cell cultures, the protective effect of clomiphene in the mouse model [ , ] could have been due to any or all of these forms. the metabolism of en-and zuclomiphene in humans is well-characterized [ , ] , and differences between the isomers have been shown in mice [ , ] . it will, therefore, be interesting to compare the efficacies of the individual clomiphene isomers in the mouse model of ebov infection. studies on their relative efficacies in the eyes and reproductive tract will be especially revealing. table in reference [ ] ; c values deduced from figure of reference [ ] ; anov., anovulating; h, hour; n/a, not applicable; p , anovulatory patient ; p , anovulatory patient ; itt, intention-to-treat (i.e., healthy males with low testosterone levels). we have shown that, like the parent mixture, the two isomers of clomiphene (and their hydroxylated metabolites) block entry and replication of ebov vlps in cell cultures. the activity of the isomers was shown in two cell types and against three filoviral gps. both isomers of clomiphene accumulate in tissues of the eye and male reproductive tract. hence we propose that clomiphene, an fda-approved drug that provided up to % protection in the mouse model of ebola virus disease, remain in consideration as an anti-ebov agent, especially for the management of post ebola virus disease syndromes, including visual problems and the risk of sexual transmission, and especially for consideration as part of a potential drug cocktail. ebolavirus evolution: past and present persistence of ebola virus in ocular fluid during convalescence long-term sequelae after ebola virus disease in bundibugyo, uganda: a retrospective cohort study ebola rna persistence in semen of ebola virus disease survivors -preliminary report molecular evidence of sexual transmission of ebola virus reversion of advanced ebola virus disease in nonhuman primates with zmapp a randomized controlled trial of zmapp™ in acute ebola virus infection hyperimmune serum from healthy vaccinated individuals for ebola virus disease? postexposure antibody prophylaxis protects nonhuman primates from filovirus disease ebola drugs still stuck in lab successful treatment of advanced ebola virus infection with t- (favipiravir) in a small animal model lipid 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drugs for inhibitors of biological threat agents development, pharmacology and clinical experience with clomiphene citrate clomiphene citrate for ovulation induction in women with oligo-amenorrhoea fertility treatment in women with polycystic ovary syndrome: a decision analysis of different oral ovulation induction agents possible hypothalamic impotencemale counterpart to hypothalamic amenorrhea? urology multiple cationic amphiphiles induce a niemann-pick c phenotype and inhibit ebola virus entry and infection ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to npc + endolysosomes is a rate-defining step ebolavirus glycoprotein directs fusion through npc + endolysosomes direct visualization of ebola virus fusion triggering in the endocytic pathway niemann-pick c is essential for ebolavirus replication and pathogenesis in vivo agonistic and antagonistic effects of clomiphene citrate and its isomers effect of estrogen agonists and antagonists on induction of progesterone receptor in a rat hypothalamic cell line clomiphene citrate and enclomiphene for the treatment of hypogonadal androgen deficiency single-dose pharmacokinetics of clomiphene citrate in normal volunteers genetic polymorphism of cytochrome p d determines oestrogen receptor activity of the major infertility drug clomiphene via its active metabolites the isomers of clomiphene citrate have dissimilar dispositions once ingested: results of a mouse ade study differential effects of isomers of clomiphene citrate on reproductive tissues in male mice testosterone restoration using enclomiphene citrate in men with secondary hypogonadism: a pharmacodynamic and pharmacokinetic study a novel life cycle modeling system for ebola virus shows a genome length-dependent role of vp in virus infectivity modeling the lifecycle of ebola virus under biosafety level conditions with virus-like particles containing tetracistronic minigenomes single-dose pharmacokinetic study of clomiphene citrate isomers in anovular patients with polycystic ovary disease dose translation from animal to human studies revisited monitoring plasma concentrations to individualize treatment with clomiphene citrate serum concentrations of enclomiphene and zuclomiphene across consecutive cycles of clomiphene citrate therapy in anovulatory infertile women pharmacokinetics of intravenous clomiphene isomers the work was supported by a grant from the nih to jmw (ro ai ) and in key: cord- -p h p bm authors: lindqvist, richard; upadhyay, arunkumar; Överby, anna k. title: tick-borne flaviviruses and the type i interferon response date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: p h p bm flaviviruses are globally distributed pathogens causing millions of human infections every year. flaviviruses are arthropod-borne viruses and are mainly transmitted by either ticks or mosquitoes. mosquito-borne flaviviruses and their interactions with the innate immune response have been well-studied and reviewed extensively, thus this review will discuss tick-borne flaviviruses and their interactions with the host innate immune response. tick-borne flaviviruses (tbfv), flaviviridae family, includes many pathogens causing severe human disease, ranging from mild fever to encephalitis and hemorrhagic fever. there are more than viruses in the genus flavivirus, and they are transmitted by arthropods such as mosquitoes (dengue virus (denv), japanese encephalitis virus (jev) and west nile virus (wnv), yellow fever virus (yfv), and zika virus (zikv) and ticks (tick-borne encephalitis virus (tbev), langat virus (lgtv), kyasanur forest disease virus (kfdv), omsk hemorrhagic fever virus (ohfv), powassan virus (powv), and louping-ill virus (liv)) [ ] [ ] [ ] [ ] [ ] . among the tbfvs, tbev, powv, and liv are encephalitic, whereas ohfv and kfdv are hemorrhagic viruses. there are general features that distinguish tbfv from mosquito-borne. one such feature is that tick-borne viruses tend to persist in certain stable foci whereas mosquito-borne viruses may suddenly emerge and they can rapidly spread to new areas and continents causing large epidemics [ ] [ ] [ ] [ ] [ ] [ ] . mosquito-borne flaviviruses are transmitted horizontally from mosquito to vertebrate to mosquito, and for some viruses, humans act as the amplifying host [ ] [ ] [ ] . mosquitoes have a short life compared to ticks, as it can take several years for a tick to develop from egg to adult [ ] . ticks also only take one blood meal at each of their life stages, larvae, nymph, and adult, this means that tick-borne viruses need to be maintained in infected ticks during a very long time [ ] . thus, replication in ticks needs to be lower compared to mosquitoes, which have higher viral turnover and a faster generation cycle, this also contributes to the high mutation rate in mosquito-borne flaviviruses, and relative stable genomes of tbfvs [ , ] . infection of tick-borne flaviviruses usually cause a short viremia in humans and larger vertebrates. this route of transmission has been described as less important in tick-borne flavivirus infection [ ] , instead tbev has been shown to be transmitted by viremic rodents and among co-feeding ticks without viremia [ ] . humans act as dead-end hosts since they do not produce high enough viremia to transmit the virus to new ticks. according to phylogenetic differences, tbev has been divided into three different subtypes, european, siberian, and far eastern. the european subtype is mainly transmitted by ixodes ricinus, whereas the siberian and far eastern subtypes are primarily transmitted by ixodes persulcatus [ , ] . tbev is found in central, eastern, and northern europe and asia ( figure ) and correlates with the presence of infected ticks. ixodes ricinus is found throughout europe, whereas ixodes persulcatus is found in eastern europe in the west, and china and japan in the east [ ] . tbev is considered one of the most important arboviruses in central and eastern european countries and in russia, with about , estimated human cases annually [ ] . in fact, over the last decade there has been an approximately % increase in the number of tbe cases in europe [ ] , and tbev is currently spreading into new regions in france, sweden, norway, and italy [ ] [ ] [ ] [ ] . this increase is thought to be due to growth in population and spread of ticks, which is promoted by factors including climate change, social and political change, and changes in the land use [ , ] . the increased expansion in europe also poses an increased risk for the population engaged in outdoor activities. tbev is a zoonotic disease and the natural cycle of tbev is dependent and maintained in a complex cycle involving ticks as the vector and reservoir of the virus and small rodents as hosts for ticks [ ] . humans are not part of the natural transmission cycle of tbev and are the incidental host when infected by a bite from an infected tick [ ] . transmission through consumption of unpasteurized milk has also been reported for tbev [ , ] , as well as transmission via solid organ transplant [ ] . during the tick bite, the virus is inoculated into the skin of the vertebrate host. the initial replication is believed to occur locally in the dendritic cells. this is followed by infection of the draining lymph nodes, resulting in the primary viremia and subsequent infection of the peripheral tissues, where further replication maintains the viremia for several days [ ] [ ] [ ] [ ] . the disease course of tbev is biphasic; the initial phase is characterized by flu-like symptoms and is followed by a second phase involving cns infection, with meningitis, encephalitis, or meningoencephalitis [ ] [ ] [ ] . the mortality rate of tbev varies from to % depending on the subtype, in which the european tbev subtype has shown lower mortality rates compared to the siberian and far eastern [ , , ] . among the patients that experience neuroinvasive tbev infection, approximately - % of the survivors suffer from long lasting neurological sequelae [ , ] . no antivirals are available for treatment of tbev infection but there is an effective vaccine [ , ] . lgtv, which is closely related to tbev, is found in south east asia and russia [ ] . lgtv has not been associated with human disease under natural infections although it shares % sequence identity with tbev [ ] . because of its avirulence in humans and close similarity to tbev, lgtv is often used as a model virus for tbev under biosafety level- conditions. powv is found in russia and north america, and is the only tbfv present in america ( figure ) [ ] . it is transmitted by ixodes scapularis, ixodes cookei, and several other ixodes tick species, to small and medium size mammals, whereas humans are accidental dead-end hosts. milk-borne powv transmission might also be possible since powv virus has been found to be secreted in milk under experimental settings [ ] . although not much is known about powv pathogenesis, recent studies in mice have found that tick saliva was important to enhance powv transmission and the outcome of disease [ ] . furthermore, it has been demonstrated that powv infects macrophages and fibroblasts in the skin, shortly after the tick bite, also, other unidentified cells were shown to be infected [ ] . interestingly, macrophages were found to be the primary target for powv in the spleen [ ] , and in the cns, which is the main target site for powv infection, neurons have been shown to be the primary target for powv in mice and humans [ , ] . during the last years there has been an increase of powv in the usa with approximately reported cases [ , ] . the recent rise in incidence could be due to increased surveillance and diagnosis of powv, or it may represent a true emergence of the disease in endemic areas, or both [ ] . the incubation period ranges from week to month. the symptoms of powv infection may include fever, headache, vomiting, weakness, confusion, seizures, and memory loss with a case fatality rate of % [ ] . approximately half of the survivors experience permanent neurological symptoms, such as recurrent headaches, muscle wasting, and memory problems (https://www.cdc.gov). there are no antiviral treatments or vaccines available against powv. liv is mainly distributed in the uk and ireland, but it has also been detected in sheep in norway and on the bornholm island in denmark ( figure ). liv is most commonly known as pathogen of sheep and red grouse although humans can also get infected [ , ] . animals develop a febrile disease, which can progress to fatal encephalitis [ ] . like tbev, the vector of liv is the tick species ixodes ricinus [ ] . ticks transmit the virus to animals, however, natural exposure to humans is rare [ ] . instead humans that are exposed to infected animals such as veterinarians, farmers, butchers, and abattoir workers, as well as laboratory scientists have acquired liv infection [ ] [ ] [ ] [ ] . in between the years of and , human cases of liv was described [ ] . in humans, liv causes a disease that closely resembles tbev, with initial flu-like symptoms which can progress to severe neurological disease. distinct but closely related viruses are also found in spain (spanish sheep encephalomyelitis virus), turkey (turkish sheep encephalitis virus), and greece (greek goat encephalitis virus) [ ] . ohfv distribution is restricted to western siberia ( figure ) [ ] . the main vector of ohfv is the meadow tick, dermacentor reticulatus, which can also transmit the virus to humans. however, humans are mainly infected after contact with infected muskrats (ondatra zibethicus) which are very sensitive to the infection and often succumb to the infection [ ] . muskrats develop high viremia which can last for several weeks. human infection occurs through contact with urine, feces, and blood [ ] . secretion of ohfv in unpasteurized goat milk has been reported but no milk-borne outbreaks have been observed [ ] . the exact number of annual cases are uncertain because of misdiagnoses and unreported cases, but cases were reported between and [ ] . ohfv may cause a biphasic disease; the initial phase is characterized by high fever, bleeding from the nose, mouth, and uterus. thirty to fifty percent of the cases experience a second phase characterized by high fever and reappearance of the symptoms from the initial phase. case fatality rates range from . to . % [ ] . no antiviral treatments are available against ohfv, instead treatment is focused on supportive care to minimize hemorrhage and other complications [ ] . kfdv is only found in india (figure ), although a similar genetic variant, alkhurma hemorrhagic fever virus (ahfv) has been detected in saudi arabia [ , ] . kfdv is mainly transmitted by ticks belonging to the genus hemaphysalis but other tick genera have also been shown to be able to transmit kfdv [ , ] . during hemorrhagic kfdv infection, the initial phase is similar to tbev infection with fever and flu-like symptoms, but it may also include bleeding from the nose, mouth or gastrointestinal tract [ , ] . the second phase, which is experienced by - % of the cases, includes neurological symptoms such as headache, mental disturbance, and tremor, however, no evidence of meninges or encephalitis have been found [ ] . there are about - reported annual cases of kfdv [ ] , with case fatality rates ranging from to % [ , ] . in a kfdv vaccine was developed, although it provided some protection, due to low efficacy, the number of cases still increased from to [ ] [ ] [ ] . treatment of kfdv is limited to supportive care [ ] . tbfvs are enveloped viruses around nm in diameter. the envelope carries two surface proteins, the envelope (e) protein and the membrane (m) protein. the latter is derived from a precursor protein, prm. the nucleocapsid (nc) lies inside the viral envelope and consists of multiple copies of capsid (c) protein and the viral genome. the tbfv genomes are single stranded, positive-sense rna of approximately , nucleotides. it has a -cap with a single open reading frame (orf). the orf is flanked by and untranslated regions (utrs). the viral protein is encoded by the orf as a single polyprotein, which is co-and post-translationally cleaved by cellular and viral proteases into individual viral proteins (three structural and seven non-structural). the polyprotein is arranged in the order -c-prm-e-ns -ns a-ns b-ns -ns a-ns b-ns - [ , ] . the first step of virus replication starts when the virus binds to its receptor and is taken up into the cell by receptor-mediated endocytosis. the attachment is mediated by viral e protein and entry receptor on the host cell. the entry receptor for tbfvs has not been identified, but attachment to heparan sulphate and glycosaminoglycan, which are present in abundance on many cell types of both vertebrates and ticks, is predicted to play a role during binding and entry [ ] . once the virus has entered the cell, it is transported to endosomes, where the acidic environment of these vesicles leads to reorganization and conformational change of the e protein, resulting in the fusion of the viral and endosomal membrane and the release of the viral capsid into the cytoplasm [ ] [ ] [ ] . the viral rna (vrna) also functions as mrna, associating with ribosomes to produce the polyprotein. the transmembrane domain of the viral protein is recognized as a signal peptide and recruits the vrna/ribosomes/nascent polypeptide complex to the er membrane, where it is co-translationally translocated into the er membrane [ , ] . the nascent polypeptide is then processed by the cellular and viral proteases into structural and non-structural (ns) viral proteins. some of the viral proteins such as ns b, ns a, and ns b integrate and alter the membrane of the er to form a membrane vesicular structure, with a small pore connecting the interior of the vesicle to the cytoplasm [ ] [ ] [ ] [ ] [ ] . these vesicles are the site for the formation of replication complexes (rc) and rna replication [ , , ] . following rna replication, genomic rna is proposed to exit the vesicle through the vesicular pore and is packaged by c proteins into the nc on the cytoplasmic side of the er membrane [ , , ] . during the process of budding of nc from the cytoplasm into the er, it acquires the lipid envelope along with e and prm proteins, which are associated with the er membrane. the mechanism that ensures efficient incorporation of nc into the er membrane with the e and prm protein is poorly understood. following budding into the er lumen, the immature virions are transported through the cellular secretory pathways in a copi and copii dependent manner [ ] to the extracellular medium. the immature virion has heterodimers of e and prm that completely cover the lipid bilayer to form a spiky proteinaceous coat. during the transport through the golgi, the e protein is glycosylated [ ] . the acidic environment of the golgi induces conformational changes in the e and prm proteins, which exposes a cleavage site on prm. cleavage of prm by the cellular protease furin results in the formation of a mature virion [ , ] . mature virions are then released by exocytosis, which completes the viral life cycle [ ] [ ] [ ] (figure ). in arthropods, such as mosquitos and ticks, rna interferences (rnai) are the most important antiviral defense and tick cells have been shown to mount rnai responses against lgtv and tbev [ ] . proteins involved in the rnai mediated response such as argonaute (ago ), ago and dicer (dcr) were subsequently identified as inhibitors of lgtv [ ] . furthermore rna-seq and mass spectrometric analysis revealed that when challenged by tbev infection, tick cells upregulated genes involved in immunity and metabolism, whereas genes involved in cellular stress were downregulated [ ] . using gene silencing approaches, this study confirmed the antiviral effect of ago and dcr in tick cells. furthermore, novel antiviral genes such as complement factor h, heat shock protein (hsp) and and trypsin was found to inhibit lgtv in tick cells [ ] . taken together, studies so far have identified activation and antiviral actions of the rnai mediated response in tick cells as well as other inhibitory proteins such as hsp , hsp , and trypsin. the innate immune response provides the first line of defense against viral infections. this defense includes physical and chemical barriers such as the skin and mucous membranes and the acidity of the stomach. they serve as an initial barrier that protects the host from infection. once these barriers have been breached, the innate immune response relies on a set of germline-encoded receptors, known as pathogen recognition receptors (prrs), which recognize pathogen specific molecular patterns that are sensed as a danger signal by the host cell [ , ] . engagement of prrs results in the activation of several defense mechanisms including, production of interferon (ifn), induction of phagocytosis, cytokines, chemokines, antimicrobial peptides, antiviral proteins, as well as activation of leukocytes and t-cells. furthermore, the innate immune response harbors cellular components, such as dendritic cells, macrophages, and natural killer (nk) cells [ ] . in isaacs and lindenmann discovered ifn as a secreted factor that interfered with viral replication [ ] . in the early pioneer work on ifn in the fifties and sixties, tbev served as a model system, and tbev was shown to induce ifn after infection and was also sensitive to pretreatment of ifns [ , [ ] [ ] [ ] [ ] . pretreatment of ifn was also found to inhibit kfdv, ohfv, and powv titers in a cells (adenocarcinomic human alveolar basal epithelial cells) [ ] , thus ifn pretreatment induced broad spectrum inhibition of tbfvs. ifn has also been found to be strongly induced in the brains of powv infected peromyscus lecuopus, which are known as a natural host of the virus [ ] . similarly, ifn was found to be induced in sheep infected with liv [ ] , and ifn-stimulated genes were induced in brains of kfdv infected mice, indicating an activated ifn response [ ] . three distinct classes of ifn, type i (ifn α and β), type ii (ifnγ), and type iii ifn (ifnλ) have been described. the expression of the ifnλ-receptors and ifnγ are restricted, but the type i ifns can be expressed by most cell types and their receptor, the interferon-α/β receptor (ifnar) is expressed on all nucleated cells [ ] . there are different type i ifns in humans; these cytokines are produced by cells upon recognition of pathogen-associated molecular patterns, which are foreign to the host cell [ , ] , and mediates antiviral activity by autocrine and paracrine signaling through ifnar [ , ] . signaling through ifnar induces an antiviral state by the expression of hundreds of interferon stimulated genes (isgs) [ , ] . the host cell detects invading pathogens through prrs, which recognize foreign molecular patterns that are generated during infection. the particular prr involved in the recognition of the pathogen depends on the infecting virus [ ] . in flavivirus infection, the most important prrs are toll-like receptor (tlr) , tlr , tlr , retinoic acid-inducible gene i (rig-i), and melanoma differentiation-associated protein- (mda- ). tlr recognizes double stranded (ds) rna, which is formed as an intermediate during flavivirus replication [ , ] . tlr and , which are located in the endosomes, recognize single stranded (ss) rnas, such as the genomic rna of flaviviruses [ , [ ] [ ] [ ] . rig-i and mda- recognize viral rna in the cytoplasm of infected cells; rig-i recognizes short dsrna and triphosphorylated and diphosphorylated ssrna, whereas mda- recognizes long dsrna [ ] [ ] [ ] . upon recognition, the different prr will recruit distinct adaptor molecules. tlr recruits tir-domain-containing adapter-inducing interferon-β (trif) [ ] , tlr and recruit myeloid differentiation primary response (myd ) [ ] , whereas the rig-i-like helicases, rig-i and mda- , recruit interferon-beta promoter stimulator- (ips- ) (also known as mitochondrial antiviral-signaling protein (mavs), virus-induced signaling adapter (visa) and card adapter-inducing ifn-beta (cardif)) [ ] [ ] [ ] [ ] . ligation of prrs and downstream recruitment of the adapter molecules result in the activation of transcription factors nf-κb and interferon regulatory factor (irf) and irf , which translocate to the nucleus and induces the expression and subsequent secretion of type i ifn [ ] . although the innate antiviral response has been well-studied in mosquito-borne flavivirus infection, the responses to tbfvs are less investigated, and most studies have used tbev which therefore will be the main focus of the remainder of this review. in tbev infection, ifnβ induction has been shown to correlate with amount of intracellular viral rna, and the ips- pathway has been shown to protect mice from lethal lgtv infection and prolong survival after tbev infection [ , ] . absence of ips- resulted in a lower systemic ifnα response, which correlated with higher viral replication in the peripheral tissues [ ] . furthermore, ips- was shown to be of particular importance for the ifn production locally within the brain, where ips- −/− mice had lower induction of ifnβ within the olfactory bulb despite higher viral burdens [ ] . furthermore, ifnβ induction was shown to be completely dependent on ips- and irf in mouse embryonic fibroblasts and viral recognition through the ips- -pathway was shown to be dependent on rig-i and not mda- in human osteosarcoma cells (u os) [ , ] . interestingly, rig-i has been shown to co-localize with stress granules (sg) during tbev infection [ ] . sg contains ribonucleoprotein aggregates with translationally stalled mrnas, s ribosomes, and several rna-binding proteins. sg function to prevent the generation of defective proteins [ ] . sg are induced during tbev infection and sg components tia- /tiar was found to bind viral rna and inhibit viral translation [ ] . little is known about the role of the tlr / -myd pathway in tick-borne flavivirus infection; however, tlr was shown to suppress lgtv replication within neurons of the brain although it did not affect pathogenesis [ ] . what we are aware of; no animal experiments have shown any significance of the tlr -trif pathway in tick-borne flavivirus infection. however, several studies have looked at the prevalence of polymorphisms in the tlr gene in tbe patients [ ] [ ] [ ] [ ] . in particular, one polymorphism has been investigated the t allele in rs . this polymorphism has been shown to reduce tlr signaling by about % [ ] . although the data regarding tlr is somewhat conflicting, grygorczuk et al. hypothesized that a functional tlr facilitates the onset of neurological disease [ ] by supporting the penetration through the blood brain barrier, but has a protective effect during the established cns infection [ ] . the differences between studies might also be connected to the different subtypes of the tbev strain and the genetic background of the studied populations [ ] . taken together, studies so far have demonstrated the importance of the rig-i-like-ips- pathway in tick-borne flavivirus infection, whereas the role of the tlr pathways remains unclear. after secretion, type i ifn signals in an autocrine and paracrine manner by binding to the heterodimeric ifnar, which consists of two subunits, ifnar and ifnar [ ] . these subunits are associated with janus activated kinases (jak) localized in the cytoplasm; ifnar interacts with tyrosine kinase- (tyk ), whereas ifnar interacts with jak . ligation of ifnar results in activation of tyk and jak , which subsequently phosphorylate signal transducers and activators of transcription (stat)- and stat- . phosphorylated stat- and stat- then form a heterodimer, which associates with irf to form ifn-stimulated gene factor- (isgf ) that binds to the ifn-stimulated response element (isre) in the promoter of many isgs to enhance the transcription of several hundreds of ifn-stimulated genes [ , , ] . together, these isgs act to coordinate an antiviral response that is able to inhibit almost any step in the viral life cycle [ ] . the ifn response in vivo in mice is very important to protect mice from lethal infection with lgtv. mice lacking ifnar all succumbed within days of infection, whereas % of wt mice ( - -week-old) survived the infection. interestingly, all ifnβ −/− mice survived the infection, demonstrating that the ifnαs can compensate for loss of ifnβ in lgtv infection. furthermore, ifnar was shown to be a critical determinant of lgtv tropism as lgtv rna was found in all organs in the absence of ifnar, whereas in wt, only low viral burdens can be detected in the olfactory bulb [ , , ] . using transgenic mice, it was further shown that the ifn response was needed both in the peripheral tissue, as well as locally in the cns, in order to clear lgtv infection [ , ] . within the cns, lgtv mainly infected neurons [ , ] , which is also the case in lethal tbev infection of humans [ ] . although neurons remain the main target cell of tbev, astrogliosis have been shown in post mortem human brains [ , ] . in vitro studies on primary cortical astrocytes show a fast up regulation and secretion of ifn after tbev infection, which is able to protect neighboring astrocytes and neurons from infection already and h post infection, respectively [ ] . astrocytes have been shown to be resistant to tbev-induced cytopathic effects [ , ] , and it was later shown that ifnar expression protected the astrocytes from the virus-induced cytopathic effects [ ] . pretreatment of cells with ifns strongly inhibits growth of most tbfvs in cell culture [ , , [ ] [ ] [ ] [ ] and this is due to the upregulation and concerted actions of several hundreds of isgs. only a few isgs have been identified to play a role in tbev and lgtv infection [ , , , , ] . one of them, the - -oligoadenylate synthetase ( - -oas) (oas) is activated by double-stranded rna, leading to the oas protein polymerization into - -linked oligoadenylates ( - as) [ , ] . these - as activate rnase l, resulting in the degradation of viral rna [ ] . several polymorphisms in the oas genes have been shown to correlate with severe forms of tbe in patients [ ] . also, the murine isoform oas b, which is often lacking in inbred mice strains, confers strain dependent resistance against neurovirulence from far eastern tbev [ ] . two other isgs which have been shown to be antivirally active against tbev are virus inhibitory protein endoplasmic reticulum associated interferon inducible (viperin) and tripartite motif- α (trim α). the trim proteins are a family of proteins able to mediate antiviral activity against many different viruses [ ] . the rodent specific trim α was identified to interact with lgtv ns in a yeast two-hybrid screen. trim α expression inhibited viral infection of lgtv and tbev by mediating lysosomal dependent degradation of ns . interestingly this mechanism was quite specific to tick-borne flaviviruses as the mosquito borne wnv was not inhibited by trim α nor did trim α interact with ns of wnv [ ] . viperin is highly conserved in evolution and was first identified as an ifn-inducible protein with antiviral activity against human cytomegalovirus in [ ] . over the last years, viperin has gained lot of attention and was shown to exhibit broad antiviral activity [ ] . viperin is also known as one of the most highly upregulated genes after viral infection [ , ] . within the family of flaviviridae, viperin has been shown to inhibit several members, such as wnv [ ] , denv [ ] , zikv [ ] , hepatitis c virus [ ] , and tbev [ , , , ] . viperin is an iron sulphur protein with three domains; a n-terminus amphipathic alpha-helix which mediates the intracellular localization to the er, a s-adenosylmethionine (sam) radical domain [ , , ] homologous to a family of proteins that use sam as a cofactor [ ] , and a highly conserved c-terminal domain, important for iron sulphur (fe/s) maturation [ , ] . although viperin is able to inhibit several different viruses, the mechanism of action, the important motif in viperin and the step of viral life cycle inhibited differs for different viruses [ ] . viperin interacts with many viral and host factors for its antiviral function. tbev replication is strongly inhibited by viperin, our research shows that viperin has no effect on the binding or entry of tbev. however, viperin targets genome replication, packaging, and release of tbev (figure ) [ , , ] . viperin specifically targets the plus-sense rna synthesis with no significant effect on the negative-sense rna of tbev during genomic replication [ ] . viperin's fe/s maturation is dependent on ciao [ , ] . the fe/s cluster and a functional sam domain of viperin is essential to inhibit the synthesis of the plus-sense rna of tbev [ ] . however, the target for the radical sam activity important for the antiviral activity of tbev is currently unknown. the structure of viperin was recently characterized [ ] , and based on similarities to other radical sam enzymes, several different hypothesis have been put forward regarding the substrate of viperin and its antiviral activity [ ] [ ] [ ] . however, none of these have shown importance in the context of mammalian viral infection. although the exact mode of action against tbev is not well understood, recent data indicates that viperin interacts with several viral proteins; both structural prm and e and non-structural ns a, ns b, and ns . these interactions lead to a viperin-ns dependent degradation of viral proteins. the degradation of tbev ns was shown to be proteasome dependent [ ] (figure ) . interestingly, ifn treatment induces an increase of tbev capsid particles and this effect was found to be dependent on viperin. viperin mediated this effect by interacting, via its n-terminus, with the cellular protein golgi brefeldin a resistant guanine nucleotide exchange factor (gbf ) [ ] . gbf is a key protein in the cellular secretory pathway and essential in the life cycle of many rna viruses, which utilize vesicular trafficking in their replication cycle and assembly process [ ] [ ] [ ] [ ] (figure ). viperin targets the ns protein for proteasomal degradation which inhibits the synthesis of + strand rna. furthermore, ns interacts with e, ns a, and ns b and these proteins are degraded by viperin in a ns -dependent manner. viperin also interferes with particle assembly by inducing secretion of c particles in a copii-dependent manner, independent of copi. viperin mediates this effect by interacting and sequestering gbf . red arrow = secretory pathway, blue arrow = "?" vesicular transport via unknown pathway. although viperin has been demonstrated to be antiviral active against many different viruses in vitro, few studies have investigated viperin's role in vivo. in tbfv infection, viperin was shown to control lgtv dissemination and replication in the brain after intraperitoneal administration, and viperin promoted survival after intracranial infection [ ] . interestingly, viperin has been shown to be expressed in the brain at the basal state [ ] , with high basal expression in primary astrocytes [ , ] . viperin's role within the brain during neurotropic lgtv infection was further mapped to certain brain regions, as viperin inhibited viral replication in the olfactory bulb and cerebrum, but not in the cerebellum or brainstem. this correlates very well with tbev infection since viperin strongly inhibited tbev replication in primary neurons and astrocytes from the cerebrum, but not in granular cell neurons isolated from the cerebellum. interestingly, cortical neurons were completely dependent on viperin for ifn-mediated inhibition of tbev, whereas in astrocytes, in which the ifn-mediated antiviral activities are strongly dependent on viperin, other isgs could partly compensate for loss of viperin [ ] . taken together, viperin has been shown to be an isg that strongly inhibits tick-borne flaviviruses in vivo and in vitro. this strong antiviral effect on tbev is mediated by targeting the virus at multiple steps of the life cycle. interestingly, viperin targeting the synthesis of plus-sense rna is dependent on the sam domain or the c-terminal domain, while the n-terminal domain of viperin is responsible for interfering with virus assembly and release. in order to establish an infection, a pathogen needs to overcome or evade the innate immune response. to breach the very first line of defense, the skin-tbfvs use the tick to deliver the virus through the skin via tick saliva. furthermore, tick saliva contains immunomodulatory compounds that enhance viral transmission and dissemination [ , ] . although there are several mechanisms in which mosquito-borne flaviviruses actively suppress the induction of type i ifn, no such mechanism has been identified in tick-borne flaviviruses so far [ ] . instead, tbev, like other flaviviruses, utilizes a passive evasion mechanism in which the virus hides its dsrna intermediates in vesicular structures inside the er membranes, and thus delaying the recognition by the cytosolic rig-i like receptors and subsequent irf phosphorylation and ifn induction ( figure a ) [ , , ] . the most conserved inhibition of the type i ifn system within the flaviviridae family is the antagonism of ifnar signaling carried out by ns ; this mechanism is conserved between several mosquito and tick-borne flaviviruses [ , [ ] [ ] [ ] [ ] [ ] . in lgtv infection, ns was shown to inhibit the jak-stat pathway and ns was shown to interact with the ifnar receptor [ ] . similarly, it was shown that ns of tbev interacts with scribble (hscrib) which mediates ns localization to the plasma membrane and this interaction enables ns to inhibit type i and type ii ifn mediated jak-stat signaling [ ] . knockdown of hscrib altered ns cellular localization and reversed the inhibition of the jak-stat signaling [ ] . further studies revealed that ns of tbev inhibited the cell surface expression of ifnar by binding to prolidase (pepd) [ ] . pepd is a peptidase that is needed for ifnar maturation and subsequent cell surface expression. ns binding of pepd prevented maturation of complex n-linked oligosaccharides on ifnar , which in turn disrupted its surface expression ( figure b ) [ , ] . in kfdv infection, ifn treatment failed to reduce viral titers when added after infection, this effect was also found to be mediated by the ns proteins antagonism [ , ] . during tbfv infection, a subgenomic noncoding rna is formed, called subgenomic flavivirus rna (sfrna) [ , , ] . it is produced as a product of incomplete degradation of genomic viral rna by cellular - exoribonuclease xrn [ ] . the ability to produce sfrna in wnv was shown to be needed for efficient viral growth in vitro and for pathogenicity in mice [ ] . interestingly, denv sfrna was found to bind to trim to inhibit rig-i-induced type i interferon expression in huh- cells [ ] . furthermore, denv and wnv sfrna was found to suppress the rnai response in both mammalian and insect cells [ ] . similarly, in tbev infection, sfrna has been demonstrated to inhibit the antiviral rnai response in tick cells [ ] . pepd is needed for maturation and subsequent transport of ifnar to the plasma membrane. ifnar and ifnar heterodimer on plasma membrane can be activated by ifnα/β which leads to the signaling cascade and phosphorylation and translocation of stat / -irf into the nucleus and upregulation of isgs. right panel: ns interferes with ifn signaling. ns protein interacts with pepd thus preventing ifnar plasma membrane localization (red t). ns also prevents stat phosphorylation (red t). arrows indicate protein transport. even though recent studies have shed light on the role of innate immunity during tick-borne flavivirus infection, much remains unknown. for example, the role of the tlr-trif and tlr-myd pathway in pathogenesis and viral recognition. furthermore, most studies were performed using lgtv and tbev, and although they are closely related to powv, liv, kfdv, and ohfv, their interactions with the innate immune response might differ. although ifn has been shown to strongly control viral tropism and pathogenesis of tick-borne flaviviruses, few antiviral isgs have been identified. viperin has been shown to be the most important isg in cortical neurons, however, other isgs that target tbev expressed in astrocytes and granular cell neurons are yet to be identified and very little is known about the early response against the hemorrhagic tbfvs. we also know very little about how the innate immune response regulates the neuroinvasion of neurotropic tbfv, and the specific interactions between the tick vector and the different viruses. emergence 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we would like to acknowledge yong-dae gwon for constructing figure , and harry tracy for critically reading the final version of the manuscript. the authors declare no conflict of interest. key: cord- -d ycc ci authors: romero-brey, inés; bartenschlager, ralf title: viral infection at high magnification: d electron microscopy methods to analyze the architecture of infected cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: d ycc ci as obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. for the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (em) methods are extremely helpful. while conventional em has given important information about virus-host cell interactions, the development of three-dimensional em ( d-em) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. during the last years several d-em methods have been developed. here we will provide a description of the main approaches and examples of innovative applications. ( -( -hydroxyethyl)- -piperazineethanesulfonic acid) and pipes (piperazine-n,n i -bis( -ethanesulfonic acid)) are, for instance, among the most widely used. (em) . adherent cells can be either chemically fixed with aldehydes (a) or fast frozen by high-pressure freezing (hpf) or plunge/jet freezing (b). (a) after chemical fixation, infected cells can be further processed as a cell monolayer (grown on glass coverslips) or be pelleted prior to further processing for em. post-fixation is done with heavy metals (e.g., osmium tetroxide (oso ) and uranyl acetate (ua)), samples are dehydrated with increasing concentrations of an organic solvent (e.g., ethanol or acetone) and embedded into a plastic resin (plastic-em; highlighted in green). coverslips must be removed from the polymerized resin block by successive immersions in liquid nitrogen and hot water; (b) for rapid immobilization of cells by hpf, they must be grown as monolayers on sapphire (or aclar, not shown) discs, which are clamped in-between two aluminium planchettes and then loaded into a hpf machine for rapid freezing. alternatively, cell pellets are directly placed into the aluminium planchettes or into capillary tubes (not shown) for freezing. frozen cells can be subjected to freeze substitution (fs), dehydration with an organic solvent and resin embedding (plastic-em; highlighted in green). alternatively, high-pressure frozen cell pellets can be further analyzed by cemovis (cryo-electron microscopy of vitreous sections) (cryo-em, highlighted in violet). cells growing on em grids can be also plunge/jet frozen and analyzed directly by cryo-em (highlighted in violet). both cryo-em approaches, which do not require further processing of the cells, allow visualizing cells in their closest-to-native status. further details about these approaches can be found in the main text. unfortunately there is not a general recipe for chemical fixation. the best results require adaptations to individual experimental conditions. therefore, we recommend consulting an em specialist or check the literature to help you to choose the optimal conditions (including type and concentration of the aldehyde; type, concentration and ph of the buffer; time and temperature for fixing), tailored for your experiments. upon fixation cells are washed with the buffer of choice, in which cells can be stored at ¥ c until further processing. note that cultured cells can be prepared for em as monolayers or as pellets ( figure a ). when the cells are further processed as pellets, they must be scraped off the cell culture dish after fixation if they are not growing in suspension. alternatively, for cryo-em cultured cells can be "trypsinized" before fixation ( figure b ). it should be kept in mind that pelleting of the cells alters their morphology and can lead to artifacts ( figure ) . therefore -whenever possible-we highly recommend the use of cell monolayers grown on flat supports such as coverslips (to be used for chemical fixation; figure a ,b), sapphire discs (for high pressure freezing; figure c ) or on em grids (for cryo-em; figure ). impact of different fixation methods on the morphology of double membrane vesicles (dmvs) induced by hepatitis c virus (hcv). (a) huh cells grown on sapphire discs were infected with hcv and processed after chemical fixation by high pressure freezing (hpf) and freeze substitution (fs); (b) huh cells were transfected by electroporation with a subgenomic hcv replicon rna and h later subjected directly to hpf-fs without chemical fixation. dmvs were also found in high abundance in these samples excluding that they are an artifact caused by chemical fixation. also note the minimal extraction of the cytosol surrounding the dmvs and their content in comparison to chemically fixed cells; (c) hcv-infected huh . cells grown on coverslips were chemically fixed and then embedded in epon; (d) huh . cells were infected with hcv and h later cells were fixed, scraped off the culture dish and sedimented by gentle centrifugation prior to embedding of the cell pellet in epon resin. note the "fried-egg"-like morphology of the dmvs observed in chemically fixed cells (c,d), in comparison to the very well-delineated membranes of cells subjected to hpf (a,b). em micrographs were taken with permission from romero-brey et al. [ ] . the main drawback of chemical fixation is that it alters the structure of the cell by forming a network of cross-linked molecules. such a network is prone to artifacts, which is a major challenge for most em-based studies (for a detailed discussion on this topic see [ ] ). nevertheless, chemical fixation has been a mainstay of em for decades as it preserves the cell morphology reasonably well. indeed dubochet and others [ , ] have shown that the major organelles of well-studied cells look essentially the same in chemically and non-chemically fixed cells. along these lines, the overall appearance of the hepatitis c virus (hcv)-induced double membrane vesicles (dmvs) is strikingly similar between specimens prepared with different methods (figure ) [ ] . as already pointed out by small years ago [ ] , the majority of ultrastructural alterations might occur during the post-fixation processing of the samples for embedding (described below), rather than during fixation. thus, a tailored protocol must be designed for the highest preservation of cells/tissue of interest, including both optimal fixation and post-fixation conditions. freezing techniques represent an alternative to the artifact-prone chemical fixation (reviewed in [ ] ). the basic principle is to arrest cells by rapid cooling, a process that takes a few milliseconds, resulting in the simultaneous stabilization of all cellular components without altering their environment. the simplest method consists of immersing cells growing on em grids in liquid ethane or propane, by means of plunge [ , ] and jet freezing [ ] [ ] [ ] [ ] [ ] , respectively ( figure b ). an inherent limitation of these rapid cooling approaches is that samples can only be vitrified to a depth of micrometers from their surface [ ] [ ] [ ] . this lies in the poor heat conductance of water: high superficial cooling rates rapidly decay within the sample, reaching a low value that causes water crystallization [ ] [ ] [ ] . ice crystals alter the cytoplasm ultrastructure by inducing phase segregation between water and solutes [ , ] . even worse, growing ice crystals might lead to the formation of holes in membranes and destroy organelles [ ] . a way of preventing ice formation is pre-incubating the biological samples with anti-freeze agents to reduce the concentration of free water, such as sucrose, glycerol, dmso or various polymers. however, the use of these cryoprotectants introduce alterations in the original cytoarchitecture [ ] . thus, the unique means to preserve cells in its native state in absence of cryoprotectants is to freeze them in such a way that the water of the living cells turn into vitreous ice [ ] . this can be achieved by high pressure freezing (hpf) [ ] , with which the vitrification depth can be increased more than -fold (up to µm) in comparison with plunge and jet freezing ( [ , ] , reviewed in [ ] ). using high pressure (~ bar) prevents the expansion of water, lowering its freezing point, increasing the freezing rate and reducing the crystallization rate of ice (reviewed in [ ] ). note that, however, for highly hydrated tissues or cell suspensions, vitrification may require indeed the use of cryoprotectans prior to high pressure freezing (e.g., soaking the samples in a non-penetrating cryoprotectant such as % dextran (w/v); [ ] ). in the case of hpf, owing to the high pressures used for freezing, cells must be grown on resistant supports like sapphire [ , ] or aclar [ ] [ ] [ ] discs ( figure b ). sapphire discs are normally carbon coated to improve cell attachment and this carbon coat serves as a predetermined breaking layer after sample embedding into resin [ ] . sapphire/aclar discs can be subsequently frozen by assembling them between two aluminium planchettes. pelleted cell suspensions resuspended in e.g., dextran, can be also placed between two planchettes for freezing. alternatively pellets can be frozen in small tubes as described by hohenberg et al. [ ] , where the cells are loaded by capillary forces. handling of infectious specimens such as viruses requires working under strict biosafety level (bsl) conditions, which is particularly important when working with human samples. since equipment for sample preparation and em analysis is rarely available in high containment biosafety laboratories, samples must be inactivated before leaving these facilities, which is achieved by chemical fixation. note, however, that few bsl laboratories in the world are equipped with freezing devices, as well as with cryo-microscopes (e.g., [ ] ), where examination of virus-infected cells can be performed in their most genuine state. chemically inactivated samples can be also subsequently subjected to cryo-fixation outside the bsl laboratory. although this double-fixation sounds redundant, as reported for the flock house virus (fhv) [ ] this combination can result in an even better preservation of the subcellular structure as compared to samples preserved by chemical fixation alone. along the same lines, we have also recently shown that this fixation technique leads to an excellent preservation of the dmvs induced by hcv [ ] (figure ). in fact, ga fixation prior to hpf (figure a ) resulted in a preservation of the dmvs close to that found in cryo-fixed cells ( figure b ) and was superior to that obtained by conventional chemical fixation alone ( figure c ,d). note that this hybrid method provides a more near-to-native morphology of the dmvs, depicting well-delineated membranes, compared to the "fried-egg"-like dmvs observed within chemically fixed cells. it is important to bear in mind, however, that this "fried-egg" appearance might be due to the performance of the dehydration at room temperature, instead of at ¥ c, that it has been shown to reduce the loss of lipids [ ] and might, therefore, result in attaining smoother membranes. this alternative dehydration protocol has been successfully used, for instance, to study the rubella virus factories [ ] . in our hands this hybrid method also caused a slight extraction of the cytosol, thus enhancing visibility of the membrane layers ( figure a ). for all these reasons, we recommend to use the combination of these methods rather than chemical fixation alone. however, a note of caution should be added that aldehyde pre-fixation might change the morphology of some cell organelles as previously reported [ ] . after chemical fixation, cells must be further processed in order to analyze them by em. due to their low electron scattering power biological samples are inherently of low contrast [ ] . therefore, heavy metals like osmium tetroxide (oso ) and uranyl acetate (ua), with high affinity to many cellular structures, are used after fixation as contrasting agents in routine em. owing to its reactivity with unsaturated acyl chains of membrane lipids oso , for instance, facilitates the retention of lipids [ , ] , in addition to its role in contrasting structures (especially membranes). similarly, silva et al. [ ] have shown that ua plays a role in protecting lipids against solvent extraction. furthermore, oso also acts as a protein fixative [ ] . however, when used at room temperature acts more like a protease [ ] . for this reason it is highly recommendable to post-fix the samples with low concentration of oso on ice (at ¥ c) [ ] . note also that due to the harmful effect of oso on antigens [ ] , osmicated cells can still be used for subsequent immunocytochemical approaches [ ] , but it is not the first choice. subsequently cells are embedded in resins. conventional plastic embedding requires resins with heat-induced (above ¥ c) polymerization such as epoxy resins, which are often used since their first introduction in the s [ ] . due to their extremely hydrophobic nature cells/tissues must be completely dehydrated in a series of ascending protein denaturing solvents (e.g., ethanol) before infiltration ( figure a ). the resulting high degree of covalent interaction between epoxy resins and the biological material make these resins not well compatible for immunocytochemistry. nevertheless, owing to their fine structural preservation epoxy resins are an excellent embedding medium for high-resolution structural preservation and d analysis. after cryo-immobilization, cells can be stored in liquid nitrogen (ln ) or immediately further processed by means of freeze substitution (fs) ( figure b ) (recently reviewed in [ ] ). in this case, frozen cells are put into a mixture of cross-linking chemicals (such as aldehydes, oso , ua or tannic acid) with an organic solvent (acetone, ethanol or methanol) at temperatures around ¡ ¥ c [ , ] . it has been also reported that membrane visibility can be improved by adding %- % water to the substitution medium [ ] . subsequently the embedding medium is introduced with the solvent, while the temperature is allowed to increase gradually. the initial polymerization of the resin is usually carried out at sub-zero temperatures. the idea behind this is that low temperatures tend to stabilize proteins during the removal of the solvent (i.e., water) minimizing putative dehydration effects [ ] and thereby protecting proteins from denaturation. for this reason low viscosity resins (e.g., lowicryl, lrwhite and lrgold resins) which enable infiltration at very low temperatures, are mostly used. these resins also have a low tendency to co-polymerize with the sample structure [ ] and are able to preserve the fluorescence, and are therefore, mostly used for immunocytochemistry [ , ] and correlative light electron microscopy (clem) [ , ] studies. after complete infiltration of the resin, the final polymerization step can be induced at room temperature with uv light ( nm). nonetheless, epoxy resins can be also used to embed freeze-substituted samples. in this case, the samples should be further dehydrated at room temperature after the substitution to be subsequently embedded and polymerized at high temperatures. in any case, long periods of infiltration are characteristic of the fs approach, which might be a practical disadvantage. however, shorter substitutions can be also carried out with satisfying results [ , , ] . for a comparative study about the use of different resins to embed cells for d-em, please see a very recent report from bruno humbel's laboratory [ ] . in order to be analyzed by tem, resin-embedded cells must be sectioned ( figure ). this is due to the inability of the electrons to penetrate the entire embedded cell. to this aim the block face containing the embedded cells must be trimmed to a smaller area (regularly~ ¢ µm; note, however, that the size of the section is limited by the size of the . mm round standard em grid and, therefore, the trimming could be made for a larger area when needed), which can then be sectioned with a diamond knife at an angle of ¥ to minimize squeezing artifacts. routine ultrathin sections ( - nm) are obtained and remain floating on the surface of the water shank of the diamond knife, from where they are collected onto an em grid. note that after acquiring the sections, they normally need to be contrasted before they can be visualized by tem. the most commonly used methodology consists of post-staining grids with ua and lead salts [ ] . however, when using the fs cocktail proposed by walther and ziegler [ ] , due to their higher contrast, sections can be directly observed by tem, without requirement for on-section staining. to its sectioning with a diamond knife equipped with a water shank; (b) the obtained ultrathin sections ( - nm, in yellow) float on the water surface from where the section ribbon can be mounted onto a formvar-coated em grid; (c) after contrasting with heavy metals, the cell sections can be examined with a conventional transmission electron microscope (tem) operated at accelerating voltages between - kv. in brief, an electron beam is generated (by a thermionic or a field emission gun) and accelerated under vacuum. the electrons are then transmitted through the cell sections. after passing through the specimen, scattered electrons are focused by electromagnetic lenses and magnified onto an imaging device such as a fluorescent screen or recorded with a digital camera (ccd, charged-coupled device, or a cmos, complementary metal-oxide semiconductor). the diagram of the tem-working principle shown on the right is adapted from briggman and bock [ ] . as stated before, the ultrastructural preservation of the cells is not only influenced by the method of fixation, but also by the subsequent processing steps (dehydration, infiltration and resin polymerization). as a consequence of the denaturing effects of these processing steps the structures are rarely preserved to the extent as they appear in vivo. to avoid these artifact-prone preparation procedures, the method of choice is the freezing or vitrification of the cells (described above) followed by their direct examination at very low temperatures by cryo-em. however, due again to the limited penetration power of electrons, only the thin cell periphery of frozen cells can be visualized by standard cryo-em [ ] . alternatively frozen cells must be sectioned in order to be observed by tem. hence frozen-hydrated sections can be analyzed by the so-called cryo-electron microscopy of vitreous sections (cemovis). in cemovis high pressure-frozen specimens ( figure b ) are cut into ultrathin sections, as described below for resin-embedded specimens (figure ), but at temperatures below ¡ ¥ c (below the de-vitrification temperature to avoid sample damage). this technique was first started with the work of fernandez-moran [ ] and was further improved among others by dubochet and coworkers [ , , ] , frederik and coworkers [ ] [ ] [ ] and sitte [ ] . more recently improvement of cryo-microtomy techniques have resulted in cemovis as a much more accessible approach ( [ ] , reviewed in [ ] ), despite of its critical challenging aspects: obtaining good quality sections (without knife marks, crevasses or ripples, produced during sectioning) that remain attached to the em grid [ ] and its subsequent imaging at low temperatures and also low electron doses. obviously, the information that we can obtain from ultrathin sections of a certain region of interest (roi) of an infected cell is limited. since ultrathin sections with a common average size of ¢ µm contain normally the profiles of several cross-sectioned cells, this limitation can be overcome even by conventional tem when analyzing intracellular events that occur with high frequency. moreover, even in case of relatively low rates of infection it is possible to find infected cells among these cell profiles when the virus-induced phenotype is rather striking and known. however, interpreting the d organization of a structure from such d sections is difficult and most often leads to a "simplification" of much more complex structures [ ] . furthermore, depending how these objects are sectioned conflicting interpretations can arise [ ] . thus, for a correct interpretation of a d structure all components must be recognizable in the section [ ] . therefore, gaining access to the ultrastructural information contained in thick sections or -ideally-in the whole volume of an infected cell is essential. several approaches have been developed to gain d information of cellular structures by em ( figure ). in the following sections these approaches and their applications to study viral infection (summarized in table ) are described. the thickness of the section of virus-infected cells is a limiting factor, especially in case of small structures such as virions that are often difficult to find. to overcome this problem thicker sections (~ nm) can be analyzed by means of electron tomography (et) ( [ ] [ ] [ ] ) ( figures a and ) . et was first used to gain structural information on viral particles [ , ] . it is based on the principle defined by radon in his seminal paper of [ ] : d information (a tomogram) can be retrieved from the projections of an object. the penetration depth of electrons through an object is directly correlated to the energy of the electrons and thus, the higher their accelerating voltage, the thicker the objects that can be analyzed [ ] . therefore the acquisition of tomograms requires not only a microscope equipped with a goniometer (that allows tilting the em grid containing the sections by ¥ - ¥ in either direction), but also requires high voltage microscopes (operated at - kv) ( figure ). the d projections of the objects obtained at different tilt angles are then reconstructed in silico to retrieve the d volume of the cell ( figure ). tilting the specimen in only one orthogonal axis (single-axis tomography) results in the so-called missing-wedge information. to partially overcome this problem the specimen can be tilted around two orthogonal axes (dual-axis tomography). the two tomograms are then computed from each tilt series and combined with general d linear transformations that can correct for distortions between the two tomograms [ , ] . although this method does not allow gaining access from all the possible tilt angles, it reduces at least the missing-wedge to a missing-pyramid, resulting in a gain of d information. despite of being a very powerful and widely used technique, the information obtained through et is from a small percentage of the whole cell volume. to obtain information from larger volumes, tomograms from consecutive sections can be joined (serial et) [ , ] (figure c ). however, due to compression happening during sectioning or material collapse occurring during electron beam exposure [ ] , which might lead to a loss of information within and between sections, reconstruction of a large volume from several tomograms is challenging. other approaches may be used instead (see below). et has become an important method for virologists and was used e.g., to visualize the remodeling of intracellular organelles and the distribution of virus particles within the host cell (reviewed in [ ] ). in fact, most of the literature pertains to the use of this approach ( table ). the bulk of these studies relates to virus-induced remodeling of cell membranes in order to build up their replication organelles, mostly positive-strand rna viruses (reviewed in [ ] ). an example of the et analysis of hcv-induced replication factories is shown below ( figure a ). [ ] . on the left: digital slice derived from stem tomography of nm thick section; on the right: d surface rendering revealing that the membrane assembly region consists of an elaborate membrane network; (c) serial sectioning of cytomegalovirus (cmv)-infected cells [ ] . on the left: single micrograph (in x-y direction) and image stack (slice through the z-axis) of the assembly complex; on the right: cellular and viral structures were segmented on all sections and superimposed on a single micrograph; (d) fib-sem of hiv-infected cells [ ] . on the left: d fib-sem image of an hiv- chronically infected h cell (left) and an uninfected astrocyte (right), co-cultured for h; on the right: d rendering of the fib-sem image stack containing the contact zone between the hiv-infected h cell and the astrocyte. the target cell extends long filopodial bridges towards the infected cell across the intercellular gap. hiv- virions are detected adjacent to the filopodial bridges; (e) cryo-et of herpes simplex virus (hsv)-infected cells [ ] . on the left: slice of the tomogram, showing secondary envelopment of capsids; on the right: d surface rendering of the whole tomogram, showing a capsid in close proximity to an enveloping vesicle. asterisks: capsids; white arrow: enveloping vesicle; arrowhead: glycoproteins; black arrows: tegument; pm: plasma membrane. all these pictures are reproduced from the original publications with permission (see acknowledgments). apart from serial et, another way of circumventing the limited thickness of et is the use of scanning transmission electron microscopy (stem) allowing the collection of tomographic datasets (figures and ) . the main advantage of using the stem modus is its scanning geometry that allows for dynamic focusing so that the imaging conditions are uniform across a tilted specimen [ ] . furthermore, its use for tomography of thick, plastic-embedded sections has revealed an improved signal-to-noise ratio (snr) and far higher contrast over conventional tem tomography [ ] [ ] [ ] [ ] . by means of stem tomography specimens up to µm can be examined [ ] [ ] [ ] ( figure b ). however, since the total thickness of a cell is - µm or more, the information retrieved from µm thick sections is still quite limited. this approach is ideal for the study of large cellular organelles or viruses, which size exceeds the thickness achieved by conventional et. thus, stem tomography has been applied to the study of large dsdna viruses, like the giant mimivirus [ ] ( figure b , table ), as well as to dissect the d architecture of the viral factories induced by african swine fever virus (asfv) [ ] or paramecium bursaria chlorella virus (pbcv- ) [ ] (table ) . the thickness limitations of the above-described methods can be overcome by analyzing consecutive thin sections (serial sections) of cells. serial sectioning, in use since [ ] , can be performed to the entire cell volume and provide, therefore, information about its architecture. it has the main advantage that it does not need special equipment, being practicable at any em facility ( figure d ). however it requires well-trained personnel and patience to acquire a long ribbon of sections of the cell of interest, without losing a single section. micrographs obtained either by tem or scanning em (sem) must be taken from every section and be aligned to obtain a stack of images (figure ). this technique can be also applied to study the anatomy of relatively large organisms like nematodes. hence, as a result of the reconstruction of about or more than micrographs [ , ] , respectively, the structure of the worm nervous system was successfully attained. a similar method, called array tomography has been developed in which arrays of serial ultrathin sections collected in glass slides are first fluorescently labeled (with antibodies or stains) and subsequently imaged by confocal microscopy to get a d distribution of antigens [ ] . interestingly the array can be repeatedly eluted, re-stained and imaged again to assess the distribution of other antigens. finally it can be stained with heavy metals and analyzed under a sem microscope. this combination method allows the correlation of volumetric imaging of antigens with ultrastructural imaging. the main drawback of this technique, however, is discontinuities between two consecutive sections due to compression artifacts generated by the sectioning or more dramatically the loss of sections. furthermore, these distortions of sections occurring during cutting, staining and imaging [ , ] hinder the subsequent alignment step, resulting in a poor resolution in the z-plane. to avoid the laborious and prone to errors manual sectioning, sections can be collected automatically for sem imaging on electron-opaque plastic tapes (atum, automatic tape-collecting ultramicrotome) [ ] . subsequently images of atum sections can be also taken in an automatic manner [ ] , allowing for high throughput image acquisition. atum-sem allows to reliably cut thousands of sections as thin as nm. figure . serial sectioning workflow. a ribbon of consecutive ultrathin sections ( - nm) of a cell can be either obtained manually (a) or automatically on a tape (atum, automatic tape-collecting ultramicrotome) (b) and subsequently analyzed by tem (c) or sem (d), respectively. generated micrographs are aligned to obtain a z-stack of this cell. note also that the same principle is used to join tomograms obtained by serial-et. although labour intensive, manual serial sectioning has been used to study virus-infected cells by means of tem (described in [ , ] ), including vaccinia virus, bunyavirus, hcv, cytomegalovirus, reovirus and a plant virus (table ) . thus, the distribution of envelopment events within the human cytomegalovirus (hcmv)-induced assembly complexes could be visualized using this technique [ ] . to this aim a total of sections with a thickness of nm were imaged, leading to a total volume of . µm ( figure c ). another example relates to a plant virus [ ] : sections (with nm thickness; a total of . µm) of cells containing cylindrical inclusions (cis) revealed that these structures form long tubes throughout the cytosol. furthermore, serial sections of varicella-zoster (vzv)-infected cells have been analyzed by sem [ ] , which allowed the reconstruction of a total nuclear volume of µm from a ribbon of consecutive serial sections with a nm thickness. creating reconstructions of cells from sections whose thicknesses exceed the limit of the penetrating power of tem can be overcome by the use of a scanning electron microscope (sem) to collect information of the block face upon removal of very thin sections ( figure e ). the working principle of these approaches is the same as with serial sectioning. however, in contrast to serial sectioning, with serial block face-sem (sbem, formerly called sbf-sem by w. denk [ ] ) and focus ion beam (fib)-sem ( figure ) the sectioning is integrated inside the sem microscope and carried out in a fully automated manner with the help of a diamond knife [ ] or a focused ion beam ( [ , ] , and others), respectively, that act as nano-scalpels. once a thin slice is made ( - nm) the block face is imaged with sem using low accelerating voltage. in this sem technique, backscattered electron detection is normally used to specifically view heavy metal-stained cellular components. however, it has been recently shown that the resolution of this approach is considerably improved when a secondary electron signal is used [ ] . . epon blocks containing the embedded cells are mounted on sem stubs with silver paint. first a trench is milled with the fib, generating a cross-section into the epon block. subsequently the newly generated block face is milled (with the fib, blue), resulting in the removal of a thin layer (as small as nm; [ ] ) and imaged (with the sem, pink) in a sequential manner. this process is repeated automatically as long as needed to obtain a tomographic dataset. further details about this method can be found in the main text. after each image is taken, a new thin slice is removed from the block face that is again imaged. the sequential "slicing and imaging" process can be repeated ad libitum allowing to produce a stack of images that can be computationally combined into a d reconstruction [ ] (figure ). the lack of compression artifacts allows a more accurate merging of sequential images to obtain large datasets. after contrast reversal the images look similar to traditional tem micrographs. the cells can be prepared as for tem. note only that for fib-sem harder and more stable resins like durcupan are recommended, to prevent damage of the sample while slicing with the powerful ion beam. an improved protocol for cell preparation to be analyzed in this way has been recently published [ ] . the embedded cells must be subsequently trimmed and mounted on a sem specimen stub with silver paint (figure ). finally, due to the non-conductive nature of biological cells, the whole specimen must be platinum or gold alloy coated to improve conductivity. in comparison with the cumbersome manual acquisition of serial sections, these approaches are far less laborious. the z-resolution of these techniques is defined by the thickness of the slices, which represents a pitfall in the case of sbem. however, recent developments in the fib-sem field allow imaging larger field of views and a section thickness of nm [ ] allowing the z-resolution of fib-sem to approach that of et. furthermore, the dual beam instrument permits targeted, site-specific imaging, which makes it ideal for correlative microscopy approaches. however, fib-sem has also some limitations: it requires a quite expensive specialized instrument and the sections cannot be re-examined again. furthermore, processes like charging and electron beam damage can result in unsuccessful imaging of the surface of interest [ ] . to our knowledge, sbem has not been applied yet to the study of viral infection. however, fib-sem, also called ion abrasion (ia)-sem, has been extensively employed for the study of hiv release and spread ( figure d ) [ ] [ ] [ ] and, more recently, also to the study of a chlorovirus [ ] ( table ) . an alternative to the analysis of resin-embedded cells (plastic-em) is the analysis of unstained, frozen-hydrated cells (cryo-em), in which the contrast is formed exclusively by the density of the biological material itself [ , ] (figure ) . however, vitrified specimens have low inherent contrast (that can be partially overcome with the use of better detectors) and are electron-dose sensitive. due to these challenging aspects, cryo-em is not as widely used as plastic-em, but in returns yields datasets with very high resolution, disclosing molecular details of the cellular landscape. as for resin-embedded cells, thick sections of frozen cells can be collected on a grid and subsequently analyzed by et. this technique, known as cetovis (cryo-electron tomography of vitreous sections) or tovis (tomography of vitreous sections), was first successfully used in ( [ ] [ ] [ ] [ ] [ ] ) ( figure f ). cryo-ultramicrotomy has shown its capability in producing samples of vitrified cells and tissues for visualization by cryo-tem and tomography [ ] [ ] [ ] . however, sectioning at cryogenic temperatures is notoriously difficult and artifact-prone: sections inevitably suffer from adverse distortions caused by the harsh mechanical interactions of a diamond knife and a moving specimen block during the cutting process [ ] . these technical limitations have prevented both cetovis, but also cemovis (described above) from becoming more commonly used approaches [ ] . cryo-fib represents an emerging alternative technique for "thinning" frozen biological specimens without the above mentioned limitations of cryo-ultramicrotomy [ ] . thus it has been recently shown that cryo-fib milling of frozen (both plunge and high pressure frozen) cells can produce homogeneously thin ( - nm), distortion free lamellae for cryo-electron tomography (cet) [ ] [ ] [ ] (figure g ). cet, following the same working principle as plastic-et, can be applied to entire vitrified cells ( figure b ), avoiding the arduous cutting step (reviewed in [ , , ] ). cet is routinely performed with plunge frozen cells [ ] (figure h ). although this enables in theory the analysis of the whole cell volume, cet can only be applied to specimen areas with a maximal thickness of~ µm, the penetration limit of electrons [ ] . therefore though many organelles and subcellular structures can be imaged directly in plunge-frozen adherent cells, in practice it is restricted to visualize the thin edges of the cell. after its first use in to address structural details of pathogen-host cell interaction [ ] , several virological studies based on this method have been reported (reviewed in [ ] ) (table ) with the herpesvirus life cycle being the most comprehensively studied [ ] . one example is the study of the envelopment of herpes simplex virus (hsv) particles [ ] (figure e ). stem tomography, as described many years ago already, can be also applied to unstained vitrified specimens [ , ] . as for standard stem tomography (described above), the increase in acceptable specimen thickness of cryo-stem tomography (also called cstet) broadens its applicability ( figure i ). due to the weak electron scattering properties of light elements, the main components of biological, unstained vitrified specimens are, in principle, not the ideal candidates for stem imaging. however, scattering is not so much weak as directed to low angles, and the spot scanning of stem can be turned into an advantage regarding sample damage [ ] . thus this approach provides information from nm frozen eukaryotic cells with a microscope operated at kv [ ] . working with higher voltage microscopes should then enable the analysis of even thicker samples. as stated by wolf and colleagues [ ] apart from the shared advantages with stem tomography of resin-embedded samples (described above), one of the main practical advantages of cstet in comparison to cet is the lack of a need for defocusing to generate contrast, thus eliminating the ensuing complications. to observe thicker samples under cryo-conditions without the need to generate sections, soft x-ray cryo-tomography can be also used ( [ ] , reviewed in [ ] ). it is a powerful method that takes advantage of the high penetration power of x-rays (up to µm) [ , ] without using any fixative or contrasting reagent [ , ] (figure j ). its intermediate resolution (typically of the order of nm, between light and electron microscopy) [ , ] in comparison with the high resolution of cryo-et, has limited its use when fine ultrastructural details are needed. however, nm spatial resolution has been already attained with this method [ ] . this technique has provided further insights into the vaccinia virus and herpesvirus infection cycles [ , ] (table ) . fib-milling can be also used (as for resin-embedded cells, described above) for serial block imaging of (high-pressure)-frozen hydrated specimens in the sem, generating large volume data for d analysis in an automated fashion [ ] ( figure k ). the lack of compression artifacts associated with cutting allows a more accurate merging of sequential images to get large datasets. another advantage of this method, a common feature to all the approaches described above with the exception of cetovis, is its speed: imaging can start immediately after freezing. however, it is important to keep in mind that prolonged electron beam irradiation should be avoided to prevent modifications occurring on the sample surface. given the challenging aspects of these cryo-methods, to our knowledge only cet and x-ray cryo-tomography have been used so far to study virus-infected cells (table ) . d-em-based techniques follow the same principle: cells are "chopped" into sections of different thickness from which we obtain a number of d projections that are assembled into a d reconstruction of the biological object. exceptions are cryo-et and soft x-ray cryo-tomography, where entire cells are imaged, although limited to thin areas. in this regard, most of the methods described here are semi-destructive methods (e.g., et, stem tomography, serial sectioning), because the sections can be analyzed again at a later time point (e.g., at different magnifications or with another type of microscope) or even further used for immunocytochemical studies (serial sectioning), with the exception of sbem or fib-sem, in which the slices are completely destroyed. however, the major advantage of the destructive sequential methods is that they do not suffer from warping, folding or loss of sections that can significantly affect the data quality and its subsequent analysis. foremost among the recent studies are those based on et, which are increasingly used to elucidate the d architecture of virus-infected cells. fib-sem represents the most modern technique, although resolution in the z-plane is still lower than with et. however, the possibility to reconstruct complete cell volumes in an automatic manner provides unprecedented insights into the morphology of inter-cellular contacts as well as large cell organelles and their remodeling upon virus infection. an important present/future direction is the use of multiple techniques to analyze the same sample. thus, correlative light electron microscopy (clem) bridges the gap between light and electron microscopy. in this way information gained by fluorescence microscopy, such as colocalization of distinct proteins or rare events occurring in cells can be visualized by em with high resolution, thus revealing the underlying subcellular structures. furthermore, with em we gain access to the so-called "space reference", i.e., not only the fluorescently labeled proteins are observed, but also their neighboring structures and even the whole cell. for all these reasons, clem is already an essential approach not only for virologists (e.g., [ , ] ), but for cell biology in general (reviewed for example in [ , [ ] [ ] [ ] [ ] ). along these lines, a recent multimodal approach named coin (correlation optical and isotopic nanoscopy) combines three different imaging techniques: super-resolution microscopy, nano-sims (secondary ion mass spectrometry) [ ] and em, to provide information about the isotopic composition of many organelles and subcellular structures [ ] . furthermore, recent strides of cryo-em (the so-called molecular microscopy) have shown that the molecular architecture of complexes can be resolved in their natural cellular environment, devoid of staining and chemical fixation artifacts. if multiple copies of these complexes are present the individual subvolumes containing them can be computationally averaged to improve the resolution to nm (recently reviewed by [ , ] ). furthermore, in combination with advanced computational methods, such as molecular identification based on pattern recognition techniques [ , ] , cryo-em is currently the most promising approach to comprehensively map the macromolecular architecture of proteins or proteins assemblies (attained by x-ray crystallography or nuclear magnetic resonance -nmr-spectroscopy) inside the cryo-em cellular tomograms (reviewed by [ ] ). in spite of all these advancements, the main drawback of em is its inability to study the dynamics of biological processes. as we have described in this review, cells must be fixed in order to be examined in the vacuum atmosphere of an electron microscope. however, some progress has been made towards observing the ultrastructure of living cells by means of in situ or environmental tem. with this technique, cells can be examined in their native liquid environment (reviewed in [ , ] . for instance, by using this technique it has been possible to observe the assembly of rotavirus particles in a microfluidic platform [ ] . the main advantage of this approach is that it circumvents the need for a dried, electron-conductive atmosphere. although one of the inherent limitations of in situ tem is that motion of the cells or viral particles in solution results in poor structural resolution [ ] , live cells engulfing nanoparticles have already been observed with this novel technique [ ] . therefore, we remain optimistic that em-based methods providing time-resolved d information will become available in the not too distant future. towards atomic resolution structural determination by single-particle cryo-electron microscopy the advent of near-atomic resolution in single-particle electron microscopy subunit interactions in bovine papillomavirus structure of the immature retroviral capsid at a resolution by cryo-electron microscopy nobel lecture. the development of the electron microscope and of electron microscopy diagnostic electron microscopy is still a 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the visibility of biological membranes is improved when the substitution medium contains water protein crystallography at sub-zero temperatures: cryo-protective mother liquors for protein crystals perspectives for achieving improved information by the observation of thin sections in the electron microscope a simple post-embedding system for the rapid demonstration of tissue antigens under the electron microscope influence of fixatives and embedding media on immunolabelling of freeze-substituted cells visualization of identified gfp-expressing cells by light and electron microscopy a single method for cryofixation and correlative light, electron microscopy and tomography of zebrafish embryos freeze substitution in hours or less correlative and integrated light and electron microscopy of in-resin gfp fluorescence, used to localise diacylglycerol in mammalian cells investigation of resins suitable for the preparation of biological sample for -d electron microscopy the use of lead citrate at high ph as an electron-opaque stain in electron microscopy volume electron microscopy for neuronal circuit reconstruction cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching electron microscopy of ultrathin frozen sections of pollen grains aspects of cryofixation and cryosectioning for the observation of bulk biological samples in the hydrated state by cryoelectron microscopy concerning the nature of the cryosectioning process surface defects on thin cryosections the ultrastructure of cryo-sections and intact vitrified cells-the effects of cryoprotectants and acceleration voltage on beam induced bubbling advanced instrumentation and methodology related to cryoultramicrotomy: a review cryo-electron microscopy of vitreous sections cryo-electron microscopy of vitreous sections improving the technique of vitreous cryo-sectioning for cryo-electron tomography: electrostatic charging for section attachment and implementation of an anti-contamination glove box identification of structure by the common-sense approach cryo x-ray nano-tomography of vaccinia virus infected cells three-dimensional analysis of a viral rna replication complex reveals a virus-induced mini-organelle electron tomography of nascent herpes simplex virus virions three-dimensional architecture of virus-packed tubule electron tomography of the contact between t cells and siv/hiv- : implications for viral entry sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum three-dimensional analysis of budding sites and released virus suggests a revised model for hiv- morphogenesis human t-lymphotropic virus- visualized at the virological synapse by electron tomography membrane remodelling during vaccinia virus morphogenesis composition and three-dimensional architecture of the dengue virus replication and assembly sites membrane rupture generates single open membrane sheets during vaccinia virus assembly association of rice gall dwarf virus with microtubules is necessary for viral release from cultured insect vector cells three-dimensional visualization of gammaherpesvirus life cycle in host cells by electron tomography the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex dengue virus-induced autophagosomes and changes in endomembrane ultrastructure imaged by electron tomography and whole-mount grid-cell culture techniques integrity of the early secretory pathway promotes, but is not required for, severe acute respiratory syndrome coronavirus rna synthesis and virus-induced remodeling of endoplasmic reticulum membranes electron tomography reveals the steps in filovirus budding structural evidence of glycoprotein assembly in cellular membrane compartments prior to alphavirus budding the transformation of enterovirus replication structures: a three-dimensional study of single-and double-membrane compartments three-dimensional analysis of the association of viral particles with mitochondria during the replication of rice gall dwarf virus ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral rna synthesis complex dynamic development of poliovirus membranous replication complexes a three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell lines three-dimensional architecture of tick-borne encephalitis virus replication sites and trafficking of the replicated rna infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes open membranes are the precursors for assembly of large dna viruses the west nile virus assembly process evades the conserved antiviral mechanism of the interferon-induced mxa protein ultrastructural characterization and three-dimensional architecture of replication sites in dengue virus-infected mosquito cells morphogenesis of endoplasmic reticulum membrane-invaginated vesicles during beet black scorch virus infection: role of auxiliary replication protein and new implications of three-dimensional architecture electron tomography of hiv- infection in gut-associated lymphoid tissue attenuated west nile virus mutant ns - qqa/ a/ a exhibits virus-induced ultrastructural changes and accumulation of protein in the endoplasmic reticulum three-dimensional visualization of the autographa californica multiple nucleopolyhedrovirus occlusion-derived virion envelopment process gives new clues as to its mechanism electron tomography analysis of tick-borne encephalitis virus infection in human neurons african swine fever virus assembles a single membrane derived from rupture of the endoplasmic reticulum membrane assembly during the infection cycle of the giant mimivirus virus-host interactions: insights from the replication cycle of the large paramecium bursaria chlorella virus the unique architecture of bunyamwera virus factories around the golgi complex d reconstruction of vzv infected cell nuclei and pml nuclear cages by serial section array scanning electron microscopy and electron tomography sequential biogenesis of host cell membrane rearrangements induced by hepatitis c virus infection reovirus forms neo-organelles for progeny particle assembly within reorganized cell membranes two and three dimensional characterization of zucchini yellow mosaic virus induced structural alterations in cucurbita pepo l. plants ion-abrasion scanning electron microscopy reveals surface-connected tubular conduits in hiv-infected macrophages d visualization of hiv transfer at the virological synapse between dendritic cells and t cells three-dimensional imaging of hiv- virological synapses reveals membrane architectures involved in virus transmission whole cell cryo-electron tomography reveals distinct disassembly intermediates of vaccinia virus native d intermediates of membrane fusion in herpes simplex virus entry cryo electron tomography of native hiv- budding sites cryo-electron tomography of marburg virus particles and their morphogenesis within infected cells cryo electron tomography of herpes simplex virus during axonal transport and secondary envelopment in primary neurons cryotomography of budding influenza a virus reveals filaments with diverse morphologies that mostly do not bear a genome at their distal end the bacteriophage t virion undergoes extensive structural remodeling during infection cryo-electron tomography: moving towards revealing the viral life cycle of rice dwarf virus visualizing virus assembly intermediates inside marine cyanobacteria electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of pathogen propulsion electron cryotomography studies of maturing hiv- particles reveal the assembly pathway of the viral core correlative vis-fluorescence and soft x-ray cryo-microscopy/tomography of adherent cells perspectives of molecular and cellular electron tomography electron tomography of molecules and cells structural studies by electron tomography: from cells to molecules reconstruction of three dimensional structures from electron micrographs electron microscopy of unstained biological material: the polytropic montage Über die bestimmung von funktionen durch ihre integralwerte längs gewisser mannigfaltigkeiten high-pressure freezing, cellular tomography, and structural cell biology double-tilt electron tomography dual-axis tomography: an approach with alignment methods that preserve resolution organization of interphase microtubules in fission yeast analyzed by electron tomography expedited approaches to whole cell electron tomography and organelle mark-up in situ in high-pressure frozen pancreatic islets a method for monitoring the collapse of plastic sections as a function of electron dose electron tomography of the supramolecular structure of virus-infected cells membranous replication factories induced by plus-strand rna viruses optimization of stem tomography acquisition-a comparison of convergent beam and parallel beam stem tomography z-contrast in biology. a comparison with other imaging modes stem tomography in cell biology stem tomography for thick biological specimens nanoscale d cellular imaging by axial scanning transmission electron tomography preparation of cryofixed cells for improved d ultrastructure with scanning transmission electron tomography development and application of stem for the biological sciences ultrastructure of retinal rod synapses of the guinea pig eye as revealed by three-dimensional reconstructions from serial sections the structure of the nervous system of the nematode caenorhabditis elegans system-wide rewiring underlies behavioral differences in predatory and bacterial-feeding nematodes array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits as-rigid-as-possible mosaicking and serial section registration of large sstem datasets fully automatic stitching and distortion correction of transmission electron microscope images automating the collection of ultrathin serial sections for large volume tem reconstructions imaging atum ultrathin section libraries with wafermapper: a multi-scale approach to em reconstruction of neural circuits three-dimensional visualization of virus-infected cells by serial sectioning: an electron microscopic study using resin embedded cells virus morphogenesis in the cell: methods and observations serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure site-specific d imaging of cells and tissues with a dual beam microscope focused ion beam-scanning electron microscope: exploring large volumes of atherosclerotic tissue three-dimensional imaging of adherent cells using fib/sem and stem multi-resolution correlative focused ion beam scanning electron microscopy: applications to cell biology focussed ion beam milling and scanning electron microscopy of brain tissue fib/sem tomography with tem-like resolution for d imaging of high-pressure frozen cells macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography mapping molecular landscapes inside cells electron tomographic analysis of frozen-hydrated tissue sections three-dimensional imaging of biological complexity electron tomography of bacterial chemotaxis receptor assemblies the molecular architecture of cadherins in native epidermal desmosomes molecular cryo-electron tomography of vitreous tissue sections: current challenges the mammalian central nervous synaptic cleft contains a high density of periodically organized complexes electron tomography of vitreous sections from cultured mammalian cells the three-dimensional molecular structure of the desmosomal plaque cutting artefacts and cutting process in vitreous sections for cryo-electron microscopy cryo-em-the first thirty years focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy focused ion beam micromachining of eukaryotic cells for cryoelectron tomography practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-tem tomography a focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms cellular nanoimaging by cryo electron tomography structure of complex viruses and virus-infected cells by electron cryo tomography a cool hybrid approach to the herpesvirus scanning transmission electron microscopy of unstained biological sections observations of unstained biological specimens using a low-energy, high-resolution stem high-resolution low-dose scanning transmission electron microscopy cryo-scanning transmission electron tomography of vitrified cells visualizing and quantifying cell phenotype using soft x-ray tomography cryo x-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging soft x-ray microscopes and their biological applications high resolution protein localization using soft x-ray microscopy a characterisation of dark-field imaging of colloidal gold labels in a scanning transmission x-ray microscope computed tomography of cryogenic biological specimens based on x-ray microscopic images x-ray tomography of schizosaccharomyces pombe soft x-ray microscopy at a spatial resolution better than nm volume imaging of cellular ultrastructure in native frozen specimens direct visualization of hiv- with correlative live-cell microscopy and cryo-electron tomography correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography bridging microscopes: d correlative light and scanning electron microscopy of complex biological structures correlated light and electron microscopy: ultrastructure lights up! nat multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism correlated optical and isotopic nanoscopy structural biology in situ-the potential of subtomogram averaging high-resolution cryo-electron microscopy on macromolecular complexes and cell organelles toward detecting and identifying macromolecules in a cellular context: template matching applied to electron tomograms identification of macromolecular complexes in cryoelectron tomograms of phantom cells cryo-electron microscopy-a primer for the non-microscopist electron microscopy of specimens in liquid in situ tem of biological assemblies in liquid visualizing viral assemblies in a nanoscale biosphere visualizing gold nanoparticle uptake in live cells with liquid scanning transmission electron microscopy the critical reading of this manuscript by rachel mellwig (embl, heidelberg, germany) is key: cord- -l lq yga authors: zhang, jing; kang, june; dehghan, shoaleh; sridhar, siddharth; lau, susanna k. p.; ou, junxian; woo, patrick c. y.; zhang, qiwei; seto, donald title: a survey of recent adenoviral respiratory pathogens in hong kong reveals emergent and recombinant human adenovirus type (hadv-e ) circulating in civilian populations date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: l lq yga human adenovirus type (hadv-e ), which is intriguingly limited to military populations, causes acute respiratory disease with demonstrated morbidity and mortality implications. this respiratory pathogen contains genome identity with chimpanzee adenoviruses, indicating zoonotic origins. a signature of these “old” hadv-e is the absence of a critical replication motif, nf-i, which is found in all hadv respiratory pathogens and most hadvs. however, our recent survey of flu-like disease in children in hong kong reveals that the emergent hadv-e pathogens circulating in civilian populations contain nf-i, indicating recombination and reflecting host-adaptation that enables the “new” hadv-e to replicate more efficiently in human cells and foretells more potential hadv-e outbreaks in immune-naïve civilian populations. special attention should be paid by clinicians to this emergent and recombinant hadv-e circulating in civilian populations. an increase in pediatric patients with influenza-like symptoms in queen mary hospital (july to october ) led to a question as to whether the putatively host-adapted human adenovirus type (hadv-e ) [ ] is circulating in hong kong. hadv-e was one of the first viral respiratory pathogens to be isolated ("r(espiratory)i(nfection)-(number) "; ) [ ] . it is intriguingly extraordinary in several aspects, the first being that it is one of only two adenoviruses for which a vaccine was developed, thrice ( , , and ), at considerable effort and expense, attesting to its potency (https: //www.historyofvaccines.org/content/blog/adenovirus-vaccines-reinstated-after-long-absence) [ ] . each vaccine cessation resulted in immediate reemergence as a major pathogen [ ] . second, hadv-e was typically and inexplicably restricted to u.s. military populations [ , , ] , with occasional yet limited infections tallied in pathogen surveys of civilian populations [ ] [ ] [ ] . third, taxonomically, hadv-e differed from other hadvs with respect to protein chemistry properties [ ] and low-resolution genomics data, including partial sequences and restriction enzyme maps [ ] . its classification, with genome sequence, as the sole hadv comprising clade or "species" e, with simian adenoviruses (sadvs), adds to its mystique [ , ] . origins included speculations of being the archetypical hadv [ ] or a hadv species b and c recombinant. genome analysis showed ri- was a chimpanzee adenovirus (chadv) [ , ] , with a genome identity of . % and . % to sadv-e and sadv-e , respectively. these are much higher than two respiratory pathogens hadv-b at . % and hadv-c at . %, with ocular pathogen hadv-d at . %. the zoonotic threat potential of adenoviruses is recognized [ ] , and hadv-e represents the first example. notably, both the hadv-e prototype and contemporaneous "vaccine" isolates contain a genome signature that is unique to chadvs and other sadvs, i.e., the absence of the nuclear factor i (nf-i) binding site that is conserved among the three replication motifs embedded in nearly all hadv inverted terminal repeats (itrs) [ ] . nf-i is a host transcription factor that binds to nt - of the hadv- origin of replication [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and is recruited by the human adenoviral replication complex [ , ] . it is firmly established as essential through in vitro and in vivo studies (see [ , ] and qtd. in), including replication reconstitution assays, as necessary for efficient adenoviral replication in human cells (see [ ] and qtd. in). recent isolates are recombinants containing this hadv replication motif [ ] , presumably permitting an expansion of the virus range into the immune-naïve populations [ ] [ ] [ ] [ ] [ ] [ ] , and should be noted as a molecular evolution example of a post-zoonotic, host-adaptation of a "novel" and "emergent" human pathogen. as noted in the literature, the itr contains critical conserved dna replication motifs that include the human transcription factor binding site, nf-i, which is shown to enhance and optimize hadv replication and growth [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . all of the hong kong isolates (red) possess this host-adapted itr, i.e., they have the nf-i binding site that is found in all human respiratory adenoviruses (yellow) (figure ). however, this motif is missing in sadv itrs (purple) [ ] , as well as the "old" hadv-e strains (green), e.g., prototype ( ) and "vaccine" ( ) strains, i.e., they have only the core origin and the nf-iii motif [ ] . the absence of the nf-i motif likely plays a role in limiting the circulation of both sadvs and prototype hadv-e in human populations, as its acquisition presumably provides efficient and enhanced replication in human cells, i.e., allowed hadv-e to adapt to the new host. this may be the "tipping point" that allows hadv-e entry into the general population that is immunologically-naïve to its epsilon antigen, as hadv-e does not normally circulate outside the u.s. military populations [ , [ ] [ ] [ ] . this is consistent with reports documenting recombination as an evolution mechanism in the genesis of emergent hadv pathogens [ ] , illustrated by hadv-d [ ] and hadv-b [ ] . hadv-b is a recent major respiratory pathogen in china, after years of quiescence, by virtue of its introduction into immunologically-naïve populations, i.e., without antibodies to its hadv-b -like epsilon antigen. another example is the reemergence of a long-dormant respiratory pathogen hadv-b into an immunologically-naïve population [ ] . consistent with this report, recombination has been reported in sadvs [ , ] and cross-species transmissions between humans and non-human simians have been reported [ ] [ ] [ ] [ ] [ ] [ ] [ ] , including tmadv across "two passages" of human hosts, with clinical manifestations [ ] , as well as seroprevalence of adenoviruses across species [ , [ ] [ ] [ ] . these support the hypothesis and data suggesting hadv-e was of zoonotic origins [ , ] . comparison of hadv-e genomes spanning years reveals highly conserved sequences. for example, the two earliest genomes, isolated within years, are . % identical to each other, differing by mismatches and two insertions (one one-base and one three-base) [ ] . one striking difference is a recombination resulting in recent hadv-e genomes acquiring a well-characterized replication-enhancing nf-i motif that distinguishes these isolates from the prototype and a contemporaneous co-circulating strain. although the acquisition of this critical replication motif alone may explain the potentially wider distribution of hadv-e , the pair-wise sequence analyses also reveal additional potentially important sequence differences in the e gene region [ ] . proteins encoded in the e region are involved in interactions with the host immune response and evasion and represent the most variable region between hadv types [ ] [ ] [ ] . this report highlights the need to re-explore the understudied e genes and their critical roles in the human immune response upon hadv infection. however, whether in concert with e gene changes or alone, the nf-i acquisition, given "occam's razor", may be the critical evolutionary modification and "tipping point" that is necessary for a wider distribution of hadv-e in immune-naïve civilian populations in light of the extensive literature reported for hadv replication requirements [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this host adaptation may be significant as hadv-e is a highly contagious pathogen with demonstrated morbidity and mortality implications and has now been reported in global civilian populations [ , , ] . during the preparation of this report, to emphasize further the potential of hadv-e emergence in the global general population, seroepidemiological data identifying hadv-e infections in china and sierra leone (africa) were published [ ] . explicitly, the authors note "an apparent increase in the frequency of human adenovirus type (hadv- ) infections among general populations has been observed over the past years" [ ] . nasopharyngeal swab specimens were collected from both outpatients and inpatients who presented with flu-like symptoms, and are archived at queen mary hospital (hong kong). adenoviruses were detected by pcr and were identified further by molecular typing using partial sequence data from the hexon and fiber genes, as previously reported [ ] . then, virus strains that were identified as type were cultured in human lung cell lines (a ), and genomic dna was extracted using the viral dna extraction kit from omega bio-tek, inc. (norcross, ga, usa), as previously noted [ ] . itrs were sequenced using genomic dna as a template, with either a '-primer "ad -itr-l" ( '-aactcttctcgctggcactcaa- ') or a '-primer "ad -itr-r" ( '-ccgcccctaacagtcgcc- '). these sanger chemistry-derived itr sequence data generated with an abi dna sequencer were aligned and parsed for sequence motifs. phylogenetic analysis was undertaken with molecular evolutionary genetics analysis software (mega) . . (https://www.megasoftware.net), for which fasta files were inputted for sequence alignments utilizing a maximum composite likelihood method to generate neighbor-joining, bootstrapped phylogenetic trees with iterations [ , ] . all other mega program parameters were set by default. bootstrap values above are considered robust. for pair-wise genome comparisons, zpicture (http://zpicture.dcode.org/) was used to provide detailed global insights. all sequences are available in genbank from which whole genome data were retrieved and aligned, and itr sequences extracted. this resulted in a data set of itr sequences ranging from to bases, as lengths were variable among the types. a subset of sequences spanning the first - bases that included the conserved hadv viral replication sequence motifs were selected for detailed analysis. genome sequence data were accessed from genbank. hadv-e genomes include ri- or "prototype" (ft leonard wood, missouri; ; ay ); cl or "vaccine" ( computational analysis 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type inverted terminal repeat promote bidirectional transcription in vitro and are important for virus growth in vivo transcription factors nfi and nfiii/oct- function independently, employing different mechanisms to enhance adenovirus dna replication conserved sequences at the origin of adenovirus dna replication outbreak of adenovirus type infection in a long-term care facility for the elderly respiratory adenoviral infections in children: a study of hospitalized cases in southern taiwan in - a swimming pool-associated outbreak of pharyngoconjunctival fever caused by human adenovirus type in beijing computational analysis identifies human adenovirus type as a re-emergent acute respiratory disease pathogen evidence of molecular evolution driven by recombination events influencing tropism in a novel human adenovirus that causes epidemic keratoconjunctivitis phylogenomic evidence for recombination of adenoviruses in wild gorillas simian adenovirus type has a recombinant genome 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circulating hadv-e strains overreliance on the hexon gene, leading to misclassification of human adenoviruses three adenovirus e proteins cooperate to evade apoptosis by tumor necrosis factor-related apoptosis-inducing ligand receptor- and - functions and mechanisms of action of the adenovirus e proteins seroepidemiological investigation of hadv- infection among healthy adults in china and in sierra leone identification and typing of respiratory adenoviruses in guangzhou, southern china using a rapid and simple method mega : molecular evolutionary genetics analysis version . for bigger datasets acknowledgments: d.s. thanks q.z. and the southern medical university for an appointment as visiting professor, and also k.y. yuen (hong kong university) for the lecture invitation that initiated this collaboration. d.s. is grateful to harold suarez del toro and the staff, particularly margarito ricardez de la cruz, of the biwa hotel in tulum (mexico) for providing a warm, tranquil, and hospitable environment that was conducive for a writing sabbatical in winter - . the authors declare no conflict of interest. key: cord- -kjkuet authors: lópez-camacho, césar; chan kim, young; blight, joshua; lazaro moreli, marcos; montoya-diaz, eduardo; t huiskonen, juha; mareike kümmerer, beate; reyes-sandoval, arturo title: assessment of immunogenicity and neutralisation efficacy of viral-vectored vaccines against chikungunya virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: kjkuet chikungunya virus (chikv) has caused extensive outbreaks in several countries within the americas, asia, oceanic/pacific islands, and europe. in humans, chikv infections cause a debilitating disease with acute febrile illness and long-term polyarthralgia. acute and chronic symptoms impose a major economic burden to health systems and contribute to poverty in affected countries. an efficacious vaccine would be an important step towards decreasing the disease burden caused by chikv infection. despite no licensed vaccine is yet available for chikv, there is strong evidence of effective asymptomatic viral clearance due to neutralising antibodies against the viral structural proteins. we have designed viral-vectored vaccines to express the structural proteins of chikv, using the replication-deficient chimpanzee adenoviral platform, chadox . expression of the chikv antigens results in the formation of chikungunya virus-like particles. our vaccines induce high frequencies of anti-chikungunya specific t-cell responses as well as high titres of anti-chikv e antibodies with high capacity for in vitro neutralisation. our results indicate the potential for further clinical development of the chadox vaccine platform in chikv vaccinology. chikungunya virus (chikv) is transmitted by aedes mosquitoes and it is an important growing human health concern in many countries, causing significant outbreaks of acute febrile illness and long-term arthropathy [ ] . its origins are traced to africa as an enzootic spillover, belonging to the semliki forest complex of the alphavirus (togaviridae) [ ] and was first isolated from a human patient in tanzania in [ ] . it currently exists as four lineages. the two african enzootic lineages are the west african (wa) and the east central south africa (ecsa) lineages. the additional two lineages originated from the ecsa lineage and are called the asian and the recently emerged indian ocean lineage [ , [ ] [ ] [ ] . chikv is composed of a positive, single-stranded genomic rna of kilobases, encoding four non-structural (ns) and five structural (s) proteins [ , ] . the non-structural proteins, nsp , nsp , nsp and nsp , are required for virus replication [ ] . a sub-genomic rna encodes the structural proteins: capsid (c), e , e , k and e , and thus a polyprotein is produced which is then processed by the capsid auto-proteinase and signalases [ ] . the chikv surface is mainly composed by e -e heterodimers [ ] where e glycoproteins mediate fusion [ ] and e glycoproteins interact with the host receptor [ ] . since its discovery the virus has spread to asia, oceania/pacific, europe, and mostly recently to the americas, where there have been more than a million reported cases since its detection in late [ ] [ ] [ ] [ ] , whilst major outbreaks continue to be recorded in the asian and african continents. although chikungunya vaccines have been under development for several decades, no licensed option is yet available and chikv still causes substantial morbidity, overwhelming public health systems and contributing to poverty [ ] . in the meantime, current control strategies rely on reducing human exposure to potentially infected mosquito vectors. several institutions are now engaged in the development of a safe and cost-effective chikv vaccine; such vaccines [ ] [ ] [ ] are based on live-attenuated or inactivated chikv, chimeric chikv, dna, subunit, virus-like particle (vlp) and viral-vectored platforms. they are mainly designed to induce humoral responses against the structural viral protein e , as well as e , due to strong correlates of protection with neutralising antibodies against structural chikv proteins in asymptomatic cases [ ] . in addition to that, passive transfer of sera from convalescent humans to mice prevented infection [ ] whilst neutralising antibodies against e and e were able to protect immunocompromised mice [ ] . in humans, an early igg neutralising response is associated with reduced clinical symptoms [ ] and asymptomatic cases have been associated with high neutralising antibody titres [ ] . here, we describe the design and development of chikv vaccines based on the clinically relevant adenoviral vector, chadox and the modified vaccinia ankara (mva) platforms [ ] [ ] [ ] [ ] [ ] . viral vector expression of the schikv proteins is able to form chikungunya viral particles, thus mimicking a real exposure of chikv, whilst being an in silico designed mosaic protein, aiming to represent all four chikv lineages. in mice, chadox vaccines candidates expressing schikv antigens are able to induce strong humoral and cellular responses upon a single and non-adjuvanted immunisation approach. importantly, we present evidence of in vitro neutralising activity in sera from vaccinated mice. finally, whilst durability of humoral responses was achieved upon a single immunisation, mva vaccines expressing schikv were produced to be used as an alternative heterologous booster regime (chadox /mva), in order to increase neutralising antibody titres. taken together, a viral vectored vaccine for chikv, based on the chadox platform is a good candidate for further pre-clinical and clinical studies. full length structural polyprotein sequences for chikv from all lineages were collected from the ncbi protein database (txid ), aligned using clustal omega [ ] , and a neighbour-joining tree created (juke-cantor, bootstraps). conservation within clades (intra-clade) and subsequently between clades was calculated using our in-house developed software based on a sliding window approach with a sequence weighting method to enable equal representation of all lineages and variants (manuscript in preparation). the structural cassette chikv sequence derived from various chikv lineages was codon optimised. to improve initiation of translation a kozak consensus sequence was included before the end of the transgene. finally, the transgene design included the required enzymatic restriction sites to allow the in-frame cloning of the transgene between the cmv promoter and the polya sequence region contained in our shuttle and expression vector (pmono). a synthetic gene cassette was produced by geneart ® (fisher scientific, regensburg, germany) and was named schikv. the schikv plasmid was used as dna template to further generate the e ,e , k,e cassette with no capsid by pcr-cloning; this variant was named schikv ∆c. forward primer: atggaggaatggtccctggctatc. reverse primer: tcatcagtgccggctgaag. the plasmid containing the schikv cassette (c,e ,e , k,e ) and the schikv ∆c (e ,e , k,e ) were digested with kpni and noti restriction enzymes (neb, ipswich, ma, usa) to allow in-frame ligation between the cmv promoter and the poly(a) regions contained in the shuttle plasmid (pmono). the recombinant dna plasmids were expanded and purified from e. coli using the midiprep kit (qiagen, hilden, germany). resulting plasmids were verified by restriction analysis and and flanking sequencing. to generate chadox vaccines, the shuttle plasmids containing attl regions sequences were each recombined with those attr regions contained in the destination vector chadox using an in vitro gateway reaction (lr clonase ii system, invitrogen tm , carlsbad, ca, usa). successfully recombined chadox schikv and chadox schikv ∆c plasmids were verified by dna sequencing using flanking primers (forward promoter primer and poly-(a) reverse primer). standard cell biology and virology techniques were performed to generate the non-replicative adenoviral vectors [ ] . to generate mva based vaccines both the schikv and the schikv cassette ∆c were digested with kpni and xhoi to allow in-frame ligation between the p . promoter and the tkr locus, contained in the entry plasmid mva. ligated dna plasmids were expanded in e. coli and a midiprep (qiagen) was used for plasmid purifications. resulting plasmids were verified by restriction analysis and and flanking sequencing, and co-transfected to produce mva schikv and mva schikv ∆c, using the methodology as previously described [ ] . control chadox and mva vectors comprised non-structural (ns) regions from chikv and they were named chadox ns and mva ns, respectively. female inbred balb/c (h- d), ( - weeks) were used for the assessment of immunogenicity (n = mice per group). mice were purchased from envigo rms inc. (bicester, g.b.). the experimental design took into account the r reduction (replacement, reduction, refinement). no randomisation was used in this work. for chimpanzee adenoviral-vectored vaccines, mice were vaccinated intramuscularly with a single dose of × infectious units (iu). for boosting, mva vaccines were administered at a dose of × plaque-forming units (pfu) per mouse. all vaccines were diluted in endotoxin-free pbs. vero cell were grown following the atcc conditions. hek- cells (atcc ® , crl- tm) were grown in dmem media supplemented with % fbs, % l-glutamine and % non-essential amino acids. baby hamster kidney- (bhk- ) cells were maintained in glasgow´s minimum essential medium (gmem) supplemented with % fetal bovine serum (fbs), % l-glutamine, % tryptose phosphate broth, mm hepes ph . , u/ml penicillin and . mg/ml streptomycin at • c and % co . the cell number and viability were calculated by trypan blue staining using the countess automated cell counter (life technologies, carlsbad, ca, usa). cell lines are described in the ncbi biosample database. the expression screening was carried out in vero cells by transduction of chadox schikv, using a moi = . three days after transduction, the cells and supernatants were harvested, the supernatant was concentrated with a kda amicon ® spin column (millipore uk, ltd). the x concentrated fraction, the non-concentrated fraction (input) and flow through fraction was also recovered. cells and supernatants samples were boiled at • c for five minutes in laemli buffer. equal amounts of total cell extract and cell supernatants were resolved by sds/page and transferred to pvdf membranes. blots were blocked with x pbs-tween- % milk and incubated with an anti-chikv e antibody (az , aalto bioreagents, dublin, ireland) at : dilution, and anti-chikv envelope seropositive mice sera ( : ), followed by incubation with hrp-conjugated secondary antibody ( : ). chemiluminescence (perkin-elmer life sciences, boston, ma, usa) was visualised using the chemidoc srs device (biorad, watford h., uk). formvar/carbon mesh cu grids were glow-discharged in air and loaded with . µl of the sample from chadox schikv vaccine stock or purified vlp of chikv by sucrose gradient ( - %). excess liquid was removed, and the grids were washed times with milliq water. finally, grids were stained with % uranyl acetate for s, excess uranyl acetate was carefully removed using filter paper. the grids were air dried and analysed with a t transmission electron microscope (fei, eindhoven, the netherlands). elispots were carried out using peripheral blood mononuclear cells (pbmcs). briefly, maip elispot plates (millipore uk, ltd) were coated with anti-mouse ifnγ antibody (mab an , mabtech), after h blocking with complete dmem media ( % fcs). isolated pbmcs (using ack buffer solution) were plated alongside with -mer specific chikv structural peptides overlapped by a.a. ( µg/ml) and . × splenocytes from naïve mice per well. after h incubation, cells were discarded, and plates washed with pbs. following this, µl of biotinylated anti-mouse ifnγ mab (mab r - a , mabtech) ( : in pbs) was added to each well and incubated for h. after washing, plates were incubated with µl of streptavidin-alp (mabtech) at : dilution in pbs for h. after another washing step, developing solution (biorad, watford h., u.k.) was used. once spots were visible, the reaction was stopped by rinsing the plates with water. spots were acquired using an elispot reader. spot forming cells (sfc)/ pbmcs producing ifnγ were calculated. for expression and purification of the chikv e protein, the codon-optimised gene of e (a.a. - ) was cloned into the phlsec vector [ ] . which is flanked by the chicken β-actin/rabbit β-globin hybrid promoter with a signal secretion sequence and a lys-his tag. in order to improve secretion of the e protein, the c-terminal region of e (a.a. - ) was deleted. the phlsec chikv e plasmid ( µg) was transfected in hek- t cells using polyethyleneimine (pei) in roller bottles (surface area of , cm ) under standard cell culture conditions. five days after transfection cells were discarded and media was filtered through . µm disposable filters. the secreted protein was purified from the supernatant by ni sepharose affinity chromatography (histrap tm , ge healthcare), using the Äkta start chromatography system and eluted with imidazole mm. finally, the eluted protein was dialysed using slide-a-lyzertm cassette (thermo fisher scientific, rockford, il, usa) against × pbs. antibody binding to chikv e was measured by an igg enzyme linked immunosorbent assay (elisa). briefly, mice sera were diluted in nunc maxisorp immuno elisa plates coated with e diluted in pbs to a final concentration of µg/ml and incubated at room temperature overnight. plates were washed times with pbs/ . % tween (pbs/t) and blocked with µl with pierce tm protein-free (pbs) blocking buffer (thermo fisher scientific, waltham, ma, usa) for h at rt. mice serum was added and serially diluted -fold down in pbs/t with µl per well as final volume and incubated for h at rt. following washing times with pbs/t, bound antibodies were detected following a h incubation with µl of alkaline phosphatase-conjugated antibodies specific for whole mouse igg (a - ml, sigma aldrich, st. louis, mo, usa). following additional washes with pbs/t, development was achieved using µl of -nitrophenylphosphate diluted in diethanolamine buffer and the absorbance values at od were measured and analysed using a clariostar instrument (bmg labtech, aylesbury, gb). serum antibody endpoint titres were defined by an absorbance value three standard deviations greater than the average od of the control. titres of neutralising antibodies were determined as described previously using chikv replicon particles (vrps) expressing gaussia luciferase (gluc) [ ] . briefly, bhk- cells were seeded in -well plates at × cells per well. the next day, vrps (moi of . ) were preincubated with -fold serial dilutions of serum samples for h at • c before the mixture was added to the -well plates. after incubation for h at • c, the inoculum was removed, cells were washed with pbs, and the medium was added. readout of secreted gaussia was performed at h post infection using a renilla luciferase assay system (promega, southhampton, uk). neutralisation potency was determined as a percentage of measured gluc activity compared to the gluc readout after vrp application without serum. results are presented as % neutralisation (nt ) titres. all animals and procedures were used in accordance with the terms of the uk home office animals act project license. procedures were approved by the university of oxford animal care and ethical review committee (ppl / ). the authors declare that the data supporting the findings of this study are shown in the article. in order to generate a structural gene cassette (c,e ,e , k, e ) from chikv, a protein sequence mosaic was constructed using full-length structural polyprotein sequences collected from the ncbi protein database. a neighbour-joining tree was created which identified three distinct clades (a, b, c; figure a ). subsequently conservation was assessed within each clade (intra-clade; figure b ) using a sliding window approach, in which a conservation value between and was assigned, being fully conserved. anything lower than the first quartile (q ) was classed as conserved. lastly, the number of windows conserved across each clade and the degree of conservation were identified and used to create a normalised mosaic consensus. figure c illustrates the level of shared conservation between clades, from to - , with - being fully conserved. the resulting sequence mosaic schikv was synthesised (figure a) . we constructed chimpanzee adenoviral vector vaccines expressing the full structural cassette (chadox schikv), the capsid-deleted structural cassette (chadox schikv ∆c) and a non-structural sequence from chikv (chadox ns), the latter being used as a control vaccine (figure b ). in addition, modified vaccinia ankara (mva) viral-vector was used to construct the mva schikv, the mva schikv ∆c and the control mva ns (figure c ). after cloning and production of chadox vaccines encoding the genomic schikv cassette, we verified the formation of adenoviral particles by transmission electron microscopy ( figure a) . because it has been described that cellular expression of the full structural chikv cassette is capable to self-assemble vlps for chikv, as well as for other alphaviruses [ ] [ ] [ ] [ ] , we collected supernatant from hek cells expressing the schikv antigen. the supernatant was then subjected to sucrose gradient showing a characteristic sedimentation band, which is representative of vlp accumulation (figure b, left) . further electron microscopy preparations revealed particles of approximately nm in size (figure b, right) , which resembled wild-type chikv particles. alternatively, we transduced vero cells with chadox schikv and chadox schikv ∆c, and verified their expression in cells and supernatants. specific detection was achieved by western blot, using an anti-chikv mice serum and an anti-e monoclonal antibody (figure c,d, respectively) . in cells transduced with chadox schikv, we detected bands at approximately kda in the supernatants (figure c, lane , , ) , with a band of approximately kda only in the concentrated supernatant (figure c, lane ) . in cellular extracts, we detected specific bands of and kda (figure c, lane ) . in cells transduced with chadox schikv ∆c we also detected bands at approximately kda in supernatants (figure c, lane , , ) , but no bands of kda or kda were found in the concentrated supernatant nor in the cell extracts. therefore, it is suggested that the kda or kda band may represent the capsid protein, as previously demonstrated [ ] , which are not present in chadox schikv ∆c-transduced cells. as a positive control, we used a commercially available chikv e protein (figure c, lane ) ; therefore, it is suggested that the kda band detected in our blots is e protein. in addition, a western blot was performed using a monoclonal antibody against chikv e (figure d ); we detected a specific band in the positive control e protein (lane ), as well as a kda band in all the supernatant fractions from both chadox schikv-and chadox schikv ∆c-transduced cells. no signal in cell extracts was detected (figure d, lane , ) , probably due to conformational epitope masking. taken together, our results suggest that our adenoviral-vectors are able to express the schikv antigens which are able to induce the formation of chikungunya viral-like particles. thus, we aim to mimic a real antigen exposure of chikv particles. to assess the specific immunogenicity of our viral-vectored vaccines, we immunised groups of balb/c animals (n = ) with a single and non-adjuvanted vaccine dose of chadox schikv, chadox schikv ∆c, and control chadox ns, respectively. two weeks after immunisation, t cell responses were quantified by ex vivo ifnγ elispot, using a specific pool of overlapping peptides spanning the full chikv structural cassette, as well as a pool of overlapping peptides related to the control ns vaccines (figure ) . balb/c mice receiving vaccines encoding schikv antigens showed high t cell frequencies after stimulation with the schikv peptide pool (chadox schikv = mean of spot-forming cells (sfc)/ peripheral blood mononuclear cells (pbmcs) and chadox schikv ∆c = mean of , sfc/ pbmcs); with no statistical difference between both groups, using one-way anova analysis (figure a ). the control group chadox ns showed low specific t cell responses against the ns peptide pool and no responses to specific schikv peptide pool. in addition, we analysed the responses towards specific peptide sub-pools for c, e , e , k, e and ns (figure b ). schikv peptide deconvolution elispot allowed the identification of an immunodominant region located in the k region which is induced upon chadox schikv and chadox schikv ∆c vaccination ( , and , sfc/ pbmcs, respectively). as expected, we detected specific cellular responses towards the capsid in animals vaccinated with chadox schikv (mean= sfc/ pbmcs) in comparison with those animals vaccinated with chadox schikv ∆c in which low background responses were recorded (figure b ). modest responses were detected in pbmcs against the e and e peptide pools. taken together, we conclude that a single injection of chadox schikv or chadox schikv ∆c is immunogenic and able to induce specific t cell responses in balb/c mice. to assess humoral responses, -well plates were coated with chikv-e protein. we performed elisa assays to identify the immunogenic properties of chadox schikv and chadox schikv ∆c, upon a single and unadjuvanted vaccination strategy ( figure a) . two weeks after a chadox schikv or chadox schikv ∆c vaccination, specific igg antibody binding to chikv e protein was detected in sera from vaccinated animals ( figure a, left) . six weeks after prime ( figure a , right) all the animals increased the levels of od binding to the e protein. of particular interest, months after a single immunisation, sera from vaccinated animals showed specific igg antibody binding to chikv e , similar to that of six weeks prime group ( figure a, bottom) . chadox ns was used as control as it does not generate antibody responses towards the schikv antigens. log reciprocal antibody titre analysis (figure b) showed that levels of anti-e antibodies increased over time for both chadox schikv or chadox schikv ∆c prime-vaccinated animals and remarkably they were maintained after months upon a single vaccination strategy. interestingly, our results indicated that the deletion of capsid in the chadox schikv ∆cvaccinated group did not impair the ability to elicit anti-e chikv antibody titres. finally, modified vaccinia ankara (mva) viral-vector technology has been shown to be a potent booster of immune responses [ , ] . therefore, we have constructed mva schikv and mva schikv ∆c to test their boosting immunogenic potential in a prime-boost heterologous strategy. mice immunised with either chadox schikv or chadox schikv ∆c were boosted with mva schikv and mva schikv ∆c at six weeks after prime, respectively; and sera was collected two weeks after boost. prime-boost vaccinated mice showed high endpoint titters of > of anti igg-e antibodies (figure b , prime-boost section). mva ns was used as control to boost animals vaccinated with chadox ns vaccines. taken together, we conclude that a single vaccination with no adjuvant strategy is able to induce early, specific and long-lasting b responses in balb/c mice towards chikv-e protein. we tested balb/c mice sera obtained after an early prime time-point and in a prime-boost vaccination to assess chikv antibody neutralisation activity in vitro. the method we used is based on the virus replicon particle-based (vrp) chikungunya virus neutralisation assay to determine nt values [ ] . naïve and control chadox ns sera were included alongside the assay. at two weeks post-vaccination, sera from the chadox schikv-and chadox schikv ∆c-vaccinated groups showed high neutralisation activity against vrp chikv with a nt titre of . x and . x respectively (figure a) . in a prime-boost regime (figure b ), chadox schikv/mva schikv showed a further . -fold increase of the nt titter ( . x ), in comparison to the prime vaccination regime of chadox schikv. a similar increase of nt titre was found in the sera from mice vaccinated with a prime-boost chadox schikv ∆c/mva schikv ∆c, with a -fold increase ( . x ), when compared to that of the nt titre from single chadox schikv ∆c vaccinated animals (figure b ). these results indicate that when the capsid is deleted from the chikv structural genes, a decrease in the antibody nt titre is observed, upon a single chadox vaccination. conversely, when a prime-boost regime is used, the deletion of the capsid is beneficial to increase the chikv nt antibody titres. taken together, our results confirm that our viral-vectored vaccines elicit functional neutralising antibodies against chikv-infective particles, tested by an in vitro model. in this work we have constructed chikv vaccines based on viral-vector platforms, such as chadox and mva vectors. the immunogen was designed based on a mosaic consensus sequence from the asian, east, central and south african and west african regions. although individual strains are antigenically related and chikv vaccines previously reported have been developed using heterologous strains [ ] , a mosaic-based approach ensures a high coverage aimed at raising neutralising antibodies against all variants circulating worldwide. here, we have developed two different vaccine immunogen candidates: a vaccine encoding the full structural chikv polyprotein (schikv) and a vaccine in which the capsid antigen was deleted (schikv ∆c). one of the functions of the capsid is to auto-cleave from the e -e - k-e polyprotein. this proteolytic process exposes a signal leader which is contained in the amino-terminal region of e and promotes the entry of the polyprotein into the endoplasmic reticulum [ ] . there are several advantages in comparing these two different antigenic cassettes. genetic vaccines, such as dna vaccines, rna vaccines, and viral-vectored vaccines, are often limited by the number of nucleotides that can be inserted into the antigenic cassette. exploring the minimum sequence length able to induce immunogenicity and efficacy (either by in vitro neutralisation or by sterile protection after a viral challenge), will inform the vaccine development of new generation of vaccine candidates against chikv. in addition, optimising the minimum number of genetic antigens could allow the inclusion of further antigens to create combined vaccines (either homologous or heterologous) to induce immune response against different pathogens, all in a single genetic vaccine approach. based on this assumption, efforts are underway to create chadox vaccines expressing shorter versions of the chikv polyprotein, such as e and e proteins from chikv and other alphaviruses. e protein is involved in receptor recognition, while e protein is involved in membrane fusion [ , ] . therefore, exploring single-protein chikv vaccines in chadox or in other viral-vectored platforms seems feasible and promising, since anti-e and anti-e antibodies are high in convalescent chikv-immune populations [ ] . however, ensuring proper antigenic folding in such developments, identical or similar to those antigenic structures in virions from chikv is paramount. here, we have demonstrated by transmission electron microscopy, that our adenoviral vaccine chadox schikv is capable of expressing chikungunya-vlps, which promotes the correct conformational antigens to be presented upon vaccination, therefore mimicking a real exposure to wild-type chikv. it will be interesting to explore and compare the neutralisation efficacy between encoded chikungunya-vlps, e or e proteins in viral-vectored vaccines. in this work we have tested the immunogenicity of our viral-vectored vaccines by assessing the cellular and humoral responses in balb/c mice. our data shows that the t cell immunogenicity elicited by chadox schikv and chadox schikv ∆c is directed primarily to a peptide region located in the k protein, by ifn-γ production. it will be interesting to explore the development of t-cell based vaccines against chikv. the role of t cell immunity in alphavirus infection, especially in chikv is not very well understood. importantly, antigen design and the modulation of its intracellular expression will play a major role at inducing t cell responses directed to the antigen. a clear example is the comparison between chikv and chikv ∆c elispots, in which t cell responses directed to capsid were detected in the chikv groups but it was ablated in the chikv ∆c groups. it will be interesting to explore the protective and immunological relevance of the t cell response directed by the capsid in pre-clinical models. although we identified k as the immunodominant region in balb/c, this observation may differ in other inbred or outbred mouse strains; or in the case that vaccines comprise only e , e or e antigens. for example, chikv-specific cd +t cells were directed mainly against e and e proteins in c bl mice [ ] [ ] [ ] . t cells induced after chikv infection in c bl/ mice were not essential to control viremia but cd t cells were involved in chikv pathology [ ] . furthermore, t cell adoptive transfer did not confer protection against chikv challenge [ ] . finally, t cell responses in human chikv infection is also poorly understood. t cell responses (cd + > cd + ) have been found in chronic and convalescent chikv patients [ ] , in which specific responses were detected for e , capsid and ns protein (nsp ). further studies using chikv challenge models would be informative to further dissect the role of cellular responses in protective efficacy using ours or other vaccine platforms. in terms of humoral responses, we have assessed the binding of antibodies from vaccinated mice by elisa, coating with e protein. e protein has been shown to be a primary target for neutralising antibodies, since e functions for recognition and attachment to cellular receptors. here, we have demonstrated that our vaccines are able to mount anti-e antibody responses. the level of antibody responses after a single immunisation of chadox schikv or chadox schikv ∆c were maintained for up to months, highlighting the suitability of our vaccine approach to promote long-lasting immune responses against chikv. nonetheless, we have tested a boosting strategy using mva chikungunya vaccines to further increase the anti-e antibody titres. although we have used an elisa assay to detect anti-e antibodies, we are possibly detecting only antibodies against monomeric e . however, our vaccine platform assembles vlp for chikv, in which conformational epitopes might be physiologically relevant to confer protective efficacy in pre-clinical models. finally, we do not rule out the possibility of inducing responses other structural components. the chadox schikv and the chadox schikv ∆c prompt responses in both, b-and t-cell compartments. in vitro neutralisation assays are an excellent standard to test the capacity of chikv sero-positive serum to neutralise chikv. here we utilised a widely used neutralisation assay, using a non-infectious chikv particle which is fully capable of packing chikv replicon rna, which carries the gaussia luciferase (gluc) gene located in the subgenomic rna region [ ] . therefore, infected cells secreting gluc can be detected as a surrogate detection of chikv infection. in this study, we tested a rather early time-point after a chadox schikv or a chadox schikv ∆c prime vaccination, with the hope to demonstrate efficacy of neutralisation after two weeks post-vaccination, as vaccines should be aimed to guarantee protection in a shortest window of time between vaccination and pathogen exposure. conversely, we have tested the neutralisation capacity upon a booster vaccination using mva schikv and mva schikv ∆c, respectively. our results demonstrate that our chadox chikv and chadox chikv ∆c were able to induce > nt titres. since the nt assay presented here has been used in several vaccine development studies for chikv, we are able to afford a comparative assessment between our nt titters and other vaccine developments. garcia-arriaza et al. reported a mva-chikv vaccine expressing the full structural cassette, in which the mean of nt titters were < in a -week prime post-immunisation strategy [ ] . hallengärd et al., developed an attenuated chikv vaccines in which they achieved > when using the vaccine ∆ nsp [ , ] . finally, chikv neutralisation titres have been assessed in human sera yielding nt titres of . x , . x and x in three different chikv patients returning from mauritius, la reunion and seychelles, [ , ] respectively. therefore, the mean of the nt titres we have achieved in both prime (> ) and prime-boost regimes (> ) reflect the capacity of our viral-vectored vaccines to induce physiologically relevant nt titres. no licensed vaccine is yet available to prevent chikv infection. from > experimental vaccines, only have reached clinical trials and three are still active and leading the field [ ] . a leading vaccine consists of a virus-like particle that presents antigens similar to chikv but without replicating, stimulating immune responses but requiring multiple injections to induce antibody titres [ ] , thus increasing costs and logistics for its use in in low-income countries. another development consists of a recombinant live attenuated measles virus (mv) expressing surface chikv antigens [ ] . the vaccine was immunogenic and safe in humans, but it also requires > doses. regarding adenoviral vectors, only one human adenovirus expressing chikv structural antigens has been published [ ] . unfortunately, human adenoviral-vectored vaccines are not suitable to vaccinate humans, due to the widespread presence of wild-type adenovirus eliciting anti-adenovirus immunity and thus decreasing vaccine efficiency [ ] ; hence the use of chimpanzee adenovirus circumvent this problem. in the ideal chikv vaccine development, safety and immunogenicity should be achieved by a single vaccine dose and no adjuvant requirement. such a simple and robust development could benefit low-income settings, in which affordability and simplification of logistics are essential to deliver the vaccine to a target population. however, in the event that a booster vaccine is needed, an mva-based vaccine for chikv could be used. given the suitably of chadox in the clinical setting, the capability to produce millions of 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island attenuated and vectored vaccines protect nonhuman primates against chikungunya virus prime-boost immunization strategies against chikungunya virus chikungunya fever in travelers returning to europe from the indian ocean region safety and tolerability of chikungunya virus-like particle vaccine in healthy adults: a phase dose-escalation trial immunogenicity, safety, and tolerability of a recombinant measles-virus-based chikungunya vaccine: a randomised, double-blind, placebo-controlled, active-comparator, first-in-man trial chimpanzee adenovirus antibodies in humans, sub-saharan africa we would like to thank the jenner institute's vector core facility for producing and supplying the recombinant viral vectors, janett wieseler for excellent technical assistance and phillip kemlo for software development. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -ikni acz authors: li, zengbin; zou, zixiao; jiang, zeju; huang, xiaotian; liu, qiong title: biological function and application of picornaviral b protein: a new target for antiviral drug development date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ikni acz picornaviruses are associated with acute and chronic diseases. the clinical manifestations of infections are often mild, but infections may also lead to respiratory symptoms, gastroenteritis, myocarditis, meningitis, hepatitis, and poliomyelitis, with serious impacts on human health and economic losses in animal husbandry. thus far, research on picornaviruses has mainly focused on structural proteins such as vp , whereas the non-structural protein b, which plays vital roles in the life cycle of the viruses and exhibits a viroporin or viroporin-like activity, has been overlooked. viroporins are viral proteins containing at least one amphipathic α-helical structure, which oligomerizes to form transmembrane hydrophilic pores. in this review, we mainly summarize recent research data on the viroporin or viroporin-like activity of b proteins, which affects the biological function of the membrane, regulates cell death, and affects the host immune response. considering these mechanisms, the potential application of the b protein as a candidate target for antiviral drug development is discussed, along with research challenges and prospects toward realizing a novel treatment strategy for picornavirus infections. the picornaviridae family consists of genera and species, mainly including enterovirus, hepatovirus, cardiovirus, aphthovirus, and rhinovirus [ ] . to date, research on picornaviruses has mainly focused on enterovirus (ev) , coxsackievirus (cv), poliovirus (pv), encephalomyocarditis virus (emcv), foot-and-mouth disease virus (fmdv), human rhinovirus (hrv), and hepatitis a virus (hav). picornavirus infections can cause enormous damage in humans and animals. the ev , cva , and cva cause hand, foot, and mouth disease in millions of children in asia-pacific region each year and can cause more serious clinical symptoms such as aseptic meningitis, acute flaccid paralysis, and neurological respiratory syndrome [ ] [ ] [ ] . picornaviruses are non-enveloped spherical viruses with an icosahedral-structured viral capsid. the picornaviral genome consists of a single-stranded positive-sense rna, which is approximately . - . kilobases in length, with a highly conserved structure, including a -noncoding region , an open reading frame, a -ncr, and a -end polya tail [ ] . the -ncr contains multiple rna secondary structural elements, including the internal ribosome entry site. the open reading frame of the viral genome consists of three regions: p , p , and p . the p region is translated and processed to form the structural proteins vp , vp , vp , and vp , which compose the capsid structure of a picornavirus. the p and p regions are separately translated to the non-structural proteins a, b, and c and a, b, c, and d, respectively. the majority of related research has focused on structural proteins of picornaviruses, such as vp , whereas the importance of the non-structural protein b has been relatively overlooked. viroporins are proteins found in a variety of viruses and are generally comprised of to amino acids. each viroporin contains a highly hydrophobic domain capable of forming at least one amphipathic α-helical structure, which oligomerizes to form transmembrane hydrophilic pores [ , ] . the b protein is a crucial component of picornaviruses that exhibits viroporin or viroporin-like activity, plays a key role in the picornavirus life cycle by inducing a series of cytotoxic reactions to promote picornaviral replication and release [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the b protein has a highly conserved sequence, which can be exploited for viral detection [ ] [ ] [ ] , vaccine development [ ] [ ] [ ] , and rna interference [ ] [ ] [ ] [ ] [ ] . in addition, the b protein exhibits a viroporin or viroporin-like activity, and thus, targeted drugs against viroporin could potentially target b protein as a novel strategy to treat or prevent picornavirus infections. however, the detailed mechanism of action of the b protein has not been elucidated to date. therefore, here, we review the recent research data on the role of the b protein in the picornaviral life cycle and discuss its possible application in antiviral therapy. the picornaviral b protein is a relatively short molecule, containing a maximum of two predicted putative transmembrane hydrophobic helices, along with n-and c-terminal domains, which are connected by a short stretch of amino acid residues. the α-helix-turn-α-helix sequence of the b protein is the basis for forming a transmembrane pore through homo-multimerization and the major determinant of the b protein function [ , , [ ] [ ] [ ] [ ] [ ] . a computational approach has demonstrated that the ev b protein is a tetramer, and b proteins with different orientations have different activities [ , ] . the b protein belongs to the type ii family of viroporins, which can be further divided into different types according to the number and orientation of the membrane-spanning domains ( figure ). in type iia viroporins, the n-and c-termini stretch to the organelle lumen, such as in the b protein of cvb [ , , , ] , whereas the n-and c-termini of type iib viroporins face the cytoplasmic matrix, such as in pv [ , , ] and fmdv [ , , ] . in addition, the c-terminus of the hav b protein has a viroporin-like activity [ ] . picornaviral b proteins target the membrane and form pores mainly through their transmembrane regions. the protein molecules are first inserted into the membrane individually and then self-interact and homo-oligomerize to form higher-order structures, which are important for the pore-forming activity, determined by the specific sequence and structure [ , , ] . the majority of b proteins are localized to organelles, with predominant co-localization with the golgi apparatus and the endoplasmic reticulum (er) in cvb , pv, and hrv ( figure ) [ ] . the hrv and fmdv b proteins are mainly localized to the er, whereas the emcv b protein is not localized to either the golgi complex or the er [ , , ] . seggewiss et al. [ ] found that the hav b protein was not localized to the er either but was involved in the amendment of the er-golgi apparatus intermediate compartment. since the protein structure determines its ultimate function and b proteins belonging to different viroporin species show both similarities and differences in their functions, much insight can be gained from research on the same viroporin b protein and on different viroporins. picornavirus infects the host cell, and then viral gene encodes b proteins. the b protein belongs to the type ii family of viroporins, including two transmembrane hydrophobic helices which are the basis for forming a transmembrane pore that results in the changes in cell membrane permeability. in type iia viroporins, the n-and c-termini stretch to the organelle lumen, whereas the n-and c-termini of type ii b viroporins face the cytoplasmic matrix. the majority of b proteins are localized to organelles, with predominant co-localization with the golgi apparatus and the endoplasmic reticulum (er), resulting in an obvious decrease in ca + of the er and golgi complex, along with a decrease in calcium uptake by the mitochondrion, and causing an influx of extracellular ca + . the b protein can induce many cellular reactions, such as changing membrane permeability, regulating apoptosis and autophagy, and affecting host immune responses. these functions are all related to changes in ion concentrations, especially of calcium ions (ca + ). therefore, here, we mostly focus on the role of ca + in these b protein activities. a common feature of infection by animal viruses is the damage to the ion balance in host cells. the picornaviral b protein may change the membrane permeability of target cells, disturbing the ion balance, especially that of ca + , in organelles, such as the er and the golgi apparatus ( figure ) [ , , ] . the changes in membrane permeability, caused by the b protein, have also been suggested to be regulated by the content of specific membrane phospholipids [ , ] . the ca + are involved in the activation of enzymes in cells and play a crucial role in viral replication and other viral biological processes [ ] [ ] [ ] . however, the role of the b protein in ca + homeostasis remains unclear. initial studies only indicated that host cells had elevated the ca + levels, owing to the expression of the b protein [ , , , , [ ] [ ] [ ] , but the mechanism was not clarified. since then, some researchers have proposed that the decrease in the concentration of ca + stored in organelles triggers the opening of specific calcium ion channels on the plasma membrane of cells, causing an influx of extracellular ca + [ ] . this idea was supported by the findings that expression of the cvb and pv b proteins resulted in an obvious decrease in the ca + concentration in the er and golgi complex, along with a decrease in calcium uptake by the mitochondria. meanwhile, the increased ca + level in the cytoplasm was suggested to be mainly due to the influx of extracellular ca + [ , , ] . similarly, the expression of the hrv b protein was shown to decrease the ca + concentrations in the er and golgi apparatus, whereas the emcv b protein only significantly reduced the ca + concentration in the er [ ] . in contrast, other studies showed that expression of the hav and fmdv b proteins elevated the cytoplasmic ca + level but did not alter the level of stored ca + in organelles, such as the er and golgi complex [ , ] . taken together, these studies suggest that there are different mechanisms by which b proteins affect the ca + concentrations, depending on the virus type. furthermore, it is unknown whether ca + directly pass through the channel formed by the b protein. pham et al. [ ] demonstrated, using a planar lipid bilayer and liposome patch-clamp electrophysiological technique, that the rotavirus non-structural protein (nsp ) viroporin region acts as a ca + conduction channel. although there is currently no direct evidence that the b protein can directly induce the observed changes in the ca + concentration in host cells upon infection, the above-reviewed studies suggest an association, and the mechanism requires further investigation. given the importance of ca + signaling for numerous cellular processes, further studies on picornaviral b protein function should include determination of the ca + concentration, which may provide more insight into the detailed function of the b protein. in particular, the b protein may change the ca + concentration to regulate autophagy and apoptosiswhich are distinct cell death mechanisms controlled by the virus to effectively evade the host immunity, thereby promoting viral replication and release [ ] [ ] [ ] [ ] [ ] . picornaviruses can form new cytoplasmic vesicles by inducing membrane remodeling, thereby promoting their own proliferation [ , , ] . the b protein is capable of binding to the membrane and inducing target membrane remodeling to form a unique membrane structure that can serve as a viral replication site. this site, known as the viroplasm, is generated from the er to accumulate all of the cellular components required for viral replication ( figure ) [ , , [ ] [ ] [ ] . the viroplasm is also the main membrane source of autophagy [ , ] . the cvb b protein is dependent on its transmembrane hydrophobic region to induce autophagy [ ] , which may be related to alterations in membrane permeability, especially with regard to the ca + concentration. moreover, at an early stage of fmdv cell infection, the virus specifically recognizes and binds to the cell surface receptors, and the b protein rapidly upregulates the autophagy pathway, leading to punctate aggregation of a large number of autophagy marker proteins, such as the microtubule-associated protein light chain (map -lc ) [ , ] . in addition, rotavirus encodes the nsp viroporin, which releases the er-stored ca + into the cytoplasm, thereby activating the ca + /calmodulin-dependent kinase kinase-β (camkk-β) signaling pathway, leading to autophagy ( figure ) [ ] . further, cvb induces autophagy in a calpain-dependent manner, causing an accumulation of lc lipids and autophagosomes [ ] . considering the ability of the b protein to alter cellular calcium homeostasis, along with its viroporin-like activity, it is feasible that the b protein may regulate autophagy mainly by changing the ca + concentration. the b protein has also been shown to regulate apoptosis through the endogenous pathway, which can be divided into er stress and the mitochondrial pathway, providing another potential mechanism of bypassing the host immune response to facilitate infection [ , , , , ] . the ca + plays a pivotal role in er stress-dependent apoptosis by regulating the flow between the er and the mitochondria [ , ] . excessive mitochondrial uptake of ca + exerts a cytotoxic effect because a high ca + concentration can open numerous mitochondrial transition pores, increase mitochondrial permeability, and destroy the mitochondrial outer membrane; consequently, cytochrome c and other proapoptotic factors are released, leading to apoptosis ( figure ) [ , , , , ] . the cvb b protein was shown to inhibit caspase activation and cell death induced by actinomycin d and cycloheximide by regulating the intracellular ca + concentration [ , ] . additionally, the b protein of hrv induced an er stress response, accompanied by an increased expression of cleaved caspase- and ccaat-enhancer-binding protein homologous protein (chop), which might have also involved a change in the ca + level [ ] . collectively, these results suggest that the b protein may regulate apoptosis by altering calcium homeostasis. furthermore, the b protein can regulate apoptosis through the mitochondrial pathway. madan et al. [ ] showed that the pv b protein interacted with the mitochondria and altered the mitochondrial morphology, in addition to the release of cytochrome c after the pv b gene expression. cong et al. [ ] reported that the ev b protein was localized to the mitochondria and induced apoptosis by directly activating the proapoptotic b-cell lymphoma -associated x (bax) protein, without a significant uptake of ca + by the mitochondria. therefore, the activation of the mitochondrial apoptotic pathway and subsequent apoptosis, induced by the ev b protein, may not involve ca + signaling. collectively, picornaviral b proteins can induce cell death in a variety of ways, with ca + playing an important role in most of these mechanisms. figure . b protein regulates autophagy and apoptosis. the b protein induces target membrane remodeling to form the viroplasm, which is generated from the endoplasmic reticulum (er). the isolation membrane is produced by the viroplasm. activation of the ca + /calmodulin-dependent kinase kinase-β (camkk-β) signal pathway is due to an increased intracellular calcium concentration. furthermore, mitochondrion takes up ca + from the er, thereby cytochrome cis released, leading to apoptosis. the host immune system is an important line of defense against pathogens, and pathogens can affect the immune system in a variety of ways. the b protein mainly affects the host immune response through inflammasome activation and by direct antagonism of the host immune response. recognition of pathogens by the immune system is mainly mediated by pathogen-associated molecular pattern receptors, known as pattern recognition receptors, including nucleotide-binding oligomerization domain (nod)-like receptors (nlrs), retinoic acid-inducible gene-i (rig-i)-like helicases, and pyrin domain-containing (nlrp ) [ ] [ ] [ ] . activation of nlrp inflammasome occurs during a period of changes in ion concentrations [ , ] . the nlrp belongs to the nlr family of inflammasomes and causes interleukin (il)- β and il- secretion via caspase- activation [ ] . the emcv, pv, ev , and hrv b proteins all activate the nlrp inflammasome but use distinct mechanisms [ , ] . the hrv and emcv b proteins can stimulate the nlrp inflammasome pathway to activate caspase- , which catalyzes the proteolysis of pro-il- β to il- β, leading to its secretion from across the plasma membrane by inducing a ca + efflux from intracellular storage ( figure ) [ , ] . wang et al. [ ] found that cvb -infected cells induced the nlrp activation in association with a k + efflux. the influenza virus m protein, which is also a viroporin, is capable of transporting na + and k + , resulting in activation of the nlrp inflammasome [ , ] . since the cvb b protein acts as a viroporin and can disrupt the intracellular ion balance [ , ] , it has been speculated that the induction of nlrp activation in cvb -infected cells may be related to the b protein. in addition to activating the inflammasome, the b protein also antagonizes the host immune response. both in vitro and in vivo studies have suggested that inhibition of protein trafficking would effectively allow viral evasion of the host immune response (figure ) [ ] [ ] [ ] . moreover, inhibition of protein transport may be related to changes in the ca + concentration [ ] . similar to the cvb b protein, the b proteins of pv, hrv and hrv , were shown to significantly inhibit the protein transport through the golgi complex, whereas the hav, fmdv, and emcv b proteins did not inhibit the protein transport [ , , ] . the fmdv b and c proteins did not block protein secretion, whereas the transport of proteins from the er to the golgi complex were blocked by the fmdv bc protein, and this effect was reproduced upon co-expression of the c and b genes [ , ] . collectively, these findings suggest that the b protein may participate in a viral evasion of the host immune response, mainly by inhibiting protein transport. figure . b protein affects the host immune response. the b protein can stimulate the nlrp inflammasome pathway to activate caspase- , which catalyzes the proteolysis of pro-il- β to il- β, which leads to their secretion across the plasma membrane by inducing a ca + efflux from intracellular storage. moreover, the b protein inhibits protein transport through the golgi protein which may be effective to evade the host immune response. the b protein has also been suggested to facilitate the viral evasion of the host immune response through other means. thus, the b protein antagonizes rig-i-mediated antiviral responses by inhibiting the expression of rig-i as an fmdv-specific reaction [ ] . the rna helicase lgp (also known as dexh-box helicase , dhx ) is a crucial factor involved in the host antiviral immune response [ ] . the fmdv leader protein (lpro), c protein, and the b protein have the ability to induce a decrease in lgp protein expression [ ] . in addition, pv b variants were shown to inhibit the antiviral interferon (ifn) system [ ] , whereas the hav b protein inhibited the synthesis of ifn-β by affecting the mitochondrial antiviral signaling protein activity, thereby antagonizing the host immune response [ ] . collectively, these evidences indicate that picornaviral b proteins can affect the host immune response, thereby promoting viral amplification or the release of viral particles. as discussed above, picornaviral b proteins have a viroporin or viroporin-like activity and play an important role in the picornaviral life cycle. therefore, many common applications targeting viroporins may be translatable to those targeting b proteins. in addition, the b protein may serve as a new target for the development of antiviral drugs. thus, further studies on the structure and function of the b protein might open up new avenues for the prevention and control of picornaviruses. owing to its highly conserved sequence, the use of the b gene as a marker could effectively improve the accuracy of virus detection. li et al. [ ] designed primers and taqman probes, based on the b and d regions, which were successfully used in real-time polymerase chain reaction to accurately detect and quantify fmdv during infection and replication. in addition, wang et al. [ ] developed a lateral-flow detection system, which could rapidly and easily detect fmdv using the b gene. in addition to gene-based detection, the virus could be detected using a b antibody. biswal et al. [ ] used an indirect enzyme-linked immunoassay based on a recombinant b protein to detect antibodies specific for fmdv. this method can be applied not only to fmdv but also to other picornaviruses, including cvb and ev . given the significant threat that picornaviruses pose to humans and animals, resulting in enormous economic damage to the livestock industry, development of picornavirus vaccines is of great significance. although inactivated virus vaccines can offer effective prevention, there are associated residual risk issues, including incomplete virus inactivation and escape during the vaccine production process [ ] [ ] [ ] . therefore, genetically engineered vaccines are considered more suitable options to overcome these shortcomings of inactivated viral vaccines. the ev vp protein is located outside the viral membrane and is thus exposed to the greatest amount of immune stress. accordingly, vp shows an extreme serological variability, thus providing the most reliable molecular epidemiological information. consequently, the vp region of ev has become a focus of vaccine research for picornavirus infections [ ] . however, dna constructs containing the vp gene of ev showed low levels of antigenicity. therefore, there is still a need to develop an effective adjuvant strategy to increase the antigenicity. one possibility in this regard is the use of recombinant vaccines incorporating the b gene to enhance the efficacy of vaccines. at present, applications of the b gene in recombinant vaccines have mainly concentrated on fmdv. the addition of a b fragment to a vaccine designed with vp as the core has been shown to effectively enhance the vaccine efficacy [ ] [ ] [ ] and reduce the dose and side effects [ ] . these effects may be similar to those leading to a greater efficacy of the adenoviral vector vaccine fused to the fmdv b protein against serotype o, which is associated with the induction of specific cd + and cd + protective t cell responses [ ] . therefore, future designs of other picornaviral genetically engineered vaccines would benefit from the addition of the b gene to increase the vaccine efficacy, including the addition of the b gene to a genetically engineered ev vaccine with the vp gene as the core. since viroporin plays an important role in all life stages of the virus, it is an attractive antiviral therapeutic target, and there have been great breakthroughs in this regard. by contrast, research and development of drugs targeting the b protein are relatively delayed. since b proteins have a viroporin or viroporin-like activity, screening for anti-picornavirus drugs among existing viroporin-targeting drugs may be a viable approach. there are four main types of inhibitors of viroporin activity, including adamantane, amiloride, alkyl iminosugar, and spirane amine [ ] . adamantane (amantadine and rimantadine) inhibits the m channel of influenza a virus by destroying the transmembrane network of hydrogen-bonded water molecules, thereby inhibiting the viral amplification [ ] . in bhk- cells infected with fmdv, the virus titer gradually decreased with an increase in amantadine concentration, which may have been due to abrogation of the pore-forming activity of the b protein and ultimate inhibition of fmdv replication [ ] . however, clinical trials showed that amantadine was not only selective for specific resistance mutations in hepatitis c virus (hcv) p [ ] , but also caused a rapid emergence of amantadine-resistant variants of influenza a virus during monotherapy for influenza [ ] . amiloride is a composite of two drugs, -(n,n-hexamethylene) amiloride and a novel inhibitor, bit , targeting hcv p and hiv- vpu, which can together block the viroporin ion channel activity or prevent ion channel formation, resulting in a potent antiviral effect [ ] [ ] [ ] [ ] . the alkyl iminosugar inhibits the formation of ion channels by targeting the hcv p viroporin [ ] . finally, spirane amines, such as bl- , also inhibit the influenza a virus m protein, with an antiviral mechanism similar to that of amantadine [ ] . there are also other drugs that act as viroporin inhibitors, including , -dibenzyl- ( h- , , , tetraazol- -yl) hexahydropyrimidine (cd), n-( -phenylethyl)- -[ -(phenylsulfonyl)- -piperazinyl]- quinazolinamine (lds ), and -methyl- , , -trihydroxyanth-raquinone (emodin), among others [ , , ] . the mechanism of action of these viroporin inhibitors is based on the inhibition of the viroporin channel activity. therefore, these drugs may have the potential to be applied for the treatment of picornavirus infections by targeting the b gene. however, this application will require further detailed investigations and drug screening. nevertheless, the b protein has the potential to widen the range of antiviral treatment strategies. furthermore, specific degradation of complementary mrna can be triggered by small interfering rnas (sirnas) or folded short hairpin rnas (shrnas) [ ] , which can be explored as an rna interference strategy, a relatively novel technology that has already been applied to treat many important pathogens, including hiv- , hepatitis b virus, and herpes simplex virus [ ] [ ] [ ] . currently, shrnas targeting the highly conserved b gene sequence are widely used in picornavirus research, including fmdv [ , ] , emcv [ ] , and cvb [ ] , and significant experimental viral suppression has been achieved. basically, rna interference against b gene affects the stability and integrity of the whole viral genome. the high nucleotide sequence conservation makes the b gene an attractive target for rna interference, which may potentially be effective against multiple picornavirus types, and open the door for additional sirna drugs. to date, there have been few studies specifically focusing on inhibitors of the b protein. xie et al. [ ] found that , -diisothiocyano- , -stilbenedisulfonic acid (dids) blocked a chloridedependent current, mediated by the ev b protein, and suppressed viral amplification. however, further research is needed to uncover the underlying mechanism. despite the many challenges in drug development, new technologies such as fourier-transform infrared spectroscopy and design of molecular dynamics analogs, as well as cryo-electron microscopy and spectroscopy, are expected to greatly contribute to the development of antiviral drugs. recent studies have gradually clarified the function and the potential of the b protein, along with increasingly recognizing its importance in the viral life cycle. however, there are still some challenges to overcome in investigations of the picornaviral b protein. in particular, its strong hydrophobicity makes it difficult to achieve soluble expression. ao et al. [ ] conjugated the small ubiquitin-like modifier (sumo) protein to the n-terminus of the fmdv b protein and successfully achieved soluble expression. therefore, this method can be tested for other picornaviral b proteins. moreover, the detailed molecular mechanism of the action of the b protein requires further study, along with the identification of interactions of b protein with host proteins, to better understand the role of the b protein in the pathogenesis of picornaviruses. in murine cells, the b protein was suggested to react with host proteins to promote rhinovirus proliferation [ ] . using a yeast two-hybrid system, the fmdv b protein was found to interact with the host elongation factor γ (eef g), and mislocalization of eef g demonstrated that the eef g deletion affected the synthesis of membrane proteins [ , ] . although a yeast two-hybrid system is a common laboratory protein-screening technique, it has a low success rate and is time-consuming. alternatively, affinity purification-mass spectrometry can be used to overcome these shortcomings, which has already been widely used in studies on dengue, zika, and ebola viruses [ , ] . at present, the development of antiviral drugs against viroporins is focused on three aspects, including viroporin and membrane fusion inhibitors, ion channel inhibitors, and targeted viroporin antibodies [ ] . with respect to the biological function of the b protein, antiviral drugs targeting the b protein could be designed based on the following three approaches: broad-spectrum screening for anti-picornavirus drugs among existing viroporin inhibitors, screening for b protein and membrane fusion inhibitors, and screening for b protein pore activity inhibitors. as discussed herein, the most important basis for the function of the b protein is that it can be polymerized into pores, thereby changing the permeability of the membrane. therefore, the design of drugs targeting b protein should be based on inhibiting polymerization of the b protein into pores, thereby reducing its effects on cellular ion homeostasis. however, these designs first require detailed determination of the refined atomic structure of the b protein, along with the expansion of screening techniques and applications of meticulous medicinal chemistry. furthermore, to develop better antiviral drugs, it will be necessary to elucidate the exact role of the b protein channel in the viral life cycle. thus, the main points of focus for research on the structure and function of the b protein toward ultimate drug development are: ( ) mechanism of increasing membrane permeability to disturb the ion balance, ( ) regulation of autophagy and apoptosis, ( ) inhibition of the host immune response, and ( ) promotion of viral replication and release. taken together, as research aimed at further elucidation of the role of the b protein progresses, along with the adoption of new technologies, it is expected that more strategies will come to light for antiviral drug development and disease control. author contributions: z.l., z.z. and z.j. wrote the manuscript; q.l. and x.h. revised for its integrity and accuracy; q.l. approved the final version of this manuscript and takes responsibility for its contents. the authors declare no conflicts of interest. human parechoviruses-biology and clinical significance examples of the two-stage membrane protein folding model. viruses viroporins: structure and biological functions membrane-active peptides derived from picornavirus b viroporin molecular characterization of the viroporin function of foot-and-mouth disease virus nonstructural protein b protein b of coxsackievirus b induces autophagy relying on its transmembrane hydrophobic sequences. viruses topology and biological function of enterovirus non-structural protein b as a member of the viroporin family single point mutation in the rhinovirus b protein reduces the requirement for phosphatidylinositol -kinase class iii beta in viral 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with foot-and-mouth disease virus nonstructural protein b: identification by yeast two-hybrid system protein interaction mapping identifies rbbp as a negative regulator of ebola virus replication comparative flavivirus-host protein interaction mapping reveals mechanisms of dengue and zika virus pathogenesis this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -afnto tz authors: baillet, nicolas; krieger, sophie; journeaux, alexandra; caro, valérie; tangy, frédéric; vidalain, pierre-olivier; baize, sylvain title: autophagy promotes infectious particle production of mopeia and lassa viruses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: afnto tz lassa virus (lasv) and mopeia virus (mopv) are two closely related old-world mammarenaviruses. lasv causes severe hemorrhagic fever with high mortality in humans, whereas no case of mopv infection has been reported. comparing mopv and lasv is a powerful strategy to unravel pathogenic mechanisms that occur during the course of pathogenic arenavirus infection. we used a yeast two-hybrid approach to identify cell partners of mopv and lasv z matrix protein in which two autophagy adaptors were identified, ndp and tax bp . autophagy has emerged as an important cellular defense mechanism against viral infections but its role during arenavirus infection has not been shown. here, we demonstrate that autophagy is transiently induced by mopv, but not lasv, in infected cells two days after infection. impairment of the early steps of autophagy significantly decreased the production of mopv and lasv infectious particles, whereas a blockade of the degradative steps impaired only mopv infectious particle production. our study provides insights into the role played by autophagy during mopv and lasv infection and suggests that this process could partially explain their different pathogenicity. lassa virus (lasv), a mammalian old-world arenavirus, is the etiological agent of lassa fever (lf), an acute and occasionally severe viral hemorrhagic fever in humans. the disease is a significant cause of morbidity ( , - , infections annually) and mortality (about fatalities annually) and is constrained to sub-saharan west africa [ ] . humans are infected after cutaneous-mucosal contact with contaminated fluids or feces from the natural host of lasv, the peridomestic rodent mastomys sp. [ ] . direct exposure to body fluids or contaminated materials from infected patients is responsible for human-to-human transmission, especially during nosocomial outbreaks, but most of the infections that occur during outbreaks result from reservoir-to-human transmission [ , ] . the absence of a licensed vaccine and efficient antiviral drug available in endemic countries renders lf a public health problem, exacerbated by the current expansion of the zone of endemicity [ ] . mopeia virus yeast two-hybrid screens were performed following the protocol described in vidalain et al. [ ] . dna sequences encoding the matrix proteins (z) of lasv or mopv were cloned by in vitro recombination (gateway technology; invitrogen, carlsbad, ca, usa) from pdonr into the yeast two-hybrid vector ppc -gw for expression in fusion downstream of the gal dna-binding domain (gal -bd). ah yeast cells (clontech; takara, mountain view, ca, usa) were transformed with viruses , , of these baits using a standard lithium-acetate protocol. spontaneous transactivation of the his reporter gene was observed in yeast cells expressing gal -bd-z. consequently, screens were performed on a synthetic medium lacking histidine (-his) and supplemented with -amino- , , -triazole ( -at) at to mm for mopv and to mm for lasv. a mating strategy was used to screen three different prey libraries with distinct characteristics: a human spleen cdna library, a mouse brain cdna library, and a normalized library containing , human orfs. all libraries were established in the yeast two-hybrid expression plasmid ppc to express prey proteins in fusion downstream of the gal transactivation domain (gal -ad). after six days of culture, colonies were picked, replica plated, and incubated over three weeks on selective medium to eliminate potential contamination with false positives. prey proteins from selected yeast colonies were identified by pcr amplification using primers that hybridize within the ppc regions flanking the cdna inserts. pcr products were sequenced, and cellular interactors were identified by multi-parallel blast analysis. pcr products were co-transformed into ah yeast cells expressing gal -bd-z constructs together with an empty ppc vector linearized downstream of the gal -ad coding sequence [ ] . homologous recombination in yeast cells between pcr products and linearized ppc vectors allows the reconstruction of the gal -ad-prey sequences. transformed cells were plated on selective -his media supplemented with at at mm for the mopv-z protein, and mm for the lasv-z protein. after five days of culture on selective medium, growing colonies were scored. veroe cells were maintained in dmem supplemented with . % penicillin-streptomycin (ps) and % fetal bovine serum (fbs). the hela and t cell lines were maintained in dmem supplemented with . % ps and % fbs. the gfp-lc hela cell line (kindly provided by dr mathias faure, centre international de recherche en infectiologie (ciri), lyon, france) was grown in rpmi supplemented with . % ps and % fbs (all from invitrogen, carlsbad, ca, usa). mopeia (an strain [ ] ) and lassa (av strain [ ] ) viruses were cultivated in veroe cells at • c in % co . viral supernatants were harvested and used as the virus stock and the absence of mycoplasma was confirmed. lasv and mopv titers were determined by plaque immunoassays as described below. all experiments with lasv were carried out in biosafety level facilities (laboratoire p jean mérieux-inserm, lyon). zm-flag and zl-flag were cloned into a phcmv vector, allowing the expression of mopv or lasv z matrix protein with a flag tag in the c-terminal position (inserted between the xmal and bamhi restriction sites). the same constructions were made for zm and zl-mcherry plasmids. tax bp plasmids were engineered into pegfp-c , allowing the expression of gfp-tagged proteins for immunofluorescence experiments. egfp-ndp was a kind gift from dr mathias faure (ciri, lyon, france). the primary antibodies used were: anti-actin (a ), anti-flag (a ), anti-atg (a ), and anti-tax bp (hpa ), all from sigma-aldrich (saint quentin fallavier, france); anti-ndp (ab ) from abcam (cambridge, ma, usa); anti-gapdh (sc- , santa cruz biotechnology, dallas, tx, usa), anti-p ( , bd biosciences, san jose, ca, usa), and anti-z (agro-bio, la ferté saint-aubin, france). the secondary antibodies used for western blotting were anti-mouse conjugated to peroxidase ( - - ) and anti-rabbit conjugated to peroxidase ( - - ), all from jackson immunoresearch (cambridge house, uk). the secondary antibody used for confocal microscopy was an anti-mouse conjugated to alexa (a ) from invitrogen (carlsbad, ca, usa). the pharmacological agent used was chloroquine (c ), purchased from sigma-aldrich. freshly passaged hela cells were added to pre-plated transfection media containing the indicated sirna at a final concentration of nm and using lipofectamine rnaimax (invitrogen, carlsbad, ca, usa), according to the manufacturer's instructions. the cells were maintained for h after sirna transfection in six-well plates at . × cells per well. the cells were then counted and infected with mopv or lasv (moi = . ) for h in dmem medium supplemented with % fbs and . % ps. infectious medium was removed before adding fresh medium (dmem % fbs, . % ps) for the indicated times of the experiment. viral rna and infectious particles were then quantified (please see corresponding paragraph). silencing rnas against ndp (sindp ) and tax bp (sitax bp ) were purchased from ambion (thermo fisher scientific, waltham, ma, usa) (references and , respectively). silencing rnas against atg (m- - ) were purchased from dharmacon (lafayette, co, usa), as well as the non-targeting control sirna (d- - - ). the efficiency of each sirna was validated by western blotting. human t cells (atcc, lgc standards, molsheim, france) were transfected with the indicated plasmids using lipofectamine (invitrogen), according to the manufacturer's protocol. at h post-transfection, cells were harvested and lysed in non-denaturing lysis buffer ( mm hepes, mm edta, mm nacl, % np , and µm pr- ( , calbiochem; merck, darmstadt, germany) supplemented with protease inhibitors ( , sigma-aldrich). lysates were clarified for min at , rpm. supernatants were then incubated with m anti-flag magnetic beads (m , sigma-aldrich) for to h at • c under agitation. the beads were washed four times in lysis buffer before boiling min in loading buffer. for western blots, equal amounts of protein were loaded and separated on - % gradient precast gels and transferred onto pvdf membranes before staining. samples were immunoblotted with the appropriate primary antibodies. protein levels were quantified by measuring the intensity of the bands by densitometry. actin or gapdh were used as positive controls of the cell extracts. all images were acquired using a confocal lsm (zeiss, oberkochen, germany) with an axioscope × oil immersion lens objective. the images were analyzed using imagej/fiji (version . m). hela or gfp-lc hela cells were cultured in -well plates with a sterile coverslip in each well. the cells were fixed in % paraformaldehyde for min and treated with glycine ( . m). for z protein-autophagosome colocalization experiments, we performed additional steps comprised of lysis ( . % triton x- ), saturation (pbs, % bovine albumin serum (bsa), % glycerol, and . % tween ), and primary antibody staining (mouse anti-flag, sigma-aldrich), followed by staining with a secondary antibody conjugated to alexa fluor . all samples were mounted with dapi (p , invitrogen). for the counting of autophagosomes, gfp-lc + vesicles were enumerated for cells for each condition. twelve-well plates were seeded with . × hela cells per well and the cells treated the day after with µm chloroquine (cq) for h before being placed on ice for min. cells were then infected with mopv at an moi of and placed on ice for min before transfer to • c (heat shock). cells were maintained for the indicated times before . % trypsin treatment (gibco; thermo fisher scientific, waltham, ma, usa) for min, in order to remove a non-internalized virus. cells were then centrifuged for min at rpm and washed two times with pbs. after centrifugation, pellets were lysed using the rneasy minikit (qiagen, hilden, germany) for quantitative viral analysis. viral rna was extracted from culture supernatants and cells using the qiaamp viral rna and rneasy minikits, respectively (all from qiagen), according to the manufacturer's instructions. quantitative pcr for viral rna was performed with the eurobiogreen lo-rox qpcr mix (eurobio, les ulis, france), using primers '-ctttcccctggcgtgtca- ' and '-gaattttgaaggctgccttga- ' for mopv and '-ctctcacccggagtatct- ' and '-cctcaatcaatggatggc- ' for lasv. vero cells were infected with sequential dilutions of supernatant and incubated at • c in % co for six days with carboxy-methyl-cellulose ( . %) (bdh laboratory supplies, poole, uk) in dmem supplemented with % fbs. infectious foci were detected by incubation with monoclonal antibodies (mabs) directed against mopv and lasv (mabs l - - a, l - - and yqb -ae , generously provided by dr p. jahrling, (usamriid, fort detrick, md usa), followed by pa-conjugated goat polyclonal anti-mouse igg (sigma-aldrich). statistical analyses were performed using graphpad prism (version . . , graphpad software, san diego, ca, usa). data were analyzed using student's t-test or the non-parametric mann-whitney test to determine the significance of differences (*p < . , **p < . , ***p < . , n.s. non-significant). we identified host cellular proteins that interact with z proteins of both viruses by using a high throughput yeast two-hybrid (ht-y h) screening protocol in which ah yeast cells were transformed with a y h vector encoding the z protein of lasv or mopv fused to the gal -bd domain (viral bait). y yeast cells were transformed with prey libraries consisting of a human spleen cdna library, mouse brain cdna library, or normalized library containing , human orfs fused to the gal -ad domain (cdna library prey). interactions between prey and bait lead to transactivation of the his reporter gene. after mating ah and y yeast on selective medium lacking histidine, the prey proteins from positive colonies were identified by pcr amplification. the pcr products were sequenced, and the cellular interactors identified by multi-parallel blast analysis. y h screens allowed us to identify, among others, two known autophagy adaptors, namely ndp and tax bp , which have been shown to interact with the z protein of mopv, but not lasv (table ) . interactions between tsg and arenavirus z protein have already been reported by others and were used here to validate our screen [ , ] . ndp and tax bp are autophagy adaptors, known as sequestosome -like receptors (slrs). slrs recognize motifs (such as ubiquitin or galectin) present on the surface of invading pathogens through a ubiquitin-binding domain (ubd) and target them into autophagosomes through a lc -interacting region (lir) for selective degradation by autophagy [ ] . we then confirmed the results by retesting each interaction in the y h system using the gap-repair procedure, in which dna sequences encoding both viral z proteins and candidate host interactors are introduced into fresh yeast cells. transformed cells were plated on selective medium lacking histidine and the growing colonies scored after five days of culture on selective medium ( figure a ). analysis of the colonies confirmed the previous results obtained by y h. these findings suggest that ndp and tax bp may play an important role in the life cycle of mopv or lasv. moreover, they also suggest that autophagy may have different roles during lasv and mopv infection as ndp and tax bp interacted only with the mopv z protein. table . selected "autophagy-linked" host protein interactors of lasv and mopv z identified by y h and validated by gap-repair. the first and second columns correspond to the canonical gene names and gene ids, respectively, for the selected interacting cellular proteins. columns and provide a summarized role of the corresponding protein in autophagy pathways and the reference, respectively. column shows the number of positive yeast colonies obtained for each cellular protein for the indicated virus z protein (retrieved from mouse brain, human spleen cdna libraries and orfeome). column shows the results obtained for the validation step by gap-repair in yeast. the "x" indicates the presence of growing colonies for the selected condition. tsg has already been described by others and served as a positive control. . all images were taken on a confocal zeiss lsm with an axioscope × oil immersion lens objective. scale bar represents µm. table . selected "autophagy-linked" host protein interactors of lasv and mopv z identified by y h and validated by gap-repair. the first and second columns correspond to the canonical gene names and gene ids, respectively, for the selected interacting cellular proteins. columns and provide a summarized role of the corresponding protein in autophagy pathways and the reference, respectively. column shows the number of positive yeast colonies obtained for each cellular protein for the indicated virus z protein (retrieved from mouse brain, human spleen cdna libraries and orfeome). column shows the results obtained for the validation step by gap-repair in yeast. the "x" indicates the presence of growing colonies for the selected condition. tsg has already been described by others and served as a positive control. ref. we next wanted to confirm the novel interactions identified by y h in mammalian cells. t cells were transfected with lasv or mopv c-terminal tagged flag z protein and/or an n-terminal egfp-fused ndp (egfp-ndp ) or tax bp (egfp-tax bp ) protein ( figure b ). cell lysates were directly analyzed by western blotting (input) or immunoprecipitated (ip) with anti-flag coupled magnetic beads followed by western blot analysis of coimmunoprecipitated egfp-tagged proteins. surprisingly, ndp and tax bp coimmunoprecipitated with the z protein of both lasv and mopv, contrary to the results of the y h screen. this discrepancy may be due to the -at concentration used, which was to times higher for the lasv z screens in the selective media of the y h and gap-repair screening. we then assessed the colocalization of z and the previously described interactors in another mammalian cell line. briefly, hela cells were transfected with the indicated plasmids before being fixed and analyzed by confocal microscopy. the z proteins of mopv and lasv colocalized with egfp-tax bp ( figure c ) and egfp-ndp ( figure d ) in the cytoplasm of transfected hela cells. overall, our findings show that both lasv and mopv z matrix proteins interact with ndp and tax bp in transfected t and hela cells. we next evaluated the functional impact of ndp and tax bp on the lasv and mopv life cycles. briefly, hela cells were transfected with control non-targeting sirna, sindp , or sitax bp . tax bp and ndp protein levels were much lower in sitax bp and sindp transfected cells, respectively, than in cells transfected with non-targeting sirnas h after transfection (figure a ). the silenced cells were then infected with lasv or mopv at an moi of . . after three days in culture, we evaluated the quantity of extracellular and intracellular rna, as well as that of viral infectious particles. there were no significant differences in the amount of intracellular viral rna ( figure b ) or infectious particles ( figure c ) between sictl, sindp , or sitax bp transfected cells for either lasv or mopv. however, there was slightly less extracellular lasv viral rna for sitax bp transfected cells than those transfected with sictl, whereas the level of mopv viral rna was unaffected ( figure d ). overall, these results suggest that ndp and tax bp are not involved in the production of infectious virus for either mopv or lasv in hela cells. we thus sought to determine whether autophagy was involved in the infection of mopv or lasv in hela cells. as ndp and tax bp interact with z and are known to be involved in the selective degradation of pathogens by autophagy, we first assessed whether mopv or lasv z protein is present in autophagosomes by transfecting mopv or lasv z protein into hela cells that stably express a gfp-fused lc (gfp-lc hela), commonly used as a marker of autophagosomes ( figure a ) [ ] . gfp-lc hela cells were treated with chloroquin (cq) for two hours before transfection with z plasmids in order to prevent the degradation of autophagosomes, thus increasing the probability to observe the possibly internalized matrix protein. strikingly, both mopv and lasv z proteins colocalized with gfp-lc puncta, suggesting a link between the autophagy machinery and mopv or lasv infection. we therefore examined the kinetics of autophagy upon infection with either virus. we infected hela cells with lasv or mopv at an moi of and assessed autophagy at various timepoints by evaluating the level of sqstm /p , a long-lived protein mainly degraded through autophagy; its degradation in cells indicating an effective autophagy flux ( figure b ) [ ] . there was a large decrease in p levels two days after mopv infection, suggesting an increase of the autophagy flux at this late timepoint. in contrast, p levels did not change in the lasv infected cells. we then sought to confirm the induction of autophagy in mopv-infected gfp-lc hela cells by counting the number of gfp-lc -labeled puncta, representing autophagosomes, over time ( figure c ). consistent with the previous findings, mopv infection induced a transient accumulation of autophagosomes two days after infection in gfp-lc hela cells. overall, these data strongly we thus sought to determine whether autophagy was involved in the infection of mopv or lasv in hela cells. as ndp and tax bp interact with z and are known to be involved in the selective degradation of pathogens by autophagy, we first assessed whether mopv or lasv z protein is present in autophagosomes by transfecting mopv or lasv z protein into hela cells that stably express a gfp-fused lc (gfp-lc hela), commonly used as a marker of autophagosomes ( figure a ) [ ] . gfp-lc hela cells were treated with chloroquin (cq) for two hours before transfection with z plasmids in order to prevent the degradation of autophagosomes, thus increasing the probability to observe the possibly internalized matrix protein. strikingly, both mopv and lasv z proteins colocalized with gfp-lc puncta, suggesting a link between the autophagy machinery and mopv or lasv infection. we therefore examined the kinetics of autophagy upon infection with either virus. we infected hela cells with lasv or mopv at an moi of and assessed autophagy at various timepoints by evaluating the level of sqstm /p , a long-lived protein mainly degraded through autophagy; its degradation in cells indicating an effective autophagy flux ( figure b ) [ ] . there was a large decrease in p levels two days after mopv infection, suggesting an increase of the autophagy flux at this late timepoint. in contrast, p levels did not change in the lasv infected cells. we then sought to confirm the induction of autophagy in mopv-infected gfp-lc hela cells by counting the number of gfp-lc -labeled puncta, representing autophagosomes, over time ( figure c ). consistent with the previous findings, mopv infection induced a transient accumulation of autophagosomes two days after infection in gfp-lc hela cells. overall, these data strongly suggest that mopv, but not lasv, induces a transient wave of autophagy two days after infection in hela cells. conversely to several viruses that take advantage of autophagy to replicate, while inhibiting autophagosome maturation, mopv (but not lasv) infection induced an increase in the autophagy flux. we investigated whether the autophagy flux is involved in mopv or lasv infectious particle production. first, we impaired the early steps of autophagy in mopv-or lasv-infected cells by reducing the expression of atg , essential for autophagosome formation ( figure a ). we confirmed that silencing of atg was efficient h after transfection, before infecting the cells with mopv or lasv at an moi of . . rt-qpcr and the titration of infectious particles in cells and/or supernatants revealed significantly less viral rna ( figure b ) and fewer infectious viral particles ( figure c ) in the supernatants of hela cells infected by mopv, and surprisingly lasv, when the expression of atg was impaired as compared to control conditions. however, the quantity of viral rna inside the cells was not affected by the silencing of atg for either virus ( figure d ), suggesting that the autophagy flux did not affect the viral entry or replication steps. altogether, these results indicate that the early stages of autophagy play an important role during the late stages of the mopv and lasv life cycle. student's t-test. (c) gfp-lc hela cells were infected with mopv at an moi of for the indicated times and fixed for confocal microscopy analysis. the images were acquired using the same microscope as in (a) with an axioscope × oil immersion lens objective. the error bars represent the standard error of the means from three independent experiments, *p < . , as determined by a student's t-test. gfp-lc dots were counted in cells per condition. conversely to several viruses that take advantage of autophagy to replicate, while inhibiting autophagosome maturation, mopv (but not lasv) infection induced an increase in the autophagy flux. we investigated whether the autophagy flux is involved in mopv or lasv infectious particle production. first, we impaired the early steps of autophagy in mopv-or lasv-infected cells by reducing the expression of atg , essential for autophagosome formation ( figure a ). we confirmed that silencing of atg was efficient h after transfection, before infecting the cells with mopv or lasv at an moi of . . rt-qpcr and the titration of infectious particles in cells and/or supernatants revealed significantly less viral rna ( figure b ) and fewer infectious viral particles ( figure c ) in the supernatants of hela cells infected by mopv, and surprisingly lasv, when the expression of atg was impaired as compared to control conditions. however, the quantity of viral rna inside the cells was not affected by the silencing of atg for either virus ( figure d) , suggesting that the autophagy flux did not affect the viral entry or replication steps. altogether, these results indicate that the early stages of autophagy play an important role during the late stages of the mopv and lasv life cycle. we next sought to confirm the impact of autophagy on infectious viral particle production by treating cells with cq. cq prevents endosomal acidification and is a well-known inhibitor of autophagosome maturation, which impairs both the fusion of autophagosomes with lysosomes and lysosomal protein degradation. viral entry of arenaviruses is characterized by internalization of the virus by endocytosis into acidified endosomes, where viral fusion occurs at low ph [ ] . we performed a viral entry assay to ensure that we were affecting autophagy rather than viral entry when using cq in the hela cells. we pretreated cells with µm cq and placed them on ice before infection with mopv at a high moi ( ) ( figure a) . we subjected the infected cells to heat shock and quantified the internalized viral rna by rt-qpcr at various timepoints. there were no differences in the quantity of internalized mopv rna between cq-pretreated and untreated cells, indicating that cq did not affect viral internalization by hela cells. arenavirus z protein is a critical regulator that mediates the budding steps prior to virus release, ensuring the packaging of all viral components required for infectious particle production [ , , ] . to this end, the z protein recruits several host proteins to facilitate efficient release of viral progeny from the plasma membrane of the infected cell. we chose to screen the lasv and mopv matrixprotein host interactors through y h screening because of its simplicity, its high throughput analysis, and the possibility to identify direct protein-protein interactions, in contrast to protein complex figure . degradative steps of autophagy increase mopv infectious particle production. (a) hela cells were pretreated with µm cq for h and placed on ice before infection with mopv at an moi of . cells were then placed on ice before proceeding to heat shock ( • c). at the indicated timepoints, attached virus was removed by trypsin treatment (tr), and viral intracellular rna harvested for rtqpcr analysis. (b) hela cells were infected with lasv or mopv at an moi of . and then treated, or not, with µm cq one day after infection for two days. cell supernatants were then harvested and titrated on vero cells. the error bars represent the standard error of the means from four independent experiments. * indicates p < . , n.s.: non-significant, as determined by the mann-whitney test. the error bars represent the standard error of the means from four independent experiments. (c) the same cells as in (b) were lysed and the quantity of z protein inside the cells has been measured by western blotting. representative results are shown, along with a graph representing the intensity of the z protein bands over actin. the error bars represent the standard error of the means from four independent experiments. n.s. non-significant; *** p < . , as determined by student's t-test. we investigated the role of the late stages of autophagy during mopv or lasv infection by infecting hela cells with lasv or mopv at an moi of . . one day after infection, cells were treated, or not, with cq at a final concentration of µm for h. three days after infection, cell supernatants and lysates were harvested. the inhibition of autophagosome maturation with cq significantly decreased the infectious particle production of mopv, but not that of lasv, when relative to control conditions ( figure b ). in addition, the treatment of cells with cq significantly decreased the level of z protein expression in mopv-but not lasv-infected cells ( figure c ). overall, these results indicate that mopv infection leads to productive autophagy which is required for the efficient production of infectious particles. in contrast, lasv infection does not trigger autophagy but basal autophagy is still important to improve the production of lasv infectious particles. arenavirus z protein is a critical regulator that mediates the budding steps prior to virus release, ensuring the packaging of all viral components required for infectious particle production [ , , ] . to this end, the z protein recruits several host proteins to facilitate efficient release of viral progeny from the plasma membrane of the infected cell. we chose to screen the lasv and mopv matrix-protein host interactors through y h screening because of its simplicity, its high throughput analysis, and the possibility to identify direct protein-protein interactions, in contrast to protein complex analysis by mass spectrometry, which does not discriminate direct from indirect partners. however, y h could represent a bias as z protein is known to localize to cell membranes. it is thus possible that some interactions between z and host cell proteins that take place in specific subcellular membranous compartments are unlikely to be recreated in the y h screen performed in this study. this method allowed us to isolate a number of host interactors and we selected from among them autophagy adaptors: ndp and tax bp . y h and gap-repair data identified ndp and tax bp as exclusive mopv z protein interactors, but immunoprecipitation assays and confocal microscopy observations revealed that the lasv z protein is also a partner of the two autophagy receptors. this difference in the detection threshold may be due to the -at concentration used ( to times higher for the lasv z screens) in the selective media for y h and gap-repair screening. -at is a competitive inhibitor of the his enzyme and is used to negatively select yeast growth when the bait protein alone (z) self-transactivates the his gene, thus limiting false-positive interactions. in our case, the self-transactivation level of the lasv z bait protein was higher than that of the mopv z bait protein. we thus used a more stringent selective medium for the lasv z constructions, which may have limited the true positive interactions between bait and prey plasmids in the yeast. we finally identified ndp and tax bp as interactors of both the lasv and mopv z proteins, as the confirmation by immunoprecipitation assays was performed under the same conditions for the matrix proteins of both viruses. other host factors interacting with lasv z protein and possibly linked to autophagy have been unraveled by others [ ] . in this study, authors have identified the ww-domain bearing protein bcl associated athanogene (bag ) as a negative regulator of lassa vlp egress. bag has several functions in cell cycle including a stress-induced role in chaperone-assisted selective autophagy (casa). although this study appears contradictory to the proviral role of autophagy described here during lasv infection, it reinforces the link existing between mammarenavirus infection and autophagy pathways. we then analyzed the functional role of ndp and tax bp in the viral life cycle. these two proteins are close paralogs involved in selective autophagy, in which specific cargo is first tagged by ubiquitination following their recognition by autophagy adaptor proteins for subsequent targeting to autophagosomes for degradation [ ] . this phenomenon has been well studied in bacteria and in several studies for viruses. for example, both tax bp and ndp can affect the replication of measles virus by interacting with viral proteins and facilitating the maturation of autophagosomes [ ] . another autophagic adaptor, p , has been shown to mediate autophagic degradation of chikungunya virus in murine cells [ ] . here, we show that ndp and tax bp are neither involved in the replication nor the release of mopv or lasv infectious particles, despite their interactions with the z proteins, as shown by rnai interference assays. these results were unexpected and suggest that autophagy adaptors could play a role during lasv or mopv infection that does not affect the viral fitness in the cell lines we used. one possible role of this interaction could be the regulation of inflammation, as tax bp and ndp have been shown to downregulate nfκb and irf signaling pathways [ , ] . the nucleoprotein of lasv is known to interfere with type i interferon (ifn-i) production through the sequestration of the ikk complex, leading to inhibition of the irf pathway [ ] . thus, both z and np could act in concert to inhibit the ikk complex, therefore controlling inflammatory response in infected cells. we extended our observations by investigating the role played by autophagy in transfected and infected cells. first, we show that the lasv and mopv z proteins colocalize with gfp-lc vesicles, arguing that autophagy likely plays a role during the course of the infection of both viruses. this observation, in conjunction with the large decrease in the quantity of z protein in mopv-infected cells treated with cq, suggests that the z protein is targeted towards autophagic degradation. such sequestration of the matrix protein in autophagosomes could have a role in z protein processing and viral assembly, as shown for gag in hiv-infected macrophages [ ] . however, sequestration of the z protein has been observed in cells over-expressing it via plasmid transfection, which may not accurately recreate the situation found in virus-infected cells. we then demonstrated that mopv, but apparently not lasv, induces a transient wave of autophagy two days after infection. these experiments were made using a high moi ( ) for each virus in order to monitor autophagy flux when most cells in the culture are infected at the same time. the correlation between p turnover and the higher number of gfp-lc vesicles indicates that mopv-induced autophagy is effective. we could not investigate whether lasv induced accumulation of autophagosomes in gfp-lc hela cells because of the lack of confocal microscopy within bsl- laboratory. however, the absence of a difference in p levels over time in lasv-infected cells suggests that lasv does not affect autophagy flux as p is generally degraded in autolysosomes. how autophagy is induced during mopv infection is unknown. as with others mammarenaviruses, mopv establishes long-term persistent infections in their natural hosts. under the experimental conditions we used in this study, mopv may transiently trigger the upr-er stress pathway, which could then induce autophagy. this phenomenon has been highlighted during hcv infection and is yet to be addressed for mopv [ ] . we demonstrated that autophagy is implicated in the release of mopv and lasv infectious viral particles, as silencing of the essential autophagy gene, atg , which prevents autophagosome formation, was sufficient to significantly decrease the amount of viral rna and the infectious titer in the supernatants of both mopv-and lasv-infected cells. in contrast, the use of cq, which inhibits autophagosome recycling, specifically impaired mopv infectious particle release. these results highlight a previously unknown role of autophagy in mopv and lasv infection. indeed, it shows that the autophagosome elongation step orchestrated by atg is a positive regulator of the late phases of mopv and lasv cycles. interestingly, the inhibition of autophagosome maturation only decreased mopv infectious particle production, highlighting the importance of the complete autophagy flux for mopv infectivity, at least in hela cells. the viral entry assays performed during mopv infection show that cq did not impact the viral entry steps, therefore indicating that the observed effects of the drug in mopv infectious particle production is linked to the inhibition of degradation steps of autophagy rather than a block of viral entry due to endosome acidification impairment. we did not perform this control with lasv, as cq did not affect lasv infectious particle production. in summary, mopv induces autophagy that must be productive to optimize the late steps of the viral cycle. in contrast to mopv, lasv does not induce autophagy, but requires the initial steps of vesicle formation to also favor the late steps of the viral cycle. given that autophagy did not appear to be blocked during lasv infection, it would be of interest to investigate whether lasv is able to escape from the last steps of autophagy degradation. we suggest that autophagy may be required for the mopv and lasv life cycle at different steps. many viruses have developed strategies to induce autophagy and hijack the process for their own benefit. this is true of the measles virus, hiv- , vesicular stomatitis virus, dengue virus, hepatitis c virus, and others [ ] [ ] [ ] [ ] [ ] . the late steps of the mopv cycle, including component assembly, genome encapsidation, and budding, may occur in the acidic mature autophagosome, such as during poliovirus infection in hela cells [ ] . lasv may take advantage of the early basal steps of autophagy to increase the processing of the matrix proteins, explaining the colocalization of lasv z protein within autophagosomes. lasv z protein could also subvert autophagosome membranes to improve viral egress. these phenomena have been observed during other viral infections. indeed, autophagy has been shown to have a dual role during hiv infection. the virus first benefits from early and non-degradative basal autophagy to increase gag processing and hiv yields in macrophages and then inhibits the degradative steps of autophagy to avoid its degradation [ ] . another virus, hepatitis b virus, induces an incomplete autophagic process in hepatoma cells that is required for hbv envelopment [ ] . in addition to mammarenavirus, the arenaviridae family includes three other genera: hartmaniviruses and reptarenaviruses are hosted by snakes and antennaviruses are hosted by fish [ ] [ ] [ ] . in contrast to mammarenaviruses and reptarenaviruses, antennavirus and hartmanivirus genomes do not contain genes encoding the z protein, indicating that the z interaction with the autophagy machinery described here is not a common feature among all arenaviruses. differences in the induction of autophagy during lasv and mopv infection could also play a role in the immunogenicity induced by these viruses. we previously showed that mopv strongly activates cd + t cells in an in vitro model of dendritic-cell (dc)-t-cell coculture, resulting in a strong proliferative response and the acquisition of effector and memory phenotypes, whereas the same responses were much weaker for lasv [ ] . autophagy plays a critical role in the adaptive immune response by delivering virus-derived peptides for presentation by major histocompatibility complex (mhc-i) molecules to cd + t lymphocytes in a process called cross presentation [ ] . as mopv induces autophagy, this process could mediate the delivery of mopv antigens to mhc-i at the dc surface and thus favor the subsequent activation of a specific cd + t-cell response and the killing of mopv-infected cells. overall, we hypothesize that, although basal autophagy is sufficient to sustain lasv infection, mopv requires an increase in autophagy flux to replicate. autophagy-mediated evolutionary pressure may have selected autophagy-inducing mopv variants with higher fitness. however, such a positive effect of autophagy on mopv replication is counterbalanced, as autophagy may also improve antigen presentation and subsequent killing of infected cells by the immune system, allowing the control of mopv replication. in contrast, lasv is not affected by this adverse effect as the virus does not induce autophagy. this hypothesis is currently under study and could highlight the key role of autophagy in the pathogenicity of lasv and could perhaps be extended to other pathogenic arenaviruses. in summary, we used y h screening to identify new targets of the mopv and lasv z matrix proteins, identifying tax bp and ndp as novel interactors of the z protein of both viruses. functional analysis of these interactors has provided insights into the role of the autophagy machinery during the course of mopv and lasv infection. autophagy is transiently induced in mopv-but not lasv-infected cells. although the early steps of autophagy play a positive role in the production of both lasv and mopv infectious particles, the degradative steps of autophagy are only required for mopv infectious particle release. these results suggest that autophagy could have different roles in lasv and mopv infection, which could explain, in part, their different pathogenicity. a prospective study of the epidemiology and ecology of lassa fever carried out primarily in the eastern province of sierra lassa 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experiments were conducted at the inserm jean mérieux bsl laboratory facility. we are very grateful to all bsl- team members for the help provided throughout this work. we thank the imaging and microscopy core facility (platim, ens, lyon) for providing access to confocal microscopy and their technical help. we thank mathias faure (ciri, lyon, france) for providing us with the gfp-lc hela cell line and the egfp-ndp -expressing vector, as well as his scientific advice. we are grateful to chloe journo (ciri, lyon, france) for providing us with the plasmids. we also thank c. clegg and g. lloyd (public health england, porton down, salisbury, uk) for the gift of mopv and s. becker for providing us with the av strain of lasv. finally, we are grateful to marielle friguet and laure diancourt for their technical support. the authors declare no conflict of interest. key: cord- - axsebya authors: lelli, davide; lavazza, antonio; prosperi, alice; sozzi, enrica; faccin, francesca; baioni, laura; trogu, tiziana; cavallari, gian luca; mauri, matteo; gibellini, anna maria; chiapponi, chiara; moreno, ana title: hypsugopoxvirus: a novel poxvirus isolated from hypsugo savii in italy date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: axsebya interest in bat-related viruses has increased considerably during the last decade, leading to the discovery of a rising number of new viruses in several bat species. poxviridae are a large, diverse family of dna viruses that can infect a wide range of vertebrates and invertebrates. to date, only a few documented detections of poxviruses have been described in bat populations on three different continents (america, africa, and australia). these viruses are phylogenetically dissimilar and have diverse clinical impacts on their hosts. herein, we report the isolation, nearly complete genome sequencing, and annotation of a novel poxvirus detected from an insectivorous bat (hypsugo savii) in northern italy. the virus is tentatively named hypsugopoxvirus (hypv) after the bat species from which it was isolated. the nearly complete genome size is , nt and it encodes genes. genome analyses suggest that hypv belongs to the chordopoxvirinae subfamily, with the highest nucleotide identity ( %) to eptesipoxvirus (eptv) detected from a microbat eptesicus fuscus in wa, usa, in . to date, hypv represents the first poxvirus detected in bats in europe; thus, its viral ecology and disease associations should be investigated further. poxviruses are dsdna viruses with large genomes ( to kb) that belong to the family poxviridae. the family is divided into the entomopoxvirinae and the chordopoxvirinae subfamilies of viruses, which infect insects and vertebrates, respectively. according to the international committee on taxonomy of viruses (ictv) release [ ] , genera have been created to classify chordopoxviruses (avipoxvirus, capripoxvirus, centapoxvirus, cervidpoxvirus, crocodylidpoxvirus, leporipoxvirus, molluscipoxvirus, orthopoxvirus, parapoxvirus, suipoxvirus, and yatapoxvirus), but other viruses remain unclassified and new genera are likely to be recognized in the future. poxviruses show a diverse host range, with some viruses having wide host tropism (e.g., orthopoxviruses) and thus being consequently associated with greater zoonotic risks [ ] , and others having strict host specificity. in recent decades, bats have been increasingly recognized as reservoirs of emerging viral infections, which has important ramifications for animal and public health [ ] . however, the majority of bat-borne viruses that can cause severe diseases in humans and other mammals, do not cause apparent clinical signs in bats. consequently, it has been assumed that bats may have a "special" relationship with viruses based on physiological, ecological, evolutionary, and/or immunological aspects, which allow them to act as special viral reservoirs with exaggerated viral richness [ ] [ ] [ ] [ ] . currently, four poxviruses from the microchiroptera and macrochiroptera suborders have been detected in bat populations on three continents (america, africa, and australia) [ ] . specifically, eptesipoxvirus (eptv) was isolated in north america in from eptesicus fuscus [ , ] ; eidolon helvum poxvirus (ehpv ) was detected in west africa in from eidolon helvum [ ] ; the pteropox virus (ptpv) was identified in northwestern australia in from pteropus scapulatus [ ] ; and a fourth poxvirus was also identified in south australia from miniopterus schreibersii bassanii in [ ] . it is remarkable that these viruses are phylogenetically divergent and are associated with variable clinical manifestations. virological investigations focused on poxviruses in bat populations may have a positive impact for future ecological studies of bat-pathogen interactions. moreover, from the perspective of the one health approach, bats could benefit from these studies, since european bat populations are currently undergoing a global decline that could be linked with so far overlooked viral infections. in this study, we report the isolation, nearly complete genomic sequencing, and annotation of a novel poxvirus detected from an insectivorous bat (hypsugo savii) in northern italy. the virus was tentatively named hypsugopoxvirus (hypv), according to the bat species from which it was isolated. phylogenetic analyses suggest that hypv belongs to the chordopoxvirinae subfamily, revealing the highest similarity ( %) with eptesipoxvirus (eptv) detected from the microbat eptesicus fuscus in wa, usa in , which is associated with bat necrosuppurative osteomyelitis in multiple joints. hypv is the first poxvirus detected in bats in europe and its viral ecology and disease associations should be investigated further. dead bats from different species were collected for virological investigations from wild animal rescue/rehabilitation centers in the context of a general surveillance project that has been implemented in northern italy since - , which focuses on the detection of emerging bat viruses [ ] [ ] [ ] . the bats were taxonomically identified based on their morphologic characteristics, according to the european bat identification keys [ ] . the carcasses were necropsied, and tissue samples were collected for further laboratory exams, particularly for viral detection and isolation. after necropsy, organ samples (lungs, heart, kidney, brain, and intestines) were mechanically homogenized in minimal essential medium ( g/ ml), which contained antibiotics. they were then centrifuged at g for min. samples were inoculated in confluent monolayers of vero and marc cells (african green monkey), incubated at • c with % co and observed daily for seven days to assess their cytopathic effects (cpes). in the absence of cpes, the cryolysates were sub-cultured twice onto fresh monolayers. cell culture supernatants showing cpe were partially purified by ultracentrifugation at , rpm for h (rotor tst kontron) through a % (w/w) sucrose cushion, and the pellet was re-suspended in pbs. this antigen was kept at − • c and then submitted for viral identification with the ngs approach and negative-staining electron microscopy (nsem) by using the airfuge (beckman instruments, palo alto, ca, usa) method [ ] . viral dna was extracted from µl of positive cell culture supernatants using a biosprint one-for-all vet kit (qiagen s.p.a., milan, italy). sequencing libraries were made with a nextera flex kit (illumina inc. san diego, ca, usa) in accordance with the manufacturer's instructions. libraries were sequenced on a miseq instrument (illumina inc. san diego, ca, usa) by using a miseq reagent kit v in a cycle paired-end run. data were assembled de novo by the clc genomic workbench v. (qiagen s.p.a., milan, italy). genome annotation and analysis was performed with tools from the bioinformatics suite developed at the viral bioinformatics resource centre [ ] . the genome annotation transfer utility (gatu) [ ] uses a reference genome to automatically annotate poxvirus genes with clear orthologs in the reference. other possible genes were presented to the annotator for further characterization and to make final annotation decisions. the case specifically concerned a juvenile hypsugo savii male that spontaneously died in a wildlife recovery center in valpredina, cenate sopra (bg), northern italy after several weeks of hospitalization. the sick bat was originally found alive on july , in telgate (bergamo province, northern italy) by a private citizen who brought it to the center. clinically, the bat had a humerus fracture, sensory depression and a lack of appetite but normal body mass. the death occurred days after admission to the center on september , ; then, the carcass was sent to the lab for necroscopy and further analyses. pathological lesions in the internal organs indicative of infectious diseases were not observed, but a soft bone callus due to pathological healing of the humerus fracture associated with osteomalacia and calcium deficiency was detected. a virus was isolated on marc cells inoculated with the organ pool composed of the bat's heart and lungs. the cpe occurred on the third day post-inoculation during the second passage and was characterized by a diffused degeneration of a monolayer with rounded cells floating in the culture medium ( figure a ,b). the cell culture supernatant showing cpe was submitted to the ngs in order to identify and characterize the unknown isolate. furthermore, nsem performed on the purified and concentrated antigen revealed the presence of viral particles that unequivocally morphologically resembled those belonging to the genus orthopoxvirus ( figure c ). the virus was tentatively named hypsugopoxvirus (hypv), according to the bat species from which it was isolated. table summarizes the basic information on the hypv identified in this study in comparison with all known poxviruses detected to date in bats worldwide. after ngs sequencing, the nearly complete viral genome of a poxvirus was obtained from one contig of , nucleotides originating from , reads with an average coverage of . . the nearly full genome sequence of the viral strain was determined and compared with those of other members of the poxviridae family available on genbank. for the nearly complete viral genome sequencing, blast analysis revealed the highest nucleotide identity ( %) to the eptesipoxvirus (eptv) strain "washington", a member of the chordopoxvirinae subfamily identified in microbats in the usa ( table ). the nearly complete genome sequence for hypv was submitted to genbank under accession number mk . [ , ] a conservative approach was taken for genome annotation to avoid over-annotating open reading frames (orfs) that were unlikely to represent functional genes. orfs less than codons or overlapping by more than % with well-characterized genes were not considered for annotation unless supported by other evidence. a total of genes were annotated for hypv, showing a percentage value of nt identity with its closest related virus eptv ranging from . % for the hypv- gene (serpin ) to % for the hypv- gene (vltf- ) ( table ) . when the seven conserved genes-rpo , rap , mrna capping enzyme large subunit, p a precursor, rpo , vetf-l, and dna primase-were considered individually, the value of nt similarity with eptv ranged from . % to . %. the above conserved genes that have been used for phylogenetic analysis in previous studies [ , ] are presented in bold in table . hypv showed nucleotide divergence from its closest relative, eptv. the smaller genome size with , nt encoding genes for hypv in comparison to , nt and genes for eptv, is likely due to the omission of the itrs from the analysis and therefore, is not possible to establish the exact length of its the viral genome. two orfs (hypv- and hypv- , table ), whose function is still unknown, appear to be unique to hypv. table . hypv genome annotation and nucleotide identities for each gene to the most similar strain eptesipoxvirus (eptv). the seven conserved genes used for phylogenetic analysis in previous studies [ , ] are presented in bold. ( - ) or "−" ( - ) ). the potential zoonotic risks associated with bats and their fascinating and special relationship with viruses have attracted the attention of many researchers worldwide. consequently, general and target surveillance on bat populations has increased in the last decade with the purpose of clarifying the genetic diversity of bat-associated viruses as well as acquiring comprehensive information on bat-pathogen interactions. in fact, viral disease prevention and biological conservation issues could both benefit from such research. virological surveillance of bat populations in italy is a relative novelty and has only recently been extensively applied, but almost immediately, a great heterogeneity of virus identifications has been observed. viruses belonging to several viral families, such as reoviridae [ ] , coronaviridae [ , [ ] [ ] [ ] [ ] [ ] , paramyxoviridae [ ] , rhabdoviridae [ , ] , and astroviridae [ ] , have been detected, allowing the identification of some novel/previously unknown viral agents. the results of the general surveillance of bats, which have been randomly applied so far as pilot virus discovery studies, may drive future activity to more specific longitudinal and target studies aimed at understanding the epidemiology of potential new pathogens. in this study, a novel poxvirus, hypv, was detected from the microbat hypsugo savii in italy. this likely represents the first poxvirus detection in bats in europe. in fact, only four poxviruses have been documented to date in bat populations worldwide, and these and these have diverse and somehow incomplete descriptions, with just some common aspects. firstly, ehpv was detected in with a high-prevalence in throat swabs from apparently healthy african megabats (eidolon helvum), and metagenomic analysis identified poxvirus sequences that were most closely related with molluscum contagiosum (mocv), a human-only pathogen [ ] . in the same year of , another bat poxvirus was incidentally detected in south australia during the investigation of an outbreak of parasitic skin disease in a population of the microbat species, miniopterus schreibersii bassanii. in one of the twenty-one bats examined, an independent (non-nematode-associated) lesion containing intracytoplasmic inclusion bodies indicative of poxvirus infection was observed, and this was confirmed with electron microscopy [ ] . between and , eptv was detected in adult big brown bats (eptesicus fuscus) with severe joint disease (tenosynovitis and osteoarthritis) at a wildlife center in northwestern united states. phylogenetic analysis revealed that eptesipoxvirus is most closely related to the cotia virus, a virus detected in sentinel suckling mice in sao paulo, brazil in [ , ] . ptpv was detected from an australian little red flying fox (pteropus scapulatus) that died following entrapment on a fence. post-mortem examination revealed multiple nodules on the wing membranes. phylogenetic analysis indicated that ptpv is not closely related to any other poxvirus isolated from bats or other species, and that it likely should be placed in a new genus [ ] . it is noteworthy that ptpv and ehpv were isolated from megabat hosts (pteropus scapulatus and eidolon helvum, respectively), whereas eptv and hypv were isolated from microbats (eptesicus fuscus and hypsugo savii, respectively). while ehpv was detected in apparently healthy bats, the other viruses were identified in sick bats and their association with the pathological condition was assumed. specifically, clinical symptoms of eptv in eptesicus fuscus manifested in the form of joint swelling and increased lethargy [ ] . ptpv-infected pteropus scapulatus presented vesicular to nodular skin lesions on the wing membranes that are typical of poxvirus infections [ ] . hypv was detected in a bat showing pathological healing of the humerus fracture associated with osteomalacia and calcium deficiency. neither symptom was directly linked to fatality and thus the capability of these viruses still needs to be ascertained, including the role of hypv in causing deadly disease in bats. the results of our study indicate that hypv presents the typical morphology of the orthopoxvirus genus and that it could be isolated in cell culture. indeed, its final identification was obtained by genomic characterization. the nearly complete genomic sequencing clearly demonstrated that hypv is a new virus that is distantly related to its closest known relative eptv (wa, usa, ) with a nucleotide identity of % (almost whole genome). indeed, the percentage value of the nt identity of hypv with eptv ranged from . % for the hypv- gene (serpin ) to % for the hypv- gene (vltf- ). regarding orfs annotation the hypv was shown to be defective in particular in the itr genes i.e., out of described in eptv, but this should be not a real structural defect but more likely due to the omission of the itrs from the analysis. on the contrary, two orfs, whose function is still unknown, appear to be unique to hypv. to conclude, a new poxvirus, hypv, was detected in bats in europe and its viral ecology and disease associations should be investigated further. author contributions: d.l. designed the study and wrote the manuscript; a.l. performed electron microscopy, participated in study coordination, and helped to draft the manuscript; c.c. and l.b. performed the next-generation sequencing and data analysis; a.m.g. and m.m. performed the sampling and data collection; g.l.c. performed the clinical investigations; a.p. and f.f. performed the necropsies and molecular tests; e.s. and t.t. were involved in the virological analysis and interpretation of the results; a.m. performed the molecular genetic studies and helped to draft the manuscript. all of the authors have read and approved the final manuscript. an increasing danger of zoonotic orthopoxvirus infections bats: important reservoir hosts of emerging virus mass extinctions, biodiversity and mitochondrial function: are bats "special" as reservoirs for emerging viruses? antiviral immune responses of bats: a review a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? host and viral traits predict zoonotic spillover from mammals poxviruses in bats . . . so what? viruses novel poxvirus in big brown bats, northwestern united states characterization of eptesipoxvirus, a novel poxvirus from a microchiropteran bat metagenomic study of the viruses of african straw-coloured fruit bats: detection of a chiropteran poxvirus and isolation of a novel adenovirus genomic characterization of a novel poxvirus from a flying fox: evidence for a new genus? outbreak of skin nodules associated with riouxgolvania beveridgei (nematoda: muspiceida) in the southern bentwing bat (miniopterus schreibersii bassanii), south australia identification of mammalian orthoreovirus type in italian bats detection of coronaviruses in bats of various species in italy isolation of a novel 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from sentinel mice in sao paulo biological characterization and next-generation genome sequencing of the unclassified cotia virus span (poxviridae) this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: special thanks to anna tirelli, loredana zingarello, giovanni bozzoni and all technicians in the izsler virology section for their valuable technical work and support in virological analysis. the authors declare no conflict of interest. key: cord- - lz tf authors: zhou, hongzhuan; su, xia; lin, lulu; zhang, jin; qi, qi; guo, fangfang; xu, fuzhou; yang, bing title: inhibitory effects of antiviral drug candidates on canine parvovirus in f cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: lz tf canine parvovirus (cpv) is a common etiological agent of acute enteritis, which occurs globally in domestic and wild carnivores. despite the widespread use of inactivated or live attenuated vaccines, the emergence of antigenic variants and the influence of maternal antibodies have raised some concerns regarding the efficacy of commercial vaccines. while no specific antiviral therapy for cpv infection exists, the only treatment option for the infection is supportive therapy based on symptoms. thus, there is an urgent medical need to develop antiviral therapeutic options to reduce the burden of cpv-related disease. in this study, a cytopathic effect (cpe)-based high-throughput screening assay was used to screen cpv inhibitors from a food and drug administration (fda)-approved drug library. after two rounds of screening, seven out of screened drugs were found to have > % cpe inhibition. three drugs—nitazoxanide, closantel sodium, and closantel—with higher anti-cpv effects were further evaluated in f cells by absolute pcr quantification and indirect immunofluorescence assay (ifa). the inhibitory effects of all three drugs were dose-dependent. time of addition assay indicated that the drugs inhibited the early processes of the cpv replication cycle, and the inhibition effects were relatively high within h postinfection. western blot assay also showed that the three drugs had broad-spectrum antiviral activity against different subspecies of three cpv variants. in addition, antiapoptotic effects were observed within h in nitazoxanide-treated f cells regardless of cpv infection, while closantel sodium- or closantel-treated cells had no pro- or antiapoptotic effects. in conclusion, nitazoxanide, closantel sodium, and closantel can effectively inhibit different subspecies of cpv. since the safety profiles of fda-approved drugs have already been extensively studied, these three drugs can potentially become specific and effective anti-cpv drugs. canine parvovirus (cpv), genus protoparvovirus, a member of the family parvoviridae (subfamily parvovirinae), is a small, highly contagious, nonenveloped, single-stranded dna virus [ ] . cpv is a major causative agent of acute gastroenteritis, leukopenia and myocarditis in dogs, and typical clinical signs include vomiting, fever, and diarrhea. generally, puppies aged weeks to months have been found to be more susceptible to cpv infection [ , ] . the cpv genome is approximately . kb in length, and contains two open reading frames (orf), which encode nonstructural proteins (ns and ns ) and two structural proteins (vp and vp ) [ ] [ ] [ ] . indicating % cpe inhibition, and od values of wells with sd infection served as a negative (virus) control indicating % cpe inhibition [ ] . the percentage inhibition was calculated using the formula: percentage cpe inhibition = (od of drug treated cells − od of negative control)/(od of positive control − od of negative control) × [ , ] . all drug plates were set-up in duplicates for primary screening, and drugs show greater than % cpe inhibition from the primary screen were used for second round of screening. the further validation of identified drugs was conducted in triplicates, and drugs show greater than % cpe inhibition were used for further analysis. dose response experiments were performed to test cc s and ec s of drugs as described above with minor modifications. for cc assays, µl of f cells ( . × cells/well) was mixed with µl prediluted drugs at final concentrations ranging from . - µm. for ec assays, µl of f cells ( . × cells/well) was pretreated with µl prediluted drugs at final concentrations ranging from . - µm for h, and then treated cells were infected with µl cpv at an moi of . . both cc and ec assays were done in triplicate. cell cytotoxicity and inhibition of cpv infection were both examined after h incubation using the transdetect ® cell counting kit (transgen biotech, beijing, china). the cc and ec values were calculated via a best-fit log (dose)-response curve-fitting in graphpad prism software (version . , la jolla, ca, usa) [ ] . absolute quantification pcr was used to evaluate the anti-cpv effect. total dna was isolated from sd infected f cells using qiaamp dna mini kit (qiagen, hilden, germany) according to the manufacture's instruction. cpv specific primers vp -f -caaatagagcattgggcttacc- ' and vp -r '-tcccatttgagttacaccacg- ' were used to amplify the -bp fragment. the obtained fragment was cloned into pmd ® -t (takara, shiga, japan), and the resulting positive clone was named pmd-vp s for further use. to evaluate the antiviral effects of the three drugs of nitazoxanide, closantel sodium and closantel, f cells were seeded in -well plates at . × cells per well and pretreated with the drugs at a final concentrations of µm, µm, and µm, respectively, for h, then treated cells were infected with cpv at moi of . as described above. cells treated with . % dmso were used as control. after h incubation, total dna was extracted from whole cell lysates by qiaamp dna mini kit (qiagen, hilden, germany). the quantitative standard curve was generated via quantitative real-time pcr of the plasmid pmd-vp s preparations at serial dilutions of , , , , , and copies/µl. each µl qpcr reaction mixture contained µl -fold diluted sample, µl superreal premix plus (sybr green) (tiangen biotech, beijing, china), and . µm of specific primers. all mixtures were then loaded into a stepone plus qpcr machine (applied biosystems, usa). the qpcr procedure was of min at • c, followed by cycles of s at • c, and s at • c. the absolute number of dna copies of the vp gene in the cells was determined according to the generated standard curve. immunofluorescence assay (ifa) was used to further evaluate the antiviral effects of identified drugs. f cells in -well plates ( . × cells/well) were pretreated with µl prediluted nitazoxanide, closantel sodium, and closantel at final concentrations of µm, µm, and µm, respectively, for h, then treated cells were infected with µl cpv at moi of . . after h postinfection, cells were fixed with % acetone, and then incubated with a : dilution of mouse anti-vp monoclonal antibody (ingenasa, madrid, spain) for min, followed by incubation with a : dilution of fluorescein isothiocyanate-conjugated goat anti-mouse igg (h+l) highly cross-adsorbed secondary antibody (invitrogen, carlsbad, ca, usa). finally, the cells were stained with , -diamidino- -phenylindole (dapi) in order to label cell nuclei in focus. after washing, the cells were examined with high content imaging system (operetta, perkinelmer, waltham, ma, usa) at × magnification. the time of addition experiment was used to test the drug inhibition stage of the cpv replication life cycle. meanwhile, the inhibitory effects of the identified drugs at different time points following the addition of the three drugs after virus infection were also evaluated by this assay. briefly, f cells were seeded in -well plates ( . × cells/well) and then infected with cpv (moi = . ). nitazoxanide, closantel sodium, closantel, or . % dmso were added at − h ( h pre-infection), h (cpv infection), . h, h, h, h or h (post-infection) to determine the inhibitory effects at different time points of drug addition [ ] [ ] [ ] . cells treated with . % dmso served as control, and the effect on drug inhibition was evaluated using transdetect ® cell counting kit (transgen biotech, beijing, china) at h postinfection, as described above. western blot was also used to evaluate the antiviral activity of nitazoxanide, closantel sodium, and closantel against different subspecies of cpv variants. f cells were seeded in -well plates at . × cells per well and pretreated with the three drugs at final concentrations of µm, µm, and µm, respectively, for h. the treated cells were infected with cpv variants sd (new cpv- a strain), sd (new cpv- b strain) and bj- (new cpv- a strain), at an moi of . as described above. cells with . % dmso were used as control. after h incubation, cells were harvested and lysed with proteinext ® mammalian total protein extraction kit (transgen biotech, china). equal amounts of cell lysates were analyzed via sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page) and then transferred onto polyvinylidene difluoride (pvdf) membranes (millipore, burlington, ma, usa). after blocking with % milk-tbs-tween for h at room temperature, anti-vp monoclonal antibody ( : dilution, ingenasa, madrid, spain), and beta-actin monoclonal antibody (ac- ) ( : dilution, thermo scientific, waltham, ma, usa) were added and incubated overnight at • c, blots were further incubated with horseradish-peroxidase(hrp)-conjugated goat anti-mouse igg for h at • c. the immunoreactive bands were detected using a supersignal™ west pico plus chemiluminescent substrate kit (thermo scientific, usa) and imaged using a chemiluminescence apparatus (proteinsimple, usa). band intensities were measured using the image j software, and viral vp protein expression was first compared with beta actin expression, and then normalized to the . % dmso-treated group. f cells were seeded in -well plates ( . × cells/well) and pretreated with µl nitazoxanide, closantel sodium, and closantel with final concentrations of µm, then infected with cpv (moi = . ). cells were incubated for h, h, h and h at • c and % co . the assay was performed in triplicates. caspase-glo / assay kit (promega, usa) was used to detect pro-or antiapoptotic effects of the identified drugs [ , ] . the luminescence of each sample was measured using a synergy h microplate reader (biotek instruments inc., winooski, usa) according to the manufacturer's instructions [ ] . the cc s and ec s of drugs were determined by a best-fit log(dose)-response curve-fitting in graphpad prism . one-way analysis of variance (anova) and dunnett's multiple comparisons test were used to analyze data. statistical significances are denoted as follows; * p < . ; ** p < . ; *** p < . ; **** p < . . in this study, a cpe-based high-throughput screening assay was used to screen cpv inhibitors from an fda-approved drug library. the timeline of drug treatment and cpv infection, as well as the flow chart of the cpe-based assay, are shown in figure a ,b. in the primary screen (first round), the z' factor was between . and . across all drug plates. as the assay quality control index z' factors were > . in all plates, it demonstrated that the cpe-based screening assay was suitable for screening anti-cpv drugs. the mean percentage cpe inhibition of each drug was plotted in figure c . viruses , , x for peer review of round), the z' factor was between . and . across all drug plates. as the assay quality control index z' factors were > . in all plates, it demonstrated that the cpe-based screening assay was suitable for screening anti-cpv drugs. the mean percentage cpe inhibition of each drug was plotted in figure c . twenty-one drugs showing > % cpe inhibition from the primary screen were used for a second round of screening, and seven drugs with percentage inhibition > % were further identified. (c) scatter plot of percentage cpe inhibition results for fda-approved drugs, numbers in x axis mean the species of the tested drugs, each number corresponds to a specific drug, and the order is the same as that provided in the manual of the fda-approved drug library, each dot shows the mean percentage cpe inhibition in the presence of μm tested drug. twenty-one drugs with > % cpe inhibitions, identified during the first round of screening, were used for the second round of screening. the drug name, catalogue number of selleck, and the final percentage cpe inhibition of the drugs are listed in table s , and the inhibitory effects of these drugs, when these drugs were added h post-virus infection are also listed in table s . seven drugs with percentage cpe inhibitions > % were selected for further cc and ec assays, and the results are shown in figures and s and are also listed in table . table . % cytotoxicity concentration (cc ), % antiviral efficacy concentration (ec ), and selectivity index (si) of identified anti-cpv drugs. flow chart of drug screen using cpe-based assay. briefly, f cells per well were pretreated with µm drugs for h, and then infected with . moi cpv, cell viability was detected at h postinfection as described above, antiviral inhibitors against cpv were determined according to the percentage cpe inhibition. twenty-one drugs showing > % cpe inhibition from the primary screen were used for a second round of screening, and seven drugs with percentage inhibition > % were further identified. (c) scatter plot of percentage cpe inhibition results for fda-approved drugs, numbers in x axis mean the species of the tested drugs, each number corresponds to a specific drug, and the order is the same as that provided in the manual of the fda-approved drug library, each dot shows the mean percentage cpe inhibition in the presence of µm tested drug. twenty-one drugs with > % cpe inhibitions, identified during the first round of screening, were used for the second round of screening. the drug name, catalogue number of selleck, and the final percentage cpe inhibition of the drugs are listed in table s , and the inhibitory effects of these drugs, when these drugs were added h post-virus infection are also listed in table s . seven drugs with percentage cpe inhibitions > % were selected for further cc and ec assays, and the results are shown in figure and figure s and are also listed in table . the top three drugs-nitazoxanide, closantel sodium, and closantel-were identified with higher percentage cpe inhibition of . ± . , . ± . , and . ± . %, respectively, at μm concentration (table s ). moreover, all three drugs showed a dose-dependent inhibition of cpv infection ( figure ). from the absolute qpcr results, dose-dependent reduction in the copy numbers of cpv viral dna were observed with increasing concentrations of nitazoxanide ( figure a ), closantel sodium, ( figure b ) or closantel ( figure c ). when cpv-infected f cells were treated with the three drugs at μm, the cpv viral dna copy numbers of ml whole cell lysates significantly reduced to . % (nitazoxanide), . % (closantel sodium), and . % (closantel) compared with the . % dmso-treated group ( figure ) . the top three drugs-nitazoxanide, closantel sodium, and closantel-were identified with higher percentage cpe inhibition of . ± . , . ± . , and . ± . %, respectively, at µm concentration (table s ). moreover, all three drugs showed a dose-dependent inhibition of cpv infection ( figure ). from the absolute qpcr results, dose-dependent reduction in the copy numbers of cpv viral dna were observed with increasing concentrations of nitazoxanide ( figure a ), closantel sodium, ( figure b ) or closantel ( figure c ). when cpv-infected f cells were treated with the three drugs at µm, the cpv viral dna copy numbers of ml whole cell lysates significantly reduced to . % (nitazoxanide), . % (closantel sodium), and . % (closantel) compared with the . % dmso-treated group ( figure ) . as shown in figure , cpv infection could be inhibited in the presence of nitazoxanide, closantel sodium, and closantel at a concentration of µm. few cells were cpv-positive when treated with µm of the drugs. almost no green signals were detected in all f cells treated with µm of the drugs. the results confirmed that these identified drugs inhibited cpv infection in a dose-dependent manner, which was consistent with the qpcr assay results. from the absolute qpcr results, dose-dependent reduction in the copy numbers of cpv viral dna were observed with increasing concentrations of nitazoxanide ( figure a ), closantel sodium, ( figure b ) or closantel ( figure c ). when cpv-infected f cells were treated with the three drugs at μm, the cpv viral dna copy numbers of ml whole cell lysates significantly reduced to . % (nitazoxanide), . % (closantel sodium), and . % (closantel) compared with the . % dmso-treated group (figure ) . as shown in figure , cpv infection could be inhibited in the presence of nitazoxanide, closantel sodium, and closantel at a concentration of μm. few cells were cpv-positive when treated with μm of the drugs. almost no green signals were detected in all f cells treated with μm of the drugs. the results confirmed that these identified drugs inhibited cpv infection in a dose-dependent manner, which was consistent with the qpcr assay results. consistent with screening results, all three drugs showed anti-cpv effects when added h before virus infection (pre-infection). the inhibitory effects of nitazoxanide, closantel sodium, and closantel were . ± . , . ± . , and . ± . , respectively, when the three drugs were added h post-virus infection ( figure ). the drugs inhibited the early processes of the cpv consistent with screening results, all three drugs showed anti-cpv effects when added h before virus infection (pre-infection). the inhibitory effects of nitazoxanide, closantel sodium, and closantel were . ± . , . ± . , and . ± . , respectively, when the three drugs were added h post-virus infection ( figure ). the drugs inhibited the early processes of the cpv replication cycle, and the inhibition effects were relatively high within h postinfection ( figure ). in addition, the anti-cpv activity of nitazoxanide was observed when the drug was added h postinfection, suggesting that nitazoxanide may partially have the ability to inhibit cpv infection at the stage of viral replication. western blot was also used to evaluate the broad-spectrum antiviral activity of identified drugs against different subspecies of three cpv variants. dose-dependent reductions in vp expression and the quantification of relative expression levels are shown in f cells treated with nitazoxanide ( figure a,d) , closantel sodium ( figure b ,e) or closantel ( figure c,f) , respectively. nitazoxanide treated at μm reduced relative expression of vp in different cpv variants to . % (sd ), . % (sd ), and . % (bj- ), respectively ( figure d ). closantel sodium treated at μm reduced relative expression of vp in different cpv variants to . % (sd ), . % (sd ) and . % (bj- ), respectively ( figure e ). closantel treated at μm reduced relative expression of vp in different cpv variants to . % (sd ), . % (sd ), and . % (bj- ), respectively ( figure f ). all the identified drugs showed inhibitory ability against cpv variants sd , sd , and bj- . western blot was also used to evaluate the broad-spectrum antiviral activity of identified drugs against different subspecies of three cpv variants. dose-dependent reductions in vp expression and the quantification of relative expression levels are shown in f cells treated with nitazoxanide ( figure a,d) , closantel sodium ( figure b ,e) or closantel ( figure c,f) , respectively. nitazoxanide treated at µm reduced relative expression of vp in different cpv variants to . % (sd ), . % (sd ), and . % (bj- ), respectively ( figure d ). closantel sodium treated at µm reduced relative expression of vp in different cpv variants to . % (sd ), . % (sd ) and . % (bj- ), respectively ( figure e ). closantel treated at µm reduced relative expression of vp in different cpv variants to . % (sd ), . % (sd ), and . % (bj- ), respectively ( figure f ). all the identified drugs showed inhibitory ability against cpv variants sd , sd , and bj- . doley et al. ( ) reported that cpv could induce caspase-dependent (involving extrinsic, intrinsic, and endoplasmic reticulum pathways) apoptosis in madin-darby canine kidney (mdck) cells [ ] . in order to evaluate whether drug-associated apoptosis was involved in the antiviral effect, we measured the caspase- activity of drug-treated cells with or without cpv infection. as shown in figure , the antiapoptotic effects were observed within h in nitazoxanide-treated f cells with or without cpv infection. therefore, nitazoxanide-associated caspase activation reduction (apoptosis) might be involved in the antiviral effect of the drug. closantel sodium-or closantel-treated cells had no pro-or antiapoptotic effects ( figure s ) ; hence, the antiviral effects of these two drugs may not be due to drug-associated apoptosis. statistical analysis was carried out using one-way anova and dunnett's multiple comparisons test. * p < . ; ** p < . ; *** p < . ; **** p < . (compared to . % dmso-treated cells without cpv figure . potential broad-spectrum anti-cpv activity of identified drugs. f cells were seeded in -well plates and pretreated with µm, µm, or µm of nitazoxanide (a), closantel sodium (b), or closantel (c) for h, respectively. then treated cells were infected with various cpv strains sd , sd or bj- . cells were harvested and lysed at hpi for western blot analysis. band intensities were then measured using software image j, and vp expression was analyzed and compared to beta actin expression. relative expression levels for nitazoxanide-(d), closantel sodium-(e), and closantel (f)-treated results are presented in bar graphs. error bars represent standard errors from three independent experiments. statistical analysis was compared to control ( . % dmso-treated cells, marked as d in the figure) and carried out using one-way anova and dunnett's multiple comparisons test. * p < . ; ** p < . ; *** p < . ; **** p < . . doley et al. ( ) reported that cpv could induce caspase-dependent (involving extrinsic, intrinsic, and endoplasmic reticulum pathways) apoptosis in madin-darby canine kidney (mdck) cells [ ] . in order to evaluate whether drug-associated apoptosis was involved in the antiviral effect, we measured the caspase- activity of drug-treated cells with or without cpv infection. as shown in figure , the antiapoptotic effects were observed within h in nitazoxanide-treated f cells with or without cpv infection. therefore, nitazoxanide-associated caspase activation reduction (apoptosis) might be involved in the antiviral effect of the drug. closantel sodium-or closantel-treated cells had no pro-or antiapoptotic effects ( figure s ) ; hence, the antiviral effects of these two drugs may not be due to drug-associated apoptosis. doley et al. ( ) reported that cpv could induce caspase-dependent (involving extrinsic, intrinsic, and endoplasmic reticulum pathways) apoptosis in madin-darby canine kidney (mdck) cells [ ] . in order to evaluate whether drug-associated apoptosis was involved in the antiviral effect, we measured the caspase- activity of drug-treated cells with or without cpv infection. as shown in figure , the antiapoptotic effects were observed within h in nitazoxanide-treated f cells with or without cpv infection. therefore, nitazoxanide-associated caspase activation reduction (apoptosis) might be involved in the antiviral effect of the drug. closantel sodium-or closantel-treated cells had no pro-or antiapoptotic effects ( figure s ) ; hence, the antiviral effects of these two drugs may not be due to drug-associated apoptosis. cpv is a widely distributed virus and contains at least three main subspecies: cpv- a, cpv- b, and cpv- c [ , , ] . currently, commercial vaccines cannot provide complete protection against all cpv variants. moreover, no effective drug is available to control cpv infection except for supportive and symptom-based care. hence, it is important to develop an alternative treatment against cpv infection. in this study, we developed a cpe-based assay to screen cpv inhibitors from a fda-approved drug library, and successfully identified three fda-approved cpv inhibitors. these drugs might provide potential treatment options for anti-cpv infections. although the selectivity index (si) of gemcitabine hcl, cladribine, gemcitabine, and trifluridine were at a higher level (table ) , the percentage cpe inhibition of the four drugs was always maintained at a lower level ( figure s ). the maximum percentage cpe inhibitions for the drugs were between . ± . and . ± . , which are relatively lower cpe inhibition levels that would not increase with increased drug concentration. therefore, nitazoxanide, closantel sodium, and closantel were selected for further study. as mentioned above, the identified drugs nitazoxanide, closantel sodium, and closantel can reduce the copy numbers of cpv viral dna to . %, . %, and . %, respectively, compared with the . % dmso-treated control ( figure ) . meanwhile, the ifa result also showed that these identified drugs inhibited cpv infection in a dose-dependent reduction manner. western blot showed that µm nitazoxanide treatment reduced relative vp expression in three cpv variants sd , sd , and bj- to . %- . %, and the reduction rates following µm closantel sodium and closantel treatment were . %- . % and . %- . %, respectively ( figure ). these results indicated that the identified drugs had significant inhibitory effects against cpv infection in f cells. in previous studies, two drugs oseltamivir and cidofovir were added in the second round of screening. as a neuraminidase (na) inhibitor, oseltamivir has been used to treat the human influenza virus. savigny and macintire ( ) used oseltamivir for cpv enteritis and found that the oseltamivir-treated group gained a significant increase of weight and had no changes in white blood cell (wbc) count compared to the control group; however, the authors also reported that no obvious advantage had been established [ ] . cidofovir is a broad-spectrum anti-dna virus drug, which had been evaluated for the treatment of human papillomavirus (hpv)-associated tumors [ ] . our cpe-based screening assay showed that the percentage cpe inhibition of oseltamivir and cidofovir were . ± . and − . ± . (table s ), respectively, and that these two drugs had no anti-cpv effects on f cells. previously, nitazoxanide was used to treat cryptosporidiosis, giardiasis, and other parasitic infections [ ] . recently nitazoxanide was reported to inhibit various dna and rna viruses, including hepatitis b virus (hbv) [ , ] , human cytomegalovirus (hcmv) [ ] , influenza a virus [ ] , hepatitis c virus [ ] , norovirus [ ] , rotavirus [ ] , japanese encephalitis virus (jev) [ ] , coronavirus [ ] chikungunya virus (chikv) [ ] , human immunodeficiency virus (hiv) [ ] , and zikv [ ] . the antiviral mechanism of nitazoxanide remains unclear for now. nitazoxanide could impair the terminal glycosylation of the influenza a hemagglutinin protein or the formation of e -e (rubella virus surface glycoproteins) complex of the rubella virus (rv), thus affecting the assembly of influenza a virus and rv, respectively [ , ] . in addition, nitazoxanide could also hinder the interactions between the proteins nsp and nsp of rotavirus or the interactions between proteins ns b and ns of zikv and dengue virus (denv ) [ , ] . mercorelli et al. ( ) also reported that nitazoxanide can inhibit the transcriptional activation properties of the hcmv immediate-early (ie ) protein [ ] . these results indicated the virus-specific effects of nitazoxanide. since nitazoxanide can inhibit the replication of various dna and rna viruses, various studies have focused on identifying host factors to explain the broad antiviral activities of nitazoxanide. ashiru et al. ( ) reported that nitazoxanide depleted intracellular ca + stores, besides the phosphorylation of pkr and eif α, further affecting n-linked glycosylation of the bovine viral diarrhea virus (bvdv) e protein and trafficking from the er to the golgi [ ] . nitazoxanide can elicit antiviral innate immunity and reduce the hiv replication by activating the interferon system and further expression of various interferon-stimulated genes (isgs) [ ] . in addition, nitazoxanide might block the production of acetyl-coa, which is a required metabolic intermediates for vaccinia virus (vacv) reproduction. in general, further studies are still required to clearly elucidate the antiviral mechanism of nitazoxanide [ ] . closantel sodium and closantel were also identified and shown to have anti-cpv activities. closantel is a salicylanilide derivative and is considered to be an anthelmintic agent in livestock [ ] . the antiangiogenesis and anticancer effects of closantel sodium and closantel have also been previously reported [ ] . however, to our knowledge, neither the antiviral activity nor the antiviral mechanisms of closantel sodium and closantel have been reported before. previous studies have shown that closantel inhibited b-raf (a serine/threonine kinase) v e [ ] , adenine nucleotide translocase (ant) [ ] , spak and osr kinase [ ] . in addition, senkowski et al. ( ) reported that closantel could inhibit mitochondrial respiration as well [ ] . these reports may contribute to further studies on the antiviral mechanism of closantel. in this study, which is aimed at identifying anti-cpv drugs for potential therapeutic use, a cpe-based assay was developed for screening cpv inhibitors from an fda-approved drug library. after screening, the top three drugs, nitazoxanide, closantel sodium, and closantel, with higher percentage cpe inhibition, were selected and further confirmed by qpcr and ifa. in addition, the identified drugs can inhibit different subspecies of cpv variants and displayed broad-spectrum antiviral activity against cpv. hence, these drugs may provide potential options for the treatment of cpv infection. the following are available online at http://www.mdpi.com/ - / / / /s , table s : percentage inhibition (%) of drugs and other tested drugs, figure s : evaluation of cytotoxicity and anti-cpv efficacy of other four drugs, figure s 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cancer growth in zebrafish models repositioning organohalogen drugs: a case study for identification of potent b-raf v e inhibitors via docking and bioassay human adenine nucleotide translocase (ant) modulators identified by high-throughput screening of transgenic yeast rafoxanide and closantel inhibit spak and osr kinases by binding to a highly conserved allosteric site on their c-terminal domains three-dimensional cell culture-based screening identifies the anthelmintic drug nitazoxanide as a candidate for treatment of colorectal cancer this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflicts of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -k wfyj b authors: paweska, janusz t.; moolla, naazneen; storm, nadia; msimang, veerle; conteh, ousman; weyer, jacqueline; van vuren, petrus jansen title: evaluation of diagnostic performance of three indirect enzyme-linked immunosorbent assays for the detection of igg antibodies to ebola virus in human sera date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: k wfyj b filovirus serological diagnosis and epidemiological investigations are hampered due to the unavailability of validated immunoassays. diagnostic performance of three indirect enzyme-linked immunosorbent assays (i-elisa) was evaluated for the detection of igg antibody to ebola virus (ebov) in human sera. one i-elisa was based on a whole ebov antigen (wag) and two utilized recombinant nucleocapsid (np) and glycoproteins (gp), respectively. validation data sets derived from individual sera collected in south africa (sa), representing an ebov non-endemic country, and from sera collected during an ebola disease (ebod) outbreak in sierra leone (sl), were categorized according to the compounded results of the three i-elisas and real time reverse-transcription polymerase chain reaction (rt-pcr). at the cut-off values selected at % accuracy level by the two-graph receiver operating characteristic analysis, specificity in the sa ebov negative serum panel (n = ) ranged from . % (gp elisa) to . % (wag elisa). diagnostic specificity in the sl ebov negative panel (n = ) was % by the three elisas. the diagnostic sensitivity in rt-pcr confirmed ebod patients was dependent on the time when the serum was collected after onset of disease. it significantly increased weeks post-onset, reaching % sensitivity by wag and np and . % by gp i-elisa. high-mortality and occurrence of ebola disease (ebod) outbreaks almost each year in the last three decades [ , ] are of great public health concern. the unprecedented and first epidemics of ebod in west africa from to [ ] [ ] [ ] [ ] and the large-scale re-emergence of ebod in the democratic republic of the congo in and [ , ] exemplify the devastating health, humanitarian, and socio-economic impacts and challenges in containing ebod outbreaks in resource-poor and politically conflicted settings [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the increasing incidence, severity and size of ebod outbreaks highlight the importance of developing and standardizing diagnostic tools for ebod rapid diagnosis and post-epidemic surveillance. the most devastating ebod outbreak to date, the west african outbreak, prompted determining seroprevalence rates, infection risk population studies, and assessing occurrence of asymptomatic infections [ ] . the purpose of this study was to evaluate and compare the diagnostic performance of ebov igg-indirect elisas based on antigens produced by classical virological and recombinant protein expression methods using human serum panels from ebov non-infected and ebov infected humans. a total of individual banked sera collected in south africa (sa) and sierra leone (sl), were used. sa sera (n = ) were originally submitted for various routine diagnostic testing to the centre for emerging zoonotic and parasitic diseases of the national institute for communicable diseases (nicd), johannesburg. these sera represented specimens collected from individuals in ebov non-endemic country and are regarded as igg ebov negative reference serum panel. ethics clearance for using sa human banked sera in the development and validation of diagnostic assays was obtained from human ethics committee, university of the witwatersrand, johannesburg, sa, clearance certification no. m , february . sl blood specimens were originally submitted for ebov rt-pcr testing to the sa modular high-biosafety field ebola diagnostic laboratory (fedl) established in august near freetown, in international response to the rapidly increasing number of ebod cases in sl [ ] . selected aliquots of processed sera were shipped on dry ice from fedl to the nicd's biosafety level (bsl- ) in total, sera obtained from sl patients suspected of having ebod between august and march [ ] were used. of those sera were from ebov rt-pcr confirmed cases for whom date of disease onset was recorded on ebod case submission form. ribonucleic acid from blood was extracted using the qiaamp viral rna kit (qiagen, hilden, germany) according to the manufacturer's instructions. rt-pcr was performed using the qiagen one-step rt-pcr kit (qiagen, hilden, germany) as per the manufacturer's instructions using previously described primers and probes targeting the ebov l gene [ ] . specimens with ct values below were considered positive for ebov rna [ ] and thus confirming ebov infectious status of a patient. serum specimens from the sl ebov rt-pcr positive patients were regarded as sl reference positive serum panel. the remaining specimens were from ebov rt-pcr negative individuals whose sera tested negative for anti-ebov igg by all igg elisas evaluated in this study using cut-offs derived from sa igg ebov negative reference serum panel. this serum panel was regarded as sl reference negative serum panel. the source of positive control serum (c++) was the imported ebod case from gabon to sa [ , ] . the case was a gabonese physician who had been brought from libreville, gabon on october and admitted to a private hospital in johannesburg where a nurse died after being infected through exposure to his blood. negative control serum (reference no. cezpd svpl / ) was obtained from south african blood service. it tested negative for anti-ebov igg and igm antibodies by in-house elisa using procedures published by ksiazek et al. [ ] . internal quality control (iqc) data were generated as described previously [ ] . upper and lower control iqc limits together with coefficients of variations ≤ % for replicates of positive internal control serum and test sera were applied as an assay acceptance criteria. diagnostic performance of three indirect elisas (i-elisa) for the detection of anti-ebov igg antibody in human sera was evaluated. these elisas were based on antigens prepared from infected cell lysate, or on recombinant antigens produced in a bacterial or mammalian expression system. the ebov whole antigen (wag) was produced as previously described with some modifications [ ] . vero c cells (atcc, manassas, va, usa) were infected with the spu / isolate of ebov ( th passage in vero cells) isolated from the serum of a nurse who contracted a fatal infection from a gabonese physician admitted to a private hospital in south africa in [ ] . after incubation at • c when cytopathic effect was observed, cells and supernatant were collected and snap freeze-thawed at − • c and • c. lysed cells and supernatant were separated by centrifugation ( , × g, • c, min). the collected supernatant was gamma irradiated with , gy to inactivate the virus. saturated ammonium sulphate solution ( %) (sigma aldrich, merck, kenilworth, nj, usa) was slowly added with constant stirring to the irradiated supernatant to a final concentration of %, and the mixture was incubated at • c overnight. the antigen-containing pellet precipitate was collected by centrifugation at , × g for min at • c and resuspended in one tenth of the original supernatant volume of phospate buffered saline (pbs) ph . . the antigen was further dialysed against pbs to remove the ammonium suphate ( - buffer changes). the wag preparation was aliquoted and stored at − • c until use. uninfected vero c cells were prepared in the same way and used as control antigen. the codon optimized nucleotide sequence for ebov nucleocapsid (np) (genbank accession number af . ) was synthesized with a c-terminal glycine linker to a × histidine tag (genscript, piscataway, nj, usa) and subcloned into the ncoi and xhoi restriction sites of the pet- b expression vector (novagen, merck, usa). the sequence verified plasmid (pet- b zebov np) was used to transform competent bl star (de ) e. coli. a starter culture was grown overnight at • c, then diluted fold and allowed to reach exponential-phase growth (od of between . - . ) before protein expression was induced using mm iptg (sigma aldrich, merck, usa) for h at • c with vigorous shaking. cells were harvested by centrifugation, resuspended in sodium phosphate buffer ( mm nah hpo , mm nacl, ph . ) and lysed using a combination of bugbuster and lysonase (novagen, merck, usa) treatment, freeze-thaw cycles and sonication. the recombinant np protein-containing soluble phase was collected by high speed centrifugation ( , × g, min, • c) and loaded onto profinity imac (biorad, hercules, ca, usa) cobalt-charged resin. the protein was left to bind overnight with gentle shaking at • c, using a ratio of ml resin to ml soluble protein fraction. contaminating proteins were removed by washing the resin twice with packed-resin volumes of sodium phosphate buffer containing . mm imidazole (ph . ), using gentle centrifugation ( × g, min, • c). the recombinant np was then eluted overnight with gentle shaking at • c in packed-resin volumes of sodium phosphate buffer containing mm imidazole (ph . ). eluted protein was dialysed into . m carbonate/bicarbonate buffer, ph . (sigma aldrich, merck, usa). to remove any residual contaminating proteins, the purified protein was passed through a sec size exclusion column (biorad, hercules, ca, usa) and the fractions containing the np were collected. collected fractions were pooled and concentrated using amicon ultra filters (millipore, merck, usa) with a kd cut-off. the purified np was quantified using the bradford concentration assay kit (pierce, thermofisher scientific, waltham, ma, usa) and aliquots were stored at − • c for later use. for the control antigen, the same process was followed using an expression vector (pet- b) without an insert. recombinant ebov glycoprotein (gp) antigen expressed in human embryonic kidney t cells was obtained from integrated biotherapeutics (rockville, md, usa). optimal immunoreagents concentrations/working dilutions for i-elisa were determined using standard checkerboard titration procedures [ ] . microtiter -wells plates (maxisorb immunoplates, nunc, roskilde, denmark) for wag, np, and gp i-elisas were respectively coated with wag and corresponding control antigen, np and corresponding control antigen or with gp. for wag and np i-elisas virus and control antigens were added to rows of the top half of the plate (rows a-d: [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and the bottom half of the plate (rows e-h: - ), respectively. for gp i-elisa all rows (rows a-h: - ) were coated with the commercial gp for which control antigen was not available. the np and corresponding control antigen were diluted : (stock concentration . mg/ml) in carbonate-bicarbonate buffer, ph . . all the other antigens were diluted in phosphate pbs without mg and ca, ph . ; wag and uninfected vero c cell culture antigen was diluted : and the gp was diluted : (stock concentration . mg/ml). each -well plate had four replicates of positive serum control (c++), two replicates of negative serum control (c−) and two replicates of conjugate control (cc). for wag and np i-elisa c++ was added to wells a - and b - coated with virus antigen and to corresponding wells e - and f - coated with a control antigen. accordingly, c− was added to c - and g - , and cc (diluent buffer) was added to d - and h - . the remaining wells (a-d - and e-h - ) were used for testing test sera in duplicate with test serum no. added to wells a and b and corresponding e and f ; adding of the remaining test sera followed the same layout principle. for gp i-elisa, not having control antigen, internal controls were placed as follows: c++ in wells a - and b - ; c− in wells c - ; cc in wells d - . the remaining wells (a-d - and e-h - ) were used for testing test sera in duplicate with test serum no. added to wells a and b and accordingly test serum no. added to wells g and h . all reagents were added to the immunoplates at a volume of µl/well unless otherwise stated. passive adsorption onto elisa plates was performed at • c overnight and all subsequent incubations (except for substrate addition) were performed at • c in a humidified chamber for h. following coating, plates were washed times with µl of pbs containing . % tween ; the same washing procedure followed each subsequent stage of i-elisas. plates were blocked with µl % non-fat milk powder in pbs. after incubation, plates were washed, and control and test sera diluted : in % non-fat milk powder in pbs (diluent buffer) were added. each test serum, negative control serum and conjugate control was tested in duplicate, and positive control was tested in quadruplicate. following incubation with the sera, the plates were washed and goat anti-human igg hrpo conjugate (invitrogen, thermofisher scientific, usa), diluted : , in diluent buffer, was added to the wells. after incubation, plates were washed and , '-azinodiethylbenzothiazoline sulfonic acid (abts) peroxidase (seracare lifesciences, milford, ma, usa) substrate added to wells. plates were incubated in the dark at room temperature (± • c) for min. reactions were stopped by the addition of % sodium dodecyl sulphate (sds) and the optical densities (od) readings were measured at nm. od readings were converted into a percentage of the positive internal control serum (pp) using the equation as previously described [ ] . briefly, a specific activity of each serum (net od) was calculated by subtracting the non-specific background od in the wells with control antigen from the od in wells with virus antigen (for np and wag i-elisa only). the mean net od readings were converted to pp using the formula: pp = (mean net of test serum/mean net od of positive control) × [ ] . cut-off values were determined as mean + standard deviations (sd) of pp values recorded in sa and sl igg ebov negative serum panels and also selected at % accuracy level by the misclassification cost term (mct) option of the two-graph receiver operating characteristics (tg-roc) analysis available from microsoft excel (redmond, wa, usa) [ , ] using sl reference igg ebov negative and positive serum panels. coefficient of variation (cv) values were calculated to measure relative variability using the formula cv = (sd of replicates/mean of replicates) × . optimisation of cut-off values by mct tg-roc was based on the following equation: to compare the diagnostic performance of the assays and agreements between results of matched test samples, data were analyzed in stata using mcnemar's test and cohen's kappa statistic (κ) [ ] . to evaluate the effect of serum inactivation on the levels of the detectable anti-ebov igg by wag, np and gp i-elisas, laboratory protocols previously shown to completely inactivate ebov virus in human serum were used [ , ] . briefly, the c++ was first diluted : in pbs containing either . % triton x- or . % tween- (sigma-aldrich, taufkichen, germany) and heated at • c for min. then, two-fold log dilutions (from : to : , ; log −log . ) of untreated and treated serum was tested by each of the i-elisas. each dilution was tested in duplicate on separate runs. a serum titer was considered the highest sample dilution at which its pp value was ≥ the i-elisa cut-off for at least % of six replicates. both within and between the runs, the c++ od readings for wag, np and gp i-elisas were within the iqc lower (lcl) and upper (ucl) control limits. within the runs, the average cv ranged between . ± . (np i-elisa) and . ± . (gp i-elisa). between the runs, the average cv ranged between . ± . (np i-elisa) and . ± . (gp i-elisa) ( table ) . both within and between the runs, the c− and cc od readings were within the iqc lcl and ucl. for wag, np, and gp i-elisa, the iqc c− od control limits ranged from − . to . , − . to . , . to . , and the iqc cc− od control limits ranged from − . to . , − . to . , and . to . , respectively. the distribution of i-elisa pp values and determination of cut-offs as mean plus three standard deviations of pp values recorded by each test in sa and sl ebov igg negative serum panels are shown in figure . selection and optimization of cut-offs by the mct tg-roc in sl serum panels is shown in figure . mct tr-roc was based on the non-parametric program option due to departure from a normal distribution of data sets analyzed. pp threshold values for each of the assays were similar irrespective of serum panels analyzed and the methods used for the determination of cut-offs: . , . ( figure a ) and . (figure a) for wag i-elisa; . , . ( figure b ) and . ( figure b ) for np i-elisa; . , . ( figure c ) and . ( figure c ) for gp i-elisa, respectively. the higher cut-off values for gp i-elisa were likely due to higher elisa noise resulting from not including a control antigen in this assay. irrespective of data set analyzed and cut-offs used, all the three assays evaluated in this study had high estimates of d-sp, ranging from . % to . % in sa, and from . % to % in sl negative serum panels. however, d-sp for gp i-elisa was generally lower in the sa negative serum panel ( table ). mct tr-roc was based on the non-parametric program option due to departure from a normal distribution of data sets analyzed. pp threshold values for each of the assays were similar irrespective of serum panels analyzed and the methods used for the determination of cut-offs: . , . ( figure a figure c ) and . ( figure c ) for gp i-elisa, respectively. the higher cut-off values for gp i-elisa were likely due to higher elisa noise resulting from not including a control antigen in this assay. irrespective of data set analyzed and cut-offs used, all the three assays evaluated in this study had high estimates of d-sp, ranging from . % to . % in sa, and from . % to % in sl negative serum panels. however, d-sp for gp i-elisa was generally lower in the sa negative serum panel ( table ) . individual results yielded by wag, np and gp i-elisas in sera from rt-pcr confirmed ebod cases at different times post disease onset and using different cut-off values are given in table . irrespective of the cut-offs used, the results were similar for the same assay, but the np i-elisa was more sensitive in detecting igg antibody during the first two weeks post disease onset compared to wag and gp i-elisas, the latter being the least sensitive. for example, when using the tg-roc derived threshold, of ebod patients bled during the two weeks post onset, ( . %), ( %), and ( . %) were positive by wag, np, and gp i-elisa, respectively. mc nemar test indicate a disagreement of diagnostic capacity (combined dse and dsp) between np and wag (p = . ) or gp i-elisa (p = . ) respectively using all sierra leone data (n = ). agreement was found between wag and gp i-elisa (p = . ; . % κ = . ± . ). most of the discrepant (non-matching) results were recorded during the first two weeks post disease onset. after two weeks post onset, detection of igg antibody and agreement between assays significantly improved. of sera tested - days post onset, all tested positive by wag and np i-eliss irrespective of the cut-off used, and depending on the cut-off, or were positive by gp i-elisa (table ) . mc nemar test indicate agreement between np, wag and gp i-elisas ranging from . % (κ = . ± . ) to % using data from non-ebod and diseased patients bled on day or later post-onset (n = ), except for gp and wag or np i-elisas with the cut-offs of respectively . , . , . (p < . ). estimates of d-se in sera collected at different times post disease onset are given in table . mean levels of igg responses measured by wag, np, and gp i-elisas in ebod rt-pcr confirmed cases bled at different times after disease onset are shown in figure . on average, the first seroconversions were detectable by np on days - , then on days - by np and wag, and from day post onset by all i-elisas. the mean levels of igg measured by all the i-elisas were not significantly different. estimates of d-se in sera collected at different times post disease onset are given in table . between - days post onset, the d-se ranged from . % (gp i-elisa) to . (np i-elisa). between - days post onset, the d-se was % for both wag and np i-elisa irrespective of the cut-off used, and ranged from . % to . % for the gp i-elisa. table . diagnostic sensitivity of a whole antigen (wag), nucleocapsid (np), and glycoprotein (gp) i-elisas for the detection of anti-igg ebov antibody in humans. cut-off pp wag i-elisa . / . ( . - . ) / ( . - ) wag i-elisa . / . ( . - . ) / ( . - ) wag i-elisa . mean levels of igg responses measured by wag, np, and gp i-elisas in ebod rt-pcr confirmed cases bled at different times after disease onset are shown in figure . on average, the first seroconversions were detectable by np on days - , then on days - by np and wag, and from day post onset by all i-elisas. the mean levels of igg measured by all the i-elisas were not significantly different. the different inactivation protocols used did not have an adverse effect on the kinetics and the detectable levels of the anti-ebov igg in c++ by either i-elisa evaluated in this study. the titers ( table ) as well as the kinetics of dose-response curves ( figure ) were similar before and after inactivation in each assay. the diagnostic decision limit or cut-off represents a serological assay test value used to dichotomize negative and positive results, and by inference, to define the infection status of an individual against a specific pathogen of disease. the relevance of data used for the determination of cut-off consequently impacts on estimates of d-se and d-sp and other measures of test performance [ ] . important consideration in determining a serological assay cut-off is to select sera from unequivocally infected individuals and sera from individuals who have never been infected with the agent in question. also, in order to account for the distribution of covariate factors (genetic, nutritional, geographical, and stage of infection) that may influence the estimates of d-se and d-sp, the target population should preferably be sampled using simple random, systematic or stratified sampling methods [ ] . these ideal conditions could not be applied during this study. an assay validation data should preferably be derived not only from testing samples from reference individuals of known history and infection status but also from the country or region in which the test is to be used. traditionally, gold standards for selection of truly infected and uninfected subjects include isolation of the agent or pathognomonic histopathological criteria. because a true gold standard is difficult to accomplish, relative standards of comparison are often necessary, and include results from other serological assays [ ] . in order to ensure ebov true infection status of individuals whose sera were used in our study, rt-pcr negative sera that tested negative for anti-ebov igg by all igg elisas using cut-offs derived from sa igg ebov negative reference serum panel were regarded as sl reference negative serum panel. serum specimens from the sl ebov rt-pcr positive patients with known dates of disease onset were regarded as sl reference positive serum panel. various statistical analyses used in our study for the selection of the cut-off values provided similar results. a cut-off value determined as two or three sd above the mean in uninfected individuals is frequently used for the interpretation of serodiagnostic tests. however, this assumes a normal distribution of the test values in population targeted by an assay, and provides only an estimate of d-sp [ ] . deviations from normality are often recorded in serological data and should be addressed in the selection of threshold values [ ] . therefore, we also used the tg-roc analysis for the selection and optimization of cut-off values to account for parametric versus nonparametric distribution of test values. all i-elisas evaluated in our study had high estimates of d-sp, but the d-se was dependent on the time when the serum was taken post disease onset. high detection rate the diagnostic decision limit or cut-off represents a serological assay test value used to dichotomize negative and positive results, and by inference, to define the infection status of an individual against a specific pathogen of disease. the relevance of data used for the determination of cut-off consequently impacts on estimates of d-se and d-sp and other measures of test performance [ ] . important consideration in determining a serological assay cut-off is to select sera from unequivocally infected individuals and sera from individuals who have never been infected with the agent in question. also, in order to account for the distribution of covariate factors (genetic, nutritional, geographical, and stage of infection) that may influence the estimates of d-se and d-sp, the target population should preferably be sampled using simple random, systematic or stratified sampling methods [ ] . these ideal conditions could not be applied during this study. an assay validation data should preferably be derived not only from testing samples from reference individuals of known history and infection status but also from the country or region in which the test is to be used. traditionally, gold standards for selection of truly infected and uninfected subjects include isolation of the agent or pathognomonic histopathological criteria. because a true gold standard is difficult to accomplish, relative standards of comparison are often necessary, and include results from other serological assays [ ] . in order to ensure ebov true infection status of individuals whose sera were used in our study, rt-pcr negative sera that tested negative for anti-ebov igg by all igg elisas using cut-offs derived from sa igg ebov negative reference serum panel were regarded as sl reference negative serum panel. serum specimens from the sl ebov rt-pcr positive patients with known dates of disease onset were regarded as sl reference positive serum panel. various statistical analyses used in our study for the selection of the cut-off values provided similar results. a cut-off value determined as two or three sd above the mean in uninfected individuals is frequently used for the interpretation of serodiagnostic tests. however, this assumes a normal distribution of the test values in population targeted by an assay, and provides only an estimate of d-sp [ ] . deviations from normality are often recorded in serological data and should be addressed in the selection of threshold values [ ] . therefore, we also used the tg-roc analysis for the selection and optimization of cut-off values to account for parametric versus nonparametric distribution of test values. all i-elisas evaluated in our study had high estimates of d-sp, but the d-se was dependent on the time when the serum was taken post disease onset. high detection rate of anti-ebov igg in rt-pcr ebov confirmed cases was only recorded after two weeks post disease onset. results of previous study in a small number of the ebod patients in kikwit, democratic republic of congo, suggested that many patients do not have antibody early in the course of their illness, and that many may die without developing detectable antibodies to ebov. therefore, measurement of igg antibody is of rather limited use in the diagnosis of acute ebod cases [ ] . i-elisa, represents one of the simplest elisa formats, but can be difficult to validate because of signal amplification of both specific and non-specific components [ ] . for these reasons, to determine the specific binding of antibody, sera should be tested with both a specific viral antigen and its corresponding control or comparison antigen to account for possible non-specific background activity. the gp i-elisa evaluated in our study was based on a commercially available recombinant gp for which a negative control could not be obtained. compared to wag and np i-elisas, which included control antigens, the higher gp i-elisa test values in ebov negative serum panels and consequently the higher pp cut-off values derived for this assay are likely due to not having a control antigen. due to inherent differences amongst assay systems, binding-antibody levels should be expressed in relative rather than absolute terms. one of the advantages of using pp values as a measure of antibody activity in the i-elisa is that this method of od readings conversion does not assume a uniform background activity, and therefore it is also more suitable for inter-laboratory standardization [ ] . the first elisas for the detection of antibodies to ebov were based on whole antigen prepared from infected cell lysate [ ] , and variations of this assay are still widely used in diagnostic and research laboratories [ ] . while whole filovirus antigens can be relatively easily produced in large volumes, their preparation pose health risks, restricting their production to biosafety level (bsl- ) facilities. these facilities are not only very expensive to construct and operate, but also are not easily available or accessible for countries where fatal filoviruses are endemic. in addition, the binding of antibodies to cellular contaminants present in ebov-infected cell lysates may lead to cross-reactivity, resulting in reduced specificity [ ] . high quality filovirus recombinant protein antigens can be safely prepared without the need for high bsl- biocontainment facilities and outside ebov endemic areas. their use in elisa has potential to reduce the risk of false positive results and allows for better standardization [ , ] . testing clinical specimens potentially containing a bsl- viral agent presents a serious biohazard. while a number of inactivation methods were shown to completely inactivate ebov [ , ] , they also markedly alter the protein components in human blood, e.g., enzymes and coagulation factors [ ] . results of viral inactivation protocols evaluated in our study indicate that they do not alter detectable levels of anti ebov-igg, thus together with recombinant antigen based elisas, provide a safe and reliable testing platform. it has previously been reported that the use of single filoviral proteins as antigens is disadvantageous for filovirus serology [ , , ] . sera from patients infected with ebov contain antibodies to several viral proteins and therefore might display reduced activity or later seroconversion in elisas based on a single recombinant antigen [ ] . results of our study indicate that anti-ebov np igg is detectable earlier then anti-ebov gp igg antibody during the first week post disease onset. this is likely due to higher abundance of this highly immunogenic protein in infected cells [ ] . long-lasting persistence of igg antibody in humans after infection with ebov [ , , ] , renders igg detection elisa a suitable tool for epidemiological investigations. evaluation of the efficacy of filovirus vaccines and therapeutics require monitoring of immune responses that correlate with protection and survival using reliable and reproducible serological methods. anti-ebov gp igg elisa was recently shown to reproducibly quantify levels of anti-ebov igg antibodies in sera from ebod survivors and immunized individuals [ ] , thus offering an important laboratory tool for assessing immunogenicity of candidate ebov vaccines. the np igg i-elisa evaluated in this study has a potential to be used for testing immunity and evaluating protection against natural ebov exposure in individuals immunized with ebov gp-based vaccines or receiving anti-ebov gp passive immunization. as highly accurate, robust and safe tests, the elisas based on recombinant ebov antigens have a potential to replace traditional serological diagnostic methods which pose health risks thus necessitating their use only in high biocontainment facilities. practically simple viral inactivation protocols evaluated in our study do not alter measurable levels of anti ebov-igg, thus together with recombinant antigen based elisas provide a safe testing application. long-lasting igg antibody makes their detection useful for epidemiological investigations. new filovirus disease classification and nomenclature ebola virus outbreaks in africa: past and present emergence of zaire ebola virus disease in guinea the international ebola emergency who ebola response team. ebola virus disease in west africa-the first months of the epidemic 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results. key: cord- -e txo z authors: ke, fei; wang, zi-hao; ming, cheng-yue; zhang, qi-ya title: ranaviruses bind cells from different species through interaction with heparan sulfate date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: e txo z ranavirus cross-species infections have been documented, but the viral proteins involved in the interaction with cell receptors have not yet been identified. here, viral cell-binding proteins and their cognate cellular receptors were investigated using two ranaviruses, andrias davidianus ranavirus (adrv) and rana grylio virus (rgv), and two different cell lines, chinese giant salamander thymus cells (gstc) and epithelioma papulosum cyprinid (epc) cells. the heparan sulfate (hs) analog heparin inhibited plaque formation of adrv and rgv in the two cell lines by more than % at a concentration of μg/ml. in addition, enzymatic removal of cell surface hs by heparinase i markedly reduced plaque formation by both viruses and competition with heparin reduced virus-cell binding. these results indicate that cell surface hs is involved in adrv and rgv cell binding and infection. furthermore, recombinant viral envelope proteins adrv- l and rgv- r bound heparin-sepharose beads implying the potential that cell surface hs is involved in the initial interaction between ranaviruses and susceptible host cells. to our knowledge, this is the first report identifying cell surface hs as ranavirus binding factor and furthers understanding of interactions between ranaviruses and host cells. interspecies transmission and infection have been reported in several viruses that infect humans [ , ] . likewise, aquatic animal viruses can also infect and cause disease in a wide range of aquatic animals [ , ] . specifically, the interspecies infection was reported following ranavirus infection [ , ] . however, the basis for the broad ranavirus host range is not clear [ , ] . ranaviruses are large double-stranded dna viruses within the family iridoviridae [ ] . ranaviruses target aquatic animals globally and have been isolated from reptiles [ , ] , amphibians [ ] [ ] [ ] [ ] , and bony fish [ , ] . among them, several isolates represent great threats to the development of the aquaculture industry and wild animal populations [ ] . aside from infecting several species within a given taxonomic class (e.g., frog virus infects diverse amphibian species), ranaviruses may also infect members of different classes (e.g., frog virus -like agents have been isolated from both amphibians and fish) [ ] . binding susceptible host cells is the first step in viral infection and is one of the key factors responsible for determining host range [ ] . diverse cellular receptors and viral envelope and capsid proteins are involved in the process. for example, vaccinia virus utilizes four viral proteins that bind to glycosaminoglycans (gags) or laminin on the cell surface [ ] [ ] [ ] [ ] [ ] . cell surface gags consist of complex linear polysaccharides, which are ubiquitously expressed in most cell types [ ] . heparan sulfate (hs), chondroitin sulfate (cs), and dermatan sulfate comprise the main types of gags on the cell surface. besides vaccinia virus, gags are involved in the binding of adenovirus [ ] , various alphaviruses [ , ] , bunyaviruses [ , ] , filoviruses [ ] , flavivirus [ ] , hepacivirus [ ] , herpesvirus [ ] , papillomaviruses [ ] , and rhabdoviruses [ ] . it has been shown that cell surface gags, especially hs, serve as initial receptors in infections with these viruses. however, there is little information on the role of hs in ranavirus binding. rana grylio virus (rgv) and andrias davidianus ranavirus (adrv) are ranaviruses isolated from diseased pig frogs r. grylio (anura amphibian) and chinese giant salamanders (cgs) a. davidianus (urodele amphibian), respectively [ , ] . the complete genomes of the two viruses have been sequenced, and several functional proteins have been characterized [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, rgv- r, a homolog of adrv- l, was identified as an envelope protein [ ] . decreased expression of rgv- r, or its frog virus (fv ) homolog fv - r impaired virus replication in cultured cells [ , ] . in this report, we examine the role of hs in ranavirus entry. furthermore, since ranavirus viral envelope proteins [ ] [ ] [ ] [ ] [ ] [ ] [ ] are likely involved in the initial interaction between virus and host, we examined the ability of rgv- r and its adrv homolog, advr- l, to bind heparin-sepharose beads. adrv isolated from diseased chinese giant salamanders (cgs) [ ] and rgv isolated from the diseased pig frog r. grylio [ ] were maintained in our laboratory and used in the present study. cgs thymus cells (gstc) [ ] and epithelioma papulosum cyprinid (epc) cells [ ] were cultured in m medium supplemented with % bovine calf serum. adrv and rgv were purified, as described previously [ ] . briefly, gstc cells were infected with adrv and rgv at a multiplicity of infection (moi) of . pfu/cell, respectively, and incubated at • c. the cell cultures were harvested when cytopathic effects (cpe) reached approximately %. the viral suspensions were frozen at − • c, thawed three times, and then centrifuged at × g for min. the resulting supernatants were ultracentrifuged at , × g (beckman, sw , brea, ca, usa) for min. the pellets were resuspended in te buffer ( mm of tris-hcl, mm of edta, ph . ) and further purified in a discontinuous sucrose gradient ( %, %, %, and %) at , × g for min. the viral bands were collected, centrifuged to remove residual sucrose, and the resulting viral pellets were resuspended in te buffer and stored at − • c. heparin is a structural homolog of highly sulfated hs and has been used as a surrogate for cell surface hs in research studies examining binding [ ] . the effect of heparin on viral plaque formation was tested in assays using gstc and epc cells for the purpose of analyzing the effect of soluble glycosaminoglycans on viral infection. the indicated cells were seeded in -well plates h prior to infection. heparin (sangon biotech, shanghai, china, from porcine intestinal mucosa, molecular weight range of - kda) was diluted in cell culture medium and incubated with adrv or rgv for h at • c. at a concentration of µg/ml, the color of the growth medium (using phenol red as an indicator) did not change, indicating a stable ph. cell culture media was removed and µl of the virus-heparin solution, containing approximately pfu of the indicated virus, was added to each well for h at • c. three replicate wells were used in each treatment. after h, the inoculum was removed, the cells were washed twice with fresh medium, and overlaid with culture media containing . % agarose. fresh medium was added to the well after agarose solidification. plaques were counted after three days of incubation at • c. based on the results obtained from the heparin treatment, two other glycosaminoglycans, heparan sulfate and chondroitin sulfate (sigma, st. louis, mo, usa), were tested by the method described above. to further investigate its role in virus binding, target cells were treated with heparinase to remove cell surface heparan sulfate. heparinase i cleaves the linkages between hexosamines and the o-sulfated iduronic acids of heparin and hs. gstc cells were seeded in -well plates h prior to infection. medium was removed before the assay, and the cells were incubated with different concentrations of heparinase i from flavobacterium heparinum (sigma) in mm of tris-hcl (ph . ), mm of cacl , mm of nacl, and . % bovine serum albumin (bsa) for h at • c and then washed twice with fresh medium. one hundred microliters of medium containing adrv or rgv ( pfu) were added and incubated for another h at • c. the supernatant was removed, the cells washed twice with fresh medium, and overlaid with medium containing . % agarose, as described above. after incubation for three days at • c, the plaques were counted. to investigate the effect of heparin on virus-cell binding, quantitative real-time pcr (qpcr) analysis that has been used in the detection of hepatitis c virus (hcv) genomes [ ] was used to determine the relative quantity of virus bound to the cell surface. both purified virions, isolated following separation on a sucrose step gradient, and a crude viral suspension, obtained by lysis of cells infected by the virus, were used in these assays. gstc cells were seeded in -well plates h prior to infection. heparin was diluted in medium and mixed with the viral suspension or purified virions for min at • c, and then µl of the mixture containing approximately pfu of the indicated virus was added to cells for h at • c, as described above. the inoculum was removed after incubation. the cells, washed twice with fresh medium, were collected by centrifugation, and dna was extracted with the takara minibest universal genomic dna extraction kit (takara, tokyo, japan). bound viral genomes, based on detection of the relative numbers of major capsid protein gene (mcp), were detected by qpcr, which was conducted using a stepone real-time pcr system (the applied biosystems, foster city, ca, usa). each qpcr mixture contained µl of dna, . µl of sybr premix ( ×), . µl of forward and reversed primers (for each primer, -cacctccatcccagtcagca- / -aatcccatcgagccgttca- ), and . µl of ultrapure water. the qpcr conditions were as follows: • c for min; cycles of • c for s and • c for min; and a melt curve analysis at • c for s, • c for min, and • c for s. the β-actin gene, used in a previous study [ ] , was used as a loading control. qpcr efficiency was evaluated with a standard curve, based on serially diluted dna samples using the β-actin gene primers, which showed that there were no obvious differences on the qpcr efficiencies among samples treated with different concentrations of heparin. for the mcp detection, mcp levels were normalized to β-actin levels in each sample. the level of bound virus (mcp level) in the treated group versus that in the control group (no heparin) was calculated by the −∆∆ct method [ ] . considering the pivotal roles that viral envelope proteins play in virus attachment and entry, we determined whether viral envelope proteins interacted with hs by monitored the binding of purified recombinant proteins to heparin-sepharose beads as described by chung et al. [ ] . previous studies demonstrated that rgv- r is amino acid (aa) envelope protein with two predicted transmembrane (tm) domains (aa - and aa - ) [ ] . adrv- l is the adrv homolog of rgv- r. the aa sequence identity between adrv- l and rgv- r is . % [ ] . in the present assay, tm helices and topology of the two proteins were predicted with the online tools hmmtop (http://www.enzim.hu/hmmtop/html/submit.html) and tmhmm (http://www.cbs.dtu.dk/services/tmhmm- . /) that presented on expasy. the primers -gtagaattcatgggagcagcggaa- and -gataagcttttatgtggtggggtccaggcc- were used for amplifying dna sequences encoding the n-terminal region ) of l and the n-terminal region of r, respectively. the other pair of primers ( -ggcgaattccccaggcccgtcaaga- / -ctataagcttttaacccctgtgggc- ) was used for amplifying the dna sequences for the c-terminal region ( - ) of l. because the amino acid sequence of the c-terminal regions of adrv- l and rgv- r are identical, only the c-terminal region of adrv- l was expressed in the present study. the resulting fragments were digested using ecor i and hind iii, and ligated into pet a or pet a vectors that had been digested with the same enzymes. successful cloning was validated by dna sequencing. for protein expression and purification, the plasmids obtained above were used to transform escherichia coli bl (de ). positive clones were cultured in lb medium and induced with . mm isopropyl-β-d-thiogalactopyranoside (iptg) for h at • c. the bacterial pellets were lysed by sonication. the recombinant protein was purified using the hisbind purification kit (novagen, billerica, ma, usa) according to the manufacturer's instructions. the purified protein was dialyzed against pbs, the concentration determined using a bca protein assay kit (beyotime, wuhan, china), and stored at − • c. heparin-sepharose beads ff and control sepharose beads (purchased from smart lifesciences, changzhou, china, particle diameter range of - µm) were equilibrated with binding buffer ( mm tris-hcl, mm sodium citrate, ph . ) before use. the purified recombinant protein ( µg) was mixed with or without heparan sulfate ( µg/ml) and incubated in binding buffer with µl of beads for h at • c. the supernatant was collected after centrifugation for min at × g. the beads were washed with binding buffer ( µl) five times, and bound protein was eluted with binding buffer containing m nacl. the samples were analyzed by % sds-page and subsequently transferred to a pvdf membrane (millipore, burlington, ma, usa). a monoclonal antibody against the his tag (santa cruz, dallas, texas, usa) was used as the primary antibody, horseradish peroxidase (hrp)-conjugated goat anti-mouse igg (h + l) (merck, kenilworth, nj, usa) as the secondary antibody, and antibody binding detected by chemiluminescence (millipore). the effect of heparin on virus infection was tested by monitoring viral plaque formation following incubation of adrv and rgv in the presence of increasing concentrations of heparin. as shown in figure , the number of plaques formed by either adrv or rgv was reduced by heparin in a concentration-dependent manner. for adrv, infectivity was reduced by approximately % by pre-exposure to heparin at . µg/ml and by more than % at µg/ml. a similar phenomenon was observed in rgv-infected cells, which exhibited a % inhibition at . µg/ml and more than % at µg/ml. moreover, inhibition was detected regardless of the cell line used. the results indicate that heparin-like gags were involved in adrv and rgv binding. for amplifying the dna sequences for the c-terminal region ( - ) of l. because the amino acid sequence of the c-terminal regions of adrv- l and rgv- r are identical, only the c-terminal region of adrv- l was expressed in the present study. the resulting fragments were digested using ecor i and hind iii, and ligated into pet a or pet a vectors that had been digested with the same enzymes. successful cloning was validated by dna sequencing. for protein expression and purification, the plasmids obtained above were used to transform escherichia coli bl (de ). positive clones were cultured in lb medium and induced with . mm isopropyl-β-d-thiogalactopyranoside (iptg) for h at °c. the bacterial pellets were lysed by sonication. the recombinant protein was purified using the hisbind purification kit (novagen, billerica, ma, usa) according to the manufacturer's instructions. the purified protein was dialyzed against pbs, the concentration determined using a bca protein assay kit (beyotime, wuhan, china), and stored at − °c. heparin-sepharose beads ff and control sepharose beads (purchased from smart lifesciences, changzhou, china, particle diameter range of - μm) were equilibrated with binding buffer ( mm tris-hcl, mm sodium citrate, ph . ) before use. the purified recombinant protein ( μg) was mixed with or without heparan sulfate ( μg/ml) and incubated in binding buffer with μl of beads for h at °c. the supernatant was collected after centrifugation for min at × g. the beads were washed with binding buffer ( μl) five times, and bound protein was eluted with binding buffer containing m nacl. the samples were analyzed by % sds-page and subsequently transferred to a pvdf membrane (millipore, burlington, ma, usa). a monoclonal antibody against the his tag (santa cruz, dallas, texas, usa) was used as the primary antibody, horseradish peroxidase (hrp)-conjugated goat anti-mouse igg (h + l) (merck, kenilworth, nj, usa) as the secondary antibody, and antibody binding detected by chemiluminescence (millipore). the effect of heparin on virus infection was tested by monitoring viral plaque formation following incubation of adrv and rgv in the presence of increasing concentrations of heparin. as shown in figure , the number of plaques formed by either adrv or rgv was reduced by heparin in a concentration-dependent manner. for adrv, infectivity was reduced by approximately % by pre-exposure to heparin at . μg/ml and by more than % at μg/ml. a similar phenomenon was observed in rgv-infected cells, which exhibited a % inhibition at . μg/ml and more than % at μg/ml. moreover, inhibition was detected regardless of the cell line used. the results indicate that heparin-like gags were involved in adrv and rgv binding. cells were infected with adrv or rgv that had been pre-incubated in the presence of different concentrations of heparin. the number of plaques obtained in the absence of heparin was set as . the data represent triplicate results and was analyzed with student's t-test. significant differences (versus virus without heparin) are marked with * (p < . ). in a second experiment, hs and cs, linear polysaccharides that constitute two major classes of cell surface gags, were monitored for their ability to reduce plaque formation. as with heparin, hs reduced the number of plaques formed by the two viruses in gstc and epc cells in a concentration-dependent manner (figure a) . for both viruses, plaque formation was inhibited by more than % in gstc cells and more than % in epc cells at µg/ml (figure a) . in contrast, a significant inhibitory effect was only seen at the highest concentration when cs was substituted for hs (figure b) . these results indicate that cell surface gags, including hs, likely play important roles in plaque formation by both adrv and rgv. cells were infected with adrv or rgv that had been pre-incubated in the presence of different concentrations of heparin. the number of plaques obtained in the absence of heparin was set as . the data represent triplicate results and was analyzed with student's t-test. significant differences (versus virus without heparin) are marked with * (p < . ). in a second experiment, hs and cs, linear polysaccharides that constitute two major classes of cell surface gags, were monitored for their ability to reduce plaque formation. as with heparin, hs reduced the number of plaques formed by the two viruses in gstc and epc cells in a concentrationdependent manner (figure a) . for both viruses, plaque formation was inhibited by more than % in gstc cells and more than % in epc cells at μg/ml (figure a) . in contrast, a significant inhibitory effect was only seen at the highest concentration when cs was substituted for hs ( figure b ). these results indicate that cell surface gags, including hs, likely play important roles in plaque formation by both adrv and rgv. if the interaction between heparin-like gags and viral particles is needed for viral infection, removal of gags should inhibit infection. to accomplish that task, heparinase i was used to remove cell surface hs. as shown in figure , the number of plaques formed by the two viruses was markedly reduced in gstc cells pretreated with heparinase i. the reduction was more than % at a heparinase concentration of . u/ml and slightly more at higher enzyme concentrations. thus, the ability of if the interaction between heparin-like gags and viral particles is needed for viral infection, removal of gags should inhibit infection. to accomplish that task, heparinase i was used to remove cell surface hs. as shown in figure , the number of plaques formed by the two viruses was markedly reduced in gstc cells pretreated with heparinase i. the reduction was more than % at a heparinase concentration of . u/ml and slightly more at higher enzyme concentrations. thus, the ability of heparinase treatment to reduce viral plaque formation supports the view that cell surface hs is a receptor for the two viruses. viruses , , of heparinase treatment to reduce viral plaque formation supports the view that cell surface hs is a receptor for the two viruses. triplicate results were analyzed by student's t-test, and significant differences are marked with * (p < . ). to further verify the role of cell surface hs on virus-cell binding, heparin was used to inhibit the binding of the two viruses competitively. the number of bound virions was determined by measurement of viral dna copy number by qpcr to monitor binding. as shown in figure , binding of either crude (viral suspension) or purified virions was inhibited by pre-exposure to heparin. binding of crude suspensions of either adrv or rgv were reduced to - % of control levels at a concentration of μg/ml. similar results were obtained with purified virions. the inhibitory effect of heparin on virus-cell binding supports the finding that cell surface hs is a viral receptor. triplicate results were analyzed by student's t-test, and significant differences are marked with * (p < . ). to further verify the role of cell surface hs on virus-cell binding, heparin was used to inhibit the binding of the two viruses competitively. the number of bound virions was determined by measurement of viral dna copy number by qpcr to monitor binding. as shown in figure , binding of either crude (viral suspension) or purified virions was inhibited by pre-exposure to heparin. binding of crude suspensions of either adrv or rgv were reduced to - % of control levels at a concentration of µg/ml. similar results were obtained with purified virions. the inhibitory effect of heparin on virus-cell binding supports the finding that cell surface hs is a viral receptor. viruses , , of heparinase treatment to reduce viral plaque formation supports the view that cell surface hs is a receptor for the two viruses. triplicate results were analyzed by student's t-test, and significant differences are marked with * (p < . ). to further verify the role of cell surface hs on virus-cell binding, heparin was used to inhibit the binding of the two viruses competitively. the number of bound virions was determined by measurement of viral dna copy number by qpcr to monitor binding. as shown in figure , binding of either crude (viral suspension) or purified virions was inhibited by pre-exposure to heparin. binding of crude suspensions of either adrv or rgv were reduced to - % of control levels at a concentration of μg/ml. similar results were obtained with purified virions. the inhibitory effect of heparin on virus-cell binding supports the finding that cell surface hs is a viral receptor. as shown above, cell surface hs is an important receptor for both adrv and rgv. here we examine the role that viral envelope proteins play in this process by monitoring the interaction of purified recombinant viral envelope protein with heparin-sepharose beads. to accomplish this, the amino terminal amino acids of adrv- l and rgv- r and the c-terminal region of adrv- l (amino acids - ) were cloned into pet a and pet a and expressed in e. coli (figure a ). because the amino acid sequences of the c-terminal regions of adrv- l and rgv- r are identical, only the c-terminal region of adrv- l was expressed. note the recombinant proteins were increased in size, due to a kda trx-his-s tag in pet a, and a kda his-t tag in pet a. as shown in figure b ,c, recombinant proteins of the expected sizes were generated using the two expression systems. subsequently, purified recombinant proteins were isolated and incubated with heparin-sepharose beads or control beads lacking heparin. the three recombinant proteins (r l-n, r r-n, and r l-c), expressed using either pet a or pet a, bound heparin-sepharose beads and were eluted in the presence of a high salt wash. in contrast, all three recombinant proteins failed to bind sepharose beads lacking heparin, and, as a result, were present in the unbound supernatant (s) fraction (figure d ). when considered together, these results support the view that hs is a cellular receptor for both adrv and rgv and the binding fashion may be similar to the interaction between r/ l and heparin that occurred in vitro. viruses , , of as shown above, cell surface hs is an important receptor for both adrv and rgv. here we examine the role that viral envelope proteins play in this process by monitoring the interaction of purified recombinant viral envelope protein with heparin-sepharose beads. to accomplish this, the amino terminal amino acids of adrv- l and rgv- r and the c-terminal region of adrv- l (amino acids - ) were cloned into pet a and pet a and expressed in e. coli (figure a ). because the amino acid sequences of the c-terminal regions of adrv- l and rgv- r are identical, only the c-terminal region of adrv- l was expressed. note the recombinant proteins were increased in size, due to a kda trx-his-s tag in pet a, and a kda his-t tag in pet a. as shown in figure b ,c, recombinant proteins of the expected sizes were generated using the two expression systems. subsequently, purified recombinant proteins were isolated and incubated with heparin-sepharose beads or control beads lacking heparin. the three recombinant proteins (r l-n, r r-n, and r l-c), expressed using either pet a or pet a, bound heparin-sepharose beads and were eluted in the presence of a high salt wash. in contrast, all three recombinant proteins failed to bind sepharose beads lacking heparin, and, as a result, were present in the unbound supernatant (s) fraction (figure d ). when considered together, these results support the view that hs is a cellular receptor for both adrv and rgv and the binding fashion may be similar to the interaction between r/ l and heparin that occurred in vitro. in this study, we tested the ability of heparin and two other gags (hs and cs) to inhibit plaque formation in fish and amphibian cell lines. these and other results showed that cell surface hs is an important receptor for the binding of adrv and rgv to target cells. as far as we know, it is the first report describing the role of cell surface hs in iridovirus infection. previously we identified rgv- r as an envelope protein [ ] . here we tested whether rgv- r, or its adrv homolog ( l), could bind hs. our data showed that recombinant r and l proteins specifically bound heparin-sepharose beads in vitro and that this binding could be inhibited by the presence of excess hs. the envelope protein r and its homologs among other members of the family constitute one of core proteins and likely function in viral entry. additional studies will be needed to identify the protein domains involved in r-heparan sulfate interaction. multiple steps are involved in virion entry and initiation of a successful infection. binding to cell surface gags, including hs, has proved to be the initial event in the entry of several mammalian viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . because gags are linked to proteins and usually exist as proteoglycans in vivo [ ] , viral envelope proteins may bind to cell surface heparan sulfate-linked proteins and facilitate attachment between the virus and potential host cells. our results suggest that binding of cell surface hs is required for initiation of productive infection by ranaviruses. however, since competition by increasing concentrations of heparin or hs, or treatment with heparinase did not completely inhibit plaque formation, it appears that hs is not the sole cellular receptor for ranaviruses. a similar phenomenon has been observed in the binding of the vaccinia virus, which uses cell surface gags and cellular matrix laminin as receptors [ ] [ ] [ ] [ ] . a recent study showed that class a scavenger receptors are utilized by frog virus , the type species of the genus ranavirus [ ] . additional ranavirus binding factors and specific cellular protein receptors may be involved in viral attachment and entry. in addition, although the host range of ranaviruses could be determined by binding to other cellular proteins, binding to cell surface gags likely plays an important role in the initial interaction between virus and host cell. it is worth noting that there are two types of virions for iridoviruses. one contains a central core surrounded by an internal membrane and a viral capsid. the other type has an outer viral envelope after the virions bud from the plasma membrane [ ] . the two types of viral particles may have different cellular receptors when they infect cells. thus, the existence of different virions that have an outer envelope or not could be another reason for the incomplete inhibition efficiency in the present study. this is a report showing that iridoviruses bind cells of different species through the interaction between cell surface gags and envelope proteins. however, it remains to be determined whether binding to gags is both necessary and sufficient for subsequent virion entry, or whether virus-gag interaction precedes binding to a second specific cellular receptor. host and viral determinants of influenza a virus species specificity broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility diversity, evolutionary contribution and ecological roles of aquatic viruses a brief review on aquatic animal virology researches in china host range, host specificity and hypothesized host shift events among viruses of lower 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virus-host interactions in aquaculture animals ranaviruses: not just for frogs virus-receptor interactions: the key to cellular invasion poxvirus cell entry: how many proteins does it take? viruses a l protein mediates vaccinia virus interaction with cell surface heparan sulfate vaccinia virus c (a l) protein on intracellular mature virus binds to the extracellular cellular matrix laminin vaccinia virus envelope d l protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells vaccinia virus envelope h l protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo glycosaminoglycans and infection heparan sulfate glycosaminoglycans are involved in adenovirus type and -host cell interactions binding of sindbis virus to cell surface heparan sulfate heparan sulfate proteoglycan: an arbovirus attachment factor integral to mosquito salivary gland ducts a haploid genetic screen identifies heparan sulfate proteoglycans supporting rift valley fever virus infection heparan sulfate proteoglycan is an important attachment factor for cell entry of akabane and schmallenberg viruses filoviruses utilize glycosaminoglycans for their attachment to target cells interaction of zika virus envelope protein with glycosaminoglycans characterization of hepatitis c virus interaction with heparan sulfate proteoglycans glycoprotein c of herpes simplex virus type plays a principal role in the adsorption of virus to cells and in infectivity human papillomavirus infection requires cell surface heparan sulfate the role of heparan sulfate proteoglycans as an attachment factor for rabies virus entry and infection identification and characterization of a novel envelope protein in rana. grylio virus grylio virus (rgv) envelope protein l: subcellular localization and essential roles in virus infectivity revealed by conditional lethal mutant rana grylio virus r encodes an envelope protein involved in virus entry sequencing and analysis of the complete genome of rana. grylio virus (rgv) characterization of the rana. grylio virus beta-hydroxysteroid dehydrogenase and its novel role in suppressing virus-induced cytopathic effect grylio virus tk and dut gene locus could be simultaneously used for foreign gene expression grylio virus (rgv) l is associated with viral matrix and exhibited two distribution patterns a conditional lethal mutation in rana grylio virus orf r resulted in a marked reduction in virion formation frog virus orf r, a putative myristoylated membrane protein, is essential for virus replication in vitro proteomic analysis of singapore grouper iridovirus envelope proteins and characterization of a novel envelope protein vp characterization of an envelope gene vp from singapore grouper iridovirus establishment of three cell lines from chinese giant salamander and their sensitivities to the wild-type and recombinant ranavirus molecular characterization of three rana. grylio virus (rgv) isolates and paralichthys. olivaceus lymphocystis disease virus (lcdv-c) in iridoviruses heparin and heparan sulfate: structure and function extensive diversification of mhc in chinese giant salamanders andrias. davidianus (anda-mhc) reveals novel splice variants analysis of relative gene expression data using real-time quantitative pcr and the −∆∆ct method class a scavenger receptors are used by frog virus during its cellular entry this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - qct authors: kummer, susann; avinoam, ori; kräusslich, hans-georg title: ifitm clusters on virus containing endosomes and lysosomes early in the influenza a infection of human airway epithelial cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qct interferon-induced transmembrane proteins (ifitms) have been shown to strongly affect influenza a virus (iav) infectivity in tissue culture. moreover, polymorphisms in ifitm have been associated with the severity of the disease in humans. ifitm appears to act early in the infection, but its mechanism of action and potential interactions with incoming iav structures are not yet defined. here, we visualized endogenous ifitm interactions with iav in the human lung epithelial cell line a and in primary human airway epithelial cells employing stimulated emission depletion super-resolution microscopy. by applying an iterative approach for the cluster definition and computational cluster analysis, we found that ifitm reorganizes into clusters as iav infection progresses. ifitm cluster formation started at - h post infection and increased over time to finally coat iav-containing endosomal vesicles. this iav-induced phenotype was due to the endosomal recruitment of ifitm rather than to an overall increase in the ifitm abundance. while the iav-induced ifitm clustering and localization to endosomal vesicles was comparable in primary human airway epithelial cells and the human lung epithelial cell line a , the endogenous ifitm signal was higher in primary cells. moreover, we observed ifitm signals adjacent to iav-containing recycling endosomes. influenza a virus (iav) is the major cause for a contagious illness of the upper and lower respiratory tract during the seasonal influenza epidemics with peaks in fall and winter for each hemisphere [ ] [ ] [ ] . besides the acute risk through circulating human specific strains, the zoonotic reservoir (e.g., birds, swine) poses a constant threat of new influenza pandemics [ ] [ ] [ ] [ ] . iav hijacks cellular import mechanisms to enter the host cell, thereby making use of multiple entry routes. the binding of iav by the sialylated receptor [ , ] is followed by clathrin-mediated endocytosis [ , ] or micropinocytosis [ ] . it is reported that iav entry is cell-type dependent [ ] and that subtypes with a filamentous particle shape preferentially enter host cells via macropinocytosis [ ] . the trimeric surface glycoprotein hemagglutinin (ha) is a key factor for several steps during viral entry via endocytosis [ , ] . irrespective of the entry mechanism, iav exploits the non-linear endosomal pathway with its multitude of branches and needs to pass different stages of the endocytic machinery, which is assembled and constantly renewed around the internalized virus particles [ ] [ ] [ ] . endosomal trafficking to the perinuclear region is crucial for ph-dependent membrane fusion [ ] [ ] [ ] , followed by the release of the viral genome into the cytosol and nuclear import [ ] . the iav genome is composed of eight single rna strands in a negative sense orientation [ ] , and each segment is complexed with the nucleoprotein np [ , ] , forming ribonucleoparticles (rnps; [ , ] ). viral genome replication, splicing and transcription then occur in the nucleus. iav replication is strongly inhibited by type interferons already at the early stage, with mx being identified as a crucial interferon-induced host protein blocking iav replication [ ] . genome-wide sirna screens have identified many host factors modulating influenza virus infection [ ] [ ] [ ] [ ] [ ] . brass et al., [ ] and shapira et al., [ ] reported the strong inhibition of iav infection by interferon-induced transmembrane proteins (ifitms). the ifitm variants - can be induced by interferon i and ii. their localization is cell type-or tissue-dependent and changes with the expression level with a preference for cytoplasmic vesicles in most monolayer cell lines [ ] [ ] [ ] [ ] [ ] . besides iav, ifitm proteins have also been reported to confer a basal resistance to members of the flaviviridae (dengue and west nile virus) [ ] , bunyaviridae (rift valley fever virus) [ ] , filoviridae (ebola virus) [ ] , coronaviridae (sars) and retroviridae (hiv) [ ] , showing an unusually broad activity against a wide variety of enveloped and some non-enveloped viruses [ ] . however, murine leukemia virus (mlv) is not restricted by ifitm despite being an enveloped virus [ ] . the relevance of the ifitm -mediated inhibition of iav infection received strong support when it was shown that ifitm polymorphisms correlated with the severity of iav disease in human infection [ ] . the relevance of the ifitm rs -c polymorphism for severe iav infection appears to be population-dependent [ ] [ ] [ ] [ ] [ ] [ ] , and a second ifitm single nucleotide polymorphism, rs -a, was also reported to influence the severity of iav infection in humans [ ] . however, the mechanism of the ifitm-mediated inhibition of iav infection remains under discussion. ifitms have been suggested to disrupt viral membrane fusion [ , ] by altering cellular membrane properties such as fluidity and curvature [ , , ] . it has also been discussed that ifitms alter the lipid/protein composition of acidic intracellular compartments such as endosomes and lysosome [ , ] . the lipid composition-based models of the ifitm function cannot easily explain the lack of antiviral effects on viruses like amphotropic mlv or arenaviruses [ , ] , which also enter by endocytosis and endosomal fusion. in the case of iav, it was recently reported that ifitm elevates the level of cholesterol on late endosomes and lysosomes, thereby restricting early iav infection [ ] . other hypotheses suggest that ifitm directly interferes with the iav fusion pore formation or redirects iav containing endosomes to a non-productive pathway [ ] . it was further demonstrated that the inhibition of the hemagglutinin-mediated membrane fusion required the amphipathic helix of ifitm [ ] . most findings about the antiviral action and localization of ifitm are based on studies using expression plasmids creating an over-expression and/or focusing at the late stages of infection (> h post-infection) [ , , ] . due to the enormous protein load in the overexpression situation, it is far more difficult to determine the subtle differences in the localization of individual molecules, and thus it is possible that these changes may be overlooked. in addition, proteins whose expression is enhanced artificially are more prone to be degraded via the lysosomal pathway. in this regard, it is difficult to judge whether this is a natural re-localization as part of the antiviral defense or just a degradation via lysosomes, particularly in the late stages of infection. we therefore investigated the role of endogenous ifitm in the viral uptake at an early stage. we analyzed the localization of endogenous ifitm through the course of iav infection using confocal and super-resolution (sted) microscopy. we observed a strong clustering of ifitm early after iav infection, which was distinct from the interferon alpha induced ifitm up-regulation. moreover, a two-color sted nanoscopy revealed a close proximity of ifitm with the iav np protein in rab positive compartments within a time range of - h after the iav addition. a and mdck cells were maintained in dulbecco's modified eagle medium (dmem) (invitrogen, karlsruhe, germany), supplemented with % foetal calf serum (fcs) (invitrogen) and penicillin ( u/ml)/streptomycin sulphate ( µg/ml) (capricorn scientific gmbh, ebsdorfergrund, germany). human small airway epithelial cells (hsaepcs) were obtained from promocell (c- ) and grown in a primary cell basal medium (promocell) without antibiotics. four days after thawing, the hsaepcs were seeded for immunofluorescence. the ifitm knock-down in a cells was induced by crispr-cas gene editing using a single guide rna targeting the polyadenylation side of ifitm [ , ] . a ifitm knock-down cells were selected by puromycin resistance ( µg/ml). the ifitm expression was checked by real-time quantitative pcr, as described by zhao et al., [ ] using the fast real time pcr system (applied biosystems, lifetechnologies, sigma-aldrich, steinheim, germany) and by immunofluorescence, as noted below ( figure s ). in general, the cells subjected to immunofluorescence were seeded in well plates on mm glass cover slips. for immunoblotting, cells were seeded in well plates. human interferon alpha (hifnα) (sigma-aldrich, steinheim, germany) was used at u/ml for h prior to infection. amantadine (sigma-aldrich) and bafilomycin a (merck, darmstadt, germany) were applied to the cells at µm and nm concentrations min prior to infection. the cells were treated with mouse anti-hifnα (catalogue no. ab , abcam, berlin, germany). influenza a virus a/hong kong/ / (hk/ / ), a/puerto rico/ / (pr/ / ) and a/regensburg/ d / (r/d / ) were recovered in a hek t-mdck-coculture using the eight plasmid reverse genetics systems, as previously described [ ] . in live-cell experiments, cell mask™ orange plasma membrane stain (catalogue no. c , life technologies, sigma-aldrich, steinheim, germany) was directly added to the cell supernatant prior to the virus purification, following the manufacturer's guidelines. the virus stocks were titrated, making use of a standard plaque assay with minor changes, as previously described [ ] . briefly, serial -fold dilutions of each virus stock were prepared in dmem supplemented with . % bovine serum albumin (bsa, sigma-aldrich, steinheim, germany), mm hepes (sigma-aldrich, steinheim, germany), penicillin ( u/ml)/streptomycin sulphate ( µg/ml) (capricorn scientific gmbh, ebsdorfergrund, germany) and tpck-trypsin ( µg/ml) (sigma-aldrich, steinheim, germany). mdck cell monolayers were incubated with µl of each dilution for h at • c in a well plate format. then, the cells were overlaid with ml of . % avicel (fmc) in aqua dest. and × dmem (merck, darmstadt, germany) in a : ratio. after days, the overlay was removed, the cells were fixed with ml/well of % ethanol for min at room temperature and stained with ml/well of . % crystal violet (sigma-aldrich, steinheim, germany) in % methanol for min at room temperature. the cells were fixed using % paraformaldehyde (pfa) in × phosphate buffered saline (pbs) for min at • c and subsequently treated with . % triton-x in pbs for min. blocking was performed using % bsa in pbs for min. the following primary antibodies were used for immunofluorescence: mouse anti-np (catalogue no. mab , merck, darmstadt, germany), rabbit anti-ifitm (catalogue no. - -ap, proteintech, manchester, united kingdom), mouse anti-eea (catalogue no. , bd transduction laboratoriestm, san jose, ca, usa), mouse anti-rab (catalogue no. ab , abcam, berlin, germany), mouse anti-rab (catalogue no. sc- , santa cruz, dallas, texas, usa), and mouse anti-lamp (catalogue no. ab , abcam, berlin, germany). the cells were incubated with the indicated primary antibodies in a blocking solution for h at room temperature, and washed three times in pbs. the fixation, cellular membrane permeabilisation, and blocking were repeated between the first and secondary antibody incubation, as described above. for sted microscopy, goat anti-mouse star red (catalogue no. - - - , abberior, göttingen, germany) and goat anti-rabbit atto (catalogue no. abin , antibodies-online gmbh) were used as the secondary antibodies. for the triple staining, including the non-diffracted nm channel, donkey anti-goat alexa (catalogue no. ab , abcam, berlin, germany) was used. the secondary antibody staining was performed for h at room temperature under exclusion of light. after repeated washing in pbs, cover slips were mounted on the objective slides using mowiol. whole-cell lysates were prepared using a × lysis buffer ( % sucrose, . % bromophenol blue, mm edta ph . , mm tris ph . , % sds, and % beta-mercaptoethanol). protein samples were resolved by . % sds-page at volt for . h. then, the proteins were transferred to immobilon-fl membranes (merck, darmstadt, germany). the membranes were blocked in li-cor blocking buffer for min at room temperature and probed with first antibodies (rabbit anti-ifitm , catalogue no. - -ap, proteintech, manchester, united kingdom, and rabbit anti-actin, catalogue no. a , sigma-aldrich, steinheim, germany, mouse anti-tubulin no. t , sigma-aldrich, steinheim, germany, or mouse anti-gapdh, catalogue no. sc , santa cruz, dallas, texas, usa) in a blocking solution diluted : at • c overnight. after three repeated washings in × tbst for min, the membranes were incubated with a secondary donkey anti-mouse antibody (catalogue no. - , li-cor) or secondary donkey anti-mouse antibody (catalogue no. - , li-cor, lincoln, nebraska usa), for h at room temperature in the dark. the protein bands were visualized using the li-cor odyssey scanner (li-cor, lincoln, nebraska usa). fluorescence imaging was performed using a two-colour-sted microscopy instrumentation (abberior instruments gmbh, göttingen, germany). for confocal and sted microscopy, a × olympus uplansapo (na . ) oil immersion objective was used. for excitation (λ= nm and λ = nm), a nominal laser power of % was applied, and for sted (λ = nm, max. power = . w), a nominal laser power of % was applied. the pixel size was set to nm (confocal) and nm (non-diffracted), respectively. minor adjustments of contrast and brightness of the acquired images as well as the richardson-lucy deconvolution with a regularisation parameter of . (stopped after iterations) were carried out with the imspector software ( . . -win -aifpgav , abberior instruments gmbh, göttingen, germany). the d object counter and coloc plugin in fiji software (https://fiji.sc/, -bit version)were used for the cluster and mander's coefficient correlation (mcc) analyses, respectively. mcc was performed using the costes regression threshold. the defined objects were assigned as a cluster being larger than nm based on sted images with approximately nm resolution. all graphs show the mean values of three independent experiments plotted with the standard error calculated as a two tailed unpaired t-test. a statistical analysis was performed using graphpad prism software (version ). to visualize the subcellular localization of endogenous ifitm , we performed immunofluorescence microscopy of the uninfected and iav infected a cells. the basal ifitm levels of the uninfected cells showed weak cytosolic signals upon immunofluorescence staining. following infection with iav a/hong kong/ / (hk/ / ) for h, most cells exhibited strong and clustered ifitm signals in the extranuclear space ( figure a ). the ifitm signal increase and clustering correlated with the overall dim np signals in the extranuclear space and no nuclear np signals in the respective cells. in contrast, the productively iav infected a cells (exhibiting a strong nuclear np signal) in the same field of view did not exhibit an increased ifitm signal intensity or clustering ( figure a , right panels). given that we used a relatively high moi of one and that dim np signals were generally detected in these cells, these observations strongly argue that iav infection was abortive in the cells exhibiting an early accumulation of ifitm . ifitm is induced by type interferon [ ] . therefore, we analyzed its distribution upon the treatment of a cells with human interferon alpha (hifnα) for h and compared this to a cells which had been pretreated with hifnα and subsequently infected with iav ( figure b ). the cells treated for h with hifnα exhibited an increased ifitm signal intensity in the extranuclear ifitm is induced by type interferon [ ] . therefore, we analyzed its distribution upon the treatment of a cells with human interferon alpha (hifnα) for h and compared this to a cells which had been pretreated with hifnα and subsequently infected with iav ( figure b ). the cells treated for h with hifnα exhibited an increased ifitm signal intensity in the extranuclear space, both in the uninfected a cells and in the iav-infected cells with a dim or absent np signal. importantly, the productively infected cells exhibiting strong nuclear np signals again exhibited a weak ifitm signal, even after the pretreatment with hifnα. comparing the non-treated and hifnα-pretreated iav-infected a cells lacking a strong nuclear np signal, we observed an increased ifitm signal in both cases, partially colocalizing with vesicular structures. vesicular localization appeared more obvious in the absence of a hifnα pretreatment with a less diffuse cytosolic signal. blocking ifnα with a neutralizing antibody during the iav infection of a cells resulted in increased infection rates, and a concomitant minimal increase of the ifitm signal ( figure c ). to determine whether an iav-induced viral membrane fusion and genome uncoating are required for the observed ifitm signal increase upon iav infection, we performed experiments in the presence of bafilomycin a , specifically inhibiting endosomal acidification, or in the presence of amantadine, specifically blocking the tetrameric m channel of iav, thereby preventing genome uncoating. in both cases, no increase in the ifitm signal intensity and no ifitm clustering were observed ( figure d ,e). these results indicate that iav-induced membrane fusion and genome uncoating are required for an ifitm signal increase in a cells. the mechanism by which ifitm impedes iav infection was divergent in previous studies, and inhibition was mostly described upon ifitm over-expression [ , , ] . it was demonstrated that an ifitm knockdown in a cells increases infection rates [ ] . to determine whether endogenous ifitm is relevant for iav infection in a cells under the experimental conditions of our study, we performed iav infection in a ifitm knock-down cells ( figure s a ,b). the ifitm expression was reduced by a factor of (on the mrna level) in these cells ( figure s c ). the iav infection rate was significantly increased in the knock-down situation compared to the a wildtype cells (figure a ). viruses , , x for peer review of space, both in the uninfected a cells and in the iav-infected cells with a dim or absent np signal. importantly, the productively infected cells exhibiting strong nuclear np signals again exhibited a weak ifitm signal, even after the pretreatment with hifnα. comparing the non-treated and hifnαpretreated iav-infected a cells lacking a strong nuclear np signal, we observed an increased ifitm signal in both cases, partially colocalizing with vesicular structures. vesicular localization appeared more obvious in the absence of a hifnα pretreatment with a less diffuse cytosolic signal. blocking ifnα with a neutralizing antibody during the iav infection of a cells resulted in increased infection rates, and a concomitant minimal increase of the ifitm signal ( figure c ). to determine whether an iav-induced viral membrane fusion and genome uncoating are required for the observed ifitm signal increase upon iav infection, we performed experiments in the presence of bafilomycin a , specifically inhibiting endosomal acidification, or in the presence of amantadine, specifically blocking the tetrameric m channel of iav, thereby preventing genome uncoating. in both cases, no increase in the ifitm signal intensity and no ifitm clustering were observed ( figure d ,e). these results indicate that iav-induced membrane fusion and genome uncoating are required for an ifitm signal increase in a cells. the mechanism by which ifitm impedes iav infection was divergent in previous studies, and inhibition was mostly described upon ifitm over-expression [ , , ] . it was demonstrated that an ifitm knockdown in a cells increases infection rates [ ] . to determine whether endogenous ifitm is relevant for iav infection in a cells under the experimental conditions of our study, we performed iav infection in a ifitm knock-down cells ( figure s a ,b). the ifitm expression was reduced by a factor of (on the mrna level) in these cells ( figure s c ). the iav infection rate was significantly increased in the knock-down situation compared to the a wildtype cells ( figure a ). the increase in the ifitm signal intensity in a cells upon iav infection ( figure a ) and interferon treatment ( figure b ) could be caused either by higher expression levels or by ifitm clustering, yielding viruses , , of a higher density of the signal. to distinguish between these possibilities, we determined the ifitm abundance by an immunoblot analysis of the a cells at different time points after iav infection or interferon treatment, respectively. no significant increase in the ifitm expression was observed during the first h of iav infection ( figure b) , indicating that the signal changes observed by fluorescence microscopy (examples are shown in figure ) during this period were caused by the re-localization of constitutively expressed ifitm rather than by an induced ifitm expression. a strong increase in the ifitm expression levels was observed at late time points ( h p.i.; figure s b ), in accordance with previous studies [ ] . an increased ifitm expression was also observed following the interferon treatment, but starting already at h after the interferon addition ( figure s ), as previously reported [ ] . accordingly, the immunofluorescence phenotype after the interferon treatment ( figure b) is likely to be caused by an increased expression rather than by clustering. the increase in the ifitm signal intensity in a cells upon iav infection ( figure a ) and interferon treatment ( figure b ) could be caused either by higher expression levels or by ifitm clustering, yielding a higher density of the signal. to distinguish between these possibilities, we determined the ifitm abundance by an immunoblot analysis of the a cells at different time points after iav infection or interferon treatment, respectively. no significant increase in the ifitm expression was observed during the first h of iav infection ( figure b) , indicating that the signal changes observed by fluorescence microscopy (examples are shown in figure ) during this period were caused by the re-localization of constitutively expressed ifitm rather than by an induced ifitm expression. a strong increase in the ifitm expression levels was observed at late time points ( h p.i.; figure s b ), in accordance with previous studies [ ] . an increased ifitm expression was also observed following the interferon treatment, but starting already at h after the interferon addition ( figure s ), as previously reported [ ] . accordingly, the immunofluorescence phenotype after the interferon treatment ( figure b) is likely to be caused by an increased expression rather than by clustering. to unravel the distribution pattern of ifitm in a cells at early stages of infection, we made use of super-resolution microscopy. the blurring effect of diffraction limited imaging can obfuscate the potential clustering of ifitm . stimulated emission depletion (sted) super-resolution microscopy was therefore applied to resolve ifitm clusters in early iav-infected a cells. cells infected with iav hk/ / were fixed at - h post infection (h p.i.), stained with anti-ifitm and subjected to confocal and sted microscopy (resolution < nm) of the same region ( figure a ). an increased ifitm signal intensity was again observed in the early phase of the iav infection, and individual ifitm clusters could be resolved by sted microscopy. we computationally analysed the ifitm subcellular localization based on raw sted images using a d cluster analysis (objects counter). the clusters were defined as extranuclear ifitm signal accumulations with a size of > nm . the threshold was chosen by an iterative approach searching for the proportion of clusters, with the critical size being altered upon the infection progression. the analysis of uninfected and hk/ / -infected a cells at different time points revealed a very low proportion of large clusters in uninfected cells or up to h p.i. at h p.i. and later, we observed a significant increase in cluster abundance ( figure b although a cells are an established model cell line for iav research, adaptation to cell-culture conditions may have occurred; hence, we verified our results in primary human respiratory epithelial cells. to determine whether iav infection also causes ifitm clustering in primary cells, we infected human small airway epithelial cells (hsaepcs) with iav pr/ / for different periods of time and performed an indirect immunofluorescence analysis for ifitm and np (from incoming iav particles) using confocal and sted microscopy ( figure , uninfected example see figure s ). the iav infected hsaepcs revealed a co-localization of ifitm and iav np signals at apparently vesicular structures as early as h p.i. (figure a , white arrowheads). at later time points ( h p.i.), this was more obvious, with a clear ifitm clustering on the np-containing vesicular structures ( figure b ). ifitm often exhibited a ring-like appearance, suggesting the coating of endosomal vesicles, and this phenotype became more obvious at later time points ( h p.i; figure c ). some ifitm -positive vesicles exhibited strong np signals (e.g., figure b ), suggesting ifitm -coated vesicles carrying multiple iav particles. a strong ifitm clustering with a ring-like appearance indicating vesicle coating was observed in both iav-infected a cells ( figure a ) and hsaepcs at h p.i. (figure c ; white arrowheads indicating ring-like structures). in the case of the a cells, the ifitm signals in uninfected cells were weak and mostly diffuse. upon iav infection, > % of cells lacking a strong nuclear np signal displayed ifitm clustering (e.g., the right cell in figure a ), while this was only observed in < % of cells with a strong nuclear np ( figure b ). given the high infection rate in this cell line and the fact that cytoplasmic np was also observed in cells lacking the bright nuclear np of replicating iav ( figure a) , we suggest that early ifitm clustering at vesicular structures correlates with an abortive infection in this cell type, and that only cells that do not induce the vesicular clustering of ifitm become infected. in general, the ifitm signals were stronger in hsaepcs compared to a cells ( figure a,c) , and ifitm clusters apparently coating cytoplasmic vesicles were also present in productively iav-infected cells exhibiting a strong nuclear np signal in this case ( figure d ). no ifitm clustering at vesicular structures was observed in hsaepcs in the absence of iav infection, while the overall ifitm signal intensity was much higher in these primary cells compared to a cells without infection ( figure s ). a strong ifitm clustering with a ring-like appearance indicating vesicle coating was observed in both iav-infected a cells ( figure a ) and hsaepcs at h p.i. (figure c ; white arrowheads indicating ring-like structures). in the case of the a cells, the ifitm signals in uninfected cells were weak and mostly diffuse. upon iav infection, > % of cells lacking a strong nuclear np signal displayed ifitm clustering (e.g., the right cell in figure a ), while this was only observed in < % of cells with a strong nuclear np ( figure b ). given the high infection rate in this cell line and the fact that cytoplasmic np was also observed in cells lacking the bright nuclear np of replicating iav (figure a) , we suggest that early ifitm clustering at vesicular structures correlates with an abortive infection in this cell type, and that only cells that do not induce the vesicular clustering of ifitm become infected. in general, the ifitm signals were stronger in hsaepcs compared to a cells ( figure a,c) , and ifitm clusters apparently coating cytoplasmic vesicles were also present in productively iav-infected cells exhibiting a strong nuclear np signal in this case ( figure d ). no ifitm clustering at vesicular structures was observed in hsaepcs in the absence of iav infection, while the overall ifitm signal intensity was much higher in these primary cells compared to a cells without infection ( figure s ). viruses , , x for peer review of the ring-like appearance of ifitm clusters suggested the vesicular recruitment of this protein to iav-carrying endosomal structures. we therefore analysed whether ifitm clusters co-localize with early endosomal (eea ), late endosomal (rab ), or lysosomal (lamp ) marker proteins at early stages of iav infection in a cells ( figure , figure s ). we observed typical staining for early and late endosomes and for lysosomes with a weak ifitm signal in uninfected cells. the ifitm signal intensity increased at h and h p.i., as seen in the previous experiments, with some apparent colocalization with the endosomal marker proteins. to determine the degree of co-localization, we calculated the mander's correlation coefficient (mcc) [ ] . only ifitm clusters defined as objects with a size of > nm were included in this analysis. we observed a highly significant increase of ifitm cluster co-localization with early endosomes at h p.i., which was subsequently lost in the course of infection ( figure a ). in contrast, a significant increase of co-localization with the late endosomal marker ( figure b ) and some increased co-localization with the lysosomal marker ( figure c ) was seen at h, but not at h p.i. these results suggest that ifitm clustering on iav carrying endosomes already occurs early after endocytosis, prior to the ph-induced fusion and release from late endosomes, and probably persists as the endosomes mature. the ring-like appearance of ifitm clusters suggested the vesicular recruitment of this protein to iav-carrying endosomal structures. we therefore analysed whether ifitm clusters co-localize with early endosomal (eea ), late endosomal (rab ), or lysosomal (lamp ) marker proteins at early stages of iav infection in a cells ( figure , figure s ). we observed typical staining for early and late endosomes and for lysosomes with a weak ifitm signal in uninfected cells. the ifitm signal intensity increased at h and h p.i., as seen in the previous experiments, with some apparent co-localization with the endosomal marker proteins. to determine the degree of co-localization, we calculated the mander's correlation coefficient (mcc) [ ] . only ifitm clusters defined as objects with a size of > nm were included in this analysis. we observed a highly significant increase of ifitm cluster co-localization with early endosomes at h p.i., which was subsequently lost in the course of infection ( figure a ). in contrast, a significant increase of co-localization with the late endosomal marker ( figure b ) and some increased co-localization with the lysosomal marker ( figure c ) was seen at h, but not at h p.i. these results suggest that ifitm clustering on iav carrying endosomes already occurs early after endocytosis, prior to the ph-induced fusion and release from late endosomes, and probably persists as the endosomes mature. endocytosed material is generally sorted to different destinations: reusable ligands and receptors are returned to the cell surface via recycling endosomes, while the material destined for degradation traffic to lysosomes [ ] . previous studies reported a substantial co-localization between virus-containing compartments and rme- that is thought to be associated with sorting and recycling endosomes [ ] . furthermore, recent reports have suggested that rab -associated recycling endosomes are required for vrnp trafficking, assembly, and virion budding at the cellular surface in the late phase of iav infection [ ] [ ] [ ] [ ] . to determine whether iav and ifitm clustering can be detected on recycling endosomes, we compared uninfected and iav-infected a cells at different time points. some co-localization of np with ifitm and rab signals was observed on apparently vesicular structures in the extranuclear space of iav infected a cells at - h p.i. (figure a-c) . however, the co-localization of ifitm and rab was already present in the uninfected a cells, with no significant change of co-localization during the course of infection, as shown by the mcc analysis ( figure s ). the uptake of iav particles into recycling endosomes is expected to lead to an abortive infection, as recycling endosomes do not undergo acidification during trafficking. it is likely that rare events of an enhanced endocytosed material is generally sorted to different destinations: reusable ligands and receptors are returned to the cell surface via recycling endosomes, while the material destined for degradation traffic to lysosomes [ ] . previous studies reported a substantial co-localization between virus-containing compartments and rme- that is thought to be associated with sorting and recycling endosomes [ ] . furthermore, recent reports have suggested that rab -associated recycling endosomes are required for vrnp trafficking, assembly, and virion budding at the cellular surface in the late phase of iav infection [ ] [ ] [ ] [ ] . to determine whether iav and ifitm clustering can be detected on recycling endosomes, we compared uninfected and iav-infected a cells at different time points. some co-localization of np with ifitm and rab signals was observed on apparently vesicular structures in the extranuclear space of iav infected a cells at - h p.i. (figure a-c) . however, the co-localization of ifitm and rab was already present in the uninfected a cells, with no significant change of co-localization during the course of infection, as shown by the mcc analysis ( figure s ). the uptake of iav particles into recycling endosomes is expected to lead to an abortive infection, as recycling endosomes do not undergo acidification during trafficking. it is likely that rare events of an enhanced ifitm and rab the ifitm -mediated inhibition of iav infection has been suggested to be caused by changes in the membrane tension resulting in a block of fusion pore formation [ ] , and may be due to constitutively expressed protein or interferon induction [ , , , ] . applying confocal and superresolution imaging of endogenous ifitm , we show that pre-existing ifitm clusters at early and late endosomal structures carry iav at early time points of infection, prior to interferon induction. as shown previously [ ] and in our study ( figure s ), interferon alpha led to increased protein levels of ifitm at later time points. on the other hand, no increase in the ifitm protein levels was observed up to h p.i. in iav infected a cells. a semi-automated image analysis of iav a/hk/ / (pdmh n ) and iav a/r/d / (pdmh n ) infected a cells revealed ifitm clustering early after infection in both cases, with few differences in kinetics, which may reflect strain specific replication kinetics [ ] . the cd domain of ifitm , which is composed of the intermembrane domain and the intracellular loop, contains two phenylalanine residues mediating the physical association between ifitms; these residues are strongly connected with the antiviral function [ ] . combining this finding with our observation of the increasing levels of larger ifitm clusters, we suggest a direct relation between cluster formation and ifitm `s antiviral activity on endosomal vesicles. the ifitm clustering on apparently vesicular structures started early after infection ( - h p.i.), with an initial co-localization of ifitm with an early endosomal marker and a later co-localization with a late endosomal marker, reflecting the early to late endosomal pathway of iav. it appears likely, therefore, that ifitm the ifitm -mediated inhibition of iav infection has been suggested to be caused by changes in the membrane tension resulting in a block of fusion pore formation [ ] , and may be due to constitutively expressed protein or interferon induction [ , , , ] . applying confocal and super-resolution imaging of endogenous ifitm , we show that pre-existing ifitm clusters at early and late endosomal structures carry iav at early time points of infection, prior to interferon induction. as shown previously [ ] and in our study ( figure s ), interferon alpha led to increased protein levels of ifitm at later time points. on the other hand, no increase in the ifitm protein levels was observed up to h p.i. in iav infected a cells. a semi-automated image analysis of iav a/hk/ / (pdmh n ) and iav a/r/d / (pdmh n ) infected a cells revealed ifitm clustering early after infection in both cases, with few differences in kinetics, which may reflect strain specific replication kinetics [ ] . the cd domain of ifitm , which is composed of the intermembrane domain and the intracellular loop, contains two phenylalanine residues mediating the physical association between ifitms; these residues are strongly connected with the antiviral function [ ] . combining this finding with our observation of the increasing levels of larger ifitm clusters, we suggest a direct relation between cluster formation and ifitm 's antiviral activity on endosomal vesicles. the ifitm clustering on apparently vesicular structures started early after infection ( - h p.i.), with an initial co-localization of ifitm with an early endosomal marker and a later co-localization with a late endosomal marker, reflecting the early to late endosomal pathway of iav. it appears likely, therefore, that ifitm clusters, initially recruited to early endosomes and then coating endosomal vesicles through their trafficking pathway, may mediate the antiviral activity of ifitm and block the release of vrnps. this hypothesis is further supported by the identification of a critical sorting signal that is essential for the ifitm localization to endosomes and its anti-viral activity [ , ] . similar to iav, arena-(including lasv, lymphocytic choriomeningitis virus, and macv) and alphaviruses (including the chikungunya virus, sindbis virus, and venezuelan encephalitis virus) also fuse from late endosomes and lysosomes at a similar acidic ph, but are not restricted by ifitm [ ] . it will be of interest, therefore, whether a similar ifitm clustering can be observed on endosomal vesicles carrying these viruses or whether they can escape from ifitm clustering. importantly, a similar phenotype was observed in human primary respiratory cells from healthy donor tissue (hsaepcs). the basal levels of ifitm were much higher in these primary cells, compared to a cells in the absence of iav infection. however, ifitm clustering was not observed in the naïve primary cell population, but rapidly increased again upon iav infection and was even stronger than in a cells. ifitm clustering on cytoplasmic vesicles was strongly induced in both a cells and hsaepcs exhibiting weak extranuclear signals for iav np, which most likely reflects an incoming input virus. in contrast, no such ifitm clusters were seen in productively infected a cells with a strong nuclear np signal, and we speculate that infection of this cell type mainly occurs in cells that have not managed to block iav entry by inducing ifitm clustering. alternatively, ifitm clusters may be abrogated once productive infection has occurred and may thus no longer be visible in these cells. live-cell microscopy using labelled ifitm will be required to distinguish between these possibilities. a different phenotype was observed in primary hsaepcs, where ifitm clustering on vesicular structures was also observed in productively infected cells with a strong nuclear signal. despite having a much higher basal level, ifitm clustering in hsaepcs does not appear, therefore, to efficiently block iav infection in these primary cells. from our findings we assume two putative and presumably additive mechanisms for ifitm mediated antiviral action: (i) the first contact with iav particles after an endocytic uptake in (early) endosomes (role of recycling endosomes is discussed below) triggers the continuous recruitment of pre-existing ifitm proteins to iav containing compartments as a fast antiviral defense in the initial phase of infection. (ii) ifitm protein levels are increased by interferon signaling, further propagating its antiviral effect and possibly synergizing with additional ifn-induced antiviral factors [ ] . continuing studies aim to identify the modalities of the direct or indirect interaction between iav and ifitm . we have to admit that deficient particles might have the potential to fuse with the plasma membrane but fail to replicate. an abortive and ineffective infection might trigger the ifitm activation and recruitment to endosomes in the same way as can be seen for an efficient iav infection. various members of the endosomal network fulfil crucial functions in the viral entry, trafficking, and genome release. rab is mainly assigned to recycling endosomes [ ] . we made the observation that rab -positive vesicles loaded with ifitm often carried iav np early during infection. previous reports using live-cell imaging suggested that ifitm mediates the directed re-localization of viral particles to lysosomes [ ] . our results argue that the internalization of iav via recycling endosomes may trap iav in a non-productive pathway, as the membrane fusion is precluded in recycling endosomes having only a mildly acidic ph of . [ ] . it is conceivable that both mechanisms exist synergistically. in general, viruses being restricted by ifitm fuse at a lower ph in late endosomes or lysosomes [ ] . ifitm on recycling endosomes is thus unlikely to restrict the endosomal entry of iav (or of other viruses described to be restricted by ifitms), but may be an important defense against other pathogens entering via recycling endosomes [ ] . supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : ifitm knock-down validation, figure s : ifitm abundance in the absence and presence of hifnα and upon iav infection ( h p.i.) determined by western blotting, figure s : ifitm proteins cover early, late and recycling endosomes, figure s : ifitm abundance stays constant in recycling endosomes up to h p.i. in iav infected a cells, figure s : indirect immunofluorescence analysis of naïve human small airway epithelial cells (hsaepcs) using anti-ifitm and anti-np. the annual impact of seasonal influenza in the us: measuring disease burden and costs influenza a pandemics of the th century with special reference to : virology, pathology and epidemiology a history of influenza characterization of a novel influenza a virus hemagglutinin subtype (h ) obtained from black-headed gulls human infection with highly pathogenic h n influenza virus detection of novel swine origin influenza a virus (h n ) by real-time nucleic acid sequence-based amplification the influenza virus enigma differential infection of receptor-modified host cells by receptor-specific influenza viruses influenza a virus entry into cells lacking sialylated n-glycans infectious entry pathway of influenza virus in a canine kidney cell line entry of influenza a virus: host factors and antiviral targets dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway differential infectious entry of human influenza a/nws/ virus (h n ) in mammalian kidney cells influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis the biology of influenza viruses changes in the conformation of influenza virus hemagglutinin at the ph optimum of virus-mediated membrane fusion endocytosis and the recycling of plasma membrane assembly of endocytic machinery around individual influenza viruses during viral entry the first five seconds in the life of a clathrin-coated pit activation of influenza virus by acidic media causes hemolysis and fusion of erythrocytes fusion mutants of the influenza virus hemagglutinin glycoprotein anti-peptide antibodies detect steps in a protein conformational change: low-ph activation of the influenza virus hemagglutinin transport of incoming influenza virus nucleocapsids into the nucleus the viruses and their replication. fields virol structure of influenza virus rnp. i. influenza virus nucleoprotein melts secondary structure in panhandle rna and exposes the bases to the solvent does the higher order structure of the influenza virus ribonucleoprotein guide sequence rearrangements in influenza viral rna? cell interferon-inducible protein mx inhibits influenza virus by interfering with functional viral ribonucleoprotein complex assembly genome-wide rnai screen identifies human host factors crucial for influenza virus replication alteration of protein levels during influenza virus h n infection in host cells: a proteomic survey of host and virus reveals differential dynamics quantitative proteomic analyses of influenza virus-infected cultured human lung cells a quantitative proteomic analysis of lung epithelial (a ) cells infected with pandemic influenza a virus using stable isotope labelling with amino acids in cell culture the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus a physical and regulatory map of host-influenza interactions reveals pathways in h n infection inhibition of proliferation by - u in interferon-alpha-responsive and non-responsive cell lines association of rat with fyn protein kinase via lipid rafts is required for rat mammary cell differentiation in vitro distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus ifitm inhibits influenza a virus infection by preventing cytosolic entry the antiviral effector ifitm disrupts intracellular cholesterol homeostasis to block viral entry the cd domain of ifitm is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication ifitm- and ifitm- but not ifitm- restrict rift valley fever virus ifitm restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion ifitm restricts the morbidity and mortality associated with influenza evaluation of ifitm rs association with severe pediatric influenza infection ifitm and susceptibility to respiratory viral infections in the community pilot screening study of targeted genetic polymorphisms for association with seasonal influenza hospital admission ifitm and severe influenza virus infection. no evidence of genetic association population genetics of ifitm in portugal and central africa reveals a potential modifier of influenza severity interferon-induced transmembrane protein inhibits hantaan virus infection, and its single nucleotide polymorphism rs influences the severity of hemorrhagic fever with renal syndrome snp-mediated disruption of ctcf binding at the ifitm promoter is associated with risk of severe influenza in humans palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm ifitm-family proteins: the cell's first line of antiviral defense ifitms restrict the replication of multiple pathogenic viruses ifitm proteins restrict viral membrane hemifusion interaction of polyene antibiotics with membrane lipids: physicochemical studies of the molecular basis of selectivity different mechanisms of cell entry by human-pathogenic old world and new world arenaviruses caveola-dependent endocytic entry of amphotropic murine leukemia virus late endosomal/lysosomal cholesterol accumulation is a host cell-protective mechanism inhibiting endosomal escape of influenza a virus ifitm requires an amphipathic helix for antiviral activity genome-scale crispr-cas knockout screening in human cells improved vectors and genome-wide libraries for crispr screening ellagic acid inhibits human pancreatic cancer growth in balb c nude mice generation of recombinant influenza virus from plasmid dna rapid accumulation of virulent rift valley fever virus in mice from an attenuated virus carrying a single nucleotide substitution in the mrna a practical guide to evaluating colocalization in biological microscopy the endocytic pathway: a mosaic of domains the ubiquitin-vacuolar protein sorting system is selectively required during entry of influenza virus into host cells rab regulates exocytosis of recycling vesicles at the plasma membrane a rab -and microtubule-dependent mechanism for cytoplasmic transport of influenza a virus viral rna apical transport of influenza a virus ribonucleoprotein requires rab -positive recycling endosome clustering of rab vesicles in influenza a virus infected cells creates hotspots containing the viral ribonucleoproteins interferon-induced transmembrane protein blocks fusion of sensitive but not resistant viruses by partitioning into virus-carrying endosomes the interferon-induced transmembrane proteins, ifitm , ifitm , and ifitm inhibit hepatitis c virus entry variation and infectivity neutralization in influenza identification of an endocytic signal essential for the antiviral action of ifitm phosphorylation of the antiviral protein interferon-inducible transmembrane protein (ifitm ) dually regulates its endocytosis and ubiquitination klf is involved in regulation of ifitm , , and genes during h n virus infection in a cells hang, h.c. ifitm directly engages and shuttles incoming virus particles to lysosomes emerging roles of recycling endosomes we thank kathleen börner (university hospital, heidelberg) for advice using crispr-cas gene editing, marino zerial (max planck institute of molecular cell biology and genetics, dresden) for the prab _egfp plasmid and jürgen stech (friedrich-loeffler-institute, insel riems) for the recovery plasmid system of influenza a/hong kong/ / , a/puerto rico/ / and a/regensburg/d / . we are grateful to vibor laketa (idip, heidelberg), bärbel glass and vera sonntag-buck (technical assistance) and carola sparn (student assistance) for excellent assistance. the authors declare no conflict of interest. key: cord- -qtwcbn m authors: gao, yaning; tai, wanbo; wang, ning; li, xiang; jiang, shibo; debnath, asim k.; du, lanying; chen, shizhong title: identification of novel natural products as effective and broad-spectrum anti-zika virus inhibitors date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qtwcbn m zika virus (zikv) infection during pregnancy leads to severe congenital zika syndrome, which includes microcephaly and other neurological malformations. no therapeutic agents have, so far, been approved for the treatment of zikv infection in humans; as such, there is a need for a continuous effort to develop effective and safe antiviral drugs to treat zikv-caused diseases. after screening a natural product library, we have herein identified four natural products with anti-zikv activity in vero e cells, including gossypol, curcumin, digitonin, and conessine. except for curcumin, the other three natural products have not been reported before to have anti-zikv activity. among them, gossypol exhibited the strongest inhibitory activity against almost all zikv strains tested, including six recent epidemic human strains. the mechanistic study indicated that gossypol could neutralize zikv infection by targeting the envelope protein domain iii (ediii) of zikv. in contrast, the other natural products inhibited zikv infection by targeting the host cell or cell-associated entry and replication stages of zikv. a combination of gossypol with any of the three natural products identified in this study, as well as with bortezomib, a previously reported anti-zikv compound, exhibited significant combinatorial inhibitory effects against three zikv human strains tested. importantly, gossypol also demonstrated marked potency against all four serotypes of dengue virus (denv) human strains in vitro. taken together, this study indicates the potential for further development of these natural products, particularly gossypol, as the lead compound or broad-spectrum inhibitors against zikv and other flaviviruses, such as denv. zika virus (zikv) is a mosquito-borne flavivirus in the same genus as other important human pathogens, including dengue virus (denv), west nile virus (wnv), yellow fever virus (yfv), japanese encephalitis virus (jev), and tick-borne encephalitis virus (tbev) [ ] . zikv was originally isolated in a rhesus macaque in [ ] , but this virus has only recently claimed worldwide attention owing to its close association with congenital zika syndrome (czs), as represented by microcephaly, fetal demise, central nervous system abnormalities, and other neurological complications [ ] [ ] [ ] [ ] [ ] . no antiviral therapeutics for the treatment of zikv-associated human diseases, particularly congenital syndrome a plaque reduction inhibition assay was carried out to measure the inhibitory activity of natural products in a natural product collection library from microsource discovery systems (gaylordsville, ct, usa) against infections of zikv and denv, as previously described [ ] [ ] [ ] [ ] . briefly, zikv (strain pan , ~ plaque-forming unit (pfu); multiplicity of infection (moi) ~ . ) was incubated with -fold serial dilutions of natural products (including curcumin, digitonin and conessine, as well as anti-zikv compound control, bortezomib) or dmso ( . % vol/vol) control at °c for h. the compound-virus mixtures were then transferred to vero e cells ( /well) and incubated at °c for h. for gossypol, zikv (strain pan , ~ . × pfu; moi ~ . ) was incubated with serial dilutions of this natural product at °c for h, and the unbound gossypol was removed by centrifugation after addition of % peg- . gossypol-treated zikv was then incubated with vero e cells at °c for h. the cells were washed thoroughly with pbs and overlaid with dmem containing % carboxymethyl cellulose and % fbs, followed by in vitro culture at °c for - days, and then stained with . % crystal violet. the inhibitory activity of all natural products tested against denv- - , as described above, was performed following the above procedures, except that llc-mk cells were used for infection, and cells were cultured at °c for - days before staining with . % crystal violet. the % inhibitory concentration (ic ) and ic of natural products were calculated based on the concentrations at % and % plaque reduction, respectively, using the calcusyn computer program, as described before [ ] . the cytotoxicity of natural products in vero e for zikv or llc-mk cells for denv- - was evaluated using the cell counting kit- (cck , sigma, st. louis, mo, usa), according to the manufacturer's instructions. briefly, -fold serial dilutions of each of the natural products ( l/well) were added to equal volumes of cells ( . × /well) in -well plates and cultured at °c for days. the cells were then incubated with cck solution and absorbance was measured at nm (a value) using a microplate reader (infinite f pro, tecan, morrisville, nc, usa). the % cytotoxic concentration (cc ) of natural products was calculated based on the percent cytotoxicity a plaque reduction inhibition assay was carried out to measure the inhibitory activity of natural products in a natural product collection library from microsource discovery systems (gaylordsville, ct, usa) against infections of zikv and denv, as previously described [ ] [ ] [ ] [ ] . briefly, zikv (strain pan , ~ plaque-forming unit (pfu); multiplicity of infection (moi) . ) was incubated with -fold serial dilutions of natural products (including curcumin, digitonin and conessine, as well as anti-zikv compound control, bortezomib) or dmso ( . % vol/vol) control at • c for h. the compound-virus mixtures were then transferred to vero e cells ( /well) and incubated at • c for h. for gossypol, zikv (strain pan ,~ . × pfu; moi~ . ) was incubated with serial dilutions of this natural product at • c for h, and the unbound gossypol was removed by centrifugation after addition of % peg- . gossypol-treated zikv was then incubated with vero e cells at • c for h. the cells were washed thoroughly with pbs and overlaid with dmem containing % carboxymethyl cellulose and % fbs, followed by in vitro culture at • c for - days, and then stained with . % crystal violet. the inhibitory activity of all natural products tested against denv- - , as described above, was performed following the above procedures, except that llc-mk cells were used for infection, and cells were cultured at • c for - days before staining with . % crystal violet. the % inhibitory concentration (ic ) and ic of natural products were calculated based on the concentrations at % and % plaque reduction, respectively, using the calcusyn computer program, as described before [ ] . the cytotoxicity of natural products in vero e for zikv or llc-mk cells for denv- - was evaluated using the cell counting kit- (cck , sigma, st. louis, mo, usa), according to the manufacturer's instructions. briefly, -fold serial dilutions of each of the natural products ( µl/well) were added to equal volumes of cells ( . × /well) in -well plates and cultured at • c for days. the cells were then incubated with cck solution and absorbance was measured at nm (a value) using a microplate reader (infinite f pro, tecan, morrisville, nc, usa). the % cytotoxic concentration (cc ) of natural products was calculated based on the percent cytotoxicity using the calcusyn computer program [ , ] . the combinatorial cytotoxicity of gossypol with other natural products to vero e cells was detected using a similar approach as described above, except for mixing gossypol with one of the natural products (curcumin, digitonin, conessine, or bortezomib) throughly before adding them to the cells. this experiment was carried out as previously described, with some modifications [ ] [ ] [ ] [ ] [ ] [ ] [ ] . briefly, vero e cells ( /well) or zikv were incubated at different infection steps as described below, with or without the tested natural products at the specified concentrations of µm for gossypol, µm for curcumin, . µm for digitonin, or µm for conessine for h before zikv infection, h after infection, or the same time during infection. anti-zikv compounds, such as temoporfin [ ] , -hydroxycholesterol [ ] , bortezomib [ ] , and nitd [ ] , were included as controls for these steps. after the culture of the zikv-or compound-treated cells at • c for - days, plaques were visualized with crystal violet staining, as described above, and the percent inhibition of natural products was calculated. specifically, the following six stages of zikv infection were tested: (a) pretreatment of zikv (pan ,~ . × pfu; moi~ . ) with natural products at • c for h before incubation with cells; (b) pretreatment of cells with natural products at • c for h before incubation with zikv (pan ,~ pfu; moi~ . ); (c) cotreatment of cells with zikv (pan ,~ pfu; moĨ . ) and natural products at • c for h; (d) cotreatment of cells, zikv (pan ,~ pfu; moĨ . ), and natural products at • c for h; (e) preincubation of cells with zikv (pan ,~ pfu; moi~ . ) at • c for h and then incubation with natural products at • c for h; and (f) preincubation of zikv (pan ,~ pfu; moi~ . ) and cells at • c for h, followed by incubation with natural products at • c for h. the binding between natural products and zikv full-length e protein (aviva systems biology, san diego, ca, usa) or envelope protein domain iii (ediii) protein (e residues - fused with a c-terminal human fc) [ ] was carried out by elisa, as previously described [ , , , ] . briefly, elisa plates were precoated with the proteins described above at • c overnight and blocked with % fat-free milk at • c for h. serial dilutions of natural products or dmso (negative control) were then added to the plates and incubated at • c for h. the plates were washed with pbs containing tween- (pbst) and incubated at • c for h with zikv ediii-specific human monoclonal antibody (mab) zka -lala ( . µg/ml) for binding to zikv full-length e and ediii proteins. the plates were washed with pbst and incubated with horseradish peroxidase (hrp)-conjugated anti-human igg-fab ( : , abcam, cambridge, ms, usa) antibody at • c for h. the , , , -tetramethylbenzidine (tmb) substrate (sigma) was added to the plates, and the reaction was stopped by n h so . absorbance at nm (a value) was measured by elisa microplate reader (tecan). ec ( % effective concentration) was calculated based on the calcusyn computer program, as described above [ ] . to determine the ability of gossypol to inhibit binding between zikv ediii protein and ediii-specific human mabs (smzab , zka -lala, zv- , or z ) or edi/ii-specific human mab control (zka ) [ ] , elisa was carried out, as described above, except that serially diluted gossypol or dmso (negative control) was added in the presence of mabs ( . µg/ml), followed by sequential incubation with hrp-conjugated anti-human igg-fab antibody and tmb substrate and detection for a value. the percent inhibition of natural products was calculated, and ic (concentration causing % reduction in ediii-mab binding) was obtained using the calcusyn computer program [ ] , as described above. the interactions between natural products and zikv full-length e were analyzed at • c using the biacore t system (ge healthcare, port washington, ny, usa), as previously described [ , ] . briefly, zikv e was immobilized on a sensor chip (cm ) using the amine coupling kit (ge healthcare). natural products at various concentrations were injected as analytes, and pbs-p ( mm phosphate buffer containing . mm kcl, mm nacl, and . % surfactant p , ph . ) was used as the running buffer. the data were analyzed using biacore evaluation software (t version . ), and the curve was fitted with a : binding model. the potential combinatorial effect of gossypol with other natural products was carried out as previously described [ , ] . briefly, zikv (strain pan , flr, or prvabc , . × pfu; moĨ . ) was incubated with serially diluted gossypol at • c for h, and the unbound gossypol was removed by centrifugation after addition of % peg- . gossypol-treated zikv was incubated with vero e cells at • c for h in the presence of dmem containing serial dilutions of each of the other three natural products identified, such as curcumin, digitonin, and conessine, or anti-zikv compound control (bortezomib). the unbound viruses and natural products were removed, the cells were cultured at • c for - days, and stained with . % crystal violet. the natural products without combinations were used as controls. the ic values of natural products were calculated using the calcusyn computer program [ ] , as described above. the natural products were then analyzed for combinatorial effects based on the combination index (ci) [ ] and ic values using the calcusyn computer program, as previously described [ , , ] . specifically, ci values < and > indicate synergy and antagonism, respectively. synergy was further identified as five different categories: ci values < . , . - . , . - . , . - . , and . - . indicate very strong synergism, strong synergism, synergism, moderate synergism, and slight synergism, respectively. fold enhancement of anti-zikv potency is expressed as the ratio of molar concentrations of natural products tested alone and in the mixture using the formula ((ic alone)/(ic in the mixture)- ). using a plaque-based assay, we initially screened natural products (at µm of concentration) from a natural product library for their inhibitory activity against infection of a recent zikv human strain (pan ). based on table , four "hit" natural products, including gossypol, curcumin, digitonin, and conessine ( figure a -d), were selected, since they demonstrated inhibitory activity against zikv infection with no obvious cytotoxicity in vero e cells when observed under a microscope. these natural products were subsequently ordered from sigma with ≥ % purity, and tested for their cytotoxicity using a cell-based cytotoxicity assay (cck kit), with cc values ranging from . to . µm (table , figure s ). among these natural products, curcumin has been previously reported to inhibit zikv infection [ ] , whereas the other three natural products have not been previously reported to have anti-zikv activity. gossypol, curcumin, digitonin, and conessine were retested to confirm their anti-zikv (strain pan ) activity, with ic values of . , . , . , and . µm, respectively ( table ). the ic values of these natural products against zikv pan strain were also calculated, equal to about . , . , . , and . µm, respectively, for gossypol, curcumin, digitonin, and conessine ( figure s ), which were slightly higher than the respective ic values, but much lower than the corresponding cc values. table . initial screening of a natural product library for identification of potential anti-zikv inhibitors. natural products from microsource discovery systems for screening primary hits (with inhibition against zikv strain pan ) a ( . ) primary hits (with high cytotoxicity in vero e cells) b ( . ) specific hits (primary hits with low cytotoxicity) ( . ) specific hits (available to purchase) ( . ) specific hits (ordered from sigma and retested to confirm anti-zikv activity) ( . ) specific hits displaying ic < cc ( . ) a a total of primary hits inhibited zikv (strain pan ) infection by more than % at µm, whereas the negative control (dmso) had inhibitory activity less than %. b observed cytotoxicity of natural products (at µm) under a microscope. the cytotoxicity was recorded as grades (−, ±, +, ++, +++, ++++), and the natural products with cytotoxicity greater than or equal to grade ++ were referred to as "high cytotoxicity in vero e cells", and discarded for further testing. viruses , , of table . initial screening of a natural product library for identification of potential anti-zikv inhibitors. no. (%) natural products from microsource discovery systems for screening primary hits (with inhibition against zikv strain pan ) a ( . ) primary hits (with high cytotoxicity in vero e cells) b ( . ) specific hits (primary hits with low cytotoxicity) ( . ) specific hits (available to purchase) ( . ) specific hits (ordered from sigma and retested to confirm anti-zikv activity) ( . ) specific hits displaying ic < cc ( . ) a a total of primary hits inhibited zikv (strain pan ) infection by more than % at μm, whereas the negative control (dmso) had inhibitory activity less than %. b observed cytotoxicity of natural products (at μm) under a microscope. the cytotoxicity was recorded as grades (−, ±, +, ++, +++, ++++), and the natural products with cytotoxicity greater than or equal to grade ++ were referred to as "high cytotoxicity in vero e cells", and discarded for further testing. the identified natural products were further studied for their broad-spectrum activity against nine additional zikv strains, including those isolated from different hosts, namely humans, mosquitos, and rhesus macaques, at different time periods in different countries, including mexico, panama, columbia, honduras, puerto rico, thailand, nigeria, and uganda ( figure ). the results showed that these natural products could inhibit infections of all nine zikv strains tested with various ic values ( table ). gossypol exhibited the most potent inhibitory activity for ic values, ranging from . to . m, against all nine zikv strains tested (table ) . it was also more potent than bortezomib, the anti-zikv previously reported compound as an active compound control, against all zikv strains tested ( table ). the ic values of these natural products against the selected zikv strain, prvabc , were calculated, among which gossypol had the lowest ic value (about . m, slightly higher than its ic value but lower than its cc value) ( figure s ), confirming its strong and broad-spectrum anti-zikv activity. the identified natural products were further studied for their broad-spectrum activity against nine additional zikv strains, including those isolated from different hosts, namely humans, mosquitos, and rhesus macaques, at different time periods in different countries, including mexico, panama, columbia, honduras, puerto rico, thailand, nigeria, and uganda ( figure ). the results showed that these natural products could inhibit infections of all nine zikv strains tested with various ic values (table ). gossypol exhibited the most potent inhibitory activity for ic values, ranging from . to . µm, against all nine zikv strains tested (table ) . it was also more potent than bortezomib, the anti-zikv previously reported compound as an active compound control, against all zikv strains tested ( table ). the ic values of these natural products against the selected zikv strain, prvabc , were calculated, among which gossypol had the lowest ic value (about . µm, slightly higher than its ic value but lower than its cc value) ( figure s ), confirming its strong and broad-spectrum anti-zikv activity. comparison of zikv e protein sequences revealed that most of the amino acid sequences were highly conserved, but that some variations occurred among the zikv strains used for evaluation of the inhibitory activity of natural products, including the pan strain tested earlier ( figure s ). the above data demonstrate that the identified natural products, particularly gossypol, were able to block infections of divergent human, mosquito, and monkey zikv strains isolated from different time periods and countries, including six recent zikv human strains, confirming their broad-spectrum anti-zikv activity. to identify which steps of zikv infection in its life cycle were blocked by these natural products, we carried out a time-of-addition experiment by incubating natural products with zikv or cells at different time points during zikv and cell interaction, and then calculated the percent inhibition based on the number of plaques formed [ , , , ] . to test whether a natural product can neutralize zikv infection or inhibit viral entry by targeting the viral proteins, zikv was pretreated with the natural product at • c before incubation with the host cells ( figure a(a) ). to evaluate whether a natural product can bind to the cellular receptors or cofactors to block virus-receptor binding, cells were pretreated with the natural product at • c before incubation with zikv ( figure a(b) ). to determine whether a natural product can inhibit attachment of zikv to target cells, but cannot block the virus-cell membrane fusion, cells were cotreated with zikv at • c in the presence of the natural product ( figure a (c)). to assess whether a natural product can inhibit attachment of zikv to target cells and subsequent virus-cell membrane fusion, the cells were cotreated with zikv and the natural product at • c ( figure a(d) ). to investigate whether a natural product can inhibit zikv fusion with the cell membrane and then entry into the cell, cells were pretreated with zikv at • c first and then incubated with the natural product at • c ( figure a (e)). to study whether a natural product can inhibit zikv infection at postentry stages (i.e., viral replication, virion assembly, or release), cells were pretreated with zikv and then incubated with the natural product at • c ( figure a (f)). after completing the above approaches, we gained insight into the potential mechanisms of the natural products responsible for inhibiting zikv (pan ) infection. after pretreatment of zikv with gossypol at • c before incubation with the target cells, zikv completely lost its infectivity, whereas it maintained its infectivity after other treatments described above ( figure b ), suggesting that gossypol can effectively neutralize zikv infection by targeting the virus, rather than the cell or cell-associated entry or replication stages. the results from curcumin revealed that about - % of zikv infection was blocked when curcumin was incubated with zikv only at • c, or coincubated with zikv and cells at • c or • c, whereas there was low to no impact on zikv infection when curcumin was pretreated with cells or postincubated with zikv-treated cells at • c and • c, respectively ( figure c ). these results suggest that curcumin inhibits zikv infection at the early stages of viral entry, particularly the viral attachment stage. pretreatment of vero e cells with digitonin and then with zikv or cotreatment of cells with zikv and digitonin at • c significantly (≥ %) blocked zikv infection, whereas preincubation of cells with zikv and then with digitonin at • c, pretreatment of cells with zikv at • c and then with digitonin at • c, or cotreatment of cells with digitonin and zikv at • c inhibited about - % of zikv infection ( figure d ). in contrast, preincubation of digitonin and zikv had no effect on zikv infection ( figure d ). these results suggest that digitonin could not directly neutralize zikv infection, but inhibited zikv infection by binding to the viral receptors or inhibiting viral entry (i.e., attachment and membrane fusion or postentry steps). the data from conessine indicated that coincubation of cells with conessine and zikv at • c or postincubation of conessine with zikv-treated cells at • c or • c resulted in - % inhibition of zikv infection, whereas pretreatment of cells with conessine before zikv incubation blocked about % of zikv infection. nevertheless, preincubation of conessine and zikv at • c or cotreatment of cells with conessine and zikv at • c had very low to no effect on zikv infection ( figure e ). these data suggest that conessine does not block zikv attachment to the host cell, but inhibits zikv infection by targeting virus-cell fusion or postentry step. the above steps of inhibition of zikv infection were further proven inhibition of zikv infection were further proven by the respective anti-zikv compound controls ( figure f-i) . therefore, the above data confirm the potent inhibitory activity of the identified natural products, particularly gossypol, in blocking zikv infection at various stages of the viral life cycle. anti-zikv inhibitor, temoporfin [ ] , was used as a control for step a. (g) an anti-zikv entry (especially in inhibition of the internalization/fusion step) inhibitor, -hydroxycholesterol [ ] , was used as a control for steps b and e. anti-zikv compound bortezomib was used as a control for step d (h), and a replication inhibitor, nitd [ ] , for step f (i). the natural product curcumin (c), which has been previously reported to inhibit the attachment of zikv to host cells [ ] , was used as a control for stage c. the percent inhibition was calculated in the presence or absence of serially diluted natural products. the data are expressed as mean ± s.e.m. (n = ). the experiments were performed in vero e cells and repeated three times, with similar results. (f) a potent anti-zikv inhibitor, temoporfin [ ] , was used as a control for step a. (g) an anti-zikv entry (especially in inhibition of the internalization/fusion step) inhibitor, -hydroxycholesterol [ ] , was used as a control for steps b and e. anti-zikv compound bortezomib was used as a control for step d (h), and a replication inhibitor, nitd [ ] , for step f (i). the natural product curcumin (c), which has been previously reported to inhibit the attachment of zikv to host cells [ ] , was used as a control for stage c. the percent inhibition was calculated in the presence or absence of serially diluted natural products. the data are expressed as mean ± s.e.m. (n = ). the experiments were performed in vero e cells and repeated three times, with similar results. to identify the potential binding regions of the natural products in zikv proteins, we first carried out an elisa-based approach by coating the plates with zikv full-length e or ediii proteins. we then tested for binding affinity using a zikv ediii-specific mab, zka -lala, for e or ediii binding. results revealed that gossypol bound potently to the full-length e and ediii proteins, with ec values of . and . µm, respectively, whereas curcumin had much lower binding affinity to zikv full-length e and ediii proteins ( figure a,b) . otherwise, digitonin, conessine, bortezomib (anti-zikv compound control), and dmso (negative control) did not bind to any zikv proteins tested ( figure a,b) . we then evaluated the binding of gossypol using an spr assay, and the result showed that it had binding affinity values of . µm to zikv e protein ( figure c ). zikv full-length e and ediii proteins ( figure a,b) . otherwise, digitonin, conessine, bortezomib (anti-zikv compound control), and dmso (negative control) did not bind to any zikv proteins tested ( figure a,b) . we then evaluated the binding of gossypol using an spr assay, and the result showed that it had binding affinity values of . m to zikv e protein ( figure c ). since gossypol bound to zikv e protein, potentially in the ediii region, we further carried out an elisa completion assay to identify its possible binding site(s) in the ediii. accordingly, zikv ediii protein was coated on the plates, and binding between zikv ediii and ediii-specific mabs (smzab , zka -lala, zv- , or z ), or a zikv edi/dii-specific mab control (zka ) was evaluated in the presence of serially diluted gossypol. the results showed that gossypol potently blocked binding of ediii-specific mabs smzab , zka -lala, zv- , or z to ediii protein in a dose-dependent manner, with ic values of . , . , . , and . m, respectively, whereas the dmso control showed no blockage of this binding ( figure d ). in the meantime, there was no binding between the control mab zka and ediii protein, so no obvious inhibition of gossypol was detected ( figure d ). the above zikv ediii-specific mabs have been previously shown to potently neutralize zikv infection [ ] and recognize epitopes, including the lateral ridge, such as residues - , - , , , , and - of zikv ediii protein [ ] [ ] [ ] . therefore, the above data suggest that gossypol most likely binds to the lateral ridge of the zikv ediii protein to block the ediii-mab binding. the ability of gossypol to inhibit the binding between zikv ediii and ediii-specific neutralizing mabs. the concentrations of zikv ediii and mabs were . and . µg/ml, respectively. the percent inhibition in the ediii-mab binding was measured in the presence or absence of serially diluted gossypol using the formula (( -(ediii-mab-gossypol)/(ediii-mab) × ), which, in turn, formed the basis for calculating % inhibitory concentration (ic ) values. zikv edi/dii-specific mab (zka ) and dmso were used as controls. the data are expressed as mean ± s.e.m. (n = ) . the experiments were repeated twice, with similar results. since gossypol bound to zikv e protein, potentially in the ediii region, we further carried out an elisa completion assay to identify its possible binding site(s) in the ediii. accordingly, zikv ediii protein was coated on the plates, and binding between zikv ediii and ediii-specific mabs (smzab , zka -lala, zv- , or z ), or a zikv edi/dii-specific mab control (zka ) was evaluated in the presence of serially diluted gossypol. the results showed that gossypol potently blocked binding of ediii-specific mabs smzab , zka -lala, zv- , or z to ediii protein in a dose-dependent manner, with ic values of . , . , . , and . µm, respectively, whereas the dmso control showed no blockage of this binding ( figure d ). in the meantime, there was no binding between the control mab zka and ediii protein, so no obvious inhibition of gossypol was detected ( figure d ). the above zikv ediii-specific mabs have been previously shown to potently neutralize zikv infection [ ] and recognize epitopes, including the lateral ridge, such as residues - , - , , , , and - of zikv ediii protein [ ] [ ] [ ] . therefore, the above data suggest that gossypol most likely binds to the lateral ridge of the zikv ediii protein to block the ediii-mab binding. the data described here demonstrate that the identified natural product gossypol bound strongly to zikv e protein, potentially the conserved ediii, thus blocking ediii-mab binding at important neutralizing epitopes and inhibiting viral entry into the target cell. these data reasonably explain the potent broad-spectrum antiviral activity of gossypol against infections of multiple zikv strains. since gossypol demonstrated the highest antiviral activity individually against all zikv strains tested, we next investigated the potential combinatorial effects of the combination of gossypol with three other natural products identified, namely curcumin, digitonin, and conessine, as well as anti-zikv compound control (bortezomib). results demonstrated significant combinatorial inhibitory effects against three zikv strains (pan , flr, and prvabc ) tested when combining gossypol with any of these natural products, and the ci values ranged from . to . µm, . to . µm, and . to . µm for zikv pan , flr, and prvabc strains, respectively (tables - ). the combinations of gossypol with each of these natural products also resulted in the highest enhancement of anti-prvabc activity among the three zikv strains tested (table ). these data show that gossypol can be combined with other inhibitors described above to further increase overall inhibitory activity against current and future emergent zikv strains. the above results on the combinatorial effects against zikv infection could not exclude the possibility that the observed decrease in ic in the presence of another natural product might result from the enhanced cell death caused by the natural product in combination. therefore, we also assessed the change of cc of the natural products alone and in combination, and compared the enhancement of cc with that of ic against zikv strain prvabc . as shown in table , the cytotoxicity of gossypol in combination with curcumin, digitonin, or conessine was not enhanced. the cytotoxicity of gossypol in combination with bortezomib was slightly increased ( . -fold), but it was much lower than the enhancement in anti-prvabc activity of gossypol ( . -fold) in combination with bortezomib. in addition, the cytotoxicity of curcumin and bortezomib, respectively, in combination with gossypol was not enhanced. the cytotoxicity of digitonin or conessine in combination with gossypol was slightly increased by about . -and . -fold, respectively, while they were still lower than the enhancement in anti-prvabc activity of digitonin ( . -fold) or conessine ( . -fold) in combination with gossypol. moreover, the ci values of gossypol with any of the four natural products tested were greater than ( table ), indicating that there was no combinatorial effect on the cytotoxicity to the test cells. these results suggest that the observed decrease in the ic values of the natural products in combination was not due to the enhancement in cytotoxicity of these natural products. note: gossypol with one of the natural products, including curcumin, digitonin, conessine (three lead natural products) or bortezomib (anti-zikv compound control), were first mixed according to the molar ratio identified in the above combinational experiment against zikv strain prvabc , and then added to vero e cells to determine cytotoxicity. the combinatorial cytotoxicity of natural products to vero e cells is expressed as cc identification of broad-spectrum anti-flavivirus inhibitors is crucial to treat infections caused by zikv and other flaviviruses, such as denv. hence, using a plaque assay similar to zikv, we evaluated the antiviral activity of natural products against infections of four serotypes of denv human strains in llc-mk cells. even though all four natural products could inhibit denv- - infections, results showed that gossypol had the highest potency against denv- , denv- , and denv- infections, with ic values of . , . , and . µm, respectively (table ) . also, the anti-denv- activity of gossypol (ic value: . µm) was only slightly higher than that of curcumin (ic value: . µm) ( table ) . the cytotoxicity of these natural products on llc-mk cells was investigated by a cytotoxicity assay, with the cc values ranging from . to . µm ( table ) . the above data indicate the potent anti-denv activity of the natural products, particularly gossypol, against infections of four denv human strains with low to no cytotoxicity. the experiments were performed on llc-mk cells, and the cytotoxicity of natural products in this cell line is expressed as cc . the inhibitory activity of natural products against infections of denv- - is expressed as ic . the data are expressed as mean ± s.e.m. (n = ). the experiments were repeated twice, with similar results. as described earlier, gossypol targeted zikv e protein, potentially ediii. although a number of variations have been identified in the amino acid sequences of e proteins of zikv and denv strains tested in this study ( figure s ), gossypol could still inhibit all zikv and denv strains tested, suggesting that it potentially targeted the conserved sequences in zikv and denv ediii proteins. our data further explain the potent, broad-spectrum activity of gossypol against infections of at least two flaviviruses, including zikv and denv. development of safe and effective antiviral therapeutics is urgently needed to treat zikv infections and zikv-caused diseases, particularly zikv-associated microcephaly, fetal death, or neurological diseases [ , [ ] [ ] [ ] [ ] . here, we have identified four small-molecule-based natural products, namely gossypol, curcumin, digitonin, and conessine, with robust anti-zikv activities in vero e cells. among them, gossypol had the greatest potency to block infections of zikv for almost all strains isolated from to from nine countries and three hosts, including six recent human strains associated with congenital zika syndrome and other neurological malformations. time-of-addition-based mechanistic studies indicated that gossypol could neutralize zikv infection by targeting the virus rather than the cell or cell-associated zikv entry or replication steps. inhibition assays revealed that gossypol strongly bound to zikv e protein, particularly the conserved ediii, and blocked the binding between zikv ediii and ediii-specific neutralizing mabs, rationalizing its efficacious and broad-spectrum anti-zikv activity. it appears that the binding between gossypol and zikv e/ediii protein might be nonspecific, since gossypol is also active against other enveloped viruses, including herpes simplex virus type , parainfluenza virus type , influenza virus, and hiv- [ ] [ ] [ ] [ ] [ ] . because of the potential toxicity and side effects of gossypol to humans [ ] [ ] [ ] , it might not be a good idea to use this natural product as a drug to treat human diseases, including zikv-associated microcephaly and other flavivirus-caused diseases. nevertheless, the goal of this study is to identify the best active, natural product, then modify it to improve its antiviral activity and drug-like properties and reduce its toxicity. due to the symmetric nature of the gossypol molecule, there will be better opportunity to defragment its structure to smaller drug-like molecules with higher activity and lower toxicity. therefore, it is suggested not to use gossypol as the lead molecule, but rather the final drug. unlike gossypol, digitonin and conessine did not neutralize zikv directly, but instead inhibited zikv infection at various stages of the life cycle, including viral attachment, membrane fusion, or postentry steps. notably, digitonin is a glycoalkaloid saponin detergent. it is widely used as a cell membrane permeabilizing agent [ , ] and a detergent for selective solubilization of membrane proteins [ ] [ ] [ ] ; thus, the antiviral activity of this natural product is likely to be very nonspecific. by comparison, conessine appears to be a good candidate with a decent anti-zikv activity and very low cytotoxicity (higher selectivity). however, it is a steroidal alkaloid [ ] , which might not be ideal for further optimization. similar to gossypol, curcumin may directly neutralize zikv infection, but it seems to mainly inhibit the early stage (attachment) of virus infection, the mechanism of which is similar to that seen in a previous report using different approaches [ ] . our data have also demonstrated strong in vitro ability of these natural products-gossypol in particular-against four serotypes of denv human strains with low to no cytotoxicity, and the ic values against denv were similar to those against zikv. we have found that gossypol bound to zikv e protein, potentially ediii, suggesting that it may recognize highly conserved regions and sites in the e proteins of zikv and denv. previously, curcumin was shown to potentially inhibit denv- infection through direct effects on viral particle production or various cellular systems [ ] . we have not found studies in the literature reporting on the antiviral activity of curcumin on other serotypes of denv or the inhibitory effects of gossypol, digitonin, and conessine on zikv and other flaviviruses. thus, the present study provides a rationale for further understanding of anti-denv and anti-zikv mechanisms of these natural products and identifying their potential binding sites in the viral proteins. except for demonstrating the individual in vitro anti-zikv activity of natural products identified in this study, particularly gossypol, we have confirmed important combinatorial anti-zikv effects of gossypol in combination with other natural products. it is interesting to note that combining gossypol with curcumin, digitonin, or conessine resulted in increased combinatorial effect against prvabc strain compared to the flr strain of zikv, potentially because gossypol had more potent anti-flr activity than anti-prvabc activity when tested individually. also, such combinations enhanced the individual antiviral activity of curcumin, digitonin, and conessine against the test viruses. we have shown that the combination of gossypol and bortezomib, a licensed proteasome inhibitor previously reported inhibiting zikv infection [ , ] , led to significant combinatorial activity against the three epidemic zikv human strains tested. therefore, our study demonstrates the possibility of combining gossypol with other natural products or compounds to enhance antiviral activity. overall, this study has evaluated the antiviral activity of four natural products in vitro, among which three are newly identified natural products with strong anti-zikv ability. future observations will be needed to evaluate the protective efficacy of these natural products individually or in combination, or using their defragmented, small drug-like molecules (in the case of gossypol) against zikv-caused congenital infection and fetal demise in available animal models. modification of the structure of gossypol and identification of its derivatives with better antiviral activity, but without cytotoxicity, would be essential for developing a safe and effective anti-zikv agent for human use. also, future studies to evaluate the antiviral activity of modified gossypol or its derivatives against other flaviviruses, both in vitro and in vivo, will be helpful for identification of an effective and safe pan-flavivirus inhibitor. taken together, the broad-spectrum ability of the identified natural products, especially gossypol, against zikv and denv infections indicates the potential for further development of these small molecules or their derivatives as the lead compounds or effective anti-flavivirus inhibitors. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . figure s : association between inhibitory activity of natural products against zikv strain pan and their cytotoxicity. figure s : association between inhibitory activity of natural products against zikv strain prvabc and their cytotoxicity. figure s : multiple sequence alignment of amino acid (aa) sequences of e protein of zikv strains and denv- - human strains used in this study. author contributions: s.j., a.k.d., and l.d. designed the study. y.g., w.t., n.w., and x.l. performed the experiments and analyzed the data. y.g. and n.w. carried out sequence alignment analyses. y.g., s.j., a.k.d., l.d., and s.c. wrote and revised the manuscript. zika virus: new clinical syndromes and its emergence in the western hemisphere zika virus (i). isolations and serological specificity zika virus infection in pregnancy: maternal, fetal, and neonatal considerations implications of zika virus 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detergents specific release of membrane-bound annexin ii and cortical cytoskeletal elements by sequestration of membrane cholesterol anti-malarial property of steroidal alkaloid conessine isolated from the bark of holarrhena antidysenterica clinical use of proteasome inhibitors in the treatment of multiple myeloma this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no competing interests. key: cord- -gs ebo authors: yin, xin; feng, zongdi title: hepatitis e virus entry date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gs ebo hepatitis e virus (hev) infection is a major cause of acute hepatitis worldwide. it is transmitted enterically but replicates in the liver. recent studies indicate that hev exists in two forms: naked, nonenveloped virions that are shed into feces to mediate inter-host transmission, and membrane-cloaked, quasienveloped virions that circulate in the bloodstream to mediate virus spread within a host. both virion types are infectious, but differ in the way they infect cells. elucidating the entry mechanism for both virion types is essential to understand hev biology and pathogenesis, and is relevant to the development of treatments and preventions for hev. this review summarizes the current understanding of the cell entry mechanism for these two hev virion types. hepatitis e virus (hev) infection is a common cause of acute hepatitis worldwide [ ] . each year about million individuals are infected with hev, resulting in~ , deaths. in recent years, increased cases have been reported in developed countries. in addition to humans, hev also infects a wide range of animal species [ ] . at least six different hev genotypes have been shown to be able to infect humans. most cases in developed countries are caused by zoonotic transmission of genotype hev through consumption of contaminated pork and game meat. in addition, genotype hev infections frequently cause persistent infections in individuals with a weakened immune system resulting in an increased risk for accelerated liver cirrhosis. hev infection-related extrahepatic manifestations including various neurologic problems have also been described [ ] . despite these public health concerns, direct antivirals are not available for hev. ribavirin, a broad spectrum nucleoside analog, has been used in treating patients with chronic hepatitis e [ , ] , but resistance mutations have been described [ , ] . there is an hev vaccine available in china that offers long-term protections against clinically symptomatic acute hepatitis caused by both genotype and hev infections [ , ] . this vaccine is currently under evaluation in a phase clinical trial in the united states (https://clinicaltrials.gov/ct /show/nct ) and in a phase clinical trial in bangladesh (https://clinicaltrials.gov/ct /show/nct ). since its discovery in the s, hev has been considered as nonenveloped [ ] . while this is true for virions in the bile and feces, it is now recognized that virions circulating in the bloodstream exist in a membrane associated, "quasienveloped" form (ehev) [ , ] . ehev particles are infectious, but they do not have classic envelope proteins thus infect cells via an unusual mechanism [ , ] . elucidating the entry mechanisms for both virion types is critical for understanding the unique life cycle of hev and its pathogenesis. knowledge obtained from studying hev entry may also aid in the development of antiviral drugs, which is crucial for patients with chronic hepatitis e experiencing ribavirin failure and in severe liver disease in pregnant women from developing countries. this review will begin with a discussion about the structural differences between the two hev virion types, then discuss the current understanding regarding the entry mechanism for each virion type. hev was discovered in by dr. mikhail balayan, a russian virologist who intentionally infected himself by ingesting pooled stool samples collected from hev-infected russian soldiers deployed in afghanistan [ ] . he subsequently developed acute hepatitis. by examining his fecal material using electron microcopy (em), dr. balayan obtained the first morphologic evidence of hev virions, which appeared as nonenveloped icosahedral particles, - nm in diameter, with a "spiky" surface [ ] . due to its similar structural appearance to the calicivirus, hev was initially misclassified within the caliciviridae family. in , hev was molecularly cloned and its full genome was sequenced [ ] . the significant sequence divergence of hev from other known families of viruses led to its reclassification into a new family, hepeviridae. the . kb single-stranded positive sense hev rna genome was shown to be polyadenylated and contain three open reading frames (orfs) (figure a ). the ' two-thirds of the genome encodes orf , a large multi-domain polyprotein that is involved in the genomic replication. the rest of the genome encodes orf and orf , both of which are translated from a subgenomic rna generated during the virus replication cycle. orf is the only capsid protein of hev. the orf protein is unique to hev and plays a role in virion egress [ ] . a new orf, orf , was recently discovered within the coding region of orf , but appears to be restricted only to genotype hev [ ] . viruses , , x for peer review of the development of antiviral drugs, which is crucial for patients with chronic hepatitis e experiencing ribavirin failure and in severe liver disease in pregnant women from developing countries. this review will begin with a discussion about the structural differences between the two hev virion types, then discuss the current understanding regarding the entry mechanism for each virion type. hev was discovered in by dr. mikhail balayan, a russian virologist who intentionally infected himself by ingesting pooled stool samples collected from hev-infected russian soldiers deployed in afghanistan [ ] . he subsequently developed acute hepatitis. by examining his fecal material using electron microcopy (em), dr. balayan obtained the first morphologic evidence of hev virions, which appeared as nonenveloped icosahedral particles, - nm in diameter, with a "spiky" surface [ ] . due to its similar structural appearance to the calicivirus, hev was initially misclassified within the caliciviridae family. in , hev was molecularly cloned and its full genome was sequenced [ ] . the significant sequence divergence of hev from other known families of viruses led to its reclassification into a new family, hepeviridae. the . kb single-stranded positive sense hev rna genome was shown to be polyadenylated and contain three open reading frames (orfs) ( figure a ). the ' two-thirds of the genome encodes orf , a large multi-domain polyprotein that is involved in the genomic replication. the rest of the genome encodes orf and orf , both of which are translated from a subgenomic rna generated during the virus replication cycle. orf is the only capsid protein of hev. the orf protein is unique to hev and plays a role in virion egress [ ] . a new orf, orf , was recently discovered within the coding region of orf , but appears to be restricted only to genotype hev [ ] . the orf capsid protein of hev contains amino acids (a.a) that are divided into structural domains: a shell (s) domain, a middle (m) domain, and a protruding (p) domain comprised of a.a. - , - , and - , respectively [ ] . each virion consists of copies of an orf dimer, forming a t = icosahedron. recombinant orf proteins expressed in the bacterial and insect cells can self-assemble into virus-like particles (vlps), of which the crystal structures have been determined [ ] [ ] [ ] [ ] . p , a t = vlp composed of a.a. - and produced in e. coli, is the main component of the vaccine hecolin currently marked in china. according to the d structure, the s domains form the base of the icosahedron, and the dimeric p domains form the "spikes" extending from the virion surface that are believed to be responsible for receptor binding and a main target for neutralizing antibodies [ ] . the enteric route of transmission, em evidence of naked virions in the feces, and the lack of coding capacity for envelope proteins all suggest that hev is a nonenveloped virus. however, recent studies show that the virus released from infected cells and circulating in the blood adopts a membrane associated, "quasienveloped" form, named "ehev" [ , , ] (figure b ). these particles band at a much lower buoyant density (~ . g/cm ) in isopycnic gradient centrifugation than the the orf capsid protein of hev contains amino acids (a.a) that are divided into structural domains: a shell (s) domain, a middle (m) domain, and a protruding (p) domain comprised of a.a. - , - , and - , respectively [ ] . each virion consists of copies of an orf dimer, forming a t = icosahedron. recombinant orf proteins expressed in the bacterial and insect cells can self-assemble into virus-like particles (vlps), of which the crystal structures have been determined [ ] [ ] [ ] [ ] . p , a t = vlp composed of a.a. - and produced in e. coli, is the main component of the vaccine hecolin®currently marked in china. according to the d structure, the s domains form the base of the icosahedron, and the dimeric p domains form the "spikes" extending from the virion surface that are believed to be responsible for receptor binding and a main target for neutralizing antibodies [ ] . the enteric route of transmission, em evidence of naked virions in the feces, and the lack of coding capacity for envelope proteins all suggest that hev is a nonenveloped virus. however, recent studies show that the virus released from infected cells and circulating in the blood adopts a membrane associated, "quasienveloped" form, named "ehev" [ , , ] (figure b ). these particles band at a viruses , , of much lower buoyant density (~ . g/cm ) in isopycnic gradient centrifugation than the traditional, nonenveloped virions (~ . g/cm ) [ ] , and are insensitive to neutralizing anti-capsid antibodies in standard neutralization assays [ ] . under em, these particles look like enveloped viruses, with hev capsids encased in limiting host-derived membranes [ ] . unlike naked hev particles, the ehev particles contain both orf and orf proteins. however, both of them are hidden within the host-derived membranes and can only be detected by specific antibodies upon disruption of the membrane by detergent [ ] . ehev is the only form detected in blood [ ] , suggesting that it mediates hev spread within the host. the biogenesis of ehev has been reviewed elsewhere [ , ] . the prevalent model involves viral hijacking of the cellular endosomal sorting complex required for transport (escrt) machinery, producing an exosome-like vesicle containing the viral capsid and orf protein. usurping escrt components is a common mechanism for the budding of classic enveloped viruses [ ] . the hev orf protein plays a key role in this process. a proline-serine-alanine-proline (psap) late domain motif located at the c-terminus of orf specifically interacts with the cellular tsg (tumor susceptibility gene- ) protein, an escrt-i protein, which then recruits escrt-ii and -iii complexes to promote the budding of the hev capsid into multivesicular bodies (mvb) [ ] . subsequent fusion of mvb with the plasma membrane leads to the release of single membrane-encased ehev particles. consistent with the biogenesis pathway for exosomes, ehev membranes contain tetraspannins such as cd , cd , and cd [ , ] . certain host proteins are likely copackaged during ehev biogenesis, but their identity and roles in the hev life cycle remain largely unexplored. a recent study used a quantitative proteomic approach to identify proteins associated with quasienveloped hepatitis a virus (ehav) [ ] . it has revealed that the ehav particles are highly enriched for components of the endolysomal system such as cd , dpp (dipeptidylpeptidase , cd ), alix, and epcam (epithelial cell-adhesion molecule), but lack lc- (an autophagosomal protein)-related peptides, consistent with an endosomal exosome-like origin of ehav. this approach may be also useful for identifying ehev-associated proteins. the nonenveloped hev particles found in the feces likely originated from ehev released through the bile canaliculi, but their membrane are stripped by the detergent action of bile [ ] . the lack of lipid membrane renders the nonenveloped virions much more stable in the environment to facilitate transmission to new hosts. the production of two virion types is integral to hev biology. thus, it is important to understand how each virion type plays a role in the infection process. the available experimental evidence suggests that they enter and infect target cells through distinct pathways. the naked, nonenveloped hev particles are present in the bile and feces of infected patients or experimentally infected monkeys [ , , ] . these particles are stable in the environment and ideal for transmission to new hosts. although hev is primarily transmitted enterically, how it penetrates the gut barrier to reach the bloodstream remains enigmatic. no compelling evidence exists that hev replicates in the human gut, although it is possible that hev infects only a rare cell type in the gut, as is the case for norovirus [ ] . regardless of the origin of the first cell to be infected, whether a hepatocyte or a yet-to-be-identified cell type in the gut, naked hev is necessary for establishing the first round of infection. despite being highly hepatotropic in vivo, under in vitro conditions, hev is able to infect a range of cell types other than hepatocytes, including a (human lung epithelial cells), caco- (human colon epithelial cells), human neuronal-derived cells, and human placenta cells [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this widened cell tropism in vitro is not unique to hev, as hepatitis a virus (hav), which is known to only infect hepatocytes, can also infect many nonhepatic cell types in vitro [ ] . it should be noted that cancer cell lines often bear genetic abnormalities and altered phenotypes as compared to their corresponding primary cells in vivo, thus results obtained from such cells must always be interpreted with caution. study of the hev life cycle has been hampered by the lack of efficient cell culture systems for hev [ ] . the particle to focus-forming unit (ffu) ratio for hev ranges from to , , prohibiting the use of single particle imaging techniques to track the hev entry process. most published studies have used fluorescently labeled vlps to study the early steps of cell entry including initial cell attachment and internalization. the recent development of hev infection systems has provided the first opportunity to study the entire hev life cycle in detail [ , ] . below we summarize the current knowledge regarding the entry process of naked hev particles. the receptor for hev is unknown. however, a number of host factors have been shown to be involved in cell attachment and/or entry of naked hev. these factors are summarized below. hsgps are glycans present on the cell surface that are involved in cell attachment of many nonenveloped and enveloped viruses. hsgps, particularly syndecans, have been shown to play a role in the binding of hev vlps to human hepatoma cells [ ] . treatment of cells with heparinase reduced vlp binding and hev infection. however, hsgps are not essential for cell attachment and infection by ehev particles [ ] . grp , also known as bip (binding immunoglobulin protein), is a molecular chaperone in the er. however, presence of grp on the cell surface has also been described and implicated in the attachment and entry of both enveloped and nonenveloped viruses [ ] [ ] [ ] [ ] . grp binds to p vlps in both co-immunoprecipitation and a cell model [ ] . asgpr is a protein receptor present on the basolateral membrane of hepatocytes that binds glycoproteins that lack sialic acid modifications. a direct interaction has been shown between the ectodomain of both asgr and asgr and hev orf by coimmunoprecipitation, pull-down, and elisa [ ] . ectopic expression of asgrp in hela cells increased hev binding, whereas depletion of asgpr in plc/prf/ cells lowered hev binding but not virion release. both anti-asgpr antibody and purified asgpr ectodomain also reduced hev binding to plc/prf/ cells. atp synthase is largely a mitochondrial protein, but a small fraction is expressed on the cell surface and is implicated in other viral infections [ ] . atp b was identified as a binding partner on the p vlp using a pull down and mass spectrometric approach [ ] . the role of atp b in hev entry was validated using antibody and sirna mediated approaches, and infectious hev from the stool of a hepatitis e patient. integrin α was recently identified as an entry factor for hev in plc/prf/ cells [ ] . hev permissive and nonpermissive subclones of plc/prf/ have been identified, and compared to each other by microarray. overexpression of integrin α in nonpermissive cells rendered cells permissive for hev, while knocking out integrin α in permissive cells abrogated permissiveness. for unknown reasons, none of these subclones supported infection by ehev particles. although these results are encouraging, independent studies will be needed to validate the role of these factors in hev and ehev cell attachment and entry in the context of infection, preferably in primary human hepatocytes. using gfp-and firefly luciferase-tagged vlps and naked hev particles, kapur et al. showed that hev is internalized by cells in a clathin-, dynamin-dependent pathway [ ] . an independent study using fitc-labeled vlps subsequently confirms these results, and additionally shows that hev-like particles initially traffic to rab -positive compartments, then to acidic lysosomal compartments where they are degraded [ ] . the same study has also identified membrane cholesterol, the pi k pathway, and actin as important factors for hev internalization and infection, but low ph is not required. more recently, using naked hev purified from cell lysates, we have shown that hev infectivity is dependent on clathrin and dynamin , consistent with others. however, we found that the infectivity of naked hev is not affected by inhibiting rab and rab , or by lysosomotropic agents such as bafilomycin and ammonium chloride [ ] . these results suggest that the uncoating of naked hev particles occurs before reaching the rab + compartment, and the co-localization of capsids to rab observed by others likely represent empty capsids that remain in the endocytic pathway after genome release. as a cytoplasmically replicating rna virus, hev has to deliver its genome across the endosomal membrane to access the cytoplasm for translation and replication. how this process takes place remains poorly understood. studies with other nonenveloped rna viruses have demonstrated that upon receptor binding the virions undergo structural rearrangements to cause the externalization of hydrophobic peptides [ ] . these hydrophobic peptides subsequently insert into the endosomal membrane, creating a channel/pore through which the genome traversed the endosomal membrane to enter the cytoplasm. viral capsids remaining in the endosome are ultimately degraded in the lysosomes. since naked hev particles do not colocalize with rab and rab , hev capsids may undergo substantial conformational changes during the uncoating process. the presence of a quasienvelope raises several questions about the entry mechanism for ehev. how does ehev bind to cells and how is its cell tropism determined? does ehev entry involve membrane fusion? and does ehev have a different uncoating mechanism from naked hev? while there are no definitive answers for these questions, available evidence suggests that the ehev quasienvelope is degraded by lysosomal enzymes prior to uncoating. if this model holds true, it would present a novel entry mechanism for viruses. since quasi-enveloped hev particles do not have viral proteins on the surface of their envelope, they must use different attachment factors and/or cellular receptors to initiate the viral entry. as with exosomes, the ehev membrane has phosphatidylserine, which may bind to its receptor such as tim- on the target cells [ , ] . our previous study found that the cellular uptake of the ehev virions is less efficient than naked hev due to inefficient cell attachment, indicating that the undetermined molecules on the ehev surface mediate the cell attachment with slower kinetics [ ] . unlike the naked hev virion, the attachment of ehev to the hepatocytes does not require hspgs [ ] . the less specific cell binding by ehev may provide an explanation for the detection of hev beyond the liver [ ] . in addition, the exosome-like nature could facilitate its penetration of immunologically privileged sites such as the central nervous system. since hev infection has been associated with various types of extrahepatic manifestations [ ] , a better understanding of the tropism and replicative capacity of ehev in relevant cells/tissues will help differentiate between virus-mediated and immune-mediated effects in these conditions and shed light on hev pathogenesis. similar to naked hev particles, ehev enters hepatocytes mainly through the clathrin-and dynamin-dependent pathway [ , ] . following the endocytic uptake, ehev particles are transported sequentially into rab + and rab + endosomal compartments, and eventually reach the lysosome, where the uncoating is thought to take place [ , ] . treatment of cells with lysosomotropic agents such as bafilomycin a and nh cl dramatically reduces ehev infectivity, indicating that the endosomal acidification is required for ehev entry. however, the low ph itself is not sufficient for ehev cell entry, since extracellular exposure to low ph did not alter the density or infectivity of ehev [ ] . the study of ehav has provided additional insights into the intracellular trafficking of quasienveloped viruses [ ] . confocal imaging experiments using fluorescently labelled ehav particles suggests that intact ehav is transported to the lysosome, but similarly labelled exosomes produced by uninfected cells do not colocalize with lysosomal markers. it is speculated that the ehav membrane may contain a signal that drives its continued trafficking towards the lysosome [ ] . whether ehev uses a similar mechanism for trafficking to the lysosome is unknown, but would be an interesting area for future investigation. the presence of a quasienvelope would require the hev capsid to penetrate two layers of membranes in order to deliver the viral genome into the cytoplasm. one possible way to achieve this is through fusion of the viral membrane with the cellular membrane. however, membrane fusion would result in the intact capsid, rather than the genome, entering the cytoplasm, likely a dead end for hev. a second, more likely scenario is by removing the quasienvelope, so that the capsid will be exposed and available for binding to a receptor. lipid membrane degradation in lysosomes is a complex process [ ] . a critical step for lipid membrane degradation is the niemann-pick disease type c (npc )-mediated extraction of cholesterol from lipids. depletion of npc reduced ehev infection by % without significantly affecting hev infectivity [ ] . moreover, cells pretreated with a specific inhibitor of lysosomal acid lipase (lal), an enzyme essential in lipid metabolism because it hydrolyzes cholesteryl esters and triglycerides in lysosomes [ ] , displayed a dose-dependent reduction in ehev infectivity, while no reduction was observed for non-enveloped hev infectivity [ ] . these results provide evidence that the ehev membrane is degraded in the endolysosomes, rather than fusing with the host membrane ( figure ) . a similar requirement of npc and lal has also been described for ehav entry [ ] . once ehev loses its membrane, the capsid would be free to interact with its receptor on the endosomal membrane. at the simplest level, the capsid binds to the same receptor for naked hev and uncoat via the same mechanism. many cell surface proteins cycle between the plasma membrane and endosomes, serving as attractive candidates if this is the case. alternatively, a different receptor may be used for ehev capsids, and it is not uncommon for viruses to switch receptors during the entry process [ , ] . an unexplored question is the role of orf in ehev entry. orf has been shown to interact with orf [ ] . thus, the presence of orf may interfere with receptor binding. in addition, orf possesses an ion channel activity that is required for hev egress [ ] . it will be interesting to know whether this activity also plays a role in ehev entry. once ehev loses its membrane, the capsid would be free to interact with its receptor on the endosomal membrane. at the simplest level, the capsid binds to the same receptor for naked hev and uncoat via the same mechanism. many cell surface proteins cycle between the plasma membrane and endosomes, serving as attractive candidates if this is the case. alternatively, a different receptor may be used for ehev capsids, and it is not uncommon for viruses to switch receptors during the entry process [ , ] . model for cellular entry of naked and quasi-enveloped hev virions. naked hev virions initially bind nonspecifically to hspgs, followed by a specific interaction with a putative cell receptor. receptor binding triggers virion internalization via clathrin-mediated endocytosis and virions subsequently uncoat in a rab − early endocytic compartment. quasienveloped hev virions attach to cells less efficiently than naked virions, but are similarly internalized via clathrin-mediated endocytosis. virions are routed through early (rab +) and late (rab +) endocytic compartments and ultimately reach the lysosome. the quasi-envelope becomes slowly degraded by lysosomal enzymes, allowing the exposure of the capsid which subsequently penetrates the endosomal membrane to release its genome into the cytoplasm. the presence of two distinct virion types has challenged our view about the fundamental biology and pathogenesis of hev. the ability of hev to infect its target cells both in the presence and absence of a viral membrane is a paradigm shift in virology and an exciting area for future investigations. the available evidence suggests that naked and quasienveloped hev particles use different mechanisms for cell entry, and that entry of ehev requires lysosomal degradation of the viral membrane. identifying the cellular receptor for both virion types will be key to elucidating their entry mechanisms. since ehev is the only form detected in the bloodstream, understanding how ehev spreads has the potential for identifying targets for intervention. with the recent development of more efficient culture systems for hev, it is anticipated that our understanding of hev entry will be improved within the next few years. funding: this work is supported by grants from the national institute of allergy and infectious diseases (ai and ai ), the gilead science research scholars program in liver disease, and internal startup funds from the nationwide children's hospital (to z.f.). the authors declare no conflict of interest. hepatitis e virus: advances and challenges the current host range of hepatitis e viruses ribavirin therapy inhibits viral replication on patients with chronic hepatitis e virus infection ribavirin for chronic hepatitis e virus infection in transplant recipients a mutation in the hepatitis e virus rna polymerase promotes its replication and associates with ribavirin treatment failure in organ transplant recipients hepatitis e virus mutations associated with ribavirin treatment failure result in altered viral fitness and 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curious and complex series of events virus-induced abl and fyn kinase signals permit coxsackievirus entry through epithelial tight junctions the phosphorylated form of the orf protein of hepatitis e virus interacts with its non-glycosylated form of the major capsid protein, orf hepatitis e virus orf is a functional ion channel required for release of infectious particles key: cord- -vmmlxr o authors: zhu, yang; ma, yuanmei; lu, mijia; zhang, yu; li, anzhong; liang, xueya; li, jianrong title: efficient production of human norovirus-specific igy in egg yolks by vaccination of hens with a recombinant vesicular stomatitis virus expressing vp protein date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: vmmlxr o human norovirus (hunov) is responsible for more than % of outbreaks of acute nonbacterial gastroenteritis worldwide. despite major efforts, there are no vaccines or effective therapeutic interventions against this virus. chicken immunoglobulin y (igy)-based passive immunization has been shown to be an effective strategy to prevent and treat many enteric viral diseases. here, we developed a highly efficient bioreactor to generate high titers of hunov-specific igy in chicken yolks using a recombinant vesicular stomatitis virus expressing hunov capsid protein (rvsv-vp ) as an antigen. we first demonstrated that hunov vp protein was highly expressed in chicken cells infected by rvsv-vp . subsequently, we found that white leghorn hens immunized intramuscularly with rvsv-vp triggered a high level of hunov-specific yolk igy antibodies. the purified yolk igy was efficiently recognized by hunov virus-like particles (vlps). importantly, hunov-specific igy efficiently blocked the binding of hunov vlps to all three types (a, b, and o) of histo-blood group antigens (hbgas), the attachment factors for hunov. in addition, the receptor blocking activity of igy remained stable at temperature below °c and at ph ranging from to . thus, immunization of hens with vsv-vp could be a cost-effective and practical strategy for large-scale production of anti-hunov igy antibodies for potential use as prophylactic and therapeutic treatment against hunov infection. the virus family caliciviridae contains five established genera (norovirus, sapovirus, lagovirus, vesivirus, and nebovirus) and at least six proposed genera (recovirus, valovirus, bavovirus, nacovirus, minovirus, and salovirus) that infect many different animal species including humans. most of these agents are enteric pathogens whose replication and chief clinical manifestations are gastroenteritis and potentially life-threatening diarrhea. examples of these viruses include human norovirus (hunov), porcine norovirus, bovine norovirus, human sapovirus, porcine sapovirus, and the recently discovered tulane virus. hunov is the major food-and water-borne virus that accounts for more than % of nonbacterial acute gastroenteritis worldwide, but this percentage may be underestimated due to in this study, we developed a highly efficient bioreactor for large-scale production of chicken egg yolk igy antibodies using rvsv-vp as an antigen. we found that hunov vp was highly expressed in chicken cells infected by rvsv-vp and that hens vaccinated rvsv-vp induced a high level of hunov-specific igy in egg yolks. interestingly, intramuscular vaccination of rvsv-vp produced approximately three times more hunov-specific igy than combination of intramuscular and nasal drop vaccination route. importantly, hunov-specific igy produced by rvsv-vp vaccination was capable of blocking the binding of hunov vlps to type a, b, and o hbga receptors. in addition, egg yolk igy antibodies remained stable at temperature below • c and at ph ranging from to . these data demonstrated that recombinant rvsv-vp is a highly effective antigen for large-scale production of hunov-specific igy for passive immunization and therapeutic agent. recombinant vesicular stomatitis virus (rvsv) expressing human nov capsid protein (rvsv-vp ) was previously constructed in our laboratory [ ] . working stocks of rvsv-vp were propagated in confluent bsrt cells. briefly, bsrt cells in t flask were infected by rvsv-vp at a multiplicity of infection (moi) of . after h of absorption, the inoculum was removed, and the cells were washed twice with dulbecco's modified eagle's medium (dmem). after addition of ml fresh dmem (supplemented with % fetal bovine serum), the infected cells were incubated at • c in co incubator. when extensive cytopathic effects (cpe) were observed, cell culture fluid was harvested, and virus titer was determined by plaque assay in vero cells. thirty-five-millimeter dishes were seeded with bsrt- or df- cells (kindly provided by dr. qingzhong yu at usda ars, athens, ga) and were infected with rvsv-vp at a multiplicity of infection (moi) of . after h of absorption, the inoculum was removed, the cells were washed twice with dmem, ml of fresh dmem (supplemented with % fetal bovine serum) was added, and the infected cells were incubated at • c. at the indicated intervals, -µl aliquots of the cell culture fluid were removed and the same amount of fresh dmem was added back to the virus-infected cells. virus titers were determined by plaque assay in vero cells. six-well plates were seeded by bsrt or df- cells ( × cells per well). after h incubation at • c in co incubator the cells were infected with rvsv-vp at an moi of . at the indicated time points, cells were lysed in lysis buffer containing % β-mercaptoethanol, . % np- , and % sodium dodecyl sulfate (sds). proteins were separated by % sds-page and transferred to a hybond ecl nitrocellulose membrane (amersham, piscataway, nj, usa) in a mini trans-blot electrophoretic transfer cell (bio-rad, hercules, ca, usa). the blot was probed with guinea pig anti-human nov vp antiserum (a generous gift from dr. xi jiang) at a dilution of : , followed by horseradish peroxidase-conjugated goat anti-guinea pig igg secondary antibody (santa cruz biotechnology, inc., dallas, tx, usa) at a dilution of : , . the blot was developed with supersignal west pico chemiluminescent substrate (thermo scientific, waltham, ma, usa) and exposed to kodak biomax mr film (kodak, rochester, ny, usa). the harvested cell lysates were also subjected to western blot using anti β-actin antibody (proteintech, rosemont, il, usa), which recognizes β-actin from multiple species, including human, mouse, hamster, and chicken. the protein bands were scanned and quantified using a typhoon phosphorimager and imagequant tl software (ge healthcare, piscataway, nj, usa). the vp protein band of each time point was normalized by the respective β-actin band. the time point ( h postinfection in bsrt cells) with highest vp expression was set as %. the percentage of vp expression at other time points was normalized by the vp expression at h postinfection in bsrt cells. the animal study was conducted in strict accordance with usda regulations and the recommendations in the guide for the care and use of laboratory animals of the national institutes of health, and was approved by the ohio state university institutional animal care and use committee (animal protocol no. a ). chickens were housed in cages inside high-security isolation rooms provided with hepa-filtered intake and exhaust air at the ohio agriculture research and development center, the ohio state university. the animal care facilities at the ohio state university are aaalac accredited. before animal study, blood samples were collected from each chicken to confirm that they were negative for hunov antibody. six, -week-old, healthy white leghorn chickens were provided by dr. lilburn, department of animal sciences, the ohio state university and were randomly divided into two groups (three chickens per group). prior to the study, these chickens were negative for vsv and hunov antibody. chickens in group i were immunized intramuscularly by injecting µl of dmem containing × pfu of rvsv-vp into three different locations of the pectoral muscle. chickens in group ii were immunized by combination of intramuscular and intranasal routes. specifically, µl of rvsv-vp ( × pfu) was injected into three different locations of the pectoral muscle, and the remaining µl of rvsv-vp ( × pfu) was used for nasal drop vaccination. at week postimmunization, chickens in groups i and ii were boosted with × pfu of rvsv-vp via intramuscular and combination of intramuscular and intranasal routes, respectively. after immunization, eggs were collected daily until week post-booster vaccination. in addition, hens were observed daily for any abnormal reactions. eggs that were collected one week before immunization were used as negative control. eggs were stored at • c before igy extraction. purification of vlps from insect cells was described previously with some minor modifications [ ] . spodoptera frugiperda (sf ) cells were infected with baculovirus expressing hunov vp at an moi of , and the infected sf cells and cell culture supernatants were harvested at days postinoculation. the vlps were purified from cell culture supernatants and cell lysates by ultracentrifugation through a % (w/v) sucrose cushion, followed by cscl isopycnic gradient ( . g/cm ) ultracentrifugation. purified vlps were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) followed by coomassie blue staining. the protein concentrations of the vlps were measured by using the bradford reagent (sigma chemical co., st. louis, mo, usa). igy was extracted and purified from egg yolks using polyethylene glycol (peg , sigma, st. louis, mo, usa) precipitation method [ ] with some modifications. briefly, egg yolks were diluted in three volumes of pbs (ph . ) and mixed, and peg was added to a final concentration of . %. after vortexing, the mixture continued to roll on a rolling mixer for min. the mixtures were centrifuged at , × g for min at • c, and the precipitated debris were removed. subsequently, peg was added to the supernatant to a final concentration of . %, and the samples were mixed on a rolling mixer for min. the mixtures were centrifuged again at , × g for min at • c. the precipitated pellets containing igy were dissolved in ml of pbs and then precipitated again with % of peg using the same procedures described above. the final pellets was dissolved in . ml of pbs, filtered through a . µm filter, and stored at − • c. the purity of the igy was determined by sds-page followed by coomassie blue staining. standard elisa measured hunov-specific igy antibody titers. briefly, -well microtiter plates were coated with µl of purified hunov vlp antigen ( ng/well) and incubated overnight at • c. after blocking with % nonfat milk, times serially diluted chicken igys were added to the antigen-coated wells and incubated at • c for h. after washing with pbst (pbs containing . % tween), goat anti-chicken igy-hrp ( : ) (santa cruz biotechnology) was added for h. plates were washed and developed with µl of , ', , '-tetramethylbenzidine (tmb), and the optical density (od) at nm was determined using an enzyme-linked immunosorbent assay (elisa) plate reader. the igys from pre-immunized chicken yolks were used as controls. to calculate the amount of total igy and hunov-specific igy, a standard curve was set-up: wells were coated with µl of serially diluted pure chicken igy (promega, madison, wi, usa) at a concentration from . µg/ml to µg/ml. after washing with pbst, µl of goat anti-chicken igy-hrp (santa cruz biotechnology) at a dilution of : ) were added and incubated at • c for h. the bound hrp was colorized by substrate reagent (kirkegaard and perry laboratories, inc., gaithersburg, maryland, usa), followed by a reading of the signal intensity at nm (epoch micro-volume spectrophotometer system, biotek, winooski, vt, usa). the resulting standard curve of absorbance was used to quantify the relative concentration of total igy and hunov-specific igy from the egg yolks by coating plates with either hunov vlp or rabbit anti-chicken igy antibodies ( µg/ml, sigma) to capture the total igy or hunov-specific igy. the purified igys from egg yolks were analyzed by sds-page. samples were boiled for min in loading buffer containing % sds, . % β-mercaptoethanol, . mm tris-hcl (ph . ), and % glycerol and loaded onto a % polyacrylamide gel. proteins were visualized by coomassie blue staining. specific reactions of the chicken igy with hunov capsid protein were examined by western blot analysis. the purified hunov virus-like particles (vlps) were separated by conventional % sds-page and transferred to a hybond ecl nitrocellulose membrane (amersham) in a mini trans-blot electrophoretic transfer cell (bio-rad). after blocking with % nonfat milk, the membrane was incubated with anti-hunov-specific igy or nonspecific igy ( : ) in % nonfat milk-pbs at • c overnight, followed by horseradish peroxidase (hrp)-conjugated goat anti-chicken igy secondary antibody (santa cruz biotechnology) at a dilution of : . the blot was developed with supersignal west pico chemiluminescent substrate (thermo scientific) and exposed to kodak biomax mr film (kodak). the saliva-based hbga binding and blocking assays were performed as described previously [ , , ] . to avoid potential hunov-specific antibodies in the saliva that may interfere with the receptor-binding assay, saliva samples were boiled before being used in the assays. the boiled human saliva samples with known hbga phenotypes (a, b, or o) were diluted -fold and coated on -well microtiter plates at • c overnight. after blocking with % nonfat milk in pbs, hunov vlps were added to a final concentration of µg/ml. the bound vlps were detected by using serially diluted igys (from : to : , ), followed by the addition of hrp-conjugated goat anti-chicken igy at a dilution of : . the color was then developed by adding tetramethylbenzidine peroxidase liquid substrate (kirkegaard and perry laboratory) and stopped after min of incubation at • c by adding mol/l sulfuric acid. optical density (od) was measured at nm with the use of an epoch micro-volume spectrophotometer system (biotek, winooski, vt, usa). hbga blocking assay was performed to determine the inhibitory activity of igy against the binding of hunov vlps to the hbga antigens [ ] . the boiled human saliva samples with known hbga phenotypes (a, b, or o) were diluted -fold and coated on -well microtiter plates at • c overnight. the hunov vlps were preincubated with serially diluted igys for h at • c, and igy-vlp solutions were added to the saliva-coated wells. plates were washed times with . mol/l sodium phosphate buffer (ph, . ). then, a guinea pig anti-hunov vlp antiserum at a dilution of : was added and incubated for h at • c. the plates were washed again, and hrp-conjugated goat anti-guinea pig igg (at dilution of : ) was added and incubated for h at • c. the color was then developed by adding tetramethylbenzidine peroxidase liquid substrate (kirkegaard and perry laboratory) and stopped after min of incubation at • c by adding mol/l sulfuric acid. the blocking rates were calculated by comparing the optical densities (ods) measured with and without blocking by the chicken igys. the igys from chickens before immunization were used as controls [ ] . blank wells were incubated with buffer instead of igy-vlp and served as negative control whereas vlp binding to carbohydrates in the absence of igy sample was used as a positive control. for the ph stability, the purified total igy solution ( ml, : , ph . ) was diluted in . mol/l sodium phosphate buffer (ph . ). the ph of the solution was adjusted using either hcl or naoh to a final ph ranging from to . the solution was incubated at • c for h, followed by neutralization by adding × pbs (ph . ) to a final ph of . the hbga blocking assays were performed to measure the activity of igy, as described above. to determine heat stability of hunov-specific igy, the purified total igy solution ( ml, : , ph . ) was treated at temperature ranging from to • c for up to min. after heat treatment, the samples were cooled quickly on ice. the hbga blocking assays were performed to measure the activity of igy as described above. quantitative analysis was performed by either densitometric scanning of autoradiographs or by using a typhoon phosphorimager and imagequant tl software (ge healthcare, piscataway, nj, usa). each experiment was performed three to six times. statistical analysis was performed by one-way multiple comparisons using spss software . (spss, chicago, il, usa). a p-value of < . was considered statistically significant. vsv replicates efficiently in a wide range of mammalian cell lines. we previously showed that rvsv-vp replicated to a high titer in bsrt cells, baby hamster kidney cells [ ] . before chicken vaccination, we first determined whether rvsv-vp replicated efficiently in df- cells, which are derived from chicken embryo. briefly, bsrt and df- cells were infected with rvsv-vp at an moi of and the kinetics of viral replication was determined at time points from to h postinfection. as shown in figure a , rvsv-vp replicated efficiently in both df- and bsrt cells. viral titer gradually increased at h postinfection and reached a peak titer of . × pfu/ml at h postinfection. however, in bsrt cells, the virus titer started to increase at h postinfection, and reached a peak titer of . × at approximately h postinfection. in addition, virus titer in df- cell decreased figure c ). we next examined the kinetics of hunov vp expression in bsrt and df- cells. briefly, bsrt and df- cells were infected with rvsv-vp at an moi of and cell lysates were harvested at the indicated times. the expression of vp was determined by western blot. β-actin was loaded as an internal control. the vp expression was detectable at h postinfection in both cell lines, and reached a peak between and h postinfection ( figure b) . hunov vp was highly expressed in both bsrt and df- cells during - h postinfection. vp expression in df- decreased after h postinfection due to the cell death. quantitation of three independent experiments showed there were no significant difference in vp expression between bsrt and df- cells at time points from to h postinfection (p > . ) ( figure c ). we previously showed that rvsv-vp was not only attenuated in vitro and in vivo, but also triggered a high level of mucosal, humoral, and cellular immunities in a mouse model [ ] . since rvsv-vp can grow in chicken cells, we hypothesis that chickens will be susceptible to rvsv-vp infection and thus produce hunov-specific igy in eggs. to do this, rvsv-vp was inoculated into chickens by two routes: intramuscular or combination of intramuscular and intranasal route. after vaccination, the safety of rvsv-vp in chickens was monitored daily. no abnormal reaction was observed in chickens vaccinated by both routes. recombinant rvsv-vp vaccination did not affect feed intake and egg production. this result suggests that rvsv-vp was safe to chickens. after rvsv-vp vaccination, eggs from each hen were collected daily. to determine whether rvsv-vp triggers hunov-specific igy, total igy was purified from each egg collected at weeks , , , and postvaccination. to examine the purity of total igy, µl of total igy from one egg collected at week postvaccination was analyzed by sds-page. as shown in figure a , two protein bands with molecular weight of and kda were observed, which were consistent with the size of heavy and light chain of igy, respectively. to next determined whether purified total igys contain hunov-specific igy. purified hunov vlps (approximately kda) were separated by sds-page ( figure b ), followed by western blotting using total igy or serum igg. as a positive control, a band of kda protein was detected when anti-hunov vp serum antibody raised in guinea pig was used ( figure c ). as a negative control, no protein bands were identified in western blot using total igy from eggs prior to rvsv-vp vaccination ( figure d ). as shown in figure e , hunov vp was detected by western blot when total igy from eggs collected at week after rvsv-vp vaccination. thus, these results confirmed that rvsv-vp vaccination triggered hunov-specific igy in egg yolks. blotting using total igy or serum igg. as a positive control, a band of kda protein was detected when anti-hunov vp serum antibody raised in guinea pig was used ( figure c ). as a negative control, no protein bands were identified in western blot using total igy from eggs prior to rvsv-vp vaccination ( figure d ). as shown in figure e , hunov vp was detected by western blot when total igy from eggs collected at week after rvsv-vp vaccination. thus, these results confirmed that rvsv-vp vaccination triggered hunov-specific igy in egg yolks. we next determined the kinetics of hunov-specific igy responses following rvsv-vp vaccination. briefly, -well plates were coated with ng of purified hunov vlp antigen in each well and were reacted with serially diluted chicken igy at • c for h. after reacting with hrp labeled goat anti-chicken igy followed by addition of substrate reagent, an elisa reader read the od value at nm. subsequently, the amount of total igy and hunov-specific igy was quantified using standard curve generated by commercially available igy. as shown in figure a , the amount of hunov-specific igy gradually increased after rvsv-vp immunization. interestingly, at weeks , , and postvaccination, the levels of hunov-specific igy in intramuscular vaccination group were significantly higher than those in the combined intramuscular and nasal drop vaccination group (p < . ). at week postvaccination, hunov-specific igy in intramuscular group reached . mg/yolk whereas only . mg/yolk hunov-specific igy was detected in intramuscular and nasal drop group. using a similar method, the amount of total igy in each egg yolk was determined. as shown in figure b , there was no significant difference in total igy between the two vaccination groups (p > . ). in addition, there was no significant difference in total igy from eggs collected before and after rvsv-vp vaccination (p > . ). under our experimental condition, the amount of total igy in each yolk ranged from . ± . to . ± . mg. hunov utilizes hbgas as attachment factors. hunov vlps possess authentic receptor binding activity that can be detected by a hbga binding assay. in this assay, human saliva containing hbga receptors (types a, b, or o) or synthetic hbgas can be recognized by hunov vlps, which can be further detected by anti-hunov serum and a hrp-conjugated second antibody. therefore, we determined whether total igy derived from rvsv-vp vaccinated hens could be used as the primary antibody for the hbga binding assay. briefly, -well plates were coated with type a, b, or o human next, we calculated the percentage of hunov-specific igy in total igy in each egg yolk. as shown in figure c , the percentage of hunov-specific igy gradually increased from . % to % in the intramuscular immunization group. in contrast, the percentage of hunov-specific igy ranged from . % to % in the combined immunization group. collectively, these results showed that both immunization routes were capable of producing hunov-specific igy, and intramuscular injection alone was more effective in triggering hunov-specific igy than combination of intramuscular and nasal drop route. hunov utilizes hbgas as attachment factors. hunov vlps possess authentic receptor binding activity that can be detected by a hbga binding assay. in this assay, human saliva containing hbga receptors (types a, b, or o) or synthetic hbgas can be recognized by hunov vlps, which can be further detected by anti-hunov serum and a hrp-conjugated second antibody. therefore, we determined whether total igy derived from rvsv-vp vaccinated hens could be used as the primary antibody for the hbga binding assay. briefly, -well plates were coated with type a, b, or o human saliva, and ng of hunov vlps were added to bind the hbga receptor. after -h incubation, a serial dilution of total igy was added followed by addition of hrp-conjugated goat anti-chicken igy. after the addition of substrate reagent, an elisa reader measured od . purified total igy from rvsv-vp vaccinated groups strongly reacted with hunov vlps in the saliva-based hbga binding assays ( figure a,b) . in contrast, control igy from pre-immunized hens was negative in this assay. overall, total igy from intramuscular vaccination group had significantly higher od values than those in combined vaccination group (p < . , compare the od value in figure a ,b). in addition, it appears that vlps had a stronger binding activity to type a saliva compared to types b and o saliva ( figure a,b) . these results demonstrated that purified total igy induced by rvsv-vp vaccination could be used as a primary antibody to measure binding of hunov vlp to all three types of saliva in hbga binding assays. saliva, and ng of hunov vlps were added to bind the hbga receptor. after -h incubation, a serial dilution of total igy was added followed by addition of hrp-conjugated goat anti-chicken igy. after the addition of substrate reagent, an elisa reader measured od . purified total igy from rvsv-vp vaccinated groups strongly reacted with hunov vlps in the saliva-based hbga binding assays ( figure a,b) . in contrast, control igy from pre-immunized hens was negative in this assay. overall, total igy from intramuscular vaccination group had significantly higher od values than those in combined vaccination group (p < . , compare the od value in figure a ,b). in addition, it appears that vlps had a stronger binding activity to type a saliva compared to types b and o saliva ( figure a,b) . these results demonstrated that purified total igy induced by rvsv-vp vaccination could be used as a primary antibody to measure binding of hunov vlp to all three types of saliva in hbga binding assays. next, we determined whether the purified igy has potential antiviral activity that can be developed as a therapeutic agent against hunov infection. recently, a hbga blocking assay, which measures the ability of antibody blocks the binding of hunov vlps to the attachment factors (hbgas), has been developed [ , ] . presumably, blockage of viral receptor binding activity will inhibit viral attachment, entry, and subsequent viral infection. thus, this assay can serve as a useful surrogate assay for serum-virus neutralization assay. briefly, -well plates were coated with a known saliva type (a, b, or o) at °c overnight. the vlps were preincubated with the serially diluted igy at °c for h before adding to the saliva-coated wells. then, a guinea pig anti-hunov vlp antiserum was added, followed by the addition of hrp-conjugated goat anti-guinea pig igg. after the addition of substrate reagent, od was measured by an elisa reader. percent of blocking activity was calculated by comparing the od values measured with or without blocking by the chicken igys. as shown in figure , total igy antibodies isolated from egg yolks of rvsv-vp vaccinated groups were capable of blocking the binding of hunov vlp to hbgas (a, b, or o next, we determined whether the purified igy has potential antiviral activity that can be developed as a therapeutic agent against hunov infection. recently, a hbga blocking assay, which measures the ability of antibody blocks the binding of hunov vlps to the attachment factors (hbgas), has been developed [ , ] . presumably, blockage of viral receptor binding activity will inhibit viral attachment, entry, and subsequent viral infection. thus, this assay can serve as a useful surrogate assay for serum-virus neutralization assay. briefly, -well plates were coated with a known saliva type (a, b, or o) at • c overnight. the vlps were preincubated with the serially diluted igy at • c for h before adding to the saliva-coated wells. then, a guinea pig anti-hunov vlp antiserum was added, followed by the addition of hrp-conjugated goat anti-guinea pig igg. after the addition of substrate reagent, od was measured by an elisa reader. percent of blocking activity was calculated by comparing the od values measured with or without blocking by the chicken igys. as shown in figure , total igy antibodies isolated from egg yolks of rvsv-vp vaccinated groups were capable of blocking the binding of hunov vlp to hbgas (a, b, or o antigen) in a dose-dependent manner with a bt (a serum dilution with % blocking activity) of approximately : , : , and : , respectively. as controls, igy purified from egg yolks of chickens before immunization did not have detectable blocking activity. again, total igy from intramuscular group had a significantly higher blocking activity compared to the intramuscular and nasal drop group (p < . ). therefore, igy from rvsv-vp vaccinated hens specifically blocked the binding of hunov vlps to the hbgas. nasal drop group (p < . ). therefore, igy from rvsv-vp vaccinated hens specifically blocked the binding of hunov vlps to the hbgas. to investigate thermal stability of igy, igy solution was incubated at temperature ranging from to °c for up to min, and the receptor blocking activity was measured by the saliva-based hbga blocking assay. as shown in figure a -c, hunov-specific igys retained wildtype level of to investigate thermal stability of igy, igy solution was incubated at temperature ranging from to • c for up to min, and the receptor blocking activity was measured by the saliva-based hbga blocking assay. as shown in figure a -c, hunov-specific igys retained wildtype level of blocking ability to all three types of saliva at the temperatures below • c. however, the blocking activity significantly impaired at the temperatures above • c (p < . ). blocking ability to all three types of saliva at the temperatures below °c. however, the blocking activity significantly impaired at the temperatures above °c (p < . ). next, we determined whether hunov-specific igy is stable in acid and alkaline environments. to do this, purified igy was diluted times in pbs solution and the ph of the solutions was adjusted with either hcl or naoh to a final ph of - . after incubation at °c for h, the solution next, we determined whether hunov-specific igy is stable in acid and alkaline environments. to do this, purified igy was diluted times in pbs solution and the ph of the solutions was adjusted with either hcl or naoh to a final ph of - . after incubation at • c for h, the solution was adjusted to neutral ph, and the receptor blocking activity was measured by hbga blocking assay. as shown in figure a -c, the blocking activity of the chicken igy remained stable at ph - for h. however, a significant decrease of blocking activity was observed when the ph is lower than or higher than . viruses , , x for peer review of assay. as shown in figure a -c, the blocking activity of the chicken igy remained stable at ph - for h. however, a significant decrease of blocking activity was observed when the ph is lower than or higher than . in this study, we developed a highly efficient bioreactor to produce hunov-specific igy in egg yolks by immunization of white leghorn chickens with recombinant rvsv-vp . we found that intramuscular immunization alone was more efficient in triggering hunov-specific igy compared in this study, we developed a highly efficient bioreactor to produce hunov-specific igy in egg yolks by immunization of white leghorn chickens with recombinant rvsv-vp . we found that intramuscular immunization alone was more efficient in triggering hunov-specific igy compared to the combination of intramuscular and nasal drop immunization in hens. we demonstrated that igy antibodies strongly reacted with hunov vlps in elisa, western blot, and hbga binding assays. the igy antibodies were capable of blocking hunov-hbga receptor interactions, and remained stable in temperature below • c and at ph ranging from to . our results suggest that chicken egg yolk igy antibodies (igy) is a promising prophylactic and therapeutic agent for hunov. replication competent rvsv-vp is an excellent antigen for production of hunov-specific igy in chickens. the natural hosts of vsv are cattle, horse, deer, and pig. however, vsv has a broad tissue tropism and can replicate to a high titer in many mammalian cell lines, insect cell, yeast, worm, and c. elegans [ , ] . in this study, we found that rvsv-vp replicated to a high titer in df- cells, a continuous cell line derived from chicken embryo fibroblasts. the virus yields in df- cells were comparable to those produced in bsrt cell. in addition, rvsv-vp produced similar amounts of vp protein in both df- and bsrt cells. prior to our study, replication capability of vsv in avian species in vivo was poorly understood. the only report of vsv infection in chickens came from the study of rvsv-based influenza virus vaccine. it was found that chickens vaccinated with rvsv expressing the ha antigen of highly pathogenic avian influenza virus (h n ) triggered a high level of serum neutralizing antibody and provided complete protection against lethal challenge of avian influenza h n [ ] . in our study, we found that hens vaccinated with rvsv-vp triggered a high level of hunov-specific igy in yolks, further supporting that chickens were susceptible to vsv infection. we also showed that immunization route affected igy production in hens. we found that intramuscular vaccination triggered a higher hunov-specific igy than combination of intramuscular and nasal drop vaccination although the total igy levels were similar between two vaccination routes. our rationale to include nasal drop vaccination is that it may trigger a higher mucosal immunity since egg yolk is developed in reproductive tract of chickens. in fact, our previous mice study showed that oral and intranasal vaccination of rvsv-vp trigged a high level of both serum and mucosal antibody [ , ] . one of the major concerns of rvsv-based vaccine is the safety, particularly since vsv is neurotropic. vsv infection in ruminant animal causes vesicular lesions in the mouth, teats, and feet. in mice, wild type vsv can cause acute brain infection and fatal encephalitis [ ] . however, insertion of hunov vp into vsv vector significantly attenuated the virus in a mouse model [ , ] . in this study, rvsv-vp did not cause any abnormal reactions, feed intake, or egg production of chickens, which further proved that rvsv-vp was attenuated in vivo. although two recent reports showed that hunov-specific igy can be induced in hen vaccinated with purified vlps or p particles from insect cells using a baculovirus expression system [ , ] , our study represents the first report of using a live attenuated recombinant virus to generate hunov-specific igy. there are many potential advantages of using live attenuated rvsv-vp for igy production. first, rvsv-vp grows to a high titer in a wide range of cell lines including chicken cells. second, replication of rvsv-vp in chickens resulted in synthesis of large amount of vlps that in turn triggered a high level of hunov antibody. third, it is an economical and time-saving approach. it does not require purification of vlps or p particles. thus, rvsv-vp is a promising vaccine antigen for large-scale production of igy in chickens. hunov-specific igy is a potential passive immunization and therapeutic agent for hunov. hunov is a leading cause of viral gastroenteritis worldwide. despite significant social, health, and economical burden it causes, no fda-approved vaccine or therapeutic strategy is available. epidemiology studies showed that hunov could cause lethal infection in humans, particularly in high-risk populations, such as infants, young children, the elderly, and immunocompromised individuals. thus, there is a need to develop a safe and effective therapeutic strategy. we found that hunov-specific igys isolated from egg yolks were biologically functional in vitro. first, hunov-specific igys can react with vlps in elisa and western blot. second, similar to serum igg, hunov-specific igys can be used as a primary antibody in hbga binding assay. third, hunov-specific igys, but not the control igy, were capable of blocking the binding of hunov vlps to type a, b, and o hbgas in human saliva. although it is unknown whether hunov-specific igy can directly neutralize the infectious hunov, blockage of virus-receptor interaction will likely block the infectivity of hunov, which will prevent hunov infection and illness. in , reeck et al. found a direct correlation between the ability of an antibody to block vlp-hbga binding and protection against hunov infection and illness in a hunov human challenge study [ ] . in addition, nurminen et al., ( ) showed that children could be protected from a gii. hunov infection due to the pre-existing hbga blocking antibodies [ ] . thus, the igys is a promising passive immunization approach to prevent and treat hunov infection and illness. future study will determine whether igy can directly neutralize infectious hunov using the recently developed b cell culture system [ ] or human enteroids culture system [ ] . the concept of igy passive immunization has been developed in rotavirus in vivo animal models. it was found that passive immunization of igy could protect neonatal calves from bovine rotavirus -induced diarrhea [ ] . in a mouse model, it was found that igy could prevent murine rotavirus infection [ ] , bovine rotavirus-induced diarrhea [ ] , and human rotavirus-induced gastroenteritis [ ] . recently, human rotavirus-specific igy administered orally as a milk supplement passively protects neonatal pigs against an enteric human rotavirus infection [ ] . porcine epidemic diarrhea virus (pedv) is an enteric coronavirus which causes severe diarrhea, vomiting, and mortality in young piglets. kweon et al. ( ) found that igy passive immunization can protect piglets against pedv infection [ ] . future experiments will investigate whether hunov-specific igy can protect gnotobiotic piglets from hunov-induced gastroenteritis and viral shedding. chicken as a "factory" for large-scale production of antigen-specific igy. antibody-based passive immunization and therapy has been shown to be an effective strategy to prevent infectious diseases in many animals [ ] . however, preparation of serum antibody from mammals is expensive and time-consuming. thus, large-scale application of serum antibody has been limited. igy egg yolk immunoglobulins derived from hyperimmunized hens represent an economically feasible and practical strategy which has been explored for the passive treatment of enteric diseases. chicken igy production is a much easier, faster, and cheaper method for polyclonal antibody production than from any other sources. it is easy to raise chickens and collect eggs without involvement of any stressful procedures (such as bleeding). white leghorn chickens are highly productive in laying eggs, and they can continuously produce eggs containing antigen-specific antibodies in their yolks for a long time period after immunization [ ] . nguyen et al. ( ) demonstrated that chicken usually lays eggs/year and each egg yolk normally contains - mg of igy, which has to % antigen-specific antibodies [ ] . in addition, extraction of antibody from egg yolk is simple and noninvasive without affecting the immunized chickens. therefore, a chicken is an excellent "factory" for igy antibody production. in order to be used as immunological supplements in infant formulas and other foods, it is important to investigate the stability of igy during storage or following processing methods, involving thermal treatments, such as pasteurization, sterilization, or spray-drying. based on the hbga blocking assay, hunov-specific igy remains stable at temperature below • c. thus, it is safe for pasteurization (below • c) of igy for human consumption. however, the receptor blocking activity of igy significantly decreased when temperature reached above • c, suggesting that igy may be denatured at this temperature. for oral administration, igy should ideally be stable in acid or alkaline environment. our results showed that the receptor blocking activity of igy decreased at ph below or above . since the stomach ph is~ - , it may be necessary to encapsulate the igy in acid resistant capsules so that it can be released in intestines for neutralizing the infectious virus particles. for example, chang et al. ( ) demonstrated that addition of sugars, glycerol, or glycine to immunoglobulin solutions was effective to protect igys. in addition, film coating with gum arabic was proven to be effective in reducing the degree of hydrolysis of igy [ ] . in summary, we have developed a highly efficient bioreactor to generate a high titer of hunov-specific igy by vaccination of hens with rvsv-vp . hunov-specific igy was biologically active in capturing hunov antigen and blocking the interaction between vlps and hbgas. this study will facilitate the large-scale production and purification of hunov-specific igy for virus detection, diagnosis, passive immunization, and therapy. noroviruses everywhere: has something changed? foodborne viruses: an emerging problem food-related illness and death in the united states human noroviruses: recent advances in a -year history foodborne illness acquired in the united states-major pathogens winter vomiting disease inactivation of caliciviruses enteric bacteria 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gastroenteritis using immunoglobulin igy antibodies protect against human rotavirus induced diarrhea in the neonatal gnotobiotic piglet disease model immunoprophylactic effect of chicken egg yolk immunoglobulin (ig y) against porcine epidemic diarrhea virus (pedv) in piglets prophylactic and therapeutic efficacy of avian antibodies against influenza virus h n and h n in mice productivity and some properties of immunoglobulin specific against streptococcus mutans serotype c in chicken egg yolk (igy) this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the ohio state university owns the patent on recombinant vesicular stomatitis virus expressing vp of human norovirus (rvsv-vp ) (patent no. ). we are grateful to members of the j. li laboratory for critically reading the manuscript. we thank xi jiang's laboratory at cincinnati children's hospital for providing human norovirus antibody and saliva samples with known hbga phenotypes for this study. we thank michael lilburn for providing hens for this study. the authors declare no conflicts of interest. key: cord- -lg gvgwt authors: zhou, shaochuan; ge, xinna; kong, can; liu, teng; liu, aijing; gao, peng; song, jiangwei; zhou, lei; guo, xin; han, jun; yang, hanchun title: characterizing the prrsv nsp deubiquitinase reveals dispensability of cis-activity for replication and a link of nsp to inflammation induction date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: lg gvgwt the papain-like cysteine protease (plp ) within the n-terminus of the porcine reproductive and respiratory syndrome virus (prrsv) nsp replicase protein specifies a deubiquitinating enzyme (dub), but its biochemical properties and the role in infection have remained poorly defined. by using in vitro assays, we found that the purified plp could efficiently cleave k and k linked polyubiquitin chains ub - in vitro although displaying a differential activity in converting the respective ubiquitin dimers to monomer. the subsequent mutagenesis analyses revealed that the requirement for plp dub activity surprisingly resembled that for cis-cleavage activity, as several mutations (e.g., d r, d r, etc.) that largely ablated the dub function also blocked the cis- but not trans-proteolytic cleavage of nsp / polyprotein. moreover, the analyses identified key mutations that could differentiate dub from plp cis- and trans-cleavage activities. further reverse genetics analyses revealed the following findings: (i) mutations that largely blocked the dub activity were all lethal to the virus, (ii) a point mutation t g that selectively blocked the cis-cleavage activity of plp did not affect viral viability in cell culture, and (iii) an e q mutation that did not affect either of the plp activities led to rescue of wt-like virus but displayed significantly reduced ability to induce tnf-α production. our findings support the possibility that the plp dub activity, but not cis-cleavage activity, is essential for prrsv replication. the data also establish a strong link of nsp to pro-inflammatory cytokine induction during infection that operates in a manner independent of plp dub activity. protein ubiquitination is an important post-translational modification that regulates a variety of cellular biological processes [ ] [ ] [ ] , i.e., signaling transduction [ ] , protein turnover [ ] , cell cycle progression [ ] , and the immune and inflammatory responses [ , , ] , etc. this modification, however, can often be reversed by deubiquitinases (dubs) through removing various ubiquitin molecules from substrates [ ] . not surprisingly, many dna and rna viruses have evolved to encode dubs to manipulate diverse cellular pathways [ ] . the currently discovered viral dubs largely resemble their cellular counterparts in structure and generally fall into two major classes: ubiquitin-specific preventing it from efficient affinity purification in large scale ( figure b , lane ). in contrast, plp ( - )-strepii was well expressed, and a substantial amount was presented in the supernatants ( figure b, lane ) . moreover, this fragment in the sonicated supernatants could be subsequently purified to homogeneity by one-step affinity purification ( figure b , lane ) with a yield of - mg per ml culture. thus, we have successfully developed a strategy to realize high-level soluble expression of prrsv plp . the dub activity of purified plp was subsequently investigated in a series of in vitro assays. we first tested its ability to hydrolyze ub-amc, an ubiquitin conjugated aminomethylcoumarin fluorophore (amc). by monitoring the release of amc, the dub activity was measured. as shown in figure a , the purified plp exhibited dub property in vitro with better activity achieved at a higher concentration ( μg). next, we examined the cleavage of a specific type of polyubiquitin chains ( figure b and c) . overall, when the total amount of processed ub monomer and dimers (ub+ub ) more recently, prrsv plp was shown to possess deubiquitinating activity in transfected ft cells and can antagonize interferon signaling through inhibiting activation of nf-κb [ , , ] , a feature that is similar to the counterparts from eav and nairoviruses of the family of bunyaviridae [ , , ] . further, deaton et al. have reported the dub activity of prrsv plp by in vitro assay and found that the e. coli purified recombinant plp (aa. - ) is able to cleave both k and k poly-ubiquitin [ ] . on the other hand, although prrsv plp was shown to have deisgylation activity in transfected human cells [ ] , the recombinant plp showed little in vitro deisgylating activity toward isg of porcine origin [ , ] , leaving in question whether it can actually efficiently cleave swine isg conjugates in primary macrophages. in any case, it is clear now that the prrsv plp possesses at least cis-, trans-cleavage, and dub activities [ , , ] . however, their biochemical properties and the contribution to viral infection have remained poorly defined. in this study, we started with characterizing the plp dub activity of hp-prrsv strain jxwn by using in vitro and cell-based assays. by using site-directed mutagenesis, we were able to identify mutations that can differentiate the dub activity from cisand trans-cleavage activities and assess their roles in the context of prrsv infection by reverse genetics analysis. our results revealed novel biochemical aspects of prrsv plp and showed that the plp dub activity, but not the cis-cleavage activity, is likely important for prrsv replication. unexpectedly, we also revealed a strong link of prrsv nsp to virus-induced inflammation that occurs in a dub-independent manner. the findings further advance our understanding of prrsv nsp function and it is the regulation of host immunity and has implications in antiviral drug and vaccine development. marc- and hek ft cells were maintained in dulbecco's modified eagle's medium (dmem) (invitrogen, carlsbad, ca, usa) with % fbs (invitrogen, ca, usa). primary porcine pulmonary alveolar macrophages (pams) were obtained from specific-pathogen-free (spf) pigs as previously described [ ] and cultured in rpmi medium (invitrogen, ca, usa) containing % fetal bovine serum (fbs) (invitrogen, ca, usa). the chinese highly pathogenic prrsv strain jxwn infectious cdna clone was described previously [ ] . mouse anti-flag monoclonal antibody (f ), mouse anti-ha monoclonal antibody (h ), mouse anti-β-actin monoclonal antibody (a ), and rabbit anti-myc polyclonal antibody (c ) were all purchased from sigma-aldrich (st. louis, mo, usa). horseradish peroxidase (hrp)-conjugated goat anti-mouse polyclonal antibodies and hrp-conjugated goat anti-rabbit polyclonal antibodies were purchased from zsgb-bio (beijing, china). all the restriction enzymes used in this study were from new england biolabs inc. (beverly, ma, usa). the prokaryotic expression plasmids expressing plp ( - )-strepii and plp ( - )-strep ii were constructed by cloning the respective region coding for nsp aa. - and aa. - from hp-prrsv strain jxwn (accession number: ef ) into the vector pet- a (novagen, madison, wi, usa) at the sites of nco i and xho i with a strep ii tag attached to c-terminus for purification purpose. the mammalian plasmid expressing plp ( - )-myc was made by cloning the same plp sequence into the vector pegfp-n (clonetech, ca, usa) at the sites of bamh i and xho i in frame with c-myc epitope tag coding sequence. the plasmids pnsp -myc, pflag-ha-nsp - , and pflag-ha-nsp - ∆ - were generated by fusion expression of the indicated epitope tags with the corresponding coding region for nsp , nsp - or nsp - lacking the first amino acids in the vector pegfp-n (clonetech, mountain view, ca, usa) at the sites of bgl ii and bamh i. all genes in the created eukaryotic plasmids are under the control of cmv promoter, and a kozak core sequence is also included to allow optimal expression. for expression of the derivatives of plp , nsp , or nsp - , the point mutation mutants were created by quikchange site-directed mutagenesis. all the constructs were generated by standard recombinant dna procedure and verified by dna sequencing. bacterial strain bl (de ) containing the plasmid for plp ( - )-strepii or its derivative was cultured overnight at • c and then inoculated at : into ml of yeast extract-tryptone medium culture ( xyt). when the bacterial density at nm reached . to . , protein expression was induced • c for h by addition of iptg (isopropyl-d- -thiogalactopyranoside) (sigma) at the final concentration . mm. the cells were harvested by centrifugation at rpm for - min and then resuspended in pbs with the protease inhibitor cocktail (sigma, p ). the cells were sonicated and lysed for min at • c on ice with % triton x- (sigma, t ). the cell lysates were cleared by centrifugation at , rpm for - min. the supernatants were incubated with ul streptactin sepharose (ge healthcare, - - ) at • c overnight on the rocker. the streptactin sepharose beads were collected by centrifugation and washed six times with tris buffer ( mm tris, ph . ). the target protein was eluted with ul elution buffer ( mm tris, . mm desthiobiotin, % glycerol, ph . ). the dub activity of purified prrsv plp was assayed in µl reaction containing indicated amount of purified plp protein, µm ub-amc (boston biochem, u- ), and × reaction buffer ( mm nacl, . mm kcl, . mm na hpo , . mm kh po , and mm dtt) in a -well black microplate. the fluorescent intensity (excitation, nm, emission, nm) of each well was observed by infinite m pro plate reader (tecan, inc). the dub activity of purified prrsv plp was assayed in µl reaction containing indicated amount of purified plp protein, . µg k -(boston biochem, uc- ) or k -linked polyubiquitin chains ub - (boston biochem, uc- ), and × reaction buffer ( mm nacl, . mm kcl, . mm na hpo , . mm kh po , and mm dtt) in a µl volume ep tube. the mixture was incubated at • c for h and then subject to sds-page analysis on % tris-hcl gel. hek t cells grown to %- % confluence in six-well plates were transfected plasmids encoding ha-ubiquitin or plp -myc or nsp , nsp - or derivatives either singly or in combination via lipofectamine plus (invitrogen, ca, usa). at h post-transfection, the cells were treated with mg- (selleck, s ) at µg/ml for h before collection. the cells were washed by pbs for twice and lysed by ripa buffer on the shaker at • c for min. the cell lysates were subject to sds-page and western blot analyses with antibodies to ha or myc epitope. the amount of actin served as a loading control. for the trans-cleavage assay, hek t cells were transfected to express plp -myc, or nsp d n, or its derivatives together with the substrate nsp - ∆ - or nsp - ∆ - g g via lipofectamine plus (invitrogen, ca, usa). for cis-cleavage assay, plasmids coding for nsp - or its derivatives were transfected singly into t cells. at h post-transfection, the expression of enzyme plp or nsp was subject to western blot analysis by using the cell lysates, whereas the cleavage of substrate was analyzed by immunoprecipitation coupled with western blot. briefly, the cells were rinsed with cold pbs and lysed with ripa buffer. the cell debris was removed by centrifugation at , rpm for min and the supernatants were precleared with the protein a/g agarose (santa cruz, ca, usa) on the shaker at • c for min. after that, the cleared supernatants were incubated with ug antibodies to flag epitope and ul protein a/g agarose (santa cruz, ca, usa) at • c overnight. the immunocomplexes were washed for thrice by cold pbs, and the proteins were separated on sds-page and transferred to pvdf membrane. for western blot analysis, the membrane was blocked by pbst with % skimmed milk for h at room temperature and incubated with indicated primary antibody (antibody to ha epitope) at • c overnight. after being washed for thrice by pbst, the membrane was incubated with indicated secondary antibody for h at room temperature. after being washed three times, the membrane was developed with ecl western blot analysis system. for mutagenesis of the infectious clone of hp-prrsv strain jxwn (ef ), the mutations were first introduced into shuttle plasmid peasy-blunt (transgen, beijing, china) contain the fragment a [ ] by site-directed quikchange mutagenesis. after verification by sequencing, digested fragment a was transferred to the infectious clone backbone as described previously [ ] . marc- cells grown on six-well plates were transfected with plasmids of either wt or prrsv mutants. the virus-induced cpe was monitored daily and the cell culture was harvested at - days post-transfection. the cell lysates were passaged blindly onto fresh monolayers for three passages. the viability of mutants was determined by rt-pcr targeting orf and immunofluorescence to n protein. the rescued viruses of passage were titrated on marc- cells. for growth kinetics analysis, marc- cells or primary pams in six-well plates were infected with plp mutants and parental virus at an moi of . . for growth on marc- cells, after h of incubation at • c, the cells were first washed with acid buffer ( mm nacl, mm kcl, mm citric acid, ph . ), followed by once rinse with dmem. at indicated time points of infection, the whole culture was harvested and freeze-thawed three times to release cell-associated viruses. samples were then titrated on marc- cells by using the standard endpoint dilution assay according to the reed-muench method [ ] . primary pams in -well plates were infected with nsp mutants or parental virus at an moi of . . at h post-infection, total rnas were extracted from lysates of infected pams with trizol reagent (invitrogen, ca, usa) according to manufacturer's protocols and dissolved into rnase free water. the concentration of rna in samples was measured with a nanodrop lite spectrophotometer (thermol scientific, usa). µg of total rnas per sample was used for cdna synthesis by fastquant rt kit (tiangen, beijing, china). we conducted qpcr assays using real-time sybr master mix kit (applied biosystem, usa) on an ab real-time pcr system (applied biosystem, usa). primary pams in -well plates were infected with nsp mutants or parental virus at indicated moi. at indicated time points post-infection, tnf-α in cell culture supernatants was quantified using a commercial sandwich enzyme-linked immunosorbent assay (elisa) according to the manufacturer's protocol (cusabio, wuhan, china). the i-tasser online service tool was used to model the structures of prrsv strain jxwn plp [ ] . the plp structures were referred to eav plp ( ium) [ ] , and then its interaction with ubiquitin was analyzed by pymol and coot software. the in vitro cleavage efficiency of k and k -linked polyubiquitin chains was expressed as the ratio of the amount of ub plus ub over total ub (ub+ub +ub +ub +ub +ub +ub ) by measuring the band density in each lance of sds-page images. the ratio of ub over ub was calculated by band density of ub over ub ). the relative dub activity of plp mutants was measured by the corresponding band density of polyubiquitin conjugates and then normalized against actin and expression level of plp . all the data are shown as mean± sem with independent experiments. statistical significance was evaluated by using a two-way analysis of variance (anova). statistical analyses were performed using graphpad prism software (version . ). the quantitation of each protein band is measured by image j software (version . . ). flanking sequence is critical for the yield and solubility of prrsv plp protease domain in e. coli the n-terminal residues of prrsv jxwn nsp was recently reported to exhibit dub activity when expressed and purified from e. coli bl cells [ ] . this fragment, however, was expressed at a very low level in our hands, preventing further efficient purification. since the downstream flanking sequence (nsp aa. - ) is critical for prrsv nsp function during infection [ ] , we hypothesized that this region might be critical for the folding of plp domain, and if so, a c-terminal extension might improve the solubility and yield of plp . accordingly, we made two additional constructs to include prrsv strain jxwn nsp region aa. - and aa. - ( figure b ). these proteins were tagged with a strep ii epitope tag at the c-terminus to facilitate purification. when expressed in e. coli bl cells, plp ( - )-strep ii mostly existed in the pellet, preventing it from efficient affinity purification in large scale ( figure b , lane ). in contrast, plp ( - )-strepii was well expressed, and a substantial amount was presented in the supernatants ( figure b the dub activity of purified plp was subsequently investigated in a series of in vitro assays. we first tested its ability to hydrolyze ub-amc, an ubiquitin conjugated aminomethylcoumarin fluorophore (amc). by monitoring the release of amc, the dub activity was measured. as shown in figure a , the purified plp exhibited dub property in vitro with better activity achieved at a higher concentration ( µg). next, we examined the cleavage of a specific type of polyubiquitin chains ( figure b ,c). overall, when the total amount of processed ub monomer and dimers (ub+ub ) was calculated, prrsv plp exhibited similar efficiency in cleaving both k -and k -linked polyubiquitin chains ub - (% cleavage efficiency = ub +ub /ub - ) ( figure b ,c). however, prrsv plp display a differential activity converting k and k linked ubiquitin dimers into monomers. the results showed that the cleavage into monomer of k -linked ub - was relatively inefficient ( figure c ) with the reaction taking place in a dose and time-dependent manner ( figure c ). for example, at a lower dose of plp ( µg), k -linked ub - was mainly cleaved into ub dimers ( figure c , lane ), even if the incubation time was extended to two hours ( figure c , lane ). however, when the dose increased ( µg), plp could hydrolyze k ub - efficiently into monomers within h ( figure c , lane ). the difference in kinetics toward cleaving k versus k ub - was further investigated in a more detailed time-dependent manner from to min at a concentration of µg for plp . the result showed that plp was able to quickly cleave both k and k ub - within minutes ( figure d ,e), but the conversion from ubiquitin dimer to monomer was several fold faster for plp k dub activity ( figure d -f). to identify critical residues that are potentially important for plp dub activity, we modeled the structure of plp core domain with the online program i-tasser [ ] by using eav plp (pdb id: ium) as a model. the homology remodeling revealed that prrsv strain jxwn plp (aa. - ) has an overall similar structure to that of the counterpart eav plp , except for two minor structural differences as indicated by the red arrows ( figure a ). to identify critical residues that are potentially important for plp dub activity, we modeled the structure of plp core domain with the online program i-tasser [ ] by using eav plp (pdb id: ium) as a model. the homology remodeling revealed that prrsv strain jxwn plp (aa. - ) has an overall similar structure to that of the counterpart eav plp , except for two minor structural differences as indicated by the red arrows ( figure a) . the bioinformatics analysis also identified an acidic cluster (d dwatded ) that has potential contact with the positively charged c-terminus of ubiquitin ( figure b ). in particular, the ub residues r and r form a positively charged surface, whereas prrsv plp residue d , d , d , e and d form a negatively charged region. the modeling showed that the charged cterminus is inserted into a grove formed by negatively charged residues d , e , and d of plp ( figure b ). further, a closer look into the contact sites revealed that both plp d and d can potentially interact with r and r respectively through potential hydrogen bond and electrostatic interactions ( figure c ). in contrast, d and d are in a position that is far away from ub cterminus ( figure c ). the bioinformatics analysis also identified an acidic cluster (d dwatded ) that has potential contact with the positively charged c-terminus of ubiquitin ( figure b ). in particular, the ub residues r and r form a positively charged surface, whereas prrsv plp residue d , d , d , e and d form a negatively charged region. the modeling showed that the charged c-terminus is inserted into a grove formed by negatively charged residues d , e , and d of plp ( figure b ). further, a closer look into the contact sites revealed that both plp d and d can potentially interact with r and r respectively through potential hydrogen bond and electrostatic interactions ( figure c ). in contrast, d and d are in a position that is far away from ub c-terminus ( figure c ). for this identified acidic cluster, the residues d , d , w , a , d , and d are highly conserved among various prrsv strains, whereas the residues t and e are highly conserved among type ii prrsv strains but are replaced with the respective residues s and y in type i prrsv strains ( figure s ) [ ] . among these residues, both w and d have been shown to be critical for the trans-cleavage of nsp / [ ] . thus, we, therefore, selected other residues (d , d , t , e , d ) for further mutational analysis with a purpose to distinguish dub from its trans-cleavage activity. the acidic residues were changed to either the residue asparagine (n) or to the opposite charge residue arginine (r), whereas t was replaced with either serine (s), glycine (g), or arginine (r). the corresponding mutants were expressed and purified from e. coli bl cells, and their enzymatic activities were tested by the in vitro dub assay ( figure a ) and the quantified relative activity was shown at the right of corresponding figures. for this identified acidic cluster, the residues d , d , w , a , d , and d are highly conserved among various prrsv strains, whereas the residues t and e are highly conserved among type ii prrsv strains but are replaced with the respective residues s and y in type i prrsv strains ( figure s ) [ ] . among these residues, both w and d have been shown to be critical for the trans-cleavage of nsp / [ ] . thus, we, therefore, selected other residues (d , d , t , e , d ) for further mutational analysis with a purpose to distinguish dub from its trans-cleavage activity. the acidic residues were changed to either the residue asparagine (n) or to the opposite charge residue arginine (r), whereas t was replaced with either serine (s), glycine (g), or arginine (r). the corresponding mutants were expressed and purified from e. coli bl cells, and their enzymatic activities were tested by the in vitro dub assay ( figure a ) and the quantified relative activity was shown at the right of corresponding figures. the results showed that the mutations of the residues d and e (e.g., d n, d r, e r, e q, etc.) did not have much effect on plp dub activity, as the corresponding mutants could efficiently cleave k or k polyubiquitin chains into monomers ( figure a , lanes , , , and ). in contrast, mutation of the residue d to either n or r largely blocked the cleavage of ub - with only a very small portion cleaved into ub dimer or monomer ( figure a , lane and ). the mutational effect of d was amino acid-dependent. the d r mutation largely blocked the plp dub activity ( figure a , lane ), whereas the mutation d n did not affect the cleavage much ( figure a, lane ) . for the residue t , substitution with r (t r) largely ablated the dub activity ( figure a , lane ), whereas the mutation to serine (t s) did not have an effect ( figure a, lane ) . the t g mutation did not have a large effect on the ability of plp to process the k polyubiquitin chains ( figure a , lane , bottom panel), but it partially crippled the ability of plp to cleave the ubiquitin dimer ( figure a , lane , top panel), suggesting a differential sensitivity. we next used a cell-based assay to further check on the mutational effect on plp dub activity within mammalian cells ( figure b ). at the same time, the mutants plp c a and d n ( figure b , lanes and ), which have been shown to be defective of trans-cleavage activity [ ] , were used as negative control. the ha-tagged ubiquitin was transiently expressed in hek t cells together with plp or plp -derived mutants. at h post-transfection, the cell lysates were collected to assess the level of ubiquitination by western blot analysis with antibodies to ha epitope ( figure b ). the results showed that the mutants d r, d n, t s, e q, and d n were dub active ( figure b ). in contrast, the mutants d r, d n, t r, and d r lost the dub activity, together with the control mutants plp c a and d n ( figure b ). on the other hand, the mutations e r and t g partially crippled the plp dub activity ( figure b, lanes and ) . overall, the cell-based results are largely consistent with the in vitro dub assay. in addition, the results of mutagenesis studies are in accordance with the bioinformatics prediction, except for several mutations (e.g., t r, e r, and d r), which will be discussed in the discussion section. we next investigated whether the same mutation affects the plp cisor trans-cleavage activity, which can be important for the processing of prrsv polyprotein nsp - during infection. to address this question, we performed the cell-based assay by co-expressing plp or its derivatives with the substrate nsp - ∆ - lacking the plp domain in transfected ft cells ( figure a ). for a negative substrate control, the plp cleavage site at the nsp - junction was mutated to make the construct nsp - ∆ - g p ( figure a ). as shown in figure a , except for the control d n, all other plp mutants were capable of efficiently processing the nsp - polyprotein, suggesting that the mutations did not affect the trans-cleavage activity of plp . thus, the mutations (e.g., d n, d r, e r, d r, t r, etc.) that affected the dub activity can be used to differentiate the dub activity from trans-cleavage activity. viruses , , x for peer review of we next investigated whether the same mutations affect the plp cis-cleavage activity. since cis-cleavage is essentially an intramolecular interaction, the mutational effect was evaluated by using the full-length nsp - polyprotein precursor as reported previously [ ] . in addition, since both cisand trans-cleavage activities can direct the nsp - cleavage [ ] , the trans-cleavage has to be silenced in the first place before proceeding to test the effect on cis-activity. here, we took advantage of the d n mutation, which is able to decouple the nsp cisfrom trans-cleavage activity by using prrsv strain vr- [ ] . consistently, the d n mutation strongly reduced the trans-cleavage activity of plp to process nsp - ∆ - in trans in the cotransfection assay ( figure a, lane ) , and the cleavage efficiency was only about - %. to make sure the full-length nsp carrying d n mutation also behaves the same as seen for the plp fragment ( figure a, lane ) , we introduced this mutation into the full-length nsp of prrsv strain jxwn . as expected, the full-length nsp carrying d n mutation also failed to process in trans the polyprotein nsp - lacking the protease domain (nsp - ∆ - ) in the co-transfected cells ( figure b, lane ) , suggesting the trans-cleavage activity is blocked. when the same mutation was introduced into the nsp - precursor (nsp - d n) , it, however, did not affect the processing in cis of nsp from nsp - d n ( figure c, lane ) . thus, this result is in agreement with the previous report based on prrsv strain vr- [ ] . next, we introduced the corresponding acidic cluster point mutations into the background nsp - d n ( figure c ), which allows only cis-processing of nsp . to our surprise, the mutations (e.g., d n, d r, t r, and d r) that largely ablated the dub activity also blocked the cis-activity of plp ( figure c , lanes , , , and , respectively). in contrast, neither d n nor d r affected the cis-cleavage activity ( figure c , lane and ). notably, the mutation t g could selectively block the plp cis-cleavage activity ( figure c, lane ) , as the mutant plp t g was still equipped with trans-cleavage activity ( figure a , lane ) and largely dub activity ( figure a, lane ) . together, these results suggest that the requirement for plp dub activity somehow is more closely related to that for cis-cleavage activity, rather than the trans-cleavage activity. in addition, t g is a critical mutation that can differentiate the cis-cleavage activity from dub and trans-cleavage activity. the mutational effect on viral replication was tested by introducing the corresponding point mutations into the infectious cdna clone of hp-prrsv strain jxwn in a dna-launched system. the recombinant plasmids were transfected into marc- cells for virus recovery. our analyses by reverse genetics of plp mutants led to the findings as follows (table ). (i) mutations (d n, d r, t r, and d r) that largely blocked the dub activity in cell-based assay were all lethal to the virus, (ii) mutations (d r, d n, t s, and e q) that did not have an effect on dub activity did not affect viral viability, (iii) the point mutation t g that selectively blocked the plp cis-activity did not affect viral viability in cell culture, and (iv) the mutation d n and e r, which did not affect either of the plp activities, somehow did not allow the rescue of viable virus. together, our results strongly suggest a dispensable role of the plp cis-activity for prrsv viability. on the other hand, the fact that the cis-cleavage activity is not required for prrsv replication suggests that the lethal phenotype of several point mutations (d n, d r, t r, and d r), which blocked both dub and cis-activity, is not due to a block in cis-cleavage activity, but may be linked to the inactivation of plp dub activity. plp activities, somehow did not allow the rescue of viable virus. together, our results strongly suggest a dispensable role of the plp cis-activity for prrsv viability. on the other hand, the fact that the cis-cleavage activity is not required for prrsv replication suggests that the lethal phenotype of several point mutations (d n, d r, t r, and d r), which blocked both dub and cis-activity, is not due to a block in cis-cleavage activity, but may be linked to the inactivation of plp dub activity. four viable mutants of passage (p ) were chosen for growth kinetics analysis in both marc- cells ( figure a ) and primary pams ( figure b ). all the mutants tested showed relatively similar growth properties to the parental virus，except for the mutant t g, which exhibited reduced growth in marc- cells by about half a log and a slight decrease in pams although being statistically insignificant ( figure b ). to test the stability, we sequenced the plp mutation region at the end of experiments or p viruses. the results showed that the mutations were stable ( figure c ). in addition, we did not see any other compensating mutations arising within nsp . since prrsv nsp is involved in antagonizing host innate immunity and the plp mutants did not have an apparent growth defect in primary pams, we tested whether mutations have an effect on inflammatory cytokine production rather than on interferon signaling. in particular, we focused on the expression and secretion of tnf-α, il- β, and il- ( figure ) , the most potent inflammatory cytokines. to this end, primary pams were infected with wt or mutant viruses with an moi of . . at h postinfection, the mrna level of tnf-α, il- β, and il- were measured by relative quantitative rt-pcr normalized against peptidylprolyl isomerase a (ppia), a molecule that has been previously used for internal control [ ] [ ] [ ] . the results showed that prrsv strain jxwn significantly upregulated the mrna expression of both tnf-α and il- β ( figure a ). on the other hand, the effect on il- was not impressive ( figure a ). interestingly, the mutant e q exhibited greatly reduced ability to induce expression of both tnf-α and il- β, and to a lesser extent by the mutant t g ( figure a ). slightly blocked viral viability, iii) the point mutation t g that selectively blocked the plp cis-activity did not affect viral viability in cell culture, and iv) the mutation d n and e r, which did not affect either of the plp activities, somehow did not allow the rescue of viable virus. together, our results strongly suggest a dispensable role of the plp cis-activity for prrsv viability. on the other hand, the fact that the cis-cleavage activity is not required for prrsv replication suggests that the lethal phenotype of several point mutations (d n, d r, t r, and d r), which blocked both dub and cis-activity, is not due to a block in cis-cleavage activity, but may be linked to the inactivation of plp dub activity. four viable mutants of passage (p ) were chosen for growth kinetics analysis in both marc- cells ( figure a ) and primary pams ( figure b ). all the mutants tested showed relatively similar growth properties to the parental virus，except for the mutant t g, which exhibited reduced growth in marc- cells by about half a log and a slight decrease in pams although being statistically insignificant ( figure b ). to test the stability, we sequenced the plp mutation region at the end of experiments or p viruses. the results showed that the mutations were stable ( figure c ). in addition, we did not see any other compensating mutations arising within nsp . since prrsv nsp is involved in antagonizing host innate immunity and the plp mutants did not have an apparent growth defect in primary pams, we tested whether mutations have an effect on inflammatory cytokine production rather than on interferon signaling. in particular, we focused on the expression and secretion of tnf-α, il- β, and il- ( figure ) , the most potent inflammatory cytokines. to this end, primary pams were infected with wt or mutant viruses with an moi of . . at h postinfection, the mrna level of tnf-α, il- β, and il- were measured by relative quantitative rt-pcr normalized against peptidylprolyl isomerase a (ppia), a molecule that has been previously used for internal control [ ] [ ] [ ] . the results showed that prrsv strain jxwn significantly upregulated the mrna expression of both tnf-α and il- β ( figure a ). on the other hand, the effect on il- was not impressive ( figure a) . interestingly, the mutant e q exhibited greatly reduced ability to induce expression of both tnf-α and il- β, and to a lesser extent by the mutant t g ( figure a ). largely blocked - : active, -: blocked, nd: not determined, asmids were transfected into marc- cells for virus recovery. our analyses by plp mutants led to the findings as follows (table ) . i) mutations (d n, d r, at largely blocked the dub activity in cell-based assay were all lethal to the virus, , d n, t s, and e q) that did not have an effect on dub activity did not affect e point mutation t g that selectively blocked the plp cis-activity did not affect l culture, and iv) the mutation d n and e r, which did not affect either of the ehow did not allow the rescue of viable virus. together, our results strongly ble role of the plp cis-activity for prrsv viability. on the other hand, the fact activity is not required for prrsv replication suggests that the lethal phenotype tations (d n, d r, t r, and d r), which blocked both dub and cis-activity, ck in cis-cleavage activity, but may be linked to the inactivation of plp dub le . the association between various plp activity and virus viability. ced production of tnf-α and il- β is strongly associated with nsp that is dub activity tants of passage (p ) were chosen for growth kinetics analysis in both marc-) and primary pams ( figure b ). all the mutants tested showed relatively similar to the parental virus，except for the mutant t g, which exhibited reduced cells by about half a log and a slight decrease in pams although being icant ( figure b ). to test the stability, we sequenced the plp mutation region at nts or p viruses. the results showed that the mutations were stable ( figure c) . not see any other compensating mutations arising within nsp . since prrsv nsp onizing host innate immunity and the plp mutants did not have an apparent rimary pams, we tested whether mutations have an effect on inflammatory n rather than on interferon signaling. in particular, we focused on the expression f-α, il- β, and il- ( figure ) , the most potent inflammatory cytokines. to this s were infected with wt or mutant viruses with an moi of . . at h postlevel of tnf-α, il- β, and il- were measured by relative quantitative rt-pcr peptidylprolyl isomerase a (ppia), a molecule that has been previously used for - ] . the results showed that prrsv strain jxwn significantly upregulated the of both tnf-α and il- β ( figure a ). on the other hand, the effect on il- was ure a). interestingly, the mutant e q exhibited greatly reduced ability to f both tnf-α and il- β, and to a lesser extent by the mutant t g ( figure a ). four viable mutants of passage (p ) were chosen for growth kinetics analysis in both marc- cells ( figure a ) and primary pams ( figure b ). all the mutants tested showed relatively similar growth properties to the parental virus, except for the mutant t g, which exhibited reduced growth in marc- cells by about half a log and a slight decrease in pams although being statistically insignificant ( figure b ). to test the stability, we sequenced the plp mutation region at the end of experiments or p viruses. the results showed that the mutations were stable ( figure c ). in addition, we did not see any other compensating mutations arising within nsp . since prrsv nsp is involved in antagonizing host innate immunity and the plp mutants did not have an apparent growth defect in primary pams, we tested whether mutations have an effect on inflammatory cytokine production rather than on interferon signaling. in particular, we focused on the expression and secretion of tnf-α, il- β, and il- ( figure ) , the most potent inflammatory cytokines. to this end, primary pams were infected with wt or mutant viruses with an moi of . . at h post-infection, the mrna level of tnf-α, il- β, and il- were measured by relative quantitative rt-pcr normalized against peptidylprolyl isomerase a (ppia), a molecule that has been previously used for internal control [ ] [ ] [ ] . the results showed that prrsv strain jxwn significantly upregulated the mrna expression of both tnf-α and il- β ( figure a ). on the other hand, the effect on il- was not impressive ( figure a) . interestingly, the mutant e q exhibited greatly reduced ability to induce expression of both tnf-α and il- β, and to a lesser extent by the mutant t g ( figure a) . similarly, none of the mutants affected the level of il- ( figure a ). we also measured the secretion of these cytokines in the infected supernatants by elisa. consistent with a reduction at the mrna level, we observed a significant drop of tnf-α in the cell culture supernatant from primary pams infected with the mutants t g and e q ( figure b ). for il- β and il- , we tried several commercial kits, and unfortunately, they did not work well in our hands. to test whether the effect was contingent on the infection dose, we chose e q and t g for further analysis (figure ) . the results showed that either moi of . , . , or could significantly reduce the secretion of tnf-α in the infected supernatants ( figure a ). when compared with wt, this reduction is by more than - % for the mutants e q and t g at h post-infection or later, moreover, the e q mutant had a greater ability to reduce the production of tnf-α ( figure a ). similarly, none of the mutants affected the level of il- ( figure a ). we also measured the secretion of these cytokines in the infected supernatants by elisa. consistent with a reduction at the mrna level, we observed a significant drop of tnf-α in the cell culture supernatant from primary pams infected with the mutants t g and e q ( figure b ). for il- β and il- , we tried several commercial kits, and unfortunately, they did not work well in our hands (data not shown). to test whether the effect was contingent on the infection dose, we chose e q and t g for further analysis (figure ) . the results showed that either moi of . , . , or could significantly reduce the secretion of tnf-α in the infected supernatants ( figure a ). when compared with wt, this reduction is by more than - % for the mutants e q and t g at h post-infection or later, moreover, the e q mutant had a greater ability to reduce the production of tnf-α ( figure a) . finally, to further evaluate whether the observed effect on pro-inflammatory cytokine production is associated with altered plp dub activity, we introduced the corresponding mutations (e q, t g, d r, and t r) into the full-length nsp rather than merely into the plp fragment ( figure b ). the deubiquitinating ability of nsp or its derivatives was then tested in transfected t cells by co-expression with ha-ubiquitin. consistent with the phenotype exhibited by the truncated nsp , the mutant nsp e q ( figure b , lane ) behaved just like wt nsp ( figure b, lane ) , and nsp t g was largely dub-active ( figure b, lane ) . in contrast, the catalytic site mutation c a blocked the dub activity of the full-length nsp ( figure b, lane ) . together, the above data provided strong evidence on a critical role of prrsv nsp in inducing tnf-α and il- β during infection, and also this property of nsp is independent of the plp dub activity. in this report, we developed a strategy to obtain high-level soluble expression of prrsv strain it is now known that prrsv plp possesses at least cis-, trans-cleavage, and dub activities and that the trans-activity requires the nsp region aa. - [ ] . in this report, we went much further to reveal several important biological aspects of this particular protease. beginning with the in vitro purification, we found that the downstream flanking sequence (nsp aa. - ) was critical for the yield and solubility of plp domain in the prokaryotic expression system ( figure b) . although a previous study reported that the prrsv strain jxwn plp (nsp aa. - ) is sufficient for dub activity in vitro, but it did not provide description about the expression level and purification of this fragment [ ] . in our hand, this fragment was unfortunately poorly expressed in e. coli bl cells despite attempts for optimization of expression conditions. although truncation is usually the way for protein expression, we used an unconventional approach to solve the problem, making a cterminal extension dramatically improved the plp yield and solubility ( figure b) . our results suggest that the flanking sequence likely plays an important role in the folding of plp core domain, and this is consistent with our previous finding that deletion of nsp aa. - is lethal to prrsv strain vr . the realization of high-level soluble expression also paves the way for future structural studies and in vitro anti-plp drug screening. previous studies have shown that prrsv plp possesses differential dub activity toward cleaving k and k ubiquitin dimers into monomer. our results are in general in agreement with this finding [ ] . different from the previous study, we found that prrsv plp is able to cleave ubiquitin dimer but just in a dose and time-dependent manner. this difference is likely attributed to the plp flanking sequence (aa. - ) (figure ), which can promote the folding of plp core domain, leading to increased cleavage efficiency toward k polyubiquitin chains. in addition, we finally, to further evaluate whether the observed effect on pro-inflammatory cytokine production is associated with altered plp dub activity, we introduced the corresponding mutations (e q, t g, d r, and t r) into the full-length nsp rather than merely into the plp fragment ( figure b ). the deubiquitinating ability of nsp or its derivatives was then tested in transfected t cells by co-expression with ha-ubiquitin. consistent with the phenotype exhibited by the truncated nsp , the mutant nsp e q ( figure b , lane ) behaved just like wt nsp ( figure b , lane ), and nsp t g was largely dub-active ( figure b , lane ). in contrast, the catalytic site mutation c a blocked the dub activity of the full-length nsp ( figure b, lane ) . together, the above data provided strong evidence on a critical role of prrsv nsp in inducing tnf-α and il- β during infection, and also this property of nsp is independent of the plp dub activity. in this report, we developed a strategy to obtain high-level soluble expression of prrsv strain jxwn plp in e. coli bl cells for assaying its dub activity in vitro. the in vitro and cell-based assays uncovered important biochemical properties of plp , including efficient cleavage ability towards both k -linked and k -linked polyubiquitin chains and a surprisingly shared requirement for both cis-cleavage and dub activity. moreover, we identified mutations that could distinguish the three activities of plp . further reverse genetics analysis revealed dispensability of cis-activity for prrsv replication. in contrast, viruses carrying point mutations that largely blocked the dub activity were not viable, pointing to a potentially critical role of plp dub in prrsv replication. during characterizing the viral mutants, we also unexpectedly discovered a dub-independent regulation of pro-inflammatory cytokine production by prrsv nsp . together, these findings further deepen our understanding of the biological properties and function of prrsv plp and nsp in virus life cycle and have implications in understanding viral pathogenesis and in vaccine development. the relevant significance or insights are discussed below. it is now known that prrsv plp possesses at least cis-, trans-cleavage, and dub activities and that the trans-activity requires the nsp region aa. - [ ] . in this report, we went much further to reveal several important biological aspects of this particular protease. beginning with the in vitro purification, we found that the downstream flanking sequence (nsp aa. - ) was critical for the yield and solubility of plp domain in the prokaryotic expression system ( figure b) . although a previous study reported that the prrsv strain jxwn plp (nsp aa. - ) is sufficient for dub activity in vitro, but it did not provide description about the expression level and purification of this fragment [ ] . in our hand, this fragment was unfortunately poorly expressed in e. coli bl cells despite attempts for optimization of expression conditions. although truncation is usually the way for protein expression, we used an unconventional approach to solve the problem, making a c-terminal extension dramatically improved the plp yield and solubility ( figure b ). our results suggest that the flanking sequence likely plays an important role in the folding of plp core domain, and this is consistent with our previous finding that deletion of nsp aa. - is lethal to prrsv strain vr . the realization of high-level soluble expression also paves the way for future structural studies and in vitro anti-plp drug screening. previous studies have shown that prrsv plp possesses differential dub activity toward cleaving k and k ubiquitin dimers into monomer. our results are in general in agreement with this finding [ ] . different from the previous study, we found that prrsv plp is able to cleave ubiquitin dimer but just in a dose and time-dependent manner. this difference is likely attributed to the plp flanking sequence (aa. - ) (figure ), which can promote the folding of plp core domain, leading to increased cleavage efficiency toward k polyubiquitin chains. in addition, we found that prrsv strain jxwn plp can equally efficiently cleave k and k -linkd ub - into dimers and monomer. we also identified critical residues for prrsv plp dub activity. d , an amino acid that is highly conserved among prrsv strains [ ] , was revealed to be a key residue for prrsv plp dub activity (figure ). either a conserved (d n) or non-conserved (d r) substitution led to a significant destruction of plp dub activity ( figure ) . the mutational effect also appears specific, as similar mutations of the nearby residue d (d n and d r) that is also highly conserved did not have an effect (figure ). this result is also in accordance with the bioinformatics prediction ( figure ) . the mutational effect of other residues is contingent on the amino acids used for substitution. for example, the mutations d r and t r largely blocked the plp activity and e r partially blocked the dub activity ( figure ). we suspect that the mutational effect of d r is most likely indirect and could result from electrostatic interaction with e to change the local structure. in a similar manner, the mutation t r can potentially interact locally with e , d , or d , leading to local conformational change of plp , which may have an adverse effect on plp function. this also partially explains why t s, e q, and d e did not have an effect. most interestingly, we found that the t g mutation can selectively block the cis-cleavage activity ( figure c ), without affecting the trans-cleavage activity ( figure a ) and only partially blocking the dub activity in either truncated ( figure b ) or full-length form ( figure b ). this is the first time to report a point mutation that distinguishes cis-cleavage from other activities and paves the way to test the importance of cis-cleavage activity in prrsv infection. it has been viewed that hydrolysis of ubiquitin substrates takes advantage of the trans-activity of viral dubs. surprisingly, we observed an intriguing relationship between prrsv plp dub and cis-activity. that is, several mutations that largely blocked the plp dub activity were found to also ablate its cis-activity, and these include the mutants d n, d r, t r, and d r (figures and c ). in contrast, none of these mutations had an effect on the plp trans-activity cleaving nsp - polyprotein ( figure a ). thus, it seems that the requirement for prrsv plp dub activity is somehow more closely related to cis-activity, suggesting that they may share some intrinsic common mechanistic details in terms of recognition and cleavage of the substrates. to our knowledge, this is the first report revealing such intimate relationship between dub and cis-activity concerning otu cysteine proteases. the molecular basis for these observations is currently not clear, but hopefully, elucidation of the three-dimensional structure of prrsv plp may help resolve the puzzle in the future. a long-asked question is whether the plp dub and cis-cleavage activities are essential for prrsv infection. the inability to differentiate the enzymatic activities by point mutations has limited the evaluation of the contribution of individual enzymatic activities. despite the reports of several studies on the plp dub activities [ , , , ] , none of them established a strong correlation between dub and viral replication, as they did not rule out the mutational effect on both transand cis-cleavage activities. by taking advantage of site-directed mutagenesis, we in the first place provided strong evidence to suggest that the plp cis-activity is not necessary for viral replication ( figure ). this is evidenced by the specific point mutation t g, which selectively blocked the plp cis-cleavage activity but allowed the successful rescue of viable virus. moreover, this mutant exhibited similar growth rate to wt in primary pams ( figure ). in contrast, the mutations (e.g., d n, d r, t r, d r, etc.) that largely disarmed the plp dub activity were lethal to prrsv in marc- cells. although these mutations also crippled the cis-cleavage activity of plp at the same time, the successful rescue of t g mutant strongly argues against an essential contribution of inactive cis-activity to the non-viability phenotype. in addition, the lethal phenotype is less likely due to a potential defect of deisgylation activity of plp , as it has been reported that the hp-prrsv strain jxwn plp used in this study has very limited deisgylation activity [ ] and that isgylation was not observed in prrsv-infected marc- cells [ ] , a cell line that was used for virus rescue. thus, there appears to exist a strong correlation between the lethal phenotype of these mutations and the essential role of plp dub activity in prrsv infection. sun et al. recently characterized the nsp otu domain of a european prrsv strain sd - (type i strain) and reported that plp inhibits nf-κb activation through its dub activity [ ] . they also mutated the residues in the acidic cluster, including the residues d , d , s , d , d , etc., corresponding to the residues d , d , t g and d in this study, respectively [ ] . consistent with our studies, the mutations d a and d a were lethal to prrsv, whereas d a, s a, and d a allowed the successful rescue of the viable virus [ ] . in these studies, the mutant d a did not affect the viral growth, whereas s a and d a crippled the virus in marc- cells [ ] . sun et al. further correlated the growth property with the ability to inhibit nf-κb activation and found that the mutations that were lethal to the virus were these capable of completely inhibiting nf-κb activation, whereas the mutations s a and d a unable to inhibit the activation as wt lead to viable virus [ ] . however, they did not further test the mutational effect of these mutants on cisand trans-cleavage activity and the dub activity. our studies here suggest that the lethal phenotype is not linked to an impairment of cisor trans-cleavage activity, but rather related to an effect on plp dub activity. however, because these are negative results, we could not rule out the possibility that the dub-inactivating mutations may have other unexpected consequences on nsp , or both, which renders the virus non-viable. on the other hand, the mutations e r and d n, which did not affect much the plp dub activity, did not allow the rescue of viable viruses. this result suggests that the mutations may have an adverse effect on other functions of nsp , highlighting the complexity of mutational effect on the function of plp or nsp . another interesting observation is that the partial ablation (t g) of plp dub activity did not affect viral viability whereas a near-complete block (e.g., d n, d r, t r, d r, etc.) was lethal to the virus. these results may suggest that there exists a threshold of dub activity required for prrsv replication, in which the dub activity may be required to remove polyubiquitins from substrates. future study will be directed to investigate detailed mechanisms by which the prrsv plp plays during infection. infections by prrsv often cause de-regulation of inflammatory cytokine production [ ] . for the chinese hp-prrsv, it inhibits inflammation in early infection, but induces inflammatory storm during the late stage in pigs, contributing to lung injury and interstitial pneumonia [ , ] . the regulatory mechanisms of this process have remained poorly understood, but it is known that several nonstructural proteins, including nsp α, nsp β, nsp , and nsp , are capable of inhibiting inflammation response [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , whereas the structural proteins n and e promote inflammation [ , ] . prrsv nsp is a type i interferon signaling antagonist [ ] and recently implicated in the modulation of pro-inflammatory cytokine production during infection [ , , ] . liu et al. [ ] reported that mutants of hp-prrsv strain bb carrying deletion of nsp region aa. - or aa. - had the reduced ability to induce the expression of inflammatory cytokines il- β, tnf-α, and il- in pams. however, the corresponding mutants also exhibited reduced growth titer by . to log when compared with the parental virus. moreover, down-regulation is quite subtle (< %). on the other hand, the single-site substitution represents a nice approach by which it can minimize the risk of gross conformational alteration as a result of mutagenesis. at the beginning of this study, we did not mean to study the role of nsp in inflammatory cytokine induction, but with two independent point-mutation mutants (t g and e q) at hand, we unexpectedly established a strong link of nsp to prrsv-induced induction of pro-inflammatory cytokines (figures and ) . the mutants t g and e q retained the similar growth rate to the parental virus jxwn in primary pams, but exhibited significantly reduced ability to induce tnf-α and il- β at both mrna and protein levels (figures and ) . moreover, this reduction rate was by more than % at hpi or later ( figure a ), suggesting a decisive role of nsp in modulating the production of tnf-α and il- β during infection. different from the previous study [ ] , we did not observe a change in expression level of il- . the crippled ability to induce tnf-α and il- β cannot be attributed to an impairment of plp dub activity, as the mutant nsp e q was fully dub active ( figure b ). in addition, because viral dubs often negatively regulate host innate immunity, it is expected that inactivation of dub activity should promote the inflammatory cytokine production as reported by several cases [ , , , , ] . our results, however, showed the opposite, the mutant t g with partially crippled dub activity showed reduced production of tnf-α and il- β (figures and ). thus, with two independent nsp mutants (t g and e q) showing the similar phenotype, our results provide 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differential tnf-alpha production induced by porcine reproductive and respiratory syndrome virus strains with different pathogenicity in vitro porcine reproductive and respiratory syndrome virus nonstructural protein antagonizes beta interferon expression by targeting the nf-kappab essential modulator we thank our colleague nianzhi zhang for his help on bioinformatics analysis of plp . the authors declare that they have no conflicts of interest with the contents of this article. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.viruses , , key: cord- -cr zskcw authors: lu, chien-yi; lin, chen-sheng; lai, hsueh-chou; yu, ya-wen; liao, chih-yi; su, wen-chi; ko, bo-han; chang, young-sheng; huang, su-hua; lin, cheng-wen title: the rescue and characterization of recombinant, microcephaly-associated zika viruses as single-round infectious particles date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: cr zskcw zika virus (zikv) is transmitted by aedes mosquitoes and exhibits genetic variation with african and asian lineages. zikv natal rgn strain, an asian-lineage virus, has been identified in brain tissues from fetal autopsy cases with microcephaly and is suggested to be a neurotropic variant. however, zikv natal rgn strain has not been isolated; its biological features are not yet illustrated. this study rescued and characterized recombinant, single-round infectious particles (srips) of the zikv natal rgn strain using reverse genetic and synthetic biology techniques. the dna-launched replicon of zikv natal rgn was constructed and contains the egfp reporter, lacks prm-e genes, and replicates under cmv promoter control. the peak in the zikv natal rgn srip titer reached . × ( ) tcid /ml in the supernatant of prm-e-expressing packaging cells h post-transfection with a zikv natal rgn replicon. the infectivity of zikv natal rgn srips has been demonstrated to correlate with the green florescence intensity of the egfp reporter, the srip-induced cytopathic effect, and zikv’s non-structural protein expression. moreover, zikv natal rgn srips effectively self-replicated in rhabdomyosarcoma/muscle, glioblastoma/astrocytoma, and retinal pigmented epithelial cells, displaying unique cell susceptibility with differential attachment activity. therefore, the recombinant zikv natal rgn strain was rescued as srips that could be used to elucidate the biological features of a neurotropic strain regarding cell tropism and pathogenic components, apply for antiviral agent screening, and develop vaccine candidates. zika virus (zikv) was first isolated in monkeys in uganda in , and then detected in aedes africanus mosquitoes in . zikv belongs to a mosquito-borne flavivirus of the family flavivirus, which spread from africa to south-eastern asia through transmission by aedes mosquitoes, such as a. to produce single-round infectious particles (srips). the packaging cells carrying the recombinant plasmid encoding the viral structural proteins are transfected with dna-launched replicons and then self-replicate viral subgenomes and express all viral proteins, allowing viral assembly and release as srips. several flavivirus, replicon-based srips, including jev, dengue virus, west nile virus, and tick-borne encephalitis virus have been generated and demonstrated as safer vaccine candidates [ ] [ ] [ ] . subsequently, replicon-based srips are suitable systems for investigating the mechanism of the zikv life cycle, zikv-host interaction, and virulence. the zikv natal rgn strain that is associated with microcephaly was recognized as the representative, epidemic, zikv asian strain in - [ ] . the zikv natal rgn strain is detected in brain tissues from fetal autopsy cases with microcephaly in the natal region of brazil; its genome has been sequenced using next-generation sequencing (genbank accession number ku ) [ ] . the zikv natal rgn strain contains a unique genotype and phenotype; therefore, this study aimed to generate srips of the zikv natal rgn strain using synthetic and reverse genetic technologies. this paper characterizes the infectivity of zikv natal rgn srips in different cell types. moreover, the paper analyzes the expression pattern of type i interferons and apoptosis-related genes induced by zikv natal rgn srips. te (human, rhabdomyosarcoma/muscle), sf (human brain, glioblastoma/astrocytoma), and arpe- (human, retinal pigmented epithelium) cells were cultured in minimum essential medium containing % fetal bovine serum, mm glutamine, mm pyruvate, and × penicillin-streptomycin at • c with % co . components of two dna segments we synthesized, i and ii, consisted of the cmv immediate-early promoter (cmvp), cdna fragments of the zikv natal rgn genome (genbank accession number ku ), enhanced green fluorescent protein (egfp), fmdv- a (f- a), hepatitis delta virus ribozyme (hdvr), and bovine growth hormone polya signal (bgh-pa), which were purchased from bio basic inc. (ontario, canada). the construction of synthesized dna fragments, plasmid information, and restriction enzyme digestion analysis of the dna fragments we purchased are shown in supplementary figures s and s and figure a . to construct the zikv natal rgn replicon, the plasmid pbr -linker, described in our prior report [ ] , was chosen as the vector to assemble fragments a, b, c, and d of the cmvp-driven zikv natal rgn replicon with the egfp reporter via the cloning sites of ecori, noti, clai, rsrii, and xhoi ( figure b ). fragments a, c, and d were generated using the platinum ® pcr supermix high fidelity reaction with the dna segments we synthesized, i and ii, as the templates (life technology, carlsbad, ca, usa). fragment b was produced through two-round pcr with the gibson assembly reaction of two first-round pcr products (b and b ) as the template (new england biolabs, ipswich, ma, usa). the pairs containing the restriction enzyme site(s) indicated, are listed in supplementary table s . the resultant plasmid carrying the cmvp-driven zikv natal rgn replicon with the egfp reporter was propagated in e. coli dh α and then sequenced by sanger sequencing assays with sequencing primers (supplementary table s ). the nucleotide and deduced amino acid sequence alignment analysis of the cmvp-driven zikv natal rgn replicon and its parent strain (genbank accession number ku ) was performed using the lasergene dnastar megalign software. figure . construction of the pbr -based zikv natal rgn replicon and pcdna . -zikv prme. two synthetic dna segments in the puc plasmid contained the entire zikv natal rgn strain genome: cmvp, egfp, fmdv- a, and bgh-pa sequences (a). four pcr products (fragments a-d) were cloned into the indicated restriction sites (ecori, noti, clai, rsrii, and xhoi) of the pbr plasmid and then assembled as the in-frame fusion of the zikv natal rgn replicon with the egfp reporter under cmv-promoter control (b). those four pcr products were analyzed using agarose gel electrophoresis (c). the pcr product of zkiv prm and e genes was cloned into restriction sites (ecori and xhoi) of the pcdna . -his-c plasmid (d). initially, te cells were transfected with pcdna . -zikv prm-e, selected with g for weeks, analyzed to examine zikv prm and e protein expression, and then recognized as the packaging cell line ( figures d, a middle, a middle). subsequently, the packaging cells were transfected with the zikv natal rgn replicon, and then cpe and the egfp reporter expressions were examined ( figure ). in the packaging cells, egfp reporter expression and cpe showed slight levels h post-transfection, but exhibited significant changes h after transfection with the zikv natal rgn replicon (figure bottom) . moreover, in vitro translation and transcription of the zikv natal rgn replicon in packaging cells were further characterized ( figure ). immunofluorescent staining indicated that the zikv e c-terminus, ns , and ns proteins were massively expressed in replicon-transfected packaging cells ( figure a bottom), but not in mock and packaging cells ( figure a top and middle). the real-time, reverse transcription pcr assay showed large amounts of positive and negative-sense zikv subgenomes in replicon-transfected packaging cells h post-transfection, but not in mock cells ( figure b top and bottom) . the results demonstrated efficient translation and replication of the zikv subgenomes in the packaging cells using the dna-launched zikv natal rgn replicon. figure . construction of the pbr -based zikv natal rgn replicon and pcdna . -zikv prme. two synthetic dna segments in the puc plasmid contained the entire zikv natal rgn strain genome: cmvp, egfp, fmdv- a, and bgh-pa sequences (a). four pcr products (fragments a-d) were cloned into the indicated restriction sites (ecori, noti, clai, rsrii, and xhoi) of the pbr plasmid and then assembled as the in-frame fusion of the zikv natal rgn replicon with the egfp reporter under cmv-promoter control (b). those four pcr products were analyzed using agarose gel electrophoresis (c). the pcr product of zkiv prm and e genes was cloned into restriction sites (ecori and xhoi) of the pcdna . -his-c plasmid (d). to detect self-amplifying rna genomes of the cmvp-driven zikv natal rgn replicon, the synthesis of positive and negative-sense rna subgenomes in vitro was examined using sybr green-based real-time pcr. total rnas of te- cells transfected with the zikv natal rgn replicon were extracted using the purelink mini total rna purification kit (thermo fisher scientific, waltham, ma, usa), reverse transcribed into cdna with specific-capture primers, and measured using real-time pcr with zikv ns -specific primer pairs (supplementary table s ), according to our prior report [ ] . relative levels of self-amplifying zikv sense and antisense genomes were normalized to glyceraldehyde -phosphate dehydrogenase (gapdh). the absolute copy number of the zikv genome was determined according to the standard curve of real-time pcr for serial dilutions of the plasmid containing the zikv ns gene at a known concentration. to explore the expression of replicon-based egfp and zikv reporter proteins, replicon-transfected cells were initially photographed using immunofluorescence microscopy; then, an immunofluorescence assay (ifa) was performed with primary antibodies against zikv ns and ns (genetex, inc., taiwan) and secondary af goat anti-rabbit igg (thermo fisher scientific). the immunofluorescent staining assay was carried out as described in our prior report [ ] . the fluorescence intensity of replicon-based egfp and zikv proteins in zikv natal rgn replicon-transfected cells was counted by image j. to establish the packaging cells expressing zikv structural proteins, the prm/m-e gene was amplified using pcr with specific primers (supplementary table s ) and synthesized dna segment i as the template. the pcr product of the zikv prm-e gene was digested with ecori and xhoi, and then cloned into the ecori and xhoi sites of the expression plasmid pcdna . -hisc. the resultant plasmid pcdna-zikv prme was transfected with te- cells at % confluence in a -well plate with lipofectamine ltx (invitrogen, carlsbad, ca, usa) according to the manufacturer's guidelines. the transfected cells were selected in the culture media with µg/ml g for weeks; the expression of zikv prm and e proteins in a stably transfected cell line (packaging cell line) was validated by real-time rt-pcr with zikv e-specific primers (supplementary table s ) and immunofluorescence staining with primary antibodies against zikv e protein (genetex, inc.), as described above. the packaging cells grown at % confluence in -well plates were transfected with or without the zikv natal rgn replicon using lipofectamine ltx. the cytopathic effect (cpe), replicon-based egfp and zikv proteins, and viral sense and antisense genomes in mock and transfected cells, were measured and h post-transfection using immunofluorescent staining assays, as described above. moreover, real-time rt-pcr assays with ns -specific primer pairs, listed in supplementary table s , were performed to examine the positive and negative-sense viral genomes in replicon-transfected packaging cells, as well as the viral genome in srips. zikv natal rgn srips in the cultured media of transfected packaging cells were concentrated using the peg virus precipitation kit (abcam, cambridge, ma, usa). the cultured media were centrifuged at × g for min at • c; then, the supernatant ( ml) was collected from each and incubated with . ml peg solution overnight at • c. the pellets of zikv natal rgn srips were harvested after centrifugation at × g for min at • c, re-suspended in µl virus re-suspension solution, and then stored at − • c. to analyze the antigenicity of zikv natal rgn srips, µl each of one-fold and -fold srip stock dilution was spotted onto a nitrocellulose membrane for the dot-blot assay with anti-zikv e antibodies. the membrane was subsequently blocked with % skim milk in tbst (tris-buffered saline, . % tween- ) buffer at • c for h, incubated overnight with anti-zkiv e antibodies (genetex, inc., taiwan), and then reacted with hrp-conjugated anti-mouse igg antibodies (invitrogen, carlsbad, ca, usa) after washing with tbst. immunoreactive signals for the e proteins of zikv natal rgn srips were amplified with ecltm western blotting detection reagents (ge healthcare, chicago, il, usa), and then imaged by the multi-function gel image system (multigel- ) (gentaur, san jose, ca, usa). the infectious titer of the zikv natal rgn srip stock was determined by a median tissue culture infectious dose (tcid ) assay. serial dilutions of the zikv natal rgn srip stock were added and incubated on the % confluent monolayer of packaging cells in -well plates. after incubating for h at • c, the cpe in each well was observed and recorded to determine the tcid titer of the zikv natal rgn srip stock. in the infectivity assay, packaging cells were cultured in -well plates overnight and infected with different doses of zikv natal rgn srips at multiplicity of infections (mois) of , . , . , and . . cpe, replicon-driven egfp, and zikv ns expression in the srip-infected cells were examined h post-infection, and fluorescence microscopy and immunofluorescent staining were performed as described above. in cell line susceptibility assays, te- , sf- , and arpe- cells were infected with zikv natal rgn srips at a moi of . tcid /cell. after incubating for h, the cells were photographed to examine cpe and explore replicon-driven egfp reporter expression using immunofluorescence microscopy. moreover, the cells were fixed with paraformaldehyde at • c for h, and then immunofluorescence staining was performed with anti-zikv ns antibodies, as mentioned above. in the attachment assay, zikv natal rgn srips at a dose of tcid /well (moi = . tcid /cell) were added onto the monolayers of arpe- , te , and sf- cells and incubated at • c for h. after washing with cold pbs, zikv natal rgn srips attached on the cell surface were harvested, and the relative levels of extracted viral genomes were quantitated using two-step, sybr, green i, real-time rt-pcr with zikv ns -specific primer pairs, as described above. all data from independent experiments were analyzed using student's t-tests or χ tests. statistical significance for each assay was judged at p < . . the zikv asian natal rgn prototype strain, a microcephaly-associated zikv, was not isolated from the brain tissues from fetal autopsy cases; therefore, this study intended to recover the recombinant zikv natal rgn strain using reverse genetics and synthetic biology techniques based on the published genome sequence (genbank accession number ku ). two synthetic dna segments in the puc plasmids comprised the entire zikv natal rgn strain genome, cmvp, egfp, fmdv- a, and bgh-pa sequences (supplementary figures s and s and figure a ,b). the synthetic dna segments were used as templates via pcr to amplify the gene fragments (a-d) that were cloned into the low-copy-number plasmid pbr to construct a dna-launched zikv natal rgn replicon under the control of the cmv promoter ( figure b,c) . fragment a consisted of cmvp, zikv '-utr, zikv c protein, the reporter egfp, and fmdv- a. fragment b containing the -residue c-terminal region of zikv e protein, ns , ns a, ns b, and the n-terminal region of ns , was produced using gibson assembly cloning ( figure c ). in order to join fragments b and c, the reverse primer for fragment b and the forward primer for fragment c contained the restriction enzyme site clai (supplementary table s ), which is recognized as the marker nt atcgat of the replicon in which thymidine at nucleotide of the zikv natal rgn genome was exchanged with adenosine, but it caused a silent mutation ( figure b) . interestingly, the rsrii restriction site within the ns b gene ( nt cggaccg ) of the zikv natal rgn genome was a unique restriction site for ligation between fragments c and d. in addition, hdvr was fused with the '-utr of zikv in the replicon to verify the accuracy of the '-end of the transcribed zikv natal rgn rna genomes ( figure b,c) . furthermore, sequencing analysis of the zikv natal rgn replicon indicated that only two amino acid substitutions appeared within the ns protein (l r and e n) (supplementary figure s ) . therefore, the dna-launched zikv natal rgn replicon was effectively constructed to generate the in-frame fusion of zikv c protein, reporter egfp, fmdv- a, the -residue c-terminal region of zikv e protein, and all non-structural zikv proteins under the control of the cmv promoter. initially, te cells were transfected with pcdna . -zikv prm-e, selected with g for weeks, analyzed to examine zikv prm and e protein expression, and then recognized as the packaging cell line ( figure d to examine the production of zikv natal rgn srips, the supernatant of replicon-transfected packaging cells was harvested h post-transfection and analyzed using dot-blotting, real-time rt-pcr, and tcid assays (figure ) . the dot-blotting assay with anti-zikv e protein demonstrated the antigenic structure of the e protein ( figure a) , and real-time rt-pcr assay elucidated the viral subgenomes of zikv natal rgn srips from the supernatant of the replicon-transfected packaging cells ( figure b) . moreover, the titer of zikv natal rgn srips in the supernatant was determined by tcid assay with the packaging cells. the titer of the zikv natal rgn srip stock was . × tcid /ml ( figure c ). to examine the production of zikv natal rgn srips, the supernatant of replicon-transfected packaging cells was harvested h post-transfection and analyzed using dot-blotting, real-time rt-pcr, and tcid assays (figure ) . the dot-blotting assay with anti-zikv e protein demonstrated the antigenic structure of the e protein ( figure a) , and real-time rt-pcr assay elucidated the viral subgenomes of zikv natal rgn srips from the supernatant of the replicon-transfected packaging cells ( figure b) . moreover, the titer of zikv natal rgn srips in the supernatant was determined by tcid assay with the packaging cells. the titer of the zikv natal rgn srip stock was . × tcid /ml ( figure c ). to examine the infectivity of zikv natal rgn srips in vitro, the packaging cells were infected with -fold dilutions (mois of , . , . , and . ) of zikv natal rgn srips to determine sripinduced cpe and srip-driven expression of egfp reporter and zikv ns ( to examine the cell line susceptibility to zikv natal rgn srips in vitro, the three cell lines te- , sf- , and arpe- were used ( figure ). zikv natal rgn srips at a moi of . tcid /cell induced more observable and higher cpe levels in te- cells than those in arpe- and sf cells (figure a top) . the cpe level induced by zikv natal rgn srips correlated with the fluorescence intensity of the egfp reporter encoded within the srip genome in the following order: te- > arpe- > sf cells infected with zikv natal rgn srips ( figure a middle) . in addition, immunofluorescent staining with anti-zikv ns antibodies indicated that zikv ns protein was expressed in these three zikv natal rgn srip-infected cell lines, in which the fluorescence intensity of the ns protein was consistent with the cpe level induced by zikv natal rgn srips ( figure a bottom). meanwhile, tcid of the srips per well was added onto the monolayer of these three cell lines to examine the attachment activity of zikv natal rgn srips ( figure b) . interestingly, the srips showed the highest level of attachment activity to te cells, in which the highest viral genome number was discovered h after attachment at °c. however, the srips showed the lowest level of attachment activity to sf cells. compared to arpe- and sf cells, it was found that te cells exhibited the highest susceptibility to zikv natal rgn srips and allowed the highest number of srips to attach to the surface. to examine the cell line susceptibility to zikv natal rgn srips in vitro, the three cell lines te- , sf- , and arpe- were used ( figure ). zikv natal rgn srips at a moi of . tcid /cell induced more observable and higher cpe levels in te- cells than those in arpe- and sf cells (figure a top) . the cpe level induced by zikv natal rgn srips correlated with the fluorescence intensity of the egfp reporter encoded within the srip genome in the following order: te- > arpe- > sf cells infected with zikv natal rgn srips ( figure a middle) . in addition, immunofluorescent staining with anti-zikv ns antibodies indicated that zikv ns protein was expressed in these three zikv natal rgn srip-infected cell lines, in which the fluorescence intensity of the ns protein was consistent with the cpe level induced by zikv natal rgn srips ( figure a bottom). meanwhile, tcid of the srips per well was added onto the monolayer of these three cell lines to examine the attachment activity of zikv natal rgn srips ( figure b) . interestingly, the srips showed the highest level of attachment activity to te cells, in which the highest viral genome number was discovered h after attachment at • c. however, the srips showed the lowest level of attachment activity to sf cells. compared to arpe- and sf cells, it was found that te cells exhibited the highest susceptibility to zikv natal rgn srips and allowed the highest number of srips to attach to the surface. the zikv natal rgn strain found in brain tissues from fetal autopsy cases with microcephaly in the natal region of brazil has not been isolated [ ] . this study, firstly, generated srips of the zikv natal rgn strain using synthetic and reverse genetic technologies (figures - and supplementary figures s -s ) . the viral yield of zikv natal rgn srips in prm-e-expressing packaging cells transfected with the zikv replicon was . × tcid /ml (figure ) , which indicated the replication capacity of the cmv promoter-launched zikv natal rgn replicon. in addition, a green fluorescence reporter, egfp, encoded within the zikv natal rgn replicon, represented an observable marker to examine replication of the zikv natal rgn replicon and srips (figures , , and ). in our laboratory, the pbr plasmid has been used to construct a jev replicon that is genetically stable in e. coli [ ] . the jev genome sequence encoding for structural proteins (c, prm/m, and e) was toxic in e. coli, which resulted in genetic instability of the jev infectious clones and replicons [ ] . similarly, construction of zikv natal rgn infectious clones was not accomplished due to the genetic instability of zikv natal rgn infectious clones, particularly within the ns gene region (supplementary figures s and s ) . however, the recombinant pcdna . plasmid containing prm and e genes and the pbr plasmid containing ' and '-utrs, c, and all ns genes, showed genetic stability. two zikv infectious clones obtained by cloning the entire fulllength native cdna have been reported [ , ] , and other zikv infectious clones were reported using strategies to avoid the toxicity of the zikv genome sequence, including in vitro assembly of viral cdna fragments and insertion of eukaryotic introns [ ] [ ] [ ] [ ] [ ] [ ] . particularly, the zikv natal rgn infectious clone was produced using bacterial artificial chromosomes (bacs) [ ] , in which a singlecopy pbelobac plasmid containing the full-length genome of the zikv natal rgn strain under the cmv promoter control was used to maintain zikv genome stability and reduce toxicity in bacteria. in addition, our prior report demonstrated that jev srips carrying the egfp reporter were a more sensitive, effective, and efficient platform for quantitating antiviral efficacy using flow cytometry compared to the traditional plaque assay [ ] . like jev srips, the zikv natal rgn srips the zikv natal rgn strain found in brain tissues from fetal autopsy cases with microcephaly in the natal region of brazil has not been isolated [ ] . this study, firstly, generated srips of the zikv natal rgn strain using synthetic and reverse genetic technologies (figures - and supplementary figures s -s ) . the viral yield of zikv natal rgn srips in prm-e-expressing packaging cells transfected with the zikv replicon was . × tcid /ml (figure ) , which indicated the replication capacity of the cmv promoter-launched zikv natal rgn replicon. in addition, a green fluorescence reporter, egfp, encoded within the zikv natal rgn replicon, represented an observable marker to examine replication of the zikv natal rgn replicon and srips ( figure , figure , and figure ). in our laboratory, the pbr plasmid has been used to construct a jev replicon that is genetically stable in e. coli [ ] . the jev genome sequence encoding for structural proteins (c, prm/m, and e) was toxic in e. coli, which resulted in genetic instability of the jev infectious clones and replicons [ ] . similarly, construction of zikv natal rgn infectious clones was not accomplished due to the genetic instability of zikv natal rgn infectious clones, particularly within the ns gene region ( supplementary figures s and s ) . however, the recombinant pcdna . plasmid containing prm and e genes and the pbr plasmid containing ' and '-utrs, c, and all ns genes, showed genetic stability. two zikv infectious clones obtained by cloning the entire full-length native cdna have been reported [ , ] , and other zikv infectious clones were reported using strategies to avoid the toxicity of the zikv genome sequence, including in vitro assembly of viral cdna fragments and insertion of eukaryotic introns [ ] [ ] [ ] [ ] [ ] [ ] . particularly, the zikv natal rgn infectious clone was produced using bacterial artificial chromosomes (bacs) [ ] , in which a single-copy pbelobac plasmid containing the full-length genome of the zikv natal rgn strain under the cmv promoter control was used to maintain zikv genome stability and reduce toxicity in bacteria. in addition, our prior report demonstrated that jev srips carrying the egfp reporter were a more sensitive, effective, and efficient platform for quantitating antiviral efficacy using flow cytometry compared to the traditional plaque assay [ ] . like jev srips, the zikv natal rgn srips carrying the egfp reporter could be applicable as a drug screening platform. therefore, the reverse genetics system for the production of zikv natal rgn srips provided an alternative, applicable, and rapid approach to studying the biological features of the unique zikv strains. the human rhabdomyosarcoma/muscle te cells were used as packaging cells expressing zikv prm and e proteins (figure ), in which transfection with the zikv natal rgn replicon significantly generated recombinant srips with the high yield of . × tcid /ml (figure ) . interestingly, te cells expressing jev c, prm, and e proteins were also used as a packaging cell line that was efficient at producing jev srips post-transfection with the jev replicon [ ] . the recombinant zikv natal rgn srips showed infectivity and self-replication in the packaging cells ( figure ). two amino acid substitutions within ns (l r and e n) (supplementary figure s ) seemed to have no influence on the activities of ns methyltransferase and rna-dependent rna polymerase. a recent study indicated that human embryonic kidney t cells had been used to establish different flavivirus prm-e-expressing packaging cells, including denv - , zikv, jev, west nile virus, yellow fever virus, and tick-borne encephalitis virus, and the cells were transfected with the denv reporter replicon to generate a chimeric flavivirus srip-based neutralization assay [ ] . therefore, recombinant zikv natal rgn srips generated in this study could be used to analyze the zikv life cycle, elucidate viral pathogenesis, screen antiviral agents, design faster and safer diagnostic tools, and even develop attenuated vaccines against zikv infection. the full-length zikv natal rgn strain genome was directly sequenced using next-generation sequencing with the total rna extracted from brain tissues from fetal autopsy cases with microcephaly (genbank accession number ku ) [ ] . in this study, recombinant zikv natal rgn srips were rescued using reverse genetic and synthetic biology techniques (figures - and supplementary figures s -s ) . three cell lines, te , sf , and arpe- , were further tested for cell susceptibility to zikv natal rgn srips ( figure ). the order of cell susceptibility to zikv natal rgn srips was te > arpe- > sf cells. the cpe level, egfp reporter intensity, and zikv ns protein expression were most obvious (> % involvement) in te cells, but were mild in arpe- cells and limited in sf cells h post-infection with srips at an moi of . tcid /cell. the results indicated that zikv natal rgn srips had a broad range of human tissue tropism with differential cell susceptibilities ( figure a ). in addition, zikv natal rgn srips had the highest attachment activity to te cells among those three cell lines ( figure b ). srip attachment to te cells was suggested to be involved in the infectivity of zikv natal rgn in te cells. this finding implied that zikv natal rgn srips had tropism for rhabdomyosarcoma/muscle and retinal pigmented epithelial cells, which was consistent with myalgia, arthralgia, retinitis, chorioretinal atrophy, and pigmentary maculopathy in symptomatic zikv infection [ , ] . therefore, the cell line susceptibility results revealed the unique features of zikv natal rgn srips and suggested the possible pathogenesis of zikv infection. in conclusion, the approach for generating recombinant zikv srips with an egfp reporter showed the genetic stability of zikv prm-e genes in the mammalian expression vector and zikv replicon sequence in the low-copy plasmid pbr , providing a faster and safer platform to study the biological aspects of zikv infection. zikv natal rgn srips were rescued by the combination of prm-e-expressing packaging cells and the zikv natal rgn replicon under cmv promoter control, which mimicked self-replicating, positive-sense, single strand rna viruses. the biological features of zikv natal rgn srips were revealed, including cpe, infectivity, cell susceptibility, and attachment activity. therefore, recombinant zikv natal rgn srips could be further used to investigate cell tropism, persistent infection, and vaccine candidates, and to establish reliable, effective, and efficient assays for screening antiviral agents and diagnosing viral infections. table s . primer pairs for real-time quantitative rt-pcr of zikv e and ns rna copies; supplemental figure s . the construction of in-frame gene fusion (a), plasmid information and restriction enzyme digestion analysis (b) of the synthesized dna segment i; supplemental figure s . the construction of in-frame gene fusion (a), plasmid information and restriction enzyme digestion analysis (b) of the synthesized dna segment ii; supplemental figure s . sequencing analysis of zikv asian natal rgn replicon with the primers listed in supplemental table s a . the amino acid substitutions were also shown in the figure. the diversification of zika virus: are there two distinct lineages? molecular evolution of zika virus during its emergence in the (th) century evolution of two major zika virus lineages: implications for pathology, immune response, and vaccine development virus pathogenesis and tissue tropism the origin and spread of a mosquito-borne virus zika virus-associated neurological disease in the adult: guillain-barré syndrome, encephalitis, and myelitis an update on zika virus infection comparative genomic analysis of pre-epidemic and epidemic zika virus strains for virological factors potentially associated with the rapidly expanding epidemic an evolutionary insight into zika virus strains isolated in the latin american region a single mutation in the prm protein of zika virus contributes to fetal microcephaly an evolutionary ns mutation enhances zika virus evasion of host 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identified in infants with presumed zika virus congenital infection all authors declare no potential conflict of interest. key: cord- -bswndfvk authors: lalle, eleonora; biava, mirella; nicastri, emanuele; colavita, francesca; di caro, antonino; vairo, francesco; lanini, simone; castilletti, concetta; langer, martin; zumla, alimuddin; kobinger, gary; capobianchi, maria r.; ippolito, giuseppe title: pulmonary involvement during the ebola virus disease date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: bswndfvk filoviruses have become a worldwide public health concern, especially during the – western africa ebola virus disease (evd) outbreak—the largest outbreak, both by number of cases and geographical extension, recorded so far in medical history. evd is associated with pathologies in several organs, including the liver, kidney, and lung. during the – western africa outbreak, ebola virus (ebov) was detected in the lung of infected patients suggesting a role in lung pathogenesis. however, little is known about lung pathogenesis and the controversial issue of aerosol transmission in evd. this review highlights the pulmonary involvement in evd, with a special focus on the new data emerging from the – ebola outbreak. ebolavirus is part of the filoviridae family, which consists of three genera: marbugvirus, cuevavirus, and ebolavirus. there are currently six known, genetically distinct, species of ebolavirus-ebola virus (ebov), sudan ebolaviurs (sudv), tai forest ebolavirus (tafv), bundibugyo ebolavirus (bdbv), reston ebolavirus (restv), and bombali ebolavirus (bomv) [ , ] . no virus has triggered fear in the general population more than the filovirus ebolavirus [ ] . ebov is categorized among the deadliest viruses, with mortality rates up to %. the zoonotic origin of outbreaks are often the result of transmission from primates, although the suspected natural reservoir for ebov, bats, is still being questioned. since it was first identified in in zaire (the actual democratic republic of congo), confirmed outbreaks, mainly in the central part of africa, have occurred, and each outbreak was accompanied by high case fatality rates up to %, including the new declared outbreak ongoing in the north kivu province of the democratic republic of the congo [ ] [ ] [ ] . the - ebola outbreak is the largest (both by number of cases and geographical extension) ebolavirus outbreak ever reported, resulting . tlr stimulates irf- and nf-κb via myd activation, leading to the release of proinflammatory cytokines and the production of ifn-α, -β, and -λ, respectively. secretion of proinflammatory cytokines and chemokines activate the immune system, through recruitment of eosinophils, neutrophils, macrophages, dendritic cells, t cells, and nk cells. most respiratory viruses have developed strategies to escape antiviral defense, mainly by interfering with the ifn system or by affecting the epithelium barrier, with the consequence of a loss of integrity and protection. furthermore, respiratory viruses can perturb (skewed or exaggerated) inflammatory responses and production of soluble mediators. ebov infection is acquired through direct contact with bodily fluids. the virus enters blood circulation through breaks in the skin and mucosa and spread to different organs, causing systemic manifestation of cardiovascular, coagulation, or inflammatory disturbances [ ] . the terminal stages of evd usually involve massive tissue injury and hemorrhage, resulting in multiorgan failure and shock, the main cause of exitus in evd patients [ ] . during evd, respiratory symptoms such as chest pain, shortness of breath, cough, and nasal discharge are signals of the multisystem involvement, but, so far, lung damage has not been directly linked to ebov replication in the respiratory tract. however, new evidences collected during the recent - ebola outbreak hypothesized shedding of the virus in the lung and identified viral replication markers in sputum samples collected from ebov infected patients [ ] . on the other hand, the high virulence of ebov is attributed in large part to the ability of this virus to interfere with the host immune response, and the high degree of variation in lung pathogenesis is usually linked to indirect damage due to endothelial and epithelial inflammation and the hyper-activation of the immune system subsequent to ebov infection. in fact, viral direct damages are always associated with indirect damages, caused by inflammatory and immune reactions elicited by the viruses through the activation of soluble mediators (cytokines and chemokines) as part of the immune response ( figure ). the acute inflammation process is characterized by increasing blood flow, which enables plasma and leukocytes to reach extra-vascular sites of injury. even though inflammation may be often restored, in evd, severe inflammation is associated with a cytokine storm and more serious pathological changes are observed. for instance, ebov in vitro infection of monocytes and macrophages triggers the robust expression of inflammatory mediators, including il- β, il- , il- , mip- a, mip- β, mcp- , and tnf-α [ , ] , whereas the dysregulation of immune mediators in humans has been associated with the secretion of other inflammatory mediators, such as il- β, il- , ccl , ccl , ccl , cxcl , cxcl , cxcl , cxcl , il , mif, spp [ ] [ ] [ ] . in addition, severe inflammatory upon entrance into the cell, viruses are recognized by the toll-like receptor (tlr) on either cell membrane or in endosomes. tlrs activate interferon regulatory factors (irfs) leading to ifn-α and ifn-β release via the toll/il- receptor domain-containing adaptor (trif). tlr stimulates irf- and nf-κb via myd activation, leading to the release of proinflammatory cytokines and the production of ifn-α, -β, and -λ, respectively. secretion of proinflammatory cytokines and chemokines activate the immune system, through recruitment of eosinophils, neutrophils, macrophages, dendritic cells, t cells, and nk cells. most respiratory viruses have developed strategies to escape antiviral defense, mainly by interfering with the ifn system or by affecting the epithelium barrier, with the consequence of a loss of integrity and protection. furthermore, respiratory viruses can perturb (skewed or exaggerated) inflammatory responses and production of soluble mediators. ebov infection is acquired through direct contact with bodily fluids. the virus enters blood circulation through breaks in the skin and mucosa and spread to different organs, causing systemic manifestation of cardiovascular, coagulation, or inflammatory disturbances [ ] . the terminal stages of evd usually involve massive tissue injury and hemorrhage, resulting in multiorgan failure and shock, the main cause of exitus in evd patients [ ] . during evd, respiratory symptoms such as chest pain, shortness of breath, cough, and nasal discharge are signals of the multisystem involvement, but, so far, lung damage has not been directly linked to ebov replication in the respiratory tract. however, new evidences collected during the recent - ebola outbreak hypothesized shedding of the virus in the lung and identified viral replication markers in sputum samples collected from ebov infected patients [ ] . on the other hand, the high virulence of ebov is attributed in large part to the ability of this virus to interfere with the host immune response, and the high degree of variation in lung pathogenesis is usually linked to indirect damage due to endothelial and epithelial inflammation and the hyper-activation of the immune system subsequent to ebov infection. in fact, viral direct damages are always associated with indirect damages, caused by inflammatory and immune reactions elicited by the viruses through the activation of soluble mediators (cytokines and chemokines) as part of the immune response ( figure ). the acute inflammation process is characterized by increasing blood flow, which enables plasma and leukocytes to reach extra-vascular sites of injury. even though inflammation may be often restored, in evd, severe inflammation is associated with a cytokine storm and more serious pathological changes are observed. for instance, ebov in vitro infection of monocytes and macrophages triggers the robust expression of inflammatory mediators, including il- β, il- , il- , mip- a, mip- β, mcp- , and tnf-α [ , ] , whereas the dysregulation of immune mediators in humans has been associated with the secretion of other inflammatory mediators, such as il- β, il- , ccl , ccl , ccl , cxcl , cxcl , cxcl , cxcl , il , mif, spp [ ] [ ] [ ] . in addition, severe inflammatory cytokines/chemokines may spill over into the circulation and result in systemic cytokine storms, which are responsible for multi-organ dysfunction and for the impairment of the vascular system and disseminated intravascular coagulation [ , ] . dendritic cells (dcs) play an essential role in the link between the innate and adaptive immune response, and their maturation is essential for the correct functionality of dcs, such as the migration, processing, and presentation of viral antigens to t-and b-cells for their activation and correct viral clearance [ , ] . ebov infection has been shown to influence these mechanisms through impairment of dcs in upregulating co-stimulatory molecules (cd , cd , and cd ) and major histocompatibility complex (mhc) class ii, as well as soluble chemokines and cytokines [ ] . ebov infection is also able to influence the adaptive immune response: severe lymphopenia and the destruction of lymphoid tissue is one of the hallmarks of ebov infection. fatal cases showed a more marked reduction of nk cells and γδ t-cell frequency, as well as a loss of peripheral blood cd + and cd + t cells [ , ] . moreover, a recent study showed that patients with fatal outcome presented lower, or often absent, levels of both ebov-specific igm and igg, which, when detected, appeared later than in survivors [ ] . overall, the alteration of the innate and adaptive response explains the paralysis of the immune system and its inability to initiate and maintain a protective immune response. at the pulmonary level, many of the pathological changes are, in fact, secondary to systemic alterations, correlating with general pathogenic mechanisms, which are the major causes of severe disease in humans, even at the respiratory level [ , ] . evd is a viral hemorrhagic fever (vhf) characterized by acute systemic manifestations with vascular damage, plasma leakage, severe inflammation, and disruption of the immune system [ ] . evd transmissibility seems to vary depending on the stage of disease [ ] . a high-level of ebov replication, associated with systemic dissemination to multiple cell types, results in a complex pathogenesis, which is linked to an increased risk of infection transmission [ ] . as stated above, these pathogenic mechanisms include detrimental immune suppression and over-activation of the immune response, disordered coagulation, and tissue damage due to direct viral and indirect host-mediated effectors. in the absence of adequate supportive care, these processes commonly result in multiple organ failure and death within about days of symptom onset in humans. it is well recognized that ebov infection is acquired by direct contact with bodily fluids. notably, studies conducted in animal models have instilled doubts about possible airborne/droplet transmission (see section . ). however, this route of infection in humans is still debated. piercy and colleagues evaluated the actual stability of the virus particles in aerosol droplets [ ] . they created ebola-containing aerosol droplets and, according to the decay rates, estimated that ebov and restv can survive in aerosols for roughly and min, respectively, at % to % relative humidity and ± • c [ ] . therefore, a key additional question to ask is whether primary pulmonary infection of ebov could be a potential scenario for the future. a fair amount of studies, based on animal experiments (table ) and clinical evidence collected during the outbreaks ( table ), suggest that pulmonary infection may be a possibility. this possibility will be fully investigated below. after its first discovery in in cynomolgus macaques imported to reston, virginia, restv was detected in domestic swine in the philippines in a co-infection with the porcine reproductive and respiratory syndrome virus (prrsv, family arteriviridae, genus arterivirus) and porcine circovirus type (pcv- ; family circoviridae) [ , ] . later on, restv was identified to cause asymptomatic infections with mild respiratory symptoms, which may result in severe mortality in cases of co-infections with other viral pathogens like viruses in the families arteriviridae and circoviridae. the virus was first isolated in lung and lymphoid tissues in the original disease investigation [ ] . however, the massive presence of the virus in the lungs may be due to the fact that restv infection in pigs has been mostly associated with other infections of the respiratory tract, which may contribute to the specific localization of the virus and the respiratory symptoms of the disease. marsh et al. [ ] conducted an experimental study to rule out the effect of other pathogens affecting pigs, using a philippines swine isolate of restv. specifically, five-week-old pigs were exposed (via the oro-nasal or subcutaneous route) to the virus, and the subsequent viral replication in internal organs and shedding of the virus from the nasopharynx was observed. the researchers detected the highest levels of virus replication in lung and lymphoid tissues, confirming previous results [ ] . the detection of restv in domestic swine raised important biosecurity concerns about the potential for the disease's emergence in humans and other livestock, mainly in animals for food consumption [ , ] . the evidence of restv seropositive individuals further increased the concern for human infections and the worries of researchers, farm owners, and the public at large (world health organization. who, , available online: https://www.who.int/csr/resources/publications/hse_ epr_ _ .pdf). interestingly, so far restv has not been seen to result in any human disease, even if there is concern that its passage through swine may allow restv to diverge and shift its potential for pathogenicity [ ] . on the other hand, several studies investigated if other ebola viruses may be transmitted through the aerosol route and may result in primary pulmonary infection [ , , ] . researchers reviewed the different animal models and offered an overview regarding the possibilities of ebola viruses causing aerosol infections in non-human primates (nhps) and other animals. experimental studies analyzed the respiratory tract involvement in filovirus infections when the animals were exposed to the virus through different aerosol routes (artificially aerosolized virus or natural aerosol transmission) [ , , , ] . in these experimental studies conducted on nhps and pigs, ebov was inoculated via the aerosol route, and, following mucosal exposure, ebov replicated, reaching high concentrations, mainly in the respiratory tract, with the development of severe lung pathology. interestingly, weingartl et al. demonstrated that piglets inoculated oro-nasally with ebov and then transferred to a different room housing macaques in an open inaccessible cage system resulted in ebov infection of all macaques, suggesting a need to revise prevention and control measures during outbreaks [ ] . viral replication was observed within alveolar spaces [ , ] , in type i pneumocytes and macrophages [ ] , and in type ii pneumocytes, bronchiolar epithelial cells, and endothelial cells [ ] , supporting the respiratory involvement. the upper and lower respiratory tract, the lymphoid tissues, and the mediastinal lymph nodes showed infection signs, as well [ ] . similarly, in experiments on cynomolgus macaques placed separately in cages with experimentally infected piglets [ ] , and on guinea pigs exposed via aerosols to a guinea pig-adapted ebov strain [ ] , viral antigens were detected within alveolar and septal macrophages, pneumocytes, epithelial cells, endothelial cells, fibroblasts, and other interstitial cells of the respiratory tree [ ] . considering the pathology of the respiratory system, the expression of disease in the lungs and the patterns of lesions seem to be influenced by the exposure routes (aerogenous or hematogenous). broncho-interstitial pneumonia, characterized by injury to both the bronchiolar and the alveolar epithelium, is commonly caused by aerogenous viral infections [ ] . moreover, such pathological features were generally not evidenced following the inoculation of ebov by other routes in nhps and laboratory animals [ , ] . as shown in animal studies, primary pulmonary infections could occur and cause active viral shedding from the respiratory tract, thus potentially setting up a cycle of ongoing respiratory transmission in humans [ , ] . overall, experimental works conducted so far have shown that ebov infection induces respiratory complications, that the virus can be shed via the respiratory secretions, and that it can cause similar pulmonary lesions both in animals exposed to aerosols and in those kept nearby in separate cages with no close contact. the pathophysiological mechanism of pulmonary disease in patients with evd is unknown. notably, autopsies were performed on a limited number of humans (about cases), primarily during the sudv and ebov evd outbreaks and revealed interesting characteristics at microscopic level. during the first known sudv outbreak, chest pain was almost universal ( % of patients), often accompanied by a dry cough. autopsies were further performed on two patients and thickening of the alveolar walls due to proliferative accumulations of alveolar cells was found [ ] . furthermore, a possible pathogenetic role of the virus in the respiratory tract was suggested by the fact that viral inclusions within alveolar macrophages and free viral particles within alveolar space were found in the lungs from fatal evd cases who showed congestion, focal intra-alveolar edema, diffuse alveolar damage, and hemorrhaging. [ , ] . one of the most common symptoms in evd patients is a cough (up to %), especially during the progression of the disease, when viral loads in serum significantly increase, and the virus is copiously emitted in most body fluids, as well as in aerosol particles of various sizes [ , ] . among the reported evd cases in the literature, respiratory symptoms were commonly reported with a wide range of symptoms, such as a cough (from % [ , ] to % [ ] ), dyspnoea or breathless (detected from % [ ] to % [ ] ), and chest pain (from . % [ ] to . % [ ] ). moreover, a who study on the first months of the epidemic in western africa found that nearly % ( out of ) of the patients experienced coughing and . % ( of ) had a bloody cough [ , ] . a study of ebov-positive patients of the - outbreak in western africa, treated in europe and usa, reported that cough and dyspnoea were present at admission in seven ( %) and five ( %) evd patients, respectively. at symptom onset, only a cough was reported in one patient. furthermore, during hospitalization, patients ( %) experienced hypoxemia while they were breathing ambient air, patients ( %) had pulmonary oedema, seven patients ( %) had pneumonia, patients ( %) had respiratory failure, and six patients ( %) had a diagnosis of acute respiratory distress syndrome (ards). of these patients, four patients ( %) received non-invasive mechanical ventilation, and seven patients ( %) received invasive mechanical ventilation [ ] . notably, the first ebov-positive patient treated in italy, mechanically ventilated for respiratory insufficiency for days, had high levels of ebov rna in the lower respiratory tract secretions. the authors concluded that the absence of other identified respiratory pathogens in broncho-alveolar lavage fluids and aspirates supported the hypothesis of a direct contribution to the lung tissues damage by ebov. notably, ebov rna was detected in bronchial aspirate fluids when the ebov rna concentration in the concomitant blood samples was barely detectable. furthermore, the blood ebov rna concentrations in the previous days were significantly lower than the concentrations detected in the bronchial aspirate samples. these findings suggest that this ebov infection is unlikely a spillover from the blood compartment, eventually accompanied by delayed clearance. instead, the most plausible explanation is that the virus actually replicated into the lower respiratory tract [ ] . in the second ebov-patient treated in italy, our group investigated the presence of ebov genetic material in the lungs and blood during the patient's treatment and recovery. the patient showed a persistence of ebov replication markers within the respiratory tract, with a prolonged detection of ebov viral rnas (negative and positive sense rnas: neg-rna and pos-rna, respectively), known to be associated with ebov replication, in the lower respiratory tract for up to five days after the ebov viral load in blood was already undetectable. these results suggest that ebov may replicate in the lungs, although it is possible that the lungs simply provided a protective environment that allowed rna to linger longer than it did in the plasma. nevertheless, the detection of pos-rna together with neg-rna in the sputum (until day and of the hospital stay, respectively) supports the concept of active viral replication within the respiratory tract, rather than plasma spill-over or prolonged rna stability [ ] . overall, the pathophysiological mechanisms of pulmonary disease in patients with evd are still uncertain, but there could be multiple contributing factors, including vascular leak from endothelial infection, cytokine dysregulation, or direct damage to ebov-infected cells (figure ). viruses , , x for peer review of of pos-rna together with neg-rna in the sputum (until day and of the hospital stay, respectively) supports the concept of active viral replication within the respiratory tract, rather than plasma spill-over or prolonged rna stability [ ] . overall, the pathophysiological mechanisms of pulmonary disease in patients with evd are still uncertain, but there could be multiple contributing factors, including vascular leak from endothelial infection, cytokine dysregulation, or direct damage to ebov-infected cells (figure ). our understanding of ebov transmission in humans mainly relies on epidemiological observations. contact with bodily fluids from evd patients remains the most likely route of transmission. notably, the number of past outbreaks and associated epidemiological studies hat carefully examine transmission patterns is small. therefore, conclusions about transmission are based on relatively limited data sets [ ] . interestingly, ( . %) of the cases in the sudv outbreak in nzara, sudan, and ( . %) of the cases during the ebov outbreak in kikwit, drc, had no direct or physical contact with an infected person or known infected dead body [ , ] , thus pointing to other possible routes of transmission, e.g., human to human respiratory tract infection through droplet and aerosols. during the - western africa epidemic, more than health care workers (hcw) were infected, with a case fatality rate of % [ ] , whereas during our understanding of ebov transmission in humans mainly relies on epidemiological observations. contact with bodily fluids from evd patients remains the most likely route of transmission. notably, the number of past outbreaks and associated epidemiological studies hat carefully examine transmission patterns is small. therefore, conclusions about transmission are based on relatively limited data sets [ ] . interestingly, ( . %) of the cases in the sudv outbreak in nzara, sudan, and ( . %) of the cases during the ebov outbreak in kikwit, drc, had no direct or physical contact with an infected person or known infected dead body [ , ] , thus pointing to other possible routes of transmission, e.g., human to human respiratory tract infection through droplet and aerosols. during the - western africa epidemic, more than health care workers (hcw) were infected, with a case fatality rate of % [ ] , whereas during the current - outbreak in drc, as of july , hcw have been already affected ( . % of total cases) [ ] . currently, full body protection is recommend by who and cdc [ , ] . all hcw involved in the care of evd patients must receive training and demonstrate competency in performing all ebola related infection control practices and procedures, specifically in proper donning and doffing ppe even if using an n mask or a powered air-purifying respirator (papr). the risk of infection via inhalation of contaminated aerosols from exposed individuals has not been documented. however, droplets containing ebov that have become aerosolized (e.g., from coughing sneezing, vomiting, invasive medical or surgical procedures, or surfaces) may have the potential to come into contact with a person's mucous membrane in their nose or mouth or non-intact skin. therefore, respiratory protection may be helpful in providing a barrier to help prevent infectious materials from contacting a wearer's mucous membranes. finally, the epidemiologic and viral evidence of ebov detection and replication in the respiratory tract raise concerns on the need of strict application of cough etiquette for patients and of droplet and/or respiratory precautions for all hcw involved in the clinical management of evd suspected and confirmed cases. acute respiratory tract infections (artis) remain a leading cause of mortality, morbidity, and economic loss, and viruses are one of the main causes of such disease. who estimates that artis cause nearly four million deaths per year, a rate of more than deaths/ , people [ ] . the microbial etiology of aris is varied, with viruses being the most common cause in humans [ ] , leading to a high level of awareness and the necessity to develop countermeasures to control them (table ) . filoviruses are not commonly considered to be viruses responsible for aris, even if respiratory symptoms may be present as a consequence of diffuse systemic alterations. interestingly, evidence collected in animal studies, in the epidemiological analysis of transmission chains, and in the most recent ebola outbreaks suggests that ebov may be able to cause primary pulmonary infection. this evidence highlights the ability of the virus to be shed in the lung, suggesting a role in lung pathogenesis. specifically, the relevant proportion of evd patients without any epidemiologic link to the exposure to contaminated biological samples or fomites, or to any contact with evd patients; the evidence of respiratory signs and symptoms commonly reported all over the clinical course; the abundance of viral antigens in the lungs in animal necropsies; the prolonged persistence of ebov detection and replication within the respiratory tract days after undetectable ebov viral load in plasma; and similar clinical patterns in several other viral respiratory tract infections are all different parameters with consistent evidence of a major role in the pathogenesis of evd in respiratory tissues [ ] . on the other hand, there is no evidence of aerosol transmission in evd. however, different studies addressing this issue have been performed [ , ] , and aerosol transmission was considered a possibility as a consequence of epidemiological observations in past outbreaks, where people showed signs of evd even in the absence of a direct or physical contact with an infected person or known infected dead body [ , ] . this hypothesis was corroborated by other studies, in which the presence of free viral particles in alveoli and within intra-alveolar macrophages demonstrated a pulmonary involvement [ ] . from a clinical point of view, the - ebov outbreak underlined the lung involvement in evd pathogenesis. in fact, only a few patients treated in europe and usa had a cough and difficulty breathing at admission. nevertheless, during the clinical progression, half of the patients experienced hypoxemia while breathing room air, one third had respiratory failure, and one fourth received invasive or non-invasive mechanical ventilation [ ] . in the italian experience at the national institute for infectious diseases "l. spallanzani" (inmi), respiratory symptoms were present in both patients, in the absence of other common respiratory pathogens [ , ] . one case required mechanical ventilation and the other presented ebov replication markers in the lungs even after clearance of the virus from the blood. the inmi experience suggests a direct role of the virus in lung pathogenesis. although lung pathogenesis in evd may be secondary to systemic alterations (correlating with general pathogenic mechanisms) the direct presence of the virus is undisputable in the lung, and its interaction with the immune system, whose hyper-activation may be the most likely explanation of the lung damage, is also indisputable. further research will be 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of the viral hemorrhagic fevers dengue viruses can infect human primary lung epithelia as well as lung carcinoma cells, and can also induce the secretion of il- and rantes pathogenesis of lassa fever. viruses pathophysiology of hantavirus pulmonary syndrome in rhesus macaques this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -qeao ghg authors: aris-brosou, stéphane; parent, louis; ibeh, neke title: viral long-term evolutionary strategies favor stability over proliferation date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qeao ghg viruses are known to have some of the highest and most diverse mutation rates found in any biological replicator, with single-stranded (ss) rna viruses evolving the fastest, and double-stranded (ds) dna viruses having rates approaching those of bacteria. as mutation rates are tightly and negatively correlated with genome size, selection is a clear driver of viral evolution. however, the role of intragenomic interactions as drivers of viral evolution is still unclear. to understand how these two processes affect the long-term evolution of viruses infecting humans, we comprehensively analyzed ssrna, ssdna, dsrna, and dsdna viruses, to find which virus types and which functions show evidence for episodic diversifying selection and correlated evolution. we show that selection mostly affects single stranded viruses, that correlated evolution is more prevalent in dna viruses, and that both processes, taken independently, mostly affect viral replication. however, the genes that are jointly affected by both processes are involved in key aspects of their life cycle, favoring viral stability over proliferation. we further show that both evolutionary processes are intimately linked at the amino acid level, which suggests that it is the joint action of selection and correlated evolution, and not just selection, that shapes the evolutionary trajectories of viruses—and possibly of their epidemiological potential. humanity is regularly reminded of the epidemiological toll of viruses, in part due to recent and ongoing viral outbreaks of influenza [ ] , ebola [ ] , and zika [ ] . thanks to recent technological and analytical developments, it is now possible to elucidate, almost in real time [ ] , their epidemiological dynamics, and unravel their evolutionary dynamics [ ] . however, while the evolutionary dynamics of rna viruses are well documented [ ] , those of other viruses are not so well known, in particular in a unifying context including major viral types such as double-stranded (ds) and single-stranded (ss) dna and rna viruses. to date, one of the most salient evolutionary features shared by all viruses is the existence of a negative correlation between mutation rate and genome size [ ] . this is a critical result as it suggests that selection is driving the evolution of mutation rates, establishing a trade-off between mutational load and availability of adaptive mutations [ ] . however, the processes driving the evolution of different types of viruses are multiple. as already argued, both ssdna and ssrna viruses share small genome sizes, high mutation rates, but also large effective population sizes, little to no gene duplication or recombination, and overlapping reading frames [ ] . this last point suggests that a less frequently explored evolutionary process in viral studies, correlated evolution, could be as critical as positive selection. correlated evolution happens when mutations at two locations in a genome occur one after the other, in a quick succession [ ] , repeatedly [ ] . typical examples include drug resistance mutations that have a fitness cost, and that require a second mutation to compensate for the first one [ ] , or trnas that require a specific base-pairing to maintain their secondary and tertiary structures, so that a mutation in the stem region necessitates a second mutation to restore the correct, functional, structure [ ] . this process is of particular interest as correlated evolution can be underlain by epistasis, which occurs when the fitness effects of these two mutations are non-additive [ ] , as in the two examples above. as such, correlated evolution can help us understand the relationship between genotype and fitness, which is a key determinant of evolutionary trajectories [ ] . to date, however, correlated evolution has only sporadically been investigated in viral evolution, and these rare instances only focused on ssrna viruses. indeed, recent work uncovered pervasive evidence for correlated evolution in influenza viruses [ , ] , and both the zika [ ] and the ebola viruses [ ] . intriguingly, in this latter case (ebola), evidence was found that sites evolving in a correlated manner could also be under positive selection-bearing the question as to how frequently these two processes, correlated evolution and positive selection, occur, possibly jointly, and if this co-occurrence is limited to ssrna viruses, or can be generalized to all viruses. to better understand the role of correlated evolution and positive selection in the evolutionary dynamics of viruses infecting humans, we constructed a nearly exhaustive viral data set spanning all dsdna, dsrna, ssrna, and ssdna viruses deposited in genbank (as of august ), and conducted an extensive survey of correlated evolution and diversifying selection in these viruses, asking more specifically about the prevalence of these two processes in each viral type, independently or jointly, with the specific hypothesis that the genes affected by both processes encode functions that are most critical to each viral life cycle. lists of dsdna, dsrna, ssrna, and ssdna viruses infecting humans were retrieved from the virusite database [ ] in august (tables s -s ); subtypes/genotypes/clades were treated as independent data sets. although some of these viruses could be segmented or not, with circular or linear genome, with positive or negative strands, and with or without overlapping reading frames, accounting for these structural features would have led to smaller and smaller data sets, precluding any statistical analysis, so that the data were not split beyond viral type. each list contained the virus names, the length of their genome, their number of protein-coding genes (cds's), and was associated with a reference coding sequence (see query_sequences.zip at [ ] ). in order to obtain corresponding sequence alignments of orthologous genes, blastn searches were performed on a custom database limited to viral genes present in the national center for biotechnology information nucleotide database with blast- . . +. for this, all gbvrl*.seq.gz files were downloaded from reference [ ], while querying virusite, and were concatenated into a single genbank file, then converted into a fasta file with readseq to specifically extract cds's [ ] . this was done to avoid retrieving ' and ' untranslated regions that would cause problems to the downstream codon analyses. blastn searches were performed for each virusite viral sequence with a stringent e-value threshold of − , keeping a maximum of sequences with at least % coverage with each query; this ensured that subtype/genotype/clade boundaries were not crossed. as the viruses retrieved from the virusite also included viruses that require a vector (e.g., arboviruses such as the dengue and yellow fever viruses), or viruses that circulate in non-human hosts but that can lead to zoonoses (e.g., the camel alphacoronavirus leading to mers [ , ] ), these stringent thresholds also ensured that sequences contained in each alignment mostly came from a single host. only data sets with at least hits were kept for downstream analyses. sequences corresponding to each accession number were retrieved from the fasta file obtained with readseq [ ] . because this file contained partial sequences, each set of retrieved sequences was first aligned with muscle . . [ ] . each alignment was then quality checked ensuring that (i) its length is a multiple of three, (ii) it starts with an atg and stops with a stop codon. alignments failing at least one condition were discarded. within each alignment, mean numbers of indelsn indels were computed for each sequence, and those containing number of indels ≥n indels + sem (standard error of the mean) were eliminated. the remaining nucleotide sequences were then re-aligned with translatorx [ ] , at the amino acid level, using the muscle aligner and their heuristics to determine the correct reading frame. alignment files were then cleaned-up with gblocks . b at the codon level using the stringent default settings [ ] . both trees and alignments are available at [ ] . to obtain gene annotations, gene ontology (go) terms were retrieved from gene sequences with hmmer go ( [ ] ), relying on the hidden markov models in [ ] (ver. febuary ). this is equivalent to performing a blast go search [ ] , but without the limitations of proprietary software. individual mapping files coming from our four viral types (ssrna, dsrna, ssdna, and dsdna viruses) were then merged to create a custom annotation file, used for go term enrichment testing with topgo [ ] , based on fisher's exact test. the reference gene list was always the entire set of genes within each viral type. phylogenetic trees were reconstructed with fasttree . . [ ] under the gtr+Γ model of evolution [ ] ; note that fasttree was recompiled locally to use double-precision arithmetics, as recommended by its authors to estimate very short branch lengths accurately. those with a nonzero tree length were midpoint rerooted using the phytools package ver. . - [ ] in r ver. . . [ ] . patterns of correlated evolution among sites were identified with the bayesian graphical model (bgm) implemented in spidermonkey [ ] (sm), which is part of hyphy ver. . . [ ] . default hyphy scripts were slightly modified to read in codon data, which were used to reconstruct mutational paths under the mg × hky substitution model [ ] at nonsynonymous sites along each branch of the estimated trees. ambiguous reconstructions were resolved by considering all possible resolutions and averaging them. these reconstructed mutational paths were then recoded as a binary matrix, with rows corresponding to branches and columns to each site of the alignment. the bgm was then used to identify the pairs of sites that exhibit correlated patterns of nonsynonymous substitutions according to their posterior probability, estimated with a markov chain monte carlo sampler that was run for steps, with a burn-in period of , steps sampling every steps for inference [ ] . patterns of episodic selection were identified based on the mixed effects model of evolution [ ] (meme), also as implemented in hyphy. the default script from ver. . . was used, still with the . . hyphy engine, to infer nonsynonymous to synonymous rate ratios ω assuming that these rates can vary across lineages and among sites. in this implementation, two categories of sites were assumed, those for which ω neg ≤ , in proportion p, and those for which ω pos > that are under positive selection, in proportion − p. evidence for selection was derived by means of a likelihood ratio test between this model, and a null model where ω pos was constrained to take its value between and . linear models were fitted through robust regressions [ ] . all r scripts and hyphy source files are available from reference [ ] (file "api_scripts.zip," in "data"). first, in order to better understand the genomic characteristics of the four types of viruses known to infect humans, dsdna (n = ), dsrna (n = ), ssrna (n = ), and ssdna (n = ), and understand how these characteristics can impact the evolutionary dynamics of these viruses (methods; figure s ), we examined the distribution of four of their genomic features. while dsdna viruses had the largest and the most variable genome lengths, ssdna were the smallest genomes by at least an order of magnitude, with both dsrna and ssrna exhibiting intermediate sizes (figure a) . because of the compact structure of these genomes, these differences were also reflected in the number of genes, protein-coding genes, and rnas encoded by the genomes of these viruses ( figure s ). in turn, these characteristics suggest that, with their large genomes, dsdna viruses potentially have a higher degree of functional redundancy than ssdna or even ssrna viruses, possibly due to duplication events [ ] , and thereby be under less stringent selective pressures than viruses with smaller genomes. on the other hand, the extent of correlated evolution and its interaction with selection is more difficult to predict. to address these questions, we first tested for the presence of episodic diversifying selection in each viral type. altogether, we found extensive differences among all four viral types in terms of the number of genes under selection (figure b ; x = . , d f = , p < . × − ), and that single stranded viruses were more subject to selection than double stranded viruses (x = . , d f = , p < . × − ). indeed, and contra our original hypothesis, there were no differences between dsdna and dsrna viruses (x = . , d f = , p = . ), or between ssdna and ssrna viruses in terms of prevalence of diversifying selection (x = . , d f = , p = . ). note that these differences cannot be attributed to genetic diversity, as dsdna and dsrna, which have similar levels of selection, have however different levels of diversity ( figure s ). however, it is unlikely that "strandedness" (single vs. double stranded genetic material) alone drives selection, even if greater instability can be postulated in single-stranded nucleic acids [ ] . indeed, strandedness is negatively correlated with genome size (t = − . , d f = . , p = . × − ), which is itself correlated with mutation rates [ , ] . as a result, episodic diversifying selection is mostly driven by structural aspects of viral genomes, which condition mutation rates across all virus types [ ] . to understand if similar aspects drive intragenic correlated evolution, we counted in the same way the number of genes for which we could find evidence for interactions. here, again, we found extensive differences among all four viral types (figure c ; x = . , d f = , p < . × − ), with no difference between dsdna and ssdna viruses (x = . , d f = , p = . ), or between dsrna and ssrna viruses (x = . , d f = , p = . ). again, diversity is not driving these differences, as both dsdna/ssdna and dsrna/ssrna have significantly different levels of diversity ( figure s ). as a result, the prevalence of correlated evolution seems to be mostly driven by the nature of viral genetic material. at least two processes can underpin correlated evolution: linkage and epistasis [ ] . as recombination is pervasive in some dsdna viruses [ ] , linkage alone may not explain the high prevalence of correlated evolution in dna viruses (close to %: figure c ). rather, this pattern suggests that intragenic constraints are higher in dna viruses for unknown structural reasons, maybe due to protein structure [ ] , or in the same way that recombination in rna viruses may be driven by mechanistic constraints associated with genome structures and viral life cycles [ ] . while previous work showed that both diversifying selection and correlated evolution can affect the same gene and even the same site in a viral genome such as ebola's [ ] , an ssrna virus, the generality of this association is still unknown. at the gene level, we found strong heterogeneity among all four viral types for gene numbers showing evidence for selection, correlated evolution or both (x = . , d f = , p < . × − ). surprisingly, ssrna viruses are those that show the least overlap between selection and correlated evolution, with only % of the genes evolving under both mechanisms (figure d ). patterns are, however, much more difficult to extract here, mostly because the number of genes involved becomes quite small, in particular for the virus type with most overlap, the dsrna viruses (figure d: . %, i.e., five genes). to assess the extent to which some of these differences at the gene level are functionally driven, we extracted the gene ontology (go) annotations, or go terms, associated with the genes analyzed, as well as those under selection, correlated evolution, or both-focusing exclusively on the virus types for which we had the largest samples sizes, dsdna and ssrna viruses (figure e) . each of the three parts of the ontology (molecular function [mf], biological process [bp], and cellular component [cc]) was first limited to the second level of go in order to derive a high-level interpretation (low-level descriptions are shown in tables s -s ). figure e shows that go terms related to catalytic activity (mf), involved in the establishment or maintenance of a certain location (bp) at the membrane level (cc) are predominantly present in both dsdna and ssrna genomes. however, only a subset of these go terms is mostly under episodic diversifying selection: helicase activity/binding (mf) and replication (bp) in the host cell nucleus (cc) in dsdna, while it is mostly peptidase activity and methylation on the viral envelope in ssrna viruses (table s ; p < . ). in spite of these differences, we note that episodic diversifying selection mostly affects genes involved in viral replication (table s ). genes that are affected by correlated evolution include: helicase activity and binding at the interface of multiple compartments in dsdna viruses, or transferase activity and protein modifications on the envelope in ssrna viruses (table s ) . again, most of these functions and processes are involved in viral replication (table s ) . at the intersection of these evolutionary processes however, the genes that are jointly affected by selection and correlated evolution are involved in structural integrity and assembly within or outside a cell at the capsid level for dsdna viruses, or in interacting with host cell surface via antigen activity on the viral envelope for ssrna viruses-functions and processes that are mostly involved in "viral stability" (cell entry, integrity, assembly, immune escape; table s ). this suggests that, despite key differences in life history strategies adopted by dsdna and ssrna, there is a certain unity across viral types, where genes involved in replication are mostly under either selection or correlated evolution, while those involved in viral stability are mostly affected by both evolutionary processes-as has been shown for the influenza [ ] and the ebola [ ] viruses (both cases involved a glycoprotein required for cell entry). because these two evolutionary processes are required by epistasis, it is possible that the genes involved in viral stability are the most likely to show evidence for non-additive fitness effects-and hence shape viral fitness landscapes. this tension between replication and stability is evocative of the existence of trade-offs between capsid stability and proliferation within a host [ ] , or fecundity and lifespan [ ] in dsdna viruses, which supports the idea that it is the joint action of selection and of correlated evolution that shapes viral life histories. the previous analyses were at the gene level. one outstanding question is whether this relationship between selection and correlated evolution also holds at the amino acid level that is, if the amino acids under selection are also involved in correlated evolution. this question was addressed in two different ways, all virus types confounded (in order to have larger sample sizes), by focusing on one process at a time, and finding at what point evidence supporting the second process becomes significant. first, we searched the list of pairs of sites evolving in a correlated manner (the sm sites) to see if at least one pair member was potentially evolving under episodic diversifying selection (the meme sites), irrespective of its probability of being a meme site. for this, we identified pairs of sm sites, that is those with a posterior probability ≥ . , and plotted their posterior probability as a function of the − log probability of each pair member to be under selection (figure a ). both the least-square (ρ slope = . , t = . , p = . ) and the robust regressions (ρ slope = . , t = . , p = . ) have positive and significant slopes, hereby demonstrating the existence of a relationship between correlated evolution and episodic diversifying selection at the amino acid level-even if most of the sm sites show weak evidence of selection (at p ≤ . , gray broken line in figure a) . thus, to validate the existence of this relationship, we then took the list of meme sites, and searched the list of sm sites to see if at least one pair member was potentially a meme site, irrespective of its posterior probability of being in an sm site pair. for this, we identified the meme sites, that is those with a p-value ≤ . (≥ on a − log scale), and plotted this (on the x-scale) vs. their posterior probability of being in an sm pair (on the y-scale; figure b ). both the least-square (ρ slope = . , t = . , p = . ) and the robust regressions (ρ slope = . , t = . , p = . ) have positive and significant slopes, further confirming the existence of a relationship between correlated evolution and episodic diversifying selection at the amino acid site level. note, however, that this latter analysis is more informative than the former, as the density also shows that most of the sites under selection are involved in weak interactions. this is intriguingly reminiscent of the involvement of weakly interacting pairs of sites in severe outbreaks or pandemics [ ] . altogether, we showed that episodic diversifying selection is mostly found in single stranded viruses, while correlated evolution is more prevalent in dna viruses. more critically, we also showed that the genes affected by each process, when acting independently, are involved in viral replication. however, the genes that are jointly affected by both processes are mostly involved in viral stability (cell entry, integrity, assembly, immune escape), and that the same amino acid sites tend to be affected by both processes. in retrospect, this tight relationship between selection and correlated evolution may not be surprising, as correlated evolution can be underlain by epistasis [ , ] , which directly involves selection (in a non-additive way). epistasis being a key determinant of fitness landscapes, and hence of evolutionary trajectories [ ] , our results suggest that, in the long-term, both processes jointly shape the life history of viruses, favoring stability over proliferation. if so, analyzing viral evolution in the joint light of selection and correlated evolution might help us better predict how viruses that affect humans might evolve [ , ] -as predicting their evolution [ ] and epidemiology [ ] has a long history fraught with mixed success. we note however that we neglected some aspects of viral structure: indeed, viruses can be segmented or not, with a circular or linear genome, with positive or negative strands, overlapping reading frames, complications that we could not consider here due to the resulting small sample sizes, even if these factors can impact the mode of evolution of viruses [ ] . future work should however strive to address these limitations. we also neglected the population genetics context in which different viruses evolve, a context that can often be correlated to structural constraints [ , ] . furthermore, as we solely focused on intragenic interactions, and not intergenic or higher order correlations, it is not impossible that we missed higher-level constraints affecting viral evolution. in particular, it is possible that intergenic correlations reveal the nature of trade-offs shaping the life history strategies in viruses [ ] . future modeling [ ] and empirical [ ] work should probably focus on elucidating not only these constraints, but also the theoretical basis connecting correlated evolution, if not epistasis, to selection in shaping the evolutionary strategies of biological replicators, as current evidence linking these processes is currently limited to the influenza [ ] and the ebola [ ] viruses. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : overview of the experimental procedures, figure s : physical characteristics of the four types of viruses, figure s : viral diversity across viral types, table s : go enrichment tests for all the meme genes of interests, table s : go enrichment tests for all the sm genes of interests, table s : go enrichment tests for all the sm + meme genes of interests. we thank three anonymous reviewers for providing us with constructive comments, as well as compute canada and ontario's centre for advanced computing for giving us access to their servers. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. the origins and evolutionary genomics of the swine-origin h n influenza a epidemic genomic surveillance elucidates ebola virus origin and transmission during the outbreak zika virus in the americas: early epidemiological and genetic findings mobile real-time surveillance of zika virus in brazil unifying the epidemiological and evolutionary dynamics of pathogens the evolution and emergence of rna viruses what does virus evolution tell us about virus origins? prevalence of epistasis in the evolution of influenza a surface proteins the unsolved challenge to phylogenetic correlation tests for categorical characters stability-mediated epistasis constrains the evolution of an influenza protein compensatory evolution in mitochondrial trnas navigates valleys of low fitness the 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high? from molecular genetics to phylodynamics: evolutionary relevance of mutation rates across viruses molecular evolution of human species d adenoviruses constraints from protein structure and intra-molecular coevolution influence the fitness of hiv- recombinants why do rna viruses recombine? viruses' life history: towards a mechanistic basis of a trade-off between survival and reproduction among phages experimental selection reveals a trade-off between fecundity and lifespan in the coliphage qß darwinian evolution can follow only very few mutational paths to fitter proteins evolutionary footprint of epistasis predicting the emergence of h n influenza viruses reveals contrasted modes of evolution of ha and na antigens forecasting national and regional influenza-like illness for the usa coordinated evolution of influenza a surface proteins deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by mxa key: cord- -kje lvgl authors: pigeyre, laetitia; schatz, malvina; ravallec, marc; gasmi, leila; nègre, nicolas; clouet, cécile; seveno, martial; el koulali, khadija; decourcelle, mathilde; guerardel, yann; cot, didier; dupressoir, thierry; gosselin-grenet, anne-sophie; ogliastro, mylène title: interaction of a densovirus with glycans of the peritrophic matrix mediates oral infection of the lepidopteran pest spodoptera frugiperda date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: kje lvgl the success of oral infection by viruses depends on their capacity to overcome the gut epithelial barrier of their host to crossing over apical, mucous extracellular matrices. as orally transmitted viruses, densoviruses, are also challenged by the complexity of the insect gut barriers, more specifically by the chitinous peritrophic matrix, that lines and protects the midgut epithelium; how capsids stick to and cross these barriers to reach their final cell destination where replication goes has been poorly studied in insects. here, we analyzed the early interaction of the junonia coenia densovirus (jcdv) with the midgut barriers of caterpillars from the pest spodoptera frugiperda. using combination of imaging, biochemical, proteomic and transcriptomic analyses, we examined in vitro, ex vivo and in vivo the early interaction of the capsids with the peritrophic matrix and the consequence of early oral infection on the overall gut function. we show that the jcdv particle rapidly adheres to the peritrophic matrix through interaction with different glycans including chitin and glycoproteins, and that these interactions are necessary for oral infection. proteomic analyses of jcdv binding proteins of the peritrophic matrix revealed mucins and non-mucins proteins including enzymes already known to act as receptors for several insect pathogens. in addition, we show that jcdv early infection results in an arrest of n-acetylglucosamine secretion and a disruption in the integrity of the peritrophic matrix, which may help viral particles to pass through. finally, jcdv early infection induces changes in midgut genes expression favoring an increased metabolism including an increased translational activity. these dysregulations probably participate to the overall dysfunction of the gut barrier in the early steps of viral pathogenesis. a better understanding of early steps of densovirus infection process is crucial to build biocontrol strategies against major insect pests. the transmission of parvoviruses predominantly occurs by horizontal routes through inhalation or oral exposure, making interaction with mucosal epithelia a crucial part of their pathogenesis (for review [ ] ). the oral route represents a major challenge for viruses as they need to overcome a diversity of barriers to invade their host. indeed, most animal epithelia are covered in their apical surface by a carbohydrate-rich meshwork of various complexity and thickness, the glycocalyx, which can be coated by an additional layer of secreted mucus [ ] . these structures constitute successive protective surfaces where viruses aggregate and either access to attachment factors and receptors at the surface of the epithelial cells or are eliminated by luminal or cilia movements [ ] . this dual fate depends on the virus affinity for glycans, which must allow to escape the trap of the mucus. the diversity of glycans present on the epithelial surfaces vary between and within species and therefore constitute an important component of the innate immunity and of the species barrier [ ] . members of the parvoviridae family are non-enveloped viruses that have a simple capsid with t = icosaedral symmetry protecting a - kb linear, single stranded (ss) dna genome [ ] . they can cause diseases of various severity in a wide range of animals. parvovirus interest in human and animal health or in biomedicine as vectors for gene transfer, has long focused research to understand their cell tropism and entry mechanisms. a number of cellular attachment factors and receptors have been characterized, mostly for vertebrates' parvoviruses. they highlight the importance of glycans in capsid recognition and binding specificity [ ] [ ] [ ] . however, how capsids interact with epithelia remains poorly known, mainly due to difficulties to reconstitute in cellular models the complexity of animal epithelial systems [ , ] . insect parvoviruses, named densoviruses, can be highly pathogenic, a feature that can both represent threats for insect mass rearing or opportunities for biocontrol against harmful insects as alternative to chemicals. developing methods against infections or tools for biocontrol requires a deep understanding of the mechanisms driving host range and pathogenesis. like their vertebrate counterparts, densoviruses are mainly transmitted orally, gut recognition and binding constitute the primary step of their pathogenesis. the mechanisms determining densovirus specificity are poorly known. depending on species, densovirus replication can be either restricted to or exclude the gut, viral particles being then transported across the epithelium by transcytosis to reach internal organs where replication goes [ ] [ ] [ ] [ ] . only one cellular receptor has been so far characterized for a "gut restricted" densovirus. it is a mucin of the gut of the silkworm bombyx mori, whose inactivation makes silkworms resistant to infection with the bombyx mori densovirus type (bmdv) [ ] . we have previously reported that gut transcytosis of the junonia coenia densovirus (jcdv) involves a gut specific receptor-dependent mechanism in caterpillars [ ] . however, the mechanisms used by viral particles to overcome the successive intestinal barriers of different structures and composition remain elusive. in insects, the intestinal tract is covered by a chitinous acellular layer, which has specific features due to the dual embryonic origin of the gut. indeed, anterior and posterior extremities of the gut are ectodermal and the acellular layer is covered by an impermeable cuticle. the midgut section is endodermal and has no cuticle but is covered in most insects by a semi-permeable membrane, named the peritrophic matrix (pm) (for review [ ] ). the pm forms a highly organized lattice of chitin fibrils associated with glycoproteins, mainly peritrophins that have a chitin-binding domain [ , ] . the midgut is thus the portal of entry for most pathogens and their interaction with the pm a critical step of their pathogenesis. the pm forms pores whose size varies between insect species (e.g., - nm in lepidoptera and up to nm in coleoptera) and developmental stages [ , ] . large entomopathogenic viruses have developed specific mechanisms to pass through extracellular matrices including virus-encoded enzymes and specific proteins that are associated with the viral particles of baculo-and entomopoxviruses [ ] [ ] [ ] [ ] . how densoviruses cope with the physical barriers that constitute the gut and in particular the pm is so far unknow. due to their small size, it was initially thought they could diffuse passively across the pores of the matrix, but measures of the size of pores, the complexity of the pm and the nature of the interactions between components make this hypothesis unlikely [ , ] . we previously reported that following oral infection, viral particles of jcdv, a type species ofthe ambidensovirus genus, aggregate on the pm of spodoptera frugiperda caterpillars as a first step of the infection process [ ] . such rapid virus concentration on a carbohydrate-rich surface suggested a lectin-like activities of the capsids. although there is no sequence similarity of the unique protein making the surface of the jcdv with lectin domains, its structure displays similarities with cellular carbohydrate binding proteins including lectins, which suggests that capsids could indeed recognize and bind carbohydrates [ ] . herein, we used a combination of approaches, including microscopy, biochemistry, proteomics and transcriptomics, to decipher the interaction of jcdv with pm major components (i.e., chitin, glycans and proteins). we found that capsids affinity for the pm might result from multiple interactions with different glycans including chitin and glycosylated proteins. in addition, we showed that jcdv early infection results in (i) an arrest of n-acetylglucosamine (glcnac) secretion by epithelial cells associated with a disorganization of the pm structure mimicking the effect of chitin-binding plant lectin; (ii) substantial changes in the expression of gut genes, which may also contribute to an early gut dysfunction and participate to viral pathogenesis. caterpillars were reared under controlled conditions ( ± • c, to % relative humidity [rh], -h light, -h dark photoperiod) on a wheat germ-based artificial diet. jcdv was amplified by oral infection. at death, larvae were crushed and virus extraction was processed by clarification and filtration on . µm to constitute a semi-purified virus stock (jcdv). to obtain a purified viral stock (p_jcdv), the semi-purified virus stock was loaded on optiprep tm (sigma-aldrich, lyon, france) density gradient and dialyzed against phosphate-buffered saline (pbs) x as previously described in [ ] . viral concentrations were estimated by quantitative pcr (qpcr) as described in [ ] and expressed as viral equivalent genomes ([veg]). virus titers were determined by the tissue culture assay method ( % tissue culture infective dose (tcid ) in the permissive ld cells as previously described [ ] . calcofluor white m r (calcofluor; f ), n-acetyl-d-glucosamine (glcnac; a ), n-acetyl-d-galactosamine (galnac; a ), d-(+)-fucose (fucose; f ), d-(+)-mannose (mannose; m ), mucin from porcine stomach (porcine mucin; m ) and wga-fitc conjugate (l ) were all purchased from sigma-aldrich (lyon, france). for in vivo bioassays, third-instar (l ) s. frugiperda caterpillars were individually infected by feeding with jcdv ( veg/caterpillar) or by intrahemocelic injection ( veg/caterpillar). each treatment was applied to a cohort of caterpillars and three independent experiments were performed. calcofluor ( . % to %) was concomitantly administrated with the virus to l or l caterpillars (as described in the figures). for competition assays, jcdv was incubated with glycans (glcnac, galnac, fucose or mannose at µm and/or mm; for one hour before feeding or injection. in all experiments, control larvae were fed or injected with pbs. caterpillars mortality was recorded each day during days and results were presented as survival rates per day. the time to death was assessed by comparing the survival curves using the kaplan meier method (graphpad prism software, version ). the significance between groups were analyzed using log-rank (mantel-cox) tests and gehan-breslow-wilcoxon tests. rabbit erythrocytes cells were diluted at % (v/v) with mm nacl and treated with . mg/ml of trypsin (sigma) for min at • c. after washes with mm nacl, µl serially two-fold dilutions of viral inocula (jcdv or p_jcdv; started from veg/µl) were mixed with µl of erythrocytes in well microtiter plates for min at • c. positive controls of hemagglutination was performed using µl of wga ( mg/ml). fifty µl of erythrocytes were subsequently added to each well for min at • c. hemagglutination was read under a light microscope. for calcofluor experiments, pms were isolated from sixth-instar (l ) s. frugiperda caterpillars, previously anesthetized on ice, then opened and washed with phosphate buffered saline (pbs) to remove the food bolus. pms isolated from caterpillars treated with calcofluor were fixed h with % paraformaldehyde (pfa), then dehydrated with increasing concentrations of ethanol ( % to %) and in : ( % ethanol and hexamethyldisilazane [hmds]) for min and finally in % hmds for min. after overnight evaporation, samples were ultimately coated with platinium and observed with a scanning electron microscope (sem) hitachi s- , with an acceleration voltage of kv and a working distance of mm. for ex vivo infection experiments, pms were isolated as described above and incubated for min at room temperature (rt) with jcdv ( veg/µl; µl), or with jcdv pre-incubated for h with glycans (glcnac, galnac, fucose or mannose; µm to mm) before infection. the pms were then washed with pbs and fixed with % pfa as above. immunolabelling of jcdv particles was performed using a specific rabbit anti-capsid antibody ( : , ; eurogentec) incubated for h at rt and an anti-rabbit secondary antibody (alexa fluor ® ; : ; invitrogen, carlsbad, ca, usa) incubated for min at rt. labeled pms were then mounted in dako (sigma) on coverslips for observation. for epifluorescence measurements, we worked on a zeiss axioimager z , equipped with suitable filters for the dyes, using a ×/ . plan-apochromat oil ph and a ×/ . plan-apochromat oil ph objectives. for structured illumination images, the zeiss apotome-slider was introduced into the field-stop plane of the microscope to improve resolution of images. we used the zen software to operate the microscope and took at least images ( fields per pm) for each treatment. all images were taken with a cmos orca flash . b&w camera. images were processed with fiji software. the intensity of fluorescence (arbitrary unit) were measured on epifluorescence images. statistical analyses were performed using the non-parametric kruskal-wallis test (graphpad prism software, version , graphpad software, san diego, ca, usa). each experiment was repeated at least three times, and each independent experiment gave similar results. l s. frugiperda caterpillars were individually infected by feeding as in section . . twenty four hours later, caterpillars were anesthetized on ice and sacrificed. in parallel, caterpillars were kept until death to insure their infection status. sacrificed caterpillars were fixed in % pfa + . % glutaraldehyde for h at • c and dehydrated by incubations in increasing concentrations of ethanol ( % to %) before progressive embedding in % unicryl resin (bb international) as described in [ ] . semi-thin sections ( µm) were cut in the central part of the caterpillars corresponding to the midgut. after min of incubation with wga-fitc ( : ), to label glcnac and the pm, or with phalloidin-fitc ( : ; sigma), to label actin cytoskeleton, and min with dapi ( µg/ml; invitrogen) to label nuclei, semi-thin sections were mounted and observed with a zeiss axioimager z microscope using the structured illumination module zeiss apotome-slider as in . . images were taken with a cmos orca flash . b&w camera using fixed parameters for all treatments and processed with fiji software. ten µl of chitin beads (new england biolab) were washed three times with pbs and added in µl of virus suspension ( veg/µl in pbs). after h of incubation with gentle rotation, beads were pulled-down by centrifugation ( × g for min à • c), washed five times with pbs, then resuspended in laemmli buffer × ( % sds, % glycerol, % -mercaptoethanol, . % bromophenol blue and . m tris hcl, ph ) and heated at • c for min before western blot analysis. two µl ( % of the pull down and % of the initial input) was loaded on - % polyacrylamide tris-hcl gel (mini-protean ® tgx™ precast gels, biorad, hercules, ca, usa) and separated by sds-page for h at v. next, samples were transferred to pvdf membrane (immobilon-p, merck) for h at ma. subsequently, the membrane was saturated with % of milk in pbs/tween . % (pbst), then incubated h at rt with the primary anti-capsid antibody ( : , ; see above). after washes in pbst, the membrane was incubated h at rt with an anti-rabbit secondary antibody hrp-conjugated ( : ; biorad, hercules, ca, usa). proteins were revealed by enhanced chemiluminescence (millipore, burlington, ma, usa) using a chemidoc imager (biorad). peritrophins were extracted from pools of five pms from sixth instar s. frugiperda in µl of laemmli buffer x, then boiled and heated at • c for min [ ] . after centrifugation at × g for min • c, the supernatant was collected and / was loaded on - % polyacrylamide tris-hcl gel and separated by sds-page as above. thirty µg of porcine mucins were also loaded on the same gel. gel was stained with page blue (thermo fisher scientific, waltham, ma, usa) to analyze total proteins or with periodic acid-schiff (pas) as described in [ ] to analyze glycosylated proteins. proteins were also transferred on nitrocellulose membranes (biorad) for h at v for viral protein overlay binding assay [ ] . briefly, the membrane was saturated with % bsa in pbst for h at rt, then incubated ovn at • c with jcdv ( veg diluted in pbst containing % bsa). after washes in pbst, the membrane was incubated with the rabbit anti-capsid antibody ( : ; see above), then the anti-rabbit secondary antibody hrp-conjugated, and proteins were revealed by enhanced chemiluminescence as above. protein bands revealed by vopba were cut in sds-page gel stained with page blue ( replicates, bands) and destained with three washes in % acetonitrile and mm triethylammonium bicarbonate (teabc). after protein reduction (with mm dithiothreitol in mm teabc at • c for min in the dark) and alkylation ( mm iodoacetamide teabc at room temperature for min) proteins were digested in-gel using trypsin ( . µg/band, gold, promega, madison, wi, usa) as previously described [ ] . digested products were dehydrated in a vacuum centrifuge and resuspended with . % trifluoroacetic acid (tfa) and % acn. the generated peptides were analyzed online by nano-flow hplc-nanoelectrospray ionization using a q-exactive plus mass spectrometer (thermo fisher scientific, waltham, ma, usa) coupled to an ultimate rslc (thermo fisher scientific). desalting and pre-concentration of samples were performed on-line on a pepmap ® pre-column ( . mm × mm, dionex, sunnyvale, ca, usa). the capillary reverse-phase column ( . mm × mm, acclaim pepmap ® c , thermo fisher scientific) fitted with an uncoated silica picotip emitter (new objective, woburn, ma, usa) was first equilibrated in solvent a ( . % formic acid) and a multistep linear gradient of acetonitrile consisting of - % of solvent b ( . % formic acid in % acetonitrile) for min, - % for min and % for min, at nl/min was used to elute peptides from. spectra were acquired with the instrument operating in the information-dependent acquisition mode throughout the hplc gradient. survey scans were acquired in the orbitrap system with resolution set at a value of , . up to twelve of the most intense ions per cycle were fragmented and analyzed using a resolution of , . peptide fragmentation was performed using nitrogen gas on the most abundant and at least doubly charged ions detected in the initial ms scan and an active exclusion time of s. for all full scan measurements with the orbitrap detector a lock-mass ion from ambient air (m/z . ) was used as an internal calibrant as described [ ] . analysis was performed using the maxquant software (version . . . ) [ ] . all ms/ms spectra were searched using andromeda against a decoy database consisting of a combination of s. frugiperda databases [ ] and classical contaminants, containing forward and reverse entities. the following settings were applied: spectra were searched with a mass tolerance of ppm (ms) and . th (ms/ms). enzyme specificity was set to trypsin. up to two missed cleavages were allowed and only peptides with at least seven amino acids in length were considered. carbamidomethylation was set as fixed cystein modification and oxidation was set as variable methionine modification for searches. fdr was set at . for peptides and proteins. sequences which found homology were annotated according to the gene ontology (go) terms and classified using blast go software (https://www.blast go.com/; [ ] ). the enrichment in go terms compared to the s. frugiperda reference (predicted proteins from the ogs . genome; gouin et al., ) was analyzed with the same software (fdr set at . ). fourth instar s. frugiperda caterpillars were orally infected or not with jcdv ( veg per caterpillar; twenty caterpillars per condition). at -day p.i. or days p.i., caterpillars were anesthetized on ice and dissected. the midguts were washed in pbs to eliminate the food bolus and the pms. trachea, malpighi tubes and visceral muscles were removed and the epithelia were incubated with . % trypsin for min to dissociate the tissues. after washes, gut cells were lysed in µl of trizol ® reagent (invitrogen) for total rna extraction according to the manufacturer's instructions. total rna amount and purity were checked by using spectrophotometrer nanodrop nd- (thermo scientific) and the integrity of total rna was analyzed by capillary electrophoresis ( bioanalyzer instrument, agilent, santa clara, ca, usa). we used digital gene expression (dge) method that generates short sequences (tags) specific for mrna [ ] [ ] [ ] . four dge libraries were constructed from midgut total rna extracted from s. frugiperda caterpillars infected (or not) for and days. sequence tag preparation was done with illumina's digital gene expression tag profiling kit according to the manufacturer's protocol (version . b) as described in [ ] . for fourth libraries, µg of total rna were incubated with oligo-dt beads. first-and second-strand cdna syntheses were performed using superscript ii reverse transcription kit according to the manufacturer's instructions (invitrogen). the cdnas were cleaved using the nlaiii anchoring enzyme. subsequently, digested cdnas were ligated with the gex adapter containing a restriction site for mmei. the second digestion with mmei was performed, which cuts bp downstream of the catg site. at this point, the fragments detach from the beads. the gex adapter was ligated to the end of the tag. a pcr amplification with cycles using phusion polymerase (finnzymes) was performed with primers complementary to the adapter sequences to enrich the samples for the desired fragments. the resulting fragments of bp were purified by excision from a % polyacrylamide tbe gel. the dna was eluted from the gel using spin-x cellulose acetate filter ( . µm), precipitated, resuspended in mm tris-hcl (ph . ) and quantified using nanodrop spectrophotometer. cluster generation was performed after applying pm of each sample to the individual lanes of the illumina g flowcell. after hybridization of the sequencing primer to the single-stranded products, cycles of base incorporation were carried out on the g analyzer according to the manufacturer's instructions. image analysis and base calling were performed using the illumina pipeline, where sequence tags were obtained after purity filtering. we could assign % of the tags (supplementary materials tables s -s ) out of which % correspond to multiple matches and were discarded from functional analysis with go. functional annotation was performed using biotag software (skuld-tech, grabels, france). the statistical value of dge data comparisons, as a function of tag counts, was calculated by assuming that each tag has an equal chance of being detected. differential expression of the tag counts of the infected vs. mock conditions was performed to obtain a list of up-and down-regulated tags for each condition. tags for which differential expression was ≥ fold change were assigned using the reference databases for s. frugiperda [ , ] . sequences with homology were annotated according to the go terms and classified using blast go software (https://www.blast go.com/; [ ] ) and represented at level of biological process and molecular function. the enrichment in go terms compared to the s. frugiperda reference (predicted transcripts from the ogs . genome) was analyzed with the same software (fdr set at . ). the junonia coenia densovirus (former jcdnv) corresponds to the complete genome of the oxford isolate (genbank accession number kc . ). early studies have estimated that the average pore size of the pm is around nm in s. frugiperda caterpillars, which might exclude nm densovirus particles [ ] . to assess the pm barrier function against densovirus infection, we disrupted its structure by feeding third instar (l ) larvae with sub lethal doses ( . %) of the chitin-binding agent calcofluor white prior jcdv infection [ ] [ ] [ ] [ ] [ ] . we then measured larval mortality rates daily. results showed that jcdv infected larvae pre-treated with calcofluor displayed a significant shorter median time to death (lt ) compared to untreated infected larvae ( vs. days p.i. for control larvae; p < . ) ( figure a and supplementary materials figure s ), supporting that the pm can limit jcdv infection. noteworthy, an increased larval mortality was also observed at late time post-treatment with calcofluor in mock-infected caterpillars compared to untreated controls ( % at days p.i.), confirming a detrimental inhibition of chitin assembly on larval development [ ] . larvae (n = ) were infected individually by feeding with jcdv ( veg/caterpillar) concomitantly with . % ( µg) of calcofluor or pbs as a control. caterpillar mortality was recorded once a day during days and results were presented as survival rates per day. three independent experiments were performed, each independent experiment gave similar results, one is represented here. the log-rank (mantel-cox) and the gehan-breslow-wilcoxon tests were used to determine statistical significance. p values of less than . were considered significant (**, p < . ). pbs refers to control (pbs-treated and non-infected) caterpillars; calcofluor refers to calcofluor-treated and non-infected caterpillars; jcdv refers to jcdv-infected caterpillars and jcdv+calcofluor to calcofluor-treated and jcdv-infected caterpillars. (b) calcofluor disrupts the pm integrity. sem images of pm ultrastructure isolated from l caterpillars fed with pbs ( %), . % ( µg) or % ( µg) of calcofluor. endoperitrophic face is shown (bars, nm). we analyzed the effect of calcofluor on the pm integrity by scanning electron microscopy (sem). because the pm of l larvae has a gel-like structure that cannot be manipulated, we took l larvae for this experiment as the pm is thick and solid at this stage and can be easily dissected. l caterpillars were fed with up to % calcofluor (not lethal at h post treatment) and pms were isolated h post-treatment and prepared for sem analysis. as shown by figure b , pms from control larvae displayed a highly organized structure, similar to pms from caterpillars treated with . % calcofluor. by contrast, pms from caterpillar fed with % calcofluor had a clear disrupted structure with enlarged pores, confirming that calcofluor binding to chitin fibrils compromised the integrity of the matrix. the rapid recognition of the pm by jcdv capsids suggests that their affinity for glycans is important for the oral infection process. to test this hypothesis, we first assayed the capsid ability to agglutinate erythrocytes, a feature displayed by vertebrate parvoviruses [ , ] . we performed a typical hemagglutination assay, adding serial dilutions of the virus inoculum to rabbit erythrocytes ( figure ). the first dilutions ( : and : ) of jcdv triggered a strong hemolysis of erythrocytes suggesting a toxic effect of the viral inoculum, ie capsids or some host-derived component associated with the inoculum. it is worthy to note that we use semi-purified inoculum as it mimics naturally occurring infections. jcdv was therefore further purified on a density gradient (p_jcdv) and similarly assayed for hemagglutination. a clear hemagglutination was obtained with p_jcdv, supporting that toxicity is likely due to a host-derived component that can be eliminated during the purification process. hemagglutination with p_jcdv was obtained up to the third dilution (hemagglutination titer of : ), which indicates a rather weak interaction of the capsids with glycans at the surface of (mammalian) erythrocytes. to better understand capsid affinity for glycans, we performed a competition bioassay using monomeric glycans as jcdv-binding competitors. jcdv binding was revealed with an immunofluorescence staining on the pms using a specific anti-capsid antibody. we quantified this fluorescence as a proxy of binding and competition. we first performed competition ex vivo on isolated pms incubated with jcdv in the presence of four monosaccharides commonly found in insects [ ] , ie n-acetyl-d-glucosamine (glcnac), which is the monomeric unit of chitin, n-acetyl-d-galactosamine (galnac), d-fucose and d-mannose (figure ). we first verified with a dot blot assay that capsids interaction with monosaccharides were not interfering with antibody recognition, which validated the competition bioassay (supplementary materials figure s ). as shown in figure , jcdv binding resulted in an intense fluorescence signal on the pms (left panel), which was similarly competed away by the four monosacharides and within a similar concentration range ( . mm to mm). we noted that fluorescence quantification did not result in a strictly linear dose-dependent effect. to further test the role of glycans in jcdv pathogenesis, we carried out these competition bioassays in vivo (figure and supplementary materials figure s ). we mixed jcdv with each monosaccharide prior infection, fed caterpillars with these inocula and calculated mortality rates daily as in figure . results showed that oral infection with mm (but not µm) of each monosaccharide significantly delayed the median time to death (lt ) of caterpillars ( vs. days; p < . for mm) ( figure a and supplementary materials figure s a ,b), further supporting that pm recognition is the first step of jcdv oral infection. to confirm these results and reveal jcdv binding and competition for binding the pm in vivo, we carried out midgut semi-thin sections and immunofluorescence as above. caterpillars were infected with jcdv mixed or not with mm glcnac and sacrificed at h p.i. for midgut isolation and preparation. as shown in figure b , we observed a red fluorescence signal in untreated infected caterpillars that typically lines the pm. in addition, labelling was also observed in the lumen, likely revealing jcdv interaction with food bolus and/or microbial components. both signals were strongly and specifically decreased following competition with glcnac ( figure b) , showing that different glcnac-containing glycans in the gut lumen can recognize the capsids. control caterpillars were fed with pbs. three independent experiments were performed, each independent experiment gave similar results, one is represented here. the log-rank (mantel-cox) and the gehan-breslow-wilcoxon tests were used to determine statistical significance. p values of less than . were considered significant (ns, non-significant; * p < . ; ** p < . ; *** p < . ). (b) immunolabeling of midgut semithin transversal sections h after ingestion of jcdv alone or jcdv ( veg/caterpillar) incubated for h with mm of glcnac before oral infection. control caterpillars were fed with pbs. the pm is shown by an arrowhead. phalloidin-fitc is in green, jcdv is in red, and nuclei are labeled with dapi (blue). bars, µm. lum, midgut lumen; hemol, hemolymphatic compartment. (c) survival curves of caterpillars (n = ) infected by injection of jcdv alone ( veg/caterpillar) or of jcdv incubated for h with mm of each glycan (glcnac, galnac, fucose or mannose) before infection. control caterpillars were injected with pbs. three independent experiments were performed, each independent experiment gave similar results, one is represented here. the log-rank (mantel-cox) and the gehan-breslow-wilcoxon tests were used to determine statistical significance, p > . were considered non-significant (ns). pbs refers to control (pbs-treated and non-infected) caterpillars; 'jcdv' to jcdv-infected caterpillars; 'jcdv + glcnac', 'jcdv + galnac', 'jc + fucose' and 'jc + mannose' refer to caterpillars infected with jcdv incubated with glcnac, galnac, fucose or mannose, respectively, before infection. last, we studied whether such "stickiness" was specifically required by the densovirus to cross the gut, i.e., for oral infection. jcdv infection of target cells (eg epidermis, trachea, hemocytes) proceeds by a receptor-dependent mechanism different from intestinal cells [ ] . these cells express glycan structures of various complexity that are attached to the cell surface or secreted and forming extracellular matrices, whose glycans might be similarly targeted by jcdv for attachment. we performed competition bioassays in vivo, bypassing the midgut by injecting caterpillars with jcdv mixed or not with mm of each monosaccharide. interestingly, the median time to death was similar for all conditions ( figure c and supplementary materials figure s c ; p > . ), showing that none of the monosaccharides competed with jcdv infection proceeding by the systemic route. altogether these results show that jcdv capsid is a carbohydrate-binding protein and this feature is required for oral infection to target the pm of caterpillars. we next wanted to determine which component of the pm, i.e., chitin and/or glycosylated proteins were involved in capsid interactions, using biochemical assays. we first tested capsid physical interaction with chitin using a pull-down assay with chitin beads. jcdv from purified or semi-purified inocula were incubated with chitin beads, pulled-down by centrifugation and subsequently revealed by western blot using a specific jcdv anti-capsid antibody. figure a shows jcdv pull-down by chitin beads and we did not observed difference between inocula (purified vs. semi-purified), which confirmed that capsids can interact directly with chitin. second, we tested virus interaction with pm proteins using a viral overlay binding assay (vobpa). total proteins were extracted from isolated pms, separated with sds-page and either stained with page blue and periodic acid schiff (pas) in order to visualize total and glycosylated proteins respectively, or blotted onto nitrocellulose membranes for vopba. we included porcine mucins as a control of highly (o-)glycosylated proteins. at the first glance, vopba revealed that jcdv binds to most if not all the pm proteins labelled by page blue and pas combined, although with different intensities ( figure b ). interestingly, no binding was observed with the porcine mucins which might support some specificity for insect glycans. more specifically, a set of jcdv-interacting proteins was identified at high molecular weights (> kda) including proteins with a pattern similar to porcine mucins. these proteins were labelled with page blue and pas or only with pas suggesting that they are mostly and probably highly glycosylated ( figure b ). interestingly, proteins at kda displayed a higher intensity as they are in a relative lower amount (according to page blue), which suggests higher affinity for jcdv. proteins interacting with jcdv were detected at - kda and - kda, and corresponding to proteins with low or no glycosylation (according to pas staining). thirty µg of porcine mucins were also loaded in the gel as a control of highly o-glycosylated proteins. proteins were then stained with page blue or periodic acid schiff (pas, pink) to visualize total or glycosylated proteins, respectively, and transferred to nitrocellulose membranes for probing with jcdv and anti-jcdv capsid antibody. proteins interacting with jcdv capsids were finally revealed by enhanced chemiluminescence (black arrowheads on the vopba jcdv membrane); the corresponding positions of these bands were reported on the page blue and pas gels and indicated as well by black arrowheads on the right of these gels. in total, bands representing jcdv interacting proteins are reproducibly obtained with vopba. noteworthy, each band probably include several proteins and/or isoform/glycoform of the same proteins. proteins corresponding to these bands were next analyzed by lc-ms/ms mass spectrometry. we only considered proteins that were shared between replicates ( figure a) , out of which were annotated in the reference genome of s. frugiperda [ ] . these proteins are pm structural proteins (i.e., peritrophins including intestinal mucins) and pm-associated proteins (enzymes, i.e., serine proteases and aminopeptidases n (apn) (supplementary materials table s ). gene ontology (go) annotation confirmed the enrichment in proteolytic activities (particularly serine-type endopeptidases) and chitin synthesis, which are consistent with the pm composition and the gut function ( figure b ). interestingly among the set of proteins > kda, we identified intestinal mucins, an atp binding cassette a type (abca ) transporter and aminopeptidases n (supplementary materials table s ); the latter being known receptors for a number of viruses and for the cry toxins from bacillus thuringiensis [ ] [ ] [ ] [ ] . [ ] . (b) go terms enrichment for the common annotated pm proteins interacting with jcdv (in green), compared to the reference in grey (predicted proteins from ogs . s. frugiperda genome) (fdr set at . ). specific enrichment in jcdv interacting proteins is considered when the green bars exceed the greys (controls). the common pm proteins were assignated to the go terms using blast go software. these results show that jcdv capsids can recognize and bind to the different components of the pm including chitin and several highly glycosylated proteins, both structural components of the pm (mucin, peritrophins) or associated proteins (enzymes). jcdv recognition and binding to glycans of the pm concentrates viral particles close to the epithelial surface, which raised questions about the mechanism involved to cross over and reach the midgut receptor(s). we hypothesized that capsids aggregation on the matrix can result in its disorganization, in a way similar to chitin-binding wheat germ agglutinin (wga) lectin or calcofluor [ ] . to test this hypothesis, we used fluorescent wga-labelling (wga-fitc) to label chitin and thus examine chitin fibrils formation and pm organization. third-instar caterpillars were fed with jcdv and then sacrificed at day p.i. to dissect and prepare midguts for semi-thin sections and wga labelling. as shown in figure , the labelling of the pm (green) lined the apical surface of the epithelium in non-infected larvae (pbs condition, figure , upper panel). in addition, we observed a specific labelling at the apex of columnar cells, probably corresponding to microvillar secretion of glcnac from these cells. by contrast, the labelling lining the epithelium appeared discontinuous following infection (figure , lower panel) , displaying a disorganized pattern reminiscent of the pm structure observed for caterpillars fed with the wga lectin [ ] . moreover, intracellular labelling was no longer observed in the sections from infected caterpillars suggesting an arrest of glcnac secretion from the cells following early infection, i.e., before we can detect virus replication in subepithelial tissues [ ] . these results thus support the hypothesis that jcdv binding on the pm and transcytosis is associated with a loss in its integrity, which might reveal gut dysfunction. to determine the midgut response following jcdv break in, we analyzed the transcriptomic response. we used digital gene expression (dge) based on the serial analysis of gene expression approach [ ] . this method involves the sequencing and quantification of end tagged short cdna fragments (i.e., tags), which enables quantitative differential gene-expression analysis. we built four cdna libraries from midguts of mock-and infected larvae (supplementary materials tables s and s ) . tag sequences were mapped to the genome and transcriptome of s. frugiperda ( [ , ] ) and to the jcdv genome. none of the tags were assigned to viral transcripts, which is consistent with jcdv pathogenesis excluding replication in midgut cells [ ] . pie charts represented go assignment corresponding to unique transcripts at and days p.i. that displayed a differential expression at least -fold up-or down-regulated (supplementary materials figure s a,b) . interestingly, the distribution of go terms was roughly similar at and days p.i., suggesting that the overall intestinal response to jcdv oral infection was poorly affected by virus replication going on in subepithelial tissues (supplementary materials figure s a ,b). we observed only go terms enrichment for the over-represented transcripts at -day p.i., more specifically in functions involved in metabolic processes including translation (i.e., regulation of biological processes, response to stimuli and signaling) at day p.i., that might indicate that jcdv intrusion induces a rapid metabolic response in the gut (figure ). interestingly, these changes did not change significantly at days p.i. suggesting that the gut response is rapidly initiated by jcdv transcytosis and was not affected by the viral replication that takes place in underlying tissues. we did not observe any significant activation of genes involved in "inflammation" nor in the canonical gut immune response. however, we observed an increased expression in cytochrome p and catalase genes that might indicate a response of the cells to the ongoing infection. table s ), we only observed few changes including a trehalose transporter and an intestinal mucin, both down-regulated from day p.i., and a chitinase up-regulated at day p.i.. except for an aminopeptidase n, no gene corresponding to the jcdv interacting proteins identified by vopba displayed a transcriptional change. we did not observe any significant activation of genes involved in "inflammation" nor in the canonical gut immune response. however, we observed an increased expression in cytochrome p and catalase genes that might indicate a response of the cells to the ongoing infection. altogether these results show that jcdv infection induces rapid changes in the gut, particularly in translation and metabolism, within h p.i.. both increased molecular activities might favor viral invasion by supporting the increased energetic demand associated with virus replication in target tissues. interestingly, the canonical midgut immune system did not detect jcdv break in and transport across the epithelium. how densoviruses cope with the forest of glycans that constitute extracellular matrices and decorates insect cell surfaces has been so far a neglected step of their early pathogenesis. results presented here show that jcdv capsids display carbohydrate-binding properties that insure recognition of the peritrophic matrix and determines caterpillars oral infection. we found that capsids can bind to the different components of the pm and their agglutination on the pm surface is associated with the disruption of its organization. furthermore, we showed that this primary step of infection of caterpillars results in a series of physiological changes in the midgut including an arrest of chitin synthesis by epithelial cells. the pm is an obligatory binding platform for capsids to avoid elimination and get closer to the epithelial cell surface where receptor recognition can occur. however, strong attachment to glycans composing the pm would trap capsids there and thus impair their physical connection with the receptor(s). therefore, a first hypothesis is that the "stickiness" of the capsids is balanced to bind and unbind glycans. we used competitions assays with monosaccharides to test this "bind and release" hypothesis and our results showed that indeed, capsids have an affinity for glycans, although the concentration range of the monosaccharides (mm) we tested likely indicates their poor affinity for the capsids. as these monosaccharides could compete capsids away from the pm further suggests that glycan-capsids interaction are probably of low affinity. the issue for bound viral particles is then to move across the pm. our experiments show that capsid binding results in a structural disorganization of the pm similar to effects induced by chitin-binding lectin wga and calcofluor. such capsid-induced disruption of the pm thus favors a second hypothesis, involving a "saturate and pass through" mechanism, where bound capsids are not released but open a way for viral particles to cross over. such "cooperative" mechanism of the capsids to overcome the pm is supported by the fact that pm disruption is enhanced by virus concentration and decreased as caterpillars age. such developmental resistance of s. frugiperda has been also reported following baculovirus infections and a high synergism with calcofluor was obtained at late instars (e.g., > -fold at th instar vs. to -fold at nd and rd instars) [ ] .the structure and the composition of the pm can vary as caterpillars grow and feed, or between populations, which might impact virus-pm interactions and consequently insect susceptibility [ , , ] . whether pm disruption results from mechanical stresses on chitin fibers similar to calcofluor and probably wga lectins or from an enzymatic activity of the capsids (i.e., of vp ) remains to be analyzed more thoroughly. understanding the early interaction of jcdv with the glycans of the pm within species, i.e., along the larval development is of importance to develop biocontrol strategies against insect pests. whether or not the pm could contribute to the species barrier against densovirus infection is unknown. a better understanding of the structure and the glycan composition of the pm in s. frugiperda together with comparative studies in different lepidopteran species are essential to go further on the role of the pm in densovirus infection. structure-fonction studies of the capsid of parvoviruses infecting vertebrates, in particular for species in the genera protoparvovirus and dependovirus, have highlighted the importance of glycans recognition on tissue tropism, pathogenicity, and host range adaption [ , , [ ] [ ] [ ] [ ] . regarding densoviruses, information and capsid structure-function studies is poor [ ] . we performed preliminary assays with a glycan array from the consortium for functional glycomics (http: //www.functionalglycomics.org/fg/, as gosselin-grenet, unpublished data). although this array represents mammalian glycans, the specific recognition of jcdv capsids by paucimannose, which are particularly abundant in insects, suggests some specificity in the interaction of the capsid with glycans. an "insect array" would be of interest to explore glycan ligands affinity and specificity for densovirus capsids. morevover, insects are particularly "handly" animal models for structure-function assays in vivo. we showed that jcdv early infection triggers an arrest of glcnac secretion, which might considerably weaken the pm and explain the disorganized pattern observed with chitin-labelling at day p.i. interestingly, these changes are observed before we could detect jcdv transcription/replication in primary targeted cells [ ] , suggesting that these effects are induced as a consequence of the transport of the viral particles across the epithelium. it has been reported that specific drugs that disorganize microtubules induce an arrest of chitin synthesis [ , ] . we speculate that jcdv transcytosis might induce some stress on the cytoskeletal network, similar to microtubules disorganizing drugs. it is worthy to note that chitin synthesis arrest was not associated with a drop in the expression of chitin synthase genes. however, we cannot exclude to have missed enzymes due to some lack in the annotation of the genome of s. frugiperda [ ] . last, our results showed that jcdv capsids can also interact with components of the luminal compartment including food and bacteria. the competition of the capsid with monosaccharides for binding the pm, suggests that food components could interfere with infection. indeed, plants contain compounds that can interfere with pm synthesis, which has been shown to consequently affect baculovirus infection [ ] (chen et al., ) . regarding bacteria, it has been shown recently that the pm controls commensal bacteria, and conversely that its synthesis and integrity can be microbiota-dependent, i.e., the gut microbiota inducing the expression of components of the peritrophic matrix [ , ] . so, it is plausible that food and microbiota can modify the outcome of the densovirus infection, either by directly competing for binding the pm, or indirectly by modulating the composition of the pm. the binding of densovirus capsids to a wide array of glycans questioned about the role of this "stickiness" in the whole infection cycle including transmission. groundbreaking articles have shown the role of microbiota polysaccharides including glcnac, on the infectivity and thermostability of picornaviruses [ ] [ ] [ ] , whose capsid share structural similarity with parvoviruses [ ] . it is tempting to speculate that densovirus "stickiness" can similarly impact their transmission, which might participate to their success if only among arthropods that occupy extremely diversified ecosystems [ ] . such consideration could also apply to parvoviruses going through faecal-oral route and environmental contamination [ , ] . more generally, stickiness is a major issue for most viruses to and mathematical models have been applied to influenza. they predict that a maximum stickiness favors a maximum fitness [ ] . however, trade-off probably exists in biological systems with an optimal "stickiness" that must be found to infect and leave a host for transmission. densoviruses can be highly pathogenic for insect pests and vectors, which have long stimulated their interest as biocontrol agents or genes vectors [ ] . they are considered today with a renewed interest as solutions to control harmful insects are lacking, which encourages efforts to understand their pathogenesis and their specificity. altogether our results suggest that pm glycans are crucial interacting components of the early jcdv pathogenesis. exploring their diversity and their complexity in insects can also provide important cues on the extend of the mechanisms that determine densovirus specificity. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s . calcofluor increases s. frugiperda caterpillars susceptibility to densovirus oral infection. figure s . virus interaction with monosaccharides did not interfere with anti-capsid antibody recognition. figure s . replicates of survival curves of caterpillars infected by jcdv. figure s . jcdv oral infection induces midgut gene expression modulation. table s . annotation of pm proteins interacting with jcdv in vopba. table s . characteristics of dge libraries generated from caterpillars orally infected with jcdv. table s . characteristics of annotated tags and 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developments calcofluor disrupts the midgut defense system in insects turnover, and functions of chitin in insects optical brightener m r destroys the peritrophic membrane of spodoptera exigua (lepidoptera: noctuidae) larvae functional implications of the structure of the murine parvovirus, minute virus of mice analysis of the cell and erythrocyte binding activities of the dimple and canyon regions of the canine parvovirus capsid diversity and functions of protein glycosylation in insects role of the phosphatidylinositol- -kinase/akt/target of rapamycin pathway during ambidensovirus infection of insect cells further characterization of aminopeptidase-n as a receptor for coronaviruses porcine aminopeptidase n is a functional receptor for the pedv coronavirus in vitro evidence supports membrane alanyl aminopeptidase n as a receptor for a plant virus in the pea aphid vector feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i effect of wheat germ agglutinin on formation and structure of the peritrophic membrane in european corn borer (ostrinia nubilalis) larvae effect of optical brighteners on the insecticidal activity of a nucleopolyhedrovirus in three instars of spodoptera frugiperda the effect of diet on midgut and resulting changes in infectiousness of acmnpv baculovirus in the cabbage looper protoparvovirus cell entry host-selected amino acid changes at the sialic acid binding pocket of the parvovirus capsid modulate cell binding affinity and determine virulence host-specific parvovirus evolution in nature is recapitulated by in vitro adaptation to different carnivore species four amino acids of an insect densovirus capsid determine midgut tropism and virulence chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases microbiota-induced peritrophic matrix regulates midgut homeostasis and prevents systemic infection of malaria vector mosquitoes pgrp-ld mediates a. stephensi vector competency by regulating homeostasis of microbiota-induced peritrophic matrix synthesis intestinal microbiota promote enteric virus replication and systemic pathogenesis interactions between enteric bacteria and eukaryotic viruses impact the outcome of infection bacteria and bacterial envelope components enhance mammalian reovirus thermostability discovery of parvovirus-related sequences in an unexpected broad range of animals transmission ecology of canine parvovirus in a multi-host, multi-pathogen system how sticky should a virus be? the impact of virus binding and release on transmission fitness using influenza as an example viral delivery of dsrna for control of insect agricultural pests and vectors of human disease: prospects and challenges we are grateful to c. gibard, r. bousquet and g. clabot for their great help with insect rearing and the piq quarantine plateform from vectopole sud. we especially acknowledge e. jublanc and c. cazevieille for their skillful technical assistance and the imaging facility mri, member of the national france-bioimaging infrastructure supported by the french national research agency (anr- -inbs- , pia «investments for the future»). we warmly thank m. agbandje-mckenna from the cfg consortium (university of florida) for the glycan array and p. clair from the qphd platform (montpellier genomix). m. younes, and d. piquemal are also acknowledged for their support in the sage analysis. special thanks to p.a. lafon for his help for statistical analyses. l. pigeyre is a doctoral fellow of the french ministry for higher education and research/ephe. key: cord- - zi bg authors: coombs, kevin m.; simon, philippe f.; mcleish, nigel j.; zahedi-amiri, ali; kobasa, darwyn title: aptamer profiling of a cells infected with low-pathogenicity and high-pathogenicity influenza viruses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: zi bg influenza a viruses (iavs) are important animal and human emerging and re-emerging pathogens that are responsible for yearly seasonal epidemics and sporadic pandemics. iavs cause a wide range of clinical illnesses, from relatively mild infections by seasonal strains, to acute respiratory distress during infections with highly pathogenic avian iavs (hpai). for this study, we infected a human lung cells with lab prototype a/pr/ / (h n ) (pr ), a seasonal h n (rv ), the pandemic h n (pdm ), or with two avian strains, an h n hpai strain or an h n strain that has low pathogenicity in birds but high pathogenicity in humans. we used a newly-developed aptamer-based multiplexed technique (somascan(®)) to examine > human lung cell proteins affected by the different iav strains, and identified more than significantly dysregulated cellular proteins. our analyses indicated that the avian strains induced more profound changes in the a global proteome compared to all tested low-pathogenicity h n strains. the pr strain induced a general activation, primarily by upregulating many immune molecules, the seasonal rv and pdm strains had minimal effect upon assayed molecules, and the avian strains induced significant downregulation, primarily in antimicrobial response, cardiovascular and post-translational modification systems. influenza a virus (iav) is a member of the family orthomyxoviridae. iav has been responsible for numerous yearly epidemics and for at least four pandemics during the past~ years; estimates are that more than million people have died from infection during this time period [ , ] . iav is a small enveloped virus with a genome of eight segments of negative-sense single-stranded rna that encode - proteins, depending on the virus strain [ ] [ ] [ ] . iav is serologically categorized by two surface proteins: the hemagglutinin (ha), of which there are currently types (h -h ), and neuraminidase (na), of which there currently are types (n - ) [ , ] . virtually all h/n combinations have been identified in water fowl [ , ] , the generally-accepted reservoir, except for the recent identification of h n and h n in bats [ ] , but only a small number of h/n types are known to circulate or have circulated in humans; h n ( "spanish flu" human lung a cells (atcc # ccl- , manassas, va, usa) were routinely cultured in dulbecco s modified mem (dmem) supplemented with . % (w/v) glucose, non-essential amino acids, sodium pyruvate, mm l-glutamine, and % fetal bovine serum (fbs; gibco, grand island, ny, usa). madin-darby canine kidney (mdck) cells (atcc # ccl- ) were cultured similarly, but in completed dmem supplemented with % fbs. cells were grown as monolayers in % co and passaged by trypsinization - times each week. , and a/anhui/ / (h n ) were amplified, concentrated by ultracentrifugation, and titered in mdck cells by a standard plaque assay or tcid procedures [ , ] . all manipulations of live h n and h n viruses were performed in a respiratory bsl- facility, using all public health agency of canada (phac) guidelines and sops. a cells were infected, or sham (mock)-infected with diluent, with each of the above viruses at an moi of when cells were~ % confluent. mock and infected cells were harvested at h post-infection (hpi). aliquots of all infections were saved for plaque titration or tcid determination to confirm infection. mock and infected cells were washed, lysed in mper solubilization buffer (pierce, waltham, ma, usa) supplemented with × halt protease inhibitor (pierce) and protein concentrations determined using the bicinchoninic acid method. cell lysate protein concentrations were adjusted to ng/µl, and µl of each was analyzed in-house on a somalogics ® -licensed somascan ® version . platform in the manitoba centre for proteomics and systems biology as described [ , ] . briefly, somascan ® is a new proteomic tool that uses somamers ® , dna slow off-rate modified aptamers ( ) . these modified nucleotides are used because they bind to specific human proteins. somamers ® bind proteins in their native state, and are measured on dna microarray chips. somamers ® measure fm-µm protein quantities. the somascan ® version . simultaneously measures distinct proteins in up to samples [ ] . two biologic replicates of pr and rv , and three biologic replicates of mock-, pdm -, h n and h n -infected samples were collected at hpi (= total samples) and were analyzed at the same time in a single somascan ® -well plate. results are reported as relative fluorescent units (rfus) for every protein, and these rfus are directly proportional to the quantities of each target protein in the original samples, confirmed by standard curve generation for every protein-somamer pair [ ] . differences between mock and influenza a virus (iav)-infected rfu values were analyzed as described below in next section. rfu values for each of the analyzed proteins in each biologic replicate were imported into excel. values were log -transformed and fold-changes determined for each protein in infected samples compared to mock samples. fold-change significances were tested both by students t-test ( tails) and by z-score analysis, as described [ , ] . briefly, all fold-changes found not to be significant by the t-test were re-tested by the z-score. this was done by determining the number of standard deviations each protein s value was from the population mean. any protein s z-score was deemed significant if the average z-score was > . σ or <− . σ, the z-score satisfied this criterion in at least replicates and trended > the same direction in one or fewer replicates. thus, z-scores had to be beyond the ± . σ limit in both of the pr and rv analyses. after compiling all proteins deemed significant, we also analyzed levels of fold-changes of the significant proteins, and as described below, applied a fold-change cut-off of . -fold dysregulation (≥ . -fold if up-regulated, or ≤ . -fold if down-regulated) to these proteins for added stringency and to maintain workable numbers of proteins for subsequent bioinformatics and pathway analyses. a cell lysates of iav-and mock-infected cells were collected hpi from two (pr and rv ) or three (mock and all other iav clones) biologic replicates and analyzed with a somalogic ® version . platform. each of protein analytes was examined and relative quantities determined. values for each of the infected samples were normalized to the mock samples from the same replicates, and these comparative values were then analyzed. more than proteins were significantly dysregulated by infection (p-value < . and/or z-score > . σ or <− . σ), and far more proteins were dysregulated by both of the h n and h n avian strains than by any of the h n strains ( table ) . most of the dysregulated proteins were affected < %; thus, we considered more stringent parameters and chose fold-change cut-offs of . -fold (=down-regulated to . of mock) along with significance. thus, proteins that were dysregulated > . -fold, but not considered significant by either p-value or z-score, because of a substantial variability in replicate values, were excluded from viruses , , of the subsequent analysis. using these parameters, we identified proteins that were significantly dysregulated by infection with any of the tested viruses (tables and ; figure a ). five or fewer proteins were significantly dysregulated by rv and the pdm strains, were dysregulated by pr , with all but one being significantly upregulated, and and were dysregulated by h n and h n , respectively, with the vast majority of these proteins downregulated. several immune-regulating proteins, including isg , oas and stat were upregulated by pr infection (table ) , consistent with our previous ms-based proteomic analyses [ ] . thrombopoietin and neural cell adhesion molecule l were the only proteins upregulated by rv and pdm , respectively. few proteins were upregulated by the avian iav strains in the panel of somamers. c-c motif chemokine (ccl ) and il- (cxcl ) were both upregulated by both h n and h n and a few other proteins were upregulated by pr and h n (table ) . pairwise comparisons revealed little correlation between the identities of proteins dysregulated by most virus strains, but an r value of . was determined when h n -dysregulated proteins were compared to h n -dysregulated proteins ( figure b ,c). many common proteins were similarly statistically downregulated by both h n and h n and some of these, including fibronectin and laminin, had been identified previously as downregulated by itraq-based quantitative ms [ ] . several immune-regulating proteins, including isg , oas and stat were upregulated by pr infection (table ) , consistent with our previous ms-based proteomic analyses [ ] . thrombopoietin and neural cell adhesion molecule l were the only proteins upregulated by rv and pdm , respectively. few proteins were upregulated by the avian iav strains in the panel of somamers. c-c motif chemokine (ccl ) and il- (cxcl ) were both upregulated by both h n and h n and a few other proteins were upregulated by pr and h n (table ) . pairwise comparisons revealed little correlation between the identities of proteins dysregulated by most virus strains, but an r value of . was determined when h n -dysregulated proteins were compared to h n -dysregulated proteins ( figure b,c) . many common proteins were similarly statistically downregulated by both h n and h n and some of these, including fibronectin and laminin, had been identified previously as downregulated by itraq-based quantitative ms [ ] . expression values for all proteins in each infection were uploaded into the ingenuity pathway analysis (ipa) tool. for this we expanded consideration of dysregulated proteins to those significantly viruses , , of dysregulated > . -fold to increase the number of analyzed molecules to nearly . as reflected by the numbers of dysregulated proteins induced by each virus (tables and ; figure a ), pr -infected a cells demonstrated a moderately global positive z-score, rv and pdm -infected cells showed no significant positive or negative z-score, and both h n -and h n -infected cells showed overall negative z-scores ( figure ) . these data imply that overall, the seasonal rv strain and the pdm strain have relatively mild effect, as measured by this small set of somamers, the pr strain has an overall activation effect, primarily of immune-modulated and cellular movement molecules, and the h n and h n strains have dramatic inhibitory effects upon multiple cellular processes. ipa network analyses also revealed differences in how each virus affected common cellular networks. the four highest scoring networks, based on numbers of focus molecules, were the cellular movement, hematological system development and function, immune cell trafficking, the antimicrobial response, cell death and survival, inflammatory response, the cardiovascular system development and function, embryonic development and organismal development and the post-translational modification, protein degradation and protein synthesis networks. the cellular movement, hematological system development and function, and the immune cell trafficking network was mildly affected by pr . no proteins in this network were affected by rv or pdm . apart from ccl and cxcl , which were both upregulated by both h n and h n , many proteins in this network were significantly downregulated by the two avian iav strains ( figure a ). the antimicrobial response, cell death and survival inflammatory response network was most significantly affected by pr infection, with numerous upregulated proteins ( figure b ). this'network also was mildly affected by rv infection, but not by pdm infection. three focus molecules in this network (cd , isg and ifnl ) were upregulated and three (igfbp , mica and pgd) were downregulated by h n infection. cd was also upregulated and mica also was downregulated by h n infection, and additionally, lgals bp and rspo were downregulated by h n infection. one or more proteins in the cardiovascular system development and function, embryonic development and organismal development network were upregulated and one or more were downregulated by every virus tested. however, many more proteins (such as fstl , igfbp , notch and stc) were downregulated by the h n and h n viruses than by any of the h n viruses. furthermore, hdl was slightly upregulated in every h n network, but downregulated in both the h n and h n networks. finally, there also were substantial differences in the post-translational modification, protein degradation and protein synthesis network. many more proteins (such as ctsa, ctsv, mfge and tnfrsf ) were downregulated by the h n and h n viruses than by any of the h n viruses. furthermore, cathepsin was slightly upregulated in every h n network, but downregulated in both the h n and h n networks. various bio-functions also were examined using the ipa ® default settings for these analyses (figure ). bio-function activation is assumed for z-scores > . σ, and bio-function inhibition is assumed for z-scores < − . σ. all of these indicated bio-functions also had significant p-values. pr activated many bio-functions, including cellular movement categories, inflammatory response, and angiogenesis, and inhibited few bio-functions; organ inflammation and anatomical organ inflammation. various bio-functions also were examined using the ipa ® default settings for these analyses (figure ). bio-function activation is assumed for z-scores > . σ, and bio-function inhibition is assumed for z-scores < − . σ. all of these indicated bio-functions also had significant p-values. pr activated many bio-functions, including cellular movement categories, inflammatory response, and angiogenesis, and inhibited few bio-functions; organ inflammation and anatomical organ inflammation. the avian iav inhibited far more bio-functions, including cell survival and viability; endocytosis and engulfment, immune response and the proliferation of numerous cell types. the avian iav strains activated few bio-functions, one of which was organismal death. we then used ipa to compare various cellular canonical signaling pathways ( figure ) . the virus strains that induced the most profound cellular responses (pr , h n and h n ) affected many canonical pathways. pr uniquely significantly activated interferon signaling, which has been reported previously [ , ] , while h n uniquely inhibited gluconeogenesis and glycolysis i, and all three strains had significant effects upon multiple pathways, including acute phase response signaling, neuroinflammation, role of pattern recognition of viruses, the complement system, and autophagy. the avian strains had the most dramatic effects upon multiple canonical pathways, including notch signaling, wnt/β-catenin signaling, epithelial adherens junction signaling and regulation of epithelial-mesenchymal transition. thus, collectively and overall, the h n and h n avian iav strains induced more profound inhibitory cellular responses than any of the h n strains, consistent with our [ ] and other′s [ ] studies. the avian iav inhibited far more bio-functions, including cell survival and viability; endocytosis and engulfment, immune response and the proliferation of numerous cell types. the avian iav strains activated few bio-functions, one of which was organismal death. we then used ipa to compare various cellular canonical signaling pathways ( figure ) . the virus strains that induced the most profound cellular responses (pr , h n and h n ) affected many canonical pathways. pr uniquely significantly activated interferon signaling, which has been reported previously [ , ] , while h n uniquely inhibited gluconeogenesis and glycolysis i, and all three strains had significant effects upon multiple pathways, including acute phase response signaling, neuroinflammation, role of pattern recognition of viruses, the complement system, and autophagy. the avian strains had the most dramatic effects upon multiple canonical pathways, including notch signaling, wnt/β-catenin signaling, epithelial adherens junction signaling and regulation of epithelial-mesenchymal transition. thus, collectively and overall, the h n and h n avian iav strains induced more profound inhibitory cellular responses than any of the h n strains, consistent with our [ ] and other s [ ] studies. influenza a virus (iav) remains a significant human pathogen that is constantly emerging and re-emerging. the virus' genetic make-up of eight segments of single-stranded rna allows for significant genetic plasticity. like other rna viruses that lack genetic proof reading, the highly errorprone rna-dependent rna polymerase leads to a high mutation rate (=antigenic drift), and the segmented genomes allows for segment mixing during co-infections, leading to emergence of new isolates (=antigenic shift). thus, vaccines need to be reformulated each year, and the process of attempting to anticipate future isolates often leads to vaccine mismatch [ ] , which may be exacerbated by an individual′s immune history [ ] . a limited repertoire of anti-viral compounds that either inhibit viral uncoating or viral release, have been approved, but viral mutation quickly arises during outbreaks [ , ] . thus, there has been increasing interest to identify host factors that the virus may require for replication and pathogenicity to complement strategies that only target the virus. a number of studies, including genome-wide rnai screens, mrna microarray screens and yeast -hybid assays have identified > cellular targets worthy of further analysis [ ] [ ] [ ] [ ] . numerous recent studies have used quantitative ms-based assays to probe the cellular proteome after perturbation by virus infection. these include -dimensional differences in gel electrophoresis ( d-dige) (ex. [ , ] ), stable isotopic labeling by amino acids in cell culture (silac) (ex. [ , , ] ), isobaric tags for relative and absolute quantitation (itraq) or tandem mass tags (tmt) (ex. [ , [ ] [ ] [ ] ), and label-free methods [ ] [ ] [ ] . each of these non-biased techniques provides information about thousands of proteins within complex mixtures, including cell extracts (reviewed in [ ] [ ] [ ] ). however, each of these provide only a small sampling of the entire cellular proteome. most of these methods detect and measure the most abundant proteins within the mixture. furthermore, there is less than % overlap between any two sample runs, whether biologic runs or technical replicates of the same sample. alternate multiplex assays that target specific proteins have been developed and include the myriadrbm ® and luminex ® platforms. many of these are limited to fewer than proteins that can be simultaneously assayed and measured, although a few, such as those offered by raybiotech ® , are reported to detect and measure several hundred influenza a virus (iav) remains a significant human pathogen that is constantly emerging and re-emerging. the virus' genetic make-up of eight segments of single-stranded rna allows for significant genetic plasticity. like other rna viruses that lack genetic proof reading, the highly error-prone rna-dependent rna polymerase leads to a high mutation rate (=antigenic drift), and the segmented genomes allows for segment mixing during co-infections, leading to emergence of new isolates (=antigenic shift). thus, vaccines need to be reformulated each year, and the process of attempting to anticipate future isolates often leads to vaccine mismatch [ ] , which may be exacerbated by an individual s immune history [ ] . a limited repertoire of anti-viral compounds that either inhibit viral uncoating or viral release, have been approved, but viral mutation quickly arises during outbreaks [ , ] . thus, there has been increasing interest to identify host factors that the virus may require for replication and pathogenicity to complement strategies that only target the virus. a number of studies, including genome-wide rnai screens, mrna microarray screens and yeast -hybid assays have identified > cellular targets worthy of further analysis [ ] [ ] [ ] [ ] . numerous recent studies have used quantitative ms-based assays to probe the cellular proteome after perturbation by virus infection. these include -dimensional differences in gel electrophoresis ( d-dige) (ex. [ , ] ), stable isotopic labeling by amino acids in cell culture (silac) (ex. [ , , ] ), isobaric tags for relative and absolute quantitation (itraq) or tandem mass tags (tmt) (ex. [ , [ ] [ ] [ ] ), and label-free methods [ ] [ ] [ ] . each of these non-biased techniques provides information about thousands of proteins within complex mixtures, including cell extracts (reviewed in [ ] [ ] [ ] ). however, each of these provide only a small sampling of the entire cellular proteome. most of these methods detect and measure the most abundant proteins within the mixture. furthermore, there is less than % overlap between any two sample runs, whether biologic runs or technical replicates of the same sample. alternate multiplex assays that target specific proteins have been developed and include the myriadrbm ® and luminex ® platforms. many of these are limited to fewer than proteins that can be simultaneously assayed and measured, although a few, such as those offered by raybiotech ® , are reported to detect and measure several hundred proteins. thus, at the time we initiated these studies, we selected an aptamer-based assay reported to reliably detect and measure more than proteins. these slow-off-rate modified aptamers (somamers ® ) were developed by somalogics, inc., (boulder, co, usa) [ ] [ ] [ ] , and have been used to examine cancer biomarkers [ ] , alzheimer s disease biomarkers [ ] and biomarkers in iav-infected clinical nasal secretions [ ] . our pilot studies with the first available version, capable of detecting proteins, reliably measured differences in influenza pr -infected a cells over a time course. we subsequently used the next-generation somascan version . , which measures > proteins, to measure zika virus-induced proteomic alterations in vero cells [ ] and in u astrocytoma cells [ ] . application of this targeted aptamer-based approach, which was designed primarily to detect low-abundance proteins, thus provides a beneficial complementary approach to the non-biased ms-based approaches that tend to measure more abundant proteins. comparative analyses of dysregulated proteins we identified in this study to proteins identified in one of our earlier ms-based studies [ , , ] showed generally good agreement. two proteins (isg and β- -microglobulin) were identified as upregulated by both methods, proteins were determined to not be significantly regulated by both techniques and no proteins were identified as significantly upregulated by one method, but significantly downregulated by the other. thus, this newer aptamer-based multiplexed system, designed primarily to detect and measure lower abundant proteins, provides complementary data to the more commonly-used ms-based approaches and to assay numerous proteins not normally detected by quantitative ms. the current study assayed - biologic replicates of five different iav strains, each compared to mock-infected samples (a total of samples). these virus strains represent a lab-adapted h n strain (pr ), a mild seasonal h n strain related to the a/new caledonia/ / -like clinical isolate (rv ), the h n pandemic strain (pdm ), and two strains that show significantly higher pathology in human patients; the h n "bird flu" and the h n strain. all tested virus strains affected the a cellular proteome, upregulating some proteins and downregulating other proteins. although none of the measurable common cellular proteins was significantly dysregulated by all five viruses, some proteins were similarly dysregulated by multiple viruses. for example, ccl was upregulated by pr , h n and h n , and pgam was significantly downregulated by pdm , h n and h n . the five viruses generated three overall patterns of dysregulation, at least as measured by the proteins that can be assayed by the somascan. pr caused an overall general activation, primarily of antimicrobial inflammatory and immune response, as reflected by a large upregulation of more than a dozen proteins, including isg , stat , oas and downregulation only of ppid. pr is a highly lab culture-adapted strain passaged multiple times in mice that is highly virulent in mice, but extremely attenuated in humans. rv and pdm had very little effect in our a cells, as we found in a previous quantitative ms study [ ] , and are relatively low virulence, despite being well adapted to growth in humans. both the h n and h n viruses, which demonstrate the third pattern (significant downregulation of many more proteins than the other strains, particularly in the antimicrobial response, cardiovascular, and post-translational modification networks), and significant downregulation of many common proteins, are poorly adapted to humans because of their receptor specificity, but when successfully delivered into the lower human respiratory tract, they can be highly virulent. this more profound proteomic effect by these avian-derived viruses also was observed in quantitative transcriptomic [ ] and ms studies [ ] . genome-wide rnai screens and mrna microarray screens identified > cellular genes and proteins influenza virus may depend upon [ ] [ ] [ ] [ ] . for example, konig and colleagues identified genes that were required for influenza virus replication as defined by replicase activity [ ] and karlas and colleagues identified genes required for replication of two different influenza viruses, including viruses , , of the pdm strain [ ] . collectively, these two rnai screens, also carried out in a cells, identified potential genes important for influenza replication, with genes found in common. the low level of overlap between these various rnai screens has been previously noted [ ] . we assessed potential overlap between genes identified in the two karlas and konig a studies with proteins we could detect and measure in a cells with our somascan ® panel. only four genes/proteins (jun, kpnb , map k and mdm ) overlapped in all three datasets, and of these, only kpnb was significantly altered according to somascan ® ; downregulated . -fold by h n infection. thirty-one additional genes identified by karlas are in the somascan ® panel; of these, were significantly dysregulated by at least one of our tested viruses. seven proteins were significantly dysregulated by both h n and h n , but < . -fold. only one protein, b m, was dysregulated > . -fold; it was upregulated . -fold and only by pr infection. forty genes identified by konig, including the four found in all three datasets, are in the somascan ® panel; of these, were significantly dysregulated by at least one of our tested viruses. fgfr was dysregulated, but less than %, by three viruses; pdm , h n and h n . only two of the proteins were dysregulated > . -fold; app was downregulated . -fold by h n and -fold by h n , and fgfr was downregulated . -fold by h n infection. the genes/proteins identified by karlas and/or konig and that were present in our somascan ® panel represent proteins involved in numerous diverse functions, including cytokines, enzymes, growth factors, transmembrane receptors and many kinases, as do many of the proteins newly identified in this somascan. thus, these different methods identify a large number of cellular genes and proteins that should be more extensively analyzed as potential targets to ameliorate influenza virus infection. some of our observed dysregulated proteins also were observed in a clinical analysis using the somascan platform. marion and colleagues found some of their most significantly differentially expressed proteins were ctsd, klk , mfge , mapk and cd [ ] . mfge (lactadherin) was significantly downregulated only by h n and h n in our study. although ctsd (cathepsin d), klk (kallikrein- ), mapk (mitogen activated protein kinase ) and cd antigen were not significantly affected by any of our tested viruses, other cathepsin isoforms, ctsv (cathepsin l ) was significantly downregulated by h n , and ctss was significantly upregulated by pr , klk was upregulated by pdm , but only . -fold, these differences probably relate to study design; marion assessed clinical nasal swabs and we examined a cell extracts. the a cell is a transformed adenocarcinoma cell line derived from an explanted tumor from a -year-old caucasian male. while this study provides some important information about how multiple iav strains induce changes in the cellular proteome of these cells, it will be important to perform similar assays in more physiologically-relevant primary cells to allow the comparison and possible identification of common cellular processes that might be amenable to therapeutic intervention. in conclusion, we used a targeted aptamer-based approach to complement earlier quantitative ms approaches to compare host cell responses to viruses that have differential host pathology. we found that the culture-adapted pr strain had an overall activation of immune molecules, the mild seasonal and pdm human strains had little effect upon the molecules targeted by the soma panel, and the avian h n and h n strains, that are much more pathogenic in humans, had the most dramatic proteomic responses, these upregulating a few tested molecules, but inhibiting many more key 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approaches, advances, and applications quantitative proteomics of complex mixtures a review on mass spectrometry-based quantitative proteomics: targeted and data independent acquisition protein signature of lung cancer tissues alzheimer's disease biomarker discovery using somascan multiplexed protein technology respiratory mucosal proteome quantification in human influenza infections zika virus infection disrupts astrocytic proteins involved in synapse control and axon guidance pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses identification of host cell factors involved in influenza a virus infection this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank neil salter for expert technical assistance and gao ang for somascan analyses. we also thank nathalie bastien and yan li for use of the phac bsl laboratory and for providing us the a/anhui/ / h n isolate. the authors declare no conflict of interest. key: cord- -y fcw dj authors: kalodimou, georgia; veit, svenja; jany, sylvia; kalinke, ulrich; broder, christopher c.; sutter, gerd; volz, asisa title: a soluble version of nipah virus glycoprotein g delivered by vaccinia virus mva activates specific cd and cd t cells in mice date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: y fcw dj nipah virus (niv) is an emerging zoonotic virus that is transmitted by bats to humans and to pigs, causing severe respiratory disease and often fatal encephalitis. antibodies directed against the niv-glycoprotein (g) protein are known to play a major role in clearing niv infection and in providing vaccine-induced protective immunity. more recently, t cells have been also shown to be involved in recovery from niv infection. so far, relatively little is known about the role of t cell responses and the antigenic targets of niv-g that are recognized by cd t cells. in this study, niv-g protein served as the target immunogen to activate niv-specific cellular immune responses. modified vaccinia virus ankara (mva), a safety-tested strain of vaccinia virus for preclinical and clinical vaccine research, was used for the generation of mva–niv-g candidate vaccines expressing different versions of recombinant niv-g. overlapping peptides covering the entire niv-g protein were used to identify major histocompatibility complex class i/ii-restricted t cell responses in type i interferon receptor-deficient (ifnar−/−) mice after vaccination with the mva–niv-g candidate vaccines. we have identified an h -b-restricted nonamer peptide epitope with cd t cell antigenicity and a h -b mer with cd t cell antigenicity in the niv-g protein. the identification of this epitope and the availability of the mva–niv-g candidate vaccines will help to evaluate niv-g-specific immune responses and the potential immune correlates of vaccine-mediated protection in the appropriate murine models of niv-g infection. of note, a soluble version of niv-g was advantageous in activating niv-g-specific cellular immune responses using these peptides. nipah virus (niv) is an emerging zoonotic pathogen of global concern that was ranked recently by the world health organization (who) as a high-priority pathogen. niv is a negative-sense, single-stranded rna virus that is a member of the genus henipavirus (family paramyxoviridae). niv was first identified during a large outbreak affecting humans and pigs in malaysia and singapore in [ ] . from onwards, seasonal outbreaks are observed almost annually in bangladesh and sporadically hohenpeißenberg, germany) and had access to food and water ad libitum. all experiments were approved by the government of upper bavaria, munich germany and were performed in compliance with the german animal welfare act ( . vet- .vet_ - - , . . ). primary chicken embryo fibroblasts (cef) were isolated from -day-old spf chicken embryos (valo, cuxhaven, germany) and grown in minimum essential medium (mem) (sigma-aldrich, taufkirchen, germany) supplemented with % heat-inactivated fetal bovine serum (fbs) (sigma-aldrich), % penicillin-streptomycin (sigma-aldrich), and % mem nonessential amino acid solution (sigma-aldrich). human hela cells (atcc ccl- ) were maintained in mem supplemented with % heat-inactivated fbs (sigma-aldrich) and the above antibiotics. df- cells (atcc crl- ) were grown in vle dulbecco's modified eagle's medium (dmem) (merck, darmstadt, germany) supplemented with % heat-inactivated fbs (sigma-aldrich), % penicillin-streptomycin (sigma-aldrich), % mem nonessential amino acid solution (sigma-aldrich), and % hepes solution (sigma-aldrich). cells were maintained at • c in a humidified % co atmosphere. the cdna that encoded for the entire amino acid sequence of the niv-g protein (nipah virus isolate ummc , genbank accession number ay . ) was modified in silico by introducing silent codon alterations to remove termination signals of vaccinia virus early transcription (tttttnt) and g/c nucleotide runs. for construction of the soluble form of niv-g protein (nivsg), the cytoplasmic and transmembrane domains were deleted and an internal leader sequence and amino acid linker sequences were added as described previously [ , ] . for vaccinia virus (vacv)-specific transcriptional regulation, we placed the nivsg gene sequences under control of the synthetic vacv early/late promoter (pmh ) [ ] and used the strong natural early vacv promoter pvgf [ ] [ ] [ ] for expression of niv-g sequences. the cdnas encoding for niv-g or nivsg including the cleavage sites for the restriction endonucleases xhol and apal were generated by dna synthesis (genewiz, leipzig, germany). cdna sequences were cloned into the mva transfer plasmid plw- [ ] , which directs insertion of heterologous sequences to a site between the open reading frames (orf) of the essential viral genes, mva r and mva l. recombinant mva viruses expressing the nivsg and niv-g proteins were generated as described previously [ , ] . to summarize, cef cells at - % confluence were infected with nonrecombinant mva (clonal isolate mva f ) at a multiplicity of infection (moi) of . and transfected with vector plasmid dna containing nivsg or niv-g gene sequences using x-tremegene™ hp dna transfection reagent (roche diagnostics, penzberg, germany). after h incubation, cells were harvested, and the recombinant viruses mva-nivsg and mva-niv-g were clonally isolated by screening for co-expression of the fluorescent protein marker gfp and plaque passaging several times. resultant vector virus isolates were quality controlled using standard protocols [ ] . polymerase chain reaction (pcr) analysis of genomic viral dna served to confirm the genetic identity and genomic stability of the mva vector viruses. the replicative capacity of the recombinant mva-niv viruses compared with nonrecombinant mva was tested by multi-step growth experiments in cef and human hela cells. to generate vaccine preparations, recombinant mva-niv viruses were amplified in cef, purified by ultracentrifugation through % sucrose cushions, and reconstituted in mm tris-hcl buffer, ph . , to make stock preparations [ ] . cef and hela cells were infected with recombinant mva-nivsg and mva-niv-g viruses at a moi of . cells infected with mva (moi ) and inoculation medium alone (mock) were used as controls. cell lysates were prepared or culture supernatants were collected at various time points after infection and stored at − • c. cell-associated and secreted proteins were resolved by sodium dodecyl sulfate (sds)-polyacrylamide ( %) gel electrophoresis (sds-page), and proteins were transferred onto nitrocellulose membranes by wet electroblotting. to investigate the glycosylation pattern, proteins were pretreated using the protein deglycosylation mix ii kit (new england biolabs, ipswich, ma, usa) according to the manufacturer's instructions prior to sds page. membranes were blocked with blocking buffer, which consisted of pbs containing % non-fat dried milk powder (carl roth, karlsruhe, germany) and . % v/v tween (sigma-aldrich) for one hour at room temperature. membranes were then probed with the primary antibody (polyclonal mouse anti-niv-g ( : ) or polyclonal rabbit anti-niv-g ( : )) diluted in blocking buffer overnight at • c. membranes were washed three times with pbs containing . % tween (pbs/t) and probed with goat anti-mouse igg conjugated to horseradish peroxidase (hrp) ( : ; agilent dako, glostrup, denmark) or goat anti-rabbit igg hrp ( : ; cell signaling technology, leiden, the netherlands). membranes were washed again with pbs/t and were developed using supersignal ® west dura extended duration substrate (thermo fisher scientific, planegg, gemany). blots were visualized using a microchemi . imager (dnr bio-imaging systems, neve yamin, israel). confluent monolayers of hela cells were infected with recombinant mva-nivsg or mva-niv-g viruses at moi . . controls included mva (moi . ) and inoculation medium alone (mock). after infection, cells were incubated for h in a • c incubator. cells were fixed with % paraformaldehyde (pfa) (sigma-aldrich) and on some occasions, permeabilized with . % triton x- (sigma-aldrich). cells were blocked in blocking buffer containing % bovine serum albumin (bsa) (sigma-aldrich). cells were stained with polyclonal rabbit anti-niv-g diluted : , in pbs containing . % bsa (pbs/bsa). cells were washed with pbs and stained with the secondary antibody goat anti-mouse igg alexa fluor (af) ( : ; thermo fisher scientific) diluted in pbs/bsa buffer. fluorescence was visualized using the keyence bz-x microscope (keyence, osaka, japan). mice were immunized with pfu in µl vaccine buffer ( mm tris and mm nacl, ph . ) of recombinant mva-nivsg, recombinant mva-niv-g or mva or vaccine buffer as a mock vaccine control via the intraperitoneal or intramuscular routes. mice received either one (prime) or two (prime-boost) immunizations over a day interval. for t cell analysis, mice were euthanized days after immunization. blood was collected on days , , and . coagulated blood was centrifuged at × g for min in minicollect vials (greiner bio-one, alphen aan den rijn, the netherlands) in order to separate serum, which was subsequently stored at − • c until further use. antigen-specific igg responses induced by immunization with the vaccine candidates were analyzed by enzyme-linked immunosorbent assay (elisa) using purified soluble recombinant niv glycoprotein g expressed in mammalian hek cells. flat bottom -well elisa plates (nunc™ maxisorp™ plates, thermo scientific) were coated with ng/well recombinant protein ( µl volume) and incubated overnight at • c. plates were washed three times with µl/well pbs/t. plates were blocked with blocking buffer containing % bovine serum albumin (sigma-aldrich) and . m sucrose (sigma-aldrich) in pbs for h at • c. plates were then washed with pbs/t as described above. sera were serially diluted in pbs containing % bsa (pbs/bsa) three-fold down the plate, starting at a dilution of : ( µl volume/well) and incubated for h at • c. after washing, plates were incubated with µl/well goat anti-mouse igg conjugated hrp ( : ; agilent dako, denmark) diluted in pbs/bsa for h at • c. plates were then washed with pbs/t as described earlier. then, µl/well , , , -tetramethylbenzidine (tmb) liquid substrate system for elisa (sigma-aldrich) was added, and plates were incubated until a color change was observed. the reaction was stopped by the addition of µl/well stop reagent for tmb substrate ( nm, sigma-aldrich). the absorbance was measured on an elisa plate reader at nm with a nm reference wavelength. total igg titers were calculated from the inflection point of the titration curve as logarithms of the reciprocal. the protein sequence for niv-g was obtained from the uniprot database (id: q ih ). using an in silico approach, we identified individual synthetic peptides that spanned the external domain niv-g protein, starting from the third amino acid of the n-terminal side to the c terminus (amino acids - ). our peptide library consisted of mer peptides that overlapped by mer. all peptides were synthesized by thermo fisher scientific as crude material (< % purity) on a - mg scale. the two-dimensional peptide pool matrix system was used for screening [ , ] . briefly, peptides were organized into two-dimensional matrix peptide pools (h -h and v -v ) containing - peptides. for mapping of potential cd t cell epitopes in positive mer peptides, every possible sequence of peptides - mer in length was determined. theoretical peptides were then analyzed for binding to the mouse major histocompatibility complex (mhc) class i allele h -b using the syfpeithi database. the peptides of each amino acid sequence length were synthesized and tested. for identification of cd t cell epitopes, mhc class ii binding predictions were performed on mer peptides found within positive peptide pools identified by ifn-γ enzyme-linked immunospot assay (elispot). using the mhc class ii binding, t cell epitope prediction resource of the immune epitope database (iedb, https://www.iedb.org/), peptides restricted to mouse mhc class ii allele h -iab were analyzed using "iedb recommended" prediction method [ ] . the most promising candidates were then chosen for further experimental epitope prediction studies. all peptides were dissolved to a concentration of mg/ml in pbs, aliquoted, and stored at − • c until use. t cell analysis by elispot was performed as described previously [ ] . briefly, spleens were collected from mice days after the final immunization. single-cell suspensions were prepared by teasing spleens through a µm cell strainer (falcon ® corning, corning, ny, usa). red blood cells (rbc) were removed using red cell lysis buffer (sigma-aldrich), and cells were washed and resuspended in rpmi- , which consisted of rpmi- (sigma-aldrich) containing % heat-inactivated fbs (sigma-aldrich) and % penicillin-streptomycin (sigma-aldrich). for experiments that required cd and cd t cell purification, splenocytes were incubated with mouse cd and cd microbeads (miltenyi biotec, bergisch gladbach, germany) and processed by negative selection using the quadromacs separator (miltenyi biotec). ifn-γ-producing cells were measured by ifn-γ elispot assay using the mouse ifn-γ elispotplus kit (mabtech, stockholm, sweden) as described in the manufacturer's protocol. in summary, × splenocytes were seeded onto -well flat bottom plates ( µl/well) (sarstedt, nümbrecht, germany), and µl/well peptide pools, subpools, or individual peptides were added (each peptide diluted to µg/ml in rpmi- ). after mixing, the splenocyte/peptide mixtures were transferred onto plates precoated with ifn-γ detection antibody and incubated for h at • c. nonstimulated cells were used as a mock control, and the positive controls cultures were treated with phorbol myristate acetate (pma) and ionomycin (both from sigma-aldrich) or vaccinia virus (vacv)-specific cd t cell epitope, b r - (tsykfesv) [ ] . after incubating, plates were processed as described in the kit manufacturer's protocol (mabtech). spots were counted and analyzed using the automated elispot plate reader (a. el. vis eli.scan and a. el. vis elispot analysis software, hannover, germany). splenocytes were diluted to × cells/ml in rpmi- , and µl/well ( × cells) was added onto a -well u-bottom plate. then, µl/well peptide diluted to µg/ml in rpmi- was added to give a final peptide concentration of µg/ml. the vacv cd t cell epitope b r - (final concentration of µg/ml) was used as a positive control along with pma ( ng/ml) plus ionomycin ( ng/ml). pbs diluted in rpmi- was used as a mock stimulated control. after plating, cells were incubated for h at • c. then, µl/well x brefeldin a, a golgi inhibitor that was prepared by diluting x brefeldin a (biolegend, san diego, ca, usa) in rmpi- , was added to give a final dilution of x brefeldin a. cells were then incubated for an additional h at • c. after incubating, plates were centrifuged ( g for min), and the supernatant was removed. samples were filtered through µm nylon mesh (sefar pty ltd., huntingwood, nsw, australia) into ml round bottom facs tubes (sarstedt). single-color controls were prepared for each facs analysis using onecomp ebeads™ compensation beads (ebioscience, thermo fisher scientific) for fluorophore-conjugated antibodies and cells for the viability dye zombie aqua. data acquisition was performed by macsquant vyb flow analyser (miltenyi biotec), and data was analyzed using flowjo (flowjo llc, bd life sciences, ashland, or, usa). data were analyzed using graphpad prism version . (graphpad software inc., san diego, ca, usa) and were expressed as mean ± standard error of the mean (sem). statistical analysis was performed using the unpaired, two-tailed t-test to compare two groups and one-way anova to compare three or more groups. the threshold for statistical significance was p < . . to generate the recombinant mva viruses delivering niv-g antigens, we used the gene from niv malaysia (isolate ummc , genbank accession number ay . ) and generated codon-optimized gene sequences encoding a full-length glycoprotein g (niv-g) or a soluble external domain g protein (nivsg). these synthetic gene sequences were placed under the transcriptional control of the vaccinia virus-specific promoters pvgf or pmh and introduced into the mva genome by homologous recombination targeting the intergenic site between the essential mva genes and l. the clonal isolation of the recombinant viruses mva-nivsg and mva-niv-g was facilitated by the co-production of the green fluorescent reporter protein (gfp), as previously described [ ] . the final recombinant viruses containing the niv-g gene sequences (mva-niv-g and mva-nivsg) were obtained after removal of the gfp reporter gene by intragenomic homologous recombination ( figure s a , marker gene deletion). to verify the identity of the desired modification, we performed the standard quality control experiments as described previously [ ] . pcr analysis of viral genomic dna confirmed the proper insertion of the recombinant gene sequences at the target site in the genome of mva (figure s b-e) and the genetic characteristics and stability of the recombinant mva viruses. we assessed the growth behavior of the recombinant viruses mva-nivsg and mva-niv-g in multi-step growth analyses in human hela cells and primary cef, which are routinely used for amplification of recombinant mva in vaccine manufacturing ( figure s f ). mva-nivsg and mva-niv-g efficiently replicated in cef and demonstrated an increase of infectivity titers that was comparable to that obtained with nonrecombinant mva. however, mva-niv-g and mva-nivsg did not productively grow in human hela cells, confirming that they had retained the characteristic replication deficiency of mva in cells of mammalian origin. these findings corroborated the expected mva phenotype and confirmed that the recombinant viruses could be handled under laboratory conditions of biosafety level . of note, we originally generated another recombinant mva virus using the synthetic early-late promoter pmh for transcriptional control of recombinant gene expression and production of the full-length niv-g protein. however, upon growth testing, this candidate virus failed to reach levels of infectious progeny in cef to be eligible for large-scale amplification as needed for vaccine production processes. our vaccine candidates, mva-nivsg and mva-niv-g, should produce recombinant niv glycoprotein g in its full-length form (niv-g) and, in parallel, the soluble form (nivsg). niv-g is a amino acid long protein consisting of an n-terminal internal domain, a transmembrane domain, and a c-terminal external domain ( figure a ). for nivsg protein, an internal leader sequence and three amino acid linkers have replaced the internal and transmembrane domains ( figure a ), as described previously [ , ] . we assessed the correct expression and studied the cellular localization of the niv-g and nivsg by immunofluorescence microscopy of mva-niv-infected cells immunolabeled with anti-niv-g antibody, followed by a fluorescently labelled secondary antibody. cell nuclei were stained with dapi ( nm). as anticipated, we observed different patterns of green fluorescence, with varying cellular localizations depending on the mva-niv construct. green fluorescence, specific for niv-g, was identified in permeabilized and nonpermeabilized cells infected with mva-niv-g ( figure b ), but not in mva-infected or mock control cells. this data confirmed that the recombinant full-length niv-g protein encoded by mva-niv-g was indeed anchored on the cell surface. in contrast, the niv-g-specific staining in cells infected with mva-nivsg virus appeared to be predominantly located within the cells and was readily detected after permeabilization. as anticipated, we failed to detect nivsg in considerable amounts without permeabilization, confirming that nivsg was not expressed on the cell surface ( figure b) . to further investigate the synthesis of niv-g proteins after infection with mva-nivsg and mva-niv-g, respectively, total proteins from infected cef and hela cell cultures were analyzed by western blot using a niv-g-specific antibody. total cell lysates or culture supernatants obtained from cef and hela cultures infected with recombinant mva virus were separated by sds-page and immunoblotted. we specifically detected a protein with an estimated molecular mass of approximately - kda in lysates from cef cells and hela cells infected either with mva-niv-g or mva-nivsg ( figure c ). in the cell lysates, the glycoprotein was first detectable at h post-infection. since the nivsg gene encoding sequences were modified to result in the secreted soluble version (sg), we performed additional western blot experiments to determine whether the protein still maintained glycosylation sites comparable to wild-type niv-g. lysates and supernatants obtained from cultures of df- cells infected with mva-nivsg were treated with enzymes that deglycosylate proteins and analyzed by western blotting. enzyme treatment resulted in a reduction in the molecular mass of recombinant nivsg protein from - kda to - kda ( figure d ), matching the expected size of unmodified g protein. this suggested that recombinant nivsg has retained the normal glycosylation pattern of wild-type niv-g. to assess the immunogenicity of the recombinant mva-niv-g/nivsg candidate vaccines, we vaccinated ifnar−/− mice with pfu via the intramuscular route at days and . serum samples were tested for niv-g-binding igg antibodies by elisa days after the first immunization (prime) and days after the second immunization (prime-boost) (figure ). even a single application of the mva-niv-g vaccines induced abundant levels of niv-g-specific igg antibodies in the mice. after booster immunization, all vaccinated animals produced even higher levels of circulating niv-g-specific antibodies, with the antibody titers increasing by approximately ten-fold. currently, there is only limited information available on niv-specific t cell immunity. in order to characterize the niv-g-specific t cell response induced by our candidate vaccines, we first aimed to identify niv-g polyprotein-specific t cell epitopes. ifnar−/− mice on a c bl/ background (mhc i = h -db/h -kb (h -b) and mhc ii = h -iab) were immunized twice with the mva-nivsg candidate vaccine via the intraperitoneal (i.p.) route, and splenocytes were prepared days after the final inoculation, cd and cd t cells were purified and restimulated with overlapping mer peptides corresponding to the niv-g protein. we pooled overlapping peptides using a twodimensional peptide matrix system ( figure a ) and screened for their ability to induce ifn-γ by elispot. ifn-γ production above background levels was observed after stimulation with out of the peptide pools tested ( figure b ). cd t cells from mice immunized with mva-nivsg, but not the mva and mock groups, showed elevated numbers of ifn-γ spot-forming counts (sfc) after stimulation with peptide pools v and h ( figure b ). in the next step, the peptides within the v and h peptide pools were used to characterize the t cell epitope specificities in more detail. for this, pools v (v a and v b) and h (h a and h b) were subdivided into subpools containing - peptides each. since two mer peptides were shared between the pools, # (g - = ytrstdnqavikdal) and # (g - = tdnqavikdalqgiq), these peptides were also tested separately. for this experiment, we used an identical immunization protocol and splenic cd t cell purification procedure. cd t cells from these mice were restimulated with the above subpools and individual peptides # and # . stimulation with the subpools h a and v a resulted in ifn-γ production above background levels in the mva-nivsg group with ifn-γ sfc mean ± sem values of ± and ± ifn-γ sfc/ splenocytes, respectively ( figure c ). subpools h b and v b, however, did not stimulate cd t cells in the mva-nivsg group. stimulation of cd t cells with individual peptides # and # , which were present in subpools h a and v a, resulted in different outcomes. ifn-γ production above the background was observed in cultures stimulated with # (mean = ± sfc/ cells), but not # , in the mva-nivsg group. these data suggested that the mer peptide # contained peptide sequences that stimulated niv-g-specific cd t cells. niv-g or niv-sg currently, there is only limited information available on niv-specific t cell immunity. in order to characterize the niv-g-specific t cell response induced by our candidate vaccines, we first aimed to identify niv-g polyprotein-specific t cell epitopes. ifnar−/− mice on a c bl/ background (mhc i = h -db/h -kb (h -b) and mhc ii = h -iab) were immunized twice with the mva-nivsg candidate vaccine via the intraperitoneal (i.p.) route, and splenocytes were prepared days after the final inoculation, cd and cd t cells were purified and restimulated with overlapping mer peptides corresponding to the niv-g protein. we pooled overlapping peptides using a two-dimensional peptide matrix system ( figure a ) and screened for their ability to induce ifn-γ by elispot. ifn-γ production above background levels was observed after stimulation with out of the peptide pools tested ( figure b ). cd t cells from mice immunized with mva-nivsg, but not the mva and mock groups, showed elevated numbers of ifn-γ spot-forming counts (sfc) after stimulation with peptide pools v and h ( figure b ). in the next step, the peptides within the v and h peptide pools were used to characterize the t cell epitope specificities in more detail. for this, pools v (v a and v b) and h (h a and h b) were subdivided into subpools containing - peptides each. since two mer peptides were shared between the pools, # (g - = ytrstdnqavikdal) and # (g - = tdnqavikdalqgiq), these peptides were also tested separately. for this experiment, we used an identical immunization protocol and splenic cd t cell purification procedure. cd t cells from these mice were restimulated with the above subpools and individual peptides # and # . stimulation with the subpools h a and v a resulted in ifn-γ production above background levels in the mva-nivsg group with ifn-γ sfc mean ± sem values of ± and ± ifn-γ sfc/ splenocytes, respectively ( figure c ). subpools h b and v b, however, did not stimulate cd t cells in the mva-nivsg group. stimulation of cd t cells with individual peptides # and # , which were present in subpools h a and v a, resulted in different outcomes. ifn-γ production above the background was observed in cultures stimulated with # (mean = ± sfc/ cells), but not # , in the mva-nivsg group. these data suggested that the mer peptide # contained peptide sequences that stimulated niv-g-specific cd t cells. graphs shows ifn-γ sfc (spot-forming counts) of stimulated cd t cells. differences between groups were analyzed by one-way anova. asterisks represent statistically significant overall differences for a specific peptide subpool or individual peptide. * p < . . to map the potential cd t cell epitope within the niv-g protein in more detail, we dissected the mer peptide # into every possible - mer sequence ( figure a ). the sequences were then analyzed for h -b binding computationally using the syfpeithi database, and the four best of each amino acid length were chosen (table ) . to test these peptides, we immunized ifnar−/− mice twice with mva-nivsg via the i.p. route and analyzed total splenocyte activation by elispot assay. of the sixteen - mer overlapping peptides tested, eight resulted in the stimulation of measurable ifn-γ in the mva-nivsg group ( figure b ). the positive peptides included three mer in length ( . , . , and . ), two mer in length ( . and . ), two mer in length ( . and . ), and one mer in length ( . ) ( figure b ). the mean ifn-γ sfc value was lower for cells stimulated with peptide . relative to peptide . . the mean values for the mva-nivsg group were ± ifn-γ sfc/ cells for peptide . and ± ifn-γ sfc/ for peptide . . comparative analysis of the positive peptide sequences demonstrated that the sequence of peptide . (rstdnqavi) was present in all positive - mer peptides (table ). in addition, the sequence of peptide . (stdnqavi) was present in the eight positive peptides. to characterize the induction of ifn-γ sfc by these peptides in more detail, ifnar−/− mice were vaccinated twice via the i.m. route, a commonly used route for human vaccinations, and in vitro stimulated splenocytes were analyzed by elispot assay. for this experiment, we chose the most promising peptides of each amino acid sequence length (peptides . , . , . , . , . , and . ). the six peptides tested significantly stimulated the activation of ifn-γ in the mva-nivsg group relative to the mva and pbs controls ( figure c ). mean sfc values, however, did not show statistically significant variations between each individual peptide. importantly, the mean sfc value for peptide . was again nonsignificantly elevated relative to peptide . (mean ± sem = ± and ± ifn-γ sfc/ cells respectively). consequently, we chose peptide . (rstdnqavi) (mean = ± ifn-γ sfc/ cells) as a potential h -b-restricted epitope candidate of niv-g ( table ). the alignment of peptide . to the full sequence of niv-g is shown in figure s . ( . , . , . , . , . , . ) . differences between groups were analyzed by one-way anova. asterisks represent statistically significant overall differences for a specific peptide. * p < . , ** p < . . peptide chosen as the most promising h -b-restricted peptide of niv-g; peptide chosen as the most promising h -iab-restricted peptide of niv-g. rows in bold indicate promising niv-g-specific t cell epitopes. to comparatively evaluate the activation of niv-g-specific immunity after i.m. vaccination with mva-nivsg and mva-niv-g using a standard dose of pfu, we stimulated splenocytes with peptide . ( table ) and analyzed cytokine production by ifn-γ elispot assay and ifn-γ plus tnf-α ics. comparisons between the two candidate vaccines showed that the mean sfc values were significantly higher for the mva-nivsg group relative to the mva-niv-g group ( figure a and figure s a ). the means of the mva-nivsg group were ± ifn-γ sfc/ cells, and the means of the mva-niv-g group were ± ifn-γ sfc/ cells ( figure c ). ifn-γ ics data showed that both the frequency and absolute number of ifn-γ+ cd t cells were significantly higher than the control background levels ( figure b ). our peptides did not induce ifn-γ production by cd t cells ( figure s b ), indicating that they indeed stimulated antigen-specific cd t cells only. when we compared our two candidate vaccines, we found that the percentage and absolute number of ifn-γ+ cd t cells was significantly higher in the mva-nivsg group. the mean frequencies of ifn-γ+ cd t cells were . % ± % for the mva-nivsg group and . % ± . % for the mva-niv-g group. mean absolute number of ifn-γ+ cd t cells were in the range of ± cells/ splenocytes and ± cells/ splenocytes for the mva-nivsg and mva-niv-g groups, respectively. taken together, our elispot and ifn-γ ics data indicate that mva-nivsg activates higher numbers of peptide-specific cd t cells than mva-niv-g. when we analyzed for coproduction of ifn-γ and tnf-α, we found that the majority of niv-g peptide-specific cd t cells from mva-nivsg and mva-nivg immunized mice were double positive for the cytokines (ifn-γ + tnf-α+) ( figure s a-c) . after showing that prime-boost immunization with our two vaccine candidates generated robust niv-g-specific cd t cell responses, we tested their immunogenicity after a single vaccination. ifnar−/− mice were vaccinated once with mva-nivsg, mva-niv-g, mva, or mock via the i.m. route and analyzed by elispot and ifn-γ + tnf-α ics as described before. our elispot results overall showed elevated sfc counts relative to the background levels of our mva and mock controls ( figure c ). statistically significant differences between the mva-nivsg and mva-niv-g were detected, with mean sfc values of ± ifn-γ sfc/ cells and ± ifn-γ sfc/ for them, respectively. facs analysis showed that the frequency and absolute number of ifn-γ+ cd t cells was higher in the experimental groups relative to the mva and mock controls ( figure d ). the mean percentage of ifn-γ + cd t cells was . % ± . % for the mva-niv-g groups, whereas for the mva-nivsg group, the mean percentage range for the peptides was . % ± . %. while mva-niv-g prime immunization induced a low frequency of ifn-γ+ cells, the mva-nivsg group showed ifn-γ+tnf-α+ secreting t cells after a single vaccination ( figure s d-f) , although percentages were lower than what was observed after prime-boost immunization ( figure s a-c) . this indicates that prime immunization with mva-nivsg was sufficient to generate a sizeable population of polyfunctional antigen-specific cd t cells. table ) and measured by elispot assay and ifn-γ ics plus facs analysis. (a,b) antigen-specific cd t cell response induced by prime-boost immunization. (a) ifn-γ sfc for stimulated splenocytes measured by elispot assay. (b) ifn-γ production by stimulated splenic cd t cell measured by ics and facs analysis. graphs show frequency and absolute number (per splenocytes) of antigen-specific ifn-γ+ cd t cells. (c,d) antigen-specific cd t cell response induced by prime immunization. (c) ifn-γ sfc for stimulated splenocytes measured by elispot assay. (d) frequency and absolute number (per splenocytes) of antigen-specific cd t cells measured by ifn-γ ics plus facs analysis. differences between individual groups were analyzed by one-way anova and tukey post-hoc test. asterisks represent statistically significant differences between two groups. * p < . , ** p < . , *** p < . . initially, we also analyzed purified cd t cell cultures from mva-nivsg immunized mice for their ability to stimulate ifn-γ by elispot using a two-dimensional pooled-peptide matrix system ( figure a , section . . ). in contrast to cd t cell-enriched splenocytes, the ifn-γ sfc signals in these cultures were lower (figures b and a) . in order to determine definitively which peptide pools were above background levels, we selected a cut off value of ifn-γ sfc/ cells ( figure a , grey line). mean ifn-γ sfc values above background were observed in out of the pools (pools h , h , v , v , v , v , v , and v ) ( figure a ). in order to identify potential cd t cell epitopes of h -iab, we performed a computational analysis of the mer peptides in the five most positive peptide pools measured by elispot assay (pools h , h , v , v , and v ). using mhc ii binding predictions obtained from the iedb online resource, we found two promising peptides. these peptides were # (lfmtnvwtppnpntv) and # (nvwtppnpntvyhcs) ( table ). next, we used ifn-γ ics to identify directly antigen-specific cd t cells after stimulation with these peptides. for this, splenocytes from mice that had been vaccinated with mva-niv-g or mva-nivsg were stimulated with peptides # and # and analyzed by flow cytometry. a small population of peptide-specific ifn-γ+ cd t cells was observed in the mva-nivsg and mva-niv-g groups ( figure b ). due to low frequencies of ifn-γ-producing cells, we chose a cut off value of . % to differentiate between positive signals and background. overall, cd t cells from the mva-nivsg group had a frequency of ifn-γ+ above the cut off relative to mva-niv-g group ( figure c ). the mean percentage of ifn-γ+ cd t cells for the two peptides was . - . % and . - . % for mva-nivsg and mva-niv-g, respectively. moreover, the frequency and absolute number of ifn-γ-producing cd t cells in peptide # stimulated cultures was significantly higher in the mva-nivsg group when compared with the mva-niv-g group (mean = ± and ± cells/ splenocytes respectively). for peptide # , mva-nivsg vaccinated mice showed a significantly elevated frequency of ifn-γ+ cd t cells only. in conclusion, our data indicate that peptide # (nvwtppnpntvyhcs) is a promising h -iab-restricted cd t cell epitope candidate of niv-g. the alignment of the peptide to the full amino acid sequence of wild-type niv-g is shown in figure s . the continuous threat of suddenly emerging niv outbreaks, particularly in bangladesh and india, demonstrate the need for countermeasure approaches ready to use in an immediate public health response. at present, there are no licensed niv vaccines for use in humans available. the existence of a niv candidate vaccine should significantly reduce the risk of infection and transmission of the virus in the case of an outbreak scenario. there are some experimental niv vaccines that have already been tested in different preclinical animal models. the major focus of these approaches was to evaluate the immunogenicity and efficacy in the context of niv challenge infection. in those studies, efficacy has been mostly associated with the generation of niv-specific antibodies, and immune monitoring is mainly relying on the detection of virus-neutralizing antibodies [ , , ] . however, there is relatively little known about the induction and the relevance of niv-specific cellular immune responses. in that context, the availability of appropriate tools to investigate the role of t cells in niv-specific immunity is an important prerequisite in the development of new vaccines and therapeutics. thus, it will be indispensable to monitor in animal models the contribution of virus-specific t cells to protective immunity but also to potential niv antigen-specific immune pathology. here, we identified a major histocompatibility complex (mhc) haplotype h -b-restricted peptide epitope in the niv-g protein by stimulating t cells from mva−nivsg vaccinated ifnar−/− mice with a two-dimensional ( d) matrix pool of overlapping peptides. ifnar−/− mice have been already established as a valuable preclinical animal model for niv infection with a ld of × pfu after intraperitoneal challenge infection [ ] . moreover, in previous studies, we have already successfully demonstrated that interferon type i receptor knockout mice (ifnar−/−) [ ] can be readily used as animal models to study the immunogenicity and protective capacity of mva immunization [ , ] . here, we wished to specifically assess the ability of mva-delivered niv-g antigen to induce the activation of cellular immune responses in mice. in general, the envelope g protein is known as the well-conserved attachment glycoprotein for both hev and niv. in a previous study, a recombinant adeno-associated virus vaccine expressing a full-length niv-g protein protected hamsters in an niv infection model [ ] . in another approach, a soluble version of hev-g has been engineered and showed an even more advantageous efficacy when tested in different preclinical animal models [ , , ] . using hevsg, a monoclonal antibody m . was derived and has already been successfully tested as a therapeutic approach in humans. to further enhance henipavirus g protein-induced immunogenicity, we also designed and tested a soluble version of the niv-g protein (nivsg) similar to the hevsg antigen used for the generation of m . . to comparatively evaluate the immunogenicity of the nivsg protein, we also generated an mva expressing full-length g. the recombinant viruses mva-niv-g and mva-nivsg produced stable amounts of niv-g antigen upon in vitro infection of human cells, indicating the unimpaired expression of the target gene under transcriptional control of the synthetic vaccinia virus-specific early-late promoter pmh or the strong natural early promoter pvgf. in the case of mva-nivsg, removal of the transmembrane domain and cytoplasmic tail resulted in the secretion of the niv-g from mva-infected cells and accumulation also in the supernatant of cell cultures, as demonstrated in western blot analysis and immunostaining. a similar result was obtained with hevsg, as expressed by conventional recombinant vacv [ ] . in contrast, the full-length mva-produced niv-g protein was not released in the supernatant, indicating the stable presentation on the cell surface through the transmembrane domains. another important aspect of proper protein expression, folding and conformational stability is influenced by the n-glycans. recent studies indicated that niv-g n-glycans reduce fusion efficiency because removal of some n-glycans caused cell-cell hyperfusogenicity and increased viral entry [ ] . glycosidase treatment of full-length mva-produced niv-g resulted in a polypeptide of kda, corresponding to the molecular mass predicted from the niv g gene encoding sequences. the glycosidase treatment of the nivsg also indicated the presence of all the n-glycans sites within the soluble version of the glycoprotein. a first in vivo evaluation in mice revealed that treatment with the mva-niv-g and mva-nivsg candidate vaccines resulted in the induction of similar levels of g-binding serum antibodies, confirming the immunogenicity of both antigen versions [ ] . in that context, the presence of binding antibodies seems to play a substantial role in the blocking of niv entry, since the mechanism of niv neutralization is complex and involves more antigenic sites than those required for simple receptor binding [ ] . however, more recent studies in different animal models suggest that cellular immune responses are also involved in mediating protection against niv infection [ , ] . this observation is further supported by studies in a pig model for niv infection showing substantial activation of cd t cells after oral infection with niv [ ] . in line with these preclinical data, humans surviving niv infection [ ] showed significant levels of proliferating (ki- +) cd t cells, indicating the presence of acute effector cells. these data emphasize that in addition to the humoral immune responses, t cells could be associated with recovery from niv infection. in a more recent study, stroh and coworkers confirmed the activation of niv-specific cd t cells in mice after vaccination with niv-like particles. these data further highlight that t cells may play a critical role in niv infection [ ] . another hypothesis is that niv-specific t cells could be involved in potential immunopathologies. in this context, data from infections with other neurotropic viruses, for example, west nile virus, demonstrated that antigen-specific t cells can open the blood brain barrier and contribute to virus infections of the brain [ ] [ ] [ ] [ ] [ ] . thus, to allow for more detailed studies characterizing t cells in niv-associated immunity or pathogenesis, it is essential to identify niv-g peptide epitopes allowing for the specific mhc-restricted antigen presentation and the activation of niv-specific t cells. we identified a nonamer epitope niv-g- . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (rstdnqavi). analysis of this peptide sequence showed that niv-g . - could be functionally conserved in hendra virus, but not cedar virus (another recognized henipavirus), g antigens ( figure s ). structural and functional analyses reveal promiscuous and species-specific use of ephrin receptors by cedar virus [ ] . this potential epitope will support a more detailed experimental characterization of t cells induced by niv infection in the mouse model and their contribution for pathogenesis and protection. in this study, we found that mva-nivsg vaccination induced significantly higher amounts of niv-g epitope-specific cd t cells compared with the mva-niv-g candidate vaccine. this was a somewhat surprising observation as the immunizations with both antigens had elicited very comparable levels of g-specific antibodies. it is tempting to speculate that nivsg, as a soluble antigen, can trigger enhanced t cell responses because it is available to two different pathways of antigen presentation. on the one hand, nivsg as intracellularly synthetized antigen is endogenously processed and presented via mhc-i on the cell surface to activate cd t cells. in addition, the nivsg is secreted in high amounts from the mva-nivsg-infected cells, and such extracellular antigen can efficiently fuel the cross-presentation pathway and thereby induce elevated cd and cd t cell immune responses [ , ] . interestingly, the mva-nivsg candidate vaccine also proved to rapidly induce niv-g epitope-specific cd t cells after single-dose application. these data are of relevance, since the epidemiology of the more recent niv outbreaks demonstrated that a potential vaccine candidate should rapidly protect. importantly, an h b-restricted epitope has been identified in ifnar−/− mice, which serve as an established small animal model for niv infection [ ] . in addition, we showed the induction of niv-g-specific cd t cells upon prime-boost immunization in the ifnar−/− with the mva-niv vaccines and using peptides for in vitro stimulation, as identified by using mhc ii binding predictions obtained from the iedb online resource (www.iedb.org). this data goes well along with the hypothesis that cd t cell responses are also significantly elevated upon infection [ ] . again, mva-nivsg vaccination results in more efficient activation of cd t cell responses. further experiments will be needed to characterize the contribution of niv g-specific cd t cells for niv infection in more detail. taken together, our findings showed the activation of niv-g-specific t cells in ifnar−/− mice following vaccination with mva-based candidate vaccines. we confirmed the identification of potential h -b-restricted niv-g cd and cd t cell peptide epitopes. in this study we also demonstrated that an mva-nivsg candidate vaccine may have superior immunogenicity, resulting in niv-specific antibodies and t cells in ifnar−/− mice. these data emphasize the promise of future studies in this animal model further evaluating the role of niv-specific t cells activated by the g- . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and g- - peptide epitopes, in both vaccine-induced protection and potential contribution to niv-induced pathologies. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : generation and characterization of recombinant mva-nivsg and mva-niv-g, figure s : ifn-γ production by cd and cd t cells stimulated by the niv-g peptide . , figure s : amino acid alignment of h -b-and h -iab-restricted peptides, figure s : quality of activated cd t cells from mice immunized with recombinant mva expressing niv-g. author contributions: g.k., s.v., c.c.b., g.s. and a.v. conceived and designed the experiments; g.k., s.v., s.j. and a.v. performed the experiments; g.k., s.v., u.k., c.c.b., g.s. and a.v. analyzed the data; g.k., g.s. and a.v. wrote the paper. all authors have read and agreed to the published version of the manuscript. nipah virus: a recently emergent deadly paramyxovirus adaptive immune responses in humans during nipah virus acute and convalescent phases of infection nipah virus infection henipavirus infection of the central nervous system clinical presentation of nipah virus infection in bangladesh emerging trends of nipah virus: a review long-term neurological and functional outcome in nipah virus infection relapsed and late-onset nipah encephalitis neuropsychiatric sequelae of nipah virus encephalitis henipaviruses: an updated review focusing on the pteropid reservoir and features of transmission changing resource landscapes and spillover of henipaviruses pathology of acute henipavirus infection in humans and animals animal challenge models of henipavirus infection and pathogenesis henipavirus infections: lessons from animal models date palm sap linked to nipah virus outbreak in bangladesh transmission of nipah virus- years of investigations in bangladesh a functional henipavirus envelope glycoprotein pseudotyped lentivirus assay system ephrin-b ligand is a functional receptor for hendra virus and nipah virus ephrinb is the entry receptor for nipah virus, an emergent deadly paramyxovirus two key residues in ephrinb are critical for its use as an alternative receptor for nipah virus quantitative analysis of nipah virus proteins released as virus-like particles reveals central role for the matrix protein functional studies of host-specific ephrin-b ligands as henipavirus receptors potent neutralization of hendra and nipah viruses by human monoclonal antibodies therapeutic treatment of nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody pathogenic differences between nipah virus bangladesh and malaysia strains in primates: implications for antibody therapy. sci a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection nipah virus: vaccination and passive protection studies in a hamster model recombinant nipah virus vaccines protect pigs against challenge 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use of recombinant adeno-associated virus-vector vaccines hendra virus and nipah virus animal vaccines receptor binding, fusion inhibition, and induction of cross-reactive neutralizing antibodies by a soluble g glycoprotein of hendra virus site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of hendra virus a recombinant subunit vaccine formulation protects against lethal nipah virus challenge in cats vaccination of ferrets with a recombinant g glycoprotein subunit vaccine provides protection against nipah virus disease for over months a hendra virus g glycoprotein subunit vaccine protects african green monkeys from nipah virus challenge protection against henipaviruses in swine requires both, cell-mediated and humoral immune response chapter five-modified vaccinia virus ankara: history, value in basic research, and current perspectives for vaccine development functional role of type i and type ii interferons in antiviral defense biochemical, conformational, and immunogenic analysis of soluble trimeric forms of henipavirus fusion glycoproteins development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus infection in an animal model kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes simultaneous high-resolution analysis of vaccinia virus and host cell transcriptomes by deep rna sequencing myristoylation increases the cd +t-cell response to a gfp prototype antigen delivered by modified vaccinia virus ankara elucidating and minimizing the loss by recombinant vaccinia virus of human immunodeficiency virus gene expression resulting from spontaneous mutations and positive selection easy and efficient protocols for working with recombinant vaccinia virus mva cells responding to the middle east respiratory syndrome coronavirus nucleocapsid protein delivered by vaccinia virus mva in mice pooled-peptide epitope mapping strategies are efficient and highly sensitive: an evaluation of methods for identifying human t cell epitope specificities in large-scale hiv vaccine efficacy trials iedb-ar: immune epitope database-analysis resource in identification of poxvirus cd + t cell determinants to enable rational design and characterization of smallpox vaccines a vlp-based vaccine provides complete protection against nipah virus challenge following multiple-dose or single-dose vaccination schedules in a hamster model hendra virus vaccine, a one health approach to protecting horse, human, and environmental health rapid expansion of cd + t cells in wild-type and type i interferon receptor-deficient mice correlates with protection after low-dose emergency immunization with modified vaccinia virus ankara critical role of perforin-dependent cd + t cell immunity for rapid protective vaccination in a murine model for human smallpox a recombinant hendra virus g glycoprotein-based subunit vaccine protects ferrets from lethal hendra virus challenge n-glycans on the nipah virus attachment glycoprotein modulate fusion and viral entry as they protect against antibody neutralization antibodies to henipavirus or henipa-like viruses in domestic pigs in ghana neutralization assays for differential henipavirus serology using bio-plex protein array systems henipavirus-like particles induce a cd t cell response in c bl/ mice pathways exploited by flaviviruses to counteract the blood-brain barrier and invade the central nervous system toll-like receptor mediates west nile virus entry into the brain causing lethal encephalitis west nile virus-induced disruption of the blood-brain barrier in mice is characterized by the degradation of the junctional complex proteins and increase in multiple matrix metalloproteinases nile virus: crossing the blood-brain barrier cd + t cells mediate recovery and immunopathology in west nile virus encephalitis structural and functional analyses reveal promiscuous and species specific use of ephrin receptors by cedar virus cross-priming of cytotoxic t cells dictates antigen requisites for modified vaccinia virus ankara vector vaccines distinct pathways of antigen uptake and intracellular routing in cd and cd t cell activation pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -bw tziup authors: perez-zsolt, daniel; martinez-picado, javier; izquierdo-useros, nuria title: when dendritic cells go viral: the role of siglec- in host defense and dissemination of enveloped viruses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: bw tziup dendritic cells (dcs) are among the first cells that recognize incoming viruses at the mucosal portals of entry. initial interaction between dcs and viruses facilitates cell activation and migration to secondary lymphoid tissues, where these antigen presenting cells (apcs) prime specific adaptive immune responses. some viruses, however, have evolved strategies to subvert the migratory capacity of dcs as a way to disseminate infection systemically. here we focus on the role of siglec- , a sialic acid-binding type i lectin receptor potently upregulated by type i interferons on dcs, that acts as a double edge sword, containing viral replication through the induction of antiviral immunity, but also favoring viral spread within tissues. such is the case for distant enveloped viruses like human immunodeficiency virus (hiv)- or ebola virus (ebov), which incorporate sialic acid-containing gangliosides on their viral membrane and are effectively recognized by siglec- . here we review how siglec- is highly induced on the surface of human dcs upon viral infection, the way this impacts different antigen presentation pathways, and how enveloped viruses have evolved to exploit these apc functions as a potent dissemination strategy in different anatomical compartments. dendritic cells (dcs) are the most potent antigen presenting cells (apcs) found in humans [ , ] and their immune function is key to initiate immunity against invading viruses [ ] [ ] [ ] . these cellular sentinels patrol distinct mucosae and upon infection, viral sensing triggers rapid innate immune responses that might initially contain viral spread. dc activation also elicits cellular migration towards secondary lymphoid tissues, where dcs acquire a fully mature phenotype and become competent for antigen presentation, activation of naïve t cells, and expansion of antigen-specific adaptive t cell responses [ , ] . despite the immune activity exerted by dcs after viral infection, it has been known for decades that viruses evolved different strategies to escape dc antiviral activity [ ] [ ] [ ] . furthermore, certain viruses exploit the immune function of dcs as a way to colonize distant tissues and effectively disseminate systemically [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the discovery of the role of the receptor siglec- /cd , a sialic acid-binding ig-like lectin- expressed by dcs, has greatly contributed to our understanding of how viruses subvert dc activity. the siglec- receptor acts as an immuno-surveillance molecule [ ] but can also be effectively hijacked by distinct enveloped viruses, which either infect dcs directly or are effectively transferred to bystander target cells that become productively infected [ ] [ ] [ ] . hence, siglec- function on dcs clearly illustrates how these apcs can trigger antiviral immunity but also enhance viral spread via this receptor. siglec- is a type i transmembrane lectin with an amino-terminal v-set domain that interacts with sialylated ligands, preferentially n-acetylneuraminic acid (neu ac) in an α - linkage [ , ] . several enveloped viruses including human immunodeficiency virus (hiv)- [ ] [ ] [ ] and ebola virus (ebov) [ , [ ] [ ] [ ] incorporate such sialylated ligands within their membranes as an integral part of the gangliosides that are dragged from the plasma membrane when viruses bud from infected cells. although siglec- affinity for sialylated ligands is in the micromolar range, high-avidity binding can be achieved upon clustering of thousands of gangliosides in the viral membrane with their receptors on the cellular membrane [ , ] . moreover, as siglec- contains ig-like c -type extracellular domains that separate the ligand-binding site from the cell surface, it is available for interaction with external ligands and not bound in cis to cell-surface molecules, which is what usually happens with shorter siglecs that are also expressed by dcs [ , , ] . in vivo, the role of siglec- during viral infection has been mostly studied in murine models, focusing on resident tissue macrophages that express this lectin and play key immunomodulatory functions. siglec- -expressing macrophages are located in the subcapsular sinus of the lymph nodes, and they protect mice against vesicular stomatitis virus (vsv) infection by containing incoming viruses. viral sensing triggers cytokine release and promotes antigen presentation to b cells [ , ] . however, studies using different retroviruses to infect mice have shown that the protective function of these macrophages can be hijacked for efficient viral infection and dissemination within tissues. indeed, robust infection of a particular retrovirus in lymphoid tissues and spleen requires siglec- -expressing macrophages [ ] . the pathogenicity of the infecting retrovirus is key to tip the balance of these siglec- -expressing macrophages in favor of the protective immune function. the antiviral response dominates when the replicating virus has an expanded tropism [ ] , as it also happens in the case of the amphotropic vsv infection [ , ] . under these pathogenic conditions, viral capture via siglec- macrophages is necessary to elicit an effective antiviral cd + t cell response via antigen cross-presentation by dcs [ ] . overall, these murine studies explain how siglec- can contain viral replication and induce antiviral immunity against highly pathogenic viruses, but also favor viral spread within tissues when retroviruses have a limited tropism. yet, how these findings correlate with the pathogenesis of different siglec- -interacting human viruses, such as hiv- or ebov, remains largely unexplored. hiv- is the causative agent of acquired immunodeficiency syndrome (aids), a pandemic that has affected more than million people worldwide [ ] , while ebov is responsible for the intermittent outbreaks that produce a filovirus-associated disease (fvd) with high fatality rates [ ] . in this review, we discuss how siglec- is induced on human dcs upon viral infection, to what degree that impacts different viral antigen presentation routes, and in which ways distant enveloped viruses have evolved to exploit siglec- function as a dissemination strategy in distinct anatomical compartments. siglec- is a receptor codified by an interferon-stimulated gene and is therefore potently upregulated on distinct human dcs, monocytes, and macrophages when these cells sense type i interferons (ifns) such as ifnα [ ] [ ] [ ] . thus, infection with viruses such as hiv- or ebov tightly upregulates siglec- expression on apcs, as they directly trigger or indirectly promote the release of type i ifns via immune activating factors (figure ). hiv- induces secretion of type i interferons (ifns) by plasmacytoid dcs (pdcs) through toll-like receptor (tlr) - and - sensing, which upregulates siglec- on dcs in a paracrine manner. in addition, lipopolysaccharide (lps) from bacterial translocation upregulates siglec- on dcs via tlr sensing and autocrine type i ifn release. (b) during ebov infection, type i ifns might also play a central role in enhancing siglec- expression on dcs, although this needs further investigation. pdcs may produce type i ifns in response to ebov infection in vivo, while bacterial translocation was suspected during a case of gram-negative septicemia in an ebov-infected patient. in parallel, viral components such as secreted ebov glycoprotein may induce activation of myeloid cells through tlr signaling, providing an alternative stimulus of autocrine type i ifns during ebov infection. while solid arrows indicate established mechanisms, dotted arrows suggest processes that require further investigation. ifnar: ifnα/β receptor; sgp: secreted glycoprotein. in the case of hiv- infection, ifnα levels are potently boosted during acute infection, and sustained-although to a lower extent-throughout the chronic stage, which is characterized by a persistent immune activation [ ] [ ] [ ] . several dc types have been identified as the sources of ifnα production during the course of hiv- infection, and therefore contribute to siglec- induction. plasmacytoid dcs (pdcs) are considered the most potent type i ifn producers in blood [ ] , and their capacity to secrete ifnα in response to hiv- sensing has been demonstrated in vitro [ ] [ ] [ ] [ ] [ ] and in vivo [ ] [ ] [ ] , both during the acute and chronic phases of the disease [ , , ] . of note, pdc activation in response to hiv- sensing induces ifnα secretion through toll-like receptor (tlr) - and - signaling [ , ] and maturation of bystander myeloid dcs [ ] , and this ifnα response is more potent on pdcs derived from females [ ] . in turn, secretion of this cytokine can directly upregulate siglec- expression on dcs [ ] ( figure a ). in addition to pdcs, myeloid dcs also secrete type i ifns, although this release is mostly and indirectly triggered by immune activating signals present during the course of hiv- infection, which can induce the expression of ifn-stimulated genes on dcs in an autocrine manner [ ] [ ] [ ] [ ] . one of those factors is bacterial lipopolysaccharide (lps), which is increased in the plasma of hiv- -infected individuals due to the bacterial translocation that takes place in the gut-associated lymphoid tissue as a consequence of the gut epithelial barrier disruption occurring early after hiv- infection [ , , ] ( figure a ). lps induces siglec- expression on dcs [ ] . moreover, plasma from hiv- -infected individuals also stimulates siglec- expression on dcs signaling via type i ifn receptor [ ] . this explains why on circulating monocytes of hiv- infected individuals, siglec- expression correlates in vivo with the levels of plasma viremia, and why these levels only diminish after introduction of combined antiretroviral treatment [ ] . moreover, both types of siglec- -inducing factors are also present throughout the course of ebov infections ( figure b ). secretion of ifnα has been detected in humans and nonhuman primate models [ , ] , especially in lethal cases [ ] , while asymptomatic ebov infections are characterized by the absence of this cytokine [ ] . although in vitro pdcs exposed to ebov do not secrete ifnα [ ] , activated pdcs have been found in ebov-infected nonhuman primates, suggesting that these cells might produce ifnα in vivo [ ] ( figure b ). aside from pdcs, myeloid cells could contribute to ifnα secretion during ebov infection, as ebov-like particles induce ifnα production by murine bone marrow-derived dcs through tlr signaling [ ] . ebov glycoprotein interaction with human monocyte-derived macrophages induced tlr -dependent ifnα secretion by these cells [ ] . moreover, a cleaved and secreted form of ebov glycoprotein signals through tlr [ ] , although ifnα secretion in response to these glycoproteins remains unexplored ( figure b ). noteworthy, lps was also found in a case of ebov infection complicated with septicemia, possibly due to bacterial translocation [ ] , which might account for indirect ifnα secretion during ebov infection as described for hiv- ( figure b) . overall, the presence of type i ifns throughout the course of these viral infections is well-established, although both hiv- and ebov have evolved particular molecular mechanisms via viral antagonistic proteins that aid to evade cellular immune sensing [ ] [ ] [ ] [ ] . intriguingly, the protective role of type i ifn responses is controversial, since the apparent antiviral function during the earliest stages of infection may, in turn, fuel pathogenesis during the later stages of viral disease. that seems to be the case not only for hiv- [ ] , but also for ebov [ ] , where clinical data collected during human outbreaks have indicated that elevated levels of circulating ifnα, as well as upregulation of type i ifn-inducible genes, correlates with fatal disease outcome [ , [ ] [ ] [ ] . thus, hiv- and ebov infections trigger an immune activation state that upregulates siglec- expression on dcs, a situation that might favor early viral dissemination events in an otherwise antiviral environment [ ] . viral sensing enhances siglec- expression on apcs, and this facilitates hiv- infection of dcs and other myeloid cells (figure a , top), such as macrophages [ ] . although dcs are generally resistant to hiv- infection, a recent study showed that siglec- mediated hiv- productive infection of a population of human dc precursors known as pre-dcs [ ] . even though all dcs express the hiv- -interacting cellular receptor cd and the viral coreceptors [ , ] , being therefore susceptible to viral infection in vitro [ ] [ ] [ ] , infectivity was less prominent than in activated cd + t cells [ ] [ ] [ ] [ ] . importantly, the activation process that enhances siglec- expression on dcs further restricts viral infection, as mature dcs are -fold to -fold less susceptible to hiv- than immature dcs [ , , [ ] [ ] [ ] . infection of dcs with hiv- also appears to be uncommon in vivo, although it has been reported for both cutaneous and mucosal dcs, including vaginal epithelial dcs [ , , ]. yet, the role of siglec- in promoting hiv- infection of these dcs remains largely unexplored. in most dc subtypes, however, the restriction factor samhd inhibits reverse transcription, thus precluding immune sensing and the synthesis of viral antigens. bottom: conversely, hiv- encodes vpx, which counteracts samhd activity allowing reverse transcription of the viral genome, that can be sensed via cgas and trigger cytokine release. newly synthesized proteins lead to the production of viral particles, but also to proteosomal cleavage the de-phosphorylated form of the host factor sterile alpha motif histidine-aspartate domain-containing protein (samhd ) is the most potent restriction factor that limits hiv- infection in dcs [ ] (figure a, top) . however, if this lack of infectivity is actually beneficial for an immune control remains controversial, as the absence of viral replication impairs viral sensing and limits presentation of hiv- -specific antigens to prime adaptive immune responses [ ] [ ] [ ] . in contrast to hiv- , hiv- naturally replicates on dcs [ ] , which depends on the counteraction of samhd by the viral antagonist vpx [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (figure a, bottom) . hiv- genome replication in infected dcs is detected by the innate cytosolic dna sensor cyclic guanosine-adenosine monophosphate synthase (cgas), which triggers immune responses upon dna sensing [ , ] (figure a, bottom) . in contrast, hiv- restriction by samhd prevents viral dna (vdna) retrotranscription in the cytoplasm, impairing the induction of antiviral type i ifn responses [ ] (figure a, top) . thus, viral replication on dcs can provide an additional source of viral components to be detected via immune sensors or presented to t cells [ , ] (figure a, bottom) , but also compromise cell viability and release inflammatory factors that fuel viral pathogenesis, as previously described for sustained or chronic type i ifn responses. while hiv- replication on dcs remains hard to identify in vivo, it has been known for more than a decade that dcs are among the first target cells encountering ebov [ ] . dcs are highly susceptible to ebov infection [ , ] , and this is a complex process that involves several host factors whose function is still being identified [ ] . indeed, siglec- expressed on activated dcs has recently emerged as a new host factor implicated in ebov attachment, a mechanism that facilitates subsequent cytoplasmic viral entry [ ] ( figure b ). initial ebov attachment to the dc surface is mediated by several receptors that recognize different elements on the viral membrane and often have a redundant activity [ ] . c-type lectin receptors (clrs) such as the dendritic cell-specific intercellular adhesion molecule- -grabbing non-integrin (dc-sign) and the liver/lymph node sinusoidal endothelial c-type lectin (lsectin) mediate viral attachment through binding to viral glycoproteins [ , ] , while receptors of the tim/tam families (comprising the t cell immunoglobulin and mucin domain receptor along with tyro-axl-mer receptors) recognize phosphatidylserine lipids present on the viral envelope [ ] (figure b ). ebov incorporates sialylated gangliosides on their membrane [ ] , and we have recently shown that these viruses are effectively recognized by the siglec- receptor [ ] (figure b ). siglec- recognition of sialylated gangliosides on ebov modulates the binding, uptake, and trafficking of filoviral particles into a sac-like virus-containing compartment (vcc) continuous with the plasma membrane ( figure b ). viruses stored in this compartment can be redirected into the classical endosomal pathway and facilitate viral entry into the cytoplasm [ ] . indeed, ebov macropinocytosis allows trafficking into late endosomes, where cleavage of viral glycoproteins by cathepsin b (ctsb) facilitates the interaction with the endosomal receptor niemann-pick c [ ] [ ] [ ] (ncp ) that triggers cytoplasmic viral entry [ ] [ ] [ ] (figure b ). thus, siglec- -mediated attachment facilitates viral access to the cell cytoplasm [ ] . while siglec- contributes to ebov entry into dcs, filoviral replication within these cells compromises immune function and prevents adaptive immune responses by limiting cytokine secretion, downregulating the expression of major histocompatibility complex (mhc) and costimulatory molecules and also by reducing the ability of dcs to stimulate t cell proliferation [ , [ ] [ ] [ ] . these results suggest that ebov suppression of dc function prevents initiation of adaptive immune responses and facilitates uncontrolled systemic virus replication [ , ] through a mechanism that is enhanced by siglec- activity [ ] . however, clinical data gathered during the west african - outbreak showed strong and sustained t cell activation [ ] , challenging the in vivo relevance of this viral dc-escape mechanism [ ] . overall, both hiv- and ebov can exploit siglec- activity to boost dc infectivity, although ebov replication is more prominent in these cells. yet, viral infection poses a difficult balance for apcs. on the one hand, infectivity triggers antiviral immunity via viral sensing and antigen presentation, but on the other hand, it also promotes cell death and suppression of immune responses through the activity of particular viral antagonist proteins. recently, it has been suggested that this apparent paradox is overcome by a division of labor between distinct dc subsets [ ] . there is therefore a dissociation between viral infection and antigen presentation, which occurs in distinct dc subpopulations. by these means, susceptible infected dcs transfer viral antigens to resistant dcs, which remain competent to launch adaptive immune responses against viral infections [ ] . while several pathways allow for antigen transfer between dcs, secretion of extracellular vesicles bearing particular antigens is among the most effective ones. although the functional paradigm of dc biology states that the particular apc that interacts with incoming viruses in the mucosa would be the one processing these viruses and then traveling to the lymphoid tissue, these cells may not always be the only ones presenting the captured antigens. rather, these pathogen-interacting dcs may transfer captured antigens to other apcs by several mechanisms, including secretion of extracellular vesicles bearing antigen-loaded fragments, which can even be already processed and presented in mhc molecules (figure , top) . by these means, the number of dcs bearing viral-specific antigens can be increased very quickly upon infection, thus amplifying the initiation of primary adaptive immune responses [ ] [ ] [ ] . importantly, to induce naïve t cell stimulation in vitro, these extracellular vesicles require a competent activated dc to deliver the co-stimulatory signals to t cells [ ] (figure , top) . thus, antigen-containing extracellular vesicles do not overcome the need for a competent apc to activate naïve t cells. among the distinct cellular receptors expressed by dcs, siglec- is key to capture secreted extracellular vesicles through the same mechanism hijacked by enveloped viruses [ ] (figure ). siglec- interacts with extracellular vesicles via recognition of sialylated gangliosides packaged on the vesicle membrane [ ] , which assemble and bud from cellular membranes as viruses do [ , ] . this result has been confirmed not only in vitro [ , ] with extracellular vesicles derived from cell lines or primary cells but also in murine models where siglec- expressed on lymphoid tissues was required to trap extracellular vesicles in vivo [ ] . upon capture of extracellular vesicles on activated dcs via siglec- , these vesicles are trafficked along with the receptor towards a sac-like compartment invagination that is continuous with the plasma membrane and allows for extracellular vesicle retention [ , ] (figure , top) . the siglec- positive compartment formed within activated dcs may serve as an antigen depot, controlling and sustaining adaptive immunity even if the source of antigen is not directly in contact with the apc, that still can trigger antigen-specific immune responses. these antigens could maintain immunity for prolonged periods, as it happens when dcs control endosomal acidification to preserve antigen cross-presentation over time [ ] . although mature or activated dcs markedly downregulate their macropinocytic capacity, these cells are still able to capture, process, and present antigens internalized via endocytic receptors [ ] , and that may also be the case for siglec- via extracellular vesicle trapping. moreover, as dcs continue to capture and present antigens after maturation in vivo [ ] , they could also initiate responses to newly encountered antigens during the course of viral infections, a process that would be boosted by siglec- expression. overall, siglec- retention of distinct viruses on extracellular vesicle-containing compartments highlight how these cells might act as "trojan horses", capturing filoviruses or retroviruses in the peripheral mucosae and carrying them to secondary lymphoid tissues, where viruses can be effectively transmitted to target cells and contribute to the systemic spread of infection [ ] [ ] [ ] , ] . recently, we have identified the presence of siglec- -expressing cells in cervical mucosa. these dcs are capable of capturing hiv- and mediate viral transmission to target cd + cells via transinfection, even in a basal state where no apparent activation is detected [ ] (figure ) . indeed, dcs directly isolated from human ectocervix displayed a basal siglec- expression that was sufficient to mediate viral transfer. however, at the endocervix, where the expression of siglec- is lower than at the ectocervix, this capacity was enhanced upon ifnα stimulation (figure ) . hiv- is mostly acquired by sexual transmission, and in the cervical mucosa, there are two major sources of antiviral type-i ifn responses after retroviral infection: resident myeloid cells [ ] and pdcs, which are the most potent producers of ifnα [ ] and are soon recruited to the cervix [ ] ( figure ) . although increased antiviral ifnα secretion could limit initial viral infection, it could promote viral capture on cervical myeloid cells via siglec- induction as well. of note, in the cervical the fate of trapped extracellular vesicles on dcs is diverse, as they provide a source not only for antigen cross-presentation to cd + t cells, but also to stimulate antigen-specific naïve cd + t cell responses in vivo [ , ] . cd + t cell stimulation can take place either by reprocessing the antigens contained in the captured extracellular vesicles or by the direct presentation of previously processed functional epitope-mhc complexes exposed in the vesicle surface [ , ] . direct extracellular vesicle antigen presentation in the absence of lytic degradation within dcs was initially described using dc populations devoid of particular mhc-ii molecules, that were still able to activate cd + t cells because the necessary mhc-ii molecules were already presenting the antigen on the extracellular vesicles trapped by those dcs [ ] . thus, extracellular vesicles displaying previously processed functional epitope-mhc complexes on their surface can be recognized, retained, and directly transferred from dcs to antigen-specific cd + t cells [ ] (figure , top) . in turn, siglec- upregulation on activated dcs, which are competent apcs, could boost extracellular vesicle uptake and magnify antiviral immune responses. intriguingly, hiv- and other retroviruses exploit this antigen dissemination pathway usually engaged by extracellular vesicles to reach cd + t cells [ ] , which are the main cellular targets of this particular retrovirus (figure , bottom left) . siglec- directs captured hiv- particles to the same vcc where ebola viral particles are retained [ ] , that is in addition the same compartment where extracellular vesicles are trapped in activated dcs [ , ] (figure , bottom left) . however, in the case of hiv- recognition, viral entry via siglec- does not lead to the productive infection of dcs as it happens with ebov, but favors the transfer of trapped viruses to bystander cd + t cells. thus, trapped viruses are efficiently transmitted across infectious synapses to susceptible lymphocytes [ , , ] ( figure ). this mechanism of viral transmission is known as trans-infection [ ] and is mediated by siglec- on activated monocyte-derived dcs, monocytes, blood conventional dcs, pre-dcs, and primary myeloid cells isolated from lymphoid and cervical tissues [ ] [ ] [ ] , , ] . filoviral trans-infection from dcs to cd + t cells is improbable as lymphocytes are largely resistant to ebov infection [ ] . nonetheless, filoviruses display a broad cell tropism, infecting hepatocytes, adrenal cortical cells, and endothelial cells, among other cellular targets [ ] [ ] [ ] [ ] . thus, aside from lymphocytes, other cellular targets could be transinfected (figure , bottom right), as it was previously shown for a human cell line binding ebov that transinfected hela cells [ ] . however, further research will be required to determine in which anatomical context dcs trapping ebov via siglec- could transfer that infectivity to susceptible cellular targets in vivo. overall, siglec- retention of distinct viruses on extracellular vesicle-containing compartments highlight how these cells might act as "trojan horses", capturing filoviruses or retroviruses in the peripheral mucosae and carrying them to secondary lymphoid tissues, where viruses can be effectively transmitted to target cells and contribute to the systemic spread of infection [ ] [ ] [ ] , ] . recently, we have identified the presence of siglec- -expressing cells in cervical mucosa. these dcs are capable of capturing hiv- and mediate viral transmission to target cd + cells via trans-infection, even in a basal state where no apparent activation is detected [ ] (figure ) . indeed, dcs directly isolated from human ectocervix displayed a basal siglec- expression that was sufficient to mediate viral transfer. however, at the endocervix, where the expression of siglec- is lower than at the ectocervix, this capacity was enhanced upon ifnα stimulation ( figure ) . hiv- is mostly acquired by sexual transmission, and in the cervical mucosa, there are two major sources of antiviral type-i ifn responses after retroviral infection: resident myeloid cells [ ] and pdcs, which are the most potent producers of ifnα [ ] and are soon recruited to the cervix [ ] (figure ). although increased antiviral ifnα secretion could limit initial viral infection, it could promote viral capture on cervical myeloid cells via siglec- induction as well. of note, in the cervical biopsy of a viremic hiv- + patient, siglec- + cells harbored hiv- -containing compartments, demonstrating that in vivo, these cells can trap viruses [ ] . interestingly, similar vcc-like structures have been detected in urethral macrophages of hiv- -infected individuals under suppressive combination antiretroviral therapy [ ] , but if siglec- is implicated in the formation of these particular structures remains to be determined. siglec- allows transferring viruses to bystander cd + t cells in the mucosa, but also the systemic viral dissemination upon dc migration to lymphoid tissues ( figure ) . indeed, dcs bearing retroviruses are found in the draining lymph nodes of distinct animal models as soon as h after vaginal challenge [ , [ ] [ ] [ ] and these findings originally led to formulation of the trojan horse hypothesis, which states that dcs can serve as vehicles transporting the virus from the entry sites to distant tissues [ , ] . . proposed mechanism of hiv- dissemination from the female reproductive tract. in the vaginal/ectocervical mucosa, basal siglec- + dcs may mediate local hiv- trans-infection to target cd + t cells. at the endocervical mucosa, lower siglec- expression is boosted in response to ifnα released by recruited pdcs sensing hiv- . siglec- + dcs may also contribute to systemic hiv- spread due to their ability to migrate to secondary lymphoid tissues, where cd + t cells accumulate. while a similar mechanism could be exploited by ebov, further work needs to address this possibility. we hypothesize that the outcome of early interactions between dcs and enveloped viruses may be key to mount effective antiviral responses, but these early encounters also foster viral dissemination to distant tissues. the emerging roles of siglec- receptor on dcs clearly exemplify how the immune-mediated activity of this lectin can be effectively hijacked by unrelated viruses such as hiv- or ebov. future work should address if other enveloped viruses causing relevant human infectious diseases may contain sialylated gangliosides on their membranes and be recognized by siglec- , as it has been already shown for the henipavirus [ ] . moreover, the precise contribution of siglec- to viral immune containment or to pathogenic viral dissemination should be carefully evaluated, as understanding both mechanisms may provide novel avenues to combat infectious agents. the identification of individuals which naturally lack siglec- expression due to the presence of an early stop codon in the siglec gene in homozygosis [ ] indicates that the role of this protein is not essential and that it may be therefore a safe therapeutic target. developing such . proposed mechanism of hiv- dissemination from the female reproductive tract. in the vaginal/ectocervical mucosa, basal siglec- + dcs may mediate local hiv- trans-infection to target cd + t cells. at the endocervical mucosa, lower siglec- expression is boosted in response to ifnα released by recruited pdcs sensing hiv- . siglec- + dcs may also contribute to systemic hiv- spread due to their ability to migrate to secondary lymphoid tissues, where cd + t cells accumulate. while a similar mechanism could be exploited by ebov, further work needs to address this possibility. sexual transmission is not considered a major route of ebov infection. however, a case of sexually transmitted ebov has been well documented [ ] . moreover, a recent mathematical model is consistent with a significant contribution of sexual ebov transmission during the - outbreak in west africa [ ] . importantly, infectious viral particles are found in semen of ebov convalescent individuals several months following symptoms onset [ ] [ ] [ ] [ ] , and seminal fluid amyloids may enhance ebov infection [ ] . as the cytokine tgf-β is abundant in semen, and it also upregulates siglec- expression on dcs [ ] , the role of this receptor should be further assessed in the context of ebov sexual transmission. moreover, dcs are early and sustained targets of ebov that can disseminate infection from the portals of viral entry to the regional lymph nodes, spleen, and liver [ ] . since siglec- is expressed in all these ebov replicating-tissues [ , ] , this receptor could also boost systemic viral spread as previously suggested for hiv- [ , , , , ] . collectively, these data indicate that siglec- on dcs may not only participate in viral transmission at the mucosa, but also promote systemic viral dissemination to secondary lymphoid tissues. we hypothesize that the outcome of early interactions between dcs and enveloped viruses may be key to mount effective antiviral responses, but these early encounters also foster viral dissemination to distant tissues. the emerging roles of siglec- receptor on dcs clearly exemplify how the immune-mediated activity of this lectin can be effectively hijacked by unrelated viruses such as hiv- or ebov. future work should address if other enveloped viruses causing relevant human infectious diseases may contain sialylated gangliosides on their membranes and be recognized by siglec- , as it has been already shown for the henipavirus [ ] . moreover, the precise contribution of siglec- to viral immune containment or to pathogenic viral dissemination should be carefully evaluated, as understanding both mechanisms may provide novel avenues to combat infectious agents. the identification of individuals which naturally lack siglec- expression due to the presence of an early stop codon in the siglec gene in homozygosis [ ] indicates that the role of this protein is not essential and that it may be therefore a safe therapeutic target. developing such pharmacological agents to block siglec- interaction with viruses could pave the way to ameliorate viral systemic dissemination. moreover, exploiting the immune-surveillance function of siglec- receptor to induce antiviral immune responses may also prove valuable. in turn, dissecting how distinct viruses exploit common molecular pathways will advance future antiviral strategies to generate broad spectrum 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dendritic cell-mediated capture and transfer of hiv- and henipavirus identification of siglec- null individuals infected with hiv- this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - zpkpdzu authors: sun, fang; xia, zhiqiang; han, yuewen; gao, minjun; wang, luyao; wu, yingliang; sabatier, jean-marc; miao, lixia; cao, zhijian title: topology, antiviral functional residues and mechanism of ifitm date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: zpkpdzu interferon-inducible transmembrane proteins (ifitm / / ) have been reported to suppress the entry of a wide range of viruses. however, their antiviral functional residues and specific mechanisms are still unclear. here, we firstly resolved the topology of ifitm on the plasma membrane where n-terminus points into the cytoplasm and c-terminus resides extracellularly. further, krrk basic residues of ifitm locating at – of the conserved intracellular loop (cil) were found to play a key role in the restriction on the zika virus (zikv) and dengue virus (denv). similarly, krrk basic residues of ifitm / also contributed to suppressing zikv replication. finally, ifitm was revealed to be capable of restricting the release of zikv particles from endosome to cytosol so as to impede the entry of zikv into host cells, which was tightly related with the inhibition of ifitm on the acidification of organelles. overall, our study provided topology, antiviral functional residues and the mechanism of interferon-inducible transmembrane proteins. human interferon-induced transmembrane protein / / (ifitm / / ) is widely expressed in different tissues and organs, and the expression level of these proteins is regulated by interferon [ ] . they contain - amino acid residues and have two transmembrane domains. the topologies of ifitm , ifitm and ifitm are similar. they all consist of five parts, including short n-terminal domain (ntd) and c-terminal domain (ctd), intramembrane domain (im ) and intramembrane domain (im ), and short conserved intracellular loop (cil) [ ] . at least three possible topological models of ifitms have been reported [ ] . one model is that n-terminus and c-terminus reside extracellularly or in intracavity, im and im stretch across the phospholipid bilayer and cil resides in the cytoplasm. the second model is that n-terminus, c-terminus, and ctd are in the cytoplasm, and im and im are in the phospholipid bilayer. the third model is that n-terminus and cil reside in the cytoplasm, and ctd resides extracellularly or in intracavity [ ] . therefore, the exact topology of ifitms is still to be investigated. ifitm proteins are involved in the regulation of many activities, such as tumorigenesis [ ] and immune signal transduction [ ] . most importantly, it has been reported that ifitm proteins have a broad antiviral spectrum in recent decades. ifitm proteins can effectively block a variety of enveloped viruses from invading host cells, such as influenza a virus (iav) of orthomyxoviridae [ ] , respiratory syncytial virus (rsv) of pneumoviridae [ ] , zikv and denv of flaviviridae [ , ] , ebolavirus (ebov) and severe acute respiratory syndrome virus (sars-cov) of [ ] . on the other hand, ifitm proteins also restrict some nonenveloped viruses, such as reovirus (rov) [ ] . additionally, ifitms have almost no inhibitory effect on certain viral infections, such as adenovirus (adv) [ ] and sendai virus (sev) [ ] . although ifitms play critical antiviral roles in the innate immune system, their antiviral functional residues are mostly still unclear. there is a report showing im of ifitm is required for both the interaction of ifitm protein and the restriction of iav and denv [ ] ; therefore, vital antiviral functional residues of ifitm are to be determined. ifitm-mediated restriction on viruses mainly occurs at the stage of virus invasion into host cells [ ] . some studies suggest that ifitm proteins may suppress the entry of viruses by inhibiting the hemifusion of viral membrane and host cell membranes [ ] or restricting the formation of fusion pores following virus-endosome hemifusion [ ] . there is also a finding that ifitm disrupts intracellular cholesterol homeostasis to block viral release into the cytosol by interacting with vesicle-membrane-protein-associated protein a [ ] ; clearly, the antiviral mechanisms of ifitms are extremely diverse and complex, and still need to be studied. in our study, immunofluorescence and confocol microscopy analysis showed that ifitm was mainly distributed on the plasma membrane adopting the topological model that n-terminus of ifitm pointed into the cytoplasm and c-terminus extended extracellularly. alanine scanning and site-directed mutations found that krrk basic residues were key for the restriction of ifitm proteins on zikv and denv. by electron microscope and flow cytometry analysis, ifitm was revealed to restrict the release of zikv from endosome to cytosol, which was tightly related to its inhibition against the acidification of organelles. hek a, hek t, huh and vero cells were purchased from the china center for type culture collection (cctcc). all cells were cultured in dmem (gibco-invitrogen, new york, ny, usa) supplemented with % fbs (gibco-invitrogen) and % penicillin/streptomycin at • c in an incubator filled with % co . zika virus envelope (e) protein antibody (gtx ) was purchased from genetex (san antonio, usa). dengue virus type - antibody (ma - ) was purchased from invitrogen (shanghai, china). abs against flag (f ) and myc (a - ) were purchased from sigma-aldrich (shanghai, china) and genscript (nanjing, china), respectively. abs against ha ( - -lg) and gapdh ( - -lg) were purchased from proteintech group (wuhan, china). anti-sodium potassium atpase (na + /k + -atpase) antibody (ep y) was kindly offered by jing yao from the college of life sciences at wuhan university. alexa fluor ( es ) and alexa fluor affinipure donkey anti-mouse ( es ) were purchased from yeasen (shanghai, china). lysosensor™ green dnd- (l ) and thermo fisher scientific page ruler prestained protein ladder ( ) were purchased from invitrogen (shanghai, china). bestar ® sybr green qpcr master mix reagent (dbi- ) was purchased from dbi ® bioscience (shanghai, china). pcdna . -flag-ifitm /ifitm /ifitm , pcdna . -flag-ifitm-mutants, pcdna . -ha-ifitm -myc, pt-dimer-rab and pt-dimer-rab were constructed by our laboratory. pegfp-lamp was kindly offered by hongbing shu and zhiyin song from the college of life sciences, wuhan university. when covering about % of the culture flask, cells were transfected with recombinant plasmids using turbofect transfection reagent (invitrogen, r ) following the instructions; then, cells were cultured for h. zikv puerto rico strain (prvabc ) cdna plasmid was kindly provided by ren sun and danyang gong at ucla. denv serotype tsv strain (denv- tsv ) was kindly provided by bo zhang from wuhan institution of virology, chinese academy of sciences. sev and adv were kindly supplied by mingxiong guo and zan huang from the college of life sciences at wuhan university, respectively. after transfecting ifitm plasmid for h, cells were incubated with zikv (moi = . ), denv (moi = . ), sev (moi = . ) and adv (moi = ) for h at • c, respectively. then, the supernatant was removed and the cells were cultured with fresh dmem supplemented with % fbs and % penicillin/streptomycin for h. next, cells were collected to be detected. cells were fixed with precooled % paraformaldehyde for min at room temperature followed by washing them in pbs twice ( min each time). then, samples were treated without . % triton x- (intact cell membranes) or with . % triton x- (permeabilized cell membranes) for min followed by washing them in pbs twice ( min each time). then, nonspecific proteins were blocked for min with % bovine serum albumin (bsa) in pbs. next, cells were incubated with primary antibody at • c overnight and washed three times in pbs ( min each time). later, cells were incubated with alexa fluor-labeled secondary antibody for h at room temperature followed by washing them in pbs three times for min each time. cell nuclei were subsequently stained with dapi fluoromount-g ® (antgene, wuhan, china, ant ). the fluorescence was observed using a confocal laser-scanning microscope (leica tcs sp , wetzlar, germany). cells were lysed with % sds. samples were boiled in boiling water for min and centrifuged at , rpm for min at • c to remove cell debris. the total protein content of the supernatants was measured using a bca protein assay kit (thermofisher scientific, shanghai, china) and was mixed with × loading sample buffer (biosharp, bl a, hefei, china). then, samples were boiled in boiling water for min. equal amounts of proteins were separated by % sds-polyacrylamide gel electrophoresis (page) and transferred to nitrocellulose membranes (nc membrane, millipore, wuhan, china). nc membranes were incubated with % nonfat milk for h at room temperature to block nonspecific proteins. then, nc membranes were incubated with primary antibodies at • c overnight, followed by incubation with secondary antibodies for h at room temperature. antibodies were used following the instructions. later, nc membranes were detected by chemiluminescence using a westernbrighttm ecl western blotting detection kit (advansta, menlo park, usa). mrna of cells was extracted with trizol reagent (takara, beijing, china), and the first-strand cdna was reversed-transcribed by using the revert aid first-strand cdna synthesis kit (thermo scientific). the cdna was quantitated by qpcr with sev and gapdh primers using the bestar ® sybr green qpcr master mix reagent (dbi ® bioscience, shanghai, china). the primers of zikv, sev and gapdh were synthesized as our laboratory previously described [ ] . qpcr experiments were performed on an abi system (shanghai, china) according to the instructions. cells were incubated with µm lysosensor dnd- at • c for min. then, cells were digested with . % trypsin and passed through nylon net to obtain a single-cell suspension. flow cytometry measuring the intensity of fitc was performed on a cytoflex flow cytometer. if ifitm and -krrk alkalize acidic organelles, the peak of fitc would shift to the left relative to the negative control. vero cells were transfected with pcdna . , pcdna . -flag-ifitm and pcdna . -flag- -krrk in a cm dish, respectively. after h, cells were incubated with zikv (moi = ) at • c for h, followed by being cultured for h at • c in an incubator filled with % co . the supernatant was removed and then cells were fixed with % glutaraldehyde at • c for min. cells were collected and centrifuged at rpm for min. after that, cells were treated with % glutaraldehyde at • c for h to be detected. em samples were commissioned to wuhan institution of virology, chinese academy of sciences, to prepare and observe. samples were examined with a tecnai em with an accelerating voltage of kv and magnifications of × , and × . adobe photoshop cs and graphpad prism were used for statistical analysis. data were usually representative and presented as the mean ± standard deviation (sd) of the three independent experiments. p values were calculated by the student t-test. statistical significance was considered at p value less than . (* < . , ** < . and *** < . ). ifitm proteins widely expressed in different tissues and organs were found to play important antiviral roles in the innate immune system. although they share a high identity of amino acid sequences ( figure a ) and are able to inhibit a wide range of viruses, ifitm / / still have different antiviral activities and spectra. to determine if the intracellular distribution of ifitm proteins influences their antiviral activities, we analyzed their intracellular location. the cdna sequences of ifitm / / were cloned from hek t cells by rt-pcr and then were inserted into pcdna . plasmid with bamhi and xhoi, where a flag-tag was added to their n-terminus ( figure b ). the overexpression level of pcdna . -flag-ifitm / / was detected, and the result showed that they were greatly expressed compared with the control ( figure c ). na + /k + -atpase is a biomarker of the plasma membrane [ ] . we analyzed the colocalization of ifitm / / and na + -k + atpase by immunofluorescence using the anti-flag antibody and na + /k + -atpase antibody coupled with alexa fluor ® . the experimental data showed that ifitm distributed on the plasma membrane and in the cytoplasm. ifitm especially had strong colocalization with na + -k + atpase ( figure d ). in comparison, ifitm and ifitm appeared to reside only in cytoplasm ( figure d ). these data indicate ifitm distributes widely on the plasma membrane and cytoplasm. the colocalization of ifitm / / and na + -k + atpase in hek t cells; pcdna . and pcdna . -flag-ifitm / / were transfected in hek t cells. after h, cells were stained with anti-flag antibody, anti-na + -k + atpase antibody and dapi. then, cells were observed using confocal microscopy. flag-ifitm / / , red; na + -k + atpase, green; dapi, blue. previously, ifitms have been reported to have three topological models on the cell membranes ( figure a ) [ ] . to determine the detailed topological model that ifitm adopted on the cell membranes, we constructed pcdna . -ha-ifitm -myc plasmid to express ifitm protein fused n-terminal ha-tag and c-terminal myc-tag for the experiments. the sketch of the amino acid sequence of ha-ifitm -myc is shown in figure b . the location of ha-ifitm -myc was analyzed using anti-ha antibody or anti-myc antibody in huh cells when the plasma membranes were intact or permeabilized. we found that ha-ifitm -myc could not be dyed using anti-ha antibody when plasma membranes were intact, but it could be observed when cell membranes were permeabilized ( figure c,d) , which suggested that the n-terminus of ifitm points into the cytoplasm. differently, ha-ifitm -myc could be observed using anti-myc antibody when cells were treated with . % were transfected in hek t cells, respectively. after h, cells were collected, and the expression level of intracellular ifitm / / was analyzed by western blotting using anti-flag antibody. (d) the colocalization of ifitm / / and na + -k + atpase in hek t cells; pcdna . and pcdna . -flag-ifitm / / were transfected in hek t cells. after h, cells were stained with anti-flag antibody, anti-na + -k + atpase antibody and dapi. then, cells were observed using confocal microscopy. flag-ifitm / / , red; na + -k + atpase, green; dapi, blue. previously, ifitms have been reported to have three topological models on the cell membranes ( figure a ) [ ] . to determine the detailed topological model that ifitm adopted on the cell membranes, we constructed pcdna . -ha-ifitm -myc plasmid to express ifitm protein fused n-terminal ha-tag and c-terminal myc-tag for the experiments. the sketch of the amino acid sequence of ha-ifitm -myc is shown in figure b . the location of ha-ifitm -myc was analyzed using anti-ha antibody or anti-myc antibody in huh cells when the plasma membranes were intact or permeabilized. we found that ha-ifitm -myc could not be dyed using anti-ha antibody when plasma membranes were intact, but it could be observed when cell membranes were permeabilized ( figure c,d) , which suggested that the n-terminus of ifitm points into the cytoplasm. differently, ha-ifitm -myc could be observed using anti-myc antibody when cells were treated with . % triton x- or not ( figure c ,d), which indicated that the c-terminus of ifitm was located outside the plasma membranes. similarly, the location of ha-ifitm / -myc was analyzed using anti-ha antibody or anti-myc antibody in huh cells when plasma membranes were intact or permeabilized. we found that neither ha-ifitm -myc or ha-ifitm -myc could be dyed using anti-ha antibody or anti-myc antibody when plasma membranes were intact, but both of them could be observed when cell membranes were permeabilized ( figure s ). these results indicated that ifitm and ifitm are also located in the cytoplasm of huh cells. these data show that ifitm adopts the topological structure (figure a , right) on the plasma membranes, where the n-terminus points into the cytoplasm and the c-terminus resides extracellularly, consistent with the topology model that ntd and cil are in the cytoplasm and ctd is extracellular or intracavity. viruses , , x for peer review of triton x- or not ( figure c ,d), which indicated that the c-terminus of ifitm was located outside the plasma membranes. similarly, the location of ha-ifitm / -myc was analyzed using anti-ha antibody or anti-myc antibody in huh cells when plasma membranes were intact or permeabilized. we found that neither ha-ifitm -myc or ha-ifitm -myc could be dyed using anti-ha antibody or anti-myc antibody when plasma membranes were intact, but both of them could be observed when cell membranes were permeabilized ( figure s ). these results indicated that ifitm and ifitm are also located in the cytoplasm of huh cells. these data show that ifitm adopts the topological structure (figure a, right) on the plasma membranes, where the n-terminus points into the cytoplasm and the c-terminus resides extracellularly, consistent with the topology model that ntd and cil are in the cytoplasm and ctd is extracellular or intracavity. an amphipathic sequence of amino acids (residues - ) of ifitm has been reported to be required for the restriction on h n iav [ ] . however, the other antiviral functional residues of ifitm are still unclear. therefore, eight alanine scan (as) mutants of the ifitm cd domain were constructed and named ( figure a ). vero cells were kidney cells of the monkey chlorocebus sabaeus from africa, which was easily infected by zikv [ ] . we observed the location of ifitm / / fused with a flag-tag in vero cells; the result showed that over-expressed ifitm was located both on cell membranes and in the cytoplasm, while ifitm and ifitm appeared to reside only in the cytoplasm in vero cells ( figure s ). then, we compared the inhibitory effects of ifitm and eight as mutants against zikv (prvabc strain) in vero cells. the result showed that ifitm inhibited % zikv e protein compared with the control, while as, as, as, as, as, as, as and as mutants limited %, %, %, %, %, %, % and % intracellular zikv e protein, respectively ( figure b,c) . relative intracellular zikv e protein was analyzed using imagej. these results suggest that as, as, as, as and as mutants of ifitm have a weaker antiviral effect than ifitm does, and these mutated amino acid residues of ifitm may be potential antiviral functional determinants. to further analyze the key functional antiviral sites of ifitm , we constructed four single-point mutants of ifitm in the cil domain named k a, r a, r a and k a and a four-point mutant named -krrk ( figure d ). then, we analyzed the antiviral effects of these five mutants. the results showed that -krrk had the least inhibitory activity against zikv in vero cells, and r a and k a appeared to have less antiviral effect than the ifitm by qpcr ( figure e ) and western blotting ( figure f,g) . however, k a and r a still shared similar anti-zikv activity with the wild type ifitm ( figure e-g) . the amino acid sequences of ifitm and ifitm in the cd domain were almost the same as those of ifitm ( figure a ). to determine if the krrk basic residues of ifitm / are also key for their antiviral functions, flag-ifitm -krrk and flag-ifitm -krrk plasmids were constructed. k , r , r and k of ifitm , and k , r , r and k of ifitm were simultaneously mutated to alanine ( figure h) . similarly, ifitm / and / -krrk were used to analyze the antiviral function against zikv. we found that both ifitm and ifitm had a great limitation on the intracellular zikv e protein, while -krrk and -krrk had less restriction than their wild types ( figure i ,j). these data show the krrk basic residues of ifitm / are also significant to suppress zikv infection. denv, like zikv, is a member of the flavivirus genus. more than million people worldwide are infected each year by any of the four-denv serotypes [ ] . to further verify the importance of the krrk basic residues of ifitm against viruses, ifitm and krrk mutants were used to detect their restriction on the denv- tsv strain in vero cells. the result showed that ifitm could decrease the expression of intracellular denv e protein compared with the control, while -krrk had almost no influence on denv replication ( figure a,b) . therefore, the krrk basic residues of ifitm also played a key role in the restriction on denv. sev, a negative single-stranded rna virus, is a member of the paramyxoviridae family. adv is a nonenveloped double-strand dna virus. we asked whether the krrk basic residues of ifitm had an influence on the infection of sev and adv. the results showed that both ifitm and -krrk hardly had inhibition on the relative intracellular sev rna, compared to the control ( figure c ). similarly, they had almost no inhibitory effect on the infection of adv (figure d,e) . in short, the krrk basic residues of ifitm / / contributed specifically to their antiviral functions. importantly, we detected the intracellular distributions of five krrk residues mutants of ifitm . the results showed -krrk, k a, r a, r a and k a all had colocalization with na + -k + atpase in hek t cells, and five mutants all could be located in the cytoplasm ( figure ). in addition, we compared the location of ifitm and -krrk in vero cells. it was found that the localization of -krrk was almost the same as that of ifitm ( figure s ). these data indicate these basic amino acids hardly influence the intracellular distribution of ifitm and do not explain the reason that the antiviral effect of -krrk is weaker than that of ifitm . basic amino acids hardly influence the intracellular distribution of ifitm and do not explain the reason that the antiviral effect of -krrk is weaker than that of ifitm . after h, the fluorescence of cells was observed using fluorescence microscopy with the magnification of × (d). fluorescence intensity was counted (e). ns = no significance. viruses , , x for peer review of figure . the colocalization analysis of ifitm mutants and na + -k + atpase in hek t cells. pcdna . and pcdna . -flag- -krrk/k a/r a/r a/k a were transfected in hek t cells, respectively. after h, cells were stained with anti-flag antibody, anti-na + -k + atpase antibody and dapi. then, cells were observed using confocal microscopy. ifitm mutants, red; na + -k + atpase, green; dapi, blue. zikv is a single-stranded rna virus, and the diameter of its mature viral particles is - nm [ ] . the life cycle of zikv can be divided into four stages, including viral entry, genome replication, viral assembly and release [ ] . zikv enters host cells by clathrin-mediated endocytosis. in this process, the viral envelope and the endosome membrane of host cells are fused under the acidic figure . the colocalization analysis of ifitm mutants and na + -k + atpase in hek t cells. pcdna . and pcdna . -flag- -krrk/k a/r a/r a/k a were transfected in hek t cells, respectively. after h, cells were stained with anti-flag antibody, anti-na + -k + atpase antibody and dapi. then, cells were observed using confocal microscopy. ifitm mutants, red; na + -k + atpase, green; dapi, blue. zikv is a single-stranded rna virus, and the diameter of its mature viral particles is - nm [ ] . the life cycle of zikv can be divided into four stages, including viral entry, genome replication, viral assembly and release [ ] . zikv enters host cells by clathrin-mediated endocytosis. in this process, the viral envelope and the endosome membrane of host cells are fused under the acidic environment of the endosome; then, the viral genomic rna is released into the cytoplasm [ ] . previous studies have shown that ifitm proteins can inhibit the entry of zikv [ ] ; however, how ifitm inhibits this stage is still unknown. here, we detected the influence of ifitm and -krrk on the release of zikv from the endosome to the cytosol. by electron microscopy, we observed ifitm prevented more virus particles from being released into the cytoplasm, compared with the negative control, while -krrk virtually unlimited the release of virus particles ( figure a ). specific results were counted and shown in figure b , suggesting that ifitm did restrict the release of zikv from the endosome to the cytosol. in addition, the colocalization of ifitm and acidic organelles was detected. rab and rab , members of the ras gtpase superfamily, are mainly located in early endosomes and late endosomes, respectively [ ] . lamp is the main constituent protein of the endosome/lysosome pathway [ ] . therefore, we detected the colocalization of ifitm and rab /rab /lamp . indeed, we found that ifitm was partially co-located with early endosomes, late endosomes and lysosomes ( figure c ). this was consistent with previous reports that exogenous ifitm can localize on cell membranes and in highly acidified late endosomes and lysosomes [ , ] . we then detected the organelles acidity of huh cells after being over-expressed by ifitm and -krrk by a flow cytometer using a nm laser for excitation. compared with the control group, the curve of ifitm shifted to the left, and the curve of -krrk was between ifitm and the control ( figure d ). these data suggested that ifitm could inhibit the acidification of organelles, and -krrk had weaker suppressive power than ifitm did. therefore, ifitm can restrict the release of zikv from endosome to cytosol, which is related with its inhibition on organelles acidification. more importantly, the krrk basic residues of ifitm are vital in these processes. viruses , , x for peer review of environment of the endosome; then, the viral genomic rna is released into the cytoplasm [ ] . previous studies have shown that ifitm proteins can inhibit the entry of zikv [ ] ; however, how ifitm inhibits this stage is still unknown. here, we detected the influence of ifitm and -krrk on the release of zikv from the endosome to the cytosol. by electron microscopy, we observed ifitm prevented more virus particles from being released into the cytoplasm, compared with the negative control, while -krrk virtually unlimited the release of virus particles ( figure a ). specific results were counted and shown in figure b , suggesting that ifitm did restrict the release of zikv from the endosome to the cytosol. in addition, the colocalization of ifitm and acidic organelles was detected. rab and rab , members of the ras gtpase superfamily, are mainly located in early endosomes and late endosomes, respectively [ ] . lamp is the main constituent protein of the endosome/lysosome pathway [ ] . therefore, we detected the colocalization of ifitm and rab /rab /lamp . indeed, we found that ifitm was partially co-located with early endosomes, late endosomes and lysosomes ( figure c ). this was consistent with previous reports that exogenous ifitm can localize on cell membranes and in highly acidified late endosomes and lysosomes [ , ] . we then detected the organelles acidity of huh cells after being over-expressed by ifitm and -krrk by a flow cytometer using a nm laser for excitation. compared with the control group, the curve of ifitm shifted to the left, and the curve of -krrk was between ifitm and the control ( figure d ). these data suggested that ifitm could inhibit the acidification of organelles, and -krrk had weaker suppressive power than ifitm did. therefore, ifitm can restrict the release of zikv from endosome to cytosol, which is related with its inhibition on organelles acidification. more importantly, the krrk basic residues of ifitm are vital in these processes. previous reports have revealed that ifitm adopts the topological model on organelle membranes where its ntd and cil are in the cytoplasm, and ctd is intracavity by using the combined electron paramagnetic resonance (epr) and solution nuclear magnetic resonance (nmr) [ ] . ifitm is high homologous with ifitm and has been reported with at least three topological models [ , ] . here, we support that ifitm adopts a topological model on cell membranes where the n-terminus points into the cytoplasm and the c-terminus reside extracellularly. this is consistent with previous reports [ ] . the anti-viral function of ifitm proteins was found initially against iav, west nile virus (wnv) and denv in [ ] . over the next years, ifitm proteins have been reported to restrict many other viruses. zikv and denv are an arthropod-borne virus (arbovirus) in the flavivirus genus of the flaviviridae family; both zikv and denv have shown a threat to global health, and the effective vaccines for them still need to be developed [ , ] . ifitm is an important downstream gene of interferon; there are reports that show the inhibitory effect of ifitm on zikv and denv replication [ , ] . in our study, we conducted a series of experiments to screen the antiviral functional residues of ifitm against zikv. we found that the krrk basic residues of ifitm were important for the restriction of ifitm on zikv and denv. it has been reported that the as and as mutants of ifitm have weaker restrictions on iav and denv replication than wild type [ ] . to further illustrate the importance of these four residues, we also compared the antiviral effects of ifitm /ifitm and their corresponding krrk mutant. indeed, the krrk basic residues of ifitm /ifitm also played a key role in their restriction on zikv. significantly, we found that ifitm can restrict the release of zikv from endosome to cytosol to prevent the entry of viral particles into host cells, which was associated with its inhibition on organelles acidification. attractively, -krrk had almost no restriction in this process. significantly, we found krrk residues of ifitm were very conservative in many species by aligning their amino acid sequences ( figure s ). we hypothesize that krrk residues of ifitm proteins of other species may play important roles in their antiviral effects. most of the previous reports have studied the effect of ifitm on viral replication, while our work mainly studied the restriction of ifitm on viruses, including zikv, denv, adv and sev. importantly, we found that the krrk basic residues of ifitm proteins played a vital role in their antiviral processes. in addition, our work provided new insights into the antiviral mechanism of ifitm related to inhibiting organelles acidification. the previous research also showed that the endosomes in hulec cells with a high basal level of ifitm were less acidic than in mdcks [ ] . the mechanism may be applicable to explain how ifitm restricts other viruses entering cells by clathrin-mediated endocytosis. however, our work could not explain why the krrk basic residues of ifitm are vital for ifitm to inhibit viral entry and suppress organelles acidification. the isoelectric point (pi) of ifitm with a flag-tag is . , predicted by the protparam website, and the ph values of the late endosome and lysosome are . - . and . - . , respectively [ ] . therefore, we hypothesize that ifitm may alkalize the acidic organelles by its own properties; besides, the pi of -krrk with a flag-tag is . predicted by the protparam website, which may explain that -krrk hardly affects the acidification of organelles. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . figure s . the location of ha-ifitm / -myc in huh cells. figure s . the localization analysis of ifitm / / in vero cells. figure s . the localization analysis of ifitm / -krrk in vero cells. figure s . the amino acid sequence alignment of ifitm across nine species. the small interferon-induced transmembrane genes and proteins a membrane topology model for human interferon inducible transmembrane protein ifitm-family proteins: the cell's first line of antiviral defense the anti-inflammatory ifitm genes ameliorate colitis and partially protect from tumorigenesis by changing immunity and microbiota the ifitm protein family in adaptive immunity palmitoylation on conserved and nonconserved cysteines of murine ifitm regulates its stability and anti-influenza a virus activity defining the range of pathogens susceptible to ifitm restriction using a knockout mouse model the ifitms inhibit zika virus replication the cd domain of ifitm is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replicatio interferon-induced transmembrane protein-mediated inhibition of host cell entry of ebolaviruses interferon-inducible transmembrane protein (ifitm ) restricts reovirus cell entry the antiviral restriction factors ifitm , and do not inhibit infection of human papillomavirus, cytomegalovirus and adenovirus ifitms restrict the replication of multiple pathogenic viruses ifitm proteins-cellular inhibitors of viral entry ifitm proteins restrict viral membrane hemifusion ifitm restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion the antiviral effector ifitm disrupts intracellular cholesterol homeostasis to block viral entry a scorpion venom peptide ev restricts viral late entry by alkalizing acidic organelles physical mapping and characterization of the human na,k-atpase isoform, atp a a sorting signal suppresses ifitm restriction of viral entry ifitm requires an amphipathic helix for antiviral activity identification of various cell culture models for the study of zika virus dengue virus infection of blood-brain barrier cells: consequences of severe disease zika virus replication in the mosquito culex quinquefasciatus in brazil development of small-molecule inhibitors against zika virus infection probing molecular insights into zika virus host interactions the autophagic roles of rab small gtpases and their upstream regulators: a review disruption of endolysosomal rab / efficiently eliminates colorectal cancer stem cells ifitm inhibits influenza a virus infection by preventing cytosolic entry the interferon-induced transmembrane proteins, ifitm , ifitm , and ifitm inhibit hepatitis c virus entry combined approaches of epr and nmr illustrate only one transmembrane helix in the human ifitm ifitm proteins mediate the innate immune response to influenza a h n virus, west nile virus and dengue virus zika virus: new clinical syndromes and its emergence in the western hemisphere dengue vaccine: hypotheses to understand cyd-tdv-induced protection ha-dependent tropism of h n and h n influenza viruses to human endothelial cells is determined by reduced stability of the ha, which allows the virus to cope with inefficient endosomal acidification and constitutively expressed ifitm ion flux and the function of endosomes and lysosomes: ph is just the start: the flux of ions across endosomal membranes influences endosome function not only through regulation of the luminal ph we thank ren sun and danyang gong of ucla for providing zikv puerto rico strain (prvabc ) cdna plasmid and bo zhang from wuhan institution of virology, chinese academy of sciences for giving denv serotype tsv strain (denv- tsv ). the authors declare no conflict of interest. key: cord- -jkzqmkz authors: thirion, laurence; dubot-peres, audrey; pezzi, laura; corcostegui, iban; touinssi, mhammed; de lamballerie, xavier; charrel, remi n. title: lyophilized matrix containing ready-to-use primers and probe solution for standardization of real-time pcr and rt-qpcr diagnostics in virology date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jkzqmkz real-time molecular techniques have become the reference methods for direct diagnosis of pathogens. the reduction of steps is a key factor in order to decrease the risk of human errors resulting in invalid series and delayed results. we describe here a process of preparation of oligonucleotide primers and hydrolysis probe in a single tube at predefined optimized concentrations that are stabilized via lyophilization (lyoph-p&p). lyoph-p&p was compared versus the classic protocol using extemporaneously prepared liquid reagents using (i) sensitivity study, (ii) long-term stability at °c, and (iii) long-term stability at °c mimicking transportation without cold chain. two previously published molecular assays were selected for this study. they target two emerging viruses that are listed on the blueprint of the who as to be considered for preparedness and response actions: chikungunya virus (chikv) and rift valley fever phlebovirus (rvfv). results of our study demonstrate that (i) lyoph-p&p is stable for at least days at °c supporting shipping without the need of cold chain, (ii) lyoph-p&p rehydrated solution is stable at + °c for at least two weeks, (iii) sensitivity observed with lyoph-p&p is at least equal to, often better than, that observed with liquid formulation, (iv) validation of results observed with low-copy specimens is rendered easier by higher fluorescence level. in conclusion, lyoph-p&p holds several advantages over extemporaneously preparer liquid formulation that merit to be considered when a novel real-time molecular assay is implemented in a laboratory in charge of routine diagnostic activity. the detection of the genome of pathogens has become the gold standard technique for direct diagnosis because of excellent sensitivity and specificity, and due to its capacity to provide a result within hours [ , ] . nonetheless, there are several factors that merit to be mastered in order to obtain results that can be steadily validated. among those factors, ensuring and maintaining the quality of the components of the reaction mix, in particular oligonucleotide primers and fluorescent probe. it is important to distinguish basic research context from diagnostic context. the latter can hardly suffer delays in result validation. it is important to underline that clinical microbiology laboratories are now frequently grouping assays for viruses, bacteria, fungi, and parasites not only for diagnosis but also for further characterization of pathogen through genotyping or resistance detection. this obviously rapidly leads to a large number of assays to run daily or weekly, and as a consequence a rather large number of potential pitfalls [ ] . although failure of one of the mix components is easily detected when the positive control does not provide adequate results, such a situation has an important impact on the laboratory throughput due to delayed results, reordering reagents, increased laboratory costs, increasing technical workload, and feeling insecure concerning the capacity of biologists to provide results and of clinicians to obtain results timely. whether this can appear as a minor problem for laboratories using few in-house assays, it can rapidly become hectic when a larger number of in-house assays are used for routine diagnostic purpose. there are several causes for failures linked to primers and/or probes such as light exposure that deteriorates fluorescence of the probe, repeated freeze-thaw cycles resulting in dna degradation, mistakes in final concentrations, or pipetting errors when the reaction mix is prepared [ ] . such problems have been at least partially solved in commercial kits through serial aliquoting and lyophilization or ambient-temperature stable reagents. ready-to-use reagents reduce the risk of human errors. lyophilized reagents are more stable than liquid formulations. the combination of both measures aims at improving the quality of the results. we describe here a process of preparation of oligonucleotide primers and hydrolysis probe in a single tube at predefined optimized concentrations (p&p for primers and probe(s)) that are stabilized via lyophilization (lyoph-p&p). we have compared the performances of two selected assays (lyoph-p&p vs. the classic protocol using frozen reagents) and have studied the long-term stability of lyoph-p&p in native and rehydrated formulations. selected assays target two emerging viruses that are listed on the blueprint of the who as to be considered for preparedness and response actions [ ] : chikungunya virus (chikv), a single-stranded positive-sense rna alphavirus, and rift valley fever phlebovirus (rvfv), a tri-segmented, single-stranded negative-sense rna phlebovirus. specific hydrolysis probe-based real-time rt-pcr (rt-qpcr) assays were selected for the detection of chikv and rvfv viruses (table ) . upon reception, lyophilized primers (eurogentec) and probes (applied biosystems, thermofisher) were regenerated in tris-hcl ( mm ph . ) buffer, to obtain (i) a µm stock solution and (ii) a µm working solution which both were stored at − • c. optimal concentrations for primers and probes have been experimentally determined and are indicated in table . rt-qpcr assays used µl of rna with the superscript ® iii platinium ® one-step quantitative rt-pcr kit (invitrogen-thermofisher scientific, waltham, ma, usa) in a final volume of µl following the manufacturer's protocol. reactions were performed on a biorad real-time thermal cycler cfx ™ and cfx manager software version . following thermal profile: min at • c ( cycle), min at • c ( cycle), ( s at • c, s at • c) ( cycles). the result was considered negative for ct values ≥ . for each rt-qpcr assay, the target sequence, preceded by the t promoter sequence, was inserted into puc plasmid (genscript). each plasmid ( µg) was regenerated, then -fold serially diluted using tris hcl buffer ( mm ph . ). a range of dilutions was submitted to m pcr (primer - : m f tgt aaa acg acg gcc agt, m r cag gaa aca gct atg acc). the pcr product from the highest plasmid dilutions giving the strongest amplification band on agarose gel was selected for subsequent transcription. synthetic rna transcripts were synthesized from µl of pcr product using megashortscript™ t transcription kit (ambion™) following the manufacturer's instructions. plasmid dna was removed with dnase (turbo dna-free™, invitrogen™), then, the rna transcript was purified using the monarch ® pcr & dna cleanup kit (biolab) following manufacturer's instructions. rna concentration (copy number per µl) was calculated for each standard from rna concentration measured using nanodrop ® from thermo scientific. the standard concentrations were . × rna copies/µl for chikv and . × rna copies/µl for rvfv. each standard was serially diluted using ave buffer-rna carrier ( ng/µl, qiagen), then each dilution was aliquoted and stored at − • c until use (ave buffer is the name provided by qiagen and contains rnase-free water with . % sodium azide). the quality of each standard was checked by submitting serial dilutions to rt-qpcr (as described above) and to qpcr (using lightcycler ® dna master hybprobe, roche). a difference in the limit of detection (lod) of at least log between rt-qpcr and qpcr, corresponding to negligible traces of remaining dna not interfering with rna quantification, was considered acceptable. the concentration of the different components of the p&p can be slightly increased (lyophilization factor) from the ones used for extemporary preparation; adequate concentrations are listed in table . the corresponding volume of p&p solution was dispensed in -ml glass vials (wheaton ® , dominique dutscher, brumath, france). two µl of sucrose m used as a stabilizing agent and, optionally, . µl of red food coloring e ( / in water) was added in order to visualize better dispensing of the p&p. the volume was adjusted to µl using molecular grade water (ultrapuretm distilled water, invitrogen). in order to prevent insufficient volume for the ultimate reactions due to pipetting errors, a safety margin was included in the calculation; for instance, to prepare a -test vial, volumes are calculated for tests. glass vials containing p&p solution to perform to reactions can be prepared using the protocols presented in table . glass vials containing p&p liquid solution (as prepared in table ) were stored at − • c for at least h prior lyophilization. frozen vials were lyophilized in a pilot bench freeze-dryer (cryotec, france) using the program# : . bar at − • c for min, . bar at − • c for min, . bar at − • c for min, and . bar at − • c for min; thereafter, program# was launched and consisted of . bar at − • c for min and . bar at − • c for min. upon completion, vacuum was broken by injection of nitrogen gas (airproducts); then, the vials were sealed and stored at − • c. all vials used for this study come from the same drying process. p&p vials containing tests were regenerated with . µl of ae elution buffer (macherey-nagel) and homogenized by multiple pipetting of a µl-volume at least times in the vials; then, rehydrated p&p was incubated at room temperature for min, after which times multiple pipetting was done again; these steps are critical to ensure adequate homogenization (table ) . after regeneration, rehydrated p&p were stored at • c for weeks. at three time points (day , day , and day ), rt-qpcr was performed using extemporaneously prepared liquid p&p. rt-qpcr was performed on three replicates for each of three dilutions ( − , − , and − ) of corresponding rna (table ). lyoph-p&p vials (not rehydrated) were placed at • c for weeks to simulate conditions that might be encountered during oversea shipping with failure of the cold chain or shipping without cold chain conditions. vials were rehydrated at day , day , and day , and results were compared with those observed at day using three replicates. the measure and comparison of the analytical sensitivity of chikv and rvfv assays were done by using synthetic standard rnas. serial five-fold dilutions of the quantitated rnas were prepared using ave buffer-rna carrier (qiagen). six decreasing concentrations ( . × − , . × − , . × − , . × − , . × − , and . × − ) were tested using three replicates for each. a ct ≥ was considered as negative. lod is considered as the lowest amount of analyte in a sample that can be detected with (stated) probability, although perhaps not quantified as an exact value. in this study the lod was defined as the number of rna copies/µl contained in the highest dilution for which the three replicates were positive. the lod is the analyte concentration that produces at least % of positive replicates. a total of clinical samples that were tested positive for chikv rna at the national reference centre for arboviruses were kindly provided by her director (dr. isabelle leparc-goffart) to be re-tested comparatively using the extemporaneously prepared liquid formulation of mix and using the lyoph-p&p as described in this study. differences between the lyoph-p&p method compared with the traditional extemporaneous preparation is illustration in figure ; figure . the results are presented in table . for chikv, results observed with the lyoph-p&p were systematically better than those obtained when using the liquid formulation extemporaneously prepared. for rvfv, the results observed with the lyoph-p&p were almost identical to those the results are presented in table . for chikv, results observed with the lyoph-p&p were systematically better than those obtained when using the liquid formulation extemporaneously prepared. for rvfv, the results observed with the lyoph-p&p were almost identical to those obtained with the liquid formulation; lyoph-p&p ct values were never higher than . ct (as compared to the liquid reference), corresponding to a theoretical difference of / log. for chikv, results observed after maintaining the lyoph-p&p at • c to mimic shipping conditions without cold chain or degraded conditions were similar, although slightly better, at days , , and compared with other conditions suggesting that the stability of freeze-dried reagents was excellent. for rvfv, the lowest ct values were observed at days , day , and day for , , and rna copies/µl, respectively; however, ct values observed between day and day were very similar suggesting that degraded shipping conditions might affect in a very limited manner the quality of the lyoph-p&p (table ). the results are presented in table . for chikv, the samples containing rna copies/µl were detected with both the liquid and the lyoph-p&p reagents. in contrast, none of the three samples containing three rna copies/µl was found positive using the liquid reagents, whereas all three samples were found positive using the lyoph-p&p; moreover, all three samples containing . rna copies/µl were also found positive with the lyoph-p&p. this denotes a better analytical sensitivity of the lyoph-p&p compared with the liquid formulation for the detection of chikv rna. for rvfv, the samples containing rna copies/µl were detected with both the liquid and the lyoph-p&p reagents. in contrast, only one out of three samples containing two rna copies/µl was found positive using the liquid reagents, whereas all three samples were found positive using the lyoph-p&p. this denotes a better sensitivity of the lyoph-p&p compared with the liquid formulation for the detection of rvfv rna. as shown in table , mean ct values and sd observed using the lyoph-p&p were lower than those obtained with the extemporaneously prepared liquid formulation in / and / samples, respectively. real-time molecular techniques are now the reference methods for the direct diagnosis of pathogens. increasingly, automation has developed in order to reduce the number of steps prone to human errors, and now the tendency is towards random access tests where all steps are automated until biological validation. however, this approach, developed by diagnostics companies such as hologic, roche, abbott, cepheid, biomerieux among others, focus on marketable tests meaning that a certain amount of assays has to be expected in the business plan before such assays are developed. commercially developed assays need to be registered by regulation agencies before they are available on the market; in many cases, this leads to delays that are not compatible with preparedness and response activities, as witnessed by the current situation with the novel coronavirus. moreover, often the development and licensing of a novel assay is conditioned by the size and volume of anticipated future market which is not necessarily considered as profitable. lastly, the price for such assay is almost always not compatible with daily use in laboratories of developing countries. obviously, a large number of microbial targets will never be addressed by such random access technologies due to their lack of marketability although they might be major human, veterinary, or plant pathogens. it is worrisome that this situation is contradictory with the principle of preparedness and response to emerging pathogens [ ] . although real-time molecular techniques are now implemented worldwide, laboratories still face technical problems due to the large number of parameters and reagents to manage, the stability of respective reagents, and the multiple steps from patient to result [ ] . among the parameters to consider in the process of clinical diagnostics, primers and probe are among those that require the largest number of steps to operate from the stage of ordering the reagents (primers and probe(s)) to the launching the pcr or rt-pcr reaction onto the thermal cycler. even in the simplest format, two oligonucleotide primers and one probe are ordered from manufacturing companies. upon reception, each of these three tubes has to be rehydrated and/ or diluted to prepare a stock solution (usually µm) and a working solution (usually µm), both stored at − • c for stability. then for each experiment, specific volumes of each of the three working solutions have to be manipulated to prepare the pcr mix solution which is then distributed into individual reaction tubes or plates. in contrast, the enzyme mix is now mostly commercialized in a x solution which requires to perform few steps until distribution into the reaction tubes. last the "to be tested" solution of total nucleic acids, rna, or dna is distributed. since the manipulation of primers and probe requires the largest number of steps, we selected it as target for simplification (figures and ) . the aim was (i) to produce a ready-to-use primers & probe mix (p&p) for each pathogen to be tested, (ii) to validate the resulting p&p in its lyophilized form (lyoph-p&p), (iii) to optimize the whole process and to make it available and usable easily to laboratories willing to adopt the same approach. the ultimate objective was to produce reagents amenable to any laboratory having the capacity to perform real-time molecular detection of pathogens for diagnostic purpose. the assays that were selected for comparative evaluation in this study have been thoroughly evaluated for the respective detection of chikv and rvfv; they have also been used in external quality assessment studies conducted by the european or international level [ , [ ] [ ] [ ] [ ] . the most important aspect was to compare the analytical sensitivity of the lyoph-p&p assays against the results obtained when the primers and probe were prepared extemporaneously using the classic liquid format. for the two assays included in this study (chikv and rvfv), the analytical sensitivity of the lyoph-p&p is not only equal to that observed with the liquid formulation, but even much better for chikv ( . copies/µl vs. copies/µl), and slightly better for rvfv (more replicates detected for the last dilution providing positive results). the results observed with clinical samples tested for chikv rna confirm the data obtained in analytical sensitivity studies. detection of chikv rna using lyoph-p&p provides results in clinical samples that are at least equal and often better than those obtained with the extemporaneously prepared liquid formulation used as reference. because of the low number of available clinical samples and due to the highly restrictive microorganisms and toxins (mot) french regulation, it was not possible to perform the parallel study for rvfv. however, such comparative studies were done for a substantial number of assays that are routinely processed in the clinical microbiology laboratory of the ihu méditerranée infection serving all beds of the public hospitals system of marseille, france (supplementary table s ). although this has to be addressed systematically when other assays will be transferred from the liquid formulation towards to lyoph-p&p, these results are very promising and should engage in this direction for the detection of other pathogens. although all experiments described here were done using the superscript ® iii platinium ® one-step quantitative rt-pcr kit (thermofischer), we have also used other enzymes such as the one-step qrt-pcr lightcycler ® multiplex rna virus master (roche) that have provided similar results (data not shown). as indicated in the table , the concentration of primers and probe (to be lyophilized) had to be adjusted sometimes in order to obtain sensitivity comparable to that observed with extemporaneously prepared liquid preparation. the correction factor was determined empirically ( . and . ); interestingly, correction was not systematically necessary, and it could also be needed for one component of the reaction only, as shown with the rvfv assay. it is important to underline that the rehydration of the lyoph-p&p must be done as recommended in the protocol. alternative protocols are likely to result in disappointing performances. despite different formats can be prepared as indicated in table ; table , the question of the stability of lyoph-p&p after rehydration is important to assess the versatility and flexibility of this solution. indeed, at laboratory level, it is likely that one or two different formats (number of tests per vial) will be either prepared or ordered; as a consequence, the time during which rehydrated material can be stored without affecting the expected performances of the assay is a key factor. interestingly, -day or -day storage at + • c had absolutely no deleterious effect on the performances; moreover, in some occurrences, sensitivity was even better after storage than after extemporaneous rehydration. stability upon + • c storage after rehydration is important for the end-users because it prevents discarding reagents; this is not only important economically, but also renders the routine activity more comfortable when a large number of different pathogens are included in detection panels. the fact that rehydrated lyoph-p&p was stable for at least days after rehydration if stored at • c is interesting because it allows to prepare or order vials containing greater number of tests without fearing the loss of material that is synonymous of increased costs. stability of rehydrated material warrants versatility of the procedures, thus allowing to prepare/order vials containing -or -reactions. the same tendency was observed for the two assays suggesting that this phenomenon is not virus-dependent and may be expected with other detection assays. assessing the stability during shipping by maintaining the lyoph-p&p at • c up to days intended to mimic degraded conditions potentially occurring during transportation at a given temperature, and also to address the possibility to perform shipping at ambient temperature. the excellent results observed at day and day support the possibility of ambient temperature shipping for this non-infectious material using rapid delivery companies such as worldcourrier, ups, dhl, fedex, or similar ones that are capable to guarantee delivery within days to almost any place in the world. again, the promising results observed with chikv and rvfv must be confirmed for supplementary assays that will be developed. as examples of its versatility, the described procedure was used to prepare lyoph-p&p with other rt-qpcr assays from the literature targeting zika, dengue, and chikungunya viruses [ ] [ ] [ ] ; the corresponding lyoph-p&p were shipped to overseas laboratories which were satisfied with the resulting performances on their own diagnostic platform (thirion, unpublished data) . the opportunity to dispense with cold chain is also important to consider for economic reasons. the last point to consider in the comparative analysis between liquid and lyoph-p&p formulations deals with the interpretation of the pcr curves. the signal observed with low-copy samples close to the lod, beyond ct , is frequently weak as shown by low rfu level (supplementary data, dataset# ); interpretation of such results is frequently difficult and as a consequence often induces repeated testing for confirmation. the stronger the intensity of the signal, the easier the discrimination between clear positives and uncertain results. a detailed analysis denotes that for low copy samples, the intensity of the signals is clearly higher with lyoph-p&p compared with the liquid formulation: approximately - rfu vs. - rfu for rvfv, and approximately - rfu vs. - rfu for chikv (suppl data, dataset# ). recently, increased robustness of real-time pcr assays has been achieved by combining two targets in a unique reaction tube in order to prevent false negative results that may arise from point mutations/deletions/insertions frequently observed with emerging pathogens, even more frequently with pathogens with rna genome [ ] [ ] [ ] [ ] . this tendency implies the need to increase the number of different primers and probe within a single assay, which renders the preparation of the reaction mix even more prone to human errors. whether or not this tendency should expand, lyoph-p&p would be even more attracting for diagnostic activities in routine clinical microbiology laboratories. the recipient laboratory will have to perform minimal validation steps before the lyoph-p&p can be included in the routine diagnostic activity. in conclusions, the advantages of lyoph-p&p reside (i) in its stability for shipping and storage, (ii) in the drastically reduced number of manipulations to prepare the ultimate reaction tube/plate to be placed in the thermocycler, (iii) in its flexibility in terms of number of reactions per prepared vial ( to , even to ). utilization of lyoph-p&p is an easy manner to transfer diagnostic capacity between laboratories. reliability of real-time reverse-transcription pcr in clinical diagnostics: gold standard or substandard? emerging technologies for the clinical microbiology laboratory syndromic panel-based testing in clinical microbiology pitfalls in pcr troubleshooting: expect the unexpected? list of blueprint priority diseases development of a taqman rt-pcr assay without rna extraction step for the detection and quantification of african chikungunya viruses the ultimate qpcr experiment: producing publication quality, reproducible data the first time geographically weighted discriminant analysis of environmental conditions associated with rift valley fever outbreaks in south rapid detection and quantification of rna of ebola and marburg viruses, lassa virus, crimean-congo hemorrhagic fever virus, rift valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-pcr first external quality assessment of molecular and serological detection of rift valley fever in the western mediterranean region international external quality assessment of molecular detection of rift valley fever virus assay optimization for molecular detection of zika virus development and validation of real-time one-step reverse transcription-pcr for the detection and typing of dengue viruses development and evaluation of a duo chikungunya virus real-time rt-pcr assay targeting two regions within the genome improved sensitivity of a dual-target hiv- qualitative test for plasma and dried blood spots improved hiv- rna quantitation by cobas ampliprep/cobas taqman hiv- test, v . using a novel dual-target approach blood screening nucleic acid amplification tests for human immunodeficiency virus type may require two different amplification targets this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank gregory molle for excellent technical assistance. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -jtaz gdp authors: wu, fangfang; zhang, shengnan; zhang, ying; mo, ruo; yan, feihu; wang, hualei; wong, gary; chi, hang; wang, tiecheng; feng, na; gao, yuwei; xia, xianzhu; zhao, yongkun; yang, songtao title: a chimeric sudan virus-like particle vaccine candidate produced by a recombinant baculovirus system induces specific immune responses in mice and horses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jtaz gdp ebola virus infections lead to severe hemorrhagic fevers in humans and nonhuman primates; and human fatality rates are as high as %– %. since the ebola virus was discovered in , the only available treatments have been medical support or the emergency administration of experimental drugs. the absence of licensed vaccines and drugs against the ebola virus impedes the prevention of viral infection. in this study, we generated recombinant baculoviruses (rbv) expressing the sudan virus (sudv) matrix structural protein (vp ) (rbv-vp -vp ) or the sudv glycoprotein (gp) (rbv-gp-gp), and sudv virus-like particles (vlps) were produced by co-infection of sf cells with rbv-sudv-vp and rbv-sudv-gp. the expression of sudv vp and gp in sudv vlps was demonstrated by ifa and western blot analysis. electron microscopy results demonstrated that sudv vlps had a filamentous morphology. the immunogenicity of sudv vlps produced in insect cells was evaluated by the immunization of mice. the analysis of antibody responses showed that mice vaccinated with sudv vlps and the adjuvant montanide isa produced sudv gp-specific igg antibodies. sera from sudv vlp-immunized mice were able to block infection by sudv gp pseudotyped hiv, indicating that a neutralizing antibody against the sudv gp protein was produced. furthermore, the activation of b cells in the group immunized with vlps mixed with montanide isa was significant one week after the primary immunization. vaccination with the sudv vlps markedly increased the frequency of antigen-specific cells secreting type and type cytokines. to study the therapeutic effects of sudv antibodies, horses were immunized with sudv vlps emulsified in freund’s complete adjuvant or freund’s incomplete adjuvant. the results showed that horses could produce sudv gp-specific antibodies and neutralizing antibodies. these results showed that sudv vlps demonstrate excellent immunogenicity and represent a promising approach for vaccine development against sudv infection. further, these horse anti-sudv purified immunoglobulins lay a foundation for sudv therapeutic drug research. abstract: ebola virus infections lead to severe hemorrhagic fevers in humans and nonhuman primates; and human fatality rates are as high as %- %. since the ebola virus was discovered in , the only available treatments have been medical support or the emergency administration of experimental drugs. the absence of licensed vaccines and drugs against the ebola virus impedes the prevention of viral infection. in this study, we generated recombinant baculoviruses (rbv) expressing the sudan virus (sudv) matrix structural protein (vp ) (rbv-vp -vp ) or the sudv glycoprotein (gp) (rbv-gp-gp), and sudv virus-like particles (vlps) were produced by co-infection of sf cells with rbv-sudv-vp and rbv-sudv-gp. the expression of sudv vp and gp in sudv vlps was demonstrated by ifa and western blot analysis. electron microscopy results demonstrated that sudv vlps had a filamentous morphology. the immunogenicity of sudv vlps produced in insect cells was evaluated by the immunization of mice. the analysis of antibody responses showed that mice vaccinated with sudv vlps and the adjuvant montanide isa produced sudv gp-specific igg antibodies. sera from sudv vlp-immunized mice were able to block infection by sudv gp pseudotyped hiv, indicating that a neutralizing antibody against the sudv gp protein was produced. furthermore, the activation of b cells in the group immunized with vlps mixed with montanide isa was significant one week after the primary immunization. vaccination with the sudv vlps markedly increased the frequency of antigen-specific cells secreting type and type cytokines. to study the therapeutic effects of sudv antibodies, horses were immunized with sudv vlps emulsified in freund's complete adjuvant or freund's incomplete adjuvant. the results showed that horses could produce sudv gp-specific antibodies and neutralizing antibodies. these results showed that sudv vlps demonstrate excellent immunogenicity and represent a promising approach for vaccine humans [ ] . furthermore, pre-existing immunity associated with live carrier vaccines is not hindered by vlp-based immunizations. previous findings showed that sudv vlps could be readily assembled by the co-expression of insect cells with baculoviruses expressing gp, np, and vp [ ] . there are currently no approved specialized drugs or vaccines to protect against sudv disease outbreaks, and thus there is an urgent need for the development of an efficacious, safe and economically viable vaccine or therapeutic antibody to control sudv infections. here, we report that production of sudv vlps has been accomplished in insect cells by the co-infection with recombinant baculoviruses rbv-gp-gp and rbv-vp -vp , and evaluate the ability of sudv-vlps to induce sudv-specific humoral and cellular immune responses in vaccinated mice. further, horses were immunized with sudv vlps, and horse serum was purified to prepare purified immunoglobulins and the purified immunoglobulins had neutralizing activity. the t and huh cells (atcc) were cultured in dulbecco's modified eagle's medium (dmem; corning-costar, coring, ny, usa) supplemented with % fetal bovine serum and penicillin-streptomycin (fbs; gibco, grand island, ny, usa). these cells were maintained at • c. the spodoptera frugiperda (sf ) cells (invitrogen, san diego, ca, usa) were cultured in suspension in sf- ii serum-free medium (invitrogen, san diego, ca, usa) supplemented with % fbs and penicillin-streptomycin. these cells were maintained at • c. female balb/c mice, - weeks old, were purchased from changchun institute of biological products (changchun, china). all experiments involving mice adhered to the principles of the welfare and ethics committee of the military veterinary research institute at the academy of military sciences. the mice were provided ad libitum access to sterilized water and food throughout the study and were vaccinated under bsl- conditions. healthy male horses - years old and - kg were provided by red hill military horse farm (changchun, china). horse studies were conducted with prior approval from the animal welfare and ethics committee of the institute of military veterinary, academy of military medical sciences (permit number scxk- - ), according to horse quarantine and immunization protocols for equine serum production. the cloning and construction of the recombinant bacmids, pfastbacdual-gp-gp, and pfastbacdual-vp -vp , were carried out. briefly, we synthesized the full-length gp figure s a) . the recombinant plasmid pfastbacdual-vp -vp (supplementary figure s b ) was constructed using the same strategy. recombinant plasmids were used to transform e. coli dh bac competent cells to generate recombinant bacmids. sf cells were transfected with recombinant bacmids using cellfectin ® ii reagent (thermo scientific, carlsbad, ca, usa) according to the manufacturer's instructions. transfected cells were incubated at • c for d and harvested, the recombinant baculoviruses rbv-gp-gp and rbv-vp -vp by collecting the tinfected-sf cells and supernatant. the sudv gp ( ~ aa) and vp proteins were generated by a prokaryotic expression system. briefly, the synthesized genes encoding the sudv gp ( - aa) and vp proteins were separately inserted into the prokaryotic expression vector pet a (+) by using bamhi and noti, in-frame with the ×his-tag on the c-and n-terminus to construct pet a (+) -gp-his-c/n and pet a (+) -vp -his-c/n. after the recombinant plasmids were confirmed by restriction digest, the two recombinant plasmids were transformed into e. coli bl (de ) (transgen biotech, beijing, china) and the transformants selected on luria-bertani (lb) agar plates with µg/ml kanamycin. a single clone was picked and inoculated into ml of lb medium with µg/ml kanamycin at • c for overnight growth. expression was induced with the addition of . mm iptg when the optical density at nm (od ) reached . . the proteins were purified using a hispurtm ni-nta spin purification kit (thermo scientific, carlsbad, ca, usa) according to the manufacturer's instructions. to visualize the expression of the purified proteins, samples were resolved on a % sds-polyacrylamide gel (sds-page) and stained with coomassie blue, and protein concentrations were determined using a bicinchoninic acid (bca) assay (thermo scientific, carlsbad, ca, usa) followed by analysis at an absorbance of nm; bovine serum albumin (bsa; sigma-aldrich, st. louis, mo, usa) was used as the protein standard. polyclonal antisera against sudv gp ( ~ aa) or vp were prepared by immunizing balb/c mice with µg purified gp or vp recombinant proteins twice at -week intervals, and harvesting the mouse serum, which were mouse polyclonal antisera against sudv gp ( ~ aa) or vp . the sf cells were infected with the fourth generation recombinant baculovirus rbac-gp-gp or rbac-vp -vp for approximately h, infected sf cells were fixed with % paraformaldehyde for min at the room temperature. infected-sf cells were washed with pbs and blocked with pbs containing % bsa (sigma-aldrich, st. louis, mo, usa). then, the cells were incubated with a : dilution of a mouse polyclonal antisera against sudv gp or a mouse polyclonal antisera against sudv vp (generated in our lab) for h at • c, and then washed with pbs and incubated with fitc-conjugated goat anti-mouse igg (bioss antibodies, beijing, china) containing % diluted evans blue for h at • c; then the infected sf cells were washed with pbst (containing . % tween- ) three times and were observed with a fluorescence microscope. the control cells were incubated with the two primary antisera at the same time. baculovirus titers were determined using a bacpaktm baculovirus rapid titer kit (takara, dalian, china). to generate sudv vlps, sf cells were co-infected with recombinant baculoviruses rbv-gp-gp and rbv-vp -vp at different ratios of : , : , : . , : , : , . : , or : . the sudv vlps were harvested at -d post-infection. to purify the vlps, culture supernatants were harvested and spun at × g. the crude vlps were then concentrated by ultracentrifugation at , × g for h, and the pellets were resuspended in pbs before purification with a - - % discontinuous sucrose gradient. bands between - % sucrose were collected and resuspended in endotoxin-free pbs, and the vlps concentrations were determined using a bca assay (thermo scientific, carlsbad, ca, usa) followed by analysis at an absorbance of nm. the vlp preparations were not tested for endotoxin after production. purified sudv vlps were processed and examined by western blotting and transmission electron microscopy. for western blotting, aliquots containing µg of total protein were diluted with reducing buffer and denatured by heating at • c for min. proteins were separated in % acrylamide gels, before they were transferred onto polyvinylidene fluoride (pvdf) membranes (merck millipore, darmstadt, germany) under denaturing conditions. for protein detection, two polyclonal antisera were used: mouse anti-sudv gp polyclonal antisera and mouse anti-sudv vp polyclonal antisera were mixed at a dilution of : as a primary antibody for blotting; a goat anti-mouse igg hrp-conjugated antibody (bioss antibodies, beijing, china) was used at a dilution of : as a secondary antibody. negative staining transmission electron microscopy (tem) was used to analyze the shape and size of purified sudv vlps. in short, µl of sucrose gradient-purified sudv vlps were fixed for min on carbon-coated formvar grids, grids were washed with µl pbs and then treated with % phosphotungstate acid for min. grids were left to air dry and observed by using a hitachi h- transmission electron microscope. a total of two batches of balb/c mice ( weeks old, female) were purchased from the changchun yisi laboratory animal technology co., ltd. (changchun, china) and immunized. in batch one, mice were randomized into four groups (n = per group) and were vaccinated intramuscularly. mice in group one were vaccinated with pbs, mice in group two were vaccinated with montanide isa vg (isa ) adjuvant (seppic, paris, france), mice in group three were vaccinated with µg of sudv vlps-only, and mice in group four were vaccinated with µg of sudv vlps mixed with an equal volume of montanide isa vg (isa ) adjuvant, and all groups were vaccinated twice at -week intervals ( figure a ). the mouse sera were collected at two-weeks after every immunization. one week after the booster immunization, splenocytes from mice of each group were isolated. in batch two, nine mice were randomly distributed into three groups (n = per group) (pbs group, isa adjuvant, µg of sudv vlps mixed with an equal volume of isa adjuvant) and were vaccinated intramuscularly. one week after the primary immunization, the inguinal lymph nodes were collected from mice. two healthy male horses (numbered # and # ), - years old, - kg in weight and without detectable antibodies against sudv detected by indirect elisa, were supplied by red hill military horse farm. the horses were multipoint injected subcutaneously in the rear area with . , . , . , . , or . mg of purified sudv-vlps for a total of times, primary immunization mixed with an equal volume of freund's incomplete adjuvant (thermo scientific, rockford, il, usa) and booster immunization mixed with an equal volume of freund's complete adjuvant, maximum immune volume does not exceed ml, with boosting at two week intervals ( figure b ). the horse sera were collected before each immunization and stored at − • c for further studies. the mice and horse serum samples were collected two weeks after each immunization. levels of sudv gp-specific antibodies were detected by indirect elisa. briefly, µl of purified -prokaryotic expressed sudv gp was coated on elisa plates at a concentration of µg/ml overnight at • c, and then the plate was blocked with % bsa for h at • c. the serum was added ( µl/well), and it was -fold serially diluted in % bsa; plates were subsequently incubated for . h at • c, and µl of hrp-labeled goat anti-mouse igg or hrp-labeled goat anti-horse igg (bioss antibodies, beijing, china), diluted : , in % bsa was added to each well. after one hour of incubation at • c, µl of , , , -tetramethylbenzidine (tmb) (sigma-aldrich, st. louis, mo, usa) substrate solution was added to each well and then was stopped with the addition of µl of . m h so . optical density (od) values were measured at a wavelength of nm (od ). after each incubation step, elisa plates were washed five times with pbst. mean od values were considered positive if the od values were more than two times the value of the negative control. a pseudotyped virus neutralization assay was performed to test the neutralzing antibodies. recombinant lentiviral vectors that express sudv glycoprotein and carry a luciferase reporter gene were produced as described previously [ ] , with some modifications. briefly, t cells were seeded in -mm culture dishes (corning, ny, usa), and h later, the % subconfluent cells were co-transfected with µg of pcdna . -gp (the glycoprotein expression vector) and µg of pnl - .luc. re vector by lipofectamine tm transfection reagent (thermo scientific, carlsbad, ca, usa). after days, the supernatant containing the pseudotyped viruses was harvested, and the titer was determined in huh cells as previously described [ ] . for testing of neutralizing antibodies, -fold serially diluted serum samples were mixed with tcid of pseudotyped viruses for . h at • c and were then added to huh cells. after h, the inoculum was removed and replaced with fresh media. then, cells were lysed at h with cell lysis buffer, that was followed by the addition of µl of luciferase substrate (promega, madison, wi, usa) to determine luciferase activity. the luciferase activity of the samples was measured with the infinite m microplate spectrophotometer (tecan, mannedorf, switzerland). the percent inhibition rate was calculated as previous showed [ ] . the experiments were independently repeated three times. one week after the booster immunization in batch one, three mice from each group were randomly selected and euthanized. their spleens were harvested into a tissue culture dish, and each spleen was roughly minced and pressed through a -ml syringe. the cells were filtered through a -µm filter (bd falcon -µm strainer) and centrifuged at rpm for min at the room temperature. the cells were processed by resuspension in a red blood cell lysis buffer and centrifuged at rpm for min at twice the room temperature. the splenocytes were harvested and washed with rpmi medium (gibco, san diego, ca, usa) containing % fbs (gibco, san diego, ca, usa). then, the splenocytes were cultured in rpmi medium containing % fbs and stimulated with or without the purified sudv-gp antigen ( µg/ml). following incubation at • c in % co for h, the frequencies of splenocytes producing ifn-γ or il- were measured using mouse elispot kits (mabtech ab, stockholm, sweden) according to the manufacturer's instructions. spot-forming cells (sfcs) were enumerated by an automated elispot reader (aid elispot reader-ispot, germany). the levels of cytokines in the supernatant of stimulated splenocytes were detected by commercial elisa. splenocytes were stimulated as described above and were then incubated for h. the cell culture supernatant was collected by centrifugation at rpm for min, and the manufacturer directions were followed to detect il- , il- , il- , ifn-γ, or tnf-α by elisa kits (mabtech ab, stockholm, sweden). inguinal lymph node samples were harvested from batch two vaccinated mice days after the primary immunization. lymphoid cells were harvested into a tissue culture dish, and were roughly minced and pressed through a ml syringe. the cells were filtered through a -µm filter (bd falcon -µm strainer). then, prepared in pbs with % fbs and were stained with equal volumes of : dilutions of anti-cd -apc and anti-cd -fitc antibodies (bd biosciences, franklin, va, usa) for min at • c; the labeled b cells were then washed twice with pbs with % fbs and analyzed using a facsaria tm cell sorter (bd biosciences, franklin, va, usa). for equine antisera, the blood was taken from the jugular vein of # immunized horses, and the sera were collected. the horse serum was diluted -fold with pbs and then added to a saturated ammonium sulfate solution until the ammonium sulfate concentration was %. the solution was allowed to stand at • c for h, and it was centrifuged at rpm for min. after removing the supernatant, the precipitate was dissolved in pbs, and saturated ammonium sulfate was added until the concentration of ammonium sulfate was %. the solution was allowed to stand at • c for h, and centrifuged at rpm for min. the precipitate was dissolved in pbs and dialyzed against pbs at • c for h to remove the ammonium salt. the treatment of all mice was in accordance with the welfare and ethical guidance of laboratory animals of china (gb - ) . the agreement was approved by the animal welfare and ethics committee of the institute of veterinary medicine of the military academy of sciences (laboratory animal care and use committee authorization, permit number jsy-dw- - ). the sequences encoding the gp ( - aa) and vp proteins of sudv were cloned into the prokaryotic expression vector pet a (+), resulting in the plasmid pet a (+) -gp-his-c/n and pet a (+) -vp -his-c/n (c and n terminal ×his-tag). correct insertion of the sequences in the recombinant plasmid was confirmed by restriction digest mapping analysis and dna sequencing. the recombinant plasmids pet a (+) -gp-his-c/n and pet a (+) -vp -his-c/n were transformed into e. coli bl (de ) cells. recombinant proteins sudv gp and vp were experessed in cells after added . mm iptg. the proteins were purified by ×his-tag affinity chromatography and detected by sds-page analysis (figure ). the purpose of expressing sudv gp and vp proteins is to prepare anti-gp polyclonal antisera and anti-vp polyclonal antisera, and these two polyclonal antisera were successfully made through immunized mice with these two proteins twice respectively. to identify the successful rescue of the recombinant baculovirus and effectively expressed the sudv gp and vp proteins, the fourth generation of recombinant baculovirus were detected by ifa. sf cells infected with recombinant baculovirus rbac-gp-gp or rbac-vp -vp were incubated with mouse anti-gp polyclonal or anti-vp polyclonal antisera respectively, and then incubated with fitc-conjugated goat anti-mouse igg. the immunofluorescence results showed that sf cells infected with recombinant baculovirus rbac-gp-gp or rbac-vp -vp were shown in specific green ( figure d ,e) and non-infected sf cells ( figure f ) did not show, which means that sudv gp and vp proteins were successfully expressed in infected sf cells. sudv vlps were generated by co-infecting with recombinant baculoviruses rbv-gp-gp and rbv-vp -vp in sf cells. the sudv vlps were harvested from the culture supernatant and purified as described in the material and methods. to further characterize the structure of the sudv vlps, we examined purified material by transmission electron microscopy. a negative staining study revealed that the sudv vlps were found to mimic the naive virus in structure and size; they were approximately nm in diameter and - nm in length ( figure a) . the identity of the protein component of the purified sudv vlps was assessed by western blot analysis with mouse anti-sudv gp polyclonal antisera and mouse anti-sudv vp polyclonal antisera. both vp and gp proteins are present in the vlps ( figure b ). these results demonstrate that sudv gp and vp efficiently assembled sudv vlps and released by insect cells. to detect a sudv-vlp-induced humoral immune response in vaccinated balb/c mice, indirect elisa and virus neutralization assay were performed to evaluate the production of sudv gp-specific antibodies and neutralizing antibodies. at the fifth week of the immunization, the sera from mice treated with µg sudv vlps mixed with an isa adjuvant group had high titers of sudv specific igg antibodies with endpoint titers up to : , ( figure c ), virus-neutralizing antibody titers averaged : and individual mice neutralizing antibody titers were up to : ( figure d) , showing significant divergence when compared to the pbs group and isa adjuvant group. but the sudv specific igg antibodies and neutralizing antibodies were not detected in the vlps-only group mice. these results demonstrate that sudv vlps at µg antigen dose cannot stimulate the production of antibodies in mice, but this antigen dose mixed with isa adjuvant can stimulate mice to produce specific igg antibodies and neutralizing antibodies. horses (b) . analysis of sudv-vlp-induced specific igg antibody response by indirect elisa at two weeks after every immunization of vaccinated mice (c). sudv neutralizing antibody titers are detected in immunized mice at two weeks after booster immunization (d). limit of detection (lod) means the minimum concentration or content that can be detected under the determined experimental conditions. error bars represent the standard deviation. the p-values were determined according to a tukey's multiple comparison test (* p < . , *** p < . ). elispot assays were used to evaluate the antigen-specific cellular immune response in vaccinated mice. since µg vlps-only did not stimulate the production of specific antibodies and neutralizing antibodies, the vlps-only group was removed in the cellular immune response experiment. the results from the il- elispot assay are shown in figure a . the sfcs produced from the splenocytes of mice immunized with µg sudv vlps mixed with an isa adjuvant were significantly higher compared with mice immunized with pbs or isa . the immunization with µg sudv vlps mixed with an isa adjuvant also resulted in increased ifn-γ responses, with sfcs production that were significantly higher than that following immunization with pbs or isa ( figure b) . these results demonstrate that µg sudv vlps mixed with an isa adjuvant enhances il- and ifn-γ responses in mice. statistical analysis between the two groups was analyzed by using tukey's multiple comparison test (* p < . , ** p < . ). to further investigate the antigen-specific cellular immune responses induced by sudv vlps, cytokines secreted by splenocytes were measured by commercial elisa kits. splenocytes were harvested from mice one week after the booster immunization and stimulated with prokaryotic-expressed sudv gp ( ~ aa). cytokines secreted into the supernatant by splenocytes were assessed. type cytokines such as il- ( figure a ), ifn-γ ( figure b ), and tnf-α ( figure c ) were significantly higher in the group immunized with µg sudv vlps mixed with an isa adjuvant than they were in the pbs and isa adjuvant group. type cytokines, such as il- ( figure d ) and il- ( figure e ), showed the same trend. these results indicate that µg sudv vlps mixed with an isa adjuvant potently enhance cellular immune response in mice. the humoral immune response in horses was assessed at , , , and weeks after immunization ( figure b ). serum samples were collected to measure the sudv gp-specific antibodies and neutralizing antibody. the level of sudv gp-specific igg antibody was detected from the second week ( figure a ). after the fifth immunization, the titer of sudv gp-specific igg antibody of horse # was : , , and for horse # was : . the results revealed that sudv vlps mixed with freund's adjuvant could stimulate horses to produce humoral immune response. regarding the research on sudv therapeutic antibodies, we selected # horse serum for further study. the # serum was extracted and purified to obtain purified igg; # horse serum and # purified igg were simultaneously subjected to virus neutralization. these findings showed that the neutralizing antibody titer of # horse serum was : , after the fifth immunization, correspondingly, the neutralizing antibody titer of purified igg was : ( figure b ). the mechanism of natural immunity against ebov remains unclear. the efficacy of different types of vaccines may vary depending on the vaccine platform: antibody is the major immune correlate factor of the rvsv-zebov vaccine [ ] . meanwhile, cd t cell responses have been attributed to protection by the ch-ad vaccine [ ] . previous vlp vaccine studies showed it is efficacious against lethal ebola challenge, however, with different adjuvants to save antigen and enhance vaccine-induced immune responses [ , ] . the impact of sudv outbreaks in recent years, and the potential for viral spread to non-endemic regions or countries, makes the development of safe and efficacious vaccines urgent. previous findings revealed that sudv vlps could be readily assembled by the co-expression of baculoviruses expressing gp, np and vp in insect cells [ ] . here, we demonstrated that co-infection of rbv-gp-gp and rbv-vp -vp recombinant baculoviruses can result in the successful assembly of vlps in insect cells, the morphology of sudv vlps is similar to a native virus, and the immunogenicity of vlps was tested with an isa adjuvant as a candidate vaccine in mice. in our study, we generated recombinant baculaviruses rbv-gp-gp and rbv-vp -vp using the pfastbacdual vector to increase the protein production utilizing the dual promoter. to determine the optimal proportion of two recombinant baculaviruses rbv-gp-gp and rbv-vp -vp , we also tried to co-infect sf cells with recombinant baculaviruses rbv-gp-gp and rbv-vp -vp at ratios of : , : , : . , : , : , . : , and : . at a ratio of . : , the morphology of vlps was similar to the native virus, as assessed by tem ( figure a) . the western blot result ( figure b ) showed that sudv gp and vp efficiently assembled sudv vlps. the immunization results depicted the failure of the group vaccinated with µg of sudv vlps-only to stimulate the production of humoral immune responses in mice, while the group vaccinated with µg of vlps mixed with an isa adjuvant could stimulate the production of humoral immunity in mice. presumably, immunization with µg of sudv vlps-only is an insufficiently low dose of an immunogen that cannot stimulate the mice to produce an immune response, and the use of an isa adjuvant with sudv vlps might cause low-dose immunogens to initiate a response, and using with adjuvant can save antigen amount ( figure c ). the same sudv vlps antigen dose ( µg) mix different adjuvants could stimulate mice to produce different degrees humoral immune responses. the isa adjuvant showed the most effective adjuvant than other adjuvants in mice. the detection of neutralizing antibody showed that sera from mice immunized with µg sudv vlps mixed with isa adjuvant could neutralize approximately % of the pseudotyped viruses at an average : dilution, while individual samples were effective with as high as a : dilution ( figure d ). both humoral and cellular responses are indispensable for providing protection. ifn-γ is a th -type cytokine involved in the antiviral action of cellular immune responses and il- is mainly produced by th cells and is associated with humoral immune responses. the sudv vlps could effectively stimulate th and th cytokine production in vaccinated mice ( figure a,b) . the levels of cytokines secreted from splenocytes, such as il- , ifn-γ, and tnf-α secreted from th cells ( figure a -c) and il- and il- were secreted from th cells ( figure d ,e), were significantly increased after vaccination. moreover, our study revealed that the b cells of mice of the group vaccinated with sudv vlps mixed with an isa adjuvant were activated at one week after the primary immunization (figure ). in addition, we have conducted research on therapeutic antibodies with the goal of preparing antibodies for the post-exposure treatment of sudv. horses were selected as immunized animals in our study because horse anti-immunoglobulins have been used in the treatment of various viral infections [ ] [ ] [ ] . sudv vlps can effectively induce humoral immune responses in horses after immunization ( figure a ). the horse anti-sudv immunoglobulin was obtained from # horse serum through crude extraction and purification. the neutralizing antibody titer of purified # igg was : , which was one time lower than that before purification ( figure b ). we tested the sudv vlps immunogenicity and neutralizing activity of hours purified igg by using pseudo typed viruses, and efficacy studies still need to be performed. these sudv vlps vaccine and horse purified igg provide ideas for the development of vaccines and therapeutic antibodies that could prevent and treat sudv infections. the ebola epidemic is still spreading, vaccination is an effective means of preventing and controlling the outbreaks, and effective antibodies represent key drugs for the treatment of ebola patients. therefore, further ebola vaccine and therapeutic antibody research are needed. the authors declare no conflict of interest. the pathogenesis of ebola virus disease ebola haemorrhagic fever in zaire report of a who/international study team changes to taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses the natural history of ebola virus in africa. microbes infect discussions and decisions of the - international committee on taxonomy of viruses (ictv) filoviridae study group the ebola outbreak, - : old lessons for new epidemics ebola and marburg hemorrhagic fever viruses: update on filoviruses ebolavirus and marburgvirus: insight the filoviridae family ebola virus entry requires the cholesterol transporter niemann-pick c live attenuated recombinant vaccine protects nonhuman primates against 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envelopes increased gene transfer in murine lung seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt a monovalent chimpanzee adenovirus ebola vaccine boosted with mva antibodies are necessary for rvsv/zebov-gp-mediated protection against lethal ebola virus challenge in nonhuman primates ebola virus-like particle-based vaccine protects nonhuman primates against lethal ebola virus challenge toll-like receptor agonist augments virus-like particle-mediated protection from ebola virus with transient immune activation quality of horse f (ab') antitoxins and anti-rabies immunoglobulins: protein content and anticomplementary activity functional and proteomic comparison of different techniques to produce equine anti-tetanus immunoglobulin f (ab') fragments comparison of five commercial anti-tetanus toxoid immunoglobulin g enzyme-linked immunosorbent assays key: cord- -ezuyjcxs authors: tomazatos, alexandru; marschang, rachel e.; maranda, iulia; baum, heike; bialonski, alexandra; spînu, marina; lühken, renke; schmidt-chanasit, jonas; cadar, daniel title: letea virus: comparative genomics and phylogenetic analysis of a novel reassortant orbivirus discovered in grass snakes (natrix natrix) date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ezuyjcxs the discovery and characterization of novel arthropod-borne viruses provide valuable information on their genetic diversity, ecology, evolution and potential to threaten animal or public health. arbovirus surveillance is not conducted regularly in romania, being particularly very scarce in the remote and diverse areas like the danube delta. here we describe the detection and genetic characterization of a novel orbivirus (reoviridae: orbivirus) designated as letea virus, which was found in grass snakes (natrix natrix) during a metagenomic and metatranscriptomic survey conducted between and . this virus is the first orbivirus discovered in reptiles. phylogenetic analyses placed letea virus as a highly divergent species in the culicoides-/sand fly-borne orbivirus clade. gene reassortment and intragenic recombination were detected in the majority of the nine letea virus strains obtained, implying that these mechanisms play important roles in the evolution and diversification of the virus. however, the screening of arthropods, including culicoides biting midges collected within the same surveillance program, tested negative for letea virus infection and could not confirm the arthropod vector of the virus. the study provided complete genome sequences for nine letea virus strains and new information about orbivirus diversity, host range, ecology and evolution. the phylogenetic associations warrant further screening of arthropods, as well as sustained surveillance efforts for elucidation of letea virus natural cycle and possible implications for animal and human health. the reoviridae family is a large and diverse group of nonenveloped, icosahedral viruses with genomes composed of - linear molecules of double-stranded rna (dsrna). reoviruses are divided between the spinareovirinae subfamily (species with turrets on the core particle) and sedoreovirinae subfamily (species with smooth, nonturreted core particles). they infect numerous host species, from plants to crustaceans, insects, aquatic and terrestrial vertebrates [ ] . among the reoviridae genera, the orbivirus genus (subfamily: sedoreovirinae) is the largest, having species recognized by the international committee on taxonomy of viruses (ictv) and a significant number of species proposals [ ] . orbiviruses are vector-borne pathogens, primarily transmitted by ticks and other hematophagous insects (mosquitoes, culicoides biting midges and sand flies). their wide host range includes wild and domestic ruminants, camelids, equids, humans, marsupials, bats, sloths and birds [ ] . the most studied orbiviruses are the culicoides-borne bluetongue virus (btv, type species), african horse sickness virus (ahsv) and epizootic hemorrhagic disease virus (ehdv), all known as important pathogens of livestock and wildlife [ ] . some orbiviruses such as tribeč virus, kemerovo, lebombo and orungo viruses have been detected in human infections and are considered human pathogens [ ] . orbiviral genomes consist of linear segments of dsrna designated by their decreasing molecular weight. they encode seven structural proteins (vp -vp ) and three to four nonstructural proteins (ns , ns , ns /ns a and ns ) [ ] . the high conservation degree of certain structural core proteins (e.g., polymerase, major core and subcore proteins) recommends them for comparative and phylogenetic analyses of different orbivirus species [ , ] . in contrast, the proteins of the outer capsid are highly variable and their specificity to the host's neutralizing antibody response can be used to distinguish between different serotypes of the same orbivirus species [ , ] . the phylogenetic clustering of orbivirus members results in clades indicating their putative or potential arthropod vectors: culicoidesor sand fly-borne (c/sbov), mosquito-borne (mbov) and tick-borne orbiviruses (tbov) [ ] . one exception to this classification is st. croix river virus (scrv), a distant member of the genus considered to be a "tick orbivirus" (tov), having no known vector [ ] . as one of europe's largest wetlands, the danube delta biosphere reserve (ddbr) located in the southeast of romania, is a very biodiverse and heterogeneous complex of ecosystems [ ] . the region is a major hub for bird migration along main african-eurasian fly corridors, with ecoclimatic conditions suitable for abundant and diverse populations of arthropod vectors [ ] [ ] [ ] [ ] , which may allow pathogen import and maintenance [ ] [ ] [ ] [ ] . during an arbovirus survey in ddbr, we identified a novel orbivirus in grass snakes (natrix natrix linnaeus ), tentatively named letea virus (leav) after the eponymous village from the study area. the aims of this study were to characterize the genome of leav and its evolutionary relationship with other members of the orbivirus genus. this is the first report of reptiles as orbivirus hosts. the present study expands our knowledge of orbivirus host range, ecology and the complete genomic data may help understand the evolutionary relationship among species of the orbivirus genus. clinical apparently healthy grass snakes (n = ) and dice snakes (natrix tessellata laurenti , n = ) were captured by hand along transects in several areas of ddbr from to , as part of an arbovirus survey ( figure , table s ). a blood sample of~ ml was collected in a ml sterile eppendorf tube from the caudal vein of adults and subadults from both species (total n = ). after clot formation and centrifugation for min at rpm, the serum was carefully transferred to cryogenic vials using a µl pipette with sterile filter tips. samples were frozen at − • c in the field, shipped to the laboratory on dry ice and stored at − • c without interruption of the cold chain. all snakes were released immediately after blood collection back into their habitats. the ddbr administration authority issued research permits for all research activities ( the protocol used to perform metagenomic and metatranscriptomic on snake sera for virus discovery has been previously described [ ] . briefly, µl sera serum samples used for deep-sequencing were filtered through a . µm filter (millipore, darmstadt, germany) in order to remove larger debris and some bacteria. the filtrates were treated with a mixture of nucleases (turbo dnase, ambion, carlsbad, ca, usa; baseline-zero, epicenter, madison, wi, usa; benzonase, novagen, san diego, ca, usa; rnase one, promega, fitchburg, wi, usa) to digest unprotected nucleic acids including host dna/rna. enriched viral particles were then subjected to rna/dna extraction by using magmax™ viral rna isolation kit (life technologies, carlsbad, california, usa) according to the manufacturer's instructions. after random rt-pcr amplification, the extracted viral rna and dna were subjected for library preparation by using a qiaseq fx dna library kit (qiagen, hilden, germany). normalized samples were pooled and sequenced on a miseq or nextseq platform. the generated raw reads were first qualitatively checked with phred quality score < trimmed and filtered to remove polyclonal and low-quality reads (< bases long) using clc workbench (qiagen, hilden, germany). the remaining filtered raw reads were de novo assembled separately using trinity v . . and clc workbench and compared with a nonredundant and viral proteome database (ncbi) using blastx with an e-value cutoff of . . the virus-like contigs and singlets were further compared to all protein sequences in nonredundant protein databases with a default e-value cutoff of . . the viral metagenomics and metatranscriptomics output have been visualized and analyzed in megan [ ] . genome finishing, sequence assembly, and analysis were performed using geneious v . . . (biomatters, auckland, new zealand) (table s ). open reading frames (orf) of the leav genome were detected with geneious v . . and orffinder (https://www.ncbi.nlm.nih.gov/orffinder/). putative functions of leav proteins were assigned by comparison to sequences in genbank, using blastx. pairwise distances for nucleotide and amino acid sequences were calculated in geneious v . . using mafft. evolutionary relationship of leav with representative members of the orbivirus genus were analyzed by inferring phylogenetic trees with amino acid and nucleotide orf sequences of conserved genes encoding the polymerase (vp ), the subcore shell protein t (vp /vp ) and the major core surface protein t (vp ) [ , ] . nucleotide and amino acid sequences were aligned with mafft in geneious v . . . phylogenetic analyses were performed with the best-fit nucleotide and amino acid substitution models selected by their lowest aic (akaike information criterion) or bic (bayesian information criterion) scores using jmodeltest v . . [ , ] and prottest v . . [ , ] , respectively. amino acid phylogenetic trees were constructed using the maximum likelihood (ml) method in seaview v [ ] with lg+i+g+f for t and t (vp ) and for vp with the lg+i+g substitution models. the robustness of tree nodes was assessed by bootstrap replicates. nucleotide phylogenies were constructed by ml ( bootstrap replicates) and by bayesian inference using the markov chain monte carlo (mcmc) method implemented in beast v . . [ ] . the output consisted of two combined mcmc chains, each of generations with sampling every steps and % burn-in. figtree v . . (http://tree.bio.ed.ac.uk/software/figtree/) was used for visualization of tree output files. nucleotide sequences of leav genes obtained in the present study were submitted to genbank and were assigned accession numbers mn -mn . the accession numbers of the other orbivirus sequences used for phylogenetic analysis are listed in table s . in order to screen leav for potential gene reassortment, we assembled complete genomes by segment concatenation and aligned them with mafft. simplot v . . [ ] was used to screen for potential reassortment between leav genomes (n = ) using a % cutoff value for tree permutation across a given genomic segment. for detection of intragenic recombination, we inspected individual gene alignments in the recombination detection program (rpd) package v . and the tests therein (bootscan, maxchi, chimaera, siscan, phylpro, seq and geneconv) [ ] . these tests were performed with default settings: a bp window size and a bonferroni correction of the p-value of . . recombination events were further considered upon detection of significant signals from at least three methods (table s ). we retrospectively and concurrently analyzed arthropods collected within the same arbovirus surveillance program at the respective sites in ddbr ( figure ), with the scope to identify a potential leav vector. in total, , culicoides ( , unfed/gravid and engorged), engorged mosquitoes and ticks were screened for detection of leav rna (tables s and s ). the unfed/gravid culicoides (n = , ) and a part of the tick samples (n = ) were screened using an rt-pcr assay. the engorged dipterans ( culicoides and mosquitoes) and the rest of the ticks (n = ) were subjected to metagenomic and metatranscriptomic analyses. the collection, processing and nucleic acid extraction from engorged mosquitoes and biting midges has been described in previous studies [ , ] . in the case of unfed/gravid culicoides midges, insects were pooled as - specimens with the rest of the process being the same as in the above-referenced work. ticks were collected from their hosts using fine tweezers and identified using morphological keys [ , ] . for homogenization, ticks were placed into a sterile ml eppendorf tube individually or as pooled nymphs, according to host, site and date of collection ( - specimens). two mm steel beads were added inside and the tube was then kept in liquid nitrogen for min. the samples were loaded into a tissue lyser (qiagen, hilden, germany) and the frozen ticks were pulverized at hz for min. to each sample we added . ml of high-glucose ( . g/l) dulbeco's modified eagle's medium (dmem) (sigma-aldrich, st. louis, usa) with l-glutamine, . % head-inactivated fetal bovine serum, µg/ml streptomycin, µg/ml penicillin and µg/ml amphotericin b. the final mix was homogenized using the tissuelyser at hz for min and clarified by centrifugation ( , rpm) for min at • c. total rna was extracted using the magmax rna/dna pathogen kit on a kingfisher flex magnetic particle processor (thermofisher scientific, usa), according to the manufacturer's instructions. in order to detect the presence of leav in the reptile and arthropod samples, we designed a specific primer pair, f: aggcaaaacagtaggatcag and r: gggctaagtggatctgaaac, which amplifies a fragment of the outer capsid protein (vp ). all pcr amplifications were performed in . µl consisting of µl rna, µl reaction mix, . µl mg so ( . µmol), µl ddh o, µl of each primer ( pmol) and . µl enzymmix. the reactions comprised a first reverse transcription at • c for min, • c for min, • c for min, followed by cycles of amplification at • c for sec, • c for sec and • c for sec. final extension was at • c for min. rt-pcr was carried out using a superscript iii one-step rt-pcr kit (invitrogen, carlsbad ca, usa). sera samples from the leav rt-pcr positive snakes were used for attempted virus isolation on c / (aedes albopictus), vero e (african green monkey kidney), bhk- (baby hamster kidney) and several reptile cell lines, including iguana heart cells (igh- , atcc: ccl- ), russell's viper heart cells (vh- , atcc: ccl- ), terrapene heart cells (th- , atcc: ccl- ) and checked for viral cytopathic effect (cpe). briefly, sera samples, undiluted and : diluted were inoculated onto the above-mentioned cell lines and were observed daily for cytopathic effects (cpe). all cultures were harvested to days later and subjected to the leav specific pcr test. this procedure was repeated until passage . of the n. natrix sera, specimens of n. natrix ( . %) were found positive for leav rna. all samples collected from n. tessellata (n = ) tested negative (table s ) , as did all arthropod samples (ticks, mosquitoes and culicoides) analyzed for the presence of leav rna. attempts to isolate the leav strains in several cell line cultures of different vertebrate and insect origins were not successful. we obtained all genomic segments of leav and assembled a total of nine complete genomes. blastx searches showed that the proteins encoded by leav genome match orbivirus homologs. each segment is monocistronic with the encoded protein spanning most of the positive strand. one exception is segment- , which additionally to vp , contains a fourth nonstructural protein (ns ) on a smaller (+ ) orf. all nine leav strains have a genome length of , nucleotides and a gc content of . - . %. gene sizes range from bp (vp ) to bp (ns ) and their coding asignments are homologous to btv [ , ] . descriptions of each leav gene with the closest relatives as retrieved by the blastx are found in table . sequencing of leav untranslated regions (utrs) revealed that the segments share seven conserved nucleotides at both and termini. the first and last two nucleotides of all leav segments are inverted complements (table ) and identical to those found in most orbiviruses [ ] . comparison of main leav protein sequences to homologs of representative orbiviruses (table ) revealed identity values of - % with culicoides-/sand fly-borne orbiviruses (c/sbov), - % with tick and tick-borne orbiviruses (tbov), and - % with mosquito-borne orbiviruses (mbov). the sequence identity between the polymerase (vp ) of leav and that of other orbiviruses was % (scrv) to % (c/sbov), above the % threshold proposed by [ ] for viruses within a single genus of the reoviridae family. analysis of the vp protein indicated that it is the t protein forming the subcore shell, homologous to the vp of btv and of other c/sbov [ ] . similarly to vp , the t is highly conserved and the level of identity relative to other orbiviruses ranged from % (scrv) to % (c/sbov). segments and encode the outer capsid proteins vp and vp in leav (table ) . vp is the most variable protein of c/sbov, located in the first line of contact with host cells and a major determinant of virus serotype [ ] . significant levels of identity of the hypervariable vp were observed only between leav and insect-borne orbiviruses (ibov, - %), while within the same group vp comparison revealed values similar to t (table ) . the orbivirus vp , t and t proteins are used in phylogenetic studies and for classification of reoviridae members at both species and genus level [ , ] . leav was placed in the c/sbov clade by all phylogenetic analyses, consistent with the levels of sequence identity revealed by comparisons with the other orbivirus proteins (figure , figure and figure s ). the phylogenetic trees based on vp and t amino acid (figure a,b) and nucleotide sequences ( figure s ) displayed a clustering typical for the orbivirus genus, with main clades indicative of their arthropod vectors [ ] . the three main branches are rooted by scrv, an orbivirus isolated from tick cells which is considered a tick-associated orbivirus (tov) [ ] . the subcore shell protein t is encoded by segment , coresponding to the vp protein of the c/sbov clade ( figure c) . as in the vp and t phylogenies, leav is basal within this clade. the main difference is that the t tree splits between two clades instead of three, having a clear separation between the orbiviruses encoding t on segment- (mbov, tbov and scrv (tov)) and those encoding t on segment- (c/sbov). putative reassortment events involving leav segments were detected by simplot (bootscan) in eight of the nine leav genomes (figure ). segment- (t ) was exchanged between nn lro and nn lro , while the other instances indicated exchanges of segment- (vp ) and - (vp ) (figure and figure s ). significant signals of recombination among leav genes (≥ methods) were found by rdp for segment- (vp ), segment- (ns ) and segment- (vp ) (figure , table s ). reptiles are known as hosts of numerous viruses. however, limited or fragmentary evidence is available regarding their role in arboviral cycles [ ] [ ] [ ] [ ] [ ] [ ] . the present study investigated the possibility that natricine snakes harbor arboviruses by screening sera collected from sympatric populations of grass snakes (n. natrix) and dice snakes (n. tessellata) from the danube delta biosphere reserve in romania. thus, we described for the first time the discovery and genetic characterization of a novel orbivirus species (letea virus, leav) infecting reptiles. inclusion and demarcation of species within the orbivirus genus considers several criteria, such as sequence identity of segments encoding the polymerase (vp ) and major subcore shell protein t , gene reassortment between close strains, high levels of serological cross-reactivity against conserved antigens like the t protein, conservation of utr terminal nucleotides, range of hosts and vectors or the clinical signs associated with orbivirus infection [ ] . we propose that leav should be included in the orbivirus genus as a separate species, based on the comparative and phylogenetic analyses reported herein. in addition to a typical orbivirus genomic architecture of linear segments of dsrna, the utrs of leav include conserved terminal sequences similar to other orbiviruses. the leav terminal nucleotides are not conserved hexanucleotides as in the case of btv or ahsv, but heptanucleotides showing little variation among the segments. distal dinucleotides at both of the utr ends are inverted complements (table ) , as shown in other orbiviruses [ , , , , [ ] [ ] [ ] . the ns is a nonstructural protein found in some orbiviruses and the last one described to date [ , ] . in leav, we found ns to be of similar size and position as in other c/sbov [ , , ] , showing also the lowest sequence identity among all compared orbiviral proteins ( table ) . the amino acid identity observed in the polymerase is above the % threshold defined by [ , ] for inclusion in the orbivirus genus ( table ). the protein sequence of leav t (vp ) showed identity levels significantly lower than the % cutoff indicated for this protein [ ] , confirming that leav is a distinct orbivirus species. furthermore, the nine different leav strains belong to the same species, as their t amino acid sequences are > % identical. additional taxonomical markers of orbiviruses are the vp (outer capsid in c/sbov) and vp (t ) proteins, determining the serotype and serogroup, respectively [ ] . the core surface protein t forms the outer layer of the viral core and is the primary antigenic constituent of virus serogroup (species) [ ] . the low amino acid identity of leav t (table ) to other t proteins confirms that this virus belongs to a distinct serogroup. vp is encoded on segment- in leav and functionally equivalent to vp of mbov and vp of tbov/tov [ ] . due to its neutralizing epitopes and role in cell attachment, the vp (oc ) protein is subjected to intense selective pressures by the host's immune responses. thus, it is one of the most variable orbiviral proteins [ ] . unsurprisingly, we found significant levels of identity for leav vp ( - %) only in comparison with ibov proteins. moreover, the high amino acid identity (> %) found between vp of the nine leav strains showed that all sequences belong to the same leav serotype. previous studies noted that the overall gc content and the utr proportion relative to the genome's length reflect three groups similar to those illustrated by phylogenetic analyses. first, the gc content is highest in tbov with - . % gc, followed by the c/sbov with . - . % gc and the mbov with . - . % gc [ , , , ] . the gc content of leav is . - . %, therefore below these intervals. second, the utrs of c/sbov span . - . % of their total genome length, in tbov the utrs are between~ . - % and in mbov~ - . % of the virus genome [ , , ] . again, leav falls outside these limits, having the proportion of utrs at . % of the genome's length, which is higher than in other orbiviruses. apart from the high mutation rate owing to a polymerase lacking proofreading activity, reassortment of cognate genomic segments is an important driver of genetic diversity in viruses with segmented rna genomes [ ] . this process can generate novel phenotypes with fundamental implications for immune escape, host or vector range, virulence and pathogenicity [ ] [ ] [ ] [ ] . the ability to reassort genomic segments is a primary criterion for inclusion in the reoviridae family [ ] and it may have contributed to the great evolutionary success of this family. most natural cases of orbivirus reassortment have been described in btv studies, mostly due to its antigenic diversity, wide geographic range, but also economic importance [ ] [ ] [ ] [ ] [ ] . additionally, reassortment has been described in ehdv [ ] [ ] [ ] [ ] , corv [ ] , cglv [ ] and banna orbivirus (baov) [ ] . we found that reassortment between leav strains involved segment- (vp ), segment- (t ) and segment- (vp ) in eight of nine genomes analyzed. we speculate on another segment exchange where leav strain nn sul received its segment- (t ) from an unidentified leav parental strain, due to the striking sequence divergence of nn sul segment- within a highly similar (> %) vp dataset (figure and figure s ). when translated to protein sequence, the identity of this segment to the rest of the leav homologs was ≥ . %, confirming the necessity of its structural conservation. intragenic recombination between segments of leav was detected in the majority of leav strains ( figure , table s ). the effective contribution of each mechanism generating diversity in segmented viruses is far from being understood. against a backdrop of rapid mutation, the formation of mosaic genes along with reassortment can have compounding effects on viral fitness. as in the case of sand fly-borne changuinola (cglv) serogroup [ ] , the strain biodiversity could be an important factor for the occurrence of rna segment/fragment exchange in leav. this is indicated by detections of reassortment and intragenic recombination in eight, respectively seven, of nine leav strains. this is all the more clear for orbiviruses with great antigenic diversity like btv [ , , ] and ahsv [ ] , but also for orthoreoviruses [ ] [ ] [ ] and rotaviruses [ ] . despite field efforts parallel to sera collection and the significant leav prevalence in grass snakes ( . %), none of the screened arthropod pools or individuals tested positive for leav rna. also, recent analyses of mosquito and culicoides host-feeding patterns in ddbr did not indicate ectothermic species as hosts of these insects (with very few exceptions provided by frogs fed upon by mosquitoes) [ ] . reoviruses known to infect reptiles belong to the turreted group of the family (subfamily spinareovirinae, e.g., reptilian orthoreovirus) and cause severe illness of digestive and respiratory organs [ ] [ ] [ ] [ ] . the grass snake and dice snake occur sympatrically across the study area. in dunărea veche and sulina we could observe some ecological features (bank structure, water body type, prey, microhabitat usage) very similar to those described for other european populations, indicating ecological partitioning in syntopic populations [ ] . although we encountered more n. tessellata in the aforementioned sites, leav was not detected in the sera of this species. infected grass snakes showed no sign of disease. to our knowledge, this is the first report of orbivirus detection in reptiles. all attempts of growing and isolating leav on insect or vertebrate cell lines showed no cytopathic effect or silent replication of the virus. most known orbiviruses grow easily in vertebrate cells in vitro, while a few are known to be restricted to insect cells [ , ] . although leav isolation could be further attempted on additional cell lines, we may speculate a shift in cell tropism underlying the inability of leav to infect certain cell types. a similar case is parry's lagoon virus (plv), a serotype of corv. in contrast to the wide vertebrate host range of corv [ ] , the antigenically related plv showed a distinct cell tropism and replicated only in insect cells [ ] . with the available data we may speculate that such shifts in cell tropism could be the result of successive changes through recombination, genetic drift and shift. reoviridae is a very successful family of segmented dsrna viruses, having wide host ranges across various econiches. the family's repertoire of evolutionary strategies also includes deletion [ , ] , gene duplication and concatemerisation [ , , ] . these strategies were also observed previously in aquareoviruses [ ] , rotaviruses [ ] , phytoreoviruses [ ] and even in a cross-family heterologous recombinant virus containing reovirus genomic components [ ] . this suggests an increased potential for "species jumps" and adaptation to new vectors and/or hosts [ , ] . earlier studies associated a conserved arg-gly-asp ( rgd ) motif on the t protein of btv with the attachment to culicoides cells [ , ] . this conserved motif was also found by later studies in some species closely related to btv [ , , ] , but we did not observed it in leav. as with other orbivirus species, this may reflect the higher divergence relative to btv. although the phylogenetic analyses indicate leav as a potentially culicoides-borne orbivirus, it is interesting to note some general inconsistencies in vector associations of ibov (c/sbov and mbov). for example, phylogenetic analyses place orungo virus (oruv), lembombo virus (lebv), pata virus (patav) and japanaut virus (japv) in the c/sbov clade, even though they were discovered in mosquitoes [ , , ] . for oruv, follow-up studies on mosquito transmission were inconclusive [ ] . interestingly, tracambé virus (trv), a serotype of the sand fly-borne cglv serogroup, was isolated from mosquitoes of the genus anopheles (reoviridae.org). some serotypes of c/sbov have been isolated from both mosquitoes and biting midges: eubenangee virus (eubv) [ , ] , palyam virus (palv) [ , ] , warrego virus warv [ ] , wongorr virus (wgrv) [ ] , and tibet orbivirus (tibov) [ ] . the associations between some viruses and more than one vector family could be the result of "species jumps" permitted by fast evolution characteristic of rna segmented viruses. such occurrences in members of the orbivirus genus would not necessarily run counter to the "coevolution" hypothesis [ , , , ] , but possibly complement it. in conclusion, a novel orbivirus (leav) was identified and characterized, expanding the known host range of orbiviruses and revealing its genetic relationship to the orbivirus genus. phylogenetic analysis indicates leav as a potentially culicoides-borne orbivirus, although this was not confirmed by the screening of culicoides midges and other arthropods from the ddbr. leav failed to replicate in vitro in several types of cells, which may warrant further attempts using additional cell types. the discovery and characterization of leav offers valuable information, expanding our knowledge about the evolution and host range of orbiviruses. the phylogenetic associations can justify further screening of arthropods and continued surveillance in order to describe the natural cycle of leav and its possible impact on vertebrate hosts. the following are available online at http://www.mdpi.com/ - / / / /s , figure s : maximum likelihood phylogenies of main conserved orbivirus genes, figure s : analysis of genetic reassortment (bootscan) performed with simplot . . using the concatenated genomic segments of letea virus, figure s : multiple alignments of letea virus (leav) genes highlighting disagreements of their nucleotide sequences (leav is indicated by the red triangle), table s : snake sampling in ddbr - , table s : summary of next-generation sequencing data for the leav genomes, table s : genbank accession numbers for orbivirus nucleotide sequences, table s : results of rdp scanning for detection of recombination in leav, table s : list of ticks screened for the presence of leav rna, table s : list of biting midges screened for the presence of leav rna. virus taxonomy: ninth report of the international committee on taxonomy of viruses zoonotic and emerging orbivirus infections yunnan orbivirus, a new orbivirus species isolated from culex tritaeniorhynchus mosquitoes in china a review of knowledge gaps and tools for orbivirus research. vector borne zoonotic dis complete sequence characterization of the genome of the st croix river virus, a new orbivirus isolated from cells of ixodes scapularis umatilla virus genome sequencing and phylogenetic analysis: identification of stretch lagoon orbivirus as a 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generating rapid genetic change of potential epidemiological importance genomic rearrangement in genome segment of rice dwarf phytoreovirus a bat-derived putative cross-family recombinant coronavirus with a reovirus gene crystal structure of the top domain of african horse sickness virus vp : comparisons with bluetongue virus vp rgd tripeptide of bluetongue virus vp protein is responsible for core attachment to culicoides cells characterization of orungo virus, an orbivirus from uganda and nigeria orungo virus: transmission studies with aedes albopictus and aedes aegypti (diptera: culicidae) replication of eubenangee virus in culicoides nubeculosus (mg.) and culicoides variipennis (coq.) phylogenetic characterization of the palyam serogroup orbiviruses the authors wish to thank andrei tomazatos and patricia iftene for their help during fieldwork and to anucha ponyiam for excellent technical assistance. the authors declare no conflict of interest. key: cord- -ok px z authors: armando, federico; gambini, matteo; corradi, attilio; giudice, chiara; pfankuche, vanessa maria; brogden, graham; attig, friederike; von köckritz-blickwede, maren; baumgärtner, wolfgang; puff, christina title: oxidative stress in canine histiocytic sarcoma cells induced by an infection with canine distemper virus led to a dysregulation of hif- α downstream pathway resulting in a reduced expression of vegf-b in vitro date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ok px z histiocytic sarcomas represent malignant tumors which require new treatment strategies. canine distemper virus (cdv) is a promising candidate due to its oncolytic features reported in a canine histiocytic sarcoma cell line (dh cells). interestingly, the underlying mechanism might include a dysregulation of angiogenesis. based on these findings, the aim of the present study was to investigate the impact of a persistent cdv-infection on oxidative stress mediated changes in the expression of hypoxia-inducible factor (hif)- α and its angiogenic downstream pathway in dh cells in vitro. microarray data analysis, immunofluorescence for -hydroxyguanosine, superoxide dismutase and catalase, and flow cytometry for oxidative burst displayed an increased oxidative stress in persistently cdv-infected dh cells (dh ond pi) compared to controls. the hif- α expression in dh ond pi increased, as demonstrated by western blot, and showed an unexpected, often sub-membranous distribution, as shown by immunofluorescence and immunoelectron microscopy. furthermore, microarray data analysis and immunofluorescence confirmed a reduced expression of vegf-b in dh ond pi compared to controls. in summary, these results suggest a reduced activation of the hif- α angiogenic downstream pathway in dh ond pi cells in vitro, most likely due to an excessive, unusually localized, and non-functional expression of hif- α triggered by a cdv-induced increased oxidative stress. neoplastic diseases are one of the major causes of death in humans and domestic animals due to disappointing results of many conventional therapies [ , ] . therefore, viral oncolysis represents an interesting potential new option in human as well as veterinary medicine [ ] [ ] [ ] . interestingly, viruses from different families, including members of the paramyxoviridae (canine distemper virus, measles virus and newcastle disease virus), poxviridae (vacciniavirus), reoviridae (reovirus serotype dearing), adenoviridae (adenovirus onyx- and h ), orthomyxoviridae (influenza virus), herpesviridae (herpes simplex virus type ), picornaviridae (coxsackievirus) and rhabdoviridae (vesicular stomatitis virus) possess oncolytic properties [ ] [ ] [ ] . canine distemper virus represents a morbillivirus closely related to human measles virus [ ] , with the latter already described as a promising oncolytic virus in human medicine that has reached the phase of clinical trials [ ] . similarly, the attenuated onderstepoort vaccine strain of canine distemper virus (cdv-ond) represents a potential oncolytic virus for the treatment of canine histiocytic sarcomas [ , ] . canine histiocytic sarcomas are malignant tumors with poor prognosis and limited therapeutic options [ , ] which originate from interstitial dendritic cells or from macrophages [ ] [ ] [ ] [ ] . since its establishment in [ ] , a canine histiocytic sarcoma cell line (dh cells) has been commercially available. dh cells can be infected by cdv-ond [ ] , and have been reported as a promising model for the investigation of viral oncolysis [ , , ] . specifically, acute infection of dh cells with cdv-ond in vitro resulted in a prominent cell death at days post infection [ ] , followed by establishment of persistent infection in tumor cells surviving the acute lytic phase [ ] . in this context, subcutaneous xenotransplantion of persistently cdv-ond infected dh cells resulted in a total regression of neoplasms in a mouse model [ ] . this promising observation was assumed to be related to a decreased vascularization of the transplants [ ] , with the underlying mechanisms not fully understood so far. therefore, additional investigations using persistently cdv-infected dh cells might represent a promising model to study virus-induced alterations of cancer hallmarks [ ] and of the tumor microenvironment [ ] avoiding the confounding effects correlated with ongoing virus-induced cytopathogenic tumor cell death associated with the acute infection [ ] . indeed, as reviewed by lapp et al. [ ] , viral oncolysis mechanisms can be distinguished between primary (i.e., direct virus-induced cytolysis and/or apoptosis) and secondary ones. the latter include a wide range of events leading to tumor cell death, such as modulation of the antiviral and antitumoral immune response, changes in the organization of the tumor-associated extracellular matrix, and alterations of the tumor-associated vasculature and angiogenesis [ , , , , , [ ] [ ] [ ] [ ] . specifically, a reduced vascularization of neoplasms often leads to intratumoral hypoxia [ ] associated with modifications especially of intracellular pathways connected with reactive oxygen species (ros) production and scavenging. ros are highly chemically reactive molecules that can induce damage to cellular macromolecules such as nucleic acid and lipids, when they outnumber scavenging systems [ ] [ ] [ ] . cdv infection can increase ros production and ros-induced damage in vitro and in vivo as shown for spontaneous cdv infection in dogs [ ] [ ] [ ] [ ] [ ] . furthermore, cdv can induce an accumulation of viral glycoproteins in the endoplasmic reticulum (er) of vero cells and primary rat neurons, resulting in increased endoplasmic reticulum stress [ ] , which has been reported as associated with an increased ros production [ ] . nevertheless, ros are physiologically involved in a plethora of different intracellular signaling pathways [ , ] , and play a key role in multiple hallmarks of cancer [ ] . hypoxia-inducible factor -alpha (hif- α) is a transcription factor that after translocation from the cytoplasm to the nucleus, forms a heterodimer with hypoxia-inducible factor -beta (hif- β), which binds to specific dna sequences known as hypoxia response elements (hres) [ , ] . this event induces the expression of numerous genes involved in different cellular responses such as angiogenesis [ , ] , which is driven by several growth factors, including members of the vascular endothelial growth factor (vegf) family. hypoxia, and to a lesser extent ros, represent the most important stimuli for hif- α stabilization and nuclear translocation [ , ] . during normoxia and redox homeostatic state [ ] , hif- α is localized within the cytoplasm and is rapidly degraded by the proteasome after hydroxylation by prolyl hydroxylases (phds) and subsequent ubiquitination by the von hippel-lindau protein (vhl) [ , , ] . in this context, hypoxia and ros directly down-regulate the activity of phds and vhl [ ] , playing therefore a key role in the inhibition of the overall hif- α degradation. in consideration of the above, the hypothesis of the present study was that a persistent cdv-ond infection of dh cells induces oxidative stress followed by a massive inhibition of hif- α degrading pathways. this in turn leads to cytoplasmic, non-functional accumulation of hif- α, which is associated with a reduced expression of hif- α downstream targets, such as vegf-b. based on the aforementioned hypothesis, the aim of the present in vitro study was to demonstrate that histiocytic sarcoma cells (dh cells) persistently infected with cdv-ond show: ( ) an increased oxidative stress status, ( ) an increased hif- α protein expression, ( ) an unusual intracellular distribution of hif- α, and ( ) a reduced expression of hif- α downstream targets, with a special focus on vegf-b. non-infected dh cells obtained from the european collection of authenticated cell cultures (ecacc no. ), and dh cells persistently infected with cdv-ond (dh ond pi) that were established as formerly described [ ] , were cultured according to standard procedures as previously reported [ ] . briefly, cells were cultured in minimal essential medium (mem) with earle's salts (paa, cölbe, germany) supplemented with % fetal calf serum (paa), % penicillin/streptomycin (paa), and % non-essential amino acids (sigma-aldrich, taufkirchen, germany). culture flasks were kept at • c in the presence of % co in a water saturated atmosphere. five formalin-fixed paraffin embedded (ffpe) cell pellets of non-infected dh cells and of dh ond pi cells were produced as previously described [ ] . briefly, cells were scraped and centrifuged at xg for min at • c. afterwards, the supernatant was removed, cells were washed in pbs and centrifuged again. following a second wash and centrifugation step, the pellet was fixed in . ml of % non-buffered formalin overnight at • c, and processed for routine paraffin embedding. in a hypothesis-driven approach, an online available microarray data set of quadruplicates of non-infected dh and dh ond pi cells (arrayexpress; http://www.ebi.ac.uk/arrayexpress; accession number e-mtab- [ , ] ) was investigated for differentially expressed genes related to ros production and scavenging, er-stress and hif- α pathway, with a special focus on the angiogenic downstream targets of the latter. this choice was justified by the results of the functional profiling of the same dataset obtained in a previous study, highlighting a down-regulation of the expression of some of the genes correlated with angiogenesis [ ] . therefore, in the current work, a list of human and murine genes and proteins was manually generated according to the literature [ , , , , [ ] [ ] [ ] [ ] , ] and translated into canine orthologous gene symbols using the web-based hgnc database (hgnc database, hugo gene nomenclature committee (hgnc), european molecular biology laboratory, european bioinformatics institute (embl-ebi), wellcome genome campus, hinxton, cambridge cb sd, united kingdom, www.genenames.org [ ] ). after filtration, the raw expression data of the selected genes were compared between non-infected dh and dh ond pi cells, employing multiple pairwise nonparametric mann-whitney u-tests. statistical analysis was performed with sas enterprise guide (sas version . ; sas institute inc, cary, nc, usa). differential expression was defined as the combination of a fold change (fc) filter (fc ≥ . or ≤ − . ) and of a statistical significance filter (mann-whitney u-test; p ≤ . ) [ ] . to facilitate the interpretation of results, each gene symbol was assigned to at least one of the following functional groups on the basis of the function(s) carried out by its corresponding protein(s): ros production; ros scavenging; er stress; hif- α activation, transcriptional activity and regulation; hif- α angiogenic downstream pathway. immunofluorescence was performed on ffpe pellets of non-infected and persistently cdv infected dh cells as previously described with minor variations [ , ] . briefly, sections were deparaffinized, rehydrated through graded alcohol and pre-treated for antigen retrieval. following blocking of unspecific bindings, sections were incubated with primary antibodies for min at room temperature. after min of incubation with the secondary antibody, nuclei were stained with bisbenzimide (sigma-aldrich chemie gmbh, taufkirchen, germany), and the slides were mounted with dako flourescence mounting medium (dako north america, inc., carpinteria, ca, usa). each reaction was carried out with corresponding positive controls (table ) . for negative controls, the first antibody was replaced with rabbit serum, balb/c ascitic fluid, or goat serum, respectively at corresponding protein concentrations. to verify the persistent infection status of dh ond pi cells (which was set as corresponding to a rate of > % infected cells), an immunolabeling with an anti-cdv nucleoprotein (cdv-np) antibody (clone d ; kindly provided by prof. a. zurbriggen, university of bern, switzerland) was performed. furthermore, pellets were stained with antibodies directed against -hydroxyguanosine/ -hydroxydeoxyguanosine ( ohg/ ohdg, in the following paragraphs simply referred to as ohdg), a marker of ros-damaged rna or dna [ ] ; superoxide dismutase (sod ) and catalase (cat), two ros scavengers; hif- α, a transcription factor; wheat germ agglutinin (wga), a cell membrane marker; cd , directed against tetraspanin- expressed on exosome membranes; and gm- , a marker for golgi apparatus. all details regarding the antibodies used are listed in table . for cdv-np, ohdg, sod , cat, hif- α, and vegf-b, the percentage of immunopositive cells for each group (non-infected dh cells and dh ond pi cells) was assessed manually by counting evenly distributed fields per pellet at a x magnification using an inverted fluorescence microscope (olympus ix- , olympus optical co. gmbh, hamburg, germany) equipped with a olympus dp camera and olympus cellsens standard software version . . additionally, for hif- α the intracellular protein distribution was assessed and calculated as percentage of cells immunopositive within the nucleus, cytoplasm and membrane. for each marker, after calculation of the median percentage of immunopositive cells per pellet, the normality of distribution of the data referring to non-infected and dh ond pi cells was evaluated with the shapiro-wilk test and followed by the mann-whitney u test for pairwise comparison. the difference of the intracellular distribution of hif- α immunopositivity within each group of cells was analyzed with the kruskall-wallis test with post-hoc dunn's test. statistical significance for each analysis was set at p-value ≤ . . all statistical analyses were performed with graphpad prism version . . for windows (graphpad software, la jolla, ca, usa, www.graphpad.com). gar-cy canine fetal brain, liver and kidney bsa, bovine serum albumin; cdv-np, canine distemper virus nucleoprotein; dag-cy , donkey anti goat cyanine -conjugated; dar-cy , donkey anti rabbit cyanine -conjugated; gam-cy , goat anti mouse cyanine -conjugated; gam-cy , goat anti mouse cyanine -conjugated; gar-cy , goat anti rabbit cyanine -conjugated; gar-cy , goat anti rabbit cyanine -conjugated; gm , golgi membrane protein of kda; hif- α, hypoxia-inducible factor α; mdck, madin-darby canine kidney cells; n/a, non applied or non applicable; pbst, phosphate buffered saline tween- ; sod , superoxide dismutase ; vegf-b, vascular endothelial growth factor-b; wga, wheat germ agglutinin; ohdg, -hydroxyguanosine/ -hydroxydeoxyguanosine. non-infected and persistently cdv-ond infected dh cells were treated with ´, ´-dichlorofluorosceindiacetate (dcf, final concentration of µm, sigma aldrich, d ) at • c and % co for min. flow cytometer (attune ® nxt acoustic focusing; laser nm ( mw), filter bl- = / ) analysis was performed measuring mean green fluorescence intensity (x-mean of bl- ) as relative ros production. respective background controls without dcf were included in all assays. threshold was adjusted to unstained cells to remove background. green fluorescence intensity (fitc) of all cells (percentage of positive cells) was recorded by flow cytometry as relative measure of ros production. the following settings were used: acquisition volume of µl/min, stop at , events on all counts; instrument settings: fsc , ssc bl (fitc). were quantified. statistical analyses of measurements were performed with graphpad prism version . . for windows (graphpad software, la jolla california usa, www.graphpad.com) using unpaired t-tests. to evaluate in more detail the intracellular localization of hif- α within dh ond pi cells, immunoelectron microscopy was performed using a % neutral buffered formalin fixed cell pellet as previously described [ ] . ultrathin sections of lr-white embedded samples were immunolabeled with an anti-hif- α antibody ( : dilution; novus biologicals) followed by a goat anti-rabbit igg nm immunogold conjugated secondary antibody (bbi solutions, crumlin, united kingdom). samples were further evaluated using a transmission electron microscope (em a, carl zeiss microscopy gmbh, jena, germany) equipped with a k-ccd-camera (trs) and using image sp professional software. the intracellular hif- α distribution was analyzed by double-labeling immunofluorescence (dl-if). therefore, hif- α was combined with wga as a marker for the cell membrane, cd as an exosomal marker, gm- as a marker for the golgi apparatus, and cdv-np. the evaluation was performed using a leica tcs sp ii fluorescence microscope (leica microsystems, bensheim, germany) with a conventional galvanometer scanner of the leica sp ii tandem scanning system and the leica application suite advanced fluorescent lite . . build (leica, biberach, germany). settings were adjusted using respective control antibodies. images were analyzed using leica las af software (version . . ). cell lysates were prepared by freezing and thawing in ml np- buffer ( mm tris-hcl, mm nacl, % np- , mm edta) with µl protease inhibitor cocktail ( . µm antipain dihydrochloride, . µm aprotinin, . µm leupeptin, . µm pepstatin a in dmso, mm pmsf, µg/ml trypsin inhibitor t ) at ph . (all reagents from sigma-aldrich, st. louis, usa). samples were analyzed by sds-page on % gels and subsequently transferred to a polyvinylidene fluoride (pvdf) membrane as described previously [ ] . immunoblotting was performed using a polyclonal anti-hif- α ( . µg/ml, cayman, ann arbor, usa) and a monoclonal anti-β-actin ( . µl/ml, santa cruz, dallas, usa) antibody, respectively. a polyclonal igg antibody from rabbit serum served as a negative control ( µg/ml, sigma-aldrich, st. louis, usa). secondary anti-rabbit or anti-mouse antibodies conjugated to horseradish peroxidase were used ( . µg/ml, thermoscientific, schwerte, germany). protein bands were visualized using supersignal™ west femto maximum sensitivity western blot chemiluminescence substrate (thermoscientific, schwerte, germany) and a chemidoc mp imaging system (bio-rad, hercules, ca, usa). quantification was performed densitometrically. obtained results for hif- α were displayed as a ratio with the corresponding amount of β-actin. statistical analyses of obtained ratios were performed with graphpad prism version . . for windows (graphpad software, la jolla, ca, usa, www.graphpad.com) using unpaired t-tests. the infection status of dh ond pi cells was assessed via immunofluorescence staining for cdv-np (supplementary figure s ). while immunoreactivity for cdv-np of non-infected dh cell pellets was negative in all cells, dh ond pi cell pellets showed a median percentage of . % (range: . - . %) infected cells (supplementary table s ) . on a molecular level, a manually generated list of canine gene symbols associated with ros production and scavenging, er stress and the hif- α pathway was analyzed using a microarray dataset of dh and dh ond pi cells. this investigation resulted in a list of genes present within the available data set (supplementary table s ). using the combination of a statistical significance filter (mann-whitney u-test; p ≤ . ) and a fold change (fc) filter (fc ≥ . or ≤ − . ), genes were differentially expressed. specifically, canine genes showed a down-regulation, whereas genes were up-regulated (table ) . table . summary of canine gene symbols related to ros production and scavenging, er-stress and hif- α pathway, differentially expressed between non-infected and persistently canine distemper virus infected dh cells, according to the combination of a fold change (fc) filter (fc ≥ . or ≤ − . ) and of a statistical significances filter (p ≤ . ). [ , ] green labeling refers to down-regulated genes; red refers to up-regulated genes. er, endoplasmic reticulum; hif- α, hypoxia-inducible factor α; ros, reactive oxygen species. "hif- α transcription & regulation" is the abbreviation for "hif- α activation, transcriptional activity and regulation" functional group; "hif- α downstream" is the abbreviation for "hif- α angiogenic downstream pathway" functional group. when specifically analyzed according to the functional grouping, genes related to ros production were up-regulated, while nine were down-regulated (table ). among the group of genes related to ros scavenging, five genes were up-and five were down-regulated (table ) . specifically, neutrophil cytosolic factor (ncf ) and thioredoxin interacting protein (txnip), belonging to ros production and ros scavenging functional groups, respectively, were the two most markedly up-regulated genes among the entire set examined. taken together, these findings should be cautiously interpreted as an increased transcription of genes which corresponding proteins are involved in increasing intracellular oxidative stress [ , , , ] . among the group of genes related to er-stress (partially overlapping with both ros production and ros scavenging functional groups), genes were up-regulated while were down-regulated. specifically, among the genes included in the er stress functional group, the xanthine dehydrogenase (xdh) was up-regulated, while among down-regulated genes were included (pdia , pdia and pdia ) out of genes related to protein disulphide isomerases, one (ero l) out of two genes related to endoplasmic reticulum oxidoreductines, and two (canx and ddit ) out of three genes previously related to er-stress induced by acute infection with cdv [ ] . taken together, these results can be cautiously interpreted as indicative of a reduced transcription of genes that are reported to correlate with er-stress [ , [ ] [ ] [ ] [ ] . the hypothesized increased oxidative stress in dh ond pi cells compared to non-infected dh cells was further investigated by means of immunoreactivity for ohdg, sod and cat, as displayed in figure , as well as by determination of oxidative burst by flow cytometry. table s ). immunofluorescence for sod displayed a significantly (p = . ) increased percentage of positive cells in dh ond pi pellets (median = . %, range: . - . %) compared to non-infected dh pellets (median = . %, range: . %- . %) (supplementary table s ). immunofluorescence for cat revealed a significantly (p = . ) increased percentage of positive cells in dh ond pi pellets (median = immunofluorescence for ohdg lacked a significant difference (p = . ) in the percentage of positive cells between non-infected (median = . %, range: . - . %) and dh ond pi pellets (median = . %, range: . - . %) (supplementary table s ). immunofluorescence for sod displayed a significantly (p = . ) increased percentage of positive cells in dh ond pi pellets (median = . %, range: . - . %) compared to non-infected dh pellets (median = . %, range: . %- . %) (supplementary table s ). immunofluorescence for cat revealed a significantly (p = . ) increased percentage of positive cells in dh ond pi pellets (median = . %, range: . %- . %) compared to non-infected dh pellets (median = . %, range: . %- . %) (supplementary table s ). the determination of oxidative burst by flow cytometry demonstrated a significantly (p = . ) increased ros production among dh ond pi cells compared to non-infected dh cells (figure ). table s ). the determination of oxidative burst by flow cytometry demonstrated a significantly (p = . ) increased ros production among dh ond pi cells compared to non-infected dh cells (figure ). despite a lack of difference in ros-induced nucleic acid damage as determined by immunofluorescence of ohdg, these results are collectively indicative of an increased oxidative stress in dh ond pi cells compared to non-infected dh cells, which might lead to an increased level of hif- α and subsequently to an inhibition of its degradation. among the gene symbols referring to the functional group "hif- α activation, transcriptional activity and regulation", three out of genes were down-regulated (table ) . specifically, downregulated gene symbols were those referring to two (engl and engl ) out of three prolyl hydroxylases and to von hippel-lindau (vhl) protein, while hif- α gene symbol (hif a) did not show any significant change (supplementary table s ) . immunoreactivity for hif- α revealed a significant (p = . ) higher percentage of positive dh ond pi cells (median = . %, range . %- . %) (supplementary table s ) compared to non-infected dh cells (median = . %, range: . %- . %), as shown in figure . in non-infected dh cells, hif- α was mainly expressed within nucleus (median = . %, range: . %- . %) and cytoplasm (median = . %, range: . %- . %) and only to a lesser extent in the membrane (median: . %, range: . %- . %), without significant differences (p ranging from . to > . ) between the three localizations. interestingly, dh ond pi cells displayed a significantly higher hif- α expression in the membrane ( figure ) compared to nuclear (p = . ; membrane median = . %, membrane range: . %- . %; nuclear median = . %, nuclear range: . %- . %) but not to cytoplasmic localizations (p = . ; cytoplasm median = . %, cytoplasm range: . %- . %). additionally, the membranous immunopositivity for hif- α in dh ond pi cells was significantly (p = . ) higher when compared to the corresponding localization in non-infected despite a lack of difference in ros-induced nucleic acid damage as determined by immunofluorescence of ohdg, these results are collectively indicative of an increased oxidative stress in dh ond pi cells compared to non-infected dh cells, which might lead to an increased level of hif- α and subsequently to an inhibition of its degradation. among the gene symbols referring to the functional group "hif- α activation, transcriptional activity and regulation", three out of genes were down-regulated (table ) . specifically, down-regulated gene symbols were those referring to two (engl and engl ) out of three prolyl hydroxylases and to von hippel-lindau (vhl) protein, while hif- α gene symbol (hif a) did not show any significant change (supplementary table s ) . immunoreactivity for hif- α revealed a significant (p = . ) higher percentage of positive dh ond pi cells (median = . %, range . %- . %) (supplementary table s ) compared to non-infected dh cells (median = . %, range: . %- . %), as shown in figure . in non-infected dh cells, hif- α was mainly expressed within nucleus (median = . %, range: . %- . %) and cytoplasm (median = . %, range: . %- . %) and only to a lesser extent in the membrane (median: . %, range: . %- . %), without significant differences (p ranging from . to > . ) between the three localizations. interestingly, dh ond pi cells displayed a significantly higher hif- α expression in the membrane ( figure ) compared to nuclear (p = . ; membrane median = . %, membrane range: . %- . %; nuclear median = . %, nuclear range: . %- . %) but not to cytoplasmic localizations (p = . ; cytoplasm median = . %, cytoplasm range: . %- . %). additionally, the membranous immunopositivity for hif- α in dh ond pi cells was significantly (p = . ) higher when compared to the corresponding localization in non-infected dh cells (supplementary table s ). summarized, these results are indicative of an increased level of hif- α in dh ond pi, which is most likely due to a decreased cytoplasmic degradation. to further characterize the intracellular localization of hif- α, immunoelectron microscopy and laser scanning confocal microscopical within non-infected dh cells, hif- α was present within nucleus and cytoplasm without significant differences between the localizations. in contrast, persistently cdv-infected dh cells displayed a significantly higher membranous hif- α expression compared to nuclear (p = . ) but not to cytoplasmic (p = . ) localizations. additionally, the membranous immunopositivity for hif- α in persistently cdv-ond infected dh cells was significantly higher compared to the corresponding localization in non-infected controls. box and whisker plots display median and quartiles with maximum and minimum values. significant differences (p ≤ . , mann-whitney utest (c,d) and kruskall-wallis test with post-hoc dunn's test (d)) are labeled by asterisks (* p ≤ . and ** p ≤ . ). within non-infected dh cells, hif- α was present within nucleus and cytoplasm without significant differences between the localizations. in contrast, persistently cdv-infected dh cells displayed a significantly higher membranous hif- α expression compared to nuclear (p = . ) but not to cytoplasmic (p = . ) localizations. additionally, the membranous immunopositivity for hif- α in persistently cdv-ond infected dh cells was significantly higher compared to the corresponding localization in non-infected controls. box and whisker plots display median and quartiles with maximum and minimum values. significant differences (p ≤ . , mann-whitney u-test (c,d) and kruskall-wallis test with post-hoc dunn's test (d)) are labeled by asterisks (* p ≤ . and ** p ≤ . ). hif- α immunoblotting confirmed the significantly increased protein expression (p = . ) in dh ond pi cells when compared to the non-infected dh cells (figure ). ultrastructural investigation of dh ond pi by immunoelectron microscopy for hif- α revealed that this protein was mostly localized in the sub-membranous compartment as well as within variably sized, round, moderately to highly electrondense vesicles ( figure ). based on the assumptions that many viruses have been shown to induce an increased production of cd + exosomes [ ] , and that viral proteins can be stored within endolysosomal system [ ] , dl-if for hif- α in association with different markers was performed and evaluated by laser scanning confocal microscopy. summarized, these results are indicative of an increased level of hif- α in dh ond pi, which is most likely due to a decreased cytoplasmic degradation. to further characterize the intracellular localization of hif- α, immunoelectron microscopy and laser scanning confocal microscopical analysis of double stainings were performed. ultrastructural investigation of dh ond pi by immunoelectron microscopy for hif- α revealed that this protein was mostly localized in the sub-membranous compartment as well as within variably sized, round, moderately to highly electrondense vesicles ( figure ) . based on the assumptions that many viruses have been shown to induce an increased production of cd + exosomes [ ] , and that viral proteins can be stored within endolysosomal system [ ] , dl-if for hif- α in association with different markers was performed and evaluated by laser scanning confocal microscopy. to verify the specificity of the membranous staining, dl-if for hif- α in association with wga was performed, confirming a membranous to sub-membranous localization of hif- α without overlapping co-staining of the two markers (supplementary figure s ) . to investigate whether hif- α was associated with exosomes, dl-if in association with cd was performed, revealing an occasional co-localization of the two markers ( figure ). to exclude an hif- α storage within the golgi apparatus, dl-if in association with gm- was performed, clearly showing that hif- α was not localized within this cell organelle (supplementary figure s ) . finally, to analyze whether hif- α was associated with cdv-np, dl-if in association with cdv-np was performed, revealing a marked and diffuse co-localization of the two markers ( figure ). in summary, these results confirmed an unexpected localization of hif- α in the sub-membranous compartment of dh ond pi cells, being occasionally associated with cd + exosomes and more frequently with cdv-np. to investigate if this unusual localization of hif- α can affect the expression of its angiogenetic downstream molecules with a special focus on vegf-b, further microarray data and immunofluorescence analyses were performed. ultrastructural investigation of dh ond pi by immunoelectron microscopy for hif- α revealed that this protein was mostly localized in the sub-membranous compartment as well as within variably sized, round, moderately to highly electrondense vesicles ( figure ). based on the assumptions that many viruses have been shown to induce an increased production of cd + exosomes [ ] , and that viral proteins can be stored within endolysosomal system [ ] , dl-if for hif- α in association with different markers was performed and evaluated by laser scanning confocal microscopy. to verify the specificity of the membranous staining, dl-if for hif- α in association with wga was performed, confirming a membranous to sub-membranous localization of hif- α without overlapping co-staining of the two markers (supplementary figure s ) . to investigate whether hif- α was associated with exosomes, dl-if in association with cd was performed, revealing an occasional co-localization of the two markers ( figure ). the intracellular hif- α localization was analyzed by double immunofluorescence with hif- α (cy , green) and cd (cy , red) in persistently canine distemper virus (cdv)-infected dh cells. both proteins were localized within cell membranes and cytoplasm. interestingly, an occasional co-expression (yellow) was noted (arrows; insert) using scanning confocal laser microscopy. (b) a double labeling directed against hif- α (cy , red) and the cdv nucleoprotein (cdv-np; cy , green) revealed a frequent co-localization (yellow) beneath the cell membrane and within the perinuclear area (insert) of persistently cdv-infected dh cells. nuclei were stained with bisbenzimide (blue). bar = µ m. to exclude an hif- α storage within the golgi apparatus, dl-if in association with gm- was performed, clearly showing that hif- α was not localized within this cell organelle (supplementary figure s ) . finally, to analyze whether hif- α was associated with cdv-np, dl-if in association with cdv-np was performed, revealing a marked and diffuse co-localization of the two markers ( figure ). the intracellular hif- α localization was analyzed by double immunofluorescence with hif- α (cy , green) and cd (cy , red) in persistently canine distemper virus (cdv)-infected dh cells. both proteins were localized within cell membranes and cytoplasm. interestingly, an occasional co-expression (yellow) was noted (arrows; insert) using scanning confocal laser microscopy. (b) a double labeling directed against hif- α (cy , red) and the cdv nucleoprotein (cdv-np; cy , green) revealed a frequent co-localization (yellow) beneath the cell membrane and within the perinuclear area (insert) of persistently cdv-infected dh cells. nuclei were stained with bisbenzimide (blue). bar = µm. among the gene symbols referring to the functional group "hif- α angiogenic downstream molecules", six out of genes were up-regulated, whereas genes were down-regulated (table ) . specifically, down-regulated gene symbols included those related to the expression of angiogenetic and anti-angiogenetic macromolecules which transcription is directly induced by the activation of the hif- α downstream pathway (i.e. vascular endothelial growth factor b-vegfb; thrombospondin -thbs ; endothelin -edn /et ; serine peptidase inhibitor e-serpine ; thrombospondin -thbs ; chemokine ligand -cxcl ; cd -nt e; basic fibroblast growth factor -fgf , adrenomedullin-adm; cd ). immunofluorescence for vegf-b revealed a significantly (p = . ) decreased percentage of immunopositive cells in dh ond pi pellets (median = . %, range: . %- . %) (supplementary table s ) compared to non-infected dh pellets (median = . %, range: . %- . %), as shown in figure . table s ) compared to non-infected dh pellets (median = . %, range: taken together, these results are indicative of a reduced activation of the hif- α angiogenic downstream pathway. this is most likely due to an excessive, unusually localized, and nonfunctional protein expression of hif- α, which might be the consequence of a decrease in its cytoplasmic degradation following a virus-induced increased oxidative stress. canine histiocytic sarcoma cells (dh ) persistently infected with cdv-ond display a complete spontaneous tumor regression when xenotransplanted subcutaneously into scid mice [ ] . considered that dh ond pi cells did not show any difference in growth and apoptotic rate compared to non-infected controls in vitro and during the initial phase after transplantation in vivo [ , , ] , it was assumed that tumor regression of dh ond pi xenotransplants was not caused primarily by direct virus-induced cell death alone. indeed, it seems more likely that secondary effects of the viral infection on the tumor microenvironment [ , ] , as similarly reported for reoviruses [ ] , account for the complete regression. specifically, it was estimated that regression of dh ond pi xenotransplants might be related to alterations in cancer-associated angiogenesis [ ] . therefore, the aim of the present in vitro study was to investigate in more detail pathways potentially involved in this regression process, taking advantage of the absence of the confounding effects correlated with ongoing tumor cell death associated with acute cdv-ond infection [ ] . furthermore, to restrict the complex interactions that occur within a living organism, a less complex, highly standardized in vitro model is assumed to facilitate the analysis of specific intracellular pathways. interestingly, the socalled "angiogenic switch" has been reported to be one of the most important hallmarks of cancer [ , ] , thus playing a central role for tumor development and expansion. in this context, the present taken together, these results are indicative of a reduced activation of the hif- α angiogenic downstream pathway. this is most likely due to an excessive, unusually localized, and non-functional protein expression of hif- α, which might be the consequence of a decrease in its cytoplasmic degradation following a virus-induced increased oxidative stress. canine histiocytic sarcoma cells (dh ) persistently infected with cdv-ond display a complete spontaneous tumor regression when xenotransplanted subcutaneously into scid mice [ ] . considered that dh ond pi cells did not show any difference in growth and apoptotic rate compared to non-infected controls in vitro and during the initial phase after transplantation in vivo [ , , ] , it was assumed that tumor regression of dh ond pi xenotransplants was not caused primarily by direct virus-induced cell death alone. indeed, it seems more likely that secondary effects of the viral infection on the tumor microenvironment [ , ] , as similarly reported for reoviruses [ ] , account for the complete regression. specifically, it was estimated that regression of dh ond pi xenotransplants might be related to alterations in cancer-associated angiogenesis [ ] . therefore, the aim of the present in vitro study was to investigate in more detail pathways potentially involved in this regression process, taking advantage of the absence of the confounding effects correlated with ongoing tumor cell death associated with acute cdv-ond infection [ ] . furthermore, to restrict the complex interactions that occur within a living organism, a less complex, highly standardized in vitro model is assumed to facilitate the analysis of specific intracellular pathways. interestingly, the so-called "angiogenic switch" has been reported to be one of the most important hallmarks of cancer [ , ] , thus playing a central role for tumor development and expansion. in this context, the present study focused on pathways correlated with increased levels of intracellular ros. these highly reactive molecules have been reported both as fundamental intermediates in physiological intracellular signaling transduction [ , ] , as well as in the regulation of different cancer hallmarks [ , , ] . specifically, together with hypoxia, ros represent one of the major activators of hif- α [ , , , , ] , a transcription factor involved in the regulation of a wide plethora of cancer features such as invasion, metastasis, and angiogenesis [ , [ ] [ ] [ ] [ ] ] . in the context of the aforementioned considerations, the present study was further directed to investigate the impact of a persistent cdv-ond infection of dh cells on cellular oxidative stress. cdv has been reported as being able to trigger an increase in ros intracellular levels, with the subsequent induction of oxidative stress in different kinds of cells such as microglia, in vitro as well as in vivo [ ] [ ] [ ] [ ] [ ] . similarly, the present study revealed increased ros levels in dh ond pi cells, as demonstrated by an increased oxidative burst, as well as suggested by increased gene transcription of txnip and ncf . specifically, the upregulation of both genes might correlate with an increased intracellular oxidative stress. indeed, ncf encodes for p phox , a protein that is involved in nadph oxidase activation [ , , ] . additionally, thioredoxin-binding protein , encoded by the txnip gene, is an important inhibitor of the thioredoxin ros scavenging system [ , ] . on the other hand, ros-induced nucleic acid damage did not differ in dh ond pi cells compared to non-infected controls. this observation might be interpreted as indicative of an increased oxidative stress associated with the neoplastic nature of dh cells rather than an effect of the viral infection. similarly, increased intracellular ros levels are described in the literature as a common feature of cancer cells [ , , ] . in addition, dh ond pi cells displayed an increased expression of sod and cat compared to non-infected controls. the overexpression of these scavenging enzymes involved in ros detoxification have been correlated with an increased oxidative stress in neoplastic [ , , ] as well as in inflammatory conditions [ ] . the results obtained by microarray analysis of genes correlated with er stress [ , , , , ] are consistent with a reduced transcription of genes correlated with this process. the data in the present study might be interpreted as suggestive of an acquired ability of dh cells to adapt to the persistent infection with cdv-ond. however, a marked protein overexpression of er-stress markers such as calnexin, calreticulin and chop/gadd have been observed in vero cell and primary rat neurons h post-infection with recombinant a / -v cdv [ ] . on the other hand, the aforementioned lack of differences in growth and apoptotic rate between non infected and dh ond pi cells [ , ] is in line with the hypothesis that a persistent infection with cdv-ond might be associated with the activation of adaptive and pro-survival pathways to contrast prolonged oxidative stress, as reported in recombinant hela cells expressing silkworm storage protein [ ] . the hypothesis of the present study is further supported by the finding of an increased expression of ros-scavenging enzymes in dh ond pi cells at both a molecular and protein level, highlighting the plasticity of cancer cells in actively contrasting excessively severe alterations in their redox potential [ , ] . the expression of hif- α was subsequently investigated due to the observation that increased oxidative stress is associated with an increased hif- α stabilization and activation [ , , , , ] . hypoxia has been widely reported as the most powerful inductor of hif- α transcriptional activity [ , , ] ; however, in the present study, cells were cultivated under normoxic conditions. therefore, hypoxia could be excluded as the cause of the increased hif- α protein expression observed in our in vitro model. consequently, it seems more plausible that the increased expression of hif- α in dh ond pi cells was induced by the increased oxidative stress level compared to non-infected controls. the down-regulation of phds as well as of vhl on a molecular level, in association with a lacking regulation of hif- α opposed to an increased expression of the corresponding protein, could imply that the increased protein expression of hif- α in dh ond pi cells does not refer to an increased synthesis, but rather to an inhibition of the degradation pathway. correspondingly, ros have been reported to be directly involved in the inhibition of the aforementioned cytoplasmic enzymes (i.e., phds and vhl) responsible for hif- α hydroxylation and ubiquitination which prelude the rapid degradation of hif- α itself by the proteasome s [ , , ] . in addition to the overall increased expression of hif- α, the present study revealed an unusual localization of the transcription factor in the sub-membranous compartment and, to a lesser extent, within cytosolic vesicles. further investigations aiming to better characterize the aforementioned vesicles, revealed a co-localization of hif- α expression with cd , a marker for the tetraspanin- expressed by exosomal membranes [ ] . interestingly, the presence of hif- α within cd + exosomes has previously been reported in epstein-barr virus-infected np cells [ ] . on the other hand, hif- α only occasionally co-localized with cd + exosomes, while it frequently overlapped with the localization of cdv-np. the measles virus n-protein, which is closely related to cdv-np [ ] , is transported within the cell through the endolysosomal system [ ] , also rendering this a possible mechanism for the canine counterpart. furthermore, this observation displays an interesting basis for future investigations on the exact sub-cellular localization of hif- α within dh ond pi cells. microarray data analysis aiming to investigate the molecular consequences of the unusual localization of hif- α and a prospective loss of function of its transcriptional activity, revealed a significant down-regulation of different genes involved in the hif- α angiogenic downstream pathway, which was further substantiated by a significantly reduced expression of vegf-b on a molecular and protein level. though vegf-b is nowadays recognized as not being directly involved in angiogenesis, this growth factor has been reported as an indirect enhancer of vegf-a (a well-known inducer of angiogenesis), as well as a key promoter of survival of different cell types (including endothelial cells, pericytes and smooth muscle cells) in several pathological conditions [ ] [ ] [ ] . as already reported in the literature [ ] , the markedly reduced expression of vegf-b in dh ond pi cells did not affect cellular growth nor the apoptotic rate [ ] . interestingly, dh ond pi cell xenotransplants displayed a significantly reduced microvessel density compared to non-infected controls [ ] . according to the results of the present study, it can be assumed that hif- α might represent an important mediator of the oncolytic effects described for the in vivo model of dh ond pi xenotransplants as reported previously in another viral oncolysis model [ ] . summarized, the results of the current in vitro study are indicative of a reduced activation of the hif- α angiogenic downstream pathway in dh cells persistently infected with cdv-ond compared to non-infected controls. this is most likely due to an excessive, unusually localized, and non-functional expression of hif- α, which might be the consequence of a decreased cytosolic degradation of this transcriptional factor following a virus-induced increased oxidative stress. future studies are warranted to better characterize the localization of hif- α and the exosomes in which it is contained, as well as to verify the presence of an increased oxidative stress and an aberrant hif- α localization in dh ond pi also in vivo. the latter approach might further substantiate the assumed correlation between reduced angiogenesis, hypoxia and tumor regression in dh ond pi xenotransplants. bar = µm, supplementary table s . summary of statistical analyses depicting median and mean percentage of immunopositive cells for each cell population (i.e. non-infected and dh ond pi cells) or for each specific intracellular localization (i.e., membrane, cytoplasm, or nucleus), with the corresponding minimum-maximum range and standard deviation for each marker investigated. the normality of distribution of each data set as well as the p-value of multiple and/or pairwise comparisons between the groups are also reported kruskall-wallis test, min-max, minimum-maximum range 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extracellular vesicles and update of the misev guidelines antigenic relationships between measles and canine distemper viruses: comparison of immune response in animals and humans to individual virus-specific polypeptides vascular endothelial growth factor-b in physiology and disease a survival, or an angiogenic factor? newcastle disease virus degrades hif- α through proteasomal pathways independent of vhl and p the authors are grateful to silke akhdar, julia baskas, petra grünig, kerstin rohn, caroline schütz, and danuta waschke for the excellent technical support. the authors want to thank also ingo gerhauser, ph.d., for his precious support with the statistical analysis. federico armando received financial support by the university of parma, parma, italy. this publication was supported by deutsche forschungsgemeinschaft and university of veterinary medicine hannover, foundation within the funding programme open access publishing. the authors declare no conflict of interest. key: cord- -cbutpjre authors: seetahal, janine f. r.; greenberg, lauren; satheshkumar, panayampalli subbian; sanchez-vazquez, manuel j.; legall, george; singh, shamjeet; ramkissoon, vernie; schountz, tony; munster, vincent; oura, christopher a. l.; carrington, christine v. f. title: the serological prevalence of rabies virus-neutralizing antibodies in the bat population on the caribbean island of trinidad date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: cbutpjre rabies virus (rabv) is the only lyssavirus known to be present within the caribbean. the island of trinidad, is richly diverse in chiropteran fauna and endemic for bat-transmitted rabies with low rabv isolation rates observed in this population. we aimed to determine the seroprevalence of rabies virus neutralizing antibodies (rvna) in light of spatio-temporal and bat demographic factors to infer the extent of natural exposure to rabv in the trinidadian bat population. rvna titers were determined by the rabv micro-neutralization test on bat samples representing species, comprising . % of local bat diversity, from locations across the island over years. rvna was positively detected in samples ( . %) representing bat species (mainly frugivorous) with titers ranging from . to iu/ml (mean . iu/ml). the analyses based on a multivariable binomial generalised linear mixed-effects model showed that bat age and year of capture were significant predictors of seropositivity. thus, juvenile bats were more likely to be seropositive when compared to adults (estimate . ; p = . ) which may suggest early exposure to the rabv with possible implications for viral amplification in this population. temporal variation in rabies seropositivity, – versus – (estimate . ; p = . ) may have been related to the prevailing rabies epizootic situation. regarding other factors investigated, rvna was found in bats from both rural and non-rural areas, as well as in both hematophagous and non-hematophagous bat species. the most common seropositive species, artibeus jamaicensis planirostris is ubiquitous throughout the island which may potentially facilitate human exposure. the findings of this study should be factored into public health assessments on the potential for rabies transmission by non-hematophagous bats in trinidad. rabies is a highly fatal but preventable zoonotic disease of major public health significance [ , ] . the causative agent rabies virus (rabv) is the type species and most ubiquitous of the lyssavirus genus [ ] . it is the only lyssavirus known to circulate in the americas [ ] . while, the major global burden of rabies is attributed to dog-mediated transmission [ ] , sylvatic-transmission is becoming increasing important in the epidemiology of rabies [ ] [ ] [ ] [ ] . this is particularly relevant in the americas with the decline of canine-transmitted cases [ ] [ ] [ ] and the recognition of distinct rabv variants in numerous bat species [ , , ] . due to the aerial nature of their reservoir, these variants are more defined by species than by geographical boundaries [ ] and in the americas, nearly distinct bat rabv variants have been found thus far [ ] . although these variants can be transmitted between bat species and to other mammals [ , ] , in latin america and the caribbean the vampire bat is the bat species most implicated as a reservoir in this region [ ] . the caribbean island of trinidad, located just km away from the northeast coast of south america, is richly diverse in chiropteran fauna with identified species, including two species of hematophagous bats [ ] . the island is enzootic for bat rabv which has been so far isolated from nine bat species [ , ] . of these, the hematophagous desmodus rotundus species is considered the most effective vector on account of its feeding practices [ ] and to date, is the only bat directly implicated in transmission of the virus on the island [ ] . some earlier unconfirmed studies have suggested that apparently healthy bats could harbour and transmit rabv for extended periods [ ] [ ] [ ] [ ] , however to date this has not been conclusively proven by modern diagnostic methods. in trinidad viral isolation from bats has been rare with a rabies positivity proportion of . % (two positive of tested between and ) obtained from samples acquired mainly through active surveillance in the bat population [ ] . despite the bias towards healthy bats inherent to this type of sampling, it has been suggested that this low proportion is a consequence of virus only being periodically imported from the south american mainland (as bats fly from mainland to island) causing vampire bat epizootics with occasional viral spill-over to the livestock population [ ] . nonetheless, over the last years, despite the apparent low levels of rabv circulation in the bat population, five significant epizootic events have occurred on the island [ ] . in light of the apparently low prevalence of virus among bats, rabies antibody levels may be used as an indicator of virus exposure to gauge the risk of virus transmission. few studies on rabies antibody prevalence have been conducted within the caribbean [ ] [ ] [ ] [ ] and the only report from trinidad (conducted in during a small epizootic event) demonstrated a seropositive proportion of . % [ ] . we therefore sought to determine the current seroprevalence of rabies virus neutralizing antibodies (rvna) in the trinidadian bat population over a period of five years in order to infer the extent of natural exposure to rabvs and the spatio-temporal dynamics of rabv infection in the bat population. we also aimed to determine whether seroprevalence varied with factors related to bat demographics and habitat, with a view to identifying potential risk factors for transmission to susceptible animal and human populations. bats were caught mainly using mist nets set at ground level at dusk and night from february to april on the island of trinidad (see table s ). bat trapping and specimen collection were carried out under special game licenses issued annually by the wildlife section, forestry division, ministry of agriculture, land and fisheries, trinidad and tobago in accordance with the government of trinidad and tobago conservation of wildlife act ( ) chapter : (section ). field and laboratory protocols were approved by the ethics committee, faculty of medical sciences, university of the west indies, st. augustine campus ( th february ). bats were transported to the laboratory in individual mesh bags for processing. all bats were apparently healthy at the time of sampling and were sampled only once. biometrics (i.e., forearm length, weight and head and body length) and the appearance of other external characteristics (e.g., tail, chin warts and nose leaf) were recorded for each animal to aid in their morphological species identification using locally developed field identification keys [ ] . juvenile bats were distinguished from adult bats by the lack of ossification in the epiphyseal joint between the metacarpal and proximal phalanx bones demonstrated upon trans-illumination of the wing. bats were humanely euthanized by anaesthetic overdose using isoflurane and organ tissues were collected by dissection and stored in-house for other parallel studies and future studies including species identification using genetic methods. blood was sampled by cardiac puncture and sera was separated by centrifugation and stored at − • c until further processing. the micro-neutralization test was developed to test smaller volumes of test serum making it an ideal comparison to the traditional rapid fluorescent focus inhibition test (rffit) [ ] . the microneutralization test was conducted following the protocol outlined in smith et al. [ ] using the cvs- rabv variant. all serum samples were screened at : dilution and positive sera were run to end-point. a cut off titer of : ( . iu/ml) was chosen based on the value for % neutralization of the challenge virus in accordance with previous studies [ , ] . the procedure for performing the micro-neutralization test involves using four-well teflon coated slides to perform serial dilutions while employing a humidity chamber throughout the procedure to avoid any evaporation. minimum essential medium (mem) ( µl) was added to each well of each slide and µl of each test serum was serially diluted. standard rabies immunoglobulin (srig) was used as positive control serum in preparing the positive control slide [ ] . cvs- was prepared for the working dilution at ffd ( % fluorescent focus forming doses) per ml and µl of this working dilution of virus, was added to each well of each test slides as well as to the positive control slide and the back-titration slide. the slides were incubated in the humidity chamber at • c with . % co for min. after completion of this incubation period, µl of mouse neuroblastoma cells (mna) were added to each well, equivalent to . × cells per well. slides were again incubated at • c for h with . % co then fixed with acetone and stained with fitc-anti-rabies immunoglobulin (fujirebio diagnostics, malvern, pa, usa) before being observed for the presence of fluorescent foci with a fluorescence microscope. the reed-muench method [ ] was used to calculate the endpoint titer, which was converted to international units per millilitre (iu/ml) based on comparison to srig diluted at iu/ml. rvna titer values were recorded originally as a continuous variable and they were further categorized as a binary variable, either as positive (≥ . iu/ml) or negative (< . iu/ml) serological status. the variables investigated in the study were related to the bat captured, i.e., sex, age and dietary habits; or to external factors such as season of capture (dry vs wet season; i.e., january-may vs june-december), year of capture, urbanization level at capture location, and district of capture. this investigation aimed to study the factors associated with the rabies serological status of the captured bats. the response variable in the analyses was positive (≥ . iu/ml) or negative (< . iu/ml) serological status according to the rvna titer obtained. initially, the analysis comprised univariable explorations to investigate the associations between the binary status (i.e., positive or negative) of each bat and the different factors investigated in the study, utilizing a binomial generalized linear model (glm). the proportion of seropositive bats and the % confidence intervals (ci) were also computed. the variables significantly associated (p < . ) in the univariable model were included in a multivariable binomial glm and a backward elimination process was followed to build the final model. then, the model evolved, allowing for random effects at the county level, utilizing a binomial generalised linear mixed-effects model (glmm). this approach allowed accounting for the potential over-dispersion present in our data due to clustering at the level of county. the goodness of fit measurement akaike's information criterion (aic), was used for comparison between nested models. additionally, substantial changes in the estimated coefficients in the models and increases in standard errors during the model building process were investigated. the wald tests were used to examine and to present the significance (p value < . ) of the variables retained in the final model. all the analyses and graphs were performed using the minitab (version ) [ ] and r statistical software environment [ ] using the libraries stats, epicalc and lme . in this study, sera samples from bats in trinidad were collected for rvna testing indicative of exposure to rabv. of these, samples representing geographical locations on the island (see figure and table s ) were of suitable quality and quantity for determination of rvna titers. twenty-one ( ) species of bats representing four of the nine families known to be present on the island, were tested from these locations. the most common species in the sample set was the desmodus rotundus (n = ; . %). after this artibeus jamaicensis planirostris (n = ; . %), carollia perspicillata (n = ; . %) followed by molossus molossus (n = ; . %) bats made up the majority of tested samples. rvna was positively detected in the sera of bats representing . % of the sample population ( % ci . , . ) with seropositive titers ranging from . to iu/ml (mean . iu/ml; sd ± . ), as illustrated in table s . bats from all counties except county nariva/mayaro tested positive for rabies antibodies. as detailed in table table s ). six species of bats accounted for the seropositive samples (table s ) and were distributed as follows: sixteen ( . %) from the species a. jamicensis planirostris, six ( . %) each from artibeus lituratus and d. rotundus, three ( . %) from c. perspicillata and one ( . %) each from phyllostomus hastatus and glossophaga soricina. most of the seropositive samples (n = ) were from frugivorous ( of ; . %) and hematophagous ( of ; . %) bat species ( figure s ). lower numbers of bats with mixed dietary preference and nectarivores were positive when compared to frugivorous and hematophagous species and no insectivorous bats were seropositive for rvna. non-hematophagous bats (n = ) demonstrated a seropositivity proportion of . % ( % ci ( . - . ); of ) compared to . % ( % ci ( . - . ); of ) for hematophagous bats (n = ), as illustrated in table . however, there was no significant association between bat diet and seropositivity for rabies antibody (p > . ) according to the results of the univariable glm. overall from the entire test population (n = ), more female bats (n = ) were tested than males (n = ). females accounted for . % ( of ) of all positives bats (n = ) as opposed to . % ( of ) for males ( figure s ). the seropositivity proportion for males was . % ( % ci ( . , . )) versus . % ( % ci ( . , . )) for females. however, there was no significant association between sex and seropositivity for rabies antibody (p-value > . ) according to the results of the univariable glm. although juvenile bats comprised only % ( of ) of the test population (n = ) they accounted for . % ( of ) of all seropositive samples (see figure s ). their seropositivity proportion was . % ( % ci ( . , . )) significantly higher (p ≤ . ) than the . % ( % ci ( . , . )) noted in adult bats. likewise, there was a statistically significant difference between the mean rvna titer status in juveniles compared to adults, with juveniles having greater risk (estimate . ; p < . ) according to the results of the univariable glm). individual seropositivity status for artibeus juvenile and dam pairs were not consistently similar. for example, in one instance, both the juvenile (t ) and dam (t ) a. jamaicensis planirostris were rvna positive with identical titer values of . iu/ml, whereas another juvenile bat (t ) of the same species with a similar titer value was associated with a seronegative dam (t ). this dam (t ) was also associated with two other pups (t and t ) both of which were seronegative. for the remaining juvenile bats, the dam-pup association was only established for one seronegative adult and juvenile a. lituratus pair, which were t and t respectively. all other juveniles were not affiliated with a dam. about two-thirds of the samples tested ( of ) were from bats sampled during the dry season and these accounted for . % ( of ) of rvna positive samples ( figure s ). the prevalence of rvna positive samples during the dry season ( . %; % ci ( . , . ); of ) was similar with that for the wet season ( . %; % ci ( . , . ); of ) with no significant difference in the seropositivity between the seasons (p > . ) according to the results of the univariable glm. the majority of tested samples ( . %; of ) were from bats trapped in rural areas where there were low human population densities (figure ). the prevalence of rvna positive samples in these areas was . % ( % ci ( . , . ); of ) compared to . % ( % ci ( . , . ); of ) in non-rural areas. seropositive bats from non-rural areas were sampled from both residential and non-residential areas, with % ( % ci ( . - . )) of the seropositive samples from non-rural areas sampled at non-residential locations. the association between seropositivity and urbanization level at the location of capture was statistically significant, with urban areas showing a greater risk in comparison to rural areas (estimate . , p < . ) according to the glm. seropositive bats were mainly from two counties, st. george east and st. patrick, with seropositivity rates of . % ( % ci ( . , . )) and . % ( % ci ( . , . )) respectively ( figure s ). other districts had lower seropositivity rates ( . - . %) and county nariva/mayaro had no positives samples (see table ). the most seropositive samples per sampled district originated in champs fleurs (table s ) . however, there was no significant difference in the seropositivity across the counties (p-value > . ) according to the results of the univariable glm. the seropositivity of bats captured by roost extraction was . % ( % ci ( . , . ); of ) versus . % ( % ci ( . , . ); of ) for those captured in the field at the feeding grounds but this difference was not found to be significant (p > . ) in the glm. the results of the final multivariable glmm retained two variables as significantly associated with the rabies serological status (see table ). regarding the bat age, the juveniles appeared to have a greater risk that adults and the samples taken during the first period of sampling ( - ) also showed a greater risk than those sampled within the second period ( - ). in comparison to viral investigative studies for rabv in bats, there have been limited investigations into the serological prevalence of rabies antibodies in bat populations. in latin america and the caribbean, the previous serological studies revealed significantly variable rates for the prevalence of rabies antibodies in bats [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] which may be attributed to differences in viral population dynamics influenced by spatio-temporal and bat demographic factors as discussed below. in an earlier trinidadian rabies seroprevalence study [ ] , only four of bat species documented on the island (i.e., . % of local bat species diversity) [ ] were tested, with only the artibeus species found to be seropositive, compared to . % ( species) in the present study. in line with studies that show a correlation between the number of bat species from which rabv has been isolated and research effort [ ] , we found six seropositive bat species and by extension more seropositive bat species and possible isolations of rabv can be expected with more extensive studies covering more bat species. in trinidad, all except phyllostomus hastatus have previously been reported to be rabv positive [ ] , while this species was found to be rabies positive in south america [ ] . it is important to note that bat taxonomic identification on the basis of morphological characteristics does not distinguish cryptic species so future work will include genetic characterization to clarify species designations. comparison of the seropositivity rate observed in the current study ( . % for the period - ) with the rate of . % reported in the study [ ] suggests that there is temporal variation in rabies seropositivity in trinidad. rabies serological studies conducted several years apart in french guiana and grenada, also showed proportion variations over time, with . % and % respectively in earlier years and . % and . % more recently [ ] [ ] [ ] [ ] . in our study, year of capture was statistically significantly associated to serological status with, a bat sampled in - having a greater risk of being seropositive than one sampled during - . in terms of the rabies epizootic situation at these time, there is evidence of viral exposure during both periods. in , the year in which % ( of ) of rvna positive bat samples in our study were sampled (see table s ), one vampire bat (n = ) was confirmed to be rabies positive [ , ] . on the other hand, in , although no bats were found to be rabid from the small number tested (n = ), bat-transmitted rabies cases were diagnosed in the livestock population clearly indicating virus was circulating in at least the vampire bat population [ ] . although not reported herein, brain samples from bats specimens sampled during the period of this study were tested for rabv and other lyssaviruses by real-time rt-pcr [ ] , however none were positive for rabv [ ] . the majority of seropositive samples in this study were < . iu/ml, consistent with natural primary exposure [ ] . some studies have shown that rabies antibody levels decline to undetectable levels between and months post-primary exposure, and up to one year after secondary exposure [ , ] . similar waning of passive immunity is thought to occur in juvenile bats [ ] . so seropositive bats found in this study may have been naturally exposed to rabv up to a year prior to capture which would mean that rabv circulated in the trinidadian bat population at the very least during the period to . only % of seropositive bats had rvna titers higher than . iu/ml, which could be attributed to immunological priming from past exposures with subsequent anamnestic responses [ , ] . so relatively high rabies antibody titers may occur without active viral infection, because of pre-existing acquired immunity from past exposures [ , ] . this phenomenon may also explain the absence of mass mortalities among bats during epizootic events. longitudinal temporal monitoring of vampire roosts in peru and french guiana [ , ] have provided insights into non-lethal rabv infection by demonstrating seroconversion with highly fluctuating individual seroprevalence rates over time, which perhaps reflects viral persistence in the roost with periodic reactivation. on the contrary, rather than single colony perpetuation of rabv, one study suggests enzootic viral persistence may occur due to movement of infectious bats among colonies, with a high frequency of immunizing non-lethal exposures [ ] . assessment of natural variations in antibody levels for individual bats can be further investigated by capture (mark) and release studies with bat recapture and successive sampling over a period of time to identify changes in rvna titer levels and determine the maintenance of immunity. this study demonstrates variation in rabies seroprevalence by district in trinidad, with st. george east and st. patrick accounting for % of all rvna positive samples, consistent with localisation of rabies livestock epizootics in these regions [ , ] . however, more structured sampling across the island, which would account for sample site variation, is necessary to confirm the observed pattern. differences between regions may be related to the influence of bat population densities on viral dynamics. in theory, large bat roosts provide ideal conditions for viral spread amongst roost mates resulting in larger numbers of bats exposed to higher amounts of virus and thus higher rates of seroconversion [ ] . nevertheless, streicker et al. [ ] found limited evidence for a relationship between vampire bat colony size and exposure to rabv. affected bats may also be more likely to forgo normal foraging behaviour in preference to staying in roost [ ] . although our results showed no relationship between seropositivity and roost versus field capture, more targeted sampling may reveal underlying associations. the fruit bat, a. jamaicensis planirostris was the species most commonly found to be seropositive on the island. this species is highly adaptable and is widespread throughout the island roosting in crevices of both homes and non-residential buildings [ ] and therefore has a high potential for human contact. in light of this, public health officials and wildlife biologists should collaborate to address potential risks for human virus exposure with minimum ecological disruption. in general, frugivorous bats had the highest rvna seroprevalence for trinidadian bats, which is similar to the situation in grenada [ , ] . as with other studies, [ , ] rvna levels varied among bat species (see table s ) within the same area, suggesting variable rabies exposure and infection dynamics. the wide range of rvna titer values observed for the hematophagous species (see table ) may suggest greater natural exposure to rabv, which may more likely result in abortive infections when compared with other species [ , ] . in the caribbean (including trinidad) despite isolation of rabv from bat species, only the desmodus viral variant has thus far been definitely identified [ ] . however, the prevalence of rvna in non-haematophagous bats (particularly artibeus species), in both past and present studies may suggest the presence of other rabies variants or may indicate transmission of the desmodus variant to these species during roost co-habitation [ ] . along these lines, and similar to the situation in other areas [ ] non-hematophagous bats may play a significant role in rabv transmission within the caribbean. the multivariable glmm analysis found that juvenile bats were more likely to be seropositive than adults. this is consistent with previous studies which suggest that bats may be exposed to rabv soon after birth [ ] , and that virus amplification within the susceptible young population can facilitate an increase in rvna seroprevalence after a birth pulse, through active immunological responses [ , ] . this in turn can facilitate long-term maintenance of the virus [ ] . the role of pre-natal exposure is debatable, as contradictory evidence has thus far been provided for in-utero rabv transfer [ , ] . alternatively, passive transfer of maternal antibodies may have resulted in seropositive juvenile bats, which may not have been naturally exposed to the virus [ ] . this may have been the case with the seropositive juvenile and dam pair in this study (t and t ). although, all attempts were made to maintain appropriate identification records for dam-offspring pairs by observational analysis of dam-pup attachment and nursing prior to and after bat capture, alloparental nursing cooperative behaviour [ , ] cannot be ruled out as an explanation for the seropositive juvenile bat paired with a seronegative dam in this study (t and t ). genetic maternity analysis, to confirm the maternal relationships between these adult-juvenile pairs [ ] , was beyond the scope of the current study, but should be employed in future studies into the relationship between maternal and juvenile rvna titers. in another study where age was noted to be a significant predictor of rvna, higher seropositivity rates in juvenile bats compared to adults were thought to reflect active infection rather than the presence of maternally derived antibodies, as titers were much higher in juveniles than the adult females and early antibody decline consistent with passive immunity was not observed [ ] . single timepoint sampling employed in the current study did not allow for assessment of antibody decline, but titer levels were noted to be comparable between the seropositive dam-pup pair. twelve out of the ( %) seropositive bats identified in this study were from one maternity roost. this finding supports the evidence for a higher viral prevalence in pregnant and lactating female bats [ , ] . the estimated prevalence of rabv in wild bat populations within endemic regions is typically < % [ , ] . over the last century, since the isolation of rabv in trinidadian bats, the rabv prevalence proportion in this population, mainly from active surveillance, has gradually decreased from . % ( s) to . % ( - s) to the most recent estimate of . % [ , [ ] [ ] [ ] . this apparent decline may reflect lower levels of viral circulation in the bat population and may be responsible for the lower seroprevalence proportion demonstrated in this contemporary study ( . %), as compared the previous study conducted in the s ( . %) [ ] . conversely, studies conducted in south america demonstrate an inverse relationship between rabv prevalence and rvna seroprevalence [ , , ] . this may be attributed to the protective effect of herd immunity and death of virus-infected individuals, with the virus less able to establish infection and sustain bat to bat transmission due to increasing numbers of immune individuals. hence it has been suggested that rabv epizootics in bat populations are unidirectionally migratory with a periodicity of at least four years between events, which allows the population to regenerate a threshold of susceptible individuals [ ] . in trinidad, bat surveillance has traditionally targeted hematophagous bats with desmodus species comprising the vast majority of tested bats [ , ] . however, the prevalence of rvna in non-hematophagous bats found in this study suggests an expansion of rabies diagnostic testing to non-hematophagous species may be warranted to obtain a comprehensive picture of rabies host diversity and viral dynamics and ultimately determine the risk of transmission [ ] . in general the trinidadian population is well aware of the risks associated with hematophagous bats due to the history of rabies on the island [ ] . however, the potential for rabies transmission by non-hematophagous bats may be overlooked and persons may not seek medical care after exposures to these bats. although the potential risk for exposure to an actively infected rabv bat is relatively low, our results indicate that the virus can be associated with non-hematophagous bats so adequate precautions must be taken when handling all bats and appropriate post exposure prophylaxis is recommended in accordance with international guidelines regardless of the species of bat [ ] . rabies serological rates from trinidad and other countries in which rabies is enzootic [ ] [ ] [ ] were comparable to those found in grenada [ , ] , which is surprising because despite the single historical isolation of rabv from an artibeus bat [ ] , the virus is not known to be enzootic in the grenadian bat population. this along with the high rvna prevalence among the artibeus bats [ , ] , which are known to have long distance flight ranges over open water [ ] may imply translocation of the virus from endemic areas such as trinidad by bat movement. taken together with the ubiquity of the artibeus species throughout the caribbean [ ] the presence or introduction of rabv into other caribbean islands previously thought to be free of bat rabies is plausible. this has direct implications for the island of tobago which is only km from trinidad, compared to km from trinidad to grenada. in general, the lack of rabid clinical syndromes in bats may not necessarily reflect the spatial range of lyssaviruses due to exposures resulting in seroconversion rather than overt disease or latent infection [ , ] . reports of serological evidence of other lyssaviruses in the old world within areas previously thought to be 'free' of these viruses [ , ] have highlighted the value of serological surveillance to determine public health risk. consequently, serological surveys for anti-rabv antibodies in bat populations within the caribbean thought to be historically "rabies-free", especially those islands closer to the american continent, may be worthwhile to establish the true geographical range of rabv in the americas. such wide scale investigations might consider prioritizing species demonstrated to have flight ranges over large distances, particularly over open water such as artibeus jamaicensis [ ] and tadarida brasilensis [ ] species. with respect to sampling effort, surveillance for antibodies may be more efficient than viral detection in the long-term, however, the presence of these antibodies do not necessarily demonstrate active infection, but reflects past exposure to a phylogroup i lyssavirus [ ] . therefore viral isolation and typing would be necessary to confirm the lyssavirus circulating in these bat populations. similarly in trinidad, although no other lyssavirus has been detected here or 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population struture of a widespread bat (tadarida brasiliensis) in an island system evidence of two lyssavirus phylogroups with distinct pathogenicity and immunogenicity this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to thank the anti-rabies unit of the ministry of agriculture, land and fisheries and the trinidad and tobago bat conservation and research unit for their support in the field. we appreciate the assistance of jasmin camacho with field sample collection and we are grateful to ron mahabir for his assistance with the production of the map for this study. we also acknowledge the department of geomatics, engineering and land management, university of the west indies, st. augustine campus and the geographic information systems unit, ministry of agriculture, land and fisheries for the provision of digital data layers for the map. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -x foqu authors: glanz, anna; chawla, karan; fabry, stephanie; subramanian, gayatri; garcia, julie; jay, bryanna; ciricillo, jacob; chakravarti, ritu; taylor, r. travis; chattopadhyay, saurabh title: high throughput screening of fda-approved drug library reveals the compounds that promote irf -mediated pro-apoptotic pathway inhibit virus replication date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: x foqu interferon (ifn) regulatory factor (irf ) is the key transcription factor for the induction of ifn and antiviral genes. the absence of antiviral genes in irf deficiency leads to susceptibility to a wide range of viral infections. previously, we uncovered a function for nontranscriptional irf (nt-irf ), rlr (rig-i-like receptor)-induced irf -mediated pathway of apoptosis (ripa), which triggers apoptotic killing of virus-infected cells. using knock-in mice expressing a transcriptionally inactive, but ripa-active, irf mutant, we demonstrated the relative contribution of ripa to host antiviral defense. given that ripa is a cellular antiviral pathway, we hypothesized that small molecules that promote ripa in virus-infected cells would act as antiviral agents. to test this, we conducted a high throughput screen of a library of fda-approved drugs to identify novel ripa activators. our screen identified doxorubicin as a potent ripa-activating agent. in support of our hypothesis, doxorubicin inhibited the replication of vesicular stomatitis virus, a model rhabdovirus, and its antiviral activity depended on its ability to activate irf in ripa. surprisingly, doxorubicin inhibited the transcriptional activity of irf . the antiviral activity of doxorubicin was expanded to flavivirus and herpesvirus that also activate irf . mechanistically, doxorubicin promoted ripa by activating the extracellular signal-regulated kinase (erk) signaling pathway. finally, we validated these results using another ripa-activating compound, pyrvinium pamoate, which showed a similar antiviral effect without affecting the transcriptional activity of irf . therefore, we demonstrate that the ripa branch of irf can be targeted therapeutically to prevent virus infection. the innate immune response is the first line of defense against microbial infection. the interferon (ifn) system represents a key antiviral innate immune response mechanism that dictates the outcome of a viral infection [ ] . ifn-β, a type-i ifn, is synthesized in the virus-infected cells by the transcriptional activity of ifn regulatory factor (irf ) [ , ] . irf remains as an inactive monomer in the cytosol of the uninfected cells; upon virus infection, it gets phosphorylated, dimerized, and translocated to the nucleus [ , ] . in the nucleus, the dimeric irf binds to the promoters of ifn-β and many ifn-stimulated genes (isgs). secreted ifns act via autocrine and paracrine signaling to amplify the transcriptional induction of hundreds of isgs [ ] . the isg-encoded protein products act on specific stages of the virus life cycle to inhibit viral replication and pathogenesis. the absence of antiviral genes in irf −/− mice causes high susceptibility to a wide range of viruses [ , ] . we have uncovered that, in addition to the transcriptional activity, irf functions in a nontranscriptional (nt) pathway in antiviral defense [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in contrast to the transcriptional pathway, nt-irf in virus-infected cells functions as a chaperone protein by translocating the pro-apoptotic protein bcl -associated x (bax) to the mitochondria, thereby causing apoptotic cell death, which we named rlr (rig-i-like receptor)-induced irf -mediated pathway of apoptosis (ripa) ( figure a ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in ripa, irf is activated by linear ubiquitination on two key lysine residues by linear ubiquitin chain assembly complex (lubac) to allow its interaction with the pro-apoptotic protein bax [ , ] . the irf /bax complex translocates to the mitochondrial membrane and stimulates the release of cytochrome c into the cytosol. cytosolic cytochrome c activates cellular caspases to trigger apoptotic cell death ( figure a ). recently, we demonstrated that ripa contributes to the optimal antiviral activity of irf . knock-in mice expressing a ripa-active nt-irf mutant (irf s /s ) can mount antiviral protection against respiratory pathogenesis by sendai virus (sev) [ ] . the sev-infected cells, in the absence of ripa, establish viral persistence [ ] . sev temporarily regulates ripa by activating the cellular survival pathways, e.g., the phosphatidylinositol -kinase (pi k)-dependent akt, which stabilizes x-linked inhibitor of apoptosis protein (xiap), an inhibitor of ripa [ ] . the inhibition of pi k rapidly triggers ripa to kill the virus-infected cells [ ] . in the time since we described a function for nt-irf , several studies have reported ripa-like activities in viral and nonviral pathogenesis. in human t cell leukemia virus (htlv )-infected primary monocytes, stimulator of interferon genes (sting)-activated irf interacts with bax to cause apoptosis. the irf /bax-mediated monocyte cell death prevents productive htlv replication [ ] . in hepatocytes, sting-activated irf causes alcoholic liver diseases (ald) during chronic ethanol administration in mice [ ] . ethanol administration triggers endoplasmic reticulum stress, which activates sting signaling to enable an interaction between irf and bax, leading to hepatocyte apoptosis. a subsequent study further revealed that carbon tetrachloride (ccl )-induced hepatotoxicity is caused by the ripa-like activity of irf , mediated by sting/irf /bax-dependent apoptotic pathway. to further investigate the role of ripa in ald, we used the irf s /s knock-in mice in a mouse alcoholic hepatitis model to show that ethanol administration activates ripa in hepatic immune cells. since the immune cells are necessary for the resolution of liver injury, our study demonstrated a detrimental role for ripa in ald pathogenesis [ ] . in contrast, irf s /s mice are protected in high-fat diet (hfd)-induced liver diseases by the resolution of hepatic inflammation [ ] . the involvement of ripa in various disease models highlights its potential as a therapeutic target. to test this, we took a pharmacological approach to isolate small molecule modifiers of ripa. in the current study, we performed a high throughput screen of a library of fda-approved compounds (prestwick chemical), and isolated a small subset of ripa-promoting compounds. using two compounds, which specifically activated ripa, but not the transcriptional function of irf , we demonstrated that therapeutic activation of the ripa branch of irf inhibits virus replication. human cell lines mda-mb- (atcc htb- ), ht (atcc ccl- ), and a (atcc ccl- ), the african green monkey cell line vero (atcc ccl- ), and mouse embryonic fibroblasts (mefs) were maintained in dmem containing % fbs, penicillin, and streptomycin. all cell lines viruses , , of used in this study were maintained in the authors' laboratory. expression vectors of human irf and irf -k were described previously [ ] , and the ligands for retinoic acid-inducible gene-i (rig-i), toll-like receptor (tlr ), and sting have been described before [ , ] . the fda-approved drug library was obtained from prestwick chemical (pc, washington, dc, usa). individual chemicals were obtained from sigma-aldrich ( , anti-ifit (described previously [ , ] ), anti-ifit (described previously [ , ] ), and anti-vsv g-protein (described previously [ , ] ). the human breast cancer cell line mda-mb- was used to screen the library of fda-approved drugs to isolate the regulators of ripa ( figure f ). the cells were seeded in -well black-bottom tissue culture plates, and the next day, the cells were transfected with polyi:c using lipofectamine to stimulate ripa. after the addition of the transfection complex, the cells were immediately treated with either dmso (vehicle) or the drug library (at µm final concentration). after h of the ripa stimulation, the cells were analyzed for caspase activity using apo-onetm homogeneous caspase- / assay (promega, san luis obispo, ca, usa) following the manufacturer's instructions. the caspase activity of the vehicle-treated well was arbitrarily considered as and all other values were normalized to this. a representative drug screening plate is shown in figure s . the normalized caspase activity was used to calculate the z-scores and the top primary hits were isolated for further validation. ripa stimulation was performed by transfecting the cells with polyi:c ( µg if not indicated otherwise in the figure legends) using lipofectamine for - h, and the cells were either analyzed for caspase activity or cleaved parp, as indicated in the figure legends. for the drug treatments, the cells were pretreated with the drugs (e.g., doxorubicin at µm, and pyrvinium pamoate at µm or as indicated in the figures) for h before ripa stimulation (polyi:c transfection). dmso was used as a vehicle control for these drugs. ht cells were transfected with either control (sc- ) or irf -specific (sc- ) crispr/cas plasmids (santa cruz) using lipofectamine (thermo fisher scientific). transfected cells were sorted for high gfp-expressers using flow cytometry, and the gfp-expressing cells were expanded to isolate individual clones. these clones were screened for irf protein levels by immunoblot, and the clones with no irf protein expression were expanded and further validated using functional assay to ensure the absence of irf -dependent ifit induction upon rlr stimulation ( figure s ). vesicular stomatitis virus (vsv) indiana strain expressing green fluorescent protein (gfp), sendai virus (sev) cantell strain (charles river laboratories, garfield heights, oh, usa), herpes simplex virus (hsv- ) kos and f strains, langat virus (lgtv), kunjin virus (kunv), and the infection procedures have been described previously [ , , ] . briefly, the cells were infected with the viruses [at a multiplicity of infection (moi) of ] in serum-free dmem for h, after which the cells were washed and replaced with normal growth medium. the virus-infected cells were analyzed at the indicated time for viral protein expression or as described in figure legends. for quantification of infectious virus particles in the culture medium, plaque assays were performed for vsv in -fold serial dilution on vero cells [ ] . hsv- titer was measured by tcid using -fold serial dilution of the culture media on vero cells [ ] . plaque assays were performed for lgtv and kunv using previously described procedures [ ] . to determine the effects of the drugs on viral replication, the cells were pre-treated with dmem containing dmso (vehicle) or individual drugs at the indicated concentrations for h before virus infection, removed during the virus adsorption, and reintroduced post adsorption. equal protein extracts from the gfp.vsv-infected cells were analyzed for gfp fluorescence using a plate reader. gfp fluorescence of vehicle-treated vsv-infected cells was set arbitrarily at and all other values were normalized to this. immunoblot analyses were performed using previously described procedures [ , ] . briefly, the cells were lysed in mm tris buffer, ph . , containing mm of nacl, . % triton x- , mm sodium orthovanadate, mm of sodium fluoride, mm of β-glycerophosphate, mm sodium pyrophosphate, protease and phosphatase inhibitors (roche). total protein extracts were analyzed by sds-page followed by immunoblot. for analyzing the ubiquitination of irf , a previously described procedure was followed [ ] . the immunoblots were quantified by image j software. total rna was isolated using trizol (thermo fisher scientific), cdna was prepared using improm-ii reverse transcription kit (promega), and the cdna was analyzed using radianttm sybr green pcr mix (alkali scientific inc., fort lauderdale, fl, usa) in roche lightcycler instrument and analyzed with the lightcycler software, version . . the expression levels of the mrnas were normalized to s rrna. for the qrt-pcr analyses of the respective genes, the following primers were used: ifit -fwd: tctcagaggagcctggctaag, ifit -rev: gtcaccagactcctcacatttgc, ifit -fwd: gaacatgctgaccaagcaga, ifit -rev: cagttgtgtccacccttcct, ifnb -fwd: cgccgcattgaccatcta, ifnb -rev: gacattagccaggaggttct, s-fwd: attgacggaagggcaccaccag, s-rev: caaatcgctccaccaactaagaacg. mda-mb- cells were grown on coverslips, infected with hsv- in the absence or the presence of doxorubicin, as described in the figure legends. the infected cells were fixed in % paraformaldehyde (electron microscopy sciences, hatfield, pa, usa, # ), permeabilized in . % triton x- (fisher scientific # - - ) and immunostained with anti-icp antibody followed by alexa. fluor-conjugated secondary antibody (invitrogen #a- ). the coverslips were mounted on microscopy slides using vectashield/dapi (vector laboratories, burlingame, ca, usa, #h- ) and analyzed using an olympus (waltham, ma, usa) confocal microscope and olympus fluoview fv software. the caspase- / activity of the cell lysates was performed using previously described procedures [ ] . briefly, the cell lysates were used for measuring caspase activity using the apo-onetm homogeneous caspase- / assay according to protocols provided by the manufacturer (promega). caspase activity in experimental samples was plotted relative to rlr-stimulated cells arbitrarily set as (as in figure d ). cell viability was measured by -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay, trypan blue exclusion, and brightfield microscopy. to measure cell viability, the cells were seeded in a -well plate, and transfected (rlr) or treated (tlr ) with polyi:c in the absence or the presence of doxorubicin or vehicle (dmso), as indicated, for h. the mtt assay was performed using previously described procedures [ ] . the absorbance of the treated cells was considered as , and the other values were normalized to this. for the trypan blue exclusion assay, the treated cells were trypsinized, resuspended in complete dmem, stained with trypan blue and the live and dead cells were counted using a hemocytometer to calculate percent viability for each treatment. the statistical analyses were performed using graphpad prism . software. the "p" values were calculated using two-tailed, unpaired student's t-tests and are shown in the relevant figures. the results presented here are the representatives of at least three biological repeats. to identify small molecule activators of the antiviral ripa branch of irf ( figure a ), we performed an unbiased high throughput screen using a library of fda-approved compounds. activation of ripa, by transfection of the rlr ligand polyi:c, caused robust cell death ( figure b , stage- , figure a ). stimulation of tlr or sting, by their cognate ligands, did not cause visible cell death ( figure b ). cleaved parp (c-parp, stage- , figure a ), a molecular marker of apoptosis, was observed in rlr-, but not sting-stimulated, cells. as expected, the apoptotic executioner caspase, caspase- (stage- , figure a ), was strongly activated only upon rlr, but not tlr or sting, stimulation for an increasing time period ( figure d ,e). therefore, we optimized conditions to screen for agents that specifically modulate ripa-induced apoptosis, without the need to separate the contribution of tlr or sting pathways. to isolate the activators of ripa, we performed a high throughput screen of a library consisting of fda-approved compounds [ ] . the library is -well formatted with each well containing an individual compound. we performed the primary screen using the strategy outlined in figure f , and the activity of caspase- was used as a readout of the ripa activity in each well. the caspase- activity of vehicle (dmso)-treated well was arbitrarily set at , and all other values were normalized to this (a sample plate is shown in figure s ). the primary screen resulted in several ripa activators, and using an arbitrary cut-off of z-scores greater than . , we obtained twenty-five candidate ripa activating compounds (figure a ,b, table s ). we noted that the ripa activators constitute only % of the library, indicating the specificities of the compounds. the primary hits were validated using a secondary screen, and the ripa-promoting activity of each compound was determined with respect to the vehicle control. the secondary screen validated twenty-four of the twenty-five compounds obtained from the primary screen ( figure c ). the secondary-validated ripa-activators consisted of compounds from a variety of therapeutic activities, e.g., anticancer, antibacterial, anti-inflammatory, antihypertensive, etc ( figure b ). we excluded act , which triggered caspase- activity nonspecifically, from the subsequent studies, and focused on doxorubicin, which has earlier been studied to activate irf [ ] . because ripa contributes to the antiviral function of irf [ ] , we hypothesized that agents that promote ripa would inhibit viral replication. for this purpose, we used vesicular stomatitis virus (vsv) as a model negative-sense rna virus. doxorubicin treatment inhibited vsv replication, analyzed by virus-encoded gfp expression ( figure a ). we validated these results by immunoblot analyses, which demonstrate that doxorubicin treatment inhibited the expression of vsv-g protein, a viral envelope glycoprotein as well as the virus-encoded gfp ( figure b ). as expected, the reduced viral protein expression led to reduced infectious virus particle release from doxorubicin-treated cells ( figure c ). we validated the results in another cell line (a ), in which doxorubicin also inhibited vsv-encoded gfp expression ( figure d ). to determine whether the antiviral activity of doxorubicin is dependent on irf , we generated irf −/− human cells (ht ) using a crispr/cas approach ( figure e , lower panel, figure s ). doxorubicin, as expected, inhibited the viral protein expression in the wt cells; however, the antiviral activity of doxorubicin was impaired in irf −/− cells ( figure e , top panel). to further validate this striking result, we used wt and irf −/− mouse embryonic fibroblasts (mefs) ( figure f , lower panel). similar to the human cells, doxorubicin inhibited vsv replication only in the presence of irf ( figure f , top panel). we further quantified, in addition to viral g protein, the expression of vsv-encoded gfp fluorescence. similar to the inhibition of g protein expression, doxorubicin inhibited gfp fluorescence in wt but not irf -deficient cells ( figure e , f). we ensured that doxorubicin did not cause substantial cytotoxicity under our experimental conditions ( figure s a , b). our results demonstrate, for the first time, that doxorubicin is a ripa-promoting agent, which inhibits viral infection in an irf -dependent manner. because ripa contributes to the antiviral function of irf [ ] , we hypothesized that agents that promote ripa would inhibit viral replication. for this purpose, we used vesicular stomatitis virus (vsv) as a model negative-sense rna virus. doxorubicin treatment inhibited vsv replication, analyzed by virus-encoded gfp expression ( figure a ). we validated these results by immunoblot analyses, which demonstrate that doxorubicin treatment inhibited the expression of vsv-g protein, a viral envelope glycoprotein as well as the virus-encoded gfp ( figure b ). as expected, the reduced viral protein expression led to reduced infectious virus particle release from doxorubicin-treated cells ( figure c ). we validated the results in another cell line (a ), in which doxorubicin also inhibited vsv-encoded gfp expression ( figure d ). to determine whether the antiviral activity of doxorubicin is dependent on irf , we generated irf −/− human cells (ht ) using a crispr/cas approach ( figure e , lower panel, figure s ). doxorubicin, as expected, inhibited the viral protein expression in the wt cells; however, the antiviral activity of doxorubicin was impaired in irf −/− cells ( figure e , top panel). to further validate this striking result, we used wt and irf −/− mouse embryonic fibroblasts (mefs) ( figure f , lower panel). similar to the human cells, doxorubicin inhibited vsv replication only in the presence of irf ( figure f, top panel) . we further quantified, in addition to viral g protein, the expression of vsv-encoded gfp fluorescence. similar to the inhibition of g protein expression, doxorubicin inhibited gfp fluorescence in wt but not irf -deficient cells ( figure e ,f). we ensured that doxorubicin did not cause substantial cytotoxicity under our experimental conditions ( figure s a ,b). our results demonstrate, for the first time, that doxorubicin is a ripa-promoting agent, which inhibits viral infection in an irf -dependent manner. the optimal antiviral action of irf depends on its transcriptional and nontranscriptional (nt, ripa) functions. we sought to determine whether doxorubicin activates either one or both functions of irf . furthermore, doxorubicin has previously been reported to induce phosphorylation and transcriptional activation of irf [ ] . thus, we examined whether doxorubicin promotes the transcriptional activity of irf by rlr-signaling in vsv-infected cells. vsv infection induced irf target gene, ifit , which was strongly inhibited by doxorubicin, examined at multiple doses ( figure a ). in contrast, doxorubicin robustly enhanced the ripa activity, i.e., the c-parp levels in vsvinfected cells ( figure a ). to solidify these results, we used a nonviral rlr agonist, polyi:c, and the irf -induced ifit was also strongly inhibited by doxorubicin in ht cells ( figure b ). similar to vsv-infected cells, doxorubicin promoted irf -induced ripa activity in these cells (c-parp, figure b ). to validate these results, we measured the transcriptional induction of additional irf induced genes, both at the protein and mrna levels. the irf -induced ifit and ifit proteins were strongly inhibited by doxorubicin at multiple times post rlr-stimulation ( figure c ). doxorubicin treatment also significantly inhibited the mrna induction of ifit ( figure d ), ifit ( figure e ), and ifn-β ( figure f ), all of which depend on the transcriptional activity of irf . collectively, our results demonstrate that doxorubicin inhibits vsv replication in the absence of irf induced antiviral gene expression. the optimal antiviral action of irf depends on its transcriptional and nontranscriptional (nt, ripa) functions. we sought to determine whether doxorubicin activates either one or both functions of irf . furthermore, doxorubicin has previously been reported to induce phosphorylation and transcriptional activation of irf [ ] . thus, we examined whether doxorubicin promotes the transcriptional activity of irf by rlr-signaling in vsv-infected cells. vsv infection induced irf -target gene, ifit , which was strongly inhibited by doxorubicin, examined at multiple doses ( figure a ). in contrast, doxorubicin robustly enhanced the ripa activity, i.e., the c-parp levels in vsv-infected cells ( figure a ). to solidify these results, we used a nonviral rlr agonist, polyi:c, and the irf -induced ifit was also strongly inhibited by doxorubicin in ht cells ( figure b ). similar to vsv-infected cells, doxorubicin promoted irf -induced ripa activity in these cells (c-parp, figure b ). to validate these results, we measured the transcriptional induction of additional irf -induced genes, both at the protein and mrna levels. the irf -induced ifit and ifit proteins were strongly inhibited by doxorubicin at multiple times post rlr-stimulation ( figure c ). doxorubicin treatment also significantly inhibited the mrna induction of ifit ( figure d ), ifit ( figure e) , and ifn-β ( figure f ), all of which depend on the transcriptional activity of irf . collectively, our results demonstrate that doxorubicin inhibits vsv replication in the absence of irf -induced antiviral gene expression. doxorubicin promoted the ripa activity, measured by robust increase in rlr-induced c-parp levels, when compared with rlr-treated cells ( figure a,b) . doxorubicin is a known inducer of the cellular apoptotic pathway [ , ] , and, as expected, also triggered apoptosis in the absence of rlr stimulation ( figure a , lane ). to inquire genetically how doxorubicin activates irf to promote ripa, we took a strategy to measure the "doxorubicin-promoted ripa activity" using the combined treatment of rlr and doxorubicin ("rlr/doxo", lane , figure a ), to avoid the individual cellular effects of rlr or doxorubicin (lanes , , figure a ). similar to c-parp, doxorubicin significantly increased the rlr-induced caspase- activity ( figure c ). the increased ripa activity by doxorubicin, as expected, led to enhanced cell death in a dose-dependent manner ( figure s c ). we examined doxorubicin-promoted ripa in wt and irf −/− cells. doxorubicin caused robust increase in rlr-induced c-parp and capsase- activation in wt cells (figures d, e) . the rlr/doxorubicininduced c-parp and caspase activity were reduced in irf −/− cells ( figure d , e). because doxorubicin specifically promotes the ripa, and not the transcriptional activity of irf , we used a nt-irf mutant, irf -k , which is functional only in the ripa, but not the transcriptional branch [ ] . the irf -k -expressing cells showed strong promotion of ripa activity upon doxorubicin treatment ( figure f ). collectively, our results demonstrate that doxorubicin inhibits the transcriptional, but promotes the ripa, activity of irf . doxorubicin promoted the ripa activity, measured by robust increase in rlr-induced c-parp levels, when compared with rlr-treated cells ( figure a,b) . doxorubicin is a known inducer of the cellular apoptotic pathway [ , ] , and, as expected, also triggered apoptosis in the absence of rlr stimulation ( figure a , lane ). to inquire genetically how doxorubicin activates irf to promote ripa, we took a strategy to measure the "doxorubicin-promoted ripa activity" using the combined treatment of rlr and doxorubicin ("rlr/doxo", lane , figure a ), to avoid the individual cellular effects of rlr or doxorubicin (lanes , , figure a ). similar to c-parp, doxorubicin significantly increased the rlr-induced caspase- activity ( figure c ). the increased ripa activity by doxorubicin, as expected, led to enhanced cell death in a dose-dependent manner ( figure s c ). we examined doxorubicin-promoted ripa in wt and irf −/− cells. doxorubicin caused robust increase in rlr-induced c-parp and capsase- activation in wt cells ( figure d ,e). the rlr/doxorubicin-induced c-parp and caspase activity were reduced in irf −/− cells ( figure d ,e). because doxorubicin specifically promotes the ripa, and not the transcriptional activity of irf , we used a nt-irf mutant, irf -k , which is functional only in the ripa, but not the transcriptional branch [ ] . the irf -k -expressing cells showed strong promotion of ripa activity upon doxorubicin treatment ( figure f ). collectively, our results demonstrate that doxorubicin inhibits the transcriptional, but promotes the ripa, activity of irf . to investigate the biochemical mechanism by which doxorubicin promotes ripa, we examined whether doxorubicin activates any specific step(s) of ripa (depicted in figure a ). we systematically analyzed both pre-and postmitochondrial steps of ripa. the ubiquitination of irf (stage- , figure a ), which is essential for activating ripa, was not promoted by doxorubicin ( figure a ). we noted that doxorubicin-treated cells exhibited shorter ubiquitin chains linked to irf than the vehicletreated cells. the results led us to examine whether doxorubicin increased the rlr-induced release of cytochrome c into the cytosol (stage- , figure a ), a step that requires the interaction of irf and bax. doxorubicin did not increase the rlr-induced release of cytosolic cytochrome c ( figure b ). these results led us to hypothesize that doxorubicin activates rlr-activated cellular signaling to promote ripa. a previous study indicated that p , which can be activated by doxorubicin [ ] , is not involved in rlr-induced apoptosis [ ] . therefore, we examined the c-jun n-terminal kinase (jnk) and extracellular signal-regulated kinase (erk) signaling pathways, which are activated upon rlr stimulation [ , ] . rlr stimulation triggered the phosphorylation of jnk (pjnk) and erk (perk), which were enhanced by doxorubicin ( figure c, d) . we then tested whether doxorubicinactivated jnk and erk are involved in promoting ripa, using pharmacological inhibitors of jnk and mitogen-activated protein kinase kinase (mek), the upstream activator of erk. inhibition of mek (u , meki), but not jnk (sp , jnki), significantly suppressed doxorubicin-promoted ripa ( figure e ). we further validated the role of erk in mouse cells expressing the nt-irf mutant (irf s /s ), which is active in ripa [ ] . in irf s /s mefs, the mek inhibitor suppressed doxorubicin- to investigate the biochemical mechanism by which doxorubicin promotes ripa, we examined whether doxorubicin activates any specific step(s) of ripa (depicted in figure a ). we systematically analyzed both pre-and postmitochondrial steps of ripa. the ubiquitination of irf (stage- , figure a) , which is essential for activating ripa, was not promoted by doxorubicin ( figure a ). we noted that doxorubicin-treated cells exhibited shorter ubiquitin chains linked to irf than the vehicle-treated cells. the results led us to examine whether doxorubicin increased the rlr-induced release of cytochrome c into the cytosol (stage- , figure a ), a step that requires the interaction of irf and bax. doxorubicin did not increase the rlr-induced release of cytosolic cytochrome c ( figure b ). these results led us to hypothesize that doxorubicin activates rlr-activated cellular signaling to promote ripa. a previous study indicated that p , which can be activated by doxorubicin [ ] , is not involved in rlr-induced apoptosis [ ] . therefore, we examined the c-jun n-terminal kinase (jnk) and extracellular signal-regulated kinase (erk) signaling pathways, which are activated upon rlr stimulation [ , ] . rlr stimulation triggered the phosphorylation of jnk (pjnk) and erk (perk), which were enhanced by doxorubicin ( figure c,d) . we then tested whether doxorubicin-activated jnk and erk are involved in promoting ripa, using pharmacological inhibitors of jnk and mitogen-activated protein kinase kinase (mek), the upstream activator of erk. inhibition of mek (u , mek i ), but not jnk (sp , jnk i ), significantly suppressed doxorubicin-promoted ripa ( figure e ). we further validated the role of erk in mouse cells expressing the nt-irf mutant (irf s /s ), which is active in ripa [ ] . in irf s /s mefs, the mek inhibitor suppressed doxorubicin-promoted ripa, analyzed by rlr-induced caspase- activity ( figure f ) and c-parp ( figure g ). together, doxorubicin-activated erk signaling is involved in promoting ripa. viruses , , x for peer review of promoted ripa, analyzed by rlr-induced caspase- activity ( figure f ) and c-parp ( figure g ). together, doxorubicin-activated erk signaling is involved in promoting ripa. to test whether doxorubicin inhibits viruses of other families, we used members of flaviviridae and herpesviridae, which are known to activate the rlr signaling pathway [ ] [ ] [ ] . we previously demonstrated that ripa can be activated by viruses with positive-sense rna or dna genomes [ ] . consistent with the vsv results (figure ) , doxorubicin treatment inhibited langat virus (lgtv) ( figure a) and kunjin virus (kunv) ( figure b ) infectious virion production. furthermore, cytosolic dna from dna viruses can activate ripa through rna polymerase iii activity [ ] . to investigate whether doxorubicin also inhibits dna virus replication, we used two strains (kos and f) of hsv- . doxorubicin strongly inhibited hsv- (kos strain) replication, indicated by the reduction in icp expression by immunoblot ( figure c ) and confocal microscopy ( figure d ). the inhibition of viral protein expression led to a significant reduction in infectious virus particle release from the doxorubicin-treated cells ( figure e ). similar to the kos strain, doxorubicin also inhibited the replication of hsv- f strain ( figure f ). collectively, our results demonstrate that the ripapromoting agent doxorubicin inhibits the replication of both rna and dna viruses. to test whether doxorubicin inhibits viruses of other families, we used members of flaviviridae and herpesviridae, which are known to activate the rlr signaling pathway [ ] [ ] [ ] . we previously demonstrated that ripa can be activated by viruses with positive-sense rna or dna genomes [ ] . consistent with the vsv results (figure ) , doxorubicin treatment inhibited langat virus (lgtv) ( figure a) and kunjin virus (kunv) ( figure b ) infectious virion production. furthermore, cytosolic dna from dna viruses can activate ripa through rna polymerase iii activity [ ] . to investigate whether doxorubicin also inhibits dna virus replication, we used two strains (kos and f) of hsv- . doxorubicin strongly inhibited hsv- (kos strain) replication, indicated by the reduction in icp expression by immunoblot ( figure c ) and confocal microscopy ( figure d ). the inhibition of viral protein expression led to a significant reduction in infectious virus particle release from the doxorubicin-treated cells ( figure e ). similar to the kos strain, doxorubicin also inhibited the replication of hsv- f strain ( figure f ). collectively, our results demonstrate that the ripa-promoting agent doxorubicin inhibits the replication of both rna and dna viruses. viruses , , x for peer review of to test the generality of the theme that ripa-promoting compounds are antiviral agents, we screened a subset of the secondary-validated ripa-promoters (act , act , act , act , act , and act ), based on an arbitrary cut-off of a . -fold increase in ripa-promotion ( figure c ), for their ability to inhibit hsv- replication ( figure a) . we confirmed the anti-hsv- activity of doxorubicin (act ), and also isolated two additional compounds (act and act ) that strongly inhibited icp expression ( figure a ). we excluded act (topotecan) from further analyses because it shares properties with doxorubicin and may activate similar pathways. we focused on the anti-helminthic drug pyrvinium pamoate (pp, act ) for validation of our hypothesis. similar to doxorubicin, pp inhibited the replication of hsv- in ht cells, in a dose-dependent manner, demonstrated by immunoblot of viral proteins icp and icp ( figure b ). pp also inhibited vsv replication, analyzed by immunoblot of the viral protein vsv-g ( figure c ) and infectious virion particle release ( figure d ). the antiviral activity of pp prompted us to test its ability to activate irf . pp, as expected, promoted the ripa branch of irf , measured by caspase activity ( figure e ) and c-parp ( figure f ). unlike doxorubicin, pp treatment did not trigger an apoptotic pathway in the absence of rlrstimulation ( figure f, lane ) . to investigate whether pp uses a mechanism similar to doxorubicin to promote ripa, we tested the mek inhibitor in pp-promoted ripa. inhibition of mek reduced rlr-induced c-parp in pp-treated cells ( figure g) . therefore, erk signaling may be a common to test the generality of the theme that ripa-promoting compounds are antiviral agents, we screened a subset of the secondary-validated ripa-promoters (act , act , act , act , act , and act ), based on an arbitrary cut-off of a . -fold increase in ripa-promotion ( figure c ), for their ability to inhibit hsv- replication ( figure a ). we confirmed the anti-hsv- activity of doxorubicin (act ), and also isolated two additional compounds (act and act ) that strongly inhibited icp expression ( figure a ). we excluded act (topotecan) from further analyses because it shares properties with doxorubicin and may activate similar pathways. we focused on the anti-helminthic drug pyrvinium pamoate (pp, act ) for validation of our hypothesis. similar to doxorubicin, pp inhibited the replication of hsv- in ht cells, in a dose-dependent manner, demonstrated by immunoblot of viral proteins icp and icp ( figure b ). pp also inhibited vsv replication, analyzed by immunoblot of the viral protein vsv-g ( figure c ) and infectious virion particle release ( figure d ). the antiviral activity of pp prompted us to test its ability to activate irf . pp, as expected, promoted the ripa branch of irf , measured by caspase activity ( figure e ) and c-parp ( figure f ). unlike doxorubicin, pp treatment did not trigger an apoptotic pathway in the absence of rlr-stimulation ( figure f, lane ) . to investigate whether pp uses a mechanism similar to doxorubicin to promote ripa, we tested the mek inhibitor in pp-promoted ripa. inhibition of mek reduced rlr-induced c-parp in pp-treated cells ( figure g) . therefore, erk signaling may be a common mechanism to therapeutically target ripa in multiple cell types. next, we inquired about the effect of pp on the transcriptional activity of irf . interestingly, although pp promoted the ripa branch of irf ( figure e,f) , it had no effect on the transcriptional activity of irf , demonstrated by qrt-pcr analyses of irf target genes ( figure h,i) . therefore, in contrast to doxorubicin, which differentially modulates irf activity by promoting ripa and inhibiting the irf transcriptional branch, pp exclusively promotes ripa without affecting irf transcriptional activity. mechanism to therapeutically target ripa in multiple cell types. next, we inquired about the effect of pp on the transcriptional activity of irf . interestingly, although pp promoted the ripa branch of irf ( figure e,f) , it had no effect on the transcriptional activity of irf , demonstrated by qrt-pcr analyses of irf target genes ( figure h,i) . therefore, in contrast to doxorubicin, which differentially modulates irf activity by promoting ripa and inhibiting the irf transcriptional branch, pp exclusively promotes ripa without affecting irf transcriptional activity. we have previously demonstrated, using genetically manipulated cells and mice, that nt-irf function via ripa contributes to the optimal antiviral activity of irf [ ] [ ] [ ] [ ] ]. in the current study, we sought to identify small molecules that activate ripa and, therefore, can limit virus replication. using a high-throughput screen of a library of fda-approved drugs, we isolated a subset of compounds with diverse therapeutic functions that promoted the activity of ripa. among the ripaactivating compounds, we focused on doxorubicin for mechanistic and functional studies. doxorubicin inhibits the replication of vsv by activating the ripa branch of irf . our in-depth biochemical studies revealed that doxorubicin inhibited the transcriptional activity of irf ; moreover, doxorubicin promoted ripa by activating the erk signaling pathway. the antiviral function of doxorubicin was expanded to the tick-and mosquito-borne flaviviruses (lgtv and kunv), and hsv- . finally, we validated our results using pyrvinium pamoate, which also inhibited the replication of vsv and hsv- by specifically promoting ripa, and not the transcriptional, we have previously demonstrated, using genetically manipulated cells and mice, that nt-irf function via ripa contributes to the optimal antiviral activity of irf [ ] [ ] [ ] [ ] ]. in the current study, we sought to identify small molecules that activate ripa and, therefore, can limit virus replication. using a high-throughput screen of a library of fda-approved drugs, we isolated a subset of compounds with diverse therapeutic functions that promoted the activity of ripa. among the ripa-activating compounds, we focused on doxorubicin for mechanistic and functional studies. doxorubicin inhibits the replication of vsv by activating the ripa branch of irf . our in-depth biochemical studies revealed that doxorubicin inhibited the transcriptional activity of irf ; moreover, doxorubicin promoted ripa by activating the erk signaling pathway. the antiviral function of doxorubicin was expanded to the tick-and mosquito-borne flaviviruses (lgtv and kunv), and hsv- . finally, we validated our results using pyrvinium pamoate, which also inhibited the replication of vsv and hsv- by specifically promoting ripa, and not the transcriptional, function of irf . therefore, we demonstrate that compounds that activate ripa have potential for antiviral action. high throughput screening to isolate novel antiviral compounds is a widely accepted strategy, and commonly utilizes virus replication as a terminal readout. we took a novel approach to isolate the antiviral agents by screening for activators of a cellular antiviral pathway. irf is the key transcription factor for the induction of ifn and antiviral isgs [ , , , ] . we have uncovered a pro-apoptotic pathway of nt-irf , ripa, in virus-infected cells [ , , ] . using cells and mice expressing a ripa active nt-irf mutant, we demonstrated the relative contribution of ripa to the antiviral response of irf [ ] . several high throughput screens have revealed small molecules that regulate the transcriptional activity of irf [ ] [ ] [ ] [ ] [ ] . however, because ripa is newly identified, there are no known regulators of this pathway. this led us to develop an unbiased screen, using a library of fda-approved drugs, to isolate ripa-activators that would function as antiviral agents. using doxorubicin, a new ripa activator, we examined both activities of irf , because an activator of either pathway would provide an antiviral response. although doxorubicin promoted ripa in multiple cell types, it inhibited the transcriptional activity of irf . therefore, doxorubicin inhibits virus replication in the absence of irf -induced antiviral genes, further emphasizing the biological significance of ripa. how doxorubicin inhibits the transcriptional activity of irf is a topic of future investigation. the dna-damaging property of doxorubicin [ ] may contribute to this activity. furthermore, whether doxorubicin, in addition to ripa promotion, can intercalate with viral genomic dna to inhibit viral replication, will require further studies. our screen isolated, in addition to doxorubicin, pyrvinium pamoate, which specifically promoted ripa, but not the transcriptional function, of irf . many viruses block the transcriptional activity of irf to dampen ifn production [ ] . in such scenarios, it will be interesting to examine whether ripa-promoting compounds inhibit virus replication. to investigate the molecular mechanisms of the doxorubicin-mediated promotion of ripa, we examined the key biochemical steps of the pathway. since viruses can block host pathways, we chose to use nonviral stimuli of rlr for the mechanistic studies, to avoid any virus-specific effects. our results indicate that doxorubicin did not enhance the irf ubiquitination nor the release of cytochrome c into the cytosol, the critical stages of ripa. interestingly, our results suggest that doxorubicin treatment may enrich a distinct pool of ubiquitinated irf in the rlr-stimulated cells. targeted proteomic analyses of this pool of irf will be done in the future to gain further insight. we also considered that doxorubicin, a dna-damaging agent, might stimulate ripa by triggering the sting signaling pathway via topoisomerase activation [ , ] . the cgas/sting pathway can also trigger transcription-independent mitotic cell death by irf activation [ ] . moreover, er stress-activated sting has been shown to trigger a ripa-like pathway in hepatocytes [ ] . however, in our test cells, mda-mb- , the sting agonists were unable to trigger apoptotic cell death, indicating a lack of sting contribution in promoting ripa. it remains to be seen whether the ripa activators also trigger additional innate signaling pathways, e.g., nlrs to amplify ripa activity. our results revealed a novel role of the erk signaling pathway in regulating ripa. sev infection activates cellular autophagy via the erk signaling pathway to trigger apoptosis [ ] . whether doxorubicin or pyrvinium pamoate use a similar mechanism to promote ripa will require additional studies. our results further revealed that doxorubicin activated both erk and jnk signaling pathways in rlr-stimulated cells; however, jnk activity was not required for promoting ripa. in the future, in-depth genetic studies will reveal the relative contribution of erk and jnk to modulate ripa. the erk signaling pathway also regulates the transcriptional activity of irf [ ] ; whether doxorubicin inhibits the transcriptional activity of irf via erk will require future investigation. in the future, in vivo studies in the context of virus-infection will be required to validate the in vitro findings regarding the erk signaling mechanism of ripa-activating drugs. a previous study indicated that p , which can be activated by doxorubicin, is not involved in dsrna-induced cell death [ ] . in previous studies, we have shown that sev-induced ripa is regulated by the pi k/akt signaling pathway [ ] . it remains to be seen, using nt-irf mutant that specifically activates ripa, whether doxorubicin also modulates the p and pi k pathways to promote ripa. our screen, using a relatively small library of compounds, revealed that a number of molecules with diverse therapeutic activities promote ripa. in the future, we will expand the screen to larger libraries to isolate additional novel ripa-activating compounds. in addition, a screen using cells expressing nt-irf mutant that specifically activates ripa may reveal additional candidates. in the future, we will use mice expressing a ripa-active irf s /s mutant to test the efficacy of the ripa-promoting drugs in mounting antiviral responses in the absence of antiviral genes. such studies will be physiologically significant by using the drug derivatives that are currently applied on patients. because doxorubicin inhibits the cellular ifn response, patients receiving this drug may be protected from the adverse effects of ifn and the ifn-induced genes. moreover, our study provides a molecular basis for the previously identified antiviral effects of the ripa-activating compounds [ , ] . a recent study showed that pp is a novel inhibitor of coronavirus replication [ ] and its antiviral property, in part, may be related to the ripa-promoting function of pp. whether activation of ripa directly inhibits virus replication or eliminates the virus-infected cells to reduce overall infection requires in-depth studies. some viruses trigger apoptosis for successful spreading and, therefore, promoting viral apoptosis to inhibit virus infection may be virus specific. finally, a strategy to repurpose ripa activators as antivirals must be met with caution, as there are a number of diseases where ripa can be detrimental (e.g., liver diseases). for instance, a ripa-like pathway in hepatocytes leads to the development of alcoholic liver diseases [ ] . moreover, we recently showed that ripa-activated hepatic myeloid cells produce inflammatory cytokines that cause hepatitis in mice [ ] . in these scenarios, it may be advantageous to investigate ripa inhibitors as therapeutic options. in summary, our results demonstrate that pharmacological activation of ripa may be a potential antiviral strategy, particularly in scenarios where the cellular ifn response is dampened. in the future, these ripa-modulating agents can be studied in additional models where ripa is involved, e.g., in fatty liver diseases. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . figure s . representative data from the primary screen. a representative primary screening plate showing the caspase- activity in each well (rows and columns are shown in blue) containing a specific drug or dmso (vehicle) control. potential activator and inhibitor drugs are shown in the sample plate. nt, no treatment. figure s . validation of irf −/− ht cells. immunoblot of ifit protein expression in rlr-stimulated wt and irf −/− (ko) ht cells to ensure the gene knockout. figure table s . caspase activity of the primary screening plates. mda-mb- cells were transfected with polyi:c in the absence or the presence of the drugs, as described in figure f . caspase- activity of each well is indicated after normalizing to the vehicle (dmso) control, which was considered as . author contributions: conceptualization, s.c. and r.c.; methodology, s.c., a.g., k.c., s.f., g.s., j.g., b.j., j.c. and r.t.t.; software, s.c., a.g., and g.s.; validation, s.c. and a.g.; formal analysis, s.c., a.g.; investigation, s.c., a.g., k.c., s.f., g.s., j.g., b.j., j.c. and r.t.t.; resources, s.c., r.c. and r.t.t.; data curation, s.c., a.g., k.c., s.f., g.s., j.g., b.j., j.c. and r.t.t.; writing-original draft preparation, s.c., a.g. and k.c.; writing-review and editing, r.c., and r.t.t.; visualization, s.c., a.g., k.c., s.f. and g.s.; supervision, s.c., r.c. and r.t.t.; project administration, s.c., r.c. and r.t.t.; funding acquisition, s.c. and r.t.t. all authors have read and agreed to the published version of the manuscript. no love lost between viruses and interferons the irf family transcription factors at the interface of innate and adaptive immune responses regulating irfs in ifn driven disease convergence of the nf-kappab and irf pathways in the regulation of the innate antiviral response interferon-stimulated genes and their antiviral effector functions inhibition of viral pathogenesis and promotion of the septic shock response to bacterial infection by irf- are regulated by the acetylation and phosphorylation of its coactivators ubiquitination of the transcription factor irf- activates ripa, the apoptotic pathway that protects mice from viral pathogenesis role of interferon regulatory factor -mediated apoptosis in the establishment and maintenance of persistent infection by sendai virus viral apoptosis is induced by irf- -mediated activation of bax irf- and bax: a deadly affair dsrna-activation of tlr and rlr signaling: gene induction-dependent and independent effects rig-i-like receptor-induced irf mediated pathway of apoptosis (ripa): a new antiviral pathway the irf- /bax-mediated apoptotic pathway, activated by viral cytoplasmic rna and dna, inhibits virus replication irf- activation by sendai virus infection is required for cellular apoptosis and avoidance of persistence phosphatidylinositol -kinase signaling delays sendai virus-induced apoptosis by preventing xiap degradation host restriction factor samhd limits human t cell leukemia virus type infection of monocytes via sting-mediated apoptosis sting-irf pathway links endoplasmic reticulum stress with hepatocyte apoptosis in early alcoholic liver disease the non-transcriptional activity of irf modulates hepatic immune cell populations in acute on chronic ethanol administration in mice nontranscriptional activity of interferon regulatory factor protects mice from high-fat diet-induced liver injury sting requires the adaptor trif to trigger innate immune responses to microbial infection the interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus. i. analysis of neutralizing epitopes with monoclonal antibodies antigenic diversification is correlated with increased thermostability in a mammalian virus traf plays a proviral role in tick-borne flavivirus infection through interaction with the ns protease inhibition of human parainfluenza virus type infection by novel small molecules herpes simplex virus type neuronal infection perturbs golgi apparatus integrity through activation of src tyrosine kinase and dyn- gtpase a new mechanism of interferon's antiviral action: induction of autophagy, essential for paramyxovirus replication, is inhibited by the interferon stimulated gene discovery of novel immunostimulants by dendritic-cell-based functional screening activation of interferon regulatory factor in response to dna-damaging agents adriamycin-induced dna damage mediated by mammalian dna topoisomerase ii identification of the molecular basis of doxorubicin-induced cardiotoxicity down-regulation of p by double-stranded rna modulates the antiviral response roles of tlr and rig-i in mediating the inflammatory response in mouse microglia following japanese encephalitis virus infection activation of c-jun n-terminal kinase upon influenza a virus (iav) infection is independent of pathogen-related receptors but dependent on amino acid sequence variations of iav ns early innate recognition of herpes simplex virus in human primary macrophages is mediated via the mda /mavs-dependent and mda /mavs/rna polymerase iii-independent pathways establishment and maintenance of the innate antiviral response to west nile virus involves both rig-i and mda signaling through ips- rig-i recognizes the region of dengue and zika virus genomes interferons and viral infections triggering the innate antiviral response through irf- activation high-throughput screening for tlr -ifn regulatory factor signaling pathway modulators identifies several antipsychotic drugs as tlr inhibitors emerging alphaviruses are sensitive to cellular states induced by a novel small-molecule agonist of the sting pathway identification of novel inhibitors of the type i interferon induction pathway using cell-based high-throughput screening a small-molecule irf agonist functions as an influenza vaccine adjuvant by modulating the antiviral immune response drug-induced histone eviction from open chromatin contributes to the chemotherapeutic effects of doxorubicin recent advances in understanding viral evasion of type i interferon topoisomerase ii inhibitors induce dna damage-dependent interferon responses circumventing ebola virus immune evasion topoisomerase inhibition promotes cyclic gmp-amp synthase-dependent antiviral responses the cytoplasmic dna sensor cgas promotes mitotic cell death erk-mediated autophagy promotes inactivated sendai virus (hvj-e)-induced apoptosis in hela cells in an atg -dependent manner disruption of erk-dependent type i interferon induction breaks the myxoma virus species barrier a derivate of the antibiotic doxorubicin is a selective inhibitor of dengue and yellow fever virus replication in vitro inhibition of herpes simplex virus replication by anthracycline compounds high-throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to thank ganes sen, eain murphy, kevin pan, jyl matson and amit tiwari for help with critical reagents and resources used for the study. the authors would like to acknowledge funding from the national institutes of health grant aa (sc), american heart association grant sdg (sc), medical research society (sc), and the university of toledo college of medicine and life sciences startup funds (sc and rtt). the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.viruses , , key: cord- -ud mf l authors: li, yingying; zhao, ling; luo, zhaochen; zhang, yachun; lv, lei; zhao, jianqing; sui, baokun; huang, fei; cui, min; fu, zhen f.; zhou, ming title: interferon-λ attenuates rabies virus infection by inducing interferon-stimulated genes and alleviating neurological inflammation date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ud mf l rabies, caused by rabies virus (rabv), is a fatal neurological disease that still causes more than , human deaths each year. type iii interferon ifn-λs are cytokines with type i ifn-like antiviral activities. although ifn-λ can restrict the infection for some viruses, especially intestinal viruses, the inhibitory effect against rabv infection remains undefined. in this study, the function of type iii ifn against rabv infection was investigated. initially, we found that ifn-λ and ifn-λ could inhibit rabv replication in cells. to characterize the role of ifn-λ in rabv infection in a mouse model, recombinant rabvs expressing murine ifn-λ or ifn-λ , termed as rb c-ifnλ or rb c-ifnλ , respectively, were constructed and rescued. it was found that expression of ifn-λ could reduce the pathogenicity of rabv and limit viral spread in the brains by different infection routes. furthermore, expression of ifn-λ could induce the activation of the jak-stat pathway, resulting in the production of interferon-stimulated genes (isgs). it was also found that rrabvs expressing ifn-λ could reduce the production of inflammatory cytokines in primary astrocytes and microgila cells, restrict the opening of the blood-brain barrier (bbb), and prevent excessive infiltration of inflammatory cells into the brain, which could be responsible for the neuronal damage caused by rabv. consistently, ifn-λ was found to maintain the integrity of tight junction (tj) protein zo- of bbb to alleviate neuroinflammation in a transwell model. our study underscores the role of ifn-λ in inhibiting rabv infection, which potentiates ifn-λ as a possible therapeutic agent for the treatment of rabv infection. rabies causes acute incurable encephalitis and is responsible for more than , human deaths each year, thus posing a severe threat to public health [ ] . rabies virus (rabv) has a non-segmented, recombinant rabv strain b c was generated from cvs-b c, which was attenuated from the challenge virus standard (cvs- ) in baby hamster kidney (bhk- ) cells [ ] . the cloned cell line bsr cells were derived from bhk- cells [ ] , which were cultured in dulbecco's modified eagle's medium (dmem) (gibco, grand island, ny, usa) containing % fetal bovine serum (fbs) (gibco, grand island, ny, usa) and % antibiotics (penicillin and streptomycin) (beyotime, wuhan, china). mouse brain capillary endothelial cell line b.end cells (atcc-crl- ) were cultured in dmem containing % fbs and % antibiotics (penicillin and streptomycin). mouse neuroblastoma (na) cells [ ] were maintained in rpmi medium (mediatech, herndon, va, usa) containing % fbs and % antibiotics (penicillin and streptomycin). african green monkey kidney cells (vero, atcc-ccl- ) were cultured in dmem containing % fbs and % antibiotics (penicillin and streptomycin). hek- t cells (atcc-crl- ) were maintained in rpmi medium supplemented with % fbs and % antibiotics (penicillin and streptomycin). na cells or vero cells were seeded into -well plates and cultured for h, and then infected with b c at a multiplicity of infection (moi) of . . at hpi, the cells were treated with or ng/ml of ifn-λ or ifn-λ (bd biosciences, san jose, ca, usa) by addition into the culture medium. at indicated time points after treatment, virus titers in the supernatant were measured. primary mixed glial cell cultures were established as described previously [ ] . briefly, brain tissues from -day-old balb/c mice were dissociated by repeated pipetting and then passed through a -nm nylon mesh (corning, ny, usa). the cells were washed once in cold pbs and cultured in dmem (with high glucose) supplemented with % fbs and % penicillin-streptomycin. the medium was changed on days , , and . on day , the flasks were shaken at rpm for h to remove any non-adherent cells (mainly microglia). the remaining adherent astrocytes were detached with trypsin-edta and then plated again for further experiments. the purity of the isolated astrocytes for further studies was greater than %, which was examined by immunohistochemistry using the anti-gfap antibody. primary microglia cells were prepared from cerebral cortices of -day-old balb/c mice as described previously [ ] . briefly, brain tissues were collected from the mice, and the cortex was dissected and minced in pbs containing . % trypsin for digestion at • c for min with a shake every min. after digestion, the dmem supplemented with % fbs and dnase i were added and incubated for min. then the isolated cells were resuspended with dmem supplemented with % fbs and the cell mass were removed by µm cell sieve filtration. finally, the isolated primary microglia cells were incubated at • c for - days and change the culture medium with fresh dmem containing % fbs at day . the purity of the isolated primary microglia for further studies was greater than %, which was examined by staining the cells with anti-iba antibody using ifa. the rrabv vector pb c was constructed by inserting the genome of cvs-b c into the mammalian expression vector pcdna . as described previously [ ] . a transcription unit containing bsiwi and nhei restriction sites was inserted between the g-and l-coding sequences by deleting the pseudogene. the rb c-ifnλ and rb c-ifnλ cdna clones were generated from pb c as previously described [ ] . briefly, the pb c vector was digested with bsiwi and nhei (neb, ipswich, ma) between the g and l genes. murine ifnλ- / cdnas were prepared by rt-pcr amplification using template rna isolated from vsv-infected mouse lung tissues [ ] . the ifnλ- and ifnλ- genes were then inserted into pb c, generating pb c-ifnλ and pb c-ifnλ , respectively. pcr primers are listed in table . the full length infectious clones (pb c-ifnλ and pb c-ifnλ ) and four helper plasmids (expressing genes n, p, g, and l from the parent virus b c) were separately transfected into bsr cells using superfect transfection reagent (qiagen, valencia, ca, usa) according to procedures described in previous studies [ ] . after incubating for days, the culture medium was harvested and then examined for the presence of rescued rrabvs using fitc-conjugated anti-rabv n antibodies, and the specific fluorescence would be observed under an olympus ix fluorescence microscope if the virus is successfully rescued. table . primers for construction and rescue of recombinant rabies viruses (rabvs) expressing murine ifn-λ. sequence ( - ) bsiwi and nhei sites are underlined. fluorescence morphologies were determined using a direct fluorescent antibody assay as previously described [ ] . na cells were infected at a low moi ( . ) with different rrabvs, overlaid with semisolid medium containing % agar, and incubated at • c for h. the agar was removed and the adhesive cells were stained with fitc-labeled rabv n-specific antibody. twenty fluorescent foci were examined to calculate the number of infected cells per fluorescent focus by using image j software [ ] . virus titers were determined using a direct fluorescent antibody assay as previously described [ ] . briefly, a series of -fold dilutions of the virus were prepared and used to inoculate bsr cells in -well microplates. the inoculations were performed in quadruplicate and then incubated at • c for h. after incubation, the cells were fixed with % ice-cold acetone and stained for h with fitc-conjugated rabv n protein-specific antibodies. antigen-positive foci were observed under an olympus ix fluorescence microscope, and virus titers were calculated and presented as focus-forming units/ml (ffu/ml). elisa was performed to quantify the amount of ifnλ- / in na cell culture supernatants. the assays were performed using commercially available mouse ifnλ- / elisa kits (raybiotech, atlanta, ga, usa), following the manufacturer's instructions. the samples (tissues or cells) were collected on ice and homogenized in trizol (invitrogen). total rna was isolated and used for qrt-pcr. briefly, complementary dna (cdna) was prepared with µg rna as template using a first-strand cdna synthesis kit (toyobo). the thermocycler conditions were used for cdna synthesis ( min at • c, min at • c, and min at • c). each qpcr was conducted in duplicate with approximately ng dnase-treated rna and nm primer pairs, using a one-step sybr green qrt-pcr mix kit (toyobo). primers are listed in table . the following conditions describe real-time pcr amplification ( s at • c; s at • c, s at • c, and s at • c for cycles; s at • c), and the cycle threshold (ct) values were recorded. a standard curve was generated from serially diluted plasmids carrying a rabv n gene and the copy numbers of viral messenger rna of rabv n gene (mrna) and viral genome rna (vrna) were normalized to µg of total rna [ ] . to quantify the level of vrna, the primer vrna-f was used for reverse transcription, while the primer n mrna-r was used for reverse transcription of n-mrna quantification. the ct value was inversely correlated with the mrna concentration, and each ct unit represented a twofold change in the mrna concentration. the mrna levels of chemokines/cytokines and tj proteins were normalized to β-actin mrna levels. the results were expressed as fold change relative to mrna levels detected in mock-infected controls. table . primers for qrt-pcr. sequence ( - ) cell pellets were lysed in ice-cold ripa lysis buffer containing protease inhibitor cocktail (composed of a proprietary mix of aebsf, aprotinin, bestatin, e , leupeptin, and pepstatin a to promote broad spectrum protection against endogenous proteases). the mixture was homogenized and centrifuged at , × g for min at • c. after centrifugation, insoluble material was removed, and total protein concentration in the supernatant was measured using a bca protein assay kit (beyotime, wuhan, china). each sample was subjected to polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane (bio-rad, richmond, ca, usa), and blocked for h at • c with % bovine serum albumin (bsa) in tris-buffered saline with . % tween- (tbst). membranes were then incubated overnight with primary antibodies. after extensive washing with tbst, the membranes were incubated with secondary antibodies. antibody binding was visualized using enhanced chemiluminescence reagents (beyotime). bands were quantified using imagej (nih, bethesda, md, usa). the values represent the relative immunoreactivity of each protein, normalized to the respective loading control. five-week-old female balb/c mice (n = ) were inoculated under isoflurane anesthesia. the groups received the following treatments: (a) inoculated intramuscularly (i.m.) with a µl volume of × ffu; (b) infected intradermally (i.d.) in both ears with a dose of . × ffu in ul dmem; (c) inoculated intranasally (i.n.) in µl of a solution containing ffu of b c, rb c-ifnλ , or rb c-ifnλ or mock-infected with dmem. body weight loss, clinical signs, and survivor numbers were recorded daily for days. the animals were scored for clinical signs as follows: , normal mouse; , disorder movement; , ruffled fur; , trembling and shaking; , paralysis; , dead. all animals were humanely euthanized at the end of the experiment. t cells ( × cells per well) were seeded into -well plates. the cells were transfected with ng of luciferase reporter plasmids (a gift of prof. xiao shaobo from huazhong agricultural university) [ ] , together with pcaggs-ifnλ or pcaggs, using lipofectamine (thermo scientific, shanghai, china). in parallel, ng of prl-tk renilla luciferase reporter plasmid (promega, madison, wi, usa) was transfected to normalize transfection efficiency. twenty-four hours after transfection, the cells were infected with b c, rb c-ifnλ , and rb c-ifnλ for h. luciferase activity in total cell lysates was measured using a dual-specific luciferase reporter assay system (promega, madison, wi, usa). primary astrocytes were mock infected or infected with rrabvs at a moi of . cell supernatants were collected at hpi. inflammatory cytokines (il- β, il- , il- a, ifn-γ, kc, tnf-α, and vegf) were quantified in the mock-and rabv-infected cell supernatants using a quantibody mouse cytokine array kit (raybiotech, norcross, ga, usa), according to the manufacturer's protocol. the array was scanned using a genepix b (molecular devices, axon instruments, silicon valley, ca, usa). data were collected using the genepix pro application at a photomultiplier tube (pmt) gain ranging from to . a gain of generated the optimal standard curve, and the results of this scan were analyzed using q-analyzer for qam-cyt- (raybiotech). a transendothelial permeability assay was conducted as previously described with minor modifications [ ] . b.end cells were cultured on transwell filters (pore size . µm) until reaching % confluence. after treatments, fitc-dextran- ( kda; sigma-aldrich, st. louis, mo, usa) was applied apically at mg/ml for min. samples were then removed from the lower chamber for fluorescence measurements with a fluorimeter (excitation, nm; emission, nm). animals were anesthetized with ether and were perfused by intracardiac injection of phosphate-buffered saline (pbs) as described previously [ ] . mouse brains were fixed with % paraformaldehyde for h at • c and then washed with pbs. brain tissues were harvested and embedded in paraffin for coronal sections. to detect rabv, nonspecific binding was blocked with % goat serum, and then sections were incubated with dapi and fitc-conjugated antibodies against the rabv n protein. to detect inflammatory cells, harvested sections were incubated with primary antibodies against cd at the concentrations indicated in the manufacturer's guidelines, and then incubated with biotinylated secondary antibodies. the sections were observed under an olympus ix fluorescence microscope. for immunofluorescence, b.end cells were seeded on coverslips. after forming a confluent monolayer, they were suspended in medium containing supernatants from infected astrocytes. the cells were subsequently fixed with % paraformaldehyde (pfa), permeabilized with . % triton x- , and then incubated with rabbit anti-zo- polyclonal antibodies. finally, they were incubated with secondary antibody conjugated with alexa fluor , and with dapi for nuclear counterstaining. cells were imaged using a laser confocal microscope (leica, germany). bbb permeability was determined by measuring sodium fluorescein uptake as described previously [ , ] . briefly, µl of mg/ml sodium fluorescein was injected intraperitoneally into each mouse. peripheral blood was collected after circulation for min, and pbs-perfused brains were then harvested. the recovered serum was mixed with an equal volume of % trichloroacetic acid (tca) and then centrifuged for min. the volume of the supernatant was adjusted to µl with m naoh and . % tca. homogenized brain samples in cold . % tca were centrifuged for min at , × g to remove debris. the supernatant was adjusted to µl with addition of m naoh. fluorescence of serum and brain homogenate samples was measured using a spectrophotometer (biotek instruments, vt, usa) with excitation at nm and emission at nm. the amount of sodium fluorescein taken up into brain tissues is calculated as (µg of fluorescence cerebrum, cerebellum, or brain stem/mg of tissue)/(µg of fluorescence sera/ml of blood) to normalize values for blood levels of the dye at the time of tissue collection. data are expressed as fold differences between the amount of tracer in tissues from virus-infected mice and the amount in tissues from the uninfected control. all data were analyzed using graphpad prism (graphpad software, lnc., san diego, ca, usa). for the percent survival tests, kaplan-meier survival curves were analyzed using the log rank test. for the other data, an unpaired two-tailed t-test was used to determine whether differences were statistically significant. data were representative of two independent experiments. for all results, the following notations are used to indicate significant differences between groups: *, p < . ; **, p < . ; ***, p < . . to test whether ifn-λ restricts rabv replication in vitro, na or vero cells were infected with b c strain, and recombinant mouse ifn-λ or ifn-λ at ng/ml and ng/ml were added to treat the rabv infected cells at h post infection (hpi), and the virus titers, the levels of mrna of rabv n gene (n mrna) and viral rna (vrna) were measured at and h after the treatment. in na cells, as shown in figure a -c, the addition of ifn-λ or ifn-λ reduced the virus titers and the levels of n mrna and vrna at both time points. notably, at h after treatment with ng/ml of ifn-λ or ifn-λ , more than -fold decreases of virus titers were observed compared with mock-treated cells. in the vero cells, an ifn-α/β independent cell line, the addition of ifn-λ or ifn-λ reduced the virus titers and the levels of n mrna and vrna at h post treatment as shown in figure d -f. at h post treatment, no significant difference was observed in the virus titers and the levels of n mrna and vrna among all. these results suggest that ifn-λ and ifn-λ inhibits rabv replication in the two cell lines used. to further characterize the role of ifn-λ in rabv infection in the mouse model, recombinant rabvs (rrabvs) expressing murine ifn-λ or ifn-λ , designated as rb c-ifnλ and rb c-ifnλ respectively, were constructed as shown in figure a , and rescued as described previously [ ] . the insertion of both genes into the rabv genome was verified by rt-pcr and sequencing (data not shown). to detect whether the rrabvs could express ifn-λ or ifn-λ , na cells were infected with the rrabvs, and ifn-λ or ifn-λ were determined by elisa. as shown in figure b , elisa results indicate that ifn-λ and ifn-λ were well expressed in rb c-ifnλ and rb c-ifnλ infected cells, respectively, in a dose-dependent manner, whereas those expressed in b c did not. moreover, viral growth curves on bsr, na, and vero cells were depicted. as shown in figure c -e, compared with parent virus b c, more than -fold losses of viral titers were observed in rb c-ifnλ or rb c-ifnλ infected bsr or na cells at and hpi, and in rb c-ifnλ or rb c-ifnλ infected vero cells at all tested time points. additionally, consistent with the results of the growth curve, western blot experiments detecting rabv n protein showed that expression of ifn-λ or ifn-λ by the rrabvs reduced rabv n protein levels in infected na cells at hpi ( figure f) . furthermore, the effects of expression of ifn-λ or ifn-λ on virus spread were measured by observing the fluorescence morphologies of rrabv-infected cells and counting the number of rabv-positive cells using fluorescence microscopy. as a result, the fluorescence foci of cells infected with rb c-ifnλ or rb c-ifnλ were significantly smaller than those infected with b c ( figure g,h) , indicating that expression of ifn-λ suppresses the cell-to-cell spread of rabv. taken together, these results suggest that expression of ifn-λ or ifn-λ inhibits rabv replication and spread in infected cells. ) . the following notations were used to indicate significant differences between groups: *, p < . ; **, p < . ; ***, p < . ; ****, p < . . to further investigate whether ifn-λ restricts rabv infection in vivo, groups of five-week-old female balb/c mice were mock-infected with dmem or inoculated with b c, rb c-ifnλ , or rb c-ifnλ by different routes. the body weight losses of mice infected with rb c-ifnλ or rb c-ifnλ by the three routes (i.m., i.d., or i.n.) were lower than those infected with b c, as shown in figure a ,d,g. similarly, the clinical scores of mice infected with rb c-ifnλ or rb c-ifnλ by either route were lower than those infected with b c ( figure b,e,h) . consistently, significantly higher percentage of survivor ratios were observed in rb c-ifnλ or rb c-ifnλ infected mice than b c infected mice ( figure c ,f,i). it is worth noting that the most striking enhancement on survival rates among the three infection routes were observed in i.n. route-that % and % of mice i.n. infected with rb c-ifnλ (p = . ) and rb c-ifnλ (p = . ), respectively, survived, while % of b c i.n. infected mice died of rabies within days ( figure i ). all the mice exhibited rabv-related symptoms and death were confirmed by rt-pcr from brain tissues. the following notations were used to indicate significant differences between groups: *, p < . ; **, p < . ; ***, p < . . to investigate the viral load in the brains of infected mice, balb/c mice were i.n. infected with ffu of rrabvs, or mock-infected with dmem, and viral burdens were analyzed in different parts of the brain by qrt-pcr at , , , and days post infection (dpi). viral copy numbers were extrapolated by quantitating mrnas corresponding to the rabv n gene. as expected, viral copy numbers in the olfactory bulb, cerebrum, cerebellum, and brain stem of rb c-ifnλ or rb c-ifnλ infected mice were significantly lower than those infected with b c ( figure a ). additionally, viral antigen (rabv n protein) in different brain tissues was also detected using immunofluorescence staining. consistent with the qrt-pcr results, almost no (or under detection limit) viral antigens were observed in all the parts of the brains of rb c-ifnλ or rb c-ifnλ infected mice, while an obviously positive fluorescent signal was observed in all parts of the brains of detected mice that were infected with b c ( figure b ). all the above data demonstrate that ifnλ significantly reduces the pathogenicity of rabv in mice. . ifn-λ restricts rabv replication in the mouse brain. five-week-old female balb/c mice were inoculated i.n. with ffu of b c, rb c-ifnλ , rb c-ifnλ , or dmem alone. (a) n mrna copy number was measured in mouse olfactory bulb, cerebrum, cerebellum, and brain stem at , , , and dpi by qrt-pcr (n = ). (b) mouse brain sections (n = ) were stained with fitc-labeled rabv n-specific antibody to detect the distribution of viral antigen in the central nervous system (cns) parenchyma (shown in green); nuclei stained with ', -diamidino- -phenylindole (dapi) are shown in blue. representative images were acquired at × magnification (scale bar = µm). error bars represent the se. the following notations were used to indicate significant differences between groups: *, p < . ; **, p < . ; ***, p < . ; ****, p < . . as a typical neurotropic virus, rabv mainly infects neurons in the brain. therefore, to define the signaling pathway by which ifn-λ restricts rabv infection, a luciferase reporter assay was carried out in na cells as described in the methods section. the results suggest that expression of ifn-λ or ifn-λ could activate ifnα , ifnβ, and isg -isre, but down-regulated the expression of nuclear factor κb (nf-κb) ( figure a) . furthermore, the mrna levels of ifn-α, ifn-β, stat , interferon-induced protein with tetratricopeptide repeats (ifit ), and ifn-inducible gtpase (iigp ) in different rrabv infected na cells were detected by qrt-pcr. significantly higher levels of ifn-α , ifn-α , ifn-β, stat , ifit , and iigp were detected in the cells infected with rb c-ifnλ or rb c-ifnλ than those in cells infected with b c. meanwhile, the mrna levels of nf-κb (p ) and tnf-α were significantly decreased in na cells infected with rb c-ifnλ or rb c-ifnλ compared with those infected with b c ( figure b) . additionally, expression of isgs (ifit and iigp ) and activation of jak-stat pathway, phosphorylation of stat , were also measured by western blotting assay at hpi, and high levels of phosphorylated stat , ifit and iigp were detected in rb c-ifnλ or rb c-ifnλ infected cell. these data demonstrate that ifn-λ enhances the expression of isgs via activating jak-stat pathway, and thus inhibits rabv replication. figure . ifn-λ activates stat / and enhances the production of isgs. (a) na cells were transfected with plasmids encoding ifn-α , ifn-β, isg -isre or nf-κb firefly luciferase reporter, respectively. after h, cells were left uninfected or were infected for h with different rrabvs. luciferase assays were performed to analyze the promoter activity of isre, ifn-α , ifn-β and nf-κb, respectively. luciferase reporter activity was expressed as fold change that normalized to renilla luciferase activity. (b) na cells were infected with b c, rb c-ifnλ , or rb c-ifnλ at a moi of . at hpi, total rna was isolated and ifn-α , ifn-α , ifn-β, stat , ifit , iigp , nf-κb (p ), and tnf-α mrna levels were analyzed by qrt-pcr. (c) protein levels of stat , ifit , ifit , iigp , and β-actin in different rrabv-infected na cells were measured by western blot at hpi. error bars represent the se (n = ). the following notations were used to indicate significant differences between groups: *, p < . ; **, p < . ; ***, p < . ; ****, p < . . as is known, astrocytes and microglia cells play an important role in innate immunity and neuroinflammation in the cns. hence, primary astrocytes and microglia cells were isolated as described in the materials and methods section and infected with different rrabvs at a moi of , and the production of several inflammatory cytokines was measured in the infected astrocytes and microglia cells. viral titers in the supernatants of astrocytes or microglia cells incubated with b c, rb c-ifnλ , or rb c-ifnλ were nearly equal ( figure a,b) . the supernatants of astrocytes were then applied to a protein array to quantify the production of specific cytokines, and the mrna levels of gm-csf, il- β, il- , kc, tnf-α, and vegf were quantified in microglia cells by qrt-pcr. as shown in figure c , pro-inflammatory cytokines, including tnf-α, il- , il- a, il- β, chemokine (c-x-c motif) ligand (cxcl /kc), and vascular endothelial growth factor (vegf, which is related to bbb opening), were significantly lower in astrocytes infected with rb c-ifnλ or rb c-ifnλ than those infected with b c. similarly, the mrna levels of gm-csf, il- β, il- , kc, tnf-α, and vegf were significantly decreased in microglia cells infected with rb c-ifnλ or rb c-ifnλ than those infected with b c ( figure d ). together, these results suggest that ifn-λ represses the production of inflammatory cytokines induced by rabv infection. figure . ifn-λ reduces the production of inflammatory cytokines in primary astrocytes and microglia cells. astrocytes and microglia cells isolated from -day-old suckling mice were infected with b c, rb c-ifnλ , or rb c-ifnλ at a moi of , and cell supernatants and lysates were collected at hpi for astrocytes and microglia cells, respectively, for the determination of indicated cytokines. the rrabv titers were measured using a focus-forming assay and are expressed as ffu/ml on astrocytes (a) and microglia cells (b). concentrations of the indicated cytokines in astrocyte supernatants were measured using a cytokine array (c) and microglia cells lysates were measured by qrt-pcr (d). error bars represent the se (n = ). the following notations were used to indicate significant differences between groups: *, p < . ; **, p < . ; ***, p < . ; ****, p < . . it is possible that the expression of ifn-λ in the cns can subdue the neuroinflammatory response and block the elevation of bbb permeability due to the low expression level of inflammatory cytokines and vegf in rb c-ifnλ or rb c-ifnλ infected astrocytes. meanwhile, the enhancement of bbb permeability that associated with rabv infection is caused by cytokines and the infiltration of inflammatory cells, as demonstrated in a previous study [ ] . to test this hypothesis, six-week-old female balb/c mice were i.c. infected with rrabvs. to exclude the effect of different virus loads on the regulation of bbb permeability and neuroinflammation, the brains of mice infected by different rrabvs were harvested to analyze viral burden by qrt-pcr. at , , and dpi, mrna levels of rabv n were nearly equal in different brain regions of mice infected by b c, rb c-ifnλ , or rb c-ifnλ ( figure a ). sodium fluorescein was injected into the mice via the tail vein for measurement of bbb permeability. at and dpi, no significant difference in the amount of sodium fluorescein uptake was detected among the three groups. at dpi, leakage of sodium fluorescein from the peripheral circulation into the cerebrum, cerebellum, and brain stem in b c-infected mice was significantly higher than those in mice infected with rb c-ifnλ or rb c-ifnλ ( figure b ). moreover, the brain sections were then stained with anti-cd antibody to observe and quantify the infiltration of cd + cells induced by different rrabvs infection. less positive signal was observed in different parts of brain from mice infected with rb c-ifnλ or rb c-ifnλ than those from mice infected with b c ( figure c ). significantly more cd + lymphocytes were found in the cerebral cortex, hippocampus, hypothalamus, cerebellum, and brain stem from the mice infected with b c than those infected with rb c-ifnλ or rb c-ifnλ at dpi ( figure d ). these results indicate that ifn-λ declines the bbb permeability to prevent excessive infiltration of inflammatory cells into the cns during rabv infection. figure . ifn-λ decreases bbb permeability and alleviates neurologic inflammation in the mouse brain. six-week-old female balb/c mice were inoculated i.c. with ffu of rb c, rb c-ifnλ , rb c-ifnλ , or dmem (mock). (a) at , , and dpi, mice (n = ) were euthanized, and the brains were harvested, and viral loads were determined by qrt-pcr. (b) the change in bbb permeability was assessed by measuring the amount of sodium fluorescein uptake in the cerebrum, cerebellum and brain stem using a spectrophotometer after intraperitoneal administration at , , and dpi (n = ). (c) for the assessment of neurologic inflammation, six-week-old female balb/c mice were inoculated i.c. with ffu of b c, rb c-ifnλ , rb c-ifnλ , or dmem (mock). at dpi, mice brains were collected and embedded in paraffin. the sections were then stained with anti-cd antibody to quantify inflammation. the scale bars represent µm (n = ). (d) cd + lymphocytes from at least three different randomly selected areas of each brain region were counted. error bars represent the se. the following notations were used to indicate significant differences between groups: *, p < . ; **, p < . . to explore the mechanism by which ifn-λ reduces bbb permeability, a transwell model was performed. mouse brain capillary endothelial (b.end ) cells were cultured in the upper chamber of a transwell insert, with supporting astrocytes in the lower chamber. the b.end cells were then treated for h with extracts from the supernatants of astrocytes infected with different rrabvs, and the expression of the tj proteins zo- and occludin were then analyzed by qrt-pcr and western blot. both mrna ( figure a ) and protein expression ( figure b ) of zo- in cells treated with supernatants from b c-infected astrocytes was significantly lower than that those incubated with the supernatants from rb c-ifnλ or rb c-ifnλ infected astrocytes. additionally, supernatant-treated cells were also stained with anti-zo- polyclonal antibodies and imaged by a laser confocal microscope to assess the integrity of zo- . the rb c-infected astrocytes supernatants caused dissociation of zo- , while those treated with rb c-ifnλ or rb c-ifnλ infected astrocytes supernatants reduced the effect ( figure c,d) . to further evaluate the integrity of the endothelial monolayer, fitc-dextran , was added to the upper chamber of a transwell insert and the fluorescence in the lower chamber was monitored. as shown in figure e , the leakage of fitc-dextran , was significantly lower when treated with supernatants from rb c-ifnλ or rb c-ifnλ infected astrocytes than that treated with b c infected astrocytes supernatant. these data suggest that ifn-λ maintains the integrity of tj proteins, resulting in the decrease in the bbb permeability during rabv infection. end cells. mouse brain capillary endothelial (b.end ) cells were co-cultured for h with extracts from supernatants of astrocytes that had been mock-infected with dmem or infected with b c, rb c-ifnλ , or rb c-ifnλ at a moi of . expression of zo- and occludin was detected by qrt-pcr (a) and western blot (b). the integrity of tj protein zo- was measured by confocal microscopy (c). relative mean fluorescence intensity for the regions of interest (roi) was determined using image j (d). b.end cells were cultured on transwell inserts and treated with extracts from supernatants of astrocytes that had been mock infected with dmem or infected with b c, rb c-ifnλ , or rb c-ifnλ at a moi of . at hpi, fitc-dextran- was added into the upper insert of the transwell and then the permeation of fitc-dextran- into the lower chamber was measured using a fluorimeter (excitation, nm; emission, nm) (e). error bars represent the se (n = ). the following notations were used to indicate significant differences between groups: *, p < . ; **, p < . ; ***, p < . ; ****, p < . . ifn-λ signaling through the ifnlr receptor on intestinal epithelial cells (iecs) induces antiviral effectors such as isgs via stat /stat /irf -mediated transcription, thereby boosting defenses against intestinal viruses such as rotavirus, reovirus, and norovirus [ ] [ ] [ ] [ ] . the neurotropic west nile virus (wnv) is inhibited in the cns by ifn-λ through a mechanism that modulates endothelial cell tight junction integrity [ ] . in our study, we found that ifn-λ curtails the replication of another neurotropic virus, rabv, in cells. moreover, ifn-λ also reduces rabv pathogenicity in infected mice by different infection routes, especially i.n. route. ifn-λ has been reported to be particularly important for innate pathogen defense at mucosal barriers [ ] , which provides a possible explanation for a better survivor rate in rrabv-expressing ifn-λ-infected mice by i.n. route. in addition, ifn-λ is expressed in a tissue-specific manner and its receptor ifnlr exhibits a much more tissue-specific expression pattern, with a preference for epithelial cells [ , ] . during intranasal challenge with rabv, the expression of ifnlr by nasal epithelial cells is necessary for the responsiveness of the tissue to ifn-λ and the suppression of viral replication in nasal mucosa. therefore, ifn-λ has evolved as a specific factor that can prevent the invasion of some neurotropic viruses through nasal epithelial cells. the ability of ifn-λ to induce isg expression in a targeted set of epithelial cells also suggests that ifn-λ promotes a focused antiviral or immunomodulatory response [ , ] . the main antiviral functions of ifn-λ have been linked to the activation of the stat / signaling pathway [ , ] . activation of stat / is required for optimal transcription of isgs, which establish antiviral defenses. ifn-α/β is also engaged in antiviral processes. cooperation between ifn-α/β and ifn-λ further increases stat / -activation. in our study, isre activity was upregulated by the expression of ifn-λ during rabv infection, indicating that ifn-λ plays a positive role in activating isre, which is involved in the downstream production of isgs [ ] . isgs such as ifit and iigp were reported to restrict rabv replication [ , ] . the two isgs were elevated after the infection of rrabvs expressing ifn-λ in our study, indicating that ifn-λ could activate the stat / signaling pathway during rabv infection, resulting in the downstream production of isgs to restrict rabv infection. encephalitis induced by lab-attenuated rabv is characterized by obvious cns inflammation [ , , ] . it has previously been reported that rabv induced inflammation in microglia cells mainly through p and nf-κb pathways [ ] . the results of luciferase reporter assay in our study implied that ifn-λ expression could suitably suppress the activation of nf-κb. subsequently, production of pro-inflammation cytokines was remarkably decreased, as expected. consistent with these observations, one previous study showed that excessive expression of ifn-λ caused by iav infection, whereas the degradation of iκb and the activation of nf-κb was significantly decreased in the infected cells. in contrast, disruption of ifn-λ signaling pathway resulted in the activation of nf-κb [ ] . on the other hand, ifn-λ that is induced in dcs and macrophages does not augment proinflammatory cytokine production during viral infection [ ] . type i ifn provides antiviral resistance but also induces proinflammatory responses essential for countering infection [ ] . previous study demonstrated that ifn-λ could upregulate suppressor of cytokine signaling (socs ) and socs , which reduce the devastating effects of excessive inflammation [ ] . consistently, in our study, it was found that ifn-λ could decrease the nf-κb activation and further alleviate inflammation via suppression of neutrophil infiltration and proinflammatory cytokines such as tnf-α, il- a, il- α, il- β, il- , il- , and vegf during rabv infection, all of which contribute to bbb breakdown. the role of ifn-λ in nf-κb activation and inflammation production in the context of rabv infection appears to be important, but the detailed mechanism of this process remains to be fully explored. although ifn-λ decreases expression of proinflammatory cytokines, it does not require the activation of inflammation to modulate bbb permeability [ ] . a previous study demonstrated that stat / signaling or protein synthesis is not required for endothelial barrier tightening [ ] . indeed, in our study, ifn-λ signaling was found to maintain the expression of tj protein zo- and protect its integrity, which tightens the endothelial barrier. the tightened barrier also prevents rabv transit across epithelial surfaces, such as the nasal mucosa barrier, which could be another explanation for the strongest attenuation of rabv pathogenicity after i.n. infection. it was reported that approximately kb of dna at gene loci for ifnl and ifnl have nucleotide sequence identity greater than % for the whole genomic region in both human and mouse, indicating that the function of ifn-λ and ifn-λ could be very similar [ ] . consistently, the results in affecting rabv replication and pathogenicity are similar between rb c-ifnλ and rb c-ifnλ in our study. additionally, non-murine cell line vero cells, which are supposed to be an ifn-α/β independent cell line, were also used in our study to exclude the possible involvement of type i ifn during the ifn-λ inhibiting rabv replication. it has been reported that both mouse ifn-λ and ifn-λ were capable of up-regulating mhc class i antigen expression in several human cell lines and induced antiviral protection in mouse b cells or human ht cells infected with vsv [ ] . these data indicate that the mouse ifn-λ could potentially be functional in cells derived from species other than mouse. in conclusion, our data indicate that ifn-λ, either ifn-λ or ifn-λ , can restrict rabv infection by inducing isgs, limiting blood-brain barrier permeability to 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protein involved in rabies virus infection glycoprotein-mediated induction of apoptosis limits the spread of attenuated rabies viruses in the central nervous system of mice attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune responses in the central nervous system rabies virus-induced activation of mitogen-activated protein kinase and nf-kappab signaling pathways regulates expression of cxc and cc chemokine ligands in microglia suppression of interferon lambda signaling by socs- results in their excessive production during influenza virus infection interferon-lambda mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness pathogenic potential of interferon alphabeta in acute influenza infection intracellular protein therapy with socs inhibits inflammation and apoptosis the role of genomic data in the discovery, annotation and evolutionary interpretation of the interferon-lambda family characterization of the mouse ifn-lambda ligand-receptor system: ifn-lambdas exhibit antitumor activity against b melanoma we thank shuaipeng he (laboratory animal center, huazhong agricultural university) for excellent mice management. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -vosu y j authors: norlander, allison e.; peebles, r. stokes title: innate type responses to respiratory syncytial virus infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: vosu y j respiratory syncytial virus (rsv) is a common and contagious virus that results in acute respiratory tract infections in infants. in many cases, the symptoms of rsv remain mild, however, a subset of individuals develop severe rsv-associated bronchiolitis. as such, rsv is the chief cause of infant hospitalization within the united states. typically, the immune response to rsv is a type response that involves both the innate and adaptive immune systems. however, type cytokines may also be produced as a result of infection of rsv and there is increasing evidence that children who develop severe rsv-associated bronchiolitis are at a greater risk of developing asthma later in life. this review summarizes the contribution of a newly described cell type, group innate lymphoid cells (ilc ), and epithelial-derived alarmin proteins that activate ilc , including il- , il- , thymic stromal lymphopoietin (tslp), and high mobility group box (hmgb ). ilc activation leads to the production of type cytokines and the induction of a type response during rsv infection. intervening in this innate type inflammatory pathway may have therapeutic implications for severe rsv-induced disease. respiratory syncytial virus (rsv) is an important and common cause of infant acute respiratory tract infections and is the primary cause for infant hospitalization in the united states each year [ ] . almost all children are infected with rsv by the age of [ ] . risk factors that predispose infants to severe rsv-associated bronchiolitis include premature birth, young age, immune deficiency, underlying heart or lung disease, as well as neuromuscular disorders [ ] . age at time of infection is another important risk factor for severe rsv-mediated bronchiolitis, with children aged between and months being at the greatest risk for complications and hospitalizations [ ] . as lasting immunity to rsv infection is not developed, individuals may experience several rsv infections throughout the course of their lives [ ] . in healthy adults, this usually manifests as a mild illness or a 'cold' [ ] . however, rsv causes severe illness in elderly patients, especially those with preexisting conditions, including individuals who are immunocompromised or those with chronic heart and lung conditions [ ] [ ] [ ] . illnesses induced by rsv infection can encompass both the upper and lower respiratory tract [ ] . several days after infection, upper respiratory tract symptoms, such as rhinorrhea and nasal congestion, typically develop. in some individuals, the disease may progress to the lower respiratory tract, resulting in coughing and wheezing, or bronchiolitis, with the severity of disease among infants being quite variable [ ] . severe rsv-induced bronchiolitis results in necrosis and the sloughing of epithelial cells into the airways, airway mucus, edema, and peribronchiolar inflammation, cumulatively resulting in airway obstruction [ ] . bronchiolitis and viral pneumonia stemming from rsv infection result in substantial morbidity and mortality in some cases. current therapeutic options are limited, and treatment is predominantly supportive [ ] . there is only one drug, ribavirin, currently fda approved for the treatment of severe rsv-induced respiratory infections. ribavirin is a guanosine analog with antiviral properties demonstrated against several viruses, including zika, hepatitis c, and rsv, making ribavirin a broad-spectrum antiviral [ ] [ ] [ ] . ribavirin functions through inhibition of viral replication [ ] . however, due to its high cost, lack of specificity, and low ability to control the symptoms, its clinical application is limited [ , ] . palivizumab is approved by the fda for immunoprophylaxis for rsv. palivizumab is a monoclonal antibody that is specific for an epitope located on the f protein [ ] . when given prophylactically, palivizumab successfully reduced hospitalizations of children caused by rsv-induced illness [ , ] . again, due to its high cost, the use of palivizumab clinically is limited and is reserved for use in high-risk populations that include infants that are premature, have low birth weight, have underlying cardiopulmonary diseases, or are immunocompromised [ ] [ ] [ ] . as a result of the lack of effective post-infection therapeutics, complications from severe rsv infection account for a substantial clinical and economic burden in developed and developing nations alike [ ] [ ] [ ] . both the innate and adaptive immune systems participate in the antiviral response to rsv. the immune response to rsv is generally classified as a type response. important aspects of the type immune response to rsv include marked production of interferon-γ (ifn-γ) by natural killer (nk) cells and natural killer t (nkt) cells of the innate immune system, and rsv-epitope specific cd + t cell mediated viral clearance [ ] [ ] [ ] . interestingly, children who developed more severe rsv-mediated bronchiolitis as infants were at elevated risk of developing asthma later in life [ ] [ ] [ ] . several factors can influence the severity of rsv infection and the development of rsv-mediated bronchiolitis, one of which includes gender [ ] . gender is also a risk factor for childhood asthma [ ] . male sex is a risk factor for severe rsv illness and boys are twice as likely as girls to develop childhood asthma; to date, bronchiolitis has only been identified as a risk factor for the development of wheeze through adolescence in boys [ , , ] . corticosteroids, a therapy often used to mitigate type immune responses in asthma, were ineffective at reducing rsv-induced disease severity and preventing hospitalizations [ ] [ ] [ ] . studies of rsv infection in mice reveal that type cytokines, such as interleukin (il)- , il- , and il- , that will be discussed below, may also be expressed during the course of infection. expression of type cytokines during rsv infection may result in airway mucus and wheezing that are associated with increased disease severity [ , ] . different strains of rsv have been shown to induce differential immune responses in mice in regard to the types of cytokines the host produces. rsv a , a strain widely used in in vitro and in vivo mouse experiments, induced an almost exclusive predominant th response in balb/c mice. however, other clinical isolates of rsv induced greater type cytokine production in balb/c mice [ ] [ ] [ ] . whether such viral strain-specific type immune responses occur in human infection is controversial and is a subject of ongoing investigation. cd + th cells are classically associated with the production of the type cytokines il- , il- , and il- . il- signaling promotes goblet cell mucin expression and the development of airway hyperresponsiveness (ahr), or narrowing of airways in response to stimuli [ , ] . il- signaling recruits eosinophils to the lungs and enhances their survival [ ] . eosinophil infiltration during rsv infection is controversial, some studies have found airway eosinophilia, while others have not [ , ] . il- signaling drives th cell differentiation and promotes b cell antibody class switch to ige [ , ] . however, group innate lymphoid cells (ilc ), a newly described cell type, are also a significant source of il- and other type cytokines in several different pulmonary diseases [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, the epithelial-derived cytokines interleukin- (il- ), interleukin- (il- ), and thymic stromal lymphopoietin (tslp), as well as the innate immune cell-derived cytokine high mobility group box (hmgb ), activate and induce ilc function, thereby promoting the progression of type -mediated pulmonary diseases ( figure ) [ ] [ ] [ ] [ ] [ ] [ ] . uncovering the contributions of these epithelial-and-innate-derived cytokines and ilc to the pathogenesis of severe rsv infection has important implications for treatment. there are currently several biologic therapies that target different mediators of innate type inflammation that are either fda approved or in clinical trials that could be repurposed for the treatment of rsv-induced bronchiolitis. one such drug is dupilumab, an il- receptor alpha (il- rα) antagonist. both the receptors for il- and il- contain the il- rα subunit, thus, blocking this subunit could reduce signaling by both il- and il- . dupilumab is fda approved for the treatment of chronic rhinosinusitis with nasal polyps, moderate-to-severe asthma, as well as eczema and moderate-to-severe atopic dermatitis in adolescents [ ] [ ] [ ] . additionally, an anti-il antibody and an anti-il receptor (st ) antibody, in separate phase ii clinical trials, have recently been evaluated for their ability to treat asthma [ ] [ ] [ ] . a second anti-il- antibody has completed a phase iia trial [ , ] . this review will focus on summarizing recently published research highlighting the contributions of ilc , il- , il- , hmgb , and tslp to rsv pathogenesis. articles were identified through a pubmed search for key terms (rsv, ilc , il- , il- , hmgb , tslp) and any combination thereof that was conducted on april , . articles published after were included in this review. is dupilumab, an il- receptor alpha (il- rα) antagonist. both the receptors for il- and il- contain the il- rα subunit, thus, blocking this subunit could reduce signaling by both il- and il- . dupilumab is fda approved for the treatment of chronic rhinosinusitis with nasal polyps, moderateto-severe asthma, as well as eczema and moderate-to-severe atopic dermatitis in adolescents [ ] [ ] [ ] . additionally, an anti-il antibody and an anti-il receptor (st ) antibody, in separate phase ii clinical trials, have recently been evaluated for their ability to treat asthma [ ] [ ] [ ] . a second anti-il- antibody has completed a phase iia trial [ , ] . this review will focus on summarizing recently published research highlighting the contributions of ilc , il- , il- , hmgb , and tslp to rsv pathogenesis. articles were identified through a pubmed search for key terms (rsv, ilc , il- , il- , hmgb , tslp) and any combination thereof that was conducted on april , . articles published after were included in this review. , and il- by these cells. these alarmin proteins released from rsv-infected epithelial cells can activate group innate lymphoid cells (ilc ) to produce the type cytokines il- and il- . il- is the most important eosinophil growth, differentiation, and survival factor. il- has many immunologic and physiologic effects, including promoting airways responsiveness and mucous cell metaplasia. ilc are a recently described cell type of the innate immune system. these cells do not express rearranged antigen receptors, unlike t and b cells, but are able to produce type cytokines at high levels when activated [ , ] . ilc are directly activated by the epithelial-derived cytokines tslp, il- , il- , and hmgb . as such, ilc express the corresponding receptors for these cytokines that include tslpr, st , il- r, and the receptor for advanced glycation end products (rage), as well as toll like receptors (tlr) and [ , [ ] [ ] [ ] [ ] . ilc and the cytokines they produce contribute significantly to the pathology of asthma and allergic diseases [ , ] . infants hospitalized with severe rsv infection had detectable ilc in their nasal aspirates and their frequency was greater than in infants with moderate disease [ ] . moreover, levels of type and epithelial-derived cytokines (il- and tslp) were significantly higher in the nasal aspirates of infants with severe disease than in those with moderate disease [ ] . furthermore, infants with lower gestational age were confirmed to be at greater risk for severe rsv infection, as were infants with higher levels of il- in their nasal aspirates, when the relationships of individual variables examined in the study and disease severity of the infants were analyzed to identify associations using bivariate logistic regression [ ] . these studies provide an important link between ilc and rsv-induced bronchiolitis in infants. stat signaling in mice is crucial for restraint of ilc responses and prevention of type pathophysiology to rsv infection [ ] . enhanced numbers of ilc were present in mice deficient in stat compared to wt , and il- by these cells. these alarmin proteins released from rsv-infected epithelial cells can activate group innate lymphoid cells (ilc ) to produce the type cytokines il- and il- . il- is the most important eosinophil growth, differentiation, and survival factor. il- has many immunologic and physiologic effects, including promoting airways responsiveness and mucous cell metaplasia. ilc are a recently described cell type of the innate immune system. these cells do not express rearranged antigen receptors, unlike t and b cells, but are able to produce type cytokines at high levels when activated [ , ] . ilc are directly activated by the epithelial-derived cytokines tslp, il- , il- , and hmgb . as such, ilc express the corresponding receptors for these cytokines that include tslpr, st , il- r, and the receptor for advanced glycation end products (rage), as well as toll like receptors (tlr) and [ , [ ] [ ] [ ] [ ] . ilc and the cytokines they produce contribute significantly to the pathology of asthma and allergic diseases [ , ] . infants hospitalized with severe rsv infection had detectable ilc in their nasal aspirates and their frequency was greater than in infants with moderate disease [ ] . moreover, levels of type and epithelial-derived cytokines (il- and tslp) were significantly higher in the nasal aspirates of infants with severe disease than in those with moderate disease [ ] . furthermore, infants with lower gestational age were confirmed to be at greater risk for severe rsv infection, as were infants with higher levels of il- in their nasal aspirates, when the relationships of individual variables examined in the study and disease severity of the infants were analyzed to identify associations using bivariate logistic regression [ ] . these studies provide an important link between ilc and rsv-induced bronchiolitis in infants. stat signaling in mice is crucial for restraint of ilc responses and prevention of type pathophysiology to rsv infection [ ] . enhanced numbers of ilc were present in mice deficient in stat compared to wt mice during the course of rsv infection [ ] . additionally, viral clearance was impaired in stat deficient mice compared to wt mice [ ] . absence of stat signaling increased lung il- expression following rsv infection and abrogation of il- attenuated the enhanced rsv-induced ilc response in animals lacking stat signaling [ ] . high levels of type i, ii, and iii interferons are produced during viral infection and all signal through stat [ ] . stat signaling is important for the generation of th polarized cells, as well as the production of ifn-γ during viral infection, both of which are essential for recovery and viral clearance [ , ] . whether stat signaling regulates rsv-induced disease and ilc activation in humans is currently unknown. however, a recent study has provided evidence that further warrants the evaluation of stat signaling in the context of severe rsv-mediated infection in humans. the study noted that infants with severe rsv-induced bronchiolitis have reduced levels of type i ifns and a reduced viral load when compared to children with mild infection [ ] . based on data from mice, this reduction in type i ifn level in infants with severe rsv-induced bronchiolitis likely results in reduced stat activation that favors the generation of type response. rsv infection also increased the expression of ox l, a marker important for the formation of th cells [ ] , on ilc [ ] . subsequent interactions of ox l+ ilc with ox expressing t cells promoted the survival and expansion of th populations [ ] . reciprocally, il- production by cd + t cells in the lungs promoted the proliferation of cytokine producing ilc during the course of rsv infection [ ] . the current evidence suggests that ilc activation may promote type immune responses that might worsen rsv-induced disease; however, there are no intervention studies to date that have tested this hypothesis, and such a study would help to elucidate the true role of ilc in rsv infection. tslp is an il- like cytokine and can influence the development of both t and b cells. tslp signals through its receptor tslpr, a heterodimer composed of the il- receptor alpha chain (il- rα) and the tslpr chain [ , ] . tslp is primarily expressed by epithelial cells; however, keratinocytes, dendritic cells, and mast cells also produce tslp [ ] . tslp induces cell proliferation, as well as the maturation of dendritic cells (dcs) [ ] . tslp is an important mediator for the development of allergic asthma. elevated amounts of tslp were present in the cells within the airway [ ] and balf [ ] of patients with asthma and in the lungs of mice with antigen-induced allergic inflammation [ ] . importantly, tslp levels were upregulated in nasopharyngeal aspirates (npas) collected at time of admission of hospitalized infants with viral bronchiolitis, % of which were hospitalized due to rsv infection, compared to samples collected from healthy infants at their primary care appointments [ ] . moreover, increased levels of tslp, as well as of il- and periostin, an extracellular matrix protein downstream of il- [ ] , were found in the npas of patients who were infected with rsv compared to noninfected controls [ ] . identification of tslp within samples collected from rsv-infected patients demonstrates its potential as a target for mitigation of rsv-induced bronchiolitis in humans. cultured primary human bronchial epithelial cells produced tslp in response to rsv infection [ ] . furthermore, cultured primary human bronchial epithelial cells from asthmatic children produced more tslp in response to rsv infection than cells collected from healthy children [ ] . the tslpr was upregulated on epithelial cells from the human lung epithelial cell line a in response to rsv infection [ ] . this study also noted that primary human bronchial epithelial cells collected from asthmatic patients had upregulated tslpr expression in response to rsv infection compared to those collected from healthy patients [ ] . together, these studies demonstrate the expression of tslp in human-derived cultured cells in response to rsv infection, further demonstrating that rsv infection induces tslp production. furthermore, a study in mice has shown that rsv-induced lung il- and airway mucus were blunted in animals that lacked the tslpr [ ] . an important question, however, is whether tslp is altering immune cell function during rsv infection. this seems to be the case, as co-culture of rsv-infected primary rat epithelial cells (praecs) with myeloid dendritic cells (mdcs) resulted in the greater maturation (enhanced expression of cd and mhcii) of the mdcs than those that were co-cultured with medium-exposed praecs [ ] . this response was abrogated when tslp was knocked down in the epithelial cells with sirna [ ] . furthermore, mdc expression of ox l, was upregulated in mdcs co-cultured with rsv-infected praecs compared to those co-cultured with medium exposed praecs [ ] . this response was again abrogated when tslp was knocked down in the epithelial cells [ ] . administration of an anti-ox l antibody during primary infection of neonatal mice resulted in reduced ahr, as well as reduced il- and il- levels in the bronchoalveolar lavage fluid (balf) from mice when they were re-infected compared to mice treated with a control antibody [ ] . similar reductions in ahr and levels of il- and il- in the balf were seen after re-infection when anti-tslp was administered prior to the primary infection of neonatal mice compared to mice administered a control antibody [ ] . moreover, administration of an anti-tslp antibody prior to rsv infection of neonatal mice resulted in a reduction in the number of dcs that accumulated in the lung, as well as their expression of ox l [ ] . these two studies demonstrate an indirect mechanism through which tslp enhances il- production mediated by ox l-ox interaction between dcs and t cells. additionally, tslp, confirmed through antibody neutralization and the use of tslpr deficient mice, directly increased the number of il- producing ilc in response to rsv infection [ ] . tslpr-deficient mice infected with rsv had reduced airway mucus visualized by periodic acid-schiff (pas) staining and reduced ahr compared to wt mice infected with rsv [ ] . moreover, the dependency of activation and accumulation of il- + ilc on tslp signaling was demonstrated across several different clinical isolates of rsv and is thus a conserved feature of rsv strains able to induce type immune responses in mice [ ] . sex differences also impact the amount of tslp present during rsv infection. neonatal male mice infected with rsv exhibited higher viral loads, as well as lower production of ifnβ, at day post-infection, leading to delayed resolution of infection compared to neonatal female mice infected with rsv [ ] . male mice had also formed bronchus associated lymphoid tissue (balt) at day post-infection that was not present in female mice [ ] . at weeks post-infection, male mice had higher levels of tslp and il- in the lungs, as well as increased airway mucus visualized by pas staining and by levels of muc ac by quantitative pcr, more lung ilc , dcs, and ox l+ cells compared to female mice [ ] . these changes in resident lung immune cell populations resulted in male mice becoming more susceptible to allergic exacerbation upon allergen challenge at weeks old compared to female mice [ ] . the increased susceptibility to allergic exacerbation at weeks post-rsv infection was reduced in male tslpr-deficient mice compared to wt males, indicating that tslp was responsible for inducing the alterations in immune profile seen in male mice [ ] . together, these data suggested an important role for tslp in the promotion of the type response to rsv infection. il- is an alarmin, or a molecule that when released can signal the presence of tissue or cell damage, and is a member of the interleukin (il- ] family of cytokines [ ] . il- is predominantly expressed and released by endothelial and epithelial cells but can be released or secreted from other cell types within the airway, including macrophages and dcs [ ] [ ] [ ] . il- binds to and signals through its receptor st (il- rl ] [ ] . il- can promote type responses [ ] , and as such, il- is an important mediator of asthma and other allergic diseases. il- can activate and induce ilc to produce type cytokines and may activate dcs to promote th polarization [ ] . il- can also activate non-type cells that exhibit st expression [ ] . clinically, increased amounts of il- and il- have been found in nasal aspirates of hospitalized infants with rsv, linking il- to type inflammation generation in response to rsv infection [ ] . in response to rsv infection, both alveolar macrophages and dcs from the lungs of infected mice became a source of il- as evidenced by flow cytometry of total homogenized lung cells [ ] . while assessing lung il- expression by flow cytometry is technically challenging given the long isolation process and resultant loss of cell viability, il- reporter mice have made possible the flow cytometric analysis of il- expression in the setting of rsv infection. for instance, our group recently showed that there was increased lung epithelial cell expression of il- twelve hours after rsv infection. this flow cytometry data correlated with lung protein levels of il- measured by elisa [ ] . upregulation of il- mrna in response to rsv infection was confirmed in vitro using the macrophage cell line raw . and the dc cell line dc . [ ] . this increase in il- production was mediated by toll like receptors (tlrs) and [ ] . furthermore, il- produced by macrophages after rsv infection was dependent upon signaling through the mapk pathway [ ] . ilc were also activated by il- in response to rsv [ ] . incubation of whole lung cell suspensions from rsv infected mice with an anti-st antibody reduced the number of il- + ilc compared to untreated cells [ ] . age at time of infection is an important risk factor for severe rsv-mediated bronchiolitis [ ] . il- is produced shortly after birth and results in a wave of ilc colonization of the lungs [ ] . activation of ilc by endogenous il- during this is important for the development of type immunity [ ] . moreover, there is evidence that demonstrates a link between the production of il- during the perinatal period and the development of asthma [ ] . correspondingly, levels of il- and il- in response to rsv infection differ greatly based on age. neonatal mice mounted rapid accumulation of il- and il- in the lungs in response to rsv infection that was not seen in adult mice [ ] . higher numbers of lung ilc were also seen in neonatal mice and not adults following rsv infection [ ] . interestingly, pretreatment of neonatal mice with an anti-il- antibody prior to rsv infection reduced levels of lung il- and ilc , and this was not seen in adult mice [ ] . development of ahr was abrogated and numbers of cd + th cells were reduced in re-infected neonates that had been pretreated with an anti-il- antibody during primary infection compared to mice that received a control antibody [ ] . st -deficient mice were protected from the development of type responses when infected with rsv [ ] . in one mouse model of rsv infection, ahr was driven by il- activated ilc producing il- as a consequence of rsv infection [ ] . all of these recent studies provide a clear link between accumulation of il- and induction of an ilc mediated type response during rsv infection. however, few studies have uncovered potential therapies to mitigate this response. one such study, identified a glucagon-like peptide receptor (glp- r) agonist, an fda approved treatment for type ii diabetes, as a potential therapy. the glp- r agonist administered during the course of rsv infection reduced rsv-induced il- upregulation in epithelial cells, as well as type immunopathology [ ] . a second study reported increased expression of xanthine oxidase in rsv-infected mice and hospitalized infants [ ] . xanthine oxidase stimulates the inflammasome ultimately resulting in the production of uric acid [ , ] . treatment of rsv infected transformed human airway epithelial cells or primary mouse airway epithelial cells in culture with a xanthine oxidase inhibitor decreased the expression of il- and tslp [ ] . additionally, treatment of mice during the course of rsv infection with a xanthine oxidase inhibitor decreased the expression of balf il- and lung il- and reduced the number of ilc present in the lungs [ ] . interestingly, interleukin- beta (il- β) expression was also increased in the balf during rsv infection and treatment of mice with an il- receptor antagonist (il -ra) reduced the expression of balf il- and lung il- , as well as the accumulation of ilc that occurred as a result of rsv infection [ ] . however, il- β has also been shown to decrease il- , il- and mucus metaplasia in a mouse model of rhinovirus (rv) infection [ ] . these opposing results could be due to differences in the microenvironment that is unique to each pathogen. in a third study, rhein, a traditional chinese medicine (lipophilic anthraquinone) found in medicinal herbs that has antiviral activities, inhibited rsv-induced activation of the nlrp inflammasome and production of lung il- in mice [ ] . high mobility group box (hmgb ) is a nonhistone chromatin binding protein that loosely binds dna [ , ] . hmgb performs several crucial functions within the nucleus. hmgb stabilizes nucleosome formation and acts as a dna chaperone to enable dna replication and repair [ , ] . hmgb is also involved in v(d)j recombination and transcription [ , ] . hmgb promotes autophagy and cell survival when present in the cytoplasm [ , ] . hmgb can be released by dcs, macrophages, and nk cells in response to inflammatory stimuli, including infection, and is also released from necrotic and dead cells [ , ] . like il- , hmgb is an alarmin and thereby mediates responses of both the innate and adaptive immune systems when released. hmgb signals through rage, as well as tlrs and [ , ] . elevated levels of hmgb have been detected in the sputum of human asthmatics [ , ] . moreover, hmgb levels were greater in the npa of infants hospitalized for severe rsv-induced bronchiolitis [ ] . several mouse studies using different inducible models of asthma demonstrated that anti-hmgb antibody therapy reduced il- , il- , and il- , as well as airway mucus compared to control antibody [ ] or mice not given antibody [ ] . initial experiments demonstrated increased levels of hmgb in lungs of rsv infected mice compared to uninfected controls [ ] . hmgb expression in the human bronchial epithelial cell line hbe was increased following infection with rsv compared to un-infected cells [ ] . hmgb was increased in the lungs of rsv-infected neonatal rats compared to mock-infected controls [ ] . treatment with glycyrrhizin, an inhibitor of hmgb , at the time of rsv infection reduced the number of rsv-infected transformed human bronchial epithelial cells [ hbe) and primary normal human bronchial epithelial cells [ ] . a separate study confirmed release of hmgb from transformed human airway epithelial cells infected with rsv, as well as demonstrated that reactive oxygen species (ros) generation triggered by rsv infection promoted hmgb release from transformed human airway epithelial cells [ ] . the study also noted that hmgb directly increased the release of cytokines, including ifn-γ, from human primary monocytes [ ] . a third study also found that rsv infection induced hmgb release from transformed human airway epithelial cells [ ] . hmgb added to cultures was sufficient to induce proinflammatory cytokine production from primary human monocytes, dcs, and eosinophils [ ] . together these studies demonstrate the capacity of rsv infection to induce hmgb release. to date, only one study has examined the effect of hmgb on type inflammation in the context of rsv infection. in this study, mice were infected with rsv and were subsequently treated with an hmgb antibody on days to post-infection [ ] . anti-hmgb therapy significantly reduced the concentrations of il- , il- , and il- in the balf at day post-infection compared to a group that was infected and treated with control antibodies [ ] . additionally, gata expression, the number of lung eosinophils, ahr, and cellular infiltration into the lung measured by hematoxylin and eosin (h&e) staining and through balf cell counts, were decreased at day post-infection in rsv-infected mice who received anti-hmgb compared to those that did not [ ] . in this model, hmgb expression was found to be primarily exist within cc + club cells inside the lung [ ] . depletion of cc + club cells by injections of naphthalene reduced levels of hmgb in the lungs and balf, as well as levels of type cytokines in the balf, compared to control animals that were infected with rsv and had received sham injections of pbs at day post rsv infection [ ] . this study demonstrates a link between hmgb and the severity of rsv infection and identifies a cell type important to its release. further studies investigating the impact of hmgb therapy during rsv infection are warranted. it is worth noting that studies have published links between type inflammation and hmgb in animals infected with pneumonia virus of mice (pvm), a murine virus that results in infection similar to that of severe rsv-induced infection in infants. the first study showed that neonatal mice deficient in rage manifested a reduced type and type ifn response, blunted peripheral dc (pdc) recruitment, higher levels of hmgb and higher amounts of airway smooth muscle mass compared to wt mice in response to pvm infection [ ] . furthermore, reinfection of rage deficient mice with pvm days after initial infection resulted in increased ahr and goblet cell hyperplasia compared to wt mice, and these endpoints were reduced with the antibody neutralization of hmgb [ ] . the second study reported that neonatal mice deficient in interferon-β promotor stimulator- (ips- ) that were pvm-infected and subsequently re-infected with pvm developed severe bronchiolitis marked by release of hmgb and il- [ ] . the third study demonstrated that pharmacological inhibition of the programmed death response (receptor-interacting serine/threonine-protein kinase (ripk ) or mixed lineage kinase domain like pseudokinase (mlkl) inhibition) both in vitro and in vivo reduced hmgb release and attenuated the development of bronchiolitis and type inflammation in vivo in irf -deficient mice, a strain of animals predisposed to developing severe bronchiolitis in response to pvm infection [ ] . pharmacological inhibition of the programmed death response in irf -deficient mice also reduced balf ldh measurements, balf dsdna measurements, mucin hypersecretion measured through percent of airway epithelial cells that expressed muc ac, eosinophilic infiltration into the balf and airway smooth muscle remodeling compared to untreated irf -deficient mice during pvm infection [ ] . together, these studies suggest that following rsv infection, hmgb contributed to the development of the type response during the course of infection. il- , also known as il- e, is a member of the interleukin- (il- ) family of cytokines and binds to its receptor, il- r, that is comprised of the il- ra and il- rb subunits. il- rb receptor expression is predominantly on epithelial cells and type cells of the immune system, including th cells and ilc , and il- is important for successful mounting of type effector responses [ ] [ ] [ ] . il- rb is noticeably upregulated in asthma and allergic disease [ ] . signaling of il- during asthma and allergic diseases leads to eosinophilia, increased airway mucus, ahr, and airway remodeling [ , ] . many cell types express il- in response to specific inflammatory stimuli including eosinophils, basophils, mast cells, epithelial cells, and th cells [ ] . il- also plays a role in rsv-mediated disease. il- is upregulated during the course of rsv infection [ ] . subsequently, neutralization of il- during the course of infection or use of il- rb deficient mice reduced airway mucus measured by pas staining, and accumulation of il- and il- measured in re-stimulated draining lymph node cell supernatants [ ] . furthermore, il- rb deficient mice were protected from allergic exacerbations resulting from antigen challenge during rsv infection [ ] . a second study also noted an increase of il- in the lungs of mice infected with rsv [ ] . mice treated with anti-asialo gm to deplete their nk cells and subsequently infected with rsv had increased levels of type cytokines in their lungs, levels of ige in their serum, and number of mucin-producing epithelial cells in their lungs compared to wt mice [ ] . the type response seen in nk cell depleted mice was attenuated by treatment with an anti-il- antibody compared to nk cell depleted mice that received an isotype control antibody, suggesting that the enhanced type response seen in nk cell depleted mice is mediated by il- [ ] . additionally, neonatal pvm-infected mice had increased lung levels of il- at day post-infection compared to un-infected mice [ ] . moreover, neonatal mice infected with pvm and later sensitized and challenged with ova had increased levels of serum ige and increased expression of il- , il- , and il- in cd t cells as evaluated by quantitative pcr compared to animals that were not infected and sensitized and challenged with ova [ ] . il- may be important for the enhanced type response found in the pvm-infected and later ova sensitized/challenged mice in this study, but this was not definitively determined. the aforementioned studies suggest a link between il- expression and the development of type inflammation during rsv infection, however, more studies are needed to truly understand the impact of il- signaling during human rsv infection. our understanding of the role the innate immune system and associated epithelial-derived cytokines in the induction of type programming in response to rsv infection has dramatically increased over the past decade, mainly due to the discovery of ilc . the data discussed in this review strongly implicate ilc , il- , tslp, hmgb , and il- as key early mediators of the type response to rsv. albeit with some discrepancies, some studies strongly implicate il- [ ] as the driver of the type response, while others implicate tslp [ ] , hmgb [ ] , and il- [ ] . it is possible that strain differences in rsv or differences in age elicit differences in the production of early mediators that then drive the type response. nevertheless, we still have a long way to go to accurately understand how and why this response is induced in response to rsv infection and its association to severe rsv-induced bronchiolitis with the possible relationship to the later development of childhood asthma. it is important to also consider that children who develop severe rsv-mediated bronchiolitis and childhood asthma may already have baseline alterations in the levels of type cytokines and numbers of cells that mediate type responses prior to infection. if this is in fact the case, then infants that are predisposed to asthma, because of genetic or other risk factors, might be more likely to develop severe rsv infections. the conundrum of whether a predisposition for allergic disease and asthma is a risk factor for severe rsv bronchiolitis, or whether severe rsv bronchiolitis is a risk factor for subsequent asthma and allergic disease, is still being investigated. regardless of the root cause of severe rsv-mediated bronchiolitis, such as host, virus, and/or environmental factors, it is worth to consider investigating emerging therapies directed at type and epithelial-derived cytokine signaling that have recently been approved, or are in the pipeline for approval, for asthma treatment as therapies through clinical trials. the potential benefit for using agents, such as dupilumab and tezepelumab, as treatments for these patients would be two-fold; it would provide concrete evidence of the contribution of innate type inflammation to disease pathogenesis and might also provide a novel therapeutic target. it is also possible that use of therapies to treat rsv-mediated bronchiolitis that target the early acting alarmins like il- may have no effect as they often will not be administered until after the peak response from these cytokines has been initiated. yet the amount of evidence related to involvement of il- in the pathogenesis of rsv-mediated bronchiolitis justifies the evaluation of those therapies. for instance, in the mouse model of rsv infection, anti-tslp antibodies administered either or hours after infection resulted in a significant decrease in il- -expressing ilc [ ] . this result suggests that treating patients after infection with biologics targeting cytokines that stimulate ilc may represent a successful strategy. immune modulation is tricky, given the extensive interplay and interdependency on the part of the immune which occurs during the clearance of infection, or really in response to any illness or disease. however, testing these antibody therapies rigorously in randomized, double-blind, placebo-controlled clinical trials may be something to consider, as they may prove to be new therapies for patients that are highly susceptible to severe infection who stand the greatest risk of sustaining lifelong 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receptor on group innate lymphoid cells can induce asthmatic characteristics human circulating group innate lymphoid cells can express cd and promote ige production elevated levels of type respiratory innate lymphoid cells in human infants with severe respiratory syncytial virus bronchiolitis stat represses cytokine-producing group and group innate lymphoid cells during viral infection the interferons and their receptors-distribution and regulation il- and ifn (alpha and gamma) exert opposite regulatory effects on the development of cytolytic potential by th or th human t cell clones reduced nasal viral load and ifn responses in infants with respiratory syncytial virus bronchiolitis and respiratory failure dendritic cell expression of ox ligand acts as a costimulatory, not polarizing, signal for optimal th priming and memory induction in vivo critical role of ox /ox l in ilc -mediated activation of cd +t cells during respiratory syncytial virus infection in mice essential role of cd + t cells for the activation of group innate lymphoid cells during respiratory syncytial virus infection in mice sensing the outside world: tslp regulates barrier immunity thymic stromal lymphopoietin thymic stromal lymphopoietin: to cut a long story short thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of th -attracting chemokines and disease severity elevated expression of il- and tslp in the airways of human asthmatics in vivo: a potential biomarker of severe refractory disease tslp and il- reciprocally promote each other's lung protein expression and ilc receptor expression to enhance innate type- airway inflammation thymic stromal lymphopoietin, il- , and periostin in hospitalized infants with viral bronchiolitis using periostin as a biomarker in the treatment of asthma thymic stromal lymphopoietin is induced by respiratory syncytial virus-infected airway epithelial cells and promotes a type response to infection respiratory syncytial virus induces functional thymic stromal lymphopoietin receptor in airway epithelial cells tslp from rsv-stimulated rat airway epithelial cells activates myeloid dendritic cells responsiveness to respiratory syncytial virus in neonates is mediated through thymic stromal lymphopoietin and ox ligand respiratory syncytial virus infection activates il- -producing group innate lymphoid cells through thymic stromal lymphopoietin sex-associated tslp-induced immune alterations following early-life rsv infection leads to enhanced allergic disease interleukin- in health and disease macrophages produce il- by activating mapk signaling pathway during rsv infection respiratory macrophages and dendritic cells mediate respiratory syncytial virus-induced il- production in tlr -or tlr -dependent manner interleukin- : its emerging role in allergic diseases resolution of airway inflammation and hyperreactivity after in vivo transfer of cd +cd + regulatory t cells is interleukin dependent respiratory syncytial virus disease is mediated by age-variable il- glucagon-like peptide receptor signaling attenuates respiratory syncytial virus-induced type responses and immunopathology natural helper cells contribute to pulmonary eosinophilia by producing il- via il- /st pathway in a murine model of respiratory syncytial virus infection first-breath-induced type pathways shape the lung immune environment activation of neonatal mouse lung ilc s by endogenous il- impacts type responses later in life perinatal activation of the interleukin- pathway promotes type immunity in the developing lung pulmonary il- orchestrates innate immune cells to mediate rsv-evoked airway hyperreactivity and eosinophilia. allergy eur uric acid pathway activation during respiratory virus infection promotes th immune response via innate cytokine production and ilc accumulation xanthine oxidoreductase regulates macrophage il β secretion upon nlrp inflammasome activation il- β prevents ilc expansion, type cytokine secretion, and mucus metaplasia in response to early-life rhinovirus infection in mice rhein suppresses lung inflammatory injury induced by human respiratory syncytial virus through inhibiting nlrp inflammasome activation via nf-κb pathway in mice high-mobility group box protein (hmgb ): nuclear weapon in the immune arsenal the role of high mobility group box in innate immunity endogenous hmgb regulates autophagy the role of high-mobility group box- (hmgb ) in the pathogenesis of asthma sputum high mobility group box- in asthmatic children: a noninvasive sensitive biomarker reflecting disease status high-mobility group box- protein from cc + club cells promotes type response in the later stage of respiratory syncytial virus infection hmgb contributes to allergen-induced airway remodeling in a murine model of chronic asthma by modulating airway inflammation and activating lung fibroblasts high mobility group box : a novel mediator of th -type responseinduced airway inflammation of acute allergic asthma respiratory syncytial virus increases the expression and release of high mobility group box- protein in the lung tissue of mice induction of high-mobility group box- in vitro and in vivo by respiratory syncytial virus respiratory syncytial virus infection triggers epithelial hmgb release as a damage-associated molecular pattern promoting a monocytic inflammatory response proinflammatory effects of respiratory syncytial virus-induced epithelial hmgb on human innate immune cell activation rage deficiency predisposes mice to virus-induced paucigranulocytic asthma the absence of interferon-β promotor stimulator- (ips- ) predisposes to bronchiolitis and asthma-like pathology in response to pneumoviral infection in mice rsv infection promotes necroptosis and hmgb release by airway epithelial cells signaling of interleukin- family cytokines in immunity and inflammation il- in allergic inflammation group innate lymphoid cells in pulmonary immunity and tissue homeostasis il- : a key requirement for the regulation of type- immunity interleukin (il)- : pleiotropic roles in asthma il- e (il- ) and il- rb promote respiratory syncytial virus-induced pulmonary disease early-life viral infection and allergen exposure interact to induce an asthmatic phenotype in mice hmgb -induced ilc s activate dendritic cells by producing il- in asthmatic mouse model respiratory syncytial virus prevents the subsequent development of ovalbumin-induced allergic responses by inhibiting ilc via the il- /st pathway key: cord- -imbpgsub authors: zhang, yun; xu, zhichao; cao, yongchang title: host–virus interaction: how host cells defend against influenza a virus infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: imbpgsub influenza a viruses (iavs) are highly contagious pathogens infecting human and numerous animals. the viruses cause millions of infection cases and thousands of deaths every year, thus making iavs a continual threat to global health. upon iav infection, host innate immune system is triggered and activated to restrict virus replication and clear pathogens. subsequently, host adaptive immunity is involved in specific virus clearance. on the other hand, to achieve a successful infection, iavs also apply multiple strategies to avoid be detected and eliminated by the host immunity. in the current review, we present a general description on recent work regarding different host cells and molecules facilitating antiviral defenses against iav infection and how iavs antagonize host immune responses. influenza a virus (iav) can infect a wide range of warm-blooded animals, including birds, pigs, horses, and humans. in humans, the viruses cause respiratory disease and be transmitted by inhalation of virus-containing dust particles or aerosols [ ] . severe iav infection can cause lung inflammation and acute respiratory distress syndrome (ards), which may lead to mortality. thus, causing many influenza epidemics and pandemics, iav has been a threat to public health for decades [ ] . the virus is an enveloped, segmented, negative-strand rna virus, belonging to the orthomyxoviriae family. the eight viral gene segments encode as many as proteins. besides polymerase basic (pb ), pb -n , pb -f , pb , polymerase acid (pa), hemagglutinin (ha), nucleoprotein (np), neuraminidase (na), matrix (m ), matrix (m ), nonstructural protein (ns ) and ns (also known as nuclear export protein, nep), new viral proteins were recently uncovered, such as pb -s [ ] , pa-x (product of ribosomal frameshifting) [ ] , pa-related proteins pa-n and pa-n [ ] , m [ ] , and ns [ ] . ha, na, and m proteins constitute surface of the iav virion, where ha is the most abundant surface protein. according to the genetic and antigenic diversity of the ha and na proteins, iavs were divided into ha and na subtypes. h n and h n subtypes were recently identified in bats [ , ] . ha is a type i glycosylated protein, which is responsible for virus entry to host cell. functional ha protein is a homotrimer structurally composed of a stem region and a globular head region in each monomer. the head region bearing n-acetylneuraminic acid (sialic acid, sa) binding pocket is critical for receptor attachment, and contains most antigenic determinants. the stem region undergoing conformational changes is responsible for low ph-triggered membrane fusion [ ] , and plays an important role in cross protection against heterosubtypic iav infection [ ] . n glycan at this region iavs can infect a broad spectrum of host species, including both wild and domestic birds, as well as many mammalian species. the virus is capable of interspecies transmission to new species. however, no interspecies transmission of the bat iavs has been reported so far [ ] . furthermore, the high frequency of mutations and recombination increases the risk of iav adaptation in humans. besides three pandemic subtypes (h n , h n , and h n ), other subtypes, including h n , h n , h n , h n , n n , and h n could cross the species barrier and cause human infections [ ] [ ] [ ] [ ] . several effect factors are essential in iav host switch events, including the receptor-binding properties of ha [ ] , as well as cellular receptors [ ] [ ] [ ] [ ] . long and his colleagues summarized the role of host factors in iavs adaption to humans, and the review is recommended here for further reading [ ] . noteworthy is the fact that most phylogenetically diverse iavs with different origins could successfully replicate in swine [ ] . since pigs have both sa α- , and sa α- , galactose receptors [ ] , they can serve as a suitable mixing reservoir for both human and avian iavs, thus raising global concern on periodic zoonotic infections. take the emergence of influenza a (h n ) pdm (ph n ) and influenza a (h n ) a/canada/ / strains for instance, both strains are swine-origin iavs and were the consequence of adaption and reassortment of several swine lineages [ , ] . furthermore, some genes of these strains originated from avian iavs [ ] . with the development of gene sequencing technology, machine learning (ml) facilitated with large genomic datasets are used in prediction about sequence changes in newly invaded viruses from other animal hosts, take the "batch-learning self-organizing map (blsom)" method for instance [ ] . ml is also applied in characterization of distinct host tropism protein signatures [ ] , and prediction of amino acid changes for interspecies transmission [ ] . these studies provided measures in identification of potential high-risk strains. in addition, the nucleotides and dinucleotide compositions of viruses play important roles in prediction of viral host species [ ] . combining gene sequencing technology viruses , , of and ml methods, researchers applied large iav genomic datasets to analyze species selection bias of iav mono-/dinucleotide composition and predict human-adaptive swine or avian iavs [ , ] . the application of multi-disciplinary subjects would provide useful information for prediction of pandemic influenza. in general, the life cycle of the iav is generally divided into four steps: virus entry into the host cell, transcription and replication of the viral genome, assembly, and virus budding. though alveolar epithelial cell is the primary target cell for iavs, different iav subtypes have different patterns of viral attachment (pva). for human iavs, alveolar type ii epithelial cells, as well as immune cells such as alveolar macrophages and dendritic cells, are major target cells for an established infection [ , ] . two seasonal iavs and pandemic h n virus, preferred to attach to ciliated epithelial cells and goblet cells in the upper respiratory tract (urt), and avian iavs, take h n for instance, attached seldom to these cells [ ] . in the lower respiratory tract (lrt), human iav h n and h n attached to more cell types than avian iav h n , a highly pathogenic avian iav (hpaiv) strain. however, h n could bind to type ii pneumocytes [ ] . considering the fact that metabolism in the type ii pneumocytes is quite active, infection of hpaivs is more likely to cause severe pneumonia [ ] . other research on low pathogenic avian iavs (lpaivs), which generally do not cause severe pneumonia, showed that these viruses usually attach to human submucosal gland cells, thus can be cleared by the mucus [ ] . iav infection starts from recognition of sa by ha protein, though in vitro research claimed that these n-linked glycans were not essential for virus entry [ ] . the cleavage of ha precursor protein ha into ha (containing receptor binding domain) and ha (containing fusion peptide) in low ph environment during ha transport is critical for virion internalization [ ] . some research showed that type ii transmembrane serine protease such as transmembrane protease serine (tmprss ), human airway trypsin-like protease (hat), transmembrane protease serine (tmprss ), homo sapiens serine protease desc and homo sapiens transmembrane protease, serine (mspl) can cleave human and avian iav ha proteins at an arginine residue [ ] . in addition, for avian iavs, ha of hpaivs can be cleaved by subtilisin-like protease, while that of lpaivs is cleaved by trypsin-like proteases [ ] or thrombin [ ] . therefore, in avian iavs, the cleavage sites are considered to be the major determinants for virus virulence [ ] , and rna folding in the cleavage region could be an important factor for virulence determination [ , ] . proteins in the vrnp complex contain different nuclear localization signals (nlss), thus helping the vrnp complex to enter the host cell nucleus via active transport, take the crm -dependent pathway for instance [ ] . the acidic environment of the endosome also activates m ion channel, hence acidifies the viral core, resulting in entrance of vrnp complex into the host cell [ ] . replication of viral genome does not require a primer but a full-length complementary rna (crna), which is essential for the newly formed vrnp complex. the viral rna polymerases first bind to the end and the end of the segmented viral rna and crna, respectively, then start replication with the help of the cap of host pre-mrnas via a pb -pb -mediated "cap snatching" mechanism [ , ] . the conserved segment-specific nucleotides at the and ends of the viral genome could modulate genome expression and replication during infection [ ] . in addition, dephosphorylation at a specific position of the h n ns protein results in attenuated virus replication [ ] . mature viral mrnas are transported to the cytoplasm by a "daisy-chain" complex and translated subsequently [ , ] . new synthesis of ha occurs on the rough endoplasmic reticulum (er). glycosylation and palmitoylation of the protein are completed later in the golgi [ , ] . after synthesis and maturation of na and m proteins, the trans-golgi network (tgn), together with coat protein i (copi) complex and gtpase rab proteins, transport the newly synthesized ha, na, and m proteins to the apical plasma membrane (pm). these proteins then assemble with viral genomic segments. the virions are finally closed and m and m proteins mediate virion budding from the apical side of the viruses , , of cells [ , [ ] [ ] [ ] [ ] . na protein cleavages the sa residues, which allows the virions to be released from the plasma membrane [ ] . since iav has a relatively small genome, host machinery is required in order to accomplish the viral life cycle. to uncover host dependency factors that are necessary for iav replication, numerous large-scale rna interference (rnai) screens and genome-wide crispr/cas screen were performed [ , [ ] [ ] [ ] [ ] . for instance, son dna binding protein was important for iav virion trafficking in an early infection stage and cdc-like kinase facilitated aiv replication [ ] . usp facilitated viral entry, whereas tnfsf (april) and tnfsf -tnfsf (twepril) helped with viral replication [ ] . using genome-wide crispr/cas screen, several genes of sialic acid biosynthesis and related glycosylation pathways were involved with h n infection [ ] , and wdr , ccdc , and tmem were essential for viral entry and regulation of v-type atpase assembly [ ] . furthermore, single-cell transcriptome sequencing (rna-seq) was applied to explore host-virus interactions, revealing a correlation between defective viral genomes and virus-induced host transcriptional programs [ ] . these data provide valuable information for developing host-targeted therapeutics. host immune system functions immediately after detection of the virus. host mucosal immune system (mis), induced after virus invasion, serves as the first line to prevent iav from adhering to the susceptible cells. in the urt, mucosal response is induced in the naso-associated lymphoid tissues (nalt), while in the lrt, it occurs in bronchus-associated lymphoid tissues (balt). host innate immunity, including phagocytic cells, interferons (ifns), proinflammatory cytokines, etc., applies multiple mechanisms in defending iav infection [ ] . host adaptive immunity, mediated by b lymphocytes and t lymphocytes, together with other immune mechanisms, reacts specifically to neutralize and eliminate the virus. on the other hand, to establish a successful infection, iavs also employ a plethora of strategies to avoid being detected or being cleared by the host immunity. notable strategies include regulation of ifn signaling [ ] , inhibition of cytokine expressions [ , ] , modulation of apoptosis [ ] [ ] [ ] , interference of autophagy [ ] , and effects on antibody production [ ] . the iav-host immunity interaction was summarized by several reviews [ , ] . upon detection of infection, innate effector cells, including natural killer (nk) cells, neutrophils, and dendritic cells (dcs), etc., are recruited to the infected sites. nk cells are large granular lymphocytes, making up % of the resident lymphocytes in the lung. after recruitment from the blood, nk cells interact with dcs and macrophages to secret various cytokines and restrict infection via lysis of the iav-infected cells. the lysis process is mediated by interaction between nk receptors p (most nkp ) and iav ha protein expressed by the infected cell [ , ] . interestingly, liver nk cells other than lung nk cells possessed a memory phenotype to protect mice against subsequent iav infection, though the lung nk cells are important in control of primary iav infection [ ] . however, nk cells are also shown to exacerbate iav pathology, since depletion of nk cells led to increased resistance to high dose h n infection in mice [ , ] . the contribution of nk cells to anti-iav defense in mouse models was later shown to be strain and dose dependent. in addition, the host genetic background also played an important role [ ] . neutrophils are key innate immune cells recruited to infection sites by cellular migration through vascular endothelium. they function in clearance of pathogens via phagocytosis, producing extracellular traps, and degranulation [ ] . in addition, they also regulate adaptive immunity via guiding influenza specific cd + t cells to the infection sites [ ] . the function of dendritic cells (dcs) is to monitor invading pathogens. after iav infection, the conventional dcs migrate from lung to lymph nodes through interaction between ccr and its ligand, and present antigens to t cells [ , ] . one study based on a mouse model showed that, during iav infection, immature and mature dcs were specialized in iav ha processing, since both types of dcs could present one epitope of h n ha (ha amino acids - ), whereas another epitope (ha amino acids - ) could only be processed by mature dcs [ ] . the complex role of dcs in initiation of robust immunity against iav infection is reviewed by waithman and mintern [ ] . t cells and b cells are critical components in adaptive immunity against iav infection. cd + t cells differentiate into cytotoxic t lymphocytes (ctls) and defend iav infection via producing cytokines and effector molecules, and cytotoxic effects (i.e., lysis) of infected cells mediated by mhc class i. cd + t cells target iav-infected epithelial cells through binding with mhc class ii molecules and contribute to b cell activation thus consequently promote antibody production. the activation of t cells and b cells in iav infection will be exposited in section . . the reaction of innate immunity is nonspecific. it is triggered by recognition of pathogen associated molecular patterns (pamps) via host pathogen recognition receptors (prrs). toll-like receptors (tlrs), retinoic acid-inducible gene-i proteins (rig-i), and nod-like receptors are common prrs, the activation of which leads to activation of innate immune signaling and further production of cytokines as well as other antiviral molecules. toll-like receptors are responsible for sensing pathogens at cell membranes, endosomes, and lysosome [ ] . tlr and tlr are shown to be involved in iav detection at endosomes [ ] . tlr recognizes double stranded rna (dsrna) which may be released by cellular stress and cell death [ ] and unidentified rna structures in phagocytosed cells infected with iavs [ ] . in macrophages and dendritic cells, tlr interacted with tir-domain-containing adapter, then activated the serine-threonine kinase iκkε (ikkε) and tank binding kinase (tbk ) to phosphorylate interferon regulatory factor (irf ), the process of which further led to expression of ifn-β [ ] . in addition, an over-reacting tlr activation promoted iav pathogenesis, which could be reduced by a single-stranded oligonucleotide (sson) functioning as a tlr inhibitor, resulting in restrained viral loads both in vitro and in vivo [ ] . tlr recognizes single stranded rna (ssrna). in plasmacytoid dendritic cells (pdcs), after activation of tlr during iav infection, irf or nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb) were activated via myeloid differentiation factor (myd ) to induce type i ifns [ ] . in avian macrophages, activation of tlr produced pro-inflammatory molecules such as interleukin (il)- β [ ] . in addition, in mouse models, tlr played an important role in activation of nk cells [ ] . it was also shown to be involved in development of adaptive immunity to prevent iav infection [ , ] . rig-i recognizes ssrnas and transcriptional products of iavs, which triggers activation of the caspase activation and recruitment domains (cards) via dephosphorylation or ubiquitination by e ligases, resulting in activation of transcription factors including irfs and nf-κb [ ] . otub played an essential role in regulation of rig-i [ ] . in addition, melanoma differentiation-associated gene (mda ) was also involved in sensing transcriptional products of iavs in the cytoplasm [ ] . for nod-like receptor family, pyrin domain containing (nlrp ) and nlr apoptosis inhibitory protein were activated after iav infection [ ] . iav m ion channel and pb -f were involved in activation of nlrp inflammasome and stimulate il- β secretion subsequently [ , ] . the role of the nlrp inflammasome in regulation of anti-iav responses is discussed in detail by sarvestani and his colleagues [ ] . delayed oseltamivir and sirolimus combined treatment could suppress nlrp inflammasome mediated secretion of il- β and il- , resulting in attenuation of h n -induced lung injury [ ] . after detecting viral components, transcription factors including nf-κb and irfs are activated, leading to transcription of ifns and pro-inflammatory cytokines. ifns bind to receptors, resulting in upregulation of multiple interferon-stimulated genes (isgs) [ ] . it is well known that type i ifns viruses , , of (ifn-α and ifn-β) and type iii ifns (ifn-λ - ) play critical roles in antiviral responses. mice failed to restrict non-pathogenic iav when both type i and type iii ifn receptors were knocked out [ ] . the expressed ifns consequentially bind to different receptors. type i ifns interact with ifn-α/β receptors (ifnar), whereas type iii ifns interact with ifn-λ receptors (ifnlr). janus kinase-signal transducer and activator of transcription (jak-stat) signaling pathway is then activated, resulting in transcription of numerous ifn-stimulated genes (isgs) [ , ] . though ifn-λs share many characteristics such as expression patterns, signaling pathways, etc. with type i ifns, they are the first ifns produced at the infected epithelial sites to block virus spread [ ] . furthermore, ifn-λs served an important role in programming dcs to direct effective t cell immunity against iav infection [ ] . isgs encode various antiviral proteins functioning in different ways to defend iav infection. for instance, mxa gtpase from the mx family could retain viral genome from entry to the cytoplasm via blocking the function of iav np. in addition, in vitro research found that avian iavs were more sensitive to mxa than human iavs [ , ] . cholesterol -hydroxylase (ch h) were identified to block iav entry via altering the cellular membrane properties to interfere with viral fusion, and amplified the activation of immune cells [ ] . guanylate-binding protein (gbp ) of ifn-inducible gtpases inhibited iav replication via binding to the viral polymerase complex [ ] . members of the tripartite motif-containing (trim) family are also involved in cellular anti-iav processes. for instance, trim could interact with iav np for ubiquitination and proteasomal degradation, thus restricting iav replication in a type i ifn and nf-κb independent manner [ ] . trim degraded iav np via polyubiquitination, thus resulting in inhibition of iav infection [ ] . trim regulated the re-localization of rig-i and was responsible for rig-i ubiquitination as well as rig-i-mediated ifn production [ ] . trim recognized iav pb protein and reduced its polymerase activity [ ] . trim targeted np for ubiquitination and degradation in vitro [ ] . for further reading on other isgs, several reviews regarding ifn responses during iav infection are recommended here [ , ] . a general description of activation of innate immunity and ifn signaling pathway after iav infection is illustrated in figure . signal transducer and activator of transcription (jak-stat) signaling pathway is then activated, resulting in transcription of numerous ifn-stimulated genes (isgs) [ , ] . though ifn-λs share many characteristics such as expression patterns, signaling pathways, etc. with type i ifns, they are the first ifns produced at the infected epithelial sites to block virus spread [ ] . furthermore, ifnλs served an important role in programming dcs to direct effective t cell immunity against iav infection [ ] . isgs encode various antiviral proteins functioning in different ways to defend iav infection. for instance, mxa gtpase from the mx family could retain viral genome from entry to the cytoplasm via blocking the function of iav np. in addition, in vitro research found that avian iavs were more sensitive to mxa than human iavs [ , ] . cholesterol -hydroxylase (ch h) were identified to block iav entry via altering the cellular membrane properties to interfere with viral fusion, and amplified the activation of immune cells [ ] . guanylate-binding protein (gbp ) of ifn-inducible gtpases inhibited iav replication via binding to the viral polymerase complex [ ] . members of the tripartite motif-containing (trim) family are also involved in cellular anti-iav processes. for instance, trim could interact with iav np for ubiquitination and proteasomal degradation, thus restricting iav replication in a type i ifn and nf-κb independent manner [ ] . trim degraded iav np via polyubiquitination, thus resulting in inhibition of iav infection [ ] . trim regulated the re-localization of rig-i and was responsible for rig-i ubiquitination as well as rig-i-mediated ifn production [ ] . trim recognized iav pb protein and reduced its polymerase activity [ ] . trim in order to counter ifn-stimulated antiviral proteins, iav viral proteins apply multiple strategies. for instance, ha protein was shown to trigger ubiquitination of ifnar to attenuate the type i ifn signaling pathway [ ] . the follow-up work showed that poly (adp-ribose) polymerase (parp ) functions as an interacting partner of ha protein to mediate the ha-induced ifnar degradation [ ] . ns is the most important ifns antagonist protein via mechanisms including inhibition of the trim -mediated rig-i ubiquitination, suppression of protein kinase r (pkr), in order to counter ifn-stimulated antiviral proteins, iav viral proteins apply multiple strategies. for instance, ha protein was shown to trigger ubiquitination of ifnar to attenuate the type i ifn signaling pathway [ ] . the follow-up work showed that poly (adp-ribose) polymerase (parp ) functions as an interacting partner of ha protein to mediate the ha-induced ifnar degradation [ ] . ns is the most important ifns antagonist protein via mechanisms including inhibition of the trim -mediated rig-i ubiquitination, suppression of protein kinase r (pkr), phosphorylation of iκb kinases (ikk) α and β in the nf-κb pathway, interruption of the phosphorylation of stat , stat , and stat [ , ] , and degradation of otub [ ] . phosphorylation of ns is crucial for its function of antagonizing ifn-β expression, since dephosphorylation at position and of the protein induced a high level of ifn-β [ ] . non-structural protein pb -f , identified from a+ open reading frame (orf) of pb gene segment [ ] , is multifunctional in deregulation of type i interferon [ , ] . it counteracted rlr-mediated activation of ifn pathway not only by targeting mitochondrial mavs [ , , ] , but also by binding to the dead-box helicase ddx to induce proteasome-dependent degradation [ ] . furthermore, pb -f interacted with mitochondrial tu translation elongation factor (tufm) to mediate formation of autophagosome, thus inducing complete mitophagy, which is critical for mavs degradation [ ] . novel pa-x protein could also modulate innate immune responses. a review regarding the function of ns and pa-x proteins in antagonizing host innate immunity is recommended here [ ] . though autophagy is essential for cellular metabolism and homeostasis, it also plays important roles in innate immune responses against pathogen infection. for cellular homeostasis, the mtor pathway is one of the most conserved autophagic pathways. the mtor complex (mtorc ) negatively regulates the ulk kinase activity, thus affecting the autophagy induction [ ] . c-jun n-terminal protein kinase (jnk ) disrupts the bcl- /beclin- complex through phosphorylation, thus regulating the autophagy induction [ , ] . jnk is also reported to upregulate beclin- expression through phosphorylation of transcription factor c-jun in vitro [ ] . in contrast to the autophagic pathways for cellular metabolism and homeostasis, less is known about autophagosome formation after iav infection [ ] . to restrict infection of multiple viruses including iavs, trim is essential to mediate autophagy via its ring e ligase and adp-ribosylation factor (arf) gtpase activity [ ] . beclin- and tufm-regulated autophagy also inhibited iav replication [ ] . in hela cells and a cells, iav infection activated jnk to induce autophagosome formation and tgf-β-activated kinase might contribute to the process [ , ] . furthermore, autophagy was involved in maintaining memory b cells to counteract iav infection [ ] . iav also utilizes autophagy to complete its life cycle. ns protein is proposed to suppress jnk -mediated autophagy induction [ ] . m could also block autophagosome maturation and mediate microtubule-associated protein light chain (lc )-bound membrane redistribution, thus allowing filamentous budding of iav [ ] [ ] [ ] . circ-gatad a (gata zinc finger domain containing a), induced by iav infection, could inhibit autophagy and promote iav replication [ ] . for a comprehensive reading on iav-induced apoptosis, a review is recommended here [ ] . upon detection of iavs, dcs trigger production of ifns and cytokines, which in turns assist maturation of the dcs into antigen presenting cells (apcs), and initiate t cell immune responses. through the activation of ag-bearing dcs, naïve cd + t cells differentiate into th , th , th , regulatory t cells (treg cells), follicular helper t cells, and killer cells. th and follicular helper t cells are the most abundant cd + t helper cells. they can secret antiviral cytokines, regulate cd + t cell differentiation, promote b cell activation, and maintain immunological memory [ , ] . th cells induced pulmonary pathogenesis and could decrease mortality of iav-infected mice [ , ] . in addition, γδ t cells, expanding in the late stage of iav infection with a t cell receptor (tcr)-independent viruses , , of manner, could efficient eliminate iav-infected airway epithelial cells, resulting in lower viral titers [ ] . new surrogate markers cd d and cd a were used to explore the kinetics of iav-specific cd t cells responses, revealing endogenous cd t cell response to primary iav infection is predominantly composed of t-bet+ cells [ ] . cd + t cells are major components for virus clearance in adaptive immunity. after activated by dcs, cd + t cells undergo rapid expansion, differentiation, and migration to the infected sites. in general, to establish effective primary cytotoxic t lymphocyte (ctl) responses, cd + t cells play an essential role, with a mouse model as an exception [ ] . ctls produce cytotoxic granules containing perforin and granzymes (gra and grb) to induce apoptosis and interrupt iav replication [ ] . in addition, ctls produce cytokines, such as tnf, fasl, and trail, which recruit death receptors to induce apoptosis [ ] . in addition, il deficiency enhanced the th and ctl responses upon iav infection [ ] . furthermore, as cd + cells could last for two years in murine models, iav-specific memory ctls reacted specific to epitopes in conserved iav proteins [ ] . in the nasal epithelia, they could prevent the spread of the virus from the urt to the lung [ ] . to establish memory cd + t cells, autophagy plays an important role [ ] , while the function of cd + t cells in memory ctl responses is "context-dependent". a recent study showed that cd + t cells promoted iav-specific ctl memory at the initial priming stage of viral infection [ ] . grant and her colleagues summarized and discussed the importance of cd + t cell immunity against iavs [ ] , and this review is recommended for further reading. with the help of cd ligand (cd l), cd + cells contribute to b cell activation [ ] . with the help of memory t cells, naïve b cells could reduce morbidity and promote recovery on heterosubtypic infection [ ] . for different types of antibodies, igg could inhibit pathogenesis, while iga functions in blocking iav transmission [ ] . in addition, iav-specific antibody-dependent cell-mediated cytotoxicity (cdcc) also plays a role in cross-protection against iav infection. a general description of adaptive immunity against primary iav infection is illustrated in figure . antigenic shift and drift, resulting in reassorted and mutated ha and/or na, are responsible for aiv escaping from host immunity [ ] [ ] [ ] . furthermore, additional glycosylation on h ha could also induce virus escape from neutralizing antibodies [ ] . apoptosis represents programmed single cell death that occurs in cell physiological remodeling, cell proliferation, or immune response to invading pathogens [ ] . besides prototypical changes, cells undergoing apoptosis can be detected through dna and biochemical assays, take the tunel and in situ end-labeling (isel) techniques for instance. two primary pathways are involved in activation of apoptosis: the intrinsic or mitochondrial pathway, and the extrinsic or death receptor pathway. the intrinsic pathway is also known as "the mitochondrial pathway", which operates in response to various intracellular stress. several factors such as nitric oxide (no), cytochrome c, and second mitochondria-derived activator of caspases (smac) can activate this pathway, and the key player of this pathway is proteins in the bcl- family, which are activated by stress signals and then release apoptotic factors via destabilizing the mitochondrial membrane [ , ] , resulting in release of mitochondrial cytochrome c. cytochrome c then binds to apoptosis protease activating factor- (apaf- ) and forms a complex with pro-caspase (then cleaved into caspase ), the function of which is to cleave its effector pro-caspase [ ] . in addition, smac, localizing in the cytosol, could initiate activation of caspase via blocking the activity of iap [ ] . the extrinsic pathway is regulated by extracellular ligands acting on transmembrane "death receptors": the first apoptosis signal (fas) receptor-fas ligand (fasr/fasl) and the tnf-αtnf receptor (tnfα/tnfr ) [ ] . in the fasr/fasl model, fas ligand binds to its receptor fasr [ ] , forming the death-inducing signaling complex (disc) with pro-caspase , resulting in activation of caspase and downstream activation of other caspases (caspase- , caspase- , and caspase- ) [ ] . in the tnfα/tnfr pathway, tnfr -associated death domain protein (tradd) is activated after binding of tnfα to tnfr , leading to recruitment of fadd and receptor interacting protein (rip) [ ] . fadd then associates with pro-caspase to form the disc, resulting in activation of caspase and apoptosis. could prevent the spread of the virus from the urt to the lung [ ] . to establish memory cd + t cells, autophagy plays an important role [ ] , while the function of cd + t cells in memory ctl responses is "context-dependent". a recent study showed that cd + t cells promoted iav-specific ctl memory at the initial priming stage of viral infection [ ] . grant and her colleagues summarized and discussed the importance of cd + t cell immunity against iavs [ ] , and this review is recommended for further reading. with the help of cd ligand (cd l), cd + cells contribute to b cell activation [ ] . with the help of memory t cells, naïve b cells could reduce morbidity and promote recovery on heterosubtypic infection [ ] . for different types of antibodies, igg could inhibit pathogenesis, while iga functions in blockin during iav infection, viruses modulate host apoptotic responses in a time-dependent manner [ ] . for instance, in order to earn enough time for replication and virion formation, iav inhibited apoptosis via upregulating the anti-apoptotic phophoinositide- -kinase-protein kinase b (pi k-akt) pathway at the beginning of infection. however, in the later phase of infection, the virus suppressed this pathway to upregulate the pro-apoptotic p pathway, thus allowing successful release of virions [ ] . several viral proteins are involved in regulation of host apoptosis. np protein induces host apoptosis to favor viral replication through interaction with ring finger (rnf ) [ ] , apoptotic inhibitor (api ) [ ] , or clusterin [ ] . pb -f also induced apoptosis and promoted viral replication through dysregulating mitochondrial potential [ ] . furthermore, m promoted apoptosis by binding to heat shock protein , thus activating caspase and the subsequent apoptosis [ ] . in addition, ns expression was reported to induce apoptosis in mdck and hela cells [ ] . however, mutant iav lacking the ns gene could induce apoptosis in cultured cells [ ] . the function of ns in inhibiting apoptosis may be explained by its ability to inhibit type i ifn [ , ] . these data demonstrate sophisticated mechanisms of iav in regulating host apoptosis. furthermore, the role of these viral proteins in apoptosis suggests that these proteins may present suitable targets for anti-iav therapies. a comprehensive review on influenza a virus-induced apoptosis discussed by ampomah and lim is recommended here [ ] . in addition, recent in vitro research found that apoptosis was induced at early iav infection stage, while later the cell death pathway was shifted to pyroptosis. the switch process was promoted by the type i ifn-mediated jak-stat signaling pathway through expression of the bcl-xl gene [ ] . during iav infection, multiple immune systems coordinate together to protect the host. accordingly, viruses antagonize the immune system through multiple measures to establish a successful infection. considering the high frequencies in genome mutations and recombination, vaccination is the most effective way to defend against the viruses via inducing cross-protective antibodies and/or enhancing immune responses. several studies in vaccine development have tried to enhance host immune responses. for instance, vaccine candidate containing ha targeted to chemokine receptor (porcine mip α) was shown to enhance t cell responses, resulting in a strong and cross-reactive cellular immunity in vaccinated pigs [ ] . another example is an attempt to intranasally administer a polyanhydride nano vaccine (iav-nanovax), which could promote robust lung-resident germinal center (gc) b cells with lung-localized iav-specific antibody responses as well as lung-resident memory cd + and cd + t cell responses [ ] . for anti-iav drugs, currently, na inhibitors (relenza tm and tamiflu tm ) are applied clinically as anti-influenza drugs [ ] . these drugs inhibit the activity of na by preventing viral budding [ ] . in addition, cap-dependent endonuclease inhibitor (baloxavir marboxil) targeting pa is also applied against influenza a and b virus infection [ ] . our progressing understanding of the iav life cycle of the virus and iav-host interaction could contribute to anti-influenza drug design. since the recognition of ha protein to sa linked glycoproteins is the first step in iav infection, effective blocking of the interaction between viral ha and sa receptor serves as a favorable target in drug design [ , ] . favipiravir, a nucleotide analogue that selectively inhibits the rna-dependent rna polymerase, is licensed in japan to be applied against emerging influenza viruses resistant to other antivirals [ , ] . oleanolic acid (oa), a kind of pentacyclic triterpene natural product, and its analogues, as well as its derivatives, were shown to bind to ha, thus blocking the attachment of iavs to mdck cells [ ] [ ] [ ] . pvf-tet is a peptide-based ha inhibitor, which was shown to sequester ha into amphisome (fusion of late endosome with autophagosome) and protected mice from the lethal iav infection [ ] . new effective drugs targeting the polymerase would be a promising strategy against iav infection, since they would directly reduce or eliminate viral replication. numerous sites, including the cap-binding site [ ] , the endonuclease [ , ] , and pa-pb inter-subunit interface [ ] can serve as potential targeting sites for new drug design. coumarin compounds, including eleutheroside b , isofraxidin, fraxin, esculetin, fraxetin, and scoparone, were investigated for their antiviral and anti-inflammatory activities against influenza virus in vitro [ ] . other candidates, such as naproxen, a non-steroidal anti-inflammatory drug, was shown to target np protein at residues f and y , thus antagonizes the crm -mediated nuclear export of np. it is suggested to have a broad-spectrum anti-influenza activity [ ] . verdinexor (kpt- ), a novel orally bioavailable drug, blocks crm -mediated nuclear export of np and repress nf-κb activation, thus reducing cytokine production and eliminating virus-associated immunopathology [ ] . for further reading on candidate anti-iv therapeutics, a review summarized by davidson is recommended here [ ] . with the increasing knowledge obtained through massive investigations on host immunity against iav infection, promoting host immune responses not limited to antibody enhancement would have good prospects not only for vaccine design, but also for development of novel antiviral agents. author contributions: manuscript preparation, y.z.; revision, z.x.; supervision, y.c.; funding acquisition, y.z. all authors read and approved the final version of the manuscript. funding: this study was supported by the "zhujiang talent program" overseas youth talent introduction program (post-doctoral program) and doctoral initiative project of natural science foundation of guangdong province ( zxxt ). the authors declare that they have no financial and personal relationships with other people or organizations that can influence the work. there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in this review. they do not have any commercial or associative interest that represents conflicts of interest in connection with the work submitted. influenza virus aerosols in the air and their infectiousness influenza: the once and future pandemic identification of a 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against rsv and influenza viruses treating influenza infection, from now and into the future key: cord- -whg w w authors: bhatta, tarka raj; ryt-hansen, pia; nielsen, jens peter; larsen, lars erik; larsen, inge; chamings, anthony; goecke, nicole b.; alexandersen, soren title: infection dynamics of swine influenza virus in a danish pig herd reveals recurrent infections with different variants of the h n swine influenza a virus subtype date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: whg w w influenza a virus (iav) in swine, so-called swine influenza a virus (swiav), causes respiratory illness in pigs around the globe. in danish pig herds, a h n subtype named h n dk is one of the main circulating swiav. in this cohort study, the infection dynamic of swiav was evaluated in a danish pig herd by sampling and pcr testing of pigs from two weeks of age until slaughter at weeks of age. in addition, next generation sequencing (ngs) was used to identify and characterize the complete genome of swiav circulating in the herd, and to examine the antigenic variability in the antigenic sites of the virus hemagglutinin (ha) and neuraminidase (na) proteins. overall, . % of the pigs became pcr positive for swiav during the study, with the highest prevalence at four weeks of age. detailed analysis of the virus sequences obtained showed that the majority of mutations occurred at antigenic sites in the ha and na proteins of the virus. at least two different h n variants were found to be circulating in the herd; one h n variant was circulating at the sow and nursery sites, while another h n variant was circulating at the finisher site. furthermore, it was demonstrated that individual pigs had recurrent swiav infections with the two different h n variants, but re-infection with the same h n variant was also observed. better understandings of the epidemiology, genetic and antigenic diversity of swiav may help to design better health interventions for the prevention and control of swiav infections in the herds. the influenza a virus (iav) is a negative-sense, single-stranded, eight-segmented rna virus belonging to the family orthomyxoviridae [ ] . the main antigenic proteins are encoded by the surface gene segments hemagglutinin (ha) and neuraminidase (na). the six internal gene segments encode for polymerase b (pb ), polymerase b (pb ), polymerase a (pa), nucleoprotein (np), matrix (m and m ), and non-structural protein (nep-ns ) respectively [ , ] . there are different ha (h to h ) and different na (n to n ) subtypes. most of these subtypes can be found in aquatic birds (h -h and n -n ), whereas only a few subtypes are found in mammals [ , ] . in pigs, circulation of iav, so-called swine influenza a virus (swiav), is currently mainly limited to three different subtypes including h n , h n and h n [ ] [ ] [ ] . avian-like swine h n swiav, was first detected in european pig herds in the late s [ , ] and caused epizootic disease outbreaks that resolved shortly within a few weeks [ , ] . however, several recent studies have shown that the dynamics of swiav have changed to a more enzootic form where the virus persists in the herds for months or even years [ ] [ ] [ ] [ ] [ ] [ ] . the altered dynamics of swiav from a short-term epizootic disease to continuous circulation in the herds, is probably a consequence of increased herd sizes and the continuous supply of naïve individuals that maintain the infection [ , ] . in european pig herds, an average prevalence of % has been estimated for swiav infection [ ] . the most prevalent subtypes identified in europe in recent years were the eurasian avian-like swine h n ( . %), the pandemic a/h n (h n pdm ) ( . %), the human-like reassortant swine h n ( %), and the human-like reassortant swine h n ( . %) [ ] . in addition, a number of studies have also shown evidence of reassortants with internal gene segments of h n pdm and the surface gene segments of predominant enzootic swiav [ ] [ ] [ ] [ ] . in denmark, the human-like reassortant h n has never been detected. however, another h n reassortant is widespread among danish pig herds. this subtype is termed "h n dk" and has the ha gene of the eurasian avian-like h n subtype and the na gene of the h n human-like swiav [ ] . in addition to the subtypes mentioned above, introductions of human seasonal iav occurs regularly, increasing the risk of novel swiav reassortants, and making the disease more difficult to control in the herds [ ] . in addition, similar to other rna viruses, swiav has a high mutation rate, which drives the viral evolution and helps the virus evade the immune system by creating novel variants with modified antigenicity [ ] [ ] [ ] . mutations in the antigenic sites (cb, sa, sb, ca and ca ) of the ha protein can lead to the virus being able to escape the binding of neutralizing antibodies [ , ] . moreover, mutations in b-cell epitopes and t-cell epitopes of other iav proteins might also impact the host immunity [ ] [ ] [ ] [ ] . finally, mutations favouring altered n-linked glycosylation (nlg) sites near/within ha and na antigenic sites can also affect the binding of antibodies [ ] . according to the danish agriculture and food council, denmark produces approximately million pigs annually from around three thousand herds of which approximately million are exported as weaners to other countries such as poland and germany [ , ] . in contrast, denmark imports a very limited number of live pigs. in denmark and other countries worldwide swiav is one of the causes of respiratory infection in pigs [ ] . swiav infections lead to destruction of the epithelial cells and impairs the immune system, thereby making the host more susceptible to infection by other viruses and bacteria. co-infection of pigs with distinct variants of swiav and other respiratory pathogens (like pasteurella multocida, mycoplasma hyopneumoniae, haemophilus parasuis, actinobacillus pleuropneumoniae, porcine circovirus type and porcine reproductive and respiratory syndrome virus) are known to cause enhanced disease compared to single pathogen infections, and are all part of the porcine respiratory disease complex (prdc), which can lead to massive economic losses [ , [ ] [ ] [ ] . the aim of the present study was to monitor the molecular epidemiology of swiav circulating in pigs between two weeks of age to weeks of age in a danish pig herd. a newly developed high-throughput real-time pcr (rtpcr) system (fluidigm, south san francisco, usa), which consists of rtpcr assays targeting selected respiratory and enteric viral and bacterial pathogens [ ] was applied for initial detection of swiav in nasal swab samples. moreover, next generation sequencing (ngs) was used to characterize the complete genome of swiav during natural infections, and to examine the genetic variability in the antigenic sites of the virus ha and na proteins. in this study, only nasal swabs were collected and thereby the study did not include the introduction of a needle, which according to the danish law on animal experimentation (lbk number of may ) is the minimum intervention that requires a specific license. a trained veterinarian was involved in the sampling and data collection, and farmer consent was obtained before the sample collection. the study was designed as an observational cohort study to monitor/screen pig health (particularly swiav infection) in a danish farrow-to-finish continuous-flow pig herd. the herd had sows and a farrowing area divided into a number of units. nursery pigs were housed at a separate site with pen places, and finisher pigs were raised at a third site with pen places. according to the danish spf (specific pathogen free) herd health declaration (seges svineproduktion ), the herd was declared positive for mycoplasma hyopneumoniae (m. hyo), actinobacillus pleuropneumoniae (app) type and and porcine reproductive and respiratory syndrome (prrs) type virus, and negative for app type , prrs type virus, brachyspira hyodysenteriae, atrophic rhinitis, demodex mites and lice. the herd had contract with a large abattoir and processing company in denmark, which in initiated the "raised without antibiotics" (rwa) concept [ ] . at present (march ), danish herds including this herd are included in the rwa program, where the producers are payed a premium price to reflect the cost of the increased workload, intensified hygiene measures and other interventions to prevent disease in the effort to reduce the use of antimicrobial agents. the herd produced and recruited gilts internally. after weaning, pigs selected for gilt recruitment were housed in a separate room at the sow site, and vaccinated with a live prrs type virus vaccine (ingelvac ® prrs vet.) at and weeks of age, and additionally with m. hyo, porcine circovirus- (pcv ) (porcilis ® pcv m hyo) and swiav (respiporc flu ) vaccines at , and weeks of age. all adult sows were vaccinated against swiav simultaneously four times a year (respiporc flu ). due to clinical signs like nasal discharge and swiav positive laboratory testing, piglets were vaccinated at four days of age with respiporc flu ( . ml, off label use). during the first week after weaning, pigs were vaccinated against m. hyo and pcv (porcilis ® pcv m hyo). the prevalence of different iav strains in danish pig herds has been reported to be around % [ ] . based on this data, the iav prevalence in this herd was assumed to be between % and %. by using a minimum sample size of , this should, at a very minimum, give at least one iav-positive sample with a high probability of getting iav-positive samples from - pigs. it was assumed from the outset of the study that some piglets may be lost to follow-up or that some may die and hence it was decided to use an initial sample size of . all live-born piglets born within three consecutive farrowing days (n = ) were ear-tagged at birth with consecutive unique id numbers. from every th piglet (id number , , etc.) nasal swabs were obtained (at five to nine day intervals) at week (n = ), week (n = ), week (n = ), week (n = ), week (n = ), week (n = ), week (n = ) and week (n = ). overall, individual piglets/pigs were sampled throughout the study (table s ). due to the variable weaning age (week or week ) of the piglets, the week sampling was done either at the farrowing unit (week f, n = ) or in the nursery unit (week n, n = ) situated at separate buffer stables at the sow site. at week , all weaned piglets were moved into separate nursery units and housed with other pigs from the same herd of the same age until approximately weeks of age. thereafter, the nursery pigs were transferred to a finisher site (~ kilometre away) where they were housed until slaughter at approximately weeks of age. this finisher site received piglets from the nursery units of the same herd. the samples were collected from the april until the september . small-or medium-sized sterile rayon swabs (medical wire, wiltshire, uk) were used to collect the nasal swab samples. the swab was inserted into one nostril of the individual piglet and was then turned a full • . samples were stored in ml phosphate buffered saline (pbs) at - • c until delivered to the laboratory within two days after sampling. at weeks and , piglets were clinically examined before obtaining the nasal swabs. clinical signs of nasal discharge and conjunctivitis were recorded for each individual pig. all samples were processed at the centre for diagnostics (cfd), technical university of denmark (dtu). the pcr analysis was carried out at cfd-dtu, while the ngs was carried out at statens serum institute (ssi), denmark. all the collected samples were vortexed for min, the rayon swab was then removed from the tube and µl of the fluid transferred to a ml eppendorf (ep) tube. the ml ep tubes were centrifuged at × g for min at room temperature. finally, µl supernatant was used for nucleic acid extraction. nucleic acid (both dna and rna) was extracted using the qiacube ht extraction robot (qiagen, hilden, germany) and the cador pathogen qiacube ht kit (qiagen) according to the manufacturer's instructions. the extracted nucleic acids were stored at − • c for further use. the extracted nucleic acids were subjected to reverse transcription using a high capacity cdna rt kit (applied biosystems, foster city, ca usa). a final volume of µl reaction mix was prepared by mixing µl of x rt buffer, . µl of mm dntp mix, µl of x random hexamer, . µl of multiscribe rt enzyme, . µl of nuclease free water and µl of extracted nucleic acids. finally, cdna synthesis was carried out in a t thermocycler (biometra, fredensborg, denmark) with the given cycling conditions: • c for min, • c for min followed by • c for min and finally paused at • c. the cdna sample was pre-amplified using x taqman preamp master mix (applied biosystems). a total volume of µl was prepared by mixing . µl of cdna with µl of x taqman preamp master mix (applied biosystems) and . µl of a nm primer mix (containing the different sets of primers used for the detection of different pathogens) as previously described [ ] . in brief, pre-amplification was carried out in a t thermocycler (biometra) using the program • c for min followed by cycles of • c for s and • c for min. finally, the amplification was paused at • c and the pre-amplified product was stored at − • c for further use. initially, the collected samples were screened for iav by using the high-throughput rtpcr platform biomark (fluidigm, south san francisco, usa) and the . dynamic array (da) integrated fluidic circuit (ifc) chip (fluidigm). a µl sample mix was prepared by mixing . µl of pre-sample mix (prepared by mixing µl of x taqman gene expression mastermix (applied biosystem) and . µl of x sample loading reagent (fluidigm) for each sample) and . µl of pre-amplified sample. similarly, µl primer/probe stock was mixed with µl of x assay loading reagent (fluidigm). three µl of the assay mix and µl of sample mix was loaded into the respective inlets of the . da ifc chip. the . da ifc chip was placed in the ifc controller rx for loading and mixing for approximately min. finally, the chip was inserted into the high-throughput rtpcr platform biomark (fluidigm) for thermal cycling with the following cycling condition: • c for min, • c for min followed by cycles of • c for s and • c for s. all samples were tested in duplicates. positive and non-template (nuclease-free water) controls were included. amplification curves and cycle threshold (ct) values were obtained on the biomark system and finally analysed using fluidigm real time pcr analysis software . . (fluidigm) as previously described [ ] . samples found positive for iav using the high-throughput rtpcr system, were selected for rna extraction. the extraction was carried out for the selected nasal swabs using the qiacube extraction robot (qiagen) and the rneasy mini kit (qiagen) according to the manufacturer's instructions as described previously [ ] . rna was eluted in µl rnase-free water and stored at − • c. detection of iav in the extracted rna was performed in the rotor-gene q (qiagen) pcr platform using a previously published rtpcr assay targeting the matrix gene of iav [ , ] . confirmed iav positive samples with ct values < (using rotor-gene pcr) were used for full genome amplification of iav. a one-tube reaction amplifying each gene segment of iav was performed using a modified version [ ] of a previously published assay [ ] . five µl of the amplified one-tube full genome iav pcr products along with µl of kb dna ladder (invitrogen, carlsbad, ca usa) were run on . % agarose gels (invitrogen) to check if the bands representing all gene segments of iav were visible on a bio-rad gel documentation system (hercules, ca, usa). only fully amplified iav pcr products were selected and were purified using a high pure pcr product purification kit (roche, mannheim, germany) following the manufacturer's protocol. the purified pcr products were sent for ngs on the illumina miseq sequencing platform at the state serum institute (copenhagen, denmark). sequence analysis was performed in clc genomic workbench . . . software (qiagen). all reads obtained from each of the samples were initially trimmed to remove short and low quality reads and primers/adaptors and then consensus sequences of each gene segment was constructed using the function "map reads to reference", using a panel of sequences representing each different lineage of each gene segment known to be present in denmark. the consensus sequences of each gene segment were then aligned using the muscle algorithm [ ] , and then examined for similarities using the function "create pairwise comparison". moreover, the function, blastn, was used to compare the generated consensus sequences with the online ncbi genbank database [ , ] . further analysis was done by selecting some of the closest swiav sequences from the ncbi genbank. phylogenetic analysis was done after aligning all sequences of each gene using clustal-w [ ] and using the maximum likelihood (ml) method with the best fitting substitution model in mega [ ] . the ha subtype numbering was done using the influenza research database (ird) tool at http://www.fludb.org [ , ] . the different amino acids present in the ha antigenic sites (cb, sa, sb, ca and ca ) were calculated by comparing our sequences with reference sequences [ , [ ] [ ] [ ] . b-and t-cell epitopes were analysed by aligning our sequences with reference sequences [ ] [ ] [ ] [ ] using clustal-w [ ] . differences present at seven antigenic sites ( , a, b, c, d, and ) of the na gene segment were calculated by comparing our sequences with reference sequences [ ] [ ] [ ] [ ] . netnglyc . (http://www.cbs.dtu.dk/services/netnglyc/) from dtu bioinformatics (department of bio and health informatics) [ ] was used for the prediction of n-linked glycosylation sites in the ha gene segments only on the n-x-s/t sequons (excluding p at x) with a score threshold > . . fisher's exact test was used to determine any association of data using the analysis tool by .xls at http://itve.dk/ [ ] . the results of the high-throughput rtpcr analysis of nasal swab samples, including an assay for detection of the matrix gene of iav, allowed for estimation of the prevalence of swiav at all sampling times. swiav was detected in pigs throughout the sampling period starting from week until week , indicating continuous circulation of iav in the herd ( table ). the prevalence of swiav increased from the first sampling (week ) ( . %) until week ( . %). among week- -old piglets, . % ( . - . % at % confidence interval) were iav-positive in the farrowing unit while % ( . - . % at % confidence interval) were iav-positive in the nursery unit (weaned at week or week ). after weaning (week ), the prevalence stabilized and then decreased, reaching the lowest prevalence at week ( . %). after the pigs had been transferred to the finisher site, the prevalence increased again, reaching . % at weeks of age. in total, . % of the pigs were iav-positive at least once during the study period (table ). occurrence of iav is defined as the detection of iav in nasal swabs in one or more consecutive weeks in the same pig, whereas recurrence is defined as the detection of iav from the same pig at two or more non-consecutive weeks [ ] . of pigs, ( . %) were found to be positive for iav at least once, whereas ( . %) pigs were found to be negative throughout the study (table s ). all the iav positive and negative pigs were housed together in mixed pens. among the positives, of the pigs ( . %) were found to have recurrent iav (table s ), whereas of pigs ( . %) were found to have only a single occurrence of iav. of these pigs, ( . %) pigs were iav-positive only at a single sampling time, whereas nine pigs ( %) tested positive at two consecutive sampling time points (table s ). among the piglets sampled in week two, ( . %) had nasal secretion and piglets ( . %) had conjunctivitis. however, only two ( . %) piglets had nasal secretion, while four piglets ( . %) had conjunctivitis in week . nasal secretion was recorded in both of the iav-positive piglets at week , while it was only present in two of the positive piglets at week . similarly, conjunctivitis was observed in one out of two positive piglets at week and was only present in one out of positive piglets at week . no association between iav infection in the individual pigs and clinical signs of nasal secretion and conjunctivitis at week and was observed (supplementary tables s , s , s and s ). fifteen samples that had a ct value < in the iav rtpcr assay were selected for one-tube full genome amplification. of these, samples (table s ) displayed clear bands representing all the iav gene segments on the agarose gel and were selected for iav whole genome sequencing (wgs) ( table s ). all the generated sequences for eight full gene segments from all the pig samples have been deposited in ncbi genbank with accession numbers mt -mt . eleven full length ( nt) ha gene segments from the study were used for phylogenetic analysis. twenty three additional ha reference sequences representing avian, human, classical swine and h n pdm subtypes were downloaded from ncbi genbank and included in the analysis. phylogenetic analysis revealed that all the ha gene segments were of eurasian avian-like h nx origin ( c. lineage using the global swine h clade classification system) [ ] . all nine h sequences obtained from pigs sampled between week and were grouped together in one cluster and had pairwise sequence differences of to . % at the nucleotide level and to . % at amino acid level. this cluster had the highest sequence identity to a/swine/germany/holdorf-idt / (h n ) (accession no.: kr ) in a blastn search with~ % identity at the nucleotide level and~ . % at the amino acid level [ ] . the two h sequences obtained from pigs sampled at week were % identical but clustered separately from the h sequences obtained from the younger pigs ( figure ). these h genes had the highest sequence identity to a/swine/denmark/ - - / (h n ) (accession no.: kr ) in the blastn search with identities of . % at the nucleotide level and . % at the amino acid level [ ] . as mentioned earlier, sequences from two pigs shedding swiav at two non-consecutive sampling times were obtained (pig id sampled at week and week , pig id sampled at week and week ; table s ). in the phylogenetic analysis, the ha sequences of pig id , obtained at weeks and were located in the same cluster and were~ . % ( / ) divergent at the nucleotide level and < % ( / ) divergent at the amino acid level. in contrast, the ha sequences of pig id at weeks and were~ % ( / ) divergent at the nucleotide level and % ( / ) divergent at the amino acid level. from the phylogenetic analysis of pig id it was clearly seen that the ha sequence obtained at the first sampling were located in the cluster defined by the viruses from the younger pigs, whereas the ha sequence obtained at the last sampling (after the pigs had been transferred to the finisher site) were located outside this cluster, thereby representing another h n variant (figure ). the majority of the differences between the two different samplings (week and week ) for pig id and week and week for pig id were located in the antigenic sites of the ha protein between amino acid - . three different amino acid differences were found in the antigenic sites (v a and k n at ca and s h at the sa region) of pig id at week and week . whereas, different amino acids differences were found in the antigenic sites of pig id at week and week (table ) . sequences from different individual piglets sampled at week have one different amino acid (s h) at the sa region and two different amino acids (v a and k n) at the ca region. similarly, sequences from pigs of weeks of age had only one different amino acid (s h) at the sa region. the two ha sequences from week were found to have identical amino acids in the antigenic sites. the cleavage site with one arginine (psiqsr: - ) and fusion peptide sequence (glfgaiagfieggwtgmidgwyg: - ) were found to be conserved in all full-length h ha sequences [ , ] . the ha region of all the ha sequences were found to be more conserved compared to the ha region, as they were only approximately - % divergent at the nucleotide level and - % divergent at the amino acid level. however, the ha region of all the ha sequences were - . % divergent at the nucleotide level and - . % divergent at the amino acid level. eleven full-length ( nt) na gene segments were used for phylogenetic analysis (figure ). twenty seven additional na reference sequences representing danish h n , european h n , h n and asian and american h n na were also used. the phylogenetic tree shown in figure revealed that the n na gene segments from the present study were most identical to the na segment of the danish h n and european h n subtypes. the n sequences obtained from pigs sampled between week and week grouped together in one cluster (danish hxn type) and had pairwise sequence differences of to . % at the nucleotide level and to . % at the amino acid level. this cluster had the highest sequence identity to a/swine/germany/holdorf-idt / (h n ) (accession no.: kr ) in the blastn search with an identity of . % at the nucleotide level and > % identity at amino acid level, when performing a pairwise comparison [ ] . in contrast, the two n sequences obtained from pigs at week were identical and were positioned apart from the cluster formed by the sequences obtained from the younger pigs. similar to the ha segment, this cluster had the highest sequence identity to a/swine/denmark/ - - / (h n ) (accession no.: kr ) in the blastn search, with an identity of . % at the nucleotide level and > % identity at the amino acid level [ ] (figure ). the n sequences obtained from pig id at weeks and were found to be < . % ( / ) divergent at the nucleotide level and . % ( / ) divergent at the amino acid level. however, the two n na sequences obtained from pig id at weeks and were found to be~ . % ( / ) divergent at the nucleotide level and~ . % ( / ) divergent at the amino acid level. amino acids present in the antigenic sites ( , a, b, c, d, and ) of the n na sequences of pig id at week and week were found to be identical. while the n sequence obtained from pig id at week was similar to the ones from pig id , the sequence from pig id at week were found to be~ . % ( / ) divergent in the antigenic sites of n . similarly, n sequences of pig id at antigenic site d were found to be most divergent, at % ( / ), whereas no changes were found in the antigenic site ( table ). all n na sequences have eight highly conserved amino acids (r , d , r , r , e , r , r and y ) at the inner shell of the na active site, which interact directly with sialic acids. similarly, ten highly conserved amino acids (e , r , w , s , d , i , e , e , n and e ) [ , , ] were also present in an outer shell of the na active site. hence, all the amino acids present at na active sites were found to be conserved. table . comparison of amino acid sequences of neuraminidase (na) antigenic sites of pig id sampled at week and week from the pig herd. amino acid positions were numbered using an n numbering system. amino acid change all the six internal gene segments (pb , pb , pa, np, m -m and nep-ns ) were compared with available online ncbi genbank reference sequences. all the internal gene segment sequences obtained from younger pigs (≤ weeks) were found to be most identical (> %) with the internal gene segments of h n pdm origin. in contrast, only four of the internal gene segments (pb , pb , pa and np) of virus sequences from older pigs (week ) were of h n pdm origin. the remaining two internal gene segments (m -m and nep-ns ) of sequences from older pigs (week ) clustered with the h n dk and european h n swiav subtypes, indicating that these viruses are the result of reassortment events between h n -like viruses and the h n pdm subtypes. in addition, respective internal gene segments obtained from pig id at week and week were compared and were found to be - . % different at the nucleotide level and - . % at the amino acid level. a similar comparison of respective internal gene segments was also done for pig id at weeks and and were found to be . - . % different at the nucleotide level, while at the amino acid level they were . - . % different ( table ). the m -m gene segments of pig id at week and were found to be . % divergent at the nucleotide level as shown in table . similarly, the nep-ns gene segments of pig id at weeks and were found to be most divergent ( . %) ( table ) and also clearly supported by two distinct clusters in the phylogenetic tree ( figure ). nep-ns gene sequences obtained from pigs sampled between week and week made one cluster close to the h n pdm subtypes. in contrast, the nep-ns sequences obtained from pigs sampled at week made a separate cluster and resembled the h n dk and european h n subtypes (figure ). table . pairwise comparison of six internal gene segments of pig id sampled at week and week both at the nucleotide and the amino acid level. week clearly supported by two distinct clusters in the phylogenetic tree ( figure ). nep-ns gene sequences obtained from pigs sampled between week and week made one cluster close to the h n pdm subtypes. in contrast, the nep-ns sequences obtained from pigs sampled at week made a separate cluster and resembled the h n dk and european h n subtypes (figure ). b-and t-cell epitopes present in all the swiav sequences between week and week were identical. one amino acid change (r k) was observed in the b-cell epitope present in the ha part of the ha gene segment after comparing ha sequences from pig id at week and week . similarly, one (e d) and four different amino acids (l f, s n, e v and s t) were found in the t-cell epitope sequence of np and ha gene segments, respectively. t-cell epitopes of other gene segments were found to be identical among the compared isolates. six to seven nlg sites were present in all the ha sequences obtained in this study of which five (at positions , , , and ) were well conserved between all analysed sequences. all the ha sequences obtained from pigs sampled between week and week had one separate nlg site at position , whereas the ha sequences obtained from pigs sampled at week have two other nlg sites at position - and at position . the nlg site at position was located near the ca antigenic site in the globular head domain of the ha protein sequence, whereas the nlg site at position - was located near the sa antigenic site (table ) . similarly, the nlg site at position was located within the sa antigenic site of the ha protein sequence. table . n-linked glycosylation (nlg) sites of h hemagglutinin (ha) gene segments obtained from all the pigs from week to week and week . "+, ++, +++" indicates the nlg potential with score threshold > . . "*" indicates that the nlg site is located in between amino acid - for h numbering system. the study was designed to describe the infection dynamics of swiav in one danish pig herd by following pigs from weeks of age until slaughter (approximately weeks of age). using a high-throughput rtpcr system, we were able to determine the prevalence of iav, and by the use of ngs, we characterized the genetic and antigenic diversity of circulating h n swiav. based on the analysis, it was found that the prevalence of iav in pigs reached a maximum around weaning ( - weeks) and then decreased until weeks and then increased again at weeks of age. at least two different h n variants were circulating in the herd, with one of the h n variants circulating at the sow and nursery sites, and the other circulating in the finisher site. finally, we also demonstrated that individual pigs could have recurrent iav infections either with a very similar h n variant (pig id ) or with two divergent h n variants (pig id ). this confirms previous findings that some pigs can have prolonged swiav infections and be subjected to re-infection, even with closely related swiav [ , ] . the understanding of the epidemiology and the genetic and antigenic diversity of swiav in pigs may help to unravel the layers of swiav infection dynamics and viral evolution from birth to slaughter, thereby helping to design better health interventions for the prevention and control of swiav in the herds. the relative low prevalence of iav before week might be due to the presence of maternally-derived antibodies (mdas), which in this herd were stimulated by the sows being vaccinated with respiporc flu four times annually [ , ] . however, the mdas wane over time, and several studies have shown results indicating that piglets are no longer completely protected from around three to four weeks of age [ , , ] . moreover, the loss of mda occurs at the same time as the piglets are weaned into the nursery, thereby mixing different litters of pigs, creating the optimal environment for iav circulation. this has also been observed in previous studies [ , , ] . similarly, a higher prevalence of iav at week also indicated that the piglet immune system did not respond to the vaccination at day , which is in accordance with a previous study showing no effect of early piglet vaccination against swiav [ ] . one of the explanations for this is that the presence of mda may hinder an active immune response in the piglets, but it could also be due to the reduced vaccine dose used ( . ml) [ ] in the piglets under study. after week , most of the weaned pigs likely developed immunity to the circulating iav subtype and at the same time no new pigs were introduced into the nursery, resulting in a lower prevalence of iav in accordance with other findings [ ] . the increase in prevalence observed at week at a separate finisher site, was most likely due to infections with a second h n dk variant that might have been circulating continuously in the finisher site. this site was managed as a multi-aged, continuous-flow pig herd. this h n dk variant from the finisher site differed significantly in antigenic regions from the h n dk variant circulating at the sow and nursery sites. mutations in the antigenic sites of eurasian avian-like swine h have previously been linked to a lack of cross protection and emphasize that the diversity within the subtypes, especially in the h avian-like viruses, have now reached a level where it makes no sense to consider viruses of the same subtypes as belonging to the same serotype [ ] . however, the lack of a significant cross-reaction should be confirmed by hi-testing, which was not performed in this study. moreover, the na gene and the internal gene cassette were also different between the two h n dk variants, which could also impact the cross-protective immunity between different swiav variants of the same subtype [ , ] . specifically, the internal gene cassette of the h n dk variant that infected the pigs in the sow and nursery sites had a complete internal gene cassette of h n pdm origin, whereas the h n dk variant circulating in pigs in the finisher site had an m -m and nep-ns gene of eurasian avian-like h nx origin. however, the protective role of immunity against the internal gene segments are still controversial [ , ] . pig id was infected twice with the same h n dk variant, which only showed minor genetic differences between samplings. however most of the differences were located in antigenic sites. hence, it can be speculated that even a small number of mutations could facilitate re-infection with the same subtype, thereby confirming the results of a previous study [ ] . in summary, it can be concluded that re-infections can occur with both similar and different variants within the same subtype. the presence of prolonged ( - weeks consecutive) and recurrent (non-consecutive) shedding of iav within week to week pigs also indicated reinfection with the same subtype and this was documented by sequencing. a number of previous studies have shown reinfection with the same strain, leading to prolonged iav shedding [ , , ] . the presence of mda may play role in the prolonged iav shedding, as mda may hinder an active immune response [ , ] . pig id was infected with two different h n dk variants, and most of the mutations were located in antigenic sites as mentioned above. similarly, the acquisition of nlg sites near/within sa and ca antigenic sites of the ha sequence may lead to a shielding effect on the antigenic sites and probably the emergence of new antigenic variants. a range of studies have shown that the shielding effect on ha antigenic sites may lead to an evolution of the ha sequence and be responsible for escaping the pre-existing immunity in the hosts [ ] [ ] [ ] [ ] . the presence of these major differences between variants within the same subtype emphasizes the presence of a massive genetic drift of eurasian avian-like h in danish herds [ ] , which in turn could have consequences for vaccine efficacy as the current swiav vaccine available against the h n subtype has not been updated since - . in our study, the presence of clinical signs of nasal discharge and conjunctivitis in pigs harbouring iav at week was not very evident. reduced levels of clinical signs could be due to the presence of mda. similarly, previous exposure or a low level of exposure to the virus might preclude clinical signs in pigs [ ] [ ] [ ] . in contrast with other studies, our study did not find any association between iav infection and nasal secretion [ , ] . however, clinical signs were only recorded at two stages (at week and week ) of the total samplings in this study. similarly, we did not find any association between iav infection and conjunctivitis, which is also supported by other studies [ , ] . in conclusion, the complexity of swiav infection dynamics in pigs from the farrowing unit to the finisher unit has been demonstrated. a high infection pressure of swiav was identified during the end of the stay in the farrowing unit and the start of the nursery unit. in addition, it has been shown that the prolonged persistence of iav in pigs could be due to re-infection with iavs that are closely related to each other. similarly, re-infections with different strains within the same lineage can also be expected, as the genetic changes affect important antigenic epitopes. supplementary materials: the following are available online at http://www.mdpi.com/ - 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influenza h n viruses a perspective on the structural and functional constraints for immune evasion: insights from influenza virus a carbohydrate side chain on hemagglutinins of hong kong influenza viruses inhibits recognition by a monoclonal antibody effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza a/h n virus hemagglutinin differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity cytokines in the pathogenesis of influenza avian and swine influenza viruses: our current understanding of the zoonotic risk this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we want to acknowledge the herd owner for providing access to his property for sampling, and the herd-veterinarian providing assistance during sampling. similarly, we want to acknowledge the laboratory technicians hue thi thanh tran and jonathan rahlff rogersen for their help with laboratory tasks. the authors declare no conflict of interest. the funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. key: cord- -so umdlt authors: paczesny, jan; richter, Łukasz; hołyst, robert title: recent progress in the detection of bacteria using bacteriophages: a review date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: so umdlt bacteria will likely become our most significant enemies of the st century, as we are approaching a post-antibiotic era. bacteriophages, viruses that infect bacteria, allow us to fight infections caused by drug-resistant bacteria and create specific, cheap, and stable sensors for bacteria detection. here, we summarize the recent developments in the field of phage-based methods for bacteria detection. we focus on works published after mid- . we underline the need for further advancements, especially related to lowering the detection (below cfu/ml; cfu stands for colony forming units) and shortening the time of analysis (below one hour). from the application point of view, portable, cheap, and fast devices are needed, even at the expense of sensitivity. the growing problem of the appearance of multidrug-resistant bacteria might completely change our lifestyle. for the last years, we have lived in a world where we have been able to kill bacteria on-demand with antibiotics. however, evolution, our lack of knowledge, and excessive use of antibiotics are slowly causing this situation to change. the gene mcr- , responsible for resistance against colistin, an antibiotic often referred to as "drug of last resort", was proved to have spread around the world [ ] and was found in several bacteria (including escherichia, salmonella, klebsiella, kluyvera, citrobacter, and cronobacter) within a few years [ ] . bacteria could gain resistance to a given antibiotic within hours [ ] . recently, the first example of the adaptation of bacteria to physical factors (nanomechanical stress) was also reported [ ] . without proper means of detection, allowing for targeted use of drugs, we will be facing a scenario where small wounds could be a potential life threat, as they were before the discovery of penicillin. the prevention of the spread of pandemics needs proper detection [ ] . to monitor and implement appropriate control measures, the use of sensitive and specific diagnostic methods is paramount. critical parameters of sensors for bacteria detection are the time of analysis, the limit of detection (lod), sensitivity, and specificity. from the application point of view, also cost, portability, ease of use, operator hands-on time, and reliability are critical. often one trait could be traded for another, e.g., the introduction of the pre-incubation step improves the limit of detection at the expense of time of analysis. conventional microbiological methods for bacteria detection, based on culturing microorganisms, are cheap and selective but also time-consuming and laborious. therefore, researchers are introducing new detection techniques. over the past decades various detection methods have been developed including (but not limited to) nucleic acid-based sensors (dna microarrays [ ] , polymerase chain reaction (pcr) and its derivatives, e.g., multiplex pcr or real-time pcr [ , ] ); immune-based sensors (e.g., enzyme-linked immunosorbent assay [ ] ) and mass spectrometry sensors (especially however, a single event generates a signal which is difficult to detect. contrary, infecting bacteria and utilizing its molecular machinery to amplify the signal (by the generation of progeny virions, introduction reporter genes, or due to release of bacterial metabolites due to lysis) offers lower detection limits, but the procedures are lengthy. bacteriophages are viruses that infect bacteria. the average size of the virion (single phage) is around nm to nm. however, the largest bacteriophages are more than nm in length [ ] . the vast majority of all known bacteriophages (above %) belong to the order caudovirales. they share a universal structure design, i.e., genetic information (dsdna) is in a capsid, to which a tail with fibers is attached [ ] . much less common are filamentous (e.g., m ) or nearly spherical (isometric) phages (e.g., ms ). unlike antibodies, phages can be quickly and cheaply produced in large quantities and properly purified. by only infecting a bacteria solution, one can obtain a large number of progeny phages. moreover, some phages are robust and retain their activity even after exposure to high temperatures [ ] , ph [ ] , and organic solvents [ , ] . thanks to the abundance of different bacteriophage types, it is theoretically possible to design biosensors to detect almost every bacterial strain. the essential traits of phages are that they are efficient and specific against host bacteria [ , ] . only recognition of a proper and viable host assures the multiplication of virions and completion of the life cycle. thus, the utilization of phages allows for the distinction between live and dead cells, a common problem in other methods. one needs to acknowledge that phages might adsorb to the surface of the dead bacterium, thus affecting the methods relying purely on the detection of capturing events [ ] . with the completion of life cycles, phages undergo evolution, so they are always up to date in the arms race against bacteria [ ] . for instance, not long after discovering crispr [ ] , anti-crispr mechanisms were also found [ ] . in general, phages are great candidates as biorecognition elements in biosensors and other assays. the most straightforward design of phage-based biosensors for bacteria detection utilizes whole virions as sensing elements. the majority of known phages belong to caudovirales order. it gives figure . summary of most often exploited designs of phage-based biosensors. methods utilizing bacteria capturing (at the surface of the sensing elements or by phage-based probes) are fast. however, a single event generates a signal which is difficult to detect. contrary, infecting bacteria and utilizing its molecular machinery to amplify the signal (by the generation of progeny virions, introduction reporter genes, or due to release of bacterial metabolites due to lysis) offers lower detection limits, but the procedures are lengthy. bacteriophages are viruses that infect bacteria. the average size of the virion (single phage) is around nm to nm. however, the largest bacteriophages are more than nm in length [ ] . the vast majority of all known bacteriophages (above %) belong to the order caudovirales. they share a universal structure design, i.e., genetic information (dsdna) is in a capsid, to which a tail with fibers is attached [ ] . much less common are filamentous (e.g., m ) or nearly spherical (isometric) phages (e.g., ms ). unlike antibodies, phages can be quickly and cheaply produced in large quantities and properly purified. by only infecting a bacteria solution, one can obtain a large number of progeny phages. moreover, some phages are robust and retain their activity even after exposure to high temperatures [ ] , ph [ ] , and organic solvents [ , ] . thanks to the abundance of different bacteriophage types, it is theoretically possible to design biosensors to detect almost every bacterial strain. the essential traits of phages are that they are efficient and specific against host bacteria [ , ] . only recognition of a proper and viable host assures the multiplication of virions and completion of the life cycle. thus, the utilization of phages allows for the distinction between live and dead cells, a common problem in other methods. one needs to acknowledge that phages might adsorb to the surface of the dead bacterium, thus affecting the methods relying purely on the detection of capturing events [ ] . with the completion of life cycles, phages undergo evolution, so they are always up to date in the arms race against bacteria [ ] . for instance, not long after discovering crispr [ ] , anti-crispr mechanisms were also found [ ] . in general, phages are great candidates as biorecognition elements in biosensors and other assays. the most straightforward design of phage-based biosensors for bacteria detection utilizes whole virions as sensing elements. the majority of known phages belong to caudovirales order. it gives promise for the possibility of simplifying the preparation process to utilize various phages for sensors preparation. such an approach dramatically expands the potential applicability of new solutions allowing for the detection of a variety of target bacteria. another significant advantage of phage-based biosensors is the possibility to isolate phages specific against any target bacteria quickly and cheaply. there is even no need to identify isolates. such an approach was used recently in several studies, and phages, which hosts are bacteria of interest, were isolated from hospital sewage water [ ] , and environmental samples [ ] [ ] [ ] . the critical decision in phage selection is the choice between temperate and lytic phages. for instance, lytic phages are a must when the release of progeny virions or bacterial metabolites are to be detected. however, the utilization of lytic phages usually limits the incubation time to below one hour. prolonged incubation results in lysis of cells captured early. such time constraints restrict the possible number of captured bacteria in case of methods requiring proximity between cells and surface to generate an analytical signal. usually, virions (viral particles) are deposited at the surface. the role of the substrate in nanotechnology is usually to provide support and increase the robustness of functional material. in the case of sensors, the surface also often takes part in the sensing process being a part of a transducer. the transducer is an element of sensor, which generates a measurable signal upon capturing target bacteria. three main designs have been explored where bacteriophages are deposited onto a solid substrate. one is based on electrochemical methods, with phages deposited on the electrodes. the second utilizes magnetoelastic sensors, where a change of mass upon bacteria capture changes the amplitude of vibrations. the third one comes from the surface-enhanced raman spectroscopy, where excited plasmons within the substrate allow for enhancement of the intensity of the recorded spectra. in electrochemical methods, the electric signal changes upon capturing of bacteria by virions deposited at the electrodes. there is an increasing number of published works utilizing the electrochemical approach to detect bacteria [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . electrochemistry offers good sensitivity, low-cost analysis, and allows for miniaturization. also, the signal in the form of electric current or voltage is easy to process. the development of bacteriophage-based electrochemical methods for bacteria detections appears in various review articles published recently [ ] [ ] [ ] . sedki et al. [ ] described setup utilizing m immobilized on the electrodes, by chemical methods, for electrochemical impedance spectroscopy. it allowed for the detection of coliforms with lod of around cfu/ml (cfu stands for colony forming units) within min. in this example, a single phage allows for the detection of multiple strains of e. coli, while not responding to non-e. coli bacteria. moon et al. have recently published a detailed review of m -based biosensors [ ] . in another interesting example, yue et al. [ ] reported a label-free electrochemiluminescent biosensor capable of detecting pseudomonas aeruginosa with lod of cfu/ml within min. the authors used the carboxyl graphene-pap composite, acting a sensing element on the glassy carbon electrode. magnetoelastic sensors are usually ribbon-like strips of amorphous ferromagnetic alloys. they vibrate under magnetic excitation. mechanical vibrations generate secondary magnetic flux that can be detected remotely. the deposition of analyte on the surface, covered with the sensing layer, changes the amplitude of the vibrations providing the analytical signal. hiremath et al., [ ] reported a sensor for the detection of methicillin-resistant staphylococcus aureus with a limit of × cfu/ml within min. in they confirmed that mrsa was detected specifically and selectively even in the presence of other competing bacteria [ ] . chen et al. [ ] , and mack et al. [ ] showed other exciting applications. both papers describe the detection of salmonella (s. enterica and s. typhimurium, respectively), at the surface of food products (chicken and lettuce, respectively). in both cases, the magnetoelastic strip was pressed against the sample to be analyzed. surface-enhanced raman spectroscopy (sers) is a technique utilizing localized surface plasmon resonance of metal surfaces to obtain ultrahigh enhancement of raman scattering. it allows for an increment of the intensity of recorded spectra by many orders of magnitude. for instance, commercially available substrates (e.g., sersitive) offer enhancement factors in the range of to for some analytes [ ] . such properties allow for the detection of analyte at an ultra-low concentration, reaching the single molecules level [ ] ; however, in the case of larger analytes, such as bacteria, the situation is more complicated. the potential of sers for bacteria detection was first proved for cells deposited directly on the sers-active substrate [ ] . one of the first examples of successful phage utilization as a sensing layer in sers was demonstrated by srivastava et al. in [ ] . they used thin silver films on a silicon platform along with t phages. the reported limit of detection of e. coli was . × cfu/ml. recently, rippa et al. [ ] developed a substrate made of plasmonic nanocavities, with a layer of immobilized bacteriophages. the authors suggested that the proposed system constitutes a novel solution for the specific detection of different species of bacteria. the same group successfully used other metastructures, appropriately functionalized with tbilisi bacteriophages, for brucella's sers-based detection. the authors performed measurements within only one hour and at the single-cell level, with bacteria deposited from a suspension of concentration higher than cfu/ml [ ] . lai et al. [ ] achieved similar lod using principal component analysis (pca) to process obtained sers spectra in the detection of bacillus spp. using gamma phages. until recently, the process of deposition of phages onto the solid substrate was poorly controlled [ , ] . this process did not affect filamentous or isometric phages but appeared crucial in tailed phages, constituting the majority of all known phages. in the case of deposition of tailed phages, entropy favors the alignment in which the long axis of the virion is parallel to the solid substrate (e.g., transducer). as this process is random, some virions may orient parallel to fibers attached to the surface. such orientations restrict the possibility of interactions between fibers and receptor binding proteins (rbps) with target bacteria (figure ). only orientation in which the long axis of the virions is vertical to the surface, and the tails are facing upward, assures that most rbps are involved in the bacteria detection process. phages that infect a bacterium are oriented nearly perpendicular to the surface of the bacterium cell wall. phages are probing a bacterium surface with fibers oriented under a large angle, typically > degrees to the surface plane. this probing is reversible. commercially available substrates (e.g., sersitive) offer enhancement factors in the range of to for some analytes [ ] . such properties allow for the detection of analyte at an ultra-low concentration, reaching the single molecules level [ ] ; however, in the case of larger analytes, such as bacteria, the situation is more complicated. the potential of sers for bacteria detection was first proved for cells deposited directly on the sers-active substrate [ ] . one of the first examples of successful phage utilization as a sensing layer in sers was demonstrated by srivastava et al. in [ ] . they used thin silver films on a silicon platform along with t phages. the reported limit of detection of e. coli was . × cfu/ml. recently, rippa et al. [ ] developed a substrate made of plasmonic nanocavities, with a layer of immobilized bacteriophages. the authors suggested that the proposed system constitutes a novel solution for the specific detection of different species of bacteria. the same group successfully used other metastructures, appropriately functionalized with tbilisi bacteriophages, for brucella's sers-based detection. the authors performed measurements within only one hour and at the single-cell level, with bacteria deposited from a suspension of concentration higher than cfu/ml [ ] . lai et al. [ ] achieved similar lod using principal component analysis (pca) to process obtained sers spectra in the detection of bacillus spp. using gamma phages. until recently, the process of deposition of phages onto the solid substrate was poorly controlled [ , ] . this process did not affect filamentous or isometric phages but appeared crucial in tailed phages, constituting the majority of all known phages. in the case of deposition of tailed phages, entropy favors the alignment in which the long axis of the virion is parallel to the solid substrate (e.g., transducer). as this process is random, some virions may orient parallel to fibers attached to the surface. such orientations restrict the possibility of interactions between fibers and receptor binding proteins (rbps) with target bacteria (figure ). only orientation in which the long axis of the virions is vertical to the surface, and the tails are facing upward, assures that most rbps are involved in the bacteria detection process. phages that infect a bacterium are oriented nearly perpendicular to the surface of the bacterium cell wall. phages are probing a bacterium surface with fibers oriented under a large angle, typically > degrees to the surface plane. this probing is reversible. researchers strive to achieve denser coverage of the surface. for instance, in recent work, farooq showed the high-density phage particles immobilization for ultra-sensitive and selective detection of staphylococcus aureus [ ] . griffith's group published first attempts to increase the number of available rbps not by increasing the number of randomly oriented virions, but through proper orientation at the beginning of s [ , ] . tolba et al. [ ] used recombinant bacteriophages, which had the outer capsid protein gene of t fused with genes coding specific binding proteins (biotin or cellulose-binding module). these binding proteins were present only at the surface of capsids. upon binding, tails and fibers remained exposed and available for bacteria. nevertheless, it was not until that the researchers quantified the effect of proper orientation. richter et al. [ ] exploited permanent dipole moment of virions to orient t phages along the electric field lines. the number of deposited phages did not change significantly upon the application of the researchers strive to achieve denser coverage of the surface. for instance, in recent work, farooq showed the high-density phage particles immobilization for ultra-sensitive and selective detection of staphylococcus aureus [ ] . griffith's group published first attempts to increase the number of available rbps not by increasing the number of randomly oriented virions, but through proper orientation at the beginning of s [ , ] . tolba et al. [ ] used recombinant bacteriophages, which had the outer capsid protein gene of t fused with genes coding specific binding proteins (biotin or cellulose-binding module). these binding proteins were present only at the surface of capsids. upon binding, tails and fibers remained exposed and available for bacteria. nevertheless, it was not until that the researchers quantified the effect of proper orientation. richter et al. [ ] exploited permanent dipole moment of virions to orient t phages along the electric field lines. the number of deposited phages did not change significantly upon the application of the electric field. however, the increase in the number of captured e. coli cells was fourfold compared to the randomly oriented layer of phages. the orientation of phages in the constant electric field was, in fact, an example of charge driven assembly. a similar approach was utilized by anany et al. [ ] who used charged cellulose membranes, or zhou et al. [ ] , who reported a carbon nanotube (cnt)-based impedimetric biosensor, in which t phages were oriented upon application of constant potential. recently, imai et al. [ ] claimed to utilize core−shell nanoparticles with the surface charge to orient phages s properly. results reported by liana et al. [ ] suggest that there is another layer of complexity in such a seemingly simple system. the authors compared amino-(positive charge in neutral ph) and carboxylic-(negative charge in neutral ph) functionalized surfaces of indium tin oxide (ito). it was found that more t virions were adsorbed on negatively charged planar ito, whereas in the case of particulate ito, better coverage was achieved for positively charged particles. the authors explained it in terms of variations of time available to bind phages on planar (longer time required) versus particulate (shorter search time) ito [ ] . anyhow, bone et al. [ ] , again proved that chemisorption allowed for much higher surface coverages. when charged surface (let it be due to chemical modification or application of constant electric field) is in the electrolyte (e.g., buffer), the electrical double layer is created. it screens the electric field, restricting its effective range to debye length. the value of debye length in standard buffer varies from below one to dozens of nm, with values in the range of few nm being most common. this length is much smaller than the size of the virion. to overcome such screening, richter et al. [ ] utilized alternating electric field (i.e., varying in time according to programmed time traces) to promote the vertical arrangement of virions. they used a trapezoidal waveform with interpulse periods with no potential applied. periods without applied electric field allowed for relaxation of the electrical double layer. such an approach resulted in a tenfold increase in the number of captured bacteria comparing to randomly oriented phages. when combined with chemical immobilization of phages, the increase was around -fold. it allowed for obtaining a limit of detection in a range of cfu/ml by using just min incubation time. very recently, xu et al. [ ] systematically studied the influence of debye length and concentrations of phage suspensions on the performance of a t bacteriophage-based micro electrochemical sensor, with a sensing layer ordered in the alternating electric field. the applied conditions were similar, as in the case of richter and coworkers [ ] . when the debye length was comparable to the phage size, the capture efficiency attains the maximum value. the obtained limit of detection was ± cfu/ml. such an approach of deliberate design of functional biomaterials is gaining importance. an example of a recent review on controlling the self-assembly of biomolecules by adjusting internal interactions of interactions and due to external stimulation was published by wang et al. [ ] . there are many more possible applications of such an approach. for example, the electric field was used to assemble m phages into colored films. due to the facile functionalization of virions, it might be used to prepare the nanodevices [ ] . in work by tronolone and coworkers [ ] the application of an ac electric field to an evaporating droplet of m bacteriophage caused the motion of the meniscus of the droplet. during the movement of the meniscus, m virions adsorbed to the surface and assembled into smectic helicoidal nanofilament. the formation of such bundles manifested as rings of color bands. the intrinsic disadvantage of the design in which the sensing layer is deposited at the surface is a relatively low probability of bacteria being in the vicinity of the immobilized phages. in the case of small molecules, even low concentrations translate to a reasonably large number of objects to be detected (e.g., for picomolar concentration, which is usually considered low, the number of molecules is around per ml). however, in the case of bacteria, the aim is to detect a few bacteria in one ml or better. a low concentration of bacteria corresponds to a significantly lower number of detectable events, i.e., capturing bacteria by phages, in a given time. one of the ways to counteract this is to incorporate the proper deposition technique. very recently, richter et al. [ ] showed the potential of using the electric field to increase the number of analyzed objects directly at the surface. another possibility is to increase the available surface area by using nanoparticles conjugated with bacteriophages. for instance, proof-of-concept studies showing the application of conjugates composed of gold nanoparticles and p b phage displaying specific peptide binding to pseudomonas aeruginosa for sers detection was reported in [ ] . in another example, gold nanoparticles were used to prepare a colorimetric sensor. due to the alteration of surface plasmon resonance properties, suspension of gold nanoparticles changes its color upon aggregation. peng and chen [ ] used chemically modified and genetically engineered m phages with exposed sh groups and displayed receptors against target bacteria (two strains of e. coli, p. aeruginosa, vibrio cholerae, and two strains of the plant pathogen xanthomonas campestris). first, modified m phages were added to the sample. after centrifugation, phages were present in pellets only if attached to target bacteria. the pellet was resuspended in buffer containing gold nanoparticles (aunps), which attached to sh groups at the surface of virions. this process resulted in aggregation and, finally, in a color change. the assay allowed for the detection of around cells (in ml of the sample) within a -min procedure [ ] . another example utilized bacteriophages immobilized at the surface of the core-shell sio @aunp nanoparticles for darkfield microscopic detection. numerous conjugates attached to the target cell resulting in apparent aggregation causing strong light scattering. the authors reported staphylococcus aureus' detection within to min with a detection limit of × cfu/ml [ ] . janczuk et al. [ ] used magnetic and fluorescent particles to create phage-based bioconjugates used as flow cytometry probes. reported lod of e. coli using t phage bioconjugates was in the range of cfu/ml, and the incubation time was min. bacteriophage modified magnetic particles were also used for isolation and separation [ ] . isolation and separation are often combined with the detection of bacteria using an auxiliary detection method, e.g., immunoassay. yan et al. [ ] used such an approach that allowed for lod of around × cfu/ml in the detection of s. aureus in apple juice within min without any pre-enrichment. liana and coworkers [ ] used bacteriophage conjugated fe o particles for the rapid capturing and isolation of e. coli. the authors focused on the optimal parameters of the operation of such a probe. they found that bacteria capturing occurred only at • c in tryptone-containing media. bhardwaj and coworkers [ , ] used metal-organic frameworks (mof) crystallites as phage carriers. first, they used irmof- (zn o(nh -bdc) ) (nh -bdc = -aminoterephthalic acid) conjugated with isolated lytic bacteriophage as a fluorescence probe. upon binding, bacteria geometrically concealed mof particles. the excitation energy which reached mof particles was thus restricted, and a loss in the fluorescence intensity was recorded. the loss in fluorescence correlated with an increasing number of bacteria. the reported lod of staphylococcus arlettae was around cfu/ml [ ] . later, the same group used nh -mil- (fe), which is similar to irmof- . nh -mil- (fe) is iron-rather than zinc-based mof but utilizes the same organic linker (nh -bdc). again, the fluorescence of the probe decreased with an increase in the bacteria concentration. thelod) of s. aureus was cfu/ml, and the assay time was around min [ ] . li et al. [ ] presented a fascinating strategy for bacteria detection. they used complex organic-inorganic particles, designed to support the cascade of three electrochemical reactions, which acted as an amplifier. cu (po ) nanoflowers were first loaded with glucose oxidase, horseradish peroxidase, and thionine. next, gold nanoparticles were incorporated into such loaded nanoflowers. t phages were attached to gold via nonspecific bonding. the bacteria detection procedure was composed of multiple steps. first bacteria were immobilized at the surface of the electrode by antibodies or antimicrobial peptide magainin i. loaded nanoflowers attached to such deposited cells upon phage binding to the surface of target bacteria. as glucose oxidase, horseradish peroxidase, and thionine appeared in the vicinity of the electrodes, electrochemical reactions occurred. first, glucose present in the buffer was oxidized. this generated hydrogen peroxide, in the second step, was used to oxidize thionine, which was regenerated at the electrode resulting in signal generation. the current increase was measured employing differential pulse voltammetry. achieved lod was in the range of cfu/ml achieved within min. instead of conjugating phages to additional functional particles or molecules, it is also possible to introduce genetic modification into the phage genome to become both sensing and signal-generating elements. the most straightforward approach is to incorporate gene coding fluorescent protein or enzymes, generating easy-to-detect products. upon infection, this gene is expressed inside the host cell, which allows for its detection. there are, however, some drawbacks of genetically modified phages. first, it is challenging to obtain modified phages, and the modification process and its optimization need to be repeated for each new target bacteria of choice. secondly, modified phages are often less infectious [ ] . finally, the environmental risks of the unpredictable effects of modified phages on the biosphere are still not assessed [ ] . a very recent review of engineered bacteriophages' practical applications was published by pizarro-bauerle and ando in [ ] . the authors showed examples of the utilization of genetically modified bacteriophages in phage therapies, medicine, animal industry, and agriculture as sources of new antimicrobials, biocontrol, and genetic engineering tools. here, we focus only on biosensing. wisuthiphaet and coworkers [ ] showed the utilization of the t -alp phage, with introduced gene coding alkaline phosphatase, which was overexpressed upon infection of e. coli. fluorescent substrate for alkaline phosphatase activity coupled with fluorescence imaging and image analysis was used for the detection of bacteria. the procedure took six hours and allowed for lod of around bacteria per gram of model beverage samples. kretzer et al. [ ] combined magnetoseparation, harnessing cell wall-binding domains from listeria phage endolysins, with a ::luxab bioluminescent reporter phage assay. thus they combined two phage-based protocols into one. the final protocol gave lod of around cells per ml of several listeria strains within six hours. nugen group showed detection of e. coli with lod of around × cfu/ml after two hours incubation with t containing nanoluc luciferase expression cassette. the method required the addition of a substrate for signal generation. modified phages were prepared by synthetic biology strategy to engineer phages using a simple in vitro method [ ] . the method used pcr fragments and in vitro dna assembly followed by rebooting through transforming into host bacteria and not dna assembly in yeast. the procedure resulted in the relatively simple and fast preparation of specific phages needed for detecting target bacteria in various applications. hinkley et al. [ ] [ ] [ ] aimed to achieve a limit of detection sufficient for the analysis of drinking water. the standard in the united states mandates a zero-tolerance of generic e. coli in ml of water. their approach was to filter ml of water sample on the cellulose filter. after to h of incubation necessary for colony development, two modified t phages with a reporter gene (luciferase or alkaline phosphatase) fused to genes for carbohydrate-binding modules (cbm) specific to cellulose were added and incubated for . h. in the final step, the enzymatic substrates were added, which allowed for the visualization of colonies. overall, the process was around two times shorter comparing to standard procedure ( h versus h) and allowed for the limit of detection of around cfu per ml [ ] . to shorten the required time of analysis, the same group proposed a t phage with the nanoluc reporter gene fused again to cbm. first, the water sample was supplemented with concentrated growth media to allow the resuscitation of e. coli. after min, genetically modified phages and microcellulose were added. during a further min of incubation, the expression of nanoluc-cbm occurred. the protein bounded to cellulose was collected by centrifugation. the luminescence was measured after the addition of the nanoglo substrate to the sample. this approach offered a limit of detection better than cfu/ml. however, the authors claimed that future improvements in the capture efficiency of the fusion reporter protein to cellulose should limit the detection of below cfu per ml [ ] . wisuthiphaet suggested that such limits of detection of these two setups could not be achieved in complex matrices due to background signals [ ] . finally, in , nugen's group reported a syringe-based biosensor using the same engineered t phage containing the nanoluc-cbm cassette, which allows for the limit of detection of around cfu of e. coli in ml of drinking water within h [ ] . some systems do not require any external substrate to allow for detection. for instance, vinay and coworkers [ ] showed detection of e. coli and salmonella enterica typhimurium using hk and p phages, respectively, with introduced the gfp gene. utilization of flow cytometry allowed for lod as low as cells/ml in seawater after one hour of incubation. the same group also reported the utilization of two engineered phages, namely hk and hk . they had an entire luxcdabe operon that encodes luciferase and the substrate generating system. the reported lod was in a range of bacteria/ml, but the main goal was to incorporate this probe into combitox. this instrument aimed to accommodate several biodetector systems to detect pollutants such as bacteria, toxins, and heavy metals [ ] . much better limit of detection was reported by kim et al. [ ] , who showed utilization of phiv phages also with luxcdabe operon. the authors showed the detection of escherichia coli o :h with lod of around cfu/ml in a pure culture within min after h of preincubation. in artificially contaminated romaine lettuce, apple juice, and ground beef, phiv lux allowed for detection limits of around cfu/cm , cfu/ml, and cfu/g, respectively. wang et al. [ ] showed the electrochemical detection of e. coli upon completion of the lytic cycle of t phages containing lacz operon encoding β-galactosidase. the endogenous and phage induced β-galactosidase was detected using differential pulse voltammetry method with -aminophenyl-β-galactopyranoside as a substrate. achieved limit of detection was in the range of cfu/ml within h. rondón and coworkers [ ] demonstrated the utilization of reporter phages in real-world applications. the authors used mcherry bomb ϕ phage for the detection of mycobacterium spp. and phenotypic determination of rifampicin resistance. the study was performed on samples collected from adult presumptive tuberculosis patients. however, the protocol took as much as three to five days. in the bacteria sensing methods mentioned above, bacteriophages acted as specific binding agents. such an approach is similar to antibody-based detection, but antibodies are much more expensive, difficult to prepare, and less stable than phages. both phage-and immune-based methods are limited by the transducers' performance, especially at an ultralow concentration of target bacteria. however, bacteriophages also offer a "built-in" amplification system. instead of detecting capturing events, it is possible to search for progeny virions released from the host cell upon completion of the phage life cycle. a large number of released virions offers a few tens up to a thousand-fold multiplication of the number of objects to be detected. however, there are some disadvantages to such an approach. first, such methods require virulent and not temperate phages. secondly, progeny phages will likely not be produced if there is already a prophage incorporated in the host's genetic material. thirdly, bacteria possess mechanisms preventing phage infections, e.g., crispr-cas [ ] . the most often used is a combination of phage amplification and detection of progeny virions employing the pcr technique. several reports utilizing such an approach were published, with time and limits of detection varying strongly. for instance, luo et al. [ ] showed the detection of acinetobacter baumannii in serum using p phages allowing for lod in the range of cfu/ml within h. later they improved the method and achieved lod of cfu/ml in sputum samples within h [ ] . garrido-maestu et al. [ ] showed the detection of cfu of salmonella enteritidis in g of chicken samples within h. extending the time of the analysis allowed sergueev and coworkers [ ] to achieve lod of around cfu/ml of brucella abortus within h in mixed cultures and blood samples. the most inspiring example was published by anany et al. [ ] , who developed a phage-based paper dipstick biosensor to detect various foodborne pathogens in food matrices. they used piezoelectric inkjet printing to prepare phage-based bioactive papers that actively lysed their target bacteria. in combination with quantitative real-time pcr, this allowed for a limit of to cfu/ml in the number of various samples with a total assay time of h. mido et al. [ ] coupled amplification of phages with immunoassay. progeny ms phages were captured by antibodies coupled on the surface of magnetic beads. upon the addition of the detector antibody (also binding to ms ) the fluorescence was measured. the fluorescence allowed for the limit of detection of around cells/ml of live e. coli cells after a h incubation. a much more straightforward method of detection of progeny virions is titration using the plaque counting method. in this method, phages are deposited onto the agar plate inoculated with bacteria. in the place where virion is present, bacteria are lysed. visible holes, plaques, appeared in the bacteria layer. said et al. [ ] used this approach to monitor the activity of a foodborne and waterborne pathogenic bacterium, salmonella typhi, under starvation conditions. phage infectivity rate was used to detect active bacteria that are not detectable by conventional methods, i.e., vbnc (viable but nonculturable) cells. in this approach, free phage concentration after incubation with samples containing vbnc cells (p n ) was compared to the initial phage titer (p ). analysis of kinetic parameters, e.g., the phage amplification rate (p n /p ) allows detecting the presence of active bacteria underestimated by using conventional methods. upon completion of the lytic cycle, not only progeny virions are released, but also the content of the cell, including essential biomarkers. these biomarkers are typical for a variety of bacteria. however, the utilization of phages allows for the specificity of such approaches. for instance, tilton et al. [ ] reported a biosensor platform based on t bacteriophage, which mediated specific lysis of target bacteria and the release of β-galactopyranoside. β-galactopyranoside catalyzed the cleavage of the substrate resorufin β-d-galactopyranoside. the cleavage resulted in the formation of highly fluorescent resorufin. the fluorescent signal was detected using an epifluorescence system. utilization of nanophotonic substrate allowed for the limit of detection of cfu/ml of e. coli in simulated spinach wash water within h. he et al. [ ] proposed a pseudomonas aeruginosa detection setup combining magnetoseparation, phage amplification, and detection of intracellular adenosine triphosphate upon cell lysis and release of progeny virions. pap phage was isolated from hospital sewage and conjugated with magnetic beads. firefly luciferase-adenosine triphosphate bioluminescence system was used to determine the concentration of p. aeruginosa. reported lod was × cfu/ml obtained within h. the procedure of isolating and amplifying phages against target bacteria from, for example, sewage water is relatively simple. one does not even need to identify the specific phage. however, there are some fundamental issues with biosensors utilizing whole virions as sensing elements. first, virions might be relatively large, and thus there is a limit of miniaturization. for instance, for magnetophoretic separation, magnetic particles conjugates to virions need to be in at least a sub-micrometer scale. otherwise, the force exerted by the external magnet is not enough to drag the conjugate or the conjugate with the attached bacteria. moreover, in many analytical techniques (e.g., surface plasmon resonance), binding events need to happen within a given distance from the transducer's surface. the relatively large size of phages might result in too long distance between the surface and the target analyte, thus hindering the analytical signal generation. the second disadvantage is related to the orientation of phages, which we did cover in section . . in short, parts of the virions that do not take part in bacteria capturing might create a steric hindrance for rbps. finally, the majority of bacteriophages ultimately cause lysis of the bacterial cells. it prohibits a more prolonged analysis in the case of samples of low bacteria concentration. it might happen that while waiting for other bacteria to attach to the sensor, the first bound bacterial cell is already destroyed. the solution might be the utilization of parts of phages for the preparation of biosensors. for instance, he et al. [ ] used recombinant tail fiber protein (p ), expressed in e. coli, for the detection of p. aeruginosa. the authors used two different approaches to detection. first, the recombinant protein was conjugated to magnetic beads. such beads were added to the sample, and target bacteria were magnetically separated. after washing, the cells were disrupted, and the concentration of atp was evaluated using the bioluminescence method. the second approach was based on p deposited onto the solid substrate. after capturing bacteria, fluorescently labeled p was added, allowing for fluorescence detection. the observed limits of detection were . × cfu/ml and . × cfu/ml for bioluminescent and fluorescent methods, respectively, within to min. similarly, wang et al. [ ] utilized bacteriophage cell-binding domain (cbd) and green fluorescent protein fused to cbd for a broad-spectrum recognition of methicillin-resistant staphylococcus aureus strains. first, cbd conjugated magnetic beads were used to separate target cells, which were later detected employing flow cytometry upon the incubation of cbd-gfp protein. the protocol allowed for lod of around cfu/ml, and the procedure took around h. gomez-torres et al. [ ] also used cbd-gfp protein and compared it with gfp-ctp l. ctp l is a bacteriophage endolysin active against clostridium tyrobutyricum. the authors were able to detect of clostridium strains, also in the form of clostridial spores. gfp-ctp l and gfp-cbd were used as biomarkers for the detection of clostridium spores in milk. an interesting example was reported by liu et al. [ ] , who used bovine serum albumin-templated co o magnetic nanozyme (co o mne) conjugated to s. aureus-specific fusion-pviii (co o mne@fusion-pviii). first, the unbound triple-functional conjugates were magnetically separated from co o mne@fusion-pviii@s. aureus complexes. next, peroxidase mimetics activity of the co o mne was exploited to detect the target bacteria with a limit of around cfu/ml. here, we review recent developments in bacteriophage-based methods for bacteria detection without omitting the fundamentals. we focus on reports published in and later, i.e., not covered by our last review [ ] . other, most recent, and general reviews on the topic were also published around that time [ , ] , and thus we believe it is justified to provide an update. the summary of the performance of the phage-based biosensors is given in table . the cbd-gfp fusion protein was used, broad host recognition due to cbd; no lysis [ ] in figure , we depict the performance of recently reported phage-based biosensors in terms of limit of detection and time of analysis. we marked cfu/ml as the limiting concentration of bacteria in blood in the case of sepsis in neonates. the second vertical line corresponds to cfu per ml of water, which is needed to analyze drinking water. a horizontal line marks another critical parameter at the time of analysis of h. it is time for medical doctors to wait for the information on which bacteria are causing sepsis before the administration of wide-spectrum antibiotics [ ] . targeted treatment is, of course, beneficial for patients, but it needs to be introduced before the bacteria can cause severe damage. as it is clear from figure , phage-based biosensors have just begun to enter the zone of fast and sensitive detection of bacteria. in figure , we depict the performance of recently reported phage-based biosensors in terms of limit of detection and time of analysis. we marked cfu/ml as the limiting concentration of bacteria in blood in the case of sepsis in neonates. the second vertical line corresponds to cfu per ml of water, which is needed to analyze drinking water. a horizontal line marks another critical parameter at the time of analysis of h. it is time for medical doctors to wait for the information on which bacteria are causing sepsis before the administration of wide-spectrum antibiotics [ ] . targeted treatment is, of course, beneficial for patients, but it needs to be introduced before the bacteria can cause severe damage. as it is clear from figure , phage-based biosensors have just begun to enter the zone of fast and sensitive detection of bacteria. table . recent advances in phage-based biosensorsʹ development bring us closer to fast and sensitive methods for bacteria detection. to achieve detection in the range below cfu/ml in time below h is still a crucial challenge. there are two main approaches to achieve this. the first is to increase the sensitivity of phagebased bioconjugates, layered sensors, and methods utilizing parts of phages without additional preincubation steps. these methods usually are relatively fast, as the event to be detected is bacteria capture. the capture usually takes only minutes. however, it is challenging to detect capturing events, especially when the concentration of bacteria is low. in such a case, not only the number of events to be detected is low, but also the time of search might be extended. the second approach utilizes phage amplification or genetically modified phages (e.g., carrying reported genes). these methods already showed some ultrasensitivity but required long incubation time as they rely strongly on the metabolism of the bacteria. we compared very recent developments described in this review in detail, with best performing phage-based biosensors reported before (see table and figure ) [ ] . the progress is visible, but there is still a definite uncharted territory for bacteria sensing at a concentration below cfu/ml in less than one hour. to the best of our knowledge, the only sensor meeting such requirements were reported in by farooq [ ] . they used phages deposited on the surface, and table . recent advances in phage-based biosensors' development bring us closer to fast and sensitive methods for bacteria detection. to achieve detection in the range below cfu/ml in time below h is still a crucial challenge. there are two main approaches to achieve this. the first is to increase the sensitivity of phage-based bioconjugates, layered sensors, and methods utilizing parts of phages without additional preincubation steps. these methods usually are relatively fast, as the event to be detected is bacteria capture. the capture usually takes only minutes. however, it is challenging to detect capturing events, especially when the concentration of bacteria is low. in such a case, not only the number of events to be detected is low, but also the time of search might be extended. the second approach utilizes phage amplification or genetically modified phages (e.g., carrying reported genes). these methods already showed some ultrasensitivity but required long incubation time as they rely strongly on the metabolism of the bacteria. we compared very recent developments described in this review in detail, with best performing phage-based biosensors reported before (see table and figure ) [ ] . the progress is visible, but there is still a definite uncharted territory for bacteria sensing at a concentration below cfu/ml in less than one hour. to the best of our knowledge, the only sensor meeting such requirements were reported in by farooq [ ] . they used phages deposited on the surface, and bacteria capturing was detected employing differential pulse voltammetry. next, lod in the range of cfu/ ml, also in the time shorter than one hour, should be realized. in the last three years, presented developments create an impression that phage-based bacteria detection is a well-established area in practical clinical diagnostics. in reality, however, there are just a handful of companies actively working on such technologies and only one (to our best knowledge) product already available on the sample detect ht system (microbiologique, seattle, wa, usa). such drastic and surprising disparity is even more puzzling when one realizes that first phage-based methods reaching the limit of detection of cfu per ml were reported already years ago [ ] . as it is continuously repeated in published works, current methods of detecting and identifying bacteria do not meet the requirements in several fields, e.g., healthcare, food industry, or biosafety. the main concern is the time of analysis, which for traditional microbiological methods can extend up to h. this time is too long when a patient's life is endangered or in case of products with a short expiry date. several less time-demanding methods were developed, such as pcr, immunomethods, mass spectrometry, spectroscopy (e.g., sers), and, discussed herein, phage-based approaches. all of them provide faster detection times ranging from a few minutes to a few hours. however, in most diagnostic laboratories around the world, slow and tedious microbiological methods are still used. thus, the time of detection is not the only important aspect. a broader and more in-depth analysis is required to understand this issue. another crucial aspect of any analytical method is the limit of detection (lod). microbiological methods provide the highest available lod of single cfu/l. such a low limit is needed in particular applications (e.g., potable water), and there are myriad examples where such a good lod is not necessary. in cases of heavily infected patients or environmental water reservoirs, concentrations of bacteria can reach even cfu/ml [ , ] . in table , it is clear that phage-based methods have lod allowing for many practical applications; thus, this cannot be the only limiting aspect. we also indicate the need to demonstrate the possibility of future biosensors to be more easily adaptable for the detection of a variety of target bacteria by merely changing the phage within the sensing element. the focus of the community is on the realization of new designs of sensing devices and methods. to demonstrate the promising features (e.g., time of analysis, lod), scientists often use a well-known phage-bacteria pair as a model system. there are only a few examples which show the utilization of more than one phage to detect various target bacteria using the same design of the biosensor. for instance, anany et al. [ ] demonstrated three different (rv , ag a, cgg - ) phages for qpcr detection of bacteria. in all three cases, the authors obtained similar lod in the range from to cfu/ml. another means of broadening the spectrum of bacteria detected by a given biosensor is to utilize phage cocktails, similar to phage therapies. the critical factor allowing for the product to reach the market is an effective transition from science to industry. the abovementioned time of analysis and limit of detection are core parameters of any analytical method, but they do not determine successful science-industry transfer. crucial aspects are, among others, repeatability, stability, portability, ease of use, selectivity, price, and ease of shipping. these parameters are often omitted in scientific work, as a single team typically does most experiments with a unique set of equipment in the laboratory's stable conditions. the development of all these additional aspects is not required for successful publication but is essential for transforming laboratory experiments to practical analytical techniques. rarely any effort is made to obtain such improvements, as they are not considered scientific anymore, but are somewhat further engineering. thus, very few ideas reported in scientific publications are ready for commercialization, and many of them require additional, often expensive development. are we there yet with technologies based on bacteriophages? after all, dozens of publications report fast, cheap, and sensitive phage-based bacteria detection spans for over a few decades. unfortunately, as with developing technology, the transition between scientific publications and widely available products is tedious. with products like sample , we indeed started this journey, and many published methods promise to repeat its success. author contributions: conceptualization, j.p., Ł.r. and r.h.; writing-original draft preparation, j.p. and Ł.r.; writing-review and editing, j.p., Ł.r. and r.h.; 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detection isolation and separation of listeria monocytogenes using bacteriophage p -modified magnetic particles t bacteriophage conjugated magnetic particles for e. coli capturing: influence of bacteriophage loading, temperature and tryptone genetically engineered phages: a review of advances over the last decade. microbiol genetically modified bacteriophages in applied microbiology engineered bacteriophages for practical applications rapid detection of escherichia coli in beverages using genetically engineered bacteriophage t ultrasensitive and fast diagnostics of viable listeria cells by cbd magnetic separation combined with a ::luxab detection utilizing in vitro dna assembly to engineer a synthetic t nanoluc reporter phage for escherichia coli detection a phage-based assay for the rapid, quantitative, and single cfu visualization of e. coli (ecor # ) in drinking water reporter bacteriophage t nlc utilizes a novel nanoluc::cbm fusion for the ultrasensitive detection of: escherichia coli in water a syringe-based biosensor to rapidly detect low levels of escherichia coli (ecor ) in drinking water using engineered bacteriophages phage-based fluorescent biosensor prototypes to specifically detect enteric bacteria such as e. coli and salmonella enterica typhimurium substrate-independent luminescent phage-based biosensor to specifically detect enteric bacteria such as e. coli sensitive detection of viable escherichia coli o :h from foods using a luciferase-reporter phage phiv lux fluoromycobacteriophages can detect viable mycobacterium tuberculosis and determine phenotypic rifampicin resistance in - days from sputum collection exploring a phage-based real-time pcr assay for diagnosing acinetobacter baumannii bloodstream infections with high sensitivity rapid ultrasensitive diagnosis of pneumonia caused by acinetobacter baumannii using a combination of enrichment and phage-based qpcr assay specific detection of viable salmonella enteritidis by phage amplification combined with qpcr (paa-qpcr) in spiked chicken meat samples highly sensitive bacteriophage-based detection of brucella abortus in mixed culture and spiked blood print to detect: a rapid and ultrasensitive phage-based dipstick assay for foodborne pathogens sensitive detection of live escherichia coli by bacteriophage amplification-coupled immunoassay on the luminex ® magpix instrument detection of active pathogenic bacteria under stress conditions using lytic and specific phage nanophotonic device in combination with bacteriophages for enhancing detection sensitivity of escherichia coli in simulated wash water highly specific bacteriophage-affinity strategy for rapid separation and sensitive detection of viable pseudomonas aeruginosa nonlytic recombinant phage tail fiber protein for specific recognition of pseudomonas aeruginosa recombinant bacteriophage cell-binding domain proteins for broad-spectrum recognition of methicillin-resistant staphylococcus aureus strains development of a specific fluorescent phage endolysin for in situ detection of clostridium species associated with cheese spoilage colorimetric assay of bacterial pathogens based on co o magnetic nanozymes conjugated with specific fusion phage proteins and magnetophoretic chromatography phage-based capacitive biosensor for salmonella detection phagomagnetic immunoassay for the rapid detection of salmonella new rapid and simple methods for detection of bacteria and determination of their antibiotic susceptibility by using phage mutants specific detection of live escherichia coli o : h using tetracysteine-tagged pp bacteriophage high-sensitivity bacterial detection using biotin-tagged phage and quantum-dot nanocomplexes prehospital antibiotics in the ambulance for sepsis: a multicentre, open label, randomised trial diagnosis of central venous catheter-related sepsis seasonal analysis of bacteriological quality of drinking water sources in communities surrounding lake bosomtwe in the ashanti region of ghana this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - ralcn p authors: schwanke, hella; stempel, markus; brinkmann, melanie m. title: of keeping and tipping the balance: host regulation and viral modulation of irf -dependent ifnb expression date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ralcn p the type i interferon (ifn) response is a principal component of our immune system that allows to counter a viral attack immediately upon viral entry into host cells. upon engagement of aberrantly localised nucleic acids, germline-encoded pattern recognition receptors convey their find via a signalling cascade to prompt kinase-mediated activation of a specific set of five transcription factors. within the nucleus, the coordinated interaction of these dimeric transcription factors with coactivators and the basal rna transcription machinery is required to access the gene encoding the type i ifn ifnβ (ifnb ). virus-induced release of ifnβ then induces the antiviral state of the system and mediates further mechanisms for defence. due to its key role during the induction of the initial ifn response, the activity of the transcription factor interferon regulatory factor (irf ) is tightly regulated by the host and fiercely targeted by viral proteins at all conceivable levels. in this review, we will revisit the steps enabling the trans-activating potential of irf after its activation and the subsequent assembly of the multi-protein complex at the ifnβ enhancer that controls gene expression. further, we will inspect the regulatory mechanisms of these steps imposed by the host cell and present the manifold strategies viruses have evolved to intervene with ifnβ transcription downstream of irf activation in order to secure establishment of a productive infection. the interferon (ifn) system provides mammalian cells with a potent framework to fend off intruding pathogens. interferons are signalling molecules first discovered more than years ago, when virus-infected cells were found to release soluble compounds that could interfere with establishment of virus infection [ ] . since this initial discovery, we have come to understand the pivotal role of interferon signalling for the immune response to invading pathogens, from conveying the very first notice of intrusion to eliciting a well-tailored immune reaction suited to thwart the infection. today, we differentiate three classes of interferons based on the receptor they employ for signal transduction. more than a dozen genes encoding ifnα subtypes and a single ifnb gene give rise to the majority of type i ifns in humans. they are the first messenger molecules released upon detection of a pathogen by infected cells and by bystanders to initiate the intrinsic defence mechanisms and to further involve dedicated cells of the immune system (recently reviewed in [ , ] ). ifnγ, the only type ii ifn, presents in the latent state, a part of the linker and the most c-terminal portion form auto-inhibitory elements (aies). phosphorylation of two serine-rich clusters (c and c ) induces conformational rearrangements of the aies and frees the iad to participate in protein-protein interactions with coactivators and for dimerisation via the phosphorylated plxis motif (p: hydrophilic, x: any amino acid). the protein further contains a nuclear localization signal (nls) and a nuclear exit signal (nes) that enable constitutive shuttling between the cytosol and nucleus. irf and irf are the most closely related members of the irf family with especially high conservation of the aie, enabling formation of functional heterodimers and providing the basis for the shared activation mechanisms as well as the similar mode of action [ ] [ ] [ ] . only in plasmacytoid dendritic cells and macrophages, irf is continuously expressed at high levels and crucial for the control of type i ifn induction following tlr and tlr engagement [ ] . since these immune cells produce the major share of type i ifns in an infected host organism, irf was termed the "master regulator" of ifn expression (reviewed in [ , ] ). in most other cell types, however, irf is expressed at very low levels in absence of stimulation [ ] . in contrast to the ubiquitous irf , participation of irf molecules seems less relevant during the very first response to virus detection at initial viral entry sites, which are usually composed of epithelial or endothelial cells or fibroblasts. nevertheless, irf is an important contributor of the type i ifn response. upon ifnα/β signalling via the interferon-alpha/beta receptor (ifnar), irf expression is highly induced as part of the positive feedback regulation of the antiviral response; in other terms, irf is the product of an ifn-stimulated gene (isg) [ ] . newly synthesized irf undergoes an activation similar to irf and in concert with irf further amplifies transcription of ifnβ. this behaviour can interfere with studies focused on irf activity or modulation thereof in later stages. irf is degraded in the course of its activity, as discussed below (see section . ), while irf , though quickly degraded, is constantly expressed during stimulation [ ] . this shifts the control of gene expression in fibroblasts, epithelial and endothelial cells from irf -mediated regulation upon initial sensing of an infection towards irf -mediated in later phases [ ] [ ] [ ] . knockout experiments in mice have shown that lack consists of an n-terminal dna-binding domain (dbd) linked by a flexible region to the c-terminal irf association domain (iad). in the latent state, a part of the linker and the most c-terminal portion form auto-inhibitory elements (aies). phosphorylation of two serine-rich clusters (c and c ) induces conformational rearrangements of the aies and frees the iad to participate in protein-protein interactions with coactivators and for dimerisation via the phosphorylated plxis motif (p: hydrophilic, x: any amino acid). the protein further contains a nuclear localization signal (nls) and a nuclear exit signal (nes) that enable constitutive shuttling between the cytosol and nucleus. irf and irf are the most closely related members of the irf family with especially high conservation of the aie, enabling formation of functional heterodimers and providing the basis for the shared activation mechanisms as well as the similar mode of action [ ] [ ] [ ] . only in plasmacytoid dendritic cells and macrophages, irf is continuously expressed at high levels and crucial for the control of type i ifn induction following tlr and tlr engagement [ ] . since these immune cells produce the major share of type i ifns in an infected host organism, irf was termed the "master regulator" of ifn expression (reviewed in [ , ] ). in most other cell types, however, irf is expressed at very low levels in absence of stimulation [ ] . in contrast to the ubiquitous irf , participation of irf molecules seems less relevant during the very first response to virus detection at initial viral entry sites, which are usually composed of epithelial or endothelial cells or fibroblasts. nevertheless, irf is an important contributor of the type i ifn response. upon ifnα/β signalling via the interferon-alpha/beta receptor (ifnar), irf expression is highly induced as part of the positive feedback regulation of the antiviral response; in other terms, irf is the product of an ifn-stimulated gene (isg) [ ] . newly synthesized irf undergoes an activation similar to irf and in concert with irf further amplifies transcription of ifnβ. this behaviour can interfere with studies focused on irf activity or modulation thereof in later stages. irf is degraded in the course of its activity, as discussed below (see section . ), while irf , though quickly degraded, is constantly expressed during stimulation [ ] . this shifts the control of gene expression in fibroblasts, epithelial and endothelial cells from irf -mediated regulation upon initial sensing of an infection towards irf -mediated in later phases [ ] [ ] [ ] . knockout experiments in mice have shown that lack of irf delays the immune response, while cells lacking irf respond early but are unable to fend off the infection without the signal amplification [ ] . accordingly, together with the hefty feed-forward amplification of ifnα/β signalling, this feature is essential for the induction of distinct and diverse cytokine subsets by irf , especially of ifnα, that differentiate the reaction and prime cellular immunity [ , ] . for this reason, viruses , , of both irf and irf are crucial for the rapid induction and potent establishment of the antiviral response [ , ] . the crucial role of irf and the posttranslational changes it undergoes upon viral infection were first reported more than years ago: upon stimulation, irf gets phosphorylated and accumulates in the nucleus where it interacts with the coactivators creb-binding protein (cbp)/p to specifically bind to virus-inducible enhancer elements and exerts transcriptional activation of the ifnb gene [ , [ ] [ ] [ ] ( figure ). since these first observations, our knowledge of the mechanism of action of irf has been greatly refined. viruses , , x for peer review of of irf delays the immune response, while cells lacking irf respond early but are unable to fend off the infection without the signal amplification [ ] . accordingly, together with the hefty feed-forward amplification of ifnα/β signalling, this feature is essential for the induction of distinct and diverse cytokine subsets by irf , especially of ifnα, that differentiate the reaction and prime cellular immunity [ , ] . for this reason, both irf and irf are crucial for the rapid induction and potent establishment of the antiviral response [ , ] . the crucial role of irf and the posttranslational changes it undergoes upon viral infection were first reported more than years ago: upon stimulation, irf gets phosphorylated and accumulates in the nucleus where it interacts with the coactivators creb-binding protein (cbp)/p to specifically bind to virus-inducible enhancer elements and exerts transcriptional activation of the ifnb gene [ , [ ] [ ] [ ] (figure ). since these first observations, our knowledge of the mechanism of action of irf has been greatly refined. by rna or dna sensors in the cytosol. activation of retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) like rig-i induces aggregation of the mitochondrial adaptor protein mitochondrial antiviral signalling protein (mavs). detection of dna by the dna sensor cyclic gmp-amp (cgamp) synthase (cgas) activates production of the second messenger cgamp, which in turn induces dimerisation of the adaptor protein stimulator of interferon genes (sting) and its translocation from the endoplasmic reticulum (er) to the golgi apparatus. higher-order structures of the adaptor molecules recruit the kinase tank-binding kinase (tbk ) which leads to their tbk -mediated phosphorylation. irf is recruited to this platform and gets phosphorylated by tbk at key residues in the aie, relieving the auto-inhibition. activated irf heterodimerises and associates with the coactivators cbp/p after translocation into the nucleus, yielding a holocomplex with trans-activation potential. in parallel, the heterodimeric transcription factors p -p (nf-κb) and atf -c-jun (ap- ) are activated and enter the nucleus. first, p -p is recruited to the enhancer element upstream of the ifnb gene, followed by atf -c-jun and the irf -cbp/p holocomplex. the assembled ifnβ enhanceosome promotes recruitment of the basal transcription machinery for the expression of ifnb . hyperphosphorylation is the first step in the activation of irf as a functional transcription factor. in unstimulated cells, the irf protein exists as two forms, an unphosphorylated (i) and a basally phosphorylated (ii) form [ ] . after viral infection, iκb kinase-epsilon (ikkε) and tank-binding kinase (tbk ), two homologs of the inhibitor of nf-κb kinase (ikk), are activated when prrs convey the sensing of virus infection to their adaptor proteins, inducing conformational changes and interactions that lead to the formation of a new interaction surface (reviewed in [ ] ). the kinase binds to this signal-induced adaptor platform and phosphorylates the plxis motif (p: hydrophilic, x: any aa) on the surface of the adaptor proteins [ , ] . irf is then recruited to the platform via its recognition site for the phosphorylated plxis motif and gets further phosphorylated, giving rise to two more protein forms (iii and iv) whose appearance correlates with cbp interaction and ifnβ induction [ , [ ] [ ] [ ] . the serine-and threonine-rich region within the c-terminal aie of irf contains two clusters of potential phosphoacceptor residues: cluster (s /s ) and cluster (s /s -s /t /s ) [ ] . first, tbk phosphorylates cluster residues of monomeric irf , and the additional negative charge induces a reorientation of the aies n-and c-terminal of the iad [ , ] . this unmasks a hydrophobic binding pocket required for further protein interactions and renders the c-terminal tail (ctt) accessible for interactions [ , ] . additionally, the conformational change is relayed to the dbd to enhance the dna-binding affinity. the residues of cluster are functionally largely redundant in terms of phosphorylation-mediated irf activation, though phosphorylation of s seems to predominate in vivo [ , ] . the alternative use of phosphorylation sites could be the reason why some studies found that mutation of irf s to alanine sometimes retained biological function (for example in [ , ] ). second, and induced by the first modification, irf gets further phosphorylated at s which is pivotal for dimerisation [ , ] . consistently, % of irf dimers generated in vitro by incubation with tbk are phosphorylated at both clusters [ ] . in contrast to cluster , added phosphate groups at cluster residues have distinct effects: addition of a phosphate group at s promotes dimerisation of irf and strengthens interaction with the coactivator cbp, while phosphorylation of s negatively affects both interactions [ , , ] . by relief of the auto-inhibition, phosphorylated irf can dimerise and associate with the coactivators cbp/p to form an active holocomplex. irf can also be rendered constitutively active by exchange of the five phosphoacceptor sites in cluster to aspartic or glutamic acid residues (irf - d or - e, respectively), and these mutants display a strong tendency to acquire the cluster phosphorylation and dimerise [ , ] . since the first description of irf dimerisation, it is generally noted as the second step of activation after phosphorylation [ ] . formation of dimers requires homotypic interactions of the iad with a second phosphorylated irf molecule. structural studies revealed that phosphorylation of irf in fact modifies a plxis motif in the ctt, similar to the motif initiating recruitment of irf to the adaptor platform [ ] . enabled by the negative charge at s after phosphorylation, the extended ctt can interact with the plxis-binding surface of a second phosphorylated irf monomer to form a domain-swapped dimer (indicated in figure ). zhao and colleagues further proposed that irf dimerises at the adaptor platform to regain stability, but this was not yet confirmed. due to repulsion caused by the newly acquired negative charges, irf proteins could also dissociate from the adaptor complex before dimerisation and converge subsequently either (i) in the cytoplasm, (ii) after translocation into the nucleus or (iii) after engagement of coactivators during recruitment to the enhancer. further, the phosphorylation-induced rearrangement of the aie unmasks a hydrophobic binding site of irf and enables the interaction with transcriptional coactivators [ , , , ] . association of irf with the histone-modifying lysine acetyltransferases (kats) creb-binding protein (cbp, or kat a) and/or p (kat b) in form of a holocomplex is pivotal for the ability of irf to trans-activate ifnb expression [ , , ] . these coactivators are large proteins that contain several folded domains and additionally a big share of intrinsically disordered regions that allow for specific binding to numerous factors upon interaction [ ] . their flexible structure enables the regulation of gene transcription by integrating the cues from several hundred transcription factors, yielding cbp/p the designation "master" coactivators of transcription. the closely related proteins cbp and p are usually regarded as functionally redundant-thus often referred to in combination as cbp/p -and due to this assumption, studies characterising irf interactions assessed association of one or the other [ ] . however, there is growing evidence that their activity is overlapping but can be distinctly involved in regulation of different pathways, as exemplified by brain development [ , ] . considering that both cbp and p can participate in holocomplex formation with irf but do so with different shares [ , ] , it would be interesting to determine whether engagement of cbp versus p is specific or coincidental. potentially, the contribution of the individual coactivators could change during the different phases of ifnb expression depending on integrated signals. to exercise its function as a transcription factor, it is pivotal that irf can reach the nucleus. in fact, the latent protein already shuttles between the cytoplasm and nucleus in unstimulated conditions driven by its nls and nes. as the influence of the nes seems to prevail the import, the main share of irf molecules localises to the cytoplasm [ ] . the constitutive export of irf was shown to involve exportin (crm ) [ , ] , and the import through nuclear pore complexes involves the importins karyopherin (kpn) subunit α (kpna ), kpna and kpna (or qip ) [ , ] . upon infection, phosphorylation of the aie and accompanying structural rearrangements enable irf to associate with cbp, and this interaction retains the activated holocomplex in the nucleus [ ] . interestingly, in vitro experiments from several groups analysing the interaction between irf and the irf-binding domain (ibid) in the c-terminus of cbp have shown that independently of dimerisation, monomeric irf can interact with cbp once the auto-inhibition is relieved [ , , , ] . moreover, in vitro analyses by chen and colleagues implied that the presence of cbp promotes oligomerisation of irf molecules with the required phosphomimetic mutations that enable dimerisation, while the mutant proteins stayed monomeric without cbp [ ] . in a cellular scenario wherein irf dimerises directly after acquiring the required modifications in the vicinity of the adaptor platform in the cytoplasm, this interaction might not be relevant, though. characterisation of the holocomplex demonstrated that the homodimer of irf is more stable than the holocomplex of irf dimer plus p [ ] , and the association of irf dimers with cbp is stronger than that of monomeric irf [ ] , viruses , , of favouring a model wherein irf first dimerises, and dimers subsequently interact with cbp. however, without determination of the precise temporal succession of interactions, this leaves the possibility that a phosphorylated irf monomer enters the nucleus and interacts with cbp/p before it encounters a second irf molecule to dimerise and swiftly induce a response, as suggested before [ ] . in the end, the coactivator needs to associate with dimeric irf because only this can induce activation of the kat activity that is later required for assembly of the pre-initiation complex, as demonstrated for p [ ] . several groups noted exceptions to the common model of irf activation followed above, drawing attention to the fact that assessment of the conventional signs like dimerisation that reflect the intermediate states is not always sufficient to infer on the downstream biological response [ , , ] . due to these discrepancies we suggest to differentiate between phosphorylated irf that is relieved of the auto-inhibition and can "actively" participate in subsequent steps and irf in the holocomplex that can exercise its trans-activation potential as a transcription factor to "actively" induce ifnb expression. in fact, some of these studies assessed the activity of irf in terms of induction of isg [ , ] , which intermingles the irf -dependent regulation of type i ifns with directly irf -regulated isg expression [ ] . to our knowledge, it remains to be determined whether the form of irf induced for up-regulation of specific isg promoters is the same as the form of irf able to induce ifnb expression. moreover, reports clearly reflect that there are different extents of irf activity, and the protein might not yet be fully activated when initiating a first wave of ifnb expression. instead, irf might acquire modifications and engage in distinct interactions in different waves of the type i ifn response until it is fully activated. in line with this, we noticed several reports referring to "full" activity of irf in later loops of the response, for example enabled by phosphorylation at both clusters of the aie or determined only in the presence of ifnar signalling [ , ] . this aligns with the kinetics of the ifnβ response we will briefly revisit below (see section . ), wherein only a part of the ifnb alleles are engaged in the early response. expression of ifnb is probably the best studied model of induced eukaryotic gene regulation. it exemplifies how factors activated by specific signals cooperate to prompt a defined expression programme, as reviewed before [ , [ ] [ ] [ ] . upon viral stimulation, a multicomponent complex of transcription factors, coactivators and architectural proteins forms at the enhancer sequence upstream of the ifnb gene promoter. this higher-order nucleoprotein complex, termed the ifnβ enhanceosome, works as a molecular switch that turns on upon virus-induced activation of a specific set of transcription factors [ , ] . when completed, the enhanceosome in turn recruits factors with chromatin-modifying activities as well as basal eukaryotic transcription factors to the nearby transcription start site (tss) to initiate gene expression. the assembly is regulated at several levels and highly specific due to an elaborate organisation of transcription factor binding motifs, the required set of activated transcription factors and architectural proteins and finely coordinated interactions of the individual participants as well as of the arising composite structures. from just slightly upstream of the tss of the human ifnb gene, a precisely arranged sequence of cis-acting dna constitutes the key enhancer element that defines ifnb expression. the ifnβ enhancer itself is accessible yet directly flanked by two nucleosomes, one of them covering the tss and a tata box [ ] . this setup strategically restricts the access of the transcription machinery in latent conditions [ , ] . the enhancer can be divided into four positive regulatory domains (prds) in the order iv-iii-i-ii that are discussed in detail in [ ] . of note, however, is the overall architecture that is essential for the precise recognition and following interplay of the array of core transcription factors [ ] . the prd farthest away from the promoter, prdiv, features motifs for binding an ap- heterodimer formed by activating transcription factor (atf ) and c-jun of the basic-region leucine zipper (bzip) family [ , ] . binding of the heterodimer over homodimers is favoured by the intrinsic architecture of the enhancer and important for subsequent interactions [ ] . the last prd in the sequence of the enhancer, prdii, is recognised by a p -p or p -p nf-κb dimer [ ] [ ] [ ] . prdiii and prdi in the middle section feature in total four overlapping irf-binding elements (irf-es) of the consensus sequence -aanngaaa- that form two composite binding sites enabling binding of two irf dimers [ , , , ] . this region was also termed p because these two prds act as a functional unit, an enhanson [ ] . recent in-depth analysis of the binding requirements of different irf members suggests that, in addition to a slight variation of the core gaaa recognition motif, the flanking regions along with the spacing in between the irf-es contribute to specific binding of distinct irf family members, here favouring recruitment of irf and irf [ , ] . the nucleotide sequence of the ifnβ enhancer causes a bent conformation of the dna helix in inactive conditions and in this way drives cooperative binding of the transcription factor heterodimers upon stimulation [ ] . after stimulus-induced activation, the transcription factors translocate into the nucleus. first, the nf-κb dimer p -p is recruited and nucleates assembly [ ] . association of nf-κb is the most limiting factor of enhanceosome assembly, and to optimise recruitment, it is initially targeted to three loci at distinct chromatin regions via alu elements, also termed nf-κb reception centres (nrcs) [ , , ] . the transcription factor thpok (t-helper-inducing poz/krüppel-like factor, also zbtb b) binds cooperatively with nf-κb to these regions, and oligomerisation of thpok mediates interchromosomal interactions of the nrcs with the ifnβ enhancer to deliver nf-κb [ , ] . alongside nf-κb, the architectural dna-binding high mobility group protein i(y) (hmg i(y), also hmga ) binds at two sites to the minor groove in prdii and straightens the dna conformation, thereby supporting interaction of nf-κb [ , [ ] [ ] [ ] [ ] . subsequently, the remaining virus-induced transcription factors are recruited. a second hmg i(y) molecule interacts with atf -c-jun to promote binding of the heterodimer to prdv by straightening the dna helix [ , , ] . additionally, nf-κb mediates capture and binding of the irf -cbp/p holocomplex [ , ] . in vitro, irf displays a surprisingly weak affinity for p , which led to the former model in which irf is the critical irf member in ifnb induction because it freely associates with the enhancer sequence [ ] [ ] [ ] . however, irf was then found to be dispensable for ifnb expression upon prr activation, which is congruent with its expression pattern as an isg [ ] [ ] [ ] . instead, when phosphorylation releases the auto-inhibition of irf and enables interaction with cbp/p , the coactivator strongly increases the affinity of irf for dna and enables association of the complex [ , , , ] . further cooperativity is mediated by the p sequence that ensures concerted binding of atf -c-jun and irf [ , ] and by cbp/p -mediated interactions within the enhanceosome complex [ , ] . clustering of irf target regions with binding motifs for nf-κb or other transcription factors along with low chromatin accessibility in steady-state conditions was recently described as a common feature of irf -dna interactions, implying an obligatory collaborative binding mode of irf that promotes access to chromatin [ ] . at the ifnβ enhancer, a total of four irf / molecules bound to four irf-es in tandem are required to induce gene expression of a luciferase-based ifnβ-reporter plasmid in the commonly used human embryonic kidney cell line hek t [ ] . structural analysis suggests that one irf dimer binds on one side of the duplex, occupying the first and the third irf-e, and the second dimer binds from the opposite side, binding to the second and fourth irf-e [ ] . remarkably, the dbds of the eight transcription factors barely interact with each other despite the great overlap of their binding elements [ ] . instead, the high level of cooperativity between binding events is achieved by the specific order of individual response elements that allows for binding-induced conformational changes of the dna to transmit allosteric effects [ , , , ] . molecular dynamics simulations by pan and nussinov further showed that the combination of overlapping recognition viruses , , of elements with an alternation between consensus and non-consensus motifs optimises the specific interactions by enhancing or restricting binding of neighbouring transcription factors. moreover, the signal integration by cbp/p that interacts with each transcription factor through different domains is essential for the functionality of the enhanceosome [ ] . in addition to the pivotal architectural role of recruiting irf and mediating interactions with the transcription machinery as coactivator, cbp/p also participates in the subsequent chromatin remodelling in its vicinity [ , , ] . as implied above, however, the precise number and identity of coactivator molecules participating in the formation of the active irf -cbp/p holocomplex as well as in the enhanceosome is unknown. potentially, they present a module for further signal integration by participating in dynamic compositions. further, also the subtle modulation by the two hmg i(y) proteins is required for the maximal level of expression [ ] , though their participation in the final assembly is controversial as previously discussed [ ] . in line with the high synergy during assembly, the ifnβ enhanceosome is extraordinarily stable and enables multiple rounds of re-initiation in vitro [ , ] . due to the binding-induced conformational changes of the dna foundation, the fully assembled structure encompasses a straight segment of dna covered by eight specifically bound transcription factors all of which interact with the essential coactivator cbp/p . this final ifnβ enhanceosome presents a single new, continuous activating surface and provides the basis for the association of the basal transcription apparatus. before transcription of the ifnb gene can commence, the chromatin landscape needs to be modified so that the pre-initiation complex can be assembled and access the tss. first, the ifnβ enhanceosome mediates the transient recruitment of a complex of p /cbp-associated factor (pcaf, or kat a) and general control nonderepressible- (gcn or, kat b) to the promoter and induces the remodelling, starting with gcn -mediated acetylation of nucleosomes and hmg i(y) in its vicinity [ , , ] . acetylation of hmg i(y) at k by gcn strengthens the association of nf-κb and thereby further stabilises the enhanceosome [ , ] . next, the modification of the histone tails of nucleosomes at h and h promotes recruitment of the chromatin remodelling brg -or brm-associated factor (baf) complex of the swi/sfn family [ , , , , ] . in parallel, general eukaryotic transcription factors (tfs) tfiia, tfiib, tfiie, tfiih, tfiif and upstream stimulatory activity (usa) cofactors assemble at the promoter along with the rna polymerase ii (rnap ii) holoenzyme [ , , ] . stimulated by the environment rich in acetylated histones, the atpase brahma-related gene (brg or smarca ) of the baf complex induces a conformational change of the nucleosome at the tss [ , ] . now, the tata box at the tss is available for binding by the tata box-binding protein (tbp) and allows association of the tfiid complex [ , ] . notably, tfiid binds here after the rnap ii complex, as opposed to the classical sequence of events in assembly of the pre-initiation complex [ ] . association of tfiid induces a bend of the dna helix so that the nucleosome covering the tata box slides bp downstream of its latent position [ , ] . with this, the arrangement of the pre-initiation complex can be completed, and ifnb transcription can begin [ , ] . notably, while the rapid induction of the type i ifn response is crucial for the host defence, the first round of ifnb expression yields only low levels of ifnβ, as discussed by ford and thanos [ ] . briefly, after stimulation of a cell population, ifnb is initially expressed only by a fraction of cells and from only one allele due to a stochastic phenomenon that indicates a limiting amount of required cellular factors [ ] [ ] [ ] . the initial recruitment of nf-κb to other loci that collect the available transcription factor and transfer it to the required binding sequence highlights the p -p heterodimer as the most limiting factor [ , ] . the weak initial signal of secreted ifnβ induces high-level expression of irf , and newly synthesized irf promotes enhanceosome assembly and ifnb transcription from the remaining allele and in more cells [ ] . finally, the weak initial ifnβ signal is then amplified to eventually induce the cytokine storm of the antiviral response. the expression of ifnb is, at least in murine cells, tightly regulated by different forms of nf-κb dimers in the initial response to infection, as reviewed by balachandran and beg [ ] . at resting conditions, p homodimers associate with the ifnβ promoter region and repress basal transcription in the absence of appropriate stimulation [ , , ] . in addition, a binding site for nf-κb regulatory factor (nrf) overlaps the nf-κb binding element in prdii and negatively affects basal transcription [ , ] . in line with a primary inhibitory effect, knockout of murine p does not affect induction of ifnb expression after virus infection [ ] . however, the p -imposed down-regulation in resting cells is competed with by p -p dimers that are constitutively activated by ikkβ and cycle between the cytoplasm and nucleus [ ] . this low level activity of p supports a basal level of ifnb expression that ensures rapid and robust responsiveness on demand, while the negative regulation by p ensures specificity [ ] . after stimulation, p -p is required to assist in the early recruitment of irf together with cbp/p to support ifnb expression [ , , ] . only at high levels of irf activity, i.e., at the peak of activity later in infection or brought about by expression of the constitutively active irf -s d, p -p is dispensable for ifnb transcription [ ] . subsequent to the initial recruitment, the increasing levels of active irf and irf can fully assume the regulation of ifnβ production, and nf-κb proceeds to regulate expression of pro-inflammatory genes [ ] . the p -p -mediated basal expression of ifnb raises the question how recruitment of the transcription machinery is achieved at resting conditions, considering that irf is missing to participate in a highly cooperative assembly as described above. moreover, since the type i ifn system is not % conserved between mice and humans, and all of these studies were carried out in murine systems, it remains to be confirmed whether the exact same mode applies for the participation of nf-κb in the human system. in addition to the proximal enhancer, dna elements further away from the promoter are involved in ifnb transcription, though again, most studies have thus far focused on the murine system. in non-infected mouse cells, a proximal region of the ifnb locus that contains binding sites for the transcription factor yin yang (yy ) was shown to mediate association with pericentromeric heterochromatin (pch), a feature related to gene silencing [ ] . after infection, the ifnb locus is repositioned away from pch, and this observation correlated with transcriptional activation of the promoter shortly thereafter. josse and colleagues suggested that this effect could be mediated by binding of yy to the proximal promoter. furthermore, yy interacts also in the absence of viral infection in mice with the signal transducer and activator of transcription (stat ), one of the main factors mediating isg expression [ ] , implying important roles of yy proteins at different stages during infection [ ] . in the human cell, yy and yy regulate enhanceosome formation from yy-binding site-containing regions far upstream of the tss of the ifnb gene at − kb and − kb, termed dnase i hypersensitive sites (hs ) and hs , respectively [ ] . yy and yy seem to modulate each other, with yy antagonising the negative effect of yy . in analogy to the murine ifnβ promoter region, this long-range interaction was suggested as requirement for the recruitment of gcn to initiate chromatin remodelling at the ifnβ promoter [ ] [ ] [ ] . a further dna element regulating ifnb transcription is the long-range enhancer l , which was first identified in human cells but is conserved in mice [ ] . l is activated upon viral stimulation and recruits irf and rnap ii dependent on the irf-e within its interferon-stimulated response element (isre). this induces expression of a non-coding rna from the enhancer, and this enhancer rna (erna) in turn supports ifnb expression. however, the mode of action of the erna was not yet determined. the rapid induction of the antiviral state is critical to overcome a nascent infection. while an insufficient response would be beneficial for the pathogen, overshooting activation of the immune system can cause severe damage to the organism. therefore, several mechanisms are implemented by the host organism to regulate irf at all levels, from moderating the potential to activate irf from its latent state, to promoting its participation in formation of the ifnβ enhanceosome, to supporting its activity during ongoing stimulation, through to terminating the response with deliberate timing. factors that contribute to the finely tuned ensemble of modulators can be continuously active, induced or inhibited upon stimulation. a wide array of molecular mechanisms that modulate the biological activity of irf are deployed, including protein-protein interactions, regulatory rnas and common as well as non-canonical posttranslational modifications. to illustrate the abundance of strategies, we will include also proteins expressed predominantly in immune cells. in particular, mechanisms aiming at dampening the activity of irf were usually identified in macrophages, highlighting the importance to keep the response primed but low until the full activation becomes necessary. since posttranslational modifications of irf and especially phosphorylation and ubiquitination were reviewed before [ , ] , we will instead focus on how the host factors engage in the different steps to modulate the biologic activity of irf ( figure ). viruses , , x for peer review of system can cause severe damage to the organism. therefore, several mechanisms are implemented by the host organism to regulate irf at all levels, from moderating the potential to activate irf from its latent state, to promoting its participation in formation of the ifnβ enhanceosome, to supporting its activity during ongoing stimulation, through to terminating the response with deliberate timing. factors that contribute to the finely tuned ensemble of modulators can be continuously active, induced or inhibited upon stimulation. a wide array of molecular mechanisms that modulate the biological activity of irf are deployed, including protein-protein interactions, regulatory rnas and common as well as non-canonical posttranslational modifications. to illustrate the abundance of strategies, we will include also proteins expressed predominantly in immune cells. in particular, mechanisms aiming at dampening the activity of irf were usually identified in macrophages, highlighting the importance to keep the response primed but low until the full activation becomes necessary. since posttranslational modifications of irf and especially phosphorylation and ubiquitination were reviewed before [ , ] , we will instead focus on how the host factors engage in the different steps to modulate the biologic activity of irf ( figure ) . in addition to the essential steps of irf activation delineated above, various protein-protein interactions and additional posttranslational modifications were demonstrated to support the biological activity after induction. before stimulation, for example, protein phosphatase (pp ) removes the phosphate groups at s and s in macrophages and consequently attenuates ifnβ production [ ] . early after tlr-or rlr-mediated stimulation, however, activity of pp is down-regulated to increase the pool of active irf and augment the immune response [ ] . also first identified in murine macrophages, the lysine methyltransferase nuclear receptor-binding set domain (nsd ) targets nuclear irf after viral stimulation and modifies irf by adding a single methyl group at k [ ] . this modification promotes the dissociation of phosphorylated irf from an isoform of pp catalytic subunit γ, thereby hindering the inhibitory effect of pp and maintaining the activated state of phosphorylated irf . similarly, type i ifn up-regulates the expression of the long non-coding rna lnclrrc -as, an antisense transcript to the gene of leucine-rich repeat-containing protein , which supports irf phosphorylation in macrophages by inhibiting an inhibitor [ ] . in the cytosol, lnclrrc -as associates with the protein phosphatase methylesterase (pme- ), which in turn promotes the interaction of pme- and the protein phosphatase a (pp a). pp a is an inhibitor of irf signalling, but lnclrrc -as mediates its demethylation and inactivation to maintain irf activity [ ] [ ] [ ] . additionally, stability of the irf protein is promoted within the loop of the type i ifn response by covalent conjugation of newly synthesised isg to irf on k , k or k mediated by herc (hect and rld domain-containing e ubiquitin protein ligase ) [ , ] . this modification, termed isgylation, disturbs the interaction between irf and peptidyl-prolyl isomerase (pin ), delays the proteolytic degradation of irf and in this way prolongs the immune response [ ] [ ] [ ] . the dual use of irf k for methylation by nsd versus isgylation by herc , both of which contribute to protein stability, hints at a complex network that also modulates the modulators. while inactive irf already shuttles between the cytosol and nucleus in latent conditions, the translocation becomes crucial after its activation in order to exercise the trans-activation activity. just recently, cai and colleagues reported that the ubiquitin specific peptidase (usp ) promotes the antiviral response from the cytoplasm by deubiquitinating importin kpna [ ] . after viral infection, kpna associates with irf for nuclear import, and usp promotes this critical step by stabilizing kpna . importin-mediated translocation is additionally regulated by the widely expressed transcription regulators yes associated protein (yap ), yap and yap : yap / / associate with latent as well as activated irf and block further interactions required for dimerisation or nuclear import [ ] . after virus infection, however, activated ikkε phosphorylates a conserved motif of the yaps, which triggers their lysosomal degradation and reliefs the yap-mediated inhibition. further, an additional phosphate group within the dbd of irf at s is important for the nuclear translocation after viral stimulation [ ] . this modification inhibits the nuclear import, and mass spectrometry revealed that about a fifth ( . %) of irf molecules carry it at latent conditions when exogenously expressed in hek . upon viral stimulation, the dual-specificity protein phosphatase and tensin homolog (pten) removes this phosphate group and consequently the negative regulation of irf [ ] . finally, to keep irf in the nucleus, dna-pk is activated in response to virus infection and phosphorylates irf at t [ ] . this phosphate moiety mediates the nuclear retention and delays proteolysis of the active transcription factor, thereby prolonging the irf -driven response. the association of cbp and irf in the nucleus is supported by the constitutively active enzyme glutaredoxin- (glrx or grx ), which acts in the cytoplasm [ ] . in resting cells, inactive irf is s-glutathionylated and this modification would impede the essential interaction of irf and cbp/p . however, glrx removes this modification after infection and thus supports irf activity. while the deglutathionylation of irf is independent of its phosphorylation or dimerisation state, the accompanying structural changes could be involved in the recruitment of glrx to remove the modification after initial activation events [ ] . within the nucleus, the interaction of irf and cbp is further promoted by a ubiquitously expressed subunit of the endosomal sorting complex required for viruses , , of transport (escrt)-ii [ ] . after virus infection, a fraction of the escrt-ii subunit ell-associated protein of kda (eap , also known as sfn ) localises to the nucleus where it interacts with activated irf and cbp and promotes binding of the holocomplex to target gene promoters. contrasting this, the protein argonaut (ago ), a component of the rna-induced silencing complex, interacts with the iad of irf and inhibits its association with cbp in the nucleus in latent conditions [ ] . upon viral infection, however, ago is exported from the nucleus, and its inhibition is revoked to promote ifnβ production during infection [ ] . a further mechanism of signalling amplification is set into motion by type i ifn-stimulated production of the e like ets transcription factor (elf ). elf is recruited to sting and activated by tbk similar to irf after viral stimulation, though without affecting irf activation itself [ ] . activated elf enters the nucleus and binds to the ifnβ promoter region via ets/irf composite binding elements (eices). dna binding of elf synergises with binding of irf , and thus promotes enhanceosome formation. the critical role of elf in the feed-forward amplification of the murine antiviral immune response was demonstrated in vivo, and notably, it is independent of the signalling in plasmacytoid dendritic cells [ ] . in contrast, a mechanism that due to the predominating expression of both factors in tissues of the immune system applies exclusively to immune cells is the association of irf together with the transcription factor pu. (also spi , spi- proto-oncogene) to the ifnβ promoter region mediated by eice [ ] . li and colleagues proposed that irf and pu. support the rapid induction of transcription by forming a scaffold at the ifnβ enhancer to facilitate recruitment of activated irf [ ] . a striking feature of irf modulation is reflected by mechanisms that are installed not to plainly heighten or terminate the biological activity of irf but to moderate its extent. some of the mechanisms delineated here affect the latent protein, others specifically target activated irf after viral stimulation and still others act on both. macrophages are a prime example in deploying numerous modulators to dampen fluctuations of irf activity until a certain threshold is passed and further to restrain the response once unleashed. for example, the ubiquitin-protein ligase e c (ube c) mediates k -linked ubiquitination of irf and irf , thereby targeting the master transcription regulators in dendritic cells for proteolysis, irrespective of their activation state [ ] . in this way, ube c helps to maintain low amounts of ifn production in resting conditions and additionally restrains the magnitude of the response after stimulation [ , ] . another e ubiquitin ligase, tripartite motif-containing (trim or ro ), was described by two groups to be important for regulation of irf but with opposing findings as already discussed by sin [ ] : higgs and colleagues reported trim -dependent mediation of irf degradation hours after infection [ ] , whereas yang and colleagues found that nine hours after infection, trim interferes with the interaction between irf and pin and in this way prevents pin -mediated ubiquitination and degradation of irf to sustain the immune response [ ] . sin suggested that, in addition to effects potentially imposed by different cell lines, the observation at different times of ongoing infection could have contributed to the contrasting findings [ ] . stability of irf is further modified by addition or removal of small ubiquitin-like modifier (sumo) proteins. in hek cells, endogenous irf is sumoylated at k and k in its dbd, and viral infection slightly increases sumoylation [ ] . the widely expressed human sentrin/sumo-specific protease (senp ) removes these sumo moieties and in this way conditions irf for k -linked ubiquitination at the same residues and subsequent degradation, thereby dampening the antiviral response [ ] . senp targets wild-type irf as well as the constitutively active irf - d mutant, suggesting that the sumoylation initially masks the protein from degradation to prolong its activity but that this modification is constantly countered by senp activity. in addition, stimulus-induced sumoylation of murine irf at k was reported to attenuate the ifnβ response irrespective of the protein's phosphorylation status, though the mediating proteins are unknown [ ] . regulation of auto-inhibition aside, phosphorylation of irf can contribute to the down-regulation of its activity also at subsequent steps. when screening the human kinome for phosphorylation events during nucleic acid sensing, meng and colleagues identified mammalian sterile -like kinase (mst , also stk ) as an inhibitor of ifnβ promoter induction and confirmed the effect of mst in knockout mice [ ] . mst suppresses activation of tbk and ikkε and thereby inhibits the phosphorylation-dependent activation of irf , but it also targets irf directly for deactivation by phosphorylating two sites of irf (t , t ). demonstrating that this effect is independent of the inhibition of the upstream kinases by mst , introduction of a single phosphomimetic mutation, t d, is sufficient to impair the ability of the constitutively active irf - d to dimerise after stimulation [ ] . a further level of negative regulation of ifnβ signalling is imposed by the ubiquitously expressed fas-associated factor (faf ), which interacts with importin (iop , also kpnb ) in resting as well as stimulated cells [ ] . after viral stimulation, irf increasingly associates with ipo for nuclear import, but this is dampened by the faf -ipo interaction which consequently reduces the translocation of phosphorylated irf and the induction of ifnb expression. in the nucleus, formation of the irf /p complex and p -mediated acetylation of irf after virus infection is assisted by bromodomain-containing (brd ) [ ] . generally, interaction of brd with p promotes recruitment of the holocomplex to the ifnβ enhancer and facilitates ifnb transcription. this supporting mechanism seems to come with a time limit, however, as analysis in macrophages revealed that viral stimulation specifically down-regulates brd abundance and thereby terminates its support [ ] . similarly, zhang and colleagues recently reported stimulation-induced non-canonical k -linked ubiquitination at three positions in the dbd of irf (k , k , k ) and demonstrated that this modification is essential for the dna-binding capacity of irf in macrophages and in hek t cells [ ] . as a counter measure, virus-infected cells additionally up-regulate expression of the ovarian tumour domain-containing deubiquitinase (otud ), and otud then removes this ubiquitination from irf , resulting in reduced irf -binding to the ifnβ promoter region at later times [ ] . to allow moderation of enhanceosome assembly after formation of the active irf -cbp/p complex, other dna-binding proteins were reported to interact with motifs contained within the ifnβ enhancer sequence. the first irf-e overlaps with a consensus binding site for an activator of pro-inflammatory responses in macrophages, nfat (nuclear factor of activated t cells ), which is conserved between human and mouse promoter sequences [ ] . nfat constitutively forms dimers that can bind to the ifnβ enhancer region and competes with irf due to the overlapping recognition sequence, resulting in limited recruitment of irf to the enhancer and thus limited promoter induction. in a similar way, the transcription regulator maf bzip transcription factor b (mafb) binds in macrophages to the ifnβ enhancer sequence mediated by ap- -like sites [ ] . in resting conditions, mafb is a weak positive regulator of basal ifnb transcription. upon stimulation and activation of irf , however, it impairs the interaction between the coactivators and antagonises enhanceosome formation. this dual role was suggested to allow the system to deal with fluctuations in irf activity [ ] . during the course of the innate immune response, the signal that started it all has to be switched off again to allow other messengers to refine the defence line. the first mechanism to terminate the irf -dependent response is set into action directly during the activation of irf by phosphorylation of the c-terminal aie [ ] . in addition to releasing the auto-inhibition, the modification represents a signal for degradation of the protein, and so activated irf has a shorter half-life than the latent form [ , ] . in this way, turnover of the activated protein by degradation rather than additional activation of repressors is an integral feature responsible for ending ifnb expression [ ] . several molecules have been reported to mediate ubiquitination of irf in order to induce its depletion after initial stimulation [ ] : pin , foxo [ ] , trim , rbck and trim (see above, section . ) all mediate k -linked ubiquitination and subsequent degradation of activated irf . for example, the peptidyl-prolyl isomerase pin specifically mediates degradation of the activated transcription factor by recognition of a further phosphorylated motif (s phos-p ) irf acquired during stimulation [ ] . additionally, viral infection promotes the nuclear localisation of the e ubiquitin ligase trim , allowing trim to bind phosphorylated irf in the nucleus and promote its k -linked ubiquitination and degradation [ ] . similarly, production of rbcc protein interacting with pkc (rbck ), another e ubiquitin ligase, is induced by viral stimulation and targets irf for ubiquitination [ ] . furthermore, caspase- (casp ) is activated by cytosolic rig-i-dependent signalling and cleaves irf at a recognition motif between dbd and iad ( sqpd ), inducing ubiquitination and degradation of the fragments [ ] . in parallel to the activation of irf , viral stimulation also activates inhibitors of irf . the transcriptional regulator krüppel-like factor (klf ), which is widely expressed in human tissues, increasingly localises to the nucleus after viral infection where it binds to the ifnβ promoter region [ ] . this reduces recruitment of irf and thus inhibits induction of ifnb expression. in immune cells, the lysine acetyltransferase kat was identified by an sirna-screen as a negative regulator of antiviral innate immunity [ ] . viral infection promotes kat -mediated acetylation of irf at k , independent of the phosphorylation and dimerisation status of irf , and this modification interferes with binding of irf to the ifnβ enhancer. a potential effect on assembly of the irf -cbp/p holocomplex was not characterised, however, leaving the exact step of interference to be determined. a further line of down-regulation is introduced by the expression of isgs that negatively modulate irf activity. considering the multitude of induced isgs [ ] , it is not surprising that they target various levels to terminate ifnb transcription. nuclear import is repressed by the increasing interaction of irf with the ifn-inducible dead-box rna helicase ddx , as this interaction competes with the association of irf and ipo [ ] . the irf -dna interaction is inhibited by interaction of newly synthesized cell growth-regulating nucleolar protein lyar (ly antibody-reactive), which is usually low expressed in most cell tissues but induced by the ifnβ response [ ] . lyar interacts specifically with the n-terminal domain of activated irf and in this way interferes with the dna-binding capacity of irf in the irf -cbp/p holocomplex. additionally, a long non-coding rna directly targets the ifnβ promoter region and interferes with transcription factor binding in a unique way [ ] : after rna deep sequencing revealed enhanced transcription of the long non-coding rna lnc-mxa from the mxa locus after viral infection, li and colleagues recently reported its mechanism of action. applying a chromatin isolation by rna purification assay and in vitro pulldown of an ifnβ promoter dsdna fragment with biotin-labelled lnc-mxa, they demonstrated that lnc-mxa forms an rna-dna triplex with the ifnβ promoter region. this changes the structure of the chromatin and interferes with binding of the transcription factors irf and nf-κb to their respective target sequence [ ] . finally, the delayed generation of prdi-binding factor (prdi-bf , or pr/set domain ) mediates recruitment of the histone h lysine methyltransferase g a for epigenetic silencing of ifnb expression [ , ] . the following host factors were additionally reported as negative modulators of irf activity, but it remains to be seen if they constantly inhibit irf or how they are regulated. for instance, calmodulin-like protein (calml or caglp) was identified to negatively modulate irf and independently of the protein's ability to bind calcium ions [ ] . calml interacts with the c-terminal aie of irf , and this interaction is strengthened when irf is phosphorylated. after virus infection, calml thereby impairs dimerisation and nuclear import of irf . the contribution of cellular flip long isoform protein (cflip l ) to regulation of the type i ifn response in several primary cancers was reported by several groups with contradicting outcomes, so gates and colleagues studied the underlying mechanism of action and concluded on an inhibitory function [ ] . when ectopically expressed in hek t cells, cflip l interacts with phosphorylated irf within the nucleus and hinders interaction with cbp and thus with the ifnβ enhancer. cflip l was then confirmed to be highly expressed in several human cancer cell lines and to interact with endogenous irf , mediating reduction of isg expression. as opposed to the major isoform (variant ) of irf that drives the type i ifn response as discussed so far, three of the five described splice-variants of human irf negatively modulate the biological activity of irf , though their regulation remains to be determined. translation of variant (irf -cl) yields a slightly longer protein ( aa) with a unique sequence of aa in the c-terminus that lacks a part of the iad including the aie of irf [ ] . irf -cl constitutively forms homodimers that localize to the cytoplasm. after viral stimulation, ikk-mediated activation induces association of irf -cl with phosphorylated irf , but the heterodimers are retained in the cytoplasm and consequently, the association with this isoform keeps irf from the induction of ifnb expression. in contrast, variant (irf a) lacks the n-terminal part of the functional dbd of irf and is consequently unable to bind to classical irf-es [ , ] . this isoform also heterodimerises with irf variant after stimulation, but in line with the absent dbd, its presence selectively inhibits virus-induced ifnb transcription. interestingly, after virus stimulation, irf a is degraded slower than phosphorylated irf and so the ratio of negative versus positive modulator increases with ongoing infection, potentially contributing to a downregulation of the type i ifn response [ ] . the fourth variant, irf e or irf -nirs, was discovered when marozin and colleagues searched for the reason of the defect in ifnb expression in hepatocellular carcinoma (hcc) cells and discovered that a truncated variant of irf was constitutively expressed in primary cells of hcc or hcc cell lines but not in primary hepatocytes [ ] . irf -nirs is produced when an in-frame exon is aberrantly skipped during splicing, leading to the generation of a protein lacking aa within the β-sandwich core of the iad. in line with this, this isoform is constitutively active and maintains the dna-binding ability. however, due to the compromised iad, irf -nirs seems unable to exercise trans-activation activity and instead competes with irf for the limited irf binding sites in the ifnβ enhancer sequence [ ] . surprisingly, given that association with the coactivator is normally essential for irf to bind to dna motifs, this implies that the interaction surface for cbp/p remains intact despite the missing adjacent structural module. in addition to the human isoforms, one variant of murine irf , mirf- a, was reported as a ubiquitously expressed negative modulator of ifnβ induction in mice [ ] . during the generation of mirf- a, an alternative donor splice site in exon is used and leads to a frameshift with a premature termination codon, yielding a shorter variant ( aa) which differs in the c-terminal region as compared to the major murine variant ( aa). mirf- a freely localises to the nucleus, binds to irf recognition sites and represses promoter activation. not long after the initial steps in the characterisation of the ifnβ enhanceosome were made, also the first viral proteins targeting its assembly were noticed [ , ] . today, a long list of viral factors targeting every step from the stimulation of prrs by nucleic acids, to induction of transcription factor activation, to activity of ifn, through to the stimulation and activity of isgs are known, and the list still grows [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the central role of irf in this pathway makes it an attractive target for viral evasion. strategies applied to interfere with the activation of irf in the cytoplasm include (i) the inhibition of irf expression; (ii) direct antagonism of essential phosphorylation events by targeting the essential kinases, their interaction with irf or dephosphorylating irf (recently joined by herpes simplex virus (hsv)- immediate early protein icp [ ] , and the nucleocapsid protein of peste des petits ruminants virus [ ] ); and (iii) mediating degradation of irf . remarkably, even nuclear phosphorylated irf can specifically be targeted for ubiquitination and proteasome-mediated degradation after activation [ ] . other viral factors aim to evade the type i ifn response more generally by limiting its induction, affecting production of ifn by transcriptional or translational shut-off or dysregulating the processing or trafficking of host mrnas [ , ] . noting that strategies to inhibit irf activation and activity applied by viruses were frequently reviewed for specific virus families as well as in broad summaries [ ] [ ] [ ] [ ] [ ] [ ] , we want to emphasize here the molecular mechanisms viruses deploy to obstruct the function of irf after its trans-activation potential is enabled by phosphorylation (figure ). obstruct the function of irf after its trans-activation potential is enabled by phosphorylation ( figure ). as the activation of irf is a multistep process, in early studies the exact level of viral interference sometimes remained elusive. dimerisation of irf was among the first steps reported to be targeted in order to antagonize its trans-activation activity. the ml protein, a splice variant of the matrix protein of thogoto virus (thov) of the family orthomyxoviridae, blocks the formation of irf homodimers irrespective of the presence of the c-terminal aie and further inhibits association of irf with cbp [ , ] . interestingly, this modulator does not affect nuclear translocation, suggesting that ml does not disable import of irf but renders otherwise activated irf monomers unable to participate in further interactions in the nucleus. the authors ruled out a lasting interaction as the activation of irf is a multistep process, in early studies the exact level of viral interference sometimes remained elusive. dimerisation of irf was among the first steps reported to be targeted in order to antagonize its trans-activation activity. the ml protein, a splice variant of the matrix protein of thogoto virus (thov) of the family orthomyxoviridae, blocks the formation of irf homodimers irrespective of the presence of the c-terminal aie and further inhibits association of irf with cbp [ , ] . interestingly, this modulator does not affect nuclear translocation, suggesting that ml does not disable import of irf but renders otherwise activated irf monomers unable to participate in further interactions in the nucleus. the authors ruled out a lasting interaction between ml and irf that could for example directly compete with binding to cbp/p because they could not detect association of ml and irf by co-immunoprecipitation [ ] . additionally, ml interferes with a later step in the induction of ifnb expression, namely, the cofactor function of tfiib at promoters that require de novo recruitment of rnap ii, which was indicated by the finding that ml also inhibited activity of the constitutively active irf - d mutant [ , ] . the leader (l) protein of the cardioviruses of the picornaviridae also interferes with irf dimerisation. in fact, at first, the l protein of encephalomyocarditis virus (emcv) was reported for its ability to generally disturb trafficking of proteins between the cytoplasm and nucleus [ ] . after infection with a related strain, mengovirus, however, irf still localized to the nucleus. to inhibit type i ifn transcription nonetheless, the mengovirus l protein antagonizes dimerisation of irf downstream of its phosphorylation [ ] . due to the observation of irf translocation without dimerisation, the authors suggested that importin-mediated transport might be a requirement for dimerisation of irf , which would take place within the nucleus, which is in line with nuclear translocation of monomeric irf , as discussed above (see section . ). the l protein from a different cardiovirus species, theiler's murine encephalomyelitis virus (tmev), also interferes with the formation of irf dimers, though it was first identified because it additionally inhibits the nuclear translocation of irf [ , ] . this function depends on the zinc finger motif of l [ ] , but whether l affects preceding phosphorylation of irf as well remains undetermined. the strong impact of l on the antiviral innate immune response is additionally accounted for by the inhibition of mrna export from the nucleus which is achieved by l-mediated phosphorylation of nucleoporin [ ] . in contrast to the yet unclear molecular mechanism of ml and l, an interesting detail of dimerisation antagonism was described for the serine/threonine kinase us of hsv- [ ] . us phosphorylates irf at s , and this hyperphosphorylation interferes with both dimerisation and nuclear translocation of irf . as also activity of irf - d could be inhibited by co-expressed us in a luciferase-based reporter assay, the effect of us seems independent of the activation status. this adds a further phosphate group to the posttranslational modifications that modulate irf activity and raises the question whether the cardiovirus l proteins might also interfere with the formation of irf dimers via small modifications, just as tmev l mediates inhibition of the nuclear pore protein by phosphorylation [ ] . the v protein of simian virus (sv ) was the first reported antagonist of irf translocation [ ] , followed by the multifunctional ns protein of influenza b virus (ibv) [ ] and the l protein of tmev [ ] . again, these early studies did not yet dissect which form of irf is targeted because the role of the essential modifications was not known in detail. later reports of viral antagonists of irf translocation then included an assessment of the activation state of irf , and some were found to specifically target irf after its activation. for example, hsv- initially stimulates activation of irf upon entry but subsequently inhibits irf activity by means of newly synthesized infected cell polypeptide (icp ) [ ] . during the viral replication cycle, a part of the multifunctional icp molecules localises to the cytoplasm and inhibits the translocation of activated irf into the nucleus in order to shorten the ongoing irf -dependent innate immune response. curiously, while this cytosolic function of icp is independent of the activity of its e ubiquitin ligase domain, the functional host proteasome is required for the localisation of icp to the cytoplasm [ ] . the inhibitory function on the innate immune response by the v protein of sendai virus (sev) was first accounted for by the interaction of v with the rna sensor melanoma differentiation-associated protein (mda ) [ ] . however, sev infection is predominantly detected by rig-i [ ] [ ] [ ] , and sev infection still inhibits ifnβ production in mda -knockout mice [ ] . for this reason, the group of sakaguchi continued their studies and discovered that the sev v protein interacts with irf as well as irf - d in the cytosol and inhibits nuclear translocation. in line with this, also the v proteins of the related measles virus (mev) and newcastle disease virus (ndv) of the paramyxoviridae interact with irf and interfere with its trans-activation activity, though mev v seems to target irf after nuclear translocation [ ] . in contrast, the non-structural protein ns of the flavivirus japanese encephalitis virus (jev) suppresses import of irf indirectly by interacting with the nuclear transport proteins kpna and kpna [ ] . this interaction competitively blocks the interaction with the native cargo of the importins, including the transcription factors irf and p . similarly, the ns / a protease of the related hepatitis c virus (hcv) triggers cleavage of importin β (ipob, also kpnb ) and in this way inhibits the transport of irf into the nucleus to interrupt ifnβ production [ ] . once irf has reached the nucleus, it interacts with the coactivator cbp/p to obtain the potential to induce transcription. when studies concerned with the molecular requirements for the induction of type i ifns were still on their way, the critical role of the coactivators for irf activity was underlined by the observation that the antagonistic activity of adenovirus (adv) protein e a on irf -mediated stimulation was dependent on the interaction of e a with cbp [ ] . the inhibition of ifnb transcription by e a could be competed with by overexpression of cbp/p but was not observed for a mutant of e a that was defective in p -binding. mechanistically, these observations implied that e a competed with irf for binding to the essential coactivator [ ] . to interfere with the productive association of irf and cbp/p , the viral antagonists ns of human respiratory syncytial virus (rsv) [ ] , e protein of human papillomavirus (hpv- ) [ , ] and the tegument protein vp of hsv- target both proteins simultaneously [ ] . in contrast, the kinase orf of murine gammaherpesvirus (mhv ) specifically interacts only with activated irf in the nucleus to interfere with the association of coactivators [ ] . this protein is highly conserved, implying similar functions of its homologue in kaposi's sarcoma-associated herpesvirus (kshv), though the conserved kinase activity is not required for this function [ ] . a striking example for the exploitation of structural homology by viruses are the viral homologues of irfs, termed virfs, which are encoded by some herpesviruses to interfere with the activity of host irfs. for details on the interplay of virfs and host irfs and their implications for the viral replication style, please refer to the recent review by myoung and colleagues [ ] . shortly, virf of kshv binds to irf as well as p and in this way obstructs formation of the active holocomplex of irf and cbp/p [ ] [ ] [ ] . additionally, kshv deploys virf to target irf -driven gene expression [ ] . similar to virf , also the virion-associated virf r of the gammaherpesvirus rhesus macaque rhadinovirus (rrv) inhibits ifnb expression by targeting cbp and competing with phosphorylated irf for binding [ ] . as tegument proteins, rrv r and also hsv- vp are released into the host cell alongside the entering virus and can directly take action to interfere with irf activity before a potent type i ifn response can be mounted. another strategy to hinder the participation of the essential coactivator cbp is applied by several rna viruses. the non-structural protein (nsp ) subunits nsp α of murine lactate dehydrogenase-elevating virus (ldv) [ ] , nsp γ of simian haemorrhagic fever virus (shfv) [ ] and nsp α of porcine reproductive and respiratory syndrome virus (prrsv) [ ] [ ] [ ] of the arteriviridae family as well as nsp of porcine epidemic diarrhoea virus (pedv) [ ] of the coronaviridae all suppress ifnb expression by specifically targeting cbp in the nucleus and mediating its proteasome-dependent degradation. this approach implies that these viruses do not require the activity of cbp to express their own genome, which is in line with their cytoplasmic replication. the final aim of irf activation is to enable its binding to recognition motifs within the ifnβ enhancer in order to allow assembly of the ifnβ enhanceosome and induce transcription. again, viral antagonists apply different mechanisms to interfere at this level. the np protein of human bocaparvovirus (bov) and us of hsv- prevent dna binding by interaction with the dbd of irf themselves [ , ] . further, nss of sandfly fever sicilian virus (sfsv) of the bunyaviridae family was recently reported to apply this strategy to prevent association of irf with the ifnβ promoter [ ] . wuerth and colleagues demonstrated that nss specifically interacts with irf via the dbd and by this masking competes for dna binding in a dose-dependent manner. several other strategies were identified in the herpesviridae family: (i) the nuclear share of the hsv- protein icp relocalises irf and cbp/p in the nucleus to special nuclear structures, thereby sequestering them away from their site of activity [ ] . additionally, this mediates deactivation and promotes degradation of irf . the icp variant of bovine herpesvirus (bhv- ) also interacts with p , but in contrast to hsv- , this interaction hijacks the acetyltransferase activity of the coactivator and activates expression from viral promoters [ ] . (ii) the kinase bglf of epstein-bar virus (ebv) directly interacts with irf and phosphorylates several residues between dbd and iad [ ] . without affecting dimerisation or association with cbp, this modification prevents binding of irf to dna. (iii) kshv latency-associated nuclear antigen (lana- ) competes with irf for binding to the prdiii-i region and in this way interferes with stable association of irf to the target dna [ ] . the dna polymerase subunit ul of hcmv was recently reported to act in a similar way [ ] : upon hcmv infection, exogenously expressed ul could be demonstrated to associate with the ifnβ promoter region in a chromatin immunoprecipitation assay and in parallel ul interfered with binding of the central transcription factors irf and p . additionally, ul interacts with both transcription factors irrespective of their phosphorylation status. (iv) the kshv protein k-bzip binds to the ifnβ promoter region itself and prevents binding of irf [ ] , but differently from lana- and ul , k-bzip weakly induces gene expression while it prevents a high and detrimental activation of the type i ifn response. in contrast, the nss protein of rift valley fever virus (rvfv) applies a more indirect approach to inhibit binding of the irf -cbp/p holocomplex [ ] . nss interacts with the host factor sin a-associated protein (sap ), which belongs to the sin a/ncor/hdacs repressor complexes. in turn, sap interacts with the transcription factor yy , and this interaction enables recruitment of nss and sap to the murine ifnβ promoter region. finally, the presence of the nss-sap -yy complex inhibits recruitment of cbp and thus transcriptional activation of ifnb . since the beginning of the detailed characterisation of irf activation more than years ago, we have gained profound insight into the mechanisms enabling the trans-activation activity of a transcription factor with low intrinsic binding affinity for its specific recognition motif and into the intricate network of interactions that allow its participation in the induction of ifnb expression. still, the fundamental principles were mostly characterised in vitro and sometimes biased from prior assumptions, rendering the sequence of events as it occurs in our cells incomplete. to reveal the full dynamics of the events summarised above, experiments with living cells will be required. the many studies reporting factors that affect irf dimerisation and/or translocation and/or trans-activation activity highlight the importance of pinpointing the modulatory mechanism as precisely as possible in future work. at the same time, the diversity of methods applied in the studies summarised here demonstrates that nowadays, dissection of the exact level of intervention with irf activity is within reach. as delineated in this review, the regulation of irf in the context of ifnb expression in itself presents us with a multi-layered circuit of promoting and dampening modulations. the ever-expanding catalogue of host and viral modulators of irf activity not only reflects the key role of this signalling pathway in the mammalian arsenal of antiviral defence mechanisms, but it further alludes to the possibility to use specific mechanisms for medical intervention. directed manipulation of host modulators could present a novel approach to stimulate the host organism to overcome an infection by its own resources or to reduce excessive ifn activity to healthy levels. more information on how to achieve this will surely emerge from the unceasing identification and characterisation of novel irf modulators deployed by host and virus. nevertheless, the multitude of additional genes targeted by irf signifies that we are still just beginning to understand the true complexity of irf fine regulation. in addition to the ongoing characterisation of the direct involvement in the expression of isgs, powerful in silico and sequencing approaches of recent years revealed a growing list of novel targets, including virus-inducible rnas [ , ] . moreover, some of the identified host modulators hint at regulation of irf activity dependent on signalling of other pathways to enable the incorporation of further virus interference. i. the interferon global virus outbreaks: interferons as st responders type i interferons: distinct biological activities and current applications for viral infection the dual nature of type i and type ii interferons shared and distinct functions of type i and type iii interferons interferon-λ orchestrates innate and adaptive mucosal immune responses targeting interferons and their pathways in systemic lupus erythematosus specificity and function of irf family transcription factors: insights from genomics interferon response of chicken embryo fibroblasts to nucleic acids and related compounds inhibition of virus multiplication by foreign nucleic acid approaching the asymptote? evolution and revolution in immunology recognition of endogenous nucleic acids by the innate immune system detection of microbial infections through innate immune sensing of nucleic acids mechanisms controlling nucleic acid-sensing toll-like receptors molecular basis of nf-κb signaling the regulation of ap- activity by mitogen-activated protein kinases the transcriptional code of human ifn-beta gene expression identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes structural and functional analysis of interferon regulatory factor : localization of the transactivation and autoinhibitory domains crystal structure of irf- reveals mechanism of autoinhibition and virus-induced phosphoactivation direct triggering of the type i interferon system by virus infection: activation of a transcription factor complex containing irf- and cbp/p regulated nuclear-cytoplasmic localization of interferon regulatory factor , a subunit of double-stranded rna-activated factor bipartite nuclear localization signal controls nuclear import and dna-binding activity of ifn regulatory factor analysis of functional domains of interferon regulatory factor and its association with irf- gene induction pathways mediated by distinct irfs during viral infection structural insights into interferon regulatory factor activation irf- is the master regulator of type-i interferon-dependent immune responses irf : activation, regulation, modification and function the multifaceted biology of plasmacytoid dendritic cells characterization of the interferon regulatory factor- and its potential role in the transcription activation of interferon a genes positive feedback regulation of type i ifn genes by the ifn-inducible transcription factor irf- differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor- selective dna binding and association with the creb binding protein coactivator contribute to differential activation of alpha/beta interferon genes by interferon regulatory factors and distinct and essential roles of transcription factors irf- and irf- in response to viruses for ifn-alpha/beta gene induction a positive feedback amplifier circuit that regulates interferon (ifn)-stimulated gene expression and controls type i and type ii ifn responses involvement of the irf family transcription factor irf- in virus-induced activation of the ifn-beta gene virus-dependent phosphorylation of the irf- transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation virus infection induces the assembly of coordinately activated transcription factors on the ifn-beta enhancer in vivo identification of distinct signaling pathways leading to the phosphorylation of interferon regulatory factor sting specifies irf phosphorylation by tbk in the cytosolic dna signaling pathway phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf activation structural basis for concerted recruitment and activation of irf- by innate immune adaptor proteins ikkepsilon and tbk are essential components of the irf signaling pathway triggering the interferon antiviral response through an ikk-related pathway interferon regulatory factor is regulated by a dual phosphorylation-dependent switch crystal structure of irf- in complex with cbp identification of the minimal phosphoacceptor site required for in vivo activation of interferon regulatory factor in response to virus and double-stranded rna phosphorylation of irf- on ser generates a hyperactive form of irf- through regulation of dimerization and cbp association ser phosphorylation of transcription factor irf- induces dimerization and association with cbp/p without overall conformational change identification of ser- of interferon regulatory factor as critical target for inducible phosphorylation that determines activation contribution of ser and ser to activation of interferon regulatory factor interferon regulatory factor and creb-binding protein/p are subunits of double-stranded rna-activated transcription factor draf analyses of virus-induced homomeric and heteromeric protein associations between irf- and coactivator cbp/p direct involvement of creb-binding protein/p in sequence-specific dna binding of virus-activated interferon regulatory factor- holocomplex transcriptional activity of interferon regulatory factor (irf)- depends on multiple protein-protein interactions role of intrinsic protein disorder in the function and interactions of the transcriptional coactivators creb-binding protein (cbp) and p target gene context influences the transcriptional requirement for the kat family of cbp and p histone acetyltransferases genome-wide assessment of differential roles for p and cbp in transcription regulation cbp/p in brain development and plasticity: disentangling the kat's cradle usp promotes irf nuclear translocation and antiviral responses by deubiquitinating the importin protein kpna transcription factor dimerization activates the p acetyltransferase differential modification of interferon regulatory factor following virus particle entry inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor transcriptional profiling of interferon regulatory factor target genes: direct involvement in the regulation of interferon-stimulated genes complex regulation pattern of irf activation revealed by a novel dimerization reporter system structure and function of the interferon-beta enhanceosome the enhanceosome virus induction of human ifn beta gene expression requires the assembly of an enhanceosome coordination of a transcriptional switch by hmgi(y) acetylation ordered recruitment of chromatin modifying and general transcription factors to the ifn-beta promoter facilitated binding of tata-binding protein to nucleosomal dna modifying gene expression programs by altering core promoter chromatin architecture dna-binding landscape of irf , irf and irf dimers: implications for dimer-specific gene regulation an atf/creb binding site is required for virus induction of the human interferon beta gene proc. natl. acad. sci. usa stability and dna-binding ability of the bzip dimers formed by the atf- and c-jun transcription factors induction of human interferon gene expression is associated with a nuclear factor that interacts with the nf-kappa b site of the human immunodeficiency virus enhancer the involvement of nf-kappa b in beta-interferon gene regulation reveals its role as widely inducible mediator of signal transduction double-stranded rna activates binding of nf-kappa b to an inducible element in the human beta-interferon promoter crystal structure of an irf-dna complex reveals novel dna recognition and cooperative binding to a tandem repeat of core sequences an atomic model of the interferon-beta enhanceosome specific enhancer selection by irf , irf and irf is determined by isre half-sites, and flanking bases, collaborating transcription factors and the chromatin environment in a combinatorial fashion virus infection induces nf-kappab-dependent interchromosomal associations mediating monoallelic ifn-beta gene expression the transcription factor thpok orchestrates stochastic interchromosomal interactions required for ifnb virus-inducible gene expression the high mobility group protein hmg i(y) is required for nf-kappa b-dependent virus induction of the human ifn-beta gene reversal of intrinsic dna bends in the ifn beta gene enhancer by transcription factors and the architectural protein hmg i(y) the mechanism of transcriptional synergy of an in vitro assembled interferon-beta enhanceosome the role of hmg i(y) in the assembly and function of the ifn-beta enhanceosome mechanisms of transcriptional synergism between distinct virus-inducible enhancer elements recruitment of cbp/p by the ifn beta enhanceosome is required for synergistic activation of transcription induction of endogenous ifn-alpha and ifn-beta genes by a regulatory transcription factor, irf- evidence for a nuclear factor(s), irf- , mediating induction and silencing properties to human ifn-beta gene regulatory elements critical role of a common transcription factor, irf- , in the regulation of ifn-beta and ifn-inducible genes regulated expression of a gene encoding a nuclear factor, irf- , that specifically binds to ifn-beta gene regulatory elements targeted disruption of irf- or irf- results in abnormal type i ifn gene induction and aberrant lymphocyte development mice devoid of interferon regulatory factor (irf- ) show normal expression of type i interferon genes assembly of a functional beta interferon enhanceosome is dependent on atf- -c-jun heterodimer orientation crystal structure of atf- /c-jun and irf- bound to the interferon-beta enhancer structure of irf- bound to the prdiii-i regulatory element of the human interferon-beta enhancer the role of response elements organization in transcription factor selectivity: the ifn-beta enhanceosome example efficient recruitment of tfiib and cbp-rna polymerase ii holoenzyme by an interferon-beta enhanceosome in vitro virus infection leads to localized hyperacetylation of histones h and h at the ifn-beta promoter deciphering the transcriptional histone acetylation code for a human gene nucleosome sliding via tbp dna binding in vivo eukaryotic transcription turns crystal structure of a yeast tbp/tata-box complex high-density nucleosome occupancy map of human chromosome p - reveals chromatin organization of the type i interferon gene cluster identification of individual interferon-producing cells by in situ hybridization stochastic regulation in early immune response chromosome-specific and noisy ifnb transcription in individual virus-infected human primary dendritic cells defining emerging roles for nf-kappab in antivirus responses: revisiting the interferon-beta enhanceosome paradigm gene repression by coactivator repulsion the specificity of innate immune responses is enforced by repression of interferon response elements by nf-κb p the transcriptional silencer protein nrf: a repressor of nf-kappa b enhancers constitutive silencing of ifn-beta promoter is mediated by nrf (nf-kappab-repressing factor), a nuclear inhibitor of nf-kappab lack of essential role of nf-kappa b p , rela, and crel subunits in virus-induced type ifn expression distinct roles for the nf-kappa b rela subunit during antiviral innate immune responses nf-kappa b rela subunit is crucial for early ifn-beta expression and resistance to rna virus replication association of the interferon-β gene with pericentromeric heterochromatin is dynamically regulated during virus infection through a yy -dependent mechanism yin yang dynamically regulates antiviral innate immune responses during viral infection enhanceosome formation over the beta interferon promoter underlies a remote-control mechanism mediated by yy and yy transcription factor yy binds to the murine beta interferon promoter and regulates its transcriptional capacity with a dual activator/repressor role binding of yy to the proximal region of the murine beta interferon promoter is essential to allow cbp recruitment and k h /k h acetylation on the promoter region after virus infection a novel virus-inducible enhancer of the interferon-β gene with tightly linked promoter and enhancer activities activation and regulation of interferon-β in immune responses post-translational regulation of antiviral innate signaling protein phosphatase pp negatively regulates the toll-like receptor-and rig-i-like receptor-triggered production of type i interferon by inhibiting irf phosphorylation at serines and in macrophage the methyltransferase nsd promotes antiviral innate immunity via direct lysine methylation of irf interferon-inducible cytoplasmic lnclrrc -as promotes antiviral innate responses by strengthening irf phosphorylation recruitment of phosphatase pp a by rack adaptor protein deactivates transcription factor irf and limits type i interferon signaling pp a facilitates porcine reproductive and respiratory syndrome virus replication by deactivating irf and limiting type i interferon production isg enhances the innate antiviral response by inhibition of irf- degradation positive regulation of interferon regulatory factor activation by herc via isg modification negative regulation of interferon-regulatory factor -dependent innate antiviral response by the prolyl isomerase pin yap antagonizes innate antiviral immunity and is targeted for lysosomal degradation through ikkε-mediated phosphorylation the tumor suppressor pten has a critical role in antiviral innate immunity interferon regulatory factor- is an in vivo target of dna-pk s-glutathionylation of irf regulates irf -cbp interaction and activation of the ifn beta pathway pivotal role for the escrt-ii complex subunit eap /snf in irf -dependent innate antiviral defense ago negatively regulates type i interferon signaling pathway by competition binding irf with cbp/p elf is critical for induction of type i interferon and the host antiviral response irf and irf cooperatively regulate rapid interferon-beta induction in human blood monocytes the ubiquitin e ligase raul negatively regulates type i interferon through ubiquitination of the transcription factors irf and irf the e ubiquitin ligase ro negatively regulates ifn-beta production post-pathogen recognition by polyubiquitin-mediated degradation of irf trim is essential to sustain ifn regulatory factor activation during antiviral response senp negatively regulates cellular antiviral response by desumoylating irf and conditioning it for ubiquitination and degradation virus infection triggers sumoylation of irf and irf , leading to the negative regulation of type i interferon gene expression mst shuts off cytosolic antiviral defense through irf phosphorylation fas-associated factor negatively regulates the antiviral immune response by inhibiting translocation of interferon regulatory factor to the nucleus bromodomain protein brd promotes ifnb transcription via enhancing irf /p complex formation and recruitment to ifnb promoter in macrophages otud negatively regulates type i ifn induction by disrupting noncanonical ubiquitination of irf the transcription factor nfat limits infection-induced type i interferon responses the transcription factor mafb antagonizes antiviral responses by blocking recruitment of coactivators to the transcription factor irf involvement of the ikappab kinase (ikk)-related kinases tank-binding kinase /ikki and cullin-based ubiquitin ligases in ifn regulatory factor- degradation negative regulation of interferon-β gene expression during acute and persistent virus infections foxo negatively regulates cellular antiviral response by promoting degradation of irf trim negatively regulates interferon-β production and antiviral response through polyubiquitination and degradation of nuclear irf negative feedback regulation of cellular antiviral signaling by rbck -mediated degradation of irf caspase- -mediated cleavage inhibits irf- protein by facilitating its proteasome-mediated degradation krüppel-like factor negatively regulates cellular antiviral immune response kat selectively inhibits antiviral immunity by acetylating irf a diverse range of gene products are effectors of the type i interferon antiviral response ddx inhibits type i interferon by disrupting assembly of irf -ipo to inhibit irf nucleus import lyar suppresses beta interferon induction by targeting phosphorylated interferon regulatory factor long noncoding rna lnc-mxa inhibits beta interferon transcription by forming rna-dna triplexes at its promoter prdi-bf recruits the histone h methyltransferase g a in transcriptional silencing identification and characterization of a novel repressor of beta-interferon gene expression the ef-hand protein calml suppresses antiviral innate immunity by impairing irf dimerization cflipl interrupts irf -cbp-dna interactions to inhibit irf -driven transcription interferon regulatory factor -cl, an isoform of irf , antagonizes activity of irf functional characterization of interferon regulatory factor a (irf- a), an alternative splice isoform of irf- dual utilization of an acceptor/donor splice site governs the alternative splicing of the irf- gene inhibition of the ifn-beta response in hepatocellular carcinoma by alternative spliced isoform of ifn regulatory factor- characterization of a novel isoform of murine interferon regulatory factor human papillomavirus e oncoprotein binds to interferon regulatory factor- and inhibits its transcriptional activity primary activation of interferon a and interferon b gene transcription by interferon regulatory factor recent advances in understanding viral evasion of type i interferon cellular sensing of viral dna and viral evasion mechanisms early ifn type i response: learning from microbial evasion strategies viral evasion strategies in type i ifn signaling-a summary of recent developments viral dedication to vigorous destruction of interferon receptors on taking the sting out of immune activation cytosolic dna-sensing immune response and viral infection intracellular sensing of viral genomes and viral evasion herpes simplex virus type immediate early protein icp inhibits ifn-β production in mucosal epithelial cells by antagonizing irf activation peste des petits ruminants virus nucleocapsid protein inhibits beta interferon production by interacting with irf to block its activation varicella-zoster virus immediate-early protein orf abrogates the irf -mediated innate immune response through degradation of activated irf viral evasion and subversion of pattern-recognition receptor signalling strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit the interferon antiviral response: from viral invasion to evasion viral suppression of the interferon system interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures ifn regulatory factors and antiviral innate immunity: how viruses can get better ten strategies of interferon evasion by viruses the molecular basis of viral inhibition of irf-and stat-dependent immune responses functional comparison of the two gene products of thogoto virus segment thogoto virus ml protein suppresses irf function the interferon antagonist ml protein of thogoto virus targets general transcription factor iib viral targeting of tfiib impairs de novo polymerase ii recruitment and affects antiviral immunity nucleocytoplasmic traffic disorder induced by cardioviruses the mengovirus leader protein blocks interferon-alpha/beta gene transcription and inhibits activation of interferon regulatory factor the leader protein of theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins inhibition of mrna export and dimerization of interferon regulatory factor by theiler's virus leader protein herpes simplex virus serine/threonine kinase us hyperphosphorylates irf and inhibits beta interferon production recovery of paramyxovirus simian virus with a v protein lacking the conserved cysteine-rich domain: the multifunctional v protein blocks both interferon-beta induction and interferon signaling the n-and c-terminal domains of the ns protein of influenza b virus can independently inhibit irf- and beta interferon promoter activation cellular localization of the herpes simplex virus icp protein dictates its ability to block irf -mediated innate immune responses analysis of interaction of sendai virus v protein and melanoma differentiation-associated gene cell type-specific involvement of rig-i in antiviral response activation of innate defense against a paramyxovirus is mediated by rig-i and tlr and tlr in a cell-type-specific manner differential roles of mda and rig-i helicases in the recognition of rna viruses inhibition of interferon regulatory factor activation by paramyxovirus v protein japanese encephalitis virus ns inhibits type i interferon (ifn) production by blocking the nuclear translocation of ifn regulatory factor and nf-κb importin beta targeting by hepatitis c virus ns / a protein restricts irf and nf-kappab signaling of ifnb antiviral response a novel mechanism for the inhibition of interferon regulatory factor- -dependent gene expression by human respiratory syncytial virus ns protein the e protein of human papillomavirus type binds to and inhibits co-activation by cbp and p herpes simplex virus -encoded tegument protein vp abrogates the production of beta interferon (ifn) by inhibiting nf-κb activation and blocking ifn regulatory factor to recruit its coactivator cbp conserved herpesviral kinase promotes viral persistence by inhibiting the irf- -mediated type i interferon response beyond viral interferon regulatory factors: immune evasion strategies functional analysis of human herpesvirus -encoded viral interferon regulatory factor and its association with cellular interferon regulatory factors and p viral interferon regulatory factor of kaposi's sarcoma-associated herpesvirus (human herpesvirus ) binds to, and inhibits transactivation of, creb-binding protein hhv- encoded virf- represses the interferon antiviral response by blocking irf- recruitment of the cbp/p coactivators inhibition of interferon signaling by the kaposi's sarcoma-associated herpesvirus full-length viral interferon regulatory factor protein a rhesus rhadinovirus viral interferon (ifn) regulatory factor is virion associated and inhibits the early ifn antiviral response biogenesis of non-structural protein (nsp ) and nsp -mediated type i interferon modulation in arteriviruses degradation of creb-binding protein and modulation of type i interferon induction by the zinc finger motif of the porcine reproductive and respiratory syndrome virus nsp alpha subunit modulation of type i interferon induction by porcine reproductive and respiratory syndrome virus and degradation of creb-binding protein by non-structural protein in marc- and hela cells suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp human bocavirus np inhibits ifn-β production by blocking association of ifn regulatory factor with ifnb promoter hsv- immediate-early protein us inhibits ifn-β production by suppressing association of irf- with ifn-β promoter nss protein of sandfly fever sicilian phlebovirus counteracts interferon (ifn) induction by masking the dna-binding domain of ifn regulatory factor recruitment of activated irf- and cbp/p to herpes simplex virus icp nuclear foci: potential role in blocking ifn-beta induction bovine herpesvirus immediate-early protein (bicp ) interacts with the histone acetyltransferase p , which stimulates productive infection and gc promoter activity epstein-barr virus bglf kinase suppresses the interferon regulatory factor signaling pathway kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen inhibits interferon (ifn) beta expression by competing with ifn regulatory factor- for binding to ifnb promoter human cytomegalovirus dna polymerase subunit ul antagonizes antiviral immune responses by suppressing irf -and nf-kappab-mediated transcription binding of kaposi's sarcoma-associated herpesvirus k-bzip to interferon-responsive factor elements modulates antiviral gene expression a sap complex inhibits ifn-beta expression in rift valley fever virus infected cells we would like to thank friedemann weber for critically reading the manuscript. we would viruses , key: cord- -n pu yeu authors: rogers, meredith c.; miranda-katz, margot; zhang, yu; oury, tim d.; uccellini, melissa b.; garcía-sastre, adolfo; williams, john v. title: stat limits host species specificity of human metapneumovirus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: n pu yeu the host tropism of viral infection is determined by a variety of factors, from cell surface receptors to innate immune signaling. many viruses encode proteins that interfere with host innate immune recognition in order to promote infection. stat is divergent between species and therefore has a role in species restriction of some viruses. to understand the role of stat in human metapneumovirus (hmpv) infection of human and murine tissues, we first infected stat (−/−) mice and found that hmpv could be serially passaged in stat (−/−), but not wt, mice. we then used in vitro methods to show that hmpv inhibits expression of both stat and stat in human and primate cells, but not in mouse cells. transfection of the murine form of stat into stat -deficient human cells conferred resistance to stat inhibition. finally, we sought to understand the in vivo role of stat by infecting hstat knock-in mice with hmpv, and found that mice had increased weight loss, inhibition of type i interferon signaling, and a th -polarized cytokine profile compared to wt mice. these results indicate that stat is a target of hmpv in human infection, while the murine version of stat restricts tropism of hmpv for murine cells and tissue. human metapneumovirus (hmpv) is a negative sense single-stranded rna virus and a member of the pneumoviridae family with its closest related human pathogen respiratory syncytial virus (rsv) [ ] . hmpv is a leading cause of severe respiratory illness in children and adults [ ] [ ] [ ] [ ] [ ] , and causes significant morbidity and mortality in immunocompromised hosts [ , [ ] [ ] [ ] . even though virtually all humans are infected with hmpv by the age of years [ , ] , reinfection with hmpv is common, which can lead to particularly poor outcomes in immunocompromised and elderly patients [ ] [ ] [ ] [ ] [ ] [ ] . due to the wide prevalence and burden of severe disease in multiple risk groups, it is essential to better understand the virus and host factors that are involved in promoting and restricting hmpv infection. many viruses encode proteins that interfere with interferon (ifn) signaling or inhibition of ifn stimulated genes (isgs) [ ] . pneumoviruses and the related paramyxoviruses antagonize host ifn signaling, specifically stat (signal transduction and activator of transcription) and/or stat . paramyxoviruses utilize alternate transcripts of the phosphoprotein [ ] , while rsv encodes the nonstructural proteins ns and ns that suppress stat /stat signaling [ ] . despite being related to rsv and paramyxoviruses, none of the nine proteins encoded by the hmpv genome have homology with known pneumovirus or paramyxovirus inhibitors of stat or stat [ , ] . in spite of this, hmpv was shown to inhibit phosphorylation of stat in cell lines and primary human epithelial cells [ ] . recent work in our lab identified the hmpv small hydrophobic (sh) protein as necessary and sufficient to inhibit phosphorylation of stat [ ] . others found no inhibition of stat by hmpv; however, these experiments used a relatively low multiplicity of infection (moi) in cell culture [ , ] . other groups have also shown roles for sh [ , ] as well as the hmpv proteins g and m - [ ] [ ] [ ] in the inhibition of innate immunity. viruses have tropism for cell types, tissues, and host species, which explains why natural infections only cause certain symptoms in certain species. tropism can be caused by a host cell lacking a viral entry receptor, or by the virus' ability to circumvent host immunity only in certain cells or species. mouse models for hmpv have been developed, but mice are only semi-permissive for hmpv, requiring a high viral inoculum for productive in vivo infection ( [ ] and unpublished observations), which may be due to species differences in the innate immune proteins antagonized by the virus. for example, human and mouse stat have~ % amino acid similarity, while human and mouse stat are relatively divergent, with only~ % similarity [ , ] . as a consequence of this sequence divergence, murine stat restricts the related human viruses rsv, hpiv , and hpiv in cell culture [ , ] . we sought to test the hypothesis that hmpv may similarly be restricted by stat in a species-specific manner. in this study, we used in vitro and in vivo approaches to determine the effect of hmpv on human and murine stat / . we found that hmpv antagonized expression and nuclear localization of both stat and stat in primate cells but not murine cells. transfection of stat -deficient u a cells with hstat or mstat revealed that suppression of both hstat and hstat were prevented by mstat . in vivo, hmpv infection of hstat knock-in (hstat ki) mice led to greater weight loss, inhibition of interferon stimulated genes, and a th -skewed cytokine profile compared to wt mice. these data indicate that mstat restricts hmpv's ability to inhibit both stat and stat and suggest that productive infection of humans by hmpv is partly due to inhibition of hstat antiviral signaling. the following cell lines were used: beas b (atcc crl- ), vero e (atcc ccl- ), nih/ t (atcc crl- ), u a (ecacc) (atcc: manassas, va, usa; ecacc: salisbury, uk). cmt / (ecacc) and u a (ecacc) cells were purchased from sigma (st. louis, mo, usa). all cell lines were maintained, infected, and transfected in dmem supplemented with % fbs. puno -hstat and puno -mstat plasmids were purchased from invivogen (san diego, ca, usa). hmpv clinical strain tn/ - (subtype a ) was grown in llc-mk cells (atcc ccl- ) and virus titers measured by plaque assay in llc-mk cells as previously described [ ] . for cell experiments, cells were inoculated with hmpv strain tn/ - at an moi of - in a -well plate. mock-infected cells were inoculated with media or llc-mk cell lysate, which had an equivalent effect on stat and stat protein levels and phosphorylation [ ] . then, - h post-infection, cells were treated with u/ml human ifnα (alpha a) (pbl; piscataway, nj, usa) for human and primate cells, or u/ml murine ifnβ (pbl) for mouse cells for - min. after treatment, media were aspirated from the tissue culture dish and cells were lysed in ripa buffer (thermofisher; waltham, ma, usa) for western blotting or fixed in % paraformaldehyde for immunofluorescence. transfections were performed using lipofectamine (life technologies; carlsbad, ca, usa) following the manufacturer's protocol with some exceptions: for each well in the -well plates, . µl lipofectamine was diluted into . µl opti-mem (not supplemented) and was mixed with µg plasmid in . µl opti-mem for a total volume of~ µl. this was incubated for min before being added to wells. fifty percent of media was replaced after h. then, - h post-transfection, cells were treated with ifnα/β as above and lysed for western blotting. cells were lysed in ice-cold ripa buffer (thermo scientific, waltham, ma, usa) containing halt protease and phosphatase inhibitors (thermo scientific). samples were centrifuged at , × g for min to pellet debris, and supernatant was used for protein analysis. total protein from cell lysate was quantified by bca assay (thermo scientific) and protein was normalized between samples. a total of - ug of protein was loaded into each well for each western blot experiment. after total protein quantification, samples were normalized and more concentrated samples were diluted in ripa buffer to achieve equal concentrations across samples. samples were diluted in × lds sample buffer (invitrogen; carlsbad, ca, usa) and × sample reducing agent (invitrogen: carlsbad, ca, usa) and boiled for min at • c. proteins were separated on a - % bis-tris polyacrylamide gel before transfer to a pvdf membrane. membranes were blocked in % bsa in tris-buffered saline with . % tween- (tbs-t) or % nonfat dry milk in tbs-t. primary antibodies against stat (cell signaling technologies (cst, danvers, ma, usa), d k y), pstat (cst, d ), stat (cst, d j l), pstat (for human, cst d p p; for mouse (polyclonal), emd millipore (burlington, ma, usa)), actin (hrp conjugated, abcam (cambridge, uk), and gfp (invitrogen, a- )) were used in a : dilution (or a : , dilution for actin) overnight with rocking at • c. after tbs-t wash, hrp-conjugated secondary antibodies against rabbit or mouse were added in % bsa-tbs-t or % milk-tbst for h. blots were washed with tbs-t and put in tbs until imaging. western blots were developed using west femto (thermo scientific) and imaged on a chemidoc xrs+ (biorad, hercules, ca, usa). band quantification was performed using image lab v . (biorad). after infection and ifn treatment, cell supernatant was removed from beas b cells and cells were fixed with % paraformaldehyde, then permeabilized with % methanol at − • c. cells were blocked with % goat serum and . % triton-x in pbs. primary and secondary antibodies were diluted in % bsa and . % triton-x in pbs. dapi ( µg/ml) was added to distinguish nuclei. antibodies used were stat (cst, d k y) stat (cst, d j l), hmpv anti-fusion protein g [ ] , secondary alexa fluor -conjugated anti-human and alexa fluor -conjugated anti-rabbit (invitrogen). all fluorescent images were equally color enhanced (increased brightness and contrast) for better visibility in publication. for quantification of nuclear stat or stat signal, original (non-color enhanced) fluorescent images were analyzed using fiji imagej [ ] [ ] [ ] . nuclei were selected from the dapi viruses , , of channel, then the mean intensity was measured in the stat or stat channel. for quantification of cytosolic protein, the cytosol was selected from the transmitted light image and the mean intensity was measured in the stat or stat channel. c bl/ mice were obtained from jackson laboratories (bar harbor, me, usa). stat −/− and stat −/− mice were from dr. david levy (new york university; new york, ny, usa) [ ] and dr. christian schindler (columbia university; new york, ny, usa) [ ] , courtesy of dr. john alcorn. hstat ki mice were from adolfo garcia-sastre (icahn school of medicine at mount sinai; new york, ny, usa) [ ] . male and female - -week-old mice were used for all experiments, with control groups age and sex matched. all mice were bred and maintained in specific pathogen free conditions in accordance with the institutional animal care and use committee of university of pittsburgh (protocol # , approved march ). for all animal experiments, mice were anesthetized with ketamine-xylazine (mouse passaging experiments) or isofluorane (hstat ki experiments) and intranasally (for mouse passaging) or intratracheally (for hstat ki) infected with - × pfu/ml hmpv tn - , or lung homogenate from a previously infected mouse, in a -µl volume. for serial passage, mice were euthanized at day post-inoculation. lungs were harvested and homogenized in - ml % opti-mem in a glass tenbroeck homogenizer as previously described [ ] . lung homogenates were clarified by centrifugation at rpm ( × g) for min. clarified lung homogenate was aliquoted into cryovials and snap-frozen in an ethanol dry ice bath before storage at − • c. for serial passage, clarified lung homogenate was pooled from - mice before inoculation into recipient mice. the left lobe of the lung was inflated and fixed in formalin, then subsequently sectioned and stained with h&e, with groups combined into a single slide. all fields of each h&e-stained slide were scanned at × magnification, and each field was scored as follows: : normal lung tissue, : % to % of tissue area with inflammation, : - % of tissue area with inflammation, : - % of tissue area with inflammation, or : - % of tissue area with inflammation [ ] . the frequency of each inflammation score was combined by group. inflammation scores were combined as follows to allow for statistical analysis by fisher's exact test: scores of - were categorized as mild/moderate inflammation and scores of - were categorized as severe inflammation. lung homogenate from the whole lung was frozen at − • c until use for quantitative rt-pcr (qpcr). rna was extracted from the lung homogenate with the purelink rna mini kit (thermo fischer scientific, waltham, ma, usa) according to manufacturer instructions and stored at − • c until further use. five microliters of extracted rna were used for qrt-pcr in -µl reaction mixtures on an abi steponeplus real-time pcr system (thermo fisher scientific) using the agpath-id one-step rt-pcr kit (thermo fisher scientific). taqman primers and probes were used according to manufacturer's instructions (all thermo fisher scientific). all values were normalized to the housekeeping gene hprt, and fold change in chemokine was measured in infected groups compared to mock-infected controls using the ∆∆ cycle threshold method. lung homogenate from the whole lung was used for cytokine analysis by cytokine & chemokine -plex mouse procartaplex panel a (invitrogen) according to manufacturer's instructions. data were analyzed using prism version . (graphpad software; san diego, ca, usa). a one-tailed student's t test was used to analyze fold change from western blots. fisher's exact test was used to analyze differences in histology inflammation scores. a two-tailed unpaired student's t test was used to analyze comparisons between two groups. for comparisons between multiple groups, a one-or two-way anova was performed. error bars on graphs represent sd. some respiratory viruses, including influenza, sars, and mers, have been serially passaged in mice to generate mouse-adapted virus strains, which have provided important tools for studying viral and host determinants of disease and tropism [ ] [ ] [ ] [ ] [ ] . we initially sought to generate mouse-adapted hmpv by serially passaging infected lung homogenate directly into a recipient mouse. however, viral titers in lung homogenate of infected c bl/ mice were not sufficiently high enough to productively infect a recipient mouse, despite repeated attempts, and serial passage in rag- −/− or ifnar −/− mice was also unsuccessful [ ] . however, hmpv replicates to significantly higher titer in ifnar −/− mice [ ] , indicating that type interferon signaling restricts hmpv in mice in vivo. we therefore inoculated stat −/− mice with hmpv strain tn/ - and found that the virus grew to significantly higher titer in stat −/− mice compared to stat −/− or wt mice ( figure a ). hmpv could be passaged mouse to mouse at low level detectable by qpcr in stat -/mice, but we chose to pursue stat based on our data showing higher replication in stat −/− mice and the fact that the stat protein is less conserved between human and mouse compared to stat . we subsequently inoculated stat −/− and wt mice with clarified lung homogenate from infected stat −/− and wt mice, respectively, and found that hmpv could be serially passaged mouse to mouse in stat −/− but not wt mice ( figure b ). these data show that serial passage of hmpv is restricted by stat in vivo, indicating that this protein and/or the ifn signaling pathway in general is a barrier to murine infection by hmpv. viruses , , of inoculated stat −/− and wt mice with clarified lung homogenate from infected stat −/− and wt mice, respectively, and found that hmpv could be serially passaged mouse to mouse in stat −/− but not wt mice ( figure b) . these data show that serial passage of hmpv is restricted by stat in vivo, indicating that this protein and/or the ifn signaling pathway in general is a barrier to murine infection by hmpv. since stat appeared to be important in restricting hmpv infection of mice, we next explored how hmpv infection affects stat and stat in human cells. previously, it had been shown by us and by others that hmpv can specifically inhibit stat phosphorylation and expression [ , ] . to further understand the effects of hmpv on stat and stat , we used a human bronchoepithelial cell line, beas b, to perform imaging studies of stat and stat in the presence or absence of hmpv. after infection with hmpv for h, beas b cells were treated with ifn to induce phosphorylation and nuclear translocation of stat and stat . after treatment with ifn, stat and stat translocated to the nucleus in mock-infected cells, whereas nuclear import was inhibited in hmpv-infected cells but not in uninfected cells in the same dish ( figure ). these data indicate that hmpv infection inhibits nuclear translocation of the stat / heterodimer in vitro, suggesting that antagonism of stat / may be a key step to promote hmpv infection. hmpv grew to significantly higher titer in stat −/− mice than in wt mice, leading us to hypothesize that hmpv inhibits stat and stat in primate cells but fails to inhibit these in murine cells. to test this hypothesis, we infected both primate and murine cell lines with hmpv and measured expression as well as phosphorylation of stat and stat after ifn treatment. we found that hmpv infection of veroe cells (primate cells that are ifn-responsive but cannot produce endogenous ifn) led to decreased total and phosphorylated stat and stat (figure ) . the decreased levels of pstat and pstat appeared to be driven by the decreased protein abundance of total stat and stat in vero cells, as quantified by the relative fold change in phosphorylated compared to total stat proteins. in contrast, when murine cell lines cmt / (c bl/ lung adenocarcinoma) and nih/ t (murine fibroblast, deficient in some steps of ifn signaling [ ] ) were infected with hmpv, we saw increased total and phosphorylated stat and stat (figure ) . these data indicate that hmpv antagonizes stat and stat in primate cells but fails to achieve this inhibition when introduced to murine cells. cells. to test this hypothesis, we infected both primate and murine cell lines with hmpv and measured expression as well as phosphorylation of stat and stat after ifn treatment. we found that hmpv infection of veroe cells (primate cells that are ifn-responsive but cannot produce endogenous ifn) led to decreased total and phosphorylated stat and stat (figure ). the decreased levels of pstat and pstat appeared to be driven by the decreased protein abundance of total stat and stat in vero cells, as quantified by the relative fold change in phosphorylated compared to total stat proteins. in contrast, when murine cell lines cmt / (c bl/ lung adenocarcinoma) and nih/ t (murine fibroblast, deficient in some steps of ifn signaling [ ] ) were infected with hmpv, we saw increased total and phosphorylated stat and stat (figure ) . these data indicate that hmpv antagonizes stat and stat in primate cells but fails to achieve this inhibition when introduced to murine cells. studies in hpiv and hpiv showed that degradation of stat or stat , respectively, required the expression of both stat and stat [ ] . to understand whether hmpv inhibition of stat and stat is dependent on expression of both proteins, we used u a and u a cells, which are specifically deficient in stat and stat , respectively [ ] . we found that hmpv infection of stat -deficient u a cells led to reduction of stat and pstat in a dose-dependent manner with increasing moi (figure a,c) . hmpv infection of stat -deficient u a cells also reduced stat and pstat , but only at a moi of ( figure b,d) . at moi = in u a cells, hmpv did not inhibit stat expression or phosphorylation. these data indicate that hmpv has the capacity to target stat and stat independently of each other; however, antagonism of stat by hmpv appears to be a more efficient process than stat inhibition. viruses , , x for peer review of studies in hpiv and hpiv showed that degradation of stat or stat , respectively, required the expression of both stat and stat [ ] . to understand whether hmpv inhibition of stat and stat is dependent on expression of both proteins, we used u a and u a cells, which are specifically deficient in stat and stat , respectively [ ] . we found that hmpv infection of stat -deficient u a cells led to reduction of stat and pstat in a dose-dependent manner with increasing moi (figure a,c) . hmpv infection of stat -deficient u a cells also reduced stat and pstat , but only at a moi of ( figure b,d) . at moi = in u a cells, hmpv did not inhibit stat expression or phosphorylation. these data indicate that hmpv has the capacity to target stat and stat independently of each other; however, antagonism of stat by hmpv appears to be a more efficient process than stat inhibition. we next asked whether the species-specific stat inhibition was specifically due to differences in the human and murine forms of stat , or whether hmpv fails to inhibit some other step in the innate immune response in murine cells. u a cells were transfected with either human or murine stat (hstat and mstat , respectively), infected with hmpv, treated with ifn, and analyzed for stat / expression and phosphorylation. we found that cells transfected with mstat were more resistant to inhibition of stat and than those transfected with hstat (figure ). at an moi of , a significant reduction of stat expression was seen in hmpv-infected cells that had been transfected with hstat , but not those transfected with mstat . however, with an increased moi of , reduced stat protein levels were seen regardless of the transfection type ( figure d ). interestingly, we found that even though stat was not required for stat inhibition (figure ) , the presence of mstat in u a cells constrained inhibition of stat by hmpv ( figure b,c) . overall, these data suggest that species-specific differences in stat limit the inhibition of both stat proteins by hmpv. to establish whether hmpv antagonizes the human form of stat compared to murine stat in vivo, we used mice engineered to express human stat in place of murine stat (hstat ki) [ ] . we infected hstat ki and wt mice with either a low ( × pfu/ml) or high ( × pfu/ml) inoculum of hmpv. at both low and high inoculum, hstat ki mice lost significantly more weight compared to wt controls ( figure a ). this correlated with a greater degree of lung inflammation ( figure b) , where hstat ki mice had a significantly increased frequency of inflammation scores that were categorized as severe (scores of or , p = . ). the weight loss and increased inflammation was not associated with higher viral titers in infected hstat ki mice; in contrast, at day five of lower inoculum infection, hstat ki mice had modestly lower viral burden compared to wt mice, though this was not replicated at higher titer ( figure c ). these data suggest that, in vivo, stat antagonism worsens disease severity but does not contribute to increased virus replication in mice. viruses , , x for peer review of to establish whether hmpv antagonizes the human form of stat compared to murine stat in vivo, we used mice engineered to express human stat in place of murine stat (hstat ki) [ ] . we infected hstat ki and wt mice with either a low ( × pfu/ml) or high ( × pfu/ml) inoculum of hmpv. at both low and high inoculum, hstat ki mice lost significantly more weight compared to wt controls ( figure a ). this correlated with a greater degree of lung inflammation ( figure b) , where hstat ki mice had a significantly increased frequency of inflammation scores that were categorized as severe (scores of or , p = . ). the weight loss and increased inflammation was not associated with higher viral titers in infected hstat ki mice; in contrast, at day five of lower inoculum infection, hstat ki mice had modestly lower viral burden compared to wt mice, though this was not replicated at higher titer ( figure c ). these data suggest that, in vivo, stat antagonism worsens disease severity but does not contribute to increased virus replication in mice. figure . hstat ki mice have greater disease severity and inhibition of isgs compared to wt mice after infection by hmpv, despite no increase in virus titer. hstat ki mice and c bl/ mice were intratracheally inoculated with hmpv at a low ( × pfu/ml) or high ( × pfu/ml) titer and weighed daily over the course of infection (a). (b) lung specimens were taken at day five postinoculation with hmpv at low titer, stained with h&e, and scored by a pathologist. histology images were captured at ×. histological scoring was calculated by percentage of inflammation per field of view, with scores of , , (mild/moderate) representing , - , and - %, and or (severe) representing - or - %, respectively. the difference between wt and hstat ki mice in the frequency of mild/moderate and severe inflammation scores was statistically significant by fisher's to better understand the ability of hmpv to antagonize interferon signaling in vivo, we quantified levels of ifnα and the isgs mx and socs in hstat ki and wt mice after hmpv infection. ifnα is produced via a feedback amplification loop of isgs during the type i ifn response [ ] , while mx and socs were selected for their roles as known isgs that have differential functions in the type i ifn response. we found that mrna expression of mx and socs were reduced in hstat ki mice compared to wt mice at h post-hmpv infection ( figure d) . additionally, protein level of ifnα declined in hstat ki mice compared to wt mice during hmpv infection ( figure e ). overall, these data indicate that hmpv is indeed able to antagonize type i ifn signaling when mstat is replaced by hstat in vivo, though this did not lead to increased virus replication. we hypothesized that the increased weight loss and inflammation despite slightly decreased viral titer in hstat ki mice was due to aberrant stat signaling in these mice. to better understand the global consequences of substituting human stat into mice in vivo, we performed multiplex cytokine analysis on lung homogenates of hmpv-infected hstat ki and wt mice at day five after high-dose inoculation, as day five represents a time point that bridges innate and adaptive immunity. overall, hstat ki mice had reduced expression of the characteristic th cytokines ifnγ and tnfα ( figure a ) and adopted a th -skewed cytokine profile demonstrated by increased concentrations of il- , il- , il- , eotaxin, and il- ( figure b ). altogether, our data suggest that the substitution of hstat for murine stat leads to skewing of both the innate and the adaptive immune response to hmpv. to better understand the ability of hmpv to antagonize interferon signaling in vivo, we quantified levels of ifnα and the isgs mx and socs in hstat ki and wt mice after hmpv infection. ifnα is produced via a feedback amplification loop of isgs during the type i ifn response [ ] , while mx and socs were selected for their roles as known isgs that have differential functions in the type i ifn response. we found that mrna expression of mx and socs were reduced in hstat ki mice compared to wt mice at h post-hmpv infection ( figure d) . additionally, protein level of ifnα declined in hstat ki mice compared to wt mice during hmpv infection ( figure e ). overall, these data indicate that hmpv is indeed able to antagonize type i ifn signaling when mstat is replaced by hstat in vivo, though this did not lead to increased virus replication. we hypothesized that the increased weight loss and inflammation despite slightly decreased viral titer in hstat ki mice was due to aberrant stat signaling in these mice. to better understand the global consequences of substituting human stat into mice in vivo, we performed multiplex cytokine analysis on lung homogenates of hmpv-infected hstat ki and wt mice at day five after high-dose inoculation, as day five represents a time point that bridges innate and adaptive immunity. overall, hstat ki mice had reduced expression of the characteristic th cytokines ifnγ and tnfα ( figure a ) and adopted a th -skewed cytokine profile demonstrated by increased concentrations of il- , il- , il- , eotaxin, and il- ( figure b ). altogether, our data suggest that the substitution of hstat for murine stat leads to skewing of both the innate and the adaptive immune response to hmpv. figure . hmpv-infected hstat ki mice adopt a th -skewed cytokine profile. hstat ki mice and c bl/ mice were intratracheally inoculated with hmpv at × pfu/ml. at day five post-infection, lungs were homogenized and cytokines were measured by multiplex cytokine assay. (a) measurement of prototypical th cytokines ifnγ and tnfα in lung homogenates of hstat ki and c bl/ mice. (b) concentration of th -related cytokines il- , il- , il- , eotaxin, and il- in murine lung homogenates. * p < . , ** p < . , unpaired t test. n = /group, one experiment. animal models are essential tools for studying infections by human viruses. the differences in host responses between humans and animals, both in vitro and in vivo, help reveal important mechanisms of how viruses interact with the immune system. we showed that hmpv can be serially passaged in stat -deficient mice, whereas serial passage in wt mice was not possible, suggesting a role for stat in mediating in vivo restriction. hmpv inhibited expression and nuclear localization of stat and stat in primate airway epithelial cells, while the virus failed to inhibit either stat in murine cells. stat inhibition occurred in a hstat -dependent manner, as transfection of mstat into human cells reduced stat and stat antagonism. furthermore, the knock-in of hstat into mice enabled hmpv to inhibit type i ifn signaling and alter the cytokine profile of infected mice. collectively, these data suggest that stat in mice represents a significant barrier to murine infection by hmpv. we observed that a relatively high moi of hmpv (three or more) was needed to inhibit stat , whereas inhibition of stat occurred at a moi of one. these effects at different mois suggest that stat inhibition by hmpv is more efficient than stat inhibition. hmpv proteins could have a higher affinity for stat compared to stat , or perhaps stat inhibition is achieved with a viral protein that is transcribed at a lower abundance. further research to elucidate which hmpv protein is responsible for antagonizing stat will increase our understanding of how hmpv inhibits different steps of the innate immune response. a limitation of the in vitro component of this work is that cell lines may have inherent differences in the strength of their innate immune signaling, in addition to the differences in the efficiency of infection and transfection between cell lines. previous work in our lab showed that autocrine and paracrine effects of ifn signaling on neighboring uninfected cells could confound the effects of hmpv infection on stat expression and phosphorylation [ ] . here, we attempted to control for this effect in primate cells by using vero e cells, which cannot produce endogenous ifn. however, no equivalent murine cell line currently exists, though we opted to use the murine nih/ t cell line for experiments in figure , as this line has some known impairments in endogenous ifn signaling [ , ] . several prior studies have described species-specific differences in stat inhibition by the flaviviruses dengue, zika, and yellow fever viruses [ ] [ ] [ ] . consequently, hstat ki mice infected with mouse-adapted zika virus exhibited increased viral replication, broader tissue spread, and enhanced transplacental spread [ ] . in contrast, we found that despite the clear preference for inhibition of hstat over mstat in vitro, hmpv-infected hstat ki mice had similar virus replication in the lung compared to wt mice. nevertheless, the presence of hstat resulted in reduced expression of ifnα and mx in these mice, consistent with inhibition of stat / signaling, although not to a sufficient level to result in a significant increase in viral replication in lung. interestingly, hstat ki mice had worse disease than wt mice when infected with hmpv, as measured by weight loss and lung inflammation. this phenotype was associated with an altered cytokine profile that favored th cytokines over th . since ifnα was significantly reduced in hstat ki compared to wt mice, it is likely that either direct or indirect inhibition of stat by hmpv drove the th phenotype in this study. ifns and il- have opposing roles in the immune response, with ifns promoting an antiviral th response and il- driving a th response characterized by asthma and allergy. this response is driven in part by ifn-mediated inhibition of il- via increased socs expression [ ] ; our data here are consistent with this previous work, as we saw increased il- and other th cytokines while ifns and socs were reduced (figures and ) . due to the dramatic skewing of the th /th axis in the presence of hstat , we hypothesize that antagonism of stat by hmpv in human infection may contribute to the association of hmpv and asthma in susceptible individuals [ ] [ ] [ ] . in addition, stat has immunoregulatory functions that may have been disrupted in the hstat ki mice [ ] [ ] [ ] . overall, these data indicate that hmpv targets expression of both stat and stat in a host-specific manner. future studies to determine the specific hmpv protein(s) and molecular interactions involved in stat inhibition will reveal mechanisms of productive infection and pathogenesis in humans. while mouse models of human diseases have strengths and limitations, these data highlight how the innate immune response to hmpv in mice may be profoundly different than it is during 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interferon against lytic virus infection, lacks ribonuclease f activity selective stat protein degradation induced by paramyxoviruses requires both stat and stat but is independent of alpha/beta interferon signal transduction use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway interferon-inducible antiviral effectors the cgas-sting signaling pathway is required for the innate immune response against ectromelia virus mouse stat restricts early dengue virus replication zika virus targets human stat to inhibit type i interferon signaling host-specific ns ubiquitination determines yellow fever virus tropism interferons inhibit activation of stat by interleukin in human monocytes by inducing socs- gene expression human metapneumovirus bronchiolitis in infancy is an important risk factor for asthma at age human metapneumovirus infection plays an etiologic role in acute asthma exacerbations requiring hospitalization in adults human metapneumovirus infection in children hospitalized for wheezing stat is an essential adaptor in usp -mediated suppression of type i interferon signaling selective loss of type i interferon-induced stat activation caused by a minisatellite insertion in mouse stat the combination of ifn beta and tnf induces an antiviral and immunoregulatory program via non-canonical pathways involving stat and irf the hstat ki mice used for this study were produced by the mouse genetics and gene targeting center of research excellence (core), which is supported by the icahn school of medicine at mount sinai and a cancer center support grant ( p ca ) from the national cancer institute/national institutes of health. committee for glaxosmithkline, neither activity involved with this publication. key: cord- -whd znan authors: han, zhenzhi; xiao, jinbo; song, yang; hong, mei; dai, guolong; lu, huanhuan; zhang, man; liang, yueling; yan, dongmei; zhu, shuangli; xu, wenbo; zhang, yong title: the husavirus posa-like viruses in china, and a new group of picornavirales date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: whd znan novel posa-like viral genomes were first identified in swine fecal samples using metagenomics and were designated as unclassified viruses in the order picornavirales. in the present study, nine husavirus strains were identified in china. their genomes share . – . % similarity, and alignment of these nine husavirus strains identified nucleotide polymorphism sites across their full-length genomes. these nine strains were directly clustered with the husavirus lineage, and their genomic arrangement showed similar characteristics. these posa-like viruses have undergone a complex evolutionary process, and have a wide geographic distribution, complex host spectrum, deep phylogenetic divergence, and diverse genomic organizations. the clade of posa-like viruses forms a single group, which is evolutionarily distinct from other known families and could represent a distinct family within the picornavirales. the genomic arrangement of picornavirales and the new posa-like viruses are different, whereas the posa-like viruses have genomic modules similar to the families dicistroviridae and marnaviridae. the present study provides valuable genetic evidence of husaviruses in china, and clarifies the phylogenetic dynamics and the evolutionary characteristics of picornavirales. the order picornavirales is composed of eight families (picornaviridae, dicistroviridae, marnaviridae, iflaviridae, polycipiviridae, caliciviridae, solinviviridae, and secoviridae) as well as other unclassified picornaviruses [ ] . picornavirales pathogens are associated with a wide range of infectious diseases, including hepatitis and hand, foot, and mouth disease (hfmd) in humans, foot-and-mouth disease (fmd) in animals (pigs, goats, cattle, and other animals), and plant diseases (e.g., tomato torrado disease and satsuma dwarf disease) [ ] . these pathogenic picornavirales usually infect a broad range of hosts, including arthropods, insects, algae, humans, monkeys, and other organisms [ ] . picornavirales have a positive sense ssrna genome between . and . kb, which encodes a polyprotein cleaved by proteases; however, some plant-infecting picornaviruses (secoviridae) possess segmented rna genomes. the genomic nucleotide sequences of picornavirales are highly divergent, and their genomic organization models are highly variable among different families. the polyprotein of picornavirales usually contains a conserved replication block of helicase, protease, and rna-dependent rna polymerase (hel-pro-pol) [ ] . there are three typical genomic organizations observed in the order picornavirales. the first genomic organization has the non-structural module (ns-module) located at the end of the genomic sequence and the structural module (s-module) located at the end, separated by an intergenic region (as in the families dicistroviridae and marnaviridae). similar genomic organization is observed in the family secoviridae, except that the two modules are located on different genomic segments. in the third genomic organization, the s-module is at the end of the genome, whereas the ns-module is at the end, as observed in picornaviridae, iflaviridae, and polycipiviridae [ , ] . with the development of deep transcriptome sequencing, more novel unclassified rna virus genomes have been identified, redefining the proposed evolutionary progress of the virosphere [ , ] . following breakthrough research, more potential viromes or viral pathogens have been identified and expanded upon [ ] [ ] [ ] . the unprecedented diversity and evolutionary scale of viromes have been analyzed and illustrated, offering deep insights into their evolutionary history [ ] . the pathogens in picornavirales have been recently expanded, with more divergent genomes identified and analyzed [ , , [ ] [ ] [ ] [ ] [ ] . the relationship between the picornavirales and their diseases is unclear, except for the culturable and disease-causing agents. the viral genomic organization patterns, host ranges, and geographic distribution of picornavirales are diverse and contribute to their pathogenicity. novel posa-like virus genomes were first identified in swine fecal samples using metagenomics and were assigned as unclassified viruses to the order picornavirales [ ] . further reports of novel posaviruses with low amino acid sequence identity revealed novel genomic organization features and phylogenetic characteristics of posa-like viruses [ , ] . in china, posaviruses were detected in fecal samples from pigs with diarrheal signs caused by unspecified pathogens [ , ] . the posa-like viruses have been detected in specimens from a broad range of hosts, such as the fish stool-associated rna virus (fisavirus), human stool-associated rna virus (husavirus), panda stool-associated rna virus (pansavirus), bat stool-associated rna virus (basavirus), and rat stool-associated rna virus (rasavirus) [ , , ] . due to high sequence similarity between posavirus strains and parasite-derived genomic sequences, it was speculated that posaviruses could not infect swine, but instead may have a dietary or environmental origin [ ] . however, the host of husavirus remained unclear, even though the virus was detected in human stool samples [ ] . most posa-like viruses were identified in the stool samples of animals, whereas limited surveys of husavirus have been reported worldwide [ , , ] . to the best of our knowledge, there are no reports of husavirus in china to date, and their genetic and phylogenetic characteristics remain unknown. in the present study, we first identified nine husavirus strains in china with high genomic similarity. the genomic characteristics, phylogenetic relationships, and genomic arrangements of these viruses revealed the detailed evolutionary lineage of husavirus. we also investigated the diversity of posa-like viruses and showed they form a separate clade within the picornavirales. outcomes of this study provide a valuable genetic evidence about husavirus in china and comprehensive information on the evolutionary characteristics of picornavirales. human stool samples were collected from healthy children. in total, fecal samples were obtained during public health surveillance. written informed consent for the analysis of their clinical samples was obtained from the parents or guardians of the children included in the present study. this study was approved by the ethics review committee (ivdc - , february ) of the national institute for viral disease control and prevention (ivdc), chinese center for disease control and prevention. all experimental protocols were approved by the ivdc, and the methods were carried out in accordance with the approved guidelines [ ] . fecal samples were processed using a previously published method [ , ] . total rna was extracted from enriched virus-like particles using a qiaamp viral rna mini kit (qiagen, hilden, germany). the extracted rna of all samples was pooled for library construction, followed by amplification using the repli-g cell wga & wta kit ( ; qiagen, hilden, germany). amplified dna was randomly fragmented by ultrasound sonication (covaris m , woburn, ma, usa) to produce bp fragments, then sticky ends were repaired and adapters were added using t dna polymerase (m , promega, madison, wi, usa), klenow dna polymerase (kp , epicentre, woburn, ma, usa), and t polynucleotide kinase (ek , thermo scientific, fermentas, glenburnie, md, usa). each viral sequencing library was prepared following the illumina truseq dna preparation protocol and was sequenced on the hiseq platform (illumina, san diego, ca, usa), with bp paired ends. the library preparation and sequencing process was performed by bgi tech (shenzhen, china). low-quality bases (phread q < ) and adaptors were trimmed using trimmomatic software (version . ) [ ] . clean reads were aligned to the human reference genome (hg ), and reads matching the human genome were discarded [ ] . the remaining reads were de novo assembled using trinity software (version . . ), and taxonomically assigned using centrifuge (version . . ) for metagenomic classification [ , ] . the assembled contigs were taxonomically assigned using the blastn algorithm (https://blast.ncbi.nlm.nih.gov/blast.cgi), with an e-value cut-off of × − . we identified the viral annotation of posa-like viruses by manually inspecting the blast results and the taxonomic results from centrifuge. to confirm the assembled contigs, clean reads were mapped to the reference genome of husavirus (genbank accession number kx . ) using bowtie (version . . . ) [ ] . finally, we manually checked the mapped results and compared them with the assembled contigs. the assembled library was used to identify husaviruses by real-time (rt)-pcr assays using previously described husavirus-specific probes and primers [ ] . after confirming husavirus in a sample, rt-pcr was performed to amplify the partial coding region using the primescript one step rt-pcr kit ver. (takara, dalian, china) with specific primers (table s , supplementary materials). the pcr products were purified using a qiaquick pcr purification kit (qiagen, hilden, germany). the abi genetic analyzer (applied biosystems, foster city, ca, usa) was used for sequencing in both directions. the acquired partial genomic sequences were analyzed using blast against the genbank database. a total of nine husavirus strains were confirmed based on their sequence information. the full-length genome sequences of nine husavirus strains were amplified using the "primer-walking" strategy, which was used to close the gaps in the sequence. briefly, the overlapping fragments representing whole genomes were amplified by rt-pcr using specific primers (table s ). the rt-pcr products were purified for sequencing using the qiaquick gel extraction kit (qiagen, hilden, germany) and the amplicons were sequenced on the abi genetic analyzer (applied biosystems, foster city, ca, usa) as described above. the end of the genome was amplified using an oligo-dt primer as reported previously [ ] . the end of the genome was amplified using the -full race kit (takara, shiga, japan) and following the manufacturer's instructions. sequencher software (version . , ann arbor, mi, usa) was used to assemble the contigs with the reference genome and to produce the consensus sequences. the open reading frame (orf) was determined using orffinder software (https://www.ncbi. nlm.nih.gov/orffinder/?tdsourcetag=s_pctim_aiomsg) for nearly full-length genomic sequences of the nine husavirus strains. combined with previous reports on the genomic organization of husaviruses, we identified the orf length and deduced the amino acid sequences. to infer the possible protein-coding domain of the novel genome, an rps-blast search against the conserved domain database (cdd) was performed [ ] . the husavirus rna-dependent rna polymerase (rdrp), helicase, c cysteine protease, and picorna-like capsid protein domains were identified. for other posa-like viral genomes, we applied a similar strategy to obtain the sequence information for major protein domains, even though some annotations of posa-like viruses failed due to the vast genomic divergence among these viruses. representative posa-like virus strains were selected based on the phylogenetic relationships of the conserved domain sequences and previous reports [ , ] . the respective protein sequences of different functional domains were extracted and incorporated into the subsequent analysis. based on the phylogenetic relationships within the rdrp domain, we extracted and analyzed the full-length genomes that were similar to husavirus ( figure c ). the full-length genomic sequences of husavirus - and the neighboring posa-like viruses were used to construct the maximum-likelihood phylogenetic tree. the obtained amino acid sequences were aligned using mafft software (version . ), with the e-ins-i algorithm [ ] . the remaining ambiguously aligned regions were removed using the trimal program [ ] . the maximum-likelihood phylogenetic tree was constructed using iq-tree software (version . . ), with bootstrap replicates, and the best amino acid substitution models were inferred with modelfinder, using bayesian information criteria [ ] [ ] [ ] . we manipulated the phylogenetic tree topology for clear display using the ggtree package [ ] . although the actual hosts of many posa-like viruses remain unknown, the hosts where they were initially identified and their regional information were included in the discriminant analysis (da). the rdrp protein sequences were used to infer the geographic and host clustering, which was implemented in the discriminant analysis of the principal component analysis (pca) using the adegenet package [ , ] . the full-length genomic sequences for the nine strains identified in the present study were deposited in the genbank nucleotide sequence database under accession numbers mt -mt , and the metagenomic data were submitted to the ncbi's sequence read archive (sra) under accession number srp . after trimming the raw reads, , , clean reads with a q larger than % were obtained. by mapping to the host genome, about % of the clean reads were removed and , , clean reads were used for de novo assembly. finally, , assembled contigs were obtained, of which were larger than kb. we compared the assembled contigs against the nucleotide database with a threshold e-value of × − , resulting in assembled contigs under viral annotation. finally, we identified two nearly full-length genome sequences for husavirus, which belong to the order picornavirales, sharing % genomic identity with strain _ (genbank accession number kx ). to confirm the identity of these assembled contigs, we mapped the clean reads back to the genome of husavirus (genbank accession number kx ). in total, clean reads were mapped to the reference genome when the repetitive reads were excluded, and the mean sequencing depth was (figure a ). we used real-time (rt)-pcr assays to detect husaviruses in all samples used to construct the library, using a previously published probe and primer [ ] . nine clinical samples were positive for husavirus, with cycle threshold (ct) values ranging from to . all patients whose clinical samples were positive were less than five years old. these children included two boys and seven girls from different counties within the same prefecture. full-length genome sequences of the nine husavirus strains were determined using sanger sequencing and a "primer-walking" strategy. all strains were - nt in length, with a poly(a) tail. alignment of the nine husavirus strains identified nucleotide polymorphic sites across the full-length genome. strains xz _xz_chn_ and xz _xz_chn_ contained a six-nucleotide deletion at position , resulting in a two amino-acid deletion at position of the protein sequence. the orf of the nine strains was - nt in length, encoding a polypeptide of - amino acids, with a -utr of nt and -utr of nt. the overall base composition of the nine strains was . - %a, . - %c, - . %g, and . - %t. the full-length genome nucleotide and amino acid similarity among the nine strains was . - . % and . - %, respectively (table s , supplementary materials). to assess the divergence between the nine strains, nucleotide variation was analyzed using the strain _ isolated from vietnam (genbank accession number kx ) as a reference strain ( figures s and s , supplementary materials) . the genome sequence of each strain had . - . % nucleotide identity and . - . % amino acid identity with the strain _ . the nine husavirus strains diverged at , , and - nt across the entire genome compared with the strain _ , indicating that evolution occurred during their circulation, despite their relatively close geographical distribution (figures s and s ) . we observed slight differences in the nucleotide sequences of the husavirus strains, implying that nucleotide substitution had occurred, although the strains were sometimes found in the same prefecture. due to the low genomic sequence similarity between the husavirus and unclassified posa-like viruses in picornavirales, it was difficult to perform a phylogenetic analysis at the full-length genome level. therefore, we identified the conserved domains of the protein sequences, which included the rna-dependent rna polymerase (rdrp), helicase, c cysteine protease, and picorna-like capsid protein domains. since some posa-like viral protein sequences could not be annotated as the major domains in the cdd, the genomes with invalid annotations were discarded. we obtained the representative lineages of the posa-like viruses from previous publications [ , ] . based on the conserved protein sequences of posa-like viruses, maximum-likelihood phylogenetic trees were constructed to explain the phylogeny of husaviruses (figure ). the posa-like viruses showed high diversity and the topology of the phylogenetic tree was variable when we used the six known families of picornavirales as the outgroup. the posa-like viruses formed a single group in the phylogenetic tree and presented complex varieties based on different domains. the nine husavirus strains in the present study clustered with husavirus (genbank accession numbers kt and kx ), and showed close phylogenetic association with posavirus (genbank accession number lc ) in all the maximum-likelihood trees except for the tree based on the helicase domain, indicating that the husavirus strains in the present study belong to the husavirus lineage (figure ). the husavirus - lineages did not cluster together in each phylogenetic tree, indicating significant divergence and an intricate evolutionary history among the husaviruses (figure , black arrows). the husavirus - lineages diverged a long time ago, even though they were identified recently in human fecal samples. the husavirus lineage is widespread globally, because the distant strains are closely clustered together. for example, the strains identified in vietnam, netherlands, china, and venezuela clustered together ( figure c,d) . we observed that the strains from china and vietnam were very similar, revealing that husavirus possibly co-circulated in tibet via china and vietnam. although several posa-like viruses were identified in the stool samples of pigs, their hosts were varied and complex, ranging from invertebrates to vertebrates ( figure ) . surprisingly, the strain hg (genbank accession number lc ) identified in sus scrofa and the strain _ identified from rats showed a close phylogenetic relationship with the known husavirus lineage ( figure c,d) . this was also observed in other posa-like viruses (e.g., fisavirus and basavirus ). collectively, our results show that these viruses have close phylogenetic relationships but they also have a varied and wide host spectrum. several major conserved domains of the husavirus - lineages are located at different genomic positions, indicating different evolutionary directions and intricate evolutionary history. the husavirus strains identified in the present study have the same genomic organization as the husavirus lineage, confirming the phylogenetic results based on the major conserved domains. the replication block of the helicase, protease, and rna-dependent rna polymerase (hel-pro-pol) was identified in every husavirus genome obtained, which is consistent with the classical conservative modules of picornavirales. we compared the representative genomic arrangements of different posa-like viruses, and found that the replication block of hel-pro-pol exists in all posa-like viruses except in the partial failed annotations of the protease domains ( figure ). two capsid protein domains were also identified in each of the representative posa-like viral genomes, which verified the common genetic characteristics of the genomic arrangements of posa-like viruses. the posa-like viral genomes had a non-structural module (ns-module) at the end and a structural module (s-module) at the end, which were similar between strains, with some deviation in the coding region ( figures b and ) . changes in the location of the main functional domains in posa-like viruses were observed, suggesting genomic rearrangements have occurred. we used the representative conserved rdrp sequences of picornavirales obtained from the genbank to assess the evolutionary history of picornavirales [ ] [ ] [ ] [ ] . the picornavirales sequences presented extremely divergent characteristics suggesting a long evolutionary time scale. posa-like viruses identified in previous reports and in the present study formed a single group and clustered with the genomes of family marnaviridae (figure ) . furthermore, the posa-like viruses had distant phylogenetic associations with other families belonging to the order picornavirales. the families iflaviridae, secoviridae, dicistroviridae, and novel branches clustered together to form a large clade. a novel group (e.g., kelp fly virus-related group), which contained the genomes of the known families polycipiviridae and solinviviridae, was identified [ , ] . the presence of clades outside those of the defined families of picornavirales allowed the identification of novel groups and the definition of their phylogenetic relationships. for instance, the unknown clades located between the dicistroviridae and marnaviridae imply that novel picornavirales may have existed, or may still exist (figure ) . as several novel genomes of picornavirales have been found, the arrangement of orfs and the order of non-structural and structural genes were investigated (figure ) . the genomic organization of picornaviridae, iflaviridae, and polycipiviridae was similar, whereas the families dicistroviridae and marnaviridae shared the same genomic module models, with the ns-module located in the end of their genomes. the genomes of family secoviridae were separated into two segments. the genomic arrangement of the posa-like viruses was similar to that of families marnaviridae and dicistroviridae, in which the former was frequently identified from marine phytoplankton (e.g., algae). the phylogeny of posa-like viruses confirmed their close relationship with the family marnaviridae, thereby providing valuable information about the origin of posa-like viruses. significant separation was observed across three major clusters (plant, invertebrate, and vertebrate groups), with the host information used as prior clusters ( figure a ). the strains from plants formed a single cluster, whereas strains from vertebrates and humans formed one cluster. the viruses from other hosts including arthropods, invertebrates, nematodes, and tunicates were clustered together, with the arthropods dominating. the groups of viruses identified in invertebrate and vertebrate hosts have close evolutionary relationships, with partial mixing. the posa-like viruses cluster within the invertebrate host group, suggesting a possible common origin. however, we did not observe a distinct divergence when location information was used as the prior cluster ( figure b ). this indicates that the individual strains from different regions are more similar than strains found in different hosts, confirming significant overlap between different regions. with the development of next-generation sequencing and its application in pathogen detection, the virosphere is being explored beyond the limits of culturable pathogens [ ] . the number of genomes belonging to the order picornavirales has sharply increased as many divergent genomic sequences and undiscovered viromes have been identified [ , ] . several members of the order picornavirales are pathogenic, and can cause devastating economic consequences [ ] [ ] [ ] . the host spectrum of the order picornavirales is wider than expected, and includes plants, algae, insects, and vertebrates. although the replication block of hel-pro-pol is conserved in picornavirales, the genomic arrangement of picornavirales seems to be extremely flexible [ ] . the order of the ns-and s-modules, as well as the arrangement of orfs, are considerably variable across different families of picornavirales. the posa-like virus isolated from pig fecal samples in was previously unclassified in the order picornavirales [ ] . although some studies had identified posa-like viruses in the stool samples of animals, information on the husaviruses remained limited [ , , , ] , with no studies reported on husaviruses in china. in the present study, we identified husavirus strains in china by rna sequencing. we found nine clinical samples positive for husavirus, and the full-length genomes were acquired from these stool samples. with different husavirus genomes identified simultaneously, our results confirm that husavirus is circulating in china. the high nucleotide and amino acid sequence similarity among these nine strains showed that they were closely associated. strains xz _xz_chn_ and xz _xz_chn_ included two amino acid deletions at the end of the coding region, and dominant divergence of the full-length genome at position - nt within the structural coding region, compared with strain _ . a similar result was also observed upon comparing the husavirus strains identified in the present study with husavirus lineage strains. the co-circulation of husavirus lineage in china and vietnam was confirmed, with some nucleotide substitutions identified between geographically close circulating strains. posa-like viruses appear to have gone through a long evolutionary progress, based on the complexity of their phylogenetic relationships. the nine strains identified in the present study clustered with the husavirus lineage, and their genomic organization was also similar to that of the husavirus lineage. although the husavirus - lineages were first found in human stool samples, they showed significant differences in their phylogenetic and genomic organization, indicating that they have diverged to some degree. the geographic distribution of husaviruses is wide, involving several distant countries; furthermore, husavirus-like viruses were identified in a broad spectrum of animals, ranging from invertebrates to vertebrates. surprisingly, the strain hg (genbank accession number lc ) from sus scrofa shared a closer phylogenetic relation with the husavirus lineage than other lineages. the conserved replication block of hel-pro-pol existed in almost all posa-like viruses sequenced in the present study, and the two capsid protein domains were also identified in all posa-like viruses, indicating a common conserved genomic arrangement. we also identified different genomic organization features of posa-like viruses, particularly in the coding region. the available posa-like virus sequences cluster near the family marnaviridae. our results confirmed the formation of a novel group within the picornavirales, and posa-like viruses could be a distinct novel family within the picornavirales [ , ] . a large number of novel branches, such as the kelp fly virus-related group, contain unclassified picornavirales genomes. the genomic organization of picornavirales is diverse, including different arrangement of orfs as well as different orders of non-structural and structural coding regions. there was no apparent association between genomic organization and host or geographical location. the kelp fly virus-related group possessed the most diverse genomic arrangements, whereas the genomes of family secoviridae were generally divided into two segments. based on discriminant analysis, we did not observe significant geographic clustering of viral lineages, whereas host-specific genomic clusters were evident. three groups of hosts were identified for which the picornavirales genomes had close evolutionary association. the posa-like viruses may have originally had an invertebrate vector although modern posa-like viruses have been identified in the stool of pigs, bats, fishes, pandas, and humans [ , ] . the lack of geographic clustering of picornavirales suggests a wide distribution and complicated diffusion. previous reports have shown that posavirus likely originated in an aquatic host, whereas fisavirus and basavirus possibly jumped to humans due to dietary or environmental contamination [ , , ] . the picornavirus sequences identified in porcine stool samples shared high identity with the cdna sequences derived from nematodes [ ] . if we infer the evolutionary relationship of posa-like viruses through genomic organization and phylogeny, posa-like viruses appear to be closely associated with the family marnaviridae. our results suggest that undigested food, which could contain invertebrates or gut parasites, might be the source of posa-like viruses, which is consistent with previous studies. to the best of our knowledge, this is the first study to report nine full-length genomic sequences of husaviruses identified for the first time in china. these husavirus strains provide the baseline data of their full-length genome features and phylogenetic characteristics. the genomic organization, entire genome features, and phylogenetic association with posa-like viruses were analyzed in detail, illuminating the dynamics of posa-like viruses. we explored the phylogenetic relationships of picornavirales and speculated the possible origins of posa-like viruses. overall, we provide comprehensive phylogenetic information to improve our understanding of the evolutionary history of picornavirales. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . figure s : sequence similarities analysis of husavirus strains with the reference strain (kx . _husavirus _isolate_ _ ). figure s : nucleotide variation across the genome of nine husavirus strains. table s : the primers used for amplification and sequencing. table s . the genomic sequence identity percentage of 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molecular sequence data management and evolutionary phylogenetics studies two methods for mapping and visualizing associated data on phylogeny using ggtree discriminant analysis of principal components: a new method for the analysis of genetically structured populations ahmed, i. adegenet . - : new tools for the analysis of genome-wide snp data detection of novel rna viruses from free-living gorillas, republic of the congo: genetic diversity of picobirnaviruses this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank the local staff for specimen collection and primary detection. we thank weifeng shi, fangluan gao, and tao hu for technological assistance. the authors declare no conflict of interest. key: cord- -o t srim authors: chaudhari, jayeshbhai; liew, chia-sin; workman, aspen m.; riethoven, jean-jack m.; steffen, david; sillman, sarah; vu, hiep l. x. title: host transcriptional response to persistent infection with a live-attenuated porcine reproductive and respiratory syndrome virus strain date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: o t srim both virulent and live-attenuated porcine reproductive and respiratory syndrome virus (prrsv) strains can establish persistent infection in lymphoid tissues of pigs. to investigate the mechanisms of prrsv persistence, we performed a transcriptional analysis of inguinal lymphoid tissue collected from pigs experimentally infected with an attenuated prrsv strain at days post infection. a total of differentially expressed genes (degs) were detected of which degs were upregulated and degs were downregulated. specifically, genes involved in innate immune responses and chemokines and receptors associated with t-cell homing to lymphoid tissues were down regulated. as a result, homing of virus-specific t-cells to lymphoid tissues seems to be ineffective, evidenced by the lower frequencies of virus-specific t-cell in lymphoid tissue than in peripheral blood. genes associated with t-cell exhaustion were upregulated. likewise, genes involved in the anti-apoptotic pathway were upregulated. collectively, the data suggested that the live-attenuated prrsv strain establishes a pro-survival microenvironment in lymphoid tissue by suppressing innate immune responses, t-cell homing, and preventing cell apoptosis. porcine reproductive and respiratory syndrome virus (prrsv) is a positive sense, single stranded rna virus that belongs to the family arteriviridae, under the order nidovirales [ ] . based on phylogenetic analysis, prrsv was originally classified into two types: type or prrsv- , which originated in europe, and type or prrsv- , which originated in north america. the international committee on taxonomy of viruses (ictv) recently updated the arterivirus taxonomic structure in which prrsv- and prrsv- are now respectively classified as two species: betaarterivirus suid and betaarterivirus suid [ ] . prrsv infects pigs of all ages; however, clinical manifestations are more severe when the virus infects pregnant sows and young pigs, causing reproductive failure and respiratory distress, respectively (reviewed in [ ] ). prrsv is endemic in most swine producing countries worldwide, causing significant economic losses to swine producers [ ] . genes involved in innate immune response and genes encoding chemokines and receptors important for t-cell homing and trafficking were downregulated. on the other hand, genes involved in the anti-apoptotic pathway and t-cell exhaustion were upregulated. functional studies revealed that the frequencies of virus-specific ifn-γ-secreting cells are lower in lymphoid tissue than in peripheral blood mononucleated cells (pbmcs). collectively, the results shed important insight into the mechanisms of prrsv persistence in the host. the attenuated prrsv strain designated con used in this study was recovered from an infectious cdna clone constructed using viral rna extracted from the attenuated con-p [ ] . marc- cells, a monkey kidney cell line [ ] , were cultured in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) and × penicillin-streptomycin ( units/ml of penicillin, and µg/ml of streptomycin (life technologies, grand island, ny, usa). pbmcs and lymph node-derived cells were cultured in complete rpmi- medium (crpmi) supplemented with % fbs, × of glutamax- (life technologies, grand island, ny, usa), units/ml of penicillin, and µg/ml of streptomycin (sigma-aldrich, st. louis, mo, usa). mouse monoclonal antibody specific to prrsv n protein sdow was purchased from the national veterinary services laboratories (ames, ia, usa). anti-porcine cd ε (clone bb - e - c ; fitc-conjugated), anti-porcine cd (clone - - ; pecy -conjugated), anti-porcine cd (clone - - ; alexa flour -conjugated), and anti-porcine ifn-γ (clone p g ; pe-conjugated), were purchased from bd biosciences (san diego, ca, usa). alexa flour -conjugated goat anti-mouse igg was purchased from invitrogen (eugen, or, usa). the pig experiment conducted in this study was approved by the university of nebraska-lincoln (unl) institutional animal care and use committee under the protocol number . ten -week-old prrsv, porcine circovirus type (pcv ), and siv negative pigs were purchased from the midwest research swine (glencoe, mn, usa). the pigs were randomly assigned to two groups of five pigs, which were accommodated in two separate rooms in the animal biosecurity level (absl- ) research facilities at unl. after week of acclimation, pigs in group were injected with dmem to serve as negative controls, whereas pigs in groups were inoculated intramuscularly with . tcid of live-attenuated prrsv strain con . whole blood samples with anticoagulant edta were collected to obtain plasma and pbmcs. plasma samples were stored at − • c for measurement of viremia and antibody response. a portion of freshly isolated pbmcs were used for measurement of t cell responses while the rest of pbmcs were cryopreserved for future analysis. at days post-infection (dpi), sample of inguinal lymph node (iln) was aseptically collected from the pigs under anesthesia. one half of the inl was stored in crpmi for lymphocyte isolation while the other half of the inl was minced and stored in trizol reagent (life technologies, carlsbad, ca, usa) for rna extraction. viral loads in plasma and tissues were measured by a commercial rt-qpcr kit (tetracore inc., rockville, md, usa). viral loads in plasma was reported as log copies per ml, whereas viral loads in tissues were reported as log copy per µg of total rna used in the rt-qpcr reaction. for statistical purposes, samples that had no detectable levels of viral rna were assigned a value of log copies. pbmcs were isolated from edta-whole blood as previously described [ , ] . single cell suspension was isolated from iln immediately after collection as follows. the tissue was processed to viruses , , of remove connecting tissue and cut into small pieces which were placed in a -µm nylon cell strainer (corning, durham, nc, usa) in the presence of crpmi. the tissue pieces were pressed against the nylon mesh by using the plunger of a ml syringe. the resulting cell suspension was collected into a ml conical tube which was passed through a -µm cell strainer one more time to remove large tissue debris. cells were pelleted by centrifugation at × g for min at room temperature and treated with a ml red blood cell (rbc) lysis buffer (life technologies, carlsbad, ca, usa). rbc lysis reaction was ceased by adding ice cold pbs containing % fbs, followed by centrifugation at × g for min at room temperature. cell pellet was resuspended in crpmi. to determine cell concentration and viability, samples of pbmc and iln-derived cells were stained with acridine orange and propidium iodide (viastaintm aopi staining solution, nexcelom, lawrence, ma, usa) and counted using an automatic cell counter (cellometer auto , nexcelom, lawrence, ma, usa). freshly isolated cells were used for elispot and flow cytometric analysis. the remaining cells were cryopreserved in % dmso, % fbs, and % rpmi- and stored in liquid nitrogen. prrsv antibody levels in plasma were measured at the veterinary diagnostic center of the university of nebraska by using the commercial elisa idexx prrs x ab test (idexx laboratories, westbrook, me, usa) following the manufacturer's instructions. the serum-virus neutralization (svn) assay was performed as previously described [ ] using plasma rather than serum. results were expressed as the log of the reciprocal of the highest dilution that showed a ≥ % reduction in the number of fluorescent foci presenting in the control wells. the frequencies of ifn-γ-secreting cells (ifn-γ scs) in pbmcs and iln-derived cells were measured by using an ifn-γ elispot assay as previously described [ , ] . briefly two replicates of , pbmcs or iln cells freshly collected from each pig were plated into two wells of a -well plate with pvdf membrane that were coated with anti-porcine ifn-γ antibody. the cells were stimulated with µl of crpmi containing . × tcid of con . for positive control, cells were plated at cells per well, followed by stimulation with a µl crpmi containing ng/ml of phorbol myristate -acetate (pma) and µg/ml of ionomycin. for negative control, cells were simply cultured in crpmi. at h post stimulation, the plate was washed with pbs-containing . % tween (pbs-t) followed by incubation with biotin-labeled antibody against porcine ifn-γ (clone p c , bd biosciences pharmingen, san diego, ca, usa). spots were developed by using alkaline phosphatase-conjugated streptavidin (southern biotech, birmingham, al, usa) in conjunction with alkaline phosphatase substrate (vector laboratories, burlingame, ca, usa). spots were counted and analyzed using an aid elispot reader version . (aid gmbh, strassberg, germany). freshly isolated pbmcs and iln cells were ex vivo stimulated with × tcid of con as described above. a cocktail solution consisting of ng/ml of pma and µg/ml of ionomycin was included as a positive control while crpmi served as a negative control. at hrs post-stimulation, µl of crpmi containing µg/ml of golgi-plug brefeldin a (bd bioscience, san jose, ca, usa) was added and further incubated for hrs. samples were centrifuged at × g for min at room temperature. cells were resuspended in flow cytometry staining buffer (facs buffer) (pbs with % fbs and . % sodium azide), and stained with anti-porcine cd ε, cd , and cd monoclonal antibodies and incubated on ice for min in the dark. cells were washed thrice using facs buffer. the cells were fixed and permeabilized with % paraformaldehyde and . % triton x- , respectively, followed by intracellular staining with an ifn-γ antibody. cells were analyzed by using a cytek dxp cytometer (cytek biosciences, fremont, ca, usa) and acquired data were analyzed using the flowjo software (bd biosciences, san jose, ca, usa) with gating based upon fluorescence minus one (fmo) control. for each sample, , events were acquired. relevant cell population was gated on cd + prior to analyzing the cd + and cd + cells ( figure s a ). ifn-γ positive cells were counted from individual cd + , cd + , and cd + cd + double positive (dp) cells ( figure s b ). total rna was isolated from iln collected from the infected and non-infected using the trizol reagent according to the manufacturer's protocol. rna was re-purified using rneasy mini kit (qiagen, hilden, nrw, germany) according to the manufacturer instructions, followed by a dnase treatment using turbo dna-free tm kit (life technologies, v.a. graiciuno , vilnius, lithuania) to remove dna contamination. purified rna was submitted to novogene bioinformatics technology co.ltd for rna sequencing. sequencing libraries were generated using nebnext ® ultratm rna library prep kit for illumina ® (neb, ipswich, ma, usa) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. briefly, mrna was purified from mg of total rna using poly-t oligo-attached magnetic beads. fragmentation was carried out using divalent cations under elevated temperature in nebnext first strand synthesis reaction buffer ( x). first strand cdna was synthesized using random hexamer primer and m-mulv reverse transcriptase (rnase h). second strand cdna synthesis was subsequently performed using dna polymerase i and rnase h. remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. after adenylation of ends of dna fragments, nebnext adaptor with hairpin loop structure were ligated to prepare for hybridization. in order to select cdna fragments of preferentially ~ bp in length, the library fragments were purified with ampure xp system (beckman coulter, beverly, ma, usa). then µl user enzyme (neb, ipswich, ma, usa) was used with size-selected, adaptor-ligated cdna at • c for min followed by min at • c before pcr. then pcr was performed with phusion high-fidelity dna polymerase, universal pcr primers and index (x) primer. finally, pcr products were purified using ampure xp system and library quality was assessed on the agilent bioanalyzer system. the libraries were sequenced on an illumina platform hiseq and approximately million raw bp/ bp paired-end) reads were generated. rna-seq reads were first trimmed with trim galore version . . [ , ] , which include filtering reads shorter than bp and low-quality ends from reads (phred score: < ) and the option of removing reads with ns. the quality of reads before and after trimming was checked with fastqc version . . [ ] . the filtered reads were aligned to the sus scrofa genome (sscrofa . ; gcf_ . ) using tophat version . . [ ] [ ] [ ] with a read mismatch of and a maximum multi hits of and further default parameters. alignment files generated by tophat were then used to generate gene counts using htseq version . . (htseq-count), with a minimum alignment quality of [ ] . a data matrix of gene counts for all samples was created using a custom python script and the data matrix was used to run the differential gene expression analysis in deseq version . . [ ] . genes with an adjusted p value smaller than . and a log fold change larger than were considered as differentially expressed. the differential expression result table generated by deseq was annotated with gene information obtained from ensembl biomart for sus scrofa, supplemented by annotation from the gtf file. to visualize the level of prrsv rna genome, reads mapped to the prrsv con genome were counted base by base, normalized to counts per million and subsequently used to generate a coverage track using deeptools version . . [ ] . gene ontology (go) in the database (http://www.geneontology.org/) is an international standardized classification system for gene function, and it supplies a set of controlled vocabulary to comprehensively describe the properties of genes and gene products. go enrichment analysis of degs was implemented by the goseq r package [ ] , in which gene length bias was corrected. go terms with viruses , , of corrected p-value less than . were considered significantly enriched by differentially expressed genes. kyoto encyclopedia of genes and genomes (kegg) is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism, and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (http://www.genome.jp/kegg/). kobas version . [ ] was used to test the statistical enrichment of differential expression genes in kegg pathway. virus-specific t-cell data were analyzed by unpaired t-test analysis using graphpad prism software version . . (graphpad software, llc, san diego, ca, usa). a p < . was considered significant. sequencing data from rna-seq were deposited in the ncbi geo and are available under accession number geo: gse all pigs inoculated with con became viremic starting from dpi. the viremia level increased and peaked at dpi ( figure a ). pigs inoculated with con did not displayed any clinical signs throughout the course of this study. at dpi, iln was collected and rna was extracted. rt-pcr analysis revealed that viral rna genome was detected in all five con -infected pigs but none of the sham-inoculated pigs ( figure b) . subsequently, the rna samples were subjected to rna-seq. an average of million paired-end reads were obtained for each rna sample (table s ). to confirm the presence of viral rna during persistent infection in pigs, the reads were mapped to the con genome. viral rna reads were only detected from iln of con -infected pigs which mapped throughout the viral genome ( figure c ). the results demonstrate that all five pigs in this study were persistently infected with the attenuated prrsv strain con . to examine the host responses to con persistent infection, rna reads were mapped to the reference pig genome (sscrofa . ; gcf_ . ). principal component analysis (pca) indicated that control and con -infected pigs formed separated clusters indicating that they had distinct transcriptional profiles ( figure a ). the infected pigs showed more variability than the control pigs. in addition, one pig in the con -infected group (con_ ) showed a unique transcriptional profile, as it associated closer to the control pigs. this association is likely due to the different genetic makeup of this pig than the other four animals within this group. direct comparison of rna transcripts between control and con -infected animals revealed a profound change in the transcriptional profiles in the iln tissue of con -infected pigs. out of genes in the annotated porcine genome, there were degs (fdr < %, |log | fold change ≥ ), of which degs were upregulated and degs were downregulated ( figure b and table s ). to examine the host responses to con persistent infection, rna reads were mapped to the reference pig genome (sscrofa . ; gcf_ . ). principal component analysis (pca) indicated that control and con -infected pigs formed separated clusters indicating that they had distinct transcriptional profiles (figure a ). the infected pigs showed more variability than the control pigs. in addition, one pig in the con -infected group (con_ ) showed a unique transcriptional profile, as it associated closer to the control pigs. this association is likely due to the different genetic makeup of this pig than the other four animals within this group. direct comparison of rna transcripts between control and con -infected animals revealed a profound change in the transcriptional profiles in the iln tissue of con -infected pigs. out of genes in the annotated porcine genome, there were degs (fdr < %, |log | fold change ≥ ), of which degs were upregulated and degs were downregulated ( figure b and table s ). the control animals are indicated by red dots labeled "dmem_ animal number," while the con -infected animals are indicated by cyan dots labeled "con_animal number"; (b) the volcano plot of differentially expressed genes between con -infected and control pigs. red dots denote significantly down-regulated genes (p < . , log fold change ≤− ), green dots indicated significantly up-regulated genes (p < . , log fold change ≥ ), and grey dots represent genes that were not differentially expressed. to understand their biological functions, degs were subjected to go enrichment analysis. go terms having a corrected value of p < . were considered statistically significant (table s ) . significantly enriched go terms were grouped under three categories: biological processes, molecular function, and cellular components. thirty highly enriched go terms in con -infected pigs are shown (figure a) . a large number of degs are enriched into the category of biological processes (cytoskeleton organization, multicellular organism development, regulation of intracellular signal transduction, cell communication, cell surface receptor signaling, regulation of programmed cell death etc.), followed by molecular function (cellular macromolecular catabolic processes, kinase activity, phosphotransferase activity, dna binding, metal ion binding) and cellular components (ribonucleoprotein complexes, bounding membrane of organelles). kegg pathway analysis revealed multiple enriched pathways including apoptosis, chemokine signaling, cellular, senescence, mitophagy, lysosome, endocytosis, and mapk ( figure b and table s ). detailed analysis of selected enriched pathways is presented in the respective sections below. to understand their biological functions, degs were subjected to go enrichment analysis. go terms having a corrected value of p < . were considered statistically significant (table s ) . significantly enriched go terms were grouped under three categories: biological processes, molecular function, and cellular components. thirty highly enriched go terms in con -infected pigs are shown (figure a) . a large number of degs are enriched into the category of biological processes (cytoskeleton organization, multicellular organism development, regulation of intracellular signal transduction, cell communication, cell surface receptor signaling, regulation of programmed cell death etc.), followed by molecular function (cellular macromolecular catabolic processes, kinase activity, phosphotransferase activity, dna binding, metal ion binding) and cellular components (ribonucleoprotein complexes, bounding membrane of organelles). kegg pathway analysis revealed multiple enriched pathways including apoptosis, chemokine signaling, cellular, senescence, mitophagy, lysosome, endocytosis, and mapk ( figure b and table s ). detailed analysis of selected enriched pathways is presented in the respective sections below. the innate immune system is the first line of defense against invading pathogens, with the major influence on the development of strong adaptive immune responses. con is capable of inducing type i ifns both in vitro and in vivo [ ] . in the current study, we did not detect differential expression of type i ifn or interferon stimulated genes (isg) rna transcripts, suggesting that the type-i ifn was not induced in iln of con-infected pigs at dpi. we observed an upregulation of rna-sensing molecules including tlr (dsrna), tlr , and tlr (ssrna) (figure a ). on the other hand, no differential expression of signaling molecules downstream of tlr pathways including interferon regulatory factors (irf) irf and irf , was observed (table s ). in addition, we observed an upregulation of tlr mrna in con -infected pigs. while the ligands and signaling pathway involving tlr remain poorly understood, it has been demonstrated that tlr can act as an anti-inflammatory receptor which can also suppress tlr -induced ifn-β production [ ] . interestingly, we observed an increased expression of cd (ox ) and its receptor cd r (figure a ). cd r is an inhibitory immune receptor that is expressed on myeloid cells and b-and t-lymphocytes [ ] , while cd is widely expressed on multiple cell types including endothelial cells, neurons, and lymphocytes [ ] . cd /cd r interaction suppresses cytokine production, and inflammatory responses [ ] . the innate immune system is the first line of defense against invading pathogens, with the major influence on the development of strong adaptive immune responses. con is capable of inducing type i ifns both in vitro and in vivo [ ] . in the current study, we did not detect differential expression of type i ifn or interferon stimulated genes (isg) rna transcripts, suggesting that the type-i ifn was not induced in iln of con-infected pigs at dpi. we observed an upregulation of rna-sensing molecules including tlr (dsrna), tlr , and tlr (ssrna) (figure a ). on the other hand, no differential expression of signaling molecules downstream of tlr pathways including interferon regulatory factors (irf) irf and irf , was observed (table s ). in addition, we observed an upregulation of tlr mrna in con -infected pigs. while the ligands and signaling pathway involving tlr remain poorly understood, it has been demonstrated that tlr can act as an antiinflammatory receptor which can also suppress tlr -induced ifn-β production [ ] . interestingly, we observed an increased expression of cd (ox ) and its receptor cd r (figure a ). cd r is an inhibitory immune receptor that is expressed on myeloid cells and b-and t-lymphocytes [ ] , while cd is widely expressed on multiple cell types including endothelial cells, neurons, and lymphocytes [ ] . cd /cd r interaction suppresses cytokine production, and inflammatory responses [ ] . the complement system is another crucial component of the innate immunity. it acts as an important link between innate and adaptive immune system. thus, viruses have developed a mechanism to modulate complement responses by down-regulating activation proteins and upregulation of regulatory proteins [ ] . in the current study, increased expression of regulatory proteins (cfh, cfi, and cd ) was observed from con -infected animals (figure b) . conversely, c q, a classical complement component involved in increasing virus neutralizing ability of antibodies [ ] was down-regulated ( figure b) . collectively, the data suggest that the con virus suppresses both complement activation and inflammatory responses at the site of persistent infection. apoptosis is a powerful innate immunity mechanism to curtail viral spread through eliminating virally infected cells. apoptosis can be triggered by both extrinsic and intrinsic stimuli [ ] . it is well documented that prrsv induces apoptosis in tissues of infected pigs, especially during an acute phase of the infection [ , , ] . in this study, expression of tnfrsf a (tnfα receptor ) gene that the complement system is another crucial component of the innate immunity. it acts as an important link between innate and adaptive immune system. thus, viruses have developed a mechanism to modulate complement responses by down-regulating activation proteins and up-regulation of regulatory proteins [ ] . in the current study, increased expression of regulatory proteins (cfh, cfi, and cd ) was observed from con -infected animals (figure b) . conversely, c q, a classical complement component involved in increasing virus neutralizing ability of antibodies [ ] was down-regulated ( figure b) . collectively, the data suggest that the con virus suppresses both complement activation and inflammatory responses at the site of persistent infection. apoptosis is a powerful innate immunity mechanism to curtail viral spread through eliminating virally infected cells. apoptosis can be triggered by both extrinsic and intrinsic stimuli [ ] . it is well documented that prrsv induces apoptosis in tissues of infected pigs, especially during an acute phase of the infection [ , , ] . in this study, expression of tnfrsf a (tnfα receptor ) gene that contains a death domain [ ] , was downregulated in the iln of infected pigs ( figure ) . similarly, expression of pro-apoptotic genes including aifm , chac , and osr , was downregulated ( figure ). on the other hand, expression of birc and bfl- /a (bcl a ), two apoptosis inhibitors that interfere with caspase activation [ ] , was upregulated. furthermore, expression of several negative regulators of apoptotic genes including bcl- -associated killer (bak ), damage induced apoptosis suppressor (ddias), x-linked inhibitor of apoptosis protein (xiap), mcl , api, bnip , and faim was upregulated. collectively, these results suggest that the pro-apoptotic signaling pathway was suppressed in iln tissue of pigs persistently infected with con . contains a death domain [ ] , was downregulated in the iln of infected pigs ( figure ) . similarly, expression of pro-apoptotic genes including aifm , chac , and osr , was downregulated ( figure ). on the other hand, expression of birc and bfl- /a (bcl a ), two apoptosis inhibitors that interfere with caspase activation [ ] , was upregulated. furthermore, expression of several negative regulators of apoptotic genes including bcl- -associated killer (bak ), damage induced apoptosis suppressor (ddias), x-linked inhibitor of apoptosis protein (xiap), mcl , api, bnip , and faim was upregulated. collectively, these results suggest that the pro-apoptotic signaling pathway was suppressed in iln tissue of pigs persistently infected with con . lymphocyte activation occurs in the secondary lymphoid organs. activated t-cells then migrate to the sites of infection where they exert their effector functions to eliminate infected cells [ ] . celladhesion molecules, chemokines, and receptors play a central role in regulating t-cell migration [ ] . prrsv mainly persists in lymphoid tissue of infected pigs [ , ] . in this study, expression of several important chemokine ligands (ccl , ccl , ccl , ccl , cx cl , and ccl ) and chemokine receptors (ccr and ccr ) which play an essential role in migration and localization of lymphocytes and antigen-presenting cells (apcs) to the lymphoid tissues was down regulated in iln of con -infected pigs (figure a ). on the other hand, expression of cd (pd- ), a marker of tcell exhaustion, was upregulated ( figure b) . likewise, expression of inhibitory receptors havcr (also known as tim ) and tgit, which transmit the inhibitory signals for t-cell differentiation and effector activities [ ] , was upregulated. we also found increased expression of other co-inhibitory molecules (btla, faslg, fas, and ido ) that are associated with the regulation of t-cell exhaustion during chronic viral infection (figure b) . together, the results suggest that t-cell migration to iln, one of the sites of prrsv persistence, might be affected because of the down regulation of important chemokines and receptors, and that the t-cells in iln might be exhausted. interestingly, markers of lymphocyte activation occurs in the secondary lymphoid organs. activated t-cells then migrate to the sites of infection where they exert their effector functions to eliminate infected cells [ ] . cell-adhesion molecules, chemokines, and receptors play a central role in regulating t-cell migration [ ] . prrsv mainly persists in lymphoid tissue of infected pigs [ , ] . in this study, expression of several important chemokine ligands (ccl , ccl , ccl , ccl , cx cl , and ccl ) and chemokine receptors (ccr and ccr ) which play an essential role in migration and localization of lymphocytes and antigen-presenting cells (apcs) to the lymphoid tissues was down regulated in iln of con -infected pigs (figure a ). on the other hand, expression of cd (pd- ), a marker of t-cell exhaustion, was upregulated ( figure b) . likewise, expression of inhibitory receptors havcr (also known as tim ) and tgit, which transmit the inhibitory signals for t-cell differentiation and effector activities [ ] , was upregulated. we also found increased expression of other co-inhibitory molecules (btla, faslg, fas, and ido ) that are associated with the regulation of t-cell exhaustion during chronic viral infection (figure b) . together, the results suggest that t-cell migration to iln, one of the sites of prrsv persistence, might be affected because of the down regulation of important chemokines and receptors, and that the t-cells in iln might be exhausted. interestingly, markers of regulatory t-cells (t reg ) were downregulated in iln of infected pigs (figure c) , suggesting that t reg might not be present in iln at dpi. regulatory t-cells (treg) were downregulated in iln of infected pigs (figure c ), suggesting that treg might not be present in iln at dpi. since the expression of chemokines and receptors important for t-cell migration was downregulated in iln, we sought to compare the frequencies of virus-specific t-cells in pbmcs and in iln using the ifn-γ sc elispot assay. the number of spots was similar when pbmcs and iln cells were stimulated with non-specific t-cell activator pha/ionomycin. however, the number of spots was significantly lower in iln than in pbmcs when the cells were stimulated with whole prrsv antigen (figure a ). the results clearly indicate that the frequencies of prrsv-specific ifn-γ scs were significantly lower in iln than in pbmcs. to further elucidate the phenotypes of prrsvspecific t-cells, we used a multi-color flow cytometric assay to identify subsets of t-cells that secrete ifn-γ in response to prrsv activation ex vivo. cd + cd + dp cells were the major t-cell population secreting ifn-γ, both in pbmcs and iln. swine have a significant population of extrathymic cd + cd + dp t cells that represent memory t-cells [ ] . similar to the elispot assay, the flow cytometric assay also indicated that the percentage of t-cell secreting ifn-γ was comparatively lower in iln than in pbmcs (figure b ). cells collected from dmem-inoculated pigs did not show any significant t-cell reactivities after ex vivo stimulation with con , both in elispot and in flow cytometry assays (data not shown). since the expression of chemokines and receptors important for t-cell migration was downregulated in iln, we sought to compare the frequencies of virus-specific t-cells in pbmcs and in iln using the ifn-γ sc elispot assay. the number of spots was similar when pbmcs and iln cells were stimulated with non-specific t-cell activator pha/ionomycin. however, the number of spots was significantly lower in iln than in pbmcs when the cells were stimulated with whole prrsv antigen (figure a ). the results clearly indicate that the frequencies of prrsv-specific ifn-γ scs were significantly lower in iln than in pbmcs. to further elucidate the phenotypes of prrsv-specific t-cells, we used a multi-color flow cytometric assay to identify subsets of t-cells that secrete ifn-γ in response to prrsv activation ex vivo. cd + cd + dp cells were the major t-cell population secreting ifn-γ, both in pbmcs and iln. swine have a significant population of extrathymic cd + cd + dp t cells that represent memory t-cells [ ] . similar to the elispot assay, the flow cytometric assay also indicated that the percentage of t-cell secreting ifn-γ was comparatively lower in iln than in pbmcs (figure b ). cells collected from dmem-inoculated pigs did not show any significant t-cell reactivities after ex vivo stimulation with con , both in elispot and in flow cytometry assays (data not shown). viruses , , x for peer review of a number of genes associated with th , or humoral response, were upregulated in the iln of con -infected pigs (figure a ). il- , rgs , and nuggc are involved in the development of germinal center (gc) and activation of b-cell follicles [ ] . tnfsf b and tnfsf are potent activators of b-cell lineage and ig class switching, respectively [ ] . b-cell surface antigens ms a (cd ), activation-induced cytidine deaminase (aicda) [ ] , and rafting family member (rftn ) are associated with b-cell receptor signaling. the upregulated expression of these genes in iln of infected animals suggested that the humoral immune response to con -infection was not affected. to corroborate transcriptome data, we measured both virus-specific antibody levels at various time points post-infection. high levels of non-neutralizing antibodies specific to viral n protein (measured by a commercial elisa) were detected in all pigs (figure b ). however, only minimal levels of virusneutralizing antibodies (titer : ) were detected in the serum of infected pigs at dpi (figure c ). together, both transcriptomic and serological data indicate that b-cell development and antibody production are not affected by con -infection. however, low levels of virus-neutralizing antibodies are likely due to the virus ability to escape antibody neutralization (see below). a number of genes associated with th , or humoral response, were upregulated in the iln of con -infected pigs (figure a ). il- , rgs , and nuggc are involved in the development of germinal center (gc) and activation of b-cell follicles [ ] . tnfsf b and tnfsf are potent activators of b-cell lineage and ig class switching, respectively [ ] . b-cell surface antigens ms a (cd ), activation-induced cytidine deaminase (aicda) [ ] , and rafting family member (rftn ) are associated with b-cell receptor signaling. the upregulated expression of these genes in iln of infected animals suggested that the humoral immune response to con -infection was not affected. to corroborate transcriptome data, we measured both virus-specific antibody levels at various time points post-infection. high levels of non-neutralizing antibodies specific to viral n protein (measured by a commercial elisa) were detected in all pigs (figure b ). however, only minimal levels of virus-neutralizing antibodies (titer : ) were detected in the serum of infected pigs at dpi (figure c ). together, both transcriptomic and serological data indicate that b-cell development and antibody production are not affected by con -infection. however, low levels of virus-neutralizing antibodies are likely due to the virus ability to escape antibody neutralization (see below). viruses , , x for peer review of prrsv persists in lymphoid tissue of infected pigs for several months [ , ] . the mechanism of prrsv persistence is not fully understood. we performed genome-wide transcriptome analysis of lymphoid tissue collected from pigs persistently infected with an attenuated prrsv strain using rna-seq technology that detects both host and viral rna. viral rna reads were detected in iln of all five infected pigs. it was reported previously that prrsv genome mainly exists in dsrna forms in lymphoid tissues during persistent infection [ ] . since only mrna (purified by using poly-t oligo-attached magnetic beads) was used for library construction and rna sequencing, the viral rna reads detected in this study must be derived from either viral genomic rna or sub-genomic mrna, not from dsrna. the viral rna reads map throughout the viral genome. however, we are not able to discern whether these reads are derived from genomic or sub-genomic mrna because we used a short-read rna sequencing platform. it would be interesting to use long-read rna sequencing to study the viral transcriptome at different states of infection in pigs [ ] . it was reported previously that no significant degs were observed in lymphoid tissue of persistently infected animals [ ] . in the current study, we identified a large number of degs in persistently infected animals (figure b ). this might be due to the difference in the experimental setup. in the previous study, pigs were infected with a wild-type prrsv- whereas in this current prrsv persists in lymphoid tissue of infected pigs for several months [ , ] . the mechanism of prrsv persistence is not fully understood. we performed genome-wide transcriptome analysis of lymphoid tissue collected from pigs persistently infected with an attenuated prrsv strain using rna-seq technology that detects both host and viral rna. viral rna reads were detected in iln of all five infected pigs. it was reported previously that prrsv genome mainly exists in dsrna forms in lymphoid tissues during persistent infection [ ] . since only mrna (purified by using poly-t oligo-attached magnetic beads) was used for library construction and rna sequencing, the viral rna reads detected in this study must be derived from either viral genomic rna or sub-genomic mrna, not from dsrna. the viral rna reads map throughout the viral genome. however, we are not able to discern whether these reads are derived from genomic or sub-genomic mrna because we used a short-read rna sequencing platform. it would be interesting to use long-read rna sequencing to study the viral transcriptome at different states of infection in pigs [ ] . it was reported previously that no significant degs were observed in lymphoid tissue of persistently infected animals [ ] . in the current study, we identified a large number of degs in persistently infected animals (figure b ). this might be due to the difference in the experimental setup. in the previous study, pigs were infected with a wild-type prrsv- whereas in this current study, pigs were infected with an attenuated synthetic prrsv strain [ , ] . besides, the previous study looked at a small set of selected genes while in this study we look at the genome-wide rna transcriptome. go terms and kegg analysis revealed that genes involved in the innate immune (complement and tlr) pathways, apoptosis, cytokine-chemokine signaling, t-cell exhaustion, and humoral responses are highly differentially expressed in prrsv-infected animals compared to control. the complement system is a constituent of innate immunity that serves by neutralizing cell-free viruses, lysing virus-infected cells, and boosting virus specific responses [ ] . it also links the innate and adaptive immune responses, enhances humoral immunity, regulates antibody effector mechanisms, and modulates t-cell function [ ] . many viruses have developed a strategy to evade the complement pathway by recruiting or enhancing the production of host transcription regulatory components. during acute infection ( dpi), prrsv significantly represses the expression of complement regulatory components (cd and c bpb) in lung tissue [ ] . early complement activation during acute infection facilitates the release of newly formed virions from infected cells, yet, chronic viral infection is reported to suppress the activity of complement activation proteins and increases the activity of regulatory proteins (reviewed [ ] ). in the current study, we found overexpression of cfh and cfi along with another regulatory factor cd (decay-accelerating factor). overexpression of cfh, a major soluble regulator of the alternative pathway, results in inhibition of c and c convertase enzymes. cfi suppresses the complement active proteins c b (opsonin) and c b via mediating cleavage to their inactive form [ , ] . regulatory factor cd is an inhibitor of c convertase which prevents c b deposition on the cell surface [ ] . apart from that, we also see the downregulation of c q, which increases neutralizing and hemagglutination inhibition activity of anti-influenza antibodies [ ] . available data from previous studies and the current study suggest that activation of complement pathway during acute infection helps to disseminate virus, while suppression of complement components like c q, c r, and c , and upregulation in regulatory components during persistent infection facilitates the virus to escape from the complement system to maintain persistence in lymphoid tissues. most naturally occurring prrsv strains suppress type i ifn production by inhibiting the activation and nuclear translocation of irf /irf and nf-kb [ , ] . deficiency of irf / results in severe mortality to infection with west nile virus (wnv), chikungunya virus (chikv), and ross river virus infection, and promotes viral persistence in the infected hosts [ ] [ ] [ ] . it has been reported that prrsv nsp β inhibits irf phosphorylation and nuclear translocation; thus, inhibiting ifn production [ , , ] . interestingly, the synthetic prrsv-con and its attenuated form con were able to induce type i ifns [ , ] . contrary to its nature to induce type i ifn, in the current study no upregulation of canonical type i ifn signaling pathway-associated genes was observed. however, expression of rna sensing molecules including tlr , tlr , and tlr was upregulated in con -infected animals. on the basis of available data, we hypothesize that reduced levels of viral replication and sequestration of viral rna in infected cells during persistent infection might limit further activation of type i ifn signaling pathway by preventing interaction with cytoplasmic pattern recognition receptors (prrs). apoptosis or programmed cell death is a potential host immune mechanism against virally infected cells to curtail the spread of newly formed viral progenies. apoptosis is induced by two distinct, yet inter-connected signaling pathways, the extrinsic and intrinsic pathways [ ] . in order to successfully establish persistent infection, viruses have developed mechanisms to inhibit apoptosis. for instance, adenovirus (e b- k), human cytomegalovirus (ul ), poxviruses (f l), and myxoma virus (m l) have an inhibitory effect on proapoptotic proteins bak/bax [ ] . it appears that prrsv can also modulate apoptosis. studies of pulmonary alveolar macrophages (pam) infected with prrsv in vitro reveal that the virus stimulates anti-apoptotic pathways early in infection while it induces apoptosis late in infection [ ] . on the other hand, the virus induces apoptosis in the tissues of infected animals during acute infection, but the frequencies of apoptotic cells reduced to normal levels observed in control, non-infected pigs from day post-infection [ ] . in this study, expression of multiple anti-apoptotic genes including xiap, bfl- /a (bcl a ), and birc was upregulated while expression of pro-apoptotic genes (aifm , chac , and osr ) was downregulated. xiap is an endogenous caspase and inhibitor [ ] . bfl- /a (bcl a ) is a transcriptional target of nuclear factor-kb (nf-kb) that suppresses caspase activation and apoptosis in response to death-inducing stimuli like tnfα [ ] . birc is an interaction partner to tnfrsf b and inhibits apoptosis by interfering with the caspase activation [ ] . the pro-apoptotic bcl- family member like bcl- -associated killer (bak ), which allows the release of cytochrome c via formation of homo-oligomers and stable insertion into outer mitochondrial membrane was significantly suppressed. thus, the data suggest that apoptosis was suppressed in the iln of con -infected animals during persistent infection. chemokine molecules ccl and ccl play a crucial role in migration, activation, expansion, and survival of antiviral t-cells. suppression of the ccr and ccl /ccl axis results in dysfunction of t-cells during viral infection [ , ] . ccl -ccr axis plays role in protection against multiple viruses, such as hiv [ ] , herpes simplex virus (hsv- ) [ ] , hsv- , hepatitis c virus [ ] , and pseudorabies [ ] . acute prrsv infection increases the expression of chemokines, leading to the infiltration of immune cells toward the sites of infection [ , ] . in this study, expression of both ccl and ccl was significantly downregulated in con -infected lymph node (figure a ). additionally, expression of different chemoattractant molecules (fractalkine/cx cl , ccl , ccl , ccl , ccl , and ccl ) and receptors (ccr and ccr ) was also downregulated. it is possible that downregulation of these chemokines and receptors might impair t-cells trafficking to inguinal lymph node, the site of prrsv persistence. our transcriptional data are supported by the functional data which demonstrate that the frequencies of ifn-γ sc in iln were significantly lower than in pbmcs (figure ) . chronic or persistent viral stimulation results in hierarchical loss of effector functions of t-lymphocytes, including proliferation, cytokine production (e.g., ifn-γ and il- ), and cytolytic responses [ , , ] . although the molecular signatures involved in t-cell exhaustion are not completely understood, the overexpression of cell surface inhibitory receptors (e.g., pd- , ctla- , and others) primarily mediates cd + t-cell dysfunction [ ] [ ] [ ] . t-cell exhaustion seems to be a common phenomenon caused by arteriviruses, as t-cell exhaustion was also reported in horses persistently infected with eav [ ] . recently, it was shown that prrsv infection alone or co-infection with porcine circovirus type (pcv ) can significantly upregulate surface expression of pd-l (cd ) on porcine monocytes-derived dendritic cells (modcs) [ ] . increased expression of pd-l on the surface of antigen presenting cells (apcs) possibly contributes to the ineffective t-cell responses during the prrsv infection. in the present study, expression of several markers for t-cells exhaustion such as pd- (cd ) and its ligand, cd (pd-l ) was upregulated in con -infected animals, suggesting that the t-cells in lymph nodes of persistently infected animals might be exhausted (figure b) . however, additional studies (e.g., t-cell cytotoxicity and cytokine production) are required to fully assess the functionality of t-cells during prrsv persistence. germinal centers (gcs) are the specialized locations in the secondary lymphoid tissues where b-cell maturation, differentiation, somatic hypermutation, and class switching of isotypes take place [ ] . in the current study, the gc development-associated genes, which includes il- produced by follicular helper t-cells [ ] , the regulator of g protein signaling (rgs ) [ ] , and gc-associated nuclear gtpase (nuggc) [ ] , were upregulated in the lymph node of con -infected animals. thus, prrsv infection does not seem to impair gc formation and b-cell activation. this is supported by the fact that all five con -infected pigs developed a robust antibody response, as measured by the idexx elisa ( figure b) . however, only minimal titers of virus-neutralizing antibodies were detected at dpi. perhaps, prrsv does not suppress the humoral immune response. instead, the virus evades from antibody-mediated neutralization through different mechanisms such as glycan shielding and decoy epitopes [ ] [ ] [ ] . in summary, we identified a robust host transcriptional response in the inguinal lymphoid tissue of pigs persistently infected with the attenuated prrsv strain con . genes involved in the innate immune responses are downregulated. similarly, chemokines and receptors associated with t-cell homing to the lymphoid tissue are downregulated. this might lead to the lower frequencies of virus-specific t-cells in lymphoid tissue than in peripheral blood. additionally, the genes involved in the anti-apoptotic pathways are upregulated. collectively, the data suggest that prrsv can create a pro-survival microenvironment at the lymphoid tissue which allows the virus to persist for an extended period. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . figure s . representative flow cytometry gating strategy used to identify ifn-γ secreting cd α + , cd α + , and cd α + cd α + (dp) t cells from pbmcs and iln. table s . summary of sequencing quality control and mapping data of samples. arterivirus molecular biology and pathogenesis ictv-international committee on taxonomy of viruses. virus taxonomy: release. ec porcine reproductive and respiratory syndrome assessment of the economic impact of porcine reproductive and respiratory 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porcine reproductive and respiratory syndrome virus pig immune response to general stimulus and to porcine reproductive and respiratory syndrome virus infection: a meta-analysis approach expression of toll-like receptor mrna and cytokines in pigs infected with porcine reproductive and respiratory syndrome virus analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus understanding prrsv infection in porcine lung based on genome-wide transcriptome response identified by deep sequencing porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus humoral immune responses and viral shedding following vaccination with modified live porcine reproductive and respiratory syndrome 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ma- cell line gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination location of t-cell epitopes in nonstructural proteins and of type-ii porcine reproductive and respiratory syndrome virus a -kda structural protein of porcine reproductive and respiratory syndrome virus encoded by orf b use of interleukin to enhance the cellular immune response of swine to an inactivated herpesvirus vaccine cross reactivity of immune responses to porcine reproductive and respiratory syndrome virus infection a wrapper script to automate quality and adapter trimming cutadapt removes adapter sequences from high-throughput sequencing reads a quality control tool for high throughput sequence data ultrafast and memory-efficient alignment of short dna sequences to the human genome differential gene and transcript expression analysis of rna-seq experiments with tophat and cufflinks accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions htseq-a python framework to work with high-throughput sequencing data moderated estimation of fold change and dispersion for rna-seq data with deseq manke, t. deeptools : a next generation web server for deep-sequencing data analysis gene ontology analysis for rna-seq: accounting for selection bias kobas . : a web server for annotation and identification of enriched pathways and diseases tlr is a negative regulator of both myd -dependent and -independent tlr signaling the inhibitory cd r is differentially expressed on human and mouse t and b lymphocytes cd and membrane protein interactions in the control of myeloid cells cd /cd r paired potent inhibitory molecules regulating immune and inflammatory responses; part ii: cd /cd r potential clinical applications complement evasion strategies of viruses: an overview. front influenza a virus m blocks the classical complement pathway through interacting with c qa apoptosis: a review of programmed cell death apoptosis in the lungs of pigs infected with porcine reproductive and respiratory syndrome virus and associations with the production of apoptogenic cytokines nf-kappab antiapoptosis: induction of traf and traf and c-iap and c-iap to suppress caspase- activation bfl- /a functions, similar to mcl- , as a selective tbid and bak antagonist activation, differentiation, and migration of naive virus-specific cd + t cells during pulmonary influenza virus infection regulation of t cell migration during viral infection: role of adhesion molecules and chemokines molecular and cellular insights into t cell exhaustion functional and phenotypic analysis of porcine peripheral blood cd /cd double-positive t cells speckled-like pattern in the germinal center (slip-gc), a nuclear gtpase expressed in activation-induced deaminase-expressing lymphomas and germinal center b cells member of the tumor necrosis factor family and b lymphocyte stimulator il- and il- collaborate to shape t-dependent antibody responses the architecture of sars-cov- transcriptome a synthetic porcine reproductive and respiratory syndrome virus strain confers unprecedented levels of heterologous protection viral mimicry of the complement system the complement system in regulation of adaptive immunity control of the amplification convertase of complement by the plasma protein beta h human complement c b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta h for cleavage of c b and c b in solution decay accelerating factor (cd ): a target for cancer vaccines? porcine reproductive and respiratory syndrome virus nonstructural protein beta modulates host innate immune response by antagonizing irf activation the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions regulation of interferon regulatory factor- by the hepatitis c virus serine protease infectious chikungunya virus in the saliva of mice, monkeys and humans induction of ifn-beta and the innate antiviral response in myeloid cells occurs through an ips- -dependent signal that does not require irf- and irf- interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus the cysteine protease domain of porcine reproductive and respiratory syndrome virus non-structural protein antagonizes interferon regulatory factor activation identification of viral genes associated with the interferon-inducing phenotype of a synthetic porcine reproductive and respiratory syndrome virus strain bax insertion, oligomerization, and outer membrane permeabilization in brain mitochondria: role of permeability transition and sh-redox regulation porcine reproductive and respiratory syndrome virus modulates apoptosis during replication in alveolar macrophages structural biology. controlling the caspases ccr and its ligands: balancing immunity and tolerance ccr coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs ccl induces mucosal homing of hiv- -specific iga-secreting plasma cells in mice immunized with hiv- virus-like particles influence of ccr ligand dna preexposure on the magnitude and duration of immunity enhancement of immune responses by co-delivery of ccl /mip- beta chemokine plasmid with hcv core dna/protein immunization genetic co-transfer of ccr ligands enhances immunity and prolongs survival against virulent challenge of pseudorabies virus molecular characterization of transcriptome-wide interactions between highly pathogenic porcine reproductive and respiratory syndrome virus and porcine alveolar macrophages in vivo molecular signature of cd + t cell exhaustion during chronic viral infection t cell exhaustion t cells and viral persistence: lessons from diverse infections pd-l expression is increased in monocyte derived dendritic cells in response to porcine circovirus type and porcine reproductive and respiratory syndrome virus infections role of nf-kappa b in cell survival and transcription of latent membrane protein -expressing or epstein-barr virus latency iii-infected cells influence of n-linked glycosylation of porcine reproductive and respiratory syndrome virus gp on virus infectivity, antigenicity, and ability to induce neutralizing antibodies immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein as well as glycoprotein identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to thank dirk anderson at flow cytometry core facility, nebraska center for biotechnology for assistance on flow cytometric analysis and the staff members of unl life sciences annex and veterinary diagnostic center for the care of animals. the use of product and company names is necessary to accurately report the methods and results; however, the united states department of agriculture (usda) neither guarantees nor warrants the standard of the products. the use of names by the usda implies no approval of the product to the exclusion of others that may also be suitable. the usda is an equal opportunity provider and employer. key: cord- -li x m authors: hung, ling-chu title: the monoclonal antibody recognized the open reading frame protein in porcine circovirus type -infected peripheral blood mononuclear cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: li x m the purpose of this study in the context of the open reading frame (orf ) protein of porcine circovirus type (pcv ) was especially its location and its relation to the capsid protein and the apoptosis protein in pcv -infected porcine peripheral blood mononuclear cells (pbmcs). to detect the orf protein, monoclonal antibodies (mabs) were generated in this study. the mab d binds to the orf peptide (residues – ) and the native orf protein in pcv -infected pbmcs, as shown by immunofluorescence assay (ifa). the data show that – % of pbmcs were positive for orf protein or p protein. further, – % of pbmcs were positive for the capsid. this study confirmed the orf protein not only colocalized with the capsid protein but also colocalized with the p protein in pbmcs. immunoassays were conducted in this study to detect the capsid protein, the orf protein, anti-capsid igg, and anti-orf igg. the data show the correlation (r = . ) of the orf protein and the capsid protein in the blood samples from the pcv -infected herd. however, each anti-viral protein igg had a different curve of the profile in the same herd after vaccination. overall, this study provides a blueprint to explore the orf protein in pcv -infected pbmcs. the porcine circovirus (pcv) is a small virus and contains closed circular single-stranded dna [ ] . previous studies indicated that porcine circovirus type (pcv ) is non-pathogenic to porcine herd [ , ] . however, porcine circovirus type (pcv )-infected pigs showed dullness, thinness, lymphadenopathy, jaundice, hepatomegaly, and other manifestations [ ] [ ] [ ] [ ] [ ] . lymphocyte depletion and apoptosis of lymphocytes were the significant histopathological lesions in lymphoid tissues of pcv -infected pigs [ , ] . pcv infection causes a reduction of t-and b-lymphocytes in the pbmc [ ] . likewise, pcv -associated disease (pcvad) manifested as severe swine herd problems [ , [ ] [ ] [ ] . recently, pcvad has become one of the significant swine diseases worldwide, and it impacts pork production [ ] . at least four commercial pcv a vaccines have been widely used to reduce viremia and pcv infectious pressure in swine herds [ ] [ ] [ ] [ ] . that might have caused the global trend shift from pcv a to pcv b first [ ] [ ] [ ] [ ] , and then pcv d (pcv b mutant strain) has become a predominant genotype globally [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the open reading frame (orf ), open reading frame (orf ), and open reading frame (orf ) genes are the three principal orfs among orfs in pcvs [ ] . the orf encodes for the replicase peptide c and peptide n were appended with an n-terminal cysteine during synthesis, which was required for conjugation with maleimide-activated carriers. animal experiments in this study were performed following the current legislation on ethical and welfare recommendations. the experiments followed the standards of the guide of the care and use of laboratory animals. all animal work was approved by the institutional animal care and use committee (iacuc) of the livestock research institute, and the iacuc of the animal health research institute. the iacuc approval numbers lriiacuc - (for swine and murine experiments), a (for murine and rabbit experiments), and a (for murine and rabbit experiments) were given in this study. antisera against pcv were generated by immunizing new zealand white rabbits (from livestock research institute, council of agriculture, taiwan). rabbits were maintained in isolation rooms, and the room temperature was at - • c. they were fed with a commercially pelleted diet (fwusow in., taiwan), and pure water was available ad libitum. then, animals were immunized with peptide immunogen (the peptide n conjugated with klh) or commercial vaccine five times at two-week intervals. for peptide immunization, an initial dose of µg of the conjugated peptide was mixed with complete freund's adjuvant (sigma-aldrich, st. louis, mo, usa) at the first injection. each rabbit was re-immunized with the same amount of immunogenic mixture with incomplete freund s adjuvant (sigma-aldrich, st. louis, mo, usa). for virus-like particles (vlp) of pcv immunization, the rabbit was injected intramuscularly in the legs with . ml of the pcv vaccine (circoflex ® , boehringer ingelheim). the blood was harvested two weeks after the last injection. the antibody titers were measured by indirect enzyme-linked immunosorbent assay (ielisa) [ ] (performed as described previously for mouse anti-pcv elisa, with the exception that peroxidase-conjugated donkey anti-rabbit igg (h + l, jackson immunoresearch, west grove, pa, usa) secondary antibody was used). hybridomas were generated following established methods, as previously described [ ] . briefly, four five-week-old, female, balb/cbyjnarl mice were purchased from a specific pathogen-free (spf) colony (the national applied research laboratories, taiwan). the mice were maintained in isolation rooms in filtertop cages, and the room temperature was at - • c. mice were fed with a commercially pelleted diet (labdiet, , st. louis, mo, usa), and pure water was available ad libitum. mice were inoculated with µg of immunogen (the peptide n conjugated with klh) emulsified in freund's adjuvant. subsequently, mice were boosted fortnightly with the same amount of immunogen. three days after the final booster, the spleens of the mice were harvested for hybridoma generation. hybridomas were screened for the secretion of anti-orf -specific mabs by ielisa [ ] using the peptide n as a coating antigen. that secondary antibody solution (hrp-conjugated goat anti-mouse iga/igg/igm, h/l chain (novus, saint charles, mo, usa) was used at : dilution for hybridoma screening. the hybridomas that secreted anti-n mabs were subcloned by limited dilution of the cells. then, ascites containing mabs were collected as previously described [ ] . subsequently, the mabs were purified from the ascites by using the nab protein g spin column (thermo scientific, rockford, il, usa), then collecting solutions were exchanged for the buffer and concentrated using the amicon ultra k (millipore, burlington, ma, usa). the concentration of mabs was determined using the nano photometer ® (implen, munich, germany). the heavy chain and the light chain of the two mabs secreted by each hybridoma were determined by using an sba clonotyping system/hrp (southern biotechnology, birmingham, al, usa). it was performed according to the manufacturer's instructions and a previously published method [ ] . briefly, the nunc maxisorp flat-bottom -well plate (thermo fisher scientific, inc., waltham, ma, usa) was coated with the capture antibody (goat anti-mouse ig, µg/ml) in . -m bicarbonate buffer (sigma-aldrich, st. louis, mo, usa) at • c overnight. after three washes with pbs containing . % tween (pbst), the plate was blocked with the blocking buffer (pbst containing % casein hydrolysate) at • c for min. after washing, µl of each culture supernatant of hybridoma were added sequentially, and the plate was incubated at • c for h. after washing, µl of hrp labeled goat anti-mouse igg , -igg a, -igg b, -igg , -iga, -igm, -κ light chain, and -λ light chain were added to appropriate wells of the plate. pbst served as background. the plate was then incubated at • c for h and after that washed with pbst. finally, µl of , -azino-bis ( -ethylbenzothiazoline- -sulfonic acid) diammonium salt (abts, sigma-aldrich, st. louis, mo, usa) buffer were added to each well of the plate. after min, the optical density (od) of each well was measured at nm using a spectramax m microplate reader (molecular devices, san jose, ca, usa). the epitope mapping of these mabs was performed by ielisa [ ] using truncated peptides (as shown in table ) as coating antigens. briefly, peptides contained the sequence of orf protein of pcv b between residues and , associated -mer peptides, truncated derivatives, and the control (peptide c ). ninety-six-well maxisorp plates were coated with each peptide ( µg/ml) in bicarbonate buffer and incubated at • c overnight. after three washes in pbst, the plates were blocked with the blocking buffer at • c for min. after washing, the culture supernatant of hybridoma was added, and plates were again incubated at • c for h. the anti-peptide n mouse serum (at a : dilution) was used as the positive control, and the anti-orf (peptide c ) mab ( h ) was served as the negative control. after rinsing three times with pbst, the secondary antibody solution was applied to the appropriate wells. peroxidase-conjugated goat anti-mouse igg (subclasses + a + b + , fcγ, jackson immunoresearch, west grove, pa, usa) was used at a : dilution for binding to the mab d . hrp-conjugated goat anti-mouse lambda light chain (novus, saint charles, mo, usa) was used at a : dilution for binding to the mab d . after h, the plates were washed three times. the colorimetric reaction was developed by using the abts solution. following a -min incubation, the od was measured at nm. both of the isotype and molecular weight of the mab d were determined by western blotting. the culture supernatant of the hybridoma (clone d ) was separated by bolttm bis-tris plus gel (invitrogen, carlsbad, ca, usa). one gel was stained with fastain protein staining solution (yeastern biotech, taiwan). the other gel was transferred to the polyvinylidene difluoride (pvdf) membranes (millipore, burlington, ma, usa) with fast semi-dry transfer buffer (thermo, waltham, ma, usa) by using a yrdimes semi-dry transfer system (wealtec, new taipei city, taiwan). the membrane was blocked with the blocking buffer for min at • c. after three washes in pbst, each strip of the membrane was incubated with one appropriate horseradish peroxidase (hrp)-conjugated antibody to detect mab at • c overnight and then at room temperature for h. peroxidase-conjugated goat anti-mouse iga/igg/igm, h/l chain (novus, saint charles, mo, usa) at : , hrp-conjugated goat anti-mouse iga (southern biotechnology, birmingham, al, usa) at : , hrp-conjugated goat anti-mouse lambda light chain (novus, saint charles, mo, usa) at : , hrp-conjugated goat anti-mouse igm, µ chain (jackson immunoresearch, west grove, pa, usa) at : , and hrp-conjugated goat anti-mouse igg (subclasses + a + b + , fcγ, jackson immunoresearch, west grove, pa, usa) at : were used in this assay. then, they were washed three times with pbst, min each wash. each strip was incubated with , -diaminobenzidine (dab, horseradish peroxidase substrate, millipore, burlington, ma, usa) buffer for min. protein molecular weight markers were included in each western blot analysis. after revelation with the dab substrate, the target protein was visualized and calculated the molecular weight by using molecular weight markers. homemade immunofluorescence assay (ifa) slides were performed as previously described [ ] . pcv -infected peripheral blood mononuclear cells (pbmcs) were collected from the pcv -infected conventional piglet. the pig serum was detected as seropositive for pcv by anti-capsid peptide (c ) specific immunoassay [ ] . the whole blood sample was confirmed as positive for pcv antigen by using the ingezim pcv das kit (ingenasa, madrid, spain). briefly, pbmcs were isolated from ethylenediaminetetraacetic acid (edta)-treated whole blood samples by ficoll-paque plus (ge healthcare bio-sciences, uppsala, sweden) and according to the manufacturer's instructions. then, mononuclear cells were drawn from the interface of the ficoll-paque plus-containing tube and resuspended in ml pbs for washing and centrifugation ( × g for min at • c). after washing three times with pbs, the cell pellet was resuspended in . ml of fetal bovine serum. an aliquot ( µl) of cell suspension was loaded onto a glass slide for the cell smear. after the cell smear, the slides were air-dried at room temperature. thereby, the pbmcs were stuck on slides. these slides were fixed with pbs containing % paraformaldehyde at • c for min and then washed three times with pbs. following that, the slides were soaked in pbs containing . % triton x- at • c for min. subsequently, slides were washed with pbs and prepared for ifa. two anti-capsid (the peptide c ) mabs ( h and b ) [ ] , one anti-pcv mab a (ingenasa, madrid, spain), one anti-p protein (wt-p ) rabbit polyclonal antibody (bioss, woburn, ma, usa), the homemade anti-orf peptide (n ) mab ( d ), and rabbit antisera (as the method mentioned above) were used in this study. the anti-capsid peptide mabs ( h and b ) recognized native capsid proteins and reacted with core peptides (p , kdpplnp ); however, the mab h bound the three minimal linear epitopes (dpplnp, dpplnpk, and lkdpplkp) and the mab b bound two minimal linear epitopes (kdpplnp and kdpplnpk). these mabs produced a variable definite staining pattern in pbmcs by ifa [ ] . the pcv -infected pbmcs slides were incubated with a : dilution of rabbit antiserum and a : dilution of mab (ascites). after incubation at • c for h, the slides were gently rinsed briefly in pbs and then soaked for min in pbs at • c. the slides were then incubated at • c with tritc-conjugated goat anti-mouse igg (subclasses + a + b + , fcγ) at a : dilution and fitc-conjugated goat anti-rabbit igg (h + l) (all from jackson immunoresearch, west grove, pa, usa, and each minimal antibody cross-reaction to other animal's serum protein) at a : dilution. after min of incubation, the slides were washed with pbs and then incubated with , -diamidino- -phenylindole, dihydrochloride (dapi, aat bioquest, sunnyvale, ca, usa) at a : dilution in pbs at room temperature for min. the slides were mounted under % glycerol and observed with an olympus bx fluorescence microscope and spot flex camera (diagnostic instrument, model . mp, usa). terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay was performed according to the manufacturer's instructions (the cell meter™ tunel apoptosis assay kit, aat bioquest ® , inc., sunnyvale, ca, usa) to detect excessive dna breakage in the pbmc. briefly, the pcv -infected pbmcs slides were incubated with the tf -dutp/reaction buffer. after min of incubation, the slides were washed with pbs. they were then incubated with hoechst solution at room temperature for min. the slides were mounted under % glycerol and observed with an olympus bx fluorescence microscope and spot flex camera. blood samples were collected from the pcv -infected herd with the following vaccination at two weeks of age with a pcv vaccine ( ml, porcilis ® pcv, msd animal health) in the conventional pig farm with the farrow-to-finish operation. pigs were selected from animals of different ages ( , , , , , , , , , and ≥ weeks of age) at one time. then, four samples were randomly chosen per age group. blood was collected in the vacutainer ® edta tubes (becton and dickinson bv) and sored at − • c. all whole-blood samples had been treated by the freeze-thaw process twice before the test. the capsid protein was detected from µl of whole-blood sample by using the ingezim pcv das kit (ingenasa, madrid, spain) according to the manufacturer's instructions, and the absorbance of each well was measured at nm. meanwhile, the homemade antigen-elisa was conducted in this study for detecting the orf protein in whole-blood specimens. it was based on the double-antibody-sandwich principle. ninety-six-well maxisorp plates were coated with µl/ml ( µl per well) of the trapping sera (rabbit anti-orf peptide) in bicarbonate buffer. the plates were incubated at • c overnight and washed three times with pbst. then, the plates were blocked with viruses , , of the blocking buffer at • c for min. after the plates were washed, µl of pbst and µl of whole-blood samples were mixed and added to the well. the plates were incubated at • c for h and then washed in pbst. then, µl/well of mab d at µg/ml were added to the plates, and the plates were incubated at • c for h. after the plates were washed, µl/well of peroxidase-conjugated goat anti-mouse igg fcγ at a : dilution were added, and the plates were incubated at • c for h. after washing, abts buffer was added to each well. following a -min incubation, the od was measured at nm. . . detection of the specific antibodies against the capsid protein or the orf protein in plasm samples from the pcv -infected herd with a pcv vaccine plasm samples were separated from the aforementioned fresh whole-blood samples and sored at− • c. the antibody titers were measured by ielisa [ ] (performed as described previously for pig anti-peptide elisa, with the exception that the capsid protein (circoflex ® , boehringer ingelheim) or peptide p was used as the coating antigen, and pig plasm at a : dilution was used). the subsequent procedures of blocking, pig antibody, washing, secondary antibody, substrate, and the od reading, were the same as described previously. the data were analyzed by using one-way analysis of variance (anova) and tukey's studentized range (hsd, honestly significant difference) multiple comparisons test using the sas enterprise guide . ® software (sas institute inc., cary, nc, usa). a p-value < . was considered significant. the orf peptide n or vlp of pcv was capable of inducing each specific antibody. the antibody titer of each post-immune serum (two weeks after the fourth booster) was detected at the dilution : ( figure s ). the binding of the rabbit antiserum to the virus was confirmed by using an immunofluorescence assay (ifa) ( figure s ). although pre-immune serum had a low background (od value less than . ) at a dilution of : , the pre-immune serum did not bind the virus as detected by ifa (data not shown). the author checked for the cross-reactivity of post-immune antiserum to other antigens. the result shows they have a minor cross-reaction at a dilution of : ( figure s ), but the od value of cross-reaction has not significantly higher than that of the negative control serum (p ≥ . ) ( figure s ). following the fusions, hybridoma supernatants were screened for the presence of anti-orf (peptide n ) specific antibodies by ielisa. among them, hybridomas supernatants reacted with the peptide n at the first screening (data not shown). after repeatedly subcloning by the limiting dilution and selection, two stable hybridomas secreting anti-orf mabs ( d and d ) were obtained. the hybridoma produced igg mab ( d ) with kappa-light chains. the other produced mab ( d ) with a lambda-light chain ( figure a) ; however, no heavy chain was detected in the supernatant of clone d by sba clonotyping system/hrp assay. the supernatant of a hybridoma (clone d ) was separated directly by bolttm bis-tris plus gel to confirm the heavy chain of this mab. the gel showed a broad staining region within - kda. the author speculated that the supernatant of a hybridoma contains abundant serum proteins from the fetal bovine serum-containing medium. that is to say, the supernatant of a hybridoma (clone d ) contains a little mab d (light chain). further, the western blotting was used to confirm the heavy chain of mab d and its molecular weight. the hrp-conjugated antibodies (goat anti-mouse iga/igg/igm, h/l chain, and goat anti-mouse lambda light chain) gave specific reactions with the supernatant. two bands were observed at about (between and ) and kda, respectively ( figure ). the lower band ( kda) implied that some degradation of this mab. however, other anti-ig antibodies (goat anti-mouse ig a (α chain), goat anti-mouse igm (µ chain), and goat anti-mouse igg (fcγ)) gave negative reactions with the supernatant. this study confirmed mab d contains a lambda light chain, but it does not include any intact heavy chain. since the molecular weight of the light chain was kda, it may be assumed that the mab d might contain a short fragment of a protein. a peptide scan analysis was performed to determine the binding site for each mab ( figure ). the mab d bound to two linear peptides (peptide n and p ). the peptide n was appended with a cysteine residue at the n-terminal of the orf peptide (residues - , p ). both of them contained the sequence of the orf protein of pcv b between residues and . however, the non-igg mab ( d ) not only reacted with peptide n but also showed minor reactions against other peptides, which compared with the mab d in the elisa test ( figure ) . interestingly, the anti-peptide n mouse serum reacted all test peptides and bound firmly to six linear peptides (peptide n , p , p , p , and p ). notably, anti-n poly antibodies reacted with these peptides (expect p ) which contained the common core peptide (p and tllhfpahfq ). nevertheless, this study did not get any mab which bound firmly to the peptide p . viruses , , x for peer review of a peptide scan analysis was performed to determine the binding site for each mab ( figure ). the mab d bound to two linear peptides (peptide n and p ). the peptide n was appended with a cysteine residue at the n-terminal of the orf peptide (residues - , p ). both of them contained the sequence of the orf protein of pcv b between residues and . however, the non-igg mab ( d ) not only reacted with peptide n but also showed minor reactions against other peptides, which compared with the mab d in the elisa test ( figure ) . interestingly, the antipeptide n mouse serum reacted all test peptides and bound firmly to six linear peptides (peptide n , p , p , p , and p ). notably, anti-n poly antibodies reacted with these peptides (expect p ) which contained the common core peptide (p and tllhfpahfq ). nevertheless, this study did not get any mab which bound firmly to the peptide p . anti-orf mabs ( d and d ) bound the linear peptide spanning from residues to . the antipeptide n mouse serum was used as the positive control, and the anti-orf (peptide c ) mab ( h ) served as the negative control. the mab binding was tested by using an ielisa. peptides contained the sequence of the orf protein between residues and , associated -mer peptides, truncated derivatives, and the control (peptide c ) (as shown in table ). the experiments were performed three times. data represent the mean ± standard error (se). for these homemade pbmcs slides, all slides were made from the same batch and contained the same proportion of pcv -infected cells. all wells contained both negative and positive cells (pcv infected cells) confirmed by ifa, as previously noted in a similar study [ ] . in this study, pcv viral protein (orf protein and capsid protein) locations were initially assessed by ifa. the previous study indicated that the mab h bound the three minimal linear epitopes (dpplnp, dpplnpk, and lkdpplkp), which were located at carboxyl-terminus (cterminus) of the capsid proteins of pcv b, pcv d, and pcv a, respectively [ ] . likewise, the mab b bound two minimal linear epitopes (kdpplnp and kdpplnpk), which were located at cterminus of the capsid proteins of pcv b, and pcv d, respectively [ ] . first, the percentage was determined by counting the number of positive cells per nuclei in an ifa slide. the data show that - % of pbmcs were positive for anti-capsid peptide mab ( h ) staining (pcv a-, pcv b-, the anti-peptide n mouse serum was used as the positive control, and the anti-orf (peptide c ) mab ( h ) served as the negative control. the mab binding was tested by using an ielisa. peptides contained the sequence of the orf protein between residues and , associated -mer peptides, truncated derivatives, and the control (peptide c ) (as shown in table ). the experiments were performed three times. data represent the mean ± standard error (se). for these homemade pbmcs slides, all slides were made from the same batch and contained the same proportion of pcv -infected cells. all wells contained both negative and positive cells (pcv -infected cells) confirmed by ifa, as previously noted in a similar study [ ] . in this study, pcv viral protein (orf protein and capsid protein) locations were initially assessed by ifa. the previous study indicated that the mab h bound the three minimal linear epitopes (dpplnp, dpplnpk, and lkdpplkp), which were located at carboxyl-terminus (cterminus) of the capsid proteins of pcv b, pcv d, and pcv a, respectively [ ] . likewise, the mab b bound two minimal linear epitopes (kdpplnp and kdpplnpk), which were located at cterminus of the capsid proteins of pcv b, and pcv d, respectively [ ] . of pbmcs were positive for anti-orf mab ( d ) staining ( figure s ). it might be due to different abundance of the protein, the degradation of the protein, different pcv strains, conformational epitopes, or linear epitopes, which interacted with the antibody. interestingly, the anti-vlp of pcv rabbit serum produced cytoplasmic staining in pcv -infected pbmcs ( figure b,f) . when pcv -infected pbmcs were co-stained with anti-vlp of pcv rabbit serum and anti-capsid peptide mabs ( b ), some vlp/capsid peptide dual-positive cells were shown ( figure d ). the overlap between the anti-vlp rabbit serum staining and the anti-orf mab ( d ) staining is presented in figure h , and the anti-orf polyclonal antibody and anti-capsid mab ( a ) overlapped the same in figure p ; however, the polyclonal antibody staining was brighter than the mab staining. this result shows orf protein overlaps the capsid protein (or vlp) of pcv ( figure h,p) . both the capsid proteins and the orf protein were detected mainly in the cytoplasm. according to the previous study, the n-terminal half of orf protein (residues - ) was discovered in the cytoplasm, and the c-terminal half of orf protein (residues - ) was majorly located in the nucleus [ ] . the medial region of orf protein (residues - ) might be primarily located in the cytoplasm in this study. of pcv ( figure h,p) . both the capsid proteins and the orf protein were detected mainly in the cytoplasm. according to the previous study, the n-terminal half of orf protein (residues - ) was discovered in the cytoplasm, and the c-terminal half of orf protein (residues - ) was majorly located in the nucleus [ ] . the medial region of orf protein (residues - ) might be primarily located in the cytoplasm in this study. a previous study suggested that orf protein led to increased p protein levels and apoptosis of the infected cells [ ] . to identify the subcellular location of the orf protein in pcv -infected pbmcs, anti-capsid, anti-orf , and anti-p antibodies were used. the p protein also colocalized with capsid peptide (figure l ), and the orf peptide ( figure x ). the p protein was mainly distributed in the cytoplasm and around the nucleus ( figure j,v) . the orf protein, as detected with mab d , seemed to colocalize quite nicely with the p protein ( figure x ). the p /orf dual-positive cell presented a segmented nucleus ( figure w,y) . the nuclei of negative cells were round or oval, and nuclear segmentation was very rare in negative cells ( figure y ). the apoptosis in pcv -infected pbmcs was observed by using a terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay (figures and s ) . the data show that - % pbmcs were positive for tunel staining. it is very similar to the percentage of anti-orf mab ( d ) staining. however, these similar percentages are not sufficient proof to show that the orf protein of pcv causes dna damage. a more careful analysis would be needed to reveal whether pcv infection causes dna damage. unfortunately, it was not easy to label both the orf protein and the signals of dna breakage simultaneously by ifa and tunel assay (data not shown). further research would be needed to determine whether the orf protein colocalizes with the signals of tunel. a previous study suggested that orf protein led to increased p protein levels and apoptosis of the infected cells [ ] . to identify the subcellular location of the orf protein in pcv -infected pbmcs, anti-capsid, anti-orf , and anti-p antibodies were used. the p protein also colocalized with capsid peptide (figure l ), and the orf peptide ( figure x ). the p protein was mainly distributed in the cytoplasm and around the nucleus ( figure j ,v). the orf protein, as detected with mab d , seemed to colocalize quite nicely with the p protein ( figure x ). the p /orf dual-positive cell presented a segmented nucleus ( figure w,y) . the nuclei of negative cells were round or oval, and nuclear segmentation was very rare in negative cells ( figure y ). the apoptosis in pcv -infected pbmcs was observed by using a terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay ( figure and figure s ). the data show that - % pbmcs were positive for tunel staining. it is very similar to the percentage of anti-orf mab ( d ) staining. however, these similar percentages are not sufficient proof to show that the orf protein of pcv causes dna damage. a more careful analysis would be needed to reveal whether pcv infection causes dna damage. unfortunately, it was not easy to label both the orf protein and the signals of dna breakage simultaneously by ifa and tunel assay (data not shown). further research would be needed to determine whether the orf protein colocalizes with the signals of tunel. b (a); mab h (i); and mab a (m)), and the orf peptide of pcv (mab d (e,u)). staining with spf mouse serum was used as the negative control (q). (b,f,j,n,v) staining with rabbit antiserum (green) was used to identify the vlp of pcv (b,f), the p (j,v), and the orf peptide of pcv (n). (c,g,k,o,s,w,y) nuclei were stained with dapi (blue). the arrow points to the positive staining cell (z) and its irregular shaped nucleus (y), and the enlarged image in the sixth row (x,w). (d,h,l,p,t,x,z) the merged images are also shown. the scale bar is μm. the viral proteins were detected in the whole-blood samples using antigen-elisa techniques. the cut-off value in the commercial pcv capsid elisa and the orf protein elisa were . and . , respectively. the capsid protein of pcv was mainly detected in pigs at , , , and ≥ weeks of age in this study farm ( figure a ). noticeably, these groups (at , , , and ≥ weeks of age) showed more elevated standard error of od value than other groups in this statistical analysis. this result implies that pigs at that age would be susceptible to pcv infection. interestingly, non-vaccinated piglets showed the highest od value of capsid protein at one week of age. after piglets were inoculated with the pcv vaccine at two weeks of age, the od value of capsid protein gradually decreased until weeks of age. however, the od value of capsid protein was raised at ≥ weeks of age (two gilts and two sows). there is evidence to suggest that the vaccine could not protect pigs against pcv infection at weeks postimmunization. the orf protein of pcv showed the same result except for od values of the orf protein were lower than the capsid protein. the correlation coefficient (r) was . . the mean od values of the orf protein were significantly higher for suckers at one week of age than postweaning ones at - weeks of age (p < . ; figure b ). there is an apparent probability that the amount of orf protein was fewer than the amount of capsid protein in pcv -infected blood from suckers at one week of age. protect pigs against pcv infection at weeks postimmunization. the orf protein of pcv showed the same result except for od values of the orf protein were lower than the capsid protein. the correlation coefficient (r) was . . the mean od values of the orf protein were significantly higher for suckers at one week of age than postweaning ones at - weeks of age (p < . ; figure b ). there is an apparent probability that the amount of orf protein was fewer than the amount of capsid protein in pcv -infected blood from suckers at one week of age. figure . assessment of the capsid protein and the orf protein in the whole-blood samples from pigs of different age groups. this study used the commercial capsid kit (a) and the orf protein elisa (b) to measure each specimen, and the absorbance was measured at or nm, respectively. the peptide n and psbt were used as a positive (pos) control and a negative (neg) control, respectively, in the orf protein elisa. the data represent the mean ± se. treatments with different letters have statistically significant differences (p < . ). figure . assessment of the capsid protein and the orf protein in the whole-blood samples from pigs of different age groups. this study used the commercial capsid kit (a) and the orf protein elisa (b) to measure each specimen, and the absorbance was measured at or nm, respectively. the peptide n and psbt were used as a positive (pos) control and a negative (neg) control, respectively, in the orf protein elisa. the data represent the mean ± se. treatments with different letters have statistically significant differences (p < . ). the anti-viral protein-specific antibodies were detected in the plasm samples using ielisa techniques. the cut-off value of in the anti-capsid igg elisa and the anti-orf peptide (p ) igg elisa were . and . , respectively. non-vaccinated suckers showed the highest mean od value of anti-capsid igg and anti-orf igg at one week of age. it may mean that non-vaccinated suckers absorbed large amounts of maternally derived antibodies after birth. notably, the non-vaccinated group showed the most elevated standard error of od value (anti-capsid igg) in this statistical analysis. a gradual decrease of the mean od value of anti-capsid igg for piglets from the suckling period (one week of age) to the weaning period (six weeks of age) was observed. from to weeks old, the mean od value of anti-capsid igg increased significantly (p < . ; figure ). the mean od value of anti-capsid igg also increased significantly for pigs from to weeks old (p < . ) and reached a plateau at weeks old. the same trend was observed in the result of the anti-orf igg elisa test, except for od values of anti-orf igg were lower than anti-capsid igg. notwithstanding, it is worth mentioning that there was a sharp decrease of the mean od value of anti-orf igg for suckers from one to three weeks old. the mean od value of anti-orf igg increased significantly for pigs from six to nine weeks old (p < . ; figure ) and reached a plateau at nine weeks old. there was a weak correlation (r = . ) between the od value of anti-capsid igg and the od value of anti-orf igg in the plasm samples. it points to the probability that the amount of anti-capsid igg reflects humoral immunity in pcv -infected pigs before and after the injection of a pcv vaccine. besides, the amount of anti-orf igg reflects humoral immunity in pigs, which were caused by pcv infecting only. the mean od value of anti-capsid igg also increased significantly for pigs from to weeks old (p < . ) and reached a plateau at weeks old. the same trend was observed in the result of the anti-orf igg elisa test, except for od values of anti-orf igg were lower than anti-capsid igg. notwithstanding, it is worth mentioning that there was a sharp decrease of the mean od value of anti-orf igg for suckers from one to three weeks old. the mean od value of anti-orf igg increased significantly for pigs from six to nine weeks old (p < . ; figure ) and reached a plateau at nine weeks old. there was a weak correlation (r = . ) between the od value of anti-capsid igg and the od value of anti-orf igg in the plasm samples. it points to the probability that the amount of anti-capsid igg reflects humoral immunity in pcv -infected pigs before and after the injection of a pcv vaccine. besides, the amount of anti-orf igg reflects humoral immunity in pigs, which were caused by pcv infecting only. figure . assessment of the anti-capsid igg and the anti-orf peptide (p ) in the plasm samples from pigs of different age groups. the experiments were repeated three times, and data represent the mean ± se. treatments with different letters at the same kind of antibody assay have statistically significant differences (p < . ). according to a previous study, the orf peptide (residues - ) of pcv interacts with the p -binding domain of ppirh [ ] . the peptide n (residues - ) of the orf protein was synthesized and used in this study. subsequently, anti-orf mabs were generated and characterized in this study. this work created one hybridoma producing the mab d with igg , and the other producing the defective-ig mab ( d ) with a lambda-light chain. the mab d (igg ) bound to the linear peptide n (c hndvyislpi tllhfpahfq kfsqpaeisdkr ) and p ( hndvyislpi tllhfpahfq kfsqpaeisdkr ). however, the defective-ig mab d minorly reacted with all truncated peptides. this finding is similar to the previous study [ ] . the defective-ig mabs likely have broad binding, moderate specificity, and low affinity with the associated peptide. there were concerns about this defective-ig mab since this seems to be very rare. further, the molecular weight of the mab d was determined by western blotting. two bands were observed figure . assessment of the anti-capsid igg and the anti-orf peptide (p ) in the plasm samples from pigs of different age groups. the experiments were repeated three times, and data represent the mean ± se. treatments with different letters at the same kind of antibody assay have statistically significant differences (p < . ). according to a previous study, the orf peptide (residues - ) of pcv interacts with the p -binding domain of ppirh [ ] . the peptide n (residues - ) of the orf protein was synthesized and used in this study. subsequently, anti-orf mabs were generated and characterized in this study. this work created one hybridoma producing the mab d with igg , and the other producing the defective-ig mab ( d ) with a lambda-light chain. the mab d (igg ) bound to the linear peptide n (c hndvyislpi tllhfpahfq kfsqpaeisdkr ) and p ( hndvyislpi tllhfpahfq kfsqpaeisdkr ). however, the defective-ig mab d minorly reacted with all truncated peptides. this finding is similar to the previous study [ ] . the defective-ig mabs likely have broad binding, moderate specificity, and low affinity with the associated peptide. there were concerns about this defective-ig mab since this seems to be very rare. further, the molecular weight of the mab d was determined by western blotting. two bands were observed at about and kda, respectively. the small one ( kda) implied some degradation of this mab. since the molecular weight of the light chain was kda, the author suggested that the mab d might contain a short fragment of a protein. interestingly, the author also tested the molecular weight of another defective-ig mab c [ ] (which against peptide c ), and two molecules at about and kda were again shown (data not shown). according to previous documents, human patients with abnormal serum immunoglobulin-free light chain production should only be due to monoclonal plasmaproliferative disorders (included multiple myeloma, light chain myelomas, and light chain amyloidosis) [ ] [ ] [ ] . arguably, the defective-ig mab ( d ) might be related to the phenotype of splenocytes, which fused with the myeloma cell. further research should be carried out using advanced techniques (such as liquid chromatography with mass spectrometry and spectroscopic techniques) to investigate the constitution of the mab d . based on previous reports, lymphocyte depletion and apoptosis in lymphoid tissues are histological hallmarks in pcv -infected pigs [ ] [ ] [ ] . interestingly, these histopathological lesions are similar to marek's disease and african swine fever [ , ] . more recent evidence shows that these viruses caused an early cytolytic infection in lymphocytes by apoptosis [ ] [ ] [ ] . therefore, this study hypothesized that lymphocyte and pbmcs lineage cells should be the primary target cells for pcv infection. to the best of the author's knowledge, the study of the orf protein by indirect immunofluorescence assay in pcv -infected pbmcs has never been performed, and only a single article mentions the transient expression of the orf in porcine pbmcs and detecting apoptosis with a tunel assay [ ] . therefore, the author explored the relation of the capsid, p protein, and the orf protein by ifa and shed light on p protein (the marker of apoptosis) accompanied the peptide (residues - , p ) of the orf protein in pcv -infected pbmcs. it is worth highlighting that - % of pbmcs were positive for some antibodies staining, including anti-orf mab ( d ) staining, anti-p protein rabbit polyclonal antibody staining, and tunel assay ( figure s ). there is a strong probability that - % of pbmcs were undergoing the process of apoptosis. however, the data revealed that the variable percentage ( - %) of pbmcs was positive for anti-capsid mabs ( h , b , and a ) staining and anti-vlp rabbit serum staining. it was because these mabs or antiserum recognized different epitopes of proteins presented in pcv -infected cells. that means that the variety of interaction between antigen-binding sites of antibodies and epitopes, which includes linear form epitopes, conformational epitopes, degradation, and different pcv strains. curiously, this study ( figure s ) showed that the percentage of pcv -infected pbmcs (by mab h staining) was about -fold the rate of orf -positive pbmcs (by mab d staining) or the percentage of p -positive pbmcs. it seems likely that not all pcv -infected cells contained orf proteins or p protein. this finding concurs with the previous study, which indicated that the apoptosis statistically decreases in the initial pcv -infected pigs [ ] . on the other hand, this study showed the orf protein colocalized with the capsid protein marker of pcv . moreover, the p protein was also colocalized with the orf protein. remarkably, the p /orf dual-positive cell presented a segmented nucleus ( figure w ). however, these p /orf dual-positive cells were very few in these samples. this finding will be confirmed by flow cytometry in the future. although a previous study indicated that pcv -infected pigs had a reduction of lymphocytes in the peripheral blood [ ] , the mechanism of lymphocyte lysis was still unclear. this study confirmed that the orf protein was related to the p protein (apoptosis marker) in pcv -infected pbmcs, while other factors (proliferative activity [ ] , caspases and [ , ] , granzymes [ ] , or corticosteroids [ , ] ) causing lymphocyte lysis or depletion need to be considered. although the experiment on apoptosis in pcv -infected pbmcs was carried out by the tunel assay (figure ) , it did not clarify the relationship between the tunel result and the orf protein in pcv -infected cells. only the ifa data confirm previous reports that exogenous orf protein was related to the accumulation of p protein [ , ] , but more proof are needed to make sure the orf protein is a major factor leading to apoptosis in pcv -infected cells [ , , ] and depletion of lymphocytes. pcv is one of the most critical pathogens in modern swine production and causes endemic disease in pig farms [ ] . the commercial vaccines were confirmed protective in the field against pcv and mainly administered to suckers in herds with pcv infection [ , , [ ] [ ] [ ] . most researchers utilized the quantitative real-time pcr to detect pcv nucleic acid, and then they evaluated the efficacy of vaccination in regard to pcv viremia in pigs [ , , ] . however, little is known about the capsid protein or the orf protein in blood from the pcv -infected herd with vaccination. although the capsid antigen-elisa could detect capsid protein, this assay could not differentiate from native viral proteins or vaccine ones in blood. the orf protein-elisa, by contrast, only identified native orf proteins in pig blood since no commercial vaccine contains the orf protein of pcv . for these purposes, this study used the commercial capsid antigen-elisa and homemade orf protein-elisa (anti-n polyclonal antibodies and mab d based) to detect viral proteins in pig blood. this study found non-vaccinated suckers had the highest of pcv proteins in blood at one week of age. the author suggests that in these suckers it was caused by pcv infection, and these viruses majorly stemmed from the sow-to-newborn transmission [ ] . after inoculated with the pcv subunit vaccine, the capsid protein, and the orf protein were gradually decreased. however, these proteins were detected again in gilts and sows (≥ weeks of age). that means pcv viral proteins (the capsid protein or the orf protein) in gilts and sows were higher than that of pigs at - weeks of age. to put it another way, vaccinated-pigs reduced viremia compared to non-vaccinated suckers, and the immunity could not continue to protect vaccinated pigs after weeks post-vaccination. since pcv is highly resistant to environmental conditions, being able to remain in the farm environment and thus represent a risk for infection maintenance [ ] , even mass pcv vaccination (without implementing further farm management practices or biosafety measures) was not able to clear out pcv infection, and the virus became detectable again when vaccination was stopped [ , ] . another key point to mention is that pcv -specific antibodies response play roles in the pcv -infected herd. it is worth noting that there were two different antibody profiles in this pcv -infected herd with the pcv vaccine. the data show that pcv -infected herd had a higher od value of anti-capsid igg at age , , and ≥ weeks, compared with - weeks. in addition, pcv -infected herd had a higher od value of anti-orf igg at age and ≥ weeks, compared with - weeks. according to previous studies, newborn suckers received colostral antibodies from seropositive sows and showed various levels of maternally derived antibodies [ , ] . these maternally derived antibodies might interfere with the viral protein-specific antibodies response while suckers immunized with the pcv subunit vaccine. it may be assumed that the artificial capsid protein (pcv vaccine) elicited anti-capsid igg producing while maternally derived antibodies progressed to degrade. the total level of anti-capsid igg decreased slowly but still maintained a high level of anti-capsid igg during the age of - weeks. this phenomenon could neutralize the pcv virus and prevent piglets from suffering pcv infection. in contrast with dual-source capsid proteins (pcv virus and subunit vaccine), the piglet's immunity response to the orf protein was only caused by pcv virus infection. the total level of anti-orf igg decreased quickly at - weeks of age. then, it increased significantly at - weeks of age. the weak correlation (r = . ) between the anti-capsid igg and the anti-orf igg is worth mentioning. it might reflect two kinds of antibody responses in virus infection and post-immunization. these data interpret the interaction of the viral protein with the host immune system. according to previous reports, orf encodes a -kda protein that is involved in viral capsid formation and contributed to self-assembled virus-like particles (vlp) [ ] . the monomer structures were assembled into a vlp model consisting of capsid subunits to form an icosahedron [ ] . vaccination with this vlp (subunit vaccine) induced both humoral and cell-mediated responses against pcv [ , ] . however, orf encodes an . -kda protein [ ] that is the non-structural protein and involved in pcv -induced apoptosis [ , , ] . until now, no report mentions that the orf protein was solely used as the vaccine against pcv , reducing viremia or pathological lesions in pcv -infected herds. these differences contributed to their application in different ways [ , ] . in this study, the percentage of orf -positive pbmcs was significantly lower than capsid-positive pbmcs. similarly, the antigen-elisa result shows that the amount of orf protein was less than the amount of capsid protein in pcv -infected blood from suckers at one week of age. that might be because mab d only recognizes the orf protein (peptide p ) for pcv b strain (genbank: aac . ), and it causes low detection. however, pcv d (pcv b mutant strain) and pcv b are still predominant genotypes in the field farms. besides the similar percentages of pbmcs were positive for these antibodies (mab d and anti-p protein rabbit polyclonal antibody) staining or tunel assay. in contrast to the orf protein, the detection of the capsid protein could be the variable result in pcv -infected pbmcs via different antibodies, due to different pcv strains [ , ] , conformational epitopes [ ] , or linear epitopes [ , ] . previous studies indicated that antibody binding residues were often on the exterior of the capsid of pcv [ , , , , , ] . although an epitope might be on the surface of the single capsid unit, it could bury in the vlp and be inaccessible to the antibody [ , ] . in general, these results confirm that mab d recognized the native orf protein in pcv -infected pbmcs. this study used mab d or its minimal linear epitope to design various immunoassays. these assays evaluate the viral load and the immune response of viral proteins stimuli. these may serve as surveillance tools for monitoring natural pcv infection in the herd. overall, the author generated anti-orf mabs against the orf peptide (residues - ) of pcv . this study confirmed the defective-ig mab ( d ) with a lambda-light chain, and its molecular weight was about kda. the mab d contained heavy chains (γ ) and kappa-light chains, and it bound to one minimal linear epitope (hndvyislpitllhfpahfq kfsqpaeisdkr). the data show that - % of pbmcs were positive for orf protein or p protein. otherwise, - % of pbmcs were positive for anti-capsid peptide mab ( h ) staining. this study confirmed the orf protein colocalized with the p protein in pcv -infected pbmcs. the author devised the orf protein-elisa (anti-orf antisera and mab d -based) to detect the orf protein in blood samples. the results show that the amount of orf protein was less than the amount of capsid protein in pcv -infected blood from suckers at one week of age. the correlation between the orf protein and the capsid protein is worth noting (r = . ). furthermore, the antibody level of anti-capsid igg and anti-orf igg could imply the immunity response of pig herd, but the sample size needs to be considered. the author declares no conflict of interest. a very small porcine virus with circular single-stranded dna characterization of novel circovirus dnas associated with wasting syndromes in pigs studies on epidemiology and pathogenicity of porcine circovirus experimental reproduction of severe wasting disease by co-infection of pigs 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produced in insect cells rüütel boudinot, s. porcine circovirus type orf protein induces apoptosis in melanoma cells antigenic subtyping and epitopes' competition analysis of porcine circovirus type using monoclonal antibodies identification of one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of porcine circovirus type fine mapping of antigenic epitopes on capsid proteins of porcine circovirus, and antigenic phenotype of porcine circovirus type in vitro and in silico studies reveal capsid-mutant porcine circovirus b with novel cytopathogenic and structural characteristics structure analysis of capsid protein of porcine circovirus type from pigs with systemic disease antibody recognition of porcine circovirus type capsid protein epitopes after vaccination, infection, and disease viruses , key: cord- -llh iq authors: stott, robert j.; strecker, thomas; foster, toshana l. title: distinct molecular mechanisms of host immune response modulation by arenavirus np and z proteins date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: llh iq endemic to west africa and south america, mammalian arenaviruses can cross the species barrier from their natural rodent hosts to humans, resulting in illnesses ranging from mild flu-like syndromes to severe and fatal haemorrhagic zoonoses. the increased frequency of outbreaks and associated high fatality rates of the most prevalent arenavirus, lassa, in west african countries, highlights the significant risk to public health and to the socio-economic development of affected countries. the devastating impact of these viruses is further exacerbated by the lack of approved vaccines and effective treatments. differential immune responses to arenavirus infections that can lead to either clearance or rapid, widespread and uncontrolled viral dissemination are modulated by the arenavirus multifunctional proteins, np and z. these two proteins control the antiviral response to infection by targeting multiple cellular pathways; and thus, represent attractive targets for antiviral development to counteract infection. the interplay between the host immune responses and viral replication is a key determinant of virus pathogenicity and disease outcome. in this review, we examine the current understanding of host immune defenses against arenavirus infections and summarise the host protein interactions of np and z and the mechanisms that govern immune evasion strategies. rna viruses, despite the limited size of their genomes, pose serious challenges to global public health [ ] [ ] [ ] . a multitude of antiviral host immune mechanisms have evolved to inhibit viral replication processes, from virus entry through to exit from the infected host cell. virus-host interactions have evolved to enable these viruses to resist or avoid host antiviral responses. these complex interactions are governed by the limited number, but multifunctional proteins that are encoded by these small viruses [ , ] . public health concerns have heightened due to the increased incidence of arenavirus epidemics in endemic countries; this is worsened by the lack of vaccines and treatments available. the ongoing, devastating outbreaks of lassa virus (lasv), the most prevalent arenavirus endemic to western africa, highlight the importance of understanding the nature and intricacy of key interactions that occur during emerging rna virus infections [ ] [ ] [ ] [ ] . severe disease associated with lasv infection is characterised by general immunosuppression that contributes to high-level viremia and reflects the ability of lasv to counteract host antiviral responses. this hallmark of infection is driven by the evasion strategies of the s segment encodes the immature glycoprotein precursor polyprotein gpc that is co-and post-translationally cleaved into gp and gp and the stable signal peptide (ssp) [ ] [ ] [ ] . these three protein subunits form the mature glycoprotein spike complex (referred to as gp) on the viral surface. gp is involved in receptor binding and entry into host cells, while gp and ssp are implicated in stabilising receptor-gp complexes and in viral fusion within host cell membranes [ ] [ ] [ ] . the s segment also encodes for a major structural component of the nucleocapsid: the nucleoprotein, np which is abundantly produced during infection. the l segment encodes for the rna-dependent rna polymerase (rdrp) that is responsible for initiating replication of the rna genome, and also encodes for the small, zinc finger matrix protein, z, that is involved in regulating viral rna synthesis, virion assembly and budding ( figure ) [ ] [ ] [ ] . notably, the genome of hartmaniviruses lacks an open reading frame for the z protein, suggesting that the replication cycle of these viruses is mechanistically distinct from other arenaviruses [ ] . indeed, it is hypothesised that like hantaviruses, gp of hartmaniviruses is able to bind to members of the endosomal sorting complex required for transport (escrt) pathway to orchestrate virus budding at the plasma membrane. this implies that gp may act as a z protein surrogate, bypassing the need for z protein expression in hartmaniviruses [ , ] . all human pathogens are members of the large mammarenavirus genus that currently encompasses recognised virus species, as defined by the international committee on taxonomy of viruses. based on phylogenetic and serological characteristics, as well as geographical prevalence, mammalian arenaviruses are further divided into old world (ow) and new world (nw) groups, representing viruses endemic to africa and the americas, respectively [ , ] . nw arenaviruses are differentiated further into clades a, b, c and a/b (recombinant species otherwise referred to as clade d) [ ] [ ] [ ] [ ] . the ow arenaviruses include the globally distributed, prototypic arenavirus lymphocytic choriomeningitis virus (lcmv) that is associated with aseptic meningitis; the highly pathogenic lasv that is associated with severe viral haemorrhagic fever (vhf) with over reported deaths annually in endemic west african countries; and the recently emerged lujo virus (lujv), associated with severe haemorrhagic fever disease in south africa and zambia [ ] . the nw group of arenaviruses also includes those that cause haemorrhagic fever in humans with associated high fatality rates of between % and %, namely: machupo virus (macv), junín virus (junv), guanarito virus (gtov), sabia virus (sabv) and chapare virus (chapv) [ , [ ] [ ] [ ] [ ] . the natural hosts for both ow and nw mammarenaviruses are the muridae family of mice, with the global spread of host habitats governing the geographical distribution of the viruses [ ] . ow mammarenaviruses are found in the murinae sub-family of mice restricted to africa. the multimammate rat mastomys natalensis and related species (hylomyscus pamfi and mastomys erythroleucus), distributed around west africa, are the main reservoir hosts of lasv. in contrast, lcmv circulates in the mus musculus host, found globally [ ] . the neotominae and sigmodontinae sub-families of mice, found in north and south america, are the natural reservoir hosts of nw mammarenaviruses, with the exception of the nw clade b arenavirus tacaribe virus (tcrv). tcrv appears to be carried by, and cause disease in, artibeus bats [ , ] . as zoonotic pathogens, mammarenaviruses cause persistent and asymptomatic infections in their natural hosts and transmission to humans usually occurs in rural endemic regions through contact with infectious murine excretions or consumption of rodent meat [ , ] . host and viral factors determine the variability in disease pathogenesis, varying from control of the viral infection and clearance by host immune responses, to a persistent infection, to severe and often fatal haemorrhagic fever. lasv infection frequently presents as an asymptomatic disease in humans; if symptoms do occur, they appear after an incubation period of around days (range - days), characteristically as non-specific flu-like symptoms including malaise, weakness, fever and headaches [ ] . in severe cases, however, patients with lassa fever (lf) can develop haemorrhagic manifestations and multi-organ failure with fatal outcome. lf can have fatality rates as high as % in healthcare settings where nosocomial transmission of lasv infections has been reported, due to exposure to highly viraemic patients under poor hygiene and sanitation conditions and the lack of proper barrier nursing and infection control [ , , ] . human-to-human transmission of nw junv and macv infections related to nosocomial outbreaks has also been reported [ ] . early diagnosis of arenavirus infection in endemic regions is critical but lacking, as symptoms are similar to other endemic diseases such as malaria, typhoid fever and other vhfs, like ebola virus disease [ ] . an approved antiviral treatment does not exist, but evidence suggests that lasv infection partially responds to treatment with the nucleoside analogue ribavirin, particularly during the early stages of infection [ ] [ ] [ ] . administration of ribavirin, however, does not significantly impact on fatality rates when compared to untreated patients, therefore there is an urgent need for improved and effective antivirals [ ] . these limited treatment options highlight the urgent need for continued research into developing antiviral therapeutics to target arenavirus infection. this is further exacerbated by the lack of approved vaccines against arenavirus vhfs. the live-attenuated candid# strain of junv has shown high efficacy against argentine haemorrhagic fever caused by the etiological agent, junv [ , ] . this vaccine, however, is not licensed outside of argentina and has been shown to be ineffective against other arenavirus infections, including macv and lasv. in , the coalition for epidemic preparedness innovations (cepi) accelerated two promising lasv vaccine candidates to the phase clinical trial stage, with data of the trials currently pending. the safety and efficacy evaluations of both the preventative lasv dna vaccine candidate ino- (nct ) and the live attenuated measles (schwartz strain) virus-based vector expressing gpc and np (mev-lasv (nct )) are the first steps towards developing a comprehensive vaccination program against lasv [ ] [ ] [ ] . a major barrier to zoonotic virus infection is the recognition of a suitable cell surface receptor on susceptible human cells. the gp subunit of several ow arenaviruses, including lasv and lcmv, and clade c nw arenaviruses, binds to the widely cell surface-expressed α-dystroglycan (α-dg) receptor ( figure ). ow arenaviruses enter via a clathrin-independent mechanism involving multivesicular body formation and sorting through the endosomal sorting complex required for transport (escrt) pathway. highly pathogenic nw arenaviruses use human transferrin receptor (htrf ), or species-specific orthologs, and enter via clathrin-mediated mechanisms. nw virus particles are delivered to eea -positive endosomes and then to late endosomal compartments in a rab and rab -dependent manner. upon exposure to low ph in the late endosome, conformational changes in the arenavirus glycoprotein lead to fusion and release of the viral genome into the cytoplasm. lassa virus (lasv) particles require a receptor switch to lysosome associated membrane protein (lamp ) in the late endosome. similarly, lujv switches to cd to mediate fusion. fusion following endocytosis of lasv particles requires a gp conformation and ph-triggered receptor switch to the endosomal cellular protein lysosome associated membrane protein (lamp ) [ , ] . similarly, ow lujv adopts an analogous two-step mechanism for entry by binding to the cell surface molecule nrp- (neuropilin- ) and switching to the tetraspanin cd in endosomal compartments, to mediate fusion with cellular membranes (figure ) [ ] . human pathogenic clade b nw arenaviruses utilise the human transferrin receptor (htrf ) for entry, whilst non-pathogenic clade b viruses engage htrf orthologs for entry [ , ] . following receptor engagement, nw virus particles fuse with cellular membranes via clathrin-mediated mechanisms and are delivered to eea -positive endosomes before moving to late endosomal compartments in a rab and rab -dependent manner [ ] . fusion for α-dg-dependent viruses, in contrast, occurs via a clathrin-independent mechanism involving multivesicular body formation and sorting through the endosomal sorting complex required for transport (escrt) pathway ( figure ) [ ] [ ] [ ] . following fusion, viral genome replication and transcription that results in -capped, non-polyadenylated messenger rnas (mrna) encoding the viral proteins, occurs in the cell cytoplasm ( figure ). here, arenaviruses encounter the next crucial barrier: the host cell's innate immune response. this is the initial, non-specific defense system against pathogen invasion that is induced prior to the activation and regulation of the adaptive immune response involving pathogen-specific antibody production and activity of cytotoxic t-lymphocyte (ctl) responses. viral infection commonly produces pathogen-associated molecular patterns (pamps) [ , ] . these unique molecular patterns, such as double stranded rna (dsrna) or -triphosphorylated rna generated during viral rna replication, are readily recognised by immune cells [ ] . arenavirus rnas possess -triphosphate containing panhandle structures that comprise the and ends of the genomic rnas as well as the structured igrs. these pamps can be sensed by host pattern recognition receptors (prrs), including retinoic acid inducible gene (rig-i)-like receptors (rlrs), toll-like receptors (tlrs) and protein kinase r (pkr). these prrs then activate downstream signalling pathways that stimulate an antiviral response in the form of the upregulation or expression of type i interferons (ifn -ifnα and ifnβ), cytokines, proapoptotic factors and the activation and maturation of innate immune cellular arms such as dendritic cells (dcs), t cells and macrophages [ ] . key to counteraction of viral infection is the ifn signalling pathway that is activated upon binding of secreted ifn to target cells. this activation cascade leads to the upregulation or expression of multiple interferon stimulatory genes (isgs). isgs can act on the host or more specifically at numerous stages of the viral life cycle, to inhibit virus growth. intriguingly, host immune responses to different pathogenic and non-pathogenic arenavirus infections, have varying implications on viral pathogenesis and thus therapeutic development. the innate immune response to ow arenavirus infection has been extensively studied during infection with the prototypic member lcmv (reviewed in [ ] ). lcmv infection leads to either an acute infection which is rapidly cleared, or a persistent infection which causes more severe disease in mouse models. the clearance of acute infection is largely due to the robustness of the ifn response early during infection that is observed to surge around - h post-infection, usually - days prior to a peak in viral titer [ , ] . the more substantial an ifn response is in the early stages of infection, the more likely it is that virus-specific cd + cytotoxic t cells will be induced and virus clearance will occur, whereas a weaker ifn response leads to the observed chronic and persistent lcmv infections in mice. target cells for lasv include monocyte-derived dcs (modcs) and macrophages, but infection does not induce ifn or cytokine responses in these cells [ ] . thus, severe cases of lassa fever in humans is characteristically associated with low levels of type i interferons and proinflammatory cytokines such as tumor necrosis factor alpha (tnf-α) and a lack of neutralizing antibody presence, given to the subsequent deficiency in stimulation of t cells, dcs and macrophages [ ] . interestingly, non-pathogenic ow mopeia virus (mopv), a close genetic relative to lasv, induces a strong initial ifn and cytokine response in infected modcs and macrophages leading to a sustained t cell activation and immune response [ , ] . pathogenic nw junv and macv infections induce robust levels of ifn and cytokines and unlike lasv do not suppress the innate immune mechanisms but shift towards a proinflammatory response involving an upregulation of ifn-α and tnf-α. these high cytokine and ifn levels are proposed to correlate with the severity of haemorrhagic disease. these differential responses are triggered by np and z protein-mediated defense mechanisms as summarised in this review. many isgs or host restriction factors, complementary to systemic innate and adaptive immune proteins, represent host cellular proteins that interfere with specific steps of the viral life cycle to inhibit infection. many of these factors are interferon inducible and can potently block viral spread [ ] [ ] [ ] [ ] [ ] . few, briefly described here, have recently been shown to limit arenavirus entry and exit processes. virus entry is a key determinant of viral host range, cellular tropism and disease outcome, hence, targeting this step of the arenavirus life cycle could have significant impact on the control of viral infection. gamma-interferon-inducible lysosomal thiol reductase (gilt) is a soluble thiol reductase that is highly expressed in the endosome and lysosomal compartments of arenavirus target cells such as epithelial cells, dcs, macrophages; and is ifn-γ inducible in other cell types [ , ] . gilt has been implicated to play a role in the processing of endocytosed immune signatures such as viral glycoproteins and in reducing the acidic nature of the lysosome to facilitate proteolysis [ , ] . using lasv gp pseudotyped lentiviral particles, chen and colleagues demonstrated that gilt suppressed the lysosomal entry pathways of lasv; postulating that its thiol reductase activity in lysosomes is required for the restriction, ultimately blocking viral fusion and genome release [ ] . one family of isgs has raised several questions about the entry mechanisms of arenaviruses. the interferon-induced transmembrane (ifitm) family of proteins display broad antiviral activity against the endocytic fusion of enveloped viruses within target cells [ , , ] . in humans, expression of ifitms , and are potently induced by ifn in most cell types but the precise mechanism by which ifitms restrict virus entry is unclear. existing explanations include direct modification of membrane content, structure, rigidity and curvature and indirect modification by altering the function of other membrane host proteins, for example the zinc metalloprotease, zmpste . these proteins are thought to block entry at the sites of fusion within endosomal compartments or at the plasma membrane [ , ] . using arenavirus-gp pseudotyped particles, trafficking and fusion of these particles was observed to occur in endosomes that lack ifitm expression ( figure ). host restriction factors involved in arenavirus infection. the ifitms block entry at the sites of fusion within endosomal compartments or at the plasma membrane. arenavirus particles, however, enter by trafficking through endosomal compartments that lack ifitm expression. fusion in late endosomal compartments is inhibited by gamma-interferon-inducible lysosomal thiol reductase (gilt) expression. viperin inhibits virion assembly by restricting the trafficking on arenavirus glycoproteins to the cell surface. viperin also inhibits np function by restricting recruitment of replication-transcription complexes orchestrated by np, to lipid droplets. the membrane protein tetherin mediates retention of budding virions at the cell surface which are then proposed to be re-internalized and delivered to lysosomes for degradation. two independent studies by suddala et al. and spence, et al. reported that trafficking of lasv-gp pseudotyped particles appeared to bypass endosomes positive for ifitm . however, analysis of lasv gp-mediated cell-to-cell fusion using an assay that rely on exposure to low ph, thus avoiding the need for endosomal trafficking, demonstrated that all three ifitms when expressed in target cells, limited lasv gp pseudotyped fusion [ , ] . this evidence suggests that, instead of being resistant to direct restriction by ifitms (particularly ifitm ), arenaviruses may employ an avoidance mechanism and use alternative endocytic pathways during entry ( figure ) [ ] . towards the end of the viral life cycle, virus assembly and budding have been shown to be targeted by the isgs, viperin and tetherin, respectively ( figure ). viperin (virus inhibitory protein, endoplasmic reticulum associated, interferon inducible) is a conserved endoplasmic reticulum protein with an amphipathic α-helix domain at its n-terminus which serves as an anchor to lipid membranes, and was proposed to inhibit junv assembly and budding [ ] . using an attenuated strain of junv (iv ), peña cárcamo and colleagues showed an induction of viperin expression in junv replicating cells. furthermore, overexpression of viperin reduced efficient virus particle production. the authors proposed that the observed viral glycoprotein mislocalisation in these cells due to lipid raft disruption accounts for the antiviral activity of viperin. further, it was suggested that a viperin-np interaction, shown by immunofluorescence and immunoprecipitation experiments, inhibits the recruitment of replication-transcription complexes (rtc) to lipid droplets which is orchestrated by np, thus inhibiting the function of np in these processes ( figure ). tetherin (also named bone marrow stromal cell antigen (bst- ) and cd ) is a type ii membrane protein inducible by both type i and type ii ifns that is constitutively expressed by plasmacytoid dcs and is expressed by activated t cells [ , [ ] [ ] [ ] [ ] . restriction of the physical release of mature virus progeny from infected cells for a broad spectrum of enveloped viruses by tetherin has been well studied and more recently, lasv, macv and junv have been included in this list. the matrix protein z forms virus-like particles (vlps) that bud from z-expressing cells in the absence of other viral proteins, thus several studies have utilised this intrinsic property to study virus budding and demonstrate an inhibition of lasv and macv vlp release from tetherin over-expressing cells [ ] [ ] [ ] [ ] . more recently, zadeh and colleagues used junv-z vlps to show an inhibition of z-mediated particle release by tetherin and showed that propagation of the candid# vaccine strain of junv was susceptible to tetherin expression [ ] . interestingly, junv infection caused an upregulation of tetherin expression in cells that correlated with increased ifn levels, and a reduction in cell surface expressed tetherin was also observed. evidence for retention and clustering of vlps at the cell surface in the presence of tetherin was visualised by electron microscopy. furthermore, a plausible role of np, in an as yet not understood mechanism in antagonising restriction that does not involve the downregulation of cell surface tetherin expression, has been proposed [ ] . as tetherin also interacts with the host cell endocytic pathway, it is further hypothesised that virions retained at the cell surface by tetherin activity are subjected to re-uptake and trafficking through the endocytic pathway to lysosomes where they are degraded ( figure ) [ ] [ ] [ ] . this is a growing area of research that requires further expansion and understanding of virus-host interactions that limit virus spread, including the identification of other restriction factors that may target replication and translation mechanisms. unravelling the evolution of np and z-mediated countermeasures against these host restriction mechanisms will make significant contributions to our knowledge about the arenavirus life cycle and has the potential to influence the development of therapeutic strategies. pathogenic and non-pathogenic ow and nw mammarenaviruses elicit immune responses that are characterised by either weak or robust ifn induction [ ] [ ] [ ] [ ] [ ] [ ] . several studies, as detailed below, have identified the mechanisms by which np proteins interfere with prr activation and the induction of innate immune signalling that account for these differences in immune response. np is located at the end of the s rna segment and is translated from antigenomic sense mrnas, transcribed directly from viral rnas, thus along with protein l, is one of the first arenavirus proteins encoded upon infection. np is the most abundantly expressed protein and orchestrates viral rna synthesis through binding to viral rna and thus facilitating transcription and replication [ ] . binding of arenavirus np to viral genomic rna is functionally essential to the formation of viral nucleocapsid complexes as well to the suppression of host immune responses ( figure , table ). ( ) irf activation is facilitated by ddx and iκb kinase ε (ikkε) together with tank binding kinase (tbk ). ddx and ikkε are both suppressed by direct binding of viral nps. ( ) irf induces ifn production through translocation to the nucleus, however this translocation is inhibited by arenavirus nps with picv directly binding to irf to inhibit activation. ( ) rig-i activation of irf also induces apoptosis through complex formation with the proapoptotic protein bax which is then translocated to mitochondria. ( ) this leads to cytochrome c production and the downstream activation of apoptosis. ( ) the ring-domain of arenavirus z protein has been implicated in abrogating apoptosis induction by relocalising proapoptotic promyelocytic leukemia protein (pml) from the nucleus where it forms pml-nuclear bodies (nbs) to the cytoplasm, here, it targets apoptotic factors such as bax and caspase activation. the nucleocapsid complexes associate with the l protein, mediating viral ribonucleoprotein (vrnp) assembly, and thus replication and transcription. np also complexes with viral and host proteins to enhance budding and assembly of virions [ ] . biochemical, mutagenesis and structural studies have shown that np is comprised of an n-terminal rna-binding domain that mediates vrnp assembly, linked via a flexible c-terminal - exoribonuclease (deddh family) domain which is highly conserved across the arenavirus family ( figure ) [ , [ ] [ ] [ ] . the n-terminal region possesses a unique fold and distinct function as a cap-binding protein that is proposed to bind ssrna through a gating mechanism of conformational changes. controversially, this is also suggested to bury the entire m gpppn mrna cap structure, with the rest of the mrna molecule outside of the binding cavity [ , ] . important to the rna-binding function of np is the capacity of this protein to self-associate. biochemical and mutagenesis assays have shown that np homo-oligomerisation is required for the replication and transcription functions in forming vrnps [ , ] . the ability of the np protein to antagonise ifn activity has been shown to be conserved by ow lcmv, lasv and nw junv, macv and pichinde virus (picv), therefore this inhibitory function is common in arenaviruses that are pathogenic and also non-pathogenic to humans [ ] . mutagenesis studies on lcmv np by martínez-sobrido and colleagues mapped this inhibitory activity to the c-terminal region of np, identifying amino acid residues , , and to a lesser extent , as critical for antagonising ifn induction [ ] . intriguingly, these lcmv mutants did not affect virus rna replication and the production of infectious virus particles, implying that these functional roles of np are distinct from its anti-ifn activity. these critical residues reside within the highly conserved dieg np motif that spans residues - of ow and nw arenaviruses, including nw tcrv np. martínez-sobrido and colleagues showed that tcrv lacks the ability to counteract the induction of ifn , with contrastingly high levels of ifn-β and isg induction observed in tcrv infected cells, despite productive viral replication and release. given that tcrv np was observed not to inhibit ifn -activity, it is likely that other residues outside of the dieg motif also contribute to the antagonistic function [ ] . indeed, the lcmv c-terminal region spanning - was found to be crucial for counteracting the ifn response and c-terminal deletions of more than residues in the presence of an intact dieg motif weakened this response [ ] . more recent, functional and structural studies on tcrv np have, however, challenged these previous reports and showed that tcrv np can inhibit the ifn pathway effectively, indicating the importance and conservation of this immunosuppressive activity by arenavirus nps [ , ] . subsequent studies observed that this immunosuppressive function of np is attributed to the c-terminal exonuclease activity of the protein [ ] . np possesses high specificity for dsrna-specific degradation in the - direction and residues recognised for their role in ifn suppression reside within the exonuclease active site. studies on non-pathogenic nw picv virus infection in cell culture and animal models, highlighted the role of the five exoribonuclease catalytic residues needed for ifn inhibition, optimal virus propagation and disease pathogenesis. several studies have demonstrated and postulated that degrading immunostimulatory viral dsrnas appears to be a common mechanism of arenaviruses, including lasv, lcmv, mopv, tcrv, and picv, to evade innate immune responses [ , [ ] [ ] [ ] [ ] . residues d , e , d , d and h (lasv protein numbering) comprise the highly conserved exoribonuclease deddh motif (exon). these, in addition to proximally located residues including g , are critical for exon activity and virus viability (figure ) [ , ] . some uncertainty exists, however, about the specificity of the exon activity amongst the arenavirus family surrounding the degradation of dsrna. recent studies by mateer and colleagues showed that np exon activity of lasv effectively degrades dsrna, whereas infection with the highly pathogenic nw arenaviruses macv and junv results in a rapid accumulation of dsrna that is not degraded to a similar degree as in lasv infected cells (figure , table ) [ , ] . mutations of the exon function of lasv np has previously been reported to lead to higher levels of ifn in dcs and also in macrophages when compared with wild-type lasv virus infection using a murine polymerase reverse genetics system [ ] . historically, immunofluorescence labelling of dsrna has been used to visualise the accumulation during rna virus infection. while this method has been effective during infection with positive-sense rna viruses with the widely used j dsrna antibody, this has not been sensitive enough for the low level of dsrna produced during infection with negative-sense rna viruses [ ] . in recent years, the d monoclonal antibody (mab) was developed, initially for use in the diagnosis of pan-enterovirus infection [ ] . this antibody has been shown to have a high affinity for dsrna and can detect the dsrna accumulating during negative-sense rna virus infection including infection with lcmv and junv [ , ] . thus, with the development of the d mab and the use of lasv, junv and macv minigenome replication systems, mateer et al., [ ] were able to visualise dsrna accumulation. they showed that lasv np exon activity was required for limiting dsrna accumulation, strengthened by the observation that mutations disrupting lasv exon activity led to dsrna accumulation. not only did this limitation occur during lasv infection, inhibition of dsrna accumulation during junv infection by co-infection with lasv was measured. interestingly, expression of lasv np alone was not sufficient to limit the accumulation of dsrna in junv infected cells, but expression of both lasv np and l proteins was required to achieve this, suggesting cooperative degradation with viral replication activity ( figure , table ) [ ] . the degradation of virus-derived dsrna may also function to suppress ifn expression by preventing pkr signalling and the downstream phosphorylation of eif α thus inhibiting host and viral protein translation ( figure ) . recently, there have been contradicting reports on the ability of junv to inhibit phosphorylation of eukaryotic translation initiation factor (eif α). a study by king et al. [ ] reports that junv np induces phosphorylation of pkr despite not leading to higher levels of phosphorylated eif α, whereas other published work suggests that junv and macv infections still result in the phosphorylation of eif α [ ] . it is important to note that the study by king et al. [ ] examined cells infected with the attenuated vaccine strain junv candid# , whereas huang et al. [ ] used the highly pathogenic junv romero strain. the latter study showed that the nw arenaviruses junv and macv, but not the ow lasv, lead to high levels of activated pkr thus corresponding with the research presented by mateer et al. [ ] . these results highlight the differences observed between the nw junv and macv infections and ow lasv infections in their ability to degrade dsrna species. the np protein of lasv limits the detection of pamp viral dsrna to prevent host recognition ( figure ) . multiple studies corroborate that this recognition is mediated through direct interaction of np with prrs, rig-i and melanoma differentiation-associated protein (mda)- , as well as downstream effectors such as iκb kinase ε (ikkε), thereby blocking irf activation [ , , , , , ] . upon recognition of viral dsrna carrying the triphosphate, rig-i and mda- undergo conformational multimerization and activation that induces production of the early ifn species through a signalling cascade, firstly through binding of mitochondrial antiviral signalling protein (mavs). formation of these molecular complexes results in the translocation of transcription factors interferon responsive factor (irf) and irf to the nucleus which, along with ap- and nuclear factor (nf)-κb, leads to the production of ifn-α, ifn-β and a number of isgs [ , ] . this signalling cascade usually establishes an antiviral state in neighbouring uninfected cells. this further stimulates immune cell activity such as natural killer (nk) cells, nkt cells, t cells, dcs and macrophages. in depth investigations using lcmv infection regulation of the ifn response, has shown that lcmv np restricts this response by binding directly to rig-i and mda- and can block the translocation of irf to the nucleus thus, reducing ifn-β induction [ , ] . similarly, co-localisation of macv and junv proteins with rig-i has been observed in junv and macv infected cells implying an upregulation of isgs indicative of ifn activation [ ] . interestingly, this co-localisation was not observed between lasv np and rig-i, implying that lasv infection does not activate the ifn-β response in a rig-i dependent manner. mutational analysis of residues within the exon motif of lcmv and lasv were able to abrogate the inhibitory effect of lcmv np on ifn but did not prevent the binding of np to rig-i or mda- , demonstrating that the np-mediated inhibition of ifn is only partially due to the interactions with rig-i and mda- [ , , ] . a crucial aspect of the induction of ifn is the translocation of irf to the nucleus (figure ) . the np proteins of both ow and nw arenaviruses including lcmv, lasv, picv, junv and macv, with the exception of tcrv, can cause a block to both the transcriptional activity and nuclear accumulation of irf . the failure of this tcrv np sequence to inhibit the translocation of irf to the nucleus correlates with the observation of the weaker ifn suppression during tcrv infection, reported by martínez-sobrido and colleagues, and may contribute to the lack of persistent infection of tcrv observed in rodent species [ , , ] . the observed inhibition of irf translocation is, however, insufficient to completely block ifn expression as demonstrated in lcmv infected mice that still elicit a strong ifn response and subsequently a robust host antiviral immune response [ ] . association of lcmv np with the kinase domain of ikkε, via the exoribonuclease domain (exon), has been reported by pythoud and colleagues, suggesting a sequestration of ikkε which subsequently prevents signalling via the mavs pathway to induce innate sensors ( figure , table ) [ ] . a amino acid protein known as pact was shown to enhance rig-i function through direct interaction [ ] . pact comprises dsrna-binding motifs (dsrbms) that bind to dsrna (dsrbm and dsrbm ) and to pkr via dsrbm [ ] . pact interacts with the c-terminal repression domain of rig-i, whilst also activating the atpase function of rig-i to potently enhance ifn production. pact potentiates the function of rig-i at a similar level to that of dsrna rig-i ligands. lasv np has been shown to block the pact-mediated regulation of rig-i function, in an exon activity-dependent manner [ ] . np does not interfere with the physical interaction between pact and rig-i, but as shao and colleagues infer, np may degrade dsrna that is associated with, and essential for the pact/rig-i complex function, thereby permitting efficient replication in infected cells in the presence of a dampened immune response ( figure , table ). this effect could be rescued in the case of a recombinant picv virus expressing an rnase-defective np mutation, where virus growth was not measured in wild type mouse embryonic cells [ ] . lasv np function has therefore evolved to counteract stimulation of ifn production, through a mechanism that indirectly blocks the activation of rig-i. arenaviruses also activate the innate immune response through the activity of tlrs following the binding of viral pamps, thus promoting an ifn response [ ] . nw junv induces activation of a tlr /tlr complex leading to the activation of transcription factors ap- and nf-κb and initiation of innate and adaptive responses [ ] [ ] [ ] [ ] [ ] [ ] . only tlr has been implicated to play a role in ow arenavirus pathogenesis and studies by hayes et al. have postulated that the observed immunosuppressive phenotype and low level proinflammatory cytokines associated with ow lasv infection is due to a suppression of tlr -dependent induction of cytokine responses [ ] . in addition to the rlrs and tlrs, accumulated dsrna during nw junv, junv candid# vaccine strain and macv infections also activates the ubiquitously expressed host dsrna sensor, pkr. in infected cells, junv np has been shown to interact with pkr suggesting a localised inhibition of pkr activity to enhance viral protein synthesis [ , ] . upon dsrna interaction and binding, pkr undergoes autophosphorylation. enzymatically activated pkr then initiates the downstream phosphorylation of eif α, thus inhibiting the translation of cellular and viral-expressing mrnas in infected cells [ ] [ ] [ ] . pkr enhances ifn production by stabilising ifn mrna and also activates the transcription factor nf-κb through phosphorylation of iκb [ , ] . interestingly, junv and macv readily activate pkr seemingly to enhance viral replication through the augmentation of ifn and isg gene translation, rather than negatively impact viral infection [ ] . similarly, ow lcmv has been shown by king and colleagues to strongly activate pkr but conversely to junv, is unable to suppress the kinase activity of pkr, given that transient eif α phosphorylation was observed during lcmv infection. this suggests that lcmv may possess alternative mechanisms to inhibit pkr activity or reflects the instability of pkr control over lcmv infection [ ] . in contrast, ow lasv infection does not regulate or stimulate pkr activation. this pathway is instead evaded by an unknown mechanism [ ] . therefore, lasv, unlike other arenaviruses, successfully avoids detection by pkr. (figure , table ). in recent years, large scale proteomics studies have aimed to provide a global picture of np host protein interactions [ , , ] . through this work, the dead (asp-glu-ala-asp)-box atp-dependent rna helicase, ddx has been identified as a host interaction partner. ddx is thought to play a complex role in host antiviral immunity, acting as a transcriptional regulator of ifn-β promoter and as a viral sensor by interacting with rig-i and mavs and a downstream signal transducer of tank-binding kinase i (tbk ) and ikkε [ ] [ ] [ ] [ ] . interestingly, reports indicate that ddx regulates the expression of pact, thereby inferring a possible strategy that viruses use to counteract antiviral innate immunity [ ] . loureiro and colleagues identified ddx as an lcmv np partner by mass spectrometry of immunoprecipitates from lcmv np overexpressing cells [ ] . subsequent infection of ddx knockout cell lines with lcmv or lasv resulted in reduced virus propagation; an activity that occurred early in infection, independently of ifn . mutagenesis studies revealed that both the atpase and helicase domains are implicated in this role. an extension to the role of ddx in suppressing ifn production may occur late in lcmv infection, where ddx abrogates ifn-β transcription. interactions of the np of nw junv, macv and tcrv with ddx have been confirmed by immunoprecipitation assays or mass spectrometry analysis, with loureiro and colleagues demonstrating that like ow arenaviruses, junv virus growth required ddx expression [ , ] . np may therefore promote viral spread by sequestering ddx from macromolecular complexes, including ikkε, rig-i or mavs, that promote ifn synthesis [ ] . in addition to the stimulation of the innate immune response upon viral infection, cells can also induce their own death in response to cell stress and damage caused by pathogen invasion, through a mechanism of programmed cell death, known as apoptosis [ ] . this effectively limits the spread of pathogens and aids in virus clearance from the host. in order to establish an effective and persistent infection, it is critical, therefore, for viruses to evolve mechanisms of evading the apoptotic pathway or even by using some of the components of this pathway to their benefit [ ] . notably, highly pathogenic junv, lasv and lcmv circumvent the induction of apoptosis throughout infection whereas the less pathogenic tcrv and the junv candid# vaccine strain induce a robust, caspase-dependent apoptosis response. it is important, however, to note that the growth kinetics of tcrv are similar to those of junv, suggesting that induction of apoptosis does not negatively affect tcrv proliferation [ , , , ] . the induction of apoptosis by junv candid# and tcrv has further been characterised to occur in a rig-i-dependent and ifn -independent manner in vitro [ , ] . while the ifn response requires irf to translocate to the nucleus, rig-i-dependent induction of apoptosis activates irf via mavs and causes irf to interact with the proapoptotic protein bax. this complex is then transported in the mitochondria which then releases cytochrome c and leads to autocatalytic cleavage of caspase- and caspase- ( figure ) [ , ] . an additional function of the arenavirus protein np has been hypothesised in relation to the induction of apoptosis in infected cells. it has been observed that the np proteins of several arenaviruses exist in a number of truncated forms [ , , ] . however, it is not known how, or indeed if these truncations are required for virus replication, assembly or budding. instead it has been suggested that these truncations are used as a mechanism of suppressing the induction of apoptosis in infected cells. cleavage of caspases is required for completion of the apoptosis pathway; in junv infected cells treated with pan-caspase inhibitors, it was observed that formation of the truncated forms of np was abrogated [ ] . further, expression of junv np alone by transfection was sufficient to induce caspase cleavage of np in a similar manner and led to the identification of several caspase cleavage target site motifs. mutation of these motifs resulted in loss of the truncated forms of junv np and consequently increased levels of apoptosis [ ] . these findings indicate a decoy function of arenavirus np as a substrate for caspase cleavage which inhibits induction of apoptosis and aids in virus dissemination. this decoy substrate hypothesis requires further investigation in the case of other arenaviruses to confirm to what extent this may be a universal strategy of apoptosis suppression by the highly pathogenic arenaviruses. indeed, it has been reported that other arenaviruses including lasv and picv produce truncated forms of the np protein whereas tcrv does not [ , , [ ] [ ] [ ] . this correlated with the observation that the highly pathogenic junv and lasv inhibit apoptosis induction while tcrv infection induces a strong caspase-dependent apoptosis response, that yet, does not impair virus replication. hence, tcrv appears to lack an np-mediated anti-apoptotic function and is likely able to modulate apoptosis using diverse strategies [ , , , ] . complementary work by wolff and colleagues showed that tcrv replication and transcription can induce caspase-dependent apoptosis that does not limit virus growth but is regulated by z protein expression [ ] . this implies that tcrv may exploit the apoptosis pathway in order to enhance virus replication and release, and possibly to evade host immune responses (section ) [ ] . the zinc-finger matrix protein z is the smallest gene product encoded by the l segment of the arenavirus genome. z proteins of ow and nw arenaviruses possess an n-terminal myristoylation site for insertion into the cell membrane, a central zinc-binding ring finger protein motif and c-terminal late-domain motifs essential for interactions with the cellular escrt machinery to facilitate virus budding (figure ) [ , , ] . the nmr structure of the z protein monomer highlights the intrinsically flexible n-and c-terminal domains that flank the ring domain that binds two co-ordinated zinc atoms. this intrinsic conformational flexibility of the small matrix protein z may play a significant role in the ability of this protein to adapt to its roles in viral assembly, immune evasion and the regulation of replication and transcription through interaction with various host and viral partners [ , , ] . the most recent structure of protein z shows that it can be crystallised in a dodecameric form. stabilisation of monomeric lasv z by mutagenesis enhances the negative regulation of replication. in contrast, stabilising the oligomeric, dodecamer state impairs the negative regulatory function, implying that the monomeric and oligomeric forms have differing and possibly opposing functions [ , ] . it will be interesting to further elucidate the molecular details of how protein interactions are modulated by these structural changes to maintain viral replication. [ ] . the n-terminal myristoylation is highly conserved amongst the ow and nw arenavirus z proteins, hence, z is strongly membrane associated and in the absence of other arenavirus proteins, can form and release enveloped vlps from the cell surface. through the recruitment of np within ribonucleoprotein complexes present and enriched at gp and gp patches at the cell surface, z drives the assembly of mature virus progeny [ ] . the ring finger protein motif of z is involved in various protein-protein interactions important for regulating multiple stages of the virus life cycle. lcmv z was shown to interact with promyelocytic leukaemia protein (pml), a regulator of cell growth, leading to its redistribution from nuclear bodies (nbs) to cytoplasmic bodies, where pml is involved in a number of apoptosis regulating pathways, implying a role in abrogating apoptosis induced upon infection (figure ) [ ] . further, pml and z bind to ribosomal p proteins (p , p and p ) in the nucleus thereby implying a role in regulating protein translation [ , ] . further, the lcmv pml-z interaction has been proposed to affect virus production in mouse embryonic fibroblasts inferring an impairment in transcription activity due to the interaction with pml [ ] . interestingly, pml is an ifn-induced protein and has been implicated to mediate anti-viral mechanisms against influenza virus replication amongst other virus families that involves the regulation of the ifn promoter and the interferon stimulated response element (isre) [ , ] . the accumulation of tcrv z protein during infection enhances the induction of apoptosis, hence it could be speculated that tcrv utilises this pathway to facilitate replication through a mechanism that involves pml-tcrv z interactions. therefore, during tcrv induced apoptosis, redistribution of pml from nbs to the cytoplasm driven by tcrv z could counteract the induction of ifn responses; a mechanism linked to the lack of impaired virus replication observed [ ] . campbell dwyer and colleagues were able to demonstrate that lcmv z also interacts with the eukaryotic translation initiation factor e (eif e) that is crucial for mrna nuclear cytoplasmic transport, for assembly of transcripts onto polysomes and for initiation of translation. by binding to eif e, z is able to suppress protein production at the post-transcriptional and post-rna transport level. therefore, preferential translation of viral transcripts over cellular mrna in a self-regulating mechanism that occurs later in the infection process may be linked to the viral persistence in chronic infections [ , ] . the proline-rich homeodomain (prh), a cellular transcription factor that regulates the development of the brain, thyroid and liver, also associates with the z protein ring domain. binding of the z protein of pathogenic and non-pathogenic strains of lcmv with prh has been observed, but prh-downregulation by pathogenic lcmv alone in human hepatic cell lines implies that z suppresses the antiproliferative effects of prh, hence stimulating cell division that is supportive of viral replication and disease pathogenesis in the absence of liver cell regeneration [ ] . whether other viral and host proteins are involved in this downregulation of prh remains unclear. the c-terminal portion of z contains small conserved tetrapeptide (p[t/s]ap-and/or ppxy-type) motifs, known as late domains, that drive virus particle release through the recruitment of escrt proteins that result in the final cellular membrane scission stage required for budding [ , , , ] . while the important role of the escrt machinery and escrt-associated host factors in arenavirus budding is well documented [ , [ ] [ ] [ ] , the exact molecular mechanism underlying escrt protein recruitment and function to promote virus release remains to be fully elucidated. for example, the p[t/s]ap and ppxy motifs vary substantially both in their number and combination between different z species, suggesting that arenaviruses have evolved different strategies to gain access to the escrt pathway [ ] . in addition, both ow and nw z proteins possess a conserved yxxl-type late domain located within the central ring domain. in the case of nw tcrv and ow mopv, the yxxl motif does not contribute to the self-budding activity of z, but it is critical for np-mediated enhancement of z-driven vlp budding as well as the incorporation of np into vlps [ , , ] . using a yeast two-hybrid screen, baillet and colleagues recently uncovered the interaction of two members of the nedd family of hect e ubiquitin ligases, itch and wwp with z proteins of mopv and lasv [ ] . they observed that itch was needed for efficient production of infectious virus particles of lcmv, lasv, lujv and mopv; and found that direct interaction with this pro-viral host factor was dependent on the ppxy late domain of lasv and mopv. thus, this host protein acts as a positive regulator of the late stages of virus infection by enhancing the processes of viral release and virus production [ ] . independently, a proteomics study conducted by ziegler and colleagues identified nedd family ubiquitin ligases, including itch and wwp , as partners of the lcmv z protein, binding specifically to the ppxy motif of lcmv z. they demonstrated that these ligases ubiquitinate lcmv z, a process that was dispensable for virus release but needed for defective interfering (di) particle release. these data infer that ubiquitination of other cellular or viral targets than the z protein by nedd ligases may be the essential link to the escrt machinery and enhancement of viral budding [ ] . alongside ubiquitination as a regulator of viral budding, ziegler and colleagues identified two phosphorylation sites, y and s (lasv numbering), located at the c-terminal tail of protein z and overlapping with the late domain region that could also influence z protein function. these sites are postulated to play a role in regulating virus budding and opens up new research questions surrounding the host protein binding repertoire of z protein, given the protein's conformational flexibility, as well as the corresponding functions related to these new findings [ ] . like np, the z protein is also able to regulate the host cell interferon system [ , ] . sequestration of eif e by the z protein as discussed previously is one such mechanism. this can lead to repression of the production of key host regulators of ifn immune responses, such as irf- that is crucial for the enhanced transcription of ifn genes, including ifn-β and ifn-α genes [ ] . similar to np, interaction of arenavirus z proteins with rig-i prevents further binding to mavs and therefore inhibits the production of ifn-β and reduces antiviral host immune responses. xing and colleagues reported the binding of all z proteins of pathogenic arenaviruses, including lasv, lcmv, junv, chapv, sabv, and gtov to rlrs, via the n-terminal domain, leading to a suppression of ifn through inhibition of the rig-i-mavs interaction (figure ) [ ] . this finding was further strengthened by swapping the n-terminal domain of the non-pathogenic picv with the n-terminal domain of lcmv or lasv z proteins. recombinant picv expressing lcmv or lasv z protein n-terminal domain was able to bind rig-i whereas the wild-type picv was not [ ] . research from the same lab also confirmed this by showing that expression of the lcmv z as a chimeric protein in the non-pathogenic picv was sufficient to bind rig-i and inhibit macrophage activity [ ] . although the inhibition of rig-i signalling by highly pathogenic arenaviruses in vitro suggests that there should be limited ifn response, it is notable that in vivo infection with some of these viruses (lcmv, junv) induces high levels of ifn , isgs and cytokines suggesting that this strategy is not completely effective or is widely varied in cell type and differs from host to host [ , , ] . activation of plasmacytoid dcs (pdc) in response to infection is important for the potent induction of ifn and the activation of other immune cell types. it has been shown that non-pathogenic mopv stimulates a strong pdc response, while pathogenic lasv weakly activates these cells and the response is short-lived [ ] . this infers that the impaired pdc activation could be a critical factor in the immunosuppression observed during lasv infection. in the study by schaeffer and colleagues, mopv z protein was detected in pdcs in a pdc /infected vero e co-culture model at a higher level than lasv z protein [ ] . thus, arenavirus z proteins may be involved in the activation of pdcs early in infection, although lasv z proteins appear to be functionally less capable of potently activating ifn pathways in pdcs. conversely, in a study that highlighted the importance of myeloid dc (mdc) activation in the immune response to lasv infection, a lasv/mopv chimera (in which the mopv z protein was swapped into the lasv genome), induced a low level ifn response, compared to wild-type lasv infection in an mdc/t cell co-culture model [ ] . this suggests that mopv z protein, unlike previous findings, is not a modulator of immunogenicity [ , ] . further, during the development of the measles virus (mev)-based lasv vaccine candidate, currently being evaluated in phase clinical trials, mateo and colleagues generated and tested mev vectors expressing lasv gpc alone or in combination with np or z protein for the induction of immune responses in macaques [ ] . the authors observed that mev-z+gpc vector induced a delayed and diminished ifn response in immunised animals, compared to other mev-lasv vector immunised animals. this lack of ifn induction had downstream effects on specific t cell stimulation and on innate and adaptive immune pathways as detected at the transcriptomic level [ ] . these findings further imply that in this study, lasv z is an inefficient activator of ifn and t cell responses and provides evidence for the multifaceted role of arenavirus z proteins in subverting host innate immune pathways. currently, very limited strategies exist to treat arenavirus infection and rely on early treatment with ribavirin and its analogues, that have very little to no impact on fatality rates [ , ] . in recent years, in addition to the search for effective, preventative vaccines, a number of reports have identified possible protein-specific inhibitors to treat arenavirus infection, but have focused on compounds that interact with the arenavirus glycoproteins and inhibit the entry processes [ ] [ ] [ ] . given the multifaceted roles that np and z possess to counter host immunity, these proteins could be targeted for the rational design of specific and efficient antiviral therapeutics. some examples include the treatment of arenaviruses with aliphatic and aromatic disulphide-and azoic-based compounds that target the zinc-binding structure of the z protein leading to the oxidation of cysteine thiolates and protein aggregation. in these studies against lcmv, junv, tcrv and picv, garcia and colleagues observed inhibition of virus infectivity and rna synthesis highlighting the potential of these z-reactive compounds. further, these compounds blocked an interaction of z with the host pml leading to the formation of nuclear bodies and a decrease in virus proliferation [ , ] . in addition, myristic acid analogs were found, in a dose-dependent manner, to inhibit virus production, affecting the localisation of the z protein and the assembly of junv virions, thus targeting protein myristoylation, essential to the function of z and its involvement in virus propagation [ ] . unlike the z protein, few np target compounds have been identified. a potent inhibitor of lcmv replication and budding, kp- , was identified from a combinatorial library of krönhke pyridines by miranda and colleagues, to block the protein-protein interactions of np and z; whether these are virus-virus or host-virus interactions remains to be determined [ ] . in summary, with the present lack of potential anti-viral drugs and effective vaccines, and the exacerbation of their need by the current lasv outbreaks in west africa, np and z remain important targets for novel therapeutic strategies. arenaviruses utilise host cellular machinery to negotiate host defenses. the remarkably multifunctional viral proteins np and z have evolved distinct and synergistic mechanisms to evade the antiviral state induced upon virus infection [ , , ] . both proteins are able to counteract rig-i mediated production of ifn thereby inhibiting protein expression and thus dampening innate immune responses; and are able to use their conformational flexibility and protein interaction promiscuity to expand host binding partners to elicit their evasion strategies [ , , ] . arenavirus infections in humans with ow and nw viruses can vary from asymptomatic to severe. importantly, differences in the interactions of np and z proteins amongst arenaviruses with the rig-i, pkr and other immune pathways discussed here, may contribute to this variation in arenavirus pathogenicity and to the molecular determinants of arenavirus virulence. given the differential immune responses induced particularly by nps of pathogenic and non-pathogenic arenaviruses, and the lack of direct correlation with disease outcome, it is clear that multiple viral and host genes and the evolution of their protein interactions determines virulence. np and z are major contributors to the shape of host antiviral mechanisms. therefore, developing np and z-specific antivirals, coupled with monitoring the evolution of these proteins as the host adapts to their evasive mechanisms, could be used to regulate the control that arenaviruses have on the host immune system, and represents a vital approach to combat these public health and socio-economic burdens [ , ] . author contributions: all authors listed compiled and wrote the manuscript agreed for publication. all authors 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virus-like particles cytoplasmic tails of bunyavirus gn glycoproteins-could they act as matrix protein surrogates? virology comparative analysis of disease pathogenesis and molecular mechanisms of new world and old world arenavirus infections interrelationships among arenaviruses measured by indirect immunofluorescence arenavirus phylogeny: a new insight the phylogeny of new world (tacaribe complex) arenaviruses. virology molecular phylogeny of the arenaviruses past, present, and future of arenavirus taxonomy genetic detection and characterization of lujo virus, a new hemorrhagic fever-associated arenavirus from southern africa multifunctional nature of the arenavirus ring finger protein z. viruses venezuelan hemorrhagic fever: clinical and epidemiological studies of cases junin virus vaccines epidemiology and pathogenesis of bolivian hemorrhagic fever new hosts of the lassa virus tacaribe virus, a new agent isolated from artibeus bats and mosquitoes in trinidad, west indies mammalian reservoirs of arenaviruses at home with mastomys and rattus: human-rodent interactions and potential for primary transmission of lassa virus in domestic spaces zoonotic diseases: who gets sick, and why? explorations from africa diagnostics for lassa fever virus: a genetically diverse pathogen found in low-resource settings containing a lassa fever epidemic in a resource-limited setting: outbreak description and lessons learned from abakaliki treatment of arenavirus infections: from basic studies to the challenge of antiviral therapy effective therapy with ribavirin lassa fever in post-conflict sierra leone argentine hemorrhagic fever vaccines dose-ranging study: safety, tolerability and immunogenicity of ino- in healthy volunteers in ghana a trial to evaluate the optimal dose of mv-lasv vaccines inducing immunity to lassa virus glycoprotein and nucleoprotein protect macaques after a single shot lamp increases the efficiency of lassa virus infection by promoting fusion in less acidic endosomal compartments virus entry. lassa virus entry requires a trigger-induced receptor switch nrp and cd are host factors for lujo virus cell entry host-species transferrin receptor orthologs are cellular receptors for nonpathogenic new world clade b arenaviruses transferrin receptor is a cellular receptor for new world haemorrhagic fever arenaviruses different mechanisms of cell entry by human-pathogenic old world and new world arenaviruses acidic ph triggers lcmv membrane fusion activity and conformational change in the glycoprotein spike old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport innate immune evasion strategies of dna and rna viruses interaction of the innate immune system with positive-strand rna virus replication organelles innate immune recognition of viral infection sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion complexities of type i interferon biology: lessons from lcmv. viruses inhibition of innate immune responses is key to pathogenesis by arenaviruses persistent virus infection inhibits type i interferon production by plasmacytoid dendritic cells to facilitate opportunistic infections lassa virus infection of human dendritic cells and macrophages is productive but fails to activate cells hemorrhagic fever-causing arenaviruses: lethal pathogens and potent immune suppressors human dendritic cells infected with the nonpathogenic mopeia virus induce stronger t-cell responses than those infected with lassa virus non-pathogenic mopeia virus induces more robust activation of plasmacytoid dendritic cells than lassa virus intrinsic immunity: a front-line defense against viral attack hiv- and interferons: who's interfering with whom? resistance of transmitted founder hiv- to ifitm-mediated restriction characterization of the alpha interferon-induced postentry block to hiv- infection in primary human macrophages and t cells tetherin inhibits retrovirus release and is antagonized by hiv- vpu enzymatic reduction of disulfide bonds in lysosomes: characterization of a gamma-interferon-inducible lysosomal thiol reductase (gilt) molecular and biochemical characterization of a novel gamma-interferon-inducible protein defective cross-presentation of viral antigens in gilt-free mice expanding roles for gilt in immunity gilt restricts the cellular entry mediated by the envelope glycoproteins of sars-cov, ebola virus and lassa fever virus ifitm- and ifitm- but not ifitm- restrict rift valley fever virus more than meets the i: the diverse antiviral and cellular functions of interferon-induced transmembrane proteins ifitm restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion hang, h.c. ifitm directly engages and shuttles incoming virus particles to lysosomes interferon-induced transmembrane protein blocks fusion of sensitive but not resistant viruses by partitioning into virus-carrying endosomes the interplay between viperin antiviral activity, lipid droplets and junín mammarenavirus multiplication interplay between ovine bone marrow stromal cell antigen /tetherin and endogenous retroviruses antiviral inhibition of enveloped virus release by tetherin/bst- : action and counteraction tetherin inhibits hiv- release by directly tethering virions to cells the interferon-induced protein bst- restricts hiv- release and is downregulated from the cell surface by the viral vpu protein infectious lassa virus, but not filoviruses, is restricted by bst- /tetherin inhibition of lassa and marburg virus production by tetherin dimerization of tetherin is not essential for its antiviral activity against lassa and marburg viruses human bst- /tetherin inhibits junin virus release from host cells and its inhibition is partially counteracted by viral nucleoprotein hiv- vpu promotes release and prevents endocytosis of nascent retrovirus particles from the plasma membrane clathrin-mediated endocytosis of a lipid-raft-associated protein is mediated through a dual tyrosine motif virus kept on a leash cap binding and immune evasion revealed by lassa nucleoprotein structure efficient budding of the tacaribe virus matrix protein z requires the nucleoprotein structure of the lassa virus nucleoprotein reveals a dsrna-specific ' to ' exonuclease activity essential for immune suppression crystal structure of the lassa virus nucleoprotein-rna complex reveals a gating mechanism for rna binding identification of two functional domains within the arenavirus nucleoprotein self-association of lymphocytic choriomeningitis virus nucleoprotein is mediated by its n-terminal region and is not required for its anti-interferon function differential inhibition of type i interferon induction by arenavirus nucleoproteins identification of amino acid residues critical for the anti-interferon activity of the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus identification of critical amino acids within the nucleoprotein of tacaribe virus important for anti-interferon activity structures of arenaviral nucleoproteins with triphosphate dsrna reveal a unique mechanism of immune suppression a vaccine platform against arenaviruses based on a recombinant hyperattenuated mopeia virus expressing heterologous glycoproteins in vitro and in vivo characterizations of pichinde viral nucleoprotein exoribonuclease functions structure of the lcmv nucleoprotein provides a template for understanding arenavirus replication and immunosuppression lassa virus nucleoprotein mutants generated by reverse genetics induce a robust type i interferon response in human dendritic cells and macrophages lassa virus, but not highly pathogenic new world arenaviruses, restricts immunostimulatory double-stranded rna accumulation during infection visualization of double-stranded rna colocalizing with pattern recognition receptors in arenavirus infected cells double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses double-stranded rna is detected by immunofluorescence analysis in rna and dna virus infections, including those by negative-stranded rna viruses a map of the arenavirus nucleoprotein-host protein interactome reveals that junín virus selectively impairs the antiviral activity of double-stranded rna-activated protein kinase (pkr) highly pathogenic new world arenavirus infection activates the pattern recognition receptor protein kinase r without attenuating virus replication in human cells inhibition of the type i interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus arenavirus nucleoprotein targets interferon regulatory factor-activating kinase ikkε lymphocytic choriomeningitis virus differentially affects the virus-induced type i interferon response and mitochondrial apoptosis mediated by rig-i/mavs arenavirus nucleoproteins prevent activation of nuclear factor kappa b arenaviral nucleoproteins suppress pact-induced augmentation of rig-i function to inhibit type i interferon production crystal structure of junin virus nucleoprotein arenaviridae exoribonuclease presents genomic rna edition capacity assays to demonstrate the roles of arenaviral nucleoproteins (nps) in viral rna synthesis and in suppressing type i interferon the c-terminal region of lymphocytic choriomeningitis virus nucleoprotein contains distinct and segregable functional domains involved in np-z interaction and counteraction of the type i interferon response exonuclease domain of the lassa virus nucleoprotein is critical to avoid rig-i signaling and to inhibit the innate immune response immune signaling by rig-i-like receptors involvement of the irf family transcription factor irf- in virus-induced activation of the ifn-beta gene induction and inhibition of type i interferon responses by distinct components of lymphocytic choriomeningitis virus persistent lcmv infection is controlled by blockade of type i interferon signaling the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response pact, a protein activator of the interferon-induced protein kinase roles of arenavirus z protein in mediating virion budding, viral transcription-inhibition and interferon-beta suppression toll-like receptors: significance, ligands, signaling pathways, and functions in mammals junín virus infects mouse cells and induces innate immune responses toll-like receptor -mediated innate immune responses against junín virus in mice lead to antiviral adaptive immune responses during systemic infection and do not affect viral replication in the brain pathogenic old world arenaviruses inhibit tlr /mal-dependent proinflammatory cytokines in vitro multiple mechanisms contribute to impairment of type interferon production during chronic lymphocytic choriomeningitis virus infection of mice plasmacytoid dendritic cells are productively infected and activated through tlr- early after arenavirus infection toll-like receptor is required for effective adaptive immune responses that prevent persistent virus infection pkr-a protein kinase regulated by double-stranded rna impact of protein kinase pkr in cell biology: from antiviral to antiproliferative action. microbiol regulation of eukaryotic protein synthesis by initiation factors double-stranded rna-dependent protein kinase activates transcription factor nf-kappa b by phosphorylating i kappa b reis e sousa, c. protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity characterization of host proteins interacting with the lymphocytic choriomeningitis virus l protein ddx suppresses type i interferons and favors viral replication during arenavirus infection human dead box helicase couples iκb kinase ε to interferon regulatory factor activation dead/h box (ddx ) helicase binds the rig-i adaptor ips- to up-regulate ifn-beta-inducing potential viral targeting of dead box protein reveals its role in tbk /ikkepsilon-mediated irf activation the dead-box helicase ddx x is a critical component of the tank-binding kinase -dependent innate immune response ddx functions in antiviral innate immunity through translational control of pact cleavage of the junin virus nucleoprotein serves a decoy function to inhibit the induction of apoptosis during infection the new world arenavirus tacaribe virus induces caspase-dependent apoptosis in infected cells rig-i enhanced interferon independent apoptosis upon junin virus infection comparison of the innate immune responses to pathogenic and nonpathogenic clade b new world arenaviruses viral apoptosis is induced by irf- -mediated activation of bax the irf- /bax-mediated apoptotic pathway, activated by viral cytoplasmic rna and dna, inhibits virus replication apoptosis during arenavirus infection: mechanisms and evasion strategies structural and cell-associated proteins of lassa virus characterization of polypeptides immunoprecipitable from pichinde virus-infected bhk- cells localization of an arenavirus protein in the nuclei of infected cells crystal structure of the oligomeric form of lassa virus matrix protein z the completed sequence of lymphocytic choriomeningitis virus reveals a unique rna structure and a gene for a zinc finger protein nmr assignment of the arenaviral protein z from lassa fever virus structural characterization of the z ring-eif e complex reveals a distinct mode of control for eif e the lymphocytic choriomeningitis virus matrix protein ppxy late domain drives the production of defective interfering particles the role of myristoylation in the membrane association of the lassa virus matrix protein z an arenavirus ring (zinc-binding) protein binds the oncoprotein promyelocyte leukemia protein (pml) and relocates pml nuclear bodies to the cytoplasm two ring finger proteins, the oncoprotein pml and the arenavirus z protein, colocalize with the nuclear fraction of the ribosomal p proteins role and fate of pml nuclear bodies in response to interferon and viral infections the lymphocytic choriomeningitis virus ring protein z associates with eukaryotic initiation factor e and selectively represses translation in a ring-dependent manner the ring domains of the promyelocytic leukemia protein pml and the arenaviral protein z repress translation by directly inhibiting translation initiation factor eif e the proline-rich homeodomain (prh/hex) protein is down-regulated in liver during infection with lymphocytic choriomeningitis virus molecular mechanism of arenavirus assembly and budding virus budding and the escrt pathway the microtubule motor protein kif a is involved in intracellular trafficking of the lassa virus matrix protein alix/aip is required for np incorporation into mopeia virus z-induced virus-like particles cellular factors required for lassa virus budding the z protein of the new world arenavirus tacaribe virus has bona fide budding activity that does not depend on known late domain motifs e ligase itch interacts with the z matrix protein of lassa and mopeia viruses and is required for the release of infectious particles nedd family ubiquitin ligases associate with lcmv z's ppxy domain and are required for virus budding, but not via direct ubiquitination of z host-driven phosphorylation appears to regulate the budding activity of the lassa z proteins of new world arenaviruses bind rig-i and interfere with type i interferon induction the z proteins of pathogenic but not nonpathogenic arenaviruses inhibit rig-i-like receptor-dependent interferon production differential inhibition of macrophage activation by lymphocytic choriomeningitis virus and pichinde virus is mediated by the z protein n-terminal domain functional role of type i and type ii interferons in antiviral defense lassa virus activates myeloid dendritic cells but suppresses their ability to stimulate t cells (favipiravir) inhibition of arenavirus replication in cell culture unique small molecule entry inhibitors of hemorrhagic fever arenaviruses targeting virulence mechanisms for the prevention and therapy of arenaviral hemorrhagic fever identification of clotrimazole derivatives as specific inhibitors of arenavirus fusion arenavirus z protein as an antiviral target: virus inactivation and protein oligomerization by zinc finger-reactive compounds an antiviral disulfide compound blocks interaction between arenavirus z protein and cellular promyelocytic leukemia protein myristic acid analogs are inhibitors of junin virus replication mining a kröhnke pyridine library for anti-arenavirus activity arenavirus variations due to host-specific adaptation key: cord- -aqwlcs g authors: uematsu, jun; koyama, aoi; takano, sayaka; ura, yukari; tanemura, miho; kihira, sahoko; yamamoto, hidetaka; kawano, mitsuo; tsurudome, masato; o’brien, myles; komada, hiroshi title: legume lectins inhibit human parainfluenza virus type infection by interfering with the entr date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: aqwlcs g three lectins with different sugar binding specificities were investigated for anti-viral activity against human parainfluenza virus type (hpiv- ). the lectins, concanavalin a (con a), lens culinaris agglutinin (lca) and peanut agglutinin (pna), inhibited cell fusion and hemadsorption induced by hpiv- . virus nucleoprotein (np) gene synthesis was largely inhibited, but fusion (f) and hemagglutinin-neuraminidase (hn) gene syntheses were not. an indirect immunofluorescence study showed that con a inhibited virus np, f and hn protein syntheses, but lca did not completely inhibit them, and that pna inhibited only np protein synthesis. using a recombinant green fluorescence protein-expressing hpiv- , without matrix protein (rghpiv- Δm), it was found that virus entry into the cells was not completely prevented. the lectins considerably reduced the number of viruses released compared with that of virus infected cells. the lectins bound to cell surface within min, and many aggregates were observed at min. con a and lca slightly disrupted actin microfilaments and microtubules, but pna had almost no effect on them. these results indicated that the inhibitory effects of the lectins were caused mainly by the considerable prevention of virus adsorption to the cells by the lectin binding to their receptors. human parainfluenza virus type (hpiv- ) is one of the major human respiratory tract pathogens of infants and children. hpiv- is a member of the genus rubulavirus in the family paramyxoviridae, and it possesses a single-stranded non-segmented and negative stranded rna genome of , nucleotides [ ] . hpiv- has structural proteins, nucleoprotein (np), v, phosphoprotein (p), matrix (m), fusion (f), hemagglutinin-neuraminidase (hn) and large (l) proteins. the gene order of hpiv- is '-(leader)-np-v/p-m-f-hn-l-(trailer)- '. all genes of hpiv- were sequenced by our group [ ] [ ] [ ] [ ] [ ] [ ] . monoclonal antibodies (mabs) were made by tsurudome, and antigenic diversity of clinical isolates was investigated [ ] . the infectious hpiv- from cdna clone was constructed by kawano, and it was shown that its growth property was the same as that of hpiv- [ ] . cytoskeleton was reported to have an important role in paramyxovirus replication. actin microfilaments are important in the hpiv- life cycle, specifically at the level of viral transport and replication [ ] . tubulin also acts as a positive transcription factor for in vitro rna synthesis by the sendai virus [ ] . however, the effect of lectins on the cytoskeleton was not reported. legume lectins are a family of carbohydrate-binding proteins, and are used as a model system for the study of protein-sugar interaction. legume lectins can be divided into five groups according to the specificity of the sugar-binding site: fucose (fuc)-specific, n-acetyl glucosamine/glucosamine (glcnac/glc) specific, glucose/mannose (glc/man) specific, galactose/n-acetylgalactosamine (gal/galnac) specific, and those that do not bind to any mono-saccharides [ ] . plant lectins have been shown to inhibit the infection of human immunodeficiency virus (hiv) [ ] [ ] [ ] and cytomegalovirus [ , ] . plant lectins were reported to inhibit hiv by the prevention of virus adsorption to the cells [ ] . however, they also prevent fusion of hiv with their target cells [ , ] . against severe acute respiratory syndrome (sars) coronavirus, the mannose binding lectins are the most effective [ ] . recently, change et al. reported that recombinant chimeric lectins consisting of mannose-binding lectins and l-ficolin are potent inhibitors of influenza a virus [ ] . in the present study, concanavalin a (con a) which binds to n-linked glycan core trimannoside mana(- - (man - ))man [ , ] , lens culinarisis agglutinin (lca) which binds to core-fucosylated bianntenary n-glycans [ , ] , and peanut agglutinin (pna) which binds to gal-β ( - )-galnac [ ] were tested for their ability of virus growth inhibition. virus rna was prepared and amplified by polymerase chain reaction (pcr). virus protein expression was observed by indirect immunofluorescence study using mabs against np, f and hn proteins of hpiv- [ ] . the inhibitory effect of lectins on hpiv- entry into the cells and replication in the cells was analyzed using a recombinant green fluorescence protein-expressing hpiv- without matrix (m) protein (rghpiv- Δm) [ ] . the number of viruses released from infected cells cultured with the lectins was determined. the binding of lectins to the cells was observed using rhodamine-labeled lectins. the effects of the lectins on actin microfilaments and microtubules were analyzed using rhodamine phalloidin and anti-tubulin α mab, respectively. the three lectins were added to the cells and they were infected with hpiv- , and cell fusion was observed at four days post infection. figure a shows uninfected cells, and figure b figure d ) and pna ( μg/ml) ( figure e ) completely inhibited cell fusion. also, hemadsorption (had) was not observed in the lectin-treated hpiv- -infected cells (data not shown). rhodamine labeled-con a ( . μg/ml), rhodamine labeled-lca ( μg/ml) and rhodamine labeled-pna ( μg/ml) completely inhibited both cell fusion and had (data not shown). the three lectins and three rhodamine labeled-lectins did not disturb normal cell morphology at the concentration used in the experiments. rna was prepared from the lectin-treated infected cells at four days post infection, and virus-synthesized rna was analyzed using hpiv- specific primers by polymerase chain reaction (pcr). the number of base pairs between forward and reverse primers of np, f and hn genes was about . there are some non-specific bands, some larger and some smaller than , and they were also observed faintly in negative control ( figure indirect immunofluorescence study was performed to investigate the effect of the three lectins on hpiv- protein expression. the lectins were added to the cells and they were infected with hpiv- . at four days post infection, the cells were fixed and stained with the mabs against np, f and hn proteins of hpiv- . figures a, b and c exhibit uninfected cells stained with the mabs against np, f and hn proteins, respectively, and no fluorescence was observed. figures d, e and f show the np, f and hn protein expression in hpiv- infected cells, respectively. in hpiv- infected cells, np, f and hn proteins were observed in almost all the cells: np protein was observed in many strong fluorescent dots mainly in the cytoplasm, while f and hn proteins were in small dots in the cytoplasm and on the cell surface. con a inhibited the expression of np ( figure g figure m ) and largely inhibited f protein expression ( figure n ), while hn protein was partly inhibited by pna ( figure o ). as shown in figure , cell fusion was not observed in lectin-treated infected cells. this is because the expressions of f and hn proteins were very little in the cells as observed in figure . the above results showed that the three lectins partly inhibited both hpiv- rna and largely inhibited protein syntheses. in the following experiment, we determined the effect of the lectins on the entry and the replication of hpiv- using rghpiv- Δm (figure ) . the lectins were added to the cell culture, and the cells were infected with rghpiv- Δm ( × tcid : multiplicity of infection . ) and cultured for four days. they were then fixed and observed under a fluorescence microscope. figure a is the organization of rghpiv- Δm. figure b is an uninfected negative control. figure c is a positive control: there are many multinucleated giant cells which have strong fluorescence, indicating that rghpiv- Δm infected the cells, replicated in the cells and caused cell-to-cell infection. figures d, e and f show the infected cells cultured with con a, lca and pna, respectively. as shown in figure , in several parts of the specimen of the infected cells cultured with the lectins, some positive cells were seen, and around them, many uninfected cells were observed. the results indicate that the lectins considerably prevented virus entry, the small amount of virus entered into cells, the virus could replicate only in a single cell, and could not infect the neighboring cells. , lca (e) and pna (f). the three lectins largely prevented the entry of hpiv- into the cells. the small amount of the virus entered into cells, the virus could replicate only in a single cell, but could not infect the neighboring cells (bar: μm). the titers of virus released from the cells cultured with and without the lectins at four days post infection were determined. without the lectins, the virus titer was × tcid /ml, and with the lectins it reduced to about x tcid /ml, indicating that the lectins largely suppressed the yield of the virus. there were no differences in the effects of the three lectins on the virus yield. the binding of the lectins to the cells was analyzed using rhodamine-labeled lectins. the labeled lectins were added to the cells, which were then infected with hpiv- and cultured for min. as shown in figure , the lectins bound to the cell surface within min of addition ( figures a, b and c for con a, lca and pna, respectively), and some aggregates were observed at min (figures d, e and f for con a, lca and pna, respectively). these results indicated that the lectins bound to the cell surface receptors, and largely prevented the binding of hpiv- to the cells non-specifically. the three lectins were added to the cell culture, and the cytoskeleton was observed under an immunofluorescence microscope at h of cultivation. figures a and e show actin microfilaments and microtubules, respectively, in llcmk cells. as shown in figure , con a ( figure b for actin and f for microtubules) and lca ( figure c for actin and g for microtubules) disrupted slightly both actin microfilaments and microtubules. however, pna exhibited no effect on the cytoskeleton ( figures d for actin and h for microtubules) . plant lectins are known to bind to the cell surface sugar moieties, and inhibit virus adsorption to the cells [ ] . mannose-and n-acetylglucosamine-specific agglutinins on hiv replication have been reported [ , ] . in addition, it was reported that these lectins were effective on cytomegalovirus [ , ] and that recombinant chimeric lectins inhibited influenza a virus [ ] . these plant lectins inhibited virus replication by preventing virus adsorption to the cells. for sars-coronavirus, the mannose-specific lectins are highly effective, but the inhibitory mechanism is different from that for other viruses. sars-coronavirus has high-mannose type glycans and the mannose-specific plant lectins can bind to the glycans and prevent the attachment of the virus to the cells [ ] . in the present investigation, we analyzed the effect of three legume lectins that have different sugar-binding specificities on the growth of hpiv- , whose receptor consists of sialic acids [ ] . the three lectins bind to each cell receptor, but not to the sialic acids which are the receptor for hpiv- , so the prevention of hpiv- adsorption to the cells must be non-specific. the lectins that bound to the sugar moieties of cell surface might prevent the binding of hpiv- by steric hindrance, not by competition. the cytoskeletons were important for virus entry and replication in cells. actin is necessary for murine leukemia virus entry into cells [ ] and for transcription/replication of measles virus [ ] and hpiv- [ ] . tubulin is also a factor necessary for the synthesis of sendai virus and vesicular stomatitis virus rnas [ ] . in the present experiment, con a and lca induced a slight morphology change in both actin microfilaments and microtubules, indicating that these may be one possibility of reduction of the numbers of the virus released from the infected cells. taken together, the lectins mainly act on virus entry by considerable prevention of virus binding to the cells. lectins: con a, lca and pna, and rhodamine labeled-con a, -lca and -pna were purchased from funakoshi (tokyo, japan). they were dissolved at mg/ml in mm phosphate buffered saline, ph . (pbs), and sterilized by filtration. virus and recombinant virus were approved by biosafety committees of suzuka university of medical science. hpiv- (toshiba strain) was used. rghpiv- Δm was constructed according to the method described previously [ ] , and it was shown that rghpiv- Δm did not produce infectious virus particles into the culture medium without addition of m protein gene in trans (data not shown). the recombinant virus induced multinucleated giant cells with strong fluorescence at four days post infection, however, no progeny virus was found in the medium (data not shown). titration of rghpiv- Δm could be carried out according to the above-mentioned nature of the recombinant virus. llcmk cells (rhesus monkey kidney cell line) were cultured in a flat-bottomed -well plate in ml culture medium. minimum essential medium α (memα: wako, osaka, japan), supplemented with % fetal calf serum (fcs) and . mg/ml kanamycin, was used. the cells were cultured at °c in a humidified atmosphere with % co . after three days, when the cells became confluent ( × cells), the medium was changed to memα with . % fcs and . mg/ml kanamycin. the lectins were added to the cells, and the cells were infected with hpiv- ( × tcid ). cytopathogenic assay: cell fusion and had were observed at four days post infection. cell fusion was observed on the cells stained with % methyl blue. a had test was carried out using sheep red blood cells (srbc). the cells were incubated with . % srbc at room temperature for min, washed four times with pbs, and had was observed under a light microscope for cell culture. rna preparation, cdna synthesis and pcr: rna was extracted from the cells ( × cells) cultured in a flat-bottomed -well plate using trizol reagent (invitrogen, ca, usa) according to the manufacturer's method. cdna was synthesized with μg of rna using superscript ii reverse transcriptase (invitrogen), with forward primers for np, f and hn genes of hpiv- [ ] . pcr was carried out with cdna using forward and reverse primers for np, f and hn genes [ ] and ex taq (takara, shiga, japan). immunofluorescence study: to detect virus proteins in the infected cells, the cells were fixed with % formaldehyde-pbs at room temperature for min, washed with pbs, and incubated with mouse mabs against np, f and hn proteins of hpiv- [ ] at room temperature for min. after washing with pbs, the cells were incubated with alexa conjugated secondary antibody to mouse iggs (invitrogen) at room temperature for min, and observed under a fluorescence microscope (olympus, tokyo, japan). to determine the binding of lectins to the cells at the early phase of infection, rhodamine labeled lectins were added, the cells were infected with hpiv- and cultured for min. they were fixed at and min after infection. actin was detected by using rhodamine phalloidin (invitrogen), and microtubule by using antitubulin α mab against sea urchin tubulin α (clone b- - - , sigma-aldrich, st louis, mo, usa). the effects of lectins on hpiv- infection were analyzed, and we showed that the lectins had a partial inhibitory effect on virus rna synthesis and they largely inhibited protein syntheses. in addition, it was shown that the lectins non-specifically prevented the entry of hpiv- into cells by binding to the cell surface lectin receptors. the viruses and their replication sequence analysis of the ' genome end and np gene of human parainfluenza type virus: sequence variation of the gene-starting signal and the conserved ' end sequence analysis of p gene of human parainfluenza type virus; p and cystein-rich proteins are translated by two mrnas that differ by two non-templated g residues complete nucleotide sequence expression of the m protein in bacteria sequence of the fusion protein gene of human parainfluenza type virus and its ' intergenic region: lack of small hydrophobic (sh) gene sequence determination of the hemagglutinin-neuraminidase (hn) gene of human parainfluenza type virus and the construction of a phylogenetic tree for hn proteins of all the paramyxoviruses that are infectious to humans characterizations of the human parainfluenza type virus gene encoding the l protein and the intergenic sequences extensive antigenic diversity among human parainfluenza type virus isolates and immunological relationships among paramxoviruses revealed by monoclonal antibodies recovery of infectious human parainfluenza type virus from cdna clones and properties of the defective virus without v-specific cysteine-rich domain involvement of actin microfilaments in the transcription/replication of human parainfluenza virus type : possible role of actin in other viruses tubulin: a factor necessary for the synthesis of both sendai virus and vesicular stomatitis virus rnas structural features of the legume lectins alpha-( - )-and alpha-( - )-d-mannose-specific plant lectins are markedly inhibitory to human immunodeficiency virus and cytomegalovirus infections in vitro the mannose-specific plant lectins from cymbidium hybrid and epipactis helleborine and the (n-acetylglucosamine)n-specific plant lectin from urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro mannose-specific plant lectins from the amaryllidaceae family qualify as efficient microbiocides for prevention of human immunodeficiency virus infection the d-mannose-specific lectin from gerardia savaglia blocks binding of human immunodeficiency virus i to h cells and human lymphocytes in vitro plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle recombinant chimeric lectins consisting of mannose-binding lectins and l-ficolin are potent inhibitors of influenza a virus compared with mannose-binding lectin structural basis of trimannoside recoginition by concanavalin a some properties of purified phytohemagglutinin from les culinaris seeds actin and myosin-driven movement of viruses along filopodia precedes their entry into cells cytoskeleton dynamics: concepts in measles virus replication and immunomodulation fucoidan inhibits parainfluenza virus type infection to llcmk cells the authors declare no conflict of interest. key: cord- -usrpodjx authors: yun, nadezhda e.; walker, david h. title: pathogenesis of lassa fever date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: usrpodjx lassa virus, an old world arenavirus (family arenaviridae), is the etiological agent of lassa fever, a severe human disease that is reported in more than , patients annually in the endemic regions of west africa with mortality rates for hospitalized patients varying between - %. currently, there are no approved vaccines against lassa fever for use in humans. here, we review the published literature on the life cycle of lassa virus with the specific focus put on lassa fever pathogenesis in humans and relevant animal models. advancing knowledge significantly improves our understanding of lassa virus biology, as well as of the mechanisms that allow the virus to evade the host’s immune system. however, further investigations are required in order to design improved diagnostic tools, an effective vaccine, and therapeutic agents. . arenaviridae family (adapted from reference [ ] the enzymatic machinery for rna synthesis in arenaviruses is contained within a single l polymerase protein. this - kda protein utilizes viral rna templates that consist of genomic rna encapsidated by the viral nucleocapsid protein np and comprises viral ribonucloprotein (rnp) [ ] . l polymerase of arenaviruses contains the sdd motif characteristic of all rna-dependent rna polymerases (rdrp). upon infection, once the virus rnp is delivered into the cytoplasm of the host cell, the l polymerase associated with the viral rnp initiates transcription from the genome promoter located at the '-end of each genomic rna segment, l and s. the ' and ' terminal nt viral promoter regions of both rna segments required for the recognition and binding by the viral polymerase [ ] exhibit a high degree of conservation among arenaviruses. the genome segments have highly complementary '-and '-ends ( nt) that have been predicted to form panhandle structures [ ] . the primary transcription results in the synthesis of mrna of viral genes encoded in antigenomic orientation, np and l polymerase, from the s and l segments, respectively. transcription terminates at the distal side of the stem-loop (sl) structure within the intergenomic region (igr). this sl structure has been proposed to stabilize the '-termini of the viral mrnas [ , ] . arenaviruses utilize a cap snatching strategy to acquire the cap structures of cellular mrnas. cap snatching is mediated by the endonuclease activity of the l polymerase that is co-factored by the cap binding activity of np [ ] [ ] [ ] . therefore, arenaviruses produce capped non-polyadenylated mrnas. subsequently, the l polymerase adopts a replicase mode and moves across the igr to generate a fulllength complementary antigenomic rna (agrna). this agrna serves as a template for the synthesis of mrnas of viral genes encoded in genomic orientation, gpc and z, from the s and l segments, respectively, and for the synthesis of full-length genomic rna (grna) [ ] . both grna and agrna of arenaviruses contain a nontemplate g residue at their '-ends [ ] . the proposed "prime and realign" mechanism includes the synthesis of a pppg p c oh dinucleotide primer from the cg nucleotides at positions + and + of the '-end genome promoter sequence, that is then realigned such that its '-terminal c oh is opposite the genome '-terminal g residue, and the realigned pppg p c oh then acts as a primer for a complementary rna strand synthesis [ , ] . the matrix protein z is not required for viral genome transcription and replication; however, z exhibits a dose-dependent inhibitory effect on viral rna synthesis [ ] [ ] [ ] . this inhibitory effect of z has been reported for new world [ ] , as well as for old world [ ] arenaviruses. in addition to the functions required to support virus replication, at least two viral proteins, np and z, have been proposed to modulate the host cell response to infection. np is the most abundant viral protein both in infected cells and in virions, comprises the main structural component of the viral ribonucleoprotein (rnp) and plays an essential role in the synthesis of viral rna [ ] . recent experimental data indicate that np is involved in virus-induced inhibition of type i ifn signaling [ ] [ ] [ ] . this activity has been mapped to the c-terminal domain of np, which has a folding that mimics that of the deddh family of '- ' exoribonucleases [ ] . functional analysis confirmed the exonuclease activity of lasv np that has been proposed to be critical for its type i ifn counteracting function [ ] . the small ring finger protein z is the arenavirus counterpart of the matrix (m) protein of other negative sense rna viruses [ ] . z protein of lcmv interacts with the promyelocytic leukemia (pml) protein and the eukaryotic translation initiation factor e (eif e) in infected cells and has been proposed to contribute to the noncytopathic nature of lcmv infection and repression of capdependent translation [ ] [ ] [ ] [ ] . lasv was first isolated in from a missionary nurse who worked in a clinic in a small town, lassa, in northeastern nigeria [ ] . the nurse presumably acquired infection from an obstetrical patient residing in lassa. she died approximately one week after the onset of symptoms. subsequently, two more nurses that attended the first patient contracted the disease, which was later named lassa fever and caused the death of one of them. infectious virus was isolated from all three cases [ ] . initially, several countries of west africa were identified to be endemic for lasv, namely sierra leone [ , ] , guinea [ , ] , liberia [ ] [ ] [ ] , and nigeria [ ] [ ] [ ] [ ] . however, a serological survey among patients admitted with a history of fever and missionaries that had experienced a febrile illness showed that lasv was also present in ivory coast, mali, and central african republic [ ] . the notion that lasv was endemic in larger areas of west africa was further supported by the results of investigation of an imported case of lassa fever in germany in . during the incubation period, the index patient traveled through several countries, namely ghana, ivory coast, and burkina faso, that were not considered to be endemic at that time [ ] . later, cases of lassa fever have been reported from burkina faso, ivory coast, ghana, senegal, gambia, and mali [ , ] . according to estimations, lasv is responsible for , - , infections and approximately , deaths annually [ ] . however, the high degree of seroprevalence of lasv-specific antibodies in the general population residing in the endemic regions, although highly variable depending on the geographical location (from . % to %) [ , , , ] , indicates that most infections are mild or possibly even asymptomatic and do not result in hospitalization. this is also supported by the findings indicating a high incidence of lasv-specific seroconversion, from % to % of the nonimmune population per year [ ] . nosocomial outbreaks are associated with higher mortality rates ranging from % to % [ , , ] . however, serosurveillance studies in hospitals dealing with suspected lassa fever cases showed that the hospital staff that routinely practiced basic hygiene measures had no higher risk of infection than the local population [ ] . infection with lasv presumably occurs through contact with body fluids or excreta, or inhalation of aerosols produced by infected animals. lasv is stable in aerosol [ ] , and animal-to-animal transmission via the airborne route has been demonstrated in the laboratory setting [ ] . hunting of peridomestic rodents and consumption of their meat is another important route of lasv transmission to humans residing in endemic areas of west africa [ ] . the multimammate mouse, mastomys natalensis, was originally identified as the primary host species for lasv [ ] . however, due to the poor understanding of the taxonomy of the genus, it is uncertain which species and particular subspecies serve as a reservoir for the virus [ ] . the studies addressing the importance of m. natalensis for the circulation of lasv in nature demonstrated that newborn animals inoculated intraperitoneally develop persistent asymptomatic infection [ ] . significant infectious virus titers were detected in many organs, tissues, and fluids including lymph node, liver, spleen, lung, blood, and brain up to days after inoculation. moreover, lasv was isolated from the urine and throat swabs of infected animals. no significant histopathological alterations were observed in these animals. interestingly, adult m. natalensis infected with lasv also developed a disseminated infection that lasted up to days. some animals cleared the virus from some organs, but there was persistence in other organs up to days when the study was terminated. the only consistent histopathological finding observed in adult animals was a moderate chronic meningoencephalitis [ ] . these data demonstrate that m. natalensis has an optimal pattern of infection and virus shedding for the maintenance of lasv in nature. the incubation period of lassa fever ranges from to days [ , ] . the clinical disease begins as a flu-like illness characterized by fever, general weakness, and malaise, which may be accompanied by cough, sore throat, and severe headache. gastrointestinal manifestations such as nausea, vomiting, and diarrhea are also common ( table ). the differential diagnosis of lassa fever based on the presenting symptoms can be problematic due to the many other acute undifferentiated febrile illnesses circulating in west africa [ , ] . although, hemorrhagic manifestations are not an important feature of lassa fever, perturbation of vascular function is likely to be central to lassa fever-associated pathobiology, since the signs of increased vascular permeability, such as facial edema and pleural and pericardial effusions, indicate a poor prognosis for the disease outcome. recovery from lassa fever generally begins within to days of disease onset. in severe cases, the condition of the patient deteriorates rapidly between the th and th day of illness with severe pulmonary edema, acute respiratory distress, clinical signs of encephalopathy, sometimes with coma and seizures, and terminal shock. bleeding from mucosal surfaces is often observed; however, it is usually not of a magnitude to produce shock by itself [ ] . sensorineural deafness is commonly observed in patients in the late stages of disease or in early convalescence in survivors [ ] . the level of viremia is highly predictive of the disease outcome. in a study involving patients with lassa fever, patients that presented with viremia < median tissue culture infectious dose (tcid )/ml on the day of hospitalization had . times greater chance of survival than those admitted with higher levels of viremia. similarly, the probability of fatal outcome in patients with serum titers > tcid /ml and serum levels of aspartate aminotransferase (ast) ≥ international units (iu)/l was times higher than that in patients not meeting either of these criteria. virtually all patients with fatal lassa fever whose sera were tested were viremic at the time of death with terminal viremia ranging from to tcid /ml [ ] . detailed studies have shown that viremia peaks between and days after the onset of symptomatic disease and is followed by pronounced clinical manifestations. patients recovering from lassa fever clear virus from blood circulation about weeks after the beginning of illness [ , [ ] [ ] [ ] . the current knowledge of lassa fever pathogenesis does not include the chain of events that take place during disease development and lead to death of severely ill patients. apparently, failure to develop the cellular immune response that would control dissemination of lasv, which is indicated by high serum virus titers, combined with disseminated replication in tissues and absence of neutralizing antibodies, leads to the development of fatal lassa fever [ ] . however, considering the high mortality and truly dramatic course of the disease, the pathological findings do not provide the basis that would explain the mechanism of disease progression and the cause of death from lassa fever. physical examination of patients after the onset of fever often reveals purulent pharyngitis, bilateral conjunctival hemorrhages, facial edema, and generalized abdominal tenderness. macroscopic pathological changes can include pleural effusions, pulmonary edema, ascites, and hemorrhagic manifestations in the gastrointestinal mucosa [ , ] . microscopic findings include hepatocellular necrosis and apoptosis, splenic necrosis, adrenocortical necrosis, mild mononuclear interstitial myocarditis without myocardial fiber necrosis, alveolar edema with capillary congestion and mild interstitial pneumonitis, lymph nodal sinus histiocytosis with mitoses, gastrointestinal mucosal petechiae, renal tubular injury, and interstitial nephritis [ , , ] . a comprehensive postmortem histopathological examination of virologically confirmed community-acquired cases of lassa fever in sierra leone revealed [ ] variable levels of hepatic necrosis involving from to % of hepatocytes. the necrotic hepatocytes were randomly distributed often forming foci of contiguous cells. mononuclear phagocytes were observed either contacting or phagocytosing necrotic hepatocytes. interestingly, this phagocytic reaction, although highly variable from case to case and even from one necrotic focus to another in the same case, demonstrated a tendency towards homogeneity of the level of involvement within a particular patient. the predominant distribution of splenic necrosis was observed in the marginal zone of the periarteriolar lymphocytic sheath. close examination of thin tissue sections revealed the presence of fibrin in addition to the debris of necrotic cells. splenic venous subendothelium appeared to be infiltrated by lymphocytes and other mononuclear cells. microscopic examination of adrenal glands showed prominent spherical, hyaline, acidophilic cytoplasmic inclusions in cells near the junction of zona reticularis and medulla. in most cases these cells appeared to be adrenocortical cells of the zona reticularis; however, some cells were of adrenal medulla origin. additionally, multifocal adrenocortical cellular necrosis was detected that was most prominent in the zona fasciculata and was often associated with focal inflammatory reaction. however, in all examined cases adrenal necrosis was mild and ≥ % of the cells of adrenal cortex appeared viable [ ] . the major and most common lesions of lassa fever in humans occur in the liver [ , , , ] . there are four principal features of lasv hepatitis can be derived: ) focal cytoplasmic degeneration of hepatocytes suggestive of phagocytosed apoptotic fragments; ) randomly distributed multifocal hepatocellular necrosis; ) monocytic reaction to necrotic hepatocytes; ) hepatocellular mitoses. these morphologic effects do not uniformly occur in all cases, but in some instances can be found simultaneously [ , ] . based on the degree of hepatic damage, three general nosopoetic phases have been proposed to divide patients with fatal lassa fever into categories with respect to pathogenic events in fatal lasv hepatitis [ ] . the first phase, active hepatocellular injury, is defined by the presence of focal cytoplasmic degeneration with < % of hepatocytes undergoing necrosis. this phase may represent the late stage of viremic spread and early cellular injury, which is, most likely, caused by direct viral action rather than mediated by a cellular immune response, since lymphocytic infiltration is not detected. the second phase, the peak of lassa hepatitis, is characterized by - % necrosis of hepatocytes, widespread focal cytoplasmic degeneration and limited phagocytic infiltration. this is suggested not only progressive hepatocellular damage, but, also, early liver recovery through phagocytic removal of necrotic hepatocytes and regeneration of new cells. the third phase, hepatic recovery, is defined by < % of hepatocellular necrosis, absence of focal cytoplasmic degeneration and clear evidence of mitoses, which indicate liver regeneration [ ] . interestingly, no correlation has been observed between the degree of hepatic necrosis and chemical indicators of liver damage, such as elevated levels of ast, alanine transaminase (alt), and lactate dehydrogenase (ldh) in serum [ , ] . overall it is apparent that the level of liver damage can vary dramatically in patients that die from lassa fever. therefore, it can be concluded that liver disease is a necessary, but not sufficient, condition in the chain of pathological events that lead to fatal outcome. lassa fever is not considered to be associated with coagulation dysfunction, e.g., neither decrease in the coagulation factors nor disseminated intravascular coagulation (dic) has been observed in infected patients. however, moderate thrombocytopenia with significantly impaired functionality of thrombocytes is detected in patients with severe lassa fever [ ] . one possible mechanism involved in lassa fever pathogenesis could be infection-triggered induction of uncontrolled cytokine expression similar to what is seen in sepsis. this hypothesis is supported by experimental data obtained from a case of fatal lassa fever imported into germany in [ ] . in this patient, who died from multi-organ failure and hemorrhagic shock, the proinflammatory cytokines, interferon γ (ifn-γ) and tumor necrosis factor α (tnf-α), rose to extremely high levels shortly before death. however, in another study no elevation of both cytokine levels was observed in the examined fatal cases of lassa fever, which suggests that the levels of ifn-γ and tnf-α are either elevated only in a fraction of patients or during a very short period that would require frequent sampling for detection [ ] . another possibility is that virus-induced immunosuppression may be involved in the pathogenesis of severe lassa fever [ ] . thus, infection with lasv fails to activate monocyte-derived dendritic cells (dc) and macrophages (mp) of human origin. infected dc fail to secrete proinflammatory cytokines, do not upregulate costimulatory molecules, such as cd , cd , and cd , and poorly induce proliferation of t cells [ , ] . importantly, human dc infected with the naturally nonpathogenic mopeia virus, a closely related arenavirus that shares % amino acid similarity with lasv and was isolated from the same rodent reservoir [ ] , induces stronger cd and cd t-cell responses than those infected with lasv [ ] . downregulation of immune responses caused by lasv infection demonstrated in vitro is also in agreement with the results of clinical observations showing that fatal outcome of lassa fever correlates with low levels or absence of interleukin (il) and ifn inducible protein (ip- ) in circulation [ ] . patients infected with lasv produce igm and igg antibody isotypes [ ] . however, since both immunoglobulin classes are detected in viremic patients, most likely the antibodies that are produced early in infection are not neutralizing. this is in agreement with reports showing that neutralizing antibodies appear months after acute infection is resolved, and the titers are often low [ ] . interestingly, the neutralizing antibody titers continue to rise even several months after convalescence has been established, which may indicate constant stimulation of b cells due to low levels of virus persistence. antibodies in seroconverted individuals are specific to gpc, np, and, likely, z protein [ ] [ ] [ ] [ ] . experiments aimed at the identification of b-cell antigenic epitopes elucidated four sites on np, two sites on gp , and six sites on gp [ ] [ ] [ ] . some antibodies are highly lasv strainspecific, while other react with a broad range of arenaviruses including african and south american members of the family [ , ] . since neutralizing antibodies appear in survivors long after virus has been cleared, it has been considered that resolution of lasv infection is mediated by cellular immunity. this hypothesis is supported by experimental data showing that cynomolgus monkeys that survive lasv infection have high concentrations of activated t lymphocytes and efficiently control viral replication. in contrast, the animals that succumb to infection display delayed, low-level t-cell activation, uncontrolled viral replication, and develop fatal disease [ ] . seropositive individuals residing in endemic areas have very strong memory cd + t-cell responses, and the antigenic epitopes are mapped to np and a amino acid region in the n-terminal part of gp that is highly conserved between old and new world arenaviruses [ , ] . importantly, a recent study showed that transgenic mice that express human major histocompatibility complex class i (mhc-i) molecules fail to control virus replication and are susceptible to lassa virus, which is in contrast to normal laboratory mice that rapidly clear virus. intriguingly, cd and cd t cell depletion in these mice prevents disease despite high levels of viremia [ ] . these data indicate that t cells are essential for rapid resolution of lasv infection; however, if the host fails to control virus replication due to inadequate activation of the immune system, t lymphocytes may play a key role in lassa fever pathogenesis. mice and guinea pigs have been evaluated as models of lasv infection [ ] . however, normal adult mice are highly resistant to peripheral routes of inoculation. mice expressing humanized mhc-i are lasv-susceptible and develop a severe illness [ ] . genotype of a particular mouse strain has significant influence on the development of pathologic manifestations in infected mice. thus, newborn mice develop an asymptomatic infection upon inoculation with lasv with high virus titers in the brain, lung, and muscle, while intracranial inoculation of adult mice results in a fatal convulsive disorder resembling classical murine lcm immunopathology. however, other investigators failed to reproduce these findings [ , , ] . pathogenicity of lasv for guinea pigs largely depends on the host strain and the virus used for inoculation. for instance, josiah strain of lasv causes a uniformly lethal disease in inbred strain guinea pigs; however, the lethality varies from approximately % to more than % in outbred hartley guinea pigs. inbred animals have higher viral titers in all target tissues than outbred guinea pigs [ , ] . lasv-infected guinea pigs destined to die develop respiratory insufficiency with pulmonary edema, alveolar hyaline membranes, myocarditis, and focal calcification of myocardial fibers and hepatocytes. terminally ill animals are viremic and contain virus in nearly every organ tested. there are two major differences between lassa fever pathogenesis in humans and guinea pigs. in humans lasv is particularly hepatotropic, and patients with severe lassa fever develop hepatocellular necrosis; however, in guinea pigs only foci of calcified hepatocytes are observed. on the other hand, lasv is myocardiotropic and myocardiopathic in guinea pigs, findings that have not been reported in humans [ ] . also, several isolates of lasv from clinical human cases are benign in guinea pigs, and some are lethal when tested in cynomolgus monkeys [ ] . therefore, in order to reliably model the pathogenesis of lassa fever in humans, studies in non-human primates are required. several non-human primate species have been evaluated as potential models for lassa fever including squirrel monkeys, capuchin monkeys, marmosets, hamadryas baboons, african green monkeys, cynomolgus monkeys, and rhesus monkeys [ , , [ ] [ ] [ ] [ ] [ ] [ ] . capuchin and squirrel monkeys seroconverted upon infection with lasv; however, the animals of both species uniformly survived low and high dose inoculations [ , ] . virus was detected in virtually all organs of infected squirrel monkeys, but the liver, lymph nodes, and kidneys were the key early targets. later in infection, the spleen, heart, and brain also showed pathological alterations. viremia persisted in all animals for up to days after infection. histopathologic changes were mild and included germinal center necrosis in the spleen and lymph nodes, myocarditis, acute arteritis, renal tubular necrosis and regeneration, chronic inflammation of the choroid plexus, ependyma, and meninges, and cerebral perivascular cuffing. hepatocytic regeneration suggesting recovery from disease-mediated liver damage was also observed [ ] . hamadryas baboons infected with lasv develop a disease that clinically resembled a severe form of human lassa fever. the animals developed fever, characteristic pathologic and hemorrhagic manifestations, and high levels of viremia [ ] . interestingly, african green monkeys and rhesus macaques challenged with low doses ( - pfu) of lasv uniformly succumbed to the infection, whereas a high challenge dose ( pfu) was only partially lethal in these animals [ ] . the gross pathological changes in lasv-infected rhesus macaques were petechiae and, in some animals, mild-to-moderate pleural effusions. viremia appeared between days and in all animals and increased progressively until the animals died or met the criteria for euthanasia. virus was detected in all organs tested, namely adrenal glands, spleen, liver, duodenum, jejunum, colon, bone marrow, lymph nodes, thymus, heart, lungs, pleural fluid, kidneys, skeletal muscle, pancreas, salivary glands, ovaries, bladder, cerebrum, cerebellum, brain stem, cerebrospinal fluid, and aqueous humor of the eye. the largest quantities of virus were generally detected in adrenal glands, spleen, liver, bone marrow, and intestines. the microscopic lesions included mononuclear pulmonary arteritis with intraluminal mononuclear cell aggregates and mononuclear cell infiltration of the subendothelium and the pulmonary arterial wall accompanied by endothelial hypertrophy and hyperplasia, hepatocellular necrosis, interstitial pneumonia, adrenal gland necrosis, encephalitis and uveitis. therefore, infected rhesus monkeys exhibited pathologic lesions similar to human lassa fever, such as the amounts and organ distribution of virus, necrosis of hepatocytes, adrenal cortical cells, and splenic marginal zone of the periaortic lymphocytic sheath, and interstitial nephritis. however, meningoencephalomeningitis, pulmonary vasculitis, systemic arteritis, and skeletal myositis were significantly more prominent in the monkey model than in human lassa fever [ , ] . the clinical manifestations of lasv-infected cynomolgus macaques included fever, weight loss, depression, and acute respiratory syndrome. other clinical features included thrombocytopenia, lymphopenia, lymphadenopathy, splenomegaly, infiltration of mononuclear cells, and pathologic alterations in the liver, lungs, and endothelium, which essentially mirrored observations from human cases [ ] . an additional feature of the disease observed in cynomolgus monkeys was multifocal to severe central nervous system lesions at terminal stages. dysregulation of the host immune response characterized by increased circulating levels of proinflammatory cytokines/chemokines including il- β, il- , mcp- , and eotaxin were detected in the infected animals. histopathologic evaluation of tissues revealed a sequence of events in lasv infection in cynomolgus macaques, which is initiated with dendritic cells in the lymphoid tissues, then progresses to infection of kupffer cells in liver and parenchymal cells in liver and adrenal glands, and endothelial cells in various organs including the central nervous system [ ] . experimental infection of common marmosets results in systemic disease from clinical and pathologic standpoints highly similar to disease observed in fatal cases of lassa fever in humans. among the main clinical features are fever, weight loss, high viremia and viral rna loads in tissues, elevated levels of liver enzymes, and severe morbidity between days and . histopathologic changes include multifocal hepatic necrosis associated with mild inflammation and hepatocyte proliferation, adrenal necrosis, lymphoid depletion, and interstitial nephritis. the necrotic hepatocellular foci observed in lasv-infected marmosets contained predominantly macrophages with the near absence of cd -, cd -, or cd -positive lymphocytes, markedly decreased expression of mhc-ii molecules, and hepatocyte proliferation. the levels of mhc-ii expression were also significantly reduced in lymph nodes. overall numbers 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arenaviruses share a highly conserved epitope in the fusion domain of the glycoprotein , which is recognized by lassa virusspecific human cd + t-cell clones lassa virus lethality for inbred mice biology and pathogenesis of lymphocytic choriomeningitis virus infection pathogenesis of lassa virus infection in guinea pigs endemic lassa fever in liberia. iii. characterization of lassa virus isolates pathology and pathogenesis of arenavirus infections experimental lassa virus infection in the squirrel monkey experimental infection of rhesus monkeys with lassa virus and a closely related arenavirus, mozambique virus experimental lassa fever in hamadryas baboons lassa virus infection in experimentally infected marmosets: liver pathology and immunophenotypic alterations in target tissues pathogenesis of lassa fever in cynomolgus macaques kinetic study of platelets and fibrinogen in lassa virus-infected monkeys and early pathologic events in mopeia virus-infected monkeys n. e. yun was supported by funding from the institute for human infections and immunity, university of texas medical branch. the authors declare no conflict of interest". key: cord- - aofwe w authors: bulow, uriel; govindan, ramesh; munro, james b. title: acidic ph triggers lipid mixing mediated by lassa virus gp date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: aofwe w lassa virus (lasv) is the causative agent of lassa hemorrhagic fever, a lethal disease endemic to western africa. lasv entry is mediated by the viral envelope glycoprotein (gp), a class i membrane fusogen and the sole viral surface antigen. previous studies have identified components of the lasv entry pathway, including several cellular receptors and the requirement of endosomal acidification for infection. here, we first demonstrate that incubation at a physiological temperature and ph consistent with the late endosome is sufficient to render pseudovirions, bearing lasv gp, non-infectious. antibody binding indicates that this loss of infectivity is due to a conformational change in gp. finally, we developed a single-particle fluorescence assay to directly visualize individual pseudovirions undergoing lasv gp-mediated lipid mixing with a supported planar bilayer. we report that exposure to endosomal ph at a physiologic temperature is sufficient to trigger gp-mediated lipid mixing. furthermore, while a cellular receptor is not necessary to trigger lipid mixing, the presence of lysosomal-associated membrane protein (lamp ) increases the kinetics of lipid mixing at an endosomal ph. furthermore, we find that lamp permits robust lipid mixing under less acidic conditions than in its absence. these findings clarify our understanding of lasv gp-mediated fusion and the role of lamp binding. lassa virus (lasv) is an arenavirus endemic to western africa. an outbreak of lassa fever in nigeria led to suspected cases and confirmed deaths in the first twenty-one weeks of [ ] . the sequelae of lassa fever include sensorineural deafness in % of survivors and a greater than % chance of maternal and/or fetal death in pregnant patients in their third trimester [ , ] . due to the deadly nature of lassa hemorrhagic fever, lasv is classified as a category a priority pathogen by the united states centers for disease control and prevention. the lasv envelope glycoprotein (gp) coordinates the fusion of the viral envelope with the endosomal membrane of the target cell [ ] . gp is synthesized as a precursor glycoprotein complex that is subsequently processed by cellular proteases into subunits gp and gp , as well as a long and stable signal peptide (ssp) [ , ] . during viral entry, gp is responsible for binding at least two identified cellular receptors, α-dystroglycan (αdg) and lysosomal-associated membrane protein (lamp ) [ , ] . studies to date suggest a model in which gp first binds α-dg on the cell surface, which causes the cell to internalize the virus into an endosome via macropinocytosis [ ] . the acidification of the late endosome causes gp to undergo an affinity switch from αdg to lamp [ ] . implicated in this affinity switch are histidines h , h , and h , which are located adjacent to the gp receptor-binding site and undergo conformational changes in response to protonation [ , ] . a crystallographic structure of gp at a neutral ph and cryo-electron tomography structures determined under acidic conditions have shown that acidic ph is sufficient to initiate conformational changes in gp [ , ] . the prevailing model of class i fusion holds that gp sits in a kinetically trapped, metastable pre-fusion conformation. the transition to the post-fusion conformation is facilitated by a trigger, which lowers the activation energy for exothermic collapse [ ] . during fusion triggering, the gp subunit undergoes putative conformational rearrangements that involve the adoption of a transient extended intermediate in which the fusion loop is inserted into the target membrane [ ] . the subsequent refolding of gp brings the target and viral membranes into close approximation, inducing fusion. like other class i viral fusogens, the formation of the six-helix bundle is thought to drive membrane fusion [ ] [ ] [ ] . previous studies in lasv and lymphocytic choriomeningitis virus (lcmv) have highlighted the importance of acidic ph in arenavirus entry [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , with some cell-cell fusion studies suggesting that a ph as low as . may be required for lasv gp-mediated fusion to occur [ , ] . of particular note, exposure to a ph consistent with the late endosome at a physiological temperature was shown to trigger the fusion of lcmv [ ] . here, we show that exposure to low ph is sufficient at a physiological temperature to trigger conformational changes in lasv gp related to membrane fusion. furthermore, we show directly by a single-particle fusion assay that exposure to ph . is sufficient to trigger lasv gp-mediated lipid mixing in the absence of receptor binding. finally, we demonstrate conclusively that lamp binding is dispensable for lasv gp-mediated lipid mixing. however, its presence in the target membrane increases the kinetics of lipid mixing, facilitating robust lipid mixing at a less acidic ph. hek t cells were cultured in dmem (thermo fisher, waltham, ma, usa) supplemented with % cosmic calf serum (ge healthcare, chicago, il, usa), l-glutamine (thermo fisher), and penicillin-streptomycin (thermo fisher) at • c and % co and were used to generate pseudovirions as described below. vero cells were cultured in dmem supplemented with % fetal bovine serum (fbs; gemini bio), l-glutamine, and penicillin-streptomycin at • c and % co and were used for infectivity assays with pseudovirions bearing gp and ha. df chicken fibroblast cells were cultured in rpmi (thermo fisher) supplemented with % fbs, l-glutamine, and penicillin-streptomycin at • c and % co , and were used for infectivity assays with pseudovirions bearing alv env, as mammalian cells are neither susceptible nor permissive to infection by alv subtype a. the pcdna plasmid encoding lasv gp from the josiah strain was provided by dr. melinda brindley at the university of georgia [ ] . the pvrc plasmid encoding iav ha (a/vietnam/ ) was provided by dr. peter kwong at the vaccine research center. ha was formed by site-directed mutagenesis as previously described [ ] . alv subtype a env was provided by dr. john coffin at tufts university. the soluble lamp expression construct was provided by dr. juha huiskonen at the university of oxford [ ] . hek t cells were transfected with µg of plasmid containing the glycoprotein of interest in a cm dish at % confluency. pei max was used for transfection at a mass ratio of : pei to plasmid viruses , , of dna. cells were infected with vsv∆g-gfp-vsvg (kerafast) h post transfection at an moi of [ ] . supernatant was collected h post infection and ultra-centrifugated over a % sucrose cushion at , × g for min. supernatant was aspirated and pellets were resuspended in phosphate-buffered saline (pbs). the pseudovirus was aliquoted and frozen at − • c until use. bald particles were generated following an identical protocol, with the exception of the omission of a glycoprotein. infections were performed in vero cells for studies with lasv gp and iav ha, and in df- cells for alv env. µl of concentrated pseudovirus were diluted in . m phosphate buffer (for ph . and . ) or . m acetate buffer (for ph . through . ) in temperature-controlled conditions. virions were then brought back to neutral ph in × volume of . m phosphate buffer at ph . while still at their experimental temperatures, and then immediately serially diluted in room temperature dmem for infection. target cells were infected with the pre-incubated virus and were incubated for one hour at • c with rocking every min. fresh dmem was then added to the cells. at h post infection, cells were collected by trypsinization and assayed for gfp expression using flow cytometry on a facscalibur. statistical significance was calculated using a two-way anova with an alpha of . . purified vsv pseudotypes were treated with either a phosphate buffer at ph . or . , or acetate buffer at ph . or . at • c or on ice for min. the virions were then brought back to a neutral ph, as described above, at their respective temperatures and bound to an elisa plate. the plate was then blocked with a % bsa solution for h and washed with pbs three times. the immobilized virions were then probed with non-neutralizing ( . e, . c) or neutralizing antibodies ( . f, . d, . h) for h, washed three times with pbs, and probed with an hrp-conjugated rabbit anti-human igg antibody (abcam, cambridge, uk) [ ] . the plate was developed with sureblue™ tmb -component microwell peroxidase substrate (seracare, milford, ma, usa) for twenty minutes and read in a synergy h biotek plate reader. hek f cells were transfected with plasmid encoding the full lumenal domain of lamp with a × his tag and grown for days. pei max was used as the transfection reagent as indicated above. supernatant was collected and circulated over ni-nta agarose beads (thermo fisher, waltham, ma, usa) overnight at • c. the beads were then washed with pbs with mm imidazole. lamp was eluted from the beads with pbs with mm imidazole. the protein was then dialyzed into pbs and stored at − • c in frozen aliquots. virions were labeled by incubating with mm did (thermo fisher, waltham, ma, usa) for two hours at room temperature. unbound did was removed by passage over a zeba desalting column (thermo fisher, waltham, ma, usa) equilibrated in pbs. labeled virus was used immediately in single-particle lipid mixing assays. the single-virion lipid mixing assay was adapted from that described by floyd et al. [ ] . pre-drilled quartz slides and glass coverslips were immersed in distilled water and sonicated for min. they were then moved to m koh and the sonication was repeated. the slides were rinsed with distilled water, before being sonicated in acetone, and rinsed again. the coverslips and slides were then allowed to dry overnight. the dry slides were then cleaned in an plasma cleaner (harrick plasma, ithaca, ny, usa) for min. flow cells were then constructed with the slides and coverslips. each flow cell was rinsed with pbs, and then µl of liposomes were injected into the flow cell by a syringe pump at µl/min. the liposomes were allowed to incubate for twenty minutes at room temperature before the channel was again rinsed with µl of pbs. labeled virions were then pumped into the channel at µl/min and allowed to incubate for min to allow virions to bind to the membrane. the channel was then rinsed with pbs again to remove unbound virus. the slide was then placed on the stage of a prism-based tirf microscope. pre-warmed buffer was pumped into the flow cell while acquiring movies at ms exposures ( hz) for a minimum of min, or until no more dequenching events could be seen. the analysis of the dequenching events was performed in matlab (mathworks, waltham, ma, usa). the interval between % loss of cf fluorescence resulting from acidification and did dequenching was extracted for each event and compiled into histograms. the histograms displayed a rise and fall in the frequency of lipid mixing events. this indicates that multiple rate-limiting steps are necessary before the arrival of lipid mixing; a single rate-limiting step would have given rise to an exponentially distributed histogram of the frequency of events. we therefore considered the kinetic scheme where a represents the pre-fusion state, b represents the state after lipid mixing, and i , i , . . . i n are the n rate-limiting intermediate states. the probability density function for this scheme, which describes the distribution of time intervals observed for the transit from a to b, is the gamma distribution where k is the rate constant and n is the number of rate-determining steps. the histograms of the time intervals were fit to the gamma distribution using a least squares algorithm in matlab. the mean time to dequenching was then determined by calculating t = n/k. we first sought to determine the ph sensitivity of lasv gp with a viral inactivation assay, a common strategy used to study the ph dependence of viral fusogen function [ ] . we therefore formed gfp-encoding vesicular stomatitis virus (vsv) pseudovirions bearing gp from the josiah strain of lasv. this pseudotyping strategy has previously been used for mutagenic studies of lasv gp, as well as for lasv vaccine trials [ , ] . vsv-gp pseudovirions were first incubated at • c for five or thirty minutes in buffer at ph values ranging from . to . . the virions were returned to a neutral ph while maintaining the temperature before they were used to infect vero cells. gfp expression was assayed using flow cytometry five hours post infection. above ph . , no loss of infectivity was seen after five minutes of incubation at • c ( figure a ). infectivity was reduced by approximately % when the virions were incubated at ph . for five minutes. a similar loss of infectivity was seen for pseudovirions carrying h hemagglutinin (ha) from influenza, which is known to be triggered by acidic ph [ , ] . when the virions were incubated at • c and ph . for five minutes, infectivity was reduced to %. infectivity was further reduced by % when incubated at ph . ( figure a ). when the incubation was extended for thirty minutes, infectivity was reduced by % at ph . ( figure b) . again, similar losses of infectivity were seen for particles carrying ha. in contrast, no loss of infectivity was seen for pseudovirions carrying the envelope glycoprotein from avian leukosis virus (alv env) following incubation for five or thirty minutes at ph . at all temperatures tested ( figure ). finally, consistent with the model in which gp is kinetically trapped in the metastable pre-fusion conformation, no loss of infectivity was observed at any ph while on ice ( figure ). these data demonstrate that incubation at ph . to . leads to loss of infectivity of gp-bearing pseudovirions at a physiologic temperature. viruses , , x for peer review of known to be triggered by acidic ph [ , ] . when the virions were incubated at °c and ph . for five minutes, infectivity was reduced to %. infectivity was further reduced by % when incubated at ph . ( figure a ). when the incubation was extended for thirty minutes, infectivity was reduced by % at ph . ( figure b) . again, similar losses of infectivity were seen for particles carrying ha. in contrast, no loss of infectivity was seen for pseudovirions carrying the envelope glycoprotein from avian leukosis virus (alv env) following incubation for five or thirty minutes at ph . at all temperatures tested ( figure ). finally, consistent with the model in which gp is kinetically trapped in the metastable pre-fusion conformation, no loss of infectivity was observed at any ph while on ice ( figure ). these data demonstrate that incubation at ph . to . leads to loss of infectivity of gpbearing pseudovirions at a physiologic temperature. virus was subsequently brought back to neutral ph and used to infect vero cells. infectivity was determined by measuring gfp expression using flow cytometry. infectivity was normalized to the percent of gfp-positive cells at the °c ph . condition for each virus. assays were performed in biological triplicate with new viral preps, and each biological replicate was performed in technical triplicate. data are presented as the mean of three biological replicates and error bars represent the standard error of the mean. an asterisk indicates a statistically significant difference as compared to the case of ph (p < . ) calculated using a one-way anova. the lack of an asterisk indicates no statistically significant difference as compared to ph . to further test whether exposure to acidic ph at a physiological temperature triggers a conformational change in lasv gp, we evaluated the recognition by neutralizing antibodies . f, . d, and . h using an enzyme-linked immunosorbent assay (elisa). these antibodies recognize epitopes that bridge both gp and gp , making them highly specific for the pre-fusion conformation of gp [ , ] . virions were incubated at ph values ranging from . to . on ice or at °c for thirty minutes. the ph was then neutralized while maintaining temperature, and virions were subsequently bound to an elisa plate at room temperature. we then probed for the presence of the pre-fusion conformation of gp with neutralizing antibodies . f, . d, and . h. irrespective of temperature, exposure to ph or led to no loss of antibody binding (figure ). at ph . , moderate figure . exposure to low ph at physiological temperature inactivates lasv gp. vsv∆g-gfp-gp viral pseudotypes were treated with either phosphate buffer at ph . or . , or acetate buffer at ph . or . at the indicated temperature for (a) min or (b) min. virus was subsequently brought back to neutral ph and used to infect vero cells. infectivity was determined by measuring gfp expression using flow cytometry. infectivity was normalized to the percent of gfp-positive cells at the • c ph . condition for each virus. assays were performed in biological triplicate with new viral preps, and each biological replicate was performed in technical triplicate. data are presented as the mean of three biological replicates and error bars represent the standard error of the mean. an asterisk indicates a statistically significant difference as compared to the case of ph (p < . ) calculated using a one-way anova. the lack of an asterisk indicates no statistically significant difference as compared to ph . to further test whether exposure to acidic ph at a physiological temperature triggers a conformational change in lasv gp, we evaluated the recognition by neutralizing antibodies . f, . d, and . h using an enzyme-linked immunosorbent assay (elisa). these antibodies recognize epitopes that bridge both gp and gp , making them highly specific for the pre-fusion conformation of gp [ , ] . virions were incubated at ph values ranging from . to . on ice or at • c for thirty minutes. the ph was then neutralized while maintaining temperature, and virions were subsequently viruses , , of bound to an elisa plate at room temperature. we then probed for the presence of the pre-fusion conformation of gp with neutralizing antibodies . f, . d, and . h. irrespective of temperature, exposure to ph or led to no loss of antibody binding (figure ). at ph . , moderate loss of binding by only . h was seen after a • c incubation, whereas full binding was maintained after incubation on ice. incubation at ph . at • c abrogated binding by approximately % in the case of . f, by more than % in the case of . d, and by % in the case of . h, whereas no loss of binding of any antibody was seen after incubation on ice. in contrast, no loss of binding after incubation at either temperature across the range of ph values was seen for the non-neutralizing antibodies . e and . c (figure ) [ ] . taken together, the infectivity and antibody binding data suggest that gp undergoes a ph-induced transition out of the pre-fusion conformation, concurrent with a loss of function. here again, only when incubations were performed at a physiological temperature did the putative transition occur at a ph consistent with the late endosome. viruses , , x for peer review of case of . f, by more than % in the case of . d, and by % in the case of . h, whereas no loss of binding of any antibody was seen after incubation on ice. in contrast, no loss of binding after incubation at either temperature across the range of ph values was seen for the non-neutralizing antibodies . e and . c (figure ) [ ] . taken together, the infectivity and antibody binding data suggest that gp undergoes a ph-induced transition out of the pre-fusion conformation, concurrent with a loss of function. here again, only when incubations were performed at a physiological temperature did the putative transition occur at a ph consistent with the late endosome. figure . the binding of neutralizing and non-neutralizing antibodies to lasv gp after exposure to acidic ph at physiological temperature. vsvΔg-gfp-gp viral pseudotypes were treated with either phosphate buffer at ph . or . , or acetate buffer at ph . or . at the indicated temperature for min. virus was subsequently brought back to neutral ph and assayed for binding by non-neutralizing ( . e, . c) and neutralizing antibodies ( . f, . d, . h) by elisa [ ] . data are presented as the mean of three biological replicates and error bars represent standard error of the mean. an asterisk indicates a statistically significant difference as compared to the case of incubation at °c and ph (p < . .) calculated using a one-way anova. the lack of an asterisk indicates no statistically significant difference. our observations thus far support a hypothesis in which acidic ph is sufficient to promote the transition of gp from the pre-fusion to the post-fusion conformation. we sought to test this hypothesis by directly visualizing the ph-induced triggering of gp-mediated membrane fusion. to this end, we developed a single-particle lipid mixing assay adapted from those used in studies of influenza [ , , ] , west nile virus [ ] , feline coronavirus [ ] , and vsv [ ] . we formed a planar lipid bilayer supported within a quartz microfluidic cell. contained within the bilayer was lipidsoluble -carboxyfluorescein (cf), a ph-sensitive dye used as a fluorescent ph indicator. vsv-gp pseudovirions were generated as described above, but with the additional inclusion of ha with a mutated furin cleavage site (ha ). this ha mutant is incapable of mediating fusion but enabled the attachment of the virions to the lipid bilayer by way of binding sialic acid, which was included in the bilayer in the form of the gd a ganglioside. this approach allowed us to consider the role of ph in gp-mediated lipid mixing independently from the engagement of a receptor in the target membrane. the viral membrane was labeled with the lipophilic fluorophore did at a concentration sufficient to partially quench fluorescence. based on the previous applications of this approach, we anticipated that the hemifusion of the viral membrane and the planar bilayer would allow the diffusion of the did into the bilayer, which would give rise to a sharp increase in fluorescence as the did dequenches. the did fluorescence then decays as the dye continues to diffuse into the planar bilayer. we bound vsv-ha -gp virions to the supported lipid bilayer in the microfluidic channel at ph . and imaged the surface using prism-based total internal reflection fluorescence (tirf) microscopy ( figure a the binding of neutralizing and non-neutralizing antibodies to lasv gp after exposure to acidic ph at physiological temperature. vsv∆g-gfp-gp viral pseudotypes were treated with either phosphate buffer at ph . or . , or acetate buffer at ph . or . at the indicated temperature for min. virus was subsequently brought back to neutral ph and assayed for binding by non-neutralizing ( . e, . c) and neutralizing antibodies ( . f, . d, . h) by elisa [ ] . data are presented as the mean of three biological replicates and error bars represent standard error of the mean. an asterisk indicates a statistically significant difference as compared to the case of incubation at • c and ph (p < . .) calculated using a one-way anova. the lack of an asterisk indicates no statistically significant difference. our observations thus far support a hypothesis in which acidic ph is sufficient to promote the transition of gp from the pre-fusion to the post-fusion conformation. we sought to test this hypothesis by directly visualizing the ph-induced triggering of gp-mediated membrane fusion. to this end, we developed a single-particle lipid mixing assay adapted from those used in studies of influenza [ , , ] , west nile virus [ ] , feline coronavirus [ ] , and vsv [ ] . we formed a planar lipid bilayer supported within a quartz microfluidic cell. contained within the bilayer was lipid-soluble -carboxyfluorescein (cf), a ph-sensitive dye used as a fluorescent ph indicator. vsv-gp pseudovirions were generated as described above, but with the additional inclusion of ha with a mutated furin cleavage site (ha ). this ha mutant is incapable of mediating fusion but enabled the attachment of the virions to the lipid bilayer by way of binding sialic acid, which was included in the bilayer in the form of the gd a ganglioside. this approach allowed us to consider the role of ph in gp-mediated lipid mixing independently from the engagement of a receptor in the target membrane. the viral membrane was labeled with the lipophilic fluorophore did at a concentration sufficient to partially quench fluorescence. based on the previous applications of this approach, we anticipated that the hemifusion of the viral membrane and the planar bilayer would allow the diffusion of the did into the bilayer, which would give rise to a sharp increase in fluorescence as the did dequenches. the did fluorescence then decays as the dye continues to diffuse into the planar bilayer. we bound vsv-ha -gp virions to the supported lipid bilayer in the microfluidic channel at ph . and imaged the surface using prism-based total internal reflection fluorescence (tirf) microscopy ( figure a) . puncta of did fluorescence indicated the presence of virions attached to the surface. pre-warmed buffers at various ph values were then introduced into the flow cell with a syringe pump with the continuous monitoring of fluorescence. viruses , , x for peer review of the introduction of ph . buffer pre-warmed to °c induced a precipitous loss of cf fluorescence, indicating the successful delivery of low ph buffer to the flow cell. following the acidification of the chamber, the did puncta exhibited the characteristic sharp increase and trailing loss of fluorescence indicative of successful lipid mixing ( figure b , movie s ). the integration of the fluorescence intensity of each punctum allowed for the generation of cf and did fluorescence traces ( figure c ). pseudovirions that had been incubated at acidic ph at °c prior to binding to the planar bilayer displayed no evidence of lipid mixing upon the introduction of acidic buffer to the flow cell. similarly, virions lacking gp and possessing only ha bound the surface but also did not display any evidence of lipid mixing, indicating that the observed lipid mixing was mediated by gp (movie s ). with a vsv core, inactive ha , and lasv gp, hemifusing to a planar lipid bilayer supported by a quartz microscope slide within a microfluidic chamber. pseudovirions were flowed onto the bilayer and allowed to attach at neutral ph by way of the ha -sialic acid interaction. did fluorescence dequenching arose due to single pseudovirions hemifusing to the supported bilayer upon the introduction of acidic ph. cf was included in the supported bilayer in order to provide a fluorescence indicator of ph. fluorescence was detected using the evanescent field generated from the simultaneous total internal reflection of -nm and -nm lasers on a custom-built prism-based tirf microscope. movies were recorded at a ms exposure time ( hz) for a minimum of frames. (b) frames from a single did dequenching event at ph acquired at the indicated time points, where time = was defined as a % decrease in cf fluorescence. (c) representative fluorescence traces (cf, red; did, blue) are from a single dequenching event. the drop in cf fluorescence arises from the acidification of the buffer in the channel. the subsequent spike in did fluorescence arises from dequenching as did diffuses into the planar bilayer. the interval (t) between the % decrease in cf fluorescence and did dequenching was extracted for kinetic analysis. to determine the kinetics of lipid mixing, we extracted the time interval between the drop in cf fluorescence and the dequenching of did fluorescence. these intervals were compiled into histograms and fit to a gamma distribution, which allowed the determination of the rate constant, the number of rate-determining steps leading to lipid mixing, and the average time to lipid mixing ( figure a , section materials and methods) [ , ] . this fitting procedure indicated that at ph . , there existed approximately . ± . rate-determining steps leading to lipid mixing, with a rate constant of . ± . s - , which yielded an average time to lipid mixing of . ± . s. increasing the ph to . and . slowed the rate constant to . ± . and . ± . , respectively, which led to corresponding increases in the time to lipid mixing ( figure b,c, movies s ,s ) . however, across all ph values considered, the number of rate-determining steps between acidification and lipid mixing was consistently approximately ( figure b ). no lipid mixing was seen above ph . on the timescale of our movies. below ph . , lipid mixing occurred too rapidly to accurately evaluate in this assay. in summary, the timing of lipid mixing occurred in a ph-dependent manner, with the kinetics decreasing with increasing ph. the number of rate-determining steps was not sensitive to ph. with a vsv core, inactive ha , and lasv gp, hemifusing to a planar lipid bilayer supported by a quartz microscope slide within a microfluidic chamber. pseudovirions were flowed onto the bilayer and allowed to attach at neutral ph by way of the ha -sialic acid interaction. did fluorescence dequenching arose due to single pseudovirions hemifusing to the supported bilayer upon the introduction of acidic ph. cf was included in the supported bilayer in order to provide a fluorescence indicator of ph. fluorescence was detected using the evanescent field generated from the simultaneous total internal reflection of -nm and -nm lasers on a custom-built prism-based tirf microscope. movies were recorded at a ms exposure time ( hz) for a minimum of frames. (b) frames from a single did dequenching event at ph acquired at the indicated time points, where time = was defined as a % decrease in cf fluorescence. (c) representative fluorescence traces (cf, red; did, blue) are from a single dequenching event. the drop in cf fluorescence arises from the acidification of the buffer in the channel. the subsequent spike in did fluorescence arises from dequenching as did diffuses into the planar bilayer. the interval (t) between the % decrease in cf fluorescence and did dequenching was extracted for kinetic analysis. the introduction of ph . buffer pre-warmed to • c induced a precipitous loss of cf fluorescence, indicating the successful delivery of low ph buffer to the flow cell. following the acidification of the chamber, the did puncta exhibited the characteristic sharp increase and trailing loss of fluorescence indicative of successful lipid mixing ( figure b , movie s ). the integration of the fluorescence intensity of each punctum allowed for the generation of cf and did fluorescence traces ( figure c ). pseudovirions that had been incubated at acidic ph at • c prior to binding to the planar bilayer displayed no evidence of lipid mixing upon the introduction of acidic buffer to the flow cell. similarly, virions lacking gp and possessing only ha bound the surface but also did not display any evidence of lipid mixing, indicating that the observed lipid mixing was mediated by gp (movie s ). to determine the kinetics of lipid mixing, we extracted the time interval between the drop in cf fluorescence and the dequenching of did fluorescence. these intervals were compiled into histograms and fit to a gamma distribution, which allowed the determination of the rate constant, the number of rate-determining steps leading to lipid mixing, and the average time to lipid mixing ( figure a , section materials and methods) [ , ] . this fitting procedure indicated that at ph . , there existed approximately . ± . rate-determining steps leading to lipid mixing, with a rate constant of . ± . s − , which yielded an average time to lipid mixing of . ± . s. increasing viruses , , of the ph to . and . slowed the rate constant to . ± . and . ± . , respectively, which led to corresponding increases in the time to lipid mixing ( figure b ,c, movies s ,s ). however, across all ph values considered, the number of rate-determining steps between acidification and lipid mixing was consistently approximately ( figure b ). no lipid mixing was seen above ph . on the timescale of our movies. below ph . , lipid mixing occurred too rapidly to accurately evaluate in this assay. in summary, the timing of lipid mixing occurred in a ph-dependent manner, with the kinetics decreasing with increasing ph. the number of rate-determining steps was not sensitive to ph. viruses , , x for peer review of we next asked whether the attachment of the virion to the target membrane by way of gp-lamp interactions would affect the kinetics of lipid mixing. to this end, the single-virion lipid mixing experiment was repeated using vsv-gp pseudovirions lacking ha , and with a soluble form of lamp immobilized on the planar bilayer by way of a polyhistidine ( × his) tag bound to a ni-nta lipid. virions were bound to the supported bilayer at ph . . as in the absence of lamp , ph . was not sufficiently acidic to trigger lipid mixing. however, rapid lipid mixing was observed at ph . in the presence of lamp (movie s ). kinetic analysis indicated a rate constant and average time to lipid mixing consistent with that observed at ph . in the absence of lamp (figure ). the number of rate-determining steps ( . ± . ) remained similar to that seen in the absence of lamp ( figure b) . thus, the interaction of gp with lamp increased the kinetics of lipid mixing to an extent equivalent to reducing the ph by approximately one unit. here again, pseudovirions that had been pre-incubated at an acidic ph at °c prior to binding to the planar bilayer in the presence of lamp displayed no sign of lipid mixing after the introduction of acidic buffer to the flow cell. the mean time to dequenching, defined as n/k for a gamma distribution. error bars reflect the % confidence intervals determined in the fitting. an asterisk indicates a statistically significant difference as compared to the case of lamp at ph (p < . .) calculated using a one-way anova. we next asked whether the attachment of the virion to the target membrane by way of gp-lamp interactions would affect the kinetics of lipid mixing. to this end, the single-virion lipid mixing experiment was repeated using vsv-gp pseudovirions lacking ha , and with a soluble form of lamp immobilized on the planar bilayer by way of a polyhistidine ( × his) tag bound to a ni-nta lipid. virions were bound to the supported bilayer at ph . . as in the absence of lamp , ph . was not sufficiently acidic to trigger lipid mixing. however, rapid lipid mixing was observed at ph . in the presence of lamp (movie s ). kinetic analysis indicated a rate constant and average time to lipid mixing consistent with that observed at ph . in the absence of lamp (figure ). the number of rate-determining steps ( . ± . ) remained similar to that seen in the absence of lamp ( figure b) . thus, the interaction of gp with lamp increased the kinetics of lipid mixing to an extent equivalent to reducing the ph by approximately one unit. here again, pseudovirions that had been pre-incubated at an acidic ph at • c prior to binding to the planar bilayer in the presence of lamp displayed no sign of lipid mixing after the introduction of acidic buffer to the flow cell. here, we demonstrate that acidic ph alone is sufficient to induce the conversion of lasv gp to a conformation that is unable to support entry into cells and unable to bind neutralizing antibodies that are specific to the pre-fusion conformation of gp. importantly, only at a physiological temperature is ph . , which approximates the ph of the most acidic endosomes [ ] , sufficiently acidic to promote this transition. no loss of infectivity or loss of antibody binding was seen when virions were incubated at room temperature or on ice at ph . . this temperature-dependent transition to an irreversible conformation is consistent with a kinetically trapped pre-fusion conformation of gp. this implies that a large activation energy limits spontaneous transition out of the pre-fusion conformation into a conformation with much lower free energy. elevated temperature increases the probability of spontaneously crossing this high activation energy, while the protonation of key residues in gp by the acidic environment likely reduces the activation energy. thus, reduced temperatures, which lower the probability of crossing the activation energy, require more acidic ph to trigger conformational changes in gp [ ] . this underscores the importance of maintaining a physiological temperature when characterizing the activity of glycoproteins such as lasv gp. by attaching virions to a target membrane by orthogonal means-through ha -mediated binding to sialic acid-we have further provided direct evidence that acidic ph is sufficient to trigger lasv gp-mediated lipid mixing in the absence of interaction with either αdg or lamp . thus, no receptor-binding event is strictly necessary to trigger gp-mediated lipid mixing. these data provide support for the previous observation that the disruption of the αdg binding site through mutagenesis only minimally affects cell-cell fusion [ ] . these data also support previous studies indicating that lamp is not necessary for gp-mediated cell-cell fusion [ ] , nor is it strictly required for entry into cells [ ] . our kinetic analysis of single-virion lipid mixing events indicates that the time from ph drop to lipid mixing decreases at more acidic ph. this provides direct evidence for the ph-induced reduction in the activation energy that kinetically traps gp in the pre-fusion conformation. interestingly, although the rate of lipid mixing increased with decreasing ph, the number of rate-determining steps remained approximately constant across the ph range tested. this indicates that the mechanism of gp-mediated lipid mixing is consistent across a range of ph values. this may be an indirect observation of intermediate conformations in gp, which may resemble those postulated for other class i viral fusogens, such as influenza ha and ebola gp [ , , , ] . a cryo-electron tomography study of gp documented a ph-induced transition of gp to a conformation distinct from the pre-and post-fusion conformations [ ] , which may reflect an intermediate conformation achieved during fusion. alternatively, the observation of multiple rate-determining steps leading to lipid mixing may be an indication that multiple gp trimers need to engage the target membrane in order to promote lipid mixing. finally, we have directly shown that the attachment of virions to the target membrane by way of binding lamp increases the kinetics of lipid mixing. when lamp is present, lipid mixing occurs at ph . at a rate approximately equal to the rate observed in the absence of lamp at ph . . thus, while lamp is not necessary for gp to promote lipid mixing, lamp increases the ph at which gp-mediated lipid mixing occurs, thus facilitating lasv entry under less harsh conditions, as previously suggested [ ] . this indicates that lamp and acidic ph synergistically reduce the activation energy that limits gp conformational changes that lead to fusion. this observation is entirely consistent with previous studies demonstrating that lamp increased the ph at which gp could mediate cell-cell fusion [ ] , and that virions enter by way of less acidic endosomes in cells expressing lamp as compared to cells lacking lamp [ ] . taken together, the present and previous studies provide a consistent understanding for the roles that endosomal ph and lamp play in lasv entry. future studies will be aimed at probing the mechanism by which acidic ph and lamp enable gp conformational changes. this will likely require a combination of structure determination and biophysical interrogations equipped to visualize gp conformational changes that lead to fusion. the following are available online at http://www.mdpi.com/ - / / / /s , movie s . representative movie of single-virion lipid mixing events that occurred upon exposure to ph . the left channel is a visualization of cf fluorescence used to mark the drop in ph. the right channel is a visualization of the did fluorescence, which was used to visualize lipid mixing. movies were recorded at ms for a minimum of frames; movie s . representative movie of the single-virion lipid mixing experiment with vsv pseudotypes bearing only ha . the movie is presented as for movie s ; movie s . representative movie of single-virion lipid mixing events that occurred upon exposure to ph . . the movie is presented as for movie s ; movie s . representative movie of single-virion lipid mixing events that occurred upon exposure to ph . the movie is presented as for movie s ; movie s . representative movie of single-virion lipid mixing events that occurred upon exposure to ph in the presence of lamp . the movie is presented as for movie s . retrospective cohort study of lassa fever in pregnancy lassa fever-induced sensorineural hearing loss: a neglected public health and social burden lassa virus glycoprotein: stopping a moving target the lassa virus glycoprotein precursor gp-c is proteolytically processed by subtilase ski- /s p the role of proteolytic processing and the stable signal peptide in expression of the old world arenavirus envelope glycoprotein ectodomain mapping of the lassa virus lamp binding site reveals unique determinants not shared by other old world arenaviruses identification of -dystroglycan as a receptor for lymphocytic choriomeningitis virus and lassa fever virus lassa virus cell entry via dystroglycan involves an unusual pathway of macropinocytosis emerging intracellular receptors for hemorrhagic fever viruses mutational analysis of lassa virus glycoprotein highlights regions required for alpha-dystroglycan utilization molecular mechanism for lamp recognition by lassa virus structural basis for antibody-mediated neutralization of lassa virus acidic ph-induced conformations and lamp binding of the lassa virus glycoprotein spike influenza hemagglutinin: kinetic control of protein function viral membrane fusion x-ray structure of the arenavirus glycoprotein gp in its postfusion hairpin conformation identification of an n-terminal trimeric coiled-coil core within arenavirus glycoprotein permits assignment to class i viral fusion proteins variations in core packing of gp from old world mammarenaviruses in their post-fusion conformations affect membrane-fusion efficiencies lamp increases the efficiency of lassa virus infection by promoting fusion in less acidic endosomal compartments role of lamp binding and ph sensing by the spike complex of lassa virus biochemical reconstitution of hemorrhagic-fever arenavirus envelope glycoprotein-mediated membrane fusion mechanism of lymphocytic choriomeningitis virus entry into cells early events in arenavirus replication are sensitive to lysosomotropic compounds different mechanisms of cell entry by human-pathogenic old world and new world arenaviruses cell entry by human pathogenic arenaviruses characterization of lassa virus cell entry and neutralization with lassa virus pseudoparticles amino acids from both n-terminal hydrophobic regions of the lassa virus envelope glycoprotein gp- are critical for ph-dependent membrane fusion and infectivity acidic ph triggers lcmv membrane fusion activity and conformational change in the glycoprotein spike direct visualization of the conformational dynamics of single influenza hemagglutinin trimers generation of vsv pseudotypes using recombinant ∆g-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines most neutralizing human monoclonal antibodies target novel epitopes requiring both lassa virus glycoprotein subunits single-particle kinetics of influenza virus membrane fusion stability of infectious influenza a viruses to treatment at low ph and heating vesicular stomatitis virus-based vaccines against lassa and ebola viruses receptor binding and membrane fusion in virus entry: the influenza hemagglutinin influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates influenza virus-membrane fusion triggered by proton uncaging for single particle studies of fusion kinetics sequential conformational rearrangements in flavivirus membrane fusion single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers mechanism of membrane fusion induced by vesicular stomatitis virus g protein endosome maturation influenza hemagglutinin is spring-loaded by a metastable native conformation conformational changes in the ebola virus membrane fusion machine induced by ph, ca + , and receptor binding we wish to thank melinda brindley, peter kwong, john coffin, and juha huiskonen for kindly providing plasmids for the expression of lasv gp, iav ha, alv env, and lamp , respectively. we are also grateful to james robinson for providing lasv antibodies. the authors declare no conflict of interest. key: cord- -tlluxd authors: welch, david title: is network clustering detectable in transmission trees? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: tlluxd networks are often used to model the contact processes that allow pathogens to spread between hosts but it remains unclear which models best describe these networks. one question is whether clustering in networks, roughly defined as the propensity for triangles to form, affects the dynamics of disease spread. we perform a simulation study to see if there is a signal in epidemic transmission trees of clustering. we simulate susceptible-exposed-infectious-removed (seir) epidemics (with no re-infection) over networks with fixed degree sequences but different levels of clustering and compare trees from networks with the same degree sequence and different clustering levels. we find that the variation of such trees simulated on networks with different levels of clustering is barely greater than those simulated on networks with the same level of clustering, suggesting that clustering can not be detected in transmission data when re-infection does not occur. to understand the dynamics of infectious diseases it is crucial to understand the structure and interactions within the host population. conversely, it is possible to learn something about host population structure by observing the pattern of pathogen spread within it. in either case, it is necessary to have a good model of the host population structure and interactions within it. networks, where nodes of the network represent hosts and edges between nodes represent contacts across which pathogens may be transmitted, are now regularly used to model host interactions [ ] [ ] [ ] . while many models have been proposed to describe the structure of these contact networks for different populations and different modes of transmission, it is not yet understood how different features of networks affect the spread of pathogens. one promising development in this field is the use of statistical techniques which aim to model a contact network based on data relating to the passage of a pathogen through a population. such data includes infection times [ ] [ ] [ ] and genetic sequences that are collected from an epidemic present in the population of interest [ ] [ ] [ ] . these data have previously been shown to be useful for reconstructing transmission histories (the distinction between a contact network and a transmission history is that a contact network includes all edges between hosts across which disease may spread, whereas the transmission history is just the subset of edges across which transmission actually occurred). infection times can be used to crudely reconstruct transmission histories by examining which individuals were infectious at the time that any particular individual was infected [ ] . genetic sequences from viruses are informative about who infected whom by comparing the similarity between sequences. due to the random accumulation of mutations in the sequences, we expect sequences from an infector/infectee pair to be much closer to each other than sequences from a randomly selected pair in the population (see [ ] for a review of modern approaches to analysing viral genetic data). the work of [ ] [ ] [ ] seeks to extend the use of this data to reconstruct a model for the whole contact network rather than just the transmission history. in theory, these statistical methods could settle arguments about which features of the network are important in the transmission of the disease and which are simply artifacts of the physical system. in this article, we focus on clustering in networks and ask whether or not networks which differ only in their level of clustering could be distinguished if all we observed was transmission data from an epidemic outbreak. the answer to this question will determine whether these new statistical techniques can be extended to estimate the level of clustering in a network. throughout, we consider a population with n individuals that interact through some contact process. this population and its interactions are fully described by a undirected random network, denoted y , on n nodes. an simple example of a network is shown in figure with illustrations of some of the terms we use in this article. y can be represented by the symmetric binary matrix [y ij ] where y ij = y ji = if an edge is present between nodes i and j, otherwise y ij = . we stipulate that there no loops in the network, so y ii = for all i. the degree of the ith node, denoted d i is the number of edges connected to i, so d i = j:j>i y ij . clustering is one of the central features of observed social networks [ , ] . intuitively, clustering is the propensity for triangles or other small cycles to form, so that, for example, a friend of my friend is also likely to be my friend. where there is a positive clustering effect, the existence of edges (i, j) and (i, k) increases the propensity for the edge (j, k) to exist, while a negative clustering effect implies that (j, k) is less likely to exist given the presence of (i, j) and (i, k). when there is no clustering effect, the presence or absence of (i, j) and (i, k) has no bearing on that of (j, k). thus clustering is one of the most basic of the true network effects-when it is present, the relationship between two nodes depends not only on properties of the nodes themselves but the presence or absence of other relationships in the network. the effect of clustering on the dynamics of stochastic epidemics that run over networks remains largely unknown, though it has been studied in a few special cases. the difficulty with studying this effect in isolation is in trying to construct a network model where clustering can change but other properties of the network are held constant. in simulations we study here, we focus on holding the degree sequence of a network constant-that is, each node maintains the same number of contacts-while varying the level of clustering. intuition suggests that clustering will have some effect on epidemic dynamics since, in a graph with no cycles, if an infection is introduced to a population at node i and there is a path leading to j then k, k can only become infected if j does first. however, where cycles are present, there may be multiple paths leading from i to k that do not include j, so giving a different probability that k becomes infected and a different expected time to infection for k. figure . an example of a network on nodes. the nodes are the red dots, labelled to and represent individuals in the population. the edges are shown as black lines connecting the nodes and represent possible routes of transmission. the degree of each node is number of edges adjacent to it, so that node has degree and node has degree . the degree sequence of the network is the count of nodes with a given degree and can be represented by the vector ( , , , , , ) showing that there are nodes of degree , of degree , of degree and so on. a cycle in the network is a path starting at a node and following distinct edges to end up back at the same node. for example, the path from node to node to node and back to node is a cycle but there is no cycle that includes node . clustering is a measure of propensity of cycles of length (triangles) to form. here, the edges ( , ) and ( , ) form a triangle with the edge ( , ), so work to increase clustering in the network. however, the edges ( , ) and ( , ) do not comprise part of a triangle as ( , ) does not exist, so work to decrease clustering. previous work on the effect of clustering on epidemic dynamics has produced a variety of results which are largely specific to particular types of networks. newman [ ] and britton et al. [ ] show that for a class of networks known as random intersection graphs in which individuals belong to one or more overlapping groups and groups form fully connected cliques, an increase in clustering reduces the epidemic threshold, that is, major outbreaks may occur at lower levels of transmissibility in highly clustered networks. newman [ ] , using heuristic methods and simulations, suggests that for sufficiently high levels of transmissibility the expected size of an outbreak is smaller in a highly clustered network than it would be in a similar network with lower clustering. these articles show that graphs with different levels of clustering do, at least in some cases, have different outbreak probabilities and final size distributions for epidemic outbreaks. kiss and green [ ] provide a succinct rebuttal to the suggestion that the effects found by [ ] and [ ] are solely due to clustering. they show that, while the mean degree of the network is preserved in the random intersection graph, the degree distribution varies greatly (in particular, there are many zero-degree nodes) and variance of this distribution increases with clustering. an increase in the variance of the degree distribution has previously been shown to lower the epidemic threshold. they demonstrate that a rewiring of random intersection graphs that preserves the degree sequence but decreases clustering produces networks with similarly lowered epidemic thresholds and even smaller mean outbreak sizes. our experiments, reported below, are similar in spirit to those of [ ] but look at networks with different degree distributions and study in detail how epidemic data from networks with varying levels of clustering might vary. ball et al. [ ] show, using analytical techniques, that clustering induced by household structure in a population (where individuals have many contacts with individuals in the same household and fewer global contacts with those outside of the household) has an effect on probability of an outbreak and the expected size of any outbreak. the probability of an outbreak, in some special cases, is shown to be monotonically decreasing with clustering coefficient and the expected outbreak size also decreases with clustering. there is no suggestion that these results will apply to clustered networks outside of this specific type of network or that they apply when degree distributions are held constant. eames [ ] also studies networks with two types of contacts: regular contacts (between people who live or work together, for example) and random contacts (sharing a train ride, for example). using simulations of a stochastic epidemic model and deterministic approximations, it is shown that both outbreak final size and probability of an outbreak are reduced with increased clustering, particularly when regular contacts dominate. as the number of random contacts increases, the effect of clustering reduces to almost zero. strong effects on the expected outbreak size in networks with no random contacts are observed for values of the clustering coefficient above about . , however, no indication of the magnitude of the variance of these effects is given. keeling [ ] reports similar results, introducing clustering to a network using a spatial technique-nodes live in a two-dimensional space and two nodes are connected by an edge with a probability inversely proportional to their distance. the clustering comes about by randomly choosing positions in space to which nodes are attracted before connections are made. the results suggest that changes in clustering at lower levels has little effect on the probability of an outbreak, but as the clustering coefficient reaches about . , the chance of an outbreak reduces significantly. as in [ ] and [ ] , while the mean degree of network nodes is held constant here, nothing is said about the degree distribution as clustering varies. serrano and boguñá [ ] look specifically at infinite power-law networks and shows that the probability of an outbreak increases as clustering increases but the expected size of an outbreak decreases. some more recent papers seek to distinguish the effects of clustering from confounding factors such as assortativity and degree sequence. miller [ ] develops analytic approximations to study the interplay of various effects such as clustering, heterogeneity in host infectiousness and susceptibility and the weighting of contacts on the spread of disease over a network. the impact of clustering on the probability and size of an outbreak is found to be small on "reasonable" networks so long as the average degree of the network is not too low. the rate at which the epidemic spreads, measured by the reproduction number, r , is found to reduce with increased clustering in such networks. in networks with low mean degree, r may be reduced to point of affecting the probability and size of an outbreak. miller [ ] points out that studies of the effects of clustering should take into account assortativity in the network, that is, the correlations in node degree between connected nodes. assortativity has been shown to affect epidemic dynamics and changing the level of clustering in a network can change the level of assortativity. to distinguish between the effects of assortativity and clustering, a method of producing networks with arbitrary degree distributions and arbitrary levels of clustering with or without correlated degrees is presented and studied using percolation methods. the effect of increasing clustering in these models is to reduce the probability of outbreaks and reduce the expected size of an epidemic. badham and stocker [ ] use simulated networks and epidemics to study the relationship between assortativity and clustering. their results suggest that increased clustering diminished the final size of the epidemic, while the effect of clustering on probability of outbreak was not very clear. like [ ] , moslonka-lefebvre et al. [ ] use simulations to try to distinguish the effects of clustering and assortativity but look at directed graphs. here, they find that clustering has little effect on epidemic behaviour. melnik et al. [ ] propose that the theory developed for epidemics on unclustered (tree-like) networks applies with a high degree of accuracy to networks with clustering so long as the network has a small-world property [ ] . that is, if the mean length of the shortest path between vertices of the clustered network is sufficiently small, quantities such as the probability of an outbreak on the network can be estimated using known results that require only the degree distribution and degree correlations. the theory is tested using simulations on various empirical networks from a wide range of domains and synthetic networks simulated from theoretical models. taken together, these studies show that clustering can have significant effects on crucial properties of epidemics on networks such as the probability, size and speed of an outbreak. these results primarily relate to the final outcome and mean behaviour of epidemics. however, if we can obtain a transmission tree for an outbreak then we have information from the start to the finish of a particular epidemic including times of infection and who infected whom. since epidemics are stochastic processes, data from a particular epidemic may differ considerably from the predicted mean. whether or not such data contains information about clustering in the underlying network is the question we seek to address here. we simulate epidemics over networks with fixed degree distributions and varying levels of clustering and inspect various summary statistics of the resulting epidemic data, comparing the summaries for epidemics run over networks with the same degree distribution but different levels of clustering. the precise details of the simulations are described in section . the results of the simulations, presented in section , show that there is likely little to no signal of clustering in a contact network to be found in a single realisation of an epidemic process over that network. we conclude that it is unlikely that clustering parameters can be inferred solely from epidemiological data that relates to the transmission tree and suggest that further work in parameter estimation for contact networks would be best focused on other properties of contact networks such as degree distribution or broader notions of population structure. we simulate multiple networks from two network models: a bernoulli model [ ] and a power-law model [ ] . under the bernoulli model (also called the erdős-rényi or binomial model), an edge between nodes i and j is present with some fixed probability ≤ p ≤ and absent with probability − p, independently of all other edges. due to their simplicity, bernoulli networks are well-studied and commonly used in disease modeling but are not generally thought to be accurate models of social systems. a bernoulli network is trivial to construct by sampling first the total number of edges in a the graph where n is the number of nodes in the network, and then sampling |y | edges uniformly at random without replacement. we set n = and p = /n = . in the simulations reported below. a power-law network is defined as having a power-law degree distribution, that is, for nodes i = , . . . , n , p (d i = k) ∝ k −α for some α > . power-law networks are commonly used to model social interactions and various estimates of α in the range . - . have been claimed for observed social networks. in the model used here, we set α = . . we simulate power-law using a reed-molloy type algorithm [ ] . that is, the degree of each node, d i , i = , . . . , n , is sampled from the appropriate distribution. node i is then assigned d i "edge stubs" and pairs of stubs are sampled uniformly without replacement to be joined and become edges. when all stubs have been paired, loops are removed and multiple edges between the same nodes are collapsed to single edges. this last step of removing loops and multiple edges causes the resulting graph to be only an approximation of a power law graph but the approximation is good for even moderately large n . we set n = and consider only the largest connected component of the network in the simulation reported below. the size of the networks considered here is smaller than some considered in simulation studies though on a par with others (see, for example, [ ] who looks a a wide range of network sizes). we choose these network sizes partly for convenience and partly because the current computational methods for statistical fitting of epidemic data to network models would struggle with networks much larger than a few hundred nodes [ ] so our interest is in networks around this size. from each sampled network, y , we generate two further networks, y hi and y lo that preserve the degrees of all nodes in y but have, respectively, high and low levels of clustering. we achieve this using a monte carlo algorithm implemented in the ergm package [ ] in r [ ] that randomly rewires the input network while preserving the degree distribution. a similar algorithm is implemented in bansal et al. [ ] . for details of the ergm model and implementation of this algorithm, we refer the reader the package manual [ ] and note that the two commands used to simulate our networks are y_hi = simulate(y˜gwesp( . ,fixed=t), theta = ,... constraints =˜degreedist, burnin= e+ ) and y_lo = simulate(y˜gwesp( . ,fixed=t), theta = - ,... constraints =˜degreedist, burnin= e+ ) we measure clustering in the resulting networks using the clustering coefficient [ ] , defined as follows. let n i = {j|y ij = } be the neighbourhood of vertex i and d i = |n i | be the degree of i. let n i = j<k∈n i y jk be the number of edges between neighbours of i. then, if d i > , the local clustering coefficient is , which is the ratio of extant edges between neighbours of i to possible edges. for d i ∈ { , }, let c i = . the (global) clustering coefficient is the mean of the local coefficients, } is somewhat arbitrary, though other possible choices, such as c i = or excluding those statistics from the mean, give similar qualitative results in our experiments. over each simulated network, we simulate a stochastic susceptible-exposed-infectious-removed (seir) epidemic. all nodes are initially susceptible to the infection. the outbreak starts when a single node is chosen uniformly at random and exposed to a disease. after a gamma-distributed waiting period with mean k e θ e and variance k e θ e , the node becomes infectious. the infection may spread across the edges of the network, from infectious nodes to susceptible nodes according to a poisson process with rate β. infected nodes recover after an infectious period with a gamma distributed waiting time with mean k i θ i and variance k i θ i . once a node is recovered, it plays no further part in the spread of the infection. the process stops when there are no longer any exposed or infectious nodes. for each pair, y hi and y lo , we start the infection from the same node. we condition on the outbreak infecting at least nodes. the parameter values are set at β = . , k e = k i = and θ e = θ i = in the simulations reported below. a transmission tree encodes all information about the epidemic outbreak it describes. as such, it is a very complicated object. to compare sets of transmission trees and decide whether there are some systematic differences between them, we rely on various summary statistics derived from the trees and compare the distribution of the summaries over the ensembles in question. the summaries we use can be divided into two groups, those relating solely to the number of infected through time and those relating to topology of the tree. the first group of summaries can all be derived from the epidemic curves, that is, the number infected as a function of time. from this, we derive scalar summaries being the total number of individuals infected, the length of the epidemic (measured from the time of the first infection to the last recovery), the maximum of the epidemic curve and the time of that maximum. we label each individual in the population (equivalently, each node in the contact network) with labels , . . . , n . a transmission tree, a distinct graph from the contact network, has a time component and can be defined as follows; an example of a transmission tree and the notation is given in figure . there are three types of nodes in a transmission tree (not to be confused with nodes in the contact network): the root node corresponding to the initial infection, transmission or internal nodes corresponding to transmission events, and leaf or external nodes corresponding to recovery events. leaf nodes are defined by the time and label pair (t i , u i ) where t ≥ is the time of the recovery event and u i is the label of individual that recovered. the internal nodes are associated with the triple (t i , u i , v i ) being the time of the event, t i , the label u i of the exposed individual and v i that is the transmitter or "parent" of the infection. the root node is like an internal node but the infection parent is given as , so is denoted (t , u , ). the branches of the tree are times between infection, transmission and recovery events for a particular vertex. for example, if the individual labelled u is infected at event (t , u, v ), is involved in transmission events (t k , v k , u), k = , . . . , m − , and recovers at (t m , u) where t i < t j for i < j and {v , . . . , u m− } are other individuals in the population, there are m − branches of the transmission tree at u defined by the intervals (t i , t i+ ], for i = , . . . , m − . we summarise the transmission tree using the following statistics: the mean branch length between internal nodes (corresponding to the mean time between secondary infections for each individual); the mean branch length of those branches adjacent to a leaf node (which corresponds to the mean time from the last secondary infection to removal for each individual); the number of secondary infections caused by each infected individual (that is, for each infected individual v we count the number of internal nodes that have the form (t i , u i , v), for some i); and, the distribution of infective descendants for each individual, v, which is defined recursively as the sum of secondary infections caused by v and the secondary infections caused by the secondary infections of v and so on. an equivalent definition is to say that number of infective descendants of v is the number of leaves that have a node of the form (t, u i , v) as an ancestor. finally, we consider the number of cherries in the tree [ ] which is the number of pairs of leaves that are adjacent to a common internal node. this simple statistic is chosen as it is easy to compute and contains information about the topology or shape of the tree. to compare the number of cherries in outbreaks of different size, we look at the ratio of extant cherries to the maximum possible number of cherries for the given outbreak. the experimental pipeline can thus be summarised as: . repeat for i = , . . . , : (a) sample a graph y i according to given degree distribution. (b) simulate two further graphs y hi i and y lo i with high clustering and low clustering, respectively, using a monte carlo sampler that rewires y i to alter the clustering level while preserving the degree of each node. we report results here for seir epidemics run over bernoulli and power-law networks. a number of smaller trials that we do not report were run: with different values chosen for the network and epidemic parameters; on networks with the same degree distributions as a random intersection graph; and, using an sir epidemic rather than an seir. the results for those smaller trials were qualitatively similar to the results reported here. the distributions of the measured clustering coefficients is shown in figure and show that the simulated networks with high and low clustering for a given degree distribution are easily distinguished from one another. the bernoulli networks with low clustering contain no triangles, so the clustering coefficient for each of these networks is zero, while for highly-clustered bernoulli networks, clustering coefficients are in the range ( . , . ). for the power-law networks, the low clustered networks have clustering in the range ( . , . ) while the highly clustered networks have clustering in the range ( . , . ). figures and show comparisons of summary statistics for networks with differing levels of clustering and bernoulli degree distributions. the summaries show some differences between the outbreaks on the differently clustered networks. in particular, the outbreaks in the highly-clustered networks spread more slowly, on average, leading to marginally longer epidemics with fewer individuals infected at the peak of the outbreak, that occurs slightly later, than we see in outbreaks on the networks with low clustering. these mean effects are in line with the predictions of [ ] . the variances of the measured statistics, however, are sufficiently large due to stochastic effects in the model that the ranges of the distributions overlap almost completely in most cases. statistics derived from the transmission tree appear to add little information, with only the number of cherries differing in the mean. figures and show the corresponding distributions for networks with power-law degree distributions. again, differences in the means between the two sets of statistics are apparent with the mean length of epidemic, total number infected and number infected at peak all lower in the epidemics on networks with high-clustering. the largest difference is found in the total number infected, where in the low-clustered networks, the range of the statistic is ( , ) while it is just ( , ) in the high-clustered networks. the primary cause here is due to the change in size of the largest connected component of the network. if we adjust for this by looking instead at the proportion of the giant component infected, the distributions again overlap almost completely with the range for the proportion infected in the low-clustered networks being ( . , . ) and ( . , . ) for the high-clustered networks. the results presented above suggest that the behaviour of an epidemic on a random network with a given degree sequence is relatively unaffected by the level of clustering in the network. some effect is seen, but it is small relative to the random variation we see between epidemics on similarly clustered networks. the results also suggest that the complete transmission tree from an epidemic provides little information about clustering that is not present in the epidemic curve. these results do not imply that clustering has little effect, rather they suggest as noted in [ ] , the apparently strong effect of clustering observed by some is more likely to due to a change in the degree distribution-an effect we have nullified by holding the degree sequence constant. these broader effects are probably best analysed on a grosser level such as the household or subgroup level rather than at the individual level at which clustering is measured. our simulation method, in which the degree sequence for each network is held constant while clustering levels are adjusted, places significant restrictions on the space of possible graphs and therefore clustering coefficients. the levels of clustering achieved in the simulations reported here (for example, having a clustering coefficient in the low-clustered bernoulli case of versus a mean of . for the high-clustered case) are not so high as those considered in the some of the simulations and theoretical work described in section , and this may partly account for the limited effect on epidemic outcomes that we find here. there is little known about the levels of clustering found in real contact networks [ ] (though one recent detailed study [ ] find values for clustering in a social contact network in the region . - . ) and no evidence to suggest that very extreme values of clustering are achieved for a given degree sequence. it is plausible, however, that the degree sequence of a social network of interest could be found-for example, via ego-centric or full-network sampling [ ] [ ] [ ] -and therefore reasonable to explore the achievable levels of clustering conditional on the degree sequence. in doing so, we separate the effects on epidemic dynamics of change in the degree sequence of the contact network from those of clustering. from a statistical point of view, these results indicate that even with full data from a particular epidemic outbreak, such as complete knowledge of the transmission tree, it is unlikely that the level of clustering in the underlying contact network could be accurately inferred independently of the degree distribution. this is primarily due to the large stochastic variation found from one epidemic to the next that masks the relatively modest effects of clustering on an outbreak. with this much stochastic noise, we suggest that it would require data from many outbreaks over the same network (that is, pathogens with a similar mode of transmission spreading in the same population) to infer the clustering level of that network with any accuracy. the results also suggest that attempting to estimate a clustering parameter without either estimating or fixing the degree sequence, as in goudie [ ] , may see the estimated clustering parameter acting chiefly a proxy for the degree sequence. it cannot be ruled out that a statistical method, which takes into account the complete data rather than the summaries we use here, or which takes data from parts of the parameter space that we have not touched on here, could find some signal of clustering from such data. in practise, however, it would be highly unusual to have access to anything approaching complete data. a more realistic data set might include times of onset and recovery from disease symptoms for some individuals in the population and sequences taken from viral genetic material. the noise that characterises such data sets already makes it difficult to accurately reconstruct the transmission tree; this extra uncertainty would likely make any inference of a clustering parameter, in the absence of other information, very difficult. i thank david hunter, marcel salathé, mary poss and an anonymous referee for useful comments and references that improved this paper. this work is supported by nih grant r -gm - . a survey of statistical network models. foundations and trends in machine learning network epidemiology: a handbook for survey design and data collection the structure and function of complex networks bayesian inference for stochastic epidemics in populations with random social structure. scand bayesian inference for contact networks given epidemic data. scand a network-based analysis of the hagelloch measles data episodic sexual transmission of hiv revealed by molecular phylodynamics integrating genetic and epidemiological data to determine transmission pathways of foot-and-mouth disease virus statistical inference to advance network models in epidemiology different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures evolutionary analysis of the dynamics of viral infectious disease collective dynamics of small world networks why social networks are different from other types of networks properties of highly clustered networks epidemics on random graphs with tunable clustering comment on "properties of highly clustered networks analysis of a stochastic sir epidemic on a random network incorporating household structure modelling disease spread through random and regular contacts in clustered populations the implications of network structure for epidemic dynamics percolation and epidemic thresholds in clustered networks spread of infectious disease through clustered populations percolation and epidemics in random clustered networks the impact of network clustering and assortativity on epidemic behaviour. theor disease spread in small-size directed networks: epidemic threshold, correlation between links to and from nodes, and clustering the unreasonable effectiveness of tree-based theory for networks with clustering on random graphs statistical mechanics of complex networks a critical point for random graphs with a given degree sequence a package to fit, simulate and diagnose exponential-family models for networks, version . - r development core team. r: a language and environment for statistical computing. r foundation for statistical computing exploring biological network structure with clustered random networks a package to fit, simulate and diagnose exponential-family models for networks distributions of cherries for two models of trees a high-resolution human contact network for infectious disease transmission using data on social contacts to estimate age-specific transmission parameters for respiratory-spread infectious agents social contacts and mixing patterns relevant to the spread of infectious diseases what does a tree tell us about a network? this article is an open access article distributed under the terms and conditions of the creative commons attribution license key: cord- -zwl safn authors: plant, ewan p.; sims, amy c.; baric, ralph s.; dinman, jonathan d.; taylor, deborah r. title: altering sars coronavirus frameshift efficiency affects genomic and subgenomic rna production date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: zwl safn in previous studies, differences in the amount of genomic and subgenomic rna produced by coronaviruses with mutations in the programmed ribosomal frameshift signal of orf a/b were observed. it was not clear if these differences were due to changes in genomic sequence, the protein sequence or the frequency of frameshifting. here, viruses with synonymous codon changes are shown to produce different ratios of genomic and subgenomic rna. these findings demonstrate that the protein sequence is not the primary cause of altered genomic and subgenomic rna production. the synonymous codon changes affect both the structure of the frameshift signal and frameshifting efficiency. small differences in frameshifting efficiency result in dramatic differences in genomic rna production and tcid( ) suggesting that the frameshifting frequency must stay above a certain threshold for optimal virus production. the data suggest that either the rna sequence or the ratio of viral proteins resulting from different levels of frameshifting affects viral replication. and protein. here, analyses using synonymous protein coding mutations demonstrate that the region of the genome that harbors the frameshift signal affects the regulation of genomic and subgenomic rna production without altering protein sequence. we discuss possible reasons for this. in an effort to further our understanding of how the sars frameshift signal functions we performed deletion and mutagenesis studies an analyzed the effects on frameshifting efficiency, rna structure and viral rna production. there are at least three different types of structures used by the coronaviruses to stimulate frameshifting. some coronaviruses, such as sars-cov, use a three stemmed pseudoknot, others such as human coronavirus e have an 'elaborated pseudoknot' or kissing stem-loops, while the avian infectious bronchitis virus utilizes a two stemmed pseudoknot [ , , ] . that these diverse coronaviruses retain some sequence between stems and yet have quite different frameshift stimulating structures is intriguing. one inference that can be made is that the rna sequence, or a structure formed by it, is involved in some other aspect of the virus lifecycle. our initial hypothesis was that a regulatory element was contained within this sequence. here we describe deletion and mutagenesis experiments with a dual luciferase reporter to show that the effect the sequence between stems and has on frameshifting efficiency is due to structural changes those mutations cause in the pseudoknot. further, we show by making synonomous mutations in the virus that different levels of frameshifting efficiency affect the production of genomic and subgenomic rna differently. this could be caused by disruption of a regulatory element or the altered ratio of proteins derived from the coding region flanking the frameshift signal. the individual residues and each stem in the sars pseudoknot make unique contributions to frameshifting efficiency [ ] [ ] [ ] . for example, while stem is essential for frameshifting, stem is not. even small changes in stem (e.g., replacing or deleting the bulged adenosine in stem with a cytosine) reduced frameshifting to levels similar to those observed with the complete disruption of stem [ ] . in contrast, altering the bulged adenosine in stem or disruption of stem promoted only modest changes in frameshifting [ ] . these observations engendered the hypothesis that stem formation may affect frameshifting by enhancing or facilitating the formation of stem . experiments were designed to test the effects of changes to the length of stem while still maintaining a stable structure. beginning with the wild-type stem containing nine paired bases and a nine residue loop, the following series of mutants were constructed. first, the loop capping stem was replaced with a shorter tetraloop; the l tetra construct contains ten paired bases and a four residue loop. this was used as the basis for construction of two additional tetraloop constructs with progressively shorter stem structures: l tetra/s - bp, containing only four paired bases in the stem, and l tetra/s - bp, which has only two paired bases. the '-uucg- ' tetraloop sequence used in the l tetra/s - bp mutant was selected because the last base-pair before the tetraloop affects its formation. the closing pair for uncg tetraloops is usually c-g compared to the g-c closing base-pair found in cuug tetraloops. all of the truncated structures promoted frameshifting at levels equal to or greater than the wild-type pseudoknot structure using a dual luciferase reporter assay ( figure ) . interestingly, the l tetra/s - bp mutant stimulated frameshifting at . % compared to the to . % observed for the other stem mutants. it is possible that the difference in the tetraloop contributed to the slight increase in frameshifting efficiency. these results, and experiments reported by brierley et al., for another coronavirus [ ] indicate that the length of stem does not play a critical role in frameshifting, suggesting that it may serve some other function. removing stem does not reduce programmed - ribosomal frameshifting (- prf) efficiency. the predicted secondary structure of the sars coronavirus pseudoknot is shown, along with a series of mutants with stem truncated. the stems are labeled s to s and the loops labeled l to l . site directed mutagenesis was used to replace the wild-type loop with a cuug tetraloop (l tetra). additional truncations to stem were made (l tetra/s - bp and l tetra/s - bp) and frameshifting efficiency analyzed by dual luciferase assay. frameshifting efficiency is expressed as a percentage with standard error as described in the experimental section. the sequence of stem itself, have similarly strong roles [ , ] . however, given the ability of tetraloop-capped stem mutants to promote high levels of frameshifting, we asked if these mutations could compensate for deletion of the bulged adenosine in stem . to this end, the bulged adenosines in stems and were deleted both individually and together in the context of the l tetra construct ( figure ). l tetra was chosen as the baseline because, among the tetraloop mutants, it most closely represents the wild-type sequence. deletion of the bulged adenosine in stem (l tetras Δ) reduced frameshifting by . % ( . % versus . %, p = . × − , figure ). when the bulged adenosine from stem was removed (l tetras Δ) a similar reduction of . % was observed ( . % versus . %, p = . × − ). interestingly, removal of both adenosines (l tetras Δs Δ) partially restored frameshifting with only a . % reduction in frameshifting ( . % versus . %, p = . × − ). bulged adenosines, which are known to bend helices [ ] , may be participating in formation of a functional pseudoknot structure. indeed, many frameshift-stimulating structures have bent conformations that are postulated to help effect frameshifting [ ] [ ] [ ] . importantly, however, the contribution of the bulges in the sars pseudoknot is not the same in the context of the native stem /loop . specifically, removing both bulged adenosines (s Δs Δ) from the wild-type backbone decreased rates of frameshifting by % ( . versus . %, p = . × − ), similar to that observed with the removal of the stem bulge alone ( figure ). together these results suggest that stabilization of stem assists in the formation or stability of stem , which in turn is essential for frameshifting. however, the actual sequence of loop or the tetraloop-capping stem also affected frameshifting efficiency, either through the stabilization of stem or the formation of stem itself, thus implicating the nucleotides in loop or the tetraloop in tertiary structure interactions. further evidence comes from the chemical protection data (below), demonstrating that the altered sequence of loop affects the structure of stems and . although secondary structure predictions indicate that the nucleotides in loop are not part of a helix, those nucleotides are susceptible to both single-and double-strand-specific nucleases [ ] suggesting that these nucleotides participate, at least part of the time, in some ordered structural state. stabilizing stem with a tetraloop preserves - prf efficiency. site directed mutagenesis was used to change loop of the wild-type sars pseudoknot into tetraloop (l tetra from figure ). the bulged adenosines in stems and were removed by site directed mutagenesis singly or together in conjunction with the tetraloop (l tetras Δ, l tetras Δ, and l tetras Δs Δ). both bulged adenosines were removed from the wild-type pseudoknot (s Δs Δ). frameshifting efficiency is expressed as a percentage with standard error as described in the experimental section. while the high degree of phylogenetic conservation of stem in many coronaviruses [ ] suggests that it serves an important function, mutations in this element have less impact on frameshifting efficiency than mutations in stem [ , , ) . prior structural analyses also suggested that the stability of the structure may contribute to frameshifting efficiency. here, specific changes were made to stem in order to directly address this issue. a negative control plasmid was constructed in which stem base-pairing was disrupted (s d) by mutating three residues in the third codon position so as to retain the amino acid sequence of the frameshift protein pp ab ( figure a) . consistent with prior studies [ , , ] , disruption of stem resulted in a % reduction in frameshifting ( % versus . %, figure a ) demonstrating that the integrity of the second stem, and hence the pseudoknot, is required for efficient frameshifting. a control plasmid, l -ucc, with a synonymous mutation in loop was also made. the agu codon was changed to ucc because the latter is present at the same position in the tgev coronavirus and would be expected to have a minimal impact. more detailed structural information about the l -ucc mutant is published elsewhere [ ] . as expected, a moderate change in frameshifting efficiency was observed ( . % versus %, figure a ). s b next, a stem mutant was designed to alter the structure of stem yet retain the same coding sequence so that the effects of these mutations on virus propagation could also be tested in vivo ( figure c ). in the s d construct, stem- base-pairing was disrupted. the observed effects in vivo should therefore reflect changes related to rna structure within the frameshifting signal, rather than changes in the function of the encoded proteins. the silent codon changes in s d promoted moderate reductions in - prf efficiency as compared to the silent s mutants ( . % versus . %; compare figures a and c. these moderate changes are similar to the l -ucc mutant and those observed for other stem mutants [ , ] . importantly these values are higher than . %, the lowest level of frameshifting previously observed for a viable sars coronavirus [ ] . to determine if the synonymous mutations altered the structure of the pseudoknot, their effects on this structure were evaluated using selective '-hydroxyl acylation analyzed by primer extension (shape) analysis (see experimental section). the negative control s d did not form a pseudoknot-like structure (data not shown). the l -ucc loop is more structured than the wild-type loop and this reduced flexibility appeared to affect the remaining structure of the pseudoknot as shown by diminished reactivity to nmia at and around the mutated bases in the loop (l ) compared to the wild-type structure ( figure b ). additionally, there was decreased reactivity with some bases in stem and in the loops l and l . these data correlate well with prior chemical analysis showing that the nucleotides in loop were susceptible to both single-and double-strand-specific nucleases [ ] . having established this baseline, s d and s Δs Δ mutants were evaluated by shape. in contrast to the l -uuc pseudoknot mutant, the s d and s Δs Δ pseudoknots were more reactive to nmia indicating that they are less structured ( figure d ). in addition to the expected changes in reactivity to the mutated bases in stem , bases in the ' region of both loop which caps stem and the loop joining stems and (l ) also showed increased reactivity for s d. the s Δs Δ mutant was also more reactive to nmia. however, the nmia reactivity pattern of the s d mutant, with more reactivity in the ' proximal end of stem and the ' portion of loop , differed from that seen for s Δs Δ in which reactivity was more clustered in the ' portion of loop and adjacent stem nucleotides. these data suggest that the native structure of loop and correct formation of stem is dependent on the presence of the bulged adenosines. although most mutations in stem have a limited impact on frameshifting there is evidence of sequence conservation between coronavirus sequences through this region [ ] . to determine the biological significance of stem , we tested a virus containing synonymous stem mutations and a virus containing synonymous loop mutations ( figure a ). rna was extracted from the infected cell lysate, amplified by rt-pcr and sequenced to confirm that the desired mutations were present. the s d virus was viable but replicates to a lower titer than the wild type ( figure b ; table ). the effect on production of genomic and subgenomic rna was quantified by rt-pcr ( figure c ). although the s d virus could be detected by rt-pcr, the tcid values were below the limit of the assay (table ; figure b ). the l -ucc mutant was detected by both methods. the rt-pcr analysis demonstrates that the genomic rna (grna) of l -ucc accumulates to wild-type levels four days post-infection ( figure c ). in contrast, genomic rna from the s d mutant only accumulated to levels approximately three orders of magnitude lower. interestingly, the genomic to subgenomic rna ratios were inverted for the s d mutant: while the wild type and l -ucc viruses had tenfold more genomic than subgenomic rna, the s d mutant had tenfold more subgenomic rna than genomic rna ( figure c ). similar changes in viral rna ratios were seen with viruses with mutated slippery sites, implicating a broader region of this part of the genome as being involved in regulation of transcription [ ] . while the changes in the previous report resulted in an altered protein sequence, it is unknown if the pseudoknot structure in the slippery site mutants was affected. the viruses used in this study all encode identical proteins indicating that protein function itself is not causing the altered transcription patterns. this suggests that the differences between the viruses are due to rna sequence and/or structural changes. because the rt-pcr quantification was performed on the supernatant from cells exhibiting cpe and expected to contain representative amounts of cellular grna and sgrna, it is possible that the rt-pcr data could be indicative of differing amounts of grna packaged in virions. the reduction in virion-associated grna levels could occur if a packaging signal was disrupted. disruption of a packaging signal would result in fewer complete virions and lower tcid and grna values as observed for the s d mutant. although the efficiency of frameshifting is similar for the l -ucc and s d mutants the grna levels differ by several log. the stabilization of l -ucc stem structure and loss of s d stem structure (see figure ) also support the idea that this region may contain a packaging signal. asterisks indicate values that were at the lower limit of the assay. (c) relative abundance of genomic and subgenomic rna in viral stocks. plaque purified virus was used to infect vero cells. four days post infection cpe was observed. media and detached cells were removed. rna was extracted from a μl aliquot using trizol. taqman analysis was used to determine the total number of genomic and subgenomic rna molecules compared to a reference rna transcribed from a sars replicon. the number of copies per ml of viral stock is shown with standard deviation. s b s b table . frameshifting efficiency affects infectivity. sars coronavirus mutants are indicated. - prf efficiencies and standard error as measured in veroe cells as described in the experimental section. the data for the wt and l -ucc viruses has previously been published [ , ] . tcid values were calculated as described in the experimental section. the error is the standard deviation of six measurements. construction of dual luciferase plasmids. the parental plasmids pjd and pjd , containing the renilla and firefly luciferase genes flanking the wild-type sars-cov frameshift signal, have been described previously [ ] . pjd is the test construct for measuring frameshifting efficiency, and pjd is a readthrough control plasmid to normalize against any defects in overall translation that the introduced sars sequence may cause. site-directed mutagenesis was used to introduce mutations into the test construct (pjd ). mutagenesis was performed using stratagene's quikchange ii kit (la jolla, ca). the mutations were confirmed by sequence analyses. construction of mutant viruses. oligonucleotide site directed mutagenesis was used to mutate sars subclone d [ ] . each mutant subclone was assembled with the other five subclones, and then t rna polymerase and a gtp cap analog were used to generate a full-length virus transcript as described previously [ ] . full-length genomic rna was transfected into vero cells resulting in a productive, lytic viral infection. the virus was allowed to grow for days at °c. viral supernatants were plated onto vero cells, and several clones were plaque purified. the plaque-purified viruses were expanded by growth on vero cells. rt-pcr was used to detect subgenomic rna, a marker of viral replication. pcr amplicons from the frameshift region were sequenced to verify that the correct mutations were introduced. virus titer was measured using a tcid assay calculated according to the method of reed and muench [ ] . all viral assays were conducted in bsl- safety facilities. the abundance of grna and sgrna was determined by quantitative real-time pcr using sybr green technology as previously described [ ] . primers complementary to grna and sgrna were used to detect rna. although the primers complementary to the sgrna can anneal to the grna, only the smallest sgrna has the ' leader and ' sequence close enough to allow pcr amplification under normal cycling conditions. rna structure probing. the structures of the pseudoknots were probed using the selective '-hydroxyl acylation analyzed by primer extension (shape) procedure of wilkinson et al. [ ] . shape analysis utilizes n-methylisatoic anhydride (nmia), which tests the local backbone flexibility of rna. more flexible nucleotides assume conformations that are reactive to nmia which irreversibly acylates the ribose '-hydroxyl groups. this then presents as a stop upstream of the modified base during primer extension. thus, a more intense band in the primer extension autoradiograph represents a more flexible region of the rna. briefly, dna was amplified from each mutant plasmid using pcr master mix (fermentas inc., glen burnie, md) and a forward-direction primer containing the t rna promoter; t forshape, '-taatacgactcactatagggaagatgcacctgatgaaatgg- ' and the reverse-direction primer; revshape, '-gcccatatcgtttcatagcttc- '. rna was synthesized from the pcr products using the ambion t megascript kit (austin, tx) as per the manufacturer's instructions. rna ( pmol) in a volume of μl was denatured at ºc and cooled on ice. reaction buffer ( μl) containing mm hepes ( -( -hydroxyethyl)- -piperazineethanesulfonic acid) ph . , mm mgcl , and mm nacl was added to each rna sample and the mixture was incubated at ºc for minutes. the reactions were split into two equal aliquots, one with μl dimethyl sulfoxide, the other with μl of mm n-methylisatoic anhydride (nmia), and incubated at ºc for minutes. the rna was then precipitated with μl h o, μl m nacl, μl mg/ml glycogen, μl mm edta ph . , and μl ethanol overnight at − ºc. after centrifugation the rna was resuspended in μl of . × te. the oligonucleotide '-gccgggcctttctttatg- ' ( pmol; integrated dna technology, coralville, ia) was labeled with μci gamma p-adenosine triphosphate (atp) using t kinase (roche, indianopolis, in) and purified through a g- column (ge healthcare). rna ( μl) was incubated with p-labeled oligonucleotide ( μl) at ºc for minutes, °c for minutes and cooled to ºc. reverse transcription reactions were performed using superscript iii enzyme (invitrogen, carlsbad, ca) at ºc for minutes. products were separated on an % polyacrylamide, . m urea gel in tbe buffer, dried and visualized by phosphorimage analysis. dual luciferase assays. for each assay cells were transfected with either a test plasmid or a control plasmid (described above) using fugene from promega (madison, wi). veroe cells were grown overnight in dulbecco's modified eagle medium supplemented with % fbs at ºc. cells were disrupted using the passive lysis buffer (promega, madison, wi) as per the manufacturer's instructions. luminescence reactions were initiated by addition of - μl of cell lysates to μl of the promega lar ii buffer and completed by addition of μl stop'n'glo reagent. luminescence was measured using a turner design td / instrument. at least three replicates were performed within each assay and all assays were repeated at least three times until the data were normally distributed to enable statistical analyses both within and between experiments. the frequency of frameshifting is expressed as a ratio of firefly to renilla luciferase from a test plasmid divided by the analogous ratio from the read-through control plasmid multiplied by %. fold change, standard error and estimates of the p-values for ratiometric analyses were performed as described previously [ ] . in line with previous studies of coronavirus frameshift signals, data presented here indicate that the sequence and length of the third stem is not critical for frameshifting efficiency [ , , ] . however, as revealed by the structural analysis, mutations in stem can cause changes in frameshift efficiency by changing stem formation. additionally, changes in stem can be detrimental to viral viability. specifically, changes that destabilize stem resulted in reductions in - prf and altered patterns of viral rna production. it remains to be determined if the latter change is due to a disruption in frameshifting or disruption of a rna regulatory element such as a packaging signal. while many sequences involved in coronavirus subgenomic rna synthesis have been elucidated, sequences and structures involved in regulating coronavirus genomic rna synthesis are less well defined [ ] . internal replication elements have been described in other plus-strand non-segmented viruses including poliovirus, hepatitis c virus and tombusviridae [ ] [ ] [ ] . each of these internal replication elements is present in the region of the viral genome encoding non-structural proteins. the tombusviridae rna-dependent rna polymerase is translated as a readthrough product and the internal replication element is downstream of the readthrough signal [ ] . although the cre element from poliovirus has been studied for several years the precise roles of these elements are not well defined [ ] . in most instances it is not known, for example, if they bind to specific viral or host proteins, or if they communicate with other elements in the genome. packaging signals for coronaviruses are also ambiguous and have been identified in the ' untranslated regions and the ' end of orf a/b [ , ] . our work demonstrated that a less structured stem in the frameshift pseudoknot led to significantly reduced amounts of grna when compared to viruses with more structured pseudoknots. however, the same change in viral rna patterns was observed with a slippery site mutant suggesting that if a packaging signal is present it extends from, or is affected by, the upstream slippery site. the correlation between stem structure and grna levels indicates that a structural requirement in stem is required for optimal rna production but we cannot, at this stage rule out a frameshifting threshold similar to that observed for the yeast double-stranded rna virus m [ ] . that is, if frameshifting drops below a certain frequency, the change in the ratio of pp a to pp ab results in a dramatic reduction in grna production. indeed, recent reports describing the function of a nonstructural protein (nsp ) encoded upstream of the frameshift signal provide an alternative hypothesis to regulatory element hypothesis investigated in this work. nsp was initially described as a non-canonical rna-dependent rna polymerase and produced as part of the pp a protein whether or not frameshifting occurs [ ] . in contrast, the canonical rdrp nsp is produced as part of the pp ab protein only if frameshifting occurs. this, in conjunction with our data suggests that the nsp polymerse might direct sgrna production and that the rdrp encoded in nsp is predominantly used for grna production. it has been hypothesized that coronavirus rna synthesis involves structurally and functionally separable rna synthesising complexes [ ] . the nsp +nsp complex from three different coronaviruses have recently been shown to have primer-independent rna polymerase activity [ , ] in contrast to the nsp rdrp which is primer dependent [ ] . currently the details regarding coronavirus rna synthesis are very limited and do not explain how the polymerases distinguish between templates or prime synthesis. te velthuis and colleagues suggested that the separate rdrps may influence plus and minus strand synthesis [ ] but our results indicate that the division might be between genomic and subgenomic rna synthesis. the rt-pcr for detection of grna and sgrna in our study generated a cdna intermediate using random hexamers and because of this the analyses performed here do not distinguish between the production of positive-and negative-strands nor between mrna and sgrna. thus, we do not know if the regulatory sequences or proteins regulating genomic rna production act upon the positive-or negative-strand replication intermediates or transcription intermediates. a slight difference in frameshifting efficiency, such as that between the l -ucc and s d mutants, would not be expected to have such significant effects on viral rna levels. similar to previously described viruses containing mutations in the slippery site of the frameshift signal [ ] , here we show that mutations to the sars-cov frameshift stimulating mrna pseudoknot can also affect the production of viral genomic rna. in both instances, the abundance of viral genomic rna was reduced to -fold lower levels than the subgenomic rna and several orders of magnitude below that of the wild-type virus. it is not clear if this result is due to a change in the abundance of grna available to be transcribed to sgrna or if it is attributable to other factors, such as interactions between the grna and other proteins or rna elements as discussed above. it is also possible that the synonymous changes altered the rate of translation and/or the folding of the rdrp [ ] which, in turn, could have affected production of genomic and/or subgenomic rna. regardless of the mechanism, our observations provide a backdrop against which new questions about coronvirus replication and transcription may be explored. discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the 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proteins and of feline coronavirus form a : heterotrimer that exhibits primer-independent rna polymerase activity the rna polymerase activity of sars-coronavirus nsp is primer dependent silent substitutions predictably alter translation elongation rates and protein folding efficiencies this work was supported by a grant from the national institutes of health to j.d.d. (ai ). the findings and conclusions in this article have not been formally disseminated by the food and drug administration and should not be construed to represent any agency determination or policy. the authors declare no conflict of interest. key: cord- -nquozaxt authors: sieg, michael; busch, johannes; eschke, maria; böttcher, denny; heenemann, kristin; vahlenkamp, annett; reinert, anja; seeger, johannes; heilmann, romy; scheffler, kira; vahlenkamp, thomas w. title: a new genotype of feline morbillivirus infects primary cells of the lung, kidney, brain and peripheral blood date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: nquozaxt paramyxoviruses comprise a large number of diverse viruses which in part give rise to severe diseases in affected hosts. a new genotype of feline morbillivirus, tentatively named feline morbillivirus genotype (femv-gt ), was isolated from urine of cats with urinary tract diseases. whole genome sequencing showed about % nucleotide homology to known feline morbilliviruses. the virus was isolated in permanent cell lines of feline and simian origin. to investigate the cell tropism of femv-gt feline primary epithelial cells from the kidney, the urinary bladder and the lung, peripheral blood mononuclear cells (pbmc), as well as organotypic brain slice cultures were used for infection experiments. we demonstrate that femv-gt is able to infect renal and pulmonary epithelial cells, primary cells from the cerebrum and cerebellum, as well as immune cells in the blood, especially cd (+) t cells, cd (+) b cells and monocytes. the cats used for virus isolation shed femv-gt continuously for several months despite the presence of neutralizing antibodies in the blood. our results point towards the necessity of increased awareness for this virus when clinical signs of the aforementioned organs are encountered in cats which cannot be explained by other etiologies. paramyxoviruses constitute a large family of single stranded, negative-sense, enveloped rna viruses that can lead to important diseases in humans and animals [ ] . historically, paramyxoviridae have been subdivided into seven genera based on biochemical properties and sds-page patterns of viral structural proteins: rubulavirus, avulavirus, respirovirus, henipavirus, morbillivirus, ferlavirus and aquaparamyxovirus. taking genome sequences and protein data into account many currently described paramyxoviruses are assigned as unclassified, e.g. rodent-borne tailam virus [ ] , nariva virus [ ] and bank vole virus [ ] , as well as paramyxoviruses detected in bats [ ] . in recent years, the genus morbillivirus has received growing attention, due to the discovery of a new feline morbillivirus (femv, formerly abbreviated as fmopv) associated with tubulo-interstitial nephritis in stray cats from hong kong [ ] . subsequently, the prevalence was reported from other countries including japan, usa, turkey, brazil, thailand, italy and germany [ ] [ ] [ ] [ ] [ ] [ ] [ ] . percentage of femv-positive urines ranged from % to % in the us [ ] and japan [ ] , respectively. seroprevalence data of femv available from hong kong and japan showed . % [ ] , . % [ ] , . % [ ] and % [ ] of investigated cats to be femv-positive using nucleo-or phosphoproteins as antigens. while some of these studies established a link between an infection with femv and the presence of kidney diseases in affected cats [ , , , , ] , others could not confirm such an association [ ] [ ] [ ] ] . these discrepancies may be due to the complexity of chronic kidney disease (ckd) pathogenesis in general, making it difficult to link cases of feline ckd to only one specific trigger [ ] . in some cats, feline morbilliviruses may induce a persistent infection of the urinary tract [ ] . so far it is not clear whether an acute or chronic infection can cause or support the development of ckd. during our current studies an unknown feline paramyxovirus was detected in urine samples from domestic cats [ ] . although this virus was initially linked to femv strains from japan, whole genome sequencing revealed a different genotype of femv, tentatively named feline morbillivirus genotype (femv-gt ). here we show that the femv-gt -gordon strain replicates in primary feline epithelial cells from different organs and is able to infect primary feline t and b cells, as well as monocytes in vitro. we demonstrate that femv-gt readily infects feline organotypic brain slice cultures with cells of the cerebrum and cerebellum being comparably susceptible. the molecular and biological characterization of femv-gt shows that the diversity of feline paramyxoviruses extends beyond the formerly known femv isolates, which must be further studied in detail. all cell lines and primary cells used were maintained at • c, % humidity and % co . llc-mk and vero ccl cell lines were purchased from the instituto zooprofilattico sperimentale della lombardia e dell'emilia romagna «bruno ubertini» (izsler), italy, whereas crfk, mdbk, mdck-ii, hek , bhk- , ma- , marc- , a , fmn-r, mgn-r, ran- -r, fln-r and ke-r were kindly provided by the friedrich-loeffler-institute (fli), germany. all cell lines were grown in dulbecco's modified eagle medium (dmem) containing . g/l glucose, % fbs, glutamax™ supplement, × mem non-essential amino acids solution and mm sodium pyruvate. fcwf cells (atcc ® crl ™) were purchased from the american type culture collection (atcc), usa and cultivated in rpmi medium containing mm l-glutamine, . g/l sodium bicarbonate, . g/l glucose, mm hepes, . mm sodium pyruvate, . mm -mercaptoethanol, % fbs or in dmem with % fbs, respectively. the organ material used in this work was provided by the institute of pathology, faculty of veterinary medicine, leipzig university and derived from dead animals euthanized for medical reasons unrelated to this study. primary feline kidney cells were isolated by adapting a previously described protocol [ ] . briefly, kidneys from dead animals were removed aseptically and stored in ice cold hank's buffered salt solution (hbss) without cacl and mgcl until further processing. kidneys were de-capsulated, bisected, and the renal cortex was removed and cut into small pieces. tissue was dissociated by collagenase ii ( mg/ml) treatment at • c for min. this step was repeated three times and the collected cell suspensions were passed through a µm cell strainer to remove cell aggregates. cells were pelleted by centrifugation at × g for min and re-suspended in complete culture medium (dmem:f [ %: %] medium containing ng/ml human epidermal growth factor, nm hydrocortisone, µg/ml insulin, µg/ml transferrin, nm sodium selenite and iu/ml penicillin-streptomycin) and seeded into cm plastic flasks. the cells were cultured at • c under % co in a humidified atmosphere until the monolayer became nearly confluent. similarly, primary feline urinary bladder cells were prepared by collagenase treatment of the urinary bladder mucosa. dissociated cells were than cultured in keratinocyte serum free medium (ksfm) including ng/ml egf, . % (v/v) of bovine pituitary extract (bpe, thermo fisher) and ng/ml choleratoxin (sigma-aldrich, st. louis, mo, usa) and iu/ml penicillin-streptomycin. to isolate primary feline pulmonary epithelial cells fresh lungs were rinsed three times with dmem through the primary bronchus to remove alveolar macrophages. pulmonary epithelial cells were dissociated from the surrounding tissue with . % trypsin and . % edta followed by an incubation for min at • c. finally, cells were collected by rinsing the lungs with dmem and a centrifugation step for min at × g. cells were seeded into a cm plastic flask and cultured in dmem containing . g/l glucose, % fbs, glutamax™ supplement, × mem non-essential amino acids and mm sodium pyruvate, iu/ml penicillin-streptomycin until the monolayer reached approximate confluence. urine samples from cats were collected as part of the routine physical examination at the department of small animals, leipzig university. the material was stored at − • c (for not more than weeks) before rna isolation was conducted. for virus isolation vero, crfk and llc-mk cells were infected with ml urine mixed with ml pure dmem (containing iu/ml penicillin-streptomycin) in cm cell culture flasks. after h incubation at • c, % co and % humidity the inoculum was replaced by ml cultivation medium (dmem, sodium pyruvate, non-essential amino acids, % fbs, iu/ml penicillin-streptomycin) and were cultured for days at the indicated conditions. the cell culture supernatant from this first passage was used to infect fresh cells. this procedure was repeated several times and the resulting cell culture supernatants were continuously tested for the presence of paramyxovirus-rna by rt-pcr. for virus infection assays different cell lines (see section . ) were infected with the llc-mk -adapted (passage no. ) 'gordon' strain of femv-gt in a -well format at an moi~ . for two hours at • c. infection medium was replaced by cultivation medium (see above) and cells were further incubated at • c, % co and % humidity for five days. percentage of virus-positive cells were semi-quantified by immunofluorescence staining targeting the femv-gt -nucleoprotein followed by microscopic analysis. viral particles were concentrated from cell culture supernatants of femv-gt -infected llc-mk via ultracentrifugation. in brief, ml virus-containing supernatant was sublayered viruses , , of with ml of a % sucrose cushion and centrifuged using an sw ti rotor (beckman coulter) at , rpm for . h at • c. the semi-purified virions were re-suspended in pbs prior to loading onto formvar-coated copper grids (plano gmbh, wetzlar, germany) and were negatively stained with % uranyl acetate. grids were viewed using an tem libra (carl zeiss, jena, germany) transmission electron microscope. detection of femv-gt in feline urine samples or in cell culture supernatants was performed as previously described [ , ] using the primer pair hen-res-mor-r and hen-res-mor-f /f . for whole genome sequencing, degenerated primers were designed based on the available femv-sequences (primer sequences are available in supplementary files, table s ). amplification was performed with the "superscript™ iii one-step rt-pcr system containing platinum™ taq high fidelity dna polymerase" (thermo fisher scientific, waltham, ma, usa). pcr products were visualized by agarose gel electrophoresis with × tris-acetate-edta buffer ( mm tris-acetate, mm edta, ph . ) containing . µg/ml of ethidium bromide. for dna-sequencing, specific pcr fragments were cut out of the gel and purified using a gel/pcr dna fragments extraction kit (geneaid, new taipei city, taiwan). sequencing was performed by sanger's dideoxy termination method (microsynth seqlab gmbh, göttingen, germany) and each pcr product was sequenced twice. nucleotide sequences were analyzed using the basic local alignment search tool (blast, www.ncbi.nlm.nih.gov/blast/), and genetic distances of whole genome sequences were calculated applying the general time reversible model with gamma distributed invariant sites (gtr + i) at the nucleotide level using the mega software. a phylogenetic tree was built by the maximum likelihood method with bootstrap replicates [ ] . sequences determined in the course of this work have been deposited in genbank, with the accession numbers mk and mk . cells were fixed with % (w/v) paraformaldehyde for min at • c, permeabilized with . % triton x- in pbs for min, blocked with % bsa-pbs for min at room temperature and probed with a cytokeratin pan type i/ii antibody cocktail (clone ae /ae , thermo fisher scientific, waltham, ma, usa) at a dilution of : . femv-gt was probed using a polyclonal rabbit antibody raised against the nucleoprotein of femv (generated in house) at a dilution of : . after overnight incubation at • c cells were washed three times with pbs and incubated with an alexa fluor -conjugated goat-anti-mouse igg (h+l) or with an alexa fluor -conjugated goat-anti-rabbit igg (h+l) secondary antibody (thermo fisher scientific, waltham, ma, usa) in % bsa-pbs at a dilution of : containing , -diamidino- -phenylindole (dapi, nm) and incubated for one hour at • c in a humidified chamber. after three pbs-washing steps images were acquired using an ax microscope (olympus, tokio, japan). virus titration was performed by the endpoint dilution assay in llc-mk cells followed by visualization of virus positive dilutions using immunofluorescence staining. viral titers were expressed as % tissue culture infective dose (tcid ) calculated after the reed-muench method [ ] . to test for femv-gt -neutralizing antibodies a serum neutralization tests (snt) was established. briefly, cell monolayers of llc-mk -cells were used at approximately % of confluence in a -well microtiter format. feline serum samples were incubated at • c for min to inactivate complement components followed by the preparation of two-fold serial dilutions in pure dmem starting with a four-fold dilution. each sample was then was mixed with~ infectious viral particles (tcid ) of femv-gt -gordon strain in an equal volume of dmem followed by an incubation period of one hour at • c. this suspension was used for infection of llc-mk -cells for two hours at • c. samples were then replaced by dmem including % fbs, sodium pyruvate, non-essential amino acids and iu/ml penicillin-streptomycin. four days later, viral plaques were visualized applying the described immunofluorescence protocol (see section . ). each experiment was performed in duplicates. neutralizing titers were defined as the reciprocal of the serum dilution at which the number of plaques was reduced to % relative to virus-only controls. feline blood samples were obtained from healthy cats by venipuncture into vacutainer tubes containing edta as an anticoagulant (bd vacutainer ® , ml, beckton dickinson, heidelberg, germany). peripheral blood mononuclear cells (pbmc) were isolated as described previously by using histopaque- (sigma-aldrich, st. louis, mo, usa) [ ] . freshly isolated cells were resuspended in rpmi medium (without supplements) and infected with femv-gt-gordon strain, moi = . for one hour at • c. the infection medium was removed by centrifugation at × g for min, cells were re-suspended and further cultured for h at • c in a -well cell culture plate (greiner bio-one, kremsmünster, austria) at a density of million cells per ml. rpmi medium supplemented with % fbs, mm sodium pyruvate, % (v/v) non-essential amino acids and iu/ml recombinant human interleukin- (thermo fisher scientific, waltham, ma, usa) was used for cultivation. mock-infected pbmc from each animal served as negative controls and were treated similarly. after the indicated incubation period cells were centrifuged at × g for min and the cell pellet washed twice with pbs. for discrimination of dead cells in the consecutive flow cytometric analysis, femv-gt -and mock-infected pbmc were stained with fixable viability dye efluor (thermo fisher scientific, waltham, ma, usa) according to the manufacturer's protocol. subsequently, cells were incubated with % (v/v) goat normal serum (thermo fisher scientific, waltham, ma, usa) for min on ice to avoid non-specific fc binding. staining of surface markers was performed using fitc-anti-cat cd (clone vpg , bio-rad, herkules, ca, usa), alexa fluor ® -anti-human cd (clone h , bio-rad, herkules, ca, usa) and pe-anti-human cd (clone b , thermo fisher scientific, waltham, ma, usa). the corresponding isotype controls were all purchased from ebioscience. surface staining was performed by incubation of cells with fluorochrome-conjugated antibodies in facs-buffer (pbs with % fbs) for min on ice. stained cells were fixed for min at • c with % (w/v) paraformaldehyde. for intracellular detection of femv-gt , cells were permeabilized using . % saponin in facs-buffer for min at room temperature (rt). the virus was labeled by incubation with a rabbit polyclonal anti-femv-nucleoprotein antibody (generated in-house) at µg/ml (in facs-saponin-buffer for min at room temperature followed by two washing steps in facs-saponin buffer. finally, cells were incubated with a : dilution of pacific blue™-anti-rabbit antibody (thermo fisher scientific, waltham, ma, usa) in facs-saponin buffer for min at rt followed by two consecutive washing steps with facs buffer. the cells were acquired with a bd lsr fortessa tm flow cytometer (becton dickinson, heidelberg, germany) and viable cells were analyzed using the flowjo . . software (treestar inc., ashland, or, usa). the gating strategy is shown in supplementary files, figure s . to investigate neuronal tropism of femv-gt we established an in vitro model of the feline brain by generating organotypic slice cultures from cerebrum and cerebellum. brains from animals euthanized for medical reasons unrelated to this study, were dissected and the cerebrum was separated from the cerebellum. following preparation the tissue was immediately submerged in ice-cold hbss and slices were generated using a vt s (leica, wetzlar, germany) microtome. due to the difference in tissue rigidity, slices of the cerebrum were cut to µm and of the cerebellum were cut to µm thickness. freshly prepared brain slices were transferred to cell culture inserts ( . µm, merck millipore) pre-equilibrated with hbss and placed into -well cell culture plates (greiner bio-one, austria). for cultivation, minimum essential medium (mem) containing % (v/v) heat-inactivated horse serum, mm l-glutamine, mm hepes, mm hbss and iu/ml penicillin-streptomycin was used and changed daily. cultures were incubated at • c, % humidity and % co . after days of cultivation each slice was infected by direct application of ffu of femv-gt -gordon strain to the tissue. negative controls (mock infections) were generated by applying an equivalent volume of dmem with % fbs to the brain tissue. days after infection slices were washed with pbs and fixed with % (w/v) paraformaldehyde for min. slices were permeabilized with . % triton x- in pbs (tpbs) for min and blocked in tpbs with % horse serum and % bsa for min. the primary antibody raised against the femv-nucleoprotein was diluted ( : ) in blocking buffer and incubated with the samples for h at • c with constant agitation. after washing with pbs a goat anti-rabbit igg (h+l) cross-adsorbed, alexa fluor ® labeled secondary antibody (thermo fisher scientific, waltham, ma, usa) was diluted ( : ) in blocking buffer, applied to the slices and incubated for two hours. nuclei were counter-stained using dapi ( nm). after two subsequent washing steps with pbs the slices were visualized using a leica sp confocal microscope. as part of the german surveillance program for paramyxoviruses in domestic cats, urine samples from animals were analyzed using the described paramyxovirus consensus rt-pcr [ ] . six samples ( . %) were positive for a previously unknown paramyxovirus, tentatively named feline morbillivirus genotype (femv-gt ). based on physical examination and urinalysis findings all six animals were diagnosed with diseases of the urinary tract (i.e. acute or chronic kidney disease; table ). no viral rna was detectable within the available serum and edta-blood samples from these urine-positive animals. two cats ('gordon' and 'tv ') were sampled for several months and were found to be positive for femv-gt -rna by pcr within the urine at all time points tested (table ) . electron microscopy analysis and successful virus isolation from fresh urine samples of these two animals revealed that intact viral particles were released. interestingly, corresponding serum samples from 'gordon' and 'tv ' showed high titers of femv-gt -specific neutralizing antibodies (titer > ). abbreviations: ckd = chronic kidney disease; flutd = feline lower urinary tract disease; iris = international renal interest society; n.a. = not available; = no follow-up urine samples were available due to euthanasia, = no serum sample available, nab = neutralizing antibodies. to characterize this virus further, isolation attempts from fresh urine samples of affected animals (appropriate samples were only available from the animals 'gordon' and 'tv ') were conducted using vero, crfk and llc-mk cells. after the third passage cell culture supernatants were tested for the presence of paramyxoviruses using rt-pcr. we successfully isolated femv-gt strains from both animals. the results revealed that all three cell lines were susceptible to femv-gt with llc-mk cells being more permissive in comparison to vero and crfk cells. these findings were also confirmed by immunofluorescence staining targeting the femv-gt -nucleoprotein ( figure ). no visible cpe was induced on any tested cell line. initial virus titer on llc-mk cells was low at passage no. viruses , , of ( × tcid /ml), but could be enhanced to × tcid /ml after passage no. . virus titer remained low (~ × tcid /ml) on crfk and vero cells independent from the passage number (tested until passage no. ). both virus strains, femv-gt -gordon and femv-gt -tv , exhibit similar growth kinetics. as a consequence, virus stock was produced on llc-mk cells and was used for further infection experiments. mk cells being more permissive in comparison to vero and crfk cells. these findings were also confirmed by immunofluorescence staining targeting the femv-gt -nucleoprotein ( figure ). no visible cpe was induced on any tested cell line. initial virus titer on llc-mk cells was low at passage no. ( × tcid /ml), but could be enhanced to × tcid /ml after passage no. . virus titer remained low (~ × tcid /ml) on crfk and vero cells independent from the passage number (tested until passage no. ). both virus strains, femv-gt -gordon and femv-gt -tv , exhibit similar growth kinetics. as a consequence, virus stock was produced on llc-mk cells and was used for further infection experiments. to exclude contamination with other feline viruses, viral stock was concentrated via ultracentrifugation and tested for feline herpesvirus (fehv), feline coronavirus (fcov), feline calicivirus (fcv) and feline parvovirus (fpv) using specific pcr systems [ ] [ ] [ ] [ ] . none of the aforementioned viruses were found to be present in the femv-gt preparations. to visualize femv-gt virions, cell culture supernatants were semi-purified and examined by transmission electron microscopy. the observed viral particles showed typical paramyxoviral morphology represented by pleomorphic, enveloped virions sized between - nm (figure a ). to exclude contamination with other feline viruses, viral stock was concentrated via ultracentrifugation and tested for feline herpesvirus (fehv), feline coronavirus (fcov), feline calicivirus (fcv) and feline parvovirus (fpv) using specific pcr systems [ ] [ ] [ ] [ ] . none of the aforementioned viruses were found to be present in the femv-gt preparations. to visualize femv-gt virions, cell culture supernatants were semi-purified and examined by transmission electron microscopy. the observed viral particles showed typical paramyxoviral morphology represented by pleomorphic, enveloped virions sized between - nm ( figure a ). both animals. the results revealed that all three cell lines were susceptible to femv-gt with llc-mk cells being more permissive in comparison to vero and crfk cells. these findings were also confirmed by immunofluorescence staining targeting the femv-gt -nucleoprotein ( figure ). no visible cpe was induced on any tested cell line. initial virus titer on llc-mk cells was low at passage no. ( × tcid /ml), but could be enhanced to × tcid /ml after passage no. . virus titer remained low (~ × tcid /ml) on crfk and vero cells independent from the passage number (tested until passage no. ). both virus strains, femv-gt -gordon and femv-gt -tv , exhibit similar growth kinetics. as a consequence, virus stock was produced on llc-mk cells and was used for further infection experiments. to exclude contamination with other feline viruses, viral stock was concentrated via ultracentrifugation and tested for feline herpesvirus (fehv), feline coronavirus (fcov), feline calicivirus (fcv) and feline parvovirus (fpv) using specific pcr systems [ ] [ ] [ ] [ ] . none of the aforementioned viruses were found to be present in the femv-gt preparations. to visualize femv-gt virions, cell culture supernatants were semi-purified and examined by transmission electron microscopy. the observed viral particles showed typical paramyxoviral morphology represented by pleomorphic, enveloped virions sized between - nm ( figure a ). in addition, characteristic free nucleocapsids were found in all virus preparations varying between - nm in diameter ( figure b ). no differences in viral morphology were detected between samples originating from urine or from cell culture supernatants. to investigate the in vitro growth spectrum of femv-gt , several cell lines of different animal origin were tested for their susceptibility and efficiency for virus production. experiments were done with the llc-mk -adapted femv-gt -gordon strain at passage no. . as summarized in table , fcwf- and llc-mk cells were most susceptible to femv-gt , with~ % of the cells being femv-gt positive by ifa five days after infection. crfk, vero, marc- and bhk- were also susceptible, but to a lesser extent ( table ) in order to determine the phylogenetic relationship between already known feline morbilliviruses and the isolated strains from this study, whole genome amplification was performed. primers were designed from conserved regions of available femv sequences. femv-gt fragments were amplified and gaps were filled by applying the primer walking strategy. near-complete genome sequences of two femv-gt strains ('gordon' and 'tv ', accession numbers mk and mk ) from domestic cats were revealed. nucleotide sequences from strains were . % identical, differing in only nucleotides mainly located in the non-coding regions of the genome. the genome organization of femv-gt resembled that of classical femv strains, encoding six ( -n-p/v/c-m-f-h-l- ) open reading frames (orfs), typical for other morbilliviruses. pairwise alignments of the orfs between femv and femv-gt demonstrated nucleotide identities ranging between . % (phosphoprotein) and . % (matrix protein) and an overall whole genome nucleotide identity of about . % (table ) . amino acid conservation between femv and femv-gt was found to be higher than % for all structural proteins, except for the phosphoprotein where the amino acid identity of the femv-gt-gordon strain to femv was only % (table ) . phylogenetic analysis based on whole genome sequences demonstrated that femv-gt is genetically closely related to (figure ) , but yet distinct from, previously described feline morbilliviruses [ ] . therefore, we suggest that femv-gt should be considered as a different genotype of feline morbilliviruses. to elucidate the target tissues of femv-gt we established protocols for the isolation of primary feline cells from various organs of cats (see section . .) experimental in vitro infection was performed using the llc-mk -adapted femv-gt -gordon strain. we initially investigated primary cells from the urinary tract and successfully established a method for the preparation and cultivation of feline to elucidate the target tissues of femv-gt we established protocols for the isolation of primary feline cells from various organs of cats (see section . ). experimental in vitro infection was performed using the llc-mk -adapted femv-gt -gordon strain. we initially investigated primary cells from the urinary tract and successfully established a method for the preparation and cultivation of feline primary kidney cells as described in materials and methods. isolated cells represented a mixture (approximately %: %) of cytokeratin-positive epithelial cells and vimentin-positive non-epithelial cells. an in-depth analysis further showed that cells also expressed the renal collecting duct cell marker aquaporin- [ ] , the distal tubulus cell marker calbindin d k [ ] and the proximal tubulus cell marker alkaline phosphatase [ ] . hence, it was concluded that the isolated primary feline kidney cells express the terminal differentiation markers also present in naïve tissues and are therefore suitable for in vitro infection experiments. the cells were infected at an moi of . for two hours and thereafter cultured for five days prior to femv-gt nucleoprotein staining. mock-infected cells served as negative controls. we observed approx. % of primary feline kidney cells to be femv-gt positive, whereas no signal was detected in the negative control (figure ) , showing that primary feline kidney cells were susceptible to femv-gt infection in vitro. no cytopathic effects (e.g. formation of syncytia) or significant cell death in comparison to the mock-infection was detected. these observations were reproduced from cell preparations of three individual animals. the nature of the femv-gt -susceptible cell types was elucidated by cytokeratin staining (marker for cells of epithelial origin). renal epithelial cells were shown to be the main target of femv-gt (figure ) , although a minority (~ %) of non-epithelial cells were also susceptible to this virus. to elucidate the involvement of adjacent organs in virus shedding, primary feline bladder epithelial cells were isolated and infected with femv-gt as described above. as shown in figure , primary feline bladder cells were successfully isolated from clinical material and stained positive for cytokeratins as expected for cell types of epithelial origin. urinary bladder cells were shown to be viable, due to trypan blue exclusion, and due to the possible cultivation for at least three passages. in contrast to kidney cells, only a few urinary bladder cells were susceptible to femv-gt . this finding was reproduced from isolated urinary bladder epithelial cells of different animals (n = ). to elucidate the involvement of adjacent organs in virus shedding, primary feline bladder epithelial cells were isolated and infected with femv-gt as described above. as shown in figure , primary feline bladder cells were successfully isolated from clinical material and stained positive for cytokeratins as expected for cell types of epithelial origin. urinary bladder cells were shown to be viable, due to trypan blue exclusion, and due to the possible cultivation for at least three passages. in contrast to kidney cells, only a few urinary bladder cells were susceptible to femv-gt . this finding was reproduced from isolated urinary bladder epithelial cells of different animals (n = ). represent µm. to elucidate the involvement of adjacent organs in virus shedding, primary feline bladder epithelial cells were isolated and infected with femv-gt as described above. as shown in figure , primary feline bladder cells were successfully isolated from clinical material and stained positive for cytokeratins as expected for cell types of epithelial origin. urinary bladder cells were shown to be viable, due to trypan blue exclusion, and due to the possible cultivation for at least three passages. in contrast to kidney cells, only a few urinary bladder cells were susceptible to femv-gt . this finding was reproduced from isolated urinary bladder epithelial cells of different animals (n = ). to determine the capability of femv-gt to infect feline immune cells we isolated pbmc from healthy cats and performed in vitro infection experiments. cats were proven to be femv-and femv-gt -negative by immunofluorescence analysis of serum samples and by rt-pcr analysis of corresponding urine samples. intracellular virus in different immune cell populations was quantified h after infection by flow cytometry (figure ). the results of these investigations demonstrated that cd + t cells, cd + b cells and fsc hi monocytes are susceptible to femv-gt infections in vitro. the percentage of virus-positive cells varied from - % for cd + t cells and from - % for both cd + b cells and fsc hi monocytes ( figure a ). in contrast, cd + t-cells were less susceptible to femv-gt ( figure b ) with percentages of virus positive cells varying between - %. in addition, flow-cytometric analysis of femv-gt infected pbmc revealed that about % of non-t and non-b lymphocytes were also positive for femv-gt . these cell types likely represent natural killer (nk) cells, but due to a lack of specific antibodies to phenotype feline nk-cells this could not be confirmed. to determine the capability of femv-gt to infect feline immune cells we isolated pbmc from healthy cats and performed in vitro infection experiments. cats were proven to be femv-and femv-gt -negative by immunofluorescence analysis of serum samples and by rt-pcr analysis of corresponding urine samples. intracellular virus in different immune cell populations was quantified h after infection by flow cytometry (figure ). the results of these investigations demonstrated that cd + t cells, cd + b cells and fsc hi monocytes are susceptible to femv-gt infections in vitro. the percentage of virus-positive cells varied from - % for cd + t cells and from - % for both cd + b cells and fsc hi monocytes ( figure a ). in contrast, cd + t-cells were less susceptible to femv-gt ( figure b ) with percentages of virus positive cells varying between - %. in addition, flow-cytometric analysis of femv-gt infected pbmc revealed that about % of non-t and non-b lymphocytes were also positive for femv-gt . these cell types likely represent natural killer (nk) cells, but due to a lack of specific antibodies to phenotype feline nk-cells this could not be confirmed. due to the fact that other morbilliviruses infect epithelial cells of the lung, feline primary pulmonary epithelial cells were also used for femv-gt infection experiments. cells were probed with a pan-cytokeratin antibody to discriminate cells of epithelial origin, alveolar macrophages and fibrocytes. representative results of distinct experiments are shown in figure . due to the fact that other morbilliviruses infect epithelial cells of the lung, feline primary pulmonary epithelial cells were also used for femv-gt infection experiments. cells were probed with a pan-cytokeratin antibody to discriminate cells of epithelial origin, alveolar macrophages and fibrocytes. representative results of distinct experiments are shown in figure . no contaminating alveolar macrophages or fibrocytes were detected in these cell preparations via ifa analysis. it can be stated that the vast majority of the cultured cells represented pulmonary epithelial cells. these primary epithelial cells were infected with femv-gt -gordon strain at an moi of . for two hours and further cultured for five days before analysis. the experiments revealed that about - % of the cultured pulmonary epithelial cells were susceptible to femv-gt . all viruspositive cells were also cytokeratin-positive. no cytopathic effect (e.g. formation of syncytia) or significant cell death was observed. we were able to cultivate brain slice cultures from adult cats for at least four weeks. when infecting these cultures with femv-gt -gordon strain, it became evident that both the cerebrum and cerebellum were highly susceptible (figure ). no contaminating alveolar macrophages or fibrocytes were detected in these cell preparations via ifa analysis. it can be stated that the vast majority of the cultured cells represented pulmonary epithelial cells. these primary epithelial cells were infected with femv-gt -gordon strain at an moi of . for two hours and further cultured for five days before analysis. the experiments revealed that about - % of the cultured pulmonary epithelial cells were susceptible to femv-gt . all virus-positive cells were also cytokeratin-positive. no cytopathic effect (e.g. formation of syncytia) or significant cell death was observed. we were able to cultivate brain slice cultures from adult cats for at least four weeks. when infecting these cultures with femv-gt -gordon strain, it became evident that both the cerebrum and cerebellum were highly susceptible ( figure ) . interestingly, infected slice cultures of the cerebellum displayed small syncytia ( - nuclei per cell) ( figure b ). this finding was the first evidence that femv-gt can induce cytopathic effects to particular cell types as it is known for other paramyxoviruses. in contrast, cpe was not visible in femv-gt -infected slice cultures of the cerebrum ( figure d ). femv-gt -infected cells from the cerebrum and the cerebellum showed large amounts of nucleoprotein in the entire cell body and cell protrusions. all infected cells retained a typical morphology of brain cells. virus-positive cells grouped together in distinctive patterns so that virus transmission via cell-to-cell contact might have occurred. all described observations were verified on at least three slice cultures from the cerebrum and the cerebellum. the specific nature of the susceptible cell types (e.g. neurons, astroglia and microglia) could not be determined definitely, but staining of the brain slice cultures with a gfap-specific antibody (glial fibrillary acidic protein, marker for glia cells) showed no co-localization interestingly, infected slice cultures of the cerebellum displayed small syncytia ( - nuclei per cell) ( figure b ). this finding was the first evidence that femv-gt can induce cytopathic effects to particular cell types as it is known for other paramyxoviruses. in contrast, cpe was not visible in femv-gt -infected slice cultures of the cerebrum ( figure d ). femv-gt -infected cells from the cerebrum and the cerebellum showed large amounts of nucleoprotein in the entire cell body and cell protrusions. all infected cells retained a typical morphology of brain cells. virus-positive cells grouped together in distinctive patterns so that virus transmission via cell-to-cell contact might have occurred. all described observations were verified on at least three slice cultures from the cerebrum and the cerebellum. the specific nature of the susceptible cell types (e.g. neurons, astroglia and microglia) could not be determined definitely, but staining of the brain slice cultures with a gfapspecific antibody (glial fibrillary acidic protein, marker for glia cells) showed no co-localization of femv-gt -positive cells with gfap-positive structures. neurons could not be stained, due to the lack of specific feline antibodies. the family of paramyxoviridae is currently divided into seven distinct genera. the number of unassigned paramyxoviruses is still increasing because well-established classification schemes, like sequence comparisons based on the viral rna-dependent rna polymerase (vrdrp) or pairwise analysis of sequence complementarity (pasc) of all orfs is often connected to several difficulties among new paramyxoviruses [ ] . feline morbilliviruses (femv) were found to be clearly distinguishable from the classical morbilliviruses, such as cdv or mev based on phylogenetic analyses [ , ] despite the fact that the size and organization of their genome reflects properties as defined for the genus morbilliviruses. here, we describe a second morbillivirus isolated from domestic cats, tentatively named feline morbillivirus genotype (femv-gt ). this virus grouped together with previously described feline morbilliviruses (femv), but could be separated from these isolates based on whole genome sequences (figure ). amino acid identities between the femv-gt and the femv structural proteins ranged between - %. these differences are comparable to the species cetacean morbillivirus (cemv) where currently five strains from different animal species exhibit a similar genetic diversity as identified for femv and femv-gt in our study [ ] . in addition, electron microscopy revealed a similar morphology of the femv-gt particle as described for femv [ ] and other morbilliviruses. we observed a wider ribonucleocapsid protein (rnp) in femv-gt preparations, but this could be due to a preparation or staining artefact, but may also be an intrinsic feature of feline morbilliviruses. therefore, we provisional propose that femv-gt should be considered as a different genotype of femv. to clarify the taxonomic status of femv-gt more information about the genetic diversity of circulating strains is needed and should be addressed to future studies. the prevalence of femv-gt was low in urine ( . %), but this may be influenced by the storage conditions. urine samples were collected as part of the routine clinical work and for practical reasons they were stored for some weeks at - • c before rna extraction could be performed. thus, rna degradation may have occurred influencing the sensitivity of the pcr analysis. two cats were femv-gt -positive for several months till years, with virus shedding in their urine emphasizing that the virus is able to establish persistent infections. this is in accordance with published data about prolonged femv-infections in domestic cats [ , , ] . interestingly, virus secretion within the urine in two persistently infected cats ('gordon' and 'tv ', table ) continued despite high titers of femv-gt -specific neutralizing antibodies in the blood. this is in contrast to other morbillivirus infections were neutralizing antibody titers higher than or prevent virus secretion in cdv [ ] and mev [ ] , respectively. the reason for this phenomenon remains speculative at the moment, but can be the result of femv-gt replication in epithelial cells of the kidney, femv-gt escape mutants or impaired t-cell response in infected animals. all femv-gt -positive cats were affected with diseases of the urinary tract, but the number of positive cases was too small to suggest the virus as the cause of the observed illnesses. an alternative explanation could be that femv-gt can only replicate on already damaged tissues of the urotract. in addition, it is known that ckd is common in geriatric cats with prevalence ranging between - % [ , ] leading to the possibility of a statistical connection between femv-gt and ckd. to further elucidate whether femv-gt infection is a cause or effect of acute and/or chronic urotract diseases, controlled animal infection experiments are required. femv-gt was propagated in various kidney cell lines from primate, rodent and feline origin although no cpe was observed in any of the tested cell lines. this is in contrast to published results from femv as this virus is able to induce syncytia formation in crfk cells [ , ] . these discrepancies could be due to lower viral titers (~ -fold) of femv-gt in comparison to femv [ , ] or may be a result of the viral adaption process to cell line not capable of expressing the wild type entry receptor which might be necessary for inducing cytopathic effects as described for mev [ ] . we showed that feline primary kidney cells are susceptible to femv-gt infection in vitro and that epithelial cells are the primary target of femv-gt in our in vitro model. whether this virus location is also reflected in natural infections needs to be clarified by (immune)-histopathology in diseased or experimentally infected cats. feline urinary bladder epithelial cells were significantly less susceptible to femv-gt . the reproducibility of the results excludes the possibility that this observation was caused by the cat's individual medical histories. data generated from in vitro infection experiments might, however, differ from the in vivo situation. the cultivation medium used for primary kidney cells, for instance, might influence the expression of terminal differentiation markers and serial passaging of these cells can induce dedifferentiation of primary distal renal tubular cells [ ] . equally, primary bladder epithelial cells might not display their physiological surface markers, so-called uroplakins, when cultured in vitro [ ] . therefore, it is possible that the femv-gt tissue tropism observed in our in vitro experiments differs from the in vivo conditions. on the other hand, femv was shown to replicate in renal tubular cells in naturally infected cats [ , , ] , supporting our results for a kidney-associated femv-gt replication. although persistent infections with femv-gt are evident, none of the analyzed blood samples harbored detectable amounts of viral rna. this implicates that viremia is present-if at all-only for a short period of time. this observation is supported by previous investigations in femv-infected cats where viremia was not detectable in the vast majority of blood samples [ , , ] . there is only one report from a shelter in thailand that found femv to be present in the blood of animals [ ] . currently, the mode of transmission of feline morbilliviruses is unknown. related viruses (e.g. canine distemper and measles virus) establish infections in their susceptible hosts by transmission via the oro-nasal route. following an initial replication in resident immune cells (tissue macrophages or dendritic cells) these viruses infect different lymphocyte subtypes resulting in a short-term viremic state [ ] . here we show that femv-gt can infect several cells of the immune system in the peripheral blood including cd + t cells, b cells, monocytes and to a significantly lesser extent cd + t cells. feline pbmc were shown to be susceptible to femv-gt infection without prior stimulation supporting the assumption that the in vitro results reflect the in vivo situation. percentage and identity of femv-gt -positive immune cells resemble findings from in vivo infection experiments with cdv in a ferret model [ ] . similar results were also observed in a macaque model of mev-infections [ ] . recently it was also shown that femv can be detected in the spleen and lymph nodes in naturally infected cats [ ] . the in vitro infection experiments using primary feline pulmonary epithelial cells, as well as cells from the cerebrum and the cerebellum revealed that they are susceptible to femv-gt . whether alveolar macrophages may also contribute to virus replication in the lung could not be investigated in this study, due to the lack of specific antibodies for phenotyping feline alveolar macrophages. alongside infections of the lung, cdv and mev are able to infect and induce severe damage to the brain under certain circumstances [ ] . although the mechanism of how cdv and mev enter the brain is not completely understood, a leukocyte-associated virus transport as a source of hematogenous brain infections is hypothesized [ ] . our in vitro findings show that such a mechanism might also be responsible for the spread of femv-gt in the body of affected animals as brain slice cultures were susceptible to the femv-gt -gordon strain. although the specific nature of the affected cell types could not be determined definitely, it is likely that neurons are a target as gfap-positive cells (representing glia cells) were found to be femv-gt -negative in our in vitro experiments. these data revealed that femv-gt is able to infect feline brain cells integrated into a physiological tissue environment. however, this needs further investigation in controlled animal infection experiments. although the observed in vitro tropism of femv-gt is in line with naturally femv-infections [ , , ] and resembles the pathology of other morbilliviruses as cdv or mev [ ] it cannot be completely excluded that these are in part the result of viral mutations emerged through the cell culture adaption process. it is well known that vero-adapted mev strains acquired mutations leading to the usage of cd as a viral entry receptor which is not a characteristic of mev wild type strains [ ] . the possibility that femv-gt have gained mutations might explain our observation that viral titer increased about -fold on llc-mk cells between consecutive passages. on the other hand, titers remained low in crfk, vero and bhk- cells independent from the number of blind passages. therefore, raising titers in the first passages of llc-mk cells can also be addressed to a low concentration of augmentable virus in urine samples. in depth sequencing approaches of femv-gt -positive urine samples and the respective cell culture adapted viral isolates will help to interpret our in vitro findings in the context of viral pathogenesis and should be conducted in future studies. nevertheless, clinicians should be aware of femv-gt when respiratory or neurological signs are encountered. although the aforementioned organs are well known as infections sites for cdv and mev [ ] , they have not been characterized as target tissues for femv in the past. until recently, the feline kidney was focused on being the target of feline morbilliviruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . our data clearly demonstrate that femv-gt can infect primary cells obtained from other organs and that the pathologic potential of femv and femv-gt might currently be underestimated. hence, further research is needed to elucidate the impact of feline morbilliviruses on animal health, especially for domestic cats. in summary, we isolated and characterized a new genotype of feline morbillivirus (femv-gt ) and showed the in vitro tropism to primary feline cells from the kidney, lung and the immune system, as well as to organotypic slice cultures from the feline cerebrum and cerebellum. our results indicate that veterinarians should be aware of this virus when symptoms of the aforementioned organs are encountered which cannot be explained by other reasons. part of the work reported in this manuscript resulted in the patent entitled "new paramyxovirus and uses thereof" with the international application no.: pct/ep / . the following are available online at http://www.mdpi.com/ - / / / /s , table s : primer sequences used for the amplification of nearly whole genome sequences of femv-gt strains. figure s : gating strategy used in flow cytometric analyses of pbmc. paramyxoviridae: the viruses and their replication complete genome sequence of a novel paramyxovirus, tailam virus, discovered in sikkim rats complete genome sequence of nariva virus, a rodent paramyxovirus genetic characterization of bank vole virus (bavv), a new paramyxovirus isolated from kidneys of bank voles in russia bats host major mammalian paramyxoviruses feline morbillivirus, a previously undescribed paramyxovirus associated with tubulointerstitial nephritis in domestic cats existence of feline morbillivirus infection in japanese cat populations chronic infection of domestic cats with feline morbillivirus, united states frequency, clinicopathological features and phylogenetic analysis of feline morbillivirus in cats in istanbul first report of feline morbillivirus in south america first evidence of feline morbillivirus detected in sheltered cats in thailand first report of feline morbillivirus in europe discovery of new feline paramyxoviruses in domestic cats with chronic kidney disease genetic diversity of feline morbilliviruses isolated in japan epidemiological and pathological study of feline morbillivirus infection in domestic cats in japan development of an elisa for serological detection of feline morbillivirus infection current understanding of the pathogenesis of progressive chronic kidney disease in cats primary kidney proximal tubule cells sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods a simple method of estimating fifty per cent endpoints simultaneous separation and purification of mononuclear and polymorphonuclear cells from the peripheral blood of cats ocular and neural distribution of feline herpesvirus- during active and latent experimental infection in cats detection of coronaviruses in bats of various species in italy development and validation of a taqman real-time reverse transcription-pcr for rapid detection of feline calicivirus epizootiologic investigations of parvovirus infections in free-ranging carnivores from germany localization and trafficking of aquaporin in the kidney immunohistochemical determination of calbindin-d k in the kidney of postnatal rats segment-specific localization of intestinal-type alkaline phosphatase in human kidney problems of classification in the family paramyxoviridae cetacean morbillivirus: current knowledge and future directions basic biological characterization of feline morbillivirus pathogenesis of canine distemper measles antibody: reevaluation of protective titers disease surveillance and referral bias in the veterinary medical database prevalence and classification of chronic kidney disease in cats randomly selected from four age groups and in cats recruited for degenerative joint disease studies morbillivirus experimental animal models: measles virus pathogenesis insights from canine distemper virus isolation and characterization of a primary proximal tubular epithelial cell model from human kidney by cd /cd double labeling normal human urothelial cells in vitro: proliferation and induction of stratification a real-time rt-pcr assay for molecular identification and quantitation of feline morbillivirus rna from biological specimens tropism illuminated: lymphocyte-based pathways blazed by lethal morbillivirus through the host immune system predominant infection of cd + lymphocytes and dendritic cells during measles virus infection of macaques morbillivirus receptors and tropism: multiple pathways for infection we thank grit müller and ines müller for assistance in sample collection and urinalyses. we thank sven reiche for providing cell lines from rodent and bat origin and uwe gerd liebert for providing access to the leica vt s microtome. the authors declare no conflict of interest. the founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. key: cord- - eq jth authors: chen, weizao; zhu, zhongyu; liao, huaxin; quinnan, gerald v.; broder, christopher c.; haynes, barton f.; dimitrov, dimiter s. title: cross-reactive human igm-derived monoclonal antibodies that bind to hiv- envelope glycoproteins date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: eq jth elicitation of antibodies with potent and broad neutralizing activity against hiv by immunization remains a challenge. several monoclonal antibodies (mabs) isolated from humans with hiv- infection exhibit such activity but vaccine immunogens based on structures containing their epitopes have not been successful for their elicitation. all known broadly neutralizing mabs (bnmabs) are immunoglobulin (ig) gs (iggs) and highly somatically hypermutated which could impede their elicitation. ig ms (igms) are on average significantly less divergent from germline antibodies and are relevant for the development of vaccine immunogens but are underexplored compared to iggs. here we describe the identification and characterization of several human igm-derived mabs against hiv- which were selected from a large phage-displayed naive human antibody library constructed from blood, lymph nodes and spleens of healthy donors. these antibodies bound with high affinity to recombinant envelope glycoproteins (gp s, envs) of hiv- isolates from different clades. they enhanced or did not neutralize infection by some of the hiv- primary isolates using ccr as a coreceptor but neutralized all cxcr isolates tested although weakly. one of these antibodies with relatively low degree of somatic hypermutation was more extensively characterized. it bound to a highly conserved region partially overlapping with the coreceptor binding site and close to but not overlapping with the cd binding site. these results suggest the existence of conserved structures that could direct the immune response to non-neutralizing or even enhancing antibodies which may represent a strategy used by the virus to escape neutralizing immune responses. further studies will show whether such a strategy plays a role in hiv infection of humans, how important that role could be, and what the mechanisms of infection enhancement are. the newly identified mabs could be used as reagents to further characterize conserved non-neutralizing, weakly neutralizing or enhancing epitopes and modify or remove them from candidate vaccine immunogens. the ability of human immunodeficiency virus type (hiv- ) to rapidly generate mutants and evade immune response is the major obstacle for development of protective, prophylactic hiv- vaccines. therefore, candidate vaccine immunogens must be capable of eliciting broadly neutralizing antibodies (bnabs) that inhibit viruses from different genetic subtypes. several human monoclonal antibodies (hmabs) such as b [ ] , x [ ] , g [ ] , f and e [ , ] exhibit potent and broad hiv- neutralizing activity in vitro and can prevent hiv- infection in animal models [ ] . these bnabs target structures on hiv- envelope glycoprotein (env) that are crucial for virus-cell fusion. therefore, envs in various formats are potential candidate immunogens and have been evaluated in animal models and human clinical trials [ ] [ ] [ ] [ ] . however, neutralization efficacy of the resulting sera as broad as that by those bnabs has not been achieved by empirically using these envs as immunogens [ ] . it has been suggested that characterization of the epitopes of the bnabs could help design vaccine immunogens that would be able to elicit these bnabs or similar antibodies in vivo [ ] . although this approach is being vigorously pursued, none of the immunogens designed has yet efficiently elicited neutralizing antibodies with broad specificity. igm is the initial antibody that the host generates when an infectious agent or antigen is encountered. activation of igm-expressing b cells provides impetus for in vivo igm-to-igg isotype switch resulting in production of high-affinity neutralizing or non-neutralizing antibodies. it is therefore important to investigate the human igm repertoire for hiv- env-specific antibodies and understand how they interact with hiv- envs and impact on viral infection which could help design effective immunogens. previous attempts to select hiv-specific antibodies by use of non-immune libraries have resulted in antibodies with modest neutralizing activity and limited breadth of neutralization [ ] . studies by several groups show that human igm antibodies play important roles not only in shaping humoral immunity against hiv- but also in inducing cell-mediated response because of their pentameric binding nature as well as their very efficient activity to activate complement. torán et al. [ ] indicated that in vivo igm-to-igg isotype switch and affinity maturation may be important for protection and long-term survival in certain hiv- -infected individuals. sheppard et al. [ ] demonstrated that complement-deactivated serum from a healthy volunteer immunized with an hiv- clade c gp exhibited igm-associated neutralizing activity that was significantly enhanced in the presence of fresh normal human serum as a source of complement. bomsel et al. [ ] showed that a human anti-hiv- gp igm monoclonal antibody blocked the transcytotic route of hiv mucosal transmission in vitro. in this study, we describe identification and characterization of several human igm-derived mabs selected from phage-displayed naive human antibody libraries constructed from healthy donors. these antibodies have high affinity and cross-reactivity with hiv- gp s from different clades, and either neutralize or enhance weakly entry of virions pseudotyped with envs from hiv- primary isolates. these data provide additional insights into the possible immune response that hiv- infection or viral env-based immunization could elicit and help in the design of candidate vaccine immunogens that elicit potent neutralizers of early transmitted virus. a large ( . × members) phage-displayed naive human antibody antigen-binding fragment (fab) library (designated m ) was constructed from peripheral blood b cells of healthy donors, spleens of healthy donors, and lymph nodes of healthy donors as described in materials and methods. the phagemid used for library construction, pzyd-n , was synthesized and briefly described previously [ ] . to approximately estimate the sequence diversity of m , clones were randomly selected from the library and sequenced. no identical sequences of heavy and light chains were found; more than % of the clones are productive. of the clones had kappa light chains and the other -lambda light chains. m was panned against several hiv- envs and human cancer-related antigens; significant enrichment was obtained with all the pannings (data not shown). these results indicate that the quality of the library is likely to be good. to identify human igm-derived antibodies specific for hiv- envs, we panned m against the gp of a clade b isolate, r (gp r ). one of the selected antibody clones, r h , contained a tga stop codon at the very beginning of the light chain due to a nucleotide deletion that resulted in a reading-frame shift. the heavy chain variable domain (vh) of r h , designated m , differs from the closest human germline sequence (vh - ) by mutations distributed in all frameworks (frs) and complementarity determining regions (cdrs) (figure a ). m contains mutations in nucleotide sequence of the variable (v) region resulting in mutations in amino acid sequence. we corrected the reading frame of the light chain by site-directed mutagenesis and the corrected antibody, designated r h m, was expressed at high levels in bacteria as an fab. fab r h m specifically bound with relatively high affinity to gp r (ec , ~ nm) and the gp of a clade c isolate, gxc (gp gxc ) (ec , ~ nm). because the antibody was initially selected as a functional heavy chain-only antibody, we hypothesized that the binding activity of r h m should be attributed mainly to the heavy chain of the antibody and the heavy chain could be paired with light chains derived from different germlines while retaining cross-reactivity with hiv- envs. we cloned m as an isolated antibody domain and found that it was highly soluble, stable, monomeric, and expressed at high levels in bacteria [ ] . in agreement with our hypothesis it specifically bound to gp r although with low affinity (ec , > nm). we further constructed a light chain-shuffling fab library ( × members) based on the heavy chain of r h . to increase the probability for selection of cross-reactive antibodies the library was panned sequentially against gp r and the gp of a clade f isolate, / / (gp / / ). five unique clones were identified that contained exclusively lambda light chains ( figure b) . they bound to gp r and gp / / with ec ranging from to nm (table ) while no significant binding to an unrelated antigen, bovine serum albumin (bsa), was observed (data not shown). one of the antibodies, m , also bound with high affinity to the envs of other three clade b isolates, gp bal, gp jrfl and gp con-s [ ] . these results suggest that m and possibly the other selected antibodies target conserved epitopes on gp . to test whether the five newly selected antibodies are capable of neutralizing hiv- primary isolates, we used viruses pseudotyped with envs from hiv- isolates representing clades a, b and c and using either ccr (r ) or cxcr (x ) or both (r x ) as a coreceptor. none of the antibodies exhibited potent broadly neutralizing activity. four antibodies (fabs m , m b, m c and m d) enhanced infection by bal even at very low antibody concentration while a positive control antibody, m [ ] , showed efficient neutralization and a negative control antibody, a , did not affect entry ( figure a ); m d enhanced infection by another clade b primary isolate, jrfl, at high concentrations ( figure b) . interestingly, the three antibodies that enhanced significantly infection by the bal weakly neutralized iiib which is a tcla clade b x isolate (figure c ). to find whether the activity of the antibodies is related to antibody size and how the viral infection could be affected by cross-linking of hiv- envs, we generated a single-chain fv fragment (scfv) (scfv m ) of m and a human igg fc-fusion protein (m fc) of scfv m ; m was selected for further characterization because its light chain was relatively less divergent from the germline ( figure b) and was the only one which did not contain any somatic mutations in the cdr of the light chain. when bal was tested, the enhancing activity of scfv m was comparable to that of fab m and slightly lower than that of m fc (data not shown). these data indicate that the antibody access to the epitope was not size-restricted and that the bivalency of the fc fusion protein strengthened the antibody activity probably due to avidity effects. the ability of m fc to modulate hiv- infection in vitro was further tested with six additional clade b isolates and an isolate each from clade a and clade c, respectively ( figure ) . enhancement was observed with jrcsf (clade b, r ) at very low m fc concentration and the enhancement was decreased with an increase in antibody concentration. m fc also conferred slight enhancement of infection by jrfl (clade b, r ) and ug . (clade a, r ) at about nm antibody concentration. it neutralized the clade b x isolates iiib and nl - . it also neutralized r , which is a clade b r isolate, displays some cd independence and is moderately neutralization sensitive, as well as gxc which is a clade c r isolate. no significant antibody activity was observed with the dual tropic clade b isolate . . these results indicate that the antibody activity in terms of neutralization or enhancement varies when different isolates are tested and is not significantly correlated with the coreceptor usage and the cd dependency of the viruses in entry. antibody, b (a) , and two cd i antibodies, m and m (b), in binding to gp bal and to gp bal -cd , respectively. an irrelevant human antibody, a , was used as a negative control. to approximately localize the antibody epitopes and begin to elucidate the underlying mechanisms of antibody neutralizing or enhancing activity, we measured the antibody binding to envs from different isolates alone and in complex with cd as well as the antibody competition with wellcharacterized antibodies. m fc bound to a single-chain polypeptide analogue of the hiv- gp -cd complex (gp bal -cd ) [ ] with threefold higher affinity (ec , ~ . nm) than to gp bal (ec , ~ nm) alone as measured by an elisa (figure a ). it did not react with scd suggesting that the antibody targeted a structure on gp that was outside the cd -binding site (cd bs) and could undergo cd -induced (cd i) conformational changes. the conformational changes on gp bal after cd binding were confirmed by a cd i bnab, m fc [ ] , which exhibited dramatically higher binding to gp bal -cd than to gp bal (figure a ). to find out whether the antibody bound to the conventional cd i epitopes, we used gp con-s , which was a synthetic env designed by aligning the consensus env sequences of group m [ ] . this env, by design, is composed by shorter v , v , v and v loops and therefore, might expose regions around the variable loops that might contain conserved neutralizing determinants. however, m epitope as a representative for cd i epitopes is still completely hidden on gp con-s because m fc did not bind in the absence of scd , it did bind with high affinity in the presence of scd (figure b) . however, m fc showed comparable binding to gp con-s with or without scd (figure b) suggesting that the antibody does not precisely target cd i epitopes but other structures on the gp . although m fc was not directed against the cd binding pocket, it strongly competed with the cd bs bnab, scfv b , which has binding surface larger than that of cd [ ] (figure a ). m fc also competed although weakly with two cd i antibodies, scfv m and m [ ] (figure b) . these results suggest that the epitope of m fc is in very close proximity to the cd bs and the coreceptor-binding site (corbs) which overlaps with cd i epitopes on gp ( figure ). figure . a schematic representation for the epitope (gray) of m on gp (blue). the red and white areas denote the cd bs and the corbs, respectively. the epitope (long dash-dotted circle on the left) of the cd bs antibody, b , is indicated in comparison with the cd -binding area. the dashed circle on the right denotes the corbs for some isolates that could partially overlap with the epitope of m . to estimate the possibility of rapid elicitation of these antibodies in vivo when an hiv- gp related immunogen is administered, we generated a germline-like scfv of m (scfv m gem) and measured its binding to envs from different isolates in the presence or absence of scd . no obvious interaction was observed with scfv m gem when several envs that bound the original m were tested (data not shown). previous studies showed that the binding activity of an antibody in the form of fab or scfv could be increased up to thousands of times when it is converted to dimeric formats such as an igg. we therefore fused scfv m gem to the fc portion of a human igg to gain possible avidity effects. still, the fc-fusion protein of scfv m gem (m gemfc) did not show measurable binding to the envs with or without scd (figure ) . these results suggest that significant somatic diversification is required for the m corresponding germline antibody to achieve recognition of the m epitope on gp . they also indicate that in some individuals certain igm antibodies could undergo somatic hypermutations to relatively high-affinity binders to the env in the absence of hiv- infection or immunization with env. figure . binding of the germline-like antibody of m to different gp s in the absence or presence of cd . the original antibody, m fc, was used as a positive control. vaccines based on recombinant envs have failed to prevent viral infection in human efficacy trials likely because of their inability to elicit neutralizing antibodies that are broad enough to combat the extremely high variability of the virus. recent success was reported but it is modest and needs to be repeated and further examined [ ] . to date only several bnabs have been isolated from hiv- patients suggesting difficulties to elicit such antibodies in vivo. however, the presence of epitopes recognized by these bnabs on envs argues that they are still a potential template for vaccine design. different strategies aiming at improving the presentation of neutralizing epitopes are rigorously pursued. analysis of the structures of envs shows that most of their surface is hidden from humoral immune responses by glycosylation and oligomeric occlusion [ , ] . the cd bs and the corbs on gp are also flanked by loop structures which may further limit the access by antibodies generated by the human immune system [ , ] . therefore, one strategy focuses on the use of modified envs, in which the variable loops have been deleted [ ] [ ] [ ] [ ] or glycosylation removed [ , ] or both [ ] in order to increase the exposure of the neutralizing epitopes. based on the finding that the neutralizing capacity of the antibodies is associated with their ability to bind to native trimeric envs on the virus but does not correlate with binding to isolated monomeric envs, another strategy focuses on preservation or re-construction of the functional trimeric envs [ , ] . the essential concept is that immunizing with a close mimic of the functional trimer will improve the chances of eliciting neutralizing antibodies. yet another strategy is to use fusion intermediates formed during virus entry [ , ] or their mimics [ ] . hiv- entry is initiated by binding of viral gp with the receptor cd and a coreceptor on the target cell surface. these interactions lead to intermediate env conformations that may include conserved structures useful for vaccine design. however, elicitation of desirable level and breath of neutralizing antibodies with the immunogens generated based on these strategies has not been achieved. while modification of envs based solely on the analysis of the interaction with neutralizing antibodies is not sufficient to create effective vaccines, we propose that the inherent capacity of the human immune system in response to hiv- infection should be explored. we have hypothesized that investigation of human igm repertoire for hiv- env-specific antibodies and understanding how they react with envs, evolve and impact on viral infection would help design effective hiv- immunogens. it has been previously found that igm-based response confers substantial effects on hiv- infection which are especially relevant in the context of acute infection [ ] [ ] [ ] . in this study, we describe the identification and characterization of several high-affinity human igm-derived mabs against hiv- from phage-displayed naive human antibody libraries constructed from healthy donors. although their potency in vitro appears minor the roles they could play in mediating hiv- infection in vivo should not be underestimated. igm antibodies are highly effective in activating complement system to destroy the virus or infected cells. the antibodies selected in this study target highly conserved regions on gp which are in very close proximity to the cd bs and the corbs. because these two regions on gp are vital for virus entry and harbor neutralizing epitopes, hiv- has evolved strategies such as steric occlusion to protect it from humoral immunity [ , ] . here we suggest that the possible high immunogenicity of the conserved non-neutralizing epitopes of these antibodies could further divert the immune system from responses to neutralizing epitopes. recent studies [ , ] showed that crossreactive non-neutralizing antibodies (igms and iggs) were elicited by immunizing mice with recombinant hiv- gp s suggesting the same possibility in humans. because the antibodies we selected are less divergent (totally about amino acid mutations in the v gene products for the heavy and light chains) (figure ) than the known bnabs which all contain extensive somatic mutations (on average > mutations) compared to germline antibodies (data not shown), they could be more easily elicited. in addition, these antibodies could directly block the access of some neutralizing antibodies generated by the human immune system, especially those targeting the cd bs or corbs. this was supported by our finding that m fc partially reversed the neutralization by a cd i antibody, m [ ] (data not shown). these results further support previous propositions to reduce the immunogenicity of unwanted epitopes when gp /gp is used as an immunogen. we found that the closest germline-like antibody to m does not bind an env (figure ) . we observed similar lack of binding of germline-like b , f and g to envs [ ] [ ] [ ] . moreover, we found that there was a lack of or no high-affinity binders to envs in the germline-like antibody repertoire of the human cord blood whereas high-affinity antibodies against other human infectious agents including sars coronavirus and henipaviruses could be easily selected (weizao chen, emily streaker and dimiter s. dimitrov, in preparation). the identified antibodies against sars coronavirus protein s exhibited nanomolar affinity and were very close to germline in sequence. by analyzing the previously identified antibody sequences we found that all of the cross-reactive neutralizing antibodies against hiv- were highly diversified from their germline sequences; in contrast, the antibodies against sars coronavirus and henipaviruses had only limited number of mutations [ ] [ ] [ ] . these results indicate that extensive somatic mutations could be required for high-affinity binding to conserved hiv- env structures. one could speculate that the maturation pathways for some hiv- antibodies are initiated by immunogens that are different from the envs; such immunogens could be used in combination with env-based immunogens to guide the immune system for elicitation of potent bnabs by additional somatic hypermutation and selection. this is in line with our proposition to elicit bnabs by using one or more primary immunogens that are different than envs but can lead to intermediates on the maturation pathways that can be subsequently further somatically hypermutated to matured bnabs [ ] [ ] [ ] . except for the conventional antigen-antibody interaction, gp s also bind to a small population of human antibodies containing products of mainly vh gene family and exhibit superantigen properties by activating the human b cells expressing such antibodies on cellular membrane [ ] . the core motif of the superantigen-binding site (sbs) on gp is a discontinuous structure spanning the v variable domain and the amino-terminal region flanking the c constant domain [ ] . the determinants on the antibody vh domains critical for gp binding are not clear yet while the putative important regions were identified in the fr and ; a modeling study showed that most of the potential contact residues were located on the face of the vhs opposite to the interface for interaction with light chain; other positions in the cdr and also influenced the binding [ ] . moreover, the gp -positive antibody vh genes showed > % similarity to the germlines suggesting that the somatic hypermutations may have adverse effects on gp binding [ ] . the antibodies described in this study contained vh - gene products ( figure a ) and therefore, their high binding could be due to the superantigen-like interactions. however, our results showed that the isolated vh of these antibodies, m , bound to gp s with affinity (ec , > nm) much lower than that (ec , - nm) of the fabs. in addition, the epitope of one (m ) of these antibodies was mapped to a structure in very close proximity to the cd bs and the corbs on gp but not to the defined sbs. these results suggest that the majority, if not all, of the binding activities of the selected antibodies should be contributed by the conventional antigen-antibody recognition. one should note that these antibodies may not represent the native ones because they were selected from phage libraries where the heavy and light chains of the antibodies are randomly recombined. however, previous studies [ ] [ ] [ ] have shown that selection of high-affinity antibodies from such libraries results in antibodies which are identical or very similar to those occurring in the host from which the libraries are made. it has been shown that at least in some systems the antigen selected antibodies such as the autoantibodies against thyroid peroxidase (tpo) from large random phage libraries have the same pairings as those from small libraries where the cognate pairing is preserved and that pairing between light and heavy chain is not promiscuous [ , ] . the study by chapal et al. [ ] directly demonstrates that antibodies derived from combinatorial libraries are likely to represent in vivo pairings, leading to high affinity antibody fragments mimicking the binding of serum autoantibodies to tpo. de wildt et al. [ ] analyzed the cognate pairings of heavy and light chain variable domains in human igg+ b cells from peripheral blood and established that the pairings are largely random. the antibodies we selected were originated from large families of genes -heavy chain from vh and light chain from vl ( figure ) . importantly, the pairings for two of them, m and m d, have been found in the very limited number ( ) of truly human antibodies previously analyzed [ ] . moreover, some (e.g., m and m b) of these antibodies are relatively close to their corresponding germlines (figure ). these data suggest the possibility of eliciting such antibodies in vivo during hiv- infection or gp -based immunization. we purchased the t cells from atcc. other cell lines and plasmids used for expression of various hiv- envs were obtained from the national institutes of health aids research and reference reagent program (arrrp). recombinant gp s were produced in our laboratories. gp bal and the single-chain fusion protein gp bal -cd were kindly provided by t. fouts (institute of human virology, baltimore; currently at profectus, baltimore, md). horseradish peroxidase (hrp)-conjugated anti-flag tag antibody and hrp-conjugated anti-human igg (fc-specific) antibody were purchased from sigma-aldrich (st. louis). m was constructed by using phagemid pzyd-n according to the reported protocols [ ] . the sources for amplification of antibody gene fragments are commercially purchased poly a+ rnas (bd biosciences, san jose, ca) from peripheral blood b cells of healthy donors, spleens of three donors and lymph nodes of healthy donors, respectively. m l was used for selection of antibodies against hiv- antigens conjugated to magnetic beads (dynabeads m- epoxy; dynal inc., new hyde park, ny) as described previously [ ] . , , . and µg of gp r were used in the first, second, third and fourth round of panning, respectively. clones that specifically bound to gp r were identified from the third and fourth round by using monoclonal phage elisa (mpelisa) as described [ ] . a light chain-shuffling fab library ( × members) was further constructed based on the heavy chain of r h . the light chain repertoire was also harvested from a naive human fab library ( × members) constructed from peripheral blood b cells of healthy donors [ ] , in addition to that from m . the new library was panned sequentially with two gp s from different clades. for the sequential panning, and . µg of gp r were used in the first and third round, respectively; antigens were alternated with and . µg of gp / / during the second and fourth round, respectively. clones that bound to both gp r and gp / / were identified from the fourth round by using mpelisa as described [ ] . the following primers were used: m f, '-tgg ttt cgc tac cgt ggc cca gcc ggc cca ggt gca gct ggtg- ' (sense); m fcr: '-gtg agt ttt gtc ggg ccc tag gac ggt cag ctt gg- ' (antisense). for construction of scfv, the full-length original or germline scfv gene fragment was synthesized (genscript, piscataway, nj), digested with sfii and cloned into pcomb x. to generate fc-fusion protein, the scfv gene was pcr (primers m f and m fcr) amplified by using the synthetic scfv gene fragment as a template. the new scfv products appended with sfii and apai restriction sites on both sides were digested and cloned into psectagb-fc. the scfv and fab were expressed in e. coli hb , as described previously [ ] . the bacterial pellet was collected after centrifugation at , × g for min and resuspended in pbs (ph . ) containing . million-unit polymixin b (sigma-aldrich). after min incubation with rotation at rpm at room temperature, it was centrifuged at , × g for min at °c. the supernatant was used for purification of scfv and fab by immobilized metal ion affinity chromatography (imac) by using ni-nta resin (qiagen, valencia, ca) according to manufacturer's protocols. fc-fusion proteins were expressed in free style cells. fectin (invitrogen, carlsbad, ca) was used to transfect free style cells according to the instructions of the manufacturer. three days post-transfection, the culture supernatant was harvested and used for purification by using nprotein a sepharose fast flow (ge healthcarezcomx, piscataway, nj). binding and competition elisas were performed as described previously [ ] . viruses pseudotyped with hiv- envs were prepared by cotransfection of - % confluent t cells with pnl - .luc.e-r-and psv d constructs encoding hiv- envs by using the polyfect transfection reagent (qiagen) according to manufacturer's instruction. pseudotyped viruses were obtained after h by centrifugation and filtration of cell culture through . µm filters. for neutralization, viruses were mixed with different concentrations of antibodies for h at °c, and then the mixture was added to about . × hos-cd -ccr (used for all r and dual tropic viruses) or hos-cd -cxcr cells grown in each well of -well plates. luminesence was measured after h by using the bright-glo luciferase assay system (promega, madison, wi) and a lumicount microplate luminometer (turner designs). mean relative light units (rlu) for duplicate wells were determined. relative infectivity (%) was calculated by the following formula: (average rlu of antibody-containing wells/average rlu of virus-only wells) × . a major finding of this study is the identification and characterization of several hiv- -specific human igm-derived mabs and their highly conserved epitopes. such antibodies were rarely investigated and reported. further characterization of these antibodies, their dynamics and epitopes could provide knowledge that in addition to its usefulness for basic understanding of immune responses to hiv- could also help in the design of candidate vaccine immunogens that elicit potent neutralizers of early transmitted virus. recognition properties of a panel of human recombinant fab fragments to the cd binding site of gp that show differing abilities to neutralize human immunodeficiency virus type broadly crossreactive hiv- -neutralizing human monoclonal fab selected for binding to gp -cd -ccr complexes human monoclonal antibody g defines a distinctive neutralization epitope on the gp glycoprotein of human immunodeficiency virus type a potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp of human immunodeficiency virus type broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type glycoprotein gp human monoclonal antibodies and engineered antibody domains as hiv- entry inhibitors extensively cross-reactive anti-hiv- neutralizing antibodies induced by gp immunization correlation between immunologic responses to a recombinant glycoprotein vaccine and incidence of hiv- infection in a phase hiv- preventive vaccine trial a clinically relevant hiv- subunit vaccine protects rhesus macaques from in vivo passaged simian-human immunodeficiency virus infection prevention of disease induced by a partially heterologous aids virus in rhesus monkeys by using an adjuvanted multicomponent protein vaccine aiming to induce broadly reactive neutralizing antibody responses with hiv- vaccine candidates antibodies, viruses and vaccines a human monoclonal antibody neutralizes diverse hiv- isolates by binding a critical gp epitope molecular analysis of hiv- gp antibody response using isotype igm and igg phage display libraries from a long-term non-progressor hiv- -infected individual a functional human igm response to hiv- env after immunization with nyvac hiv c intracellular neutralization of hiv transcytosis across tight epithelial barriers by anti-hiv envelope protein diga or igm human domain antibodies to conserved sterically restricted regions on gp as exceptionally potent cross-reactive hiv- neutralizers construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain a group m consensus envelope glycoprotein induces antibodies that neutralize subsets of subtype b and c hiv- primary viruses expression and characterization of a single-chain polypeptide analogue of the human immunodeficiency virus type gp -cd receptor complex improved breadth and potency of an hiv- -neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning crystal structure of a neutralizing human igg against hiv- : a template for vaccine design vaccination with alvac and aidsvax to prevent hiv- infection in thailand the antigenic structure of the hiv gp envelope glycoprotein structure of an hiv gp envelope glycoprotein in complex with the cd receptor and a neutralizing human antibody the ability of an oligomeric human immunodeficiency virus type (hiv- ) envelope antigen to elicit neutralizing antibodies against primary hiv- isolates is improved following partial deletion of the second hypervariable region immunogenicity and ability of variable loop-deleted human immunodeficiency virus type envelope glycoproteins to elicit neutralizing antibodies immunogenicity of dna vaccines expressing human immunodeficiency virus type envelope glycoprotein with and without deletions in the v / and v regions selective modification of variable loops alters tropism and enhances immunogenicity of human immunodeficiency virus type envelope influence of n-linked glycans in v -v region of human immunodeficiency virus type glycoprotein gp on induction of a virus-neutralizing humoral response role of n-linked glycans in a human immunodeficiency virus envelope glycoprotein: effects on protein function and the neutralizing antibody response modified hiv envelope proteins with enhanced binding to neutralizing monoclonal antibodies immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type gp improved elicitation of neutralizing antibodies against primary human immunodeficiency viruses by soluble stabilized envelope glycoprotein trimers crosslinked hiv- envelope-cd receptor complexes elicit broadly cross-reactive neutralizing antibodies in rhesus macaques purified complexes of hiv- envelope glycoproteins with cd and ccr (cxcr ): production, characterization and immunogenicity mutagenic stabilization and/or disruption of a cd -bound state reveals distinct conformations of the human immunodeficiency virus type gp envelope glycoprotein broadly reactive monoclonal antibodies to multiple hiv- subtype and sivcpz envelope glycoproteins. virology production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type envelope glycoproteins in a mouse model expressing human immunoglobulins all known cross reactive hiv- neutralizing antibodies are highly divergent from germline and their elicitation may require prolonged periods of time germline-like predecessors of broadly neutralizing antibodies lack measurable binding to hiv- envelope glycoproteins: implications for evasion of immune responses and design of vaccine immunogens maturation pathways of cross-reactive hiv- neutralizing antibodies immunoglobulin vh gene products: natural ligands for hiv gp identification of the b cell superantigen-binding site of hiv- gp structural basis of the gp superantigen-binding site on human immunoglobulins influenza virus hemagglutinin-specific antibodies isolated from a combinatorial expression library are closely related to the immune response of the donor original and artificial antibodies thyroid peroxidase autoantibodies obtained from random single chain fv libraries contain the same heavy/light chain combinations as occur in vivo recombinant thyroid peroxidase-specific autoantibodies. i. how diverse is the pool of heavy and light chains in immunoglobulin gene libraries constructed from thyroid tissue-infiltrating plasma cells recombinant thyroid peroxidase-specific autoantibodies. ii. role of individual heavy and light chains in determining epitope recognition analysis of heavy and light chain pairings indicates that receptor editing shapes the human antibody repertoire construction of a large naive human phage-displayed fab library through one-step cloning potent neutralization of hendra and nipah viruses by human monoclonal antibodies we thank xiaodong xiao in our group for helpful discussion. key: cord- -l v x authors: hooper, chantelle; debnath, partho p.; biswas, sukumar; van aerle, ronny; bateman, kelly s.; basak, siddhawartha k.; rahman, muhammad m.; mohan, chadag v.; islam, h. m. rakibul; ross, stuart; stentiford, grant d.; currie, david; bass, david title: a novel rna virus, macrobrachium rosenbergii golda virus (mrgv), linked to mass mortalities of the larval giant freshwater prawn in bangladesh date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: l v x mass mortalities of the larval stage of the giant freshwater prawn, macrobrachium rosenbergii, have been occurring in bangladesh since . mortalities can reach % and have resulted in an % decline in the number of hatcheries actively producing m. rosenbergii. to investigate a causative agent for the mortalities, a disease challenge was carried out using infected material from a hatchery experiencing mortalities. moribund larvae from the challenge were prepared for metatranscriptomic sequencing. de novo virus assembly revealed a kb single-stranded positive-sense rna virus with similarities in key protein motif sequences to yellow head virus (yhv), an rna virus that causes mass mortalities in marine shrimp aquaculture, and other viruses in the nidovirales order. primers were designed against the novel virus and used to screen cdna from larvae sampled from hatcheries in the south of bangladesh from two consecutive years. larvae from all hatcheries screened from both years were positive by pcr for the novel virus, including larvae from a hatchery that at the point of sampling appeared healthy, but later experienced mortalities. these screens suggest that the virus is widespread in m. rosenbergii hatchery culture in southern bangladesh, and that early detection of the virus can be achieved by pcr. the hypothesised protein motifs of macrobrachium rosenbergii golda virus (mrgv) suggest that it is likely to be a new species within the nidovirales order. biosecurity measures should be taken in order to mitigate global spread through the movement of post-larvae within and between countries, which has previously been linked to other virus outbreaks in crustacean aquaculture. the giant river prawn (macrobrachium rosenbergii) is a species of global aquaculture importance, with culture rapidly expanding from < t in to > , t worldwide in [ ] . production, particularly in developing parts of asia, provides direct and indirect employment, a food source to alleviate poverty, and export income in a high value international market [ ] . despite the exponential growth of the prawn industry, disease has been a significant problem, and when mortalities occur they are usually high, causing widespread impacts to the socioeconomic balance in these countries [ ] . bangladesh is considered to be a favourable environment for farming m. rosenbergii, named 'golda' by bangladeshi farmers, due to its vast inland freshwater river systems with adjacent brackish areas and a suitable climate for culturing tropical and sub-tropical aquaculture species [ ] . in , bangladesh was the world's second largest producer of m. rosenbergii, producing , t [ ]. despite this promising production figure, m. rosenbergii hatcheries have been experiencing mass mortalities of larvae, resulting in the number of hatcheries actively producing larvae declining by almost % over the past decade as they are unable to complete the production cycle and it becomes uneconomical to continue. between and , over hatcheries were operating in bangladesh, producing up to two hundred million post-larvae (pl) per year; however, in production had decreased to . million pl/year from only hatcheries [ , ] . hatcheries experiencing mortalities reported that larvae had abnormal shapes, reduced appetite, issues with moulting and a gradual whitening of the body, finally resulting in either disappearance from the culture system, or mortality [ ] . to compensate for the loss of hatchery-produced post-larvae, wild post-larvae are being caught from the vast river systems in the south of bangladesh, violating a government ban on the capture of wild pl [ ] . ahmed and troell [ ] reviewed the potential for negative environmental impact associated with fishing for wild pl and identified two main issues; high levels of invertebrate and fish bycatch associated with fishing for larvae (and a subsequent decline in biodiversity) and a reduction in numbers of larvae returning to the freshwater environment as adults to spawn (reducing natural m. rosenbergii abundance). as fishing for wild post-larvae increases due to the hatcheries being unable to complete production cycles, it is likely that the negative environmental impact caused by fishing will become more pronounced. a number of surveys have been carried out by several organisations to determine possible causes for the mortalities observed in hatcheries in bangladesh. the surveys identified many shortcomings, including deficiencies in water quality, water filtration systems, temperature fluctuations due to weather conditions, feeding practices, probiotic use and poor-quality inputs that had been exposed to formalin and bleaching [ ] . however, despite these constraints, hatcheries were successfully completing production cycles until , suggesting that a new factor has arisen, which could be disease-related. biosecurity has been highlighted as a major issue, in relation to the spread of mortalities throughout the hatcheries of bangladesh. as the culture of m. rosenbergii has increased globally, the incidence of diseases and emergence of novel pathogens has increased in parallel [ ] . diseases known to affect different life stages of the giant freshwater prawn include numerous pathogenic bacteria, viruses and fungi. opportunistic bacteria such as aeromonas spp., pseudomonas spp. and vibrio spp. can cause infections in all life stages of m. rosenbergii culture (reviewed in pillai and bonami [ ] ). spiroplasma eriocheiris [ ] has been shown to cause mortalities in later life stages of m. rosenbergii. macrobrachium rosenbergii nodavirus (mrnv) [ ] , in association with extra small virus (xsv) [ ] , causes white tail disease (wtd), a disease listed by the world organisation for animal health (oie). the viruses have caused devastation in the prawn hatchery industries of thailand [ ] , china [ ] , india [ ] , taiwan [ ] and indonesia [ ] . other viruses causing mortalities include macrobrachium rosenbergii taihu virus (mrtv), a novel dicistrovirus associated with larval mortalities in china [ ] , infectious hypodermal and haematopoietic necrosis virus (ihhnv) [ ] , and decapod iridescent virus (div ) [ ] . other pathogens have been identified to infect m. rosenbergii without large mortalities including: macrobrachium parvo-like virus (mpv) [ ] and white spot syndrome virus (wssv) [ , ] . with the lack of immortal shrimp cell lines, disease challenges can be used to identify pathogenic agents infecting m. rosenbergii. this technique was recently used to determine that mrnv alone can cause mortalities in m. rosenbergii in the absence of xsv [ ] . alam et al. ( ) attempted to determine the causative agent(s) associated with the larval mortalities in bangladesh hatcheries using a screen for vibrio spp., which are known to cause disease in larval stages of prawn culture [ ] . however, this study was unable to find a strong correlation between the vibrio species present and the levels of mortalities seen in larvae. larvae were also screened for the presence of mrnv and xsv, but neither of these were detected. therefore, no significant pathogens have yet been associated with the large-scale larval mortalities in bangladesh. the clinical signs of disease observed in these larvae associated with the current large-scale mortalities do not precisely fit those described for infection with any of the known larval prawn pathogens; therefore, an approach to detect novel pathogens was needed to determine if there was a pathogenic cause for the mortalities. in this study we ( ) carried out an experimental challenge trial using larvae sourced from a hatchery displaying disease, ( ) generated and analysed metatranscriptomic libraries obtained from pools of larvae from experimental trials to identify any potential pathogens present in moribund animals, and ( ) screened for pathogens identified as a risk in other hatcheries that had been experiencing mortalities to determine whether these agents were present. larvae were collected from six hatcheries in southern bangladesh over the production seasons of and . batches of approximately - whole larvae were fixed in rnalater. in the same years, adult m. rosenbergii were collected from seven rivers, also in southern bangladesh (figure ). to produce materials for disease challenge, moribund larvae were collected from a hatchery that was experiencing mortalities at the time of the study. larvae were ground using a sterile pestle and mortar, suspended in phosphate buffered saline (pbs) and filtered through a . µm syringe filter. larvae from a healthy hatchery that had not experienced mortalities in past production cycles were also collected and prepared in the same manner as a control treatment. experimental larvae were obtained from bangladesh fisheries research institute's (brfi) domesticated f broodstock. after hatching, larvae were reared up to stage three, before splitting into three groups for different experimental tank exposures. experimental larvae were directly immersed into filtered challenge medium from moribund larvae for min prior to being transferred to a fibreglass tank. experimental larvae were fed artemia that had been immersed in challenge medium from moribund larvae for min. experimental larvae were immersed into filtered challenge medium from healthy larvae for min prior to being transferred to a fibreglass tank. for all experiments, ± larvae were used, and experiments were carried out in l fibreglass tanks heated to a constant temperature of • c with a submerged thermostat heater. larvae were fed three times per day ( g/ l) with red jungle brand ® artemia (ocean star international, snowville, ut, usa). twenty to thirty larvae were collected every day for the first five days of the experiment and on days seven and ten. rna was extracted using ribozol™ (vwr, radnor, pa, usa). for larvae, approximately larvae were added to ml ribozol, and for adults, approximately mg of each tissue type (pleopod, hepatopancreas, gill, gut) was pooled and added to ml of ribozol. rna extraction was carried out following the manufacturer's protocol, with a final resuspension in µl molecular grade water (fisher scientific, waltham ma, usa). to remove any dna co-extracted with rna, a dnase step was carried out using dnase (sigma aldrich, st. louis, mo, usa), following the manufacturer's protocol. first strand synthesis was performed in µl reaction volumes using µl of mmlv-rt × buffer (promega, madison, wi, usa), . µl of dntps ( mm) (promega), . µl of recombinant rnasin (promega), µl of random hexamer primers (promega) and µg of rna in . µl. prior to the addition of µl of mmlv-rt enzyme (promega), the reaction was incubated at • c in order to denature double stranded rna. following the addition of the mmlv-rt, the reaction was incubated at • c for h. second strand synthesis was performed immediately after first strand synthesis. single-stranded cdna was incubated at • c for min, followed by • c for min. following incubation, µl sequenase buffer (thermo fisher, waltham, ma, usa), . µl sequenase (thermo fisher) and . µl molecular grade water (thermo fisher) were added. following this, reactions were incubated at • c for min, • c for min and • c for min. during incubation at • c, . µl : (sequenase:sequenase dilution buffer (thermo fisher)) diluted sequenase was added to reactions and incubated at • c for min and • c for min prior to transfer to ice. three pools of cdna were constructed from in vivo tank experiments: ( ) challenge days - larvae from immersion and feeding challenges using filtered medium from moribund larvae ( ) challenge days - larvae from immersion and feeding challenges using filtered medium from moribund larvae and ( ) larvae from a hatchery that had been experiencing mass mortalities. these pools were prepared for metatranscriptomic sequencing using the illumina compatible nextera xt library preparation kit (illumina, san diego, ca, usa) and sequenced on an illumina miseq using v chemistry (illumina). the generated raw illumina paired-end sequence reads were trimmed to remove adaptor and low quality sequences using trim galore! v . . [ ] . trimmed sequences were quality-checked using fastqc v . . [ ] before reads from individual pools were assembled using both rnaspades v . . [ ] and the iterative virus assembler (iva) v . . [ ] . assembled contigs were subsequently annotated using the blastp algorithm of diamond v . . [ ] and the full ncbi non-redundant (nr) protein database (downloaded november ), and the results were visualised using megan community edition v . . [ ] . paired reads from all samples were then mapped to the assembled contig representing the viral genome sequence using bwa-mem v . . and samtools v . with default parameters [ , ] . the output from bwa-mem was visualised with integrative genomics viewer (igv) v . . [ ] . assembly quality and accuracy were assessed with qualimap v . . [ ] . predicted open reading frames (orfs) were identified using four different tools: prokka v . [ ] fgenesv (softberry.com), genemarks v . [ ] and vgas [ ] . orfs that were supported by two or more programs were analysed further. supported orfs were annotated using ncbi blastp and the full ncbi protein sequence database (downloaded december ) and protein motifs were identified by hhpred [ ] (default parameters) and interproscan [ ] . predicted protein motifs were aligned against known nidovirus protein motifs using mafft [ ] and multiple sequence alignments (msas) were visualised in esprict v . [ ] (default parameters). transmembrane regions were identified using tmhmm v . [ ] (default parameters). ribosomal frameshift identifiers were found using fsfinder using the virus genome settings [ ] . secondary structure of the untranslated region (utr) was predicted using mfold [ ] (default parameters). phylogenetic analyses was performed using the rna-dependent rna polymerase (rdrp) protein domain as in saberi et al. [ ] , conserved in known and proposed nidovirales, on nidovirales with representatives from arteriviradae, roniviridae, mesoniviridae, mononiviridae, euroniviridae, coronavirinae and torovirinae families and subfamilies. two representatives from the astroviradae order of viruses were also aligned as an outgroup. msas were performed using default parameters in mafft [ ] . maximum likelihood phylogenetic analyses were carried out using raxml blackbox v. [ ] (generalised time-reversible (gtr) evolutionary model; all parameters estimated from the data). a bayesian consensus tree was constructed using mrbayes v. . . [ ] . two separate multi-core markov-chain monte carlo (mc ) runs with randomly generated starting trees were carried out for million generations each with one cold and three heated chains using a gtr model. all parameters were estimated from the data. the trees were sampled every generations and the first , generations discarded as burn-in. all phylogenetic analyses were carried out on the cipres server [ ] . primers specific to the predicted enveloping protein orf of mrgv were designed using primer v . . [ ] using default settings and an amplicon length of - bp: mrgv_f : -tttgcccaggttaattgccc- and mrgv_f : -acaagtgccagtgagacgta- , producing an amplicon of bp. pcr amplification was performed in µl reactions using µl × green gotaq flexi buffer (promega), . µl mgcl ( mm) (promega), . µl of each primer ( µm), . µl dntps ( mm) (promega), . µl gotaq polymerase (promega), . µl molecular grade water and . µl of template cdna. initial denaturation was carried out at • c for min, followed by cycles of • c for s, • c for s and • c for s. this was followed by a final extension at • c for min. amplicons were purified with wizard ® sv gel and pcr clean-up system (promega) and sequenced via the eurofins tubeseq service. primers were tested on larval cdna from five hatcheries and cdna from wild adult m. rosenbergii sampled from the river networks surrounding the hatcheries. cdna from penaeus monodon tissues infected with yellow head virus (yhv) were tested as a negative control. pcr screens for specific pathogens were carried out with the following primers: mrnv using mrnv af and mrnv ar primers [ ] ; xsv using xsv-external forward and reverse primers [ ] ; mrtv using mrtv f and mrtv r primers [ ] ; wssv using f , r , f and r primers [ ] ; penaeus monodon nudivirus (pmnv) using f and r primers [ ] ; spiroplasma eriocheiris using f and r primers [ ] ; and yhv using yc-f a, yc-f b, yc-r a, yc-r b, yc-f a, yc-f b, yc-r a and yc-r b primers [ ] . all pcr reactions were performed as above using the cycling conditions specified in the original publications. at the end of the -day disease challenge, mortalities exceeding % had occurred in all three experimental groups, including larvae exposed to an extract produced from a hatchery that at the time had not experienced mortalities ( figure ). however, it was later discovered that the healthy hatchery experienced mass mortalities to a similar level of other hatcheries in the south of bangladesh shortly after they were sampled for the experimental material. as the challenge progressed, reduction in swimming ability, feeding and growth were observed in all treatments. moribund larvae appeared white in colour compared to healthy larvae. a total of , , , , , and , , illumina read-pairs were generated for libraries , and , respectively, and after quality-trimming and filtering, , , , , , and , , read-pairs remained. contigs assembled using rnaspades, both separately and by combining all three libraries, were annotated using diamond in blastx mode. rnaspades assembly of combined libraries produced , contigs; contigs, of average length bp, had similarity in protein sequence to yhv or gill-associated virus (gav), but when the trimmed reads were aligned against the yhv genome (accession number gca_ . ), no alignment was seen. de novo virus assembly using iva from library three produced contigs, and two non-overlapping contigs, of lengths , and , had blastx similarity to yhv. iva assembly using a pool of all three libraries produced a full genome consensus sequence of . kb, with an average coverage of ×. the two contigs produced by iva assembly of library three mapped to the . kb consensus sequence. the full genome sequence is deposited under accession number mt on genbank. henceforth, we refer to this novel virus as macrobrachium rosenbergii golda virus (mrgv). mapping trimmed and quality-filtered reads from each individual library back to the consensus genome gave coverage of . × for library one (pooled challenged larvae from days - ), . × for library two (pooled challenged larvae from days - ) and . × coverage for library three (pooled larvae from a hatchery experiencing mortalities). to compare the efficiency of the rnaspades assembly, assembled contigs from individual and pooled libraries were mapped back to the consensus genome. eleven contigs from library one mapped to mrgv with . × coverage, contigs from library two mapped with . × coverage, contigs from library three mapped with . × coverage and contigs from the pooled libraries mapped with . × coverage. rnaspades contigs from libraries two and three gave good coverage over the whole genome but failed to assemble the and ends. a comprehensive table of assembly statistics is in supplementary table s . fgenesv predicted five protein-coding orfs in the positive sense direction, four of which were supported by genemarks, prokka and vgas (figure ), which were investigated in more detail. the two longest orfs, orf a and orf b, showed homology to the replicase polyproteins of yellow head virus (evalues of × − and ), whereas orf showed homology to glycoproteins associated with species in the negarnaviricota phylum (realm riboviria), including a number of species in the genus orthobunyavirus (e value of × − ). orf had no significant similarity to any known proteins. hhpred produced confident predictions (≥ %) for a picornain-like protease and endopeptidase enzyme in orf a, a coronavirus-like rna-dependent rna polymerase (rprp), a metal-binding helicase and a - exoribonuclease (exon) in orf b. hhpred did not detect any zinc-binding domains (zbds); however, when orf b was run against the interpro database using interproscan , a coronavirus-specific zbd was identified. in orf , hhpred identified an enveloping glycoprotein associated primarily with viruses of the order bunyavirales. both hhpred and interproscan were unable to identify any other protein domains; therefore, predicted mrgv orfs were aligned against the nidovirus domains identified in saberi et al. [ ] . msa identified a further three protein motifs: a nidovirus rdrp-associated nucleotidyltransferase (niran) and s-adenosylmethionine (sam)-dependent n -and -o-methyltransferases (n-mt and o-mt, respectively). as nidovirales typically translate orf a and orf b consecutively to produce pp ab by − ribosomal frame shift [ ] , fsfinder [ ] was used to identify − frameshift sites in the overlap between orf a and orf b. both major elements of − frameshifting were identified in the overlap: a slippery site at position , nt with the sequence "gggtttt", proceeded by a stem-loop stimulatory structure located a few nucleotides downstream between positions , and , nt. analysis of the utr secondary structure following the final stop codon of the final orf revealed a thermodynamically stable rna hairpin secondary structure (figure ) . the predicted hairpin structure of mrgv is stabilised by three helices with a nt hairpin loop (∆g = − . ), in comparison to the gav -utr structure, which forms a nt hairpin loop stabilised by four helices (∆g = − . ), previously identified in wijegoonawardane et al. [ ] . a bayesian consensus tree was produced based on the rdrp protein domain universally conserved in nidoviruses, including the rdrp sequence generated in this study for mrgv, and the only nidovirus protein domain able to align to the rdrp of astrovirales (outgroup). all families within nidovirales were monophyletic in our analyses. mrgv branched as sister to gav and yhv within the roniviridae clade ( figure ). other invertebrate-infecting nidovirales shown in figure include charynivirus- infecting the crab charybdis, and paguronivirus- infecting hermit crabs [ ] , all branching within the euronivirdivae clade. all invertebrate-infecting nidoviruses branched together with maximal bayesian posterior probability. as mrgv branches within roniviridae, which have a distinct genome organisation to other nidoviruses [ ] , mrgv orf and orf were aligned against the corresponding orfs of gav. this alignment ( figure ) showed similarities in protein sequences of the three transmembrane helices of orf of gav to the predicted transmembrane helices of mrgv. sequence similarities were also seen in orf of gav to orf of mrgv ( . % residue similarity), as well as gp ( . % residue similarity) and gp ( . % residue similarity) protein coding regions of orf in comparison to the aligned regions in mrgv orf . the virus was not detected by specific pcr in cdna from any adult tissues from wild river populations across the two-year sampling period ( animals) and s. eriocheiris was the only pathogen detected in adult m. rosenbergii ( / ). however, mrgv was detected in larval cdna from all three hatcheries sampled in production cycles and all five hatcheries sampled in production cycles with no other pathogens detected by pcr in hatchery larvae. mrgv was also detected in brfi 'control' hatchery larvae prior to challenge, potentially explaining the low survival of treatment larvae. no histopathological signs of infection were seen in the larvae. we report and analyse the complete genome of a novel single-stranded positive-sense rna virus infecting cultured m. rosenbergii from hatcheries in southern bangladesh. the novel virus, macrobrachium rosenbergii golda virus, has a similar genome arrangement to viruses of the order nidovirales, and appears to be most closely phylogenetically related to yellow head virus and gill-associated virus, both infecting penaeid shrimp. mrgv was detected by specific pcr in larvae from three hatcheries in , two of which were experiencing mass mortality events when sampled, and one which was sampled just prior to a mass mortality event. in , two of the hatcheries that had experienced mass mortalities the previous year were re-sampled during mass mortality events and were again positive for mrgv; both of these hatcheries underwent two production cycles, both suffering mass mortality events and both were pcr positive for mrgv. in addition to the two hatcheries that were re-sampled in , a further three hatcheries, none of which had been sampled in , were sampled in during mass mortality events. these further three hatcheries were also pcr positive for mrgv. given the number of hatcheries affected by mass mortalities linked to mrgv, its temporal prevalence and spatial spread, with one of the hatcheries over km in distance to the nearest hatchery that was pcr positive for mrgv, we suggest that this novel virus represents a very significant threat to m. rosenbergii aquaculture within bangladesh, and may be a significant factor in the collapse of larval production in the industry of bangladesh since . all pcr screens of larvae collected from hatcheries experiencing mass mortalities for pathogens known to infect the larval stage of m. rosenbergii were negative: mrnv and xsv, the causative agents of white tail disease [ , ] ; mrtv, a virus associated with mass larval mortalities in china [ ] , spiroplasma eriocheiris [ ] , and wssv-shown to be able to infect m. rosenbergii experimentally [ ] . larvae were also screened for yhv and pmnv, viruses known to infect all life stages of marine shrimp species [ , ] ; these screens were all negative. furthermore, no sequences were assignable to any other known pathogens of m. rosenbergii in our metatranscriptomic data. the absence of these pathogens and no obvious bacterial cause observed by alam et al. [ ] strongly suggests that the survival problems in bangladesh hatcheries are not due to a currently known pathogen and that mass mortalities were linked either to hatchery practice factors or/and the emergence of a novel pathogenic agent. despite numerous problems identified in hatchery practices, hatcheries were successfully producing until , and since then, hatchery practices have not changed, suggesting that this is not the main source of mortality events. pcr screens of adult m. rosenbergii cdna from rivers used to collect berried females as the supply of broodstock for the hatcheries were also all negative for mrgv, suggesting that broodstock may not be the entry route of mrgv into the hatcheries. adults were also negative for all other pathogens as above, except for s. eriocheiris. all larvae sampled from hatcheries experiencing mortalities were negative for s. eriocheiris and no reads for any spiroplasma species were detected in metatranscriptomic analysis of the moribund larvae, suggesting that s. eriocheiris is also not causing the mortalities in hatcheries. nidoviruses (order nidovirales) are enveloped positive-sense rna viruses infecting a range of hosts including both vertebrates and invertebrates [ ] . the invertebrate nidoviruses are composed of families: mesoniviridae, roniviridae, mononiviridae and euroniviridae, with the latter two families discovered within the last four years [ , , ] . roniviridae comprises one genus, okavirus, composed of two closely related crustacean-infecting viruses: gav and yhv [ ] . both gav and yhv are associated with disease in marine shrimp farming, with the former initially associated with mid-crop mortality syndrome (mcms) in penaeus monodon in australia [ ] and the latter first associated with yellow head disease (yhd) in p. monodon in thailand [ ] . prior to this study, no nidoviruses had been discovered in macrobrachium rosenbergii. when amino acid sequences of mrgv were screened against the ncbi non-redundant protein database, there was weak similarity to yhv and even weaker similarity to other nidoviruses. however, the only significant matches in pp a and pp ab were to this order; therefore, we searched for protein motifs shared among other nidoviruses. through database searches we identified five protein motifs present in all known nidoviruses: a protease, an rna-dependent rna polymerase, a zinc-binding domain, a helicase and a methyltransferase-exoribonuclease complex. a further three domains were found by sequence alignment of mrgv to the protein motifs of niran and (sam)-dependent n -and -o-methyltransferases (n-mt and o-mt, respectively). we also identified nucleotide sequences within the overlap of orf a and orf b suggestive of a − ribosomal frameshift. the slippery sequence "gggtttt" proceeded downstream by a putative stem-loop stimulatory rna structure suggests that pp a and pp ab are translated continuously by programmed ribosomal frameshift, a characteristic of nidoviruses [ ] . the genome of okavirus, the genus to which gav and yhv belong, have a unique genome architecture to that of vertebrate nidoviruses-orf , encoding a nucleocapsid protein, is located upstream of the glycoprotein gene (orf ) [ ] . this structure is also seen in mrgv, as well as sequence similarity of orf and orf to the corresponding orfs in gav. a characteristic of positive-sense rna viruses is the presence of a secondary rna structure at the utr; this is present in other members of the nidoviruses including coronavirdae and arteriviradae [ ] . these utr structures and/or specific sequences have been shown to be critical to polymerase recognition and minus-strand genomic rna synthesis [ ] . a utr secondary structure has been identified in gav and yhv, and appears to be well conserved, with complementary base-changes implemented in yhv genotypes to retain a conserved stem-loop structure stabilised by four helices [ ] . in this study we used mfold to predict the secondary rna structure of mrgv in the utr of the genome following the final stop codon of the orfs. mfold predicted a stem loop structure of a similar size to that of gav and yhv, with three helices stabilising a nt hairpin loop. it is hypothesised that the utr rna structure in gav and yhv may act as a polymerase recognition site for minus-strand rna synthesis [ ] , and given the similarities in the utr rna structure in mrgv to gav and yhv, this structure in mrgv may have a similar function. based on multiple sequence alignment of the rdrp protein motifs of a representation of families within nidovirales, mrgv groups phylogenetically with family roniviridae, which, thus far, exclusively comprises gav and yhv. we hypothesise that mrgv also belongs to roniviridae; however, further sampling is needed to obtain additional material for electron microscopy to visualise the virus in situ in order to confirm that virion morphology conforms with the rod-shaped characteristics of the family [ ] . our study has also highlighted that choice of assembly tool is important in assembling viruses. rnaspades was not able to assemble the complete mrgv genome, even when libraries were pooled to give higher coverage than single libraries alone, whereas iva was able to assemble the full genome from the same pool of libraries. it is likely that iva was able to assemble the full genome as it is capable of assembling rna sequences at highly variable depths [ ] , a useful tool in the case of the nidoviruses, which are prone to variability in sequencing depth due to the production of sub-genomic mrnas (sgmrnas). the presence of sgmrnas were not assessed in this study but given that rnaspades could not assemble the full genome, it is likely that these sgmrnas do exist. future work includes identifying the presence or absence of sgmrnas and potential transcription regulation sites (trss). samples for histology or electron microscopy matching those used for molecular analyses were not available from the disease challenge studies carried out here. therefore, there is currently no information about histopathological signs of disease caused by mrgv. histological samples were taken from the larvae sampled from hatcheries in , however these animals were likely not to be moribund and were probably in the early stages of infection by mrgv and thus, though infected, no pathology was seen. furthermore, rna viruses can only be visualised indirectly in histology sections (normally associated with damaged host cells and nuclei). further sampling is ongoing in order to visualise the virus infecting larval tissue(s) using transmission electron microscopy. the challenge experiment data from this study were unable to determine the route of entry of mrgv into the hatchery system, as it was later determined that control animals were sourced from a hatchery assumed to be free of mrgv, but which after sampling larvae for the challenge experienced mass mortalities to a similar level as seen in other hatcheries in southern bangladesh. larvae were sampled from the apparently unaffected hatchery and fixed before challenging; these animals were screened by pcr for all pathogens as above and were only positive for mrgv, thus suggesting that animals were infected with mrgv prior to the challenge, explaining the high levels of mortality in 'control' animals. this confounding situation nonetheless further suggests the role and ubiquity of mrgv in mass mortality events across multiple hatcheries in bangladesh. future work to identify how the virus is entering the hatcheries is ongoing to suggest preventative methods that could be implemented to ensure biosecurity. the use of metagenomic techniques to identify both novel rna and dna viruses is becoming more common in aquaculture, with new viruses identified in economically-important species including fish, crustaceans and molluscs (reviewed in munang'andu et al. [ ] ). metatranscriptomics has recently been used to identify another novel rna virus in m. rosenbergii, crustacea hepe-like virus (chev ), associated with animals exhibiting growth retardation in china [ ] . the identification of mrgv is the first study, to our knowledge, to use a metatransciptomic approach to investigate the mass mortalities experienced in hatchery-reared prawn larvae in bangladesh. previous studies have investigated possible agents involved in mortality events using pcr screens and microbial culture, but required the agent to have gene sequences sufficiently similar to known pathogens to amplify with pcr primers or culturable on media, respectively, thus limiting the detection of more genetically divergent pathogens, possibly including many viruses. the metatranscriptomic approach used in this study to discover the agent involved in the bangladesh mortalities was able to identify and characterise a novel pathogen that would likely have not been identified by most 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on virus discovery and diagnostic role of viral metagenomics in aquatic organisms a novel hepe-like virus from farmed giant freshwater prawn macrobrachium rosenbergii this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - gyrmh authors: liu, shan-lu; saif, linda title: emerging viruses without borders: the wuhan coronavirus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gyrmh the recently emerged coronavirus in wuhan, china has claimed at least six lives as of january and infected hundreds if not thousands of individuals. the situation has drawn international attention, including from the virology community. we applaud the rapid release to the public of the genome sequence of the new virus by chinese virologists, but we also believe that increased transparency on disease reporting and data sharing with international colleagues are crucial for curbing the spread of this newly emerging virus to other parts of the world. the recently emerged coronavirus in wuhan, china has claimed at least six lives as of january and infected hundreds if not thousands of individuals. the situation has drawn international attention, including from the virology community. we applaud the rapid release to the public of the genome sequence of the new virus by chinese virologists, but we also believe that increased transparency on disease reporting and data sharing with international colleagues are crucial for curbing the spread of this newly emerging virus to other parts of the world. it is now clear that the mysterious respiratory illness in wuhan is caused by a new type of coronavirus distantly related to the sars coronavirus (sars-cov) [ , ] . we applaud the rapid release to the public of the genome sequence of the new virus by chinese virologists [ ] , as this represents an important first step in curbing the spread of the new virus to other parts of the world. viruses emerge and re-emerge globally without consideration for borders. in the recent past, we have witnessed outbreaks of sars, ebola, chikungunya, and zika [ ] . with each new outbreak, lives are lost, and the world is placed on high alert. lessons have been learned from the initial coverup and misidentification of the sars pathogen in [ , ] , and the recent slow response of the world health organization to the - ebola outbreak [ ] . from this perspective, it is understandable that chinese scientists cautiously released the identity and genome sequence of this new virus [ ] . however, transparency on disease reporting to the public and data sharing with international colleagues must continue. with an increasing number of new cases of infection by the new coronavirus reported in china and neighboring countries, such as japan and thailand [ ] , human-to-human transmission should be thoroughly investigated. the international virology community, which includes many chinese american virologists residing in north america, is concerned about this virus. it is imperative that animal reservoirs that transmitted the virus to humans be identified as quickly as possible to aid in control of this disease. it is thus urgent that the results of animal testing from the seafood market in wuhan, where the virus was initially isolated, be released as soon as possible. this is particularly crucial given the rapidly approaching lunar chinese new year, which will take place on january , during which tens of millions of people will travel, and large amounts of animal meats, some of which may contain this virus, will be consumed. it is noteworthy that recently the local health administration in wuhan issued guidance regarding the prevention and treatment of the pneumonia caused by this coronavirus [ ] . it is also encouraging that the world health organization has issued detailed guidance on the clinical management of the disease in relation to this outbreak [ ] . viruses spread irrespective of borders-they jump from animals to humans, and they move from one country to another. controlling the spread of emerging and re-emerging viruses requires international efforts and collaboration. we anticipate that scientific data and reagents will be shared publicly and fairly, and most importantly, that the scientific collaborations between the us and china, including the study of emerging viruses and infectious diseases, will continue unabated despite some turmoil in other aspects of the us-china relationship. new virus discovered by chinese scientists investigating pneumonia outbreak new-type coronavirus causes pneumonia in wuhan: expert. available online novel coronavirus genome evolution and emergence of pathogenic viruses: past, present, and future beijing doctor alleges sars cases cover-up in china china accused of sars cover-up global health security demands a strong international health regulations treaty and leadership from a highly resourced world health organization confirm new cases of chinese coronavirus clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected the authors wish to thank eric freed, thomas gallagher and stanley perlman for helpful discussion. we also thank colleagues of the chinese american virology community for many stimulating debates and discussions. key: cord- -wsmnlnlk authors: grädel, carole; terrazos miani, miguel a.; baumann, christian; barbani, maria teresa; neuenschwander, stefan; leib, stephen l.; suter-riniker, franziska; ramette, alban title: whole-genome sequencing of human enteroviruses from clinical samples by nanopore direct rna sequencing date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: wsmnlnlk enteroviruses are small rna viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. in routine diagnostic laboratories, enteroviruses are identified by pcr-based methods, often combined with partial sequencing for genotyping. in this proof-of-principle study, we assessed direct rna sequencing (drs) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. the approach was complemented by sequencing the corresponding viral cdna via illumina miseq sequencing. drs of total rna extracted from three different enterovirus-positive stool samples produced long rna fragments, covering between % and . % of the most similar reference genome sequences. the identification of the enterovirus sequences in the samples was confirmed by short-read cdna sequencing. sequence identity between drs and illumina miseq enterovirus consensus sequences ranged between % and %. here, we show that nanopore drs can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains. enteroviruses (evs) present a major burden for human health and health care systems worldwide, with large outbreaks consisting of hundreds of thousands of hospitalized cases occurring periodically [ ] . evs are associated with a wide variety of symptoms, ranging from mild respiratory diseases to severe neurological infections, leading potentially to death [ ] . these single-stranded rna viruses that belong to the picornaviridae family possess a relatively small genome size ranging from . to . kb. for routine diagnostics, ev presence is generally determined by real-time, reverse-transcription pcr (qrt-pcr) assays, complemented by partial sequencing of the vp -vp coding regions for genotyping [ , ] . due to the lack of proofreading mechanism in rna replication, ev genomes are highly variable and the likely subject of within-and between-genome recombinations [ , ] . therefore, when diagnostic tests solely rely on pcr using conserved primer sequences, false-negative results cannot be excluded. there is thus a need to generalize the assessment of ev diversity and evolution via a whole-genome approach, rather than from the limited information gained from sequencing single genomic regions. the main limitation towards a general adoption of whole-genome sequencing (wgs) for better ev characterization and better understanding of their evolution and epidemiology is mostly of a technological nature: wgs approaches are more expensive, technically demanding and hence time consuming than standard molecular assays. as such, they are generally not routinely used in diagnostic laboratories. in the case of rna viruses, the challenge is even more exacerbated because various molecular steps (rna purification, extraction, cdna synthesis, and optionally amplification) add to the turnaround time and final cost of the assays. clinical wgs applications are mostly based on sanger sequencing and on second-generation sequencing platforms, with illumina miseq/hiseq (illumina, san diego, ca, usa) and ion torrent machines leading the market, as benchtop sequencing technologies enable high-throughput sequencing at an affordable price per sample when samples are multiplexed [ ] . third-generation sequencers, i.e., smrt technology (pacific biosciences, menlo park, ca, usa) and nanopore sequencing (oxford nanopore technologies (ont), oxford, uk), have recently emerged as complement to or replacement of second-generation sequencers, by enabling the sequencing of single, long dna molecules, a feature that is particularly attractive in the context of sequencing full-length viral genomes [ ] . nanopore sequencing technology has been successfully applied to genome sequencing of rna viruses. based on viruses propagated on cell lines, viral transcriptomes have been examined by nanopore sequencing, for instance for porcine circovirus [ ] , herpes simplex virus [ ] , hepatitis c virus [ ] , cultured influenza virus a, and human cytomegalovirus (hcmv) [ ] . additionally, nanopore sequencing has been used successfully to sequence whole ev genomes from viral cdna extracted from cell cultures: in their proof-of-concept study [ ] , the authors demonstrated that nanopore sequencing could be applied for rapid routine whole-genome sequencing of ev with sufficient accuracy compared to sanger sequencing. metagenomic nanopore sequencing of influenza virus was performed directly from randomly amplified viral cdna obtained from clinical respiratory samples [ ] . furthermore, ont has presented a new, potentially revolutionary nanopore sequencing application, which allows sequencing of rna molecules directly, i.e., without the pre-requirement of cdna synthesis or pcr amplification [ ] . this method, direct rna sequencing (drs), yields full-length, strand-specific rna sequences and enables the direct detection of nucleotide modifications in native rna molecules. drs has been used in several studies, including human poly(a) transcriptome [ ] and dna virus transcriptome, such as hsv herpes simplex virus type (hsv- ) during productive infection of primary cell [ ] . drs was also used for sequencing rna genomes of, e.g., pseudorabies virus propagated on immortalized porcine kidney epithelial cell line [ ] , influenza a virus (h n ) from infected chicken eggs [ ] , human coronaviruses viral rnas produced in cell cultures [ ] , and many other examples of complete sequencing of multiple, single-stranded rna (ssrna) viruses obtained with or without poly(a)-tailing of rna viruses obtained from cell cultures [ ] . here, in a proof-of-concept study, we apply nanopore drs to ev-positive stool samples and show that whole rna genomes of enteroviruses can be retrieved with enough genomic information for the characterization of the infectious agents. we further analyze the metatranscriptomic data provided by the drs approach and compare it with that obtained by illumina miseq sequencing of the same samples. in this study, we used three independent patient stool samples (e , e , e ), which were sent for enterovirus diagnostics to the clinical diagnostic laboratory of the institute for infection diseases, bern, switzerland. the samples were collected in (e , e ) and (e ) and had a cycle threshold (ct) value of . (e ), . (e ) and . (e ) via real-time pcr using enterovirus-specific primers (see description below). ethics approval was granted on march by the swiss ethics committee on research involving humans to conduct sequencing of enteroviruses in clinical samples stored in the ifik biobank (basec-nr: req- - ). after homogenization with a sterile pipette, approximately . g of stool sample was added to ml of transport medium [ ] containing - glass beads (diameter mm; merck ag, zug, switzerland). after s of vortexing, the suspension was centrifuged for min at g and the resulting supernatant was filtered through a pore size of . µm with % penicillin/streptomycin (biochrom, berlin, germany). this preparation was further extracted on the easymag platform (biomérieux, geneva, switzerland) for real-time pcr and with trizol ls reagent (thermo fisher scientific, reinach, switzerland) for illumina miseq sequencing ( figure ). after homogenization with a sterile pipette, approximately . g of stool sample was added to ml of transport medium [ ] containing - glass beads (diameter mm; merck ag, zug, switzerland). after s of vortexing, the suspension was centrifuged for min at g and the resulting supernatant was filtered through a pore size of . µm with % penicillin/streptomycin (biochrom, berlin, germany). this preparation was further extracted on the easymag platform (biomérieux, geneva, switzerland) for real-time pcr and with trizol ls reagent (thermo fisher scientific, reinach, switzerland) for illumina miseq sequencing ( figure ). figure . analytical workflow. the workflow used for diagnostic assays is indicated by greyed boxes and that followed for ngs techniques by white boxes. a total of µl of trizol ls reagent (thermo fisher scientific) was added per µl of stool suspension in pbs. following homogenization by pipetting, the sample was transferred to phasemaker tubes (thermo fisher scientific). after incubation for min, µl of chloroform was added, and the tubes were shaken vigorously by hand for s. after min incubation, the sample was centrifuged for min at , g, at °c. the aqueous phase was transferred to a new tube and µg of carrier rnase-free glycogen (thermo fisher scientific) was added, followed by µl isopropanol. after incubation for min, the sample was centrifuged for min at , g, at °c, and the supernatant was discarded. the total rna precipitate was resuspended in ml of % ethanol. the sample was vortexed briefly, and then centrifuged for min at × g, at °c. the supernatant was discarded and the rna pellet was air-dried for min, before being resuspended in µl of rna storage solution (thermo fisher scientific). after incubation at °c for min, the rna sample was either used directly in downstream applications, or stored at − °c. alternatively, total nucleic acid extraction was performed with nuclisens easymag from µl of the routine diagnostic stool preparation, to which . µl carrier rna (qiagen ag, hombrechtikon, switzerland) was added and eluted in µl. this extract was used for real-time pcr and sanger genotyping. we used the who-recommended protocol for pre-treating stool samples for enterovirus rna isolation (enterovirus surveillance guidelines, [ ] ) adapted from nix et al. [ ] as follows: a low amount ( . to g) of stool sample was added to pbs (thermo fisher scientific) up to ml volume, to which . g of glass beads ( mm diameter, merck) and . ml of chloroform (applichem, aesch, switzerland) were added. the mixture was shaken vigorously using a tissuelyser (qiagen ag) for min at maximum speed. the suspension was centrifuged at × g for min at °c, and approximately ml of the supernatant was transferred to a new . ml tube and continued with rna extraction. for drs of sample e , rna was extracted using easymag, while for e and e , the trizol ls method was used (see supplementary material table s ). a total of µl of trizol ls reagent (thermo fisher scientific) was added per µl of stool suspension in pbs. following homogenization by pipetting, the sample was transferred to phasemaker tubes (thermo fisher scientific). after incubation for min, µl of chloroform was added, and the tubes were shaken vigorously by hand for s. after min incubation, the sample was centrifuged for min at , × g, at • c. the aqueous phase was transferred to a new tube and µg of carrier rnase-free glycogen (thermo fisher scientific) was added, followed by µl isopropanol. after incubation for min, the sample was centrifuged for min at , × g, at • c, and the supernatant was discarded. the total rna precipitate was resuspended in ml of % ethanol. the sample was vortexed briefly, and then centrifuged for min at × g, at • c. the supernatant was discarded and the rna pellet was air-dried for min, before being resuspended in µl of rna storage solution (thermo fisher scientific). after incubation at • c for min, the rna sample was either used directly in downstream applications, or stored at − • c. alternatively, total nucleic acid extraction was performed with nuclisens easymag from µl of the routine diagnostic stool preparation, to which . µl carrier rna (qiagen ag, hombrechtikon, switzerland) was added and eluted in µl. this extract was used for real-time pcr and sanger genotyping. we used the who-recommended protocol for pre-treating stool samples for enterovirus rna isolation (enterovirus surveillance guidelines, [ ] ) adapted from nix et al. [ ] as follows: a low amount ( . to g) of stool sample was added to pbs (thermo fisher scientific) up to ml volume, to which . g of glass beads ( mm diameter, merck) and . ml of chloroform (applichem, aesch, switzerland) were added. the mixture was shaken vigorously using a tissuelyser (qiagen ag) for min at maximum speed. the suspension was centrifuged at × g for min at • c, and approximately ml of the supernatant was transferred to a new . ml tube and continued with rna extraction. for drs of sample e , rna was extracted using easymag, while for e and e , the trizol ls method was used (see supplementary material table s ). one-step rt-pcr was performed with the agpath-id one-step kit (ambion, reinach, switzerland) using published primers and probes [ ] , following the protocol described previously [ ] . primers and probes were synthesized at microsynth ag, balgach, switzerland. genotyping of the samples was performed by vp amplicon sanger sequencing as described previously [ , ] and carried out at microsynth. for nanopore sequencing, we followed the manufacturer's instructions of the protocol for kit sqk-rna (version drs_ _v _revn_ dec ), but substituted superscript iii with superscript iv (thermo fisher scientific). the input rna and amounts loaded on flow cells for each experiment are specified in table s . quantification was performed using a qubit rna hs assay kit (rna) or dna hs assay (cdna) kit on a qubit fluorometer . (thermo fisher scientific). the reverse-transcribed and adapted rna was loaded onto r . . flowcells and sequenced on minion sequencer. each drs run was conducted with a new, previously unused flowcell. the following three specific primers for coxsackievirus a were designed for this project to hybridize to coxsackievirus a sequence kj : r_a : -cccgtttctgccgctt- adapted from primer by oberste et al. [ ] ; r_a : -atatctctgaatttctcatt- adapted from primer hev. c.d by bessaud et al. [ ] ; ev- utr _a _rc: -catattcacgaccagattcctggtg- (this study). all primers were synthesized at microsynth. first-strand synthesis was performed with the superscript iv first-strand synthesis system as follows: for reverse transcription, a reaction mixture was prepared containing . µl of the specific primers ev- utr _a _rc, r_a , r_a ( µm), or µl of random hexamers ( µm) or µl of oligo(dt) ( µm) together with µl of mm dntp mix and filled with rna extract and depc-treated water to a total volume of µl. after heating at • c for min and snap cooling, a mixture of µl ssiv buffer, µl mm dtt, µl ribonuclease inhibitor and µl superscript iv reverse transcriptase ( u/µl) were added. the samples were incubated for min at • c and min at • c (preceded by min at • c when containing random hexamer primers). subsequently, µl of e. coli rnase h ( u/µl) was added and incubated at • c for min. to this mixture, µl of second-strand synthesis reaction buffer nebnext (new england biolabs (neb), ipswich, ma, usa), µl nebnext second-strand synthesis enzyme mix and µl of nuclease-free water were added. the reaction mixture was incubated at • c for min. clean up with µl of agencourt ampure xp magnetic beads (beckman coulter, nyon, switzerland) was performed according to the manufacturer's instructions, with an elution volume of µl for samples e and e and µl for sample e . for the first sample e , an additional end-prep step was performed, but that step was later removed in the protocol as it was deemed unnecessary: to µl of the elution, µl of ultra ii end-prep buffer (neb), µl of ultra ii end-prep enzyme mix (neb), and µl of nuclease-free water were added and the mixture was incubated for min at • c and min at • c. after an additional purification step using µl of agencourt ampure xp magnetic beads, the cdna was eluted in µl of nuclease-free water. libraries were prepared from unamplified cdna using nextera xt dna library prep kit (e ) and nextera dna flex library prep kit (e , e ) (illumina), and sequenced using an illumina miseq benchtop sequencer generating × bp paired-end reads (v ), according to the manufacturer's protocols. sequencing was performed at the next-generation sequencing platform of the inselspital, bern, switzerland. raw fast files produced by minion sequencing were basecalled under the high accuracy mode using the ont basecaller guppy version . . with the parameter: "guppy_basecaller --input_path path --recursive --save_path path --qscore_filtering --min_qscore --flowcell flo-min --kit sqk-rna --cpu_threads_per_caller -num_callers ". statistics for nanopore sequencing output are summarized in table s . we did not perform any adapter trimming due to the inaccuracy of the rna basecaller when calling dna adapter sequences, which results in unreliable identification [ ] , but do not expect it to impair our downstream analysis, as basecalled nanopore reads were classified using blastn against the ncbi's nucleotide (nt) database (downloaded . . ), using blast version . . . blast results were classified using megan (v. . . , [ ] ), with the blast rma parameters "c false --m --ms --me . --mpi --top --supp . --sup --alg naive --mrc --mrefc --ram readcount --a t nucl_acc tax-jul .abin". rna sequences were mapped to the best-scoring reference genome (whole-genome reference with highest bit-score in blastn output) using minimap (version . -r -dirty, [ ] ). for illumina miseq data (table s ) , adapter trimming was performed with bbduk.sh from the bbmap package (v . , [ ] ), and human and rrna reads were removed by mapping reads against the corresponding databases (grch genome, cdna and ncrna, and silva lsu ssu https://www.arb-silva.de/documentation/release- /) using minimap [ ] . non-human, non-rrna reads were then assembled using spades (parameters: -k --rna --only-assembler; version . . ; [ ] ). the resulting contigs were subsequently analyzed using blastn and megan as described for nanopore data. sequence identity between illumina miseq and nanopore drs enterovirus genome consensus sequences was calculated using legacy blast . . . coverage information was generated using bbmap and plots created using r statistical computing environment (version . . ). after removal of any human reads, all illumina miseq sequencing data (fastq), raw and basecalled ont data (fast and fastq, respectively), and sanger sequences were deposited in the european nucleotide archive (ena) under the project reference prjeb . drs was performed with enterovirus-positive stool samples with similar viral load (ct values between and ) in three independent experiments, consisting of samples from three different patients. the samples were prepared using the who-recommended protocol for viral enrichment using chloroform/bead treatment, followed by rna extraction using trizol or easymag (table s ) . we sequenced the total polyadenylated rna using drs on a minion nanopore sequencer. the runs were continued until there was only negligible sequencing output or until the maximum recommended duration of the run was reached. for validation purposes, all three samples were also subjected to cdna sequencing using illumina miseq from samples prepared by the routine diagnostic procedure (figure ). the cdna was produced with either genotype specific primers (e ), or with both oligo-dt primers and random hexamers (e , e ). drs reads from extracted rna were obtained up to h after the beginning of the sequencing run, and sequencing was stopped after h. out of a total output of , raw nanopore reads, reads were successfully basecalled into rna sequences, with an average length of bases (range - bases). more than % of basecalled rna reads ( reads) were taxonomically classified using blastn against the ncbi's nucleotide (nt) database. the large majority (> %) of those hits matched eukaryotic sequences, and only . % bacterial ( reads) and . % viral sequences ( reads) ( table ). the majority of eukaryotic reads were assigned to the yeast species saccharomyces cerevisiae ( reads; %). all other eukaryotic species had less than five reads assigned. within the reads assigned to bacteria, the only species with notable number of reads were escherichia coli ( reads), faecalibacterium prausnitzii ( reads) or bacteroides vulgatus ( reads), all of which are known to be commensal species in the human gut. as far as viral sequences were concerned, all ( . % of ) reads matched the enterovirus genus, species ev-a ( table ). they were on average bases long (range - bases). alignment of the rna sequences to the best-scoring reference genome (coxsackievirus a kj ; figure ) demonstrated that the long reads covered almost the entire genome of coxsackievirus a , with one single read covering alone . % of the reference genome (with only bases missing at the end). the top two longest rna sequences ( and bases long) were obtained within the first hour of sequencing, and most of the larger sequences (> kb) were obtained within the first h of sequencing. after h of sequencing, sequences matching enterovirus were all < bases long. classical amplicon sequencing of the vp region was attempted for sample e , but it did not yield good results. in order to confirm genotype identification obtained via drs and to obtain a highly accurate whole-genome enterovirus sequence, we also subjected sample e to cdna sequencing using illumina miseq. miseq sequencing library preparation was performed with stool material and following the routine diagnostic pre-treatment (as opposed to chloroform/bead treatment) given that low amount of patient stool sample was available (figure ). for this sample, an enterovirus-targeted approach was chosen to produce the cdna by using genotype-specific primers given that low number of reads was obtained by drs. after removal of reads mapping to human genome and bacterial rrna, metatranscriptomic reads were assembled into contigs and further subjected to blastn similarity analysis against sequences in the nt database, and the top hits were taxonomically summarized using megan. most contigs of sample e were classified as bacteria ( ) or viruses ( ) ( table ). such differences in species composition when compared to the results of the drs run may be explained by the genotype-specific approach used for cdna synthesis and different pre-treatment of the sample. based on the promising results obtained for sample e , the drs transcriptomic approach was repeated on two other clinical samples: for the drs of rna extracted from sample e , the total yield of the sequencing run was , reads ( . mb) after h of sequencing. a total of , reads passed basecalling, with an average length of . bases (range of - , ). only % ( , ) of the basecalled sequences were taxonomically classified using blastn. interestingly, there was a drastic difference in read composition as compared to the first sample (e ), and the majority of reads classified as of bacterial origin ( . %, , ), and only very few reads matched to eukaryotic ( , . %) or archaeal ( , . %) species. for bacteria, drs reads were assigned to a large variety of species. the most prominent phyla were bacteroidetes ( , reads) and proteobacteria ( reads). ) . for e , the coverage plot was produced by using the assembled contig sequence of the illumina sequencing run (oligo-dt primers) as reads did not map well to any reference ev genome sequence. for samples e and e , illumina miseq reads based on cdna produced with oligo-dt and random hexamers are indicated by light and dark grey lines, respectively. all viral sequences matched again uniquely to ev sequences. however, megan analyses revealed the presence of two enterovirus species consisting of ev-a ( contigs; contigs lengths ranging from to bases) and ev-b ( contigs; contigs lengths ranging from to bases) in the cdna sample, of which the majority of contigs were assigned to coxsackievirus a and echovirus ( table ). mapping of the viral sequences to a vp database confirmed the presence of these two genotypes and the identification was verified by using the online rivm bioinformatic platform [ ] . alignment of the reads to the closest reference genome sequences revealed that the coverage of cv-a was % with an average depth of . (± . ), while for echovirus , only % of the reference genome was covered (figure ) . the consensus sequence for cv-a obtained by illumina miseq was . % identical ( / ), with % gaps ( / ), to the consensus sequence obtained by drs sequencing. based on the promising results obtained for sample e , the drs transcriptomic approach was repeated on two other clinical samples: for the drs of rna extracted from sample e , the total yield of the sequencing run was , reads ( . mb) after h of sequencing. a total of , reads passed basecalling, with an average length of . bases (range of - ). only % ( , ) of the basecalled sequences were taxonomically classified using blastn. interestingly, there was a drastic difference in read composition as compared to the first sample (e ), and the majority of reads classified as of bacterial origin ( . %, , ), and only very few reads matched to eukaryotic ( , . %) or archaeal ( , . %) species. for bacteria, drs reads were assigned to a large variety of species. the most prominent phyla were bacteroidetes ( reads) and proteobacteria ( reads). a total of viral reads, corresponding to . % of classified reads, were found. as seen for the previous sample, only matches to the enterovirus genus were found. although many more viral reads were found in comparison to sample e , they were, with an average of bases, much shorter, and with a range of - . no reads spanned the complete genome sequence of any known ev species or genotype, and altogether could cover % of the reference genome sequence (figure ). short fragment lengths might result from increased fragmentation of the viral rna during extraction or library preparation. due to the directional sequencing used in the drs process of nanopore sequencing, higher coverage at the ' end of the sequence may be obtained, and fragments without poly-a tail cannot be sequenced. therefore fragmented, incomplete rna molecules may hinder the recovery of the end of the rna genome. nevertheless, although the covered region did not span the vp region, we were able to identify the enterovirus as echovirus by genomic similarity to the best-scoring reference genome sequence. this species identification was independently confirmed by a standard genotyping approach that uses sanger-based sequencing of the partial vp amplicon. the cdna from sample e was also sequenced by illumina miseq, although in this case with non-specific primers for cdna synthesis for a more unbiased approach towards ev detection and not to miss possible ev co-infections. both oligo-dt and random hexamer primers were used to ensure a better chance of obtaining the full genome, with the idea that oligo-dt may lead to results more comparable to those of drs due to polyadenylation requirements, and random hexamers may offer better chance of obtaining a well-covered ' end of the enterovirus genome. the distribution of contigs assigned on the domain level did not vary much between random hexamer and oligo-dt produced cdna: most contigs were assigned to bacteria ( . % or . %, respectively), with only a small minority mapping to eukaryotes ( . %, . %) and viruses ( . %, . %). within bacteria, most contigs were assigned to the phyla proteobacteria ( , , , ), followed by bacteriodetes ( , ) and actinobacteria ( , ). taking a closer look at all the detected viral genotypes (table ) , we found that, besides echovirus , there were also contigs mapping to other virus genotypes, such as rhinovirus a, and a variety of other bacterial or plant viruses, albeit only with few short contigs. the echovirus genome, however, was well covered with either approach (figure ) . comparison of the drs nanopore sequencing consensus to the one obtained from illumina miseq showed . % identity ( / ), with . % gaps ( / ). the drs run of the rna extract from sample e was stopped after the maximal run duration of h. notably, this run had much higher output compared to the other two samples, resulting in a total of . m sequenced reads ( . gb), from which . m reads passed basecalling. the average length was also considerably higher than in previous runs with bases (range - bases). although the rna input material used for library preparation was measured to be of a similar amount for all three samples ( ng), cdna measurement ( ng) before loading on the flow cell indicated that the initial rna concentration might not have been the same. overall, % of basecalled reads ( . m reads) were taxonomically classified using blastn. of those, . % ( , , ) mapped to eukaryotic sequences. as seen for sample e , the vast majority of eukaryotic reads mapped to saccharomyces cerevisiae ( . %), while . % ( , ) were classified as of bacterial origin, and only a negligible amount as archaea ( , . %). viral sequences comprised . % ( ) of the reads, the majority of which were classified as plant viral pathogens (table ) , mostly belonging to the tomato mosaic virus ( reads, % of viral reads). other, more rarely detected species, included melon necrotic spot virus ( ) , and cactus virus x ( ) . in total, reads mapped to enteroviruses. although sequences were mapped to several genotypes, mapping the reads to the best-scoring reference genome and to a vp database identified genotype echovirus as the most likely and only ev genotype present in the sample. although there were relatively few hits to enteroviruses considering the high total output of the run, long sequences (average . , range - bases) were observed, which covered up to . % of the reference genome sequence (figure ). illumina sequencing of the cdna from sample e produced using oligo-dt primers and random hexamers showed similar distributions on the domain level: the majority of contigs belonged to bacterial species ( . % and . %, respectively, for oligo-dt and random hexamer approaches), with a minority of reads mapping to eukaryotes ( . %, . %) and viruses ( . %, . %). this major shift from yeast to bacteria as compared to the drs approach, which was also observed for sample e , could be explained by the different pre-treatment using filtration with a pore size of . µm. additionally, comparison of number of contigs and nanopore reads are of limited value when not considering coverage depth. for e , the genome of echovirus genotype, whose presence was also confirmed by sanger sequencing of the vp gene, was well covered with both types of cdna synthesis ( figure ). some contigs also mapped to another genotype, echovirus , but these were only short contigs, which may have been wrongly taxonomically assigned due to the short sizes of the illumina reads. otherwise, we also found a variety of viral sequences, with the most prominent being the tomato mosaic viruses and other plant pathogens, reflecting the results of the drs run. the consensus sequence identity for echovirus sequenced by drs to illumina miseq was calculated as . % ( / ), with % gaps ( / ). using rna extracted from clinical samples, we were able to repeatedly identify human enteroviruses in stool samples from three independent patients by nanopore-based drs in this proof-of-concept study. beyond identifying ev species and genotypes correctly, we showed that the approach may also provide rich metatranscriptomic information on sample composition for all life domains. clear differences in overall species compositions, with either yeast or bacteria dominating the majority of obtained reads, were observed between samples. viral reads constituted between . % and . % of total passed reads. by complementing drs data with illumina miseq sequencing data for the same samples, we were able to validate the obtained enterovirus sequences and added further metatranscriptomic information. illumina miseq sequencing revealed a higher diversity in viral species in these samples (table ) , which was not captured by the drs approach. in all samples, % to . % of the ev reference genome sequences were covered. the average identity of the drs consensus was - % compared to the illumina miseq consensus sequence. other studies using drs have achieved similar values with - % identity when sequencing viruses from cell culture [ , ] , or up to % consensus identity for influenza a [ ] . for applications with samples with low target concentration such as patient samples, getting enough sequencing depth to obtain accurate consensus sequences constitutes a challenge for the method. the resulting consensus sequences might therefore be of limited use for applications which require high accuracy, such as phylogenomic analysis. however, for the purpose of genotype identification, the approach is sufficiently accurate, especially as it can provide long reads, which can facilitate mapping to the correct reference, and expedite downstream bioinformatic analyses. furthermore, different ev genotypes have < % nucleotide identity in the vp region [ , ] , which makes vp -based genotype identification possible even with the current high error rate of drs (modal read accuracy of > % [ ] ) when considering read length above few hundred bases. our wet laboratory approaches were refined during the course of the study while analyzing further samples, and the experiments had to be adapted to limited patient material availability. the good overall agreement in ev detection between the two ngs approaches suggests that the variability observed in number of reads and composition between samples may be attributable to the natural biological variability that may exist between the sampled patients. yet, finer differences in composition between samples may be explained by the sequencing technology and by the different pre-treatments of the samples: on the one hand, smaller cdna molecules produced by the wet laboratory procedure (cdna synthesis, bead cleaning), followed by short-read sequencing, may be easier to detect, than larger, intact rna molecules via drs. on the other hand, unreliable mapping and poor taxonomical identification may be produced by short reads aligning to genomic regions that are conserved among genotypes, but also by long, error-prone nanopore reads [ ] aligning incorrectly to reference sequences. therefore, further large-scale comparisons of the two ngs approaches would be needed to confirm or reject the hypothesis about the effect of read length on organism detectability in metagenomic or metatranscriptomic studies. while enteroviruses were detected in all tested samples using drs, it should be noted that these were all samples with relatively high viral load, i.e., low ct values. with a range of only - total reads mapping to enteroviruses for the three clinical samples, the approach was close to miss ev detection in those samples. in the case of sample e , this sensitivity issue could explain why co-infection by another enterovirus was not detected by drs, but only by the short-read sequencing-based approach. in a recent study on the use of drs for identification of porcine reproductive and respiratory syndrome virus, viruses could be detected in spike-in samples with . × viral copies and in clinical samples with . × viral copies [ ] . another study has previously reported low sensitivity of nanopore sequencing in a viral metagenomic approach in patient samples with low viral titers, even when sequencing viral genomes via sispa-based amplification [ ] . an improvement in sensitivity would be necessary before attempting to sequence samples with low viral load in general. a limiting factor of the drs approach was the amount of polyadenylated rna used for library preparation, as in two of the sequencing runs, the full flowcell sequencing capacity was not used and the sequencing was stopped before maximal run duration, because low amounts of rna library were loaded on the flowcell. if a sufficient amount of patient samples is available to obtain enough rna, increasing the input concentration to fulfill the recommended input of at least ng polyadenylated (as opposed to total) rna might have provided better coverage of the target genome sequences. furthermore, drs sequencing is expensive if only one sample is loaded per flowcell. recent progress with barcoding for drs [ ] might, however, reduce this limitation in the future. additionally, results might be improved with adaptations in viral enrichment steps. we used a chloroform/bead pre-treatment in drs samples for enrichment of viral capsid and although it might be efficient in enriching viral reads, it could also reduce the overall amount of rna extracted. as the maximum sequencing yield of the flowcell was not always reached, perhaps milder enrichment methods might be preferable, especially if the broader diversity of pathogens is to be studied. currently, the drs method is restricted to sequencing of polyadenylated rna. however, performing polyadenylation of the total rna fraction could expand its applications. another characteristic of the drs method is the expected sequencing bias towards the ' end of the polyadenylated molecules [ ] . hence, coverage of the ' region of the ev genomes was generally low, which may be problematic given that the vp region is the main region used for ev genotyping (e.g., located at position - in echovirus kt . ). thus, correct genotyping would require around bases long rna reads from the ' towards the ' end of the genome sequence. we encountered lesser coverage of the ' region of the genome with sample e , although in that instance, we were still able to identify the same genotype as found by the gold-standard vp pcr-based approach. however, unambiguous identification relies on the capsid region and its presence is crucial in case of recombinations or co-infections. therefore, great care must be applied during extraction and library preparation to avoid shearing or degrading extracted rna. the known high variability of ev genome sequences makes it necessary to have suitable alternatives for genotype identification available if pcr-based approaches fail. in such cases, drs of patient samples could be a valuable alternative, as the sequencing is primer independent and the sequences of long, native rna molecules are recovered. additionally, as is the case with all nanopore sequencing, the data is available in real time, which can provide faster identification and a sequencing-on-demand approach to molecular assays. overall, our proof-of-concept study demonstrated the possibilities offered by the drs technique for genotype identification of human enteroviruses directly from clinical samples. the method requires further optimization to improve the overall sensitivity and to lower costs for possible applications in routine diagnostics. however, with the rapid advancement of the technologies, those issues will likely improve in the near future. supplementary materials: the following are available online at http://www.mdpi.com/ - 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- journal: viruses doi: . /v sha: doc_id: cord_uid: vdeffy l middle east respiratory syndrome coronavirus (mers-cov) is a highly pathogenic virus with a crude mortality rate of ~ %. previously, we established a human dpp transgenic (hdpp -tg) mouse model in which we studied complement overactivation-induced immunopathogenesis. here, to better understand the pathogenesis of mers-cov, we studied the role of pyroptosis in thp- cells and hdpp tg mice with mers-cov infection. we found that mers-cov infection induced pyroptosis and over-activation of complement in human macrophages. the hdpp -tg mice infected with mers-cov overexpressed caspase- in the spleen and showed high il- β levels in serum, suggesting that pyroptosis occurred after infection. however, when the c a-c ar axis was blocked by an anti-c ar antibody (ab), expression of caspase- and il- β fell. these data indicate that mers-cov infection induces overactivation of complement, which may contribute to pyroptosis and inflammation. pyroptosis and inflammation were suppressed by inhibiting c ar . these results will further our understanding of the pathogenesis of mers-cov infection. middle east respiratory syndrome coronavirus (mers-cov), the second highly pathogenic coronavirus to emerge after severe acute respiratory syndrome coronavirus (sars-cov), causes severe acute respiratory failure and extra-pulmonary multi-organ damage accompanied by severe systemic inflammation [ ] [ ] [ ] . however, the pathogenesis of mers-cov still needs to be explored. complement activation and pyroptosis are two proteolytic cascades that defend the host against dangerous pathogens. they are important parts of the innate immune system and have some similar characteristics, including pore-formation and proinflammatory characteristics. pyroptosis is a lytic and inflammatory mode of regulated cell death catalyzed by the caspase family [ ] . activation of caspase- relies on assembly of inflammasome complexes, which contain nlrp b, nlrc , nlrp , and aim . different inflammasomes are activated by different pathogen-associated molecular patterns (pamps) or danger-associated molecular patterns (damps) via particular pattern recognition receptors (prrs). the best-characterized inflammasome is the nlrp inflammasome, which responds to a variety of bacterial, viral, and fungal agents [ , ] , damps (e.g., atp, monosodium urate crystals, and amyloid-β aggregates) [ , ] , and even environmental and industrial particles such as silica and asbestos [ ] . the nlrp inflammasome comprises the nlrp scaffold, the asc (pycard) adaptor, and pro-caspase- . the activated nlrp inflammasome promotes transformation of pro-caspase- to its active form, which proteolytically cleaves gasdermin d, pro-il- β, and pro-il- to yield their bioactive forms. the n-terminal domain of cleaved gasdermin d perforates the cell membrane, resulting in osmotic lysis [ ] , whereas mature il- β and il- act as proinflammatory cytokines [ ] . the complement system is an ancient molecular cascade; indeed, homologs have been found in sea urchin [ ] and mosquitoes [ ] . complement is activated via three pathways: the classical, lectin, and alternative pathways. during the process of activation, an enzyme named c convertase cleaves c to c a and c b, which are recruited to the c convertase to form the c convertase. c convertase catalyzes cleavage of c to c a and c b to initiate the terminal complement pathway, resulting in formation of the membrane attack complex, which has pore-forming properties. during this process, two split products, c a and c a (known as anaphylatoxins), promote inflammation or serve as chemoattractants by engaging their cognate receptors [ , ] . in a previous study we demonstrated that aberrant complement activation contributes to severe outcomes in hdpp transgenic mice infected with mers-cov, and that preventing over-activation of the complement system may be an effective clinical therapy for mers [ ] . here, we examined the role of pyroptosis in the pathogenesis of mers, along with the relationship between pyroptosis and complement. the results may help us to better understand the mechanism underlying severe outcomes after mers-cov infection. all animal experiments were approved by the institutional animal care and use committee (iacuc) of the beijing institute of microbiology and epidemiology (iacuc permit no: bime - ; permit date: march ). animal studies were carried out in strict accordance with the recommendations set out in the guide for the care and use of laboratory animals. human monocytic cells (thp- ) were purchased from the american type culture collection (manassas, va, usa, atcc number: tib- ) and cultured in rpmi medium supplemented with % heat-inactivated fetal bovine serum (fbs), u/ml penicillin, µg/ml streptomycin sulfate, × glutamax-i (l-glutamine alternate), and . mm -mercaptoethanol. thp- (differentiated) macrophages were obtained by exposing the cells to nm phorbol- -myristate- -acetate (pma) for h, followed by culture for a further h in complete growth medium without pma. mers-cov (hcov-emc/ strain) was propagated and titrated on vero cells in an approved biosafety level laboratory. thp- differentiated macrophages cultured in -cm flasks were infected with mers-cov at a multiplicity of infection of . . the virus was adsorbed at • c for h and unbound virus was washed away. hdpp -tg mice ( weeks old, female) [ ] were maintained in a pathogen-free facility and housed in cages containing sterilized feed and drinking water. following intraperitoneal anesthetization with sodium pentobarbital ( mg/kg body weight), mice were inoculated intranasally with mers-cov ( . % tissue culture infectious dose (tcid )) in µl dulbecco's modified eagle's medium (dmem). mice in the sham group received the same volume of dmem. for the experiments of c ar inhibition, mice were received an intravenous (i.v.) injection ( µg/kg) of a monoclonal ab (mab) specific for mouse c ar (hycult biotech, uden, the netherlands) to block the interaction of c a to c ar or an injection of phosphate-buffered saline (pbs) (sham treatment control) at the same time as mers-cov inoculation. all infectious experiments related to mers-cov were performed in an approved biosafety level facility. thp- differentiated macrophages were lysed in trizol™ reagent (life technologies, carlsbad, ca, usa) at h post-infection with mers-cov. total rna and proteins were isolated according to the reagent user guide. mice were euthanized by overdose inhalation of carbon dioxide at different time points after infection with mers-cov. lungs were harvested and total rna was extracted and purified using an rneasy extraction kit (qiagen, hilden, germany). to detect expression of inflammasomes and complement components in mers-cov-infected thp- differentiated macrophages and hdpp -tg mice, µg of total rna from cells or the lung of mice we used as template for first-strand cdna synthesis. the resulting cdna was subjected to quantitative pcr using power sybr ® green pcr master mix (life technologies, carlsbad, ca, usa) to determine the relative abundance of inflammasome and complement components. the forward and reverse primers used for each component are listed in the table . the relative amount of each gene was obtained by normalization against an endogenous control gene (gapdh) and calculated using the comparative −∆∆ct method. sham-infected monocytes and sham-infected hdpp -tg mice were used as respective calibrators. an identical amplification reaction comprising (i) polymerase activation and dna denaturation at • c for min, (ii) cycles each of the denaturation at • c for s, and (iii) an annealing/extension step at • c for s, was used for each gene analyzed. proteins isolated from thp- monocytic cells and thp- differentiated macrophages were electrophoresed in a % sds-page gel and transferred to a pvdf membrane (ge healthcare, dassel, germany). pvdf membranes were then blocked for h at room temperature in % non-fat cytokines in mouse serum were measured using a milliplex mouse cytokine/chemokine magnetic panel kit (merck millipore, burlington, ma, usa). a panel of inflammatory cytokines (il- β, il- , tnf-α, and ifn-γ) was detected according to the manufacturer's protocol. sections of paraffin-embedded spleen and lung tissues ( µm thick) were prepared and stained to detect antigen expression. briefly, retrieved sections were incubated overnight at • c with the following antibodies: mouse anti-caspase- mab (adipogen, san diego, ca, usa), polyclonal rabbit anti-cd (abcam, cambridge, ma, usa), and polyclonal anti-ifn-γrα (santa cruz biotechnology). biotinylated immunoglobulin g was then added, followed by an avidin-biotin-peroxidase conjugate (beijing zhongshan biotechnology co., ltd., beijing, china). immunoreactivity was detected using , diamino benzidine (dab). slides were counter-stained with hematoxylin. statistical analyses were performed using graphpad prism software, version . (graphpad software, san diego, ca, usa). student's t test was used to compare two groups with respect to relative expression of mrna and cytokine levels in serum. p values < . were considered significant. unlike abortive infection of sars-cov in human macrophages, mers-cov can establish a productive infection in macrophages and induce production of proinflammatory cytokines and chemokines [ ] . many rna viruses, such as ev , h n , h n influenza a virus, and zika virus, can infect macrophages and trigger il- β secretion via the nlrp inflammasome [ ] [ ] [ ] [ ] . to evaluate the response of macrophages to mers-cov infection, we inoculated thp- monocytic cells and thp- differentiated macrophages with mers-cov or rpmi medium (sham-infection). we then examined expression of nlrp , pro-caspase- , and pro-il- β h later by rt-qpcr. as shown in figure , mers-cov infection induced relatively higher expression of pro-caspase- ( figure a ) and pro-il- β ( figure b ), but not nlrp ( figure c ), in both thp- monocytes and macrophages. expression of pro-il- β in monocytes increased by -fold, whereas that in macrophages increased by -fold (on average). we verified expression of caspase- , il- β, and mers nucleocapsid protein (np) by western blotting ( figure d ). mers-cov-infected thp- macrophages expressed higher levels of pro-caspase- , pro-il- β, and activated il- β (p ) than sham-infected thp- macrophages or mers-cov-infected thp- monocytes. mers np was detected in both mers-cov-infected thp- monocytes and macrophages. these results indicate that mers-cov infection induces high levels of proinflammatory il- β secretion and thp- macrophage pyroptosis. to determine whether mers-cov infection induces pyroptosis in mice, we used rt-qpcr to detect mrna encoding nlrp , pro-caspase- , and pro-il- β in lung tissue from hdpp transgenic mice at day post-mers-cov infection. although there was no significant difference in expression of nlrp and pro-caspase- between the sham-infected and mers-cov-infected groups ( figure a ,b), expression of pro-il- β mrna was significantly higher after mers-cov infection ( figure c ). in addition, we measured the concentration of il- β in serum. the results showed that mers-cov infection induced production of il- β ( figure d ). furthermore, we examined expression of caspase- in the lung and spleen at day post-mers-cov infection by ihc. in line with the mrna results, there was no significant difference in expression of caspase- in the lung of sham-infected and mers-cov-infected mice. however, the spleens of mice infected with mers-cov showed higher expression of caspase- than those of mice in the sham group ( figure e ). the results indicated that mers-cov infection could induce pyroptosis in mice. (d) samples of total protein were subjected to western blotting to detect pro-caspase- , pro-il- β, activated il- β, and mers np. to determine whether mers-cov infection induces pyroptosis in mice, we used rt-qpcr to detect mrna encoding nlrp , pro-caspase- , and pro-il- β in lung tissue from hdpp transgenic mice at day post-mers-cov infection. although there was no significant difference in expression of nlrp and pro-caspase- between the sham-infected and mers-cov-infected groups (figure a,b) , expression of pro-il- β mrna was significantly higher after mers-cov infection ( figure c ). in addition, we measured the concentration of il- β in serum. the results showed that mers-cov infection induced production of il- β ( figure d ). furthermore, we examined expression of caspase- in the lung and spleen at day post-mers-cov infection by ihc. in line with the mrna results, there was no significant difference in expression of caspase- in the lung of sham-infected and mers-cov-infected mice. however, the spleens of mice infected with mers-cov showed higher expression of caspase- than those of mice in the sham group ( figure e ). the results indicated that mers-cov infection could induce pyroptosis in mice. il- β plays an important role in mediating autoinflammatory diseases and in generating inflammatory responses to infection [ ] . therefore, to assess the inflammatory responses in mice, we measured tnf-α, ifn-γ, and il- in serum at day post-mers-cov infection. as shown in figure a -c, serum from mice in the mers-cov-infected group contained more tnf-α, ifn-γ, and il- than that from sham-infected mice. ihc examination of cd and ifn-γ receptor expression also suggested greater macrophage infiltration and activation in the lung and spleen of mice at days post-mers-cov infection ( figure d ). these results indicate that mers-cov infection causes systemic inflammation, as reported in clinical mers patients and mers-cov infected animal models [ , ] . il- β plays an important role in mediating autoinflammatory diseases and in generating inflammatory responses to infection [ ] . therefore, to assess the inflammatory responses in mice, we measured tnf-α, ifn-γ, and il- in serum at day post-mers-cov infection. as shown in figure a -c, serum from mice in the mers-cov-infected group contained more tnf-α, ifn-γ, and il- than that from sham-infected mice. ihc examination of cd and ifn-γ receptor expression also suggested greater macrophage infiltration and activation in the lung and spleen of mice at days post-mers-cov infection ( figure d ). these results indicate that mers-cov infection causes systemic inflammation, as reported in clinical mers patients and mers-cov infected animal models [ , ] . the complement system links activation of toll-like receptors to transcription of il- β mrna [ , ] . it has been studied that intracellular c is converted to biologically active c a and c b by the protease cathepsin l [ ] , and c a activates nlrp and triggers il- β production in human monocytes by regulating efflux of atp [ ] . in addition, c a is believed to induce a proinflammatory or anti-inflammatory response when ligated to c ar or c ar respectively [ ] [ ] [ ] . thus, we used rt-qpcr to examine expression of complement components and their receptors c ar, c ar , and c ar . as shown in figure , c and c ar expression by both thp- monocytes and macrophages was highly upregulated (by - -fold) after mers-cov infection. c ar was upregulated, whereas c ar was downregulated, after mers-cov infection. the complement system links activation of toll-like receptors to transcription of il- β mrna [ , ] . it has been studied that intracellular c is converted to biologically active c a and c b by the protease cathepsin l [ ] , and c a activates nlrp and triggers il- β production in human monocytes by regulating efflux of atp [ ] . in addition, c a is believed to induce a proinflammatory or anti-inflammatory response when ligated to c ar or c ar respectively [ ] [ ] [ ] . thus, we used rt-qpcr to examine expression of complement components and their receptors c ar, c ar , and c ar . as shown in figure , c and c ar expression by both thp- monocytes and macrophages was highly upregulated (by - -fold) after mers-cov infection. c ar was upregulated, whereas c ar was downregulated, after mers-cov infection. our previous study demonstrated that mers-cov infection results in dysregulated host immune responses and severe tissue damage [ ] and inhibiting c ar alleviates mers-cov infection-induced tissue damage by regulating host immune responses [ ] . here, we used an anti-c ar ab to block the c a-c ar axis. the antibody was administered at the same time as mers-cov infection. we then measured expression of caspase- in the spleen and il- β in the lung and serum. compared with the pbs-treated group, mice receiving the anti-c ar ab expressed less caspase- in the spleen at day post-mers-cov infection ( figure a ). although there was no significant difference between the two groups with respect to pro-il- β mrna expression ( figure b ), serum levels of il- β were lower in the anti-c ar ab-treated group than in the pbs-treated group at day ( figure c ) and day [ ] post-mers-cov infection. these results suggest that complement inhibition decreased the expression of pyroptosis indicators, il- β and caspase- , in mice infected with mers-cov. our previous study demonstrated that mers-cov infection results in dysregulated host immune responses and severe tissue damage [ ] and inhibiting c ar alleviates mers-cov infection-induced tissue damage by regulating host immune responses [ ] . here, we used an anti-c ar ab to block the c a-c ar axis. the antibody was administered at the same time as mers-cov infection. we then measured expression of caspase- in the spleen and il- β in the lung and serum. compared with the pbs-treated group, mice receiving the anti-c ar ab expressed less caspase- in the spleen at day post-mers-cov infection ( figure a ). although there was no significant difference between the two groups with respect to pro-il- β mrna expression ( figure b ), serum levels of il- β were lower in the anti-c ar ab-treated group than in the pbs-treated group at day ( figure c ) and day [ ] post-mers-cov infection. these results suggest that complement inhibition decreased the expression of pyroptosis indicators, il- β and caspase- , in mice infected with mers-cov. our previous study demonstrated that mers-cov infection results in dysregulated host immune responses and severe tissue damage [ ] and inhibiting c ar alleviates mers-cov infection-induced tissue damage by regulating host immune responses [ ] . here, we used an anti-c ar ab to block the c a-c ar axis. the antibody was administered at the same time as mers-cov infection. we then measured expression of caspase- in the spleen and il- β in the lung and serum. compared with the pbs-treated group, mice receiving the anti-c ar ab expressed less caspase- in the spleen at day post-mers-cov infection ( figure a ). although there was no significant difference between the two groups with respect to pro-il- β mrna expression ( figure b ), serum levels of il- β were lower in the anti-c ar ab-treated group than in the pbs-treated group at day ( figure c ) and day [ ] post-mers-cov infection. these results suggest that complement inhibition decreased the expression of pyroptosis indicators, il- β and caspase- , in mice infected with mers-cov. at day after mers-cov infection, we measured proinflammatory cytokines (ifn-γ, tnf-α, and il- ) in serum. ifn-γ levels in mice treated with the anti-c ar ab were much lower than those in the pbs-treated group ( figure a ). to further evaluate the effect of complement inhibition on the local inflammation at later time after mers-cov infection, we examined expression of cd and ifn-γ receptor in lung and spleen at days post-mers-cov infection. ihc revealed that macrophage infiltration and activation were lower in the anti-c ar ab-treated group ( figure d ). taken together, these results suggest that inhibiting complement dampens the over-activated inflammatory response in mice infected with mers-cov. at day after mers-cov infection, we measured proinflammatory cytokines (ifn-γ, tnf-α, and il- ) in serum. ifn-γ levels in mice treated with the anti-c ar ab were much lower than those in the pbs-treated group ( figure a ). to further evaluate the effect of complement inhibition on the local inflammation at later time after mers-cov infection, we examined expression of cd and ifn-γ receptor in lung and spleen at days post-mers-cov infection. ihc revealed that macrophage infiltration and activation were lower in the anti-c ar ab-treated group ( figure d ). taken together, these results suggest that inhibiting complement dampens the over-activated inflammatory response in mice infected with mers-cov. macrophages play important roles in host defense by clearing dead cells, ingesting and destroying microbes, and presenting antigens to t lymphocytes. in addition, macrophages produce the full array of complement components [ ] and prrs, which are closely associated with inflammasome activation and pyroptosis. the accumulated studies indicate that macrophages play an important role in the pathogenesis of sars and mers [ , ] . macrophages infected with mers-cov secrete proinflammatory cytokines and chemokines [ ] . widespread distribution of these macrophages throughout many organs is one of the reasons underlying multi-organ damage and systemic inflammation after virus infection. pyroptosis or inflammasome activation plays an important role in virus-mediated pathogenesis. for example, abortive hiv- infection in quiescent macrophages play important roles in host defense by clearing dead cells, ingesting and destroying microbes, and presenting antigens to t lymphocytes. in addition, macrophages produce the full array of complement components [ ] and prrs, which are closely associated with inflammasome activation and pyroptosis. the accumulated studies indicate that macrophages play an important role in the pathogenesis of sars and mers [ , ] . macrophages infected with mers-cov secrete proinflammatory cytokines and chemokines [ ] . widespread distribution of these macrophages throughout many organs is one of the reasons underlying multi-organ damage and systemic inflammation after virus infection. pyroptosis or inflammasome activation plays an important role in virus-mediated pathogenesis. for example, abortive hiv- infection in quiescent lymphoid cd t cells leads to cd t cell pyroptosis independent of the nlrp inflammasome [ ] . ev d and zikv ns activate the nlrp inflammasome by interacting with nacht and the lrr domain of nlrp [ , ] . the pb -f protein of avian influenza a virus h n or h n induces inflammation by activating the nlrp inflammasome [ , ] , and deficiency of nlrp or caspase- protects mice against h n infection-associated morbidity and mortality [ ] . here, we demonstrate that the dysregulated macrophage-mediated immune responses after mers-cov infection may contribute to a severe outcome. several studies demonstrate relationships between complement and inflammasomes. for example, c -/-mice display reduced inflammasome activation in an intracerebral hemorrhage (ich) model [ ] . engagement of c a and c ar on cd + t cells generates reactive oxygen species, which are a classical damp, thereby triggering inflammasome assembly [ ] . in monocytes, c a alters metabolic programming and increases atp efflux, leading to nlrp activation and il- β generation via the receptor p × [ ] . thus, the complement system is considered to be an essential regulatory component of the cellular alarm system that controls inflammasome activation [ ] . here, we inhibit mers-cov infection-induced inflammation and pyroptosis by blocking the c a-c ar axis with an anti-c ar antibody. in our study, we first demonstrated that virus infection leads to pyroptosis by measuring activation of caspase- and il- β in human macrophages infected with mers-cov ( figure ). next, we found that mers-cov infection induced transcription of pro-il- β in the lung and expression of caspase- in the spleen. activated caspase- could be released into the extracellular space and delivered to the lung via exosomes [ ] or the systemic circulation; it then cleaves pro-il- β into its bio-activated form, which is secreted into the serum ( figure d ). we cannot exclude the possibility that pyroptosis occurs in the spleen because we did not detect expression of pro-il- β. meanwhile, examination of cd and ifn-γ receptor expression revealed macrophage infiltration and activation in the lungs and spleen ( figure d ). thus, pyroptosis could occur in both organs. in our previous study, we showed that inhibiting c ar increased splenic cell regeneration and decreased splenic cell apoptosis, thereby alleviating mers-cov infection-induced tissue damage [ ] . the spleen happens to be a large reservoir for myeloid lineage cells such as macrophages, dendritic cells and cd + t cells in which pyroptosis mainly occurred [ , , ] . here, we studied and demonstrated that inhibiting c ar suppressed caspase- activation ( figure a ) and macrophage infiltration and activation in the spleen ( figure d ), and thereby reduced the secretion of il- β into the serum ( figure c ) and the systemic inflammation ( figure ). however, in the lung tissue, there was no significant difference of pro-il- β transcription between the ab and sham-treated groups ( figure b ), which may due to the limited macrophages, the main cell type in which pyroptosis occurred, when compared to that in spleen. although many studies have focused on the link between complement and pyroptosis, the pathways that link them remain unclear. here, our results showed that mers-cov infection induces pro-il- β transcription, and complement activation, which leads to pyroptosis in macrophages. induction of pyroptosis was related with complement activation and may be promoted by ligation of c a and c ar , which was confirmed by the blockade of anti-c ar antibody (figure ) . in summary, these data indicate that mers-cov infection induces overactivation of complement, which may contribute to pyroptosis and inflammation. pyroptosis and inflammation were suppressed by inhibiting c ar . these research will further our understanding of the pathogenesis of mers-cov infection. occurred, when compared to that in spleen. although many studies have focused on the link between complement and pyroptosis, the pathways that link them remain unclear. here, our results showed that mers-cov infection induces pro-il- β transcription, and complement activation, which leads to pyroptosis in macrophages. induction of pyroptosis was related with complement activation and may be promoted by ligation of c a and c ar , which was confirmed by the blockade of anti-c ar antibody (figure ) . clinical course and 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influenza a virus infection results in lethal inflammation in the mammalian host via the nlrp -caspase- inflammasome zika virus infection induces host inflammatory responses by facilitating nlrp inflammasome assembly and interleukin- beta secretion immunological and inflammatory functions of the interleukin- family clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission crosstalk pathways between toll-like receptors and the complement system cholesterol crystals induce complement-dependent inflammasome activation and cytokine release intracellular complement activation sustains t cell homeostasis and mediates effector differentiation c a modulates il- beta secretion in human monocytes by regulating atp efflux and subsequent nlrp inflammasome activation the chemotactic receptor for human c a anaphylatoxin evidence for a functional role of the second c a receptor c l an anti-inflammatory function for the complement anaphylatoxin c a-binding protein, c l extrahepatic complement biosynthesis: where, when and why? multiple organ infection and the pathogenesis of sars cell death by pyroptosis drives cd t-cell depletion in hiv- infection activation of the nlrp inflammasome by iav virulence protein pb -f contributes to severe pathophysiology and disease pb -f peptide derived from avian influenza a virus h n induces inflammation via activation of the nlrp inflammasome nlrp is required for complement-mediated caspase- and il- beta activation in ich intracellular complement activation-an alarm raising mechanism? inflammasome-derived exosomes activate nf-kappab signaling in macrophages structure and function of the spleen key: cord- -uf sx qi authors: dijkman, ronald; mulder, h. lie; rumping, lynne; kraaijvanger, ilse; deijs, martin; jebbink, maarten f.; verschoor, ernst j.; van der hoek, lia title: seroconversion to hcov-nl in rhesus macaques date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: uf sx qi hcov-nl is a recently identified respiratory virus. its pathogenesis has not been fully unraveled because an animal model is currently lacking. here we examined whether rhesus macaques encounter hcov-nl infections during life, by examining the levels of antibodies to hcov-nl in time. the animals were followed for up till years, and in three animals we observed a steep rise in antibodies during follow up, indicative of a natural infection with hcov-nl . hcov-nl was identified in in a child with bronchiolitis in the netherlands [ ] , yet worldwide screenings revealed that the virus can be detected throughout the globe in children and adults with respiratory tract infections [ ] [ ] [ ] [ ] [ ] [ ] [ ] . people acquire their first infection with hcov-nl open access early in life [ ] , and during this first infection the chance to develop larynchotracheabronchitis (croup) is high [ ] . with increasing age people experience repeated infections (unpublished findings lvdh), and in case of underlying disease an hcov-nl infection can require hospitalization. on the whole we can say that within a few years a vast amount of data on this new virus has become available, except that the pathogenesis of hcov-nl remains unknown. the lacking of an animal model system is the problem. in addition, an animal model system is needed to fulfill the koch's postulates and link infection to a disease. to date, only a limited number of primary or immortalized cells from either human or non-human primate origin support propagation of hcov-nl [ , [ ] [ ] [ ] . the first hcov-nl isolate that was cultured was propagated on tertiary monkey kidney cells of a cynomolgus monkey (latin: macaca fascicularis) and subsequently passaged to llc-mk cells, a cell line derived from the kidney of a rhesus macaque (latin: macaca mulatta). it is of interest to investigate whether rhesus macaques can be infected by hcov-nl . this can either be investigated by direct inoculation of rhesus macaque with hcov-nl . alternatively, one can establish whether rhesus macaques that are in close contact to humans have experienced a natural hcov-nl infection by investigating the nl -directed antibody titers in time. a sudden rise in coronavirus antibody titers is a strong indication for a coronavirus infection. we have shown this for children who acquire their first hcov-nl infection during their first years of life [ ] . at later ages repeated infections with human coronaviruses can occur [ ] , depending on the levels of protective antibodies. protective antibodies peak shortly after infection and gradually decrease during the following years, with accompanying susceptibility to a reinfection with time. in the current study we use the kinetics of the antibody response to hcov-nl to determine whether rhesus macaques encounter natural infections with hcov-nl , or related coronaviruses. the serological assays were performed with the n-protein of hcov-nl which is an immunodominant protein, although there is some homology with the n-protein of the closest relative of hcov-nl , hcov- e. to ascertain the most specificity, the c-terminal region (amino acids to ) of the protein -which has the most difference between nl -n and e-n -was used. in addition competition experiments with homologous and heterologous proteins were included in each experiment to ascertain the specificity of an antibody response. the first indication of infections with hcov-nl -like viruses in rhesus macaques was found when we tested serum samples of monkeys (table and figure ). five samples showed high levels of antibodies to the hcov-nl protein. the same samples did not have elevated levels of antibodies to hcov- e, indicative that the response was specific (data not shown). furthermore, competition experiments with the nl -protein and the e-protein revealed that only the homologous protein could compete with binding to the nl -protein on the elisa plate (data not shown), suggesting that the antibodies are directed to an hcov-nl -like virus. on the x-axis the rhesus macaque id is indicated. on the y-axis relative luminescence signal (rlu) in an elisa detecting antibodies to the c-terminal nucleocapsid protein is shown. rhesus macaques of which longitudinally collected serum samples were analyzed are indicated with a "*". longitudinal serum samples were available of three rhesus macaques with the highest antibody levels to hcov-nl (numbers , and ). these monkeys had been sampled on a regular basis, with on average one serum sample every one or two years. sampling started when the macaques were already a few years old, except for one which was followed from months of age. in all three animals a sudden rise in antibody titers was noticed ( figure , panel a, b and c). rhesus macaque number was followed from (starting at the age of years) with high levels of nl -directed antibodies. a strong peak was observed in , indicating that the animal experienced an additional hcov-nl infection (at years of age). animal number started with low titers to hcov-nl (age years) but after some years very high titers to hcov-nl were visible (age years). unexpectedly, these high antibody levels remained for the complete follow-up period (last sample collected at the age of ). this is suggesting that the adaptive immune response to hcov-nl is constantly activated, and one could speculate that this animal is experiencing a chronic hcov-nl infection. unfortunately, additional samples like throat swabs or faeces to test for chronic infections were not available. the third animal (number ) was followed from early age on, starting with low levels of nl -antibodies. between the years / a steep rise in antibody levels was noted, whereas a second rise occurred after the winter of / (at years and almost years of age respectively). the animals were all checked for antibodies to hcov- e, but for none of the three monkeys reactivity was detected ( figure ). this is in accordance with the first screening of the animals, which already showed low levels of hcov- e antibodies in these animals. reactivity of rhesus macaque number sera towards hcov-nl infected (hcov-nl positive) and non-infected (control) llc-mk cells, at the age of (preseroconversion) and (postseroconversion) years. the images represent the cell nucleus (blue) and rhesus macaque antibodies (green) signals. to confirm our findings we determined whether the post-seroconversion samples also react with whole virus by performing an immunofluorescence assay with hcov-nl infected cells. in all cases with low hcov-nl antibodies we saw no reactivity with the infected cells. whereas all samples obtained after seroconversion showed clear reactivity with whole virus (shown for animal number in figure ). with the assay used we cannot obtain evidence that it were hcov-nl infections that these monkeys have encountered. however, one can conclude that it were infections with hcov-nl , or a virus which is serologically closely related. it can never be excluded that there is a coronavirus infecting these rhesus monkeys that is closely related to hcov-nl (but has not been identified yet). sampling of the respiratory tract on a regular basis throughout the years and eventually identify the pathogen that is eliciting the antibody response can answer this question, but is not an easy task. to our opinion the most likely explanation would be that natural infection with hcov-nl occurs. the macaques described in this study live in a closed system with close contacts to humans (feeding, cleaning of cages, etc.). through this contact they can encounter the virus as infections of humans with hcov-nl occurs frequently in winter seasons [ ] . as the cells of this monkey species are susceptible to infection (llc-mk cells, a kidney epithelial cell line) it is very likely that in vivo infections can occur as well. the animals of our study were clinically examined on a routine yearly basis for colony health surveillance, but subclinical infections with coronaviruses will go unnoticed during this check up and in the periods between the examinations. thus it can not be determined whether the seroconversion or reconversion to hcov-nl was accompanied with minor clinical signs. future studies should be designed to investigate viral infection with human viruses and the accompanying clinical signs to further unravel this subject. here we show that it is not necessary to sacrifice monkeys by experimental infecting. a well-designed follow up through the years with serum and respiratory samples collected on a regular basis would be sufficient to unravel the potential of the human respiratory viruses to infect rhesus macaques. once a cohort has set up and samples have been collected for a few years, all newly identified respiratory viruses can be screened with the appropriate serological assays, and infection related to clinical symptoms. natural infections of rhesus macaques with viruses that are known to be of human origin are not unusual. it has been described that smallpox, measles, rubella or parainfluenzavirus and cause natural infection of rhesus macaques and the closely related cynomolgus macaques [ , ] . furthermore, experimental infection with several viruses showed that the animals are susceptible to various human respiratory viruses like respiratory syncytial virus and sars coronavirus [ , ] . with the knowledge that natural infection of hcov-nl is likely to occur, one should keep in mind that this has implications for animal model experiments. in case rhesus macaques are experimentally infected with hcov-nl this might be a reinfection, in case the animal has encountered the virus previously, or the first infection, in case the animal is still young and was not naturally infected through human contact. the clinical signs during the first infection might be of a more severe nature in comparison with a recurrent infection. this should be taken into account in case a study is conducted to reveal the pathogenesis of the virus in an animal model system. cross-sectional and longitudinal serum specimens were collected from rhesus macaques with an indian, burmese or chinese genetic background. the rhesus macaques were either imported or born in the netherlands in one of the breeding colonies. the animals have not been isolated but remained in a breeding colony and were not used for immunization with antigens or adjuvant, or any other study. all serum specimens were stored at - o c and heat-inactivated at o c for minutes prior to analysis. the generation of the plasmid construct was performed as described [ ] . for hcov-nl the following primer combination was used ' nl _n _ct ( ' -caccaaacctaataagcctct ttctcaac - ') and ' nl _nexp ( ' -ttaatgcaaaacctcgttgac - '), whereas for hcov- e the primer combination ' e_ n_ct ( ' -caccccttctcgtaatcagagtcct - ') and ' e_nexp ( ' -ttagtttacttcatcaattat - ') was used. the generated pet _nl _ct and pet _ e_ct plasmids were sequenced and shown to be % identical to the virus reference sequences of hcov-nl (amsterdam- ) and hcov- e (inf- ), respectively. subsequent expression and purification of the hcov-nl and hcov- e recombinant carboxyl-terminal nucleocapsid proteins was performed as previously described [ ] . ninety-six-well elisa plates (greiner bio-one) were coated overnight at o c with μg/ml of expressed recombinant c-terminal n protein of hcov-nl or hcov- e. the proteins were diluted in . m carbonate buffer ph . . unspecific binding sites were blocked with pbs + . % tween (pbst) supplemented with % skim milk (fluka) for one hour at room temperature (rt). longitudinal and cross-sectional sera were diluted : , in pbst containing % skim milk and incubated on the plate for hours at rt. after washing, alkaline phosphatase conjugated anti-monkey igg antibody (sigma aldrich) diluted ( : , ) in % skim milk pbst was added. following one hour at rt the plates were washed and signal was developed with μl of lumi-phos plus (lumigen). measurements were done with a glomax tm plate luminometer (promega). all sera were tested in duplicate. rhesus macaque sera were diluted ( : ) in pbst containing % skim milk and twofold serial dilutions ranging from to μg/ml of either expressed recombinant c-terminal n protein of hcov-nl , n protein of hcov- e or lacz protein and incubated for hour at rt. prior to incubation, the mixtures were briefly homogenized by vortexing. no centrifugation was performed. following the pre-incubations the samples were measured by hcov-nl or hcov- e elisa, as described above. llc-mk cells were seeded on coverslips in -well plates at a density of .  cells / well and cultured at °c with % co in minimal essential medium (mem; parts hanks' mem and part earle's mem) supplemented with % heat-inactivated fetal calf serum (paa laboratories), penicillin ( u/ml), and streptomycin ( g/ml). cells were infected with hcov-nl at a multiplicity of infection of . and incubated at °c with % co . after days cells were fixated with . % pfa in pbs for minutes at room temperature (rt). the unspecific binding sites were blocked overnight at °c with incubation buffer ( % bsa (sigma aldrich) in pbs with . % saponin and mm ammonium chloride). for detection of hcov-nl proteins cells were double stained for hour at rt in incubation buffer with rabbit derived anti-s -nl sera ( : ) and rhesus macaque sera ( : ) as primary antibodies. donkey derived, dylight labeled, anti-rabbit igg (h+l) and donkey derived, dylight labeled, anti-human igg (h+l) ( : ) (jackson immunoresearch) were applied as secondary antibodies for hour at rt in incubation buffer, followed by nuclear dna staining with hoechst . fluorescent images were acquired on a leica tcs sp aobs spectral confocal microscope with a x hcx pl apo . oil objective. in the early days of human coronavirus identification (the s) it was possible to experimentally infect volunteers with the viruses which were isolated from persons with common colds. nowadays, with the recent epidemic of sars-cov, it is very difficult to attract volunteers for such studies. not only has the public become aware of the potential dangers of a coronavirus infection, the recently identified viruses (hcov-nl and hcov-hku ) have been isolated from children or adults with severe lower respiratory tract infection, and not simple common colds. consequently, the only possibility to connect the recently identified coronaviruses to a certain disease is via associations with disease or animal experiments. to date no animal model system has been described for hcov-nl . although all signs suggest that rhesus macaques might be a good model system -since cells of rhesus macaques can be in vitro infected by hcov-nl -nobody has reported infection of these animals yet. we show here that there are clear signs that rhesus macaques acquire natural infections with hcov-nl , or a serologically very closely related coronavirus. in future animal experiments to be carried out with rhesus macaques the history of an animal should be known to determine whether the experimental infection occurs in an hcov-nl naïve animal or an animal that has encountered the virus during life, as clinical symptoms might differ greatly in first or recurrent infection. identification of a new human coronavirus new human coronavirus, hcov-nl , associated with severe lower respiratory tract disease in australia human coronavirus nl infection in canada human coronavirus nl infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong detection of human coronavirus nl in young children with bronchiolitis a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl in children hospitalized with respiratory tract infections in belgium detection of human coronavirus-nl in children in japan human coronavirus nl and e seroconversion in children croup is associated with the novel coronavirus nl plaque assay for human coronavirus nl using human colon carcinoma cells systematic assembly of a full-length infectious clone of human coronavirus nl identification of cell lines permissive for human coronavirus nl the time course of the immune response to experimental coronavirus infection of man natural transmission of smallpox from man to performing monkeys. an ecological curiosity exposure to human respiratory viruses among urban performing monkeys in indonesia experimental respiratory syncytial virus infection of four species of primates macaque model for severe acute respiratory syndrome ronald dijkman and lia van der hoek are supported by vidi grant . . from the netherlands organization for scientific research (nwo) and by sixth framework grant lshm-ct- - from the european union. we are grateful to a.c. van der kuyl for useful discussions and sample selection. key: cord- -dbexsugq authors: wu, yang; zhang, hongling; shi, zhaorong; chen, jianfei; li, mingwei; shi, hongyan; shi, da; guo, longjun; feng, li title: porcine epidemic diarrhea virus nsp antagonizes interferon signaling by rna degradation of tbk and irf date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: dbexsugq porcine epidemic diarrhea virus (pedv) causes a porcine disease associated with swine epidemic diarrhea. the type i interferon (ifn-i or ifn α/β) is a key mediator of innate antiviral response during virus infection. different antagonistic strategies have been identified and determined as to how pedv infection inhibits the host’s ifn responses to escape the host innate immune pathway, but the pathogenic mechanisms of pedv infection are not fully elucidated. our preliminary results revealed that endogenous tank-binding kinase (tbk ) and interferon regulatory factor (irf ), the key components in the ifn signaling pathway were downregulated in pedv infected ipec-j cells by itraq analysis. in this study, we screened nsp as the most important viral encoded protein involved in tbk and irf reduction. endoribonuclease (endou) activity has been well determined for coronavirus nsp . three residues (h , h , and k ) of pedv nsp were identified as critical amino acids for pedv endou but not d , which was not well correlated with published results of other coronaviruses, such as severe acute respiratory syndrome virus (sars-cov). moreover, pedv nsp can directly degrade the rna levels of tbk and irf dependent on its endou activity to suppress ifn production and constrain the induction of ifn stimulated genes (isgs), by which pedv antagonizes the host innate response to facilitate its replication. collectively, these results have confirmed that pedv nsp was capable of subverting the ifn response by the rna degradation of tbk and irf . porcine epidemic diarrhea (ped) is caused by porcine epidemic diarrhea virus (pedv), which has a positive-strand rna genome of kb in length in the genus alphacoronavirus, family coronaviridae and order nidovirales [ ] [ ] [ ] . the disease is characterized by severe enteritis, vomiting, watery diarrhea, dehydration, and a high mortality rate among swine [ ] . two-thirds of the genome (orf a and b) encode a large replicase polyprotein, whereas the remainder of the genome encodes for structural proteins and accessory proteins [ , ] . after pedv virus entry into the host cells, orf a codes for a large polyprotein a (pp a), while orf b is expressed as the pp ab fusion protein via the ribosomal frameshifting. by the proteinase activity of nsp (a papain-like protease) and nsp (a main protease), these polyproteins are proteolytically processed to nonstructural proteins (nsp to ) , which mediate the replication of the viral rna genome and synthesis of a nested set of subgenomic mrnas [ ] . in late , a newly emerging pedv variant was reported with more virulence and higher mortality in suckling piglets, compared to the classical pedv that was first discovered in europe in [ ] . to date, the newly emerging pedv variant is recognized as the major infectious pathogen for swine diarrhea-associated diseases in swine-raising farms in china and it has spread to other countries worldwide, causing a high number of pig deaths and significant economic impacts [ ] [ ] [ ] [ ] [ ] . during viral infection, the innate immune response is activated, leading to the induction of the type i interferon (ifn-i or ifn α/β). ifn-i is the potent cytokine of critical importance in controlling viral infections and priming adaptive immune responses [ ] . following production, ifn-i initiates a positive feed-back loop by binding to their cognate receptors on the cell surface in an autocrine and paracrine manner [ , ] and activating jak protein tyrosine kinases (jak and tyk ) which phosphorylate signal transducers and activators of transcription stat and stat . stat and stat together with interferon regulatory factor (irf ) form a transcription factor complex termed ifn-stimulated gene factor (isgf ). then, isgf is translocated into the nucleus and binds to the ifn-stimulated response elements (isre) to induce the expression of ifn-stimulated genes (isgs), which establish an antiviral state [ , ] . however, many viruses, including coronaviruses, have evolved mechanisms to evade the host immune system [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . previous studies suggest that pedv can restrain host innate immune response by different strategies, such as by degradation or cleavage of key factors essential the ifn signaling pathway [ , ] , competitive interaction between viral encoded proteins and modulators for ifn production [ , ] , or localization changes of antiviral components [ ] . whether there are other mechanisms utilized by pedv to circumvent the host response remains unclear. our previous results have demonstrated that differentially expressed proteins were identified in pedv infected ipec-j cells by the analysis of isobaric tags for relative and absolute quantitation (itraq). we identified differentially expressed cellular proteins, of which eight were upregulated and downregulated. these differentially expressed proteins were involved in apoptosis, signal transduction, and stress responses. in our analysis, tbk and irf , two important modulators in the activation of the interferon signaling pathway were downregulated post pedv infection. in this study, we screened the pedv viral proteins involved in the reduction of tbk and irf expression. among the identified viral proteins, nsp was recognized as the most effective viral protein contributing to the reduction of both tbk and irf expression after the co-transfection of tbk or irf with individually encoded pedv proteins in vitro. it was confirmed that the two major well-known degradation systems, namely the ubiquitin-proteasome system or autophagy, were not involved in pedv nsp mediated reduction of tbk and irf . in contrast, pedv nsp was capable of suppressing tbk and irf expression by endoribonuclease-dependent degradation of tbk and irf rna. this resulted in a decrease of ifn and isg production, resulting in pedv host innate immune escape. ipec-j cells (porcine small intestine epithelial cell clone j ; atcc), vero e (african green monkey kidney cell line; atcc), and hek cells (human embryonic kidney epithelial cells; atcc) were cultured in dulbecco's minimum essential medium (dmem) (life technologies, usa) supplemented with % heat-inactivated fetal bovine serum (fbs) (gibco, usa), u/ml penicillin, µg/ml streptomycin at • c in an incubator with % co (thermo scientific, usa). pedv strain cv (genbank accession number kt ) was prepared and titrated as previously described [ ] . vesicular stomatitis virus that expresses the green fluorescence protein (vsv-gfp) was preserved in harbin veterinary research institute, harbin, and stored at − • c. the full-length sequence of tbk and irf were constructed into the pcaggs-ha vector to obtain recombinant plasmids, pcaggs/ha-tbk and pcaggs/ha-irf , respectively. the recombinant pcaggs plasmids containing individual pedv viral protein (nsp - , nsp - , s, e, m, and n) with a flag fusion tag were kindly provided by prof. yue wang from harbin veterinary research institute. mutagenesis of the pedv nsp constructs (h a, h a, d a and k a) were performed by using site-directed mutagenesis kit (takara, china). recombinant prokaryotic expression plasmids were obtained following cloning of the individual gene of pedv nsp and its mutant into the ecor i and xho i sites of pgex- p- plasmid vector (ge healthcare life sciences). recombinant pgem-t/tbk and pgem-t/irf plasmids were generated to serve as dna templates for an rna transcription assay in vitro by amplifying the full length sequence of tbk or irf into pgem-t easy vector by the t-a ligation method. the specific primers used for the construction of target plasmids are listed in table and all the constructed plasmids were verified by dna sequencing. the listed antibodies were used in this study including tbk rabbit monoclonal antibody (mab) (cell signaling technology), irf rabbit mab (cell signaling technology), and phospho-irf (ser ) ( d g), rabbit mab (cell signaling technology), anti-flag mouse mab (sigma), anti-ha mouse mab (sigma), irdye-conjugated secondary antibody (li-cor biosciences) , and β-actin mouse mab (sigma). monolayers of vero e and ipec-j cells were infected with pedv strain cv at multiplicity of infection (moi) of . for h at • c. unbound virus was removed, and cells were maintained in complete medium for various time points until samples had been harvested. some cell samples were treated with proteasome inhibitor mg (sigma) at the concentration of µm, autophagy inhibitor -methyladenine ( -ma, sigma) at mm, or carrier control dmso during some transfection assays as previously described [ ] . hek cells were transfected with indicated plasmids using x-tremegene transfection reagent according to manufacturer's instruction (roche, usa). at h post transfection, cell samples were collected and lysed in ripa buffer (beyotime, nantong, china) for the western blot analysis of targeted proteins. immunofluorescence assays (ifa) were performed as described previously with slight modification [ ] . briefly, hek cells were co-transfected with tbk , irf together with nsp , nsp mutants, or empty vector control followed by the collection of supernatants for each treatment at h post transfection. ipec-j cells were treated with the collected supernatants with three-fold dilution for h followed by inoculation of vsv-gfp at moi of . . the fluorescence was visualized at hpi with an olympus inverted fluorescence microscope equipped with a camera. western blot analysis was performed as previously described with a slight modification [ ] . treated samples were lysed in radioimmunoprecipitation assay (ripa) buffer (haigene, china) containing protease inhibitor cocktail and phosphatase inhibitors (roche, switzerland), separated by sds-page under reducing conditions, and transferred onto a pvdf membrane (merck millipore, usa). after blocking, the membranes were incubated with a primary antibody and then incubated with an appropriate irdye-conjugated secondary antibody (li-cor biosciences, lincoln, ne). the membranes were scanned using an odyssey instrument (li-cor biosciences) according to the manufacturer's instructions. linearized dna was prepared by digestion with restriction endonuclease sal i prior to in vitro transcription to produce rna of defined length. in vitro transcribed rna of tbk and irf were generated from recombinant pgem-t/tbk or pgem-t/irf plasmid as template, respectively, using the ribomax™ large scale rna production systems (promega, usa). transcribed rna was purified by removal of the dna template and proteases following transcription reaction as the manufacturer's instruction (promega, usa) and stored for nuclease assay at - • c. for protein expression, individual plasmid of pgex- p- -pedv nsp , pgex- p- -pedv nsp mutant derivatives (h a, h a, d a, and k a), or pgex- p- empty vector was transformed to escherichia coli bl (de ) cells, respectively. the glutathione s-transferase (gst) fusion proteins were expressed following isopropyl-β-d-thiogalactopyranoside (iptg) inductions and purified by affinity chromatography using glutathione immobilized to a sepharose matrix per the manufacturer's instruction (ge healthcare life sciences, usa). the endoribonuclease activity assay was done as previously described [ ] . briefly, nuclease reactions contained µg of purified wild-type pedv nsp protein, pedv nsp mutant protein, or gst tag protein as control, and µg tbk or irf rna transcribed and purified in vitro. reactions were performed in mm hepes-koh (ph . )/ mm nacl/ mm mncl / mm dtt. following incubation at • c for h, the reactions were extracted using phenol-chloroform-isoamyl alcohol and analyzed by agarose-formaldehyde gel electrophoresis. for northern blot, total rna was harvested by using trizol reagent (invitrogen, usa) and analyzed by agarose-formaldehyde gel electrophoresis. rnas were transferred to a . -µm nylon membrane and probed with biotin-labeled dna probes generated with the specific primers (table ) using the north south tm biotin random prime dna labeling kit (thermo scientific). the membrane was imaged an odyssey instrument (li-cor biosciences) followed by incubation with irdye -conjugated streptavidin. quantitative rt-pcr analyses were carried out as described previously with a slight modification [ ] . at indicated time points post transfection or pedv infection, total rna was extracted from cells and subjected to quantitative rt-pcr using specific primers as listed in table . relative gene quantification was performed by the (-delta delta c(t)) method [ ] . collected virus samples were frozen and thawed three times and clarified by centrifugation at × g for min prior to titration. tcid assays were performed in vero e cells following the method of reed & muench as previously described [ ] . briefly, cell monolayers were inoculated with serial dilutions of each virus stock and incubated for days prior to observation of the presence of cytopathic effect. variables are expressed as mean ±s.d. data were statistically analyzed by using graphpad prism v . software. statistical analyses were performed using student's t test. a p value of < . was considered significant. ipec-j cells were infected with pedv or left untreated as control and cell samples were collected at h and h for itraq analysis as previously described [ ] . our preliminary results revealed that endogenous tbk and irf were downregulated at h and h in pedv infected ipec-j cells by itraq analysis (data not shown). subsequently, ipec-j cells were infected with pedv at moi of . and the mrna levels were determined by quantitative pcr. as shown in figure a , tbk mrna levels were significantly decreased at h and h post infection (hpi). in contrast, irf mrna levels were first increased at hpi and then decreased at hpi, indicating no obvious changes in irf mrna levels following pedv infection ( figure b) . to further confirm the results, hek cells were inoculated with pedv at moi of . and cell samples were collected for endogenous tbk and irf detection at indicated time points post infection. consistent with the results from ipec-j cells by itraq, endogenous tbk and irf were evidently reduced at hpi and hpi in hek cells ( figure c ). these findings suggest that a reduction of endogenous tbk and irf may be achieved by downregulating the mrna transcriptional levels of tbk and irf post pedv infection. to explore which viral protein contributes to the reduction of tbk and irf , hek t cells were co-transfected with tbk or irf and each pedv encoded protein. at h post transfection, cell samples were collected and lysed for detection of tbk or irf expression. several viral proteins were involved in the reduction of tbk or irf expressions to varying extents, e.g., nsp , nsp , and nsp for tbk and nsp , nsp , nsp , and nsp for irf , among which nsp can evidently downregulate the expression of either tbk or irf compared with other viral proteins (figure a,b) . moreover, endogenous tbk and irf were also reduced followed by the ectopic overexpression of nsp at h post transfection ( figure c ). therefore, we mainly focused on pedv nsp as the research target in this study to investigate its role in modulating tbk and irf expressions. table . three independent experiments were performed in triplicate, and values are the means ± sd for all three experiments. *, p < . . (c) hek cells were inoculated with pedv at moi of . for h and h followed by verification of endogenous tbk and irf proteins by western blot analysis. within eukaryotic cells, there are two major intracellular protein degradation pathways: the ubiquitin-proteasome system and autophagy [ ] . the proteasomal degradation pathway has high selectivity and the proteasome generally recognizes ubiquitinated substrates [ ] . by contrast, autophagy is a highly conserved process for degrading redundant cellular components by encircling them with membrane followed by a fusion of the vesicle with lysosomes [ ] . therefore, to determine the mechanism that might be responsible for the depletion of tbk and irf by nsp , the expression levels of tbk and irf proteins were examined in cells treated with a protease inhibitor mg [ , ] . as shown in figure a ,c, treatment with mg cannot block the downregulation of tbk and irf in hek t cells with co-transfection of tbk or irf together with nsp , thus not suggesting the proteasome-mediated degradation of tbk and irf by nsp . additionally, we tested the possible role of autophagy in the reduction of tbk and irf by treating cells with -ma, which is commonly used to inhibit autophagy [ , ] . we observed that -ma treatment did not inhibit tbk or irf downregulation in hek t cells co-transfected with tbk or irf along with nsp ( figure b,d) . these data indicate that downregulation of tbk and irf by nsp is not through the ubiquitin-proteasome system and autophagy. coronavirus nsp has been reported as a uridine-specific endoribonuclease and nuclease activities as well as crystal structure have been well identified as previously described [ , [ ] [ ] [ ] [ ] . four inactive mutants of sars-cov nsp , including h a, h a, d a, and k a, have been identified to lose the cleavage activity for substrate rna due to loss of endoribonuclease activity, indicating that these four residues were critical for maintaining the nuclease activity of sars-cov nsp [ ] . based on amino acid sequence alignment of pedv nsp with other coronavirus orthologs as well as xendou from x. laevis, the four mentioned residues were also conserved in pedv nsp and were denoted in red with the corresponding position in pedv nsp sequence below ( figure e ). here, we asked whether tbk or irf downregulation by pedv nsp is dependent on its endoribonuclease activity. to determine whether the amino acid residues are also required for pedv nsp endonuclease activity, four mutants (h a, h a, d a, and k a) were constructed by mutating corresponding residue of pedv nsp to alanine. hek t cells were co-transfected with tbk or irf together with nsp or constructed mutants following the detection of tbk and irf by western blot. it was demonstrated that nsp and d a mutant can obviously reduce the expression levels of tbk and irf post transfection. in contrast, the reduction of tbk or irf expression was blocked when hek t cells were co-transfected with tbk or irf and the remaining mutants (h a, h a, and k a), indicating that residues of h , h , and k but not d are critical for the endoribonuclease activity of pedv nsp ( figure f,g) . . pedv-induced reduction in tbk and irf expression is due to pedv nsp endoribonuclease activity, but not proteasome or autophagy-mediated mechanisms. (a,c) hek t cells were co-transfected with nsp (flag tagged) and tbk or irf (ha tagged) and were treated with the proteasome inhibitor mg ( µm) or carrier control dmso. detergent lysates were collected and subjected to reducing sds-page and immunoblotting with anti-flag and ha antibodies. (b,d) hek t cells were co-transfected with nsp (flag tagged) and tbk or irf (ha tagged) and were treated with -ma ( mm) for further culture. at h post transfection, cell lysates were subjected to blotting with corresponding antibody. (e) alignments of the nsp orthologs from several coronaviruses and x. laevis endou. the amino acid sequence of the pedv nsp (pedv, kt ) was aligned with orthologs of severe acute respiratory syndrome coronavirus (sars, kf ), murine hepatitis virus (mhv, kf ), avian infectious bronchitis virus (aibv, nc_ ) and xenopus laevis (xendou, bc ) using dnastar software. gaps in the sequence alignment are denoted by hyphens. residues with red are the conserved residues critical for nsp activity as previously described. the number below the red residue indicates its corresponding position at pedv nsp protein, respectively. (f,g) four mutants (h a, h a, d a and k a) of pedv nsp were constructed by mutating the corresponding conserved residue mentioned in (e) into alanine using site-directed mutagenesis kit. hek t cells were then co-transfected with tbk or irf and wild-type pedv nsp , the constructed nsp mutant (h a, h a, d a or k a) or empty vector. at h post transfection, cell samples were subjected to immunoblotting with antibodies to flag, ha or β-actin (loading control). combined with the previous results, we hypothesized that pedv nsp contributed to reduction of tbk and irf expression by targeted mrna level degradation in an endou activity dependent manner. to test this hypothesis, hek t cells were co-transfected with tbk or irf and pedv nsp as well as the constructed mutants followed by quantitative analysis of tbk or irf mrna level with the primers listed in table . as shown in figure a , the relative mrna level of tbk was significantly more decreased in pedv nsp and d a transfected cells than in other mutants and empty vector transfected cells, which suggested that pedv nsp can reduce tbk expression by downregulating the tbk mrna levels dependent on its endou activity. similar results were obtained that nsp can reduce irf expression by decreasing the irf mrna levels in an endou dependent manner ( figure b ). in addition, the mechanism was further verified by northern blot assay using the specific probe as designed in table , following co-transfection with tbk or irf and pedv nsp as well as the constructed mutants in hek t cells. consistent with the quantitative results by real time pcr assay, tbk or irf rna was evidently more reduced in nsp and d a mutant transfected cells than in mutant h a, h a, k a, or empty vector control transfected cells ( figure c,d) . these data demonstrate that pedv nsp can reduce tbk and irf expression by the targeted degradation of tbk and irf mrna. table . three independent experiments were performed in triplicate, and values are the means ± sd for all three experiments. *, p < . . (c,d) hek t cells were co-transfected with tbk or irf and pedv nsp expression plasmid (pedv nsp , h a, h a, d a and k a) or empty vector. at h post transfection, rna were extracted from the collected cell samples followed by rna detection by northern blot assay using the designed specific probes (table ) as described in the materials and methods. we next investigated whether pedv nsp can directly degrade mrna of tbk and irf . to this end, wild-type and four mutant versions of pedv nsp were produced as gst-tagged proteins and purified under mild conditions by the addition of reduced glutathione to the elution buffer as the manufacture's instruction (ge healthcare life sciences). as shown in figure a , recombinant wild-type pedv nsp and four mutants (h a, h a, d a, and k a) were purified successfully at the expected molecular weight of kda by sds-page analysis. the tbk and irf sequences were amplified by pcr and subsequently cloned into the pgem-t easy vector containing a t rna polymerase promoter upstream of the multiple cloning region to construct the recombinant plasmids of pgem-t/tbk and pgem-t/irf , respectively. in vitro-transcribed rnas of tbk and irf were synthesized from the constructed recombinant dna templates (pgem-t/tbk and pgem-t/irf ) by the ribomax™ large scale rna production systems (promega, usa). the gst-purified pedv nsp proteins were incubated with the generated tbk and irf mrna as substrate in presence of mn + , a known cofactor for the endoribonuclease activity. [ , ] . it was revealed that synthesized tbk and irf mrna were effectively reduced post incubation with wild-type pedv nsp and d a mutant, but not with the other mutant derivatives (h a, h a, and k a) , demonstrating the pedv nsp can directly degrade tbk and irf mrna dependent on its endoribonuclease activity ( figure b,c) . (b,c) targeted rna was transcribed and purified in vitro as described in the materials and methods. endoribonuclease activity assay was performed by incubating the transcriptional rna (tbk or irf ) with purified recombinant pedv nsp proteins (pedv nsp , h a, h a, d a or k a) or gst tag protein as a control at • c for h followed by rna detection of tbk or irf rna as described in the materials and methods. type i ifns are transcriptionally regulated, and are induced following recognition of pathogen components during infection. tbk and irf are the key effectors during viral infections to induce ifn production [ ] . following stimulation with virus components including dsrna, irf becomes phosphorylated by the serine-threonine kinases tank-binding kinase- (tbk ) or the inducible iκb kinase (ikk-i/ikkε) [ , ] . irf- then dimerizes, translocates into the nucleus, and combines with the co-activator cbp/p to activate the expression of ifnβ [ ] . to determine whether pedv nsp modulates the phosphorylated irf , cells were co-transfected with tbk and irf along with pedv nsp as well as its mutant derivatives (h a, h a, d a, and k a) and then collected to examine the phosphorylated irf levels by western blot. as anticipated, the tbk stimulation led to the irf phosphorylation in empty vector transfected cells. however, the irf phosphorylations were significantly inhibited in nsp and d a mutant transfected cells compared to the remaining mutants (h a, h a, and k a) transfected cells, suggesting that pedv nsp impeded irf expression as well as irf phosphorylation dependent on its endou activity ( figure a ). vesicular stomatitis virus (vsv) is frequently used for the assessment assay of ifn activity [ ] . to determine the role of nsp in regulation of ifn production, hek t cells were co-transfected with tbk and irf together with wild-type nsp , individual nsp mutant or empty vector and cell supernatants were collected at h post co-transfection for determining the status of ifn secretion. ipec-j cells were treated with the supernatant followed by inoculation of vsv-gfp at moi of . . fluorescence was visualized at h post infection with an olympus inverted fluorescence microscope equipped with a camera. vsv-gfp infection was evident in treatments with supernatant from nsp and d a mutant transfected cells, and conversely vsv-gfp infection was obviously inhibited in treatments with supernatant from cells transfected with the remaining mutants (h a, h a, and k a) or empty vector, demonstrating that nsp was an antagonistic protein in ifn production ( figure b ). moreover, we continued to investigate the effects of collected supernatant on pedv infection in vero e and ipec-j cells. vero e cells were treated with the individual collected supernatant as mentioned above prior to pedv infection, cell samples were then collected and subjected to virus titration by tcid assay. pedv infection was significantly enhanced in cells treated with supernatant from wild-type nsp and d a mutant transfected cells than that from mutant h a, h a, k a or empty vector transfected cells ( figure c ), confirming that pedv nsp can evade the host antiviral response by antagonizing ifn production. meanwhile, a pedv infection assay was further performed in ipec-j cells, a target cell line for pedv infection in vivo. similar results were obtained in that pedv nsp can facilitate pedv infection, based on pedv genomic quantitation by real-time pcr assay instead of by tcid assay due to its low susceptibility to ipec-j cells ( figure d ). these data collectively demonstrate that nsp can promote pedv infection by limiting ifn secretion dependent on its endoribonuclease activity. ifn-i is the key innate immune cytokine produced by cells to trigger antiviral function [ , ] . therefore, we assessed the effect of nsp on the ifn mediated antiviral response signaling pathway. here, hek cells were co-transfected with tbk and irf together with wild-type nsp , nsp mutants, or empty vector control and cells samples were collected to investigate the effects of nsp on induction of innate antiviral molecules. quantitative rt-pcr showed that mrna levels of immune related molecules, such as ifnβ, tnfα, oas , isg , isg , and isg , were significantly disrupted by nsp and mutant d a post transfection compared to that by empty vector control. however, the disruptions were impeded by the other nsp mutants that impaired the endoribonuclease activities (figure ) , suggesting that pedv nsp restrains cellular antiviral activity and thus facilitates pedv infection. table . the results are representative of three independent experiments (mean ± sd). *, p < . . the p value is calculated using student's t-test. the host innate immune system is the first line of defense against virus invasion through production of ifns as well as various other cytokines. innate immune responses are activated through host pattern recognition receptors (prrs), which recognize pathogen-associated molecular patterns [ ] . ifns exert antiviral effects through inducing the expression of hundreds of isgs [ , , ] . however, during coevolution with their host, viruses always evolve diverse strategies to escape and even inhibit host ifn responses [ , , [ ] [ ] [ ] [ ] , ] . pedv has acquired multiple mechanisms that avoid the action of ifn by preventing the binding of viral products to cellular sensors. it was revealed that pedv n protein antagonized ifn production by preventing tbk from interaction with irf [ ] . of the several known viral evasion strategies, the cleavage of crucial innate immune molecules, including adaptors, kinases, and transcriptional factors, are considered to be a particularly powerful way for viruses to escape the innate immune response. for example, the c-like protease of pedv and porcine delta coronavirus (pdcov), disrupts type i ifn signaling by cleaving the nf-κb essential modulator (nemo) [ , ] . in addition, pdcov nsp antagonizes type i ifn signaling by cleaving stat , an essential factor for ifn responses [ ] . furthermore, the ubiquitination and deubiquitination are highly regulated post-translational modification processes in modulating the antiviral innate immune response. within the cells, polyubiquitination plays several different roles depending upon the attachment position on the target proteins, and linked polyubiquitin chains regulate the proteasomal degradation of target proteins [ , , ] . multiple ubiquitin ligases and ubiquitin-binding scaffold proteins contribute to the positive regulation of the ifn response, such as rig-i, traf , traf , and tbk . previous studies have indicated that pedv plp significantly inhibits the ubiquitination of rig-i and sting, which is essential for the activation of type i ifn signaling [ ] . meanwhile, the proteasomal degradation of target proteins for the ifn response can also be achieved by viruses through the removal of k polyubiquitin chains [ ] [ ] [ ] . pedv-induced stat degradation inhibits type i interferon signalling in a proteasome-dependent manner [ ] . however, viruses are not just limited to the mentioned strategies to antagonize ifn responses. in this study, we first identified that pedv nsp was capable of subverting the ifn response by the rna degradation of tbk and irf , which differentiated from the strategies utilized by other coronavirus orthologues previously described. the functions of nsp of coronaviruses (nsp in arteriviruses), an endoribonuclease encoded by nidoviruses, have received more attention. previous studies showed that the nsp encoded by sars-cov [ ] , mhv [ , ] , pedv [ ] , pdcov [ ] , and the nsp encoded by prrsv [ , ] can antagonize antiviral innate immune responses by utilizing the different mechanisms involved, e.g., by mediating the evasion of viral dsrna by host for mhv and hcov- e [ , ] , by suppressing both mavs and rig-i expression for prrsv [ ] , by impairing the activation of transcription factor nf-κb for pdcov [ ] , or by inhibiting mavs-induced apoptosis for sars-cov [ ] . pedv nsp of is a -residue polypeptide that results from the cleavage of pp ab at sites nlq↓gle and qlq↓ase by the main protease nsp . several recent studies have focused on the structural and functional characterization of coronavirus nsp due to its potential importance as a drug target. it has been reported that the endou activity of pedv nsp is not required for pedv replication in vero cells. however, the endou activity is involved in the suppression of host ifn response in epithelial cells and macrophages in vitro, and subsequently can facilitate pathogenesis development in vivo by enhancing viral replication and shedding [ ] . although previous studies have reported pedv nsp as a key virulence factor that suppressed ifn responses in vitro and facilitated pedv replication, the underlying mechanism remains unknown. in this study, we found that endogenous tbk and irf were downregulated post pedv by previous itraq assay and western blot analysis. nsp was selected as the investigation candidate due to its evident effect on tbk and irf reduction post co-transfection with each viral encoded protein. it was exhibited that pedv nsp was capable of downregulating the expression of tbk and irf proteins by the degradation of the rna of tbk and irf in an endoribonuclease activity dependent manner, while residues of h a, h a, and k a were critical for the endoribonuclease activity of pedv nsp , but not d a. the reason that results in these differences remains unclear. whether these structure differences result in different mechanisms used to antagonize ifn production remains a subject of further study (figure ) , elucidating a novel antagonistic mechanism utilized by pedv to counter the antiviral response. in summary, our data reveal that pedv nsp acts as an ifn antagonist to inhibit immune response by the rna level degradation of tbk and irf , key ingredients involved in the ifn signaling pathway dependent on its 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porcine epidemic diarrhea virus and the role of viral protein nsp in irf signaling porcine epidemic diarrhea virus-induced epidermal growth factor receptor activation impairs the antiviral activity of type i interferon tight junction protein occludin is a porcine epidemic diarrhea virus entry factor major genetic marker of nidoviruses encodes a replicative endoribonuclease modulation of cd expression by metalloprotease adam regulates porcine reproductive and respiratory syndrome virus entry analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method itraq-based proteomic and bioinformatic characterization of human mast cells upon infection by the influenza a virus strains h n and h n autophagy fights disease through cellular self-digestion the v protein of simian virus inhibits interferon signalling by targeting stat for proteasome-mediated degradation hepatitis c virus expression suppresses interferon signaling by degrading stat the ubiquitin-proteasome pathway is required for processing the nf-kappa b precursor protein and the activation of nf-kappa b -methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes mutational analysis of the sars virus nsp endoribonuclease: identification of residues affecting hexamer formation rna recognition and cleavage by the sars coronavirus endoribonuclease crystal structure and mechanistic determinants of sars coronavirus nonstructural protein define an endoribonuclease family coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type i and type iii interferon responses purification, cloning, and characterization of xendou, a novel endoribonuclease involved in processing of intron-encoded small nucleolar rnas in xenopus laevis ten strategies of interferon evasion by viruses ikkepsilon and tbk are essential components of the irf signaling pathway ifn-regulatory factor -dependent gene expression is defective in tbk -deficient mouse embryonic fibroblasts establishing a safe, rapid, convenient and low-cost antiviral assay of interferon bioactivity based on recombinant vsv expressing gfp immunomodulatory functions of type i interferons regulation of type i interferon responses how cells respond to interferons a positive feedback amplifier circuit that regulates interferon (ifn)-stimulated gene expression and controls type i and type ii ifn responses ifnbeta-dependent increases in stat , stat , and irf mediate resistance to viruses and dna damage rna-virus proteases counteracting host innate immunity porcine deltacoronavirus nsp inhibits interferon-beta production through the cleavage of nemo the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase ubiquitin-mediated modulation of the cytoplasmic viral rna sensor rig-i pvhl negatively regulates antiviral signaling by targeting mavs for proteasomal degradation induction of otud by viral infection promotes antiviral responses through deubiquitinating and stabilizing mavs early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication coronavirus nonstructural protein mediates evasion of dsrna sensors and limits apoptosis in macrophages porcine deltacoronavirus nsp antagonizes interferon-beta production independently of its endoribonuclease activity porcine reproductive and respiratory syndrome virus nsp antagonizes type i interferon signaling by targeting irf nonstructural protein of porcine reproductive and respiratory syndrome virus suppresses both mavs and rig-i expression as one of the mechanisms to antagonize type i interferon production mavs-mediated apoptosis and its inhibition by viral proteins this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we want to thank jin tian for his excellent technical assistance on this project. the authors declare no conflict of interest. key: cord- -c ftvgio authors: mackay, ian m.; arden, katherine e.; speicher, david j.; o’neil, nicholas t.; mcerlean, peter k.; greer, ristan m.; nissen, michael d.; sloots, theo p. title: co-circulation of four human coronaviruses (hcovs) in queensland children with acute respiratory tract illnesses in date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: c ftvgio acute respiratory illnesses (aris) with unconfirmed infectious aetiologies peak at different times of the year. molecular diagnostic assays reduce the number of unconfirmed aris compared to serology- or culture-based techniques. screening of inpatient and outpatient respiratory specimens spanning late autumn through to early spring, , identified the presence of a human coronavirus (hcov) on occasions ( . % of all specimens and . % of all respiratory virus detections). prevalence peaked in august (late winter in the southern hemisphere) when they were detected in . % of specimens tested. hcov-hku and hcov-oc comprised . % of all hcovs detected. positive specimens were used to develop novel reverse transcriptase real-time pcrs (rt-rtpcrs) for hcov detection. an objective clinical severity score was assigned to each positive hcov patient. severity scores were similar to those from a random selection of young children who were positive for respiratory syncytial virus at a different time but from the same specimen population. during the cooler months of , sensitive and specific rt-rtpcrs identified the concurrent circulation of all four hcovs, a quarter of which co-occurred with another virus and most of which were from children under the age of two years. acute respiratory illnesses (aris) are a frequent cause of paediatric morbidity and a common reason for outpatient visits and hospitalisations. among children, rna viruses are the most frequent cause of "colds" and "influenza-like" illness (ilis); usually self-limiting upper respiratory tract illnesses (urtis) [ ] . virus detections are also often associated with lower respiratory tract illness (lrti; [ ] ) although their replication in these tissues is seldom identified. human rhinoviruses (hrv), respiratory syncytial virus (hrsv), influenzaviruses (ifvs) adenoviruses (hadv), metapneumovirus (hmpv) and parainfluenza viruses (hpiv) are among the most frequently sought respiratory viruses in the clinical microbiology laboratory [ ] [ ] [ ] [ ] [ ] . many peak at distinct times of the year. however, even when bacterial pathogens are added to this viral panel, - % of suspected infections remain without laboratory confirmation [ ] [ ] [ ] . the inclusion of more viruses into the diagnostic algorithm is recommended to support and confirm a clinically diagnosed infectious aetiology [ ] . nonetheless, extended testing panels are most often used by research projects and the human coronaviruses (hcovs) are one group of pathogens often overlooked by routine testing. two of the enveloped positive sense rna hcovs, hcov- e and hcov-oc , have been known for more than years [ ; ] . infections by these two viruses can be difficult to distinguish from ili in a population vaccinated for ifv [ ] . both hcovs have also been identified in non-respiratory specimens [ ] . recently three new hcovs, all detected in patients with aris were described; the severe acute respiratory syndrome coronavirus (sars-cov) in , hcov-nl in and hcov-hku in [ ] [ ] [ ] . some studies have noted that genus alphacoronavirus species hcov-nl and hcov- e and the genus betacoronavirus species hcov-hku and subspecies hcov-oc may circulate annually, but the prevalence varies significantly [ ] . only hcov-hku has a seroprevalence below % in adults; the reason for this apparently reduced exposure may be related to a cross-protective antibody effect afforded by prior infection with hcov-oc [ ; ] . rt-pcr methods more frequently detect hcovs than in vitro culture because they are difficult to grow without a source of primary tissue [ ] [ ] [ ] . studies that definitively link the presence of viral rna to human disease, akin to those conducted using infection of human volunteers are lacking. this has hampered the assignment of specific disease associations for these and other newly identified respiratory viruses. screening for all hcovs in a multi-year population sampling is infrequent so studies that directly compare the impact of hcov infections are rare [ ; - ] . despite a sizable historical role in the common cold [ ] the hcovs are also found in patients with more severe cases of ari [ ; - ] including lrti and pneumonia in adults and the elderly [ ; ; ] . hcovs are also found in cases of bronchial hyper-responsiveness in susceptible individuals [ ; ] and nosocomial respiratory viral infections [ ] [ ] [ ] . we have previously identified instances of hcov-hku from samples collected during the winter of [ ] . here we expanded upon that investigation, using newly designed reverse transcriptase real-time pcr (rt-rtpcr) assays to screen for all four non-sars-covs in a hospital-based, predominantly paediatric population with ari. the clinical status of patients with single and multiple hcov detection was also compared. we expanded our previous study of specimens using pcr assays to screen for hcovs. we previously identified that a pan-hcov rt-pcr often failed to detect hcov-hku and hcov-oc although it was useful for hcov- e and hcov-nl detection [ ; ] . we therefore included a specific hcov-hku assay [ ] and developed an rt-rtpcr for hcov-oc detection (this study). when enough specimen extract remained, positives were confirmed using nucleotide sequencing ( figure ). newly developed hcov-hku , hcov-nl and hcov- e rt-rtpcrs confirmed previous and new (table ; this study) conventional rt-pcr hcov positives [ ; ] . previous sequencing of the b region had confirmed hcov identity where specimen remained [ ; ] . the rt-rtpcrs did not produce any positive results (defined in the experimental section) when amplifying clinical samples positive for hrsv (n = ), hadvs (n = ), hrvs (n = ) ifvs (n = ) or hpiv-positive (n = ) specimens. the hcov rt-rtpcrs have since been used in several other studies (including many more positives for each of these respiratory viruses listed) and no cross-reactions have been noted. the analytical sensitivity of each assay was determined to be  copies ivtrna/ μl reaction. overall instances of a coronavirus ( . % of tested specimens) were detected. the majority of which were hcov-hku (n = ; . % of all hcov), followed by hcov-oc (n = ; . %), hcov-nl (n = ; . %) and hcov- e (n = ; . %). hcov detection peaked in late winter (august in the southern hemisphere) when sample numbers were lowest ( figure ). most ( . %) of the hcov detections were from patients two years old or less with children (age  years) comprising . % of the positive patients. hcov types were equally distributed between males and females except for hcov-nl which was only detected in males (p = . ; % confidence interval). hcov-oc was detected in all age groups, was the only hcov detected in neonates (n = ) and was detected in adults ( . % of all viruses detected in patients over years old) more than any other respiratory virus ( . %). the average age of positive patients was highest for hcov-oc among the four hcovs ( figure ; table ). each hcov was involved in more co-detections than the average level of co-detections for the study population ( . %; table ) and more frequently (hcov- e, . %; hcov-oc , . %; hcov-hku , . %) than most other respiratory viruses sought (hrsv, . %; hadv, . %; hpiv , . %; hmpv, . %). clinical reviews were obtained on hcov positives, representing patients ( . % of hcov detections; table ). in one patient both hcov-hku and hcov-oc were co-detected (sequencing confirmed that both viruses were present; data not shown). this male toddler ( months old), also positive for hmpv, had been admitted three months prior with bronchiolitis. for the current presentation, following seven days of symptoms including cough, otitis media, a measurable fever and rhinorrhoea, salbutamol and antibiotics were administered. the average clinical severity score among patients positive for any hcov was . with cases ( . % of hcov-positive reviews) admitted to hospital ( were only positive for an hcov). of these admissions, ( . %) remained for  hours, with of these sole hcov detections. there was no difference in severity score between the four hcov groups, either between samples with a single detection and co-detections considered together, p = . , or between those with single detections, p = . , or those with co-detections, p = . (kruskal-wallis test). the highest average severity score ( . ) was observed from hcov-nl -positive patients who were also positive for another virus. most hcov cases met gaunt et al.'s criteria [ ] for urti (n = ; . %) or lrti (n = ; . %; table ). data could not be obtained for four ( . %) hcov-positive cases. antibiotics were given to cases ( . %). hcov-hku detections, whether single or co-detections, were most often made from patients with urti or lrti ( . %; figure ). most single hcov-oc detections were in patients with chronic disease ( . % of detections). most hcov- e detections were accompanied by another virus and no hcov-nl detections were made in chronically diseased or immunocompromised patients. [ ] . the number of hcov detections is shown to the left of each bar. ic-immunocompromised; lrti-lower respiratory tract illness; urti-upper respiratory tract illness. the most commonly noted additional clinical features among patients only positive for a hcov (n = ; . % of hcov cases) were cough (n = ; . %), fever (n = ; . %) and vomiting (n = ; . %; table ). among hcov co-detections the most common features were otitis media (n = ; . % of hcov cases), cough (n = ; . %), fever (n = ; . %) and vomiting (n = ; . % of hcov-positives). in the comparison population of hrsv-positive children whose charts were reviewed the average severity score was also . . the obvious differences between hrsv and hcov hospitalisations were that slightly fewer hrsv cases were admitted than in the hcov group ( . % vs. . %), hospitalisation times were shorter ( . % vs. . % inpatients at hours) and fewer patients required mechanical ventilation ( . % vs. . %). however, more hrsv-positive children had a measurable fever (n = ; % compared to . % of hcov-positive patients). table . clinical features of patients positive for an hcov. the study comprised respiratory specimens from individuals who had presented to queensland hospitals with signs and symptoms of ari during may to september . this included previously described specimens [ ] . specimens were predominantly nasopharyngeal aspirates (npa; . %) collected either from outpatients or admitted patients. specimens were selected by season, without prior knowledge of patient details or viral diagnostic status. the subjects ranged in age from three days to . years (mean = . years, median = . years, mode = . years), with infants (one to twelve months old) comprising . % of the study population. prior to this retrospective study, nucleic acids had been extracted from specimens, tested for common respiratory viral pathogens and stored at - c. the clinical microbiology laboratory assays included culture-amplified direct fluorescent assay and subsequent pcrs to detect hrsv, hadv, hpiv- , and and influenza viruses a & b (ifav, ifbv) [ ] . an additional pcr assay was used to detect hmpv [ ] . no virus was detected in specimens ( . %). additional rt-rtpcr testing for human rhinoviruses (hrvs) was based on a modified, previously described method [ ; ] , only applied to the hcov-positive samples. the rt-pcrs used to initially screen specimens included a mix of conventional (for hcov- e, hcov-nl and hcov-hku ; described previously [ ; ] ) and rt-rtpcrs (hcov-oc ; described in this study). the positives patients from these assays were subject to retrospective medical chart review. the positive specimens, when extract remained, were used for the validation of newly designed rt-rtpcrs for the detection of hcov- e, hcov-nl and hcov-hku . single-target rt-rtpcr assays were developed to maximise sensitivity, using in-house oligonucleotides specific to hcov- e, hcov-hku , hcov-oc or hcov-nl (table ) . reaction mixes were combined with μl of purified rna and subjected to rt (onestep rt-pcr kit, qiagen, australia) for min at c followed by a min incubation at c. pcr was performed for cycles of c for sec, c for sec (rotor-gene , or q, qiagen, australia). synthetic t -tagged hcov-specific in vitro transcribed rnas (ivtrnas) were created by adapting a previously described approach [ ] . we used oligonucleotides which bound at (hcov-hku ) or outside (all other hcovs) the diagnostic rt-rtpcr target area (t oligonucleotides; table ). rna was subjected to two treatments with dnase (turbo, life technologies) followed by column purification (high pure viral nucleic acid kit, roche diagnostics). these stock rnas were used to determine the analytical sensitivity of each assay after testing a -fold dilution series. the last dilution to yield a positive result (defined below) was taken as the limit of analytical sensitivity. the ivtrna copy numbers were calculated using the optical density to approximate the mass of rna in the stock solution at nm, determining the number of moles by establishing the molecular weight of each hcov-specific ivtrna and then multiplying by . × (avogadro's number). a positive diagnostic result was defined by the presence of a sigmoidal curve that crossed an arbitrary threshold of . , before cycles, during an experiment in which duplicate non-template controls did not cross the threshold. nucleotide sequencing reactions were performed using μl of amplicon with the abi prism™ bigdye cycle sequencing kit (perkin elmer applied biosystems division, usa). sequences were determined using an applied biosystems xl capillary electrophoresis genetic analyser. nucleotide sequences were aligned using geneious pro v [ ] and presented in a neighbourjoining tree prepared in mega [ ] . nucleotide sequences deposited into genbank as a result of this study include jq -jq . medical records of patients with specimens positive for hcov by conventional rt-pcrs [ ; ] were reviewed to determine the severity of the ari using a validated, objective scoring system [ ] . the severity score is based on the patient's need for hospitalisation, supplemental fluids and degree of respiratory support. if other clinical information such as cough, wheeze, fever and pre-existing conditions was documented, this was also recorded. a secondary dataset of clinical information (n = ) was also collected from age-matched patients (less than three years of age) positive for hrsv who had also presented with ari between july and december . we retrospectively investigated an australian paediatric, hospital-based winter population for the presence of hcovs and developed four novel rt-rtpcrs. studies identifying the co-circulation of all four non-sars-covs are uncommon [ ; - ] . a paucity of such data hinders direct comparison of the epidemiology and clinical features of individuals infected by the different hcovs. some assays designed to detect all four hcovs are too insensitive for practical use in a clinical microbiology laboratory [ ; ] . others, using similar measures of assay sensitivity to those employed here, have proven sensitive and specific when used to screen focused or hospitalized disease populations [ ; ; ] . the appearance of commercial assay alternatives is progressing but large scale or routine use is rare [ ] . the incidence of . % in our samples is higher than reported by a three-year study (< % prevalence; [ ] ) and among those hospitalized with pneumonia or ari and/or fever [ ; ; ] reflecting our more focused winter study period and mixed inpatient and outpatient hospital population; the known period of peak hcov prevalence. similar to others, we observed a high number of co-detections ( . - . %) among the hcovs compared to the overall study population ( . %) [ ; ] . others have commented that hcovs impart a limited impact due to their low prevalence, relatively higher proportion of involvement in co-detections and low secondary attack rate in households [ ] . our retrospective study also observed that clinical features and the average severity scores from patients with hcov were very close to those from a similarly aged set of children who were positive for hrsv. hcov-oc was detected in patients with a range of clinical conditions but predominated among those with chronic conditions and manifested as both urti and lrti. hcov-hku dominated detections overall and specifically in those with urti, lrti and in those with vomiting, cough, fever and otitis media. our findings are similar to those from hong kong where hcov-hku , previously identified in patients with pneumonia, was associated with fever and febrile convulsion [ ] . however, hcov-hku whether as a single or co-detection had the lowest average severity score, a metric that was focussed on respiratory symptoms. dijkman et al. proposed that hcov-oc seroconversions dominated those of hcov-hku (and that hcov-nl dominated hcov- e) because of cross-protective neutralizing antibodies elicited by the first infection with hcov-oc or hcov-nl [ ] . our study found that most hcov-hku detections (peaking in august) were made after the hcov-oc detections (peaking in july). although the numbers in our winter study were smaller than those of dijkman et al.'s and the differences in peak detection were not great, our testing of all inpatient and outpatient samples from queensland hospital presentations between may and september may have better resolved and reflected hcov activity than did the outcome of a five-year average of testing results from paediatric inpatients below the age of two years [ ] . extending serosurveys to other populations may resolve this discrepancy. neither hcov-nl nor hcov- e occurred in patients with rash, immunocompromise, diarrhoea or vomiting or in those with chronic disease. hcov-nl was most common among those with cough (including croup and prolonged cough) and lrti which is in keeping with its association with croup [ ] . vomiting and diarrhoea occurred most often in hcov-hku cases supporting other recent findings for this hcov [ ] . several study limitations should be highlighted. this population does not represent clinically well subjects (i.e., those who do not feel ill enough to warrant a visit to their hospital or outpatients clinic) so no conclusions about the impact of these viruses in the community can be drawn beyond the extrapolation that because our patient population is sampled from the community, it is reasonable to assume that these viruses have been circulating concurrently. it is additionally worth noting that all respiratory viruses have so far been found to some extent in clinically well populations. this finding does not bestow unimportance or reduced pathogenicity upon the viruses, just as a statistically significant higher proportion in the number of detections in an ill population versus a control group cannot guarantee causality upon the virus. because human immunity plays a significant regulatory role in every virus challenge a control population is only the first of many steps to identify a disease association. another useful step is a birth cohort followed for several years and sampled at regular short intervals regardless of disease state. we have such a cohort underway. because this is a cross-sectional study, we could not define what was happening to the hcovs during the "off-season". we did not test all specimens for bocaviruses or hrvs so the overall number of co-detections may be under-reported. we did not expect the predominance of males among the hcov-nl cases, a pattern of gender distribution which differs from the other hcovs. it is possible this is a type error associated with multiple tests. however, as there were only four tests and a small p-value, it is possible this indicates a true virus effect, which requires further investigation for confirmation. hcov-hku was the most frequently detected hcov and involved in the broadest array of clinical outcomes. hcov-oc should be considered in the diagnosis of acute adult respiratory disease while hcov-nl may play a role in more severe disease involving multiple viruses. because hcov- e occurs infrequently and mostly in co-detections, its role in aris beyond the common cold remains hard to define. despite reports of a role for hcov- e in ari in immunocompromised patients [ ] , we did not have many cases of immunocompromise in our population and so we may have missed this role in our study. we have presented the design and application of four sensitive, specific and novel rt-rtpcrs that allow us to differentiate between peak hcov prevalence and that of other circulating respiratory viruses. we observed hcov-specific differences in co-detection frequencies, monthly peak prevalence and the sex of the positive patients. even though many differences were noted in this cross-sectional study, future respiratory virus studies are needed to confirm and better define these differences through frequent analysis 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fever, cough and wheeze during real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses newly identified human rhinoviruses: molecular methods heat up the cold viruses frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections a simple method for preparing synthetic controls for conventional and real-time pcr for the identification of endemic and exotic disease agents bioedit: a user-friendly biological sequence alignment editor and analysis program for windows / /nt mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods design and validation of consensus-degenerate hybrid oligonucleotide primers for broad and sensitive detection of corona-and toroviruses human coronavirus infections in rural thailand: a comprehensive study using real-time reversetranscription polymerase chain reaction assays human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls comparison of the filmarray respiratory panel and prodesse real-time pcr assays for the detection of respiratory pathogens impact of human coronavirus infections in otherwise healthy children who attended an emergency department human coronaviruses are uncommon in patients with gastrointestinal illness the study was funded by queensland children's medical research institute project grants # and # sponsored by the children's hospital foundation queensland. the project was approved by the human research ethics committee, queensland children's health services, queensland, australia (#hrec/ /qrch/ and hrec/ /qrch/ ) and the medical research ethics committee, university of queensland (# and # ). the authors do not have a commercial or other association that might pose a conflict of interest. key: cord- -cik wmlk authors: ban, junsu; lee, na-rae; lee, noh-jin; lee, jong kil; quan, fu-shi; inn, kyung-soo title: human respiratory syncytial virus ns targets trim to suppress rig-i ubiquitination and subsequent rig-i-mediated antiviral signaling date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: cik wmlk respiratory syncytial virus (rsv) causes severe acute lower respiratory tract disease. retinoic acid-inducible gene-i (rig-i) serves as an innate immune sensor and triggers antiviral responses upon recognizing viral infections including rsv. since tripartite motif-containing protein (trim )-mediated k -polyubiquitination is crucial for rig-i activation, several viruses target initial rig-i activation through ubiquitination. rsv ns and ns have been shown to interfere with rig-i-mediated antiviral signaling. in this study, we explored the possibility that ns suppresses rig-i-mediated antiviral signaling by targeting trim . ubiquitination of ectopically expressed rig-i- cards domain was decreased by rsv infection, indicating that rsv possesses ability to inhibit trim -mediated rig-i ubiquitination. similarly, ectopic expression of ns sufficiently suppressed trim -mediated rig-i ubiquitination. furthermore, interaction between ns and trim was detected by a co-immunoprecipitation assay. further biochemical assays showed that the spry domain of trim , which is responsible for interaction with rig-i, interacted sufficiently with ns . suppression of rig-i ubiquitination by ns resulted in decreased interaction between rig-i and its downstream molecule, mavs. the suppressive effect of ns on rig-i signaling could be abrogated by overexpression of trim . collectively, this study suggests that rsv ns interacts with trim and interferes with rig-i ubiquitination to suppress type-i interferon signaling. respiratory syncytial virus (rsv) belongs to the family pneumoviridae and contains a negative-sense single-stranded rna genome. rsv infection is a leading cause of severe acute lower respiratory tract disease and related hospitalization in children and the elderly [ , ] . despite the global burden from rsv infection, there are no available rsv-specific vaccines or effective therapeutic agents at present. the cellular innate immune system utilizes various sensors including retinoic acid inducible gene-i (rig-i) and toll-like receptors (tlrs) to detect viral infection and activate antiviral immune signaling pathways [ , ] . among these, rig-i and toll-like receptor (tlr ) have been implicated in early antiviral immune responses against rsv infection in airway epithelial cells [ ] . deficiency cells were collected and lysed in triton x- cell lysis buffer ( mm tris-hcl, mm nacl, mm edta, . % triton x- ) containing protease and phosphatase inhibitor cocktail (thermo scientific, waltham, ma, usa). lysates were incubated overnight at • c with the corresponding antibody. after the binding reaction, protein a/g resin (sigma, st. louis, mo, usa) were added to samples and further incubated for h at room temperature. the resin was then washed four to five times with cell lysis buffer. anti-flag antibody conjugated resin (sigma) and anti-v antibody conjugated resin (sigma) were also used for co-ip in a similar manner. precipitated proteins were eluted with sds sample buffer ( mm tris-hcl, % sds, % glycerol, % mercaptoethanol, . % bromophenol blue) and subjected to sds-page and immunoblotting. to precipitate gst-tagged fusion proteins, the cell lysates were incubated with glutathioneconjugated beads (sigma) at rt for - h. after incubation, the beads were washed four to five times with cell lysis buffer, followed by sds-page and immunoblotting. after polyacrylamide gel electrophoresis, target proteins on the gel were transferred to a pvdf membrane (millipore, st. charles, mo, usa). the membrane was incubated overnight at • c with the indicated antibody in % bovine serum albumin (bsa, rmbio, missoula, mt, usa) solution for binding. after extensive washing with pbst, the membrane was then incubated with the horseradish peroxidase (hrp) conjugated secondary antibody at rt for h. luminata forte (millipore) was used as the hrp substrate. anti-v rabbit antibody (cell signaling, denvers, ma, usa, # ), anti-v mouse antibody (e-bioscience, san diego, ca, usa, # - - ), anti-ha mouse antibody (santacruz, santa cruz, ca, usa, #sc- ), anti-flag mouse antibody (sigma, #f ), anti-ubiquitin (p d ) mouse antibody (cell signaling, # ), anti-gst antibody (abcam, cambridge, uk, # ) and hrp conjugated antibodies (cell signaling, # , # ) were used. hek t cells in -well plates were transfected with interferon-β firefly luciferase ( . µg/well) and thymidine kinase (tk) renilla reporter plasmids ( . µg/well) were transfected along with other constructs as indicated [ ] . after h, transfected hek t cells were lysed in passive lysis buffer (promega, madison, wi, usa) at rt for min. cell lysates were then analyzed by a dual-luciferase assay according to the instruction (promega). all assays were performed in triplicate and repeated at least three times. total rnas were extracted using trizol (thermo scientific, # ) according to the instruction. cdnas were generated from rnas ( µg) using superscript iii reverse transcriptase (thermo scientific, # ) and oligo (dt) primers. real-time pcr was conducted using synthesized cdna ( µl). the mrna levels of interferon-β and interferon-stimulated gene (isg ) were determined using the following primers and normalized to those of β-actin: interferon-β-forward; -aagagttacactgcctttgccatc- , interferon-β-reverse; -cactgtctgctggtggagttcatc- , isg -forward; -cctctgagcatcctggt- , isg -reverse; -aggccgtactcccccag- , β-actin-forward; -tggaatcctgtggcatccatgaaac- , β-actin-reverse; -taaaacgcagctcagtaacagtccg- . hek t cells were seeded onto fibronectin coated coverslips in a -well plate. after h, cells were transfected with flag-tagged rsv ns and v -tagged trim and incubated for h. then, cells were fixed with % paraformaldehyde followed by blocking and permeabilization in viruses , , of permeabilization buffer ( . % bsa, . % triton x- in pbs) at rt in min. the cells were incubated with anti-flag mouse and anti-v rabbit antibodies in . % bsa solution at • c overnight. next day, the cells were extensively washed and incubated with fitc-and pe-labeled secondary antibodies at rt for h. the co-localization between rsv ns -flag and trim -v was analyzed by nanoscope k -fluo confocal microscope. data were presented as the mean ± sem. statistical comparisons between the control and treated groups were performed using the student's t-test. a value of p ≤ . was considered to be significant. to confirm the suppression of rig-i-mediated signaling by ns , rsv ns was transfected along with constitutively active rig-i- cards (rig-in). ectopically expressed ns inhibited interferon-β promoter activity that was induced by rig-in as determined by the luciferase assays in hek t cells, confirming that ns itself is capable of inhibiting rig-i-mediated antiviral signaling ( figure a ). consistently, rig-in-mediated induction of interferon-β and isg mrna synthesis was significantly hampered by ectopic expression of ns ( figure b ). similar results were obtained from experimental settings using a and hep- cells, suggesting that ns is able to suppress rig-i-mediated type-i interferon production in various cells including airway epithelial cells ( figure c ,d). incubated with anti-flag mouse and anti-v rabbit antibodies in . % bsa solution at ·°c overnight. next day, the cells were extensively washed and incubated with fitc-and pe-labeled secondary antibodies at rt for h. the co-localization between rsv ns -flag and trim -v was analyzed by nanoscope k -fluo confocal microscope. data were presented as the mean ± sem. statistical comparisons between the control and treated groups were performed using the student's t-test. a value of p ≤ . was considered to be significant. to confirm the suppression of rig-i-mediated signaling by ns , rsv ns was transfected along with constitutively active rig-i- cards (rig-in). ectopically expressed ns inhibited interferon-β promoter activity that was induced by rig-in as determined by the luciferase assays in hek t cells, confirming that ns itself is capable of inhibiting rig-i-mediated antiviral signaling ( figure a ). consistently, rig-in-mediated induction of interferon-β and isg mrna synthesis was significantly hampered by ectopic expression of ns ( figure b ). similar results were obtained from experimental settings using a and hep- cells, suggesting that ns is able to suppress rig-imediated type-i interferon production in various cells including airway epithelial cells (figs. c and d). because trim -mediated rig-i card ubiquitination is an essential step for the successful induction of rig-i-mediated antiviral responses, we explored the possibility that rsv suppresses rig-i activation by inhibiting the trim -mediated rig-ubiquitination. first, we have tested whether rig-i ubiquitination is suppressed by rsv infection. as seen in figure a , ubiquitination of ectopically expressed gst-rig-in is decreased by the presence of rsv (multiplicity of infection = ), suggesting that rsv is capable of suppressing rig-i ubiquitination to modulate rig-i signaling. next, we have tested whether rig-i ubiquitination is affected by the presence of ns . as shown in figure b , both ns and ns suppressed the trim -mediated ubiquitination of rig-in. increasing amounts of ns resulted in an augmented effect on reducing the ubiquitinated form of rig-in ( figure c ). the results clearly showed that ns is capable of interfering with trim -mediated rig-i ubiquitination to suppress rig-i-mediated interferon production. were repeated at least three times. the results show the most representative data from a single experiment conducted in triplicate. because trim -mediated rig-i card ubiquitination is an essential step for the successful induction of rig-i-mediated antiviral responses, we explored the possibility that rsv suppresses rig-i activation by inhibiting the trim -mediated rig-ubiquitination. first, we have tested whether rig-i ubiquitination is suppressed by rsv infection. as seen in figure a , ubiquitination of ectopically expressed gst-rig-in is decreased by the presence of rsv (multiplicity of infection = ), suggesting that rsv is capable of suppressing rig-i ubiquitination to modulate rig-i signaling. next, we have tested whether rig-i ubiquitination is affected by the presence of ns . as shown in figure b , both ns and ns suppressed the trim -mediated ubiquitination of rig-in. increasing amounts of ns resulted in an augmented effect on reducing the ubiquitinated form of rig-in ( figure c ). the results clearly showed that ns is capable of interfering with trim -mediated rig-i ubiquitination to suppress rig-i-mediated interferon production. interaction between rig-in and ns was examined using a gst-pulldown assay to test whether ns interacts with rig-in similar to ns . however, we could not detect any interaction (data not interaction between rig-in and ns was examined using a gst-pulldown assay to test whether ns interacts with rig-in similar to ns . however, we could not detect any interaction (data not shown). therefore, interaction between trim and ns was investigated to determine whether ns targets trim to suppress rig-i ubiquitination. obvious interaction between ectopically expressed viruses , , of trim and ns was detected by the co-immunoprecipitation assay, whereas interaction between ns and trim was not detected ( figure a) . furthermore, endogenous trim was also co-precipitated with ns , supporting that rsv ns targets trim ( figure b ). co-localization of ns and trim in the cytoplasm was detected by confocal microscopic observation ( figure c ). to further dissect the interaction between ns and trim , we determined the domain of trim that is responsible for this interaction using truncated trim domains. flag-tagged ns was expressed along with the ring domain, b-box/ccd domain, or spry domain of trim and co-immunoprecipitation assays were performed. as depicted in figure d , the spry domain sufficiently interacts with ns , whereas the ring or b-box/ccd domains do not show any interaction. in addition, the spry deleted trim mutant failed to interact with ns , indicating that the spry domain is required for interaction with ns ( figure e ). figure a , figure a ). to further confirm the effect of ns on rig-i activation, the effect of ns ectopic expression on rig-in interaction with mavs was examined. flag-tagged mavs-card-prd and gst-tagged rig-in was co-expressed with increasing amounts of v -tagged ns followed by a co-immunoprecipitation assay using an anti-flag antibody. as seen in figure b ,c, the clear interaction between rig-in and mavs-card-prd was decreased by ns expression in a dose-dependent manner. these results suggest that rsv ns expression diminishes the interaction between rig-i and mavs by interfering with trim -mediated rig-i ubiquitination. since trim oligomerization is crucial for its e -ligase activity, it has been tested whether ns suppresses trim e -ligase activity by interfering the oligomerization. as seen in figure d , expression of ns did not affect the interaction between interaction between ha-trim and v -trim was analyzed by co-ip and immunoblotting using indicated antibodies. all experiments were conducted at least three times with similar results. to confirm that interaction between ns and trim contributes to the interferon-suppressive effect of ns , the effect of trim overexpression on the suppression of rig-i signaling by ns was investigated using interferon-β luciferase promoter assays. as shown in figure a , activation of interferon-β promoter activity by polyi:c transfection was suppressed by ns expression. the effect of ns was abrogated by the overexpression of trim ( figure a) . similarly, suppression of rig-in-induced interferon-β promoter activity by ns was also reversed by the ectopic expression of trim ( figure b ). these results indicate that ns interaction with trim contributes to its rig-i suppressive activity. co-ip and immunoblotting using indicated antibodies. all experiments were conducted at least three times with similar results. to confirm that interaction between ns and trim contributes to the interferon-suppressive effect of ns , the effect of trim overexpression on the suppression of rig-i signaling by ns was investigated using interferon-β luciferase promoter assays. as shown in figure a , activation of interferon-β promoter activity by polyi:c transfection was suppressed by ns expression. the effect of ns was abrogated by the overexpression of trim ( figure a) . similarly, suppression of rig-in-induced interferon-β promoter activity by ns was also reversed by the ectopic expression of trim ( figure b ). these results indicate that ns interaction with trim contributes to its rig-i suppressive activity. many viruses possess defensive mechanisms against the host immune system for their effective replication. the ns and ns proteins of rsv have been shown to effectively inhibit the host immune system. ns proteins target diverse proteins related to type-i interferon induction and signal transduction. for example, rsv ns upregulates socs and socs and triggers stat degradation [ ] . rsv ns also inhibits the interferon alpha response by targeting the interferon alpha receptor [ ] . ns also degrades stat to downregulate interferon-mediated jak-stat signaling responses [ ] . in addition, ns and ns exert suppressive activities on the rig-i-mediated antiviral signaling pathway, which is a crucial response against rsv infection. previous studies suggest that ns and ns inhibit rig-i-mediated signaling by inducing degradation of key molecules such as irf / [ , ] . in this study, we explored the possibility that these proteins interfere with rig-i signaling by directly inhibiting rig-i activation. indeed, we could demonstrate that ns and ns suppressed trim -mediated ubiquitination of rig-i, which is a crucial step for rig-i activation. previously, ns has been shown to interact with rig-i, whereas the interaction between ns and rig-i could not be detected [ ] . thus, suppression of rig-i ubiquitination by ns may be due to its interaction with many viruses possess defensive mechanisms against the host immune system for their effective replication. the ns and ns proteins of rsv have been shown to effectively inhibit the host immune system. ns proteins target diverse proteins related to type-i interferon induction and signal transduction. for example, rsv ns upregulates socs and socs and triggers stat degradation [ ] . rsv ns also inhibits the interferon alpha response by targeting the interferon alpha receptor [ ] . ns also degrades stat to downregulate interferon-mediated jak-stat signaling responses [ ] . in addition, ns and ns exert suppressive activities on the rig-i-mediated antiviral signaling pathway, which is a crucial response against rsv infection. previous studies suggest that ns and ns inhibit rig-i-mediated signaling by inducing degradation of key molecules such as irf / [ , ] . in this study, we explored the possibility that these proteins interfere with rig-i signaling by directly inhibiting rig-i activation. indeed, we could demonstrate that ns and ns suppressed trim -mediated ubiquitination of rig-i, which is a crucial step for rig-i activation. previously, ns has been shown to interact with rig-i, whereas the interaction between ns and rig-i could not be detected [ ] . thus, suppression of rig-i ubiquitination by ns may be due to its interaction with rig-i. we also could not detect interaction between ns and rig-i, indicating that ns may utilize a separate molecular mechanism to suppress rig-i ubiquitination. in addition, a previous study showed that ns co-localizes with mavs and inhibits rig-i interaction with mavs [ ] . considering that rig-i ubiquitination is required for its oligomerization and interaction with mavs, it is conceivable that ns suppresses rig-i interaction with mavs by interfering with rig-i ubiquitination, as shown in the current study. thus, it was tempting to test the hypothesis that ns targets trim , an e -ubiquitin ligase responsible for rig-i ubiquitination. moreover, several viruses utilize their proteins to interfere with trim activation and subsequent rig-i activation. for instance, the influenza ns binds to trim and inhibits trim multimerization [ ] . the sars coronavirus nucleocapsid protein binds to trim spry and disrupts rig-i ubiquitination [ ] . in this study, the interaction between ns and trim was demonstrated. since several studies have shown that trim migrates to the mitochondria upon viral infection to interact with mavs [ ] , the interaction between mavs and ns might be a result of the interaction between ns and trim . as expected, ns inhibited the interaction between rig-i card and mavs. these results clearly indicate that ns is capable of suppressing rig-i activation by targeting trim and inhibiting rig-i ubiquitination through trim . we have shown that ns could suppress the interferon-β promoter activity induced by rig-in and mavs, indicating that ns is also capable of suppressing downstream signaling. this can be explained by previous studies showing that ns can trigger the degradation of critical downstream molecules such as irf . nonetheless, we have shown that ns can suppress trim -mediated ubiquitination of rig-in without affecting its amount and that its effect on rig-in-mediated interferon-β promoter activity can be reversed by an excessive amount of trim . these results indicate that inhibition of trim -mediated rig-i ubiquitination by ns contributes to the suppression of rig-i signaling, at least in part. interestingly, the biochemical domain mapping study revealed that the spry domain of trim is responsible for interaction with ns . given that the spry domain is responsible for interaction with rig-i, a possible molecular mechanism is that ns binds to trim and sequesters it to prevent its interaction with rig-i. however, we could not observe a significant reduction in interaction between rig-i and trim by ns (data not shown). further studies are needed to identify the detailed molecular mechanism of rsv ns -mediated trim dysfunction. rsv ns is known to bind with rig-i card and disrupts its activation [ ] and we showed that rsv ns interacts with trim to decrease card ubiquitination. in a previous study, rsv ns and ns have been shown to exist as homo-or heterodimers in mammalian cells. the rsv ns -ns dimer was detected in mitochondria, suggesting that ns and ns can cooperate to suppress rig-i signaling [ ] . it is also possible that the dimer of rsv ns and ns interacts with trim and rig-i card through each ns protein interacting with trim or rig-i card. although we could not observe a significant synergistic effect of ns and ns co-expression in terms of suppression of rig-i ubiquitination ( figure c) , the ns and ns interaction may have additional functions in rig-i signaling. for instance, it is possible that the ns /ns complex induces rig-i degradation followed by suppression of rig-i k -ubiquitination for complete inhibition of the rig-i signaling pathway. collectively, this study demonstrates that rsv ns interacts with trim and suppresses trim -mediated rig-i activation to evade rig-i-mediated antiviral responses. further studies including structural analysis of protein interaction and analysis of the interaction modes between rig-i, trim , ns and ns need to be conducted. the burden of respiratory syncytial virus infection in young children global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis pathogen recognition and innate immunity intracellular pattern recognition receptors in the host response retinoic acid-inducible gene i mediates early antiviral response and toll-like receptor expression in respiratory syncytial virus-infected airway epithelial cells mavs and myd are essential for innate immunity but not cytotoxic t lymphocyte response against respiratory syncytial virus the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses trim ring-finger e ubiquitin ligase is essential for rig-i-mediated antiviral activity martinez, i. trim in the regulation of the antiviral innate immunity influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i modulation of host immunity by human respiratory syncytial virus virulence factors: a synergic inhibition of both innate and adaptive immunity respiratory syncytial virus ns protein degrades stat by inducing socs expression respiratory syncytial virus non-structural protein facilitates virus replication through mir- a-mediated inhibition of interferon-alpha receptor respiratory syncytial virus nonstructural proteins upregulate socs and socs in the different manner from endogenous ifn signaling human respiratory syncytial virus nonstructural protein ns antagonizes the activation of beta interferon transcription by interacting with rig-i respiratory syncytial virus ns protein colocalizes with mitochondrial antiviral signaling protein mavs following infection viral degradasome hijacks mitochondria to suppress innate immunity multiple functional domains and complexes of the two nonstructural proteins of human respiratory syncytial virus contribute to interferon suppression and cellular location a novel p mitogen activated protein kinase (mapk) specific inhibitor suppresses respiratory syncytial virus and influenza a virus replication by inhibiting virus-induced p mapk activation roles of rig-i n-terminal tandem card and splice variant in trim -mediated antiviral signal transduction a novel mechanism for the inhibition of interferon regulatory factor- -dependent gene expression by human respiratory syncytial virus ns protein effects of nonstructural proteins ns and ns of human respiratory syncytial virus on interferon regulatory factor , nf-kappab, and proinflammatory cytokines the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim -mediated rig-i ubiquitination mavs ubiquitination by the e ligase trim and degradation by the proteasome is involved in type i interferon production after activation of the antiviral rig-i-like receptors this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest.viruses , , key: cord- - s vh authors: delvecchio, rodrigo; higa, luiza m.; pezzuto, paula; valadão, ana luiza; garcez, patrícia p.; monteiro, fábio l.; loiola, erick c.; dias, andré a.; silva, fábio j. m.; aliota, matthew t.; caine, elizabeth a.; osorio, jorge e.; bellio, maria; o’connor, david h.; rehen, stevens; de aguiar, renato santana; savarino, andrea; campanati, loraine; tanuri, amilcar title: chloroquine, an endocytosis blocking agent, inhibits zika virus infection in different cell models date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: s vh zika virus (zikv) infection in utero might lead to microcephaly and other congenital defects. since no specific therapy is available thus far, there is an urgent need for the discovery of agents capable of inhibiting its viral replication and deleterious effects. chloroquine is widely used as an antimalarial drug, anti-inflammatory agent, and it also shows antiviral activity against several viruses. here we show that chloroquine exhibits antiviral activity against zikv in vero cells, human brain microvascular endothelial cells, human neural stem cells, and mouse neurospheres. we demonstrate that chloroquine reduces the number of zikv-infected cells in vitro, and inhibits virus production and cell death promoted by zikv infection without cytotoxic effects. in addition, chloroquine treatment partially reveres morphological changes induced by zikv infection in mouse neurospheres. zika virus (zikv) is an arthropod-borne virus, transmitted by aedes mosquitoes, that belongs to the flavivirus genus, which also includes other pathogens such as west nile virus (wnv), yellow fever virus (yfv), japanese encephalitis virus (jev), and dengue virus (denv). zika virus was first isolated from a sentinel monkey in the zika forest in uganda in [ ] . since then, zikv has been isolated from humans and mosquitoes throughout africa and southeast asian countries. phylogenetic analysis of the nonstructural protein encoding region has disclosed three zikv lineages: east african, vero cells (atcc, manassas, va, usa) are derived from the kidney of african green monkey and were grown in dmem high glucose (gibco, thermo fisher scientific, waltham, ma, usa) supplemented with % fetal bovine serum (fbs) (gibco). human brain microvascular endothelial cells (hbmec) were a kind gift from dr. julio scharfstein (federal university of rio de janeiro, rio de janeiro, brazil), and hbmec isolation was performed as previously described [ ] . these cells were cultured in dmem high glucose supplemented with % fbs. the c / cell line is derived from aedes albopictus. c / cells (atcc, manassas, va, usa) were grown in leibovitz l- medium (gibco) supplemented with . g/l tryptose phosphate broth (sigma aldrich, boston, ma, usa), mm glutamine (gibco), . % sodium bicarbonate (gibco), x non-essential amino acids (gibco) and % fbs. neural stem cells (nscs) were derived from human induced pluripotent stem cells (ipscs). ipscs were provided by the biobank of ipscs of the brazilian ministry of health (conep b- # . / - ). according to the supplier, fibroblasts were reprogrammed using the protocol developed by paulsen et al. [ ] , and transduced with the cytotune-ips sendai kit (thermo fisher scientific, waltham, ma, usa). ipscs presented a normal karyotype and the expression of pluripotency markers. these cells were cultured with e culture media (gibco) on a matrigel (bd biosciences, san jose, ca, usa) coated surface. ipsc colonies were manually passaged every - days until they reached %- % confluence and were maintained at • c in humidified air with % co . to produce nscs, human ipscs were exposed to the serum-free neural induction medium (gibco), containing neurobasal medium (gibco) and the pluripotent stem cell neural induction supplement (gibco), according to the manufacturer's protocol [ ] . briefly, the medium was changed every other day until day , when initial nscs are split and grown on neural expansion medium (advanced dmem/f and neurobasal medium ( : ) with neural induction supplement; gibco). nscs were used after four passages in neural expansion media. mouse central nervous system (cns) cells were harvested from swiss mouse embryos at embryo day (e ) and grown for h as free floating neurospheres in neurobasal culture media (gibco) supplemented with x b (gibco). chloroquine diphosphate was kindly supplied by farmanguinhos (fiocruz, rio de janeiro, brazil). the lyophilized powder was diluted in double distilled water to mm. the chloroquine solution was filtered through a . µm membrane and stored at − • c. zikv strain mr (uganda/africa, accession no.: nc . , kindly provided by dr. davis ferreira, federal university of rio de janeiro, rio de janeiro, brazil) was isolated from a rhesus monkey and injected intracerebrally on swiss mice for several passages [ ] and zikv br (recife/brazil, zikv pe/ , accession no: kx . , kindly provided by dr. marli tenório cordeiro, centro de pesquisas aggeu magalhães, recife, brazil) was isolated from a patient presenting classical symptoms of zikv infection [ ] . these viruses were propagated in vero and c / cells, respectively. briefly, the cells were infected with zikv at a multiplicity of infection (moi) of . and incubated at • c. after h, the inoculum was removed and replaced with dmem high-glucose (vero) and leibovitz l- (c / ) growth media supplemented with % fbs. after to days, the conditioned medium was harvested, centrifuged at × g, and sterile-filtered to remove cells and cellular debris. virus stocks were stored at − • c. virus titers were determined by plaque assay performed on vero cells. virus stocks or samples were serially diluted and adsorbed to confluent monolayers. after h, the inoculum was removed and cells were overlaid with semisolid medium constituted of alpha-mem (gibco) containing % carboxymethyl cellulose (sigma aldrich) and % fbs (gibco). cells were further incubated for days when cells were fixed in % formaldehyde. cells were stained with % crystal violet in % ethanol for plaque visualization. titers were expressed as plaque forming units (pfu) per milliliter. vero cells, hbmec, and nsc were infected with zikv mr or zikv br strain at an moi of . after h, the inoculum was removed and medium containing chloroquine ( . to µm) was added to the cells. after to days, cells were fixed with % paraformaldehyde (sigma aldrich) in phosphate buffered saline (pbs) for min at room temperature and washed with pbs. cells were permeabilized with . % triton x- (sigma aldrich) in pbs, washed with pbs, and blocked with pbs with % fbs. cells were incubated with g , a pan-flavivirus antibody raised against the zikv envelope e protein produced in g - - hybridoma (atcc), diluted : in pbs with % fbs. cells were labeled with donkey anti-mouse alexa fluor antibody (thermo scientific, waltham, ma, usa) diluted : in pbs with % fbs, and were analyzed by flow cytometry in a bd accuri c (becton, dickinson and company, franklin lakes, nj, usa) for zikv infection. vero, hbmec, and nsc were seeded on black -well plates with clear bottoms and infected with zikv mr or zikv br strain at an moi of for h. neurospheres were seeded on coverslips and infected with zikv mr with . × pfu after infection, the viral inoculum was removed and cells were incubated with medium containing chloroquine ( . to µm) for to days, depending on cell type. cells and neurospheres were fixed with % paraformaldehyde in pbs for min at room temperature. the fixative was removed and the samples were washed three times with pbs. blocking of unspecific binding of the antibody and permeabilization were performed with pbs supplemented with % bovine serum albumin (bsa, sigma aldrich) and . % triton x- for min at room temperature. incubation with anti-map antibody was performed following the manufacturer's instructions (abcam, cambridge, cambridgeshire, uk, # ; : ) and the g antibody was diluted : in pbs with % bsa and incubated with cells for h. after washing three times with pbs, cells were incubated with secondary antibodies coupled to alexa fluorochromes (thermo fisher scientific) for min and washed five times. coverslips with neurospheres were mounted with prolong gold mounting medium (thermo fisher scientific). samples were imaged using either leica sp or leica spe (leica biosystems, wetzlar, hesse, de) confocal microscopes and a nikon te (tokyo, japan) inverted microscope coupled to a leica dfc fx camera (leica biosystems, wetzlar, germany). vero, hbmec, and nsc were exposed to zikv mr at an moi of . after h, inoculum was removed and chloroquine-containing medium ( . to µm) was added to the cells. five days post-infection, µl of celltiter blue reagent (promega, madison, wi, usa) was added in each well, incubated for - h, and fluorescence was measured ( / nm), except for nscp cells when celltiter blue was added at three days post-infection. mean fluorescence intensity (mfi) and standard deviation were displayed. in order to calculate the half maximum effective concentration (ec ) that protects cells from death caused by zikv infection, mfi values from zikv mr control were subtracted from every condition and then these values were normalized over the mock control. these data were plotted and hill parameter sigmoidal regression was performed on sigma plot v. . (systat software inc., san jose, ca, usa). vero cells, hbmec, and nsc were incubated with medium containing chloroquine ( . to µm) for days (nsc) or days (vero and hbmec). celltiter blue reagent (promega) was added in each well, incubated for - h, and fluorescence was measured ( / nm). in order to calculate the % cytotoxicity concentration (cc ), mfi values were normalized over the untreated control. these data were plotted and hill parameter sigmoidal regression was performed on sigma plot v. . (systat software inc.). vero cells were inoculated with zikv mr at an moi of for h at • c. cells were washed three times with cold pbs to remove unbound virus and treated with µm chloroquine that was added at different time points: , . , , , and h post-infection. conditioned media were collected at h post-infection to analyze the production of virus particles through viral rna content or the amount of infectious virus particles. viral rna was extracted and quantitative reverse transcription polymerase chain reaction (rt-qpcr) was performed. the titer of infectious virus particles was determined by plaque assay. vero cells were infected with zikv mr or zikv br strain at an moi of for h at • c. virus input was washed three times with cold pbs and cells were treated with chloroquine ( . to µm) for h, and then the supernatant was collected and the rna was extracted and analyzed by relative quantification by rt-qpcr. the supernatant was also evaluated by plaque assay to quantitate the infectious virus particles. viral rna was extracted from µl supernatant of infected cells using qiamp minielute virus spin (qiagen, hilden, düsseldorf ,de), following the manufacturer's recommendations. zikv detection was performed using one step taqman rt-qpcr (thermo fisher scientific) on a real-time pcr system (applied biosystems) with primers and the probe described elsewhere [ ] . threshold cycle (ct) was determined and ∆ct (ct chloroquine treated − ct untreated) was calculated. the fold reduction of virus particles' release, including defective viral particles, were calculated by ∆ct . mean and standard deviation (sd) were calculated for each assay. one way analysis of variance (anova) was conducted using the non-parametric test (kruskal-wallis) followed by dunn's multiple comparisons test. a p-value of < . was considered significant. all analyses were performed on graphpad prism v. (graphpad software, san diego, ca, usa). the sample size is provided in the respective figure legends. we characterized the antiviral properties of chloroquine in vero cells, a model widely used to study viral infections. vero cells were infected with zikv mr at an moi of (i.e., pfu/cell) and were then treated for days with chloroquine in concentrations ranging from . to µm. viral infectivity was assessed using the g antibody, which detects flavivirus envelope e protein. we observed that chloroquine treatment decreased the number of zikv-infected cells in a dose-dependent manner. flow cytometry analysis showed a reduction of % and % in zikv-infected cells when cultures were treated with µm and µm chloroquine, respectively, compared to untreated infected cells ( figure a ). immunofluorescence staining corroborated these results ( figure b ) and additionally, chloroquine decreased the production of infectious ( figure c ) and total ( figure d ) virus particles, including defective viral particles, by zikv-infected cells. to confirm that viral inhibition is independent of chloroquine cytotoxicity, the viability of uninfected cells treated with chloroquine ( . to µm) for days was analyzed. chloroquine did not impact cell viability at concentrations of µm or lower ( figure e ). we further analyzed whether chloroquine treatment could protect vero cells from zikv infection as assessed by cell viability. chloroquine, ranging from . to µm, increased cell viability from % up to % ( figure f ). microcephaly cases and neurological disorders have only been associated with infection with strains of zikv from the asian lineage, detected in french polynesia and in the americas [ , , ] . to determine the inhibition spectrum of chloroquine against zikv infection, vero cells were infected with the brazilian isolate of the asian lineage (zikv br). chloroquine decreases the percentage of cells infected with the brazilian isolate from % to % and % at . and µm, respectively ( brazilian strain at an moi of and exposed to chloroquine for h. the supernatant was collected and viral rna was relatively quantified over the untreated infected control (b) or infectivity was analyzed by immunofluorescence with g antibody (c). the dashed line represents fold reduction on virus production of . data are represented as mean ± sd from two independent experiments. statistical significance was assessed by kruskal-wallis test and multiple comparisons with infected and untreated control corrected by dunn's test (* p < . ). inhibition of viral infection mediated by chloroquine can occur in both the early and later stages of the viral replication cycle [ , ] . to evaluate which step of the viral cycle was susceptible to inhibition, chloroquine was added to vero cells at different time points post-infection with zikv mr . the supernatant was collected h post-infection and virus production was evaluated by relative quantification of viral rna over the untreated control by rt-qpcr. virus titers were also determined by plaque assay in vero cells. incubation of vero cells with chloroquine at h postinfection had a greater impact on the production of zikv particles, decreasing viral rna -fold over the controls ( figure a ). the addition of chloroquine from min to h post-infection was able to reduce virus release - fold over untreated, infected-cells. however, chloroquine added at h post-infection had only a minor effect on viral production ( figure a) . these results were confirmed by quantification of zikv infectious particles released after chloroquine treatment ( figure b ). these data confirm that chloroquine interferes with the early stages of the zikv replication cycle. inhibition of viral infection mediated by chloroquine can occur in both the early and later stages of the viral replication cycle [ , ] . to evaluate which step of the viral cycle was susceptible to inhibition, chloroquine was added to vero cells at different time points post-infection with zikv mr . the supernatant was collected h post-infection and virus production was evaluated by relative quantification of viral rna over the untreated control by rt-qpcr. virus titers were also determined by plaque assay in vero cells. incubation of vero cells with chloroquine at h post-infection had a greater impact on the production of zikv particles, decreasing viral rna -fold over the controls ( figure a ). the addition of chloroquine from min to h post-infection was able to reduce virus release - fold over untreated, infected-cells. however, chloroquine added at h post-infection had only a minor effect on viral production ( figure a) . these results were confirmed by quantification of zikv infectious particles released after chloroquine treatment ( figure b ). these data confirm that chloroquine interferes with the early stages of the zikv replication cycle. reduce virus release - fold over untreated, infected-cells. however, chloroquine added at h post-infection had only a minor effect on viral production ( figure a) . these results were confirmed by quantification of zikv infectious particles released after chloroquine treatment ( figure b ). these data confirm that chloroquine interferes with the early stages of the zikv replication cycle. considering that zikv infects hbmecs [ ] , we investigated whether chloroquine could inhibit viral infection of these cells. chloroquine reduced % and % of the number of zikv mr -infected hbmecs at and µm, respectively ( figure a,d) . these concentrations are non-cytotoxic ( figure b ), and protected approximately % of hbmecs from zikv infection as demonstrated by the increase in cell viability ( figure c ). considering that zikv infects hbmecs [ ] , we investigated whether chloroquine could inhibit viral infection of these cells. chloroquine reduced % and % of the number of zikv mr -infected hbmecs at and μm, respectively ( figure a,d) . these concentrations are non-cytotoxic ( figure b) , and protected approximately % of hbmecs from zikv infection as demonstrated by the increase in cell viability ( figure c ). neural stem cells are key cells in the process of corticogenesis, giving rise to the three main cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. depletion of the nsc pool is one of the main mechanisms responsible for primary microcephaly [ ] . to evaluate if chloroquine could protect these cells from zikv mr infection, nscs were exposed to up to μm chloroquine for days. chloroquine treatment decreased the number of nscs infected with zikv mr by %, and, through cell viability assessment protected % of nscs from zikv infection, without cytotoxicity effects ( figure a-d) . similar results were observed when nscs were infected with the brazilian strain ( figure e ). neural stem cells are key cells in the process of corticogenesis, giving rise to the three main cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. depletion of the nsc pool is one of the main mechanisms responsible for primary microcephaly [ ] . to evaluate if chloroquine could protect these cells from zikv mr infection, nscs were exposed to up to µm chloroquine for days. chloroquine treatment decreased the number of nscs infected with zikv mr by %, and, through cell viability assessment protected % of nscs from zikv infection, without cytotoxicity effects ( figure a-d) . similar results were observed when nscs were infected with the brazilian strain ( figure e ). neuroprogenitor-enriched neurospheres, when subjected to differentiation culture conditions, can generate neurons. our group recently demonstrated that zikv infection affected neurosphere size, neurite extension, and neuronal differentiation [ ] . as we previously observed, neurospheres infected with zikv strain mr showed convoluted and misshapen neurites. neurite extension was evaluated in chloroquine treated cultures by microtubule-associated protein (map ) staining and phase contrast microscopy and although many neurospheres were severely impacted by the infection, many others displayed the same general characteristics of mock-infected spheres indicating that chloroquine treatment rescued the neurite extension phenotype (figure a-c) . furthermore, zikv infection decreased when neurospheres were treated with . μm chloroquine, as evaluated by g staining (figure d-f) . data are represented as mean ± sd from two to three independent experiments. statistical analysis was performed with kruskal-wallis test and multiple comparisons with infected and untreated control corrected by dunn's test (* p < . ). neuroprogenitor-enriched neurospheres, when subjected to differentiation culture conditions, can generate neurons. our group recently demonstrated that zikv infection affected neurosphere size, neurite extension, and neuronal differentiation [ ] . as we previously observed, neurospheres infected with zikv strain mr showed convoluted and misshapen neurites. neurite extension was evaluated in chloroquine treated cultures by microtubule-associated protein (map ) staining and phase contrast microscopy and although many neurospheres were severely impacted by the infection, many others displayed the same general characteristics of mock-infected spheres indicating that chloroquine treatment rescued the neurite extension phenotype (figure a-c) . furthermore, zikv infection decreased when neurospheres were treated with . µm chloroquine, as evaluated by g staining (figure d-f) . chloroquine is known to be a non-specific antiviral agent, but its effect on the zika virus replication has not been evaluated yet. this is the first report of inhibitory effects of chloroquine on zikv replication, which, given the ongoing epidemics, may become interesting both for the scientific knowledge of the virus and for the clinical perspective. although zika virus was first identified in uganda in , from january to april , zikv transmission has been reported in countries and territories [ ] . the zika virus disease is in general mild, but the recent positive correlation between infection, congenital malformations, and neurological damage in adults has intensified the need for therapeutic approaches. prophylactic treatments for women intending to get pregnant in epidemic areas and travelers going to affected countries would represent relevant tools to reduce zikv transmission and avoid the spread of the disease by travelers. moreover, a drug that blocks placental transfer of the virus could decrease the figure . chloroquine inhibits zikv infection in mouse neurospheres. mouse neurospheres were infected with zikv mr ( . × pfu and were treated with chloroquine for days. neurospheres were analyzed by phase contrast microscopy (a-c), and triple stained for envelope viral protein (green), microtubule-associated protein (map- , red), a neuron-specific protein, and dapi (blue) (d-f). chloroquine is known to be a non-specific antiviral agent, but its effect on the zika virus replication has not been evaluated yet. this is the first report of inhibitory effects of chloroquine on zikv replication, which, given the ongoing epidemics, may become interesting both for the scientific knowledge of the virus and for the clinical perspective. although zika virus was first identified in uganda in , from january to april , zikv transmission has been reported in countries and territories [ ] . the zika virus disease is in general mild, but the recent positive correlation between infection, congenital malformations, and neurological damage in adults has intensified the need for therapeutic approaches. prophylactic treatments for women intending to get pregnant in epidemic areas and travelers going to affected countries would represent relevant tools to reduce zikv transmission and avoid the spread of the disease by travelers. moreover, a drug that blocks placental transfer of the virus could decrease the chance of vertical transmission in viremic pregnant women as was shown for hiv-infected pregnant women treated with antiretroviral therapy [ ] . here we demonstrated that chloroquine decreases the number of zikv-infected cells and protected cells from zikv infection as measured by cell viability at non-cytotoxic concentrations (figures , and ) . the ec or concentration of chloroquine that protected % of the cells from zikv infection assessed by cell viability, was . - . µm depending on the cell model and the cc ranged from . to . µm ( table ) . the values of ec obtained for zikv mr are lower than those obtained for denv inhibition (~ µm) and hiv inhibition ( µm) [ , ] . furthermore, we observed similar zikv inhibitory effects of chloroquine when tested on different zikv lineage infections (figures and ) , supporting the idea that chloroquine could help to manage recent infections caused by asian zikv lineage. although chloroquine has shown antiviral activity against a large spectrum of viruses in vitro, few clinical studies have been performed to evaluate chloroquine effects on patients with viral infections. two clinical trial studies of chloroquine have been conducted to assess chloroquine treatment in patients infected with denv [ , ] . one of the trials evaluated the benefits of chloroquine treatment for days in patients infected with denv and showed no reduction in the duration or intensity of denv viremia or nonstructural protein (ns ) antigenemia clearance [ ] . however, a trend towards a reduction in the number of dengue hemorrhagic fever cases was noticed in the chloroquine-treated group [ ] . a more recent clinical trial of chloroquine administration to denv-infected patients, also for days, showed that % of the patients in the chloroquine-treated group reported feeling less pain and showed improvement in the performance of daily chores during treatment [ ] . moreover, the symptoms returned after medication withdrawal. however, chloroquine treatment did not reduce the duration and intensity of the fever or duration of the disease [ ] . the antiviral effect of chloroquine may be insufficient to produce a decrease in viral load or improvement of the disease progression when chloroquine/hydroxychloroquine is used in monotherapy. however, chloroquine may produce a significant antiflaviviral effect when used in combination therapies, as recently shown in a clinical trial of hydroxychloroquine plus ribavirin and interferon alpha in individuals infected with hepatitis c virus (hcv) [ ] . in regard to the potential antiviral therapeutic combinations for zika, a freshly published screening of drugs already approved for other clinical indications has resulted in the identification of more than candidate drugs [ ] . of note, one of these is mefloquine, a compound related to chloroquine. in terms of safety for pregnant women, however, mefloquine is included in the b category, i.e., a drug for which the animal reproduction studies have failed to demonstrate a risk to the fetus and there are no adequate and well-controlled studies in pregnant women. be that as it may, the aforementioned study corroborates our results using chloroquine, and provides new anti-zikv drugs that could be tested in combination with chloroquine. differing from mefloquine administration, the use of chloroquine during pregnancy was thoroughly evaluated and when prophylactic doses of chloroquine were administered for malaria ( mg/week), no increment in birth defects was observed [ ] . higher concentrations of chloroquine ( mg to mg/day) were administered to pregnant women with severe diseases, such as lupus or rheumatoid arthritis. in these studies, few cases of abortion and fetal toxicity were observed. however, fetal toxicity or death could not be discarded as direct consequences of the disease itself. in addition, all term deliveries resulted in healthy newborns [ , ] . chloroquine has been successfully tested in animal models. twice daily administration of chloroquine ( mg/kg) has been shown to increase the balb/c mice survival rate to %- % after infection with ebola virus (ebov) [ ] . in a c bl/ mice model for coronavirus infection, chloroquine ( mg/kg) protected % of -day-old suckling mice infected with human coronavirus oc (hcov-oc ) when administered to pregnant mice day prepartum [ ] . a survival rate of % was observed in balb/c mice infected with avian influenza h n virus treated with chloroquine at mg/kg/day [ ] . these results suggest that chloroquine has the potential to inhibit zikv in in vivo mouse studies. chloroquine is widely distributed to body tissues as well as its analogue hydroxychloroquine. the concentration of hydroxychloroquine in the brain is - times higher than in the plasma [ ] . the concentration of chloroquine in the plasma reached µm when a daily intake of mg was prescribed to arthritis patients [ ] . chloroquine is able to cross the placental barrier and is supposed to reach similar concentrations in both maternal and fetal plasma [ ] . concentrations of chloroquine, similar to the ec values calculated here (table ) , are achieved in the plasma in current chloroquine administration protocols and might reach the brain. different mechanisms for the chloroquine inhibition of viral infection have been described [ ] [ ] [ ] . we observed a strong reduction in the release of zikv particles when the drug was added at h post-infection (figure ) , suggesting a higher impact on early stages of infection, possibly during fusion of the envelope protein to the endosome membrane. chloroquine inhibits acidification of the endosome, consequently inhibiting the low ph-induced conformational changes required for the fusion of the envelope protein of flaviviruses with the endosomal membrane [ ] . chloroquine was also effective in decreasing virus release, although less pronouncedly and not statistically significant, when added after the early stages of virus infection (from . to h post-infection), suggesting that later stages of the zikv replication cycle might also be affected (figure ) . zikv was detected in the cerebrospinal fluid of zikv-infected adult patients that manifested meningoencephalitis, indicating that zikv invades the central nervous system through yet unknown mechanisms. transcytosis through the endothelial cells of the blood brain barrier is a known mechanism of viral access to the central nervous system [ , ] . here we demonstrated, by different methodologies, that chloroquine protects hbmec, an immortalized cell line widely used as in vitro model of the blood-brain barrier [ ] , from zikv infection (figure ) . recent studies showed that neural stem cells are highly permissive for zikv infection and one of the mechanisms proposed for the cause of microcephaly would be the depletion of the stem cell pool induced by zikv [ , , ] . our data showed that chloroquine inhibits the infection of human neural stem cells ( figure ). using the mouse neurospheres model to study neural stem cell differentiation into neurons, another process that might be disturbed in microcephaly, we observed that chloroquine inhibited the infection of neuronal progenitors and partially protected the ability of these cells to extend neurites ( figure ). the protective effect of chloroquine on stem cells and committed progenitors is potentially a groundbreaking feature of this compound, as it would be prescribed to women at childbearing age that are traveling to affected countries and women planning pregnancy in endemic areas. this would decrease the chances of infection and thus fetal damage, especially to the developing brain. our results suggest that the chloroquine concentrations inhibiting zikv replication in vitro may overlap the highest drug concentrations detected in humans [ ] . we therefore suggest that the therapeutic potential of chloroquine for zika be subjected to further study. zika virus. i. isolations and serological specificity genetic and serologic properties of zika virus associated with an epidemic, yap 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dengue-related symptoms hydroxychloroquine augments early virological response to pegylated interferon plus ribavirin in genotype- chronic hepatitis c patients a screen of fda-approved drugs for inhibitors of zika virus infection safety of chloroquine in chemosuppression of malaria during pregnancy antimalarial drugs, systemic lupus erythematosus and pregnancy a systematic screen of fda-approved drugs for inhibitors of biological threat agents antiviral activity of chloroquine against human coronavirus oc infection in newborn mice anti-malaria drug chloroquine is highly effective in treating avian influenza a h n virus infection in an animal model recent developments in the understanding of the pharmacokinetics and mechanism of action of chloroquine dose refinements in long-term therapy of rheumatoid arthritis with antimalarials transfer of chloroquine and desethylchloroquine across the placenta and into milk in melanesian mothers mechanism of borna disease virus entry into cells weak bases affect late stages of mayaro virus replication cycle in vertebrate cells effects of chloroquine on viral infections: an old drug against today's diseases? rodenhuis-zybert, i.; wilschut, j. flavivirus cell entry and membrane fusion human immunodeficiency virus- uses the mannose- -phosphate receptor to cross the blood-brain barrier mechanism of west nile virus neuroinvasion: a critical appraisal brain-region-specific organoids using mini-bioreactors for modeling zikv exposure chloroquine and beyond: exploring anti-rheumatic drugs to reduce immune hyperactivation in hiv/aids acknowledgments: this work was supported by conselho nacional de desenvolvimento e pesquisa (cnpq), key: cord- -cz t n authors: jansen van vuren, petrus; wiley, michael; palacios, gustavo; storm, nadia; mcculloch, stewart; markotter, wanda; birkhead, monica; kemp, alan; paweska, janusz t. title: isolation of a novel fusogenic orthoreovirus from eucampsipoda africana bat flies in south africa date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: cz t n we report on the isolation of a novel fusogenic orthoreovirus from bat flies (eucampsipoda africana) associated with egyptian fruit bats (rousettus aegyptiacus) collected in south africa. complete sequences of the ten dsrna genome segments of the virus, tentatively named mahlapitsi virus (mahlv), were determined. phylogenetic analysis places this virus into a distinct clade with baboon orthoreovirus, bush viper reovirus and the bat-associated broome virus. all genome segments of mahlv contain a ' terminal sequence ( '-gguca) that is unique to all currently described viruses of the genus. the smallest genome segment is bicistronic encoding for a kda protein similar to p membrane fusion protein of bush viper reovirus and an kda protein similar to p non-structural protein of baboon orthoreovirus. this is the first report on isolation of an orthoreovirus from an arthropod host associated with bats, and phylogenetic and sequence data suggests that mahlv constitutes a new species within the orthoreovirus genus. bats have been increasingly associated with emerging and re-emerging viruses. the likelihood of possible transmission of these pathogens to humans is ever increasing as a result of human encroachment on animal habitats, climate change and change of human behaviour. pathogens of particular public health importance are filoviruses [ , ] , coronaviruses [ , ] , paramyxoviruses [ , ] and lyssaviruses [ , ] . other viruses, without a known human disease link, have also been detected recently [ ] [ ] [ ] . some human pathogens, such as rift valley fever virus, that have been detected in bats were likely a result of coincidental infection and do not constitute proof that bats play a role as reservoirs [ ] . bats are parasitized by a number of ectoparasites, including mites, bat flies, ticks and fleas, and often by some or all of these simultaneously [ ] . the bat flies are members of two families in the diptera order, namely, the streblidae and nycteribiidae, and are highly host-specific obligate ectoparasites of bats [ ] [ ] [ ] . both bat fly families are hematophagous and potentially capable of (qiagen). cellular debris was removed by centrifugation at , ˆg for min, and the supernatant used for subsequent virus isolation and nucleic acid extraction procedures. viruses , , x mm stainless steel beads (qiagen). cellular debris was removed by centrifugation at , × g for min, and the supernatant used for subsequent virus isolation and nucleic acid extraction procedures. the wells of -well tissue culture plates (nunc) were seeded with vero e cells and grown to %- % confluency in eagle's minimum essential medium (emem, lonza) supplemented with antibiotics (penicillin/streptomycin/amphotericinb, lonza) and % foetal calf serum at °c and % co . culture medium was removed and the monolayers in individual wells inoculated with μl of ectoparasite pool homogenates (one pool representing parasites from one bat). after one hour adsorption at °c, the inoculum was removed and fresh emem containing antibiotics and % foetal calf serum added. the -well plates were incubated for days and cytopathic effects (cpe) monitored. a second and third blind passage was performed for all samples by inoculating and incubating monolayers as described above with μl of undiluted supernatant from the preceding passage. supernatants were collected from all wells displaying cpe after three blind passages, a / dilution prepared in emem, and ml of this used to inoculate a cm tissue culture flask. if the same cpe was noted in the sub-cultured cm flask, a / dilution of this supernatant was prepared and used to inoculate a cm tissue culture flask for preparation of stock virus. stock virus titres were determined by standard tissue culture infectious dose (tcid ) titrations on -well microtitre plates as described previously [ ] . the wells of -well tissue culture plates (nunc) were seeded with vero e cells and grown to %- % confluency in eagle's minimum essential medium (emem, lonza) supplemented with antibiotics (penicillin/streptomycin/amphotericinb, lonza) and % foetal calf serum at ˝c and % co . culture medium was removed and the monolayers in individual wells inoculated with µl of ectoparasite pool homogenates (one pool representing parasites from one bat). after one hour adsorption at ˝c, the inoculum was removed and fresh emem containing antibiotics and % foetal calf serum added. the -well plates were incubated for days and cytopathic effects (cpe) monitored. a second and third blind passage was performed for all samples by inoculating and incubating monolayers as described above with µl of undiluted supernatant from the preceding passage. supernatants were collected from all wells displaying cpe after three blind passages, a / dilution prepared in emem, and ml of this used to inoculate a cm tissue culture flask. if the same cpe was noted in the sub-cultured cm flask, a / dilution of this supernatant was prepared and used to inoculate a cm tissue culture flask for preparation of stock virus. stock virus titres were determined by standard tissue culture infectious dose (tcid ) titrations on -well microtitre plates as described previously [ ] . for electron microscopy specimen preparation, %- % confluent vero e monolayers in cm flasks were inoculated with stock virus and monitored for cpe. at the first sign of cpe, culture supernatant was collected, cleared of cellular content by centrifugation ( ˆg for min), and subsequently fixed in an equal volume of . % glutaraldehyde in . m hepes buffer (ph . ) for visualization of virus particles by negative staining. a beckman airfuge ® (beckman coulter, brea, ca, usa) was used to concentrate all samples ( min at kpa), after which droplets of sample were adsorbed to . % formar-coated copper grids for a minimum of min, rinsed twice in deionised, distilled water and stained briefly in % phosphotungstic acid (ph . ). for ultramicrotomy, the remaining infected monolayers were flooded with the same fixative overnight, then routinely processed (postfixation in % buffered osmium tetroxide, graded ethanol dehydration, infiltration with a low viscosity resin (agar scientific, stansted, uk) and overnight polymerisation at ˝c). seventy nm sections were cut on a leica em-uc , double stained with saturated uranyl acetate and lead citrate, and viewed at kv on a biotwin spirit (fei company, hillsboro, or, usa). imaging was done with an olympus quemesa ccd camera (olympus, tokyo, japan). single-primer amplification (sispa), rapid amplification of cdna ends (race), next-generation sequencing (ngs) and bioinformatics stock virus culture supernatant was added to trizol-ls (life technologies, waltham, ma, usa) at a ratio of µl supernatant to µl trizol-ls. rna was extracted using a column based kit (direct-zol rna kit, zymo research, irvine, ca, usa). to increase sensitivity, rrna was depleted using the same method as described previously [ ] . rnas were converted to cdna and amplified using sispa as described previously with modifications [ ] . to enhance coverage of the terminal ends, an oligo containing three rgtp at the ' end (gccggagctctgcagatatcggccattat ggccrgrgrg) was added during first-strand cdna synthesis and the reverse transcriptase was changed to maxima h minus (thermo scientific, waltham, ma, usa), which has terminal transferase activity that enables addition of the rgtp containing oligo to the ' end during cdna synthesis. amplicons were sheared and libraries prepared using the illumina truseq dna library preparation kit (illumina, san diego, ca, usa). sequencing was performed either on an illumina miseq (illumina) or nextseq (illumina) using either a ˆ or ˆ version kit. illumina and sispa adapter sequences were trimmed from the sequencing reads using cutadapt- . . [ ] , quality filtering was conducted with prinseq-lite (-min len -derep -lc method dust-lc threshold -trim ns left -trim ns right -trim qual right ) [ ] and reads were assembled into contigs using ray meta with kmer length = [ ] . resultant contigs were aligned to the ncbi sequence database using blast. the mega (version ) program was used to prepare alignments (clustalw) of nucleic acid segment sequences, deduced amino acid sequences, phylogenetic trees and pairwise distance calculations [ ] . the publicly available reovirus sequences used in the analysis were obtained from ncbi-nucleotide (genbank). nucleotide sequences from a small number of viruses from each genus in the reoviridae family were used to prepare a maximum likelihood tree showing the placement of mahlv in the family based on the full rna-dependent rna polymerase (rdrp) encoding segment. maximum likelihood trees were prepared using amino acid sequences of all open reading frames from all segments, showing the placement of mahlapitsi virus (mahlv) in the orthoreovirus genus relative to other viruses in this genus for which sequence is available on genbank. virus sequence accession numbers are summarised in table . the evolutionary history was inferred by using the maximum likelihood method based on the jtt matrix-based model [ ] . the tree with the highest log likelihood is shown. the percentage of trees in which the associated taxa clustered together is shown next to the branches ( bootstrap iterations). initial tree(s) for the heuristic search were obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using a jtt model. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site all positions containing gaps and missing data were eliminated. evolutionary analyses were conducted in mega [ ] . open reading frames were located and deduced protein amino acid sequences prepared by using the clc genomics workbench (qiagen). putative functions of the new virus deduced proteins were determined by blastx similarity searches to sequences available on genbank. the ectoparasite pool homogenate used for virus isolation was used as dna source for phylogenetic confirmation of species. dna was extracted using trizol (invitrogen, waltham, ma, usa) and the method as described by the manufacturer. amplification of the cytochrome c oxidase subunit i (coi) gene was performed with barcoding primers as described by tortosa et al.: lco and hco [ ] . polymerase chain reaction was carried out in µl reactions containing µl mytaq red mix ˆ(bioline, london, uk), µl of forward and reverse primer ( µm), dna template ( µl) and nuclease free water ( µl). amplification steps were ˝c for min, cycles of ˝c for s, ˝c for s and ˝c for s, and ˝c for min. pcr product was purified using the minelute kit (qiagen). purified pcr amplicon products were then sequenced at the nicd core sequencing facility (nicd, sandringham, south africa). replication of mahlv was evaluated in two cell lines: vero e (source african green monkey kidney) and c - (source aedes albopictus mosquitoes). cells were grown to %- % confluency in cm flasks, supernatant removed and respective flasks inoculated with ml of ´ , ´ , ´ and ´ dilutions from stock virus ( ˆ tcid /ml) in emem. after h adsorption at ˝c (veroe ) or ˝c (c - ), the inoculum was removed, cells washed with ml phosphate buffered saline (pbs) and fresh emem, antibiotics and % foetal calf serum (hyclone, logan, ut, usa) added. cultures were incubated for days at ˝c (veroe ) or ˝c (c - ) while . ml aliquots of supernatant were collected from each flask directly after inoculation and addition of fresh medium (day ), followed by day , and . rna was extracted from µl of the serial supernatant collections (qiamp viral rna kit, qiagen) and subjected to taqman real-time rt-pcr. a taqman real-time rt-pcr was developed to detect the rdrp gene of mahlv. primers and probe sequences are: forward morv_ f ( '-tagtggttcgtatgcgtggt- '), reverse morv_ r ( '-aacagccattcaatctcagg- ') and probe morv_ p (fam-ggcacatatccctcaactgg-bhq), with the number in the oligonucleotide name indicating the nucleic acid position in the segment encoding rdrp. real-time rt-pcr was performed on the extracted rna using the qiagen one-step rt-pcr kit (qiagen) on a smartcycler (cepheid, sunnyvale, ca, usa) with the following program: reverse transcription ( ˝c for min), hot-start taq activation ( ˝c for min) and cycles of amplification ( ˝c for s; ˝c for s plus signal acquisition; ˝c for s). rna extracted from diluted stock mahlv (final ˆ tcid /ml) was used as a qualitative positive control in each run. from a total of bat ectoparasite pools subjected to virus isolation by three blind passages, two yielded an agent that caused obvious cytopathic effects in the form of syncytia (giant cell) formation by three or four days post inoculation (d.p.i.) (figure ). the parasite pool that yielded mahlv isolate was collected from an apparently healthy adult female rousettus aegyptiacus bat captured at mahune cave in may . cpe in vero cells were noted after two blind passages, and the supernatant collected on day five from passage four in a cm flask containing infected vero cells yielded ˆ . tcid /ml of the unknown virus. the second parasite pool that yielded mahlv isolate - was collected from an apparently healthy juvenile male rousettus aegyptiacus bat captured at mahune cave in june . cpe in vero cells were noted after three blind passages, and the supernatant collected on day five from passage five in a cm flask containing infected vero cells yielded ˆ tcid /ml of the virus. these supernatants, passage four of and passage five of - , were used for subsequent identification by tem and ngs. the ectoparasites from which the viruses were isolated were morphologically identified as bat flies, eucampsipoda africana theodor (diptera: nycteribiidae) ( figure ) [ ] . sequencing of the cytochrome c oxidase subunit i gene (coi) and alignment to sequences available on genbank, followed by phylogenetic analysis (figure ) confirms that the bat flies in this study are closest related to eucampsipoda spp. identified before. initial screening of negatively-stained culture supernatants revealed the presence of rounded icosahedrons lacking envelopes, which resembled non-rotavirus-like virions of the reoviridae ( figure ). two-layered capsids with an outer diameter of - nm (n = ) and an inner core of - nm, possessed clearly defined solvent channels radiating outwards through the clustered capsomers of the outer capsid layer ( figure ). although the dimensions of the negatively-stained, inner capsid layer were comparable to those recorded after processing for ultramicrotomy, the outer layer was slightly larger, occasionally measuring up to nm in diameter. the coi partial sequence from the bat fly sequenced from this study is indicated by the red star. initial screening of negatively-stained culture supernatants revealed the presence of rounded icosahedrons lacking envelopes, which resembled non-rotavirus-like virions of the reoviridae ( figure ). two-layered capsids with an outer diameter of - nm (n = ) and an inner core of - nm, possessed clearly defined solvent channels radiating outwards through the clustered capsomers of the outer capsid layer ( figure ). although the dimensions of the negatively-stained, inner capsid layer were comparable to those recorded after processing for ultramicrotomy, the outer layer was slightly larger, occasionally measuring up to nm in diameter. (a) negatively-stained particle with two distinct layers; (b) icosahedral negatively-stained particle; (c) resin-embedded, sectioned viral particle in cytoplasm of infected vero e cell. white arrows indicate some of the characteristic spaces between the finger-like, capsomeric projections surrounding the solvent channels through the outer capsid layer. evident in ultrathin sections of infected vero e cells were multinucleate cells with extensive nuclear lobing, and cytoplasmic inclusion bodies associated with developing, double-shelled virus particles ( figure ). tem observations therefore suggested that the isolated virus belonged to the reoviridae, sub-family spinareovirinae. (a) negatively-stained particle with two distinct layers; (b) icosahedral negatively-stained particle; (c) resin-embedded, sectioned viral particle in cytoplasm of infected vero e cell. white arrows indicate some of the characteristic spaces between the finger-like, capsomeric projections surrounding the solvent channels through the outer capsid layer. evident in ultrathin sections of infected vero e cells were multinucleate cells with extensive nuclear lobing, and cytoplasmic inclusion bodies associated with developing, double-shelled virus particles ( figure ). tem observations therefore suggested that the isolated virus belonged to the reoviridae, sub-family spinareovirinae. evident in ultrathin sections of infected vero e cells were multinucleate cells with extensive nuclear lobing, and cytoplasmic inclusion bodies associated with developing, double-shelled virus particles ( figure ). tem observations therefore suggested that the isolated virus belonged to the reoviridae, sub-family spinareovirinae. an unbiased next-generation sequencing approach using sispa amplification confirmed the presence of a novel orthoreovirus. initial sequencing results of both isolates yielded enough sequence coverage to identify all segments. a polyetheleneglycol (peg) precipitated preparation of isolate - yielded the most viral specific reads and formed contigs aligning to othoreoviruses using blastn and blastx. both the ' and ' ends were missing for all the segments, so to obtain complete genomes for each isolate, rrna depletion and a combination of sispa and rapid amplification of cdna ends (sispa-race) was done. read numbers were also increased by running samples on an illumina nextseq . both an increase in the percentage of viral reads aligning to the genome segments and an increase in coverage of the ends were observed. presence of mahlv in the original homogenates from which the isolates were obtained was confirmed by sispa amplification and ngs directly from the homogenates. expectedly, only a low number of reads from both homogenates mapped to the mahlv sequence due to the high amount of host sequence obscuring viral specific sequences combined with likely low viral load in the homogenates and the relatively low sensitivity of the sispa method. a maximum likelihood tree, constructed with nucleic acid sequence data for the rna-dependent rna polymerase (rdrp) encoding segments of representative viruses from the different genera within reoviridae (figure ) shows the placement of both isolates amongst other orthoreoviruses in the family. maximum likelihood trees were prepared using the deduced amino acid sequences from the open reading frames (orf's) of all the virus' segments and those of other viruses in the orthoreovirus genus (figures - ) . a distinct clade is formed by mahlv, bush viper reovirus, baboon orthoreovirus and broome virus within the genus. the above-mentioned clade is visibly distinct from others composed of bat-associated viruses; the nelson bay orthoreovirus and bat-derived mammalian orthoreoviruses. the closest relative of mahlv, based on sequence homology of a conserved core protein, is bush viper reovirus (lambda b nucleic acid identity- . %; rdrp amino acid identity- . %) while the closest bat-associated virus is broome virus (lambdab nucleic acid identity- . %; rdrp amino acid identity- . %) ( table ) . homology of the divergent major outer capsid protein of mahlv to known orthoreoviruses is much lower: sigma b nucleic acid identity- . %- . %; amino acid identity- . %- . % (table ) . the genome segments of mahlv were named according to the nucleotide length, which is consistent with the nomenclature of other orthoreoviruses [ ] . a summary of the mahlv genome is given in table . the total genome size is , nucleotides and predicted to encode eleven proteins, seven of which are structural. all ten genome segments of mahlv contain an identical ' terminal sequence, ucauc- ', which is conserved between all known species of orthoreovirus, and an identical ' terminal sequence, '-gguca which is unique to mahlv. non-coding regions (ncrs) are present at both ends of the genome segments, with the ' ncrs being shorter in nucleotide length than ' ncrs. the nucleotide sequences of the two isolates of mahlv, and - , are not identical. nucleotide homology of the rdrp encoding segment between the two isolates is . % ( . % deduced amino acid sequence), and . % ( . % deduced amino acid sequence) for the sigma b encoding segment. putative protein functions were determined by blastx similarity searches to sequences available on genbank, revealing putative functions known for other orthoreoviruses. the segments l , l , m , m , s , s and s each contain a single start aug codon in close proximity to the ' end. the l segment of mahlv contains two aug start codons in close proximity to the ' end, at positions and . agcaugg) . the open reading frame initiating at position is nucleotides in length and putatively encodes for an outer capsid protein involved in membrane penetration during infection (mub). the second aug initiates a nucleotide open reading frame but the deduced amino acid sequence does not match any viral protein of note on genbank. the deduced sigma a protein of mahlv (segment s ) contains the fusogenic orthoreovirus-wide conserved arginine amino acid at position . the s segment is bicistronic and encodes for a kda protein similar to p membrane fusion protein of bush viper reovirus and a non-overlapping kda protein similar to p non-structural protein of baboon orthoreovirus without a known function ( table ). the isoelectric point of mahlv p is acidic ( . ), similar to that of p of broome virus and baboon orthoreovirus and contrary to that of other orthoreoviruses. the first ten amino acids in the putative kda protein of mahlv are identical to the first ten amino acids in the p fusion protein of broome virus and represent the myristoylation consensus sequence required for fusion activity of the protein. the s segment does not encode a cell attachment protein, an observation also characteristic of broome virus and baboon orthoreovirus. . . . . . . . . mahlv replicated efficiently in vero cell culture, with the inoculum containing a high dose of virus ( tcid /ml) leading to rapid monolayer destruction after inoculation, with a peak in virus rna (measured by real-time rt-pcr) by day , followed by a decrease on day . the inoculums containing a lower virus dose ( and tcid /ml) resulted in a peak of rna detection on day . all three above-mentioned inoculum doses yielded detectable virus rna by day after inoculation. the inoculum containing tcid /ml virus did not generate detectable viral rna until day , and was still showing an upward trend on day (last sampling day). the virus did not replicate in the insect cells (c - ) up to day , but adaptation to these cells through serial passaging was not attempted. the role of bats in harbouring pathogens of public health and veterinary importance is becoming an increasingly popular topic of research within the field of emerging and zoonotic diseases. the most notable viruses in which natural transmission from bats have been implicated include filoviruses, coronaviruses, paramyxoviruses, herpesviruses, lyssaviruses and bunyaviruses. the implication of bats in transmission or maintenance of some of these viruses is very circumstantial and often based only on serological evidence. more convincing evidence for others is based on detection of viral nucleic acid and isolation of live virus, although this does not conclusively prove that a vertebrate host is a reservoir. finding pathogens in bats leads to the questions of how they are transmitted between bats, and from bats to incidental hosts such as humans. one possible transmission mechanism could be bat-associated hematophagous arthropods, such as the bat flies, but migration of parasites between bats is not well understood and would require further entomological investigation to better understand [ ] . two isolates of a novel fusogenic orthoreovirus were cultured and we determined their full genome sequences, which were compared to currently known viruses in the genus. the two isolates are not identical but similar enough to suggest that they are merely two isolates of the same virus. this suggests that there are multiple variants of the virus present in the host population. members of a species within the orthoreovirus genus are usually identified by a number of characteristics: amino acid and nucleotide sequence identity, organization of the polycistronic genome segment and host species [ ] . for conserved core proteins, an amino acid identity > % for homologous proteins indicates that two viruses belong to the same species, while identity < % indicates a possible new species. when comparing the amino acid sequence of more divergent outer capsid proteins, > % identity indicates one species and < % indicates different species. nucleic acid sequence identity of homologous segments of > % indicates the same species and < % a new species. the nature of conserved genome segment termini sequences of orthoreoviruses is also useful for virus classification [ ] . the divergence of mahlv sequence from other known orthoreoviruses combined with a unique conserved ' genome segment end and a unique host species, suggests that this is a new virus species in this genus. along with broome virus and baboon orthoreovirus, mahlv is the third orthoreovirus that lacks an identified cell attachment protein in the s segment. this unique characteristic further strengthens the phylogenetic classification which places these viruses in a separate clade and suggests that entry of these viruses into cells is mediated differently than for other orthoreoviruses. taking the abovementioned criteria and the sequence characteristics of the novel virus described here into consideration, we propose that mahlapitsi virus constitutes a new species within the orthoreovirus genus. to our knowledge this is the first description of an orthoreovirus in africa with an indirect link to bats. considering the rich diversity of bat species found on the continent and increased scientific interest in this field, this is unlikely to be the only such virus to be isolated from bat ectoparasites in years to come. however, to our knowledge this is the first orthoreovirus to be isolated from an arthropod host, since all currently known viruses in this genus are associated with vertebrates. mahlv did not replicate on c - cells in this study but aedes albopictus, from which the c - cell line is derived, is classified in a completely different dipteran family, the culicidae, likely pointing to a cell receptor incompatibility. another possible explanation could be the temperature at which insect cells are cultured compared to mammalian cells, which might be incompatible with this virus. the arthropod-borne nature of mahlv transmission needs further investigation, especially to establish whether nycteribiid flies are only involved in mechanical or possibly biological transmission, and if the virus is even transmitted to bats. various other genera in the reoviridae family contain vector-borne viruses, including banna virus (seadornavirus), colorado tick fever virus (coltivirus) and bluetongue virus (orbivirus). our isolation of mahlv from arthropods might direct some attention to the possible role of insects in the transmission of currently known orthoreoviruses, or possibly the presence of other yet unknown viruses in various arthropods. we have no information on the geographical range of mahlv, but the wide distribution of rousettus aegyptiacus in africa and the middle east [ ] , and the strict host preference and specificity of bat flies [ ] [ ] [ ] , dictate that their ranges will overlap. we have no data to suggest that mahlv has any human health implication, but this warrants further investigation. the virus grows to high titers in vero e cells which suggests that it may infect vertebrates, although growth in in vitro systems cannot be translated directly into replication in a vertebrate host. it is important to note, also, that this was after blind passage and cell culture adaptation (cpe noted after two-three blind passages). any risk of human infection for now, however, is only likely in individuals who come into close contact with wild egyptian fruit bats and their ectoparasites. although highly host-dependent, the bat flies have been noted to leave their bat hosts and crawl on bat researchers (personal observation). respiratory disease has been noted in humans infected with melaka, kampar and nelson bay orthoreovirus, including limited human-to-human transmission [ , , , ] . thus the potential for mahlv to infect humans, and spread between humans, cannot be excluded until further investigation is done, especially considering that the virus grows very efficiently on a monkey-derived cell line. in conclusion, we have identified a novel orthoreovirus which we propose should constitute a new species within the genus. two virus strains were isolated from ectoparasitic bat flies collected from egyptian fruit bats from a south african cave roost. this represents the first isolation of an orthoreovirus from arthropods and the first african virus in this genus with an indirect link to bats. fruit bats as reservoirs of ebola virus marburg virus infection detected in a common african bat bats are natural reservoirs of sars-like coronaviruses middle east respiratory syndrome coronavirus in bats, saudi arabia nipah virus: a recently emergent deadly paramyxovirus isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus isolation of rabies virus from an insectivorous bat (tadarida mexicana) in california fatal case of human rabies (duvenhage virus) from a bat in kenya: the netherlands detection of a novel herpesvirus from bats in the philippines a novel 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member of the pteropine orthoreovirus species isolated from fruit bats imported to italy structure and cytopathic effects of nelson bay virus a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans identification and characterization of a new orthoreovirus from patients with acute respiratory infections isolation and characterization of three mammalian orthoreoviruses from european bats isolation and identification of a natural reassortant mammalian orthoreovirus from least horseshoe bat in china broome virus, a new fusogenic orthoreovirus species isolated from an australian fruit bat isolation and genomic characterization of a novel orthoreovirus from a brown-eared bulbul (hypsipetes amaurotis) in japan discovery of an orthoreovirus in the aborted fetus of a stellar sea lion (eumetopias jubatus) high similarity of novel orthoreovirus detected in a child hospitalized with acute gastroenteritis to mammalian orthoreoviruses found in bats in europe reptilian reovirus: a new fusogenic orthoreovirus species investigation of a potential zoonotic transmission of orthoreovirus associated with acute influenza-like illness in an adult patient imported case of acute respiratory tract infection associated with a member of species nelson bay orthoreovirus standards for sequencing viral genomes in the era of high-throughput sequencing isolation of genetically diverse marburg viruses from egyptian fruit bats virological and serological findings in rousettus aegyptiacus experimentally inoculated with vero cells-adapted hogan strain of marburg virus selective depletion of rrna enables whole transcriptome profiling of archival fixed tissue viral genome sequencing by random priming methods cutadapt removes adapter sequences from high-throughput sequencing reads quality control and preprocessing of metagenomics datasets scalable de novo metagenome assembly and profiling molecular evolutionary genetics analysis version . the rapid generation of mutation data matrices from protein sequences an illustrated catalogue of the rothschild collection of nycteribiidae (diptera) in the british museum (natural history), with keys and short descriptions for the identification of subfamilies, genera, species and subspecies sequence at both termini of the genes of reovirus serotype (strain dearing) mapping the zoonotic niche of marburg virus disease in africa the authors would like to thank the following individuals for their contributions towards fieldwork and technical assistance: busi mogodi, justice kgatitsoe, antoinette grobbelaar, terence scott, joe kgaladi, marinda mortlock, marike geldenhuys, jessica coetzer, andre coetzer. we would like to thank dorothy southern and alfred musekiwa for proofreading the manuscript.the project is jointly funded by the following grants awarded to: janusz t. paweska the grant holders acknowledge that opinions, findings and conclusions or recommendations expressed in any publication generated by gdd and nrf-supported research are those of the authors and that the gdd and nrf accept no liability whatsoever in this regard.author contributions: petrus jansen van vuren and janusz paweska conceived and designed the experiments; all authors were involved in some aspect of performing the experiments and interpretation of the data; petrus jansen van vuren, janusz paweska, monica birkhead, michael wiley and gustavo palacios analysed the data; petrus jansen van vuren wrote the paper with inputs from all other authors; janusz paweska, petrus jansen van vuren, wanda markotter and gustavo palacios contributed funding. all authors read and approved the final manuscript. the authors declare no conflict of interest.viruses , , key: cord- -qnn hp e authors: cong, yingying; verlhac, pauline; reggiori, fulvio title: the interaction between nidovirales and autophagy components date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qnn hp e autophagy is a conserved intracellular catabolic pathway that allows cells to maintain homeostasis through the degradation of deleterious components via specialized double-membrane vesicles called autophagosomes. during the past decades, it has been revealed that numerous pathogens, including viruses, usurp autophagy in order to promote their propagation. nidovirales are an order of enveloped viruses with large single-stranded positive rna genomes. four virus families (arterividae, coronaviridae, mesoniviridae, and roniviridae) are part of this order, which comprises several human and animal pathogens of medical and veterinary importance. in host cells, nidovirales induce membrane rearrangements including autophagosome formation. the relevance and putative mechanism of autophagy usurpation, however, remain largely elusive. here, we review the current knowledge about the possible interplay between nidovirales and autophagy. nidoviruses rank among the most complex rna viruses and their molecular genetics clearly discriminates them from other rna virus orders [ ] . still, our knowledge about their life cycle, mostly unveiled with studies on coronaviruses (covs), is very limited [ , , , ] . to enter cells, nidoviruses bind to cell surface receptors, an event that precedes the fusion of the viral and cellular membranes ( figure , step ), which is presumably mediated by one of the surface glycoproteins [ , ] . the fusion takes place either at the plasma membrane or in the endosomes and releases the nucleocapsid into the host cell cytoplasm [ ] (figure , step ). after genomic rna uncoating from the nucleocapsid, two large replicase open reading frames (orfs), orf a and orf b, are translated by host ribosomes to yield two large polyprotein precursors that undergo autoproteolytic processing to eventually produce the non-structural (nsp) proteins. the nsp proteins interfere with the host defenses but also induce the formation of double-membrane vesicles (dmvs) and convoluted membranes, on which they collectively form the replication-transcription complexes (rtcs) [ , ] (figure , steps , , and ). these complexes mediate the synthesis of the genomic rna and a nested set of subgenomic rnas that directs the translation of the structural proteins (the nucleocapsid n protein, the membrane m protein, the envelope e protein and the spike s protein) and some accessory proteins, like the hemagglutinin esterase in the case of severe acute respiratory syndrome (sars)-cov or mouse hepatitis virus (mhv) [ ] [ ] [ ] (figure , steps and ). newly synthesized genomic rnas associate with the cytoplasmic nucleocapsid proteins to generate the so-called ribonucleoprotein complexes [ , ] . the viral structural envelope proteins are inserted into endoplasmic reticulum (er) and targeted to the site of virus assembly, the er, or the golgi, where they interact with the ribonucleoprotein complex to initiate the budding of virus particles into the lumen of the membrane compartment [ , , ] (figure , steps , and ) . newly formed virions then egress the host cell through secretion via the exocytic pathway [ , ] (figure , step ). viruses , , of they interact with the ribonucleoprotein complex to initiate the budding of virus particles into the lumen of the membrane compartment [ , , ] (figure , steps , and ). newly formed virions then egress the host cell through secretion via the exocytic pathway [ , ] (figure , step ). generalization of nidovirales life cycle, based on the information acquired studying arteriviruses and coronaviruses. infection starts with the binding of the viral particle to a cell surface receptor and subsequent cell entry through membrane fusion in endosomes upon endocytosis (step ). translation of the released genomic rna (grna) yields replicase polyproteins (step ), i.e., polyprotein a (pp a) and polyprotein ab (pp ab), which undergo autoproteolytic processing to generate nonstructural proteins that assemble into replication-transcription complexes (rtcs). the rtcs are part of a complex membranous network composed of double membrane vesicles (dmvs) and convoluted membranes (step ). the rtcs first engage in minus-strand rna synthesis to produce both single strand full-length and subgenomic (sg) minus-strand rnas (step ). subsequently, they use sg mrnas as templates for the production of the grna and plus-strand sg mrnas required to express the structural protein genes (step ). newly synthesized s, e, and m structural proteins are inserted in the endoplasmic reticulum (er) (steps and ), whereas the n nucleocapsides are translated and oligomerize in the cytosol, where they interact with rtcs and associate with the grna to form the ribonucleoprotein complexes (step ). virion assembly takes place in the er and/or golgi (step ), and involves the inward budding of the limiting membrane of these compartments, which is triggered by the interaction between the structural proteins and the ribonucleoprotein complexes. mature virions are released extracellularly by exocytosis (step ). within the term autophagy are grouped all those catabolic pathways mediating the delivery of cytoplasmic material into the mammalian or plant/yeast vacuole for degradation. there are three major types of autophagy, i.e., macroautophagy, microautophagy, and chaperone-mediated autophagy [ ] . macroautophagy (hereafter referred to as autophagy) is conserved among eukaryotes that allows the turnover of excess or damaged cellular components, including long-lived arteriviruses and coronaviruses. infection starts with the binding of the viral particle to a cell surface receptor and subsequent cell entry through membrane fusion in endosomes upon endocytosis (step ). translation of the released genomic rna (grna) yields replicase polyproteins (step ), i.e., polyprotein a (pp a) and polyprotein ab (pp ab), which undergo autoproteolytic processing to generate nonstructural proteins that assemble into replication-transcription complexes (rtcs). the rtcs are part of a complex membranous network composed of double membrane vesicles (dmvs) and convoluted membranes (step ). the rtcs first engage in minus-strand rna synthesis to produce both single strand full-length and subgenomic (sg) minus-strand rnas (step ). subsequently, they use sg mrnas as templates for the production of the grna and plus-strand sg mrnas required to express the structural protein genes (step ). newly synthesized s, e, and m structural proteins are inserted in the endoplasmic reticulum (er) (steps and ), whereas the n nucleocapsides are translated and oligomerize in the cytosol, where they interact with rtcs and associate with the grna to form the ribonucleoprotein complexes (step ). virion assembly takes place in the er and/or golgi (step ), and involves the inward budding of the limiting membrane of these compartments, which is triggered by the interaction between the structural proteins and the ribonucleoprotein complexes. mature virions are released extracellularly by exocytosis (step ). within the term autophagy are grouped all those catabolic pathways mediating the delivery of cytoplasmic material into the mammalian or plant/yeast vacuole for degradation. there are three major types of autophagy, i.e., macroautophagy, microautophagy, and chaperone-mediated autophagy [ ] . macroautophagy (hereafter referred to as autophagy) is conserved among eukaryotes that allows the turnover of excess or damaged cellular components, including long-lived proteins and organelles, to maintain cellular energy levels and ensure survival [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this process is characterized by double-membrane vesicles called autophagosomes, which sequester the cytoplasmic components targeted to destruction and deliver them into lysosomes for degradation [ ] [ ] [ ] [ ] [ ] (figure ). the process can be divided into three different steps: the initiation step, when the phagophore or isolation membrane is formed, the elongation step, during which the phagophore expands and close to generate an autophagosome, and the maturation step, where the autophagosome fuses with the lysosome (figure ). autophagy is regulated by several signaling cascades, including the one centered on the mammalian target of rapamycin (mtor) [ , , ] . autophagosomes are formed through the concerted action of the autophagy-related (atg) genes [ , ] . the proteins encoded by the atg genes have been classified in five functional groups. the unc- like autophagy activating kinase (ulk) kinase complex, the class iii hvps phosphatidylinositol -kinase complex, and the atg cycling system, on one hand, play a key role in the initiation of autophagy and phagophore formation. the atg and microtubule-associated protein light chain (lc ) conjugation systems, on the other hand, mediate the elongation and closure of the phagophore. the first of these atg protein complexes responds to upstream regulatory signals, such as the inactivation of mtor, and key in initiating the formation of an autophagosome, is the ulk kinase complex, which is composed of ulk or ulk , atg , fak family kinase-interacting protein of kda (fip ), and atg [ , ] . the class iii hvps phosphatidylinositol -kinase, which is part of a complex including hvps , atg l , and beclin , generates the pool of phosphatidylinositol -phosphate (ptdins p) on autophagosomal membranes that facilitates the recruitment of ptdins p effectors such as double fyve-containing protein (dfcp ) and the human wd-repeat protein interacting with phosphoinositides (wipi) proteins [ ] [ ] [ ] [ ] [ ] [ ] . the hvps -containing complex as well as the transmembrane protein atg , are two other important factors during the early stage of autophagosome biogenesis [ , ] . subsequently, two ubiquitination-like systems, which ultimately recruit members of the lc /atg protein family onto autophagosomal membranes through their conjugation to phosphatidylethanolamine, are essential for the completion of the forming autophagosomes [ ] . finally, the fusion of autophagosomes first with endosomes and then with lysosomes, leads to the formation of amphisomes and autolysosomes, respectively, where the degradation of the autophagy cargoes take place [ ] . proteins and organelles, to maintain cellular energy levels and ensure survival [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this process is characterized by double-membrane vesicles called autophagosomes, which sequester the cytoplasmic components targeted to destruction and deliver them into lysosomes for degradation [ ] [ ] [ ] [ ] [ ] (figure ). the process can be divided into three different steps: the initiation step, when the phagophore or isolation membrane is formed, the elongation step, during which the phagophore expands and close to generate an autophagosome, and the maturation step, where the autophagosome fuses with the lysosome (figure ). autophagy is regulated by several signaling cascades, including the one centered on the mammalian target of rapamycin (mtor) [ , , ] . autophagosomes are formed through the concerted action of the autophagy-related (atg) genes [ , ] . the proteins encoded by the atg genes have been classified in five functional groups. the unc- like autophagy activating kinase (ulk) kinase complex, the class iii hvps phosphatidylinositol -kinase complex, and the atg cycling system, on one hand, play a key role in the initiation of autophagy and phagophore formation. the atg and microtubule-associated protein light chain (lc ) conjugation systems, on the other hand, mediate the elongation and closure of the phagophore. the first of these atg protein complexes responds to upstream regulatory signals, such as the inactivation of mtor, and key in initiating the formation of an autophagosome, is the ulk kinase complex, which is composed of ulk or ulk , atg , fak family kinaseinteracting protein of kda (fip ), and atg [ , ] . the class iii hvps phosphatidylinositol -kinase, which is part of a complex including hvps , atg l , and beclin , generates the pool of phosphatidylinositol -phosphate (ptdins p) on autophagosomal membranes that facilitates the recruitment of ptdins p effectors such as double fyve-containing protein (dfcp ) and the human wd-repeat protein interacting with phosphoinositides (wipi) proteins [ ] [ ] [ ] [ ] [ ] [ ] . the hvps -containing complex as well as the transmembrane protein atg , are two other important factors during the early stage of autophagosome biogenesis [ , ] . subsequently, two ubiquitination-like systems, which ultimately recruit members of the lc /atg protein family onto autophagosomal membranes through their conjugation to phosphatidylethanolamine, are essential for the completion of the forming autophagosomes [ ] . finally, the fusion of autophagosomes first with endosomes and then with lysosomes, leads to the formation of amphisomes and autolysosomes, respectively, where the degradation of the autophagy cargoes take place [ ] . it has long been believed that the atg proteome is exclusively involved in autophagy, but recent findings have revealed that single atg genes or functional clusters of atg genes fulfill important cellular functions outside the context of their role in autophagy [ ] [ ] [ ] . some of these functions have it has long been believed that the atg proteome is exclusively involved in autophagy, but recent findings have revealed that single atg genes or functional clusters of atg genes fulfill important cellular functions outside the context of their role in autophagy [ ] [ ] [ ] . some of these functions have been discovered by studying host-pathogen interactions [ , , ] . for example, atg but no other atg proteins play a unique role in the defense against mycobacterium infection [ ] . more recently, it has been shown that fip and atg participate in the controlling of picornaviral infections outside the context of autophagy [ ] . here, we will review the current knowledge on the interplay between nidovirales and autophagy. there are currently no data available for several viruses and few nidovirales families. thus, this compendium will focus on the documented viruses in the arterivirus and coronavirus families (summarized in table ). green shading indicates a pro-viral role for autophagy, grey shading indicates conflictual reports and blue shading highlights an unconventional role of autophagy-related (atg) proteins. the table also provides the information about how autophagy has been experimentally manipulated in the studies addressing the role of this pathway with the indicated virus, the used cell types, and the references. -ma: -methyladenine; bafa : bafilomycin a , lc : microtubule-associated protein light chain , mefs: mouse embryonic fibroblasts. the two most studied arteriviridae are prrsv and the equine arteritis virus (eav). the prrsv strain, which was historically first characterized and is commonly referred to as atypical (i.e., ap prrsv), causes the abortions in - % of the sows, and fever and anorexia leading to the death of - % of them [ ] . however, in , the emergence of a novel virulent highly pathogenic prrsv (hp prrsv) strain, carrying mutations in nsp β, nsp , and orf genes, caused higher morbidity ( %) and mortality ( %) rates in piglets and sows [ ] . the equine arteritis virus, in contrast, infects horses and donkeys, and can cause abortions in pregnant females and mortality in neonates [ ] . the first study on the role of autophagy in arterivirus life cycle was carried out with the hp prrsv strain [ ] . infected cells displayed the presence of a higher number of autophagosome-like double-membrane vesicles, an accumulation of green fluorescent protein (gfp)-lc -positive puncta and higher levels of lipidated lc , indicating an induction of autophagy [ ] . inhibition of autophagy with either -methyladenine ( -ma), a non-specific hvps inhibitor, or depletion of atg or beclin , led to a significant reduction in both expression of prrsv nsp and prrsv titer. conversely, induction of bulk autophagy using rapamycin (mtor inhibitor) resulted in an enhancement of viral replication [ ] , a result that later was confirmed by others [ ] [ ] [ ] [ ] . in one of these subsequent studies, pujhari et al. showed that while the virus titer in the supernatants of infected cells treated with rapamycin was higher than in the control, intracellular levels of prrsv n protein or nsp assessed by flow cytometry were lower [ ] . this latter result is the opposite of the ones obtained by the other studies where rapamycin treatment led to an up-regulation of nsp expression [ ] [ ] [ ] [ ] . two subsequent works reached the same conclusion of autophagy having a beneficial role in prrsv life cycle [ , ] . liu et al. confirmed autophagy induction by both ap and hp prrsv strains [ ] . interestingly, they also observed a decrease in the virus titer in cells treated with bafilomycin a (bafa ), a drug inhibiting either autophagosome-lysosome fusion or lysosomal degradation, which suggested that autolysosomes might serve as replication platforms for prrsv [ ] . in contrast, sun et al. showed, using confocal microscopy, that the hp prrsv infection inhibits the fusion between autophagosomes and lysosomes [ ] . this result indicates that prrsv might trigger an incomplete autophagy and implicates that this could be beneficial for the virus life cycle. to gain insights into a possible molecular connection between autophagy and prrsv, they also transfected cells with either nsp or nsp , which encode two transmembrane components of arterivirus rtcs that play a central role in dmvs formation [ ] . interestingly, they found the co-localization between endogenous lc and ectopically expressed nsp , but not nsp , by confocal microscopy and fractionation on continuous density gradients, suggesting that the accumulated autophagosomes during prrsv could represent the replicative platforms [ ] . thus, it still remains to be firmly demonstrated whether the results obtained with the ectopic expression of nsp recapitulates a situation occurring in cells exposed to prrsv. recently, three additional research articles provided evidences that autophagy but also apoptosis are induced by prrsv in host cells [ , , ] . wang and collaborators investigated apoptosis and autophagy activation in the thymus of piglets infected with the hp prrsv strain because they previously showed that this virus leads to thymic atrophy and thymocyte apoptosis [ ] . their investigation concluded that the hp prrsv could induce apoptosis in bystander cells and autophagy in both infected and bystander cells in the thymus of infected piglets [ ] . in a follow-up study, another laboratory found that hp prrsv replication was attenuated in autophagy deficient marc- cells and potentiated by inhibiting apoptosis using z-vad-fmk, a caspase pan-inhibitor [ ] . interestingly, hp prrsv replication could be restored in the autophagy deficient cells by blocking apoptosis, suggesting a functional interplay between autophagy and apoptosis during prrsv replication. subsequently, zhou et al. confirmed the activation of autophagy and a subsequent induction of apoptosis over the course of a prrsv infection in marc- cells [ ] . in their study, inhibition of autophagy by -ma caused a significant increase in prrsv-induced apoptosis, also unveiling a potential connection between both mechanisms. in line with this conclusion, they also observed an increase in the expression of bcl- -associated death promoter (bad), a pro-apoptosis protein, and beclin , an autophagy regulator. interestingly, co-immunoprecipitation and confocal microscopy experiments revealed the formation of a bad-beclin complex in infected cells [ ] . beclin knockdown significantly decreased viral replication and prrsv-induced autophagy as expected, while knocking down of bad resulted in an induction of autophagy and enhanced viral replication [ ] . the authors concluded that the enhancement of autophagy could promote prrsv replication by postponing apoptosis through the formation of the bad-beclin complex [ ] . in a study exploring a potential connection between eav and autophagy, monastyrska et al. found that the dmvs induced by this virus are decorated with lc but the eav life cycle proceeded unaffected in cells lacking atg [ ] . although autophagy was not required, depletion of lc markedly reduced eav replication and it could be fully restored by expression of a non-lipidable form of lc [ ] . similar to mhv, eav-induced dmvs were also positive for edem (er degradation enhancing alpha-mannosidase like protein ) leading to the conclusion that eav might also hijack the er-derived membranes of edemosomes to ensure its replication [ , ] . it still needs to be investigated, however, whether other atg proteins are dispensable for eav life cycle. furthermore, it is unclear whether both autophagy and apoptosis are induced over the course of an eav infection as observed for prrsv. the most studied alphacoronaviruses (alpha-covs) are the transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv), which both infect suckling piglets and lead to a high mortality rate [ , ] . recurrent outbreaks of pedv have occurred across asia and the usa, causing significant economic losses [ ] . alpha-cov also includes human pathogens such as hcov- e and hcov-nl , which are associated with respiratory tract infections such as the common cold to bronchiolitis [ , ] . despite their medical and veterinary relevance, however, the exact mechanisms of alpha-cov replication and pathogenesis are not well characterized yet. sun et al. recently performed a high throughput mass spectrometry analysis in pedv-infected vero cells [ ] . their goal was to identify which cellular proteins are differentially expressed during viral infection to better understand the impact of pedv on host cells. interestingly they found that autophagy might be among the altered pathways as sequestrosome (sqtsm /p ) and lc expression levels were upregulated. a subsequent study thus focused on the potential interplay between autophagy and pedv [ ] . the authors found that pedv infection induces autophagy in vero cells using different assays such as lc -positive puncta formation, transmission electron microscopy (tem) and western blot assessment of both lc lipidation and sqstm /p turnover. in line with these observations, -ma treatment or the ablation of either beclin or atg , reduced the production of infectious viral particles. treatment of the infected cells with rapamycin, however, did not change the viral titer, probably because of the multiple effects of this compound on the cell physiology. altogether, these data showed that autophagy induction during pedv infection could be beneficial for the virus. two more recent studies have addressed the link between autophagy and tgev replication but they have reached opposite conclusions [ , ] . they both demonstrated that autophagy is induced in tgev-infected cells using methods such as tem, lc puncta formation and western blot analysis of both lc lipidation and sqstm /p degradation. in their article zhu et al. also showed that the selective degradation of mitochondria by autophagy, i.e. mitophagy, might be induced by tgev as they observed in infected ipec-j cells a reduced mitochondrial mass, a light oxidative stress, and mitochondria in autophagosome-like vesicles [ ] . in support of this notion, the authors also found that tgev n protein and gfp-lc localize to mitochondria. interestingly, induction of mitophagy by mitochondria depolarization using carbonyl cyanide m-chlorophenyl hydrazone (cccp) increased the viral titer, suggesting that this pathway might be beneficial for viral replication. similarly, induction of bulk autophagy using rapamycin also led to more production of progeny virus [ ] . conversely, incubation with -ma or atg depletion inhibited viral replication assessed by n protein expression and viral titers. zhu et al. thus concluded that autophagy, and mitophagy in particular, plays an important role in tgev life cycle [ ] . this conclusion, however, differs from the one reached in a parallel study. guo and collaborators found that both hvps and lysosomal inhibitors increased both the number of cells infected by tgev and the viral titer, while rapamycin had an opposite effect [ ] . moreover, silencing lc , atg , or atg expression in st cells promoted tgev replication, showing that autophagy has an antiviral function [ ] . the apparent discrepancies between these two studies could be explained by the use of different tgev strains (shxb versus h ) and/or cell lines (ipec-j versus st). further investigations are thus needed to conclusively determine whether autophagy plays a role in tgev life cycle. it will be particularly important to establish this in tissues that are normally infected by pedv. the first investigations on the interplay between cov and autophagy focused on mhv, a betacoronavirus (beta-cov) that is often used as a model virus to study the mechanism of cov infections. as a result, there is a relatively large amount of data about various aspects of mhv life cycle. importantly, the genus beta-cov also includes the highly pathogenic human viruses sars-cov and mers-cov, two viruses that cause acute respiratory symptoms and they are on the who list of viruses likely to cause future epidemics (figure ). like other cov, mhv replication takes place on interconnected structures formed by convoluted membranes and dmvs, with the latter being reminiscent of autophagosomes [ ] . this structural similarity prompted the investigation of a possible interplay between autophagy and cov replication. interestingly, the first two studies on the importance of atg proteins during mhv replication reached conflicting conclusions. prentice et al. argued that components of the autophagy machinery are required for mhv replication while zhao et al. demonstrated that autophagy was dispensable for the same process [ , ] . in particular, both teams monitored viral replication in murine embryonic fibroblasts (mefs) knocked out for atg . the first group found that both mhv replication and dmv formation was impaired in atg −/− knockout cells, while the second did not observe any effect on mhv life cycle in absence of atg [ , ] . the fact that mhv infection does not require intact atg machinery was also later confirmed by another group using atg −/− mefs [ ] . data from both laboratories, however, established that the viral rtcs are co-localizing with endogenous lc , which on one hand was in agreement with observations gained from sars-cov, but on the other hand was conflicting with results obtained with mhv by a third group [ , ] . these apparent discrepancies were subsequently explained by showing that endogenous lc but not ectopically expressed gfp-lc co-localizes with cov rtcs [ ] . data from different groups strongly support an er involvement in convoluted membranes and dmvs biogenesis, as those structures can be found connected to the er and transmembrane nsp proteins can be glycosylated and localize to the er when individually expressed [ , ] . the rtcs and dmvs, however, do not co-localize with marker proteins of the er, ergic, and the golgi [ , ] and disruption of the secretory pathway has no major effect on cov replication [ ] . this indicates that dmvs' biogenesis might not involve the secretory pathway. in contrast, the er-associated degradation (erad) tuning pathway, a vesicular transport route out of the er, has been shown to be important for cov infection [ ] . erad allows for the degradation of misfolded er proteins and it is negatively regulated during normal growing conditions, in absence of stress. this tuning down of the erad activity is mediated at least in part by small vesicles called edemosomes, which specifically capture key positive erad regulators such as edem and os- , and degrade them in compartments of the endolysosomal system [ ] . interestingly, edemosomes are decorated with non-lipidated from of lc (also called lc -i) and their formation might require selected components of the atg machinery, such as atg [ , ] . reggiori et al. eventually revealed that dmvs were associated with lc -i and positive for both edem and os- , suggesting that mhv might actually highjack part of the membranes of the erad tuning pathway [ ] . although expression of a non-lipidable lc impaired dmvs biogenesis and viral replication, absence of edem and os- had no effect. thus, the authors hypothesized that one or more nsp proteins might associate with components of the machinery of edemosomes, such as a cargo receptor or a coat protein, to subvert these vesicles and generate the dmvs. lc -i could be such a candidate but no interaction between lc -i and mhv nsp proteins was detected using the yeast two-hybrid assay [ ] . how mhv highjacks edemosomes and what the exact role of lc -i is in this process are questions that remain unanswered. overall, beta-cov interactions with autophagy and atg proteins appear to be complex. although mhv hijacking of lc -positive edemosomes for its replication appears to be clearly established, this finding has not yet been extended to other beta-cov or to other cov in general. co-localization between sars-cov rtcs and endogenous lc , however, has been reported [ ] . beta-cov do not require canonical autophagy for their life cycle [ ] [ ] [ ] but it cannot be excluded a priori that they could need a non-canonical form of autophagy, independent from atg and atg [ ] . furthermore, while autophagy might be induced during infection or transient expression of single viral proteins [ , ] , there is currently no evidence that this is directly regulated by beta-cov. indeed, autophagy stimulation could be part of a cellular response caused by either the presence of toxic exogenous proteins or er stress induced by the massive production of viral proteins [ ] [ ] [ ] . a recent study concluded that expression of nsp fragments derived from several cov, which comprise the papain-like protease domain and one transmembrane segment, induce autophagy through direct binding to lc and beclin [ ] . this conclusion, however, has to be carefully considered since the authors of this study observed gfp-lc puncta formation in cells ectopically expressing the nsp fragment but they did not examine whether these puncta are indeed autophagosomes. moreover, the relevance of the use of a truncated form of nsp in absence of a cov infection remains to be determined. additional studies are thus needed to address the questions if and, eventually, how beta-cov trigger autophagy. gammacoronaviruses (gamma-covs) are viruses that mainly infect poultry but are also transmissible to humans. they replicate in the respiratory tract and thus cause respiratory defects. the infectious bronchitis virus, which causes major loses in the poultry industry, is the model virus for gamma-cov. similarly to other cov, the ibv genome encodes several nsp proteins that help with replication and interfere with host cell functions. cottam et al. have reported that infections with ibv trigger the formation of endogenous lc -positive puncta in host cells [ , ] . interestingly, they noted that a fraction of these puncta partially co-localized with double stranded viral rna. by screening several ibv nsp proteins, they found that ectopically expressed nsp localized with the er and was able to autonomously induce the formation of gfp-lc puncta. this raised the question whether the gfp-lc puncta induced by nsp were edemosomes [ ] . in contrast to edemosomes, however, formation of these gfp-lc -positive vesicles required atg and lc lipidation, suggesting that they are canonical autophagosomes [ , , ] . interestingly, nsp from mhv and sars-cov also induced gfp-lc puncta formation indicating that the nsp -dependent mechanism for autophagy induction might be conserved among covs. cottam et al. also argued that nsp might reduce the expansion of the autophagosomes as well, while maturation into autolysosomes is still possible [ ] . these results have been obtained using ectopic expression of nsp and thus the relevance of nsp -mediated induction of autophagy during cov infection remains to be explored. the investigation of the potential interplay between nidovirales and autophagy is still at its beginning. nonetheless, it can already be firmly concluded that nidovirales infections trigger autophagy in host cells. several viral families and virus species such as torovirinae, mesoviridae, and roniviridae, have yet to be investigated while for others, such as prssv and tgev, opposite conclusions have been reached regarding whether autophagy induction is beneficial or detrimental for the viral life cycle. the apparent contradictory results could be due to the use of different cell lines and tissues, and/or virus strains. some of these discrepancies could also be due to potential noncanonical functions of atg proteins as was shown for mhv. further investigations are therefore needed to reconcile these results. another drawback of several of the studies cited in this review is the extensive use of drugs to modulate autophagy during viral infection. as none of the employed compounds inhibits autophagy specifically, they can have adverse effects on cellular or viral biology, making the interpretation of the results difficult. the genetic ablation of atg proteins is a better option but it must be kept in mind that these factors are also part of other pathways [ , ] . as a result, it is crucial to compare the results obtained by depleting more than one atg protein. moreover, the few studies that have depleted atg proteins have blocked the initial steps of the autophagic pathway ( figure and table ) without analyzing the steps following the completion of an autophagosome. this is relevant since some viruses such as influenza a or epstein-barr virus, have been shown to manipulate this part of the pathway and therefore it is critical to investigate whether it could also be the case for nidovirales [ , ] . it also remains unclear which step of the virus life cycle is impacted, as most studies relied on assays quantifying general parameters such as the viral protein levels, the number of infected cells, and /or the number of produced infectious viral particles. results that were obtained by studying viruses from other orders have revealed that autophagy and atg proteins can practically play a key role in every step of a virus life cycle, from entry to assembly and egression [ ] . while it is indisputable that large part of the investigated nidovirales induces autophagy in host cells, it still remains unclear whether this is due to a subversion of autophagy by the virus or whether it is a physiological response to the cellular stress caused by either the infection or the transfection of single viral proteins. future research should 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coronavirus membrane-associated papain-like proteases induce autophagy through interacting with beclin to negatively regulate antiviral innate immunity coronavirus nsp restricts autophagosome expansion lc -associated phagocytosis (lap): connections with host autophagy. cells matrix protein of influenza a virus blocks autophagosome fusion with lysosomes epstein-barr virus blocks the autophagic flux and appropriates the autophagic machinery to enhance viral replication manipulation or capitulation: virus interactions with autophagy. microbes infect author contributions: y.c., p.v. and f.r. organized all related references and wrote the manuscripts. all authors read and approved this manuscript. the authors declare no conflict of interest. key: cord- -lq knxgf authors: takano, tomomi; satoh, kumi; doki, tomoyoshi; tanabe, taishi; hohdatsu, tsutomu title: antiviral effects of hydroxychloroquine and type i interferon on in vitro fatal feline coronavirus infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: lq knxgf feline infectious peritonitis (fip) is a viral disease with a high morbidity and mortality by the fip virus (fipv, virulent feline coronavirus). several antiviral drugs for fip have been identified, but many of these are expensive and not available in veterinary medicine. hydroxychloroquine (hcq) is a drug approved by several countries to treat malaria and immune-mediated diseases in humans, and its antiviral effects on other viral infections (e.g., sars-cov- , dengue virus) have been confirmed. we investigated whether hcq in association with interferon-ω (ifn-ω) is effective for fipv in vitro. a total of μm of hcq significantly inhibited the replication of types i and ii fipv. interestingly, the combination of μm of hcq and ( ) u/ml of recombinant feline ifn-ω (rfifn-ω, veterinary registered drug) increased its antiviral activity against type i fipv infection. our study suggested that hcq and rfifn-ω are applicable for treatment of fip. further clinical studies are needed to verify the combination of hcq and rifn-ω will be effective and safe treatment for cats with fip. coronaviruses are single-stranded positive-sense rna viruses in the subfamily orthocoronavirinae of the family coronaviridae [ ] . coronaviruses are important pathogens causing life-threatening infectious disease in mammals and birds [ ] . in humans, outbreaks of the novel coronavirus ( -ncov, official name is severe acute respiratory syndrome-related coronavirus : sars-cov- ) occurred worldwide [ ] . feline infectious peritonitis (fip) is a fatal, immune-mediated disease caused by feline coronavirus (fcov) [ ] . fcov is a member of the species alphacoronavirus- , genus alphacoronavirus, in the subfamily orthocoronavirinae. it is divided into two serotypes based on the amino acid sequence of the spike (s) protein, serotype i fcov, and serotype ii fcov [ ] . the majority of fcov infections are subclinical (avirulent fcov is known as feline enteric coronavirus: fecv; type i and type ii fecv) [ ] . however, several mutations occurred in the s protein, leading to development of the virulent type called feline infectious peritonitis virus (fip virus, fipv; type i and type ii fipv) [ , ] . fipv infection typically causes a fatal disease in cats known as fip. the hallmark pathological findings of fip in cats are serous fluid in peritoneal and pleural cavities, and pyogranulomatous lesions in the internal organs and brain [ ] . the absorbance of formazan produced was measured at nm using a -well spectrophotometric plate reader, as described by the manufacturer. percentage cell viability was calculated using the following formula: cell viability (%) = [(od of compound-untreated cells -compound-treated cells)/(od of compound-untreated cells)] × . the % cytotoxicity concentration (cc ) was defined as the cytotoxic concentration of each compound that reduced the absorbance of treated cells to % when compared with that of the untreated cells. confluent fcwf- cell monolayers were cultured in medium with or without compounds at the indicated concentrations in -well multi-plates at • c for h or h. cells were washed and the virus (moi . ) was adsorbed into the cells at • c for h. after washing, cells were cultured in . % carboxymethyl cellulose (cmc)-mem or mem with or without compounds. in the case of cells cultured in cmc-mem, the cell monolayers were incubated at ºc for h, fixed, and stained with % crystal violet solution containing % buffered formalin, and the resulting plaques were then counted. the percentage of the decrease or increase in plaques was calculated using the following formula: percentage of the plaque reduction (%) = [(plaque number of compound-treated cells)/(plaque number of compound-untreated cells)] × . the ec was defined as the effective concentration of compounds that reduced the virus titer in the culture supernatant of infected cells to % when compared with that of the virus control. in the case of cells cultured in mem, the culture supernatants were collected h post-infection, and virus titers were measured by the tcid assay. the nucleocapsid (n) protein levels of fipv-infected fcwf- cells were determined by an immunofluorescence assay (ifa), as described previously [ ] . briefly, fipv-infected cells were washed pbs and fixed with % paraformaldehyde at rt for min. the cells were incubated with mab yn- (fipv n protein-specific mab) at • c for min. after washing, the cells were incubated with goat anti-mouse-igg conjugated to fluorescein (jackson immunoresearch, pa, usa) at • c for min. after washing, the cells were stained with , -diamidino- -phenylindole (dapi; dojindo laboratories, kumamoto, japan) at rt for min. the stained cells were analyzed using leica dm b microscope and las x integrated imaging system (leica microsystems, wetzlar, germany). data from only two groups were analyzed using the student's t-test (welch's t-test) and those of multiple groups were analyzed by one-way anova followed by tukey's test using microsoft excel software and open-source statistical graphpad prism (graphpad software, ca, usa). a p-value of < . was considered statically significant. cytotoxicity assay was performed to clarify the non-toxic concentration of cq and hcq against fcwf- cells (figure ). the cc of cq and hcq was . µm ( figure a ) and . µm ( figure b) , respectively. hcq was less toxic ( . %) than cq in feline cells. the antiviral activity of compounds against both serotypes of fipv was evaluated using plaque inhibition assay. the ec of cq and hcq against fipv-i ku was . µm ( figure a ) and . µm ( figure b ), respectively. the ec of cq and hcq against fipv-ii - was . µm ( figure a ) and . µm ( figure b) , respectively. therefore, the antiviral effects of hcq on fipv were comparable to those of cq. based on these results, hcq was suggested to be a safer anti-fipv drug than cq. the antiviral activity of hcq and rfifn-ω in fcwf- cells for a prolonged time ( h) was investigated. to evaluate and assess the antiviral effects of hcq ( μm) and rfifn-ω ( u/ml), we used strains of serotype i fipv (fipv-i ucd , fipv-i ucd , and fipv-i ku ) and strain of serotype ii fipv (fipv-ii - ). the virus titers of both serotypes of fipv significantly decreased in the culture supernatant of cells pretreated with hcq or rfifn-ω ( figure ). we evaluated the antiviral effects of the combination of hcq and rfifn-ω. the combination of these drugs strongly suppressed the replication of viruses in fcwf- cells. when both μm hcq and u/ml of rfifnω were added, fcwf- cell viability was . ± . %. the antiviral activity of hcq and rfifn-ω in fcwf- cells for a prolonged time ( h) was investigated. to evaluate and assess the antiviral effects of hcq ( µm) and rfifn-ω ( u/ml), we used strains of serotype i fipv (fipv-i ucd , fipv-i ucd , and fipv-i ku ) and strain of serotype ii fipv (fipv-ii - ). the virus titers of both serotypes of fipv significantly decreased in the culture supernatant of cells pretreated with hcq or rfifn-ω ( figure ). we evaluated the antiviral effects of the combination of hcq and rfifn-ω. the combination of these drugs strongly suppressed the replication of viruses in fcwf- cells. when both µm hcq and u/ml of rfifn-ω were added, fcwf- cell viability was . ± . %. the antiviral activity of hcq and rfifn-ω in fcwf- cells for a prolonged time ( h) was investigated. to evaluate and assess the antiviral effects of hcq ( μm) and rfifn-ω ( u/ml), we used strains of serotype i fipv (fipv-i ucd , fipv-i ucd , and fipv-i ku ) and strain of serotype ii fipv (fipv-ii - ). the virus titers of both serotypes of fipv significantly decreased in the culture supernatant of cells pretreated with hcq or rfifn-ω ( figure ). we evaluated the antiviral effects of the combination of hcq and rfifn-ω. the combination of these drugs strongly suppressed the replication of viruses in fcwf- cells. when both μm hcq and u/ml of rfifnω were added, fcwf- cell viability was . ± . %. we investigated whether hcq and rfifn-ω, which acted on fipv in fcwf- cells for a short time ( h), exhibit antiviral activity. as shown in figure , type i fipv and type ii fipv replication was significantly inhibited by hcq and rfifn-ω. interestingly, the combination of these drugs strongly decreased the replication of type i fipvs in fcwf- cells, but not type ii fipv. viruses , , x for peer review of we investigated whether hcq and rfifn-ω, which acted on fipv in fcwf- cells for a short time ( h), exhibit antiviral activity. as shown in figure , type i fipv and type ii fipv replication was significantly inhibited by hcq and rfifn-ω. interestingly, the combination of these drugs strongly decreased the replication of type i fipvs in fcwf- cells, but not type ii fipv. we evaluated the antiviral activity of hcq and rfifn-ω against type i fipv after viral infection. hcq and rfifn-ω were added to the cells h after inoculation. as shown in figure , type i fipv replication was significantly inhibited by hcq and rfifn-ω, and the combination of these drugs strongly decreased the replication of virus. we investigated the expression of viral proteins in order to evaluate the antiviral effects of the combination of hcq and rfifn-ω on fipv. the n protein levels of fipv-i ku- were specifically decreased in fcwf- cells pre-treated (short-time exposure) and post-treated with hcq and rfifn-ω we evaluated the antiviral activity of hcq and rfifn-ω against type i fipv after viral infection. hcq and rfifn-ω were added to the cells h after inoculation. as shown in figure , type i fipv replication was significantly inhibited by hcq and rfifn-ω, and the combination of these drugs strongly decreased the replication of virus. viruses , , x for peer review of we investigated whether hcq and rfifn-ω, which acted on fipv in fcwf- cells for a short time ( h), exhibit antiviral activity. as shown in figure , type i fipv and type ii fipv replication was significantly inhibited by hcq and rfifn-ω. interestingly, the combination of these drugs strongly decreased the replication of type i fipvs in fcwf- cells, but not type ii fipv. we evaluated the antiviral activity of hcq and rfifn-ω against type i fipv after viral infection. hcq and rfifn-ω were added to the cells h after inoculation. as shown in figure , type i fipv replication was significantly inhibited by hcq and rfifn-ω, and the combination of these drugs strongly decreased the replication of virus. we investigated the expression of viral proteins in order to evaluate the antiviral effects of the combination of hcq and rfifn-ω on fipv. the n protein levels of fipv-i ku- were specifically decreased in fcwf- cells pre-treated (short-time exposure) and post-treated with hcq and rfifn-ω we investigated the expression of viral proteins in order to evaluate the antiviral effects of the combination of hcq and rfifn-ω on fipv. the n protein levels of fipv-i ku- were specifically decreased in fcwf- cells pre-treated (short-time exposure) and post-treated with hcq and rfifn-ω ( figure ). in contrast, post-treatment with hcq and rfifn-ω slightly affected the protein levels of fipv-ii - in fcwf- cells. viruses , , x for peer review of ( figure ). in contrast, post-treatment with hcq and rfifn-ω slightly affected the protein levels of fipv-ii - in fcwf- cells. fip is a fatal coronaviral infection of cats. several drugs have been identified aiming at the treatment of fip, but no commercial drugs can be used to treat fip by veterinarians. we have searched for a drug applicable to treat fip among commercial drugs [ , ] . cq is an antimalarial drug and improved symptoms of cats with fip [ ] . however, increased liver enzymes were observed in some cats treated with cq. increased liver enzymes are observed in cats with fip, but the possibility of cq-induced liver disorder was also suggested. if there is a drug with cytotoxicity weaker than that of cq that exhibits comparable antiviral effects, it may be applicable as a therapeutic drug for fip. we focused on hcq, which is -aminoquinoline similar to cq [ ] . the cytotoxicity of hcq has been reported to be lower than that of cq in mouse, rat, and dog [ ] . in addition, hcq has been demonstrated to have antiviral effects on sars-cov- infection equivalent to those of cq in vitro [ ] . we confirmed that hcq has anti-fipv activity equivalent to that of cq. moreover, cytotoxicity of hcq setting the criterion to cc was one-third or lower than that of cq. accordingly, hcq is applicable to fip treatment as a substitute for cq. hcq at μm significantly inhibited the replication of both serotypes of fipv. to our knowledge, the pharmacokinetics of hcq in cats have not been analyzed. thus, it is necessary to refer to pharmacokinetic data of hcq in dogs. the tolerated dose of intramuscular injection of hcq is mg/kg [ ] . in dogs treated with mg/kg of hcq, the plasma hcq level reaches . μm ( μg/l) [ ] , i.e., it is difficult to make the plasma hcq level reach μm in dogs. however, it has been reported that the tissue hcq levels in the liver, spleen, kidney, and lung increased to a level several hundred-times higher than the plasma level [ ] . therefore, hcq administration to cats with fip within the low dosage may be expected to yield sufficient therapeutic effects. on the other hand, it is unclear whether the pharmacokinetics described above can apply to cats. cytochrome p (cyps) are involved in the metabolism of hcq [ ] . generally, cyp activities could be lower in cats than in dogs [ ] . on the basis of this fact, the blood concentrations of hcq in cats will be higher than those in dogs. therefore, pharmacokinetic studies are still needed to use hcq in cats. the antiviral agent rfifn-ω has a wide safety range and is practically used to treat feline viral infection in veterinary practice. many points are unclear as to whether rfifn-ω is effective as a therapeutic drug for fip. fip is a fatal coronaviral infection of cats. several drugs have been identified aiming at the treatment of fip, but no commercial drugs can be used to treat fip by veterinarians. we have searched for a drug applicable to treat fip among commercial drugs [ , ] . cq is an antimalarial drug and improved symptoms of cats with fip [ ] . however, increased liver enzymes were observed in some cats treated with cq. increased liver enzymes are observed in cats with fip, but the possibility of cq-induced liver disorder was also suggested. if there is a drug with cytotoxicity weaker than that of cq that exhibits comparable antiviral effects, it may be applicable as a therapeutic drug for fip. we focused on hcq, which is -aminoquinoline similar to cq [ ] . the cytotoxicity of hcq has been reported to be lower than that of cq in mouse, rat, and dog [ ] . in addition, hcq has been demonstrated to have antiviral effects on sars-cov- infection equivalent to those of cq in vitro [ ] . we confirmed that hcq has anti-fipv activity equivalent to that of cq. moreover, cytotoxicity of hcq setting the criterion to cc was one-third or lower than that of cq. accordingly, hcq is applicable to fip treatment as a substitute for cq. hcq at µm significantly inhibited the replication of both serotypes of fipv. to our knowledge, the pharmacokinetics of hcq in cats have not been analyzed. thus, it is necessary to refer to pharmacokinetic data of hcq in dogs. the tolerated dose of intramuscular injection of hcq is mg/kg [ ] . in dogs treated with mg/kg of hcq, the plasma hcq level reaches . µm ( µg/l) [ ] , i.e., it is difficult to make the plasma hcq level reach µm in dogs. however, it has been reported that the tissue hcq levels in the liver, spleen, kidney, and lung increased to a level several hundred-times higher than the plasma level [ ] . therefore, hcq administration to cats with fip within the low dosage may be expected to yield sufficient therapeutic effects. on the other hand, it is unclear whether the pharmacokinetics described above can apply to cats. cytochrome p (cyps) are involved in the metabolism of hcq [ ] . generally, cyp activities could be lower in cats than in dogs [ ] . on the basis of this fact, the blood concentrations of hcq in cats will be higher than those in dogs. therefore, pharmacokinetic studies are still needed to use hcq in cats. the antiviral agent rfifn-ω has a wide safety range and is practically used to treat feline viral infection in veterinary practice. many points are unclear as to whether rfifn-ω is effective as a therapeutic drug for fip. the combination of hcq and rfifn-ω blocked virus production in type i fipv-infected cells, but although the duration of activity was only h, the antiviral activity of these drugs decreased in type ii fipv-infected cells. we previously demonstrated that types i and ii fipv enter the cytosol through late and early endosomes, respectively [ ] . we also reported that type ii fipv strongly inhibited type i ifn expression [ ] . based on this knowledge and our current study, type ii fipv may show less effect on the antiviral activity of hcq and type i ifn, compared to type i fipv. there are some reports about the relationship between hcq and type i ifn. wang et al. reported that hcq inhibited dengue virus infection in all serotypes in vitro [ ] . they suggested that the induction of interferon or related protein is an antiviral activity mechanism of hcq. on the other hand, inhibition of type i ifn production in hcq-treated cells has been reported [ ] , being contradictory to other findings. we confirmed that potent antiviral activity was induced by the combination of hcq and rfifn-ω (type i ifn). although negative action on type i ifn may have been induced by hcq, type i ifn added at the same time may have canceled this. to demonstrate this, further investigation is necessary. in this study, we confirmed that hcq is a safer anti-fipv drug than cq. in addition, we demonstrated that the combination of hcq and rfifn-ω increases the antiviral activity. our study revealed that these fip therapeutic drugs are applicable to veterinary practice. it should be noted that in vitro data do not always translate into in vivo efficacy. therefore, a deeper understanding of pharmacokinetics of the combination between hcq and rfifn-ω will be needed in cats. an overview of their replication and pathogenesis the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- feline coronaviruses: pathogenesis of feline infectious peritonitis molecular cloning and sequence determination of the peplomer protein gene of feline infectious peritonitis virus type i natural feline coronavirus infection: differences in cytokine patterns in association with the outcome of infection spike protein fusion peptide and feline coronavirus virulence mutation in spike protein cleavage site and pathogenesis of feline coronavirus efficacy and safety of the nucleoside analog gs- for treatment of cats with naturally occurring feline infectious peritonitis efficacy of a c-like protease inhibitor in treating various forms of acquired feline infectious peritonitis effect of chloroquine on feline infectious peritonitis virus infection in vitro and in vivo animal toxicity and pharmacokinetics of hydroxychloroquine sulfate hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro hydroxychloroquine-inhibited dengue virus is associated with host defense machinery regulation of type i interferon responses homogeneous production of feline interferon in silkworm by replacing single amino acid code in signal peptide region in recombinant baculovirus and characterization of the product treatment of canine parvoviral enteritis with interferon-omega in a placebo-controlled challenge trial feline calicivirus infection: abcd guidelines on prevention and management the use of recombinant feline interferon omega therapy as an immune-modulator in cats naturally infected with feline immunodeficiency virus: new perspectives use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis orf -encoded accessory protein a of feline infectious peritonitis virus as a counteragent against ifn-α-induced antiviral response a study on the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection in feline macrophages by monoclonal antibodies antiviral activity of itraconazole against type i feline coronavirus infection therapeutic effect of an anti-human-tnf-alpha antibody and itraconazole on feline infectious peritonitis pharmacologic actions of -aminoquinoline compounds mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology cytochrome p -mediated hepatic metabolism of new fluorescent substrates in cats and dogs endocytic pathway of feline coronavirus for cell entry: differences in serotype-dependent viral entry pathway differential induction of type i interferon by type i and type ii feline coronaviruses in vitro hydroxychloroquine is associated with impaired interferon-alpha and tumor necrosis factor-alpha production by plasmacytoid dendritic cells in systemic lupus erythematosus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors are grateful to their colleagues. the authors declare no conflict of interest. key: cord- -aty vrat authors: frabutt, dylan a.; zheng, yong-hui title: arms race between enveloped viruses and the host erad machinery date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: aty vrat enveloped viruses represent a significant category of pathogens that cause serious diseases in animals. these viruses express envelope glycoproteins that are singularly important during the infection of host cells by mediating fusion between the viral envelope and host cell membranes. despite low homology at protein levels, three classes of viral fusion proteins have, as of yet, been identified based on structural similarities. their incorporation into viral particles is dependent upon their proper sub-cellular localization after being expressed and folded properly in the endoplasmic reticulum (er). however, viral protein expression can cause stress in the er, and host cells respond to alleviate the er stress in the form of the unfolded protein response (upr); the effects of which have been observed to potentiate or inhibit viral infection. one important arm of upr is to elevate the capacity of the er-associated protein degradation (erad) pathway, which is comprised of host quality control machinery that ensures proper protein folding. in this review, we provide relevant details regarding viral envelope glycoproteins, upr, erad, and their interactions in host cells. despite their vast diversity, animal viruses can be simply divided into two categories: non-enveloped viruses and enveloped viruses [ ] . while non-enveloped viruses are wrapped with naked shells made of viral capsid proteins, enveloped viruses are covered with a lipid-bilayer, which is called a viral envelope. the viral envelope is obtained from progenitor host cells during the budding process, which can be a portion of plasma membrane or intracellular membrane. on the surface of the enveloped viruses, there are peplomers that project from the viral envelope, and play a critical role in viral infection. these peplomers are also described as spikes, which are made of viral envelope glycoproteins. envelope spikes serve to identify and bind to viral receptors on the host cell surface, allowing viral entry into cells and the initiation of infection by mediating virus-cell fusion. thus, the infectivity of enveloped viruses is absolutely dependent on the integrity of the viral envelope, and the functionality of the viral glycoproteins found therein. enveloped viruses are more stable than non-enveloped viruses under physiological conditions, at the expense of their sensitivity to high-temperature, low-ph, desiccation, or detergent-treatment, which limits their ability to withstand severe environments [ ] . the entry of enveloped viruses requires the formation of a fusion pore between the viral envelope and the cell membrane, through which the viral genome is released into the cell. this fusion process is triggered by interactions between viral glycoproteins on the viral envelope and viral receptors on the cell surface, which can occur directly at the plasma membrane at neutral ph or in endocytic compartments at either low or neutral ph [ ] . in addition, enveloped viruses can also enter cells through direct cell-to-cell contacts via virological glycosylation, which is one of the most common post-translational modifications in eukaryotic cells, is required for protein folding and maintaining protein structure. viruses have taken advantage of this benefit at nearly every step of the viral life cycle [ ] . n-glycosylation significantly promotes their folding and solubility, enhances subsequent trafficking of these viral proteins to their destinations, and ensures that they are properly processed and incorporated into virions. nevertheless, glycosylation can have distinct effects that are both advantageous and detrimental to viral fitness. for example, if glycosylation occurs close to the glycoprotein processing sites, it may block the precursor cleavage by proteases and inhibit viral infection [ ] ; if glycosylation occurs adjacent to the receptor-binding site, it may enhance the binding affinity and promote viral infection [ , ] . in addition, the high density of glycans on virions may form a shield to impede antibody attack and promote immune evasion. however, these glycans can also become epitopes for stimulating neutralizing antibodies and the innate immune response, making viruses more vulnerable to immune clearance [ ] . thus, there are multiple selective pressures on viral envelope glycosylation that can influence the pattern of glycosylation in order to achieve the optimal fitness in their hosts [ ] . viruses are obligate intracellular parasites, and their glycoprotein biosynthesis and modification rely entirely on host cell machinery in the secretory pathway. therefore, viral and host proteins are glycosylated in a similar manner by the same mechanism. although glycans can be attached to polypeptide structures via several different mechanisms, asparagine n-linked glycosylation represents a fundamental and well characterized post-translational modification in eukaryotic organisms [ ] . n-linked glycosylation starts from the membrane of the endoplasmic reticulum (er), where the tetradecasaccharide precursor is assembled. this precursor consists of two n-acetylglucosamine (glcnac), nine mannose (man, are α , -linked), and three terminal glucose (glc) residues distributed on three extended man branches: a, b, and c (glc man glcnac ) ( figure a ) [ , ] . when nascent polypeptides enter the er lumen, the precursor is en bloc attached to asn residues of a nascent polypeptide in a consensus asn-x-(ser/thr) motif. after the attachment, these precursors are processed by a series of enzymes in both the er and the golgi apparatus to remold the core oligosaccharide into diverse n-linked glycan structures ( figure b ). the first step in this process is the sequential removal of the two outermost glc residues on branch a. the first glc residue is removed by glucosidase i (gi), resulting in the di-glycosylated oligosaccharide glc man glcnac , which is recognized by an er transmembrane lectin malectin [ ] . the second glc residue is then removed by glucosidase ii (gii), resulting in the mono-glucosylated oligosaccharide glc man glcnac , which is recognized by two other er lectins, the membrane-bound calnexin (cnx) and/or soluble calreticulin (crt). interaction with these two chaperones segregates the newly formed glycoprotein and provides access to protein disulfide isomerases (pdis) such as erp , which promotes disulfide bond formation, resulting in protein folding into a native conformation. once a protein is properly folded, gii cleaves the last glc residue on branch a, which releases the protein from the cnx/crt cycle. the er class i α-mannosidase (ermani) then cleaves the outermost man residue on branch b on native proteins, resulting in the oligosaccharide man glcnac . these high-man glycans are then recognized by lectins including er-golgi intermediate compartment- (ergic- ), vesicular integral membrane protein of kda (vip ), and vip -like (vipl), which promote trafficking from the er to the golgi [ ] . the remaining man residues are cleaved by the golgi mannosidases, and the glycan remolding process is continued through the remainder of the n-glycosylation pathway, which generates functional glycoproteins that are delivered to the cell surface ( figure b ). in addition to these chaperones and enzymes that promote protein folding, the er is also equipped with a unique quality control mechanism that extracts and degrades proteins that are not correctly folded or assembled into their native conformation, which is called er-associated protein degradation (erad) [ ] . in fact, the folding efficiency of glycoproteins in the er is very low, which requires cycles of association and dissociation from cnx/crt to ensure proper glycoprotein maturation. if glycoproteins with the man glcnac oligosaccharide display non-native conformations, they are reglucosylated by the udp-glc:unfolded glycoprotein glucosyltransferase (ugt or uggt), and are subject to additional rounds of re-engagement with the cnx/crt machinery until folding is achieved. however, if a certain time frame for the folding is exceeded, proteins may never fold properly. misfolded proteins are sequestered into coat protein complex ii (cop-ii) -dependent, highly mobile er-derived quality control vesicles (qcvs), where ermani is enriched ( figure b ) [ ] . because ermani is able to excise all α , -man residues when it is expressed at much higher levels in vitro [ ] , the enzyme may catalyze extensive demannosylation, resulting in the production of low-man oligosaccharide man glcnac -containing glycoprotein precursors. the removal of the a branch terminal man residue, which is the acceptor for glc transferred by uggt, disables these proteins from reengagement with cnx/crt and re-entering into the folding cycle. importantly, the low-man n-glycans represent a tag for defective glycoproteins, targeting them to erad [ ] . figure b ) [ ] . because ermani is able to excise all α , -man residues when it is expressed at much higher levels in vitro [ ] , the enzyme may catalyze extensive demannosylation, resulting in the production of low-man oligosaccharide man glcnac -containing glycoprotein precursors. the removal of the a branch terminal man residue, which is the acceptor for glc transferred by uggt, disables these proteins from reengagement with cnx/crt and re-entering into the folding cycle. importantly, the low-man n-glycans represent a tag for defective glycoproteins, targeting them to erad [ ] . , and endoplasmic reticulum (er) stress pathways. nascent polypeptides are translocated through sec into the rough er, where the core oligosaccharide is transferred from a dolichol phosphate onto asparagine residues in asparagine-x-serine/threonine (nxs/t) motifs (i). the two terminal glucose residues on the core oligosaccharide are trimmed by glucosidase i, (gi) (ii), and gii (iii), respectively, allowing for the association with the chaperones, membrane-bound calnexin (cnx) and and/or soluble calreticulin (crt), which promote folding to a native conformation. eventually, the last terminal glucose residue will be trimmed by gii, and the glycoprotein will attain a native conformation (iv), or misfold (vii). glycoproteins that reach a native conformation will have the terminal α , -man residue on the b branch removed by er class i α-mannosidase (ermani) (v), as a signal to allow it to traverse the canonical secretory pathway for surface presentation or secretion (vi). polypeptides unable to reach a native conformation (vii) will engage in multiple rounds of the cnx/crt cycle, facilitated by reglucosylation of the terminal glucose by udp-glc:unfolded glycoprotein glucosyltransferase (uggt) (viii), and trafficking between quality control vesicles (qcv) (ix) and the the er-derived quality compartments (erqc) (x) under er stress. terminally misfolded glycoproteins will be demannosylated to remove all α , -man residues (xi), followed by association with lectins osteosarcoma amplified (os ) and xtp -transactivated gene b protein (xtp -b) for erad (xii). figure . (a) schematic presentation of the n-linked core oligosaccharide structure. the core is composed of two n-acetylglucosamine (glcnac, blue), nine mannose (man, red), and three glucose (glc, yellow) residues. a, b, and c are three oligosaccharide branches. (b) schematic description of n-glycosylation, endoplasmic reticulum-associated protein degradation (erad), and endoplasmic reticulum (er) stress pathways. nascent polypeptides are translocated through sec into the rough er, where the core oligosaccharide is transferred from a dolichol phosphate onto asparagine residues in asparagine-x-serine/threonine (nxs/t) motifs (i). the two terminal glucose residues on the core oligosaccharide are trimmed by glucosidase i, (gi) (ii), and gii (iii), respectively, allowing for the association with the chaperones, membrane-bound calnexin (cnx) and and/or soluble calreticulin (crt), which promote folding to a native conformation. eventually, the last terminal glucose residue will be trimmed by gii, and the glycoprotein will attain a native conformation (iv), or misfold (vii). glycoproteins that reach a native conformation will have the terminal α , -man residue on the b branch removed by er class i α-mannosidase (ermani) (v), as a signal to allow it to traverse the canonical secretory pathway for surface presentation or secretion (vi). polypeptides unable to reach a native conformation (vii) will engage in multiple rounds of the cnx/crt cycle, facilitated by reglucosylation of the terminal glucose by udp-glc:unfolded glycoprotein glucosyltransferase (uggt) (viii), and trafficking between quality control vesicles (qcv) (ix) and the the er-derived quality compartments (erqc) (x) under er stress. terminally misfolded glycoproteins will be demannosylated to remove all α , -man residues (xi), followed by association with lectins osteosarcoma amplified (os ) and xtp -transactivated gene b protein (xtp -b) for erad (xii). ermani containing qcv are rapidly recycled through autophagy/lysosome pathways (xiii). without interactions with client glycoproteins, edemosome components are degraded through an autophagy-like mechanism (xiv). viruses can hijack edemosomes to form double membrane vesicles (dmvs) that serve as platforms for their replication (xv). with only one-tenth of the total cell volume, the er is responsible for the synthesis of the vast majority of the secreted or membrane proteins, which account for one-third of total cellular proteins. therefore, the er has extremely high protein concentrations ( mg/ml), which renders this organelle very susceptible to protein aggregation [ ] . in addition, the protein folding is error prone, and this process can be further compromised by physiological and pathological perturbations. moreover, genetic mutations may prohibit proteins from being folded properly. all these factors may cause the accumulation of unfolded or misfolded proteins. when the level of these aberrant proteins exceeds the folding and clearance capacity of the er, known as er homeostasis, it leads to a cellular stress response termed "er stress", which in turn activates the unfolded protein response (upr) to restore the er homeostasis [ ] . er stress is sensed by three er transmembrane receptors: double-stranded rna (dsrna)-activated protein kinase (pkr)-like er kinase (perk), inositol-requiring enzyme (ire ), and activating transcription factor (atf ). perk and atf are in association with another er chaperone, the binding immunoglobulin protein (bip, or grp ), when the cell is not under stress. bip preferentially binds to misfolded proteins and dissociates from perk and atf under er stress, resulting in their activation and upr to mitigate this stress [ , ] . ire is activated by the direct binding of unfolded proteins [ ] . ire then activates the transcription factor x-box binding protein (xbp- ), which in turn up-regulates er chaperones to assist in the folding capacity of the er as well as erad components to boost protein degradation. perk phosphorylates the eukaryotic initiation factor (eif)- α and halts protein translation, and atf up-regulates protein expression to boost the er protein folding capacity and erad. however, if these objectives are not achieved within a certain time span or if the disruption is prolonged, upr also activates pathways leading to cell death. although perk activation causes global inhibition of protein translation by blocking eif- α activity, it paradoxically enhances translation of the transcription factor atf . atf then trans-activates the ccaat/enhancer-binding protein-homologous protein (chop), which is a pro-apoptotic transcription factor, resulting in cell death by apoptosis [ ] . erad is a protein quality control mechanism conserved in all eukaryotic cells, which is an important arm of upr, necessary to alleviate er stress [ ] . erad results in the selective dislocation of unfolded and misfolded proteins from the er to the cytosol via specific membrane machinery. erad targets are subsequently degraded by the cytosolic ubiquitin proteasome system (ups) [ ] . quality control of functional proteins produced from the er is also critical for maintenance of the er homeostasis by eliminating unfolded and misfolded proteins. thus, erad is a central element of both the secretory pathway and upr, which targets a number of physiological and pathological substrates such as the t cell antigen receptor (tcr) [ ] , -hydroxy- -methylglutaryl coenzyme-a (hmg-coa) reductase (hmgcr) [ ] , squalene monooxygenase (sqle) [ ] , inositol , , -trisphosphate (ip ) receptor [ ] , diacylglycerol acyltransferase (dgat ) [ ] , heme oxygenase- (ho- ) [ ] , alpha- antitrypsin [ ] , and cystic fibrosis transmembrane regulator (cftr) [ ] . so far, more than human diseases have been attributed to this pathway [ ] . although the vast majority of secreted proteins are glycosylated, the er is responsible for the folding and assembly of both glycosylated and non-glycosylated proteins into functional complexes, which are subjected to erad quality control if they are misfolded. the process of erad can be divided into three steps: substrate recognition, retrotranslocation, and ubiquitylation/proteasomal degradation. in fact, extensive excision of α , -man residues from n-glycans sends an important signal to trigger misfolded glycoprotein degradation, which is dependent on class i mannosidases [ ] . class i mannosidases belong to the glycoside hydrolase family (gh ), which are exo-acting α , -mannosidases that are divided into three subfamilies [ ] . the first subfamily consists of ermani, which is supposed to cleave the outmost α , -man residue on the b branch from n-linked glycans in the er. the second subfamily consists of three golgi α-mannosidase i, including golgimania, golgimanib, and golgimanic, which cleave the remaining three α , -man residues in the golgi complex for n-glycan maturation. the third subfamily consists of the er degradation-enhancing α-mannosidase-like proteins (edem) , , and . although some edem orthologs in lower eukaryotes have detectable α , -mannosidase activity, such activity has not been reported for any mammalian edem proteins in vitro. nevertheless, there is evidence suggesting that these edem proteins should have enzymatic activity in vivo [ , ] . indeed, the extent of man excision determines the fate of a glycoprotein, which could be either targeted to erad for degradation or sent to the golgi for normal trafficking. ermani exhibits a slow rate of enzymatic activity, which allows nascent proteins to perform multiple rounds of reglucosylation and achieve proper folding [ ] . properly folded glycoproteins should have one man residue trimmed off from n-glycans by ermani. these glycoproteins then interact with the high-man binding lectin, er-golgi intermediate compartment kda protein (ergic- ) [ ] , for trafficking from the er to the golgi ( figure b) . however, if glycoproteins are misfolded terminally, the remaining three α , -man residues are excised from these molecules, which targets misfolded proteins for degradation [ ] . it is still not completely understood how misfolded glycoproteins are subjected to such extensive demannosylation in the er and then targeted for erad. although ermani alone may be able to complete this task, there is evidence suggesting that additional gh enzymes are involved. elevation of the golgi mannosidases has been shown to accelerate erad, so these enzymes may possibly be responsible for such extensive excision, likely by trafficking back to the er via an unknown mechanism [ ] . in fact, the localization of the golgimania has recently been observed in qcv with other canonical erad machinery such as ermani, and overexpression and knockdown can, respectively, increase or retard trimming of misfolded glycoproteins from man glcnac to man glcnac in vitro [ ] . in addition, upon er stress, qcv converge to form the er-derived quality compartments (erqc), where edem proteins are also sequestered ( figure b ) [ ] . edem and edem boost mannose trimming when overexpressed [ , , ] . in addition, using a genomic knockout approach, it has been recently proposed that edem plays a central role in the trimming of the outmost man residue on the b branch, whereas edem and edem should be responsible for trimming of the remaining α , -man residues. accordingly, a "double check" model for misfolded glycoproteins has been proposed, which suggests that edem catalyzes the first step of man trimming, and edem and edem contribute the second step [ ] . under these joint actions, all four α , -man residues are removed from the oligosaccharide, which is then recognized by the lectins osteosarcoma amplified (os ) and xtp -transactivated gene b protein (xtp -b) via the mannose -phosphate receptor homology (mrh) domain ( figure b) [ , ] . misfolded proteins are targeted to specific translocation channels (retrotranslocons) for retrotranslocation in an energy-dependent manner. this process is facilitated by p , a member of the atpases associated with the diverse cellular activities (aaa) family, by catalyzing atp hydrolysis [ ] . the p atpase is recruited by the ubiquitin-like (ubx)-domain-containing protein ubxd , an er-membrane protein that plays a role in erad [ ] . it is still mysterious how these retrotranslocons are formed and how integral membrane and lumenal erad substrates are exported across the er membrane through these retrotranslocons. the first candidate channel is composed of the sec complex, which is comprised of α, β, and γ subunits. the α subunit crosses the membrane times, and forms a channel with the other subunits. the sec heterotrimeric channel is the main translocon involved in co-translational protein transport into the er [ ] . however, there is evidence suggesting that the sec translocon is also involved in retrotranslocation of erad substrates, implying the non-specificity and bi-directional property of this channel [ ] . the second candidate is a member of the derlin family (derlin- , - , and - ) [ , ] . derlins are integral membrane proteins that likely span the er membrane four times and contain a rhomboid-like domain [ ] . the third candidate includes the erad-specific e ligases. they have a large number of transmembrane domains, which are not only responsible for polyubiquitylation, but could act as potential exit channels for erad substrates [ ] . in saccharomyces cerevisiae, there are two major types of really interesting new gene (ring)-finger e ligase complexes, hrd and doa , which mediate erad by targeting discrete substrates [ ] . hrd was the first e enzyme identified in the erad pathway during the study of hmg-coa reductase degradation (hrd) [ ] . hrd has transmembrane helices in its n-terminal transmembrane domain and a catalytic ring domain in the soluble c-terminal region extended to the cytosol. using an elegant erad assay reconstituted in vitro, the hrd -mediated formation of ubiquitin-gated protein-conducting membrane channels has been demonstrated [ , ] . hrd has two mammalian orthologs named hrd and gp , and its functioning e enzyme is known as ubc , which also has two mammalian orthologs, ube g and ube g [ ] . hrd is unstable, and must be stabilized by its co-factor hrd in an equimolar ratio [ ] . the mammalian ortholog of hrd is sel l, which is required for erad substrate retrotranslocation [ ] . hrd interacts with der , and the der -hrd interaction is bridged by another integral membrane protein usa , which is also required for hrd oligomerization [ ] . the usa mammalian ortholog is called herp, which also interacts with hrd and derlin- and plays an important role in erad [ ] . doa was identified in degradation studies of mating type (mat)-α - (doa), which is a yeast transcription factor. doa is a~ kda protein that has transmembrane domains, which requires both ubc and ubc as e enzymes. the doa mammalian ortholog is teb (march ), which functions in the erad pathway with similar subcellular distribution and topology [ ] . the ubc e enzyme has two mammalian orthologs, ube j and ube j ; both are involved in erad [ , ] . in saccharomyces cerevisiae, erad is executed synergistically by hrd and doa with minimal redundancy because they exhibit different substrate specificity. doa mainly triggers ubiquitylation of specific soluble proteins and membrane proteins with degrons exposed to the cytosol; a process referred to as erad-c [ , ] . hrd interacts with two other types of substrates, whose degradation is termed erad-l and erad-m. erad-l includes soluble lumenal proteins in the er and transmembrane proteins with degrons exposed to the er lumen; erad-m includes transmembrane proteins with degrons embedded into the er membrane [ ] . simultaneous inactivation of both genes has been shown to increase the sensitivity to heavy metal-induced cellular stress and exhibit an elevated upr. the regulation of protein folding and the functional relation between erad and the upr are much more complex in mammalian cells. in saccharomyces cerevisiae, the cnx cycle does not exist due to the lack of uggt. in addition, protein synthesis is tightly controlled at the translational level by determination of the stoichiometry to avoid surplus production, resulting in minimal dependence on the post-translational regulation of protein expression. moreover, although the yeast ermani ortholog mns p and edem ortholog htm p are indispensable for erad, only one edem ortholog is present in yeast [ , ] . because overly active erad may interfere with the regular protein folding process in the er, mammalian cells have evolved mechanisms to tightly regulate this quality control device by a combination of compartmentalization and tuning. edem is segregated into er-derived, lc -i-associated vesicles, which are called edemosomes, where edem , os- , and sel l are concentrated when they lack client glycoproteins to dislocate ( figure b) [ ] . notably, unlike chaperones and the other enzymes, many erad regulators including ermani, edem , os- , herp, and sel l are short-lived proteins, and ermani, edem , sel l, and os- are targeted to the lysosomal pathway for degradation [ , ] . thus, edemosomes are called erad tuning vesicles, which deliver their content to lysosomes for disposal via an autophagy-like pathway to reduce the erad capacity under natural conditions [ ] . additionally, lysosomal inhibitors are able to cause the accumulation of an aggregating mutant of dysferlin in the er when compared to the wild-type, which was used as evidence to propose that large protein aggregates are disposed of via an autophagy/lysosomal pathway, dubbed erad ii [ ] . however, under er stress, most of these factors are highly induced, including the edem proteins, but not ermani [ , , ] . under stress, qcv are recruited to the erqc, resulting in the accumulation of ermani and its glycoprotein substrates [ ] . moreover, many other erad components, including edem , hrd , derlin- , sec β, and herp, are also concentrated in erqc. importantly, it has been found that edem stabilizes ermani and increases its protein expression at steady-state levels [ ] . such enrichment of these critical components accelerates efficient assembly of the erad machinery, potentiating the degradation of misfolded glycoproteins and alleviating er stress. during infection, viruses are able to hijack the host translational machinery and saturate the er with viral proteins. not only do viruses use the er to generate their glycoproteins, but some even utilize the er as their site to assemble progeny particles [ ] . such accumulation of viral proteins in the er places a heavy demand on the protein folding machinery, which may cause er stress, and in turn, activate the upr, resulting in restoration of the er homeostasis or apoptosis. so far, at least viruses have been found to be able to induce er stress, and activate the three upr stress signaling pathways [ ] . enveloped viruses may bud through the plasma membrane or an intracellular compartment. in addition, their envelope glycoproteins are targeted to the er for post-translational modifications and folding. not surprisingly, many viral envelope glycoproteins are significant inducers of the upr, which includes hcv [ ] , hepatitis b virus (hbv) [ ] , coronaviruses [ ] , chikungunya virus (chikv) [ ] , and retroviruses [ ] . as introduced earlier, the upr utilizes three different mechanisms to alleviate er stress: reducing global protein translation, increasing the er folding capacity, and enhancing erad by activating the perk, atf , or ire -xbp pathways, respectively. viral infections may activate these pathways, resulting in the inhibition or enhancement of viral replication (table ) . for example, the perk-mediated global translation shutdown is a very effective antiviral mechanism, and a similar shutdown by pkr has been used in the interferon pathway to defend against viral infection [ ] . conceivably, viruses have evolved a number of strategies to circumvent the detrimental effect of upr to establish productive infection. hcv is still able to produce viral proteins even when the cellular translational machinery is shut down, because these viruses have their own internal ribosome entry site (ires) to recruit and assemble the ribosomal initiation complex for protein expression [ ] . epstein-barr virus (ebv), herpes simplex virus (hsv), and african swine fever virus (asfv) can counteract the perk-mediated eif α phosphorylation by activating an eif α phosphatase pp [ ] [ ] [ ] . in another example, the hcv e protein directly interacts with perk to prevent er stress sensing by acting as a pseudo-substrate to block perk activity [ ] . in addition to combating the upr, viruses also take advantage of the upr pathways to benefit their replication. for example, influenza a virus (iav) replication is promoted by activation of the ire -xbp pathway [ ] ; atf activation promotes asfv, lymphocytic choriomeningitis virus (lcmv), denv, human cytomegalovirus (hcmv), and japan encephalitis virus (jev) replication [ , ] , and atf activation enhances hiv- replication [ ] . thus, despite the detrimental effects, viruses have evolved to manipulate host upr signaling pathways to promote viral infections. below, we will focus on the roles of erad played in virus replication, which is the main target of this review. as introduced earlier, erad transports unfolded/misfolded proteins from the er into the cytosol for proteasomal degradation. conceivably, viruses can manipulate and exploit this cellular machinery to degrade several important host factors to promote their propagation. herpesviruses have evolved multiple mechanisms to suppress the host immune response via erad. major histocompatibility complex (mhc) molecules play an indispensable role in triggering an immediate immune response to inhibit virus infections. herpesviruses inhibit mhc class i (mhc-i) expression by targeting these molecules to erad for degradation. for example, hcmv produces two transmembrane proteins, us and us , and each is sufficient to bind to mhc-i heavy chains, causing their dislocation from the er to the cytosol for degradation [ ] . notably, us and us use different mechanisms to degrade mhc-i. us -dependent mhc-i degradation is mediated through an interaction with the e ligase, trc . this us /trc complex has been implicated in the degradation of other membrane proteins including multiple alpha-integrins, the interleukin receptor (il- r), thrombomodulin (thbd), protein tyrosine phosphatase receptor type j (ptprj), and cd [ ] . although the signal peptide peptidase (spp) has been shown to bind to trc , the us /trc complex maintains its mhc- degradation activity in spp−/− knockout cells, suggesting that spp binding is not related to mhc- degradation [ , ] . recent reports now regard the us /trc complex as a multifunctional hub that is able to degrade a multitude of targets in order to further hcmv immune evasion [ , ] . a complex formed between us , derlin- , and the e ligase, tmem , mediates mhc-i degradation via us [ ] . initial reports concerning us found an association with sel l and assumed that us mediated mhc- degradation could be sel l/hrd dependent. recent literature has confirmed that while the us /tmem complex degrades mhc- , us itself is degraded through a sel l/hrd axis in the absence of the client mhc- [ , ] . recruitment of p by ubxd is also crucial for us -mediated mhc-i degradation [ ] . with regard to us , hcmv utilizes erad to dispose of mhc-i and its own effector protein using discrete axes for ubiquitination. mouse gammaherpesvirus (mhv ) uses another mechanism to inhibit mhc-i. mhv produces a protein termed mk , which is a ring-finger e ligase anchored on the er membrane. mk interacts with mhc-i heavy chain molecules, and it also associates with the transporter associated with antigen processing (tap), p , derlin- , and the e ube j . association with ube j results in an interesting pattern of ubiquitination of non-lysine residues (the mk /ube j complex can ubiquitinate serines as well as lysines) that leads to rapid degradation of the mhc-i by proteasomes [ , ] . thus, herpesviruses have evolved numerous strategies to block the mhc antigen presentation and evade the host immune response to establish a persistent infection. primate lentiviruses also harness the erad pathway to promote their replication via downregulation of their receptor cd . cd downregulation prevents superinfection and promotes viral release by interrupting viral receptor-envelope interactions on the plasma membrane, leading to a controlled and productive viral infection and immunodeficiency [ ] . these viruses produce two accessory proteins, nef and vpu, to trigger cd degradation via two distinctive mechanisms [ ] . nef uses the endocytic pathway to redirect cd from the cell surface, or to interfere with the transport of newly synthesized cd from the trans-golgi network (tgn) to the cell surface, resulting in cd dislocation to endosomes and degradation by lysosomes [ ] . however, vpu interacts with cd in the er and induces cd proteasomal degradation via erad [ ] . vpu is a small transmembrane protein encoded by hiv- and some simian immunodeficiency virus (siv) isolates. vpu forms ion conductive membrane pores; it also interacts with β-transducin repeat-containing proteins (βtrcp), which are f-box/wd repeat-containing proteins that are part of the skp -cul -f-box (scf) e ubiquitin ligase complex [ ] . the vpu-induced cd degradation is strictly dependent on the scf-β-trcp complex [ ] . notably, this e ligase complex is not associated with the er membrane, and therefore does not normally function in erad. however, the degradation also requires the cytosolic atpase p and its cofactors ufd l and npl , which are key components of the erad machinery, suggesting that cd is degraded via erad [ ] . nevertheless, the degradation is not dependent on hrd , sel l, and ubc . in addition to degradation, viruses may harness erad components to benefit their replication. first, erad can promote viral protein expression. mouse mammary tumor virus (mmtv) is a betaretrovirus, which expresses the rem protein in the er. rem has a n-terminal -amino acid signal peptide (sp), which is cleaved off by signal peptidase and retrotranslocated in a p -dependent manner [ ] . rem sp then promotes the nuclear export of viral unspliced rnas to the cytosol for protein expression. similarly, hepatitis e virus (hev) orf is an n-linked glycoprotein, but functions as the major capsid protein. although orf is expressed in the er, it depends on erad components to exit from the er to the cytoplasm without being polyubiquitylated [ ] . second, erad can promote virus entry. polyomaviruses (pyv) enter cells through the er and then replicate in the nuclei [ ] . to get from the er to the nucleus, these viruses can cross the er membrane into the cytosol via the erad retrotranslocons [ ] . an example of this is mouse pyv, which uses derlin- , whereas simian virus (sv ) uses derlin- and the sel l complex for dislocation [ , ] . in addition, the proteasome machinery is also required for the human bk pyv exit from the er [ ] . third, erad can promote virus replication. the replication of positive-strand rna viruses normally involves the formation of double-membrane vesicles (dmvs) and convoluted membranes (cms) by rearrangement of cellular membranes, which segregates and protects viral proteins and genomes from the host's innate immune response. as introduced earlier, the erad activity can be adjusted by erad tuning vesicles termed edemosomes ( figure b) , which display non-lipidated lc and segregate the erad factors edem , os- , and sel l from the er lumen [ ] . by comparing the similarity between dmvs and edemosomes, it has been discovered that mouse hepatitis virus (mhv), equine arteritis virus (eav), and jev indeed replicate in these erad tuning vesicles [ ] . thus, these viruses can subvert edemosomes as their replication vesicles to promote infection [ ] . although erad has been frequently manipulated by a number of viruses to promote infection or attenuate immune responses, it may also function directly as an antiviral device to protect host cells from infection. because viral envelope glycoprotein production and folding take place in the er, these viral proteins may become the primary targets for erad, resulting in the inhibition of viral infection. primate lentiviruses, including hiv and siv, have low levels of envelope glycoproteins on their surface, and the average copy number is~ env trimers per virion [ , ] . in contrast, ifa, sendai virus, hsv, and moloney murine leukemia virus (momulv) have much more envelope glycoproteins on their surfaces [ ] [ ] [ ] [ ] . the exceptionally low number of env spikes may protect hiv- from host immune responses [ ] since almost % of env proteins are retained in the er and are degraded [ ] [ ] [ ] . this degradation mechanism was not clear until we recently reported that hiv- env glycoproteins are targeted for erad. from completely unrelated studies, we isolated hiv- non-permissive (np) and permissive (p) t cell clones n -np and n -p from the original cem.nkr human t cell line [ ] . our initial analysis uncovered that hiv- replication is restricted from the second round of the viral life cycle in n -np cells, resulting in~ -fold inhibition when compared to n -p. further transcriptome analysis by microarrays revealed that n -np cells overexpress the mitochondrial translocator protein (tspo), which strongly inhibits hiv- env expression [ ] . tspo interacts with the mitochondrial permeability transition pore (mptp) complex, which includes the outer membrane protein voltage-dependent anion channel (vdac) protein, the inner membrane protein adenine nucleotide translocase (ant), and the mitochondrial matrix protein cyclophilin d (cypd) [ ] . tspo binds to vdac and contributes to the regulation of the mitochondrial membrane permeability by the mptp complex [ ] . our results suggested that tspo overexpression could reduce the oxidative redox status in the er, which interferes with the env oxidative folding process, resulting in env degradation. consistently, the rapid env degradation in n -np cells was rescued by kifunesine, an effective inhibitor of glycoside hydrolase family (gh ) enzymes [ ] , suggesting that hiv- is degraded via erad in n -np cells. to further explore the env degradation mechanism, we investigated which of those four er-associated gh enzymes was responsible for the env degradation. notably, when ermani, edem , edem , and edem were ectopically expressed in t cells, only ermani strongly inhibited env expression in a dose-dependent manner. in addition, when the endogenous ermani was knocked out by crispr/cas , tspo was no longer able to suppress the env expression [ ] . these results demonstrated that ermani should be responsible for the initiation of hiv- env degradation via erad. human ermani is a -amino-acid, . -kda, type ii membrane protein, which is divided into an n-terminal cytoplasmic domain (cd), transmembrane (tm) helix, lumenal 'stem' region, and a catalytic domain [ , ] . using an immunoprecipitation assay, we found that hiv- env interacts with the catalytic domain of ermani [ ] . the structure of this catalytic domain shows an (αα) -barrel composed of consecutive helices [ ] . in the catalytic domain, there are seven residues that are critical for ermani function. c and c form a highly conserved disulfide bond and were reportedly critical for protein folding [ ] , whereas e , d , and e were proposed as catalytic residues [ ] . r c and e k mutations are found in nonsyndromic autosomal-recessive intellectual disability (ns-arid) disease [ ] , and the r c mutation is also found in the congenital disorders of glycosylation [ ] . all these residues are required for hiv- env degradation, suggesting that the mannosidase activity is important for the ermani activity. ermani also targets the terminally misfolded human alpha -antitrypsin variant null (hong kong) (nhk) for degradation via erad, but neither its catalytic activity nor its catalytic domain is required for this degradation, suggesting that different mechanisms are involved in hiv- env and nhk degradation [ ] . we have also found that the viral protein r (vpr) of hiv- enhances viral replication in monocyte-derived macrophages (mdms) and dendritic cells (mddcs) by rescuing env from erad degradation through the erad (ii) autophagy pathway. compounds known to facilitate glycoprotein folding (pk and as o ) and inhibit er α-mannosidases crucial for erad (kifunensine), and those that block lysosomal proteases (bafilomycin) rescued envelope expression and infectivity in a ∆vpr background to that of wild-type virus [ ] . as aforementioned, unlike ermani, whose expression is not responsive to upr, the expression of the edems is induced upon upr via the ire /xbp activation pathway, which boosts erad and alleviates er stress. although ectopic expression of edems did not inhibit hiv- env expression [ ] , these proteins inhibit the expression of some other envelope glycoproteins. hbv expresses three surface glycoproteins, the large (l), middle (m), and small (s), which are translated from different initiation codons within the same open reading frame (orf) and share the tetra-spanning transmembrane domains in the s protein. the n-terminus of the m and l protein contain additional pres and pres -pres domains, respectively. the common s domain has an n-glycosylation site, and the m pres domain has another site. overexpression of the surface proteins is sufficient to activate the ire /xbp pathway and elevate edem , edem , and edem expression. importantly, edem overexpression destabilizes s, m, and l, and edem silencing stabilizes their expression [ ] . in addition, the autophagy/lysosomal pathway, but not the proteasomal pathway, is involved in the degradation of hbv surface glycoproteins, further complicating our understanding of the viral protein degradation process via erad [ ] . hcv has two n-glycosylated envelope proteins e and e on the surface of virions, which are type i transmembrane proteins expressed from a common viral polyprotein precursor. hcv infection strongly induces the activation of the ire stress sensor, resulting in elevation of edem , edem , and edem , but not the ermani expression. both edem and edem , but not edem , interact with e , and overexpression of these two proteins induces e polyubiquitylation and degradation. conversely, knockdown of edem expression or treatment with kifunesine increases e expression, and also reduces the interaction of edem and edem with sel l [ ] . taken together, these results strongly suggest that edem proteins are able to extract viral polypeptides from the er quality control cycle, and degrade them via erad. however, since none of these proteins can target the jev e protein to erad for degradation, not every viral glycoprotein is recognizable by these proteins [ ] . in vivo experiments on patients with chronic liver injury were unable to identify up-regulation of upr and erad elements in diseased versus control patients [ ] . erad has also been implicated in the degradation of hcmv glycoproteins, gh and gl, via the s proteasome. hcmv produces at least unique glycoproteins, with four homologues to the hsv glycoproteins, gh, gb, gl, and gm [ ] . the glycoproteins, gh and gl, are constituents of the gcii type complexes found on the surface of hcmv virions. the gcii trimeric complex between gh, gl, and go can initiate ph independent fusion [ ] . in addition, a pentameric complex between gh, gl, and the gene products u , u , and u is able to mediate entry into different cell types via ph-dependent receptor-mediated endocytosis; a process that requires the trimeric gh/gl/go complex [ ] . although previous studies have shown that the glycoprotein gl stabilizes the expression of gh and potentiates its surface localization [ ] , recent work revealed that gh is degraded via erad in the absence of gl [ ] . replacement of the cytoplasmic tail of gh with that of the human cd protein subverted gh degradation via erad, potentiating surface expression. current studies describe two paradigms for erad to target viral glycoproteins for degradation: ermani-mediated, which targets hiv- env, and edem-mediated, which can target hcv and hbv surface glycoproteins. gh family members share a common catalytic mannosidase homology domain of~ -residues [ ] , and the three catalytic residues e , d , and e found in ermani are all conserved in these proteins [ ] . nevertheless, there is little protein sequence homology beyond this domain among these proteins. unlike ermani, all three edems are er-lumenal proteins, although the signal sequence of edem is resistant to cleavage [ ] . edem has two novel features including an additional protease-associated domain of unknown function and a kdel signal for er retention [ ] . whether or how the coordination between the edems and ermani facilitates erad is still a convoluted issue. due to lysosomal degradation mediated by the n-terminal cytoplasmic tail, ermani is expressed at very low basal levels in cells, and its expression is not induced by upr [ ] . such proteolytically driven checkpoint control of ermani expression may contribute to establish glycoprotein quality control at a baseline level, which maintains er homeostasis without activation of ire /xbp . however, if this basic mechanism fails to restore er homeostasis, ire /xbp is induced to elevate expression of the edems, which will increase erad. unlike hcv and hbv, hiv- induces upr, but barely activates the ire /xbp pathway, which may explain why hiv- env is not directly targeted by edem proteins [ ] . nevertheless, these two different arms of erad do not exclude the role of the edems in ermani-mediated degradation. edems may accelerate the release of terminally misfolded glycoproteins from the cnx/crt cycle, and thereby help ermani to conduct more extensive demannosylation [ ] ; and the association of edem with sel l may further accelerate the cytosolic delivery of misfolded proteins [ ] . moreover, edem may form a complex with ermani, which stabilizes ermani by the suppression of its proteolytic degradation [ ] . discrepancies concerning the localization of ermani with various labs determining colocalization with the er, golgi, or er-golgi intermediate compartments and quality control vesicles, lends credence to both current theories that ermani is either a golgi checkpoint in quality control that will return misfolded proteins back to the er for further processing, or that it resides in quality control vesicles with glycoprotein substrates as part of the cnx/crt cycle [ , ] . it is well established that viruses have evolved to manipulate host upr and erad to optimize their replication, whether they are 'tuning' host quality control to ensure the proper folding of their envelope glycoproteins, circumventing erad in order to prevent degradation of their viral envelope glycoproteins, or hijacking erad to dispose of host proteins. there are still many questions left to be answered, including the identities of the dislocons that each envelope glycoprotein is targeted to, the motifs or patterns that allow α , -mannosidases to differentiate between native and misfolded glycoproteins, why some viral proteins are disproportionately targeted (hcmv gh), and the roles that the upr and erad play in vivo during viral infections. these exciting areas merit more extensive studies. virus entry: molecular mechanisms and biomedical applications the cell biology of receptor-mediated virus entry recruitment of hiv and its receptors to dendritic cell-t cell junctions penetration of nonenveloped viruses into the cytoplasm virus and cell fusion mechanisms both e protein glycans adversely affect dengue virus infectivity but are beneficial for virion release a systematic study of the n-glycosylation sites of hiv- envelope protein on infectivity and antibody-mediated neutralization playing hide and seek: how glycosylation of the influenza virus hemagglutinin can modulate the immune response to infection the hepatitis c virus glycan shield and evasion of the humoral immune response comprehensive functional analysis of n-linked glycans on ebola virus gp tracking global patterns of n-linked glycosylation site variation in highly variable viral glycoproteins: hiv, siv, and hcv envelopes and influenza hemagglutinin glycosylation affects cleavage of an h n influenza virus hemagglutinin and regulates virulence importance of hemagglutinin glycosylation for the biological functions of influenza virus interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics the hiv glycan shield as a target for broadly neutralizing antibodies virus glycosylation: role in virulence and immune interactions vertebrate protein glycosylation: diversity, synthesis and function roles of n-linked glycans in the endoplasmic reticulum n-glycan-based er molecular chaperone and protein quality control system: the calnexin binding cycle malectin: a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein n-glycosylation the role of lectin-carbohydrate interactions in the regulation of er-associated protein degradation the long road to destruction mammalian er mannosidase i resides in quality control vesicles, where it encounters its glycoprotein substrates the specificity of the yeast and human class i er alpha , -mannosidases involved in er quality control is not as strict previously reported endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to man - glcnac the unfolded protein response: integrating stress signals through the stress sensor ire alpha endoplasmic reticulum stress sensing in the unfolded protein response dynamic interaction of bip and er stress transducers in the unfolded-protein response the unfolded protein response: from stress pathway to homeostatic regulation unfolded proteins are ire -activating ligands that directly induce the unfolded protein response er-stress-induced transcriptional regulation increases protein synthesis leading to cell death endoplasmic reticulum-associated degradation regulation of endoplasmic reticulum-associated protein degradation (erad) by ubiquitin. cells how early studies on secreted and membrane protein quality control gave rise to the er associated degradation (erad) pathway: the early history of erad sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase doa /teb . elife , , e when worlds collide: ip( ) receptors and the erad pathway regulation of diacylglycerol acyltransferase protein stability by gp -associated endoplasmicreticulum-associated degradation ubiquitin-proteasome system mediates heme oxygenase- degradation through endoplasmic reticulum-associated degradation pathway selective inhibition of endoplasmic reticulum-associated degradation rescues deltaf -cystic fibrosis transmembrane regulator and suppresses interleukin- levels: therapeutic implications the delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology flagging and docking: dual roles for n-glycans in protein quality control and cellular proteostasis family alpha-mannosidases in n-glycan processing edem initiates mammalian glycoprotein erad by catalyzing the first mannose trimming step edem , a soluble edem homolog, enhances glycoprotein endoplasmic reticulumassociated degradation and mannose trimming in vitro mannose trimming property of human er alpha- , mannosidase i ergic- and traffic in the secretory pathway endoplasmic reticulum (er) mannosidase i is compartmentalized and required for n-glycan trimming to man - glcnac in glycoprotein er-associated degradation stimulation of erad of misfolded null hong kong alpha -antitrypsin by golgi alpha , -mannosidases mannosidase ia is in quality control vesicles and participates in glycoprotein targeting to erad edem regulates er-associated degradation by accelerating de-mannosylation of folding-defective polypeptides and by inhibiting their covalent aggregation glycoprotein folding and the role of edem , edem and edem in degradation of folding-defective glycoproteins os- and grp deliver mutant alpha -antitrypsin to the hrd -sel l ubiquitin ligase complex for erad the mrh domain suggests a shared ancestry for the mannose -phosphate receptors and other n-glycan-recognising proteins in vitro analysis of hrd p-mediated retrotranslocation of its multispanning membrane substrate -hydroxy- -methylglutaryl (hmg)-coa reductase derlin- and ubxd are engaged in dislocation and degradation of lipidated apob- at lipid droplets structure of the native sec protein-conducting channel proteasome s rp binding to the sec channel plays a key role in erad a membrane protein required for dislocation of misfolded proteins from the er a membrane protein complex mediates retro-translocation from the er lumen into the cytosol derlin- is a rhomboid pseudoprotease required for the dislocation of mutant alpha- antitrypsin from the endoplasmic reticulum protein quality control in the er: balancing the ubiquitin checkbook the ubiquitylation machinery of the endoplasmic reticulum role of s proteasome and hrd genes in the degradation of -hydroxy- -methylglutaryl-coa reductase, an integral endoplasmic reticulum membrane protein autoubiquitination of the hrd ligase triggers protein retrotranslocation in erad key steps in erad of luminal er proteins reconstituted with purified components selective ubiquitylation of p and cdt by ubch and ube g ubiquitin-conjugating enzymes via the crl cdt ubiquitin ligase complex endoplasmic reticulum degradation requires lumen to cytosol signaling. transmembrane control of hrd p by hrd p association of the sel l protein transmembrane domain with hrd ubiquitin ligase regulates erad-l usa functions as a scaffold of the hrd-ubiquitin ligase the ubiquitin-domain protein herp forms a complex with components of the endoplasmic reticulum associated degradation pathway membrane topology of the yeast endoplasmic reticulum-localized ubiquitin ligase doa and comparison with its human ortholog teb (march-iv) ube j ubiquitinates hydroxylated amino acids on er-associated degradation substrates hrd and ube j target misfolded mhc class i heavy chains for endoplasmic reticulum-associated degradation distinct ubiquitin-ligase complexes define convergent pathways for the degradation of er proteins the yeast erad-c ubiquitin ligase doa recognizes an intramembrane degron htm p, a mannosidase-like protein, is involved in glycoprotein degradation in yeast the saccharomyces cerevisiae processing alpha , -mannosidase is localized in the endoplasmic reticulum, independently of known retrieval motifs segregation and rapid turnover of edem by an autophagy-like mechanism modulates standard erad and folding activities human endoplasmic reticulum mannosidase i is subject to regulated proteolysis disposal of cargo and of erad regulators from the mammalian er two endoplasmic reticulum-associated degradation (erad) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome erad(i) and autophagy/lysosome erad(ii) a novel er alpha-mannosidase-like protein accelerates er-associated degradation a novel stress-induced edem variant regulating endoplasmic reticulum-associated glycoprotein degradation glycan regulation of er-associated degradation through compartmentalization the mammalian upr boosts glycoprotein erad by suppressing the proteolytic downregulation of er mannosidase i how viruses use the endoplasmic reticulum for entry, replication, and assembly the expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis unfolded protein response in hepatitis c virus infection modulation of the unfolded protein response by the human hepatitis b virus coronavirus infection, er stress, apoptosis and innate immunity differential unfolded protein response during chikungunya and sindbis virus infection: chikv nsp suppresses eif alpha phosphorylation hiv infection and antiretroviral therapy lead to unfolded protein response activation signal integration via pkr the pathway of hcv ires-mediated translation initiation the lmp oncogene of ebv activates perk and the unfolded protein response to drive its own synthesis herpes simplex virus infection activates the endoplasmic reticulum resident kinase perl and mediates eif- alpha dephosphorylation by the gamma( ) . protein the african swine fever virus dp l protein recruits the protein phosphatase catalytic subunit to dephosphorylate eif alpha and inhibits chop induction but is dispensable for these activities during virus infection protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis c virus envelope protein e through the eukaryotic initiation factor alpha kinase perk influenza a viral replication is blocked by inhibition of the inositol-requiring enzyme (ier ) stress pathway the atf branch of unfolded protein response and apoptosis are activated to promote african swine fever virus infection er stress, autophagy, and rna viruses short communication: activating transcription factor (atf ) promotes hiv type activation pb -f attenuates virulence of highly pathogenic avian h n influenza virus in chickens dengue virus modulates the unfolded protein response in a time-dependent manner cytomegalovirus downregulates ire to repress the unfolded protein response the sars coronavirus a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type interferon receptor the hcmn gene products us and us target mhc class i molecules for degradation in the cytosol plasma membrane profiling defines an expanded class of cell surface proteins selectively targeted for degradation by hcmv us in cooperation with ul cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins signal peptide peptidase is required for dislocation from the endoplasmic reticulum the trc e ligase ubiquitinates mhc class i molecules before dislocation from the er tmem is a derlin- associated erad e ligase essential for virus-induced degradation of mhc-i identifying the erad ubiquitin e ligases for viral and cellular targeting of mhc class i sel l nucleates a protein complex required for dislocation of misfolded glycoproteins mhc class i ubiquitination by a viral phd/lap finger protein role of cd receptor down-regulation during hiv- infection mechanisms of cd downregulation by the nef and vpu proteins of primate immunodeficiency viruses a novel human wd protein, h-beta trcp, that interacts with hiv- vpu connects cd to the er degradation pathway through an f-box motif hiv- vpu -an ion channel in search of a job multilayered mechanism of cd downregulation by hiv- vpu involving distinct er retention and erad targeting steps retroviral rem protein requires processing by signal peptidase and retrotranslocation for nuclear function cytoplasmic localization of the orf protein of hepatitis e virus is dependent on its ability to undergo retrotranslocation from the endoplasmic reticulum the polyomaviridae: contributions of virus structure to our understanding of virus receptors and infectious entry murine polyomavirus requires the endoplasmic reticulum protein derlin- to initiate infection simian virus depends on er protein folding and quality control factors for entry into host cells coronaviruses hijack the lc -i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication how viruses hijack the erad tuning machinery electron tomography analysis of envelope glycoprotein trimers on hiv and simian immunodeficiency virus virions distribution and three-dimensional structure of aids virus envelope spikes retrovirus envelope protein complex structure in situ studied by cryo-electron tomography three-dimensional structure of herpes simplex virus from cryo-electron tomography paramyxovirus ultrastructure and genome packaging: cryo-electron tomography of sendai virus zernike phase contrast electron microscopy of ice-embedded influenza a virus why hiv virions have low numbers of envelope spikes: implications for vaccine development model for intracellular folding of the human immunodeficiency virus type gp secretion of a truncated form of the human immunodeficiency virus type envelope glycoprotein biosynthesis, cleavage, and degradation of the human immunodeficiency virus envelope glycoprotein gp a novel hiv- restriction factor that is biologically distinct from apobec cytidine deaminases in a human t cell line cem the mitochondrial translocator protein, tspo, inhibits hiv- envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway the mitochondrion in apoptosis: how pandora's box opens tspo interacts with vdac and triggers a ros-mediated inhibition of mitochondrial quality control elucidation of the molecular logic by which misfolded alpha -antitrypsin is preferentially selected for degradation ermani (endoplasmic reticulum class i alphamannosidase) is required for hiv- envelope glycoprotein degradation via endoplasmic reticulum-associated protein degradation pathway identification, expression, and characterization of a cdna encoding human endoplasmic reticulum mannosidase i, the enzyme that catalyzes the first mannose trimming step in mammalian asn-linked oligosaccharide biosynthesis cloning and expression of a specific human alpha , -mannosidase that trims man( )glcnac( ) to man( )glcnac( ) isomer b during n-glycan biosynthesis structural basis for catalysis and inhibition of n-glycan processing class i alpha , -mannosidases role of the cysteine residues in the alpha , -mannosidase involved in n-glycan biosynthesis in saccharomyces cerevisiae. the conserved cys and cys residues form an essential disulfide bond mutations in the alpha , -mannosidase gene, man b , cause autosomal-recessive intellectual disability somatic overgrowth associated with homozygous mutations in both man b! and sec a. cold spring harb. mol. case stud a golgi-localized mannosidase (man b ) plays a non-enzymatic gatekeeper role in protein biosynthetic quality control hiv- vpr increases env expression by preventing env from endoplasmic reticulum-associated protein degradation (erad) activation of erad pathway by human hepatitis b virus modulates viral and subviral particle production role of the endoplasmic reticulum-associated degradation (erad) pathway in degradation of hepatitis c virus envelope proteins and production of virus particles no evidence of the unfolded protein response in patients with chronic hepatitis c virus infection human cytomegalovirus glycoproteins human cytomegalovirus penetrates host cells by ph-independent fusion at the cell surface human cytomegalovirus gh/gl/go promotes the fusion step of entry into all cell types, whereas gh/gl/ul - broadens virus tropism through a distinct mechanism a novel herpes simplex virus glycoprotein, gl, forms a complex with glycoprotein h (gh) and affects normal folding and surface expression of gh human cytomegalovirus gh stability and trafficking are regulated by er-associated degradation and transmembrane architecture characterization of early edem protein maturation events and their functional implications role of edem in the release of misfolded glycoproteins from the calnexin cycle edem recognition and delivery of misfolded proteins to the sel l-containing erad complex golgi localization of ermani defines spatial separation of the mammalian glycoprotein quality control system the authors declare no conflict of interest. key: cord- -bh csogu authors: fathima, sumana; lee, bonita e.; may-hadford, jennifer; mukhi, shamir; drews, steven j. title: use of an innovative web-based laboratory surveillance platform to analyze mixed infections between human metapneumovirus (hmpv) and other respiratory viruses circulating in alberta (ab), canada ( – ) date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: bh csogu we investigated the proportions of mono vs. mixed infections for human metapneumovirus (hmpv) as compared to adenovirus (adv), four types of coronavirus (crv), parainfluenza virus (piv), rsv, and enterovirus/rhinovirus (erv) in alberta, canada. using the data integration for alberta laboratories (dial) platform, , respiratory specimens at provlab between july and june were selected and included in the study. using the respiratory virus panel these specimens tested positive for one or more respiratory virus and negative for influenza a and b. from our subset hmpv was the fourth most common virus (n= , ) with ( %) identified as mixed infection using dial. mixed infection with hmpv was most commonly found in infants less than months old and erv was most commonly found in mixed infection with hmpv ( / , %) across all age groups. the proportion of mixed-infection vs. mono-infection was highest for adv ( %), followed by crv e ( %), crv hku ( %), crv nl ( %), crv oc ( %), piv ( %), rsv ( %), hmpv ( %) and erv ( %). hmpv was significantly more likely to be identified in mono infection as compared with adv, crv, piv, and rsv with the exception of erv [p< . ]. respiratory tract infections are a global public health concern and in canada is the eighth leading cause of death in [ ] . human metapneumovirus (hmpv) is an rna virus belonging to paramyxoviridae family. the virus was identified by researchers in the netherlands in as an important cause of respiratory infections that affect all age groups [ ] . a study from saskatchewan, canada by liu et al. [ ] , using elisa showed that seroprevalence for hmpv approaches % by young adulthood. this virus can affect any age group but several studies have shown that hmpv is a leading cause in lower respiratory tract infections in children [ ] [ ] [ ] but it also affects the elderly [ ] . clinical manifestations are similar to respiratory syncytial virus (rsv) primarily leading to pneumonia and bronchiolitis [ ] . in canada, outbreaks associated with hmpv have been reported in alberta, british columbia and quebec mainly in long term and senior care facilities [ ] [ ] [ ] . other common respiratory viruses in these settings include influenza a (flua), influenza b (flub), parainfluenza virus (piv), enterovirus/rhinovirus (erv), adenovirus (adv), and coronavirus (crv), which all can cause lower respiratory tract infections [ , ] . provincial laboratory for public health (provlab) in alberta provides testing for all respiratory virus pathogens for the province of alberta and surrounding northern territories (excluding yukon) of canada. the diagnostic testing algorithm for respiratory virus at provlab changed during the h n pandemic. from april , respiratory specimens arriving at provlab were screened by an inhouse real-time reverse-transcriptase (rt)-pcr for influenza a [ ] . specimens that were positive for influenza a were also subtyped for seasonal h , seasonal h and pandemic h n ( ) genes by rt-pcr. a cost effective approach was adopted in june , to only test specimens negative for both influenza a and b by either singleplex or multiplex real time pcr assays using the respiratory virus panel (rvp) classic assay, a multiplexed assay which detects multiple respiratory viral pathogens including flua, flub, piv, erv, adv, types of crv, rsv, and hmpv [ ] . exceptions to this testing policy include samples submitted from a provincial influenza-like-illness surveillance program (tarrant viral watch) and some samples from patients with severe illness and admission to the intensive care units. in alberta, a unique platform was developed for laboratory-based surveillance called data integration of alberta laboratories (dial). dial is a secure web based platform, which is used to extract, interpret, collate and analyze respiratory virus testing data from provlab, laboratory information system (lis) in real time [ ] . dial has an automatic engine that extracts raw specimen-based laboratory data from provlab lis, including patient demographics, information of physician and submitting agencies, and test data for specific targets. the second and most important component of dial is a built-in automated interpretation engine (aie) which provides clinically relevant interpretation and final target-specific classifications for each specimen. finally, dial also has an analytical engine which allows users to select and create specific data sets for various targets by different factors, e.g., patient demographics, geographic distribution, time periods, testing methods and perform different types of analysis including graphical presentations, tables, maps, rate calculation and trending analysis. in the case of respiratory specimens from provlab, dial's aie was designed to assign positive and negative classifications for each respiratory virus as well as a summary classification that classifies each specimen as: ) only positive for one specific virus, ) mixed infection with more than one respiratory virus or ) negative for all respiratory viruses. using these final classifications, positive and negative specimens for each virus can easily be selected in dial for further analysis. in this study we used dial to select specimen-based data and investigated the proportions of mono vs mixed infections for hmpv as compared to adv, crv, erv, piv and rsv for a period of three years, july to june . in order to create a uniform dataset for this study, we excluded all samples that tested positive for influenza a or b by the in-house real-time pcr assays and included only samples that had undergone rvp testing. using dial, , specimens were identified as positive for one or more respiratory virus during the study period. a total of , were excluded from this study with , tested positive for influenza a, , for influenza b, for both influenza a and b and specimens not tested by rvp even though they were negative for influenza. for the , rvp positive specimens included in the study, , ( %) were received between july and june , , ( %) between july and june and , ( %) between july and june . mixed infection (having more than one virus identified) was found in , ( %) specimens and , ( %) had mono-infection (having only one virus identified). the majority of the specimens were collected from the upper respiratory tract as nasopharyngeal/nasal/throat swabs or nasopharyngeal aspirates % (n= , ), % (n= ) as respiratory samples with unspecified source, % (n= ) were from the lower respiratory tract e.g., endotracheal aspirates or bronchaveolar lavage, and remaining as unknown sample types and a few tissues and sterile body fluid. the age distribution of specimens submitted and tested positive for hmpv is summarized in table . the highest number of specimens received was from patients less than months old and the proportion of specimens tested positive for hmpv ranged from - % among the different age groups (p< . , chi square test). overall, mixed infection was detected in % of specimens tested positive for hmpv. using specimens from the youngest age group (less than six months old) as the reference age group, there was significant difference for the proportion of mixed hmpv infection among the various age groups (p< . , binary logistic regression) ( table ) . mixed infection with hmpv was most commonly found in specimens from patients younger than six months and rarely in specimens submitted from older than years old. among the specimens with mixed infections with hmpv, the three most commonly found virus was erv, rsv and piv, which also were the three most common viruses detected in all the specimens ( table ). in comparison with adv, four types of crv, piv, and rsv, hmpv had a significantly lower proportion of mixed infection specimens [χ with bonferroni's correction, p=< . ] and there was no significant difference of mixed infection comparing hmpv and erv [χ with bonferroni's correction, p= . ]. the age distribution of virus found in mixed infection with hmpv is summarized in table . erv was the most commonly found virus in hmpv mixed infection across all age groups. peak hmpv activity was observed in february , june and november , whereas peak erv activity was found in the month of september in three consecutive years ( , and ) ( figure ). specimens with mixed infection with these two viruses followed the trend and circulatory pattern of hmpv. * influenza a and b positive samples and samples not tested by rvp were excluded from the analysis † some specimens in these subsets had more than one virus identified as co-existing with hmpv * five specimens with unknown age group were not tabulated † some specimens in these subsets had more than one virus identified as co-existing with hmpv the objective of this study was to describe mono and mixed infections of hmpv in our jurisdiction for a three year period. all specimen data was acquired using provlab's dial application and we were able to identify that hmpv was more likely to occur in mono infection ( %) than mixed ( %). the results in this study were similar to a previous pilot study in alberta which identified hmpv mixing with other pathogens in % of specimens ( / ) [ ] . in other populations, namely hospitalized patients, mixed hmpv infections were in the minority. our study also determined that in hmpv mixed infections, the pathogen most likely to co-exist with hmpv is erv ( %). in contrast, other studies have identified rsv as the leading cause of mixed infection with hmpv [ ] [ ] [ ] . there may be several reasons for the difference in findings including the type of diagnostic assays used and the detection of various respiratory targets, clinical setting, patient age, and timing of specimen collection. many studies have focused on populations mostly consisting of hospitalized pediatric patients and these studies have shown that rsv was the leading pathogen identified in these young children who were between months to years old [ ] [ ] [ ] . in contrast, our study examined all age groups, including community-based and hospital-based specimens. erv and hmpv remained closely linked across all age groups. mixed infection with hmpv was most commonly found in the age group less than months old. moreover, erv was still the dominant virus found to be mixed with hmpv. our data set included specimens collected during the second phase of h n pandemic in alberta and northern territories, which made up of more than one third of the specimens included in this study. we found % of the samples between july and june tested positive for erv, significantly higher than that found in the other two time periods, july to june ( %) and july to june ( %) (p< . , binary logistic regression). other studies have also shown that during the pandemic erv was a very common circulating virus followed by crv [ , , ] . moreover, this linkage between erv and hmpv could also be due to the inclusion of a very large group of viruses identified as erv since rvp could not distinguish between various strains of rhinovirus and enterovirus. there might also be factors related to host immune responses and viral infection kinetics [ ] [ ] [ ] [ ] . we have only examined the seasonality of hmpv and erv and the mixed infection between these two viruses because of the relatively lower number of hmpv mixed infections for the other six viruses. annual variations in peak hmpv activity was observed in our study with one of the peak months occurring in the summer of , which was different from other studies in various canadian provinces showing peaks of hmpv in the winter (january to april) [ ] [ ] [ ] . as expected, the mixed infection of hmpv and erv followed the circulatory pattern of hmpv but differed from erv. although our findings have shown some trends with hmpv and its ability to exist as mono versus mixed infection, there are some important limitations in this study. firstly, influenza a and b positive samples were excluded mainly because provlab does not routinely test influenza positive specimens for hmpv since the pandemic . excluding influenza viruses from our database may have impacted viral co-infection rates identified in our study. on the other hand, a recent study in england showed a lower prevalence of influenza a (h n ) in patients positive for hmpv, adv, rsv, piv and rhinovirus with statistical significance for this relationship with hmpv and rhinovirus [ ] . another limitation was that the analysis by age group was based on specimen-based data only with duplicate samples from some individuals. moreover, only limited information is usually provided on the requisition submitted with the specimens so we were not able to study clinical manifestations of mono-infection versus mixed infections and explore different settings despite the limitations stated, this study has considerable public health implications. this study helps us better understand the frequency of hmpv mono versus mixed infection over a period of three years. better understanding of what mechanisms or conditions support mono versus mixed infections and the difference in clinical presentations and prognosis is needed. the prevalence of this virus supports research efforts to develop vaccine strategies which may become available in the future [ ] . this also brings us to question whether testing algorithms at provlab need to be changed to better understand the interaction between hmpv and influenza a and b. since hmpv has been shown to be an important pathogen, it should be included in ongoing surveillance and public health strategies. enhanced surveillance programs will help us better understand hmpv-associated diseases and maintain our awareness of trends in mono and mixed infections. provlab provides respiratory virus testing for the province of alberta and surrounding northern territories (excluding yukon) of canada. since june , all respiratory specimens arriving at provlab was screened by an in-house (rt)-pcr for influenza a [ ] . specimens tested positive for influenza a were also subtyped for seasonal h , seasonal h and pandemic h n ( ) genes by rt-pcr. a multiplex assay to detect both influenza a and b was implemented in february . only specimens tested negative for both influenza a and b were tested using the rvp classic assay, a multiplexed assay which detects multiple respiratory viral pathogens including flua, flub, piv, erv, adv, four types of crv, rsv, and hmpv [ ] . exceptions to this testing policy included samples submitted from a provincial influenza-like-illness surveillance program (tarrant viral watch) and some samples from patients with severe illness and admitted to the intensive care units. specimens tested by rvp and tested positive for one or more respiratory virus excluding influenza a and b between july and june identified using the dial application was included in this study. dial provided classification of the specimens by each respiratory virus target as well as mono versus mixed infection. dial also allowed a user to select specimens based on the type of testing and over various geographic and time periods and age groups. statistical analysis of the proportion of mono versus mixed infections for the different virus was performed using pearson chi-squared (χ ) test with bonferroni's adjustment for multiple analyses. the proportion of hmpv as mixed infection by age group and erv positive specimens by analysis of age group and time periods was analyzed using binary logistic regression. the spss software version . (ibm® spss® statistics, ibm corp., usa) was used for statistical analysis with the level of significance set at p< . . in this study we used dial to provide user-defined data sets of respiratory virus testing data and found that over a period of years, july to june , hmpv is significantly more likely to be identified in mono infection ( %) as compared with adv, four types of crv, piv, and rsv with the exception of erv. the three viruses most likely to be found in mixed infection with hmpv in this study were erv, piv and rsv with a higher proportion of mixed infection found in young infants. leading causes of death in canada a newly discovered human pneumovirus isolated from young children with respiratory tract disease seroprevalence of human metapneumovirus (hmpv) in the canadian province of saskatchewan analyzed by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay epidemiological and clinical features of hmpv, rsv and rvs infections in young children human 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china multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children rate and influence of respiratory virus co-infection on pandemic (h n ) influenza disease the viral etiology of an influenza-like illness during the pandemic nonstructural proteins of respiratory syncytial virus suppress premature apoptosis by an nf-kappab-dependent, interferon-independent mechanism and facilitate virus growth suppression of the induction of alpha, beta, and lambda interferons by the ns and ns proteins of human respiratory syncytial virus in human epithelial cells and macrophages respiratory infections by hmpv and rsv are clinically indistinguishable but induce different host response in aged individuals correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection human metapneumovirus: a new player among respiratory viruses seasonality and clinical features of human metapneumovirus infection in children in northern alberta human metapneumovirus infection in the canadian population newly discovered respiratory viruses: significance and implications this article is an open access article distributed under the terms and conditions of the creative commons attribution license the authors would like to acknowledge the staff of the provincial laboratory of public health who performed all laboratory testing and the canadian network for public health intelligence program for their development work on the dial platform. the authors declare no conflict of interest. key: cord- -rgrt e r authors: yan, bingpeng; chu, hin; yang, dong; sze, kong-hung; lai, pok-man; yuan, shuofeng; shuai, huiping; wang, yixin; kao, richard yi-tsun; chan, jasper fuk-woo; yuen, kwok-yung title: characterization of the lipidomic profile of human coronavirus-infected cells: implications for lipid metabolism remodeling upon coronavirus replication date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: rgrt e r lipids play numerous indispensable cellular functions and are involved in multiple steps in the replication cycle of viruses. infections by human-pathogenic coronaviruses result in diverse clinical outcomes, ranging from self-limiting flu-like symptoms to severe pneumonia with extrapulmonary manifestations. understanding how cellular lipids may modulate the pathogenicity of human-pathogenic coronaviruses remains poor. to this end, we utilized the human coronavirus e (hcov- e) as a model coronavirus to comprehensively characterize the host cell lipid response upon coronavirus infection with an ultra-high performance liquid chromatography-mass spectrometry (uplc–ms)-based lipidomics approach. our results revealed that glycerophospholipids and fatty acids (fas) were significantly elevated in the hcov- e-infected cells and the linoleic acid (la) to arachidonic acid (aa) metabolism axis was markedly perturbed upon hcov- e infection. interestingly, exogenous supplement of la or aa in hcov- e-infected cells significantly suppressed hcov- e virus replication. importantly, the inhibitory effect of la and aa on virus replication was also conserved for the highly pathogenic middle east respiratory syndrome coronavirus (mers-cov). taken together, our study demonstrated that host lipid metabolic remodeling was significantly associated with human-pathogenic coronavirus propagation. our data further suggested that lipid metabolism regulation would be a common and druggable target for coronavirus infections. coronaviruses are enveloped viruses with a large single-strand, positive-sense rna genome [ , ] . as of today, there are a total of six coronaviruses that are known to infect humans, including human coronavirus oc (hcov-oc ), human coronavirus e (hcov- e), severe acute respiratory syndrome coronavirus (sars-cov), human coronavirus hku (hcov-hku ), human coronavirus nl (hcov-nl ), and the middle east respiratory syndrome coronavirus (mers-cov) [ ] . these human-pathogenic coronaviruses cause a broad range of clinical manifestations. hcov-oc , hcov- e, hcov-hku , and hcov-nl cause mild, self-limiting upper respiratory tract infections. in contrast, sars-cov and the recently emerged mers-cov may cause severe pneumonia with acute respiratory distress syndrome, multi-organ failure, and death in both immunocompetent and immunocompromised hosts [ ] [ ] [ ] [ ] . lipids play crucial roles at various stages in the virus life cycle. first, lipids can serve as the direct receptors or entry co-factors for enveloped and non-enveloped viruses at the cell surface or the endosomes [ , ] . second, lipids and lipid synthesis play important roles in the formation and function of the viral replication complex [ , ] . third, lipid metabolism can generate the required energy for efficient viral replication [ ] . moreover, lipids can dictate the proper cellular distribution of viral proteins, as well as the trafficking, assembly, and release of virus particles [ , ] . in this regard, the host lipid biogenesis pathways play indispensable roles in modulating virus propagation. as in other viruses, lipids play key roles in the life cycle of coronaviruses. coronaviruses confiscate intracellular membranes of the host cells to generate new compartments known as double membrane vesicles (dmvs) for the amplification of the viral genome. dmvs are membranous structures that not only harbor viral proteins but also contain a specific array of hijacked host factors, which collectively orchestrate a unique lipid micro-environment optimal for coronavirus replication [ ] . a recent study indicated that a key lipid processing enzyme, cytosolic phospholipase a α enzyme (cpla α) that belongs to the phospholipase a (pla ) superfamily, was closely associated with dmvs' formation and coronaviruses' replication [ ] . the viral protein and rna accumulation, as well as the production of infectious virus progeny, were significantly diminished in the presence of cpla α inhibitor [ ] . at the same time, phospholipase a group iid (pla g d), an enzyme that predominantly contributes to anti-inflammatory/pro-resolving lipid mediator expression, contributed to worsened outcomes in mice infected with sars-cov by modulating the immune response [ ] . however, to date, the change and modulating effects of the specific lipids involved in lipid rearrangement upon coronavirus infection remains largely unexplored. to obtain a comprehensive and unbiased profile of perturbed lipids upon coronavirus infection, we performed mass spectrometry (ms)-based lipidomics profiling on coronavirus-infected cells using hcov- e as a model virus. specific lipids including glycerophospholipids and fatty acids (fas) upon virus infection were identified, which represented the lipid species that were rearranged by hcov- e infection. further pathway analysis revealed that the linoleic acid (la) and arachidonic acid (aa) metabolism axis was the most perturbed pathway upon hcov- e infection. importantly, supplement of additional la and aa to coronavirus-infected cells significantly inhibited virus replication of both hcov- e and the highly virulent mers-cov, suggesting that the la-aa metabolism axis is a common and essential pathway that could modulate coronavirus replication. in this regard, temporal modulation of the host lipid profile is a potential novel strategy to combat emerging human coronaviruses. high performance liquid chromatography (hplc)-grade methanol, acetonitrile, chloroform and -propanol were purchased from merck (darmstadt, germany). hplc-grade water was prepared using a milli-q water purification system (millipore, burlington, ma, usa). analytical grade acetic viruses , , of acid and commercial standards used for biomarker identification were purchased from sigma-aldrich (st. louis, mo, usa). internal standards (is) including arachidonic acid-d , (s)-hete-d , leukotriene-b -d and platelet-activating factor c- -d (paf c- -d ) were purchased from cayman chemical (ann arbor, mi, usa) [ ] . huh- and veroe cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with % heat-inactivated fetal bovine serum (fbs), u/ml penicillin, and g/ml streptomycin ( % co at • c). mers-cov (emc/ strain) was kindly provided by professor ron fouchier (erasmus medical center, rotterdam, the netherlands). mers-cov and hcov- e were cultured in veroe cells in serum-free dmem supplemented with u/ml penicillin and g/ml streptomycin as we described previously [ ] [ ] [ ] . the supernatants were harvested when cytopathic effects (cpe) were observed and centrifuged to generate the viral stocks. the viral stocks were titrated by plaque assay on veroe cells and stored at − • c as previously described [ , ] . briefly, confluent veroe cells were infected with -fold serial viral dilutions. the cells were incubated with diluted viruses at • c for h and subsequently overlaid with % low-melting-point agarose (promega, madison, wi, usa). the cells were fixed with % formaldehyde as the plaques were observed and then stained with . % crystal violet. all experiments involving live mers-cov followed the approved standard operating procedures of the biosafety level facility as previously described [ ] [ ] [ ] [ ] . huh- cells were seeded into -well plate to reach % confluency and infected with mers-cov or hcov- e at multiplicity of infection (moi) of . or , respectively. after h of inoculation, the cells were washed with phosphate-buffered saline (pbs) and maintained in lipids-supplemented medium at the indicated concentrations for h. aa, la, oleic acid (oa), and palmitic acid (pa) were dissolved in ethanol and ethanol was used as a negative control. the lipids were purchased from cayman chemical (ann arbor, mi, usa). the supernatants and cell lysates were collected at h post-infection. the viral genome copy numbers were determined by reverse-transcription quantitative polymerase chain reaction (rt-qpcr) as previously described [ ] [ ] [ ] . confluent huh- cells were mock infected or infected with hcov- e at moi of and incubated in dmem medium. at hpi, cells were collected for cellular lipid extraction. the lipid extraction was performed for liquid chromatography-mass spectrometry (lc-ms) analysis according to a previously described protocol with slight modifications [ , ] . inactivation of virus infectivity was confirmed before further processing as we previously described with some modifications [ ] . briefly, µl of ice-cold mm ammonium bicarbonate solution was added to dissociate cells. two millilitres of chloroform/methanol (v/v : ) containing is were added, followed by vortexing and centrifugation at rpm for min at • c. the bottom phase was transferred to glass vials and dried using a vacuum concentrator for storage at − • c. the dried samples were reconstituted in µl solvent mixture containing methanol/ -propanol/water (v/v/v : : ) for lc-ms analysis. after centrifugation at , rpm for min at • c, supernatants were transferred to lc vials for lc-ms analysis. the lipid extract was analyzed using an acquity uplc system coupled to a synapt g -si high definition mass spectrometry (hdms) system (waters corp., milford, ma, usa). the chromatography was performed on a waters acquity beh c column ( . µm, . × mm, i.d., . mm, waters corp., milford, ma, usa). the mobile phase consisted of (a) . % acetic acid in water and (b) acetonitrile. gradient elution applied for ultra-high performance liquid chromatography-mass spectrometry (uplc-ms) analysis was described in table s . the column and autosampler temperature were maintained at • c and • c, respectively. the injection volume was µl [ ] . the mass spectral data were acquired in both positive and negative modes. the capillary voltage, sampling cone voltage and source offset were maintained at . kv, v, and v, respectively. nitrogen was used as desolvation gas at a flow rate of l/h. the source and desolvation temperatures were maintained at • c and • c, respectively. mass spectra were acquired over the m/z range of to . the synapt g -si hdms system was calibrated using sodium formate clusters and operated in sensitivity mode. leucine enkephalin was used as a lock mass for all experiments. ms/ms acquisition was operated in the same parameters as ms acquisition. collision energy was applied at the range from to ev for fragmentation to allow putative identification and structural elucidation of the significant lipids. acquisition of the raw data was performed using masslynx software version . (waters corp., milford, ma, usa) and raw data were converted to the common data format (netcdf) files using conversion software databridge (waters corp., milford, ma, usa). the netcdf data were subsequently deconvolved into a usable data matrix using the xcms software (http://metlin.scripps. edu/download/) [ ] and the grouping of features was performed using the camera r package [ ] . preprocessed data were then exported as a .csv file for further data statistical analysis. metaboanalyst . (http://www.metaboanalyst.ca) and simca-p v . (umetrics, umeå, sweden) were used for univariate and multivariate statistical analysis, respectively [ ] . for univariate analysis, statistical significance of features was determined between the mock and hcov- e infected group using the student's t-test and fold change. the p-value < . and fold change > were used as criteria for significant features selection. for multivariate analysis, the features were subjected to pareto scaling firstly then orthogonal partial least squares discriminant analysis (opls-da) was performed as a supervised method to find important variables with discriminative power. the opls-da model was evaluated with the relevant r and q . the variable importance in projection (vip), which reflects both the loading weights for each component and the variability of the response explained by this component, was used for feature selection [ ] . ms/ms fragmentation was performed on the significant features with high abundances. the significant features identification were carried out by searching accurate ms and ms/ms fragmentation pattern data in the metlin database (metabolomics database, http://metlin.scripps. edu/), human metabolome database (http://www.hmdb.ca/), and lipd maps (lipidomics gateway, http://www.lipidmaps.org/). for confirmation of lipid identity using authentic chemical standard, ms/ms fragmentation pattern of the chemical standard was compared with that of candidate lipid under the same lc-ms condition to reveal any matching [ , ] . to investigate how coronavirus perturbs host lipid metabolism, we performed lipidomics analysis on hcov- e-infected huh cells and compared the results with those of the mock-infected cells. the preliminary features list included precursor ions, adducts and isotope ions, which were imported into the metaboanalyst and simca-p software for further analysis. the r x/ r y, represented the x/y variables explanation rate of the opls-da model, were . % and . %, respectively. the predicted component, as estimated by cross-validation, was . (q ). these cross-validated parameters were satisfactory for opls-da mode (supplementary figure s a) . at the same time, the permutation test ( times) also indicated that the validated model was satisfied (supplementary figure s b) . overall, our results demonstrated that these significant lipid features could be selected by the validated statistical model for subsequent identification. a total of (positive mode) and (negative mode) ion features were selected according to the omics-based statistical analysis method. these ion features were significantly discriminative between hcov- e-infected and mock-infected cells. to observe the discrimination trend in more detail, a hierarchical clustering analysis was performed based on the degree of similarity of lipid abundance profiles to show the overview trend of all significant ion features. as indicated in figure , most of the significant features from both negative mode ( figure a ) and positive mode ( figure b ) expressed an up-regulation trend after hcov- e infection compared with the mock infection controls. furthermore, to identify lipids specific to hcov- e infection, these significant features were grouped and annotated using the camera software, and the potential precursor ions were used to perform further ms/ms experiments for obtaining their fragmentation patterns. finally, a total of lipids were identified, which could be classified into three lipid classes, including lysophosphatidylcholine (lysopc), lysophosphatidylethanolamine (lysope) and fatty acid (fa). the chromatogram peak heights of these identified lipids were generated by lc-ms raw data and the ratio between infected and mock-infected cells was determined. as demonstrated in figure , we found a consistent up-regulation trend of the identified lipids in hcov- e-infected cells. in particular, lysopc was the predominant lipid class of all identified, accounting for approximately % of all identified lipids with significant elevation (figure a ). at the same time, arachidonic acid (aa), which belongs to the fa class, showed the highest increase in fold-change among all identified lipids with a maximum of . -fold increase ( figure b ). in addition, the level of lysopes ( figure c ) was also up-regulated with a maximum fold change of . , which was comparatively less than that of the lysopcs and fas. the identities of lysopc ( : / : ), platelet-activating factor c- (paf c- ), lysope ( : / : ), aa, la, pa and oa were confirmed by matching the retention time (rt) and ms/ms fragmentation patterns of the authentic chemical standards that distinguish between hcov- e-infected cases and non-infected controls ( figure ). the detailed information of the identified lipids was listed in table based on the list of significantly up-regulated lipids after hcov- e-infection, metaboanalyst (http://www.metaboanalyst.ca) was applied to investigate which pathway might be markedly perturbed. the result of the pathway analysis was graphically presented in figure . from the enrichment analysis results, the la metabolism pathway and fa biosynthesis pathway had a statistically significant raw p-value (raw p < . , as shown in the y-axis). pathway impact results indicated that the la metabolism and aa metabolism pathways presented higher impact than the other pathways, as indicated in the x-axis value. combining the above two analysis results, we postulated that the la metabolism pathway to be a markedly perturbed pathway that correlated with the lipid rearrangement process induced by hcov- e infection. from the enrichment analysis results, the la metabolism pathway and fa biosynthesis pathway had a statistically significant raw p-value (raw p < . , as shown in the y-axis). pathway impact results indicated that the la metabolism and aa metabolism pathways presented higher impact than the other pathways, as indicated in the x-axis value. combining the above two analysis results, we postulated that the la metabolism pathway to be a markedly perturbed pathway that correlated with the lipid rearrangement process induced by hcov- e infection. to better understand the current pathway analysis results and the cellular lipid signaling response upon hcov- e infection, we constructed a global la pathway map based on the pathway information in the kyoto encyclopedia of genes and genomes (kegg) database (https://www.genome.jp/kegg/) and literature mining ( figure ). upon hcov- e infection, the glycerophospholipids, as main components of the cell membrane, were metabolized to lysophospholipids and fas after cpla enzyme activation. lysophospholipids such as lysopcs and lysopes were correspondingly increased after hcov- e infection. moreover, lysopcs could be further metabolized to platelet-activating factor. fas were also released from glycerophospholipids but only la and aa could initiate downstream pathways to generate corresponding metabolites. the up-regulation of both lysophospholipids and fas were partially confirmed by authentic standards. furthermore, to investigate the downstream pathways trend of fas, the authentic standards were also applied in lc-ms method to confirm whether these downstream lipids were changed correspondingly. as illustrated in figure , aa is a downstream lipid of la and the origin lipid of aa metabolism pathway. the identity of aa was confirmed by authentic standard (supplementary figure s d) , which was found to be significantly up-regulated. therefore, combining pathway analysis and the authentic standards verification results, our data to better understand the current pathway analysis results and the cellular lipid signaling response upon hcov- e infection, we constructed a global la pathway map based on the pathway information in the kyoto encyclopedia of genes and genomes (kegg) database (https://www.genome.jp/kegg/) and literature mining ( figure ). upon hcov- e infection, the glycerophospholipids, as main components of the cell membrane, were metabolized to lysophospholipids and fas after cpla enzyme activation. lysophospholipids such as lysopcs and lysopes were correspondingly increased after hcov- e infection. moreover, lysopcs could be further metabolized to platelet-activating factor. fas were also released from glycerophospholipids but only la and aa could initiate downstream pathways to generate corresponding metabolites. the up-regulation of both lysophospholipids and fas were partially confirmed by authentic standards. furthermore, to investigate the downstream pathways trend of fas, the authentic standards were also applied in lc-ms method to confirm whether these downstream lipids were changed correspondingly. as illustrated in figure , aa is a downstream lipid of la and the origin lipid of aa metabolism pathway. the identity of aa was confirmed by authentic standard (supplementary figure s d) , which was found to be significantly up-regulated. therefore, combining pathway analysis and the authentic standards verification results, our data suggested that the la-aa metabolism axis was the most significantly perturbed pathway and might be associated with lipids rearrangement or other processes in hcov- e infection. suggested that the la-aa metabolism axis was the most significantly perturbed pathway and might be associated with lipids rearrangement or other processes in hcov- e infection. to investigate the potential implication of the perturbed la-aa metabolism axis in hcov- e infection, we treated hcov- e-infected huh cells with la and aa and included pa and oa for comparison. the la and aa were mapped and played a vital role in the perturbed la-aa metabolism axis ( figure ). in contrast, pa and oa were not mapped in the perturbed pathway and may only be produced from glycerophospholipids due to cpla enzyme activation. huh- cells were infected with hcov- e and treated with aa, la, pa, or oa. the cell lysates and culture supernatants were harvested at h post-infection to determine the viral genome copy number by rt-qpcr. as shown in figure , la and aa consistently inhibited the replication of hcov- e as evidenced by the decrease in virus genome copies in both cell lysate ( figure a ,c) and supernatant samples ( figure b-d) . in contrast, pa inhibited hcov- e replication only when supplied at high concentration while hcov- e replication was largely independent of oa ( figure a-d) . to further investigate if the modulatory effects of la and aa were conserved among other human-pathogenic coronaviruses, we evaluated the effects of these lipids on the replication of the recently emerged and highly virulent mers-cov. our data demonstrated that la and aa potently suppressed mers-cov replication in a similar manner as hcov- e ( figure e,f) . overall, our results demonstrated that exogenously supplied la and aa could interfere with the optimal to investigate the potential implication of the perturbed la-aa metabolism axis in hcov- e infection, we treated hcov- e-infected huh cells with la and aa and included pa and oa for comparison. the la and aa were mapped and played a vital role in the perturbed la-aa metabolism axis ( figure ). in contrast, pa and oa were not mapped in the perturbed pathway and may only be produced from glycerophospholipids due to cpla enzyme activation. huh- cells were infected with hcov- e and treated with aa, la, pa, or oa. the cell lysates and culture supernatants were harvested at h post-infection to determine the viral genome copy number by rt-qpcr. as shown in figure , la and aa consistently inhibited the replication of hcov- e as evidenced by the decrease in virus genome copies in both cell lysate ( figure a ,c) and supernatant samples ( figure b-d) . in contrast, pa inhibited hcov- e replication only when supplied at high concentration while hcov- e replication was largely independent of oa ( figure a-d) . replication of human-pathogenic coronaviruses, which suggested that the la-aa metabolism axis was significantly involved in the propagation of these viruses. to further investigate if the modulatory effects of la and aa were conserved among other human-pathogenic coronaviruses, we evaluated the effects of these lipids on the replication of the recently emerged and highly virulent mers-cov. our data demonstrated that la and aa potently suppressed mers-cov replication in a similar manner as hcov- e ( figure e,f) . overall, our results demonstrated that exogenously supplied la and aa could interfere with the optimal replication of human-pathogenic coronaviruses, which suggested that the la-aa metabolism axis was significantly involved in the propagation of these viruses. in this study, a ms-based lipidomics approach was established to characterize the host cell lipid changes upon coronavirus infection. univariate and multivariate statistical analyses were applied in data processing for the selection of significant lipid features. a total of lipids including lysophospholipids and fas were identified and were consistently up-regulated in hcov- e-infected cells. seven representative lipids were confirmed by authentic standards, including lysopc ( : / : ), paf c- , lysope ( : / : ), aa, la, pa and oa. subsequent pathway analysis indicated that the la-aa metabolism axis, consisting of la and aa as important precursor lipids, was substantially perturbed after hcov- e infection. moreover, we demonstrated that exogenously supplied la and aa were capable of inhibiting the replication of hcov- e and the highly pathogenic mers-cov, which suggested the la-aa metabolism axis to be a conserved and essential pathway in the propagation of human coronaviruses. a total of lipids including lysopcs, lysopes and unsaturated/saturated fas were identified to be significantly upregulated after hcov- e infection. twenty of these ( . %) lipids were lysopc and lysope. lysopc is the most abundant lysophospholipid in humans, with a high plasma concentration of several hundred micromoles. in addition, lysopc was a potent inhibitor and could reversely arrest pore expansion during syncytium formation mediated by diverse viral fusogens [ ] . another lysophospholipid, lysope, is present at low concentrations in vivo but they induce various cellular responses such as activation of mitogen-activated protein kinase (mapk) and neuronal differentiation when applied to cells in vitro [ ] . among the identified fas, the la and aa both belong to polyunsaturated omega- fatty acid and are essential fatty acids. in addition, la is the metabolic precursor of aa, both of which are key components of the cell membrane. la and aa also play fundamental roles in the biological function of many tissues by modulating enzymes, ion channels, receptors, as well as inflammation [ ] . coronavirus replication is associated with intracellular membrane rearrangement and depends on the formation of double membrane vesicles (dmvs) and other membranous structures as replicative organelles [ ] . the cell membrane components consist mainly of glycerophospholipid components such as phosphatidylcholine (pc), phosphatidylethanolamine (pe), lysophosphatidylcholine (lysopc), and lysophosphatidylethanolamine (lysope). a specific phospholipids composition is required by different viruses to form the optimal replicative organelles best suited for their replication [ ] . moreover, the lysopc/pe was produced from pc/pe by cpla activation, which simultaneously generated corresponding fatty acid moiety. in this regard, cpla activation is commonly believed to be beneficial for virus replication [ , ] . in our study, we found that a number of lysophospholipids and fas downstream of cpla activation, were upregulated upon hcov- e infection. the upregulation of these lipid species including la and aa were believed to promote efficient coronavirus replication. however, when we evaluate this hypothesis by exogenously supplementing additional la and aa to hcov- e-or mers-cov-infected cells, we noticed a significant reduction in virus replication. taken together, our data suggested that coronavirus infection did not randomly perturb the cellular lipid compositions. instead, we speculate that coronaviruses precisely modulate and rearrange the host lipid profile to reach an intricate homeostasis optimized for its replication. any exogenous manipulation that disrupts the equilibrium may interfere with the optimal replication of the viruses. alternatively, supplementing la and aa might disturb the la-aa metabolism axis and result in feedback reversion of lysophospholipids into phospholipids through land's cycle [ ] , thus limiting virus replication. in addition, la and aa are polyunsaturated fatty acids that are biological signaling precursors. they can be metabolized to important eicosanoids and metabolites, which play multiple roles in the host immune response and the pathogenesis of viral infections [ ] [ ] [ ] . however, previous study had suggested that arachidonic acid (aa) downstream metabolites show no evidence of anti-coronaviral activity as observed through special inhibitors of cyclooxygenases (cox) / and -lipoxygenase (lox), which are two key enzymes requiring aa as a precursor. the results indicated aa downstream products may not have a significant effect on coronaviruses replication, at least in vitro [ , ] . in this regard, the function of the downstream metabolites of la and aa may play key roles in the pathogenesis of coronaviruses in vivo. in the present study, we revealed that the cellular lipid profile was rearranged upon hcov- e infection. a total of lipids including lysopcs, lysopes and fas were upregulated. among them, la and aa, which were mapped into the la-aa metabolism axis, demonstrated strong modulatory effects on the replication of hcov- e and the highly pathogenic mers-cov. in this regard, our data suggested that optimal coronavirus replication required a specific composition of cellular lipids and any disruption could decrease the efficiency of coronavirus replication. thus, the ms-based lipidomics strategy could be used to monitor virus-specific lipid requirement, to discover the perturbed pathways and identify novel lipids to interfere with virus replication. in further studies, combining lipidomics data with biological and immunological data may help to elucidate specific pathogenic mechanisms and identify novel treatment strategies for virus infections. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : gradient elution program applied for uplc-ms analysis, figure s : opls-da model validation and permutation test. the funding sources had no role in study design, data collection, analysis or interpretation or writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. is the discovery of the novel human betacoronavirus c emc/ (hcov-emc) the beginning of another sars-like pandemic? 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developing seeds the arachidonic acid metabolome serves as a conserved regulator of cholesterol metabolism the role of arachidonic acid in the regulation of nitric oxide synthase isoforms by hiv gp protein in astroglial cells. free radic an insight into the role of arachidonic acid derived lipid mediators in virus associated pathogenesis and malignancies l-lysine acetylsalicylate + glycine impairs coronavirus replication key: cord- - vmdqky authors: bordi, licia; sberna, giuseppe; lalle, eleonora; piselli, pierluca; colavita, francesca; nicastri, emanuele; antinori, andrea; boumis, evangelo; petrosillo, nicola; marchioni, luisa; minnucci, giulia; d’agostini, elena; castilletti, concetta; locatelli, franco; zumla, alimuddin; ippolito, giuseppe; capobianchi, maria rosaria title: frequency and duration of sars-cov- shedding in oral fluid samples assessed by a modified commercial rapid molecular assay date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: vmdqky background: rt-pcr on nasopharyngeal (nps)/oropharyngeal swabs is the gold standard for diagnosis of sars-cov- infection and viral load monitoring. oral fluid (of) is an alternate clinical sample, easy and safer to collect and could be useful for covid- diagnosis, monitoring viral load and shedding. methods: optimal assay conditions and analytical sensitivity were established for the commercial simplexa™ covid- direct assay adapted to of matrix. the assay was used to test of and nps specimens collected in parallel from hospitalized patients; bronchoalveolar lavage (bal) specimens from a subgroup of severe covid- cases were also analysed. results: using simplexa™ covid- direct on of matrix, % analytical detection down to tcid /ml (corresponding to × ( ) copies (cp)/ml) was observed. no crossreaction with other viruses transmitted through the respiratory toute was observed. parallel testing of of and nps samples showed highly concordant results (κ = . ; % ci = . – . ), and high correlation of ct values (r = . ; p < . ). high concordance and elevated correlation was observed also between of and bal. prolonged viral rna shedding was observed up to days from symptoms onset (dso), with % and % positivity observed in of and nps samples, respectively, collected between and dso. conclusions: simplexa™ covid- direct assays on of have high sensitivity and specificity to detect sars-cov- rna and provide an alternative to nps for diagnosis and monitoring sars-cov- shedding. since the global pandemic spread of sars-cov- [ ] [ ] [ ] , a priority focus has been on development of rapid and sensitive diagnostic assays using easily obtainable clinical samples. diagnostic testing for sars-cov- infection by viral rna detection in respiratory specimens is required for decision making for clinical management, infection control or public health measures, triage and isolation in healthcare facilities. the who currently recommends rt-pcr testing using nasopharyngeal (nps) and oropharyngeal swabs (ops) as gold standard for sars-cov- diagnosis and for monitoring viral load [ , ] . of has been suggested as an alternate clinical sample, easy and safer to collect, minimizing exposure of healthcare workers and could be useful for making a diagnosis and measuring sars-cov- viral load and viral shedding during the course of the illness and convalescence [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . to et al., demonstrated that sars-cov- was present in of specimen of out of patients, with viral load being higher during the first week after symptoms onset and declining thereafter, being detectable until days after symptoms onset (dso) [ , ] . in another study, sars-cov- rna was detected in of of one patient for prolonged period, up to dso [ ] . we evaluated the use of commercial simplexa™ covid- direct assay on of samples from hospitalized covid- patients, for identification of sars-cov- rna, duration of viral shedding, and determining the assay specificity and sensitivity on of samples compared to nps and bal samples. of specimens were collected from patients hospitalized at national institute for infectious diseases "lazzaro spallanzani" (inmi) in rome. the median age of patients was years (iqr: - ), males ( . %) and females ( . %). a total of of of samples were collected in parallel with nps and results were compared; bal samples were also collected and analysed concomitantly with nps and of samples from a subgroup of patients attending the intensive care unit, showing more severe presentation (pao /fio < ), of whom subsequently died. in this study, patients admitted with suspect of covid- but with definitive diagnosis other than covid- , were considered as negative controls. number of patients and clinical characteristics are described in table . nps were immediately put into sterile tubes containing - ml of viral transport media, like copan utm ® universal transport medium; from a subgroup of patients with more severe manifestations also bal samples were collected. as far as of is concerned, most specimens were collected by passive drooling, spontaneusly produced without external stimuli; for some patients, to obviate the scarce salivation, sublingual of was collected using sterile pipettes. all of were collected neat, without any type of diluent and at least min after drinking or eating or washing theeth. the sars-cov- isolate -ncov/italy-inmi [ ] was propagated in vero e cells (c ; african green monkey kidney cells). cells were maintained in dulbecco's minimal essential medium viruses , , of (dmem) containing % foetal bovine serum (fbs) and · mg/ml gentamycin at • c with % co and fbs concentration was reduced to % for viral propagation. the infectious titre of the viral stock used in the study, performed by reed and muench method on veroe cells, was tcdi /ml. the evaluation of the corresponding concentration of rna copies (cp)/ml in the viral stock preparation was performed as follows: sars-cov- rna was extracted from the isolate and amplified by real-time quantitative rt-pcr (qrt-pcr) in rotor-geneq real-time cycler (qiagen, hilden, germany) using realstar ® sars-cov- rt-pcr kit . (altona diagnostic gmbh, hamburg, germany). a standard curve prepared through serial dilutions of corman's e-sars-cov- gene [ ] , obtained by european virus archive -globalevag has been used to determine the concentration of the virus stock, corresponding to rna cp/ml. to establish the analytical sensitivity, sars-cov- particles from the viral stock were spiked into a pool of of coming from healthy donors, mixed together and diluted : with . % nacl isotonic solution. serial ten-fold dilutions from to − tcdi /ml were prepared to be tested in triplicates. when established the last dilution with % of positive results, obtained at tcid /ml, five replicates of serial : dilutions were performed until reaching . tcid /ml. the results were used to obtain the limit of detection (lod) by probit analysis. to assess analytical specificity, of from healthy donors were mixed together, diluted : with . % nacl isotonic solution, aliquoted in different tubes; each tube was spiked with different respiratory viruses and loaded on mdx instrument. the following viral stocks were used: measles virus, edmonton strain, titer . tcid /ml; influenza b virus, b/shandong/ / strain, ha titer : ; influenza a h n virus, a/pt. chalmers/ / strain, ha titer : ; adenovirus , adenoid strain, titer . tcid /ml; human coronavirus oc , pt isolate, titer . tcid /ml; human coronavirus e, pt isolate, titer . tcid /ml. simplexa™ covid- direct assay is a real-time rt-pcr system that enables the direct amplification of coronavirus sars-cov- rna from several specimens, without sample processing like rna extraction. in the simplexa™ covid- direct assay (diasorin molecular llc, cypress, ca , u.s.a.), fluorescent probes are used together with corresponding forward and reverse primers to amplify two different regions of the sars-cov- genome: orf ab and s gene; an rna internal control is used to detect rt-pcr failure and/or inhibition. for testing with the simplexa™ covid- direct assay, one vial of reaction mix was thawed for each sample followed by loading µl of sample (of) that was previously diluted : with . % nacl and µl of reaction mix to their specific wells on a direct amplification disk (dad). the dad was then loaded onto the liaison ® mdx instrument (diasorin molecular), which is a compact and expandable thermal cycler with an extremely small footprint and the capability to connect up to four instruments with a single laptop. upon completion of the run, the software automatically calculates and provides easy to understand results with the ability to check amplification curves after a run. samples with ct values < were considered positive, according to test procedure indications; for statistical calculations, an arbitrary value of ct was assigned to negative samples. data management and analyses were performed using ibm spss statistics version (ibm corp., armonk, ny, usa), stata version (stata corp lp, college station, tx, usa) or graphpad prism version . (graphpad software, la jolla, ca, usa). descriptive analysis was performed to characterize patients enrolled in the study and above described. median values and interquartile ranges (iqr) were used to describe numerical variables, while counts and percentages were employed for categorical variables. the analytical sensitivity (sars-cov- copy number and tcdi at a % viruses , , of detection proportion) was calculated by probit analysis, using the medcalc statistical software (medcalc software ltd., ostend, belgium), on the basis of results obtained by several replicates of serial dilutions of the -ncov/italy-inmi spiked into of matrix. the evaluation of the qualitative concordance between results was performed using the weighted cohen kappa statistics [ ] and its % confidence interval (ci); agreement was evaluated as: poor (less than . ), moderate ( . - . ), substantial ( . - . ), and almost perfect if greater than . . linear regression analysis, adjusted for gender and age, was used to evaluate the relationship between the two quantitative results. to account for the possible correlation, that may arise from multiple samples belonging to the same patient, robust standard errors were computed. this work can be considered exempt from continuous review by an institutional ethical review board, because it comprises secondary use of completely anonymized specimens. the analytical sensitivity (i.e., the limit of detection, lod, corresponding to the concentration of sars-cov- -rna detected with response probability of % for either s or orf ab) was determined by probit regression model, and resulted to be . (ci: . - . ) tcid /ml for s and . (ci: . - . ) tcid /ml for orf ab, corresponding to . (ci . - . ) logrna cp/ml and . (ci . - . ) logrna cp/ml, respectively ( figure ). analytical sensitivity was similar to that obtained for nps in a previous study from our group, using the same virus isolate and the same experimental methods, being . (ci: . - . ) tcid /ml for s and . (ci: . - . ) tcid /ml for orf ab corresponding to . (ci: . - . ) log cp/ml and . log (ci: . - . ) log cp/ml for s and orf ab, respectively (around cp/ml) [ ] . however, a similar analytical sensitivity ( cp/ml) was also described for nps in two additional assays widely used in sars-cov- molecular diagnosis: realstar ® sars-cov- rt-pcr kit . (altona diagnostics, hamburg, germany: https://www.fda.gov/media/ /download) and cdc covid- rt-pcr panel assay (idt, coralville, ia: https://www.fda.gov/media/ /download) [ ] . viruses , , x for peer review of software (medcalc software ltd., ostend, belgium), on the basis of results obtained by several replicates of serial dilutions of the -ncov/italy-inmi spiked into of matrix. the evaluation of the qualitative concordance between results was performed using the weighted cohen kappa statistics [ ] and its % confidence interval (ci); agreement was evaluated as: poor (less than . ), moderate ( . - . ), substantial ( . - . ), and almost perfect if greater than . . linear regression analysis, adjusted for gender and age, was used to evaluate the relationship between the two quantitative results. to account for the possible correlation, that may arise from multiple samples belonging to the same patient, robust standard errors were computed. this work can be considered exempt from continuous review by an institutional ethical review board, because it comprises secondary use of completely anonymized specimens. the analytical sensitivity (i.e., the limit of detection,lod, corresponding to the concentration of sars-cov- -rna detected with response probability of % for either s or orf ab) was determined by probit regression model, and resulted to be . (ci: . - . ) tcid /ml for s and . (ci: . - . ) tcid /ml for orf ab, corresponding to . (ci . - . ) logrna cp/ml and . (ci . - . ) logrna cp/ml, respectively ( figure ). analytical sensitivity was similar to that obtained for nps in a previous study from our group, using the same virus isolate and the same experimental methods, being . (ci: . - . ) tcid /ml for s and . (ci: . - . ) tcid /ml for orf ab corresponding to . (ci: . - . ) log cp/ml and . log (ci: . - . ) log cp/ml for s and orf ab, respectively (around cp/ml) [ ] . however, a similar analytical sensitivity ( cp/ml) was also described for nps in two additional assays widely used in sars-cov- molecular diagnosis: realstar ® sars-cov- rt-pcr kit . (altona diagnostics, hamburg, germany: https://www.fda.gov/media/ /download) and cdc covid- rt-pcr panel assay (idt, coralville, ia: https://www.fda.gov/media/ /download) [ ] . results obtained for of spiked with h-cov e, h-cov oc , adv, flua; flub and mv confirmed high specificity, and lack cross-reactivity with other viruses transmitted through the respiratory toute. results obtained for of spiked with h-cov e, h-cov oc , adv, flua; flub and mv confirmed high specificity, and lack cross-reactivity with other viruses transmitted through the respiratory toute. the first performance evaluation on clinical specimen was done by testing consecutive of samples, including samples from sars-cov- -negative patients, with the simplexa™ covid- direct assay and comparing results with that obtained using rt-pcr method established by corman vm. et al. [ ] as reference assay. analysis showed a substantial concordance in sars-cov- rna detection between the two assays (κ = . ; % ci = . - . ). of the samples, resulted positive to both tests, resulted positive only using simplexa and resulted negative to both assays. notably, the discordant results positive with simplexa™ covid- direct assay but negative with corman's method, came from patients with clinically confirmed covid- . the ct values obtained in the two assays show good correlation (r = . ; p < . ) in linear regression analysis, figure a . viruses , , x for peer review of detection between the two assays (κ = . ; % ci = . - . ). of the samples, resulted positive to both tests, resulted positive only using simplexa and resulted negative to both assays. notably, the discordant results positive with simplexa™ covid- direct assay but negative with corman's method, came from patients with clinically confirmed covid- . the ct values obtained in the two assays show good correlation (r = . ; p < . ) in linear regression analysis, figure a . the performance of simplexa™ covid- direct assays on clinical specimens was further established by testing in parallel nps and of samples for the presence of sars-cov- rna. concordance analyses performed on samples, showed a total of concordant and discordant results, with κ = . ; % ci = . - . . linear regression analysis adjusted for cluster of repeated measures, sex and age, performed on samples for which ct values were available for both matrices, showed elevated correlation of ct values among nps and of (r = . ; p < . )( figure b ). an even higher correlation was obtained excluding repeated measures and considering only first results from each patient (r = . and p < . ), thus confirming high correlation of ct values among nps and of. for samples we were able to analyse the presence of viral rna both in of ( figure a ) and nps ( figure b ) samples, considering the days from symptoms onset (dso). for asymptomatic individuals, dso has been calculated from the time of notification to the surveillance system. presence of rna in both matrices was observed during the first dso ( % of; % nps), remaining stable between and dso with similar frequency ( % of; % nps) and was still observed until dso ( % of; % nps). among the analysed samples, were from severe patients (red symbols) while the remaing samples were from paucisymptomatic and asympomatic patients (black symbols). moreover, statistical comparison between of and nps has been performed, showing no significant difference (p > . ) between median ct values in of and nps, neither in total nor according to different dso intervals (table ). the performance of simplexa™ covid- direct assays on clinical specimens was further established by testing in parallel nps and of samples for the presence of sars-cov- rna. concordance analyses performed on samples, showed a total of concordant and discordant results, with κ = . ; % ci = . - . . linear regression analysis adjusted for cluster of repeated measures, sex and age, performed on samples for which ct values were available for both matrices, showed elevated correlation of ct values among nps and of (r = . ; p < . )( figure b ). an even higher correlation was obtained excluding repeated measures and considering only first results from each patient (r = . and p < . ), thus confirming high correlation of ct values among nps and of. for samples we were able to analyse the presence of viral rna both in of ( figure a ) and nps ( figure b ) samples, considering the days from symptoms onset (dso). for asymptomatic individuals, dso has been calculated from the time of notification to the surveillance system. presence of rna in both matrices was observed during the first dso ( % of; % nps), remaining stable between and dso with similar frequency ( % of; % nps) and was still observed until dso ( % of; % nps). among the analysed samples, were from severe patients (red symbols) while the remaing samples were from paucisymptomatic and asympomatic patients (black symbols). moreover, statistical comparison between of and nps has been performed, showing no significant difference (p > . ) between median ct values in of and nps, neither in total nor according to different dso intervals (table ) . results obtained from statistical analysis confirmed a comparable trend in the two matrices of and nps, with ct median values lower in the first dso (corresponding to higher viral load) progressively increasing at and > dso, as expected. concerning gender, median ct values in both district were slightly higher in males (median ct in of: . ; median ct in nps: . ) than in females (median ct in of: , ; median ct in nps: . ), despite the differences were not significant. concerning age, ct values both in of and nps were significantly lower in patients < years (i.e., according to the median value of patient's age) ( table ) . the data obtained in individuals with repeated measures have been separately shown in figure a : of and figure b : nps confirming in both matrices the general trend to progressive decrease of viral rna concentration (i.e., increase of ct values) observed in figure and in table . results obtained from statistical analysis confirmed a comparable trend in the two matrices of and nps, with ct median values lower in the first dso (corresponding to higher viral load) progressively increasing at and > dso, as expected. concerning gender, median ct values in both district were slightly higher in males (median ct in of: . ; median ct in nps: . ) than in females (median ct in of: , ; median ct in nps: . ), despite the differences were not significant. concerning age, ct values both in of and nps were significantly lower in patients < years (i.e., according to the median value of patient's age) ( table ) . the data obtained in individuals with repeated measures have been separately shown in figure a : of and figure b : nps confirming in both matrices the general trend to progressive decrease of viral rna concentration (i.e., increase of ct values) observed in figure and in table . viruses , , x for peer review of analyses of a subgroup of severe patients for whom repeated parallel nps, of and bal samples were available ( figure ) showed % positivity (ct values < ) in all district during the first dso; % positivity in of, % in nps and % in bal between and dso and % of positivity in all matrices > dso ( figure a ,c,e). elevated concordance was observed for virus detection in the various matrices (nps vs of: κ = . , % ci= . - . ; bal vs of: κ = . , % ci= . - . ; of vs bal: κ = . , % ci= . - . ), and highly significant correlation between the ct values obtained on the three matrices (nps vs of: r = . , p < . ; bal vs of: r = . , p < . ; nps vs bal: r = . , p < . ) ( figure b,d,f) . statistical comparison between of, nps and bal has been performed, showing no significant difference between median ct values in the three district, neither in total nor according to different dso intervals (table ) . nevertheless, when considering the earlier time interval ( - dso), median ct values in balwere lower respect to nps and of, although the difference was not statistically significant (p > . ). analyses of a subgroup of severe patients for whom repeated parallel nps, of and bal samples were available ( figure ) showed % positivity (ct values < ) in all district during the first dso; % positivity in of, % in nps and % in bal between and dso and % of positivity in all matrices > dso ( figure a ,c,e). elevated concordance was observed for virus detection in the various matrices (nps vs. of: κ = . , % ci = . - . ; bal vs. of: κ = . , % ci = . - . ; of vs. bal: κ = . , % ci = . - . ), and highly significant correlation between the ct values obtained on the three matrices (nps vs. of: r = . , p < . ; bal vs. of: r = . , p < . ; nps vs. bal: r = . , p < . ) ( figure b,d,f) . statistical comparison between of, nps and bal has been performed, showing no significant difference between median ct values in the three district, neither in total nor according to different dso intervals (table ). nevertheless, when considering the earlier time interval ( - dso), median ct values in balwere lower respect to nps and of, although the difference was not statistically significant (p > . ). we re-analyzed data of figure a ,c,e only for individual repeated measures, in order to show interpersonal and intrapersonal variability ( figure ). we re-analyzed data of figure a ,c,e only for individual repeated measures, in order to show interpersonal and intrapersonal variability ( figure ). the data obtained in individuals with repeated measures in the three matrices have been separately shown in figure a : of; figure b : nps; figure c : bal confirming in both matrices the general trend to progressive decrease of viral rna concentration (i.e., increase of ct values) observed in figure and in table . table . there are three important findings from our study. first, our results indicate that simplexa™ covid- direct assay applied to of has high analytical sensitivity and specificity, similar to that observed for nps; in addition the rate of detection of sars-cov- in of by the simplexa™ covid- direct assay is similar to that of a standard test, based on corman's protocol, and ct values from both tests are highly correlated. second, results from testing on paired of, nps and bal samples by simplexa™ covid- direct assay showed almost perfect concordance for virus detection, and high correlation of ct there are three important findings from our study. first, our results indicate that simplexa™ covid- direct assay applied to of has high analytical sensitivity and specificity, similar to that observed for nps; in addition the rate of detection of sars-cov- in of by the simplexa™ covid- direct assay is similar to that of a standard test, based on corman's protocol, and ct values from both tests are highly correlated. second, results from testing on paired of, nps and bal samples by simplexa™ covid- direct assay showed almost perfect concordance for virus detection, and high correlation of ct values. third, this assay detected prolonged oral shedding of sars-cov- dso, which continued at least as long as nasopharyngeal shedding did. hence, from these results, if appears that the use of this commercial assay to detect sars-cov- rna in of is of potentially high clinical utility for diagnosis and virological monitoring purposes. key advantages of the simplexa™ covid- direct assay are simple operation procedures, with an all-in-one reagent mix and high-speed of detection in just over an hour, which is significantly faster than the up to seven hours required by traditional extraction followed by amplification technologies, currently used to detect sars-cov- rna in of samples [ ] [ ] [ ] [ ] [ ] [ ] ] . moreover, the test does not require extra-equipment (i.e centrifuges or an extraction system) and technical laboratory infrastructure, being suitable for the field settings and for near-to-patient diagnosis. the only limitation of the assay is the small number of samples which can be tested in a run, since each instrument can support a ring of maximum eight position. several recent studies have showed that of could be an appropriate sample for diagnosis of sars-cov- [ , ] . the meta-analysis by czumbel et al. on the reliability and consistency of sars-cov- viral rna detection in of specimens found % ( %ci = %- %) sensitivity for of tests and % ( %ci %- %) sensitivity for nps in previously confirmed covid- infected patients [ ] . diagnostic testing for sars-cov- rna detection in clinical specimens supports decision making for clinical, infection control or public health management. sars-cov- detection is essential for patient care, triage and isolation in healthcare facilities. of specimen offers an option for self-sampling, especially in situations where other specimens are difficult to obtain. sars-cov- rna detection in of can also be used for screening of close contacts for asymptomatic infection and disease as part of contact tracing or outbreak investigations, local surveillance programmes and for screening specific groups like healthcare and social workers. it could also be useful for early control of viral transmission to vulnerable persons living in closed institutions and long-term care facilities. apart from real-time use for medical or public health case management and transmission control, tests using of as a specimen for virus detection can be used to surveillance and determining incidence and prevalence of infection and disease. in a situation where nps or other above mentioned specimen is not acceptable, of could be considered a valuable alternative specimen. on th may, , the u.s. food and drug administration had authorized the first diagnostic test with the option of using home-collected of samples for covid- testing issuing an emergency use authorization (/media/ /download) (eua) to rutgers clinical genomics laboratory for their covid- laboratory developed test. the simplexa™ covid- direct assay on of to detect sars-cov- rna has high sensitivity, and provides an additional alternative for diagnosis and monitoring sars-cov- shedding. further evaluation of the simplexa™ covid- direct assay for home based self-use, surveillance purposes to monitor the epidemiologic situation in terms of incidence and prevalence of infection and disease in the community are required. author contributions: l.b.: designed the study, analysed data, wrote manuscript, performed laboratory testing; g.s.: assisted in designing the study, performed laboratory testing and analysed data; e.l.: assisted in designing the study, discuss results and read manuscript; p.p.: performed statistical analysis; f.c., c.c.: performed preparation of viral stock; g.m., e.d.: contributed to establish conditions to apply to the simplexa™ covid- direct assay; e.n., a.a., e.b., n.p., l.m.: provided clinical samples and discuss results; m.r.c.: analysed data, discuss results, read and revised manuscript; f.l., a.z., g.i.: discuss result, read and revised manuscript. all authors have read and agreed to the published version of the manuscript. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a novel coronavirus from patients with pneumonia in china clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in laboratory testing for coronavirus disease (covid- ) in suspected human cases. interim guidance sars-cov- ) discharge criteria for confirmed covid- cases e when is it safe to discharge covid- cases from the hospital or end home isolation? available online viral dynamics of sars-cov- in saliva from infected patients saliva sampling and its direct lysis, an excellent option to increase the number of sars cov diagnostic tests in settings with supply shortages saliva as a noninvasive specimen for detection of sars-cov- clinical evaluation of self-collected saliva by quantitative reverse transcription-pcr (rt-qpcr), direct rt-qpcr, reverse transcription-loop-mediated isothermal amplification, and a rapid antigen test to diagnose covid- comparison of sars-cov- detection in nasopharyngeal swab and saliva saliva is a reliable tool to detect sars-cov- saliva sample as a non-invasive specimen for the diagnosis of coronavirus disease : a cross-sectional study covid- pandemic and role of human saliva as a testing biofluid in point-of-care technology consistent detection of novel coronavirus in saliva temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study a case report of sars-cov- confirmed in saliva specimens up to days after onset: proposal of saliva specimens for covid- diagnosis and virus monitoring molecular characterization of sars-cov- from the first case of covid- in italy the measurement of observer agreement for categorical data rapid and sensitive detection of sars-cov- rna using the simplexa™ covid- direct assay comparing the analytical performance of three sars-cov- molecular diagnostic assays saliva as a candidate for covid- diagnostic testing: a meta-analysis key: cord- -q tk tq authors: baker, kate s.; murcia, pablo r. title: poxviruses in bats … so what? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: q tk tq poxviruses are important pathogens of man and numerous domestic and wild animal species. cross species (including zoonotic) poxvirus infections can have drastic consequences for the recipient host. bats are a diverse order of mammals known to carry lethal viral zoonoses such as rabies, hendra, nipah, and sars. consequent targeted research is revealing bats to be infected with a rich diversity of novel viruses. poxviruses were recently identified in bats and the settings in which they were found were dramatically different. here, we review the natural history of poxviruses in bats and highlight the relationship of the viruses to each other and their context in the poxviridae family. in addition to considering the zoonotic potential of these viruses, we reflect on the broader implications of these findings. specifically, the potential to explore and exploit this newfound relationship to study coevolution and cross species transmission together with fundamental aspects of poxvirus host tropism as well as bat virology and immunology. been identified in bat populations for which the zoonotic potential is unknown, including novel influenza types and hepadnaviruses [ , ] . as a result, there has been well-grounded speculation that owing perhaps to physiological, ecological, evolutionary, and/or immunological reasons, bats may have a "special" relationship with viruses [ , , ] and be particularly good viral reservoirs with exaggerated viral richness [ ] . indeed, a recent intensive study found that a single bat species likely carries ≥ different viral species from only nine viral families [ ] . as well as the obvious first step of considering the zoonotic potential of newly identified bat viruses, further exploring the impacts of these findings and the opportunities they present for multiple research fields is necessary to capitalize on these discoveries. poxvirus infections have recently been identified in bats, comprising part of the increase in viral families newly identified in this taxonomic order. here, we review the current evidence of poxvirus infections in bats, present the phylogenetic context of the viruses within the poxviridae, and consider their zoonotic potential. finally, we speculate on the possible consequences and potential research avenues opened following this marrying of a pathogen of great historical and contemporary importance with an ancient host that has an apparently peculiar relationship with viruses; a fascinating and likely fruitful meeting whose study will be facilitated by recent technological advances and a heightened interest in bat virology. there are three documented detections of poxviruses in bat populations under distinct circumstances (summarized in table ). the viruses were detected in animals from both bat suborders on three different continents. they had varied clinical impacts on their hosts and were phylogenetically dissimilar. genetic sequence of one bat poxvirus was detected at high prevalence during active surveillance on apparently-healthy african straw-colored fruit bats (eidolon helvum) [ ] . metagenomic analysis of pooled throat swabs collected from e. helvum in ghana in contained poxvirus sequences most closely related with molluscum contagiosum (mocv) a human-only pathogen ( figure ). detected sequences were distributed across the mocv genome and reconstructed sequences relating to viral genes were deposited in genbank as being derived from eidolon helvum poxvirus [ ] . retrospective analysis of throat swabs from individual bats revealed a high prevalence of this virus in the apparently healthy study population with % (n = / ) of swabs containing poxviral dna. notably, the detection of true poxvirus sequences in this metagenomic study, in which sequences related to multiple genes distributed throughout the genome were found and reconfirmed in individual throat swab samples, is distinct from the detection of poxvirus-like sequences described in other metagenomic studies performed on pooled bat feces, whose presence was ultimately attributed to the presence of other (non-pox) viruses or viral elements integrated into host genomes [ , ] . between and , a poxvirus associated with pathology (tenosynovitis and osteoarthritis) was detected in six adult big brown bats (eptescicus fuscus, a microbat) sampled at a wildlife center in the north western united states [ ] . the clinical illness of the bats was progressive and ultimately led to their euthanasia. histopathological examination of the joint lesions was indicative of poxvirus infection, which was confirmed by electron microscopy. the virus was successfully isolated on an african green monkey cell line (bsc ) and the genome was partially characterized (seven full protein coding sequences). phylogenetic analysis revealed that the novel eptesipox virus was most closely related with cotia virus, a virus detected in sentinel suckling mice in sao paulo, brazil in ( figure ) [ , ] . finally, a bat poxvirus was again detected in a clinical setting, in south australia in . the virus was identified as an incidental infection during investigation of an outbreak of parasitic skin disease in a population of southern bentwing bats (miniopterus schreibersii bassanii, a critically-endangered microbat species) [ ] . bats presented with white nodular skin lesions that contained encysted nematodes. however, in one of the twenty-one bats examined, an independent (non-nematode associated) lesion contained intracytoplasmic inclusion bodies indicative of poxvirus infection, which was confirmed with electron microscopy [ ] . no further confirmation or characterization of the virus was reported, and both the epidemiology and consequent conservation implications of poxviral disease for this species remain unknown. the three detections of poxviruses in bat populations are distinct and inherently incomplete stories with very few common threads; high-prevalence detection in throat swabs from apparently healthy african megabats, severe joint disease in several north american microbats and, negligible though comorbid skin disease in an endangered australasian microbat. further to their varied clinical impact, the partial genetic characterization of the former two viruses shows that these viruses are genetically diverse. the two viruses are most closely related with the very distinct poxviruses, molluscum contagiosum virus and cotia virus respectively (figure ), and although only partially genetically characterized, a small ( amino acids) region of overlap in their rap proteins has only % amino acid identity (please see table s in the supplementary files). that this is as far as these new viruses can be contrasted demonstrates the dearth of information currently available for further investigation of poxviruses in bats. the finding of poxviruses in bats is not unique among wildlife taxa (in fact it would have been more surprising had they not been found to carry poxviruses) and there is no reason to believe they would have greater zoonotic potential than other animal poxviruses. poxviruses with varying zoonotic potentials have been found in a broad range of wildlife taxa including hundreds of bird species, reptiles, marine mammals, macropods, marsupials, monotremes, ungulates, equids, and primates [ , , , [ ] [ ] [ ] [ ] and there is currently insufficient evidence available to determine what the zoonotic potential of bat poxviruses might be on this spectrum. for example, although eidolon helvum poxvirus is closely related to mocv, a human-only contagion, poxvirus-associated lesions mirroring mocv-disease have also been found in horses, donkeys and a red kangaroo [ ] [ ] [ ] . similarly, the discovery of eptesipox virus in north american brown bats is analogous to the discovery of the other north american poxviruses found in voles, skunks, raccoons and squirrels, which are also detected at high prevalence in their reservoir hosts [ , ] . notably however, in the initial eptesipox virus report, the authors comment that poxvirus infection manifesting as musculoskeletal disease (osteomyelitis) has also been reported in human varv and vaccinia virus (vacv) infections [ ] . however, given that no bat poxviruses identified to date are orthopoxviruses, and the little information available, it is clear that much more detail is needed before the potential threat of bat poxviruses to man can be commented on. notably however, the two hosts in which poxviruses have been identified are widely distributed across their respective continents (africa and north america) and both habit urban areas, so have ample opportunities for contact with potential spillover hosts (i.e., humans and domestic animal species). to determine the zoonotic risk posed by bat poxviruses there are, as for other novel viruses, a number of obvious and relatively straightforward investigations that can be done. full genomic characterization of these viruses to identify known and putative poxvirus host range genes (discussed further below) would be an obvious step. similarly, testing the in vitro host range of isolated viruses such as eptesipox virus would help inform whether human and further animal cell lines are permissive for infection (i.e., that they contain the necessary host factors to support infection and do not contain antiviral components that restrict infection). serological and clinical surveillance of human populations for poxvirus infections in geographical regions near detection sites, and/or overlapping with bat home ranges would be a direct approach that would provide samples useful for evaluating multiple candidate zoonoses. whether bat poxviruses pose a zoonotic threat will likely comprise part of the future research agenda as these investigations are prudent for the discovery of all novel viruses. however, our current knowledge on bat poxviruses does not allow us to make firm predictions about their ability to infect humans. irrespective of their potential role as zoonotic agents however, the study of poxviruses in bats opens unique avenues of highly relevant research for multiple research fields beyond the individual host-pathogen relationships. further field (in situ), in vitro and in silico studies could elucidate the possible coevolution, cross species infections and mechanisms of host range restriction of bat poxviruses, the implications of which are relevant for bat ecologists, virologists and emerging infectious disease specialists (including those with a specific interest in bats) alike. it is likely that comparative phylogenetics of bats and poxviruses would inform and deepen our understanding of origins and evolution of both elements. bats and poxviruses are diverse host and pathogen taxa respectively and given their . million years of likely co-existence [ ] , there is surely a vast amount of knowledge to be gained by studying the phylogenetic relationships between bats and poxviruses. further sampling of bat populations for poxviruses would undoubtedly dramatically expand the poxvirus phylogeny, as has occurred subsequent to the study of other viral taxa in bat populations [ ] [ ] [ ] [ ] [ ] [ ] [ ] . comparative phylogenetics of bats and their poxviruses could differentiate between ancient co-speciation, or a more recent introduction and dissemination, of poxviruses among bat species. the two thus far partially characterized bat poxviruses are quite distinct from each other and are both relatively basal (i.e., have older most recent common ancestors with other extant viruses) in the poxvirus phylogeny when compared with other mammalian-infecting poxviruses. it is possible that if evidence of coevolution between bats and poxviruses were present, as has been suggested for the north american poxviruses [ ] , this could inform the phylogenies of both bats and poxviruses which are complicated by convergent evolution and horizontal gene transfer respectively [ ] [ ] [ ] . in addition to allowing the study of co-evolution, such studies provide the context for the identification of cross-species infections. with concerted research effort to identify reservoir species of bat poxviruses and cross species infections of poxviruses in bats could be identified and would have important implications for both bat and zoonotic-disease specialists. continued serological and molecular studies of naturally infected bat populations would allow the clinical effect and ecological impact of cross species poxvirus infections in bats to be assessed. we already noted that poxvirus infections across species barriers can devastate wildlife populations (e.g., squirrelpox, see introduction), an effect so severe that it was used to control introduced rabbit species in australia in the s [ ] . white nose syndrome, a fungal pathogen causing massive die offs in north american bat populations, is an unfortunate contemporary example of the severe ecological impacts that emerging pathogens can have on bat populations [ , ] . hence, from an ecological perspective if a bat poxvirus, e.g., eptesipox virus with its severe disease manifestations, were an emerging cross-species infection it would be useful to identify this rapidly, especially in already endangered species as is the case of the southern bentwing bat in which a poxvirus was reported. further to the conservation implications of such research, combining data regarding cross species infection and ecological aspects of host taxa (e.g., behavior, habitat, range overlap, host relatedness) will likely inform key concepts of virus sharing among bat species, as has been done with lyssaviruses [ , ] . given the heightened interest in bat virology, further analysis of bat poxviral isolates from both within-and cross-species infections will allow for a deeper understanding of the extent and mechanisms of poxvirus host restriction. many bat cell lines have now been developed [ ] [ ] [ ] [ ] [ ] , and at least one of these allows productive poxvirus infection [ ] . such tools will allow the in vitro refinement of host range definitions beyond detection in the field. furthermore, full genome sequencing information of poxviruses (now a comparatively easy and cost effective task) would facilitate the in silico identification of poxvirus host range gene orthologues, as recently done by bratke and colleagues who performed a systematic survey for the presence of known poxviral host range genes on among chordopoxviruses [ ] . furthermore, applying new bioinformatics tools to genomic sequence information and host range data could facilitate the identification of novel host-range determinants, perhaps even unique to bat poxviruses [ , ] . in addition, with the aforementioned in vitro tools in place, hypothetical host range genes can be validated, advancing our fundamental knowledge of poxvirus host range restriction. finally, and most speculatively, the identification of genes involved in poxvirus host range restriction in bats may represent a unique opportunity to study bat immunology, which may have broader implications for their confirmed roles as zoonotic reservoirs. since genes that interplay with the host innate immune system, not those involved with cell entry, are typically responsible for host range determination in poxviruses [ , ] , the identification of bat-unique poxvirus host range genes could facilitate the cognate identification of (possibly novel) host immune factors. this is particularly important for bats as they potentially have antiviral immunity distinct from our own, which seemingly allows them to harbor numerous human pathogens viruses asymptomatically [ ] . some preliminary evidence of this distinction existing for poxviruses is that in the single described report of infection of bat cell lines with poxviruses, bat cells were found to behave very differently from other mammalian cell lines, being susceptible to a highly attenuated strain of vaccinia virus [ ] . with several bat genomes recently sequenced [ ] and the capabilities of newer proteomic approaches, it is realistic that novel non-orthologous innate immune factors of bats (if they exist) could be identified. that these novel immune factors might then be candidate therapeutics against a range of viral zoonoses for which bats are the natural reservoir is an exciting, if not fantastical, point to ponder. recent advances in the study of bats and their viruses as well as the current biotechnological revolution leave us in a position to explore 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characterisation of pteropid bat cell lines authentication of the r e fruit bat cell line establishment of cell line from embryonic tissue of pipistrellus ceylonicus bat species from india & its susceptibility to different viruses type i interferon reaction to viral infection in interferon-competent, immortalized cell lines from the african fruit bat eidolon helvum genome-wide association study identifies vitamin b biosynthesis as a host specificity factor in campylobacter comparative analysis of bat genomes provides insight into the evolution of flight and immunity the authors thank daniel streicker and gustavo delhon for their helpful comments on the manuscript. both authors reviewed the literature, wrote, and edited the manuscript. the authors declare no conflict of interest. key: cord- -eksl pp authors: sun, heting; li, yuanguo; jiao, weiyi; liu, cunfa; liu, xiujuan; wang, haijun; hua, fuyou; dong, jianxiu; fan, shengtao; yu, zhijun; gao, yuwei; xia, xianzhu title: isolation and identification of feline herpesvirus type from a south china tiger in china date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: eksl pp in , an fhv- -like virus was isolated from a tiger that presented with clinical signs of sialorrhea, sneezing and purulent rhinorrhea. isolation was performed with the fk cell line, and the virus was identified by pcr, transmission electron microscopy (tem), and the phylogenetic analysis of the partial thymidine kinase (tk) and glycoprotein b (gb) genes. a total of bp of the tk gene and bp of the gb gene were amplified from the trachea of the tiger by pcr/rt-pcr method. phylogenetic analysis showed that the isolate belonged to the same cluster with other fhv- strains obtained from genbank. herpes-like viruses with an envelope and diameters of approximately nm were observed in the culture supernatants of fk cells inoculated with samples from the tiger. the fhv- infection was confirmed by an animal challenge experiment in a cat model. our finding extends the host range of fhv- and has implications for fhv- infection and south china tiger conservation. feline herpesvirus type (fhv- ; felid herpesvirus (fehv- ), family herpesviridae, subfamily alphaherpesvirinae, genus varicellovirus) is an important pathogenic agent that causes feline viral rhinotracheitis, which is a highly infectious upper respiratory tract infection of felids [ ] . this infection is often fatal to kittens, but adult cats usually survive and exhibit lifelong latency [ , ] . since the first strain of fhv- was isolated in america, infected felids have been reported in many countries, including canada, switzerland, the united kingdom, holland, hungary and japan [ ] . there have been no documented reports on fhv- in the past few years, although the distribution in china was confirmed by serological survey and virus isolation in domestic cats [ ] . the south china tiger (panthera tigris amoyensis) is a tiger unique to china and is the most endangered tiger subspecies, as free ranging individuals have not been found in its historic distribution areas for many years [ ] . to aid in the recovery of wild populations, it is common to re-introduce captive individuals into their native range; however, there are fewer than captive south china tigers in china. furthermore, only a few captive tigers are suitable for reintroduction, and infectious diseases threaten captive tigers. any sign of disturbance or trouble with the captive populations, in particular any risk of infectious disease, will greatly concern the stakeholders. in june , a south china tiger in shenzhen wildlife zoo presented with sneezing, purulent rhinorrhea, which ended with its death, although treatment including antibiotics had been tried. in the present study, we used molecular methods, virus isolation, tem examination and an animal challenge experiment to diagnose the cause of death of the south china tiger, and for the first time, we confirmed the infection with fhv- in the captive tiger population in china. the age (agarose gel electrophoresis) results showed that a target fragment of bp in length was amplified by pcr/rt-pcr, from dna/rna extracted from trachea samples of the dead tiger [ ] . as indicated, the tested specimens were positive for fhv- but negative for other tested pathogens, including canine/feline distemper virus (cdv/fedv) and feline calicivirus (fcv). benefitting from the clinical diagnosis, the authors were able to narrow the range of the laboratory examinations, and, based on the positive result, the subsequent isolation and identification methods focused on fhv- . the glycoprotein b (gb) gene and thymidine kinase (tk) gene have been selected for the study of molecular phylogeny [ , ] . a bp sequence was obtained, and alignment analysis determined that the tk gene cloned in this study shared a high identity (from % to %) with that of other fhv- isolates (figure ) . a bp fragment of the gb (glycoprotein b) gene was also cloned, and was found to share % identity with that of other fhv- isolates. therefore, its phylogenetic tree was omitted here. the two sequences have been deposited in genbank whose accession numbers are ** and **, for tk gene and gb gene fragment separately. the phylogenetic tree based on the tk gene sequences showed that the isolate investigated in this study, was closely related to the ten isolates of fhv- ( figure ), a result consistent with the alignment analysis. all isolates of fehv- appear to be relatively similar, as they antigenically belong to one serotype [ ] . the tk gene is a conserved gene, and the target fragment is located in its highly conserved region. therefore, the above results are strong evidence for the presence of fhv- . phylogenetic tree based on the nucleotide sequences of the tk gene. the tree was constructed with mega [ ] . the isolate of this study is indicated by a solid triangle. figure a ,b). subsequently, their nostrils were completely blocked with purulent secretions resulting from thin nasal discharge ( figure c ). after the th day, the cats recovered completely and exhibited no signs of infection. similar signs and similar clinical courses were observed in the cats in the eg (exposed group) and in the cat of the control group, but the initial clinical signs manifested on the th day and the th day post-inoculation, respectively. the changes in the cat's body temperatures generated a curve with a single peak. for the ig group cats, pyrexia began at day post-inoculation and continued for days, with a peak of . °c . for the eg group and control group cats, fever was detected on the th day and the th day, respectively ( figure ). figure . temperature changes of cats infected with fhv- . for the ig group cats, positive results appeared on the th day post-inoculation. pcr was used to detect fhv- dna extracted from the specimens, which included nose washing fluid, eye swabs and swallow swabs. positive results were found on the th day and th day for the eg group cats and the control cat, respectively (table ) . these results revealed a baseline of virus shedding in the infected cats after they were inoculated or were exposed to fhv- . the incubation period of fhv- infection as indicated by this challenge test is approximately days, which is consistent with that reported previously [ ] . additionally, clinical signs, including pyrexia, inappetence, and serous ocular and nasal discharge, were observed in the cats [ ] , and a long period of virus shedding was observed [ ] . although most fhv- strains produce a relatively uniform disease seen primarily in the respiratory tract, pancreatitis and generalized disease may be seen occasionally in debilitated animals or in neonatal kittens [ ] [ ] [ ] . the fhv- described in this study, was isolated from a captive tiger that exhibited respiratory signs that were suspected to be due to rhinotracheitis. by pcr/rt-pcr, the only virus detected in the trachea homogenates was fhv- , which was confirmed afterwards by virus isolation, the tem examination of cell cultures showing cpe, and a challenge experiment in cats. furthermore, the full genome of the virus isolated in cell culture is being sequenced, and the obtained sequences are %- % homologous with that of pcr products either for tk gene or for gb gene. challenged cats exhibited uniform clinical symptoms and shed fhv- virus. other agents causing respiratory disease were excluded as possible pathogens. the tiger was born in shenzhen wildlife zoo, and had chances to contact stray cats and other felids during its lifetime. on its infection, the authors supposed two possible sources. one was that the tiger got the virus in captivity perhaps due to stray cats in the zoo, because fhv- was shed in ocular, nasal, and oral secretions, and transmission was primarily by direct contact with an infected cat. the other was an endogenous infection originating from a latent viral infection. as with other alphaherpesviruses, latency is common, and periodic viral reactivation is sometimes associated with the reoccurrence of clinical signs [ ] . to validate the first supposition, a retrospective investigation was conducted after the virus isolation and identification. although negative pcr results were found for the swab samples collected from stray cats and captive leopards and tigers, the contagious source could not be easily obviated. it is very much regrettable that neither prior sera nor post mortem sera were stored enabling the seroconversion analysis, making our second supposition to be theoretic. fhv- is relatively fragile in the environment and is highly susceptible to the effects of common disinfectants [ ] . even though negative pcr results of the retrospective survey indicated that widespread infection was unlikely to occur in the zoo, serological data of felid animals would be obtained in the next research to assess the epidemic risk for tigers better and more precisely. currently available evidence indicates that the host range of fhv- includes several members of the felid such as cheetahs [ ] , lions [ ] , and wild and domestic cats [ ] , but to our knowledge, fhv- infection has not been reported in tigers previously. this report describes the first occurrence of fhv- in a south china tiger in china and extends the species range, confirming that this virus poses a risk to this species. because of the clinical signs, the tiger was initially diagnosed with rhinotracheitis, and only trachea samples were obtained for laboratory examination after it died. the viral pathogen of felidae include canine/feline distemper virus (cdv/fedv), feline calicivirus (fcv) and fhv- , we first considered these three agents. polymerase chain reaction (pcr) and reverse transcription polymerase chain reaction (rt-pcr) were used to detect these viruses. the pcr/rt-pcr results indicated the presence of fhv- . the weighted trachea specimen was ground (parameter: tps) into a homogenate with a beveller (type: qiagen, spoorstraat, netherlands), and then the products were ground for minutes (parameter: tps) again, to which serum-free mem was added. the supernatant obtained from a -minute centrifugation (at °c and rpm) was fractionated into two centrifuge tubes for later tests. viral genomic rna and dna were extracted from the supernatant obtained in section . using a multisource genomic dna/rna miniprep kit (axygen, hangzhou, china), subjected to rt-pcr or pcr and screened for the three potential pathogens ( table ) . the target fragments observed by agarose gel electrophoresis (age) were extracted by a dna gel extraction kit (axygen, hangzhou, china), and were cloned into a pgem-t easy vector with routine methods [ ] . the sequencing of the cloned plasmid was performed by bgi sequencing, and the results were submitted to genbank for alignment analysis. the phylogenetic tree was constructed using mega- software [ ] . briefly, fk- cell monolayers (from toronto university of canada and stored in this lab) in costar flasks were inoculated with ml supernatant (obtained in section . ) treated with antibiotics at final concentration u/ml. after gentle rotation, the flasks were incubated for h at °c in % co to allow for attachment; then, the supernatants were removed, and the monolayers were washed three times with mem without fbs. after washing, ml dmem with % fbs was added, and the flasks were then incubated for days at °c in % co . if the cells did not exhibit a cytopathic effect (cpe) by the fourth day, the incubated monolayers were subjected to another two passages, and the culture supernatant ( μl) was collected for pcr/rt-pcr. the monolayers were not discarded, unless the pcr/rt-pcr results were negative and no cpe was observed in the third fk- passage. if % of cells exhibited cpe and the pcr/rt-pcr results were positive, the cells were frozen for further analysis. the cultured cells were harvested and fixed in . % glutaraldehyde for transmission electron microscopy (tem) examination (jem- , tokyo, japan). five healthy domestic cats free of fhv- and fhv- antibodies, aged months old and weighing from . kg to . kg, were divided into three groups. because the transmission of fhv- is largely by direct contact with an infected animal, the cage for cats no. and no. , which made up the ig (inoculated group), was placed at the bottom, the cage for cats no. and no. , which made up the eg (exposure group), was placed in the middle, and the cage for cat no. , the control cat, was placed at the top. under anesthesia, the two cats of the ig were inoculated by intranasal and ocular routes with . ml sample/cat containing tcid of the sz strain, and the other cats were mock-inoculated with . ml pbs/cat. each cat's temperature was taken, and clinical signs were observed daily. from the fourth day post-inoculation, nose, eye and throat secretions were collected for pcr to detect fhv- infection. in this study, the authors described the first occurrence of feline herpesvirus type (fhv- ) in a south china tiger in china. the tk gene sequences of the fhv- isolate were highly similar to those of other strains. challenge experiments found that the isolate caused typical clinical signs, a long period of virus shedding and efficient transmission in cats. the study expands the species range for fhv- infection and provides new epidemiologic data. immunogenic proteins of feline rhinotracheitis virus a review of feline viral rhinotracheitis (feline herpesvirus i infection) association of bartonella species, feline calicivirus, and feline herpesvirus infection with gingivostomatitis in cats isolation and identification of feline herpesvirus type dramatic decline of wild south china tigers panthera tigris amoyensis: field survey of priority tiger reserves detection of feline calicivirus, feline herpesvirus and chlamydia psittaci mucosal swabs by multiplex rt-pcr/pcr molecular phylogenetic analysis of felid herpesvirus characterization of the feline herpesvirus genome and molecular epidemiology of isolates from natural outbreaks and latent infections mega- , molecular evolutionary genetics analysis version . the dose response of cats to experimental infection with feline viral rhinotracheitis virus experimental feline viral rhinotracheitis in the germfree cat experimental induction of feline viral rhinotracheitis (fvr) virus re-excretion in fvr-recovered cats fatal generalized feline viral rhinotracheitis in a young adult cat the association of a herpesvirus with generalized disease in a kitten pancreatitis associated with a feline herpesvirus infection feline infectious respiratory disease virucidal efficacy of four new disinfectants infectious disease surveillance in captive and free-living cheetahs-an integral part of the species survival plan prevalence of antibodies to feline parvovirus, calicivirus, herpesvirus, coronavirus, and immunodeficiency virus and of feline leukemia virus antigen and the interrelationship of these viral infections in free-ranging lions in east africa feline viruses in wild cats from scotland a novel nested pcr for the diagnosis of calicivirus infections in the cat detection of canine distemper virus from samples of dead animals by rt-pcr human mitochondrial thioredoxin reductase we thank aje journal experts for editing the manuscript. this work was supported by the national key technologies r&d program (grant no. the authors declare no conflict of interest. key: cord- - pf nsw authors: harwig, alex; landick, robert; berkhout, ben title: the battle of rna synthesis: virus versus host date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: pf nsw transcription control is the foundation of gene regulation. whereas a cell is fully equipped for this task, viruses often depend on the host to supply tools for their transcription program. over the course of evolution and adaptation, viruses have found diverse ways to optimally exploit cellular host processes such as transcription to their own benefit. just as cells are increasingly understood to employ nascent rnas in transcription regulation, recent discoveries are revealing how viruses use nascent rnas to benefit their own gene expression. in this review, we first outline the two different transcription programs used by viruses, i.e., transcription (dna-dependent) and rna-dependent rna synthesis. subsequently, we use the distinct stages (initiation, elongation, termination) to describe the latest insights into nascent rna-mediated regulation in the context of each relevant stage. all living organisms are fully equipped to synthesize messenger rna (mrna) using their endogenously encoded multi-subunit rna polymerases (rnaps). however, many viruses form an exception to this dogma and employ a parasitic lifestyle that exploit parts of the host transcription pathways. this co-option allows viruses to dramatically downsize their genomes to the minimum number of genes essential for successful infection. as a result, viruses and their hosts have been involved in an eternal battle of adaptation and counter-adaptation for rna synthesis since far before the emergence of humans [ ] [ ] [ ] . during this time, viruses coevolved many pathways to transcribe their own genetic material, meanwhile avoiding counter-adaptations of the host [ ] . weapons in the arsenal of the virus include virally-encoded proteins and micro rnas (mirnas), which alter transcription pathways in both host and virus via many different mechanisms. these mechanisms have been reviewed extensively elsewhere (for example [ ] [ ] [ ] [ ] ). however, one major, yet underappreciated, component of viral gene regulation occurs via nascent rnas. rnas have many attractive properties that can be used by viruses to manipulate gene regulation pathways. first, rnas, like dnas, can specifically pair with complementary viral or host sequences using minimal investment of genetic material, thus allowing further minimization of the viral genome. encoding a dna-binding protein, on the other hand, consumes significantly more genetic information. a typical dna-binding domain recognizes~ base pairs (bp) or more of dna and comprises at least amino acids (aa), thereby requiring bp of encoding dna [ ] . in contrast, rna requires only the number of complementary nucleotides as the dna/rna region it recognizes. this can be beneficial for the virus where space constraints are dictated by the size of the viral capsid. second, rnas are less immunogenic compared to proteins. whereas hosts developed many pathways to respond to modules that make up the rna-exit channel are depicted in pink and blue, respectively. the first repeat of the rnapii c-terminal domain (ctd) is shown as a string of amino acids with their respective numbers. next, the stages of the transcription cycle are shown, with the viral interfering mechanisms. first, general transcription factors assemble at the promoter and direct rnapii towards the transcription start site. transcription factor (tf) iih phosphorylates the rnapii at ser (black asterisk). tfiih opens the dna template, forming the transcription bubble, permitting rnapii to begin transcribing rna in its active center (yellow dot). upon transcribing the first - nucleotides, most rnap will pause. transcription factor recruitment regulates these transcription kinetics and phosphorylate rnapii at ser . when the correct signal is encountered, rnapii will terminate transcription and release the rna. some viruses encode endonucleases (yellow sphere with scissor) that can cleave rnas, causing rna degradation. initiation is promoted by factors that recruit rnap to the promoter, melt the dna duplex and load the template dna strand into the active site of rnap. for rnapii, these steps require the combined action of basal transcription factors (tf) (figure ) [ , ] . the minimal pre-initiation complex (pic) includes rnapii and six general transcription factors: tfiia, tfiib, tfiid, tfiie, tfiif, and tfiih. first, tfiid containing the tata-binding protein (tbp) is recruited to tata box containing promoter sequences ( figure ) [ ] [ ] [ ] . the binding of tfiid to the tata box in the promoter region of the gene initiates the recruitment of tfiib, tfiie, tfiif and tfiih, which position the double-stranded (ds) dna above the cleft within rnapii [ ] [ ] [ ] . local unwinding of the dsdna by the ssl helicase subunit of tfiih delivers the template strand into the rnapii active center and creates a dna bubble downstream of the tata box [ ] . efficient dna opening requires tfiie and tfiih, but these factors are not required for low levels of transcription [ ] . the tfiik kinase module of tfiih will subsequently phosphorylate rnapii on the ser of its ctd repeats, facilitating promoter clearance and starting the elongation stage [ ] . whereas dna viruses only need to generate mrna, rna viruses without a dna stage have to synthesize both vrna and mrna. the vrna is generated through a replication intermediate, named antigenome which serves as a template for vrna synthesis. ssrna viruses evolved modules that make up the rna-exit channel are depicted in pink and blue, respectively. the first repeat of the rnapii c-terminal domain (ctd) is shown as a string of amino acids with their respective numbers. next, the stages of the transcription cycle are shown, with the viral interfering mechanisms. first, general transcription factors assemble at the promoter and direct rnapii towards the transcription start site. transcription factor (tf) iih phosphorylates the rnapii at ser (black asterisk). tfiih opens the dna template, forming the transcription bubble, permitting rnapii to begin transcribing rna in its active center (yellow dot). upon transcribing the first - nucleotides, most rnap will pause. transcription factor recruitment regulates these transcription kinetics and phosphorylate rnapii at ser . when the correct signal is encountered, rnapii will terminate transcription and release the rna. some viruses encode endonucleases (yellow sphere with scissor) that can cleave rnas, causing rna degradation. initiation is promoted by factors that recruit rnap to the promoter, melt the dna duplex and load the template dna strand into the active site of rnap. for rnapii, these steps require the combined action of basal transcription factors (tf) (figure ) [ , ] . the minimal pre-initiation complex (pic) includes rnapii and six general transcription factors: tfiia, tfiib, tfiid, tfiie, tfiif, and tfiih. first, tfiid containing the tata-binding protein (tbp) is recruited to tata box containing promoter sequences ( figure ) [ ] [ ] [ ] . the binding of tfiid to the tata box in the promoter region of the gene initiates the recruitment of tfiib, tfiie, tfiif and tfiih, which position the double-stranded (ds) dna above the cleft within rnapii [ ] [ ] [ ] . local unwinding of the dsdna by the ssl helicase subunit of tfiih delivers the template strand into the rnapii active center and creates a dna bubble downstream of the tata box [ ] . efficient dna opening requires tfiie and tfiih, but these factors are not required for low levels of transcription [ ] . the tfiik kinase module of tfiih will subsequently phosphorylate rnapii on the ser of its ctd repeats, facilitating promoter clearance and starting the elongation stage [ ] . whereas dna viruses only need to generate mrna, rna viruses without a dna stage have to synthesize both vrna and mrna. the vrna is generated through a replication intermediate, named antigenome which serves as a template for vrna synthesis. ssrna viruses evolved sophisticated approaches to generate both rna species from one template. using respiratory syncytial virus (rsv) as a model, it was illustrated that rdrp has two rna synthesis activities which determine nascent rna fate. rsv is a −ssrna virus and the major cause of respiratory tract disease in infants and young children, causing between , and , deaths per year worldwide [ ] . to differentiate between rd-mrna synthesis and vrna replication, rdrp initiates at one of two different start sites within the promoter sequences at the end of the rsv rna genome. synthesis of the antigenome starts at the expected + site. however, the more abundant rna species in infected cells was found to initiate at the + site [ ] . rdrp's that initiate at the + site are unable to enter a stable elongation mode and release the rna after approximately - nucleotides. many de novo transcription initiation events by rdrp are known to generate abortive transcripts during the initiation phase of rna synthesis [ ] [ ] [ ] . however, the function of this small rna is yet unknown. it has been proposed that synthesis of this small abortive rna allows the polymerase to break contacts with the promoter [ ] . after releasing the small rna, the rsv polymerase can now scan the template and locate the conserved gene start signal (~ - nt) of the first gene, however the mechanism behind this transition is unknown [ ] . here the polymerase will reinitiate rna synthesis, cap the end of the mrna and commit the rdrp to rd-mrna synthesis. the choice between the + and + initiation site by rsv rdrp seems to be determined by the loading of a specific nucleotide and subsequent creation of a dinucleotide primer complementary to the specific initiation site [ ] . remarkably, even when the priming site is mutated or absent, the enzyme is able to selectively synthesize the original dinucleotide priming site at the end of the viral rna, explaining the strict conservation of the -and -end dinucleotides of both +ss and −ssrna viruses [ ] . it has been proposed that a domain, unique in de novo initiating rdrp's, named "priming loop" (t to a ) regulates the accessibility of the active site of rdrp [ ] . an essential role during initiation is played by the residue h of the priming loop, by providing a platform to which the base of the priming atp could establish a stacking interaction [ ] . these conservational mechanisms, mediated by the polymerase alone have been suggested to be used by several ssrna viruses [ , ] . -terminal capping of eukaryotic mrnas is probably the first co-transcriptional rna processing event. formation of the -terminal cap structure (m gppp[ ]n-; where n is the first nt of the nascent rna; figure a ) in eukaryotic mrnas, in which -methylguanosine (m g) is linked to the initiator nucleoside of mrna through the - triphosphate bridge, is mediated by a series of enzymatic steps and is required for the efficient translation and stability of mrnas. eukaryotes and some dna viruses (e.g., vaccinia virus) and dsrna viruses (e.g., reovirus) obtain this cap by processing the -triphosphate end (pppn-) of nascent rna transcripts into a diphosphate (ppn-) using an rna -triphosphatase (rtpase) ( figure b ). subsequently, the guanosine monophosphate (gmp) moiety of a guanosine triphosphate (gtp) is transferred to the diphosphate by a gtp:rna guanylyltransferase (gtase) to generate the cap core structure (gp-ppn-). this core structure is then methylated at the guanine- n position by s-adenosyl-l-methionine (adomet)-dependent cap (guanine-n ) methyltransferase (mtase) to produce the final cap structure (m gpppn-). higher eukaryotes and some viruses include an additional methylation step in which the oh position of the penultimate ribose is methylated by cap ribose- -o-mtase (m gpppnm-). however, ssrna viruses (e.g., influenza, ebola, measles) do not have a dna template and thus cannot rely on the cellular capping enzymes. for this reason, rna viruses have evolved capping mechanisms that are different from capping during cellular transcription. most +ssrna viruses (e.g., flaviviruses and nidoviruses) encode capping enzymes that selectively cap the newly synthesized viral mrnas [ ] [ ] [ ] . for instance, the flavivirus-encoded nonstructural protein (ns ) contains a triphosphatase that releases the terminal phosphate from the -triphosphate end of nascent + stranded rna, forming a diphosphorylated ppn-rna [ ] . subsequently, a gmp moiety from gtp is transferred to the end of the ppn-rna through the gtase activity of the viral ns mtase. the gpppa-capped rna is further methylated by the ns mtase to form the final m( )gpppa-and m( )gpppnm-rna products [ , ] . however, both the process of discrimination between −rna and +rna -ends for capping and the precise timing of the capping process remain largely undefined [ ] . on the other hand, unsegmented −ssrna viruses (including rabies, measles, and ebola) carry an rdrp termed "l" and a phosphoprotein "p", which share extensive amino acid sequence similarity among all these viruses [ ] . p is an essential co-factor that targets the l polymerase to the vrna [ ] [ ] [ ] . here, the l polymerase covalently links with the -monophosphorylated viral mrna-start sequence to form an intermediate complex ( figure c ) [ ] . subsequently, the rna:gdp polyribonucleotidyltransferase activity of the l polymerase transfers the gdp moiety of gtp to the monophosphorylated viral mrna forming the viral mrna cap. thus, in contrast to gtases which transfer a monophosphate to the diphosphate end of rna, the l polymerase-mediated capping reaction transfers a diphosphate to the monophosphate end of rna. another solution to the capping problem of the negative-sense rna viruses comes from the influenza virus in the form of "cap-snatching" from nascent host pre-mrnas [ ] . to synthesize viral mrna and vrna from each of the eight single-stranded viral rna segments, influenza uses a virally encoded heterotrimeric rdrp called flupol ( figure d ). flupol consists of three virally encoded subunits: polymerase basic (pb ), pb , and polymerase acidic (pa) arranged in a globular structure [ ] [ ] [ ] . each genome segment is circularized by the base-pairing of the near complementary and ends, and individually packaged into viral ribonucleoprotein particles (vrnps) together with one copy of flupol. flupol is bound to the duplex at the point of circularization, tightly docked into the end of the vrna by a pocket formed between the pa and pb subunits, creating an intramolecular stem-loop ('hook'; figure a ) [ , ] . the segment of the negative-sense vrna enters the active-site cavity of flupol, where it serves as a template ( figure e ) [ , ] . after entry into the cell, the vrnps are released and transported into the nucleus. after arrival in the nucleus, the c-terminal domain of the flupol pa subunit will specifically target and bind the phosphorylated ser of actively transcribing rnapii ctd [ ] ( figure e ). subsequently, the pb cap-binding domain associates with the end of the cellular mrna [ ] . the pa endonuclease domain next cleaves the mrna. this creates a capped - nt small rna primer that is loaded into the active site of flupol [ ] [ ] [ ] (figure e ). processive rd-mrna synthesis is initiated by the addition of an ntp to the end of the capped primer, complementary to the second or third residue in the vrna template. during elongation, the -capped viral mrna is separated from the vrna template by threading into their respective exit channels. after elongation, the viral mrna is polyadenylated at an oligo-u stretch near the vrna end [ ] . the capped viral mrna can now be used to translate all the necessary viral proteins, whereas the vrna can be packaged, re-used as template, or degraded. small host non-coding rnas (snrnas), especially u and u snrna, are the preferred targets for cap-snatching, rather than mrnas or pre-mrnas [ ] [ ] [ ] . why the virus prefers to use these snrnas as targets has yet to be experimentally established, but it has been proposed that the selective de-capping of u and u rnas in combination with the binding of the viral ns protein to u snrna may serve to inhibit host pre-mrna splicing [ ] . this will allow the pb -cap domain to bind the cap and to direct the nascent rna towards pa-endo. after cleavage the nascent rna will be loaded into the active domain of flupol where it will be used to prime the ′ end of the vrna, allowing elongation of the rna primer from ′ to ′ direction (indicated with black arrow). elongation is the repeated addition of a nucleoside monophosphate (nmp) to the ′ end of the growing rna chain. progressive rna synthesis during the elongation stage can be affected by several hurdles, including transcriptional pause sites, that rnapii must overcome [ ] . these purple) is circularized with the extreme end docked in a pocket formed by pa-c and pb and the end loaded in the flupol active center (yellow star); (e) the first step in cap-snatching is the recognition and binding of flupol to the phosphorylated ser of rnapii through its pa-c domain. this will allow the pb -cap domain to bind the cap and to direct the nascent rna towards pa-endo. after cleavage the nascent rna will be loaded into the active domain of flupol where it will be used to prime the end of the vrna, allowing elongation of the rna primer from to direction (indicated with black arrow). elongation is the repeated addition of a nucleoside monophosphate (nmp) to the end of the growing rna chain. progressive rna synthesis during the elongation stage can be affected by several hurdles, including transcriptional pause sites, that rnapii must overcome [ ] . these transcriptional pauses can be used to create a time window for co-transcriptional events such as splicing, nucleotide modification, or rna processing. pauses also yield opportunities to regulate transcription in response to intra-and extra-cellular cues [ ] . at most eukaryotic promoters, major transcriptional pauses typically occur within - nt after transcription initiation [ ] . these promoter-proximal pauses are induced by a myriad of factors, including negative elongation factor (nelf) and nascent rna structures. these pauses give opportunities for the organism to regulate transcription at an early time point. a promoter-proximal pause will often allow the nascent rna to fold a structure that can serve as a protein-recruiting scaffold [ ] . additional transcription factors (tfs) can be recruited during this step. three distinct modes of action can be envisaged whereby tf-interacting rnas might affect transcription. they could act as (i) scaffolds to recruit one or more tfs [ ] [ ] [ ] ; (ii) competitors, regulating the binding or release of a tf [ ] ; or (iii) transacting guides for interacting tfs by base-pairing with dna or rna in the vicinity of their target sites [ ] . the best-studied case of a tf-recruiting rna scaffold formed by promoter-proximal pausing is the human immunodeficiency virus type (hiv- ) trans-acting region (tar; figure a ) [ ] . as a retrovirus, hiv- contains an rna genome that is reverse transcribed into dna upon entry of the host cell. this dna integrates into the host genome as a provirus where it can be actively transcribed or enter a latent state. as in all retroviruses, the located long terminal repeat will act as the viral promoter. the viral promoter can be subdivided into the enhancer and core regions. the enhancer region contains binding sites for nf-κb [ , ] , nuclear factor of activated t-cells (nfat) [ ] , activator protein (ap ) [ , ] and variable other tfs dependent on the hiv- subtype [ ] . this enhancer upregulates hiv- transcription, but removal of its binding sites is not deleterious in certain cell lines [ ] . in contrast, the core promoter is essential for transcription and contains three binding sites for sp [ ] and a tata box (catataa) element [ ] . rnapii, phosphorylated at ser by tfiih, will initiate transcription from this core region [ , ] (figure b ). however, most rnapii complexes will pause after transcribing -nt, yielding short transcripts [ , ] . this is caused by the combined action of nelfs/ , -dichloro- -β-d-ribofuranosylbenzimidazole (drb) sensitivity inducing factor (dsif) [ , ] , the local rna structure [ , ] , microprocessor [ ] and sequences within template dna that promote transcriptional pausing [ ] . the position of the promoter-proximal paused rnapii on the end of the short transcript allows the nascent tar transcript to fold into an initial, semi-stable rna structure [ ] (figure b ; paused tar). this nascent hairpin allows the initial rnapii complex to backtrack [ ] , such that the end of the nascent transcript is displaced from the rnapii catalytic center [ ] (figure b ). as a result, rnapii is paused until it is reactivated, which may include refolding into the most stable tar configuration. the folding of the tar hairpin is key to the regulation of hiv- transcription [ ] as it is used as a scaffold to recruit essential transcription factors, including the - amino acid (aa) viral trans-activator protein (tat) [ ] ( figure c ). the interaction between tat and tar is necessary to recruit positive transcription elongation factor b (p-tefb) from the sk small nuclear ribonucleoprotein (snrnp) [ ] . p-tefb consists of cyclint (cyct ; figure c ) tightly associated with cdk kinase and is sequestered in an inactive form by the sk snrnp. tat recruits p-tefb by outcompeting the sk snrnp in several ways. first, the tar-binding arginine-rich motif (arm) region of tat ( figure c ; aa [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] shows high similarity to the sk-binding of sk snrnp component hexamethylene bisacetamide-induced protein (hexim ) [ ] . second, the hexim -bound sk stem-loop (sl ) is highly similar to the consensus minimal structure of the hiv- tar element [ ] , and third, both tat and hexim bind cyct in a mutually exclusive manner due to the shared cyct binding site [ , ] . however, the tat activation domain (figure c ; ad; aa - ) has a -fold higher affinity for cyct [ ] . tat will thus bind to sk and cyct by displacing hexim (figure b ) [ ] . as soon as tar forms in the nascent viral transcript emerging from rnapii, the tat:p-tefb complex will bind to tar, resulting in the release of the inhibitory sk snrnp. this activates p-tefb, allowing it to phosphorylate nelf, dsif, and ser on the rnapii ctd, thus flipping the switch between rnapii pausing and elongation [ ] . interestingly, the human t-lymphotropic virus type transcriptional activator tax also utilizes p-tefb for viral transcription and displaces p-tefb from sk snrnp through binding cyct [ , ] , suggesting that p-tefb liberation from sk snrnp could be a common theme developed by different viruses to support their replication in host cells. viruses , , of phosphorylate nelf, dsif, and ser on the rnapii ctd, thus flipping the switch between rnapii pausing and elongation [ ] . interestingly, the human t-lymphotropic virus type transcriptional activator tax also utilizes p-tefb for viral transcription and displaces p-tefb from sk snrnp through binding cyct [ , ] , suggesting that p-tefb liberation from sk snrnp could be a common theme developed by different viruses to support their replication in host cells. instead of using promoter-proximal paused transcripts as cis-acting scaffolds to recruit tfs, viruses can also use noncoding rnas. a recent example described that the epstein-barr virus (ebv) uses a virally encoded noncoding rna named ebv-encoded rna (eber ) that acts as a transacting guide to promote its own transcription [ ] (figure ). ebv is a human γ-herpesvirus that persists as a mini-chromosome in the nucleus of b lymphocytes [ ] . herpesviruses are large enveloped ds dna viruses that encode more than genes. they are classified into three subfamilies (α-, β-, and γ-herpesviruses). ebv is the causative agent of mononucleosis and is associated with several types of tumors, including lymphomas and carcinomas [ ] . two long ncrnas called eber ( nt) and eber ( nt) are expressed in infected cells at high levels (eber : ~ copies; eber : ~ . × ) [ , ] and localize exclusively to the nucleoplasm [ ] . due to their high copy number, instead of using promoter-proximal paused transcripts as cis-acting scaffolds to recruit tfs, viruses can also use noncoding rnas. a recent example described that the epstein-barr virus (ebv) uses a virally encoded noncoding rna named ebv-encoded rna (eber ) that acts as a trans-acting guide to promote its own transcription [ ] (figure ). ebv is a human γ-herpesvirus that persists as a mini-chromosome in the nucleus of b lymphocytes [ ] . herpesviruses are large enveloped ds dna viruses that encode more than genes. they are classified into three subfamilies (α-, β-, and γ-herpesviruses). ebv is the causative agent of mononucleosis and is associated with several types of tumors, including lymphomas and carcinomas [ ] . two long ncrnas called eber ( nt) and eber ( nt) are expressed in infected cells at high levels (eber :~ copies; eber :~ . × ) [ , ] and localize exclusively to the nucleoplasm [ ] . due to their high copy number, these ncrnas were used as diagnostic tool for ebv infection [ ] , but their function remained unknown. employing a technique named 'capture hybridization analysis of rna targets' (chart), lee et al. [ ] found that eber localizes to the terminal repeats (trs) in the latent ebv genome. trs are tandem direct repeats of approximately bp that flank both ends of the linear genome present in virions and are the site of circularization to form the viral episome after its entry into the host cell [ ] . eber localizes in the vicinity of a binding site for the paired box protein (pax ) host transcription factor [ ] . this co-localization suggested that these factors collaborate in some way, and lee et al. show that eber interacts with pax , albeit indirectly through an as-yet unidentified bridging factor. pax is known to downregulate viral latent membrane protein / a/ b (lmp / ) gene expression and entry into ebv lytic replication [ , ] . top candidates for this bridging factor include three host proteins: splicing factor proline and glutamine rich (sfpq), non-pou domain-containing octamer-binding protein (nono), and rna-binding motif protein (rbm ) [ ] . sfpq and rbm contact eber directly as shown by rna-protein crosslinking, and binding studies show that sfpq and nono associate with pax ( figure ). eber depletion abrogates pax recruitment to the tr, but pax depletion has no effect on eber localization, suggesting that this ncrna is used to recruit pax to the tr. indeed, eber base pairs with nascent transcripts generated from the tr regions, thus potentially targeting the interacting pax tf to its consensus sequence sites within the trs and facilitating its binding. lee et al. [ ] found that eber localizes to the terminal repeats (trs) in the latent ebv genome. trs are tandem direct repeats of approximately bp that flank both ends of the linear genome present in virions and are the site of circularization to form the viral episome after its entry into the host cell [ ] . eber localizes in the vicinity of a binding site for the paired box protein (pax ) host transcription factor [ ] . this co-localization suggested that these factors collaborate in some way, and lee et al. show that eber interacts with pax , albeit indirectly through an as-yet unidentified bridging factor. pax is known to downregulate viral latent membrane protein / a/ b (lmp / ) gene expression and entry into ebv lytic replication [ , ] . top candidates for this bridging factor include three host proteins: splicing factor proline and glutamine rich (sfpq), non-pou domaincontaining octamer-binding protein (nono), and rna-binding motif protein (rbm ) [ ] . sfpq and rbm contact eber directly as shown by rna-protein crosslinking, and binding studies show that sfpq and nono associate with pax ( figure ). eber depletion abrogates pax recruitment to the tr, but pax depletion has no effect on eber localization, suggesting that this ncrna is used to recruit pax to the tr. indeed, eber base pairs with nascent transcripts generated from the tr regions, thus potentially targeting the interacting pax tf to its consensus sequence sites within the trs and facilitating its binding. the last stage of transcription is termination, which leads to the release of the rna transcript and the dissociation of rnap from the template dna. upon encountering sequence-encoded termination signals (e.g., the canonical polyadenylation signal, aauaaa), factors will be recruited that cleave and modify the rna ′ end. eukaryotic mrna undergoes multiple types of modifications at its ′ end throughout its life cycle [ ] . for example, newly synthesized mrna acquires a long poly (a) tail (up to ∼ nt) through canonical transcription-coupled polyadenylation, which facilitates mrna export from the nucleus [ , ] . in the cytoplasm, poly(a) tails are associated with poly(a)-binding proteins (pabpcs) that stabilize the mrna by acting as a safeguard against multiple decay machineries and promote protein synthesis [ , ] . mrnas carrying short poly(a) tails (< nt) are recognized and poly-uridylated by terminal uridylyl transferases (tutases) and , which labels the mrna for degradation [ ] . many +ssrna viruses lack a poly (a) tail at the ′ end of their genome, but are still efficiently translated. for instance, flaviruses (e.g., dengue virus, west nile virus) have a capped rna genome the last stage of transcription is termination, which leads to the release of the rna transcript and the dissociation of rnap from the template dna. upon encountering sequence-encoded termination signals (e.g., the canonical polyadenylation signal, aauaaa), factors will be recruited that cleave and modify the rna end. eukaryotic mrna undergoes multiple types of modifications at its end throughout its life cycle [ ] . for example, newly synthesized mrna acquires a long poly (a) tail (up to ∼ nt) through canonical transcription-coupled polyadenylation, which facilitates mrna export from the nucleus [ , ] . in the cytoplasm, poly(a) tails are associated with poly(a)-binding proteins (pabpcs) that stabilize the mrna by acting as a safeguard against multiple decay machineries and promote protein synthesis [ , ] . mrnas carrying short poly(a) tails (< nt) are recognized and poly-uridylated by terminal uridylyl transferases (tutases) and , which labels the mrna for degradation [ ] . many +ssrna viruses lack a poly (a) tail at the end of their genome, but are still efficiently translated. for instance, flaviruses (e.g., dengue virus, west nile virus) have a capped rna genome which contains conserved sequences at the and ends, allowing for circularization and efficient translation [ , ] . other examples that follow the same strategy include rotaviruses [ ] , barley yellow dwarf virus [ ] , and possibly hepatitis c virus [ ] . additionally, premature transcription termination events are being uncovered that allow viruses to produce another layer of transcripts encoded by the same genome. by using these sub-genomic transcripts, viruses can create regulatory rna molecules that undergo a different fate than the main vrna. this can for instance be mirna production by retroviruses [ ] or the above described eber small ncrnas produced by ebv. retroviruses have to produce an alternative transcript as a mirna-source to prevent cleavage of the vrna genome during mirna processing by the microprocessor complex [ , ] . most of these sub-genomic transcripts are rnapiii-encoded [ , ] , but can also result from transcription termination of promoter-proximal paused rnapii [ ] . this nascent rna will subsequently be exported to the cytoplasm, where it can enter the rnai pathway at the dicer step. viruses can downregulate host transcription by targeted mrna degradation, to benefit their own gene expression. in this way, the host transcription machinery is deprived of targets and the virus can redirect the machinery to its own viral expression. as a beneficial side-effect to the virus, lowering host transcription will also reduce the effectiveness of the immune system. therefore, during infection with many human viruses, the accumulation of viral proteins is accompanied by a progressive global reduction in the production of host proteins, a phenomenon that has been termed "host shutoff". viral factors that can induce host shutoff include severe acute respiratory syndrome (sars) coronavirus nsp [ , ] , herpes simplex virus (hsv- ) vhs [ , ] , vaccinia encoded decapping nucleases d and d [ ] [ ] [ ] , and two recently described viral endonucleases: kaposi's sarcoma-associated herpesvirus (kshv) sox [ , ] and influenza a virus (iav) pa-x protein [ ] . like all influenza viruses, iav is a segmented negative strand rna virus. the pa-x protein-encoding gene overlaps with the open reading frame (orf) for the pa flupol subunit [ ] . ribosomal frameshifting on a rare cgu codon results in a fusion protein with the -aa n-terminal mrna endonuclease domain of pa and an alternative c-terminal x domain of either or aa [ , ] . consequently, pa-x lacks the ctd of pa responsible for its interaction with the pb subunit of flupol, and was found to share sequence similarity with the above described viral host shutoff proteins that trigger rna degradation. kshv is a γ-herpesvirus that causes tumors of endothelial cells and b cells. during lytic infection with kshv, global degradation of host rna is triggered by a virally encoded protein named shutoff and exonuclease (sox) [ , , ] . sox cleaves most cellular mrnas at random positions [ , , ] . although the α-herpesvirus vhs and γ-herpesvirus sox proteins are not orthologs, they have a similar mechanism of action [ ] ; both are rna endonucleases that cut host mrnas into fragments. the initial cleavage is followed by degradation of the rna body by the cellular - exonuclease xrni and potentially other enzymes [ , ] . this mechanism is shared by other viruses with shutoff-rnases like pa-x for iav and nsp for sars [ , , ] . moreover, all known viral host shutoff-rnases, degrade mrnas transcribed by the cellular rnapii complex and spare ncrnas transcribed by rnapi and rnapiii [ , , ] . notably, viral mrna transcription escapes from decay-induced repression, and this escape also relies on xrni. however, the mechanisms that allow viral mrnas, unlike cellular mrnas, to escape from xrni cleavage are unknown. a complex interplay between viruses and the host transcription machinery that is mediated by nascent rna has recently become increasingly apparent. here, we explored a few of the myriad pathways by which viruses (i) use their own nascent rna-in cis or in trans-as a scaffold to recruit host transcription factors; (ii) use host-capped rnas as primers for rna synthesis; (iii) use termination of nascent rna synthesis to create sub-genomic mrnas, and (iv) and down-regulate host nascent rna production for their own benefit. viral rna synthesis pathways are promising targets for antiviral development because these mechanisms are essential for virus replication. however, in order to identify additional weak spots in viral infection programs, it is important to understand how viruses regulate their gene expression and to disentangle the complex ways in which these pathways are intertwined with host transcription. many antivirals display adverse side-effects because the viral target molecule and process was incompletely understood. the recently described new insights into the roles of nascent rna in viral gene regulation are important steps in the path to new, more effective antivirals with fewer side-effects. genetic conflicts: the usual suspects and beyond paleovirology-modern consequences of ancient 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functional domains structure and functional analysis of the rna-and viral phosphoprotein-binding domain of respiratory syncytial virus m - protein the respiratory syncytial virus m - protein forms tetramers and interacts with rna and p in a competitive manner crystal structure of a nucleocapsid-like nucleoprotein-rna complex of respiratory syncytial virus unconventional mechanism of mrna capping by the rna-dependent rna polymerase of vesicular stomatitis virus influenza virus rna polymerase: insights into the mechanisms of viral rna synthesis structural insight into cap-snatching and rna synthesis by influenza polymerase structure of influenza a polymerase bound to the viral rna promoter crystal structure of the rna-dependent rna polymerase from influenza c virus single-molecule fret reveals the pre-initiation and initiation conformations of influenza virus promoter rna an in vitro fluorescence based study of initiation of rna synthesis by influenza b polymerase rna-free and ribonucleoprotein-associated influenza virus polymerases directly bind the serine- -phosphorylated carboxyl-terminal domain of host rna polymerase ii influenza a virus preferentially snatches noncoding rna caps sequencing the cap-snatching repertoire of h n influenza provides insight into the mechanism of viral transcription initiation deep sequencing reveals the eight facets of the influenza a/hongkong/ / (h n ) virus cap-snatching process polyadenylation of influenza virus mrna transcribed in vitro from model virion rna templates: requirement for conserved sequences the influenza virus ns protein binds to a specific region in human u snrna and inhibits u -u and u -u snrna interactions during splicing mammalian net-seq reveals genome-wide nascent transcription coupled to rna processing stable pausing by rna polymerase ii provides an opportunity to target and integrate regulatory signals promoter-proximal pausing of rna polymerase ii: emerging roles in metazoans setx, xrn , and rrp co-operate to induce premature termination of transcription by rnapii sk small nuclear rna binds to and inhibits the activity of cdk /cyclin t complexes long noncoding rna as modular scaffold of histone modification complexes the sk small nuclear rna inhibits the cdk /cyclin t kinase to control transcription polycomb proteins targeted by a short repeat rna to the mouse x chromosome ebv noncoding rna binds nascent rna to drive host pax to viral dna tat trans-activates the human immunodeficiency virus through a nascent rna target an inducible transcription factor activates expression of human immunodeficiency virus in t cells specific nf-κb subunits act in concert with tat to stimulate human immunodeficiency virus type transcription host control of hiv- parasitism in t cells by the nuclear factor of activated t cells functional roles for the tata promoter and enhancers in basal and tat-induced expression of the human immunodeficiency virus type long terminal repeat human immunodeficiency virus type subtypes have a distinct long terminal repeat that determines the replication rate in a host-cell-specific manner functional differences between the long terminal repeat transcriptional promoters of human immunodeficiency virus type subtypes a through g the hiv- tat protein has a versatile role in activating viral transcription activation of the aids retrovirus promoter by the cellular transcription factor, sp . science hiv- core promoter lacks a simple initiator element but contains a bipartite activator at the transcription start site promoter clearance by rna polymerase ii anti-termination of transcription within the long terminal repeat of hiv- by tat gene product transcriptional pausing at + of the hiv- nascent rna modulates formation of the tar rna structure dsif and nelf interact with rna polymerase ii elongation complex and hiv- tat stimulates p-tefb-mediated phosphorylation of rna polymerase ii and dsif during transcription elongation negative elongation factor (nelf) coordinates rna polymerase ii pausing, premature termination, and chromatin remodeling to regulate hiv transcription roles of rna:dna hybrid stability, rna structure, and active site conformation in pausing by human rna polymerase ii a pause sequence enriched at translation start sites drives transcription dynamics in vivo rna polymerase switches between inactivated and activated states by translocating back and forth along the dna and the rna the tar hairpin of human immunodeficiency virus type can be deleted when not required for tat-mediated activation of transcription rna-mediated displacement of an inhibitory snrnp complex activates transcription elongation a human immunodeficiency virus type tat -like arginine-rich rna-binding domain is essential for hexim to inhibit rna polymerase ii transcription through sk snrna-mediated inactivation of p-tefb controlling cellular p-tefb activity by the hiv- transcriptional transactivator tat identification of a cyclin t-binding domain in hexim and biochemical analysis of its binding competition with hiv- tat bensaude, o. maq and sk rna interact with cdk /cyclin t complexes in a transcription-dependent manner tat competes with hexim to increase the active pool of p-tefb for hiv- transcription human t-lymphotropic virus type tax protein complexes with p-tefb and competes for brd and sk snrnp/hexim binding tax interacts with p-tefb in a novel manner to stimulate human t-lymphotropic virus type transcription epstein-barr virus: years on epstein barr virus-associated tumours: an update for the attention of the working pathologist viral noncoding rnas: more surprises genome-wide analyses of epstein-barr virus reveal conserved rna structures and a novel stable intronic sequence rna epstein-barr virus noncoding rnas are confined to the nucleus, whereas their partner, the human la protein, undergoes nucleocytoplasmic shuttling laboratory assays for epstein-barr virus-related disease covalently closed circular duplex dna of 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conserved in poxviruses vaccinia virus d protein has mrna decapping activity, providing a mechanism for control of host and viral gene expression characterization of a vaccinia virus mutant with a deletion of the d r gene encoding a putative negative regulator of gene expression lytic kshv infection inhibits host gene expression by accelerating global mrna turnover viral nucleases induce an mrna degradation-transcription feedback loop in mammalian cells selective degradation of host rna polymerase ii transcripts by influenza a virus pa-x host shutoff protein an overlapping protein-coding region in influenza a virus segment modulates the host response ribosomal frameshifting used in influenza a virus expression occurs within the sequence ucc_uuu_cgu and is in the + direction evolutionary conservation of the pa-x open reading frame in segment of influenza a virus host shutoff is a conserved phenotype of γ herpesvirus infection and is orchestrated exclusively from the cytoplasm crystal structure of a kshv-sox-dna complex: insights into the molecular mechanisms underlying dnase activity and host shutoff coordinated destruction of cellular messages in translation complexes by the γ herpesvirus host shutoff factor and the mammalian exonuclease xrn a common strategy for host rna degradation by divergent viruses the novel influenza a virus protein pa-x and its naturally deleted variant show different enzymatic properties in comparison to the viral endonuclease pa we thank michael bellecourt, tatiana mishanina and indroneil ghosh for helpful comments on the manuscript. this research was partially supported by nih grant gm .author contributions: all authors wrote, read, and approved the final manuscript. the authors declare no conflict of interest. key: cord- -huyyw z authors: moshe, adi; gorovits, rena title: virus-induced aggregates in infected cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: huyyw z during infection, many viruses induce cellular remodeling, resulting in the formation of insoluble aggregates/inclusions, usually containing viral structural proteins. identification of aggregates has become a useful diagnostic tool for certain viral infections. there is wide variety of viral aggregates, which differ by their location, size, content and putative function. the role of aggregation in the context of a specific virus is often poorly understood, especially in the case of plant viruses. the aggregates are utilized by viruses to house a large complex of proteins of both viral and host origin to promote virus replication, translation, intra- and intercellular transportation. aggregated structures may protect viral functional complexes from the cellular degradation machinery. alternatively, the activation of host defense mechanisms may involve sequestration of virus components in aggregates, followed by their neutralization as toxic for the host cell. the diversity of virus-induced aggregates in mammalian and plant cells is the subject of this review. increasing evidence suggests that the assembly of many mammalian viruses occurs at specific intracellular sites, which have been termed "virus factories". the ultrastructure of the factories has been determined for a number of rna and large dna viruses that assemble in the cytoplasm, at the microtubule organizing center (mtoc) [ ] [ ] [ ] . in the case of dna viruses that replicate in the nucleus, the identity and structure of virus assembly sites are not clear, likely due to the complexity and the dynamic nature of the nuclear architecture. virus inclusions in nuclei are often formed in promyelocytic leukemia nuclear bodies and in nuclear aggresomes [ ] . in plant cells, both rna and dna viruses are associated with large inclusions detected in the cytoplasm and nucleus, however, their role in virus propagation or oppositely in virus restraint is less investigated than in infected mammalian cells. in general, mammalian and plant viruses make use of aggregates as scaffolds for anchoring the replication complex, increasing the local concentration of viral and host components required for replication and assembly, and shielding the process of replication from host defense. alternatively, these aggregates may be part of an innate cellular response that recognizes virus components and targets them for storage and degradation. to understand the aggregation processes accompanying virus infection, it is important to discover the origin of the cellular components that gives rise to the virus-induced inclusions and smaller aggregates, and to identify the molecular motors that are involved in their trafficking from the site of origin to the final destination. virus aggregates often result in rearrangement of cellular membrane compartments and/or cytoskeleton. the functions of these organelles are carefully regulated in cells. changes in cellular architecture may constitute responses to the stress associated with virus infection. throughout this review we suggest that the line that separates viral aggregates as storage of dead-end material from a functional viral factory is rather artificial. viruses may target key stages in the regulatory pathways that control organelle structure and function to generate sites that are essential for replication and assembly. the same structures can be associated with cellular defenses against infection and cell stress. given the co-evolution of viruses with their host cells, changes in cell structure induced during infection are likely to involve a combination of the two strategies. numerous viruses assemble and replicate in large insoluble inclusion bodies. in the case of mammalian viruses, these inclusions, called "virus factories" or "viroplasm", are generally localized near the mtoc and maintained by dynein microtubule motor proteins (reviewed in [ ] ). virus factories concentrate viral components needed for the genome replication and morphogenesis of new virus particles. the same structures also contain cellular chaperones/heat shock proteins (hsps), proteases, and the elements of s proteasome degradation machinery, which might enable cellular protective mechanisms to target viral compounds for degradation. in this regard, virus factories are functionally comparable with the cellular aggresomes, where aggregated toxic proteins are immobilized for subsequent proteasomal or autophagic degradation [ ] . hence, for many viruses, transport to the mtoc for storage and eventual degradation is a means to protect cells from infection. lately, two newly discovered cellular compartments named the "er-associated compartment" (erac) and the "juxtanuclear quality control compartment" (junq) have been shown to have common features with aggresomes. in addition, distinct cytoplasmic inclusions, coined 'insoluble protein deposit' (ipods), result from the accumulation of aggregation-prone mostly non-ubiquitinated substrates that are sequestered to protect the cell from the consequences of their potential toxicity [ ] . given that aggresomes, junq, ipods can be found in yeast and mammalian cells, it is reasonable to hypothesize that the mechanisms promoting cellular aggregation and inclusion formation are conserved across kingdoms. the same statement could be applied for virus-induced aggregation even though analogy between virus aggregates, junq and/or ipod has not been demonstrated. once in the cell, viruses may appear to the host as foreign or misfolded proteins, stimulating an aggresome response. many viral core particles have a size ( to nm) similar to the aggregates that are transported to aggresomes by dynein motors [ ] . in the case of large viruses such as poxviruses and african swine fever virus (collectively named nucleocytoplasmic large dna viruses, or ncldv), replication and assembly take place in viral factories that contain viral dna and structural proteins and resemble pericentriolar aggresomes. this raises the possibility that the aggresome pathway is used by these viruses to generate sites for replication and assembly [ , ] . mtoc-dependent development of virus factories was found also for mammalian rna viruses of types i/togaviruses around lysosomes, type ii/flaviviruses, rna viruses iii/bunyaviruses, coronaviruses, and arteriviruses (reviewed in [ ] ). in general, cytoplasmic virus factories are considered as gathering points for coordinated genome replication and capsid protein assembly into virions. at the same time, these subcellular domains could protect host cells from toxic viral proteins degraded by s proteosome or via autophagy pathways [ ] (see scheme in figure ). mtocs are not found in plant cells. therefore, viral proteins are not gathered according to the aggresome-like pathway for microtubule dependent aggregation of toxic or misfolded proteins. nonetheless, several studies demonstrated the possible involvement of an aggresome-like pathway in movement protein (mp) accumulation during rna virus infections. plasmid encoded protein mp of potato leaf roll virus (plrv) developed large aggregates in cells treated with the s proteosome inhibitor clasto-lactacystin b-lactone [ ] . tobacco mosaic virus (tmv) -kda mp was shown to become polyubiquitinated during virus infection and, subsequently, to enter the s proteasome degradation pathway [ ] . tmv infection mobilized perinuclear and cytoplasmic endoplasmic reticulum (er) membranes to form virus replication complexes (vrcs) where the tmv mp was localized ( figure ). vrcs moved to adjacent cells through plasmodesmata as large bodies; vrcs contained the components necessary to initiate a rapid spread of infection. vrc cell-to-cell spread was blocked by inhibitors of actin and myosin. the proposed model implied that tmv cell-to-cell spread was achieved by vrcs dispersed throughout the cortical region of the cell [ ] . the association of positive-stranded rna viruses' replicating machineries with intracellular membranes is a feature common in animals, plants and insects (reviewed in [ ] ). intracellular membranes are used by different viruses to anchor their replication complexes. for example, er appears to be re-configured in such a way that the surface of the membrane is used as a scaffold, where viral replication complexes are juxtaposed with capsid proteins specifically delivered to these locations [ ] . complex interactions between viral and host proteins in these structures, different from mammalian virus factories, served for the regulation of potato virus x (pvx) multiplication. pvx replicase was detected in large membrane bound containers that developed from the er (approximately nm in diameter) together with the virus mp tgbp [ ] . tgbp caused the increased expression of the transcription factor bzip , which functions as an er resident sensor of stress [ ] . consequently, the expression of er resident chaperones, such as bip, pdi, and skp , which is a component of scf ubiquitin ligase complexes, are affected by pvx tgbp , while silencing bzip expression in protoplasts greatly inhibits pvx replication [ ] . in general, plant positive strand rna viruses are associated with membranes originating from the er and from organelles. these membranous complexes protect the virus replication and translation machineries from degradation by proteases and nucleases, and also protect the viral rna from silencing (reviewed in [ ] ). herpes simplex virus type- , adenovirus type (ad ), and sv accumulate at nuclear sites named promyelocytic leukemia nuclear bodies (pml, previously termed nd ) [ ] [ ] [ ] [ ] . in uninfected cells, the nucleus contains five to thirty pml/nd bodies, which may serve as scaffolds for mobilizing a variety of proteins involved in transcriptional regulation, chromatin organization, and dna repair. many dna viruses appropriate the cellular dna repair proteins for their replication [ ] and use pml/nd s for capsid assembly. in the infected cells, virus inclusions also appear as subnuclear structures called nuclear aggresomes [ ] . they contain proteasome subunits, ubiquitin, and molecular chaperones; they specialize in the containment and/or removal of protein aggregates. nuclear aggresomes lie adjacent to pml/nd s and to sites of virus replication ( figure ). in nuclear aggresomes, misfolded proteins are not discarded by autophagy, which makes them favorite sites for virus replication in the nucleus [ ] . recent studies revealed that nuclear aggresomes could also mobilize cellular proteins that inhibited virus replication [ ] . evidence has accumulated showing that not only dna viruses, but also rna viruses belonging to several different families relate to pml/nd s. human foamy virus, human t-cell leukemia virus type , human immunodeficiency virus type and many other mammalian rna viruses were found to interact with the subnuclear structure pml/nd s [ ] . it is important to note that the major nd components constitute host factors with antiviral activities. almost all herpesviruses, for which replication has been linked to nd , have evolved regulatory proteins that are capable of inactivating nd components or disturbing the integrity of the whole subnuclear structure [ ] . moreover, an enhanced infectivity of human cytomegalovirus was observed in the absence of either one of the basic nd components [ ] . herpesviruses are able to induce additional types of nuclear inclusion bodies. the tegument proteins vp and vp / are localized in aggregates that align closely but do not overlap with pml/nd s [ ] . it is not known whether these different structures relate to each other, whether they are homogenous accumulations of the individual herpesvirus protein(s), or whether they are simply dead-end aggregates of viral proteins. different aggregates are induced by small dsdna polyomaviruses (infectious pathogens of mammals and birds) [ ] . the polyomavirus-specific structures/virus factories are not dependent on the presence of the pml protein, and virus replication is not affected in knockout mice [ ] . the role of pml/nd s in polyomavirus replication is complex and pml/nd associated proteins may provide the necessary architectural foundation for the structures that appear to be polyomavirus factories. cajal bodies (cbs) could be considered as a part of the nuclear inclusion family (figure ). cbs are structures present in the nucleus of plant and animal cells. they contain small nuclear/nucleolar ribonucleoprotein particles (snrnp, snornp) and other proteins such as collin and fibrillarin. they are involved in maturation of spliceosomal snrnps and snornps [ ] . infection of hela cells by ad , a double-stranded dna virus, leads to cb fragmentation into smaller foci, organized in ring structures termed rosettes. cb rosettes localize to the periphery of viral e a- k-containing replication centers and disappear at later stages of infection, suggesting a role in adenovirus late gene expression [ ] . in plants, the ssrna groundnut rosette virus (grv) protein orf is responsible for long distance movement via the phloem. grv-orf is produced in the cytoplasm, moves into the nucleus where it recognizes cbs, and, consequently, forms multiple cb-like structures (cbls) and promotes their fusion with the nucleolus. it interacts with host proteins, one of which is fibrillarin, normally found in cb and nucleolus. these complexes migrate to the cytoplasm, where fibrillarin, orf and viral rna accumulate to form viral filamentous ribonucleoprotein (rnps) cytoplasmatic inclusions that protect viral rna from degradation. these rnps cytoplasmatic aggregates are able to move through the phloem [ ] . viruses often disrupt host cell gene expression. on the other hand, cellular anti-viral response may involve inhibition of viral gene expression via rna silencing or translational arrest. degradation, inhibition and storage of mrna and mrna-protein complexes (mrnp) are important for cellular homeostasis. while translation of mrnps takes place in polyribosomes, degradation and storage often occurs in cytoplasmic aggregates [ ] . hence it is expected that viruses interact with stress-related cytoplasmic rna aggregates, which are important elements of anti-viral defense and at the same time are attractive targets for viral countermeasures [ ] . processing bodies (pbs) are cytoplasmic foci in eukaryotic cells that are involved in mrna decapping, degradation and storage. they contain rnps, decapping proteins and proteins involved in rna-induced silencing such as argonaute (ago) [ ] . plant pbs have many proteins also found in yeast and mammalian pbs. in arabidopsis, pbs contain the decapping proteins atdcp and atdcp [ ] , and the cytoplasmic component of ago [ ] . mammalian pbs were shown to contain a cellular protein named moloney leukemia virus (mov ) [ ] . the overexpression of mov reduced hiv infectivity by interrupting early stages of post-entry replication, while mov silencing decreased hiv infectivity. therefore, it has been suggested that a basic level of the pb machinery is needed for hiv- rna processing and assembly; however, conversely, an increased expression of the pb component mov restricts viral replication [ ] . for some plant viruses, accumulation of viral components in pbs may serve as viral replication sites. for example, brome mosaic virus rnas accumulate in pbs, confirming the importance of pbs in the formation of replication complexes [ ] . in mammals, upon a large diversity of stress including viral infection, cells inhibit translation by converting active translation initiation complexes into inactive mrnps. these complexes are shuttled to cytoplasmic stress granules (sg) for storage ( figure ). indeed, active viral infection is not commonly seen if sgs are present, suggesting a role for sgs in inhibition of viral infection. it appears that there are different mechanisms for sg formation, but commonly, sg development is triggered by problems in the initiation of protein translation. pbs may promote sg assembly, and mrnp and other proteins may shuttle between the two structures [ , ] . many viruses are known for their ability to block sg formation or, alternatively, induce sg disassembly, thereby allowing rapid translation of viral mrnas. for example, junin virus blocks sg formation by interrupting the phosphorylation of eif [ ] . some viruses appear to mediate sg response to create virus replication/translation sites. upon infection of vaccinia virus, cytoplasmic aggregates emerge and share some properties of sgs, such as the sg marker eif e. they do not contain stalled mrnps but contain viral mrna. these aggregates are in close proximity to viral replication factories and may form a site for translation separated from replication [ ] . in plants, sgs were first observed in the cytoplasm of tomato cells upon heat stress and were termed heat shock granules (hsgs) [ ] . in addition to mrnp complexes, hsgs contain heat stress-induced proteins belonging to the hsp family [ ] . hsg-like structures may play a role in the rna-dependent rna polymerase (rdr )-mediated sirna pathway. accordingly, rdr -mediated dsrna is produced in cytoplasmic suppressor of gene silencing (sgs )/rdr bodies, separated from pbs [ ] . under stress conditions that trigger sgs formation, sgs /rdr bodies co-localize with sgs markers such as eif e, suggesting that these structures function as sgs [ ] . p protein of rice stripe virus (rsv) was found to be localized in sgs /rdr -bodies [ ] . sgs /rdr -bodies play a role in the multiplication of the plant dna geminivirus tomato yellow leaf curl virus (tylcv). tylcv v protein was shown to be a suppressor of plant rna silencing [ , ] . v interacts with sgs in sgs /rdr bodies, impairing sgs function in the rna silencing pathway, resulting in suppression of rna silencing and enabling viral infection [ ] . the appearance of large inclusion bodies containing virions in geminivirus-infected plants is a characteristic known for many years [ ] . the kinetics of formation of dna virus-related aggregates/inclusion bodies of different sizes and its relation to pathogenesis is the subject of recent investigation. the geminivirus indian cassava mosaic virus av protein implicated in viral movement was shown to form cytoplasmic and nuclear inclusion bodies [ ] . similar inclusions were generated by abutilon mosaic virus [ ] . other plant dna viruses also form aggregates of different sizes. the caulimovirus cauliflower mosaic virus (camv) firstly produced small electron-dense inclusion bodies (edibs), which were later exported to a single massive elib in each infected cell [ ] [ ] [ ] . camv elib's formation was microtubule-dependent, even though disruption of microtubules by oryzalin did not totally abolish elib development [ ] . despite the obvious resemblance of elibs formation to that of aggresomes, e.g., microtubule-dependent formation and the "one-per-cell" distribution, elibs differ from virus factories because mtoc is absent in plant cells. edibs, but not elibs, contained camv multifunctional protein p , virus particles and the virion-binding protein piii. the occurrence of elibs was shown to be essential for successful transmission of camv by their aphid vector, but was not required for the other viral functions. camv mutants that did not form elibs were fully infectious [ ] . it must be emphasized that the functions of viral inclusions other then virus factories is poorly understood, especially in plants. tylcv induce aggregates of various size in phloem-associated cells of infected tomato leaves. at early stages of infection, immunodetection of tylcv coat protein (cp) under the fluorescent microscope showed discrete punctate spots in the cytoplasm. at the later stages, signals of increasing size localized first in cytoplasm then in the nucleus (figure ). tylcv genomic dsdna replicative form together with cp-dna complexes were found exclusively in the nuclear aggregates [ ] . moreover, the nuclear inclusion contained infectious particles that could be transmitted by the insect whitefly vector, causing the tylcv disease in new tomato plants. several cellular proteins, such as hsp /hsc , hsp , and ubiquitin were detected in the large cytoplasmic and nuclear cp-inclusions [ ] . in tomato plants resistant to the virus, the formation of large cp aggregates is delayed and most aggregates are of small size [ ] . the molecular basis of natural tylcv resistance is unknown (reviewed in [ ] ), however the responses to virus infection of resistant and suscaptible tomato plants at the level of metabolites and proteins patterns are significantly different [ ] . in the field, the timing of infection of susceptible seedlings is critical: deleterious effects of tylcv (symptoms and arrest of growth) are preeminent when infection occurs during the first three weeks after planting; if infection is delayed, the plants will develop almost normally and will yield [ ] . resistant tomatoes may initiate a protective mechanism which leads to the accumulation of sequestering units in which the virus cp is captured in small/midsized aggregates by host compounds, disabling its capacity to participate in the formation of large inclusions in cytoplasm and consequently in nuclei to develop new virions. such delay of viral spread allows the plant to grow enough to sustain pathogenesis. the similarity between tylcv cytoplasmic large inclusions with perinuclear virus factories or similarity of virion-containing nuclear inclusions with nuclear aggresomes or pml/nd s is debatable. the absence of mtoc in plants, a required virus factory's component, has been discussed already; pml/nd s have not been detected so far in plant cells. at present, we are unable to name the tylcv-induced inclusions, but their abundance (especially of nuclear aggregates) correlates with efficient virus multiplication. alternatively, the maintenance of small aggregates containing tylcv cp is a characteristic of tylcv-resistant tomatoes [ ] . for a long time, electron microscopy studies revealed virus particles arranged in aggregates of different sizes in infected cells. in the current review, we have summarized the multiplicity of virus-induced aggregates, their function, localization, size and content. virus-induced aggregation in plants is much less known than aggregation of mammalian viruses, even though cylindrical inclusions induced by the potyvirus tobacco etch virus [ ] , and crystalline arrays caused by tmv [ ] , were described decades ago. moreover, the appearance of viral aggregates has been widely used to diagnose viral diseases (see "extension plant disease clinic", university of florida [ ]). another typical characteristic of plant virus invasion known for a long time is the alteration of the morphology of host organelles and membranes, for example by cowpea chlorotic mottle virus [ ] and cymbidium ringspot virus [ ] . in some instances, tubules containing virus-like particles were identified in or near the cell walls of infected cells (by cowpea mosaic virus, for example [ ] ). virus-induced membrane structures, mostly shown for plant rna viruses, house the rna replication complex and may be compared with virus factories in infected mammalian cell [ , ] , even though plant cells lack mtoc, the most characteristic component of virus factories. interestingly, these structures were identified as a degrading center for a plant virus movement protein, and thus may be involved in the viral cycle. recent studies in plant virology emphasize the absolute requirement for the formation of virus inclusions or virus factories for successful virus multiplication (reviewed in [ ] ). the exact name of certain structures does not seem to be as important as the definition of their role in the virus cycle. for example, in the case of the plant dna virus camv, small multiple aggregates (edibs) showed characteristics of virus factories, while single large cytoplasmic aggregates (elibs) resembled aggresomal structures and were shown to be important for aphid transmission, but not for camv infection in the plant cell [ ] . furthermore, camv replication and accumulation in edibs were not dependent on microtubule cytoskeleton functioning in contrast to known mammalian virus factories [ ] . virus-induced aggregates play a dual role in virus propagation in the infected cells. the recruitment of host cellular proteins into cytoplasmic and nuclear inclusions to facilitate virus replication has been described for many viruses (see above); on the other hand, the same viruses could be captured in cellular protective aggregative structures. for example, replication sites of dna vaccinia virus and asfv can be targeted by sg components and mx proteins, respectively [ , ] . mx proteins are interferon (ifn)-induced members of the dynamin superfamily of large gtpases. in general, mx gtpases appear to detect viral infection by sensing nucleocapsid-like structures. as a consequence, these viral components are trapped and sorted to locations where they become unavailable for the generation of new virus particles. mouse mx and human mxa proteins aggregate into punctate granula in the nucleus or cytoplasm, respectively, of ifn-treated cells. the aggregation of mx proteins prevents their rapid degradation and provides a storage structure from which active molecules can be recruited for prolonged periods of time [ ] . in these cases, virus induced aggregations play a protection role. the other well-defined example is the antiviral defense mechanism of pml/nd bodies [ ] . pml/nd components are constitutively expressed, allowing immediate antiviral activity of these molecules unlike interferon-induced antiviral properties of mx proteins. in the case of tylcv, two different types of virus-induced aggregates have been described [ ] . large nuclear inclusions contained dna-cp complexes and infectious particles were transmitted to test plants by insect vectors, in contrast to cytoplasmic small/midsized aggregates. in cytoplasmic aggregates, tylcv cp could be trapped and sorted to locations where the main viral protein became unavailable for the generation of new virus particles. differential states of aggregation could be a part of the plant immune response, reflecting different inclusion types, similar to nuclear aggresome and cytoplasmic ipods. the plant host quality control machinery recognizes tylcv compounds as foreign structures and directs them to huge insoluble nuclear inclusions, which the virus uses in its own favor: to house a large complex of proteins of both viral and host origin to promote virus replication and assembly. alternatively, virus protein(s) is (are) captured and redirected by the plant protective system to sg-like structures or ipod-like compartments, where they are sequestered and neutralized as protein aggregates. in future research, identification of putative cellular factors within small or large tylcv aggregates or involved in their arrangement will help to better understand their role in plant protection, and possibly elucidate novel plant cell mechanisms, whether related to ipod and aggresome assembly. understanding the phenomenon of virus aggregation and of the response of cells under attack, whether facilitating or inhibiting virus replication, may help to develop novel therapeutic approaches against virus infections in animal and plant cells. controlling viral diseases of agriculture crops is a major challenge to achieve food security in developed as well as in developing countries. the authors declare no conflict of interest. virus factories: associations of cell organelles for viral replication and morphogenesis a guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication virus factories, double membrane vesicles and viroplasm generated in animal cells aggresomes and pericentriolar sites of virus assembly: cellular defense or viral design? aggresomes and autophagy generate sites for viral infection aggresomes, inclusion bodies and protein aggregation misfolded proteins partition between two distinct quality control compartments viral stop-and-go along microtubules: taking a ride with dynein and kinesins intracellular trafficking of potato leafroll virus movement protein in transgenic arabidopsis degradation of tobacco mosaic virus movement protein by the s proteasome tobacco mosaic virus infection spreads cell to cell as intact replication complexes cytoplasmic viral replication complexes analysis of potato virus x replicase and tgbp subcellular locations identification of an arabidopsis transmembrane bzip transcription factor involved in the endoplasmic reticulum stress response the unfolded protein response is triggered by a plant viral movement protein wrapping membranes around plant virus infection properties and assembly mechanisms of nd , pml bodies, or pods cellular proteins localized at and interacting within nd /pml nuclear bodies/pods suggest functions of a nuclear depot promyelocytic leukemia nuclear bodies are predetermined processing sites for damaged dna interactions between dna viruses, nd and the dna damage response using or abusing: viruses and the cellular dna damage response nuclear aggresomes form by fusion of pml-nb-associated aggregates increased susceptibility of cytoplasmic over nuclear polyglutamine aggregates to autophagic degradation intrinsic cellular defense mechanisms targeting human cytomegalovirus interplay between herpesvirus infection and host defense by pml nuclear bodies evidence for a role of the cellular nd protein pml in mediating intrinsic immunity against human cytomegalovirus infections sequential localization of two herpes simplex virus tegument proteins to punctate nuclear dots adjacent to icp domains functional reorganization of promyelocytic leukemia nuclear bodies during bk virus infection virion assembly factories in the nucleus of polyomavirus-infected cells cajal bodies: a long history of discovery the role of cajal bodies in the expression of late phase adenovirus proteins cajal bodies and the nucleolus are required for a plant virus systemic infection plant stress granules and mrna processing bodies are distinct from heat stress granules how do viruses interact with stress-associated rna granules? on track with p-bodies characterization of arabidopsis decapping proteins atdcp and atdcp , which are essential for post-embryonic development processing bodies and plant development perturbation of the p-body component mov inhibits hiv- infectivity interactions between brome mosaic virus rnas and cytoplasmic processing bodies viral modulation of stress granules junin virus infection impairs stressgranule formation in vero cells treated with arsenite via inhibition of eif α phosphorylation formation of antiviral cytoplasmic granules during orthopoxvirus infection formation of cytoplasmic heat shock granules in tomato cell cultures and leaves transient expression and heat-stressinduced co-aggregation of endogenous and heterologous small heat-stress proteins in tobacco protoplasts sgs and rdr interact and colocalize in cytoplasmic sgs /rdr -bodies cytoplasmic arabidopsis ago accumulates in membrane-associated sirna bodies and is required for ta-sirna biogenesis xie, l. p of rice stripe virus (rsv) interacts with ossgs and is a silencing suppressor suppressor of rna silencing encoded by tomato yellow leaf curl virus-israel interaction with host sgs is required for suppression of rna silencing by tomato yellow leaf curl virus v protein virus-like particles in tomato plants affected by the yellow leaf curl disease tissue and cell tropism of indian cassava mosaic virus (icmv) and its av (precoat) gene product immunogold labeling of the abutilon mosaic virus in ultrathin sections of epoxy resin embedded leaf tissue intracellular distribution of viral gene products regulates a complex mechanism of cauliflower mosaic virus acquisition by its aphid vector electron-lucent inclusion bodies are structures specialized for aphid transmission of cauliflower mosaic virus a role for plant microtubules in the formation of transmission-specific inclusion bodies of cauliflower mosaic virus splicing of cauliflower mosaic virus s rna serves to downregulate a toxic gene product progressive aggregation of tomato yellow leaf curl virus coat protein in systemically infected tomato plants, susceptible and resistant to the virus the robert h. smith faculty of agriculture, food and environment biotic and abiotic stress response in tomato breeding lines resistant and susceptible to tomato yellow leaf curl virus stress responses to tomato yellow (leaf curl virus tylcv) infection of resistant and susceptible tomato plants are different behavior of tolerant tomato breeding lines (lycopersicon esculentum) originating from three different sources (l. peruvianum, l. pimpinellifolium and l. chilense) upon early controlled inoculation by tomato yellow leaf curl virus cylindrical inclusions in the cytoplasm of leaf cells infected with tobacco etch virus the hexagonal lattice spacing of intracellular crystalline tobacco mosaic virus an ultrastructural study of inclusions and disease development in plant cells infected by cowpea chlorotic mottle virus the fine structure of cymbidium ringspot virus infections in host tissues. iii. role of peroxisomes in the genesis of multivesicular bodies structure in cells of vigna unguiculata infected with cowpea mosaic virus cellular remodeling during plant virus infection inhibition of a large double-stranded dna virus by mxa protein human mxa protein: an interferon-induced dynamin-like gtpase with broad antiviral activity research in the authors' laboratory at the institute of plant sciences and genetics in agriculture, the hebrew university of jerusalem, was supported by a grant from the u.s. agency for international development, middle east research and cooperation (merc) program to h.c. (geg-g- - - - ), project m - . key: cord- - gzont l authors: guo, nan; zhang, bingzhou; hu, han; ye, shiyi; chen, fangzhou; li, zhonghua; chen, pin; wang, chunmei; he, qigai title: caerin . suppresses the growth of porcine epidemic diarrhea virus in vitro via direct binding to the virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gzont l porcine epidemic diarrhea (ped) has re-emerged in recent years and has already caused huge economic losses to the porcine industry all over the world. therefore, it is urgent for us to find out efficient ways to prevent and control this disease. in this study, the antiviral activity of a cationic amphibian antimicrobial peptide caerin . against porcine epidemic diarrhea virus (pedv) was evaluated by an in vitro system using vero cells. we found that even at a very low concentration, caerin . has the ability to destroy the integrity of the virus particles to block the release of the viruses, resulting in a considerable decrease in pedv infections. in addition, caerin . showed powerful antiviral activity without interfering with the binding progress between pedv and the receptor of the cells, therefore, it could be used as a potential antiviral drug or as a microbicide compound for prevention and control of pedv. alphacoronavirus of the family coronaviridae with single-stranded positive-sense rna [ ] . it is the causative agent of an acute infectious enteric disease known as porcine epidemic diarrhea (ped) that is clinically manifested by severe watery diarrhea, vomiting, and dehydration in the suckling piglets [ ] . with the high mortality in the piglets, ped infection finally caused enormous economic losses to the global swine industry, especially after its recent re-emergence caused by variant pedv strain throughout the world [ , ] . however, the classical ped vaccines could not provide appropriate protection against the variant pedv infection. considering this, relevant studies of new antiviral materials are needed to prevent and control emerging or re-emerging infectious diseases such as ped [ ] . antimicrobial peptides (amps) are important components of the nonspecific immune system of animals to eradicate invaders [ ] , and the skin secretion of anuran amphibians are rich sources for collecting amps [ ] [ ] [ ] . amps have a wide spectrum of antimicrobial activity against microorganisms such as bacteria, viruses, fungi, and parasites [ , ] , but are very friendly to host cells [ ] [ ] [ ] . moreover, amps can work as growth and health promoters to improve the performance of pigs by enhancing the immune status, improving the intestinal health, and alleviating the toxic effects of deoxynivalenol in pigs [ ] . there are also research findings that amps can modulate immune responses like chemokines, cytokine production, and pro-inflammatory responses [ , ] . caerin . is a peptide from the granular glands within the skin of the australian green tree frog with -residues (gllsv lgsva khvlp hvvpv iaehlnh ). nuclear magnetic resonance (nmr) of caerin . in the membrane mimetic environments showed that it has two α-helices and a flexible hinge region composed of two prolines [ ] . both prolines are essential for the antimicrobial activities [ ] . some studies have reported that caerin . exhibits antibacterial and antiviral properties by forming pores on the membrane to destroy the integrity of the particles [ , ] . meanwhile, it has been confirmed that caerin . has a complete inhibitory effect against hiv by preventing viral fusion to target cells and disrupting the hiv envelope, and, remarkably, that caerin . is also highly effective in inhibiting the transfer of hiv from dendritic cells (dcs) to t cells even when dcs are continuously exposed to peptides for h after virus capture, and that caerin . has a bacteriostatic activity against escherichia coli and bacillus subtilis [ , ] . although the mechanism that suppresses the activities of some bacteria and viruses has been illustrated clearly, it remains unknown whether caerin . can inhibit the growth of pedv. so, this study is aimed to investigate the function of caerin . . for this purpose, we investigated the inhibitory ability and antiviral mechanisms of caerin . against different pedv strains in vero cells, which will provide an insight into amps' antiviral mechanisms and its application as antiviral drugs or as drug loading compounds. vero cells (african green monkey kidney cell lines) were propagated at • c in a % co humidified incubator using dulbecco's modified eagle medium ((dmem), gibco, langley, va, usa) containing % fetal bovine serum (invitrogen, carlsbad, ca, usa). cells were infected with three different pedv strains: yn (accession no. kf ), cv (accession no. kt ), and dr (accession no. jq ). the yn strain ch/ynkm- / was isolated from a suckling piglet suffering from acute diarrhea. the cells infected with pedv strains were cultured in dmem supplemented with µg/ml trypsin. caerin . and n-terminus fitc labeled caerin . (purity: both > %) were chemically synthesized by bioyeargene (wuhan, china). caerin . was initially dissolved in . % acetic acid to reach the concentrations of mg/ml and mg/ml as stock solutions, respectively and stored at − • c until further use [ ] . the cytotoxicity of caerin . in vero cells grown in -well culture plates was assessed by mtt ( -( , -dimethyl- -yl)- , -diphenyltetrazolium bromide, mg/ml) assay. the cells were incubated with caerin . at different concentrations for h, then µl/well mtt was added and cultured for additional h. the supernatant was then removed and dimethylsulfoxide (dmso) was added to culture plates ( µl per well), then the plates were shaken at room temperature (rt) for min. finally, the optical density (od) value was measured at the wavelength of nm [ ] . the plaque forming assay was performed on vero cells cultured in -well plates. pedv ( pfu/well) was incubated using different concentrations of caerin . for h at • c before infection and then incubated pedv was added on vero cells covering % monolayer. after h of viral adsorption, the supernatant was removed, and the cells were rinsed with pbs and overlaid with µg/ml trypsin supplemented with sodium carboxymethyl cellulose-containing medium. the plates were fixed with % formaldehyde after h infection, and then stained with crystal violet solution [ ] . vero cells cultured in -well plates were washed with pbs for times and inoculated respectively with single medium, or single pedv, or pedv pre-incubated with different concentrations of caerin . . the cells were rinsed with pbs after h infection, the cells were fixed with % formaldehyde for min at rt. then the cells were incubated with an anti-pedv monoclonal antibody (made in our laboratory) for h and fluorescein isothiocyanate (fitc)-conjugated goat anti-mouse antibody ( : dilution) for h at • c. the immunofluorescence images were taken with a nikon eclipse ti microscope (nikon, tokyo, japan) [ ] . vero cells were cultured in -well plates and infected with pedv, and treated with caerin . at different time points. the cells were rinsed three times with pbs at h post infection (hpi), and treated with µl/well lysis solution containing protease inhibitors (pmsf). then µl of sodium dodecyl sulfate (sds) loading buffer was added to the cell extracts and the samples were boiled for min. the proteins were separated by % sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) and then transferred into pvdf membranes (millipore, mississauga, on, canada). membranes were blocked with % skimmed milk for h at • c and then incubated with primary antibodies over night at • c. the blots were then incubated with corresponding horseradish peroxidase (hrp)-conjugated secondary antibodies (abclonal, wuhan, china). the membranes were washed times at each step. the protein bands were visualized using the clarity™ western ecl blotting substrate (bio-rad, hercules, ca, usa). the protein blots were quantified by image j software (national institutes of health, bethesda, md, usa). pedv-infected cell culture was prepared and centrifuged at • c for min at × g to get rid of the cell debris. the supernatants were ultracentrifuged at • c for h at , × g. then the purified virus pellets were re-suspended with pbs and treated with caerin . ( µg/ml) for h at • c and the viruses treated in the same manner were used as positive control. afterwards, the cocktail was dripped on the copper grid and negatively stained with phosphotungstic acid (pta). finally, the samples were examined using the electron microscope (hitachi h , tokyo, japan) [ ] . pedv suspensions containing different concentrations of caerin . were pre-incubated for h at • c, and were serially diluted before they were inoculated on the % confluent vero cell monolayers grown in the -well plates, followed by washing times with pbs. about hpi, the inoculates were removed and the cells were washed again with pbs for times and incubated for another days with dmem containing trypsin ( µg/ml). the infectivity was calculated by tcid (tissue culture infectious dose ) following the reed-muench method established by l. j. reed and h. muench [ ] . the vero cell monolayers were infected with pedv ( pfu). at hpi, the cells were washed twice with pbs and then treated with different concentrations of caerin . , and at hpi, the culture supernatants and the cells were collected respectively [ ] . the cell samples were frozen and thawed times to completely release the intracellular virus particles. both the extracellular and intracellular virus yields were determined by tcid . the vero cells in -well plates were inoculated with pedv in the presence or in the absence of caerin . at different time points: a. h before infection (pre); b. incubation of caerin . and pedv for h before infection (co); c. hpi (post). the supernatants and the cells were collected together at different time points and the samples were treated in the same way mentioned above in an infectious virus yield reduction assay. on the other hand, the inhibitory ability of caerin . against pedv was examined using a -fold higher concentration for further understanding antiviral activities in the pre and post treatment situations. finally, the cell lysates were collected in a parallel set for western blot assay. cells were pre-chilled at • c for h before infection and were chilled again after inoculated with pedv for h, then the cells were washed with ice cold pbs times, replenished with dmem containing trypsin, were incubated at • c for h, and the inhibitory effects were determined by tcid . caerin . was separately added at two time points: together with pedv at the time of viral absorption, and after the cells were washed at the time of viral entry to reach a highest final concentration of µg/ml. the cell lysates were processed in the same procedures with western blot analysis [ ] . vero cells were seeded onto some glass coverslips and were separately treated with fitc-caerin . ( µg/ml), pedv ( pfu), and both fitc-caerin . ( µg/ml) and pedv ( pfu), then incubated at • c for h. cells were fixed with % formaldehyde followed by permeabilization for min with . % triton x- , and were blocked with % bsa for h. the cells were washed times with pbs at each step. cell nuclei were counterstained with . % , -diamidino- -phenylindole ((dapi), invitrogen), and the antibodies used here were: pedv-s monoclonal antibody (made in our laboratory) and alexa fluor -conjugated affinity pure donkey anti-mouse igg (h+l) (ant gene, wuhan, china). the samples were examined using a confocal microscope (lsm meta, carl zeiss, munich, germany) [ ] . all experiments were performed with three independent experiments, and the calculated results were presented as mean ± standard deviation (sd). statistical analyses were performed using student's t-test. graph pad prism . was used to analyze the statistics in this study. the statistical significances were defined as p < . (*), and the higher significance was denoted by p < . (**) and p < . (***). the result of cytotoxicity assay indicated that cell viability increased with the decrease of the concentration of caerin . , that the viability of cells remained over % when treated with caerin . at the concentrations not exceeding µg/ml, and that the cell morphology was not affected even at the concentration µg/ml with a cell viability value of %, as seen in figure . thus, µg/ml caerin . was chosen as the highest final concentration in most of the subsequent experiments. we used µg/ml, µg/ml, µg/ml, and . µg/ml of caerin . in the ifa assay and in the addition assay. different incubating conditions of pedv in the presence of caerin . can directly affect the antiviral activity of caerin . . in this experiment, we determined the titer of pedv under different incubating temperatures and time durations. the results showed that the most suitable incubating temperature was °c, shown in figure a , and time length was h, shown in figure b . low temperature and inadequate incubation time would obviously affect the inhibition activity of caerin . against pedv. different incubating conditions of pedv in the presence of caerin . can directly affect the antiviral activity of caerin . . in this experiment, we determined the titer of pedv under different incubating temperatures and time durations. the results showed that the most suitable incubating temperature was °c, shown in figure a the results are presented as the mean ± sd of three independent experiments. the statistical analysis was performed using graph pad prism . . significance was defined as p < . (*), and higher significance was defined as p < . (**). to fully understand the inhibitory effect of caerin . against pedv-yn strain, several experiments were performed including cytopathic effects (cpe) observation, plaque reduction assay, tcid , and ifa. the cpe observation results showed that there were remarkable differences between the cells infected with pedv in the absence or presence of caerin . . caerin . -treated cells were in a relatively normal status, and the cpe appeared in caerin . -treated group about h later than in the pedv control group, as seen in figure a . with the addition of caerin . , the number of plaques declined at a surprising speed and the diameters decreased notably. similar results can be observed even with pfu of pedv treated by caerin . . the cells infected with pfu pedv exactly remained the same as the cells in negative control group, as seen in figure b . caerin . significantly inhibited the multiplication of pedv in a dose-dependent manner, demonstrating that pedv infection to vero cells was blocked to a large extent. the results of tcid , shown in figure c , and ifa, in figure d , indicated that the titer and the fluorescence intensity of pedv decreased significantly with the increase in the concentration of caerin . . to fully understand the inhibitory effect of caerin . against pedv-yn strain, several experiments were performed including cytopathic effects (cpe) observation, plaque reduction assay, tcid , and ifa. the cpe observation results showed that there were remarkable differences between the cells infected with pedv in the absence or presence of caerin . . caerin . -treated cells were in a relatively normal status, and the cpe appeared in caerin . -treated group about h later than in the pedv control group, as seen in figure a . with the addition of caerin . , the number of plaques declined at a surprising speed and the diameters decreased notably. similar results can be observed even with pfu of pedv treated by caerin . . the cells infected with pfu pedv exactly remained the same as the cells in negative control group, as seen in figure b . caerin . significantly inhibited the multiplication of pedv in a dose-dependent manner, demonstrating that pedv infection to vero cells was blocked to a large extent. the results of tcid , shown in figure c , and ifa, in figure d , indicated that the titer and the fluorescence intensity of pedv decreased significantly with the increase in the concentration of caerin . . the inhibitory effects of caerin . against another two different pedv strains were examined to evaluate the anti-pedv potential of caerin . based on ifa. as shown in figure , vero cells were infected with pedv ( pfu) pre-incubated with different concentrations of caerin . . compared with the virus control, treatment group exhibited excellent inhibitory effects in a dose dependent manner even at extremely low concentrations of caerin . . these results revealed that the intracellular viruses evidently reduced in the presence of caerin . in a dose dependent manner. groups (pedv) were not treated by caerin . . the results of three independent experiments are presented as the mean ± sd. the statistical analysis was performed using graph pad prism . . the significance was defined as p < . (*), and the higher significance was defined as p < . (**), p < . (***); (d) fluorescence intensity of pedv in the presence of caerin . at different concentration. the inhibitory effects of caerin . against another two different pedv strains were examined to evaluate the anti-pedv potential of caerin . based on ifa. as shown in figure , vero cells were infected with pedv ( pfu) pre-incubated with different concentrations of caerin . . compared with the virus control, treatment group exhibited excellent inhibitory effects in a dose dependent manner even at extremely low concentrations of caerin . . these results revealed that the intracellular viruses evidently reduced in the presence of caerin . in a dose dependent manner. to better understand the inhibitory effects of caerin . against the progeny virus production of pedv, we added caerin . at hpi to pedv infected cells. and both the intracellular and extracellular virus titers were determined separately by using tcid , as shown in figure a . the progeny virus titer decreased significantly as compared to that of virus control in a dose dependent manner, as seen in figure a . on the other hand, extracellular virus titers were also determined at different time points and different caerin . concentrations. the results showed that the increase of extracellular virus titer slowed down under the treatment of caerin . , as seen in figure b . incubated with caerin . at the concentrations of . (c), (d), (e) and (f) μg/ml for h. scale bar, μm. to better understand the inhibitory effects of caerin . against the progeny virus production of pedv, we added caerin . at hpi to pedv infected cells. and both the intracellular and extracellular virus titers were determined separately by using tcid , as shown in figure a . the progeny virus titer decreased significantly as compared to that of virus control in a dose dependent manner, as seen in figure a . on the other hand, extracellular virus titers were also determined at different time points and different caerin . concentrations. the results showed that the increase of extracellular virus titer slowed down under the treatment of caerin . , as seen in figure b . in the fluorescent confocal experiment, the fluorescence of caerin . and pedv around cell nucleus could be observed when we incubated them with vero cells respectively. additionally, the distribution of caerin . and pedv were almost the same, so the results of confocal laser scanning microscopy verified that caerin . could combine with pedv, and that the antiviral function of caerin . in the cell plasma could be observed especially in the areas showing severe cpe, as seen in figure . in the fluorescent confocal experiment, the fluorescence of caerin . and pedv around cell nucleus could be observed when we incubated them with vero cells respectively. additionally, the distribution of caerin . and pedv were almost the same, so the results of confocal laser scanning microscopy verified that caerin . could combine with pedv, and that the antiviral function of caerin . in the cell plasma could be observed especially in the areas showing severe cpe, as seen in figure . in caerin . -pedv pre-incubation process, the morphological changes of pedv particles were observed clearly. so caerin . could destroy the structure of the virus to block the infection of the host cells, as seen in figure a , resulting in the inhibition of viral release, which in turn could reduce the transmission of the progeny virus among the adjacent cells. as shown in the growth curve, pedv co-incubated with caerin . showed the lowest titer, while pedv was only slightly inhibited in preincubation process of caerin . -cells and the delayed usage of caerin . , as seen in figure b . in a western blot experiment, the protein band could barely be observed in pedv-caerin . co-incubation group. however, no obvious differences in pedv-n protein expression were observed among the cell-caerin . pre-incubation group, the post treatment group, and pedv control group, as seen in figure c . to further explore whether caerin . could bind to some virus receptors in host cells to interfere with the attachment and entry process, we kept the cells at °c to maintain the attaching status. caerin . did not show obvious inhibitory effects during the attachment and entry process. there were no significant differences in virus titers between the virus control groups and caerin . treated groups during both attachment period and entry period, even if the concentration of caerin . was increased tenfold, as seen in figure d . the western blot assay did not exhibit any obvious differences in protein expression among attachment period treated group, entry period treated group, and the pedv control group, shown in figure e , either. in caerin . -pedv pre-incubation process, the morphological changes of pedv particles were observed clearly. so caerin . could destroy the structure of the virus to block the infection of the host cells, as seen in figure a , resulting in the inhibition of viral release, which in turn could reduce the transmission of the progeny virus among the adjacent cells. as shown in the growth curve, pedv co-incubated with caerin . showed the lowest titer, while pedv was only slightly inhibited in pre-incubation process of caerin . -cells and the delayed usage of caerin . , as seen in figure b . in a western blot experiment, the protein band could barely be observed in pedv-caerin . co-incubation group. however, no obvious differences in pedv-n protein expression were observed among the cell-caerin . pre-incubation group, the post treatment group, and pedv control group, as seen in figure c . to further explore whether caerin . could bind to some virus receptors in host cells to interfere with the attachment and entry process, we kept the cells at • c to maintain the attaching status. caerin . did not show obvious inhibitory effects during the attachment and entry process. there were no significant differences in virus titers between the virus control groups and caerin . treated groups during both attachment period and entry period, even if the concentration of caerin . was increased tenfold, as seen in figure d . the western blot assay did not exhibit any obvious differences in protein expression among attachment period treated group, entry period treated group, and the pedv control group, shown in figure e , either. figure b . gapdh (glyceraldehyde- phosphate dehydrogenase) was used as a loading control; (d) tcid showed that caerin . had no obvious inhibitory effects during the attachment and entry process. caerin . was added at pedv attachment and entry periods, then the titers of pedv were detected; (e) western blot analysis of the expression level of pedv-n protein when caerin . was added in different ways as shown in figure d . gapdh was used as a loading control. pedv is one of the most important pathogens leading to diarrhea in pig industry. the aim of this study is to find whether caerin . has the antiviral activity against the three pedv strains and to reveal the antiviral mechanism of caerin . . according to published data, caerin . is a cationic peptide with two α-helices and has proved to be an effective agent against hiv by damaging the virus envelope and stopping the infection of hiv to t cells [ , ] . some α-helical peptides such as melittin and cecropin have been reported to exert antiviral activities by direct disruption of virus membranes or inhibition of the virus replication [ ] . the current study found that caerin . was able to destroy the structure of viral particles and reduce the viruses capable of infecting the cells and decrease their titers almost up to logs. this is the first attempt to reveal that caerin . has strong antiviral activity against pedv infection. figure b . gapdh (glyceraldehyde- -phosphate dehydrogenase) was used as a loading control; (d) tcid showed that caerin . had no obvious inhibitory effects during the attachment and entry process. caerin . was added at pedv attachment and entry periods, then the titers of pedv were detected; (e) western blot analysis of the expression level of pedv-n protein when caerin . was added in different ways as shown in figure d . gapdh was used as a loading control. pedv is one of the most important pathogens leading to diarrhea in pig industry. the aim of this study is to find whether caerin . has the antiviral activity against the three pedv strains and to reveal the antiviral mechanism of caerin . . according to published data, caerin . is a cationic peptide with two α-helices and has proved to be an effective agent against hiv by damaging the virus envelope and stopping the infection of hiv to t cells [ , ] . some α-helical peptides such as melittin and cecropin have been reported to exert antiviral activities by direct disruption of virus membranes or inhibition of the virus replication [ ] . the current study found that caerin . was able to destroy the structure of viral particles and reduce the viruses capable of infecting the cells and decrease their titers almost up to logs. this is the first attempt to reveal that caerin . has strong antiviral activity against pedv infection. some amps including cecropin d (cd-prrsv, salps-aiv) were reported to have blocked viral attachment and invasion by interacting with the receptors of the host cells [ ] . based on these findings, so we tried to keep pedv infection within the periods of the attachment and entry through the temperature control, and the results showed no evident differences in protein expression among attachment period treated group, entry period treated group, and the pedv control group. the number of virus infecting cells did not diminish. this means that caerin . did not interfere with the attachment and entry process. considering the fact that caerin . does not compete with the virus to conjugate with the cells, and the fact that viral attachment-entry processes are very fast and caerin . will not have enough time to act against the viral membrane, it can be concluded that caerin . has less significant antiviral activities in the pre-incubation of caerin . -cells progress. to further understand the mechanism in the post-treatment process, we examined the antiviral activity during the replication period. the results showed that caerin . did not have obvious antiviral effect during viral replication period, but it can control the infection progress by blocking the release of pedv particles to reduce viral transmission among the adjacent cells. antiviral activities of caerin . is expected to be improved through the combination usages with other different amps or other antiviral agents like graphene oxide (go), which could make caerin . a good weapon to deal with the diseases caused by viruses including pedv [ ] . as a matter of fact, we did a combination treatment with piscidin which was also proved to be effective against pedv [ ] , but the results were not as good as expected based on our unpublished work. the mechanisms still remain to be further investigated. although the joint use of piscidin and caerin . did not work as well as expected, other candidates can be explored for the prevention of pedv and other pathogens in drug combination studies. on the other hand, some related research work has been done in our laboratory. our other unpublished work has preliminarily testified the extraordinary ability of caerin . against some other viruses including prv (pseudorabies virus) which is a herpesvirus with double-stranded dna that can infect many mammal species. therefore, many potential functions of caerin . are still to be explored in the future. in this study, we investigated the antiviral activities of caerin . against pedv strains and its antiviral mechanism. the results show that caerin . can maintain the integrity of the host cells since it has very low cytotoxicity, and that it exhibits excellent virucidal activity in a dose-dependent manner even at very low concentrations. caerin . can reduce the viruses infecting the cells by destroying the integrity of the viral membrane, resulting in the decrease in the virus replication and protein expression. caerin . can also interfere with the virus release process essential for the inhibition of intracellular infection. therefore, caerin . is an excellent antiviral material against pedv. in conclusion, our study 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structure might facilitate the processes necessary for genome replication and packaging and shield viral components from host innate immune defenses. the biogenesis of the hcv mw is a complex process involving a concerted effort of hcv nonstructural proteins with a growing list of host factors. although a comprehensive understanding of mw formation is still missing, a number of important viral and host determinants have been identified. this review will summarize the recent studies that have led to our current knowledge of the role of viral and host factors in the biogenesis of the mws and discuss how hcv uses this specialized membrane structure for its replication. hepatitis c virus (hcv) is a globally prevalent human pathogen. more than million people are chronically infected worldwide, among whom many will develop cirrhosis and hepatocellular carcinoma. hcv is an enveloped, single-stranded positive-sense rna virus classified in the hepacivirus genus within the flaviviridae family. the . kb genome contains one open reading frame (orf) that is flanked by non-translated regions, which are necessary for viral rna translation and replication [ ] [ ] [ ] . a single polyprotein is translated from the orf, which is co-and post-translationally processed by cellular and viral proteases to generate ten mature proteins: core, e , e , p , ns , ns , ns a, ns b, ns a, and ns b. the structural proteins (core, e , and e ) are incorporated into virus particles, whereas the nonstructural proteins p to ns b coordinate the intracellular processes of the virus life cycle [ ] [ ] [ ] . while p and ns are dispensable for genome replication, they are required for particle assembly [ , ] . ns through ns b are necessary and sufficient for hcv genome replication. as we will discuss extensively in this review, hcv replication is associated with the induction of host membrane alterations that are thought to support sites of viral rna replication. the induction of altered host membranes for viral replication is characteristic of all positive sense rna viruses [ ] . a negative sense replicative intermediate synthesized from the positive sense rna genome serves as template for the generation of progeny positive sense rna genomes. the newly-synthesized positive sense rna can either enter a new translation/replication cycle or be packaged into virions [ ] . this review will summarize our current knowledge on hcv-induced membrane alterations, as well as the role of viral nonstructural proteins and host factors in this process. hcv-induced membrane alterations, as well as the role of viral nonstructural proteins and host factors in this process. even before the identification and molecular cloning of hepatitis c virus, electron microscopy (em) studies of liver tissue from chimpanzees infected with "non-a, non-b hepatitis" demonstrated membrane alterations in hepatocytes [ , ] . the successful isolation of the first hcv cdna clone [ ] enabled studies to determine the effects of expressing viral proteins in hepatocytes in cell culture. egger et al. reported that expression of the entire hcv polyprotein in u -os human osteosarcoma cells was associated with the formation of membrane alterations described as vesicles within a membranous matrix, which collectively was termed "membranous webs" (mws) [ ] . in this study, expression of ns b alone also induced membrane alterations similar to those seen with the whole viral polyprotein. the subsequent establishment of the replicon model of hcv replication made it possible to visualize hcv induced membrane alterations in the context of viral genome replication. by using cell lines harboring persistent subgenomic replicons, gosert et al. [ ] found altered membrane structures similar in ultrastructural morphology to those observed by egger et al. [ ] ; furthermore, by using immunogold em, the authors reported that the membranous web could be labeled by antibodies against each of the hcv nonstructural proteins. at the light microscope level, immunofluorescence labeling for ns or ns a was visible as dot-like cytoplasmic structures [ ] [ ] [ ] [ ] . a later study of cells containing a subgenomic hcv replicon reported that the membrane alterations induced by hcv replication consisted in part of double-membrane vesicles (dmvs; figure ) with a diameter around nm that were also positive on immunoelectron microscopy with antibodies against ns a and double-stranded rna (dsrna) [ ] . this was a notable observation, as a number of other rna viruses also induce dmvs in infected cells [ , ] . the morphology of mws described above was confirmed and extended by using the cell culture infectious, full-length jc clone of hcv in conjunction with high pressure freezing with freeze substitution and electron tomography [ ] . in this study, romero-brey et al. found that hcv infection was initially associated with accumulation of dmvs with an average diameter of nm. the kinetics of dmv accumulation correlated with viral rna replication; at later time points of infection, multi-membrane vesicles (mmvs) with larger diameter (~ nm) became more predominant [ ] . d reconstructions of em tomographic series showed that most of the dmvs were tightly opposed to er membranes, and some of them were identified as protrusions from the er membrane into the cytosol, suggesting that mws originate from er membranes [ ] . in addition, most of the dmvs were closed structures, with only a minority possessing either a visible opening towards the cytosol or a short neck-like structure connecting the dmv to the er membrane bilayer [ ] . the identification of sites within hcv proteins that tolerate the insertion of heterologous sequences, such as epitope tags and even fluorescent proteins (for example, domain iii of ns a tolerates the insertion of green fluorescent protein (gfp)), made it possible to study the dynamics of mw in live cells. ns a-gfp was found in the cytoplasm as brightly fluorescing dots and in a reticular staining pattern [ ] , similar to the distribution of ns a observed in fixed and immunostained replicon cells. live cell imaging revealed two populations of ns a-gfp foci in cells: larger, relatively static structures and smaller structures with saltatory microtubule-dependent movements over long distances, both of which contain other hcv replicase components [ ] . however, the relationship of the two structures to one another is not well understood; for example, it is not known whether the smaller structures are precursors of or arise from larger static structures, or whether either structure participates preferentially in genome replication versus particle assembly. another approach to visualizing hcv replication organelles dynamics employed snap (a mutant of the human dna repair protein o -alkylguanine-dna alkyltransferase) tagging of ns a [ ] , which permitted labeling of temporally-distinct populations of ns a. this approach revealed that ns a synthesized - h before imaging was located on structures distinct from those associated with ns a synthesized - h before imaging, which provides an upper bound for the duration of viral polyprotein translation at a given replication organelle. a fundamental shortcoming of light microscopy is its limited resolution. correlative light electron microscopy (clem), which integrates the molecular specificity of fluorescent light microscopy with the resolution of em, has been employed to study the ultrastructural morphology of mws [ ] and has also been used to analyze the effect of ns a small molecule inhibitors [ ] or ns a mutants [ ] on the morphology of mws. the imaging techniques to study mw morphology and dynamics summarized above have been complemented by biochemical studies. unfortunately, in vitro reconstitution of a functional hcv replicase is still beyond our technical reach. early studies showed that crude membrane fractions isolated from hcv subgenomic replicon cells or selectively permeabilized replicon cells could use endogenous replicon rna as a template to synthesize new viral rna [ , , [ ] [ ] [ ] [ ] . viral rna synthesis was found to be resistant to protease and nuclease treatment, suggesting that replication occurs in membrane structures [ ] . additionally, membrane fractions containing hcv rna and nonstructural proteins were found to have properties similar to detergent-resistant membranes (drms) in that they were resistant to solubilization by cold triton x- and that these drms co-fractionated with cellular markers of drms on density gradient centrifugation [ , ] . the specialized lipid and protein composition of the hcv replication organelle might, therefore, facilitate the generation of membrane curvature necessary for dmv formation [ ] . in addition, as drms are important platforms for cellular membrane trafficking and signal transduction [ , ] , it is conceivable that this property of hcv replication membranes may also regulate signal transduction and membrane trafficking at viral replication sites. while the viral rna is largely resistant to nuclease treatment, only a small fraction ( %- %) of viral ns protein is resistant to protease treatment [ , ] . a likely explanation for this observation is the large excess of hcv protein compared to positive-and negative-strand hcv rna; indeed, a quantitative analysis of hcv rna and protein content of replicon cells estimated that there is a -fold excess of hcv protein over hcv rna [ ] . in addition to subcellular fractionation, attempts have been made to isolate mws or replicase components by affinity capture of tagged ns proteins [ ] [ ] [ ] or viral rna with associated proteins [ , ] . one of these studies using epitope-tagged ns b identified dmvs in the affinity-purified fraction and the presence of replicase activity associated with dmvs [ ] , providing direct evidence that dmvs are a site of hcv rna synthesis. although hcv has long been known to induce membrane rearrangements, it is only recently that some of the mechanisms that are responsible for the formation of these structures have begun to be unraveled. despite the substantial progress that has been made during the past few years, we are still far from understanding this complex process in detail. in the following sections, we summarize what is known about the role of both viral and cellular proteins in hcv-induced membrane reorganization. by using hcv polyprotein overexpression, egger et al. [ ] first showed that expression of viral nonstructural proteins resulted in membrane alterations that morphologically resemble those observed in replicon cells, demonstrating that viral rna replication is not required for membranous web formation. by expressing individual hcv proteins, these authors found that expression of ns b was sufficient to induce membrane alterations resembling mws. later studies have shown that the n-terminal alpha helix ah [ ] as well as c-terminal sequences [ ] are important for hcv replication, ns b oligomerization, and dmv morphogenesis. mutations of either of the two positively-charged lysine residues flanking the n-terminal alpha helix ah abrogate hcv replication and are associated with the formation of significantly larger dmvs when expressed in the context of ns - b using a non-replicative system [ ] . subsequent studies, however, have provided evidence that ns a is the only hcv ns protein capable of forming dmvs when expressed in isolation [ , ] , albeit much less efficiently than when expressed in the context of ns - b. in contrast, expression of ns b alone leads to the exclusive formation of single-membrane vesicles rather than dmvs [ ] . the amino-terminal "domain " of ns a is necessary and sufficient for the formation of dmvs when expressed in the context of the ns - b polyprotein [ ] . this work also identified roles of other hcv ns proteins in efficient dmv formation, notably the ns helicase domain, and the expression of the ns - a protease in cis with ns b- b. finally, mutations that accelerate the normally slow polyprotein cleavage kinetics at the ns b- a junction or constructs that do not express any ns b- a precursor impair or abrogate dmv formation [ ] , suggesting that a ns b- a precursor is somehow essential for dmv biogenesis. further evidence for a functional interaction between ns b and ns a comes from the identification of mutations in ns a that rescue mutations flanking ns b ah [ ] or help rescue a ns b c-terminal mutant [ ] . overall, these studies suggest that most, if not all, nonstructural proteins are required to work in concert for the efficient formation of dmvs. in addition to viral proteins, an increasing list of host factors has also been shown to contribute to membranous web formation. here, we well briefly discuss the mechanisms of several selected host factors in mw formation. several rna interference screens have identified the cellular lipid kinase pi ka (also known as phosphatidylinositol -kinase iii alpha, pi kiiiα, and pik ca) as essential for hcv replication [ ] [ ] [ ] [ ] [ ] . inhibition of pi ka, either by rna interference [ , [ ] [ ] [ ] or by pharmacologic inhibitors [ ] , leads to accumulation of large 'clusters' of ns a-positive membranes at the light microscopic level. at the ultrastructural level, these 'clusters' correspond to clusters of dmvs with reduced diameter [ ] , suggesting that pi ka is essential for the proper formation and/or integrity of mws. ns a and ns b can interact with pi ka and ns a activates its lipid kinase activity, giving rise to elevated intracellular phosphatidylinositol -phosphate (pi p) levels [ , ] . pi p has a highly negatively-charged headgroup and has been reported to cause membrane curvature at physiologically relevant concentrations [ ] , so local production of pi p at nascent replication organelles might facilitate membrane curvature and dmv formation. another function of pi p is to recruit specific viral and/or host proteins with pi p-binding domains [ ] . in particular, two pi p effectors, oxysterol-binding protein (osbp) and four-phosphate adaptor protein (fapp ) are essential for hcv replication [ , ] , and inhibition of either osbp or fapp results in altered mw morphology [ , ] . interestingly, osbp and fapp are both lipid transfer proteins (ltps), which are responsible for non-vesicular sterol and glycosphingolipid trafficking, respectively [ ] ; both of these lipids are important components of drms generally and, likely, also of hcv replication organelles specifically. inhibition of osbp leads to reduced trafficking of cholesterol to hcv replication organelles [ ] . similarly, the ltp ceramide transfer protein cert has also been reported to be involved in the hcv life cycle [ ] . these findings suggest a model in which pi p recruits ltps such as osbp and fapp to hcv replication organelles, which in turn result in the trafficking of cholesterol and glycosphingolipids to hcv replication membranes. dmvs are highly-curved structures; it is likely that proteins and/or lipids with membrane-deforming properties are involved in mw biogenesis. one such protein with membrane-deforming activity, proline-serine-threonine phosphatase-interacting protein (pstpip ), has been identified as a host factor essential for hcv viral replication and mw formation [ ] . pstpip is a member of the pombe cdc homology (pch) family proteins with membrane-deforming properties, likely mediated by their f-bar domain. pstpip co-fractionates with detergent-resistant membranes regardless of the presence of hcv, interacts with both ns b and ns a, and co-localizes with ns a on mws by immunoelectron microscopy. mutations in pstpip predicted to ablate its membrane-deforming function rendered it less effective in rescuing hcv replication in cells silenced for endogenous pstpip relative to expression of wild-type protein. another member of the pch family, bridging integrator (bin ), has been reported to be possibly involved in the hcv life cycle through an interaction with ns a [ ] , though it is not known whether bin is essential for viral replication or participates in mw biogenesis. the precise contribution of pch family proteins and other membrane-deforming proteins to mw formation remains to be determined. as a positive-sense rna virus, hcv is not known to require the nucleus for any step in its infection cycle. however, putative nuclear localization signals and nuclear export signals have been reported in hcv proteins (reviewed in [ ] ), and some reports have described localization of core protein and ns a to the cell nucleus during viral infection [ ] [ ] [ ] [ ] . furthermore, hcv and other positive-sense rna viruses appear to interact with nucleocytoplasmic transport factors [ ] . more specifically, hcv infection has been reported to directly interact with and relocate nuclear transport components, including karyopherins and nucleoporins, to sites enriched for hcv replication and assembly [ ] . furthermore, knockdown of a few of these karyopherins and nucleoporins impairs viral replication and/or virion assembly [ ] . these findings raises the intriguing hypothesis that relocation of nuclear transport components to the hcv replication organelles might influence membrane curvature and/or transport factors across membranes of dmvs and other replication organelle structures. however, this remains to be functionally demonstrated. autophagy is a cellular response to a variety of stimuli, including nutrient depletion, hormone treatment, and viral or bacterial infection in eukaryotic cells [ ] . one of the most distinguishing features of autophagy is the formation of double-membrane vesicles called autophagosomes, which engulf cytoplasmic macromolecules and damaged organelles and deliver them to lysosomes for degradation and recycling. while the cellular origin of the autophagosome membrane is not completely established, the endoplasmic reticulum may be one of its membrane sources [ ] . given these similarities between cellular autophagosomes and the dmvs seen in hcv infection, multiple studies have examined the possibility that dmv formation in hcv infection exploits the cellular autophagocytic machinery (reviewed in [ ] ). several studies have reported that the expression of hcv replicons e.g., [ , ] or hcv infection [ ] induces the accumulation of autophagosomes in cultured cells. other studies have demonstrated that ectopic expression of hcv ns b or ns a is also sufficient to induce autophagic vesicles and upregulate markers of autophagy induction, such as lipidated lc [ , ] , though whether these findings reflect effects of protein overexpression is unclear. in addition, an important question that arises from these experiments is whether autophagy and autophagosome induction are byproducts of hcv infection or whether autophagy itself is necessary for hcv infection and replication. while multiple studies have indicated that autophagy is somehow important for productive hcv infection, there is controversy regarding the precise steps of hcv infection that are facilitated by autophagy. several groups have reported that autophagy plays an important role in hcv rna replication [ , [ ] [ ] [ ] [ ] . however, dreux et al., found that autophagy specifically modulates the onset of translation of incoming hcv rna and, therefore, the initial establishment of hcv replication [ ] , and this observation was supported by another study [ ] . in addition to a possible role for autophagy in establishing hcv replication, tanida et al. observed that the release of hcv core and infectious particles from infected cells is reduced when autophagy is inhibited, and they proposed that in addition to facilitating the initiation of viral replication, autophagy proteins also contribute to hcv particle assembly and/or egress [ ] . we still do not understand the molecular mechanisms by which the autophagy machinery supports either of these processes; in particular, why autophagy should be required only for the establishment of hcv replication but not for the maintenance of ongoing replication remains to be elucidated. the biogenesis of membranous web is a complex process involving a concerted effort of hcv nonstructural proteins and a growing list of host protein and lipid factors. twenty-seven years after the molecular cloning of hcv, we all still far from understanding the molecular processes that lead to mw formation in the hcv-infected cell. based on our current state of knowledge, we will discuss candidate general mechanisms that direct mw formation. it is generally believed that hcv induced mws are derived primarily from the host cell er membrane [ , , , ] . as mmvs appear later in hcv infection than dmvs, after the peak of hcv rna replication [ ] , and as several other rna viruses also induce the formation of dmvs, most investigators have focused on the mechanisms of dmv morphogenesis rather than on mmvs. studies of other viruses that also induce the formation of er-derived dmvs have led to the proposal of several models for the formation of virus-induced double-membrane vesicles, including but not limited to a protrusion and detachment model, a double-budding model, and a model of exvagination, followed by invagination [ , , ] (figure ). these models are not necessarily mutually exclusive and, in theory, could operate simultaneously in infected cells. the first 'protrusion and detachment model' invokes local bending/deformation of part of an er cisterna with tight apposition of the two lipid bilayers, followed by pinching off and sealing to form a double-membrane vesicle. in the 'double budding model', a single-membrane vesicle buds by invagination into the er lumen, from which it is subsequently released by a second budding event into the cytosol to give rise to a dmv. in the last model, exvagination or tubulation of the er membrane is followed by partial invagination to form a cup-like structure that is then sealed to form a dmv. in the case of hcv, d reconstruction of electron tomographic images reveals that virus-induced dmvs are exvaginations connected via a short neck-like structure to the er membrane bilayer, and most dmvs are linked to the er only via their outer membrane [ ] . reconstruction of electron tomographic images reveals that virus-induced dmvs are exvaginations connected via a short neck-like structure to the er membrane bilayer, and most dmvs are linked to the er only via their outer membrane [ ] . at this point in time none of these models has been convincingly demonstrated to be a mechanism of dmv formation. however, kinetic analysis of the ultrastructural membrane alterations following hcv [ , ] infection have identified single-membrane vesicles early in hcv infection, while similar studies of enterovirus-infected cells have identified single-membrane tubules [ ] , which could be precursors of dmvs and thus might argue for models a and/or c presented above. the membranous web appears to be the site of hcv rna genome replication. by immunofluorescence microscopy, newly synthesized viral rna metabolically labeled with -bromouridine '-triphosphate (brutp) co-localizes with ns a protein as a marker of the membranous web [ , , ] . furthermore, negative strand hcv rna, which is a necessary intermediate of hcv replication, has been detected at ns a-positive foci by confocal microscopy [ ] . the precise localization of the hcv replicase complex at the different membranous structures that make up the membranous web (e.g., smvs, dmvs, and mmvs) has not yet been unequivocally determined, as localization of nascent hcv rna or the hcv negative strand has yet to be clearly demonstrated at the ultrastructural level e.g., [ , ] . this may be due to poor accessibility of the interior of membrane structures to rna labeling reagents and/or to incompatibility between em sample preparation techniques that preserve ultrastructural detail and currently available rna labeling methods. we will discuss three lines of evidence that hcv rna replication occurs in association with dmvs. at this point in time none of these models has been convincingly demonstrated to be a mechanism of dmv formation. however, kinetic analysis of the ultrastructural membrane alterations following hcv [ , ] infection have identified single-membrane vesicles early in hcv infection, while similar studies of enterovirus-infected cells have identified single-membrane tubules [ ] , which could be precursors of dmvs and thus might argue for models a and/or c presented above. the membranous web appears to be the site of hcv rna genome replication. by immunofluorescence microscopy, newly synthesized viral rna metabolically labeled with -bromouridine '-triphosphate (brutp) co-localizes with ns a protein as a marker of the membranous web [ , , ] . furthermore, negative strand hcv rna, which is a necessary intermediate of hcv replication, has been detected at ns a-positive foci by confocal microscopy [ ] . the precise localization of the hcv replicase complex at the different membranous structures that make up the membranous web (e.g., smvs, dmvs, and mmvs) has not yet been unequivocally determined, as localization of nascent hcv rna or the hcv negative strand has yet to be clearly demonstrated at the ultrastructural level e.g., [ , ] . this may be due to poor accessibility of the interior of membrane structures to rna labeling reagents and/or to incompatibility between em sample preparation techniques that preserve ultrastructural detail and currently available rna labeling methods. we will discuss three lines of evidence that hcv rna replication occurs in association with dmvs. first, immunoelectron microscopy using an anti-dsrna monoclonal antibody suggests that dsrna labeling is associated with dmvs in hcv-replicating cells [ , ] and with dmvs immunoisolated from hcv-infected cells [ ] . while it is assumed that dsrna-containing replicative intermediates are formed as a result of viral negative-strand rna synthesis and thus indicative of sites of viral rna replication, it is likely that some fraction of dsrna-containing foci is not actively engaged in rna synthesis and instead represents inactive replication complexes or products of replication. second, analysis of the kinetics of hcv rna and dmv accumulation in acutely infected cells has shown a correlation between the two, while the appearance of mmvs lags significantly behind both [ ] , suggesting that dmvs might be the principal site of hcv rna replication and that mmvs are not a major site of hcv rna replication. there are two caveats to this interpretation: a role for single-membrane vesicles (smvs) in hcv replication was not specifically evaluated in this study, and correlation between dmv and viral rna accumulation does not exclude the possibility that dmvs serve as storage sites for replication-inactive hcv rna molecules that have been synthesized elsewhere. a similar study of the kinetics of poliovirus-induced membrane alterations found that the appearance of smvs correlated best with the exponential phase of viral rna synthesis, while dmvs appeared only later in infection [ ] . third, as already mentioned above, perhaps the most direct evidence that dmvs are sites of hcv rna synthesis is a study of replicon cells expressing epitope-tagged ns b [ ] . dmvs are presented in affinity-purified membranes from these cells, and about half of these dmvs can be labeled by brutp in in vitro replicase assays. however, the immunogold labeling of brutp was observed both on the exterior and in the interior of dmvs, leaving unresolved the question of whether the hcv replicase is located on the interior or on the exterior of the dmv. in favor of the former model is the observation that hcv rna is sensitive to nuclease digestion only in the presence of detergents e.g., [ , , ] , and this is also true of in vitro replicase activity of membranes isolated from hcv replicon cells [ ] . on the other hand, replicase localization within a membrane-enclosed compartment raises the question of how ribonucleotides and other molecules necessary for rna synthesis gain access to the replicase complex and how progeny rna genomes exit the membrane structure. in an ultrastructural study using cryoelectron tomography of hcv-infected cells, only about % of all dmvs had an identifiable opening connecting the interior to the cytosol [ ] . the outer membrane bilayer of % of dmvs was contiguous with the er membrane, but the inner bilayer appeared to be closed. a spherical dmv of nm in diameter [ , ] , assuming a ribonucleoside tri-phosphate (rntp) concentration of mm [ ] and no exchange with the cytosol, will contain only about molecules of each rntp, which is enough to synthesize only about one complete hcv genome. therefore, any model of hcv replicase localization within a dmv or membrane structure of comparable volume must also allow for a mechanism for rntp replenishment. it may be that only the minority of dmvs with an opening to the cytosol are engaged in active genome replication. another possibility is that protein channel(s) in both dmv membrane bilayers mediate the entry of rntps and other necessary factors, though this has not been experimentally demonstrated. an alternative model is that the hcv replicase complex is located on the cytosolic surface of dmvs or another membrane compartment. this would be analogous to the poliovirus replicase complex, which is associated with the cytosolic face of virus-induced vesicles [ ] . coronaviruses also generate dmvs during infection [ , ] ; the nonstructural protein (nsp ) of murine hepatitis coronavirus has been shown to localize to the cytosolic face of dmvs [ ] . although this would appear to be inconsistent with the known nuclease resistance of hcv rna and hcv replicase activity, it would resolve the problem of accessibility of rntps and other factors to the replicase complex. membrane association of the hcv replicase complex has also been shown to shield viral rna from innate immune recognition. the hcv rna genome and its replicative intermediates are thought to encode potent pathogen-associated molecular patterns (pamps) recognized by host cell pattern recognition receptors (prrs) such as retinoic acid-inducible gene (rig-i) and melanoma differentiation-associated protein (mda ) (reviewed in [ ] ). recently, neufeldt et al. showed that both rig-i and mda are excluded from the hcv replication organelles, and that addition of a nuclear localization signal to rig-i or mda resulted in their replicase complex localization and stimulation of immune response [ ] . these results would appear to support a model of hcv replicase and viral rna localization inside dmvs, and suggest that enclosure of the hcv replicase and hcv rna within membranous structures restricts access of prrs to hcv-encoded pamps and by doing so, protect viral rna from innate immune recognition. despite important recent advances in our understanding of the molecular requirements of the hcv replication process, many important questions remain unresolved. it is still not clear how viral proteins and host factors work in concert to alter the host er membranes to initiate the formation of mws. it is also not known whether the replication of hcv occurs on the exterior or in the interior of dmvs. advances in microscopy techniques, including cryo-electron tomography, 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virus-induced membranous web and associated nuclear transport machinery limit access of pattern recognition receptors to viral replication sites the authors declare no conflict of interest. the following abbreviations are used in this manuscript: key: cord- -bxpzo xu authors: aydin, malik; naumova, ella a.; paulsen, friedrich; zhang, wenli; gopon, felix; theis, christian; lutz, sören; ehrke-schulz, eric; arnold, wolfgang h.; wirth, stefan; ehrhardt, anja title: house dust mite exposure causes increased susceptibility of nasal epithelial cells to adenovirus infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: bxpzo xu adenovirus (adv) infections in the respiratory tract may cause asthma exacerbation and allergic predisposition, and the house dust mite (hdm) may aggravate virus-induced asthma exacerbations. however, the underlying mechanisms of whether and how adv affects asthmatic patients remains unclear. to address this question, we investigated nasal epithelial cells (naepcs) derived from a pediatric exacerbation study cohort for experimental analyses. we analyzed twenty-one different green-fluorescent protein- and luciferase-tagged adv types in submerged d and organotypic d cell culture models. transduction experiments revealed robust transduction of adv type (adv ) in naepcs, which was associated with an increased uptake of adv in the presence of hdm. in healthy and asthmatic naepcs exposed to hdm before infection, we observed a time- and dose-dependent increase of adv uptake associated with upregulation of entry receptors for adv . furthermore, electron microscopic and histologic analyses of d cell cultures revealed an impairment of the respiratory cilia after hdm exposition. this ex vivo pilot study shows the impact of adv infection and hdm exposition in a primary cell culture model for asthma. currently, more than human adenoviruses (adv) (http://hadvwg.gmu.edu/) have been identified. they have been phylogenetically divided into seven species (a to g) based on hemagglutination features, oncogenic potential in rodents, dna homology, and genome organization [ , ] . adenoviruses are non-enveloped viruses, which contain a double-stranded dna viral genome of an approximate length of - kbp [ ] . the capsid consists of capsomeres, and the virus shape is icosahedral with hexon-, penton-, and fiber proteins including shaft and knob [ ] . for adv cell entry, several cellular receptors have been described, including the coxsackie andadenovirus receptor (car), cd , sialic acid, desmoglein- (dsg- ), and heparan sulfate proteoglycan (hspg) [ ] . adv are known as pathogens, but they have also been explored as viral vectors in gene therapeutic applications. in clinics, human adv have become increasingly important in recent years. they cause different clinical symptoms in a wide range of diseases, e.g., pneumonia, conjunctivitis, gastroenteritis, or myocarditis [ ] [ ] [ ] [ ] . threatened groups include children younger than five years of age or immune-deficient patients after transplantation. in addition, adv have also been causatively associated with pneumonia outbreaks in us-military bases [ ] . several adv can be isolated from patients with lung infections [ ] , and here we addressed the question of whether this is associated with asthma exacerbation. there is strong evidence that asthma exacerbations are associated with virus-mediated upper and/or lower respiratory infections [ ] , and therefore there is a broad interest in studying the role of viruses in asthma pathogenesis and exacerbation [ ] [ ] [ ] [ ] . it was described that predominantly rhinovirus (rv), and other viruses, particularly adenoviruses (adv), cytomegalovirus, bocavirus, coronavirus, herpes simplex virus, influenza virus, parainfluenza virus, respiratory syncytial virus, or enteroviruses, may be involved in asthma development [ ] . in addition to viruses, house dust mite (hdm) as a major allergen is strongly associated with asthma and presents an important risk factor for virus-induced asthma exacerbation [ , ] . although various studies have investigated the molecular roles of some respiratory viruses in allergic pathways [ , ] , the relationship between adv infection and hdm sensitization in asthma exacerbation has not been sufficiently analyzed. here, we aimed at analyzing this relationship in primary nasal epithelial cells (naepcs) as an ex vivo cell culture model, to better study allergies and the immunology of asthma [ ] [ ] [ ] . to analyze adv infection in the context of hdm sensitization, we utilized primary nasal naepcs derived from our pediatric exacerbation study cohort in submerged d and organotypic d cell culture models. for this approach, we applied twenty-one previously described green-fluorescent protein (gfp)-and luciferase-tagged adv types [ ] , encompassing adv of all seven species. moreover, we performed electron microscopic and histologic analyses of d cell cultures to study the impairment of respiratory cilia after hdm exposure. we found that hdm exposure may increase adv infection in vitro, and that major adv surface receptors may play a role. we established a pediatric exacerbation study network in two children's hospitals in germany. participating study centers were wuppertal (witten/herdecke university, germany) and niederberg/velbert (teaching hospital of the university hospital essen, germany). pediatric subjects with a chronic bronchitis/wheeze ( months to ≤ years of age) or asthma (> years to years of age) suffering from acute exacerbation episodes were recruited. in addition, healthy controls (age range > months to years of age) were recruited for comparison. a detailed study description and protocol is currently in preparation for publication. for the experimental approach used here, only naepcs from asthmatics and healthy donors were used (n = per each group as biologic with each n = technical replicates). for the control experiments, cells from healthy donors were compared with cells from asthmatic subjects (treated and untreated). only asthmatics with an hdm sensitization (released ige levels in serum, positive immunocap ® results for dermatophagoides pteronyssinus, cap class > ) were chosen for this work. furthermore, only healthy donors without any positive sensitization to dermatophagoides pteronyssinus were selected. clinically, asthmatic patients showed an immunologic reaction to dermatophagoides pteryonossinus, and the healthy donors did not present any allergic reactions. for this prospective study, all analyzed biomaterials and data involving human participants were collected in concordance with the ethical standards and with the helsinki declaration and its later amendments or comparable ethical standards. ethics approval was obtained from the local ethics committees (witten/herdecke university ( / ) and medical chamber (Ärztekammer nordrhein, nr ), germany). the study was retrospectively assigned the human study at german clinical trials register (drks) with the registration number drks . all study cohort relevant data analyses were pseudonymously performed. all participated subjects or their legal custodians/parents provided a written informed consent. this article does not contain any animal studies. the naepcs were obtained by performing a nasal brushing (cytobrush eswab ® copan italia) from both nostrils, resuspended in warm begm ® medium (purchased from lonza, basel, switzerland), and shaken for s. approximately, , - , cells per nasal brushing procedure (including both nostrils) were collected, the total number was approximately × cells at passage (p ). to eliminate possible contamination with erythrocytes and fibroblasts, lysing procedures were performed before experimental set-up. after centrifugation at × g for min, the cell number was calculated using a neubauer counting chamber. cells were incubated in collagen i pre-coated t flasks (greiner bio-one, austria) for up to a maximum of two passages (p ) at • c and % co atmosphere. at p , the cells were seeded using an organotypic d air liquid interface (ali) cell culturing technique adapted to the instructions of stemcell tm technologies (https://www.stemcell.com/). for this, , cells were seeded in collagen i pre-coated transwells using pneumacult tm ali proliferation medium added to the basal and apical chambers, and the medium was changed every single day. at day , the medium in both chambers were removed, and the cells received pneumacult tm ali differentiation medium at the basal chamber. the medium was changed every to days assuring airlifting for up to weeks until the pseudostratified morphology was reached, which was confirmed by ciliary beats and production of mucus (pneumacult tm ali and differentiation medium were purchased from stemcell tm technologies, canada). naepcs were characterized through flow cytometry using cd -apc (miltenyi biotec, germany), cd (epcam)-pe (miltenyi biotec, germany), and anti-cytokeratin-fitc (miltenyi biotec, germany), followed by fixation and permeabilization using inside stain kit (miltenyi biotec, germany) according to manufacturer's instructions. the selected antibodies for the characterization of naepcs were adapted in a previously published study [ ] . the adv receptors, cd -apc (miltenyi biotec, germany), cxadr/car-pe, and dsg- -pe (affymetrix, thermo fisher scientific) were chosen for flow cytometry analyses. the specimens were fixed with . m cacodylate buffer containing . % glutaraldehyde, % polyvinylpyrrolidone, and mm nano for min. at • c. samples were washed in . m cacodylate buffer without glutaraldehyde and subsequently incubated in a solution containing % arginine-hcl, glycine, sucrose, and sodium glutamate for h at room temperature (rt). the specimens were rinsed in distilled water, followed by immersion in a mixture of each % tannic acid and guanidine-hcl for h at rt. the samples were rinsed again in . m cacodylate buffer and incubated in a % oso solution for min at rt. after three rinsing steps with . m cacodylate buffer, the specimens were dehydrated, dried in liquid co , and finally examined with a zeiss sigma sem (zeiss, oberkochen, germany) scanning electron microscope using kv acceleration voltage after sputtering with gold palladium. as detectors, the in-lens and se detectors were used. the specimens were processed for tem according to a previously published protocol [ ] . in brief, samples were fixed in ito's fixative ( . % glutaraldehyde, . % paraformaldehyde, and . % picric acid) dissolved in phosphate buffered saline (pbs) (ph = . ) and embedded in epon. semi-thin sagittal sections of µm were cut with a microtome (ultracut e; reichert jung, vienna, austria) and subsequently stained with toluidine blue. sections were viewed with an epifluorescence microscope (aristoplan; ernst leitz, wetzlar, germany) and photographed (keyence biorevo bz microscope). ultrathin sections were stained with uranyl acetate and lead citrate and viewed with a transmission electron microscope (em ; carl zeiss meditec gmbh, oberkochen, germany). the naepcs were seeded in collagen coated wells (day ). at day , the culture medium was changed. for exposition experiments, dermatophagoides pteronyssinus (citeq biologics, netherlands, product code: . . ) was used in different concentrations ( µg/ml, µg/ml, and µg/ml) for two time points. the first time point included an exposition duration of h. the read-out parameters will be presented in the course of the manuscript. after h exposition with dermatophagoides pteronyssinus, the supernatant was removed, fresh begm ® cell culture medium was added, and naepcs were incubated for h. after this incubation time, a second exposition with dermatophagoides pteronyssinus was performed for h, and after that, the read-out parameters were measured (see below). these two time points were presented as h and h throughout the manuscript. in addition, to easily follow the manuscript, the term hdm was used for dermatophagoides pteronyssinus throughout the manuscript. we used different recombinant adv types deleted for the early gene region e , which was replaced by a transgene expression cassette encoding the reporter genes luciferase and gfp [ ] . in brief, recombinant viruses were amplified in permissive cell lines and purified using cesium-chloride gradients as described before [ ] . we explored n = different adv types derived from different species to transduce naepcs. twenty-four hours before transduction, we seeded × naepcs per well in collagen i pre-coated wells plates. gfp and luciferase expressing adv types were added to each well using viral particles per cell (vpc). twenty-four hours post transduction of naepcs, we determined luciferase activity within the transduced cells using the nano-glo ® luciferase assay system from promega. to perform the luciferase assay, we removed µl supernatant, added nano-glo ® luciferase assay substrate and nano-glo ® luciferase assay buffer (dilution: : ) to cells, and incubated for up to min at • c and % co until cells were detached. subsequently, luciferase expression levels were quantified using a tecan elisa reader. statistical analyses were performed using graphpad prism version . . for windows, graphpad software, la jolla california usa, www.graphpad.com. data were presented as mean and standard error of the mean (sem) or as absolute values with percentages or fold changes (n = to ). comparisons between two groups were performed with unpaired/paired, two-tailed, and t-tests. comparisons between more than two groups were performed with one-way-anova and holm-sidak's multiple comparison posttest. the significance levels were set at * p < . , ** p < . , and *** p < . . to prove the purity of the cultured cells derived from our pediatric exacerbation study cohort, flow cytometric analyses were performed for each culture. as presented in figure a , the isolated cells were cd neg epcam pos pan-cytokeratin pos . in addition, the morphology of the cultured cells was analyzed through raster electron microscopy (rem) as well as histology, which revealed the purity of the cultures (figure b,c) . to address the question whether naepcs were submissive to adv infection, we transduced cultured naepcs in a monolayer with different luciferase and gfp expressing adv types ( figure ) at vpc. twenty-four hours post-transduction, the luciferase activity of infected cells was determined. permeabilization, anti-cytokeratin-fitc was used for intracellular staining. (b) after passage (p ), the cells were seeded for organotypic d air-liquid interface cultures. nasal mucus secretion and ciliary beats were observed through light microscopy after to weeks of culturing. final specimens were then processed for raster electron microscopic imaging. (c) histologic analyses confirmed the morphology of the nasal epithelial cell population ( x magnification). to address the question whether naepcs were submissive to adv infection, we transduced cultured naepcs in a monolayer with different luciferase and gfp expressing adv types ( figure ) at vpc. twenty-four hours post-transduction, the luciferase activity of infected cells was determined. as shown in figure , adv type , followed by , , , and , showed the highest transduction rates in naepcs if directly compared to other adv types. therefore, adv , as a common respiratory virus and the most analyzed virus in terms of gene therapeutic approaches, was then used for further analyses. to determine the infections rates of adv in naepcs, we transduced naepcs with adv using different vpc and measured the luciferase expression level and visualized gfp pos cells through immunofluorescence microscopy. twenty-five hours post-transduction, there was a clear correlation as shown in figure , adv type , followed by , , , and , showed the highest transduction rates in naepcs if directly compared to other adv types. therefore, adv , as a common respiratory virus and the most analyzed virus in terms of gene therapeutic approaches, was then used for further analyses. to determine the infections rates of adv in naepcs, we transduced naepcs with adv using different vpc and measured the luciferase expression level and visualized gfp pos cells through immunofluorescence microscopy. twenty-five hours post-transduction, there was a clear correlation between the virus dose, the luciferase expression levels, and the gfp + -expressing cell numbers. a cytopathic effect of infected cells associated with adenovirus infection was not observed (figure a,b) . it is assumed that patients with allergies and asthma suffer from increased virus infections [ ] . therefore, we studied the virus transduction efficiency of adv in hdm-provoked naepcs. different concentrations of hdm ( µg/ml, µg/ml, and µg/ml) were used to provoke naepcs ex vivo, and the schematic outline of this experiment is shown in figure . between the virus dose, the luciferase expression levels, and the gfp + -expressing cell numbers. a cytopathic effect of infected cells associated with adenovirus infection was not observed (figure a,b) . between the virus dose, the luciferase expression levels, and the gfp + -expressing cell numbers. a cytopathic effect of infected cells associated with adenovirus infection was not observed (figure a,b) . it is assumed that patients with allergies and asthma suffer from increased virus infections [ ] . therefore, we studied the virus transduction efficiency of adv in hdm-provoked naepcs. different concentrations of hdm ( μg/ml, μg/ml, and μg/ml) were used to provoke naepcs ex vivo, and the schematic outline of this experiment is shown in figure . during the nasal brushing procedure, we collected the cells in cell culture medium, centrifuged at x g for min, and washed with pbs. the cell pellet was resuspended in cell culture medium, and the cells were seeded in collagen i pre-coated t flasks. after passage (p ) was reached, naepcs were collected and seeded in collagen i pre-coated well plates. different hdm concentrations were used ( μl/ml, μg/ml, μg/ml). the transduction concentration of adv was set at virus particle per cell (vpc). this figure was generated using biorender.com luciferase assays were performed one and three days post-transduction correlating with adv transduciton rates. there was a trend of increased adv transduction in healthy control cells and in cells derived from asthmatics. especially on day after hdm exposure, we observed an enhanced adv mediated luciferase activity in hdm-provoked naepcs from asthmatics, compared to naepcs derived from healthy cells ( figure ). during the nasal brushing procedure, we collected the cells in cell culture medium, centrifuged at × g for min, and washed with pbs. the cell pellet was resuspended in cell culture medium, and the cells were seeded in collagen i pre-coated t flasks. after passage (p ) was reached, naepcs were collected and seeded in collagen i pre-coated well plates. different hdm concentrations were used ( µl/ml, µg/ml, µg/ml). the transduction concentration of adv was set at virus particle per cell (vpc). this figure was generated using biorender.com luciferase assays were performed one and three days post-transduction correlating with adv transduciton rates. there was a trend of increased adv transduction in healthy control cells and in cells derived from asthmatics. especially on day after hdm exposure, we observed an enhanced adv mediated luciferase activity in hdm-provoked naepcs from asthmatics, compared to naepcs derived from healthy cells ( figure ). to shed light on the underlying mechanism for enhanced adv uptake, major adv receptor expression levels, in particular, car, cd , and dsg- , were characterized on naepcs through flow cytometry. in comparison to hdm-provoked naepcs, we observed a significant increase of car and cd expression levels on naepcs of asthmatics as well as on naepcs of healthy donors. thus, we speculate that an increased adv receptor expression level may explain the enhanced adv transduction efficiency of naepcs after hdm exposition (figure a,b) . in contrast, there was no figure . the effect of hdm stimulation and adv infection on naepcs. naepcs were stimulated at different time points (day and day ) with different hdm concentrations ( µg/ml, µg/ml, and µg/ml), and the cells were subsequently transduced with adv at virus particle numbers per cell (vpc). twenty-four hours post-transduction, luciferase assays were performed. we observed an increased adv transduction efficiency in pre-stimulated naepcs with hdm, particularly at day in asthmatic specimens. this was in contrast to samples of healthy controls. values were normalized and presented as fold change given as mean and standard error of mean (sem). to shed light on the underlying mechanism for enhanced adv uptake, major adv receptor expression levels, in particular, car, cd , and dsg- , were characterized on naepcs through flow cytometry. in comparison to hdm-provoked naepcs, we observed a significant increase of car and cd expression levels on naepcs of asthmatics as well as on naepcs of healthy donors. thus, we speculate that an increased adv receptor expression level may explain the enhanced adv transduction efficiency of naepcs after hdm exposition (figure a,b) . in contrast, there was no significant difference in the expression level of dsg- on hdm-treated naepcs, compared to healthy naepcs controls (figure c ). the mean-pe values for car were set as absolute numbers. as shown, the asthmatic group had significant differences at car expression levels after hdm stimulation, particularly at day compared to day . (b) the mean-apc values for cd were set as absolute numbers. as shown, the healthy control group showed significant differences between time points and concentration levels, compared to the asthmatic group (c) desmoglein- -pe was not significantly different in terms of time points and concentration levels in healthy controls and asthmatics. to analyze whether hdm influences the barrier function on naepcs that may lead to enhanced adv transduction, we provoked d naepcs cultures with hdm and performed raster-em on days (d ) and day (d ). raster-em scan provided interesting insights into the irritated epithelium after high dosage of hdm, in particular at d (figure a ). measuring the lengths and thickness of the ciliary, there was no statistical differences for the different groups (figure b) . histologic analyses confirmed an increased mucus production and an irritated basal layer after hdm treatment (figure c ). furthermore, we observed a different tight junction conformation in organotypic d naepcs cultures of asthmatics compared to healthy controls. the tight junction conformation in untreated asthmatic samples was tightly packed and was presented in a higher number than the untreated control samples (figure d,e) . here, we characterized the effects of hdm exposition on adv infection in an ex vivo cell culture models of naepcs of exacerbated pediatric asthmatics and healthy controls from our pediatric exacerbation study. with this experimental approach, we successfully characterized the effects of hdm exposition on human adv infection in naepcs ex vivo. using a library of luciferase-and gfp-tagged advs, we analyzed adv infection rates in naecps in the presence of hdm as a common allergen. we found that adv luciferase expression levels were significantly increased days after hdm exposure in asthmatics, compared to healthy controls, hinting towards the hypothesis that replication of adv may be influenced by hdm stimulation. however, this hypothesis needs to be further analyzed by performing adv genome replication studies in naecps. it was described previously that virus infection can synergize with allergens in the induction of asthma exacerbations. the first study describing the interaction between rv infection and hdm exposure was performed by bossios et al. ( ) [ ] . they observed an increase in cytokine levels in immortalized human bronchial epithelial cell line when rv and hdm were applied or when cells were provoked with hdm before rv infection [ ] . interestingly, akbarshahi et al. ( ) found a similar effect by showing that hdm exposure affected the antiviral response provoked by virus infections [ ] . furthermore, golebski et al. ( ) described a relationship between hdm and poly i:c stimulation in terms of gene and protein expression [ ] . the results of our study broaden the preceding findings, as we have observed an increased uptake of adv after hdm stimulation potentially mediated by enhanced major adv receptor expression. our results represent the first in vitro evidence that hdm stimulation may increase susceptibility to adv infections. adenovirus receptors, including car, cd , or dsg- , are required for uptake of adv into the respective target cell. adv mostly interacts with car, whereas cd and dsg- receptors are used by adv , , , and others. car is localized on the basolateral and apical surface of the epithelial cells. lung epithelial cells are interconnected by tight junctions. by this formation, the basolateraly-located car receptors may protect the cell from the interaction with adv [ ] [ ] [ ] . the flow cytometric analyses of hdm-provoked naepcs showed higher levels of car on naepcs. in contrast to the work of excoffon et al., our data do not include the analyses of a car localization shift but include the adenoviral susceptibility of hdm-provoked cells [ ] . we speculate that an upregulation of adv receptors upon hdm inhalation may lead to increased susceptibility to adv infection in patients with allergic rhinitis or asthma exacerbation in vivo. however, to support this hypothesis, additional in vivo experiments are needed. we furthermore noticed a low but measurable increase of cd receptor expression levels on naepcs after hdm exposition. contrary to our results, tsai and colleagues ( ) observed decreased cd levels and increased apoptosis in primary nasal mucosa samples from adult mild asthmatics when cultured with hdm extracts [ ] . naepcs gained increased attention as a model to study asthma development, to study immune responses during virus infection in asthma patients, and to define biomarkers specific for asthma and virus infections [ ] [ ] [ ] . their innate immune reactions such as toll-like receptor pathways, secretion of il- , or thymic stromal lymphoprotein boost adaptive immune responses and start a broad range of stimulation processes, e.g., activation of naïve t cells [ ] . by using naepcs from healthy and allergic subjects, vroling et al. showed that both groups had differences in the chemokine, growth factor, and transcription factor levels [ , ] . thus, naecps represent a valuable alternative as an ex vivo model, compared to tracheal or bronchial epithelium of the small airways. this further supports the use of naecps in our study to explore the relationship between asthma development, hdm exposure, and adv infections. future studies should analyze immune responses in these cells after hdm exposure and adv infection. note that the used set-up of the present study was completely based on a primary human biomaterial database. this is in contrast to other published studies, which exemplarily used cancer cell lines to correlate their experimental findings with clinical observations. in summary, this work provides novel insights into the mechanism of adenoviruses on the airway epithelium of asthmatics. to the best of our knowledge, this is the first ex vivo study presenting the impact of hdm sensitization on adv infection in an in vitro exposition model. our study may open new paths for the interaction between allergens and virus infections, and may provide a basis for further potential gene therapeutic approaches in treatment of childhood asthma exacerbation. this work analyzed the interaction of the hdm exposition in the presence of adv infection in an in vitro model based on naepcs. we demonstrate that a pre-stimulation with hdm may induce an increased adv infection rate at least partially mediated by increased car receptor expression. moreover, electron microscopy and histologic imaging revealed an effect on the cilia in organotypic d cell cultures when exposed to hdm that may be associated with an increased adenoviral transduction. to the best of our knowledge, this pilot work contributes novel aspects to the hypotheses regarding whether an allergic predisposition may increase an adenovirus infection. molecular evolution of human adenoviruses lessons learned from adenovirus genetic content and evolution of adenoviruses human adenovirus: viral pathogen with increasing importance epidemiology, clinical presentation and respiratory sequelae of adenovirus pneumonia in children in kuala lumpur genomic foundations of evolution and ocular pathogenesis in human adenovirus species d role of coxsackievirus and adenovirus receptor (car) expression and viral load of adenovirus and enterovirus in patients with dilated cardiomyopathy adenovirus diseases: a systematic review and meta-analysis of case reports adenovirus-associated deaths in us military during postvaccination period regional, age and respiratory-secretion-specific prevalence of respiratory viruses associated with asthma exacerbation: a literature review rhinovirus infections change dna methylation and mrna expression in children with asthma house dust mite impairs antiviral response in asthma exacerbation models through its effects on tlr home environmental interventions for house dust mite childhood asthma and infection: virus-induced exacerbations as determinants and modifiers viruses and bacteria in acute asthma exacerbations-a ga( ) len-dare systematic review comparison of expression profiles induced by dust mite in airway epithelia reveals a common pathway primary nasal epithelium exposed to house dust mite extract shows activated expression in allergic individuals the immunology of asthma an engineered virus library as a resource for the spectrum-wide exploration of virus and vector diversity novel flow cytometry approach to identify bronchial epithelial cells from healthy human airways serum-induced keratinization processes in an immortalized human meibomian gland epithelial cell line response to infections in patients with asthma and atopic disease: an epiphenomenon or reflection of host susceptibility? rhinovirus infection and house dust mite exposure synergize in inducing bronchial epithelial cell interleukin- release high degree of overlap between responses to a virus and to the house dust mite allergen in airway epithelial cells regulation of the coxsackie and adenovirus receptor expression is dependent on cystic fibrosis transmembrane regulator in airway epithelial cells effects of cigarette smoke on barrier function and tight junction proteins in the bronchial epithelium: protective role of cathelicidin ll- isoform-specific regulation and localization of the coxsackie and adenovirus receptor in human airway epithelia complement regulatory protein cd induces autophagy against oxidative stress-mediated apoptosis in normal and asthmatic airway epithelium nasal epithelial cells can act as a physiological surrogate for paediatric asthma studies we thank the center for biomedical education and research, school of life sciences (zbaf), and its members for their intellectual input during seminars and lectures of the zbaf ph.d. program. we thank manuela besser for the technical preparation of few d cultures for histologic analyses. we appreciate the support of susanne haussmann from the witten/herdecke university, germany; elke kretschmar from the friedrich-alexander-university erlangen, germany; and the colleagues, including nurses, physicians, and technicians of the emergency room of the children's hospital of the helios university medical hospital of the witten/herdecke university, germany. in addition, we thank the children and parents who participated in this study. finally, figure was created with biorender.com. funding: this research received funding from the internal research promotion of the faculty of health at witten/herdecke university, germany (iff - ). the funding organizations had no role in the design and conduction of the study; sample collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; or decision to submit the manuscript for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. the authors have declared that they have no competing interests. this work has not been published before, and it is not under consideration for publication elsewhere. the manuscript has been approved for publication by all co-authors. key: cord- -whfyczcj authors: seissler, tanja; marquet, roland; paillart, jean-christophe title: hijacking of the ubiquitin/proteasome pathway by the hiv auxiliary proteins date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: whfyczcj the ubiquitin-proteasome system (ups) ensures regulation of the protein pool in the cell by ubiquitination of proteins followed by their degradation by the proteasome. it plays a central role in the cell under normal physiological conditions as well as during viral infections. on the one hand, the ups can be used by the cell to degrade viral proteins, thereby restricting the viral infection. on the other hand, it can also be subverted by the virus to its own advantage, notably to induce degradation of cellular restriction factors. this makes the ups a central player in viral restriction and counter-restriction. in this respect, the human immunodeficiency viruses (hiv- and ) represent excellent examples. indeed, many steps of the hiv life cycle are restricted by cellular proteins, some of which are themselves components of the ups. however, hiv itself hijacks the ups to mediate defense against several cellular restriction factors. for example, the hiv auxiliary proteins vif, vpx and vpu counteract specific restriction factors by the recruitment of cellular ups components. in this review, we describe the interplay between hiv and the ups to illustrate its role in the restriction of viral infections and its hijacking by viral proteins for counter-restriction. the human cell is in a continuous arms race with various viruses. this has led to the coevolution of cellular restriction factors on the one hand and viral proteins for counter-defense on the other hand. restriction factors are generally induced as a result of an interferon response-they use unique mechanisms to impair specific steps of the replication cycle and they exhibit a dominant and autonomous effect. in this continuous fight, the ubiquitin-proteasome system (ups) plays a central role on the cellular as well as on the viral side. the cell expresses restriction factors, some of which are themselves components of the ups, targeting viral proteins for degradation and thereby inhibiting some crucial steps of the viral life cycle. however, viruses have evolved to use the ups to their own benefits, subverting components of the ups to degrade restriction factors, thereby protecting themselves from the cellular defense machinery to allow their dissemination. in this review, we will describe the mechanisms by which the human immunodeficiency viruses (hiv- and ) use and subvert the ups in the continuous battle between cellular defense and viral counter-defense. the ubiquitin-proteasome system (ups) is an important pathway in the cell, ensuring regulation of the protein pool in the cytoplasm as well as in the nucleus. the ups is constituted by three main components: the proteasome holoenzymes, several ubiquitin ligases and a large variety of deubiquitinating enzymes (dubs) [ ] . ubiquitin is a ubiquitously expressed and well-conserved the ups plays a central role in many viral infections (reviewed in [ ] [ ] [ ] ), with five main modes of action on the viral life cycle: ( ) some cellular e ubiquitin ligases recognize viral proteins and induce their ubiquitination, which can have a positive effect on viral replication. for instance, ubiquitination of the p domain of the hiv- gag polyprotein is important for the interaction of p with the escrt machinery. however, the mono-ubiquitination of lysine residues within the p domain (k and k ) does not seem to be sufficient to facilitate budding of new virions, the latter being also dependent on the cumulative ubiquitination of nc-p (nucleocapsid-peptide ) domain [ ] [ ] [ ] [ ] . ubiquitination of the hiv- accessory protein tat by cellular e ligases stimulates transcription of viral rna [ , ] . ( ) ubiquitination of viral proteins can also induce their degradation, thereby blocking the viral life cycle. this is a strategy used by certain restriction factors. the polymerase pb (protein binding ) of the influenza a virus (iav) for example is ubiquitinated (k -linked ubiquitin) by the cellular e ubiquitin ligase trim (tripartite motif-containing protein ), followed by its degradation by the proteasome [ ] . this seems to be a general mechanism as pb proteins derived from various iav serotypes (h n (hemagglutinin neuraminidase ), h n , h n or h n ) associate with trim in multiple cell types and this suggests that pb has not yet adapted to avoid trim targeting [ ] . the human herpesvirus type i (hsv- ) capsid protein vp has also been shown to be degraded by the ubiquitin proteasome system, leaving the viral genome exposed to innate immune sensors [ ] . interestingly, trim α was reported to inhibit hsv- and - replication at an early stage of the infection cycle [ ] , suggesting a role for this or related protein in cytosolic sensing of herpesvirus capsids. ( ) certain viruses have evolved to recruit the cellular e ligases to induce the degradation of cellular proteins that might have harmful effects on the viral life cycle. for instance, the protein e of human papillomavirus (hpv) recruits the cellular e ubiquitin ligase e -ap to induce ubiquitination and degradation of p , thereby allowing viral replication [ , ] . the nsp (non-structural rna binding protein ) protein of rotaviruses subverts the skp -cul -fbox (scf) e ligase to induce the ubiquitination and degradation of β-trcp (β-transducin repeat containing protein). β-trcp is by itself a substrate adaptor of an e ligase and its degradation leads to accumulation of the nf-κb inhibitor iκb, resulting in inhibition of the nf-κb induced antiviral responses [ , ] . these mechanisms are important for hiv replication and will be detailed in section . ( ) other viruses directly encode their own e ligases. kaposi sarcoma herpesvirus (kshv) protein k and k (ring-ch family of ligases) ubiquitinate mhc-i (major histocompatibility complex i), resulting in its down-regulation from the cell surface through a clathrin-dependent sorting pathway to an endolysosomal compartment [ , ] . this endolysosomal sorting requires k -linked instead of k -linked polyubiquitin chains [ ] . another well-known example is the icp protein (infected cell protein ) of hsv- , an e ubiquitin ligase which induces the degradation of the nd (nuclear domain ) nuclear body components pml (promyelocytic leukemia protein) and sp through the ups, thereby avoiding antiviral sensing [ , ] . icp has also been shown to have a ring-independent e ligase activity that polyubiquitinates the e enzyme cdc . icp influences many cellular pathways and is required for the activation of most viral and many cellular genes, for reactivation from latency and suppression of innate immunity [ ] . ( ) finally, ubiquitin modifications can be reversed by the isopeptide-bond specific proteolytic activity of dubs. in addition to cellular dubs, it has been reported that various virus families code their own dubs (coronavirus, herpesvirus etc.) to evade host antiviral immune response and promote virus replication (for a recent review see [ ] ). for instance, in the herpesviridae family, a variety of dubs play an important role in the virus life cycle (e.g., ul usp (ubiquitin ligase ubiquitin specific protease) of hsv- , tegument protein pul of human cytomegalovirus (hcmv)). regarding hiv- , a recent study reported that several cellular dubs (usp and usp , ubiquitin specific protease family) play an important role in its replication by regulating gag processing and thus the infectivity of released virions and simultaneously the entry of gag into the ups and mhc-i pathway [ ] . moreover, this study showed that treatment with dub inhibitors targeting usp causes a general gag processing defect, indicating that usp interacts with gag and prevents its entry into the ups. similarly, proteasome inhibitors have been shown to impact hiv- replication by reducing the release and maturation of infectious particles [ , ] or by suppressing its transcription [ ] . taken together, these studies suggest a potential antiretroviral activity of dub and proteasome inhibitors. the importance of the ups in antiviral restriction will be discussed here using hiv as an example. hiv- and are retroviruses of the genus lentivirus. their genome is composed of two (+) single stranded rnas encoding the gag, pol and env polyproteins, which correspond to the structural (matrix, capsid, nucleocapsid and p ), enzymatic (protease, reverse-transcriptase and integrase) and envelope (transmembrane and surface) viral proteins. in addition, the genome of these two viruses express two regulatory (tat and rev) and four auxiliary (nef, vpu/vpx, vpr and vif) proteins, which regulate several steps in the viral life cycle [ , ] . the main difference between hiv- and hiv- is the lack of the vpu protein in the former, which is replaced by vpx [ ] . following viral attachment and entry into the target cell, the dimeric viral genomic rna is partially uncoated and transported to the cell nucleus. concomitantly, reverse transcription of the viral genomic rna takes place to form the pre-proviral dna, which is then integrated into the cellular genome. the integrated provirus mediates the synthesis of new full-length viral rna (or unspliced rna), which will be used as genomic rna encapsidated into viral particles and as mrna for structural and enzymatic proteins and mono-and multi-spliced viral mrnas, which encode the viral envelope and the regulatory and auxiliary proteins in the infected cell. finally, the components of the viral particle assemble at the plasma membrane, where new viral particles bud, maturate and disseminate to other host cells in the infected organism ( figure ) [ , , [ ] [ ] [ ] [ ] . during its life cycle, hiv is subjected to different cellular restriction factors (figure ), the first line of defense of cellular immunity. the newly discovered serinc (serine incorporator ) and serinc proteins target the very beginning of the viral life cycle by inhibiting correct fusion of the viral envelope with the plasma membrane, thereby preventing the virus from entering into a new host cell [ , ] . ifitm (interferon-induced transmembrane) proteins , and also target the viral entry into the cell by inhibiting viral fusion with target cells. the exact mechanism of restriction is yet a matter of debate, as well as whether ifitm incorporation in virions or its expression in target cells is responsible for the antiviral effect. ifitm proteins might act on env to inhibit its functions in viral fusion and it has been shown that some mutations in the env protein can indeed confer resistance to ifitm restriction [ ] [ ] [ ] [ ] [ ] [ ] . once the virus has entered the cell, trim α (tripartite motif-containing protein α) can inhibit the early steps of the viral life cycle in a species-specific manner by accelerating viral uncoating [ ] [ ] [ ] . the viral capsid protein also seems to be the target of myxovirus resistance (mx /b), a restriction factor that inhibits nuclear import and subsequent integration of the provirus through an unknown mechanism. some mutations in the capsid protein have been shown to confer resistance to mx and particularly some mutations located at the site of interaction with cyclophilin a, an important host factor for hiv- infectivity [ ] [ ] [ ] [ ] [ ] [ ] . samhd (sterile alpha motif and histidine aspartate domain-containing protein ) also targets the early phase of viral infection: this deoxynucleotide-triphosphohydrolase inhibits reverse transcription by depleting the pool of cellular dntps (deoxy nucleotide triphosphates) [ , ] . during reverse transcription of the viral rna, the restriction factor apobec g (apolipoprotein b mrna editing enzyme, catalytic polypeptide-like g, or a g) and other factors from the apobec family, can induce g to a hypermutations, which prevent production of functional viral proteins [ ] [ ] [ ] . the amount of viral proteins that are produced in an infected cell can be limited by schlafen (slfn ). due to the bias of hiv- towards a/u rich codons, the virus stimulates production of corresponding trnas by the cell to increase viral translation, a mechanism that seems to be partly counteracted by slfn , which binds trnas in a codon-specific manner [ ] [ ] [ ] . the final steps in the viral life cycle can be targeted by tetherin/bst (bone marrow stromal antigen ), which inhibits release of new viral particles from the host cell [ ] [ ] [ ] and march (membrane-associated ring-ch protein), which decreases incorporation of envelope proteins into newly produced virions, thereby decreasing their infectivity [ ] . two of these restriction factors, trim α and march , use the ups to exert their restricting activity. hiv is able to counteract restriction factors using its accessory proteins ( figure ): nef prevents serinc incorporation into virions by mediating its relocalization to late endosomes through interaction with the clathrin adaptor ap- [ , ] . vif counteracts a g by inducing its proteasomal degradation as well as by reducing its transcription and translation [ , [ ] [ ] [ ] . vpx (and vpr of certain simian immunodeficiency virus (siv) strains) counteracts samhd by inducing its proteasomal degradation [ , , ] . vpu (env for hiv- and nef or vpu for siv) counteracts bst /tetherin by sequestering it away from sites of viral budding [ , , ] . amongst these accessory proteins, vif, vpx and vpu hijack the ups to exert their counter-defense. in the following section, we will discuss in detail the restriction factors as well as the viral proteins which use the ups for their respective mechanisms. infected cell can be limited by schlafen (slfn ). due to the bias of hiv- towards a/u rich codons, the virus stimulates production of corresponding trnas by the cell to increase viral translation, a mechanism that seems to be partly counteracted by slfn , which binds trnas in a codon-specific manner [ ] [ ] [ ] . the final steps in the viral life cycle can be targeted by tetherin/bst (bone marrow stromal antigen ), which inhibits release of new viral particles from the host cell [ ] [ ] [ ] and march (membrane-associated ring-ch protein), which decreases incorporation of envelope proteins into newly produced virions, thereby decreasing their infectivity [ ] . two of these restriction factors, trim α and march , use the ups to exert their restricting activity. hiv is able to counteract restriction factors using its accessory proteins ( figure ): nef prevents serinc incorporation into virions by mediating its relocalization to late endosomes through interaction with the clathrin adaptor ap- [ , ] . vif counteracts a g by inducing its proteasomal degradation as well as by reducing its transcription and translation [ , [ ] [ ] [ ] . vpx (and vpr of certain simian immunodeficiency virus (siv) strains) counteracts samhd by inducing its proteasomal degradation [ , , ] . vpu (env for hiv- and nef or vpu for siv) counteracts bst /tetherin by sequestering it away from sites of viral budding [ , , ] . amongst these accessory proteins, vif, vpx and vpu hijack the ups to exert their counter-defense. in the following section, we will discuss in detail the restriction factors as well as the viral proteins which use the ups for their respective mechanisms. one example of the cell using the ups to restrict hiv is trim α, an e -ubiquitin ligase that interacts with the viral capsid after its entry into the cell. trim α mediates a species-specific block: hiv- is restricted by the trim α proteins of old world monkeys like rhesus or cynomolgous one example of the cell using the ups to restrict hiv is trim α, an e -ubiquitin ligase that interacts with the viral capsid after its entry into the cell. trim α mediates a species-specific block: hiv- is restricted by the trim α proteins of old world monkeys like rhesus or cynomolgous monkeys, while the trim α of human or new world monkeys have no or only a very weak effect on hiv- [ , , , ] . trim α thereby constitutes one of the factors responsible for the interspecies barrier. the restriction of hiv- by trim α is mediated by the interaction of the trim α spry (spia and ryanodine receptor) domain ( figure a ) with the viral capsid in the cytoplasm of newly infected cells [ ] . this interaction leads to premature decapsidation of the viral core. moreover, viral capsid and integrase proteins are degraded ( figure c ) and the reverse transcription of the viral genome is inhibited in the presence of a restricting trim α. these effects seem to be mediated by the ups, since treatment with proteasome inhibitors restores a normal decapsidation rate and reverse transcription. it has also been shown that the proteasome co-localizes with trim α and viral cores in the cytoplasm [ , ] . trim α is also degraded by the proteasome but only in the presence of susceptible viral cores [ ] , suggesting that trim α recruits the proteasome to the viral cores and induces their degradation. this mechanism seems to be mediated by the e -ubiquitin ligase activity of trim α, through its ring domain ( figure a ) [ , ] . nevertheless, trim α inhibits integration of the proviral dna independently of the proteasome, suggesting that trim α uses an additional, yet uncharacterized, strategy to block viral infection ( figure c ) [ , ] . finally, the association of trim α with the viral capsid enhances its e -ubiquitin ligase activity, which, in conjunction with the e enzyme ubc /uev a (ubiquitin-conjugating enzyme /ubiquitin-conjugating enzyme variant a), leads to the synthesis of free k -linked ubiquitin chains, thus stimulating tak (transforming growth factor β-activated kinase ) and finally activating ap and nf-κb signaling ( figure c ) [ , ] . this indicates that trim α, in addition to its direct antiviral activity, also functions as a sensor that induces a general antiviral state of the cell. figure a ) with the viral capsid in the cytoplasm of newly infected cells [ ] . this interaction leads to premature decapsidation of the viral core. moreover, viral capsid and integrase proteins are degraded ( figure c ) and the reverse transcription of the viral genome is inhibited in the presence of a restricting trim α. these effects seem to be mediated by the ups, since treatment with proteasome inhibitors restores a normal decapsidation rate and reverse transcription. it has also been shown that the proteasome co-localizes with trim α and viral cores in the cytoplasm [ , ] . trim α is also degraded by the proteasome but only in the presence of susceptible viral cores [ ] , suggesting that trim α recruits the proteasome to the viral cores and induces their degradation. this mechanism seems to be mediated by the e -ubiquitin ligase activity of trim α, through its ring domain ( figure a ) [ , ] . nevertheless, trim α inhibits integration of the proviral dna independently of the proteasome, suggesting that trim α uses an additional, yet uncharacterized, strategy to block viral infection ( figure c ) [ , ] . finally, the association of trim α with the viral capsid enhances its e -ubiquitin ligase activity, which, in conjunction with the e enzyme ubc /uev a (ubiquitin-conjugating enzyme /ubiquitin-conjugating enzyme variant a), leads to the synthesis of free k -linked ubiquitin chains, thus stimulating tak (transforming growth factor β-activated kinase ) and finally activating ap and nf-ϰb signaling ( figure c ) [ , ] . this indicates that trim α, in addition to its direct antiviral activity, also functions as a sensor that induces a general antiviral state of the cell. (d) march (red) mediates intracellular retention of envelope proteins (env, brown), leading to reduced env incorporation into virions, thereby decreasing infectivity. march has recently been identified as a restriction factor of hiv- , expressed by differentiated myeloid cells like monocyte derived macrophages and dendritic cells [ ] . march significantly reduces infectivity of virions produced from march -expressing cells by decreasing the number of env-proteins incorporated into budding virions. march is a transmembrane e -ubiquitin ligase, possessing an n-terminal, cytoplasmic ring domain ( figure b ), known to downregulate multiple cellular proteins from the plasma membrane by ubiquitination followed by degradation in the endo-lysosomal pathway [ ] [ ] [ ] [ ] . in the case of hiv- restriction, it has been shown that march interacts with env and causes its downregulation from the cell surface. the ring-domain of march is necessary for this mechanism, suggesting that ubiquitination plays a role. however, env does not seem to be degraded in the endo-lysosomal pathway like cellular proteins targeted by march but seems rather to be retained in intracellular compartments. march thus sequesters env away from hiv- budding sites, thereby reducing env incorporation into newly formed virions, making them less competent for infection of new target cells ( figure d ) [ ] . the family of apolipoprotein b mrna-editing enzyme, catalytic polypeptide-like (apobec /a ) proteins, is a family of cytosine deaminases (a a to a h) which induce transition of cytosine to uracil on single-stranded dna, with a preferential recognition of cc sequence motifs by a g and tc motifs by the others [ ] [ ] [ ] . a g ( figure a ) has been the first member of this family to be identified as a potent antiviral factor. it is incorporated into budding hiv virions and is thereby carried over into the next infected cell [ ] . during reverse transcription of the viral genomic rna, the single stranded negative sense dna is sensitive to the cytosine-deaminase activity of a g, leading to c to u transitions [ , ] . these mutations can either be recognized by uracil dna glycosylases, like the virion-associated ung (uracyl n-glycosylase ), leading to the degradation of the provirus by abasic site endonucleases [ ] , or they can be conserved in the provirus. due to the sequence preference of a g, these mutations very frequently introduce new stop codons in the viral genome, thus leading to the expression of non-functional mutated or/and truncated viral proteins ( figure c ). hiv- counteracts a g with its vif protein, which prevents a g incorporation into virions by inducing its degradation through the proteasome [ ] . to do so, vif recruits an scf-like e -ubiquitin ligase, composed of cullin , rbx , elongin b and c. in this complex, vif possesses the role of a substrate adaptor, directly interacting with a g through its n-terminal domain ( figure b ), thereby recruiting it for ubiquitination ( figure c ) [ ] . the recruitment of cullin is mediated by the zinc-binding domain of vif [ ] and cullin in turn recruits the e -ubiquitin-conjugating enzyme rbx . the recruitment of elongin b and c is mediated by the bc-box domain of vif ( figure b ), which can be negatively regulated by phosphorylation. in this complex, not only a g but also vif is ubiquitinated, which might contribute to the transport of a g to the proteasome [ ] . the cellular protein hdac (histone deacetylase ) has been shown to play a role in this process, by inducing vif degradation through autophagosomes as well as by protecting a g from ubiquitination and degradation [ ] . the expression level of vif is also regulated by mdm (mouse double minute homolog), an e -ubiquitin ligase that can induce the ubiquitination of vif and its proteasomal degradation [ ] . cbf-β (core binding factor β), a co-factor of the runx transcription factor family, is recruited by vif and ensures its stability by inhibition of mdm binding [ ] . cbf-β is also necessary to allow assembly of the scf-like e -ubiquitin ligase mediated by vif, resulting in the inability of vif to induce ubiquitination and degradation of a g in the absence of cbf-β [ , ] . moreover, by sequestering cbf-β in the e -ubiquitin ligase complex, vif indirectly causes a decrease in a g transcription as the a g gene is regulated by the runx transcription factor family, which requires cbf-β as cofactor ( figure c ) [ ] . degradation of a g through the ups has been known for a long time as the main mechanism for hiv- to counteract cellular restriction; however it has been shown that vif can also inhibit a g translation [ , , ] and this inhibition significantly contributes to the counteraction mechanism ( figure c ) [ , , ] . while a g is the main member of the a -family that efficiently restricts hiv, a d, f and h also showed a restricting activity towards hiv- in the absence of vif, even though to a lesser extent than a g [ ] . vif is also able to recruit these a proteins by different motifs of its n-terminal domain ( figure b ), thus inducing their degradation by the proteasome similarly to a g [ ] [ ] [ ] . translation [ , , ] and this inhibition significantly contributes to the counteraction mechanism ( figure c ) [ , , ] . while a g is the main member of the a -family that efficiently restricts hiv, a d, f and h also showed a restricting activity towards hiv- in the absence of vif, even though to a lesser extent than a g [ ] . vif is also able to recruit these a proteins by different motifs of its n-terminal domain ( figure b ), thus inducing their degradation by the proteasome similarly to a g [ ] [ ] [ ] . sterile alpha motif and histidine-aspartate domain-containing protein (samhd , figure a ) is a dgtp-regulated deoxynucleoside-triphosphohydrolase that catalyzes the hydrolysis of dntps to deoxynucleosides and inorganic triphosphate [ , ] . in non-cycling myeloid cells as well as in resting cd + t cells, this restriction factor causes a block in the early steps of the hiv- life cycle [ ] by depleting the intracellular pool of dntps [ ] , which leads to abortion of the viral genomic rna reverse transcription and accumulation of defective viral cdna ( figure c ) [ ] . this block strongly (c) the mechanism of apobec g restriction and vif counteraction. apobec g (red) is incorporated into virions and induces hypermutations of the provirus leading either to its degradation or production of truncated viral proteins. vif (blue) decreases a g transcription , inhibits its translation (red t bar) and induces its degradation by the proteasome . sterile alpha motif and histidine-aspartate domain-containing protein (samhd , figure a ) is a dgtp-regulated deoxynucleoside-triphosphohydrolase that catalyzes the hydrolysis of dntps to deoxynucleosides and inorganic triphosphate [ , ] . in non-cycling myeloid cells as well as in resting cd + t cells, this restriction factor causes a block in the early steps of the hiv- life cycle [ ] by depleting the intracellular pool of dntps [ ] , which leads to abortion of the viral genomic rna reverse transcription and accumulation of defective viral cdna ( figure c ) [ ] . this block strongly affects infectivity of hiv- in these cell types but has no effect on hiv- infectivity [ ] . indeed, hiv- possesses the viral protein x (vpx, figure b ) which alleviates the post-entry block mediated by samhd by inducing its degradation by the proteasome. vpx has been found to recruit the cul a-ddb -dcaf (ddb and cul associated factor ) e ubiquitin ligase through a direct interaction with its substrate recognition protein dcaf (ddb and cul associated factor ) [ ] while also interacting with the c-terminal domain of samhd , thereby loading samhd onto the e complex and inducing its ubiquitination followed by its proteasomal degradation ( figure c ). the nuclear localization of samhd is required for its vpx-induced proteasomal degradation, suggesting the nuclear ups is important in this mechanism [ , ] . degradation of samhd leads to an increase in cellular dntp levels and the efficiency of proviral dna synthesis [ ] . in this manner, the vpx protein allows hiv- to efficiently infect human dendritic and myeloid cells and it significantly increases the infection by hiv- [ ] . vpx therefore seems to be an important protein for viral replication, however it is present exclusively in hiv- and some siv strains. in these lineages, the vpx gene has evolved from vpr which is present in all hiv and siv strains and whose main function is the induction of cell cycle arrest [ ] [ ] [ ] [ ] [ ] . vpx and vpr share many similarities, like for example their interaction with the same cul a e ubiquitin ligase [ , ] . interestingly, the vpr protein of some siv strains has been shown to induce proteasomal degradation of samhd , thereby compensating for the lack of vpx. indeed it seems that the ability to degrade samhd has first been acquired by the vpr protein in certain lentiviral strains before the evolution of a separate vpx gene which has subsequently conserved the function of samhd antagonism [ , ] . nevertheless, many lineages, like hiv- for example, lack an anti-samhd activity. hiv interestingly, samhd seems to be regulated in cells by phosphorylation mediated by cdk -(cyclin-dependent kinase ) dependent cdk , which links its activity to cell cycle control. indeed, samhd is phosphorylated in cycling cells which blocks its activity as a dntp hydrolase [ ] . this correlates with the permissiveness of cycling cells for hiv- infection as opposed to non-cycling cells. moreover, hiv infection is made possible despite the lack of a viral factor counteracting samhd by different cellular proteins: cd for example has recently been shown to favor hiv- infection by interacting with samhd and stimulation of its proteasome-dependent degradation [ ] . cyclin l also induces samhd proteasomal degradation through interaction with samhd and dcaf , a mechanism interestingly similar to the one used by vpx [ ] . affects infectivity of hiv- in these cell types but has no effect on hiv- infectivity [ ] . indeed, hiv- possesses the viral protein x (vpx, figure b ) which alleviates the post-entry block mediated by samhd by inducing its degradation by the proteasome. vpx has been found to recruit the cul a-ddb -dcaf (ddb and cul associated factor ) e ubiquitin ligase through a direct interaction with its substrate recognition protein dcaf (ddb and cul associated factor ) [ ] while also interacting with the c-terminal domain of samhd , thereby loading samhd onto the e complex and inducing its ubiquitination followed by its proteasomal degradation ( figure c ). the nuclear localization of samhd is required for its vpx-induced proteasomal degradation, suggesting the nuclear ups is important in this mechanism [ , ] . degradation of samhd leads to an increase in cellular dntp levels and the efficiency of proviral dna synthesis [ ] . in this manner, the vpx protein allows hiv- to efficiently infect human dendritic and myeloid cells and it significantly increases the infection by hiv- [ ] . vpx therefore seems to be an important protein for viral replication, however it is present exclusively in hiv- and some siv strains. in these lineages, the vpx gene has evolved from vpr which is present in all hiv and siv strains and whose main function is the induction of cell cycle arrest [ ] [ ] [ ] [ ] [ ] . vpx and vpr share many similarities, like for example their interaction with the same cul a e ubiquitin ligase [ , ] . interestingly, the vpr protein of some siv strains has been shown to induce proteasomal degradation of samhd , thereby compensating for the lack of vpx. indeed it seems that the ability to degrade samhd has first been acquired by the vpr protein in certain lentiviral strains before the evolution of a separate vpx gene which has subsequently conserved the function of samhd antagonism [ , ] . nevertheless, many lineages, like hiv- for example, lack an anti-samhd activity. hiv interestingly, samhd seems to be regulated in cells by phosphorylation mediated by cdk -(cyclin-dependent kinase ) dependent cdk , which links its activity to cell cycle control. indeed, samhd is phosphorylated in cycling cells which blocks its activity as a dntp hydrolase [ ] . this correlates with the permissiveness of cycling cells for hiv- infection as opposed to non-cycling cells. moreover, hiv infection is made possible despite the lack of a viral factor counteracting samhd by different cellular proteins: cd for example has recently been shown to favor hiv- infection by interacting with samhd and stimulation of its proteasome-dependent degradation [ ] . cyclin l also induces samhd proteasomal degradation through interaction with samhd and dcaf , a mechanism interestingly similar to the one used by vpx [ ] . in the absence of vpu, newly formed virions remain tethered to the plasma membrane of their host cell after budding and are eventually endocytosed and degraded [ ] . the cellular restriction factor responsible for the block of virion release is tetherin/bst- . bst- is found as a disulfide-bond-linked dimer which is anchored into the plasma membrane by two domains: a transmembrane domain close to its n-terminus and an extracellular c-terminal glycosyl-phosphatidylinositol (gpi)-anchor ( figure a ) [ ] . these two domains mediate virion-tethering to the host cell, one remaining in the plasma membrane and the other one being inserted into the viral envelope ( figure c ). it has been shown that this tethering involves approximately a dozen of bst- dimers and that among the two membrane-associated domains, the gpi-anchor is preferentially incorporated into budding virions [ ] . the extracellular domain of bst- thereby acts like a molecular ruler, maintaining the virus at a constant distance of the plasma membrane, preventing it from disseminating to other target cells [ ] . the viral protein vpu counteracts bst- by direct interaction of their transmembrane domains embedded in the plasma membrane [ ] . the exact mode of action of vpu is still a matter of debate, but it seems clear now that vpu sequesters bst- away from virion budding sites, thereby preventing it from incorporation into the viral envelope ( figure c ) [ , , , ] . several studies have shown that in the presence of vpu, newly synthesized bst- is sequestered in intracellular compartments, particularly the trans-golgi-network ( figure c ). this finally results in the downregulation of surface levels of bst- , thereby allowing normal rates of virion release in the presence of vpu [ , , , ] . bst- is constitutively regulated by ubiquitination and lysosomal degradation mediated by the cellular e ubiquitin ligases march and nedd (neural precursor cell expressed developmentally down-regulated protein ) [ ] . it is still a matter of debate however, whether vpu also uses the endo-lysosomal system for bst- counteraction. the e -ubiquitin ligase adaptor β-trcp is known to be recruited by the cytoplasmic dsgxxs motif of vpu ( figure b ) [ ] , which might lead to ubiquitination of bst- followed by its degradation in the endo-lysosomal system ( figure c ) [ , ] . even though the interaction of vpu with β-trcp, as well as the capacity of β-trcp to recruit an e -ubiquitin ligase seem to be required for bst- counteraction by vpu [ , , ] , conflicting data have also been reported [ ] [ ] [ ] . certain components of the autophagy pathway, as well as clathrin adaptors ap- and and components of the escrt system might also be involved in the downregulation of bst- by vpu, which would corroborate transport of bst- in the endosomal system [ , [ ] [ ] [ ] . however, degradation of bst- might not be absolutely required for viral counteraction of bst- , since vpu is capable of intracellular sequestration of bst- independently of its degradation [ , ] . the guanylate binding protein (gbp ) has very recently been discovered as a new restriction factor of hiv- infection, that interferes with viral env proteins, thereby decreasing infectivity of produced virions [ , ] . as vpu and env are expressed from the same transcript by leaky scanning, the loss of vpu expression can in this case lead to an increase of env expression, as observed in the macrophage tropic ad isolate [ ] , allowing the virus to partly overcome gbp restriction. surprisingly, such vpu mutants seem to occur frequently despite the presence of bst- . hiv- and siv are also counteracted by bst- proteins expressed by their respective host species in a species-dependent manner, but some of them lack vpu to counteract this mechanism. it has been shown that the hiv- env protein can enhance virion release in the presence of bst- thereby substituting for vpu [ , ] . certain siv strains, like sivagm, sivblu and sivmac also lack the vpu gene and rely on the accessory protein nef to counteract bst- . other siv strains like sivmon, sivmus, sivgsn and sivden express vpu and use it to counteract bst- . even though sivgor and sivcpz express vpu, nef seems to take over the role of bst- counteraction. this gives interesting clues about the evolution of hiv and siv strains [ ] [ ] [ ] . vpu can also induce bst- degradation in the endo-lysosomal pathway . the ups is hijacked by hiv and plays an important role for the viral defense against multiple cellular restriction mechanisms. apart from restriction factors, several other cellular proteins can also be targeted by hiv through the ups. the viral auxiliary protein vpu for example possesses the ability to associate with the cul -skp e ubiquitin ligase through interaction with its substrate receptor β-trcp. this association not only seems to play a role in the counteraction of bst- but has also been shown to induce degradation of the hiv receptor cd . indeed, vpu induces cd ubiquitination followed by its extraction from the endoplasmic reticulum (er) [ , [ ] [ ] [ ] [ ] [ ] . the mechanism used by vpu to induce cd depletion involves the cellular er-associated degradation (erad) pathway, which operates as a quality control mechanism to dispose of unwanted er membrane proteins into the cytosol for subsequent proteasomal degradation. the dislocation of protein from the membrane is achieved by the recruitment of the vcp-ufd l-npl (valosin-containing protein -ubiquitin fusion degradation protein -nuclear protein localization protein ) complex through recognition by ufd l of k -linked poly-ubiquitin chains on the cd cytosolic tail. interestingly, the degradation of cd depends also on ubiquitination of serine/threonine residues [ , [ ] [ ] [ ] [ ] . the atpase activity of vcp then drives dislocation of cd from the er membrane into the cytosol and eventually its degradation in proteasomes. the multiple levels at which vpu acts to prevent export the ups is hijacked by hiv and plays an important role for the viral defense against multiple cellular restriction mechanisms. apart from restriction factors, several other cellular proteins can also be targeted by hiv through the ups. the viral auxiliary protein vpu for example possesses the ability to associate with the cul -skp e ubiquitin ligase through interaction with its substrate receptor β-trcp. this association not only seems to play a role in the counteraction of bst- but has also been shown to induce degradation of the hiv receptor cd . indeed, vpu induces cd ubiquitination followed by its extraction from the endoplasmic reticulum (er) [ , [ ] [ ] [ ] [ ] [ ] . the mechanism used by vpu to induce cd depletion involves the cellular er-associated degradation (erad) pathway, which operates as a quality control mechanism to dispose of unwanted er membrane proteins into the cytosol for subsequent proteasomal degradation. the dislocation of protein from the membrane is achieved by the recruitment of the vcp-ufd l-npl (valosin-containing protein-ubiquitin fusion degradation protein -nuclear protein localization protein ) complex through recognition by ufd l of k -linked poly-ubiquitin chains on the cd cytosolic tail. interestingly, the degradation of cd depends also on ubiquitination of serine/threonine residues [ , [ ] [ ] [ ] [ ] . the atpase activity of vcp then drives dislocation of cd from the er membrane into the cytosol and eventually its degradation in proteasomes. the multiple levels at which vpu acts to prevent export of cd from the er underscore the importance of ensuring complete depletion of cd from the plasma membrane for progression of the infection [ , , , ] . other targets of vpu-induced ubiquitination and degradation include the cell surface glycoprotein icam- and the amino acid transporter snat- , both involved in immune signaling [ , ] . it is well established that the viral auxiliary protein vpr associates with the cul a-ring e ligase through interaction with its substrate recognition subunit dcaf . this complex has been shown to induce ubiquitination followed by proteasomal degradation of the dna glycosylase ung . thereby vpr reduces encapsidation of ung , ultimately contributing to the protection against the restriction factor a g. ung recognizes c to u mutations induced by a g and generates abasic sites, leading to degradation of viral dna. indeed a virus lacking vif can be partially rescued by vpr-mediated reduction of ung compared to viruses lacking both vif and vpr [ ] [ ] [ ] . moreover, it has recently been shown that vpr can also induce the degradation of a g itself through the ups [ ] . vpr seems to also enhance hiv- production in macrophages by ups-mediated degradation of the cellular protein dicer, which is involved in rna silencing [ ] . the main function of vpr known to date is the induction of a cell cycle arrest at the g phase. the association of vpr with the cul a e ubiquitin ligase has been shown to be important for this process, although the exact mechanism is still unknown [ ] [ ] [ ] [ ] . cell cycle arrest seems to involve vpr association with the slx -slx -mus -eme complex, leading to slx (structure-specific endonuclease subunit) activation and ultimately proteasomal degradation of mus (crossover junction endonuclease) and eme (essential meiotic structure-specific endonuclease ) [ , ] . vpr also induces the degradation of multiple other cellular proteins such as the dna translocase hltf (helicase-like transcription factor) [ ] , the dna replication factor mcm (mini chromosome maintenance ) [ ] , as well as the chromatin associated proteins zip (leucine zipper), szip and class i hdacs (histone deacetylase ) [ , ] . the ups plays an important role in viral infections in general and especially in the process of viral restriction and counter-restriction. in this continuous battle between the virus and the cell, the ups constitutes an efficient tool for both sides. several hiv auxiliary proteins have evolved the ability to interact with components of the ups, subverting it for its own means. this allows the targeting of a multitude of different cellular proteins through a single platform. this strategy is not limited to hiv, but is used by a plethora of different viruses to ensure various aspects of their life cycles. overall, the specific degradation of certain cellular proteins in the ups allows viruses to generate a favorable environment for their own replication. the almost universal role of the ups in counteraction of cellular restriction factors by hiv makes the ups an interesting target for antiviral therapy. one of the main difficulties in therapy-design against hiv is the rapid evolution of the virus, which easily escapes therapeutic molecules by mutation of the targeted viral proteins. targeting the human ups represents a promising antiviral strategy because it would allow to avoid the escape through mutations [ , ] . a better knowledge on how the virus hijacks the ups and which components are involved in viral replication is crucial in this attempt. structure and function of viral deubiquitinating enzymes the ubiquitin-proteasome pathway ubiquitin: same molecule, different degradation pathways ubiquitination in the antiviral immune response function and regulation of cullin-ring ubiquitin ligases components of ubiquitin-protein ligase system. resolution, affinity purification, and role in protein breakdown protein ubiquitination involving an e -e -e enzyme ubiquitin thioester cascade a uniform isopeptide-linked multiubiquitin chain is sufficient to target substrate for degradation in ubiquitin-mediated proteolysis a multiubiquitin chain is confined to specific lysine in a targeted short-lived protein the logic of the s proteasome involvement of the proteasome in various degradative processes in mammalian cells ubp deubiquitinase controls conformational dynamics and substrate degradation of the s proteasome redundant roles of rpn and rpn in recognition of ubiquitinated proteins and cellular homeostasis crystal structure of the human s proteasome in complex with carfilzomib the escrt complexes: structure and mechanism of a membrane-trafficking network k -linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, escrt-i the ubiquitin-conjugating system: multiple roles in viral replication and infection viral avoidance and exploitation of the ubiquitin system hiv- , ubiquitin and ubiquitin-like proteins: the dialectic interactions of a virus with a sophisticated network of post-translational modifications ubiquitin is covalently attached to the p gag proteins of human immunodeficiency virus type and simian immunodeficiency virus and to the p gag protein of moloney murine leukemia virus cumulative mutations of ubiquitin acceptor sites in human immunodeficiency virus type gag cause a late budding defect tsg and the vacuolar protein sorting pathway are essential for hiv- budding ubiquitination of hiv- and mulv gag a non-proteolytic role for ubiquitin in tat-mediated transactivation of the hiv- promoter pja ubiquitinates the hiv- tat protein with atypical chain linkages to activate viral transcription trim senses and restricts influenza a virus by ubiquitination of pb polymerase the trimendous role of trims in virus-host interactions proteasomal degradation of herpes simplex virus capsids in macrophages releases dna to the cytosol for recognition by dna sensors simian trim α proteins reduce replication of herpes simplex virus the e oncoprotein encoded by human papillomavirus types and promotes the degradation of p the hpv- e and e -ap complex functions as a ubiquitin-protein ligase in the ubiquitination of p rotavirus nsp inhibits nfκb activation by inducing proteasome-dependent degradation of β-trcp: a novel mechanism of ifn antagonism putative e ubiquitin ligase of human rotavirus inhibits nf-κb activation by using molecular mimicry to target β-trcp downregulation of major histocompatibility complex class i molecules by kaposi's sarcoma-associated herpesvirus k and k proteins kaposi's sarcoma-associated herpesvirus k utilizes the ubiquitin-proteasome system in routing class major histocompatibility complexes to late endocytic compartments herpes virus induced proteasome-dependent degradation of the nuclear bodies-associated pml and sp proteins pml contributes to a cellular mechanism of repression of herpes simplex virus type infection that is inactivated by icp inhibitors of deubiquitinating enzymes block hiv- replication and augment the presentation of gag-derived mhc-i epitopes proteasome inhibition interferes with gag polyprotein processing, release, and maturation of hiv- and hiv- retroviruses have differing requirements for proteasome function in the budding process proteasome inhibitors block hiv- replication by affecting both cellular and viral targets hiv- : fifteen proteins and an rna hiv- replication hiv interaction with human host: hiv- as a model of a less virulent infection on the whereabouts of hiv- cellular entry and its fusion ports hiv- reverse transcription hiv dna integration. cold spring harb hiv- assembly, budding, and maturation. cold spring harb serinc and serinc restrict hiv- infectivity and are counteracted by nef hiv- nef promotes infection by excluding serinc from virion incorporation ifitm proteins incorporated into hiv- virions impair viral fusion and spread the ifitm proteins inhibit hiv- infection the v loop of hiv- env determines viral susceptibility to ifitm impairment of viral infectivity ifitm proteins are incorporated onto hiv- virion particles and negatively imprint their infectivity ifitm proteins restrict hiv- infection by antagonizing the envelope glycoprotein resistance of transmitted founder hiv- to ifitm-mediated restriction ring domain mutations uncouple trim α restriction of hiv- from inhibition of reverse transcription and acceleration of uncoating specific recognition and accelerated uncoating of retroviral capsids by the trim α restriction factor the cytoplasmic body component trim α restricts hiv- infection in old world monkeys mx is an interferon-induced inhibitor of hiv- infection human mx is an interferon-induced post-entry inhibitor of hiv- infection restriction of hiv- requires the n-terminal region of mxb as a capsid-binding motif but not as a nuclear localization signal the highly polymorphic cyclophilin a-binding loop in hiv- capsid modulates viral resistance to mxb host and viral determinants of mx antiretroviral activity host and viral determinants for mxb restriction of hiv- infection samhd is the dendritic-and myeloid-cell-specific hiv- restriction factor counteracted by vpx samhd restricts the replication of human immunodeficiency virus type by depleting the intracellular pool of deoxynucleoside triphosphates isolation of a human gene that inhibits hiv- infection and is suppressed by the viral vif protein the cytidine deaminase cem induces hypermutation in newly synthesized hiv- dna broad antiretroviral defence by human apobec g through lethal editing of nascent reverse transcripts codon-usage-based inhibition of hiv protein synthesis by human schlafen non-human primate schlafen inhibits production of both host and viral proteins hiv- modulates the trna pool to improve translation efficiency hiv- vpu promotes release and prevents endocytosis of nascent retrovirus particles from the plasma membrane tetherin inhibits retrovirus release and is antagonized by hiv- vpu the interferon-induced protein bst- restricts hiv- release and is downregulated from the cell surface by the viral vpu protein march inhibits hiv- infection by reducing virion incorporation of envelope glycoproteins the antagonism of hiv- nef to serinc particle infectivity restriction involves the counteraction of virion-associated pools of the restriction factor the antiretroviral enzyme apobec g is degraded by the proteasome in response to hiv- vif transcriptional regulation of apobec antiviral immunity through the cbf-β/runx axis hiv- vif blocks the antiviral activity of apobec g by impairing both its translation and intracellular stability vpx relieves inhibition of hiv- infection of macrophages mediated by the samhd protein the ability of primate lentiviruses to degrade the monocyte restriction factor samhd preceded the birth of the viral accessory protein vpx vpu binds directly to tetherin and displaces it from nascent virions early replication block of human immunodeficiency virus type in monkey cells restriction of hiv- (subtype b) replication at the entry step in rhesus macaque cells recruitment and dynamics of proteasome association with rhtrim α cytoplasmic complexes during hiv- infection trim α associates with proteasomal subunits in cells while in complex with hiv- virions proteasomal degradation of trim α during retrovirus restriction trim α-mediated ubiquitin chain conjugation is required for inhibition of hiv- reverse transcription and capsid destabilization fates of retroviral core components during unrestricted and trim -restricted infection proteasome inhibition reveals that a functional preintegration complex intermediate can be generated during restriction by diverse trim proteins an innate immune sensor for the retrovirus capsid lattice ring dimerization links higher-order assembly of trim α to synthesis of k -linked polyubiquitin inhibition of mhc class ii expression and immune responses by c-mir ubiquitination by the membrane-associated ring-ch- (march- ) ligase controls steady-state cell surface expression of tumor necrosis factor-related apoptosis inducing ligand (trail) receptor the e ubiquitin ligase march negatively regulates il- β-induced nf-κb activation by targeting the il rap coreceptor for ubiquitination and degradation march ubiquitin ligases alter the itinerary of clathrin-independent cargo from recycling to degradation family-wide comparative analysis of cytidine and methylcytidine deamination by eleven human apobec proteins apobec f properties and hypermutation preferences indicate activity against hiv- in vivo dna deamination mediates innate immunity to retroviral infection virion-associated uracil dna glycosylase- and apurinic/apyrimidinic endonuclease are involved in the degradation of apobec g-edited nascent hiv- dna induction of apobec g ubiquitination and degradation by an hiv- vif-cul -scf complex a zinc-binding region in vif binds cul and determines cullin selection phosphorylation of a novel socs-box regulates assembly of the hiv- vif-cul complex that promotes apobec g degradation the hdac /apobec g complex regulates hiv- infectiveness by inducing vif autophagic degradation mdm is a novel e ligase for hiv- vif core binding factor β protects hiv, type accessory protein viral infectivity factor from mdm -mediated degradation vif hijacks cbf-β to degrade apobec g and promote hiv- infection t-cell differentiation factor cbf-β regulates hiv- vif-mediated evasion of host restriction hiv- vif binds to apobec g mrna and inhibits its translation translational regulation of apobec g mrna by vif requires its 'utr and contributes to restoring hiv- infectivity human and rhesus apobec d, apobec f, apobec g, and apobec h demonstrate a conserved capacity to restrict vif-deficient hiv- the activity spectrum of vif from multiple hiv- subtypes against apobec g, apobec f, and apobec h suppression of apobec -mediated restriction of hiv- by vif regulation of apobec f and human immunodeficiency virus type vif by vif-cul -elonb/c e ubiquitin ligase hiv- restriction factor samhd is a deoxynucleoside triphosphate triphosphohydrolase aicardi-goutieres syndrome gene and hiv- restriction factor samhd is a dgtp-regulated deoxynucleotide triphosphohydrolase tight interplay among samhd protein level, cellular dntp levels, and hiv- proviral dna synthesis kinetics in human primary monocyte-derived macrophages samhd restricts hiv- infection in resting cd (+) t cells the human immunodeficiency virus type vpx protein usurps the cul a-ddb dcaf ubiquitin ligase to overcome a postentry block in macrophage infection role of samhd nuclear localization in restriction of hiv- and sivmac the vpx lentiviral accessory protein targets samhd for degradation in the nucleus ddb and cul a are required for human immunodeficiency virus type vpr-induced g arrest hiv- vpr-mediated g arrest involves the ddb -cul avprbp e ubiquitin ligase premature activation of the slx complex by vpr promotes g /m arrest and escape from innate immune sensing slx -slx protein-independent down-regulation of mus -eme protein by hiv- viral protein r (vpr) complex evolutionary history of primate lentiviralvprgenes evolutionary toggling of vpx/vpr specificity results in divergent recognition of the restriction factor samhd cell cycle control and hiv- susceptibility are linked by cdk -dependent cdk phosphorylation of samhd in myeloid and lymphoid cells cd association with samhd enhances hiv- reverse transcription by increasing dntp levels cyclin l is a critical hiv dependency factor in macrophages that controls samhd abundance structural basis of hiv- tethering to membranes by the bst- /tetherin ectodomain mechanism of hiv- virion entrapment by tetherin hiv- vpu protein antagonizes innate restriction factor bst- via lipid-embedded helix-helix interactions vpu antagonizes bst- -mediated restriction of hiv- release via β-trcp and endo-lysosomal trafficking antagonism of tetherin restriction of hiv- release by vpu involves binding and sequestration of the restriction factor in a perinuclear compartment characterization of e ligases involved in lysosomal sorting of the hiv- restriction factor bst a novel human wd protein, h-β trcp, that interacts with hiv- vpu connects cd to the er degradation pathway through an f-box motif hiv- accessory protein vpu internalizes cell-surface bst- /tetherin through transmembrane interactions leading to lysosomes vpu directs the degradation of the human immunodeficiency virus restriction factor bst- /tetherin via a {β}trcp-dependent mechanism hiv- vpu neutralizes the antiviral factor tetherin/bst- by binding it and directing its β-trcp -dependent degradation hiv- vpu utilizes both cullin-ring ligase (crl) dependent and independent mechanisms to downmodulate host proteins serine phosphorylation of hiv- vpu and its binding to tetherin regulates interaction with clathrin adaptors β-trcp is dispensable for vpu's ability to overcome the cd /tetherin-imposed restriction to hiv- release berlioz-torrent, c. lc c contributes to vpu-mediated antagonism of bst /tetherin restriction on hiv- release through a non-canonical autophagy pathway hiv- vpu antagonizes cd /tetherin by adaptor protein- -mediated exclusion from virus assembly sites berlioz-torrent, c. the escrt- component hrs is required for hiv- vpu-mediated bst- /tetherin down-regulation guanylate binding protein (gbp) is an interferon-inducible inhibitor of hiv- infectivity identification of potential hiv restriction factors by combining evolutionary genomic signatures with functional analyses regulation of virus release by the macrophage-tropic human immunodeficiency virus type ad isolate is redundant and can be controlled by either vpu or env the envelope glycoprotein of human immunodeficiency virus type enhances viral particle release: a vpu-like factor? the human immunodeficiency virus (hiv) type envelope protein is a functional complement to hiv type vpu that enhances particle release of heterologous retroviruses nef proteins from simian immunodeficiency viruses are tetherin antagonists species-specific activity of siv nef and hiv- vpu in overcoming restriction by tetherin/bst the evolution of pandemic and non-pandemic hiv- strains has been driven by tetherin antagonism human immunodeficiency virus type vpu protein induces rapid degradation of cd cd glycoprotein degradation induced by human immunodeficiency virus type vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway requirements for the selective degradation of cd receptor molecules by the human immunodeficiency virus type vpu protein in the endoplasmic reticulum multilayered mechanism of cd downregulation by hiv- vpu involving distinct er retention and erad targeting steps transmembrane domain determinants of cd downregulation by hiv- vpu cd and bst- /tetherin proteins retro-translocate from endoplasmic reticulum to cytosol as partially folded and multimeric molecules hiv- vpu downmodulates icam- expression, resulting in decreased killing of infected cd (+) t cells by nk cells cell surface proteomic map of hiv infection reveals antagonism of amino acid metabolism by vpu and nef hiv- vpr loads uracil dna glycosylase- onto dcaf , a substrate recognition subunit of a cullin a-ring e ubiquitin ligase for proteasome-dependent degradation the ddb -dcaf -vpr-ung crystal structure reveals how hiv- vpr steers human ung toward destruction human immunodeficiency virus type vpr induces the degradation of the ung and smug uracil-dna glycosylases the hiv- accessory protein vpr induces the degradation of the anti-hiv- agent apobec g through a vprbp-mediated proteasomal pathway the hiv- protein vpr targets the endoribonuclease dicer for proteasomal degradation to boost macrophage infection hiv- vpr degrades the hltf dna translocase in t cells and macrophages hiv- vpr protein enhances proteasomal degradation of mcm dna replication factor through the cul -ddb [vprbp] e ubiquitin ligase to induce g /m cell cycle arrest hiv- vpr induces the degradation of zip and szip, adaptors of the nurd chromatin remodeling complex, by hijacking dcaf /vprbp hiv- vpr protein induces proteasomal degradation of chromatin-associated class i hdacs to overcome latent infection of macrophages the ubiquitin-proteasome system in hiv replication: potential targets for antiretroviral therapy inhibition of vpx-mediated samhd and vpr-mediated host helicase transcription factor degradation by selective disruption of viral crl (dcaf ) e ubiquitin ligase assembly key: cord- -gosqpg k authors: martínez, josé l.; arias, carlos f. title: role of the guanine nucleotide exchange factor gbf in the replication of rna viruses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gosqpg k the guanine nucleotide exchange factor gbf is a well-known factor that can activate different adp-ribosylation factor (arf) proteins during the regulation of different cellular vesicular transport processes. in the last decade, it has become increasingly evident that gbf can also regulate different steps of the replication cycle of rna viruses belonging to different virus families. gbf has been shown not only to facilitate the intracellular traffic of different viral and cellular elements during infection, but also to modulate the replication of viral rna, the formation and maturation of viral replication complexes, and the processing of viral proteins through mechanisms that do not depend on its canonical role in intracellular transport. here, we review the various roles that gbf plays during the replication of different rna viruses. eukaryotic cells contain a collection of diverse organelles that perform specialized functions depending on the enzymatic activities of their components. although these organelles are spatially separated, there is a dynamic interaction between them by different trafficking events, some of which are mediated by vesicle transport [ ] . this transport is responsible for the proper intracellular distribution of proteins and lipids and is essential to sustain the correct function and structure of cell organelles [ ] [ ] [ ] [ ] [ ] [ ] . in vesicle transport, the proteins and lipids to be transported are selectively concentrated on specialized membrane sites of a donor compartment and included into spherical carrier vesicles coated with specific proteins. the vesicles detach from the donor membrane and travel through the cytoplasm until they finally fuse with their target compartment to release their content [ ] [ ] [ ] . based on the protein elements that form the coat, three important transport vesicles have been extensively described in which the function depends on either the coatomer protein i (copi), copii, or clathrin [ , ] . these vesicles mediate different steps of the secretory pathway of the cell. copii regulates the anterograde transport from the endoplasmic reticulum (er) to the golgi apparatus [ , ] , whereas copi facilitates the transport between the golgi cisternae as well as the retrograde transport from golgi to the er [ ] [ ] [ ] . on the other hand, clathrin-coated vesicles direct the transport from the golgi to the plasma membrane, as well as between the plasma membrane and endosomes [ ] ( figure ). recently, besides its canonical functions in the secretory pathway, the copi machinery has been shown to participate in the maturation of early endosomes and in recycling proteins to the plasma membrane [ , ] , as well as in the maturation of phagosomes [ , ] and peroxisomes [ ] . furthermore, multiple reports suggest that copi-vesicles are also involved in transport events related to the maturation and function of lipid droplets (lds) [ ] [ ] [ ] . the mechanism of copi vesicle formation is a complex process regulated by the activation of the small gtpase arf (adp-ribosylation factor ) (figure ). this protein belongs to a family of low molecular weight guanine-nucleotide-binding proteins that can be divided into arf proteins of class i (arf , arf , and arf ), class ii (arf and arf ) and class iii (arf ), depending on their sequence identity. arf proteins cycle between an inactivated cytosolic form, bound to gdp, and an active membrane-associated state, when bound to gtp. arf proteins are highly conserved and are present in all eukaryotes examined to date, except for arf , which has not been reported to be present in humans. these proteins have been shown to play a number of regulatory roles related not only with membrane traffic, but in lipid metabolism, organelle morphology, and cellular signaling [ ] [ ] [ ] . given that arf and arf are the most abundantly expressed arf isoforms in cells, class i arfs have been assumed as the principal actors in vesicle transport. however, in recent years it has been demonstrated that although the different arf proteins play overlapping and redundant roles in cell transport, specific arfs display a unique profile of activities to control specific points in the secretory and endocytic pathways. in addition, it has also been observed that in some cases the control of vesicle transport can depend on the cooperation of two particular arfs [ ] . the mechanism of copi vesicle formation is a complex process regulated by the activation of the small gtpase arf (adp-ribosylation factor ) (figure ). this protein belongs to a family of low molecular weight guanine-nucleotide-binding proteins that can be divided into arf proteins of class i (arf , arf , and arf ), class ii (arf and arf ) and class iii (arf ), depending on their sequence identity. arf proteins cycle between an inactivated cytosolic form, bound to gdp, and an active membrane-associated state, when bound to gtp. arf proteins are highly conserved and are present in all eukaryotes examined to date, except for arf , which has not been reported to be present in humans. these proteins have been shown to play a number of regulatory roles related not only with membrane traffic, but in lipid metabolism, organelle morphology, and cellular signaling [ ] [ ] [ ] . given that arf and arf are the most abundantly expressed arf isoforms in cells, class i arfs have been assumed as the principal actors in vesicle transport. however, in recent years it has been demonstrated that although the different arf proteins play overlapping and redundant roles in cell transport, specific arfs display a unique profile of activities to control specific points in the secretory and endocytic pathways. in addition, it has also been observed that in some cases the control of vesicle transport can depend on the cooperation of two particular arfs [ ] . mechanism of copi transport. copi vesicle formation starts when the small gtpase arf , bound to gdp (arf -gdp), associates with the golgi-specific brefeldin a (bfa) resistance factor (gbf ), a guanine nucleotide exchange factor (gef) that catalyzes the activation of arf by promoting the exchange of gdp for a gtp molecule (step ). this exchange induces a conformational change in arf that leads to the exposure of a myristoyl group that allows the association of this protein with the membrane [ ] (step ). the membrane-bound arf -gtp then promotes the recruitment of the preformed copi coat formed by seven subunits, called α, β, β', δ, ε, γ and ζ, and the arf-gtpaseactivating protein (arfgap ) (step ). the formation of the arf -gtp-copi-arfgap complex in the membranes stimulates the binding and concentration of different cargoes (step ) and induces the bending of the membrane into a vesicle (step ). once the vesicle is completed, it buds from the membrane with a coat of copi (step ). finally, the coat proteins are disassembled when the gtpase activity of arf is enhanced by arfgap , leading to the hydrolysis of the gtp molecule, promoting the release of arf -gdp, the copi subunits, and arfgap from the vesicle to produce the free carrier vesicle used in the vesicular transport (step ) [ , ] . in addition to arf proteins, the participation of specific arf gefs has also been shown to play an essential role in the regulation of the vesicle transport. for instance, the activation of arf can be driven either by gbf to promote copi transport, or by the brefeldin a-inhibited gefs and (big and big ) to initiate clathrin transport; thus, it is thought that arf is committed to each transport pathway depending on the interaction with a specific gef. in order to achieve this regulation, the different gefs have been shown to be restrained to particular sites where a specific transport is needed; for example, gbf is located at sites where copi exerts its functions, either in the cis-golgi cisternae, the ergic or the lds [ , ] , whereas big and big are located at the trans-golgi network (tgn), where clathrin works. in this way, each transport pathway depends on the precise temporal and spatial interaction of a specific gef with a combination of small gtpases. gbf is the gef that specifically regulates the copi vesicle transport. based on its size, gbf belongs to the group of large arf gefs, which also contains big and big . this group of gefs shares a catalytic sec domain that regulates the activation of arf proteins through binding to the arf-gdp form, inducing its conformational change, and promoting the exchange of gdp for gtp that leads to the dissociation of the protein from gbf [ , ] . through this mechanism, gbf can activate arf , arf , and arf [ ] [ ] [ ] . in addition to its sec domain, gbf contains five non-catalytic domains: figure . mechanism of copi transport. copi vesicle formation starts when the small gtpase arf , bound to gdp (arf -gdp), associates with the golgi-specific brefeldin a (bfa) resistance factor (gbf ), a guanine nucleotide exchange factor (gef) that catalyzes the activation of arf by promoting the exchange of gdp for a gtp molecule (step ). this exchange induces a conformational change in arf that leads to the exposure of a myristoyl group that allows the association of this protein with the membrane [ ] (step ). the membrane-bound arf -gtp then promotes the recruitment of the preformed copi coat formed by seven subunits, called α, β, β', δ, ε, γ and ζ, and the arf-gtpase-activating protein (arfgap ) (step ). the formation of the arf -gtp-copi-arfgap complex in the membranes stimulates the binding and concentration of different cargoes (step ) and induces the bending of the membrane into a vesicle (step ). once the vesicle is completed, it buds from the membrane with a coat of copi (step ). finally, the coat proteins are disassembled when the gtpase activity of arf is enhanced by arfgap , leading to the hydrolysis of the gtp molecule, promoting the release of arf -gdp, the copi subunits, and arfgap from the vesicle to produce the free carrier vesicle used in the vesicular transport (step ) [ , ] . in addition to arf proteins, the participation of specific arf gefs has also been shown to play an essential role in the regulation of the vesicle transport. for instance, the activation of arf can be driven either by gbf to promote copi transport, or by the brefeldin a-inhibited gefs and (big and big ) to initiate clathrin transport; thus, it is thought that arf is committed to each transport pathway depending on the interaction with a specific gef. in order to achieve this regulation, the different gefs have been shown to be restrained to particular sites where a specific transport is needed; for example, gbf is located at sites where copi exerts its functions, either in the cis-golgi cisternae, the ergic or the lds [ , ] , whereas big and big are located at the trans-golgi network (tgn), where clathrin works. in this way, each transport pathway depends on the precise temporal and spatial interaction of a specific gef with a combination of small gtpases. gbf is the gef that specifically regulates the copi vesicle transport. based on its size, gbf belongs to the group of large arf gefs, which also contains big and big . this group of gefs shares a catalytic sec domain that regulates the activation of arf proteins through binding to the arf-gdp form, inducing its conformational change, and promoting the exchange of gdp for gtp that leads to the dissociation of the protein from gbf [ , ] . through this mechanism, gbf can activate arf , arf , and arf [ ] [ ] [ ] . in addition to its sec domain, gbf contains five non-catalytic domains: the n-terminal dimerization and cyclophilin binding (dcb) domain, the homology upstream of sec (hus) domain, and three c-terminal downstream of sec (hds - ) domains [ , ] (figure ). the functions of these non-catalytic domains are not well understood, but given that gbf cycles between a soluble and a membrane-bound state, similar to arf proteins, it has been proposed that these domains are important for the specific interaction of gbf with cellular membranes [ , [ ] [ ] [ ] . [ , ] (figure ). the functions of these non-catalytic domains are not well understood, but given that gbf cycles between a soluble and a membranebound state, similar to arf proteins, it has been proposed that these domains are important for the specific interaction of gbf with cellular membranes [ , [ ] [ ] [ ] . the multidomain structure of gbf allows this protein to engage in numerous interactions with other proteins, leading gbf to participate in many different cellular processes and even in the process of infection of some bacteria [ , ] . in addition to its canonical role in vesicular transport, gbf has also been shown to participate in the function of lds and mitochondria, as well as in a clathrin-independent endocytosis pathway and the chemotaxis of migrating neutrophils [ ] . many of these functions have been elucidated through the use of the fungal toxin brefeldin a (bfa) and the small-molecule golgicide a (gca), which inhibit the catalytic activity of this nucleotide exchange factor. these inhibitors bind to the protein-protein interface in the complex formed by the sec domain of gbf and arf-gdp, and block the conformational changes of arf proteins that allow the exchange of gdp for gtp [ ] . this inhibition leads to the release of copi from the membranes with the consequent inhibition of the vesicle transport, and also primes the disassembly of the cisternae of the golgi apparatus. moreover, it has also been observed that bfa and gca induce the swelling of the er compartment, likely due to the fusion of the cis-golgi with the er. both bfa and gca share many similitudes in their effects; however, while bfa has been shown to inhibit the activity of gbf , big , and big gefs, gca does not affect other gefs as it is a specific inhibitor of gbf [ , , , ] . these inhibitors have been useful tools for understanding the participation of gbf in different cellular functions. moreover, in recent years, the use of these drugs has unveiled the essential role that gbf plays in the replication of several rna viruses that belong to different families (table ) , strongly suggesting that gbf can exhibit diverse functions that go beyond its role in vesicle transport. in this review, we explore the current knowledge of the role gbf plays in the replication of rna viruses and present an overview of the different viral functions that depend on the activity of this factor. the multidomain structure of gbf allows this protein to engage in numerous interactions with other proteins, leading gbf to participate in many different cellular processes and even in the process of infection of some bacteria [ , ] . in addition to its canonical role in vesicular transport, gbf has also been shown to participate in the function of lds and mitochondria, as well as in a clathrin-independent endocytosis pathway and the chemotaxis of migrating neutrophils [ ] . many of these functions have been elucidated through the use of the fungal toxin brefeldin a (bfa) and the small-molecule golgicide a (gca), which inhibit the catalytic activity of this nucleotide exchange factor. these inhibitors bind to the protein-protein interface in the complex formed by the sec domain of gbf and arf-gdp, and block the conformational changes of arf proteins that allow the exchange of gdp for gtp [ ] . this inhibition leads to the release of copi from the membranes with the consequent inhibition of the vesicle transport, and also primes the disassembly of the cisternae of the golgi apparatus. moreover, it has also been observed that bfa and gca induce the swelling of the er compartment, likely due to the fusion of the cis-golgi with the er. both bfa and gca share many similitudes in their effects; however, while bfa has been shown to inhibit the activity of gbf , big , and big gefs, gca does not affect other gefs as it is a specific inhibitor of gbf [ , , , ] . these inhibitors have been useful tools for understanding the participation of gbf in different cellular functions. moreover, in recent years, the use of these drugs has unveiled the essential role that gbf plays in the replication of several rna viruses that belong to different families (table ) , strongly suggesting that gbf can exhibit diverse functions that go beyond its role in vesicle transport. in this review, we explore the current knowledge of the role gbf plays in the replication of rna viruses and present an overview of the different viral functions that depend on the activity of this factor. flaviviruses are enveloped, positive-sense, single-stranded rna viruses with a genome of approximately . - kb in length. the genome is organized as a long open reading frame (orf) encoding a polyprotein that is cleaved into three structural (c, prm, and e) and seven nonstructural (ns , ns a, ns b, ns , ns a, ns b, and ns ) proteins. during infection, flaviviruses induce a profound rearrangement of cellular membranes to provide platforms for viral replication. it has been observed that rna replication occurs in vesicular structures called vesicle packets derived from membranes of the er [ ] . although the assembly process of flaviviruses is unclear, it has been suggested that during rna replication, the viral capsid protein (c protein), located on the surface of lds, recruits the viral genome for formation of the nucleocapsid [ ] . the newly formed nucleocapsid then buds into the er lumen, acquiring during this process the structural glycoproteins e and prm, as well as the membrane envelope. finally, as the novel viral particle travels through the secretory pathway to be released by exocytosis, the virus experiments a maturation process in which the prm protein is cleaved by the furin protease to yield the mature m protein [ ] . in recent years, increasing evidences have shown that gbf is an important cellular factor involved in the replication of several flaviviruses, including dengue virus (denv) [ , [ ] [ ] [ ] , yellow fever virus (yfv) [ ] , tick-borne encephalitis virus (tbev) [ ] , and kunjin virus (kun) [ ] . in the case of denv, it was shown that bfa and gca reduce the abundance of viral rna, and depletion of gbf was sufficient to cause this reduction and to inhibit the viral production [ , ] . also, bfa has been shown to retard the mortality of mice infected with denv, but not to alter the final mortality outcome [ ] . in support of the participation of gbf in the denv lifecycle, it was found that the viral rna decrease induced by gca could be reverted by overexpression of either wild-type gbf (gbf -wt) or a drug resistant gbf mutant [ , ] . the mechanism through which gbf favors the replication of denv rna is unknown; however, the fact that this factor directly interacts with the ns protein, the viral rna-dependent rna polymerase (rdrp) of denv, suggests that gbf might be directly involved in the rna replication process through regulation of the activity of ns [ ] ; alternatively, it has also been proposed that gbf might modulate the replication of viral rna by participating in the biogenesis of the viral vesicle packets [ ] . in support of this latter idea, it has been reported that bfa can prevent the membrane rearrangement induced by kun, when the drug is added at early times of infection [ ] . altogether, these findings suggest that gbf might engage the nascent replication complexes of flavivirus (vesicle packets) through its interaction with ns ; once in these sites, gbf could regulate the formation of these viral organelles through the activation of arf proteins and the recruitment of cellular effectors that ultimately could lead to the creation of a microenvironment that allows the proper activity of ns . in addition to the role of gbf in rna replication, it has been reported that depletion of this factor or its pharmacological inhibition with bfa could also affect the denv assembly by blocking the accumulation of the c protein into the lds, which depends on the catalytic activity of gbf [ ] . moreover, considering that depletion of other elements involved in copi transport, such as the cop-β subunit or the simultaneous depletion of arf and arf , largely reduce the accumulation of the c protein in lds, it was suggested that the dynamic delivery of the c protein from er to lds is regulated by the copi complex through the gbf -mediated activation of arf and arf . on the other hand, and similar to denv, in tbev-infected cells, the inhibition of gbf also showed an alteration in the transport of the structural prm, e, and c proteins [ ] . of interest, the increment of the c protein transport and the inhibition of viral progeny production has also been recapitulated by treating tbev-infected cells with interferon (inf ), through the induction of the expression of the viperin (virus inhibitory protein, er-associated, ifn-inducible) protein [ ] ; viperin also has antiviral effects on other flaviviruses such as denv [ ] and west nile virus (wnv) [ ] . the antiviral activity of viperin seems to be the result of the inhibition of the gbf activity, since overexpression of this factor was shown to counteract the effects of viperin on the tbev infection [ ] . on the other hand, it has been shown that the simultaneous depletion of arf and arf decreases the secretion of denv and yfv particles, without affecting the constitutive secretory pathway [ ] . the fact that these arf proteins can be selectively activated by gbf , and that the block in the secretion of denv particles can be replicated by treatment of cells with bfa, suggest that the secretion of denv particles to the extracellular medium is gbf -mediated and is dependent on the activation of arf and arf . however, this requirement is not shared by all flaviviruses since the replication of wnv is not affected by depletion of these arfs [ ] . although many aspects of the pestiviruses replication cycle remain unknown, a recent study described that replication of the classical swine fever virus (csfv) depends on the activity of gbf . it was described that both bfa or gca are able to reduce the viral titers and the viral rna level, and depletion of gbf is sufficient to cause this effect [ ] . it seems that gbf could facilitate pestiviruses replication by regulating the copi-mediated transport, since knocking down the expression of rab , a regulatory element of the retrograde transport mediated by copi [ ] , has been shown to significantly decrease the viral titer and the rna level of csfv. of interest, this transport does not seem to rely on the activation of arf , since depletion of this factor did not affect csfv replication [ ] . the replication of bovine viral diarrhea virus (bvdv), the prototype member of the pestivirus genus, was also demonstrated to be strongly inhibited in the presence of bfa [ ] , suggesting that the dependence on gbf could be a feature shared among different pestiviruses. infection by hepatitis c virus (hcv), the only member of the hepacivirus genus, induces an extensive remodeling of intracellular membranes to produce a membranous web composed of er membranes, lds, and double-membrane vesicles containing hcv nonstructural proteins (ns , ns a-b, ns a-b) as well as the hcv rna [ , ] . although the biogenesis of this membranous web is not well understood, the ns b and ns a proteins appear to play a major role in the induction of membrane rearrangements [ , ] . once the web is formed, the ns b protein (rdrp) directs the replication of the viral positive-sense rna genome. detailed knowledge about the individual steps of hcv particles assembly is lacking; however, it is generally assumed that nucleocapsid formation and budding are spatially and temporally linked events occurring in an er-derived compartment [ , , ] . in this regard, it has been reported that gbf facilitates multiple steps of hcv infection, including the replication of viral rna [ , ] . a decrement in the levels of gbf was found to diminish the expression of ns a, a multifunctional protein that modulates the viral polymerase ns b; however, most of the effect on virus replication seems to be related to the role of gbf in the formation and function of the membranous web where the replication complexes of hcv assemble [ ] . gbf inhibition neither disrupts the preformed membranous webs of hcv nor blocks the formation of novel membranous web structures, but it rather affects the maturation of these viral organelles, which exhibit a smaller and less organized structure during the inhibition of this factor [ , ] . in this regard, it was reported that the inhibition of gbf induces a change in the intracellular localization of ns a and ns (viral protease) from their usual location in the replication complexes to the rims of lds [ ] , suggesting that gbf could mediate the transport of non-structural viral proteins and perhaps cellular proteins to these sites. in support of this possibility, it has been found that while a bfa-resistant gbf mutant could revert the effects of bfa on hcv, an inactive mutant or a truncated form of gbf lacking the catalytic sec domain were unable to maintain the replication of hcv in the presence of bfa [ , ] ; this suggests that the role of gbf depends on its capacity to activate arf proteins. however, in a contrasting observation, the expression of ns a was reported to downregulate the activation of arf [ ] . arf activation has also been shown to be related with the viral assembly of hcv through modulation of the recruitment of the adipose differentiation-related protein (adrp) to lds. adrp, a member of the perilipin family, is a major protein associated with lds in various cell types. this protein has been proposed to play a positive role for hcv rna replication while performing a negative function for hcv assembly [ ] . although the role of this protein in rna replication is unknown, it has been observed that adrp shields the recruitment of the hcv core protein into lds, a step essential for virus morphogenesis [ , ] . in addition, the association of adrp with lds has been shown to be dependent on the activation state of arf . altogether, these results suggest that during hcv infection the activation of arf induced by gbf promotes the exportation of ns a and ns from the lds to the sites where replication complexes are located, to favor the replication of the viral rna and, at the same time, induces the release of adrp from lds to favor the morphogenesis of the virus particles. since bfa treatment has also been shown to inhibit the secretion of hcv viral particles, leading to their progressive intracellular accumulation within the er [ ] , it seems that gbf is important for both the assembly and exit of the newly formed hcv virions. similar to arf , the simultaneous depletion of arf and arf , in which the activation also depends on gbf , has been reported to reduce the rna replication of hcv probably through affecting the morphology of ld but not the secretion pathway [ , ] . altogether, these results suggest that the role of gbf is not restricted to maintain a single type of transport but rather to coordinate different transport pathways that promote the infection of hcv. moreover, they also indicate that the role of gbf in the ld transport, but not in the secretory pathway, is important for hcv infection. in agreement, several works have reported that hcv replication can be completely inhibited at bfa concentrations that do not affect the secretion of proteins [ , , ] . in addition to the role of gbf in the transport of cellular and viral proteins relevant for rna replication and viral assembly, it has also been observed that this factor can regulate the lipid composition of the membranous web induced by hcv. inhibition of gbf activity and arf activation abolishes the accumulation of phosphatidylinositol -phosphate (pi p) in the membranes of the hcv replication complexes [ ] , a process that has been shown to promote the replication of the viral rna. although it is not clear how gbf and arf prompt the accumulation of pi p, recent findings suggest that these factors might induce the transport to the hcv replication sites of the type iii phosphatidylinositol -kinases α (pi kiiiα) and β (pi kiiiβ) enzymes, which are responsible in the golgi apparatus for the generation of pi p. although the precise role of pi p in hcv replication remains unknown, it has been proposed that these lipids might be important to generate membranes sites that permit the association of proteins to the replication complex of hcv, but it could also impact the organization and kinetics of the transport pathways modulated by hcv [ , , ] . in a mechanism apparently different from the regulation of protein traffic, it has been found that gbf might also modulate the processing of the hcv polyprotein by regulating the protease activity of the ns -ns a complex; this regulation process requires the interaction between the sec domain of gbf and the protease domain of ns . moreover, the protease activity of the ns -ns a complex has been shown to depend on the catalytic activity of gbf [ ] . the genus enterovirus (ev) includes a collection of small non-enveloped viruses with a small positive-sense single-stranded rna genome. these viruses have been classified into serologically distinct groups, comprising groups of enterovirus (ev-a to l), and three groups of rhinovirus (rv) (rv-a to c). the replication cycle of ev starts with the attachment of the virus to specific cell host receptors. after virus internalization, the viral genome is released into the cytoplasm to direct the synthesis of a single polyprotein, which is auto-processed by viral proteases a and c to produce three structural proteins (vp , vp , and vp ) and seven nonstructural proteins ( a- c and a- d), as well as some stable protein precursors ( bc, ab, and cd) that can also participate in the replication cycle. as the infection progresses, ev promotes the creation of viral cytoplasmic replication complexes formed by clusters of tightly associated vesicles of heterogeneous size, where the viral rdrp d protein directs the replication of the genomic rna. the novel genomic rna then is either translated or packaged into preassembled procapsids (composed of vp , vp , and vp ). finally, the newly ev procapsids are secreted into the extracellular medium through a process in which vp is cleaved into vp and vp , to form the mature, infectious virus particles. although many of the details of the ev replication cycle have been described, the role of host factors in the replication complexes of ev are less characterized. one of the most intriguing features of ev replication is the participation of the cellular secretory pathway in the infectious process. the inhibition of this pathway by bfa and gca revealed that the rna replication of many members of ev genus, such as poliovirus, cvb , coxsackievirus b (cvb , member of ev-b group), coxsackievirus a (cva , member of ev-c group), enterovirus (ev , member of ev-a group), echovirus (e , member of ev-b group), and bovine enterovirus type (bev , member of ev-f group) are sensitive to these drugs [ , , , , [ ] [ ] [ ] [ ] [ ] . furthermore, the replication of human rhinovirus (hrv , member of rv-a group) as well as human rhinovirus (hrv , member of rv-b group) was also inhibited by bfa and gca [ ] . the reason for this blockage is unknown, although it has been reported that these inhibitors do not alter the formation of the replication complexes of ev, but rather inhibit the proper functioning of these viral organelles, which are unable to support the viral rna synthesis [ ] . also, it has been shown that the blockage of rna replication produced by bfa and gca is related to the activity of gbf since the overexpression of gbf prevents the effects of these drugs on ev replication [ , , , ] . moreover, gbf knockdown confirms that the reduction of this factor reduces the ev rna replication [ , [ ] [ ] [ ] [ ] ] . although the precise role of gbf in ev replication is unknown, further analysis of poliovirus, cvb , and ev infection revealed that the n-terminal region of gbf interacts with the viral a protein; this interaction, in turn, stimulates the association of gbf with the membranes where the ev replication complexes are located [ , , , [ ] [ ] [ ] [ ] , inducing ultimately a steady increase in the activation of arf , arf , and arf [ , , ] . although it is generally accepted that gbf interacts with the a protein, the importance of this interaction for the replication of the ev rna remains to be elucidated. the use of different gbf mutants revealed that mutations in this protein that abolish the interaction with a reduce the ability of this cellular factor to support ev rna replication [ , ] . however, it has also been found that an ev mutant strain with a a protein incapable of interacting with gbf replicates to a similar extent as the wild type virus does [ , , ] , suggesting that the interaction gbf - a is dispensable for the ev replication. in this regard, it has also been found that even though gbf can be recruited to the replication complexes formed by hrv and hrv , the a protein of hrv only shows a slight interaction with gbf , whereas the a protein of hrv did not interact at all [ , ] , indicating that the recruitment of gbf to the replication complexes of ev is not dependent on its interaction with a. reconciling these findings is complicated; however, it is possible that mutations in gbf that alter its interaction with a might also abolish the ability of gbf to interact with other cellular factors needed for the proper function of this gef in the rna replication of ev, an effect that is not observed when the a protein is mutated. furthermore, taking into account that the expression of a can inhibit selectively some steps of the secretory pathway [ , , ] and that overexpression of gbf has been shown to counteract these effects [ ] , it seems that the interaction of a with gbf is essential to block the secretory pathway, rather than to participate in the replication of ev. therefore, it is possible that a functions as a competitive inhibitor that binds gbf in order to hinder its activity in the secretory pathway. the relevance of the arf activation in the replication cycle of ev is also unclear. the depletion of arf proteins has been shown to reduce the replication of ev , cvb , and cvb [ , , ] , indicating that these factors are important for ev replication. however, the expression of a truncated mutant composed of the n-terminal dcb and hus domains of gbf , but lacking the sec domain, has been shown to be sufficient for the rescue of poliovirus and cvb rna replication in the presence of bfa [ , ] . thus, it has been proposed that although gbf is essential for ev replication, the increment of arf activation detected during infection of some members of ev genus might be a side effect produced by the increased association of gbf with membranes [ ] . these results led to propose that the n-terminus of gbf might play an essential role in rna replication by stimulating the recruitment of cellular factors that are essential for the function of the replication complexes of ev; however, the identity of these effectors remains to be elucidated. recent findings have shown that poliovirus triggers the lipid membrane synthesis of replication complexes by mobilizing the neutral lipids stored in lds, in an adipose triglyceride lipase (atgl) and hormone-sensitive lipase (hsl) dependent fashion [ ] . these observations, together with described functions of gbf in lds morphology, and its role in prompting the transport of atgl to lds [ , ] , seem to indicate that gbf might support the function of ev replication complexes through regulating the lipid flux from lds. in this regard, it has been observed that bfa inhibits the increment of lipid synthesis induced by poliovirus infection [ ] . in contrast to the strong evidence regarding the relevance of gbf for the replication of several enteroviruses, this dependency is not a common feature shared by all members of the picornaviridae family. encephalomyocarditis virus (emcv, member of the cardiovirus genus), the foot-and-mouth disease virus (fmdv, member of the aphthovirus genus), as well as the aichi virus (aiv, member of kobuvirus genus) have been shown to be resistant to the effects of bfa, indicating that gbf is not involved in their replication cycle [ , , , ] . on the other hand, the dependency on gbf does not seem to be limited to the ev genus, since the replication of parechovirus (parv , member of the parechovirus genus) is partially sensitive to bfa [ ] . this genus includes a large number of mosquito-borne viruses that can infect mammals, birds, fish, reptiles, and amphibians. alphaviruses consist of enveloped viral particles that contain a single-stranded positive-sense rna genome that encodes two orfs. the first orf directs the synthesis of four non-structural proteins (nsp - ), whereas the second orf codes for the structural capsid protein (cp) and two surface envelope glycoproteins (e and e ) [ ] . once in the cytoplasm, the viral genome is translated to produce a single polyprotein formed by the four nonstructural proteins. the auto-proteolytic processing of this polyprotein renders the individual nsp - proteins that assemble to form a replicase complex. the replicase complex, together with the genomic rna and host proteins, then assemble at the plasma membrane to form viral replication compartments called spherules that direct the replication and transcription of the viral rna. furthermore, as the infection progresses, the internalization of the spherules from the plasma membrane has been shown to create large cytopathic type vacuoles (cpv- ) that can also direct the replication and transcription of viral rna. either from spherules or cpv- , a subgenomic rna is transcribed from the second orf to produce a single polyprotein composed of the structural proteins cp, e , and e . the auto proteolysis of this polyprotein releases cp in the cytosol and permits the translocation of e and e into the er, where they are post translationally modified to yield the final glycoproteins that are then transported by the exocytic pathway to the plasma membrane. concomitant with these processes, the formation of new icosahedral nucleocapsids occurs in the replication complexes that associate to plasma membrane sites decorated with e /e and then bud to the extracellular medium, producing the mature enveloped particles. [ , ] . given the involvement of the secretory pathway in the proper processing and transport of e /e to the plasma membrane, this transport has been thought to be important only for virus particle maturation. however, in recent years it was found that the inhibition of protein transport by bfa or gca could disturb the rna replication and protein synthesis of sindbis virus (sinv) [ , ] , the prototype member of this genus, as well as of chikungunya virus (chikv) [ ] . this alteration was found to be associated with a post entry virus replication step that depends on the activity of gbf , since depletion of this factor was sufficient to reduce the viral rna replication [ ] . given that alphavirus replication has been shown to induce the formation of membrane vesicles in order to create the viral replication complexes (spherules and cpv- ) that direct the synthesis of the viral rna, these results led to propose that the function of gbf could be to facilitate the efficient replication of the alphavirus rna genome by promoting the formation of the membrane vesicles required for this process [ ] . in support of this model, it has been found that the alphavirus rna replication depends on the presence of arf , arf , arf , and arf [ , ] , as well as on a functional copi complex [ ] . thus, these results indicate that gbf might induce the formation of membrane vesicles through the activation of the copi transport mediated by different arf proteins. in agreement with the importance of gbf early in infection, it was found that the inhibitory effect of bfa on the replication sinv is lost when this drug is added at late times post infection. moreover, these data also suggest that the activity of gbf is dispensable for the function of the alphavirus replication complexes since bfa does not affect the replication of the viral rna once these organelles are formed [ , ] . on the other hand, taking into account that arf and arf participate in maintaining the ld morphology, but are not important for protein transport [ ] , the involvement of these arf proteins in alphavirus rna replication might suggest that gbf and copi could also participate in the viral rna replication through the function of lds. although the possible relation between lds and alphavirus replication has never been tested in mammalian cells, the finding that sinv infection induces the accumulation of lds in mosquito aag cells as well as in the midgut of aedes aegypti females [ ] seems to support the idea that alphavirus might rely on lds for its replication. the family hepeviridae englobes a group of hepatitis e viruses (hev) that are non-enveloped and contain a positive-sense single-stranded rna genome that contains two orfs. the first orf encodes a non-structural polyprotein (orf protein) that exhibits several functions, such as methyltransferase, protease, rna helicase, and rdrp, while the second orf codes for the capsid protein (cp). due to the low efficiency of these viruses to grow in cell culture, their replication process is poorly understood, although it has been proposed that it might be similar to that of togaviruses [ ] . in fact, a recent report revealed that similar to viruses in the togaviridae family, the rna replication of hev was sensitive to treatments with bfa and gca, and that gbf plays an essential role in this process although, unlike togaviruses, the inhibition of gbf activity by bfa does not affect the formation of the hev replication complexes. the members of this genus are enveloped viruses that contain a large positive-sense, single-stranded rna genome with several orfs that direct the synthesis of nonstructural proteins (nsp to nsp ), four structural proteins (s, m, e, and n) and, in some viruses, a group of up to eight accessory proteins that are key for the efficient replication of the virus in natural conditions [ ] . once released in the cytoplasm, the viral rna directs the synthesis of the nonstructural proteins that form the rdrp complex, which transcribes the full-length, negative-sense antigenomic rna. the coronavirus antigenome is then used for the production of the full-length genomic rna copies and a set of subgenomic mrnas of different lengths that direct the synthesis of the structural and accessory proteins. following their synthesis, the structural proteins s, e, and m are inserted into the er and then transported along the secretory pathway up to the ergic, where the nucleocapsid, formed by the new rna genome bound to n proteins, buds to form the mature virions. following assembly, virions are finally released to the extracellular medium by exocytosis. during coronavirus infection, all rna synthesis processes occur in a replication-transcription complex (rtc), composed of viral and host proteins that are associated to a reticulo-vesicular network (rvn) made of modified intracellular membranes and double-membrane vesicles (dmvs) apparently derived from the er [ , ] . recent evidence has indicated that the secretory pathway participates in the formation and function of both rtc and rvn. it has been reported that the inhibition of the secretory pathway by bfa or gca strongly reduces the rna replication of different coronavirus strains such as the human coronavirus e (hcov- e, alphacoronavirus), the mouse hepatitis coronavirus (mhv, betacoronavirus), and the severe acute respiratory syndrome coronavirus (sars-cov, betacoronavirus) [ , , ] . these effects were shown to be related with the specific inhibition of the activity of gbf [ , ] . the precise role of gbf in coronavirus rna replication is not clear; however, given that the inhibition of this factor by bfa does not alter the rna-synthesizing activity of rtcs, but rather decreases the number of these organelles, as well as the number of dmvs in mhv-and sars-cov-infected cells, it has been proposed that the gbf activity might be required for the formation of the virus-induced rvn [ , ] . altogether, coronavirus infection seems to redirect the vesicle transport mediated by gbf toward the rtcs to maintain the correct morphology of these viral organelles. in support of this proposal, the silencing of arf proteins or subunits of the copi complex has shown to induce a similar inhibitory effect on mhv and hcov- e replication, similar to that observed with the gbf depletion [ , ] . on the other hand, the finding that overexpression of the dominant-negative mutant arf -t n strongly inhibits mhv replication [ ] , together with the fact that this arf mutant has been shown to inhibit the ergic to er transport [ ] , lead to suggest that gbf might regulate the maturation of rvn through the retrograde vesicular transport of membrane lipids from the ergic to the er [ ] . the vesiculovirus genus comprises a distinct monophyletic group of enveloped bullet-shaped viruses that contain a negative-sense, single-stranded rna genome that codes for five structural proteins: the nucleocapsid protein (n), the phosphoprotein (p), the matrix protein (m), the glycoprotein (g), and the rdrp (l). after virus entry into the cell, the viral nucleocapsid is released into the cytoplasm. once free, the virion polymerase complex, composed by the n, p, and l proteins drives the transcription of the viral mrnas, which then direct the synthesis of the different viral proteins. once sufficient quantities of the n and p protein have been produced, the virion polymerase complex starts the synthesis of a full-length complementary antigenome rna of positive sense. the antigenome then serves as a template to synthetize new viral negative-sense genome molecules, which can be used for a second round of transcription or can be assembled into new viral particles. the union of the n protein to the newly genomic rna compromises these molecules to the formation of helical nucleocapsids that interact with the m protein and are transported to cell membrane sites enriched by the spike g glycoprotein. budding of the nucleocapsid through these sites leads to the formation of the final enveloped virus [ ] . given that the transport of the g protein to the cell membrane depends on a functional secretory pathway, the vesicular transport was thought to only play a role in the final steps of vesiculovirus replication. however, studies with the vesicular stomatitis virus (vsv), the prototypic member of this genus, revealed that this transport was also important for the synthesis of the viral rna. it was observed that bfa or gca treatments induce a substantial reduction in the levels of viral mrna, and the antigenome rna of vsv [ , , ] . the action of these inhibitors was shown to be related to the gbf protein [ , ] . although the exact role of gbf in rna synthesis is unknown, taking into account that the depletion of arf or the overexpression of the dominant-negative mutant arf -t n decrease the abundance of viral rna products and reduce the vsv gene expression [ ] , it seems that the role of gbf in the process might be related to vesicle transport. on the other hand, a structure-based computational analysis of possible protein-protein interactions between human proteins and the proteins of chandipura virus (chpv), another member of the vesiculovirus genus, which has emerged as a pediatric encephalitic virus in india [ , ] , revealed that gbf and arf exhibit a high potential for interaction with the g and l proteins of this virus [ ] . although the association of gbf and arf with the g protein was rather expected, given the association of the g protein with the secretory pathway, the potential interaction of gbf and arf with the polymerase l of chpv is a surprising finding that might indicate that gbf could directly regulate the activity of the l protein during viral rna replication; the interaction between gbf and the l protein was not corroborated by biochemical assays. on the other hand, considering that the microtubule-dependent transport of viral mrnas from the vsv inclusions to the cytoplasm has been proved to be essential for their efficient translation [ ] , it might also be possible that gbf promotes the protein expression of vesiculoviruses by facilitating the transport of viral mrnas out of the inclusions through the activation of arf and the subsequent recruitment of copi elements. in support of this model, it has been reported that vsv infection can be inhibited by microinjection of an antibody against cop-β that blocks the transport between the er and golgi [ ] . influenza viruses are enveloped viruses that contain a segmented negative-sense, single-stranded rna genome that can be formed by seven or eight rna segments, depending on the virus strain. the genome of these viruses codes for three proteins that form the viral rdrp (pa, pb , pb ), a nucleoprotein (np) that is associated with each genome segment, a hemagglutinin (ha) protein that facilitates virus attachment, and the matrix protein (m ) that acts as an adaptor protein between the viral ribonucleoproteins (rnps) and the membrane envelope. moreover, some members also code for a neuraminidase (na) that contains sialidase activity essential for the releasing of the mature viral particles, and a small integral membrane matrix protein (m ), a proton selective ion channel. in addition to structural proteins, these viruses may also code for two nonstructural proteins (ns , ns ). after cell entry, the rnps are released into the cytoplasm; once free, the rnps are then imported to the cell nucleus, where the viral rdrp synthesizes the mrnas that direct the synthesis of the viral proteins in the cytoplasm of the cell. during their synthesis, the proteins ha, na, and m are inserted into the er membrane, and are transported to the sites of viral assembly in the plasma membrane. concomitant with this process, the np, pa, pb , and pb proteins are imported into the nucleus and bind the newly synthesized rna genome segments to form the novel rnps that are then exported to the cytoplasm and subsequently transported to the sites of viral assembly in the plasma membrane. finally, the budding of rnps in the enriched ha, na, and m membrane sites produces the new influenza virus infectious particles [ , ] . two models have been proposed to explain the rnp traffic to the plasma membrane, and in both cases the interaction of the rnps with rab is involved [ ] [ ] [ ] . recently, it was also reported that, in addition to rab , the activity of gbf was important for the assembly process of influenza a virus (iav) [ , , ] . the block on iav assembly was found to be related with a decrement on the cell surface of the ha, na, and m viral proteins, as well as with disruption of the transport of rnps to the plasma membrane [ ] [ ] [ ] ] , indicating that gbf facilitates the assembly of iav by regulating the transport to the plasma membrane of the viral elements that form the iav virion. besides its role in regulating the transport of viral proteins to the plasma membrane, it has been shown that gbf interacts specifically with the protein m [ ] . although the biological importance of this interaction is unknown, the observation that iav induces the disruption of the golgi apparatus [ ] makes it possible that through the m -gbf interaction the gbf is hijacked from the secretory pathway (as observed for the a protein of picornavirus), to promote the transport of ha, na, m proteins, and the rnps. moreover, the observation that the inhibition of rnp transport by bfa induces the accumulation of rnps in tubulated structures in the perinuclear region [ ] , suggest that m might recruit gbf from the golgi apparatus to the tubulate er network to promote the transport of ha, na, m , and rnps to the plasma membrane. in support of the role of gbf to stimulate the transport of these viral elements, it has been found that arf can colocalize with rnps of iav [ ] , suggesting that gbf might mediate the traffic of iav rnps by maintaining an active copi transport. in support of this idea is the discovery that rnps travel to the plasma membrane in a microtubule dependent fashion [ , ] , as well as the close relationship between the movement of copi vesicles and microtubules, which seem to indicate that copi vesicles might be important to the transport of rnps. in addition, copi was also suggested to be related with the internalization process of iav, since its depletion was shown to reduce this process [ , ] . however, given that these results could not be recapitulated by bfa and gca treatments, it was presumed that this effect might be due to the long-term inhibition of protein and lipid transport to the plasma membrane [ ] . the evaluation of the effects of rnai depletion of copi on iav replication assays that bypass the virus internalization process would help to bring light into this controversial matter. the participation of gbf in the replication of negative-sense rna viruses is not restricted to iav. the replication of human parainfluenza virus type (hpiv , member of paramyxoviridae family), as well as that of the lymphocytic choriomeningitis virus (lcmv, the prototypic member of the arenaviridae family) have also shown to be dependent on the gbf activity. depletion of this factor by rnai or its pharmacological inhibition by bfa or gca, has shown to significantly reduce the protein synthesis of these viruses [ ] . however, whether the role of gbf might be related directly to viral protein synthesis or results from defects in the previous steps of the viral cycle, remains to be elucidated. on the other hand, although hpiv and lcmv were shown to depend on gbf , the finding that arf depletion reduces the lcmv protein expression, but does not affect that of hpiv , suggests that the role of gbf in lcmv could be related to the copi transport, whereas for hpiv gbf might play an alternative function independent of vesicle transport, although the possibility that hpiv could depend on other arfs cannot be ruled out. rotaviruses are non-enveloped viruses formed by three concentric layers of proteins that surround a genome composed of segments of double-stranded rna (dsrna) that code for six nonstructural (nsp - ) and six structural (vp - , vp , and vp ) proteins. the inner core of rotavirus is composed by the protein vp , which encloses the replication machinery formed by the dsrna segments, the rdrp vp , and the protein vp . the intermediate layer, formed by vp , surrounds the viral cores to form double-layered particles (dlps). finally, the addition of the glycoprotein vp and the spike protein vp onto the dlps forms the infectious triple-layered particles (tlps). the replication of rotavirus occurs in cytoplasmic non-membranous electron-dense inclusions termed viroplasms. replication and packaging of the viral genome into newly synthesized dlps take place in these inclusions. the novel dlps then bud into the lumen of er through membrane sites modified by the presence of nsp and vp , acquiring a transitory lipid envelope bearing the nsp , vp , and vp proteins. as the new dlps move into the lumen of the er, the transient lipid envelope is removed by an unknown process in which nsp is eliminated while vp and vp are assembled to produce the final infectious tlps [ ] . although many of the steps of rotavirus replication have been elucidated in recent years, the precise mechanism of the final events of rotavirus assembly is not well understood. it is known that nsp (a transmembrane er glycoprotein) plays a crucial role in the last steps of rotavirus assembly. the interaction of nsp with vp and vp [ ] , together with the binding of nsp to the vp protein on the surface of dlps, has been proposed to drive the incorporation of the outer layer proteins to form the final tlps. however, whether specific cellular pathways or cell proteins are involved in the assembly process remains unknown. in recent years, the use of genome-wide rnai screens has shown that the vesicle transport mediated by the copi/arf machinery is relevant for virus replication, since knocking down the expression of some of the proteins that integrate such machinery significantly impairs the yield of rotavirus progeny [ , ] ; similar results are obtained when the vesicle transport is inhibited by bfa treatment of rotavirus-infected cells [ , ] . this inhibition is related to the activity of gbf , since interfering specifically with this factor by gca, or depletion of this nucleotide exchange factor by rnai, significantly reduced the production of new viral infectious particles [ ] . in addition, the block in the production of infectious virus was associated with the assembly of the virus surface proteins vp and vp , since both bfa and gca block the assembly of the viral surface proteins onto dlps, preventing the production of mature infectious tlps [ ] . the restriction in the assembly of tlps was shown to be the result of a defective trimerization of vp , required for its assembly onto dlps, rather than a block in the budding of dlps into the er lumen. it has been shown that the inhibition of gbf by either bfa or gca, or the depletion of this factor by rnai, alters the electrophoretic mobility of the viral glycoproteins vp and nsp [ , ] . although the altered posttranslational modification of vp was initially considered to explain its lack of trimerization, increasing evidence has pointed out that the alteration of nsp is rather responsible, since depletion of this protein or alteration of its posttranslational processing with tunicamycin, an inhibitor of n-glycosylation, affects the trimerization of vp and restricts the maturation of the transient enveloped viral particles to tlps. [ , , ] . in support of this observation, it was recently demonstrated that a recombinant vp protein overexpressed in transfected cells formed trimers only when co-expressed with nsp [ ] . therefore, it is likely that the altered post-translational modification is suffered by nsp when the expression of gbf is knocked down, affects its role in facilitating (directly or indirectly) the trimerization of vp . it cannot be discarded, however, that the altered glycosylation pattern of vp in the presence of the pharmacological inhibitors could also have an impact in its trimerization. the altered electrophoretic mobility of vp in the presence of drugs or in cells depleted of gbf has been shown not to be related to n-glycosylation, o-glycosylation, or phosphorylation [ ] . given that gbf functions at the er-golgi interface, mediating the retrograde vesicular transport, and that rotaviruses mature in the er, it seems likely that golgi-er transport is the relevant process required for the correct maturation of rotavirus. however, considering that lds have been proposed to play a significant role in the replication cycle of these viruses, it remains possible that the protein transport between the er and lds is also relevant, although, a so far uncharacterized function of gbf cannot be discarded. on the other hand, in support of the requirement of an active gbf transport during rotavirus infection, it was found that catalytically inactive mutants of gbf failed to support replication of the virus in the presence of bfa, suggesting that gbf activity is essential for rotavirus progeny production. however, given that knocking down the expression of arf was found not to affect the production of infectious virus, it is likely that rotavirus replication does not depend on the gbf /arf transport but might require the activation of other gbf substrates, such as arf or arf , that could compensate for the reduced level of arf . moreover, it was also found that while the c-terminal domain of gbf is dispensable for the viral replication, the first n-terminal amino acids of this factor are essential to support replication of the virus [ ] . the requirement of the n terminus of gbf for rotavirus replication has also been reported to be essential for the replication of cvb and poliovirus (see above). thus, these results might indicate that gbf could bind a rotavirus protein, similar to what has been observed with the picornavirus a protein. however, an interaction of gbf with a cellular factor might also be important for virus replication. further experiments have to be carried out to explore in more detail the mechanism through which gbf supports the replication cycle of rotavirus replication. as intracellular pathogens, the infection of viruses depends on their capacity to replicate successfully within the host cell. the restricted coding potential imposed by the small size of viral genomes had led the viruses to hijack essential systems of cellular functions to facilitate their replication. in this regard, emerging data have shown that many cellular factors are susceptible to be reprogrammed during viral infections to perform novel functions that benefit these pathogens. one of these cellular elements is the guanine nucleotide exchange factor gbf , which has proved to be essential for the efficient replication of many rna viruses. it has been shown to be required for different stages of the virus lifecycles, including the formation and maturation of viral replication complexes as well as the coordination of rna genome replication, virus assembly, and release. the role of gbf has been found, principally, to depend on its ability to activate distinct arf proteins, to regulate different transport pathways that permit the communication between the viral replication organelles and cellular organelles (er and lds) involved in virus infection. however, for many viruses, the role of gbf in virus replication has also been shown not to depend on the catalytic activity of the protein. although it is not well understood how gbf could exert this non-catalytic activity, the multidomain structure of the protein seems to play an essential role by permitting gbf to engage in the interaction with many cellular and viral proteins that seems to regulate, directly or indirectly, viral replication. the relevance of gbf for the replication of several rna viruses identifies this factor as a potential candidate for antiviral therapies to control a broad-spectrum of viral infections. however, given the essential role of gbf in the secretory pathway, and in other so far ill-characterized functions of the cell, the development of safe and effective antiviral therapies that could help to deal with viral infections, without harming the host cells, represents a challenging mission. in conclusion, the data discussed in this review bring to light a new diverse range of functions of gbf that might explain how this factor serves for so many processes in 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viral rna apical transport of influenza a virus ribonucleoprotein requires rab -positive recycling endosome influenza virus genome reaches the plasma membrane via a modified endoplasmic reticulum and rab -dependent vesicles involvement of vesicular trafficking system in membrane targeting of the progeny influenza virus genome. microbes infect human host factors required for influenza virus replication fields virology rotavirus proteins vp , ns , and vp form oligomeric structures a systems survey of progressive host-cell reorganization during rotavirus infection genome-wide rnai screen reveals a role for the escrt complex in rotavirus cell entry effect of brefeldin a on rotavirus assembly and oligosaccharide processing dissecting rotavirus particle-raft interaction with small interfering rnas: insights into rotavirus transit through the secretory pathway effects of tunicamycin on rotavirus morphogenesis and infectivity this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -g os m authors: martins-da-silva, andrea; telleria, erich loza; batista, michel; marchini, fabricio klerynton; traub-csekö, yara maria; tempone, antonio jorge title: identification of secreted proteins involved in nonspecific dsrna-mediated lutzomyia longipalpis ll cell antiviral response date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: g os m hematophagous insects transmit infectious diseases. sand flies are vectors of leishmaniasis, but can also transmit viruses. we have been studying immune responses of lutzomyia longipalpis, the main vector of visceral leishmaniasis in the americas. we identified a non-specific antiviral response in l. longipalpis ll embryonic cells when treated with non-specific double-stranded rnas (dsrnas). this response is reminiscent of interferon response in mammals. we are investigating putative effectors for this antiviral response. secreted molecules have been implicated in immune responses, including interferon-related responses. we conducted a mass spectrometry analysis of conditioned medium from ll cells and h after dsrna or mock treatment. we identified proteins. at h, proteins had an abundance equal or greater than -fold change, while the levels of proteins were reduced when compared to control cells. at the h time point, these numbers were and , respectively. the two most abundant secreted peptides at h in the dsrna-transfected group were phospholipid scramblase, an interferon-inducible protein that mediates antiviral activity, and forskolin-binding protein (fkbp), a member of the immunophilin family, which mediates the effect of immunosuppressive drugs. the transcription profile of most candidates did not follow the pattern of secreted protein abundance. insect-borne diseases afflict billions of people throughout the world. these illnesses are caused by different pathogens such as helminths, protists, bacteria, and viruses, which are the etiologic agents of diseases such as filariasis, leishmaniasis, babesiosis and dengue. among the most important insect vectors are mosquitos and sand flies [ , ] . phlebotomine sandflies from the genera phlebotomus and lutzomyia are the main vectors of leishmania in the old and new world, respectively. in the old world, these dipterans are also proven virus vectors of phlebovirus, vesiculovirus and orbivirus [ ] [ ] [ ] . despite the lack of evidence proving the vectorial capacity of new world sandflies in the transmission of viruses to humans, the occurrence of diverse virus species naturally infecting lutzomyia spp. populations has been demonstrated [ ] [ ] [ ] [ ] . among insect transmitted diseases, the arboviruses are among the most devastating infectious illnesses in our planet. chikungunya virus (chikv), west nile virus (wnv) and dengue virus (denv) are three of a large number of human pathogenic arthropod-borne viruses [ ] . also, the world recently witnessed the reemergence of zika virus (zikv), which is associated with severe human neurological pathologies in adults and fetuses [ ] . the predominant strategy for controlling vector-borne diseases consists of targeting the vectors, mostly using insecticides. the deleterious side effects of insecticide usage are widely known. among these are consequences to ecosystems, animal and human health, and development of resistance, pointing to the urgency of developing new control strategies. the understanding of biological mechanisms involved in the relationship between pathogens and vectors can provide new approaches for the development of control strategies. the infection of aedes aegypti with wolbachia [ ] is an excellent example of a novel arbovirus control strategy. our group has been studying the interaction and immune responses of the sand fly lutzomyia longipalpis (diptera: psychodidae: phlebotominae), the most important vector of american visceral leishmaniasis [ ] , when infected with different pathogens [ ] [ ] [ ] . the establishment of continuous insect cell lines has been an important tool for the study of insect biology. cells from drosophila melanogaster (s ), aedes albopictus (c / ), and anopheles gambie (sua b), among other insect cell lines, have been employed to scrutinize diverse features of insect immunity [ ] [ ] [ ] . we have detected complex immune responses in the lutzomyia longipalpis ll embryonic cell line [ ] when challenged by different pathogens [ ] . we have also identified a non-specific antiviral response in these cells when transfected with non-specific double-stranded rna (dsrna) [ ] , in a way similar to interferon response in mammals [ ] . after dsrna transfection, regardless of nucleotide sequence, there was no replication of a west nile reporter virus that expresses luciferase and lacks structural genes, as indicated by a lack of luciferase production [ ] . we are presently searching for molecules responsible for this response. secreted proteins have a key role in biological processes involving cell signaling. naïve cells can be modulated by viral or host components transferred from neighboring infected cells via the release of extracellular vesicles and secretion of soluble molecules, leading to activation of host immune response and changes in the secreted protein repertoire [ ] [ ] [ ] . in order to investigate the effect of polyinosinic:polycytidylic acid (poly i:c) transfection on the protein secretion profile of l. longipalpis ll cells, we carried out mass spectrometry (ms) analyses comparing the protein composition of conditioned medium from poly (i:c) and mock-treated ll cells. l. longipalpis embryonic ll cells obtained from dr. robert b. tesh (university of texas medical branch, galveston, tx, usa) were grown at • c in l medium (sigma, mendota heights, mi, usa) supplemented with % fetal bovine serum (fbs; hyclone, logan, ut, usa), % tryptose phosphate broth, and % antibiotics (penicillin u/ml and streptomycin mg/ml, sigma). for mock transfection, ng/ml lipofectin transfection reagent (invitrogen, carlsbad, ca, usa) were added to l medium (leibovitz) containing % tryptose phosphate broth (tpb). the transfection mix contained the mock transfection mix added with ng/ml of polyinosinic:polycytidilic acid (poly i:c), a synthetic analog of double-stranded rna (invitrogen). these mixtures were added to × ll cells for h and supernatant was aspirated and stored. new medium was added and supernatant was aspirated and stored after h. for rna preparation, transfected and mock-transfected cells were collected after , , , and h incubations, as described above. cell viability was verified by trypan blue staining. more than % of cells were viable in all experiments. protease inhibitor cocktail × (sigma) was added to the media. dead cells and large debris were pelleted by centrifugation at × g for min. supernatants were centrifuged again at , × g for min to exclude remaining cell debris and microvesicles. the proteins in the supernatant were then precipitated using trichloroacetic acid (tca). one volume of tca % (sigma) was added to four volumes of conditioned medium and incubated for min at − • c. the samples were centrifuged at , × g for min. the supernatant was removed and the protein pellet was washed with cold acetone, vortexed and centrifuged at , × g for min. the material was submitted to mass spectrometry. the pellets were suspended in m urea, m thiourea, and mm -( -hydroxyethyl)- piperazineethanesulfonic acid (hepes). the proteins were reduced with mm dithiothreitol (dtt) and mm ammonium bicarbonate (abc), and alkylated with . mm iodacetamide and mm abc. before trypsinization, the samples were purified with detergent removal spin columns (thermo scientific). then, the samples were digested in mm abc with trypsin (promega, cat. v , madison, wi, usa) in a : trypsin to protein mass ratio by incubation at • c for h. after trypsinization, trifluoroacetic acid (tfa) was added to a final concentration of . %. peptides were desalted with homemade c spin columns. the peptides were analyzed in triplicate by liquid chromatography tandem-mass spectrometry (lc-ms/ms) in a thermo scientific easy-nlc system coupled to a ltq orbitrap xl etd (mass spectrometry facility rpt h/carlos chagas institute-fiocruz, curitiba, pr, brazil). peptide separation was carried out in -cm ( -µm inner diameter) fused silica, in-house packed with reversed-phase reprosil-pur c -aq -µm resin (dr. maisch gmbh, ammerbuch-entringen). chromatography runs were performed in a flow rate of nl/min from to % mecn in . % formic acid in a -min gradient, and a voltage of . kv was applied for peptide ionization. the mass spectrometer operated in a data-dependent acquisition mode. survey full-scan ms spectra (at a - m/z range) were acquired in the orbitrap analyzer with resolution of , at m/z (after accumulation to a target value of , in the c-trap). the ten most intense ions were sequentially isolated and fragmented in the linear ion trap using collision-induced dissociation at a target value of , . the "lock mass" option was enabled at . m/z in all full scans to improve mass accuracy of precursor ions [ ] . protein identification was performed with maxquant algorithm [ , ] version . . . . default parameters of the software were used for all analysis steps, unless stated otherwise. proteins were searched against an l. longipalpis protein sequence database (containing , protein sequences from the vectorbase protein database, downloaded on december ) and common contaminants, besides their respective reverse sequences to estimate the false discovery rate (fdr). carbamidomethylation of cysteine was set as a fixed modification, while methionine oxidation and n-terminal acetylation (protein) were allowed as variable modifications. in addition, an fdr threshold of . was applied at both peptide and protein levels. protein quantification was performed using a label-free approach, where the peptide peaks were detected as three-dimensional features-retention time versus signal intensity (extracted ion chromatogram, xic) versus mass/charge-and were aligned and compared across the runs, as previously described [ ] . the amino acid sequences of identified secreted proteins were subjected to bioinformatic analyses. the protein signal peptide (sp) from proteins was estimated using the software tool prediction of signal peptide "predsi" at http://www.predisi.de./home.html [ ] , under standard configuration. protein-protein interactions were performed using the search tool for the retrieval of interacting genes/proteins (string) database v . at http://string.embl.de/ [ ] . three biological replicates of × cells were transfected. the supernatant was removed , , , and h post-transfection and the cells, after being washed three times with pbs, were resuspended in ml of trizol (ambion, waltham, ma, usa). the rna was extracted following the trizol manufacturer's instructions. total rna was precipitated with isopropanol, resuspended in water and stored at − • c. rna was treated with dnase i (promega) and cdna was synthesized from µg of total rna using superscript iii first-strand synthesis (invitrogen). real-time polymerase chain reaction (qpcr) was performed using sybr green pcr master mix (applied biosystems, foster city, ca, usa) and the primers listed in table . expression levels were determined through (-delta delta c(t)) method (ddct) [ ] , normalized using rp gene expression [ ] , yielding the relative expression value. statistical analyses were done using graphpad prism software, version . (graphpad software, inc., san diego, ca, usa). for comparison of the transfected and mock samples at different times after transfection, an unpaired two-tailed student's t test was performed. * p ≤ . , ** p ≤ . , *** p ≤ . . table . list of primers used for qpcr. hemocytin basic transcription factor btf -f agtgcataagcaggcaacacca btf -r tacgtcgccaccgaaatgagtt juvenile hormone-inducible protein jhip-f ccttgctgagctccttgagaaact jhip-r tacacatggccattcccatcttcc eukaryotic translation initiation factor etif -f tcgataggcatctcacgtttccac etif -r tcttccccactgtatccaggatgt transmembrane protease serine -like repressor splicing factor rrm-f aatttgggaagctcaata rrm-r gaggatgtcgcaagccttct forskolin-binding protein proliferating cell nuclear antigen pcna-f catgaatctcgaccaggagcactt pcna-r tcacggcaaatgcgtgcaaa tubulin-specific chaperone a tbca-f cgtacgaaaaggaagcagatcagca tbca-r cttccttccggatcacgtgttcat ribosomal protein rp -f gaccgatatgccaagctaaagca rp -r ggggagcatgtggcgtgtctt comparative analysis of expression values was performed to verify the correlation between mrna levels of ll cells (samples collected , , and h post transfection) evaluated by qpcr, and secreted protein levels (samples collected or h post transfection) evaluated by mass spectrometry. linear regression, pearson's correlation coefficient (r), and goodness of fit (r ) were calculated using graphpad prism software (version . ). immune responses are triggered through the recognition of pathogen-associated molecular patterns (pamps) by host pattern recognition receptors (prrs). binding of pamps leads to the activation of signaling pathways: rnai, janus kinase/signal transducers and activators of transcription (jak/stat), immune deficiency (imd) and toll [ ] [ ] [ ] [ ] . the major insect innate immune response triggered by virus infection is normally the small interfering rna pathway. rnai is a molecular mechanism in which the presence of intracellular dsrna leads to the production of small rnas that suppress the expression of genes with matching sequences [ ] . nevertheless, alternative mechanisms exist. in previous work, our group identified an antiviral response when the ll embryonic l. longipalpis cell lineage was transfected with dsrna, which included the synthetic poly i:c. after dsrna transfection, cells did not support wnv virus like particle (vlp) replication, as evidenced by lack of luciferase reporter gene expression. this block in replication may be due to a cellular antiviral response triggered by dsrna. [ ] . a similar non-specific antiviral response was observed in the marine shrimps litopanaeus vannamei and penaeus monodon [ , ] . more recently, the occurrence of dsrna-mediated non-specific antiviral response was also described in two members of the apidae family: the honey bee apis melifera, and the bumblebee bombus terrestris [ ] [ ] [ ] . this non-specific response differs from what is seen in drosophila, where transfection with virus-specific dsrna triggers an rna interference (rnai)-mediated response against the virus, whereas non-related dsrnas fail to activate the antiviral response [ ] . in vertebrates, intracellular dsrna is recognized by the prr retinoic acid-inducible gene (rig-i) and the melanoma differentiation-associated protein (mda ). these are also known as rig-i-like receptors (rlrs) [ , ] . these receptors recruit the mitochondrial antiviral-signaling (mavs) protein that mobilizes kinases and ubiquitin ligases, leading to the activation of transcription factors, such as interferon receptor factor (irf- ) and nuclear factor κb (nf-κb) [ , ] . the transcription factors induce type i interferon (ifn) and pro-inflammatory cytokine production. dsrna, including poly i:c, is also detected by toll-like receptor , initiating the type-i interferon (ifn-α, β) signaling pathway via a toll/interleukin- receptor (tir)-domain-containing adapter-inducing interferon-β (trif) signal, which activates interferon receptor factor (irf- ) and nuclear factor κb (nf-κb), leading to ifn-β expression [ , ] . to investigate variations in the exoproteome profile of dsrna-transfected l. longipalpis ll cells, we carried out mass spectrometry (ms) analyses comparing the protein composition of conditioned medium from poly i:c and mock-treated cell cultures at two time points ( and h); in total proteins were identified (table s ) . only . % ( ) of all identified secreted proteins presented a signal peptide (sp). among proteins with ≥ -fold positive change variation, this percentage decreased to . %, with only six proteins with sp: at h and at h (table s ). the lack of signal peptide in the majority of secreted proteins suggests that unconventional protein secretion pathways are the main secretory mechanisms in ll cells, as already seen in other systems [ ] . this could be due at least partly to exosomal transport. we identified some of the known exosome markers among our secreted proteins, based on the exo carta exosome marker list (www.exocarta.org/exosome_markers). among these were glyceraldehyde- -phosphate dehydrogenase (gapdh), enolase (eno), fructose-bisphosphate aldolase (aldoa), moesin (msn), phosphoglycerate kinase (pgk), cluster of differentiation (cds) , and , the heat shock proteins (hsps) and , and annexins (table s ). these were not differentially expressed with a greater than -fold change and were present in both mock and poly i:c transfection samples. we performed an interaction analysis of the ll dsrna-transfected exoproteome. the network analysis of proteins presenting different abundance between mock and transfected groups (table s ) predicted that proteins (table s ) were connected by edges ( figure a) forming an intricate network. the prevised protein interaction (ppi) enrichment p-value was . , meaning that secreted proteins have more relations among themselves than what would be expected for a random set of proteins of similar size drawn from the genome. exoproteome functional enrichment analysis identified enhanced interactions related to specific biological functions. these interactions have a significant false discovery rate (fdr) of less than . . among these gene ontology (go) categories and pathways, we can quote: biological process go: (peptide metabolic process) fdr: we also carried out string interaction analysis ( figure b ) of proteins with ≥ -fold change variation detected on mass spectrometry analysis. from the protein sequences (table s ) shown in figure b ,c, were identified in the string database (table s ) and from those, (table s ) ( figure b) were predicted as connected at least to one protein. this interaction analysis displayed a significant ppi enrichment p-value of . × − . in this interactome we can distinguish two major protein groups: the first one with members is related to oxidative stress modulation and includes phospholipid scramblase (plscr ), thioredoxin (txn), glutaredoxin (glrx ), superoxide dismutase (sod ), and forskolin-binding protein (fkbp ), among others. the second group is comprised of proteins linked with peptide synthesis and cytoskeleton organization. among these are the riboproteins rps and rplp , the eukaryotic translation initiation factor (eif d), moesin (msn), cortactin protein (cttn), and others. two biological functions were enhanced in the ≥ -fold change secreted protein cluster: cellular component: go: (extracellular exosome) fdr: . × − and molecular function: go. (rna binding) fdr: . × − . these same biological function enrichments are found when we study the whole exoproteome. the complete fdr-filtered (p < . ) lists are shown in table s . the measurement of relative protein abundance revealed that the secretion of most proteins did not vary significantly between test and control supernatants (figure a ). only . % ( ) of all identified proteins at h presented variation of ≥ -fold change, and . % ( ) presented variation of ≤ -fold change ( figure b ). these percentages change to . % ( ) and . % ( ) , respectively, at h after dsrna transfection ( figure c ). we focused on the proteins with ≥ -fold change variation, which represent . % of the total identified proteins ( figure c ). the proteins with intensified secretion in the first h had their relative amounts decreased at h. most proteins more abundant at h had a basal detection level at h. interestingly only one protein, the enzyme carboxylesterase, presented secretion levels with ≥ -fold change at both time points. in insects this enzyme is related to detoxifying processes, including insecticide resistance [ ] . several proteins with higher secretion at h or h are related to immune responses. among the proteins with higher secretion at h we can highlight eight proteins related to immune response. phospholipid scramblase (plscr ) had a positive variation with a greater than -fold change. scramblases are conserved in all eukaryotic organisms. they are multiple palmitoylated, endofacial membrane proteins originally identified based on their capacity to promote accelerated transbilayer phospholipid movement in response to ca + [ ] . plscr was identified as an interferon and growth factor-inducible protein necessary for the ifn-dependent induction of gene expression and antiviral activity [ ] . in human and mouse plasmacytoid dendritic cells (pdcs), which are professional interferon-producing cells specialized in recognizing viral rna and dna through the endosomal toll-like receptors (tlrs) tlr and tlr , respectively, plscr was described as a tlr -binding protein that plays a significant role in type- interferon responses in pdcs by regulating tlr expression and trafficking [ ] . the direct interaction of plscr with human t-cell leukemia virus type- (htlv- ) leads to a reduction of virus titers [ ] . these reports demonstrate the significant, but not fully understood, role of phospholipid scramblase in antiviral response. another interesting protein with a greater than -fold increased secretion at h was the hexameric chaperone complex prefoldin (pfd). subunits of prefoldin have been found to be associated with cellular response to viral infection. the pfdn expression was positively modulated in both vaccinia virus-infected hek cells and in the rabv-infected n a cells [ , ] . pfdn was found directly bound to the hepatitis c virus (hcv) f protein [ ] . the string interactome maps were generated using default settings (medium confidence of . , with criteria for linkage: neighborhood, genefusion, co-occurrence, co-expression, experimental evidence, existing databases, and textmining). proteins were represented as nodes and their functional links were defined by solid lines. the thickness of the lines signifies the level of confidence of the reported association. the non-connected nodes were suppressed. no additional interplay proteins were added to the networks. the protein symbols and node colors are listed in table s . still within the proteins with higher secretion at h, we found forskolin-binding protein (fkbp ), an immunophilin family member, with a secretion level increase of greater than -fold. this is a highly conserved group of proteins which binds to immunosuppressive drugs such as fk , rapamycin, and cyclosporine [ , ] . fkbps are involved in several biochemical processes and participate in the immune response against viral infection. mitochondrial fkbp was identified as a tumor necrosis factor (tnf) receptor-associated factor (traf ) and tnf receptor-associated factor (traf ) binding protein. binding of fkbp to traf proteins facilitates the type-i interferon response induced by dsrna transfection or newcastle disease virus (ndv) infection in murine fibroblasts. depletion of fkbp reduced the expression of type inf in these cells [ ] . one interesting protein with a greater than -fold enhanced secretion at h is coatomer protein i (copi). copi-coated vesicles are associated with transport from the golgi to the endoplasmic reticulum [ ] , endocytosis [ ] , maturation of autophagic vacuoles [ ] , expression of cell surface receptors, and modulation of the plasma membrane lipid composition [ ] . studies demonstrated the involvement of copi subunits in diverse aspects of virus infection [ ] [ ] [ ] . depletion of the copi β subunit in human /ace cells led to a decrease in the severe acute respiratory syndromecoronavirus (sars-cov) replication [ ] . the string interactome maps were generated using default settings (medium confidence of . , with criteria for linkage: neighborhood, genefusion, co-occurrence, co-expression, experimental evidence, existing databases, and textmining). proteins were represented as nodes and their functional links were defined by solid lines. the thickness of the lines signifies the level of confidence of the reported association. the non-connected nodes were suppressed. no additional interplay proteins were added to the networks. the protein symbols and node colors are listed in table s . still within the proteins with higher secretion at h, we found forskolin-binding protein (fkbp ), an immunophilin family member, with a secretion level increase of greater than -fold. this is a highly conserved group of proteins which binds to immunosuppressive drugs such as fk , rapamycin, and cyclosporine [ , ] . fkbps are involved in several biochemical processes and participate in the immune response against viral infection. mitochondrial fkbp was identified as a tumor necrosis factor (tnf) receptor-associated factor (traf ) and tnf receptor-associated factor (traf ) binding protein. binding of fkbp to traf proteins facilitates the type-i interferon response induced by dsrna transfection or newcastle disease virus (ndv) infection in murine fibroblasts. depletion of fkbp reduced the expression of type inf in these cells [ ] . one interesting protein with a greater than -fold enhanced secretion at h is coatomer protein i (copi). copi-coated vesicles are associated with transport from the golgi to the endoplasmic reticulum [ ] , endocytosis [ ] , maturation of autophagic vacuoles [ ] , expression of cell surface receptors, and modulation of the plasma membrane lipid composition [ ] . studies demonstrated the involvement of copi subunits in diverse aspects of virus infection [ ] [ ] [ ] . depletion of the copi β subunit in human /ace cells led to a decrease in the severe acute respiratory syndrome-coronavirus (sars-cov) replication [ ] . we identified two juvenile hormone-inducible proteins at h with . -and . -fold increases. despite the absence of information on the possible involvement of juvenile hormone-inducible proteins in the insect antiviral response, the juvenile hormone itself and retinoic acid are terpenoids, with similar structure and biological effects in mammalian and insect cells [ ] . retinoic acid-inducible genes are related to type-i ifn response in mammals [ , ] . viruses , , of we identified two juvenile hormone-inducible proteins at h with . -and . -fold increases. despite the absence of information on the possible involvement of juvenile hormone-inducible proteins in the insect antiviral response, the juvenile hormone itself and retinoic acid are terpenoids, with similar structure and biological effects in mammalian and insect cells [ ] . retinoic acidinducible genes are related to type-i ifn response in mammals [ , ] . another virus infection-related protein found was kinesin, an atpase protein belonging to the class of motor proteins. kinesins move along microtubule (mt) filaments, and are power-driven by the hydrolysis of adenosine triphosphate (atp) [ ] . recently this protein has been associated with the intracellular transport of different viruses. hiv- trafficking to the nucleus is kinesin-and dynein motor-dependent [ ] . kinesin was also identified as a chandipura virus matrix binding protein [ ] . kinesin from lepidopteran trichoplusia ni binds directly to nucleocapsid proteins of the autographa californica multiple nucleopolyhedrovirus (acmnpv) [ ] , and the α herpersviral envelope protein pus uses kinesin to promote the anterograde axonal transport of herpes simplex virus (hsv- ) [ ] . these results demonstrate that kinesin protein interacts directly with the virus particle. the insect protein canopy is a homolog of the human protein associated with toll-like receptor (tlr) (prat ). prat is required for cell surface expression of the toll family of receptors and for the intracellular nucleic acid-sensing tlr / . this protein is essential for tlr-dependent immune response [ ] . the . -fold increased secretion of this prat homolog signals the participation of toll-like receptors in the ll antiviral non-specific immune response. the barrier to autointegration factor (baf) dna binding protein had an increase of . -fold at h. this protein is described as a potent antiviral effector against poxyviruses, retroviruses and the herpes virus. the baf antiviral property is regulated by kinases and phosphatases, with dephosphorylation activating its antiviral properties [ ] . notwithstanding the higher number of positively modulated secreted proteins at h when compared with h, the number of proteins described in immune response to viruses in interferon-like responses was smaller than what was seen at h. among them we quote the protein hemocytin, with a secretion increase of . -fold. hemocytin is an insect humoral lectin homologous to mammalian von willebrand factor, which is involved in platelet adhesion to subendothelium [ ] . hemocytin expression is induced by pathogens. lectins are adhesive molecules which bind carbohydrates and among other functions are involved in hemolymph bacterial clearance [ ] . also of interest is the occurrence of glutaredoxin (fold change of . ), thioredoxin (fold change of . ) and superoxide dismutase (fold change of . ), antioxidant enzymes involved in the control of oxidative stress at h. reactive oxygen species (ros) production, generated due to microbial invasion, has long been known to exert an antimicrobial effect. the activation of the antiviral and inflammatory signaling pathways has also been linked with the production of ros [ , ] . due to the high chemical reactivity of ros, cells possess scavenger antioxidant mechanisms that maintain redox homeostasis [ ] . real time pcr (qpcr) analysis of genes coding for significantly positive-or negatively-modulated secreted proteins revealed the existence of some genes with transcriptional profile correlated with the amount of protein detected in the supernatants ( figure a ). there were seven molecules that presented a correlation between mrna and secreted protein levels (represented in figure a ). three of them, hemocytin (hmc), lecithin cholesterol acyltransferase (lcat), and scramblase (scr), showed coordinated mrna and secreted protein levels. when mrna levels changed at or h post transfection (either by up-or down-regulation), we detected a corresponding change in secreted protein levels or h after the mrna level changes. hemocytin (hmc), an insect humoral lectin which plays an important role in a non-specific self-defense mechanism [ ] , had increased mrna and secreted protein levels at h and h. this indicates that the transcription, translation and secretion of this molecule are correlated and the prolonged increase of its expression suggests this molecule may be important for the ll cells response to poly i:c transfection. two other molecules, lecithin cholesterol acyltransferase (lcat) (a plasma enzyme which esterifies cholesterol) [ ] , and scramblase (scr) (a regulator of transbilayer lipid asymmetry in eukaryotic cell membranes) [ ] , were reduced both in mrna and secreted protein levels at or h post-transfection. these two molecules are involved in lipid metabolism or membrane formation and the reduction of their expression may suggest that ll cells reduce vesicle formation h after transfection challenge. three other molecules showed increased mrna levels at or h post transfection and increased secreted protein levels at h, with posterior decrease at h. one was the eukaryotic translation initiation factor (etif ), which in humans is directly recruited by the hepatitis c virus (hcv) internal ribosome entry site (ires) to promote the translation of viral proteins [ ] . in ll cells it showed increased mrna levels at h and h, and increased secreted protein levels h after mrna increase. curiously, h post transfection the secreted protein levels decreased drastically. juvenile hormone-inducible protein (jhip) showed increased mrna levels after h and increased secreted protein levels h after mrna increase. curiously, at h post transfection the secreted protein levels decreased drastically regardless of changes in mrna levels prior to this timepoint. basal transcription factor (btf ) has been reported to play a significant role in the transcriptional regulation related to eukaryote growth and development [ ] . in ll cells it showed increased mrna levels at h and increased secreted protein level h after mrna increase. at h post transfection the secreted protein levels decreased drastically regardless of changes in mrna levels prior to this time point. transketolase mrna levels increased at h post transfection and the secreted protein levels also increased at this same time point, suggesting a late effect. curiously, regardless of the maintenance of mrna levels from h to h, the secreted protein levels were considerably low at h post transfection. changes in transketolase activity are involved in a number of tumors and degenerative diseases [ ] . based on the data presented in figure a , we observed that in the majority of the molecules (six out of seven) there was a correlated change of secreted protein levels h to h after changes in mrna levels. the remaining genes investigated showed no correlation between the mrna levels and the detection of proteins in the supernatants. ( figure b ). the linear regression of mrna levels plotted against secreted protein levels revealed a weak correlation (figure ). the pearson correlation coefficient (r) and goodness of fit (r ) calculated for mrna levels ( h, h, h and h) versus secreted protein ( h and h) are shown in figure and table . the non-correlation between transcription levels and amounts of secreted protein indicates that secretion is regulated by some not-yet disclosed mechanism, involving transcription, translation, storage, sorting, and transport, as already reported for other systems [ ] . this non-correlation was also observed when the exoproteome of wrl- human hepatic cells infected with the chikungunya virus was analyzed [ ] . we observed that the majority of analyzed genes were transcribed in the first h ( figure a,b) . the results obtained with the signal peptide prediction, the secreted protein interaction network, the identified biological functions enriched pathways, and the relatively small number of proteins with significant abundance variation reveal the existence of finely-tuned protein secretion modulation in response to dsrna treatment. there is also a strong indication for the participation of the exosomal secretory pathway as the major ll cell secretion mechanism in this response. the increased secretion of proteins such as phospholipid scramblase, fkbp, juvenile hormone-inducible proteins, and protein canopy in the first h indicate that this non-specific antiviral response is in a way similar to an interferon-like response mediated by the toll pathway. this response occurs preferentially in the first hours after transfection. the increase of antioxidant enzymes at h indicates that the cells are working to control the oxidative stress level in the culture. we are presently in the process of validating these findings by silencing specific genes with subsequent analysis of nonspecific antiviral response. (table ) and samples collected at different time points posttransfection. quantification was normalized relative to the house keeping gene rp , and relative gene expression expressed as fold change was calculated relative to the mock group. bars represent mean with standard error (sem) of two biological replicates. tables represent protein fold change corresponding to each gene name. * p ≤ . , ** p ≤ . , *** p ≤ . . 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rna screen identifies proviral and antiviral host factors in severe acute respiratory syndrome coronavirus replication, including double-stranded rna-activated protein kinase and early secretory pathway proteins activation of mammalian retinoid x receptors by the insect growth regulator methoprene regulation of antiviral innate immune signaling by stress-induced rna granules myosin v motor proteins: marching stepwise towards a mechanism hiv- capsids bind and exploit the kinesin- adaptor fez for inward movement to the nucleus host interactions of chandipura virus matrix protein trichoplusia ni kinesin- associates with autographa californica multiple nucleopolyhedrovirus nucleocapsid proteins and is required for production of budded virus the basic domain of herpes simplex virus pus recruits kinesin- to facilitate egress from neurons a protein associated with toll-like receptor (tlr) (prat a) is required for tlr-dependent immune responses the barrier to autointegration factor: interlocking antiviral defense with genome maintenance cloning and expression of the gene of hemocytin, an insect humoral lectin which is homologous with the mammalian von willebrand factor immune proteins and their gene expression in the silkworm, bombyx mori hsv infection induces production of ros, which potentiate signaling from pattern recognition receptors: role for s-glutathionylation of traf and reactive oxygen species activate nf-κb (p ) and p and induce apoptosis in rvfv infected liver cells the nrf cell defence pathway: keap -dependent and -independent mechanisms of regulation establishment and application of a high throughput screening system targeting the interaction between hcv internal ribosome entry site and human eukaryotic translation initiation factor exploring the roles of basal transcription factor in eukaryotic growth and development structure and functioning mechanism of transketolase control of cystic fibrosis transmembrane conductance regulator membrane trafficking: not just from the endoplasmic reticulum to the golgi differential analysis of the secretome of wrl cells infected with the chikungunya virus the authors declare no conflict of interest. key: cord- -o qbqxhx authors: cavalcante, liliane t. f.; muniz, cláudia p.; jia, hongwei; augusto, anderson m.; troccoli, fernando; medeiros, sheila de o.; dias, carlos g. a.; switzer, william m.; soares, marcelo a.; santos, andré f. title: clinical and molecular features of feline foamy virus and feline leukemia virus co-infection in naturally-infected cats date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: o qbqxhx feline foamy virus (ffv) and feline leukemia virus (felv) belong to the retroviridae family. while disease has not been reported for ffv infection, felv infection can cause anemia and immunosuppression (progressive infection). co-infection with ffv/felv allows evaluation of the pathogenic potential and epidemiology of ffv infection in cats with felv pathology. blood and buccal swab samples from cats were collected in rio de janeiro. plasma was serologically tested for felv. dna extracted from peripheral blood mononuclear cells and buccal swabs was used to pcr detect ffv and felv. a qpcr was developed to detect and measure ffv proviral loads (pvls) in cats. felv qpcr was performed using previous methods. the median log pvl of ffv mono-infected individuals was lower than found in ffv/felv co-infected cats in buccal swabs (p = . ). we found % of cats had detectable buccal ffv dna in ffv mono-infected and ffv co-infected felv-progressive cats, while in felv-regressive cats (those without signs of disease) % of cats had detectable buccal ffv dna (p = . ). our results suggest that regressive felv infection may reduce ffv saliva transmission, the main mode of fv transmission. we did not find evidence of differences in pathogenicity in ffv mono- and -dually infected cats. in summary, we show that fvs may interact with felv within the same host. our study supports the utility of cats naturally co-infected with retroviruses as a model to investigate the impact of fv on immunocompromised mammalian hosts. foamy viruses (fv) are complex retroviruses that belong to the retroviridae family and comprise a unique genus within the spumaretrovirinae subfamily that naturally infect many vertebrates, including feline, simian, bovine, and equine [ ] . fv infection in humans is invariably associated with zoonotic stomatitis. the risk of developing lymphoid malignancy is estimated to be to -fold greater in fiv-infected only cats compared with uninfected cats [ ] . several studies have investigated co-infection in domestic cats [ , , ] . other studies have investigated the virologic factors involved in retrovirus co-infection [ ] . but they mostly focused on fiv/felv co-infections. a study by yamamoto et al. showed that a pre-existent felv infection enhanced the expression of fiv in the body and increased the severity of transient primary and chronic secondary stages of fiv infection [ ] . only a single study has evaluated the presence of all three retroviruses in cats [ ] , but was limited only to prevalence estimates and not their possible interactions or associations with disease. in another study, felv and fiv co-infection was examined and showed a greater risk for lymphoid cancers in dually infected cats [ ] . the combination of susceptibility to multiple retroviral infections and varied possible disease outcomes makes domestic cats an attractive animal model to study the epidemiology and pathogenesis of co-infection by distinct retroviruses. we examined blood and buccal swab specimens of domestic cats in brazil for detection and quantification of each feline virus to evaluate their potential association with disease and transmissibility in animals with single or multiple retroviral infections. this project was approved by the ethics committee on animal use of the center for health sciences at federal university of rio de janeiro (ceua-ccs) (protocol ibo - / ). blood and buccal swab samples from domestic cats were collected between and from three different veterinary clinics in rio de janeiro (n = ) or captured within the grounds of fundação jardim zoológico da cidade do rio de janeiro (riozoo, n = ), an urban area in the north zone of the city. cats coming from veterinary clinics were selected for this study once they were suspected of felv infection, while cats from riozoo were selected randomly, independently whether they looked sick or not. the cats from clinics were not previously vaccinated for felv, while for captured animals we are not sure about their vaccination status although the chances they have been vaccinated are minimal, since brazilian public policies do not include felv vaccination. upon presentation at the clinics or after capture, the cats were weighed and anesthetized with the administration of g of ketamine or . µg of xylazine per kilogram of body weight. after anesthesia, - ml of whole blood was collected from the jugular vein by experienced veterinarians and placed into an edta blood tube and stored at room temperature until processing. buccal swabs were collected from cats by rubbing the upper, bottom and side of the oral cavity with a sterile cotton swab. hemogram tests displaying blood cell counts of animals were performed by a veterinarian diagnostic laboratory. hemogram results were kindly provided by dr. carlos gabriel dias. cats with low erythrocytes (below milions/µl), hemoglobin (below g/dl) or hematocrit (below %) levels were considered anemic. twenty-eight cats were followed three years after the initial collection to determine their life status (alive or dead). for felv-infected cats, the time elapsed from infection was estimated based on the diagnosis date at the veterinary clinics. for cats whose infection status was unknown before sampling, the time elapsed from infection was considered using the sampling date. cats were classified according to gender, age, outdoor access, household type, neuter status and health status. for age, cats were classified as: (a) kitten, up to months old; (b) young, between - months of age and (c) adult, above one year of age. for feral cats without a known birthdate, age was estimated by veterinarian evaluation. household status was classified as: (a) single, cats living alone at home; (b) multicat, cats living with up to cats in the same house; (c) shelter, cats living in a shelter or house with more than cats; and (d) feral, cats that live or have lived in the streets before. cats classified as single, multicat and shelter were also categorized for outdoor access. cats were considered sick when any clinical signs, including respiratory tract disease (rtd), progressive weight loss (pwl), ocular secretion (os), anemia, cachexia, ulcers, hair loss, presence of ectoparasites, tooth loss, prolapses, scabies, pemphigus foliaceous, pyometra, sporotrichosis, diarrhea, neurological symptoms, abortion, kidney disease, stomatitis and neoplasia, or hematological changes were reported. healthy cats were those without any reported clinical signs at the time of specimen collection. blood samples were centrifuged at × g for min to separate plasma from cells. plasma aliquots were stored at − • c for fiv/felv serologic testing. peripheral blood mononuclear cells (pbmcs) were obtained from the cellular fraction by ficoll-paque plus (ge healthcare, waukesha, wi, usa) centrifugation and were stored at − • c until genomic dna (gdna) extraction. gdna from pbmc and buccal swabs was extracted using the purelink ® genomic dna mini kit (invitrogen, carlsbad, ca, usa) and was quantified using a nanovue plus™ nanodrop (ge healthcare). pcr amplification of cytochrome b oxidase gene (cytb) sequences was used to verify dna integrity of the extracted material as previously described [ ] . plasma from domestic cats was tested for fiv/felv by two commercial immunochromatographic tests: the fiv ac/felv ag test alere ® (bionote inc., gyeonggi-do, korea) or quickvet fiv/felv ubio (biotechnology systems, kerala, india) kits at a veterinary clinic and ufrj, respectively. these tests simultaneously detect an felv antigen and an fiv antibody. assay sensitivity of % and . % for felv and % and . % for fiv, and specificity of % and . % for felv and % and . % for fiv were established by alere ® and ubio, respectively. nested or semi-nested pcr was performed to diagnose ffv infection by detection of four different ffv genome sequences, including the long terminal repeat (ltr, -bp) [ ] , integrase (int, -bp) [ ] , gag/polymerase (pol) ( -bp) [ ] and envelope (env, -bp) [ ] . three new pcr primers for this study (ffv ltr out, ffv_f and ffv_r ) were designed using an alignment of the four available complete ffv genomes from domestic cats (genbank accession numbers aj , aj , nc and y ). primer sequences are provided in table . primers used for detection of the fiv reverse transcriptase (rt) portion ( -bp) of the pol gene and of felv env sequences ( -bp) ( table ) were published previously ( [ ] ), respectively. for all published primers, the pcr conditions were followed according to those previous studies [ , [ ] [ ] [ ] [ ] [ ] . pcr conditions using newly designed primers were performed using × pcr buffer, mm mgcl , mm dntps, pmol forward and reverse primers, unit of taq platinum polymerase (invitrogen) and - nanograms of dna in a total reaction volume of µl. amplicons from pcr-positive samples were purified using the pcr dna mini kit (real genomics rbc, banqiao, taiwan) following the manufacturer's protocol and were sequenced in an abi xl platform (thermo scientific, waltham, ma, usa). a new quantitative pcr assay was developed to simultaneously detect and measure ffv proviral loads in infected cats. generic pol primers and probe were designed using an alignment of complete ffv genomes at genbank. the forward and reverse primers ffvpf -cat gtt gtc agc acc aag tat ac- and ffvpr -tgc aag aga gca agt tct tct tc- , respectively, and probe ffvpfp -fam-ttg gaa ttg att gta att tac cat ttgc-bhq - were used to detect a -bp pol sequence. cycling conditions included an initial step of • c for min, followed by cycles of s at • c and s at • c with a final hold step at • c using a bio-rad icycler cfx (bio-rad laboratories, hercules, ca, usa). for assay validation, human blood donor pbmc dnas and five different human cell lines (mcf- , cemx , jurkat, lncap and hela) were used as negative controls to assess assay specificity. specificity and sensitivity were also determined using pbmc lysates from us domestic cats previously found to be ffv-negative (n = ) or positive (n = ) by western blot analysis. these cat specimens were available from a previous study investigating the zoonotic potential of feline retroviruses [ ] . the limit of detection (lod) of the assay was determined by testing of - replicates (depending on the copy number) ten and fivefold serial dilutions of plasmids containing a cloned fragment of the ffv int sequence ranging from to a single copy diluted in µg of negative human gdna. once the potential lod was determined using replicates of the -fold dilutions, we used a larger number of five-fold plasmid dilution replicates to further refine the lod. we tested ten copy replicates, five copy replicates and single copy replicates. standard curves were constructed using - copies of plasmid dna. the number of provirus copies/cell was estimated using the formula: number of copies detected/(dna quantification in picograms/ pgs cell) where the mass for each domestic cat cell genome was considered as six picograms dna per cell [ ] . the amount of dna in nanograms was measured by spectrophotometry. the felv qpcr test was performed as previously described [ ] using a unique region within the u of the felv ltr with a reported detection limit of copies per reaction. specificity of the assay was % by obtaining negative results for pbmc dna from pathogen-free, felv-negative cats. assay sensitivity was also % by confirmation of infection in felv p -positive cats. cats that were experimentally infected and that became persistently antigenaemic as determined by p elisa were considered positive. the primers and probe felv-u -exo-f -aac agc aga agt ttc aag gcc- (forward), felv-u -exo-r -tta tag cag aaa gcg cgc g- (reverse) and felv-u -probe -fam-cca gca gtc tcc agg ctc ccc a-tamra- were used to detect a -bp ltr sequence. standard curves were performed with a plasmid containing the target region in a serial dilution range of - copies. the cycling conditions consisted of an initial step of gotaq (promega, madison, wi, usa) activation at • c for min followed by cycles of s at • c and min at • c using an abi real-time cycler (applied biosystems, foster city, ca, usa). the number of pvl copies/cell was calculated similarly to the ffv pvl, except the pvl was divided by two to compensate for the presence of two ltr copies per integrated provirus. we classified ffv-infected cats that were tested for both oral swab and pbmc tissues by nested pcr and/or qpcr. ffv-positive cats without virus detected in saliva were considered as latent infections, and were classified as non-transmissible. ffv-positive cats with virus detected in oral swabs were considered as potential ffv transmitters to other cats and were classified as transmissible. cats testing qpcr positive for felv were classified into two groups based on vl and serostatus: progressives (those with high pvl, most seropositive) and regressives (those with low vl, most seronegative). the mann-whitney test was used for statistical comparisons of pvl between groups. kaplan-meier survival curves were generated for survival analysis. linear regression was used to evaluate a correlation between the ffv and felv vls. odds ratios (or), % confidence intervals ( % ci) and p-values were determined to analyze risk factors for felv and ffv co-infection, including sex, age, whether neutered, and household status. a multivariate analysis was conducted by adjusting the model for variables that showed significant associations with ffv/felv co-infection, i.e., that showed p-values below . . to test the different proportion of non-and transmissible cats, probabilities (p) were calculated using the chi-square test. all statistics were performed using medcalc v. . . . or in the r environment and p-values ≤ . were considered significant. all domestic cats sampled in this study were from fundação jardim zoológico da cidade do rio de janeiro (riozoo) and from veterinary clinics throughout the city of rio de janeiro. cats were classified according to gender, age, outdoor access, sexual sterilization, household type, and health status ( table ) . we found an approximately equal proportion of male and female cats and a higher number of adult individuals ( %). twenty of male cats ( %) were neutered, while of females ( %) were spayed. most cats ( %) were classified as feral and of the cats residing in any household ( multicats, shelters and singles), the majority ( %) had no outdoor access. the majority of cats ( %) were also sick at the time of sample collection. blood dna specimens from all cats were tested by nested pcr and serology for diagnosis of fiv and felv infection. buccal swab genomic dna (gdna) specimens available from cats were also analyzed using pcr testing. animals were considered pcr-positive when at least one viral sequence was detected in pbmcs and/or buccal swab samples. we found three fiv-positive animals, of which / ( . %) were positive only by serology and / ( . %) was pcr-positive only. of two serologically fiv-positive animals, one was a house cat and the other was a feral cat. the fiv pcr-positive only cat was feral (table ) . we found / ( %) animals reactive in the felv p antigen test in plasma and an equal number of pcr-positive animals but cats did not always have concordant p and pcr results. specimens from five cats were pcr-positive only and five different cats were felv p -reactive only. cats positive using at least one assay (serology or pcr) were classified as infected. among the household cats (single, multi-cat and shelter) and feral cats, felv-positive results in both pcr and serological tests tended to be higher in households, % ( / ) compared to feral cats % ( / ) (p = . ; fisher´s exact test; table ). two cats were dually infected with both fiv and felv. the pbmc and buccal swab gdna samples were screened by nested pcr for amplification of four different ffv genomic sequences (table ). overall, we found a ffv pcr prevalence of % ( / ). we found more cats with gag/pol pcr-positive results ( / , % in pbmcs and / , % in buccal swab gdna) compared to other viral targets. of the ffv-positive animals, had both pbmc and buccal swab gdna specimens available for nested ffv pcr testing. twenty-nine animals were pcr-positive ( %) in the pbmc compartment, while only ( %) were positive in the buccal swab (p = . ). comparing both tissue compartments, animals were positive in both pbmc and buccal swab, while cats were positive only in pbmc and four were positive only in the buccal swab. no significant differences were found when comparing feral to household cats that were ffv pcr-positive in either the pbmc or the buccal swab compartments (p = . and p = . , respectively). ( ) a cats with at least one virus fragment detected. b in three cases, a limited number of samples were tested due to material availability. we also developed a new quantitative real-time pcr (qpcr) assay to simultaneously detect and measure ffv proviral load (pvl) in cats. testing of replicates containing serial dilutions of ffv integrase (int)-containing plasmids showed that / replicates ( %) containing five copies/ug dna tested positive, whereas / ( %) replicates containing a single ffv copy were qpcr positive. hence, we set detection limit of the ffv qpcr assay at five ffv copies/ug gdna or about . × − copies/ cells. ffv int sequences were not detected in pbmc gdna from us human blood donors or in five different human cell lines. the sensitivity of the qpcr assay was also evaluated using pbmc lysates from us domestic cats for which the infection status was previously determined by wb testing [ ] . detection of ffv correlated with the wb and/or nested pcr results in . % ( / ) of the samples. the average and median ffv pvls were . × and . × copies/ cells, respectively. two wb-positive but qpcr-negative samples also tested negative for β-actin, indicating possible dna degradation or pcr inhibitors in those samples. five of the ffv wb-negative animals had low pvls ( - copies/ cells), indicating possible infection during the seroconversion window period or latent infection with a revertant antibody response. one cat with an indeterminate wb result (seroreactivity to a single gag protein) also tested qpcr negative, suggesting possible nonspecific seroreactivity in this animal. pbmc gdna was further available from of brazilian cats testing positive using the nested pcr assays. of these cats, were qpcr-positive with a median pvl of − . log copies/cell ( , copies/ cells) and an average of − . log copies/cell ( , copies/ cells) ( figure a ). of cats with available buccal swab gdna for qpcr testing, were positive with a median pvl of − . log copies/cell ( , copies/ cells). of the cats testing negative by nested pcr, had pbmc gdna available for qpcr testing. of these cats, were qpcr-positive with a median pvl of − . log copies/cell ( , copies/ cells). buccal swab gdna from nested pcr-negative animals identified seven qpcr positive cats with a median pvl of − . log copies/cell ( , copies/ cells). there was no pvl differences between animals that were previously positive or negative for ffv using the nested assays (p > . for both tissues). interestingly, the pvl was higher in buccal swab than in pbmc specimens (p = . ). fifty-three of the pbmc samples tested ( %) were qpcr-positive versus % ( / ) of buccal swab samples. for ffv-monoinfected cats, we did not find differences between pvl in pbmcs (median pvl of − . log copies/cell; n = ) and in buccal swabs (median pvl of − . log copies/cell; n = ) (p = ). by using qpcr, the total number of ffv-positive cats increased from ( %) to ( %). of these qpcr-positive cats, ( . %) were monoinfected with ffv. analysis of cats classified as potentially transmissible and non-transmissible found no statistical difference between ffv pvls. by using qpcr, the total number of ffv-positive cats increased from ( %) to ( %). of these qpcr-positive cats, ( . %) were monoinfected with ffv. analysis of cats classified as potentially transmissible and non-transmissible found no statistical difference between ffv pvls. of felv-positive cats diagnosed by serological and/or molecular assays, with available pbmc gdna were felv qpcr-positive with a median pvl of . log copies/cell ( . × copies/ cells) ( figure b) . buccal swab gdna was available for cats, of which were felv of felv-positive cats diagnosed by serological and/or molecular assays, with available pbmc gdna were felv qpcr-positive with a median pvl of . log copies/cell ( . × copies/ cells) ( figure b) . buccal swab gdna was available for cats, of which were felv qpcr-positive with a median pvl of − . log copies/cell ( . × copies/ cells). the felv pvl was approximately × higher in pbmc than in buccal specimens (p = . ). our pvl data are correlated with felv outcome (progressives of group i, those cats with high vl, mostly seropositive) and regressives (group ii, those with low vl, mostly seronegative). group i (n = ) had a higher pvl with an average of . log felv copies/cell ( . × copies/ cells) and median pvl of . felv log copies/cell ( . × copies/ cells) whereas group ii (n = ) cats had a lower median pvl of − . felv log copies/cell ( . × copies/ cells) (p < . ). in this case, there was no difference between serological status of the two groups: in group i, % (n = ) were felv-seronegatives while in group ii, % (n = ) had that status (p = . ). we also tested the of the cats with negative felv serology and/or molecular assays with available pbmc gdna. we found felv-positive cats by qpcr with a median of − . log copies/cell ( . × copies/ cells). this median pvl is almost × lower than the median pbmc pvl in felv-seropositive cats (p < . ). pvls of felv-seronegative cats were similar to those measured in the group ii felv-positive cats (p = . ), indicating that the felv-seronegative cats with low pvls likely had a regressive infection. testing of buccal swab gdna samples identified four qpcr-positive cats with a median felv pvl of − . log copies/cell ( . × copies/ cells). however, there was no difference between buccal swab pvls in felv-seronegative and seropositive cats (p = . ). cats with progressive and regressive infections differed in the qpcr detection of felv in buccal tissues. of regressive animals, only five ( %) had detectable pvl in buccal cells, while all ( %) of animals with progressive infection had detectable pvl in that compartment (p < . ). nonetheless, buccal swab pvls of progressive and regressive animals were similar (p = . ). by using qpcr, the total number of felv-positive cats increased from ( %) to ( %). of the qpcr-positive cats, eight ( %) were monoinfected with felv, while ( %) were co-infected with both ffv and felv and two ( %) were infected with all three feline retroviruses. we found % ( / ) of the cats in our study to be ffv-monoinfected, % ( / ) with felv monoinfection, % ( / ) with dual ffv/felv co-infection, % ( / ) with dual ffv/fiv co-infection, % ( / ) with triple ffv/felv/fiv co-infection and % ( / ) with no evidence of any feline retrovirus. no fiv monoinfections or dual felv/fiv co-infections were identified. therefore, subsequent statistical analyses were based on the three groups consisting of ffv and felv monoinfections and ffv/felv dual infections, since these infections were more common in our population. we found an approximate : ratio between females and males in all three ffv and felv infection groups (table ) . kittens, young, and adult cats were present in all three groups, except for the felv-monoinfected group which only contained adults. the majority of cats living in multi-cat homes or shelters were ffv/felv-co-infected ( % and %, respectively). the majority of cats with outdoor access and poor health were also co-infected with ffv and felv (table ), but these differences were not statistically significant. in captive animals, monoinfection of ffv was . % ( / ) while in feral animals the prevalence was % ( / ) (p < . ). however, the total prevalence of ffv (mono and dual-infection with felv) was % in captive animals and % in feral animals (p = . ). felv monoinfection prevalence was lower in feral ( %) compared to captive ( %) cats, and similarly the total prevalence of felv was lower in feral cats ( %) than in the captive cats ( %) (p = . ). odds ratios were determined to evaluate whether ffv infection was a risk factor for felv infection. infection with ffv did not increase the chances of having felv infection (or = . , p = . ). among ffv-infected cats, odds ratios were also calculated to evaluate risk factors involved with felv co-infection (table ). neutered ffv-infected cats were more likely to be co-infected than non-neutered counterparts. cats living in shelters showed higher rates of co-infection than those cats in other housing types. non-feral cats living alone, in a shelter or multicat household had a higher risk of co-infection than feral cats (or = . , p = . ). no associations of co-infection were found with either age or sex. we also evaluated risk factors for ffv co-infection of felv-infected cats. no significant differences were observed when considering the same factors analyzed for ffv-infected cats (or = . , p = . ). since the street and neuter variables showed significance at the univariate level, a multivariate analysis was performed with adjustment for those two variables. while the effect of neutering was lost (or = . ; p = . ), street cats remained significantly less prone for co-infection with ffv and felv than non-street cats (or = . ; p = . ). when we compared median log pvls of ffv mono-and ffv/felv co-infected individuals ( figure ) we found higher ffv pvls in buccal swab specimens in co-infected cats (p < . ; figure a ). in contrast, differences in pbmc pvls between mono-and co-infected cats were not significant (p = . ). however, pvls in the buccal specimens were significantly higher than those in pbmc of co-infected cats (p < . ). no differences were found in felv pvls between buccal swabs and pbmcs or between felv mono-and co-infected cats (p = and p = . , respectively; figure b ). we also evaluated pvls of cat samples that were tested for both ffv and felv, but did not observe a correlation between felv and ffv pvls in pbmcs among cats with progressive or regressive infection ( figure a) . similarly, there was no correlation between the ffv and felv pvls in buccal specimens ( figure b ) except for felv-regressive individuals (r = . ; p = . ; figure b ), which showed a negative association. the percentage of potentially transmissible ffv with detectable pvls in buccal swabs in monoinfected cats ( . %) was very similar to that in felv progressive cats ( . %) (figure ). the opposite was found when comparing ffv-monoinfected cats and felv regressives (p = . ). for cats with ffv and felv positive results for both buccal swab and pbmc specimens, we compared the ffv pvls between ffv-monoinfected and ffv/felv-co-infected animals and also the pvls between felv progressive and regressive animals. however, we did not observe a correlation between pvls in buccal swab or pbmc in either situation (data not shown). for cats with ffv and felv positive results for both buccal swab and pbmc specimens, we compared the ffv pvls between ffv-monoinfected and ffv/felv-co-infected animals and also the pvls between felv progressive and regressive animals. however, we did not observe a correlation between pvls in buccal swab or pbmc in either situation (data not shown). for cats with ffv and felv positive results for both buccal swab and pbmc specimens, we compared the ffv pvls between ffv-monoinfected and ffv/felv-co-infected animals and also the pvls between felv progressive and regressive animals. however, we did not observe a correlation between pvls in buccal swab or pbmc in either situation (data not shown). to investigate possible disease associations in cats with mono or dual ffv and felv infection we analyzed clinical signs reported in these animals at the time of sampling. we did not detect any differences among the proportions of major clinical signs (rtd, pwl and os) in ffv-monoinfected, or felv-monoinfected regressive or progressive cats ( table ). five of seven ( %) cats with ffv/felv-progressive infection were anemic at the time of data collection by hemogram testing while none of the ffv/felv-regressive cats were anemic ( / ; p = . ). less common clinical signs detected were cachexia ( / ), ulcers ( / ), hair loss ( / ), presence of ectoparasites ( / ), tooth loss ( / ), prolapses ( / ), scabies ( / ), pemphigus foliaceous ( / ), pyometra ( / ), sporotrichosis ( / ), diarrhea ( / ), neurological ( / ), abortion ( / ), kidney disease ( / ), stomatitis ( / ) and neoplasia ( / ). however, the low frequency of these clinical signs did not permit an analysis of their association with retrovirus infection of these cats. we analyzed ffv pvls in cats with or without each reported major clinical signs, but did not observe differences between pvls in cats with or without pwl (p = . ), rtd (p = . ) or os (p = . ). we also did not find any ffv pvl differences in cats with or without any clinical signs (p = . ), including anemia and death (p = . and p = . , respectively). for felv-positive cats (either by serology or pcr), anemia at the time of sampling was correlated with higher pbmc pvl, with a median of . log felv copies/cell ( , , copies/ cells) compared to − . log felv copies/cell ( , copies/ cells) in cats without anemia (p = . , mann-whitney u test). death was also correlated with higher pbmc pvl, with a median of . log felv copies/cell ( , , copies/ cells) among cats who died during follow-up compared to a median of − log felv copies/cell ( , copies/ cells) at sampling time among those who were still alive at the end of follow-up (p = . , mann-whitney u test). we followed cats from entry into the study until months later. among those, three were ffv-monoinfected, and were felv progressives and five were felv regressives, irrespective of their ffv infection status. after months of follow-up, all three ffv monoinfected cats were alive. after months, . % ( / ) of cats with progressive infection had died (figure ), of which three were feral ( %) and nine were captive ( %), were adults ( %) and one was young ( %) whereas the th cat was lost to follow-up at months. in contrast, all regressive cats were alive after months (p = . ). among regressive cats, two were feral ( %) and three were captive ( %), while four were adults ( %) and one was a kitten ( %). there was no difference in age or household status in these cat groups. similar rates of ffv infection were found in both groups; / ( . %) in the first group and / ( . %) in the second. in this study, we evaluated fv infection in cats co-infected with other retroviruses known to impair the feline host immune system with pathologic consequences. retroviral infection of domestic cats was used as a model to investigate possible outcomes associated with mono or dual infections because biological materials such as blood and buccal swabs are usually collected from domestic cats at veterinary clinics for periodic clinical examination and pets can also be easily followed. moreover, domestic cats can be infected with felv, a retrovirus historically considered to be the causative agent of more clinical syndromes than any other single microbial agent in cats, affording an assessment of dual retroviral infections on disease outcomes in cats [ ] . unfortunately, the low fiv prevalence in our study population did not permit an evaluation of dual or triple infection with that retrovirus. in this study we found that both ffv and felv qpcr testing were more sensitive than their respective nested pcr assays for detecting ffv and felv, respectively. even though the felv and ffv nested pcr assays in our study targeted multiple viral regions, none were able to detect % of the qpcr-positive samples. these results likely reflect viral sequence variation at the primer locations and the target viral genomic fragment size for which detection of smaller amplicon sizes, as those in the qpcr tests, is typically more sensitive. we have obtained similar results when comparing nested pcr and qpcr testing of new world primates (nwp) [ ] . in this study, about one fourth of ffv-infected cats were pcr-positive in only the pbmc compartment. these animals may represent recent infections, in which the virus has not yet disseminated to the oral tissues. this hypothesis is supported by a previous report showing a delay in the appearance of sfv in the saliva of cynomolgus macaques (macaca fascicularis) infected through blood transfusion from an sfv-infected animal [ ] . epstein-barr virus (ebv) shares some features with fv, including a high incidence of infection, a salivary mode of transmission and replication in the oropharyngeal tissues. as it is well established for ebv, it is possible that migratory cells, such as macrophages and other leukocytes initially infected by fv, eventually traffic to the oropharyngeal tissues and spread infection to less differentiated epithelial cells [ ] . a recent study with nwp showed an average of − . log sfv copies/cell in pbmc [ ] whereas in old world primates (owp) the pvl average was − . log sfv copies/cell among rhesus macaques [ ] . we found a × higher ffv pvl (− . log copies/cell) in the same tissue in this study, we evaluated fv infection in cats co-infected with other retroviruses known to impair the feline host immune system with pathologic consequences. retroviral infection of domestic cats was used as a model to investigate possible outcomes associated with mono or dual infections because biological materials such as blood and buccal swabs are usually collected from domestic cats at veterinary clinics for periodic clinical examination and pets can also be easily followed. moreover, domestic cats can be infected with felv, a retrovirus historically considered to be the causative agent of more clinical syndromes than any other single microbial agent in cats, affording an assessment of dual retroviral infections on disease outcomes in cats [ ] . unfortunately, the low fiv prevalence in our study population did not permit an evaluation of dual or triple infection with that retrovirus. in this study we found that both ffv and felv qpcr testing were more sensitive than their respective nested pcr assays for detecting ffv and felv, respectively. even though the felv and ffv nested pcr assays in our study targeted multiple viral regions, none were able to detect % of the qpcr-positive samples. these results likely reflect viral sequence variation at the primer locations and the target viral genomic fragment size for which detection of smaller amplicon sizes, as those in the qpcr tests, is typically more sensitive. we have obtained similar results when comparing nested pcr and qpcr testing of new world primates (nwp) [ ] . in this study, about one fourth of ffv-infected cats were pcr-positive in only the pbmc compartment. these animals may represent recent infections, in which the virus has not yet disseminated to the oral tissues. this hypothesis is supported by a previous report showing a delay in the appearance of sfv in the saliva of cynomolgus macaques (macaca fascicularis) infected through blood transfusion from an sfv-infected animal [ ] . epstein-barr virus (ebv) shares some features with fv, including a high incidence of infection, a salivary mode of transmission and replication in the oropharyngeal tissues. as it is well established for ebv, it is possible that migratory cells, such as macrophages and other leukocytes initially infected by fv, eventually traffic to the oropharyngeal tissues and spread infection to less differentiated epithelial cells [ ] . a recent study with nwp showed an average of − . log sfv copies/cell in pbmc [ ] whereas in old world primates (owp) the pvl average was − . log sfv copies/cell among rhesus macaques [ ] . we found a × higher ffv pvl (− . log copies/cell) in the same tissue compartment in cats. at oral sites, the average nwp sfv pvl was − . log copies/cell [ ] while in owps the pvl ranged from − . to − log sfv dna copies/cell [ ] . we found similar ffv pvls in the cat oral cavity (− . log copies/cell). unlike the previous work with primates [ ] , we did not find significant differences between the pvl in pbmc and in oral tissues of cats. these data suggest that fv pvl distribution at distinct anatomical sites can vary in different natural hosts. we also showed that buccal swabs can be useful as non-invasive biological materials for ffv diagnosis, as recently shown for sfv [ ] . non-feral cats were almost five times more likely to be co-infected with ffv/felv than feral cats. we hypothesize that being confined in a household or shelter environment increases the risk of co-infection since these cats may have more intimate contact during grooming or sharing feeding bowls. we also considered possible recruitment bias since clinic cats were suspected to be ill as explained in methods. these cat behaviors are reported as the most likely routes of felv transmission [ ] , as opposed to biting, which has been more strongly associated with sfv transmission [ ] . in our study the ffv prevalence was not different between captive and feral adult cats, while in sfv studies of owps the prevalence is higher in adult captive (~ %) [ ] than in wild-born primates (~ %) [ ] , whereas the sfv prevalence in nwp were found very similar in captive and wild animals ( - %) [ ] . these differences can likely be explained by some captive cats having outdoor access and in shelters it is not uncommon for the introduction of new individuals from the street, both increasing the likelihood of exposures to potentially infected cats. using qpcr, we identified two distinct cat groups based on felv pvl; a regressive group with low pvls (− . to . log copies/cell) and a progressive group with higher pvls ( . to . log copies/cell). our results support previous studies showing that pvls measured by qpcr can be used to identify a regressive infection outcome in which plasma viremia has been cleared [ ] . moreover, the felv pvl we detected is within the range reported by others for experimentally and naturally infected cats [ ] . however, we show for the first time that naturally-infected cats with progressive felv infection can result in death after months of follow-up, although the cause of death cannot be exclusively attributed to felv infection. other studies showed the same outcomes only for experimentally infected cats [ , ] . in one of those studies, progressive and regressive cats were followed for weeks post transfusion and the authors found that % ( / ) of regressive cats remained healthy while % ( / ) of progressive cats developed non-regenerative anemia and multicentric t-cell lymphoma [ ] . to the best of our knowledge, our study is the first to observe the influence of another retrovirus on fv pvl in buccal swabs. a previous co-infection study conducted in the rhesus macaque model has shown that a pre-existing sfv infection can influence the biology and the outcomes of an siv infection, including higher plasma siv vl, a decreasing trend in the cd + t-cell counts and a greater number of animal deaths [ ] . however, sfv pvls were not assessed in that study. herein, with the determination of pvls of two feline retroviruses (ffv and felv), it was possible to observe that felv progressive or regressive infections were not able to reactivate ffv from latency in pbmc. however, a recent study showed that ffv pvl in blood was positive correlated in cats with status of felv progressive, presence of felv-b subtype and positive status for feline coronavirus (fecov), demonstrating a mechanism of complex interaction that need to be clarified [ ] . furthermore, co-infected cats with progressive felv infection are likely more prone to become ffv-transmissible and vice-versa as evidenced by the higher pvls in these oral compartments. as the proportion of potential-transmissible ffv infections was similar between ffv monoinfections and in cats with progressive felv infections, but lower in regressive felv co-infections, we postulate that the latency mechanism in regressive felv could also contribute to reduction of the establishment of ffv colonization of the oral cavity. these findings may also be a consequence of an altered disease susceptibility caused by the cats' virome composition. the virome influences the phenotype of the host in a combinatorial manner by interacting with other components of the microbiome and by interacting with individual variations in host genetics. together these interactions may influence a range of phenotypes as we observed in the distinct behavior of felv progressive and regressive infections as also in the felv regressive correlation with reduced ffv saliva transmission [ ] . the microbiome composition may also allow the stimulation of specific immune cell subsets, which may carry the ffv proviral dna, thus indirectly increasing the ffv pvl due to the increased number of cells. we also found that ffv pvl was higher in buccal tissues of ffv/felv co-infected cats than among ffv monoinfections. however, we identified a negative correlation between the pvls of both viruses circulating in this tissue. a previous study has reported felv as a cofactor for fiv infection by enhancing the expression and spread of fiv in the host and by increasing the severity of immunodeficiency caused by fiv by an unknown mechanism [ ] . another study evaluated fiv/felv interactions in in vitro and in vivo experiments, but did not find evidence of direct viral interactions [ ] . since felv is mainly transmitted through saliva [ ] and superficial differentiated epithelial cells of the oral mucosa are the major cell types in which ffv replicates [ ] , we hypothesize that both viruses can infect the same compartment. just like the tax protein of htlv- can activate hiv expression in vitro via the ltr [ ] , felv may enhance ffv replication in the oral cavity by a similar mechanism, or related to regulatory protein synergism, or some other means. a simplified model of this hypothesis is presented in figure . any potential interaction mechanism between these two viruses, and their infectiousness, remains to be determined and any contributions of infectious dna-containing viral particles compared to integrated proviral dna sequences was not measured in our study due to the small volumes of materials obtained for analysis. however, we identified a negative correlation between the pvls of both viruses circulating in this tissue. a previous study has reported felv as a cofactor for fiv infection by enhancing the expression and spread of fiv in the host and by increasing the severity of immunodeficiency caused by fiv by an unknown mechanism [ ] . another study evaluated fiv/felv interactions in in vitro and in vivo experiments, but did not find evidence of direct viral interactions [ ] . since felv is mainly transmitted through saliva [ ] and superficial differentiated epithelial cells of the oral mucosa are the major cell types in which ffv replicates [ ] , we hypothesize that both viruses can infect the same compartment. just like the tax protein of htlv- can activate hiv expression in vitro via the ltr [ ] , felv may enhance ffv replication in the oral cavity by a similar mechanism, or related to regulatory protein synergism, or some other means. a simplified model of this hypothesis is presented in figure . any potential interaction mechanism between these two viruses, and their infectiousness, remains to be determined and any contributions of infectious dna-containing viral particles compared to integrated proviral dna sequences was not measured in our study due to the small volumes of materials obtained for analysis. although we found evidence for increased buccal ffv pvls in cats co-infected with felv with the potential for increased transmissibility, we did not observe significant disease associations in these cats. a small number of dually infected cats with felv-progressive infection had anemia but this trend was not highly significant. we also did not find large numbers of fiv-infected cats to evaluate the effect of fv co-infection on disease outcomes. additional studies with larger numbers of naturally or experimentally infected cats and longer periods of follow-up may be needed to determine these potential associations. our study also reinforces the importance of felv qpcr testing to define felv infection outcomes, enabling differential clinical treatment of cats that were negative by serological methods. for example, blood transfusion from regressive cats containing latent feline leukemia provirus caused infection and disease in naïve recipient cats [ ] . moreover, felv regressive cats with undetectable antigenemia, but that are immune to vaccination, are often equivocally vaccinated [ ] . figure . hypothetical pathway to progressive or regressive outcomes via dual infection with feline foamy virus (ffv) and feline leukaemia virus (felv) and potentially active viral replication measured by proviral load (pvl). felv progressive and regressive outcome determined by pbmc tissue (blood) pvls were correlated to different ffv and felv pvl patterns of detection in the oral cavity. figure . hypothetical pathway to progressive or regressive outcomes via dual infection with feline foamy virus (ffv) and feline leukaemia virus (felv) and potentially active viral replication measured by proviral load (pvl). felv progressive and regressive outcome determined by pbmc tissue (blood) pvls were correlated to different ffv and felv pvl patterns of detection in the oral cavity. although we found evidence for increased buccal ffv pvls in cats co-infected with felv with the potential for increased transmissibility, we did not observe significant disease associations in these cats. a small number of dually infected cats with felv-progressive infection had anemia but this trend was not highly significant. we also did not find large numbers of fiv-infected cats to evaluate the effect of fv co-infection on disease outcomes. additional studies with larger numbers of naturally or experimentally infected cats and longer periods of follow-up may be needed to determine these potential associations. our study also reinforces the importance of felv qpcr testing to define felv infection outcomes, enabling differential clinical treatment of cats that were negative by serological methods. for example, blood transfusion from regressive cats containing latent feline leukemia provirus caused infection and disease in naïve recipient cats [ ] . moreover, felv regressive cats with undetectable antigenemia, but that are immune to vaccination, are often equivocally vaccinated [ ] . in summary, we provide evidence that fvs may interact with other retroviruses infecting cats. our study supports that domestic cats naturally infected by ffv and felv can be used as a model to investigate the potential impact of fv on immunocompromised mammalian hosts. the results of these animal model studies may help inform the design of clinical and research investigations into the potential for sfv to cause disease in infected humans. the tools developed in our study will be useful to further explore consequences and interactions of multiple feline retrovirus infections of cats. virus taxonomy identification of a human population infected with simian foamy viruses frequent simian foamy virus 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outcomes in a domestic cat breeding colony: relationship to endogenous felv and other chronic viral infections evaluation of in vivo and in vitro interactions of feline immunodeficiency virus and feline leukemia virus activation of the hiv- ltr by t cell mitogens and the trans-activator protein of htlv-i this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank the veterinary clinicians who collected cat specimens and associated clinical data over the study period. this work is part of the requirements for the phd thesis of l.t.f.c. at the department of genetics, universidade federal do rio de janeiro, brazil. use of trade names is for identification only and does not imply endorsement by the u.s. department of health and human services, the public health service, or the cdc. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the cdc, or any of the authors' affiliated institutions. the authors declare no conflict of interest. key: cord- -ntq f ix authors: mao, he-ting; wang, yan; cai, juan; meng, jun-ling; zhou, yu; pan, yu; qian, xiao-ping; zhang, yu; zhang, jun title: hace negatively regulates virus-triggered type i ifn signaling by impeding the formation of the mavs-traf complex date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ntq f ix during virus infection, the cascade signaling pathway that leads to the production of proinflammatory cytokines is controlled at multiple levels to avoid detrimental overreaction. hace has been characterized as an important tumor suppressor. here, we identified hace as an important negative regulator of virus-triggered type i ifn signaling. overexpression of hace inhibited sendai virus- or poly (i:c)-induced signaling and resulted in reduced ifnb production and enhanced virus replication. knockdown of hace expression exhibited the opposite effects. ubiquitin e ligase activity of the dead mutant hace /c a had a comparable inhibitory function as wt hace , suggesting that the suppressive function of hace on virus-induced signaling is independent of its e ligase activity. further study indicated that hace acted downstream of mavs and upstream of tbk . mechanistic studies showed that hace exerts its inhibitory role on virus-induced signaling by disrupting the mavs-traf complex. therefore, we uncovered a novel function of hace in innate immunity regulation. to initiate an effective antiviral response, rna viruses are recognized by pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and rig-i-like receptors (rlrs), and then trigger multiple signaling pathways to promote the production of proinflammatory cytokines, including type i ifns [ ] [ ] [ ] [ ] . aberrant overreaction may lead to proinflammatory diseases. therefore, the intensity and duration of the signaling are finely modulated at multiple steps of the signaling cascades [ , ] . in recent years, we have had great interest in the identification of the essential regulators in this signaling pathway. this will provide potential therapeutic intervention and targets for infection, inflammation or autoimmune diseases in the future. rlrs are cytosol sensors, which include rig-i, melanoma differentiation factor (mda ) and laboratory of genetics and physiology (lgp ) [ , ] . all three rlrs possess a dexd-box rna helicase domain for rna binding [ ] . except lgp , both rig-i and mda also contain a caspase recruitment domain (card) that is indispensable for downstream protein-protein interactions. upon viral infection, the activated rig-i undergoes self-dimerization and structural changes that permit the card domain of rig-i to interact with the card domain of downstream essential adaptor protein mavs (also known as ips- /cardif/visa) [ ] [ ] [ ] [ ] . mavs has a transmembrane domain (tm), that guides it to the outer mitochondrial membrane. besides, mavs contains three traf-interacting motifs (tim), two included in the n-terminal proline-rich region (pro), the other one located in the c-terminal region [ , ] . upon rna virus infection, the downstream tumor necrosis factor (tnf) receptor-associated factors (trafs) are recruited to mavs, and the mavs complex is formed [ , ] . this is a crucial step to initiate type i ifn signaling. traf , , and are all mavs binding partners through different tim. the mavs-traf complex provides the essential platform for downstream tbk -dependent irf or irf activation. traf bridges the upstream mavs and downstream kinase tbk and assembles the active mavs-traf -tbk signaling complex [ , ] . therefore, the regulation on the mavs-traf signalosome may be very important for the pathway. hace (hect domain and ankyrin repeat-containing e ubiquitin protein ligase ) is a hect-type ubiquitin e ligase. the functions of hace have not been fully understood. until now, the identified ubiquitinated substrates of hace include active rac [ , ] , optineurin (optn) [ ] and rab gtpases [ ] . the catalytic cysteine (c ) of hace is essential for its e ligase activity [ , , ] . mutation of c to serine or alanine will abolish its e ligase activity. hace gene is located on chromosome q , a prominent tumor-suppressor region [ , ] . the tumor suppressive function of hace is also characterized. hace is downregulated in multiple cancer types due to allelic loss or promoter methylation, such as wilms' tumor, gastric cancer, lymphoma, hepatocellular carcinoma, breast cancer, neuroblastoma, advanced colorectal cancer, etc. [ ] [ ] [ ] [ ] [ ] [ ] [ ] . hace -deficient mice developed spontaneous, late-onset cancer [ ] . re-expression of hace in human tumor cells directly abrogates in vitro and in vivo tumor growth, which is dependent on its e ligase activity. the mechanical analysis for its growth control shows that hace modulates the expression level of cyclin d , then reducing cell cycle progression [ ] . moreover, in breast cancer, hace ubiquitinates and promotes the degradation of rac , then leading to impaired rac signaling [ ] . in contrast, hace deficiency results in enhanced rac signaling, contributing to breast cancer progression [ ] [ ] [ ] . in lung cancer, hace ubiquitinates optn and targets it for autophagic degradation. the hace -optn axis synergistically suppresses the growth and tumorigenicity of lung cancer cells [ ] . moreover, hace is also involved in other biological processes or pathological conditions. for example, hace mediates resistance to oxidative stress [ ] . hace regulates golgi membrane fusion in cells [ ] . it has protective roles in the pathology of neurodegenerative diseases, such as huntington disease [ ] . it also provides cardiac protection in response to hemodynamic stress [ ] . however, the functions of hace in immune responses are not investigated. in recent years, ubiquitination has been reported as an important post-transcriptional modification to control the duration and intensity of antiviral immune responses [ ] . both hect and ring domain e ubiquitin ligases are identified as essential regulators in this pathway. for example, rnf is reported to ubiquitinate and degrade mda- , rig-i and mavs [ ] . the hect domain containing ubiquitin ligase aip can ubiquitinate and degrade mavs in collaboration with pcbp [ ] . our group previously showed that smurf promotes the ubiquitination and degradation of mavs, as well [ ] . in the search for unknown ubiquitin e ligases involved in antiviral signaling, some ubiquitin e ligases were used for the dual reporter luciferase assay. then, hace was suggested as a potential candidate in the regulation of this pathway. in this study, we demonstrate for the first time that hace contributes to negative regulation of the virus-induced type i ifn signaling via disrupting the mavs-traf complex. hace suppressed virus-induced type i ifn signaling independently of its ubiquitin e ligase activity. this study highlights the importance of hace in the modulation of virus-induced type i ifn response. hek t and hek cells were cultured with high-glucose dmem (life technologies, new york, ny, usa) medium plus % heat-inactivated new-born bovine serum and supplemented with antibiotics ( u/ml penicillin, µg/ml streptomycin). cells were grown at ˝c in a humidified atmosphere with % co . mouse anti-flag (m ) (sigma-aldrich, st. louis, mo, usa), mouse anti-hemagglutinin (ha) (merck millipore, darmstadt, germany), anti-gapdh (bioworld, atlanta, ga, usa), anti-hace (abcam, cambridge, uk) and anti-gfp (neobioscience, shenzhen, china) were from the indicated manufacturers. mammalian expression plasmids for human ha-tagged hace and flag-tagged rac were constructed by inserting the open reading frame of hace or rac into the n terminal ha or flag-tagged prk vector. the mammalian expression plasmid for hace /c a was constructed by site-directed mutagenesis. all of these vectors were verified by sequencing. pcdna -flag-tbk was a gift from tom maniatis. pef-bos-flag-rig-i was a gift from takashi fujita. pcdna -flag-mavs was a gift from zhijian chen. the prl-tk-renilla luciferase plasmid was from promega (madison, wi, usa). ifn-β and isre luciferase reporter plasmids were provided by hong-bing shu. all small interfering rnas (sirnas) (gene-pharma, shanghai, china) were transfected by permute (ucallm, jiangsu, china) at nm according to the manufacturers' instructions. to determine the efficiency of protein knockdown, at h post-transfection, cells were harvested, lysed and immunoblotted with rabbit anti-hace ab. the sequences of the individual sirnas were as follows: nonspecific control, -uucuccgaacgugucacgu- ; hace # , -uauagcgcugaugucaaca- ; hace # , -ggucuguuucugaacuacu- [ ]. the luciferase assay was performed as described [ ] . cells ( . ˆ ) were seeded on -well plates and transfected the next day using vigofect (vigorous biotechnology, beijing, china) with ng isre luciferase reporter, or ifn-β reporter and ng prl-sv plasmid, or with indicated plasmids. in the same experiment, when necessary, an empty control plasmid was added to ensure that each transfection received the same amount of total dna. then, h after transfection, cells were infected with sev at the multiplicity of infection (moi) of or transfected with poly (i:c) (invivogen, san diego, ca, usa) using lipofectamine (invitrogen, carlsbad, ca, usa) for h, and luciferase activity was measured with the dual-luciferase reporter assay system (promega) according to the manufacturer's instructions. firefly luciferase activity was normalized based on renilla luciferase activity. all reporter assays were performed in duplicate and repeated at least three times. the representative results are shown in each figure. total rna was isolated using trizol reagent (life technologies). cdna was synthesized using a reverse transcription system (promega) according to the manufacturer's instructions. quantitative real-time polymerase chain reaction (pcr) was carried out with the power sybr green pcr master mix (bio-rad, berkeley, ca, usa). each reaction was in duplicate. the amounts of hifnb were amplified using the following primers: ifnb -f: -attgcctcaaggacaggatg- and ifnb -r: the cells were infected by vsv-gfp virus at moi of . then, the vsv genome was quantified by real-time pcr using the following primers: vsv-f: -acggcgtacttccagatgg- ; vsv-r: -ctcggttcaagatccaggt- . hek t cells were transfected with indicated plasmids for h. then, the cells were lysed in lysis buffer containing a proteinase inhibitor mixture (roche, indianapolis, in, usa) and pmsf. cell lysates were incubated with µg/ml anti-ha ab or anti-flag or control ig (igg) and protein a-sepharose (ge healthcare, ge healthcare, calbiochem, sweden) and resolved by sds-page. the blot was then probed with anti-flag or anti-ha ab. irdye -conjugated anti-igg or hrp-conjugated anti-igg was used as a secondary ab, and proteins were identified using the odyssey imaging system or detected by the ecl assay. statistical analysis was carried out with spss . . all data are shown as the mean˘sd. the mean values from each group were compared by student's t-test. in all tests, p-values of less than . were considered statistically significant. by a small-scale screening of unknown ubiquitin e ligases in the regulation of virus-induced type i ifn signaling by the dual-luciferase reporter, we identified hace as a potential negative regulator in this pathway. then, we tried to systematically investigate whether hace is indeed involved in the regulation of virus-induced ifn signaling. as shown in figure a , overexpression of hace inhibited sev-induced activation of both isre (an interferon stimulated response element) and ifn-β promoter in a dose-dependent manner in hek t cells. in addition, activation of the isre promoter primed with the synthetic rna duplex poly (i:c) was also inhibited by overexpression of hace ( figure b) . to further support these results, the amount of ifnb was measured at various time points by reverse transcription (rt)-pcr during the twelve hours of infection by sendai virus. hace suppressed sev-induced transcription of endogenous ifnb gene ( figure c ). vsv is another representative rna virus for rig-i signaling studies. it is easy to detect the virus replication. consistent with the suppressive function of hace on virus-induced signaling, the replication of vsv was enhanced when hace was overexpressed ( figure d ). these data together suggested that hace negatively regulates virus-induced type i ifn signaling. next, to investigate the functions of endogenous hace on virus-induced type i ifn signaling, we knocked down the expression of hace in hek cells. two sirna oligos against hace were used, and the knockdown efficiency was monitored. as shown in figure a , both # and # sirna oligos can remarkably reduce the expression of endogenous hace in hek cells. compared to control cells, knockdown of hace expression augmented sev-induced ifnb gene transcription ( figure b ) and inhibited vsv replication ( figure c ). thus, hace is a negative regulator in virus-induced type i ifn signaling. statistical analysis was carried out with spss . . all data are shown as the mean ± sd. the mean values from each group were compared by student's t-test. in all tests, p-values of less than . were considered statistically significant. by a small-scale screening of unknown ubiquitin e ligases in the regulation of virus-induced type i ifn signaling by the dual-luciferase reporter, we identified hace as a potential negative regulator in this pathway. then, we tried to systematically investigate whether hace is indeed involved in the regulation of virus-induced ifn signaling. as shown in figure a , overexpression of hace inhibited sev-induced activation of both isre (an interferon stimulated response element) and ifn-β promoter in a dose-dependent manner in hek t cells. in addition, activation of the isre promoter primed with the synthetic rna duplex poly (i:c) was also inhibited by overexpression of hace ( figure b) . to further support these results, the amount of ifnb was measured at various time points by reverse transcription (rt)-pcr during the twelve hours of infection by sendai virus. hace suppressed sev-induced transcription of endogenous ifnb gene ( figure c ). vsv is another representative rna virus for rig-i signaling studies. it is easy to detect the virus replication. consistent with the suppressive function of hace on virus-induced signaling, the replication of vsv was enhanced when hace was overexpressed ( figure d ). these data together suggested that hace negatively regulates virus-induced type i ifn signaling. next, to investigate the functions of endogenous hace on virus-induced type i ifn signaling, we knocked down the expression of hace in hek cells. two sirna oligos against hace were used, and the knockdown efficiency was monitored. as shown in figure a , both # and # sirna oligos can remarkably reduce the expression of endogenous hace in hek cells. compared to control cells, knockdown of hace expression augmented sev-induced ifnb gene transcription ( figure b ) and inhibited vsv replication ( figure c ). thus, hace is a negative regulator in virus-induced type i ifn signaling. hace has been documented to act as a ubiquitin e ligase. so far, only a few ubiquitinated substrates have been identified, including rac , optineurin (optn) and rab gtpases [ , , ] . the catalytic cysteine (c ) is indispensable for its e ubiquitin ligase activity [ , , ] . then, we tried hace has been documented to act as a ubiquitin e ligase. so far, only a few ubiquitinated substrates have been identified, including rac , optineurin (optn) and rab gtpases [ , , ] . the catalytic cysteine (c ) is indispensable for its e ubiquitin ligase activity [ , , ] . then, we tried to determine whether the e ligase activity of hace is required for the inhibition of virus-induced type i ifn signaling. it is well-known that the catalytic cysteine (c ) of hace is indispensable for its e ligase activity [ ] . therefore, we constructed a mutant hace in which the amino acid cysteine was mutated into alanine ( figure a) . consistent with the previous report, wt hace can promote the degradation of rac , whereas the hace /c a mutant lost the ability to degrade rac , indicating the lost e ligase activity of hace /c a ( figure b ). reporter assays showed that sev-induced or poly (i:c)-induced activation of isre and ifn-β promoter activities were inhibited by hace /c a to a similar degree as wt hace (figure c,d) . these data indicate that hace inhibits virus-induced type i ifn induction independently of its e ligase activity. upon viral infection, recognition of viral rna by rig-i induced a downstream signaling cascade, including mavs, tbk , irf [ ] . we next sought to determine a step within the signaling pathway that hace targets. as shown in figure , isre and ifn-β promoter activity were activated by transfection of an active form of rig-i (rig-in), mavs, tbk and an activated form of irf (irf / d), respectively. co-expression of hace inhibited rig-in, mavs-induced isre or ifn-β reporter activation ( figure a,b) . on the other hand, it had no apparent effects on tbk or irf - d-induced isre or ifn-β reporter activation. these data suggested that hace acted downstream of mavs and upstream of tbk in the virus-induced signaling. hek t cells were coexpressed with rac and increasing amounts of ha-tagged wt hace or hace /c a. twenty-four hours after transfection, the cells were harvested and detected by western blot; (c) hace /c a still has the ability to inhibit sev-induced isre or ifn-β activation. hek t cells were seeded on -well plates and were transfected the next day with mock control, hace or hace /c a expressing vector, together with isre or ifn-β reporter vector. twenty-four hours later, cells were infected with sev or left uninfected for h before luciferase assays were performed; (d) hace /c a inhibited isre and ifn-β promoter activation by transfected poly (i:c) in hek t cells. data are representative of at least three independent experiments. * p < . ; *** p < . . upon viral infection, recognition of viral rna by rig-i induced a downstream signaling cascade, including mavs, tbk , irf [ ] . we next sought to determine a step within the signaling pathway that hace targets. as shown in figure figure a,b) . on the other hand, it had no apparent effects on tbk or irf - d-induced isre or ifn-β reporter activation. these data suggested that hace acted downstream of mavs and upstream of tbk in the virus-induced signaling. as suggested by figure , hace modulated the virus-induced signaling downstream of mavs and upstream of tbk . then, we tried to investigate the exact mechanisms underlying the suppressive function of hace . we coexpressed hace with mavs, traf and tbk . unexpectedly, hace cannot interact with either mavs or tbk . it interacted with traf ( figure a ,b). hace is a hect ubiquitin e ligase. then, we tested whether hace can promote the degradation of traf . as shown in figure c , hace cannot degrade traf . this is consistent with the results above ( figure c ,d) that the hace /c a mutant has a comparable inhibitory function to wt hace on virus-induced signaling. it has been reported that the mavs and traf complex is essential for virus-induced signaling [ , ] . then, we detected the impact of hace expression on the mavs-traf complex. with the increase of the expression level of hace , the formation of the complex of mavs-traf was severely impaired ( figure d ). these data indicate that hace may negatively regulate the virus-induced signaling by disrupting the mavs-traf complex. seeded on -well plates and transfected the next day with indicated signaling molecules, ifn-β or isre luciferase reporter and prl-sv plasmid and increasing doses of hace for h. the luciferase activities were quantified by normalizing with renilla luciferase activities. data are representative of at least three independent experiments. * p < . ; ** p < . ; *** p < . . in the present study, we provide evidence for the first time that hace is an important negative regulator of virus-induced type i ifn signaling. additionally, this suppressive function is e ligase activity independent. hace plays its suppressive role downstream of mavs and upstream of tbk . co-immunoprecipitation assays showed that hace did not interact with mavs or tbk . unexpectedly, it binds traf , which interacts with mavs and forms a platform for rna virus signaling. hace does not promote the degradation of traf . this is consistent with the data that hace negatively regulates the virus-induced signaling independent of the e ligase activity. further studies shown that hace can disrupt the mavs-traf complex. this provides a mechanistic explanation for the suppressive function of hace on virus-induced innate immune response. hace is a hect-type e ligase. the most studied function of hace is its involvement in tumor development. this function is e ligase dependent [ ] . hace can also function in an e ligase independent manner. for example, hace can repress the transcriptional activity of rarα and rarβ . mutation of the putative catalytic cysteine (c ) does not alter the repressive effect of hace on the transcriptional activity of rarβ [ ] . hace can mediate p -dependent selective autophagic turnover of ubiquitinated proteins. this process is achieved by protein-protein interaction through its ankyrin repeat domain and is independent of its e ligase activity [ ] . under stress conditions, hace cleared the ubiquitinated proteins in an e ligase activity independent manner. here, we demonstrated that hace suppressed virus-induced signaling independently of its e ligase activity, suggesting that hace exerts diverse biological functions by different mechanisms. several viral or cellular negative regulators may hijack traf or the traf complex to mediate immune evasion. herpes simplex virus ubiquitin-specific protease ul deubiquitinates traf [ ] . a few sars coronavirus proteins are identified as viral negative regulators, which target the traf signalosome [ ] . sars coronavirus papain-like protease binds and disrupts the sting-traf -tbk complex [ ] . sars coronavirus m protein or open-reading frame- b impedes the formation of the traf /tank/tbk /ikkε complex or the mavs/traf /traf complex, respectively [ ] . besides, some studies reported endogenous physiological negative regulators that target traf or the mavs-traf complex. the linear ubiquitin assembly complex (lubac) downregulates virus-mediated ifn induction by targeting nemo for linear ubiquitination. then, linear ubiquitinated nemo is associated with traf and disrupts the mavs-traf complex, which inhibits ifn activation [ ] . mip-t , a ciliary protein, is also a traf binding protein, which acts as a cellular inhibitor in virus-induced ifn production. mip-t impedes the formation of multiple traf signaling complex, such as the mavs-traf complex, the traf -tbk or traf -ikkε complex [ ] . the ubiquitin e ligase triad a targets traf for degradation to negatively regulate the rig-i signaling [ ] . here, we elucidate a novel role of hace in virus-induced signaling, which also targets the central mavs-traf complex. the interaction of proteins with traf family members were mediated by the traf interaction motif. we also analyzed the structure of hace and found that there is a potential tim between amino acid to amino acid (fkplellwh); we mutated central amino acid ple to aaa, and then performed the luciferase assays. the results showed that this mutant lost the ability to suppress virus-induced signaling, indicating that the suppressive function of hace is dependent on the complete traf interaction motif. further studies will focus on the link of the spontaneous mutation of hace with inflammation or tumor development, which will be intriguing. in this study, we identified a novel function of hace on virus-induced type i ifn signaling, which targets the mavs-traf complex and impedes the assembly of the mavs-traf complex. activation of host pattern recognition receptors by viruses innate recognition of viruses innate immune response to viral infection interferon induction by rna viruses and antagonism by viral pathogens regulation of rig-i-like receptor signaling by host and viral proteins activation and regulation of pathogen sensor rig-i. cytokine growth factor rev recognition of viruses by cytoplasmic sensors recognition of viral nucleic acids in innate immunity rig-i-like receptors and negative-strand rna viruses: rlrly bird catches some worms ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction visa is an adapter protein required for virus-triggered ifn-beta signaling identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-κb and irf cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus regulation of antiviral responses by a direct and specific interaction between traf and cardif a functional c-terminal traf -binding site in mavs participates in positive and negative regulation of the ifn antiviral response the e ubiquitin-ligase hace catalyzes the ubiquitylation of active rac ubiquitylation of active rac by the e ubiquitin-ligase hace ubiquitylation of autophagy receptor optineurin by hace activates selective autophagy for tumor suppression ubiquitylation and activation of a rab gtpase is promoted by a β ar-hace complex the e ligase hace is a critical chromosome q tumor suppressor involved in multiple cancers hace : a novel repressor of rar transcriptional activity constitutional translocation breakpoint mapping by genome-wide paired-end sequencing identifies hace as a putative wilms tumour susceptibility gene differential expression of a novel ankyrin containing e ubiquitin-protein ligase, hace , in sporadic wilms' tumor vs. normal kidney hace is a tumor suppressor gene candidate in natural killer cell neoplasms hace , a potential tumor suppressor gene on q , is not involved in extranodal natural killer/t-cell lymphoma pathophysiology aberrant methylation of the hace gene is frequently detected in advanced colorectal cancer methylation of the hace gene is frequently detected in hepatocellular carcinoma methylation of hace in gastric carcinoma loss of the e ubiquitin ligase hace results in enhanced rac signaling contributing to breast cancer progression the tumour suppressor hace controls cell migration by regulating rac degradation rac in human breast cancer: overexpression, mutation analysis, and characterization of a new isoform hace reduces oxidative stress and mutant huntingtin toxicity by promoting the nrf response the ubiquitin ligase hace regulates golgi membrane dynamics during the cell cycle hace -dependent protein degradation provides cardiac protection in response to haemodynamic stress smurf negatively modulates rig-i-dependent antiviral response by targeting visa/mavs for ubiquitination and degradation negative regulation of the rig-i signaling by the ubiquitin ligase rnf pcbp mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip e ligase wwp negatively regulates tlr -mediated innate immune response by targeting trif for ubiquitination and degradation immune signaling by rig-i-like receptors mammalian hect ubiquitin-protein ligases: biological and pathophysiological aspects herpes simplex virus ubiquitin-specific protease ul inhibits beta interferon production by deubiquitinating traf sars-coronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/traf /traf signalosome sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex we are grateful to takashi fujita for the rig-i expressing vector, tom maniatis for the tbk and ikkε vector and zhijian chen for the mavs (visa) vector. we also thank hong-bing shu, hong tang the authors declare no conflict of interest. key: cord- - tre rn authors: premanand, balraj; zhong wee, poh; prabakaran, mookkan title: baculovirus surface display of immunogenic proteins for vaccine development date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: tre rn vaccination is an efficient way to prevent the occurrence of many infectious diseases in humans. to date, several viral vectors have been utilized for the generation of vaccines. among them, baculovirus—categorized as a nonhuman viral vector—has been used in wider applications. its versatile features, like large cloning capacity, nonreplicative nature in mammalian cells, and broad tissue tropism, hold it at an excellent position among vaccine vectors. in addition to ease and safety during swift production, recent key improvements to existing baculovirus vectors (such as inclusion of hybrid promoters, immunostimulatory elements, etc.) have led to significant improvements in immunogenicity and efficacy of surface-displayed antigens. furthermore, some promising preclinical results have been reported that mirror the scope and practicality of baculovirus as a vaccine vector for human applications in the near future. herein, this review provides an overview of the induced immune responses by baculovirus surface-displayed vaccines against influenza and other infectious diseases in animal models, and highlights the strategies applied to enhance the protective immune responses against the displayed antigens. the existing battle between the human immune system and pathogens has a long-lasting history and is destined to continue. due to the advancement in the field of infectious diseases and the discovery of effective vaccines, therapies in recent history have aided in controlling the spread of diseases. however, the looming danger of re-emerging pathogens poses a serious issue and threatens to tilt the newfound equilibrium. among the types of vaccines available, inactivated vaccines are generally considered safe and stable. given their inactive nature, these vaccines can be given to people with weakened immune systems without the serious complication of opportunistic infection. however, the immune responses induced by inactivated vaccines are mainly humoral, and repeated immunization is required to improve immunity to the targeted disease. moreover, an inactivated vaccine may not retain the correct antigenic conformation and thus induces poor immune response against the targeted antigen [ ] . in contrast, live attenuated vaccines are more effective in inducing protective immune responses in vaccinated individuals. the major risks of live attenuated vaccines are the possibility of reversion and recombination with circulating strains, as well as the incompatibility of the vaccine with the immunocompromised, the elderly, the chronically ill, and the pregnant [ ] . recombinant subunit vaccines, on the other hand, can generally be used regardless of health status but possess other disadvantages. some of their disadvantages include the need for supporting adjuvants to improve immunogenicity, and purification issues due to the hydrophobic nature of antigens, which complicates the purification process, reducing the cost-effectiveness of vaccine production [ ] . dna vaccine utilizes genetically engineered dna capable of inducing humoral and cell-mediated immune responses against parasites, bacteria, and viruses [ , ] . modification of elements in the dna, such as promoters or enhancers, can enhance the expression of the encoded protein in vaccine recipients. moreover, a mixture of plasmids encoding a plethora of immunogenic genes can potentially be used to generate broad-spectrum vaccines. however, dna vaccine may induce antibodies against dna, resulting in autoimmune responses and the development of immunologic tolerance in recipient host [ ] . since , viral vaccine vectors have emerged, and such vectors have a more favorable safety profile than live attenuated virus vaccines, while having better immunogenicity than inactivated vaccines. viral vaccine vectors present the desired antigens in their native conformation, resulting in a stronger immunogenic response, while maintaining a higher level of foreign gene expression in vivo compared to dna vaccines [ , ] . however, some considerations exist for the use of viral vectored vaccines in humans: ( ) pre-existing vector immunity may have a serious impact on vaccine vector efficacy and ( ) increased transgene size could cause genetic instability and decrease viral yield. to overcome the issues of pre-existing vector immunity, vaccine vectors utilizing recombinant viruses of nonhuman origin have been developed to avoid neutralization of viral vector by pre-existing antibodies. a class of virus infecting insect cells, known as baculoviruses, has been developed for use as nonhuman viral vectors. among the various baculoviruses, autographa californica multicapsid nucleopolyhedrovirus (acmnpv) is the most widely studied. acmnpv is a large, double-stranded dna virus with a genome size of . kbp and contains open reading frames (orfs) [ ] . acmnpv has a biphasic life cycle resulting in the production of two forms of virus, budded virus (bv) and occlusion-derived virus (odv). the odv is responsible for the primary infection of the host insect, while bv is released from the host cells for cell-to-cell infection. moreover, the bv is produced during early stages of infection, whereas the odv is produced during late stages of viral infection, becoming concentrated in the nucleus and occluded within polyhedra [ ] . generally, acmnpv expresses each gene at specific time phases during its replication cycle. the genes encoding proteins that are responsible for viral replication and/or late gene expression (e.g., dnapol, lef - ) are expressed at an early time; structural protein genes (e.g., vp , p . , and e ) are expressed at late times; some genes are expressed at both early and late phases (e.g., ie , pp , and gp ); and p and polyhedrin (polh) are highly expressed at late and very late times of infection [ ] . based on published reports, the acmnpv promoters polyhedrin (polh) or p have been extensively used for expression of foreign proteins. in addition to the conventional acmnpv and other viral promoters, polh and p showed high levels of recombinant protein expression [ ] . interestingly, the combination of some of these promoters exhibited higher levels of protein expression compared to standard late promoters alone [ ] [ ] [ ] [ ] . moreover, it is well known that the polyhedrin promoter is active in the late stage of infection, and budding baculoviruses in the early stage of infection are unable to efficiently incorporate the target protein on the baculovirus envelope. hence, the use of immediate-early promoter may improve incorporation of target protein into the viral envelope. its salient features, including the ease of high-titer virus production, capability for simultaneous delivery of multiple genes, potential transduction ability, as well as its nonpathogenic and nonreplicative nature in humans, have led to the use of acmnpv in the production of complex eukaryotic protein in vitro, ex vivo, or in vivo [ ] [ ] [ ] and also for the vaccine development using surface display technology [ ] . in this review, we describe the immune responses elicited by recombinant baculovirus displayed vaccines against various infectious diseases and explore the unique strategies used to enhance the protective immunity against baculovirus displayed antigens (table ) . nt-not tested. since , influenza virus has been considered as an ever-present threat to humans, causing annual epidemics and infrequent pandemics, resulting in emergence of new virulent strains that pose a sustained alarm as a public health emergency [ ] . currently, the best method for the prevention and control of influenza virus infections is via vaccinations. however, a traditional influenza virus vaccine, while effective at managing infections, faces severe challenges. limited vaccine production capacity, long production time, poor growth properties of selected vaccine strains, and necessity of high-level biocontainment facilities are some major issues limiting vaccination production. particularly, uncertainty in prediction of emerging pandemic viral subtypes poses an obstacle in the prompt production of vaccines during an influenza pandemic. thus, a novel platform for the rapid development of vaccines, which overcomes limitations of currently available traditional vaccines, is required for swift response to influenza outbreak. hemagglutinin (ha) is a major surface glycoprotein of the influenza virus and is the main target for generating protective immunity against influenza virus [ ] . known to be a key determinant of host specificity, introduction of new ha subtypes was found to be associated with influenza strains responsible for pandemic outbreaks [ ] [ ] [ ] [ ] . hence, ha could serve as an important ideal target for development of influenza vaccines. recombinant subunit vaccines present one such method for targeting ha. recombinant subunit vaccines targeting ha were developed using an insect cell expression system and have been extensively evaluated in humans and animals as influenza vaccines. previously, a trivalent recombinant ha influenza vaccine-flublok ® , produced in insect cells using a baculovirus expression vector-was approved by the us fda. however, the need for a high dose with adjuvants and vaccine purification issues due to the hydrophobic nature of ha pose major setbacks in the development of recombinant subunit vaccines. an alternative strategy in vaccine development is the use of the baculovirus surface display technique. with baculoviral vaccines, targeted protein can be expressed on the surface of the virus without affecting the replication of the virus. moreover, insect cells possess the necessary cellular machinery to perform extensive post-translational modifications, such as glycosylation [ ] , phosphorylation [ ] , and disulfide bond formation [ ] , essential for the expression of many complex eukaryotic proteins, which require such modifications to maintain structural integrity and biological activities. furthermore, it has been reported that baculoviruses have strong adjuvant properties and are capable of inducing humoral and cellular immune responses against vaccine antigens [ ] . glycoprotein (gp ) is a major envelope protein of acmnpv which is essential for viral infection in insect cells. through the addition of gp domains (signal sequences, transmembrane and cytoplasmic regions), foreign proteins such as glutathione-s-transferase, human immunodeficiency virus (hiv) gp protein, rubella virus envelope protein, and synthetic igg binding domains can be displayed on the baculoviral envelope [ ] [ ] [ ] . alternatively, certain membrane proteins can be displayed on the budded virus itself without the need for gp domains. for example, vesicular stomatitis virus (vsvg) protein and measles virus receptor can be independently displayed on the viral envelope [ , ] . similarly, ha protein can be incorporated into the viral envelope without gp domains. the different regions of gp , such as the signal sequences, transmembrane domain, and cytoplasmic domain, have been widely used for the expression of secretory proteins in baculovirus/insect cell expression systems, and have been exploited to display foreign proteins or peptides on viral envelopes [ , ] . in a case study, the efficiency of h ha incorporation into the baculoviral envelope was shown to differ between recombinant baculoviruses carrying ha with different cytoplasmic domains (ctds), despite equal expression of recombinant ha. bac-ha , which expresses histidine-tagged ha with gp -ctd (cytoplasmic domain of gp ), incorporates ha to the viral envelope more efficiently than bac-ha, which expresses ha with ha-ctd (cytoplasmic domain of ha). moreover, bac-ha elicited higher hemagglutination inhibition titer (hi) compared to bac-ha. the result indicates the importance of the cytoplasmic domain and the degree of palmitoylation on the domain in the incorporation of ha on the viral envelope and on vaccine potential [ ] . in a second study, a modified bac-ha vaccine was developed by employing baculovirus p and cytomegalovirus (cmv) promoters with ha, which demonstrated surface display and endogenous expression of ha after transduction. an immunization study on the vaccine vector system revealed superior or equivalent immune responses against ha compared to other vaccine forms. the immune response is mediated by interaction with antigen-presenting cells (apcs) via the major histocompatibility ii (mhc-ii)-mediated antigen presentation pathway [ ] . in general, vaccines targeting respiratory viral infections, like influenza, act by inducing neutralizing antibodies that neutralize virions or block viral attachment and entry into host cells. however, currently available conventional vaccines induced humoral responses against only homologous strains and provided inadequate protection against heterologous strains. in contrast, t-cell-mediated cellular immune responses against conserved internal regions of antigens mediate protective immunity against heterologous strains [ , , ] . in previous studies, stimulation of helper t-lymphocytes (th cells) and cytotoxic t-lymphocytes (ctls) by nucleoprotein amino acids np - and np - provided complete cross-protection in challenge studies [ , ] . sridhar et al. [ ] observed that individuals having a higher number of pre-existing cd + t-cells against conserved cd epitopes showed milder illness after infection with pandemic h n influenza virus [ ] . furthermore, wilkinson et al. [ ] showed that, in the absence of antibody responses, pre-existing cd + t-cells respond to influenza internal proteins, reduce the severity of illness, and demonstrate lower shedding of virus [ ] . recently, zhang et al. [ ] developed a recombinant baculovirus-based vaccine containing three tandem copies of the highly conserved extracellular domain of influenza m protein (m e) (bv-dual- m e); a second vaccine had an additional mucosal adjuvant heat-liable enterotoxin b (bv-dual- m e-ltb). mice inoculated with bv-dual- m e-ltb vaccine induced higher levels of mucosal antibodies and more efficient cellular immunity against different h n clades (clade , . . . , . . , and ) compared to non-adjuvanted bv-dual- m e vaccine. interestingly, it was discovered that mucosal immunity alone was insufficient for protection from lethal h n challenge, whereas adjuvanted vaccine provided enhanced protection against similar challenge through cd + t-cell response. to validate the role of cd + t-cells in enhancing protective immunity, mice with depleted cd + or cd + t-cells were inoculated with bv-dual- m e-ltb. mice with depleted cd + t-cells displayed reduced lung viral titers, but no significant effect on survival rate was observed. comparatively, depletion of cd + t-cells or both cd + t-cells and cd + t-cells markedly increased lung viral titers and decreased survival rate. further research on enhancing ha-specific immune responses utilized a baculovirus vaccine vector with additional elements to improve immunogenicity. these elements included cag promoter, mhc class i signal sequence (mhciss), mhc class i trafficking domain (mitd), and woodchuck hepatitis virus posttranscriptional regulatory element (wpre). cag promoter enables robust expression of ha, while mhciss and mitd enhance mhc class i and ii antigen presentation, which stimulates the epitope-specific cd + and cd + t-cells. wpre, on the other hand, elevates the ha gene expression and increases immunogenicity. as shown in the study, mice immunized with baculovirus possessing the additional elements induced higher levels of ha-specific igg and igg a, higher hemagglutination inhibition titers, and better th and ifn-γ + /cd + t-cell responses compared to baculoviruses without the additional elements [ ] . furthermore, the role of wpre and itrs (inverted terminal repeats) in enhancement of ha expression and their effect on induction of humoral and cellular immune responses was studied. two series of baculovirus that use different promoters to drive the expression of ha were generated: bv-s series (bv-s-ha, bv-s-itrs-ha, bv-s-con-ha), which utilizes the cmv promoter for the expression of ha, and bv-a series (bv-a-ha, bv-a-itrs-ha, bv-a-con-ha), which utilizes white spot syndrome virus (wssv) immediate-early promoter one (ie ) for the expression of ha. results from immune studies indicate that baculoviral vaccine with wssv ie promoter induces a higher level of humoral and cellular immunity compared to similar baculoviral vaccine utilizing the cmv promoter [ ] . in another study, recombinant baculovirus vectors with an egfp expression cassette under p and cmv promoters were examined. the baculoviral vectors were modified to express ha using baculovirus polyhedrin (polh) promoter (vac-ha) or using dual-promoter with polh and cmv promoter (vac-ha-dual). while ha was expressed efficiently in sf insect cells for both constructs, mice vaccinated with vac-ha induced higher levels of igg and iga compared to mice vaccinated with vac-ha-dual. in addition, stimulation of splenocytes in immunized mice with ha-specific peptide triggered th immune response for vac-ha-immunized mice, whereas vac-ha-dual-immunized mice showed th -biased immune response. moreover, vac-ha-dual vaccine was more effective in inducing ha-specific cd + and cd + t-cell response in vitro [ ] . it has been reported that the signal peptide of membrane proteins is important in directing the protein to the endoplasmic reticulum membrane and in protein trafficking [ ] . the transmembrane (tm) domain plays a role in protein trafficking, membrane anchoring, membrane fusion, and viral budding [ , ] , whereas the ct domain is crucial for envelope incorporation, virus budding, interaction with the components of viral core [ , ] , and incorporation of influenza ha into the baculovirus envelope [ ] . supporting these observations was the research by tang et al. [ ] on the functional role of signal peptide (sp), ctd regions of gp , and tm of ha in the incorporation of ha into the baculovirus envelope. the study demonstrated enhanced ha incorporation and increased hi titers for baculovirus with gp sp, ctd, and ha tm. in stark contrast, recombinant baculoviruses containing different combinations of gp or ha domains (gp sp or ha-sp with ha-tm and ha-ctd; gp -tm or ha-tm with ha-sp and gp ctd; gp sp or ha-sp with gp -tm and gp -ctd) showed the opposite effect on ha incorporation and hi titers. based on the results, the second generation of tetravalent baculoviral vaccine was developed using gp -sp, ha-tm, and gp -ctd. the vaccine displays has from four subclades of h n influenza viruses and induces strong humoral and cellular immunity against homologous and heterologous h n strains [ ] . in another study, efficiency of wssv ie promoter-mediated h ha expression by recombinant baculovirus was examined [ ] . also, they confirmed that ha expressed by the baculovirus was able to successfully translocate to the plasma membrane of the infected cells and observed that surface-displayed ha was able to sustain its antigenic conformation and authentic cleavage [ ] . it has been reported that baculovirus pseudotyped with vesicular stomatitis virus glycoprotein (vsvg) from vesicular stomatitis virus (vsv) effectively increased transduction levels in mammalian cells [ ] and has the capability to increase the amount of expressed protein displayed on the surface of the entire viral envelope [ , ] . in addition, recombinant baculovirus which displays ha and co-expresses vsvg under wssv ie showed ha displayed on the baculovirus surface has high ha activity. intranasal or intramuscular immunization of chickens with baculovirus containing wssv ie induced strong ha-specific antibodies and hi titer compared to baculovirus with cmv promoter. nevertheless, co-expression of vsvg by both viruses contributed to increased anti-ha antibody level and hi activity [ ] . similarly, recombinant baculovirus pseudotyped with vsvg (bv-g-ha) was shown to successfully transduce into mammalian cell, mediate gene delivery, and enable efficient expression of h ha. an immunization study revealed that the vaccine candidate induced higher levels of h ha-specific antibodies and cellular immunity compared to the mice vaccinated with µg of dna vaccine. furthermore, immunized chickens, administered with varying doses, demonstrated complete protection from a lethal dose of highly pathogenic h n avian influenza virus [ ] . however, the vaccine did not efficiently protect mice against an evolutionarily distant strain [ ] , possibly due to a high degree of genetic divergence of the influenza virus [ ] . for example, current monovalent inactivated whole virus vaccine can induce neutralizing antibodies against the homologous strain but shows lower magnitude of response against heterologous strains [ ] . hence, it is necessary to develop a vaccine that expresses antigens covering the major circulating viruses [ ] . therefore, to broaden the protective efficacy of monovalent bv-g-ha vaccine, a pseudotyped baculovirus-based bivalent vaccine that encodes ha from clade . . and clade influenza viruses was developed. mice immunization with pseudotyped baculovirus-based bivalent vaccine induced significantly higher humoral and cellular immune responses and provided complete protection against h n viruses from clade . . and clade compared to other candidates used in the study [ ] . similarly, the cross-protective efficacy of wssv ie promoter-controlled bivalent baculovirus surface-displayed ha of a/indonesia/ / and a/anhui/ / (bivalent-bacha) was tested in another study. mice orally vaccinated using live bivalent-bacha vaccine showed both systemic and mucosal immunity, unlike mice immunized subcutaneously with live or inactivated bivalent vaccine, which induced only robust systemic immunity. the level of systemic immunity induced by oral bivalent vaccine in immunized mice was lower compared to subcutaneous immunization. however, mice orally immunized with bivalent-bac-ha vaccine demonstrated strong ha-specific mucosal iga response. in addition, oral vaccination induced potent cross-clade neutralizing antibodies against distinct clades ( , . . , . . . , . . , . . , , , and ) of h n viruses which are absent in monovalent inactive whole h n vaccination, whereas mice subcutaneously vaccinated with live or inactivated bivalent-bacha vaccine showed low neutralization titer against clade h n compared to other clade of viruses. enzyme-linked immunospot assay revealed that mice immunized orally or subcutaneously with live bivalent vaccine triggered both ifn-γ-secreting th cells and il- -secreting th cells, while mice immunized subcutaneously with inactive adjuvanted bivalent vaccine stimulated only the il- response. moreover, mice vaccinated orally or subcutaneously with live bivalent bac-ha vaccine showed complete cross-protection against clade and clade . . . h n viruses, suggesting that this bivalent vaccine can, potentially, protect against cross-clade h viruses during pandemic situations [ ] . since baculovirus has been proved to efficiently express foreign genes and is capable of transduction in a variety of cell lines, researchers utilized this virus as a potential vaccine vector against various infectious agents. furthermore, additional elements, like vsvg protein, were included by researchers to increase the efficiency of transduction, gene delivery, and expression in the recipient host [ ] . while recombinant baculoviral vector expressing both vsv-g and influenza ha was shown to evoke both humoral and cellular immune responses and provided effective protection against lethal virus challenge in mouse and chicken hosts [ ] , the high cytotoxicity of vsv-g protein [ ] and its immediate inactivation by serum complement systems impedes the use of the element in a vaccine delivery vehicle [ ] . recent reports have shown that human endogenous retrovirus (herv) envelope-coated recombinant baculovirus-based human papillomavirus l vaccine significantly improved the expression of l in mammalian cells and induced humoral immune responses which are comparable with the level obtained using commercial cervical cancer virus-like particles vaccine [ ] . further, it has been reported that herv envelope-coated baculovirus-based ha of the h n pandemic influenza vaccine induced a strong cellular immune response compared to the commercial vaccine [ ] . in another study, prabakaran et al. [ ] developed a recombinant baculovirus displayed ha vaccine under the control of wssv ie promoter (bacha) against the pandemic h n virus. the vaccine showed successful ha expression on its envelope, and mice vaccination studies showed that both the live and adjuvanted with inactive form of recombinant baculovirus induced ha-specific antibody responses and offered complete protection against lethal viral infection [ ] . studies have shown that high neutralizing antibodies generated against influenza virus target specific regions in the globular head of influenza ha. moreover, the ha stalk domain is well conserved and antibodies targeting this region are capable of neutralizing the heterologous influenza viral subtypes [ ] [ ] [ ] [ ] [ ] [ ] . for example, chimeric ha constructs expressing the globular head and stalk regions from different subtypes induced antibodies that showed broad protection [ , , ] . similarly, mice immunized with recombinant baculovirus displayed ha of the pandemic h n influenza virus induced ha-stalk-specific antibodies and demonstrated protective immunity against homologous and heterologous h n subtype viruses [ ] . in recent decades, avian h influenza virus was responsible for numerous influenza outbreaks among poultry industries in europe and north america. since , none of these poultry-adapted viruses has evolved to widely infect humans or cause a pandemic. however, there are cases where the virus acquired the ability to infect mammals, as evidenced by the largest outbreak of highly pathogenic h n (nl/ . ) in the poultry industry of the netherlands, where human infections were reported [ ] . h n viruses isolated from the patients were shown to possess replicative potential in human conjunctiva and ability for human-to-human transmission. interestingly, the virulence of the virus in chickens and turkeys increased with acquisition of additional amino acids at the hemagglutinin (ha) cleavage site [ ] . in , human infections with avian influenza a h n subtype were first reported in china and caused several waves of human infection. it was speculated that the virus possesses high mutagenicity, which contributed to its ability to infect humans. higher mutagenicity may be the cause of enhanced pathogenicity, as supported by recent human cases, where the isolated virus was found to have mutations associated with reduced susceptibility to neuraminidase inhibitors used in antiviral treatment [ ] . since h subtype viruses pose a major challenge for public health and the poultry industry, numerous vaccines have been generated against these subtype viruses using various platforms. recently, baculovirus displayed ha vaccine against h n (nl ) was developed and its efficacy was tested via intranasal (i.n.) or subcutaneous (s.c.) administration in mice. it was observed that intranasally immunized mice with live baculovirus displayed ha vaccine induced higher or comparable humoral, mucosal, and cellular immune responses compared to other vaccine candidates, irrespective of immunization route. additionally, the immunization provided % complete protection, whereas no protection was observed in mice vaccinated with other vaccine candidates [ ] . in another study, recombinant baculovirus-based vaccine (bacha) against avian h n virus with high pandemic potential was generated, and its immunogenicity and cross-protective efficacy was evaluated in mice. they found that live bacha i.n. immunized mice elicited high levels of ha-specific iga mucosal immune response and humoral immune response, comparable to mice administered subcutaneously with live bacha. on the other hand, subcutaneous immunization with adjuvanted inactive bacha stimulated robust humoral immune response and induced higher neutralizing antibodies against h n viruses and other h subtype (h n and h n ) viruses compared to other vaccine candidates. in addition, subcutaneous immunization with inactive whole h n vaccine protected % against rg-h n challenge but demonstrated insufficient protection against rg-h n (nl ) subtype, whereas mice vaccinated subcutaneously with live or inactive recombinant baculovirus against h n (nl ) protected only - % of mice against h n challenge. furthermore, mice immunized i.n. with live bacha of h n or h n showed complete protection against both h n and h n infection in a challenge study [ ] . it has been reported that the differences between the immunogenicity of ha of h n (nl ) and ha of h n might be due to the amino acid changes at a t, which introduced a potential glycosylation site at position n, masking the epitopes of h ha (nl ) [ , ] . to account for this difference in immunogenicity, kumar et al. [ ] compared the ha of h n and h n viruses and selected three positions in their study: (a) , (b) , and (c) , which are located within or near the receptor-binding site of h n ha protein. by site-directed mutagenesis, they generated six mutant constructs by single or double amino acid substitution at these three positions (t a, t a, i v, t a- a, t a-i v, and i v-t a) . among the generated mutant constructs (bac-ha m ), mice vaccinated with constructs t a, t a-i v, and i v-t a induced higher hemagglutination inhibition and neutralization titer that neutralizes both h n and h n viruses compared to mice immunized with bacha or other mutant vaccines. the result shows that the substitution of amino acids responsible for forming potential glycan motif-epitope masks would improve immunogenicity of the vaccine candidates. in another study, mice i.n. immunized with baculovirus-displaying ha of low pathogenic influenza h subtype triggered higher ha-specific humoral, mucosal, and cellular immunity compared to mice immunized with inactivated influenza virus. moreover, regardless of immunization route, it conferred % protection against lethal homologous mouse-adapted h n virus [ ] . moreover, recombinant baculovirus with cmv-polyhedrin dual promoter for expressing chimeric ha of h n was shown to efficiently express ha in both mammalian and insect cells, induce strong immune response, and provide % protection against lethal h n viral challenge in mice, unlike other vaccine candidates observed [ ] . shrimp immunity relies on an innate immune mechanism, and their lack of adaptive immunity hinders vaccine development against shrimp pathogens. though limitations exist in developing shrimp vaccines, the presence of quasi-immune responses after natural or experimental infection with white spot syndrome virus (wssv) in p. japonicas and the resistance against experimental rechallenge with wssv due to neutralizing factors in the survivors' plasma [ , ] encouraged the researchers to continue the development of vaccines against the wssv virus. several attempts have been made to develop vaccines by targeting the immunogenic vp protein of wssv virus [ ] [ ] [ ] [ ] syed musthaq et al. [ ] , developed a recombinant-based vaccine (bac-vp ) that displays recombinant vp (rvp ) on its envelope. confocal and transmission electron microscopy analysis confirmed that rvp was able to localize on the plasma membrane of infected insect cells, and the virus also successfully acquired rvp on its envelope from the insect cell membrane during the budding process. approximately . µg/ml of vp protein was anchored on the bac-vp envelope ( viral particles). furthermore, its protective efficacy against wssv infection was tested via injection, oral, or immersion methods in shrimp. the bac-vp -injected shrimp successfully transcribed and translated vp and showed survival rates of . % and . % against wssv virus challenge at and days postvaccination, respectively [ ] . on the other hand, the transcriptional levels of lipopolysaccharide and β- , -glucan binding protein (lgbp) and signal transducer and activator of transcription (stat) genes (which are crucial for triggering innate immune responses) [ ] were altered at different days postinfection (dpi) in shrimp vaccinated with bac-vp by oral or immersion routes. the expression level of stat gene in the control group was downregulated at various dpi as wssv infection progressed, but in the vaccinated group, stat gene level decreased until dpi and started to increase at dpi and dpi. moreover, the levels of lgbp gene in the control group was upregulated as infection progressed, whereas the vaccinated group showed an increasing level at early infections, which later decreased as wssv infection cleared. these results confirm that oral vaccination of bac-vp triggers the innate immune responses and, possibly, was responsible for higher survival rates of . % and . % upon wssv challenge at and days postvaccination. also, shrimp vaccinated with bac-vp immersion vaccine showed % and . % survival rates compared to % mortality obtained with the control group in a challenge study [ ] . porcine reproductive and respiratory syndrome virus (prrsv), classical swine fever virus (csfv), and porcine circovirus type (pcv ) are the major pathogens continuously affecting the pig industry and cause significant economic losses worldwide. to date, researchers all over the world have contributed various forms of vaccines utilizing different strategies targeting these pathogens. virus-based vectored vaccines were developed using modified vaccinia virus ankara, canine adenovirus- , pseudorabies virus, and semliki forest virus [ ] [ ] [ ] [ ] . however, these vectors are of mammalian origin and pose a biohazard threat. in contrast, recombinant baculoviruses possess good safety profiles and have been utilized to develop vaccines against prrsv, csfv, and pcv viruses. using gp sp, tm, and ct domains, xu et al. [ ] developed recombinant baculovirus-based bivalent vaccine vector (bacsc-dual-gp -cap) that surface-displayed gp (major immunogenic inducer of neutralizing antibodies against prrsv infection) and pcv (major immunogenic capsid (cap) protein). a swine vaccination study demonstrated that this vaccine candidate stimulated potent gp and cap-specific neutralizing antibodies and induced higher lymphocyte proliferation responses compared to control groups, which indicates that the bivalent vaccine can potentially be used as a vaccine against a mixed prrsv and pcv infections. in another study, the orfs of prrsv were successfully displayed on the baculovirus envelope using the gp sp, h n tm, and gp ctd regions. the antibody responses elicited by the vectored vaccines were sustained for up to days after the booster, whereas the titers in the inactivated prrsv-vaccinated group were reduced dramatically at this time point. the enhanced immune responses could be due to the in vivo transduction of the prrsv genes by baculovirus vaccine, which activates the endogenous antigen-processing and -presenting pathways. amongst the vaccines developed in this study, baculoviruses that displayed orf a, orf , and orf induced higher antibody responses in mice compared to other vaccine candidates [ ] . a recombinant baculovirus-based vaccine that contains the pcv cap and vsvg protein (bv-gd-orf ) was developed. the expression of pcv was placed under the control of cmv and polh promoters, whereas expression of vsvg was placed under the p promoter. due to vsvg-mediated transduction and the successful expression of the pcv cap protein in transduced cells, bv-gd-orf behaved as a subunit/dna vaccine and induced robust cap protein-specific humoral and cellular immune responses [ ] . studies reported that e or e rns glycoprotein are the main immunogenic proteins of csfv, which can induce neutralizing antibodies and provide protective immunity against csfv. xu et al. generated recombinant baculoviruses that surface-displayed either e (bacsc-e ) [ ] or e rns (bacsc-e rns ) [ ] as fusion forms with gp domains. the presence of the gp domains did not alter the viral titers of the recombinant viruses. moreover, a mice vaccination study revealed that bacsc-e or bacsc-e rns induced stronger e -or e rns -specific antibodies, respectively, compared to other vaccine candidates, suggesting that the vaccine candidates could potentially be applied in the treatment of csfv infection. respiratory syncytial virus (rsv) is a common contagious virus that causes acute lower respiratory tract infection in infants, young children, elderlies, and immunocompromised individuals [ ] . currently, there is no approved vaccine available to treat this infection in humans. studies have reported that immunization with recombinant viral vector expressing rsvg protein showed immune pathological issues by enhancing th skewing of the cd + t-cell response [ ] [ ] [ ] . countering the issue, zhang et al. [ ] developed recombinant baculovirus vaccines that showed reduced immune pathological conditions in mice. four vaccine constructs were developed: bac-tf (displays the f ectodomain (tf) on the envelope), bac-cf (expresses full-length f protein in transduced mammalian cells), bac-cf/tf (displays tf on the envelope and expresses f in cells), and bac-cf/tf -visa (contains additional virus-induced signaling adaptor (visa gene). bac-cf and bac-cf/tf showed an increased mixed th /th cytokine response, higher levels of igg a/igg antibody responses, and reduced immunopathology upon rsv challenge compared to bac-tf . moreover, co-expression of visa reduced the th immune responses induced by bac-cf/tf and protected lungs from inflammatory immune response upon a subsequent rsv challenge. therefore, the inclusion of visa elements could be an effective alternate vaccine strategy against rsv infection [ ] . rabies virus (rabv) is highly infectious and poses a constant threat to humans and animals. traditionally, vaccine development against rabv mainly focused on inducing neutralizing antibodies [ ] [ ] [ ] . however, studies have shown that, in addition to humoral immunity, cellular immune responses are important for providing protective immunity against rabv [ , , ] . previously, a recombinant baculovirus viral vector vaccine against rabv glycoprotein (rvg) was constructed (bv-rvg/rvg). the vector expresses two types of rvg antigen (baculovirus-expressed rvg and in vivo expressed rvg) to enhance specific immune responses against the rvg antigen. based on immunization study, bv-rvg/rvg-immunized mice displayed higher humoral and cellular immune responses and offered complete protection against rabv compared to mice immunized with other vaccine candidates. the enhanced immunogenicity could be due to immediate recognition of rvg incorporated into bv-rvg/rvg by immunocompetent cells, resulting in presentation to dendritic cells (dcs) and, subsequently, activation of b cells to secrete viral-specific neutralizing antibodies. furthermore, cmv-mediated in vivo rvg expression by bv-rvg/rvg activated the endogenous antigen presentation pathway, inducing persistent and specific immune responses [ ] . in another study, feng et al. [ ] displayed the entire ectodomain of sars-cov spike protein (s) on the baculovirus envelope using gp signal peptide and vsv-g membrane anchor. in mice, the vaccine induced specific neutralizing antibodies against s protein in the absence of adjuvants [ ] . in a subsequent study, researchers developed recombinant baculovirus vaccines against avian infectious bronchitis virus (ibv), japanese encephalitis virus (jev), and bovine herpesvirus- (bhv- ) with the gp domains (sp, tm, and ctd). chickens immunized with baculovirus displayed s protein of ibv vaccine induced more humoral and cellular immune responses and showed better protection ( %) compared to other vaccines studied [ ] . on the other hand, glycoprotein e of jev displayed on the baculovirus envelope induced strong neutralizing antibodies in mice and swine and offered % protection in vaccinated mice against a lethal jev challenge [ ] . similarly, bhv- envelope glycoprotein (gd) displayed on the baculovirus envelope elicited strong neutralizing antibody against bhv- in mice [ ] . enterovirus (ev ) immunogenic capsid protein (vp ) is a non-membrane protein which requires additional regions, like sp, tm, and ctd of gp , ha, or other domains for successful incorporation into envelope, while membrane proteins like ha or na (neuraminidase) can be translocated to the plasma membrane and incorporated onto the envelope during viral budding. meng et al. [ ] generated recombinant baculoviruses, with wssv ie (bac-pie -gp -vp ) or polyhedrin promoter (bac-pph-gp -vp ), which surface-displayed chimeric vp (vp was fused in between gp sp and mature domain). transmission electron microscopy analysis confirmed that vp was successfully anchored on the recombinant baculoviral envelope. bac-pie -gp -vp -immunized mice were found to induce slightly higher vp -specific antibodies with potent neutralizing activity (can neutralize c subgenogroups and heterologous subgenogroups) than bac-pph-gp -vp and to show neutralizing activity comparable with inactivated ev vaccine. subsequently, premanand et al. [ ] generated the recombinant baculovirus (bac-vp ) that surface-displayed the chimeric vp using minimal fusion partners, like gp sp ( aa), h n -ha transmembrane domain ( aa), and gp ctd ( aa) to facilitate better incorporation of vp and higher baculovirus titers. expectedly, bac-vp contains -fold higher vp ( ng/µg) on its envelope compared to vp on bac-pie -gp -vp envelope ( ng/µg). bac-vp -immunized mice elicited stronger vp -specific antibody responses and cross-neutralization activity against homologous or heterologous strains compared to bac-pie -gp -vp and bac-pph-gp -vp vaccines. moreover, bac-vp provided % protection against a mouse-adapted lethal strain of ev [ ] . in both studies mentioned above, vp was anchored on the viral envelope similar to type transmembrane protein with unrestricted n-terminus exposure. mimicking other types of transmembrane protein, kolpe et al. generated recombinant baculovirus (bac-na-vp ) which surface-displayed vp using influenza a neuraminidase (na), resulting in a type ii transmembrane-anchored pattern with a distal c-terminus. mice immunization studies revealed that bac-na-vp induced both humoral and cellular immune responses and provided % protection of suckling balb/c mice against mouse-adapted ev -b strain challenge [ ] . infectious bursal disease virus (ibdv) is the causative agent of infectious bursal disease, which affects chickens and causes considerable economic loss for the poultry industry [ , ] . it has been reported that live attenuated vaccines elicited lower level of antibodies and failed to protect the chickens against ibdv [ ] . to target this issue, xu et al. [ ] generated a recombinant baculovirus that surface-displayed his -tagged vp protein using the gp tm and ct domains (bacsc-vp ). bacsc-vp -immunized chickens demonstrated higher levels of neutralizing antibodies and better protective immunity against a very virulent ibdv strain than control groups. furthermore, ibdv-specific lymphocytes were observed in bacsc-vp -vaccinated chickens. also, the major immunogenic capsid protein vp of canine parvovirus type (cpv- ) and immunogenic proteins σc and σb of avian reovirus (arv) have been utilized to develop a baculovirus-based vaccine against cpv- and arv, respectively. fusion of gp domains (sp, tm, and ctd) with the capsid proteins allows their incorporation into the baculoviral envelope. an animal vaccination study revealed that baculovirus that surface displayed vp of cpv- induced higher neutralizing antibodies in mice compared to other vaccines tested [ ] . moreover, recombinant baculoviruses that displayed immunogenic proteins of either σc or σb of arv elicited strong antibody titers with higher neutralizing activities compared to antibody responses by other vaccine candidates [ ] . malaria, a global disease caused by plasmodium parasites, is endemic in tropical and subtropical regions and results in up to million deaths annually. a recombinant protein-based subunit vaccine containing a part of a major surface protein from plasmodium falciparum circumsporozoite protein (pfcsp) provided short-lived protective immunity, and the effective period was affected by age group and malaria transmission intensity [ , ] . another vaccine plasmodium falciparum sporozoite (pfspz) was reported to provide protection against homologous and heterologous human malaria infection [ ] [ ] [ ] . aiming to curb the spread of malaria, the who has set to a goal of % vaccine efficiency by the year . novel and alternate strategies will be required to ensure the development of more effective vaccines against malaria infection. to develop a vaccine with dual functions (such as a malaria vaccine that can act as a liver-directed gene delivery vehicle), yoshida et al. [ ] developed the recombinant baculovirus-based vaccine that displayed rodent malaria parasite plasmodium berghei circumsporozoite protein (pbcsp). in the immunization study, the vaccine induced both humoral and cellular immune responsea and provided % protection against sporozoite challenge [ ] . subsequently, a series of baculovirus-based vaccines expressing pfcsp (with deleted glycosylphosphatidylinositol gpi anchor region) under the control of polh or cmv promoter was developed. acnpv.cs vector expresses the cs protein in apcs under the control of the cmv promoter to enable (preferentially) induction of cd + t-cells upon mhc class i presentation. acnpv.cs vector expresses a cs protein with gp as a fusion form using the polyhedrin promoter in insect cells, which allows the incorporation of cs on the baculovirus envelope during the budding process (mimicking the p. falciparum sporozoite). uptake of acnpv.cs into apcs triggers cd + cells via mhc class ii presentation and induces a potent humoral immune response. acnpv.cs-cs combines expression and presentation of recombinant cs antigens in mammalian cells to induce both cs-specific cd + and cd + t-cells. a mouse vaccination study showed that the acnpv.cs-cs vaccine induced efficient humoral and cellular immune responses with intramuscular immunization compared to other vaccine candidates [ ] . recombinant baculovirus dual expression-based malaria vaccine, which drives pbcsp expression by polh and cmv promoters, was developed. mice immunized with the vaccine induced both th and th responses after intramuscular inoculation and provided complete protection against sporozoite challenge [ ] . another baculovirus-based vaccine candidate which displays the major surface antigen of p. falciparum, ppfs , was developed as a transmission-blocking vaccine against human malaria. both i.m. and i.n. immunized mice showed high levels of pfs -specific antibodies and effective transmission-blocking response, with an % (intranasal) and % (intramuscular) reduction in oocyst intensity [ ] . in another case study, a recombinant baculovirus-based plasmodium vivax transmission-blocking vaccine using autographa california nuclear polyhedrosis virus (acnpv-dual-pvs ) was developed. acnpv-dual-pvs not only displayed pvs on the acnpv envelope in native conformation, but also expressed pvs upon transduction of mammalian cells. acnpv-dual-pvs induced pvs -specific antibodies in intranasally or intramuscularly immunized mice. moreover, in a rabbit immunization study, the vaccine induced significant transmission-blocking response, regardless of immunization route (subcutaneous, intramuscular, and intranasal) [ ] . yoshida et al. [ ] developed a baculovirus-based nasal drop vaccine (acnpv-pymsp surf ) that surface-displayed the carboxyl terminus of merozoite surface protein (pymsp ) from plasmodium yoelii. intranasal and intramuscular immunization of mice with acnpv-pymsp surf induced mixed th /th immune responses and provided % protective efficacy. however, intranasal immunization elicited higher pymsp -specific antibody titers and stronger natural boosting after a challenge study [ ] . in another study, a baculovirus dual-expression system-based pfcsp protein (bdes-pfcsp) vaccine was generated. based on an immunization study, the vaccine was found to induce mixed th /th immune responses in mice, generate a higher level of pfcsp-specific antibodies, and confer significant protection against malaria. in addition, sera from immunized monkeys prevented sporozoite invasion in hepg cells [ ] . a multistage malaria vaccine was developed using the baculovirus platform to target and inhibit the plasmodium parasite in its pre-erythrocytic and sexual stages. the generated baculovirus displayed correct folding of both p. vivax circumsporozoite protein (pvcsp) and p (pvs ) protein in fusion form (bdes-pvs -pvcsp) and acted as a pre-erythrocytic and a transmission-blocking activity vaccine. the bdes-pvs -pvcsp vaccine induced higher antibody levels against pvs and pvcsp while providing % protection against malaria and % transmission-blocking activity [ ] . in a recent study, human decay-accelerating factor (hdaf) was incorporated into a baculovirus vector, which effectively displayed hdaf and pfcsp on the surface of the baculoviral envelope to prevent serum complement inactivation. following intramuscular immunization, the modified vaccine conferred higher protective efficacy ( %) compared to control group ( %) in a challenge study [ ] . mucosal surfaces (respiratory, gastrointestinal, and urogenital tracts) provide the major route of entry for various microorganisms, and the mucosal immune system acts as first line of defense against microbial infection [ ] . therefore, triggering of the mucosal immune system is important in preventing infection, and this can be achieved by mucosal vaccinations via intranasal, oral, pulmonary, rectal, or vaginal routes of administration. among these routes, oral or intranasal immunizations are the two most viable options for vaccine delivery. the nasal route, in particular, offers several advantages, like the possibility of self-administration, the induction of both mucosal and systemic immunity in the gastric mucosa, respiratory tracts and genital tracts, as well as the lower doses of antigen and adjuvant required compared with oral vaccination. several nonhuman viral vectors have been developed in recent years, among which is the baculoviral vector. baculovirus has been reported to possess strong adjuvant properties in mammals, promoting enhanced humoral, mucosal, and cellular immune responses against co-administered antigens. supporting this are previous reports which indicated that mice immunized with baculovirus genomic dna and ovalbumin showed humoral and cytotoxic t-lymphocyte responses against co-administered antigens. the abundance of cpg motifs in the baculovirus improves its ability to induce innate immune responses through the tlr /myd -dependent endosomal recognition pathway and dna-dependent activator of interferon regulatory factor-mediated pathway [ ] . other studies have shown that intranasal inoculation of mice with wild-type baculovirus provided protection against h n , h n , and other influenza b strains [ ] , which highlighted the potential of the baculoviral vector in the development of vaccines against mucosal acquired pathogens. intranasal immunization of mice with live recombinant baculovirus (bac-ha) vaccine induced significantly higher mucosal iga immune response against h n -ha compared to other intranasally delivered vaccines. the higher immune response could be attributed to the efficient transduction of baculoviral vector or the strong adjuvant property of baculovirus due to the presence of unmethylated cpg motifs. in addition, i.n delivery of bac-ha stimulated higher amounts of ifn-γ and il- compared to s.c immunization of live bac-ha, which is similarly reported in other studies [ , ] . the enhancement of cytokine levels could be due to recall response in splenocytes or due to the presence of nasopharynx-associated lymphoid follicles in the nasal mucosa that trigger antigen-specific th lymphocytes, ctls, and iga immune responses. a challenge study in mice revealed that i.n immunized mice with live bac-ha showed % protection against lethal viral challenge compared to other vaccines, irrespective of route of administration. even though s.c immunized mice achieved a higher level of igg antibodies, the incapability of bac-ha in protecting against lethal viral challenge could be due to lack of mucosal iga, which is more crucial in clearing upper respiratory tract infection than igg [ ] . in another study, a recombinant baculovirus-based vaccine against another pandemic potential avian h n virus was generated (bac-ha). they found that mice intranasally immunized with live bac-ha elicited higher levels of ha-specific mucosal and humoral immune responses. mice vaccinated subcutaneously with live or inactive baculovirus displayed ha of h n (nl ) only protected - % of mice against h n challenge, but mice immunized intranasally with live bac-ha of h n or h n showed complete protection against both h n and h n infection in a challenge study. the result indicates the importance of additional mucosal immunity in recovery and protection during h subtype infection [ ] . the inclusion of adjuvant improves the immune response induced by the vaccine, and the cause of the better immune response in mucosal adjuvants, in particular, is due to the induction of protective local and systemic immune responses against various infectious diseases [ ] . also, intranasal immunization of recombinant baculovirus-displaying ha of h n virus induced high levels of mucosal iga and serum igg immune responses in mice. combination of recombinant mucosal adjuvant ctb further enhanced serum igg and iga responses compared to unadjuvanted log or log ha titer bac-ha alone. log ha titer live bacha with µg of rctb conferred % protection against homologous and heterologous h n strains [ ] , suggesting that the combination of safe adjuvants with vaccines would be ideal for optimal efficacy of intranasal vaccines. oral administration is considered as another efficient route for delivering the mucosal vaccines. it is non-invasive, safe, and shows good patient compliance. for example, oral polio vaccine against highly contagious poliovirus helped to eradicate poliovirus infection in most parts of the world [ ] . moreover, studies have shown that oral vaccination can prevent infection of lungs and is capable of inducing mucosal immune responses (iga) in the respiratory tract, providing protection against influenza viruses. prabakaran et al. [ ] reported that live recombinant baculovirus displaying ha of h n virus (bac-ha) was able to express ha in epithelial cells of intestines of orally vaccinated mice and induced strong systemic and mucosal immune responses. mice immunized with live bac-ha of h n without any inclusion of adjuvants conferred % protection against homologous and heterologous h n strains, while immunization with inactivated bac-ha resulted in a lower survival rate of . %. the reduced immune responses of the inactivated bac-ha could be due to loss of transduction ability, whereas the live bac-ha was able to bind to the receptors that are expressed in the intestinal cells, resulting in gene delivery and stimulation of cell-mediated immune responses [ ] . moreover, the formulation of inactivated vaccine can have a huge impact on its immunogenicity. in one study, the immunogenicity lost by inactivated baculovirus displayed ha (bac-ha) vaccine was recovered by encapsulation using reverse micelle of phosphatidylcholine adjuvant. mice orally immunized with encapsulated inactive bac-ha induced systemic and mucosal immune response comparable to live bac-ha [ ] . however, there are limitations in the use of mucosal adjuvants in humans. aside from effectiveness of the adjuvant, the major concern in using mucosal adjuvants is its possible detrimental impact on humans. therefore, adjuvant used in mucosal immunization has to be proven safe for use. premanand et al. [ ] utilized naturally occurring lipid-based vesicles (bilosomes) as a mucosal adjuvant to coat the recombinant baculovirus (bac-vp ). an oral immunization study revealed that bac-vp , associated with bilosomes, induced higher vp -specific immune responses and conferred complete protection in a challenge study [ ] . to overcome the hurdles of selecting safe mucosal adjuvants, data analysis can be applied to search for suitable mucosal adjuvants and to understand their mechanism of action. in addition to new technologies, novel concepts are welcomed in the development of suitable mucosal adjuvants. finally, considerations on the delivery route and the appropriate combination of vaccine and adjuvant are essential in ensuring optimal effectiveness of vaccination in humans. baculovirus surface-displayed vaccines have been proven effective in inducing protective immune responses in animal models. however, further advancement in the context of immunogenicity will be made by utilizing hybrid promoters for the enhanced expression and anchoring of complex protein on recombinant baculoviral envelopes. in addition, inclusion of novel regulatory elements and molecular adjuvant property-carrying motifs in the vector will certainly boost the immunogenicity of antigen displayed on the baculovirus envelope. the main obstacle in implementing baculovirus surface-displayed vaccine as a human vaccine candidate is the lack of safety guidelines for human immunization. however, international organizations, like organization of economic co-operation and development in [ ] directorate-general in [ ] , concluded that no adverse effects of baculovirus on human health have been observed in safety tests, and they are not pathogenic, carcinogenic, or genotoxic in mammalian cells. however, environmental and ecological impacts of the baculovirus in invertebrates will be reduced by modification of baculovirus essential genes required for replication and budding, allowing the development of a 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expressing vp of infectious bursal disease virus induce protection against bursal disease in chickens first results of phase trial of rts,s/as malaria vaccine in african children seven-year efficacy of rts,s/as malaria vaccine among young african children attenuated pfspz vaccine induces strain-transcending t cells and durable protection against heterologous controlled human malaria infection safety and efficacy of pfspz vaccine against plasmodium falciparum via direct venous inoculation in healthy malaria-exposed adults in mali: a randomised, double-blind phase trial protection against plasmodium falciparum malaria by pfspz vaccine inside the mucosal immune system involvement of the toll-like receptor signaling pathway in the induction of innate immunity by baculovirus baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice single intranasal mucosal mycobacterium bovis bcg vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis comparative evaluation of intranasal and subcutaneous route of immunization for development of mucosal vaccine against experimental tuberculosis mucosal adjuvants: opportunities and challenges oral vaccines: directed safe passage to the front line of defense reverse micelle-encapsulated recombinant baculovirus as an oral vaccine against h n infection in mice consensus document on information used in the assessment of environmental applications involving baculovirus; series on harmonization of regulatory oversight in biotechnology number european commission's health & consumer protection directorate-general we are grateful for the financial support received from temasek life sciences laboratory, singapore. the authors declare no conflict of interest. key: cord- -i loz xb authors: li, tongya; ke, zunlong; liu, weiyong; xiong, ying; zhu, ying; liu, yingle title: human hepatitis b virus core protein inhibits ifnα-induced ifitm expression by interacting with baf date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: i loz xb human hepatitis b virus core protein (hbc) is a structural protein of the hepatitis b virus (hbv) and contributes to hbv regulation of host-cell transcription. however, the mechanisms of transcriptional regulation remain poorly characterized. to dissect the function of hbc, a yeast two-hybrid was performed to identify hbc-binding proteins, and the c-terminal of brg /hbrm-associated factors (baf c) was identified. then, the existence of hbc interactions with baf c and full-length baf was confirmed via co-immunoprecipitation assays in t, hepg and hepg -ntcp cells. furthermore, we show that the binding between hbc and baf was of vital importance to hbc mediated downregulation of interferon-induced transmembrane protein (ifitm ) expression, and the mechanisms for the downregulation were disclosed as follows. basal level of ifitm expression depends on baf , rather than the jak–stat pathway. the interaction of hbc with baf disturbs the stability of the polybromo-associated baf (pbaf) complex and results in the suppression of iftm transcription. finally, the antiviral effects of ifitm on cell proliferation and hbv replication were found to be partially restored when hbc was co-transfected with baf . collectively, our findings indicate that hbc plays a role in hbv resistance against the antiviral activities of ifnα, providing details about hbv evasion of host innate immunity. the human hepatitis b virus (hbv) is a double stranded dna virus in the hepadnaviridae family [ ] . hbv infection could cause acute and chronic hepatitis b (chb), which can progress to cirrhosis and hepatocellular carcinoma, leading to high mortality rates worldwide. antiviral therapy with interferon aims to induce permanent immune control of hbv infection through stimulation of the hosts' innate immune response. nevertheless, experimental data from hbv infected chimpanzees and urokinase-type plasminogen activator/severe combined immunodeficiency (upa-scid) mice have shown that hbv infection does not induce an intrahepatic innate immune response that can be detected [ , ] . this is because early in infection it acts like a stealth virus, remaining undetected and spreading until the onset of the adaptive immune response several weeks later [ ] . besides acting as a stealth pathogen, recent developments have shown that hbv can avoid recognition by the host innate immune system. however, the precise mechanisms are largely unknown. human hepatitis b virus core protein (hbc) is amino acids in length and dimeric in solution [ ] . hbc dimers assemble into t = ( copies) or t = ( copies) capsids of hbv, with t = capsids being the predominant form in vivo [ ] . hbc is composed of an assembly domain (aa - ) and a nucleic acid-binding domain (aa - ) (figure a) , moreover, it not only acts as a structural protein of hbv, but also works as an essential regulator in viral replication [ , ] . the nucleic acid-binding harbors a nuclear localization sequence (nls), which mediates the transport of hbc into the nucleus [ , ] . in vitro studies have shown that hbc binds directly to the covalently closed circular dna (cccdna) upon entering the nucleus, such as the camp response element of hbv, enh i [ ] , and the nuclear factor kappa b binding site of hbv, enh ii [ ] , to regulate hbv transcription. in addition, hbc appears to regulate the activities of host cells by interacting directly with the host genome [ ] . however, the details of hbc function in host transcriptional regulation are not well understood. human swi/snf (mating-type switching (swi) and sucrose non-fermenting (snf)) complexes regulate the expression of numerous interferon (ifn)-inducible genes by mediating atp-dependent chromatin remodeling, exposing the binding sites to the transcriptional machinery. swi/snf complexes are critical for proliferation, differentiation, tumorigenesis, and dna repair [ ] . there are two forms of swi/snf complexes: brg /hbrm-associated factors (baf) and polybromo-associated baf (pbaf). only pbaf can facilitate the ligand-dependent transcriptional activation by interacting with the nuclear receptors [ ] . baf and pbaf complexes share most of their subunits and are distinguished by the presence of two specific subunits-baf and baf -both of which only exist in pbaf [ ] . furthermore, baf , but not baf , is essential for the stability of pbaf, and the depletion of baf leads to the complete inactivation of pbaf [ ] . baf is encoded by arid . besides the n-terminal at-rich interactive domain (arid), baf contains multiple lxxll motifs, which have been shown to participate in the regulation of protein-protein interaction [ ] . additionally, baf has been reported to be a potential tumor suppressor and involved in the ifn signal pathway [ ] . ifns are essential components of the innate immune response and act as the first line of defense against invading microorganisms or pathogens. interestingly, hbv escapes the host innate immune response merely by preventing the induction of ifns [ ] . ifns modulate host defenses against microbial infection through the induction of ifn-stimulated genes (isgs) by the janus kinase (jak)-signal transducer and activator of transcription (stat) signaling pathway. among these isgs, interferon-induced transmembrane protein (ifitm) , , and , which are a cluster of genes encoding membrane proteins, exhibit antiviral capabilities mainly through the inhibition of virus entry [ ] . ifitm restricts the infection of various viruses, including type human immunodeficiency virus [ ] , hepatitis c virus [ ] , severe acute respiratory syndrome (sars) coronavirus [ ] , and influenza a virus [ , ] . however, whether ifitms inhibit hbv infection has not been reported. in this study, we start with the discovery that hbc can interact with baf . then, we focus on functions of the interaction and disclose that overexpressed hbc downregulates the baf -dependent expression of ifitm via disruption of pbaf complex stability. finally, our data demonstrates that the antiviral effects of ifitm on cellular proliferation and hbv replication are partially restored when hbc is co-expressed with baf in hbv-infected cells. these findings enrich details about how hbv counteracts human natural immunity, revealing a potential target for novel therapeutic strategies of hbv infection. baf c ( - nt of arid ) was amplified from the fragment screened by the yeast two-hybrid system using primers-sense, '-gatccatggcaaactcgacggggaa- ' and antisense, '-aattctcactgcagcatttctga- '-and inserted into a pcmv-flag vector (stratagene, san diego, ca, usa). baf (full-length arid ) and hbc cdnas (taxonomy id: ) were synthesized by genscript co. ltd. (nanjing, china). pgc-fu-flag and the phbv . vector were kindly gifted by prof. r xiang (xiangya school of medicine of central south university, china). the pgc-fu-flag vector was ligated and constructed with baf at restriction enzyme site agei. the full-length of hbc was cloned into the pgbkt (clontech, clontech laboratories, inc., mountain view, ca, usa) vector with primers-sense, '-acttccagacttctagggagac- ' and antisense, '-ctgccctgtgacggaattga- '-and cloned into the pcmv-ha vector (clontech) with primers-sense, '-gatccatggacattgaccactataaa- ' and antisense, '-tcgacctaacattgagattcccgaga- '. the matchmaker gal two-hybrid system (clontech) was used for the screening of a human fetal brain cdna library (clontech) with pgbkt -hbc as bait. co-transformants were selected on synthetic dropout (sd), media lacking leucine, and tryptophan (sd/-leu/-trp) and were validated by growth on sd, media lacking leucine, tryptophan, adenine, and histidine (sd/-leu/-trp/-ade/-his) and containing -bromo- -chloro- -indolyl-α-d-galactoside (x-α-gal). then, positive colonies were sequenced (invitrogen, carlsbad, ca, usa). hepg cells and t cells, purchased from cctcc, were cultured in dulbecco's modified eagle medium (dmem, gibco, thermo fisher scientific, waltham, ma, usa), supplemented with % (v/v) fetal bovine serum ((fbs, gibco), % penicillin and . mg/ml streptomycin in a humidified incubator maintained at • c with % co . hepg . . cells, obtained from prof. r xiang, were cultured in medium (gbico) in the presence of g ( µg/ml, sigma-aldrich, st. louis, mo, usa) in a humidified incubator maintained at • c with % co . hepg -ntcp cells and hepaad cells, provided by prof. y zhu (wuhan university, china), were cultured in dmem (gibco), supplemented with % heat-inactivated fetal calf serum (gibco), u/ml penicillin, and µg/ml streptomycin sulfate at • c in % co . all the transfection reactions were performed on indicated cells ( × ) in log phase, using lipofectamine (invitrogen) according to the manufacturer's protocol. co-transfection was performed using a total of µg of plasmids or vectors in a : (w/w) ratio: the pcmv-ha-hbc or pcmv-ha vectors were co-transfected with pcmv-flag-baf c or pgc-fu-flag-baf into indicated cells. the medium was refreshed with serum-free dmem/ h after transfection. the supernatant was harvested h post incubation for co-immunoprecipitation or western blot analyses. the supernatants of hepaad cells were concentrated -fold by ultracentrifugation as hbv inoculums. hbv stock titer (genome equivalents [geq] per milliliter) was measured by using qpcr. for infection, hepg -ntcp cells of a low passage number were seeded onto precooling collagen i-coated plates and incubated in dmem for h, then the medium was replaced by primary hepatocyte maintenance medium (pmm) with % fbs (gibco) for h. after this, cells were infected with geq per cell of hbv in pmm containing % (w/v) polyethylene glycol (peg ) for h. after the virus-containing medium was removed, cells were washed several times and cultured in fresh pmm. the medium was changed every other day [ , ] . pmm is williams' e medium supplemented with an insulin-transferrin-selenium solution (thermo, waltham, ma, usa), ng/ml of human epidermal growth factor (egf, peprotech, rocky hill, nj, usa), mm l-glutamine (thermo), µg/ml of hydrocortisone, % dimethyl sulfoxide (dmso), ng/ml of dexamethasone, µg/ml of streptomycin, and u/ml of penicillin. after transfection, cells were washed with ice-cold pbs after h, harvested by scraping, then lysed using an ripa lysis buffer ( mm tris-hcl [ph . ], mm nacl, % np , . % sds) supplemented with a protease inhibitor cocktail (thermo scientific). after centrifugation at , rpm at • c for min, supernatants were incubated with primary antibody or igg (santa cruz, santa cruz, ca, usa) and protein g agarose beads (ge healthcare, chicago, il, usa) at • c overnight. immunoprecipitates were washed with a washing buffer ( mm tris-hcl [ph . ], mm nacl) three times, then whole cell lysate and immunoprecipitated fractions were used for western blot analysis. the following primary antibodies were used: anti-ha (catalog no. h , sigma-aldrich), anti-flag (catalog no. f , sigma-aldrich), anti-baf (catalog no. a - a, bethyl), anti-hbc (catalog no. b , dako). in order to make sure the loading amounts of the protein were comparable, the protein concentration was quantified by bradford assay [ ] . thirty micrograms of protein was separated by sds polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (pvdf, thermo scientific) membranes. after blocking with % gelatin in tbst, the membranes were incubated with the indicated primary antibodies at • c overnight. the membranes were washed three times, incubated with secondary antibodies ( : ) conjugated with horseradish peroxidase (hrp) for h at room temperature, and visualized with the ecl system (biorad, hercules, ca, usa) according to manufacturer's instructions. blots were probed with hrp-conjugated secondary antibodies. the following antibodies were used: anti-ifitm , , , (catalog no. , , , cell signaling technology, danvers, ma, usa), anti-baf (catalog no. a - a, bethyl), anti-stat (catalog no. , cell signaling technology), and anti-phospho-stat (catalog no. , cell signaling technology), and β-actin (catalog no. - -ig, proteintech group). the gray density of the western blots was measured by using imagej software (national institutes of health, bethesda, md, usa). rt-pcr assays were performed to determine the relative mrna levels. total rna was extracted by trizol (invitrogen) according to the manufacturer's instructions. the quantity of the rna samples was detected by nanodrop (thermo scientific). one microgram of rna was reverse transcribed using random hexamer primers (fermentas, waltham, ma, usa) and m-mulv reverse transcriptase. the levels of ifitms mrna and intracellular hbv genomic dna were determined by rt-qpcr analysis using sybr green premix (takara, tokyo, japan) on a lightcycler® ii (roche, basel, switzerland) system. the expression of the target genes was normalized to glyceraldehyde -phosphate dehydrogenase (gapdh) by the ∆∆ct method. β-actin was used as control. primers were as follows: ifitm , forward: '-ccccaaagccagaagatgcacaaggag- ', reverse: '-cgtcgccaaccatcttcctgtccctag- '; ifitm , forward: '-catcatcatcccagtgttgg- ', reverse: '-gataaagggctgatgcagga- '; ifitm , forward: '-caaggaggagcacgagg- ', reverse: '-ttgaacagggaccagacg- '; β-actin, forward: '-ctcttccagccttccttcct- ', reverse: '-agcactgtgttggcgtacag- '; gapdh, forward: '-gatggcaagatcttctgcgtg- ', reverse: '-ccgtcgactcacaggaaatagtcggc- '. the sirnas were designed using oligo . software, and synthesized by genscript co. ltd. (nanjing, china) as follows: siifitm : sirna : '-aaaccuucacucaacacuuccuu- ', sirna : '-aaacuuaagagaaauacacacuu- '; sihbc: sirna : '-aaacuuuacugggcuuuauucuu- ', sirna : '-aagagaaacuguccuugaguauu- '; viruses , , of sicontrol: '-guauauaagcaagcauuacuu- '. all the sirnas were transfected using rnaimax (invitrogen) according to the manufacturer's instructions. one hundred microliters of hepg or hepg . . cells of the same passage number were seeded into -well plates at a density of × cells per well. after culturing for h, cells were transfected with pgc-fu-flag vector, sirna, or pgc -fu-flag-baf and incubated for h, then treated with , , , , , or u/ml ifnα for h. afterwards, cell viability was assessed using the mtt assay. the medium was refreshed and mg/ml -( , -dimethlthiazol- -yl)- , -diphenyltetrazolium bromide (mtt, sigma-aldrich) was added at µl per well. after incubating for h, the medium was removed, and cells were lysed in µl dmso. finally, absorbance was measured by a microplate reader (thermo scientific) at nm. to ensure the results of the mtt assays, a trypan blue exclusion assay was performed. after the transfection and treatment of ifnα, cells were harvested. then trypan blue (sigma-aldrich) was added to the cell suspension to a final concentration of . % (w/v), and the mixture incubated at room temperature for min. ten microliters of the suspension was transferred to a hemocytometer and viable cells were counted. the test was repeated at least three times. hepg cells were transfected with phbv . and pgc-fu-flag-baf , or together with pcmv-hbc-ha, in a : ratio. after treatment with ifnα for h, the supernatant was collected to detect the levels of the hepatitis b surface antigen (hbsag) and hepatitis b e antigen (hbeag) using a commercial elisa kit (neobioscience, hangzhou, china) and the hbv dna copy number was quantified by real-time pcr with a commercial pcr-fluorescence quantification kit (bioer, hangzhou, china). for the extraction of the nucleic acid, hepg cells were collected at h post-transfection and lysed in a precooling lysis buffer ( . % np- , mm tris-hcl [ph . ]). after centrifugation at , × g for min, the nuclei were pelleted, and the supernatant was adjusted with mm mgcl and treated with dnase i for h at • c to remove the free dna. the mixture was incubated at • c for min in the presence of mm edta to inactivate the enzymes, then cultured with proteinase k in the presence of % sds to digest proteins. at last, the nucleic acids were purified by phenol-chloroform extraction and ethanol precipitation. for the extraction of extracellular encapsidated hbv dna, free dna and enzymes of µl cell culture supernatant were removed according to the methods mentioned above. then, the mixture was added to µl of a lysis buffer ( mm edta, mm tris-hcl, . % sds, and mm nacl) containing proteinase k and incubated at • c overnight. after this, hbv dna was isolated by phenol-chloroform extraction and ethanol precipitation. hbv dna was subjected to real-time by pcr using primers ( '-agaaacaacacatagcgcctcat- ' and '-tgccccatgctgtagatcttg- ') and probe ( '-tgtgggtcaccatattcttggg- '). data were presented as mean ± standard deviation (sd). all experiments were repeated three times. the statistical analysis was assessed using the student's t-test. differences were considered statistically significant at p < . (**). to dissect the function of hbc (figure a ), we screened a human fetal brain cdna library for novel hbc-interacting proteins using a yeast two-hybrid system. co-transformants were selected on sd/-leu/-trp and were validated by growth on sd/-leu/-trp/-ade/-his/x-α-gal ( figure b) . then, positive colonies were sequenced. finally, the c-terminal of baf (baf c, aa - ) was identified as one of the strongest binding partners of hbc in ah . baf c contains znf domains and can bind to dna, rna, or proteins [ ] (figure a ). because this interaction might play an important role in hbv-infected hepatocytes, our studies focused on the function of hbc interaction with baf . to verify the two-hybrid results, co-ip assays were performed. first, baf c was co-transfected with either empty vectors or hbc into t cells, then the whole cell lysate was immunoprecipitated by an anti-flag antibody and then subjected to western blot by anti-ha antibodies to detect the interacting proteins. the results indicate that the expressed c-terminal of baf co-precipitated with hbc ( figure c ). then, hbc interaction with full-length baf was further assessed in hepg cells. ip was performed against ha-tagged hbc and the co-precipitation was detected with the anti-flag antibody. the data showed that overexpressed baf co-precipitated with hbc ( figure d ). to investigate the endogenous interaction of baf and hbc, hepg -ntcp cells were infected with or without hbv by inoculation with or without the supernatants of hepaad cells. exogenous expression of sodium taurocholate co-transporting polypeptide (ntcp) in human hepg cells (hepg -ntcp) rendered them susceptible to hbv/hdv infection [ ] . hepg -ntcp cells were infected with hbv by inoculation with or without the supernatants of hepaad cells [ , ] . the lysates of hepg -ntcp cells were immunoprecipitated by hbc antibodies or baf antibodies, then subjected to western blot by baf antibodies or hbc antibodies to detect the interacting proteins ( figure e ). the result indicated that endogenous baf also co-precipitated with endogenous hbc, as seen in overexpressed proteins. collectively, the co-ip results confirm the binding between hbc and baf . and can bind to dna, rna, or proteins [ ] (figure a ). because this interaction might play an important role in hbv-infected hepatocytes, our studies focused on the function of hbc interaction with baf . to verify the two-hybrid results, co-ip assays were performed. first, baf c was cotransfected with either empty vectors or hbc into t cells, then the whole cell lysate was immunoprecipitated by an anti-flag antibody and then subjected to western blot by anti-ha antibodies to detect the interacting proteins. the results indicate that the expressed c-terminal of baf co-precipitated with hbc ( figure c) . then, hbc interaction with full-length baf was further assessed in hepg cells. ip was performed against ha-tagged hbc and the co-precipitation was detected with the anti-flag antibody. the data showed that overexpressed baf coprecipitated with hbc ( figure d) . to investigate the endogenous interaction of baf and hbc, hepg -ntcp cells were infected with or without hbv by inoculation with or without the supernatants of hepaad cells. exogenous expression of sodium taurocholate co-transporting polypeptide (ntcp) in human hepg cells (hepg -ntcp) rendered them susceptible to hbv/hdv infection [ ] . hepg -ntcp cells were infected with hbv by inoculation with or without the supernatants of hepaad cells [ , ] . the lysates of hepg -ntcp cells were immunoprecipitated by hbc antibodies or baf antibodies, then subjected to western blot by baf antibodies or hbc antibodies to detect the interacting proteins (figure e ). the result indicated that endogenous baf also co-precipitated with endogenous hbc, as seen in overexpressed proteins. collectively, the co-ip results confirm the binding between hbc and baf . hbc-interacting partners were tested for β-galactosidase activity on an sd/-leu/-trp/-ade/-his/x-α-gal plate. vector: pgadt ; positive control: pgbkt - and pgadt -t co-transformant; negative control: pgbkt -lam and pgadt -t transformant. (c) t cells were co-transfected with pcmv-flag-baf c and pcmv-ha vector or pcmv-ha-hbc, and co-ip assays were performed with anti-flag antibody or control igg. immuno-complexes were detected by western blot assays using the anti-ha antibody or anti-flag antibody (control). (d) hepg cells were co-transfected with the pgc-fu-flag-baf and pcmv-ha vectors or pcmv-ha-hbc, and co-ip assays were carried out with anti-ha antibody or igg. immuno-complexes were detected by western blot assays using the anti-flag antibody or anti-ha antibody (control). (e) hepg -ntcp cells were incubated with supernatants isolated from the supernatants of hepaad cells cultures (containing hbv) at geq for h or not (mock). co-ip assays were performed with hbc antibody or baf antibody, immuno-complexes were detected by western blot assays using baf antibody or hbc antibody. input control assays were performed in whole cell lysates (wcl). because baf was reported to be a critical regulator of ifn signaling [ ] , we first focused on the effect of baf on the expression of ifitms, which are ifnα effectors. the impact of overexpressed baf on ifitm , , and was examined via protein ( figure a ) and mrna (figure b ) levels. the results demonstrated that baf up-regulated ifitm protein expression (figure a) and significantly increased the mrna expression ( figure b ). however, no apparent effect of baf was observed on the expression of ifitm and ifitm (figure b) . furthermore, we observed that the ifitm expression level was suppressed . -fold when endogenous baf was silenced in hepg cells (figure c) . the data suggest that baf specifically mediates basal level expression of ifitm . next, the effect of the hbc interaction with baf on ifitm expression was further investigated. we co-transfected hbc and baf into hepg cells, treated the cells with u/ml ifnα, and detected the expression of ifitm by western blot (figure d ). the results indicate that baf enhanced ifitm expression, while the enhancement was partially reduced when hbc was co-transfected. to further examine the effect of ifnα on hbc function, we treated the transfected cells with ifnα at various concentrations from u/ml to u/ml for h and examined the ifitm mrna levels ( figure e ). as expected, hbc impaired the enhancement of ifitm mrna expression by baf . however, compared to the control, expression was inhibited to a similar degree upon ifnα stimulation with different concentrations. taken together, the data demonstrates that hbc can down-regulate ifitm expression via binding to baf , whereas the binding is irrelevant to the stimulation of ifnα. ifnα at various concentrations from u/ml to u/ml for h and examined the ifitm mrna levels (figure e ). as expected, hbc impaired the enhancement of ifitm mrna expression by baf . however, compared to the control, expression was inhibited to a similar degree upon ifnα stimulation with different concentrations. taken together, the data demonstrates that hbc can down-regulate ifitm expression via binding to baf , whereas the binding is irrelevant to the stimulation of ifnα. furthermore, the mechanism about how hbc regulated ifitm expression was explored. since the jak-stat signaling pathway has been reported to be essential to ifnα-induced antiviral activities [ ] , we examined whether it was involved in baf -mediated ifitm expression. since the phosphorylation of stat is a necessary step for jak-stat signaling, we used western blot assays to detect phosphorylated stat (pstat ) in hepg cells with or without baf (figure a) . the results showed that pstat appeared only upon ifnα stimulation, independent of the presence of baf . it has been shown that baf is essential for the stability of pbaf, and the depletion of baf leads to the complete inactivation of pbaf [ ] . hence, we predicted that the interaction of hbc with baf would interrupt the stability of pbaf complexes, leading to potential baf -dependent ifitm expression. to verify this hypothesis, co-ip assays were carried out to analyze the effect of hb on baf -baf immuno-complexes in hepg cells (figure b) . ip was performed against flag-baf and the co-precipitation of baf was probed. it demonstrated that hbc co-transfection along with baf profoundly reduced the co-precipitation of baf -baf immuno-complexes when compared with the baf transfected alone condition. endogenous baf -baf interaction was further assessed in hbv infected hepg -ntcp cells (figure e) . the co-ip assay showed that the concentration of baf -baf complexes was reduced more than -fold compared to the non-infected mock control. these results imply that hbc de-stabilizes the pbaf complex by preventing the baf -baf interaction, probably by competitive binding to baf . we next determined whether overexpression of hbc regulated ifitm transcription in vitro. flag-baf was transfected with or without ha-hbc into hepg cells. cells were later treated with ifnα ( u/ml) and total mrna was extracted to detect transcription levels of ifitm , , and by rt-qpcr. the data indicates that, upon ifnα stimulation, the suppression of ifitm expression by hbc is specific (figure c ) and statistically significant (figure d ) in vitro. collectively, the data demonstrates that hbc interacts with baf , and the hbc-baf interaction prevents the baf -baf interactions that might abolish pbaf complex stability, which in turn regulates the suppression of ifitm transcription. the pcmv-flag vector and pcmv-flag-baf were transfected into hepg cells, with or without ifnα ( u/ml) treatment for h. phosphorylation of stat was detected via western blot using antibodies against phosphorylated stat (pstat ). meanwhile, stat and baf were detected using anti-flag and anti-stat antibodies as control. (b) hepg cells were co-transfected with pgc-fu-flag-baf and pcmv-ha vector or pcmv-ha-hbc, with or without ifnα ( u/ml) treatment for h. co-ip assays were performed with anti-flag antibody, then baf -baf immuno-complexes were detected by western blot assays using anti-baf antibody or anti-flag antibodies (control). input control assays were performed in wcl using anti-ha or anti-baf antibodies. meanwhile, total mrna was extracted to examine the effect of baf and hbc combination on ifitm , , transcriptions by rt-pcr (c). β-actin was detected as control. ifitm transcription level was determined using rt-qpcr and normalized to the gapdh mrna level. (d) the flag-baf transfection with ifnα treatment was designated as . *, p < . ; **, p < . . nd = not detected. (e) hepg -ntcp cells were incubated with supernatants isolated from the supernatants of hepaad cells cultures (containing hbv) at geq for h or not (mock). co-ip assays were performed with baf antibodies, and immuno-complexes were detected by western blot assays using baf antibodies or baf antibodies. input control assays were performed in wcl using hbc antibody or baf antibody. finally, we evaluated the effect of hbc on the antiviral activities of iftim in hbv-infected cells. firstly, the impact of hbc on the anti-proliferation action of ifitm was investigated via sirnamediated knockdown in hepg cells and hepg . . cells. proliferation of indicated cells was assessed by mtt assays and viable cell counting. indeed, in hepg cells and hepg . . cells, the data from direct cell counting were consistent with those obtained from mtt assays (figure a, c, d, e) . in hepg cells, the cell viability increased with the time of ifnα stimulation (figure a) and decreased with the concentration of ifnα (figure c) . unter treatment of u/ml ifnα, baf enhanced the inhibitory effect of ifnα, and overexpression of hbc partially recovered the cell the pcmv-flag vector and pcmv-flag-baf were transfected into hepg cells, with or without ifnα ( u/ml) treatment for h. phosphorylation of stat was detected via western blot using antibodies against phosphorylated stat (pstat ). meanwhile, stat and baf were detected using anti-flag and anti-stat antibodies as control. (b) hepg cells were co-transfected with pgc-fu-flag-baf and pcmv-ha vector or pcmv-ha-hbc, with or without ifnα ( u/ml) treatment for h. co-ip assays were performed with anti-flag antibody, then baf -baf immuno-complexes were detected by western blot assays using anti-baf antibody or anti-flag antibodies (control). input control assays were performed in wcl using anti-ha or anti-baf antibodies. meanwhile, total mrna was extracted to examine the effect of baf and hbc combination on ifitm , , transcriptions by rt-pcr (c). β-actin was detected as control. ifitm transcription level was determined using rt-qpcr and normalized to the gapdh mrna level. (d) the flag-baf transfection with ifnα treatment was designated as . *, p < . ; **, p < . . nd = not detected. (e) hepg -ntcp cells were incubated with supernatants isolated from the supernatants of hepaad cells cultures (containing hbv) at geq for h or not (mock). co-ip assays were performed with baf antibodies, and immuno-complexes were detected by western blot assays using baf antibodies or baf antibodies. input control assays were performed in wcl using hbc antibody or baf antibody. finally, we evaluated the effect of hbc on the antiviral activities of iftim in hbv-infected cells. firstly, the impact of hbc on the anti-proliferation action of ifitm was investigated via sirna-mediated knockdown in hepg cells and hepg . . cells. proliferation of indicated cells was assessed by mtt assays and viable cell counting. indeed, in hepg cells and hepg . . cells, the data from direct cell counting were consistent with those obtained from mtt assays (figure a,c,d,e) . in hepg cells, the cell viability increased with the time of ifnα stimulation (figure a) and decreased with the concentration of ifnα (figure c) . unter treatment of u/ml ifnα, baf enhanced the inhibitory effect of ifnα, and overexpression of hbc partially recovered the cell viability (figure a) , however, the sirna mediated knock down of iftim increased the cell viability robustly, about two-four-fold compared to the control (figure c) . the results suggest that ifitm makes major contributions to ifnα inhibitory activities for hbv replication. next, we focused on the role of hb. in hepg . . cells, which contain a stably integrated hbv genome and produce endogenous hbc, overexpression of baf promoted the inhibition of ifnα (figure d ), however when hbc was silenced by sihbc, cell viability was reduced substantially (figure e) . consequently, the results demonstrate that hbc antagonizes the suppression of iftim and the proliferation of hbv-infected cells in vitro. next, the hbc effects on the inhibition of ifitm on hbv replication were measured. in figure e , phbv . , which contains . -fold hbv genome and can produce endogenous hbc, was co-transfected with empty vector flag-baf or ha-hbc into hepg cells. after treating the cells with u/ml ifnα for h, supernatants were collected to determine the hbv replication level by detecting the quantitation of hbsag, hbeag, and hbv dna copy numbers (figure f ). in the control, empty vector and phbv . were co-transfected, and it was observed that ifnα stimulation inhibited the hbv replication level significantly. in contrast to the control, when baf was overexpressed, the concentration of hbsag and hbeag in the supernatant was reduced about one-fold, and hbv dna copies were also suppressed up to five-fold. as expected, the ifnα inhibition was restored when hbc was co-transfected with baf . notably, hbsag and hbeag levels increased to similar levels as the control, with a slight elevation in the hbv dna copy numbers. collectively, the results illustrate that hbv evades ifnα mediated antiviral activity in vitro by downregulating the expression of ifnα effector. however, overexpressed hbc had little observed impact on the recovery of hbv dna levels, implying that ifitm is not the only primary ifnα-inducible isg that suppresses hbv dna replication. hbv replication is not directly cytotoxic to cells, while the host immune responses in infected hepatocytes are the main determinants of hepatocellular injury and hbv pathogenesis. innate , then treated with ifnα in indicated concentration for h. afterwards, the cell viability was measured using mtt assay and direct cell counting via trypan blue exclusion assay (a,c-e). (f) hepg cells were transfected with phbv . and pgc-fu-flag-baf , or co-transfected with pcmv-ha-hbc, then treated with u/ml ifnα for h. hbv replication levels were determined in the supernatants by measuring hbsag and hbeag concentration using elisa and hbv dna copy numbers using rt-qpcr. the empty vector and phbv . were co-transfected in the non-ifnα induced condition as control. the control without ifnα treatment was designed as % (a,c-f). *, p < . , **, p < . , ns = non-significant. hbv replication is not directly cytotoxic to cells, while the host immune responses in infected hepatocytes are the main determinants of hepatocellular injury and hbv pathogenesis. innate immunity is important in controlling viral spread immediately after infection and initiates efficient development of an adaptive immune response. the early phase of a viral infection is mainly characterized by the production of cytokines, type i ifn, and natural killer (nk) cells [ ] . type i ifns can be triggered directly by virus replication through the detection of viral rna or dna, and play a critical role in the immune recognition of hbv. they can be triggered directly by viral replication via detecting the presence of viral rna or dna and induce a large number of effectors working in combination to achieve a fully functional antiviral state [ , ] . ifn effectors target different steps of the viral life cycle, limiting the propagation and spread of the virus and restricting viral infection. therefore, interferon therapy remains one of the therapeutic strategies for hbv infection. in fact, the majority of chb patients treated with ifnα cannot acquire a long-lasting sustained response, especially those with a high viral load. data from animal models have shown that ifn effectors were rapidly induced upon infection with hbv [ ] . however, the hbv-induced ifn responses are weak [ ] , and that is why acute hbv infections usually show a lack of clinical symptoms. studies have shown that the clinical outcomes in chb patients are primarily determined by the interaction between hbv replication and host immune responses [ ] . as an important effector of type i ifns, mxa exhibits strong anti-viral activity against hbv, however, hbv down-regulates mxa expression significantly via the interactions between hbc and the interferon-inducible mxa promoter [ , ] . in addition, reports have indicated that under the stimulation of ifnα, the ifn-signaling pathway is generally blocked, however the formation of stat and isgf were evenly enhanced in hepg . . cells [ ] . here, our study shows that hbc inhibits the ifnα-induced ifitm transcription through disturbing the stability of pbaf complex in vitro, independent of the jak-stat pathway (figure a ). moreover, hbc exhibits anti-apoptosis activity by inhibiting trail-induced expression of the pathway-related death receptor in hepatocytes [ ] . hbc acts as a tumor suppressor as well, by blocking the transcription of the human tumorigenesis-associated genes, ifnβ and p [ , ] . notably, our data indicates that the inhibitory effect of ifitm on hbv dna replication seems weak. a potential explanation is hbv integration into the host genome. the hbv genome can present as non-integrated cccdna, which serves as a template for replication and can persist even after hbsag loss [ , ] . hbv manages to escape immunological recognition in early infection, remaining undetected and spreading for nearly five weeks [ ] . the use of cccdna as a transcriptional template in the nucleus likely contributes to hbv's capacity to limit detection in hepatocytes [ ] . in addition, hbv evades the host response through a complex combination of processes that include signaling interference, effector modulation, and continual viral genetic variation [ ] . reports have shown that hbv polymerase and hbx protein directly inhibit the cellular machinery that detects replication intermediates [ , ] . here, our study provides novel evidence that hbv counteracts ifnα antiviral activities through effector modulation, i.e., the downregulation of ifitm expression by hbc. the mechanism of the downregulation is illustrated as follows. under normal conditions, ifnα induction activates pbaf complexes, which consist of baf , baf , and other subunits. the activation of the pbaf complex triggers histone sliding on chromatin dna, leading to the exposure of interferon-stimulated response elements (isre). in this way, pbaf complexes facilitate the initiation of ifitm transcription (figure a) . actually, baf is essential for the stability of the pbaf complex. under hbv-infected condition, the interaction of hbc with baf competes against the binding between baf and baf , resulting in the loss in pbaf complex stability. thus, the efficient process of the pbaf complex is interrupted, leading to the suppression of ifitm transcription (figure b ). it provides details for the mechanism of hbv escape from ifnα-induced immune elimination. moreover, our future work will continue to explore the intrahepatic effect of hbc on hbv replication using hbv-transgenic mice. were evenly enhanced in hepg . . cells [ ] . here, our study shows that hbc inhibits the ifnαinduced ifitm transcription through disturbing the stability of pbaf complex in vitro, independent of the jak-stat pathway (figure a) . moreover, hbc exhibits anti-apoptosis activity by inhibiting trail-induced expression of the pathway-related death receptor in hepatocytes [ ] . hbc acts as a tumor suppressor as well, by blocking the transcription of the human tumorigenesis-associated genes, ifnβ and p [ , ] . taken together, our data demonstrate that hbc downregulates ifitm expression through interactions with baf , rather than the jak-stat pathway. the results also show that hbc partially restores ifitm antiviral effects on cell proliferation and hbv replication in infected cells in vitro by disturbing the stability of pbaf. it enriches the mechanism that hbv counteracts the innate immunity of ifnα, revealing a potential target for novel therapeutic strategies of hbv infection. nuclear import of hepatitis b virus capsids and genome genomic analysis of the host response to hepatitis b virus infection efficacy of nvr - , alone and in combination with pegylated interferon, vs entecavir in upa/scid mice with humanized livers and hbv infection innate immune responses in hepatitis b virus (hbv) infection full-length hepatitis b virus core protein packages viral and heterologous rna with similarly high levels of cooperativity morphological irregularities in dane particle cores hepatitis b virus core protein phosphorylation sites affect capsid stability and transient exposure of the c-terminal domain nuclear export and import of human hepatitis b virus capsid protein and particles the hepatitis b virus (hbv) core protein enhances the transcription activation of cre via the cre/creb/cbp pathway hepatitis b viral core protein activates the hepatitis 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virus ifitm proteins mediate the innate immune response to influenza a h n virus, west nile virus and dengue virus hepatitis b and d viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding ifn-alpha inhibits hbv transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccdna minichromosome viruses and interferon: a fight for supremacy interferon-stimulated genes: a complex web of host defenses temporal analysis of early immune responses in patients with acute hepatitis b virus infection innate and adaptive immune responses in chronic hepatitis b virus infections: towards restoration of immune control of viral infection mxa inhibits hepatitis b virus replication by interaction with hepatitis b core antigen human mxa protein participates to the interferon-related inhibition of hepatitis b virus replication in female transgenic mice interferon-alpha response in chronic hepatitis b-transfected hepg . . cells is partially restored by lamivudine treatment hepatitis b virus core protein inhibits trail-induced apoptosis of hepatocytes by blocking dr expression transcriptional repression of the human p gene by hepatitis b viral core protein (hbc) in human liver cells persistence of cccdna during the natural history of chronic hepatitis b and decline during adefovir dipivoxil therapy hepatitis b virus infection and the immune response: the big questions viral clearance without destruction of infected cells during acute hbv infection this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we are grateful to r xiang for kindly providing hepg . . and phbv . plasmids, a francis and r dillard for their invaluable comments on the manuscript. the authors declare no conflict of interest. key: cord- -fuz r vj authors: al ali, sally; baldanta, sara; fernández-escobar, mercedes; guerra, susana title: use of reporter genes in the generation of vaccinia virus-derived vectors date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: fuz r vj vaccinia virus (vacv) is one of the most extensively-studied viruses of the poxviridae family. it is easy to genetically modify, so it has become a key tool for many applications. in this context, reporter genes facilitate the study of the role of foreign genes introduced into the genome of vacv. in this review, we describe the type of reporter genes that have been used to generate reporter-expressing vacv and the applications of the recombinant viruses obtained. reporter-expressing vacv are currently employed in basic and immunology research, in the development of vaccines and cancer treatment. since the first description of recombinant dna techniques, many advances have been achieved in the field of molecular biology and genetic modification. currently, there is a wide variety of tools that allow the genetic modification of animals, plants, bacteria and viruses [ ] [ ] [ ] [ ] . the genetic modification of viruses has become one of the best strategies for introducing nucleic acids into different cells, tissues or even in in vivo models, given the high transfection efficiency and ease of carrying it out, compared to chemical or physiological methods [ , ] . after the description of recombination events in cells infected with vaccinia virus (vacv) and through recombinant dna technology [ , ] , vacv has become a suitable model for the generation of recombinant virus vectors [ ] . at first, the main purpose for introducing foreign genes into virus genomes was basic research about the biology of the viruses both in vitro and in vivo. however, with the latest technical advances and the higher understanding of the vacv viral cycle, virus genetic modification is getting a wider spectrum purpose. thus, they can also be used for the development of vaccines or as oncolytic agents. this review aims to highlight the main aspects of the genetic modification of vacv and the generation and application of reporter-expressing virus in this model. vacv is the prototype member of the poxviridae family, so most research of poxvirus has been focused on its use [ ] . vacv is a large dna double-stranded virus, with a complex envelope. it was the live vaccine used to eradicate smallpox and nowadays is also used as a viral vector for recombinant vaccines and cancer therapy [ , ] . the vacv genome is one of the largest of all dna viruses, with a size of kbp and about encoding genes [ ] . the genome has a high genetic compaction, with a few intergenic and small non-coding regions. the coding regions are continuous, thereby not given to splicing [ , ] . vacv have a complete replicating cycle inside the cytoplasm of the host cell, even though it is a dna virus (figure ) [ ] . this fact determines the genetic characteristics of the virus, being completely independent of the replication and transcription machinery of the host cell. once the virion infects the host cell, the viral core is uncoated, and nearly early viral genes are transcribed [ , ] . early genes produce the required enzymes for catalyzing the viral core breakdown, viral dna replication and the modulation of the host antiviral response [ ] . viral dna begins to replicate inside the infected host cell using viral enzymes at h post-infection. as soon as the viral replication starts, transcription of downstream genes encoding for regulatory proteins that induce the expression of the late genes occurs. late genes encode for proteins and enzymes required for the assembly of new viral particles. after dna and all viral proteins are synthesized, the process known as morphogenesis begins, which results in the formation of the new virions [ , ] . these can be retained inside the cell until cellular lysis or released to the environment by other mechanism [ , ] . several features of the biology of vacv make it suitable for its use as a vector in biological experiments, vaccine design or cancer therapy. the complete cytoplasmic replication of vacv facilitates the expression of foreign genes inserted in the viral genome and its detection or isolation [ , ] . usually, bacterial or non-mammalian viral vectors fail to make the expressed proteins to perform its full activity as antigens. however, vacv has the ability to transcribe its genes using its own transcription factors and enzymes. that means that if a foreign gene is inserted directly to a vacv promoter element, it will be transcribed with foreign proteins reaching high levels of expression in the infected cell. moreover, this replication cycle is an appropriate model for molecular and genetic investigations of cis and trans factors that are mainly required for gene expression [ , ] . furthermore, since vacv remains in the cytoplasm, the risk of insertional mutagenesis and oncogenesis, the main problems encountered in gene therapy using integrative viruses, disappears. in some cases, patients treated with retroviral vectors have developed cancer years after they have been treated [ , ] . vacv can replicate in different cell lines, primary cell cultures, and also grows in several animal species, such as mice, guinea pigs, rabbits, etc. [ ] . this broad host range allows infection of cell lines with recombinant viruses for large-scale expression of heterologous proteins, which reduces its cost several features of the biology of vacv make it suitable for its use as a vector in biological experiments, vaccine design or cancer therapy. the complete cytoplasmic replication of vacv facilitates the expression of foreign genes inserted in the viral genome and its detection or isolation [ , ] . usually, bacterial or non-mammalian viral vectors fail to make the expressed proteins to perform its full activity as antigens. however, vacv has the ability to transcribe its genes using its own transcription factors and enzymes. that means that if a foreign gene is inserted directly to a vacv promoter element, it will be transcribed with foreign proteins reaching high levels of expression in the infected cell. moreover, this replication cycle is an appropriate model for molecular and genetic investigations of cis and trans factors that are mainly required for gene expression [ , ] . furthermore, since vacv remains in the cytoplasm, the risk of insertional mutagenesis and oncogenesis, the main problems encountered in gene therapy using integrative viruses, disappears. in some cases, patients treated with retroviral vectors have developed cancer years after they have been treated [ , ] . vacv can replicate in different cell lines, primary cell cultures, and also grows in several animal species, such as mice, guinea pigs, rabbits, etc. [ ] . this broad host range allows infection of cell lines with recombinant viruses for large-scale expression of heterologous proteins, which reduces its cost in comparison to other production systems [ , ] . additionally, vacv enables high production titers, so it is an advantage in the manufacturing of a large amount of vaccines [ ] . although the vacv genome is large and compact, it can tolerate the deletion of certain viral sequences and the insertion of exogenous genetic material [ ] . a vacv vector has a transgene capacity of approximately - kb, higher than other viral vectors, including adeno-associated virus ( . kb), adenovirus ( - kb) and retrovirus ( - kb) [ ] . thus, vacv is an excellent candidate vector in the design of polyvalent vaccines with antigens from several pathogens or different antigens from the same pathogen [ , ] . finally, as far as its use as a vaccine vector is concerned, vacv is clearly immunogenic effective, strong evidence being the eradication of smallpox in [ ] . vacv is also safe and easy to inoculate, since it can be administrated intradermally or with an air gun without medical training. in some organisms, it has been found that it can cause problems by preexisting immunity, but the probability of having post-vaccination complications, such as progressive vacv infection or encephalitis, is significantly low [ ] . nowadays, due to the better knowledge of the vacv biology and the immune response generated after vaccination, vaccines based on this virus are becoming safer [ ] . in addition, it is important to remark that vacv vectors are very stable and can be lyophilized and kept frozen for several years, facilitating its transport and storage [ ] . to get recombinant vacv expressing foreign genes, the main method used is homologous recombination ( figure ) [ ] . first, it is necessary to construct a plasmid that contains the gene or transgene of interest. after that, the cells have to be infected with the virus and subsequently transfected with the plasmid that contains the transgene. an alternative method could be used, employing two viruses, one defective for some genes and one wild-type acting as a helper [ , ] . for both methods, the recombinant viruses are produced by homologous recombination inside the infected cell. in comparison to other production systems [ , ] . additionally, vacv enables high production titers, so it is an advantage in the manufacturing of a large amount of vaccines [ ] . although the vacv genome is large and compact, it can tolerate the deletion of certain viral sequences and the insertion of exogenous genetic material [ ] . a vacv vector has a transgene capacity of approximately - kb, higher than other viral vectors, including adeno-associated virus ( . kb), adenovirus ( - kb) and retrovirus ( - kb) [ ] . thus, vacv is an excellent candidate vector in the design of polyvalent vaccines with antigens from several pathogens or different antigens from the same pathogen [ , ] . finally, as far as its use as a vaccine vector is concerned, vacv is clearly immunogenic effective, strong evidence being the eradication of smallpox in [ ] . vacv is also safe and easy to inoculate, since it can be administrated intradermally or with an air gun without medical training. in some organisms, it has been found that it can cause problems by preexisting immunity, but the probability of having post-vaccination complications, such as progressive vacv infection or encephalitis, is significantly low [ ] . nowadays, due to the better knowledge of the vacv biology and the immune response generated after vaccination, vaccines based on this virus are becoming safer [ ] . in addition, it is important to remark that vacv vectors are very stable and can be lyophilized and kept frozen for several years, facilitating its transport and storage [ ] . to get recombinant vacv expressing foreign genes, the main method used is homologous recombination ( figure ) [ ] . first, it is necessary to construct a plasmid that contains the gene or transgene of interest. after that, the cells have to be infected with the virus and subsequently transfected with the plasmid that contains the transgene. an alternative method could be used, employing two viruses, one defective for some genes and one wild-type acting as a helper [ , ] . for both methods, the recombinant viruses are produced by homologous recombination inside the infected cell. another way to generate recombinant viruses is the method described by falkner and moss [ ] , denominated transient dominant selection (tds), which allows the introduction of site-directed mutations into the vacv genome. generally, the recombinant viruses obtained by this method are rescued by metabolic selection, using the guanine phosphoribosyltransferase gene (gpt) from escherichia coli as a marker. the presence of the protein encoded by gpt allows the recombinant viruses to grow in the presence of mycophenolic acid, xanthine and hypoxanthine [ ] . subsequently, after this first metabolic selection, a second recombination event must occur to eliminate the selection marker, maintaining the mutation introduced into the vacv genome ( figure ) [ ] . in contrast to the method described above, in the tds technique the marker should not be flanked by homologous regions of the vacv genome [ ] . alternatively, puromycin resistance could be used as a selection marker in tds, increasing the recombinant viruses' generation efficiency [ ] . another way to generate recombinant viruses is the method described by falkner and moss [ ] , denominated transient dominant selection (tds), which allows the introduction of site-directed mutations into the vacv genome. generally, the recombinant viruses obtained by this method are rescued by metabolic selection, using the guanine phosphoribosyltransferase gene (gpt) from escherichia coli as a marker. the presence of the protein encoded by gpt allows the recombinant viruses to grow in the presence of mycophenolic acid, xanthine and hypoxanthine [ ] . subsequently, after this first metabolic selection, a second recombination event must occur to eliminate the selection marker, maintaining the mutation introduced into the vacv genome ( figure ) [ ] . in contrast to the method described above, in the tds technique the marker should not be flanked by homologous regions of the vacv genome [ ] . alternatively, puromycin resistance could be used as a selection marker in tds, increasing the recombinant viruses' generation efficiency [ ] . two important aspects to be considered when obtaining recombinant poxvirus are the vacv genome insertion sites and the reporter genes introduced. the vacv genome has about seven known insertion sites where foreign genes can be inserted ( figure ) [ ] . the insertion site choice depends mainly on the future application of the recombinant viruses. it may also be important in the later selection of the recombinant viruses obtained. for instance, inserting the gene of interest in the thymidine kinase (tk) locus confers a detectable phenotype (tk-): the recombinant viruses are able to grow in the presence of -bromo- '-deoxyuridine (brdu), a synthetic analog of thymidine [ , ] . another important site of insertion that allows a subsequent selection is the vacv hemagglutinin (ha) gene as the recombinant viruses can be easily recognized by their disability to bind erythrocytes in a hemagglutination test [ ] [ ] [ ] . two important aspects to be considered when obtaining recombinant poxvirus are the vacv genome insertion sites and the reporter genes introduced. the vacv genome has about seven known insertion sites where foreign genes can be inserted ( figure ) [ ] . the insertion site choice depends mainly on the future application of the recombinant viruses. it may also be important in the later selection of the recombinant viruses obtained. for instance, inserting the gene of interest in the thymidine kinase (tk) locus confers a detectable phenotype (tk-): the recombinant viruses are able to grow in the presence of -bromo- '-deoxyuridine (brdu), a synthetic analog of thymidine [ , ] . another important site of insertion that allows a subsequent selection is the vacv hemagglutinin (ha) gene as the recombinant viruses can be easily recognized by their disability to bind erythrocytes in a hemagglutination test [ ] [ ] [ ] . vacv has five more places of insertion: the bamhi site of the hindiii-f dna fragment [ ] ; the vacv growth factor gene (vgf), located in both inverted terminal repeats (itrs) [ ] ; the n and m genes located on the left side of the vacv genome [ ] ; the m subunit of the ribonucleotide reductase (rr) gene in the hindiii-i dna fragment [ ] ; and the a l gene encoding the kda fusion protein, in the large hindiii-a dna fragment [ ] . it is noteworthy that some strains of vacv have only one copy of vfg, such as vacv lister variants [ ] . recombinant production using these insertion sites, although successfully occurring, requires the use of a marker gene or other strategies for later selection of the recombinant viruses. due to these limitations, the tk gene is the most common site of insertion in the vacv genome [ ] . some authors have used temperature-sensitive vacv strains, allowing the recombinant viruses to be selected in culture at ˝c [ ] . however, the most common way for an easy identification of recombinant viruses is the use of reporter genes as selectable markers, which will be discussed in section . [ ] . vacv has five more places of insertion: the bamhi site of the hindiii-f dna fragment [ ] ; the vacv growth factor gene (vgf), located in both inverted terminal repeats (itrs) [ ] ; the n and m genes located on the left side of the vacv genome [ ] ; the m subunit of the ribonucleotide reductase (rr) gene in the hindiii-i dna fragment [ ] ; and the a l gene encoding the kda fusion protein, in the large hindiii-a dna fragment [ ] . it is noteworthy that some strains of vacv have only one copy of vfg, such as vacv lister variants [ ] . recombinant production using these insertion sites, although successfully occurring, requires the use of a marker gene or other strategies for later selection of the recombinant viruses. due to these limitations, the tk gene is the most common site of insertion in the vacv genome [ ] . some authors have used temperature-sensitive vacv strains, allowing the recombinant viruses to be selected in culture at °c [ ] . however, the most common way for an easy identification of recombinant viruses is the use of reporter genes as selectable markers, which will be discussed in section . [ ] . in spite of the promoter or the regions between the promoter and coding region, the insertion site also influences foreign gene expression and virus virulence [ , , ] . insertion into the tk, vgf, rr or a l genes has an impact on viral replication in vivo, but not in vitro [ , ] . moreover, the method described in figure requires the use of special cell lines or mutagenic selective agents, such as tk-/-cell lines and brdu [ ] . for this reason, different strategies and new insertion sites are being studied to ensure the correct expression of the transgenes in vitro and in vivo [ , , , ] . reporter-expressing viruses are recombinant viruses expressing a reporter gene [ ] . in some cases, the reporter gene is located downstream of a viral promoter, to study biological pathways or, fused with other viral or foreign genes. as reporter genes are expected to be easily detected, they are the best indicators for screening successfully recombinant viruses. the reporter gene should be chosen considering the non-endogenous activity in the cell type, tissue or organism used to culture the viruses [ ] . reporter genes have additional applications in vitro and in vivo, as the reporter gene acts as a substitute of the gene of interest. moreover, reporter genes facilitate the use of tissue-specific and pathway-specific promoters, as well as regulatory promoter elements as biomarkers for a particular event route. furthermore, it is important that the existence of the reporter gene should not affect the normal physiology and general characteristics of the transfected cells [ ] [ ] [ ] . table presents an overview of the reporter genes commonly used in the generation of recombinant vacv. in spite of the promoter or the regions between the promoter and coding region, the insertion site also influences foreign gene expression and virus virulence [ , , ] . insertion into the tk, vgf, rr or a l genes has an impact on viral replication in vivo, but not in vitro [ , ] . moreover, the method described in figure requires the use of special cell lines or mutagenic selective agents, such as tk-/cell lines and brdu [ ] . for this reason, different strategies and new insertion sites are being studied to ensure the correct expression of the transgenes in vitro and in vivo [ , , , ] . reporter-expressing viruses are recombinant viruses expressing a reporter gene [ ] . in some cases, the reporter gene is located downstream of a viral promoter, to study biological pathways or, fused with other viral or foreign genes. as reporter genes are expected to be easily detected, they are the best indicators for screening successfully recombinant viruses. the reporter gene should be chosen considering the non-endogenous activity in the cell type, tissue or organism used to culture the viruses [ ] . reporter genes have additional applications in vitro and in vivo, as the reporter gene acts as a substitute of the gene of interest. moreover, reporter genes facilitate the use of tissue-specific and pathway-specific promoters, as well as regulatory promoter elements as biomarkers for a particular event route. furthermore, it is important that the existence of the reporter gene should not affect the normal physiology and general characteristics of the transfected cells [ ] [ ] [ ] . table presents an overview of the reporter genes commonly used in the generation of recombinant vacv. elisa: enzyme-linked immunosorbent assay. cat was the first reporter gene used in transcriptional assays in mammalian cells. cat is an enzyme from escherichia coli that detoxifies the antibiotic chloramphenicol, which inhibits protein synthesis in bacteria [ ] . particularly, cat links acetyl-coenzyme a (acetyl-coa) groups to chloramphenicol, preventing it from blocking the s ribosomal subunit. this gene is not found in eukaryotes, so eukaryotic cells do not present any basal cat activity [ ] . the reaction catalyzed by cat can be quantified using fluorogenic or radiolabeled substrates, such as h-labeled acetyl-coa and c-labeled chloramphenicol. cat can be detected either by thin-layer chromatography, autoradiography or enzyme-linked immunosorbent assay (elisa) [ ] . there is a strong link between cat gene transcript levels and enzymatic activity, which is easy to quantify. thus, cat has become a suitable reporter gene for investigating transcriptional elements in a wide variety of experiments implicating animal and plant cells, as well as viruses [ ] . there are some disadvantages of using the cat system, such as the higher amount of cells required when compared to other assays, like the luciferase assay (detailed in section . . ). in addition, the cat system is not suitable for use with weakly-expressed genes and cat promoter activity quantification takes longer than other reporter systems [ ] . finally, this reporter gene has another important limitation due to the use of radioisotopes [ ] . the first study using lacz as a reporter gene was published in , and since then, it has become one of the most commonly-used reporter genes in molecular biology [ ] . although β-galactosidase catalyzes the cleavage of the disaccharide lactose to form glucose and galactose, it recognizes several artificial substrates, which has promoted its use as a reporter gene [ ] . thus, β-galactosidase can hydrolyze substrates such as ortho-nitrophenyl beta-galactoside (onpg), -bromo- -chloro- -indolyl beta-d-galactopyranoside (x-gal) and , -cyclohexenoesculetin beta-d-galactopyranoside (s-gal), resulting in a yellow, blue or black product precipitate, respectively [ , ] . furthermore, expression of the lacz gene can be stimulated with isopropyl beta-d-thiogalactopyranoside (iptg), which is a highly stable synthetic and non-hydrolyzable analog of lactose [ ] . one of the applications of the lacz reporter gene is the selection of transformed bacterial colonies. the recombinant (white) and non-recombinant (blue) bacteria are discriminated based on the interruption of the lacz gene by the insert dna or gene of interest using x-gal as a substrate [ ] . other uses are the visualization of the β-galactosidase expression in transfected eukaryotic cells or the selection of the recombinant virus by viral plaque screening [ ] . finally, lacz is used to detect β-galactosidase activity in immunological and histochemical experiments [ ] . one of the main advantages of using this reporter gene system is its low cost, since it does not require specific devices to detect the colorimetric reaction or to identify its expression. another escherichia coli-derived hydrolyzing enzyme gene that lends a reporter assay is gus. the β-glucuronidase protein catalyzes the breakdown of complex carbohydrates, such as glycosaminoglycans. this reporter gene system has been widely used in transgenic plants, and it has also been successfully used in mammalian cells for vacv recombinant virus selection [ ] . for the β-glucuronidase (gus) assay -methylumbelliferyl beta-d-glucuronide (mug) or -bromo- -chloro- -indolyl beta-d-glucuronide (x-gluc) can be used as substrates. they respectively lead to a fluorogenic or a blue product after cleavage [ , ] . monitoring β-glucuronidase activity through a gus assay allows the determination of the spatial and temporal expression of the gene of interest [ ] . the most known fluorescent protein is green fluorescent protein (gfp), which was cloned from the species of jellyfish aequorea victoria. because of the great impact of fluorescent proteins in molecular biology applications, the nobel prize in chemistry was awarded to osamu shimomura, martin chalfie and roger y. tsien for the discovery and development of gfp [ , ] . gfp is the most used reporter gene; however, genetic engineering has developed a wide variety of color mutants, such as red fluorescent protein (rfp) or yellow fluorescent protein (yfp) among others [ ] . fluorescent proteins tolerate n-and c-terminal fusions to a wide-range of proteins, have been expressed in most known cell types and are used as a non-harmful fluorescent marker in living cells and organisms. the use of fluorescent proteins allows a variety of applications: cell lineage tracker , reporter for gene expression assays or measure of protein-protein interactions. additionally, cell-fixation is not needed to examine its expression, and the probability of artifacts is quite small compared to immunocytochemical methods which require cell fixation [ ] . one of the disadvantages of these proteins is their size. therefore, in some cases, they can affect the in vivo function of fused proteins or genes of interest. nevertheless, one limitation of using gfp is its low sensitivity [ ] , another is that its signal cannot be exogenously amplified [ ] . the first luciferase (luc), from the firefly photinus pyralis, was cloned in and luc has been widely used as a reporter gene. later, it was also described in bacteria and dinoflagellates [ ] . luciferases are enzymes that catalyze a chemical reaction resulting in the production of light. firefly luciferase oxidize the d-luciferin, in the presence of oxygen and adenosine triphosphate (atp) as the energy source. as in β-galactosidase assays, an exogenous substrate is needed, and it may be a disadvantage [ ] . in other systems, such as the luciferase identified in bacteria (luxcdabe operon), the enzyme catalyzes the oxidation of long-chain aldehydes and flavin mononucleotides (fmnh ) in the presence of oxygen to yield green-blue light [ ] . although in bacteria this operon encodes all components necessary for light emission, it is limited in mammalian cells. therefore, the exogenous substrate has to be added to improve the reaction [ ] . besides the different substrates required, each luciferase system is categorized by having specific kinetics, with a particular detection and sensitivities that require adjusting the experimental design [ , ] . the use of luciferase is extremely widespread in biological systems studies and includes cell proliferation assays, protein folding/secretion analyses, in vivo imaging and control of in vivo viral spreading [ , [ ] [ ] [ ] . the main advantage of using this system is its high sensitivity when compared to other systems, such as cat. additionally, the luc system is more direct, rapid and suitable when it comes to weakly-expressed genes, and it can be used to quantify gene activity. one disadvantage of the luc system is the requirement of ultrasensitive charge-coupled device (ccd) cameras to detect gene expression [ ] . reporter-gene assays have helped the pox virologists in basic research, for example for the study of the location, structure and function of many vacv proteins during the infection cycle and their interaction with proteins of the host cell [ , ] . as shown in dvoracek and shors [ ] , the gus reporter gene was used for deleting the d r viral protein and selecting the recombinant viruses, with the aim to understand the role of this protein in the viral life cycle. in addition, the lacz gene has typically been used mainly for the selection of recombinants [ ] . moreover, several studies have reported the different transgenes' insertion points and vacv promoters in which the recombinant virus production was enhanced. these studies are essential for improvement of the development of vaccines based on recombinant vacv [ , ] . on the other hand, fluorescent markers such as the gfp, yfp or luciferase are also useful for labelling vacv replicative strains. these viruses have allowed the study of processes like the input and output morphogenesis in virus-infected cells [ , , [ ] [ ] [ ] [ ] . in these studies, fluorescence of certain viral proteins allows us to study their interaction with other viral or cellular proteins [ ] . furthermore, vacv is a clear example of how viruses have developed strategies to evade the immune response [ ] . in this field, the generation of recombinant vacv with reporter genes is also useful to discern the molecular mechanism by which vacv proteins manipulate the immune system of the host. thus, in unterholzner et al. [ ] , the generation of a gfp-labeled recombinant vacv revealed that the c viral protein acted as an immunomodulatory agent, blocking the expression of type i interferon. another major application of reporter-expressing vacvs is the design of high-throughput assays. the generation of lacz or gfp expressing recombinant virus can be used to optimize antibody neutralization assays [ , ] . lastly, vacv and reporter genes have been used to study proteins from other viruses, particularly rna viruses, such as influenza or severe acute respiratory syndrome-associated coronavirus (sars-cov) [ ] . to genetically modify these viruses, rna must be reverse transcribed to cdna, since this is particularly unstable in plasmids, making vacv a good tool for functional studies of proteins from such viruses [ ] . there are several in vivo applications for recombinant reporter-expressing viruses. for example, in virulence studies, the use of labeled viruses allows us to follow the viral pathogenicity and detect in which organs the viral replication and dissemination occur [ , ] . for example, zaitseva et al. [ ] used the recombinant vacv western reverse strain (wr)-luc to analyze the viral spread in vivo for several days reducing the number of mice used. moreover, vacv is an effective enhancer for both humoral and cell-mediated immunity; it is used as a vector to study the immune system and the expression of proteins' antigenicity of other pathogens. furthermore, vacv is used to explore the immunopathological mechanisms, to know which epitopes or antigens presented by a pathogen have the ability to induce the host-immune response, and to demonstrate the specific role of a particular antigen during the pathogenic process [ , , ] . despite the examples mentioned above, the most common uses of recombinant vacv in vivo are the production of prophylactic vaccines and treatments against cancer [ , ] . table shows some of the vaccines based on vacv, with the reporter gene and the insertion site employed indicated in each case. in these vaccines, vacv acts as a vector capable of delivering antigens from other organisms [ ] . while in many recombinant vaccines a viral antigen has been inserted, some of them have also been developed against bacteria [ ] or protists [ , , ] . these vaccines simulate infection by the pathogen from which the antigens are and elicit the immune response, by producing antigens for different pathogens. in several vaccines, mainly against human immunodeficiency virus (hiv) or influenza, genes of immunomodulatory cytokines are added for coexpression with the antigen, improving the immunogenicity of the vaccines [ , , ] . as summarized in table , most of the transgene insertion sites are within the tk or the ha genes, making the selection of recombinants easier, as explained above. however, in several vaccines, besides using this strategy, a reporter gene is used as well. the use of reporter genes facilitates the preliminary tests of the vaccine on animal models. moreover, especially in vaccines used in animals, the reporter gene makes it possible to distinguish between vaccinated and infected animals [ ] . for example, vacv has been used for nearly twenty years to eradicate rabies from wildlife as an oral-based vaccine. in this case, the recombinant vacv expresses the rabies virus glycoprotein and has been used to vaccinate raccoons, red foxes, skunks and coyotes in the united states and europe. this battle has successfully purged rabies in some parts of europe and the united states [ ] . streptococcus pyogenes m protein tk gene not mentioned [ ] another application for vacv vectors is in cancer treatment, known as oncolytic virotherapy [ ] . this is the use of replication-competent viruses to selectively attack and destroy cancer cells, without harming healthy host cells [ ] . examples of recombinant vacv used are summarized in table . a promising study is the use of oncolytic vacv as a vector for the human sodium iodide symporter (hnis) gene in prostate cancer therapy, which has been demonstrated to restrict tumor growth and to increase survival in mice [ ] . vacv is also a promising therapeutic agent for pancreatic cancer [ ] , cholangiocarcinoma [ ] and colorectal cancer [ ] . it is worth mentioning that many of the viral vectors developed to treat tumors have several common characteristics. generally, vacv oncolytic vectors have a deletion in the tk gene, essential for the pyrimidine synthesis pathway, which forces the virus to replicate in cells displaying a high amount of nucleotide pools, enhancing the viral tropism to cancer cells. others have a deletion in the vgf gene, preventing non-infected cells from proliferation [ ] . furthermore, as in the development of vaccines, viral vectors are "armed" with genes that enhance the antitumor activity, the virus tropism or the immunoreactivity, to promote better tumor destruction, such as granulocyte-macrophage colony-stimulating factor (gm-csf) or erythropoietin genes (enhanced virus; table ). another particular feature is that many of these recombinants carry reporter genes, and thus viral replication can be monitored by non-invasive imaging methods [ , , , ] . the main limitation of using vacv as a vector is the short-term gene expression, since it is a lytic virus killing the infected cells. thus, gene expression will not last for more than - h post-infection [ , ] . additionally, although for some applications it is an advantage, since vacv replicates completely in the infected cell cytoplasm, it is hard to use vacv to engineer nuclear gene replacement [ ] . the other main disadvantage is the limited immunogenicity in individuals vaccinated against smallpox. this pre-existing immunity reduces the effectiveness of vaccines based on vacv, although some trials have overcome this problem by mucosal vaccination with vaccinia vectors [ ] . the vacv safety profile should be considered because it has progressive complications especially with immunocompromised individuals [ ] . these limitations primarily affect in vivo applications of vacv recombinants in vaccine development, so several attenuated strains of vacv are being generated [ ] . as for other viruses, the development of vaccines or oncolytic therapies based on vacv requires the understanding of its pathogenesis and biology. despite improvements in the vectors' design, such as the use of different promoters or insertion sites, homologous recombination has been almost exclusively the way to obtain vacv recombinants [ ] . homologous recombination requires the use of markers or reporter genes for selecting recombinants, which offers many disadvantages. apart from the physical space needed for the marker gene, which is limited in therapeutic virus, the use of certain markers can introduce mutations or generate artifacts that are only found after an analysis of the generated virus. sometimes, these problems cannot be detected in vitro, but are very important to overcome when these vectors are used in vivo on animal models [ , ] . in recent years, some strategies have been developed to avoid these risks using markers, or at least to remove them from the final recombinant vacv. rice and colleagues [ ] described a double selection method to improve the selection of recombinant vacv, so that a reporter or marker gene is not necessary. a helper virus is used to rescue a recombinant vacv and is subsequently grown in non-permissive cells to the helper virus; allowing the selection of a large percentage of recombinant virions. however, the method that has certainly had an enormous importance in the modification of genomes is the clustered regularly interspaced short palindromic repeats (crispr)/crispr-associated protein (cas ) system. briefly, the crispr/cas system consists of an endonuclease (cas ) employing a guide rna to generate a break in a target place of the genome, later to be repaired, either randomly or precisely using a specifically designed "restful" template [ ] . the effectiveness of this system has been proven in different organisms, including viruses, such as herpes simplex virus (hsv) [ ] , hepatitis b virus (hbv) [ ] and hiv [ ] . currently, this technique is starting to be used also in vacv [ ] . for example, this system has achieved the deletion of vacv virulence genes, such as a l and n l. a l and n l are vacv intracellular proteins that inhibit nuclear factor-kappa b (nf-kb) activation, so it is undesirable that they were present in vacv vectors with therapeutic purposes [ ] . furthermore, given the efficiency of the method, "reparative" vectors with excisable marker genes have been designed. therefore, recombinant viruses are effectively isolated, but eventually, the marker gene is eliminated [ ] . given the simplicity of recombinant vacv by the crispr/cas system generation, an exponential increase of applications with better markers for basic research or without selectable markers for clinical application is expected [ , ] . in conclusion, the development of recombinant viruses is a promising therapeutic advance in the biomedical field. in this sense, the use of reporter-expressing vacvs has become a fundamental tool for a number of applications, in basic research, vaccine design and cancer therapy. as many of these trials are still experimental, more information is required regarding the side effects of the viral treatment. continuing efforts are necessary to develop new reporter-expressing vacvs that are safer and more effective for future therapies. author contributions: sara baldanta wrote the paper and created the figures, saly al ali and the rest of the authors also wrote the paper. the authors declare no conflict of interest. western reserve strain x-gal -bromo- -chloro- -indolyl beta-d-galactopyranoside x-gluc -bromo- -chloro- -indolyl beta-d-glucuronide yfp yellow fluorescent protein. genetic engineering of mammals genetic engineering of crops: a ray of hope for enhanced food security precision genome engineering in lactic acid bacteria developments in viral vector-based 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application of crispr/cas technology to hbv we thank all of the pox virologist who contributed to this study. this work is supported by grant fis - and reference saf - -r to sg. key: cord- -oyevbxtc authors: su, airong; wang, huanru; li, yanlei; wang, xiaohui; chen, deyan; wu, zhiwei title: opposite roles of rnase and kinase activities of inositol-requiring enzyme (ire ) on hsv- replication date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: oyevbxtc in response to the endoplasmic reticulum (er) stress induced by herpes simplex virus type (hsv- ) infection, host cells activate the unfolded protein response (upr) to reduce the protein-folding burden in the er. the regulation of upr upon hsv- infection is complex, and the downstream effectors can be detrimental to viral replication. therefore, hsv- copes with the upr to create a beneficial environment for its replication. upr has three branches, including protein kinase rna (pkr)-like er kinase (perk), inositol-requiring enzyme (ire ), and activated transcription factor (atf ). ire α is the most conserved branch of upr which has both rnase and kinase activities. previous studies have shown that ire α rnase activity was inactivated during hsv- infection. however, the effect of the two activities of ire α on hsv- replication remains unknown. results in this study showed that ire α expression was up-regulated during hsv- infection. we found that in hec- -a cells, increasing rnase activity, or inhibiting kinase activity of ire α led to viral suppression, indicating that the kinase activity of ire α was beneficial, while the rnase activity was detrimental to viral replication. further evidence showed that the kinase activity of ire α leads to the activation of the jnk (c-jun n-terminal kinases) pathway, which enhances viral replication. taken together, our evidence suggests that ire α is involved in hsv- replication, and its rnase and kinase activities play differential roles during viral infection. herpes simplex virus type (hsv- ), a member of the herpesviridae family, is one of the most prevalent human pathogens. hsv establishes latent infection in neuronal cell bodies, and becomes reactivated when triggered by environmental or physiological factors [ , ] . the replication of herpes virus is temporally regulated by immediate early (ie), early and late genes. although the clinical manifestations by viral infection are generally benign and self-limiting, severe or even life-threatening cases such as herpes encephalitis (hse) are observed occasionally. there are no effective drugs that can completely eradicate the virus [ ] ; therefore, studies on the interaction between the virus and its host are necessary, and may shed light on antiviral strategy. endoplasmic reticulum (er) is a membranous network of branching tubules and flattened sacs, running through the cytoplasm of all eukaryotic cells, and continues with the nuclear envelope. approximately one-third of the total cellular proteins are translocated into the lumen of the er for post-translational modification and correct folding [ ] . misfolded or unfolded proteins are transported out of the er and degraded through er-associated degradation (erad). when a large amount of newly synthesized proteins are transported into er and are beyond the folding capability of er, er stress occurs. er stress is an evolutionarily conserved cellular stress condition, which plays important roles in diverse biological processes, including metabolism, tumorigenesis and viral infection [ ] [ ] [ ] [ ] . er stress response can transmit stress signal from er to nucleus by triggering signal transductions known as the unfolded protein response (upr). upr consists of three independent er membrane transducers, including protein kinase rna (pkr)-like er kinase (perk), activated transcription factor (atf ), and inositol-requiring enzyme (ire ). triggering of these transducers could lead to the modulation of protein translation, protein folding, and degradation to either reestablish the homeostasis or activate apoptosis when the stress persists [ , ] . virus infections could induce upr by accumulating unfolded or misfolded proteins in the er lumen when a burst of viral protein synthesis occurs. on the other hand, upr was shown to influence the replication of a number of viruses including hepatitis c virus (hcv) [ ] , japanese encephalitis virus (jev) [ ] and hsv- [ ] , etc. the ire α/xbp (x-box binding protein ) branch is the most conserved signaling pathway in upr from yeast to humans. ire has two isoforms in mammalian cells: ire α and ire β. ire α is expressed in most tissues and cells, while the expression of ire β is primarily restricted to intestinal epithelial cells [ ] . ire α possesses both kinase and endoribonuclease (rnase) activity at its c-terminal cytosolic moiety [ ] , and its n-terminal luminal domain acts as a sensor of unfolded or misfolded proteins under stress conditions [ ] . bip (binding immunoglobulin protein), also known as glucose-regulated protein (grp ), constitutively binds to ire α to prevent its activation under nonstress conditions. in response to er stress, bip binds to the unfolded proteins and releases from ire α, leading to ire α activation [ ] . xbp mrna is the first discovered substrate for the ire α rnase. the activation of ire α results in its dimerization and autophosphorylation, leading to the cleavage of xbp mrna via an unconventional splicing reaction [ ] . expression of the spliced xbp (xbp s) acts as a transcription factor for the expression of genes which are critical for efficient protein folding, maturation, and degradation [ ] . besides of xbp pre-mrna, rnase activity of ire α also mediates the degradation of a select subset of cellular mrnas, that is termed regulated ire -dependent decay (ridd) [ ] . ridd cleaved pre-mrnas at a site different from the xbp spliced site, and either maintains er homeostasis or induces apoptosis [ ] . if the er stress is not mitigated, the activated ire α can also lead to the activation of jnks (c-jun n-terminal kinases) by recruiting and clustering the tnf receptor-associated factor (traf ), and then triggers apoptosis [ ] . ire α was reported to be involved in many viral infections. hassan et al. reported that influenza a viral replication activated the ire branch of upr [ ] . the ire α pathway is also activated during a murine coronavirus mouse hepatitis virus (mhv) infection [ ] . another study observed that hepatitis c virus suppressed the ire α-xbp pathway during infection [ ] . human cytomegalovirus (hcmv) is a betaherpes virus, which can also inhibit the xbp target gene edem (er degradation enhancing α-mannosidase-like protein) to benefit viral replication [ ] . the investigation of cellular upr during hsv- replication has been mainly focused on the perk signaling pathway [ , , ] . although studies have shown that rnase activity of ire α was inactivated during hsv- infection [ , ] , it remains unknown about the effect of ire α rnase activity on viral replication. stress-induced jnks activation was reported to be important for the efficiency of hsv- viral replication since jnk inhibitors could reduce the viral yield by % [ , ] . since activated ire α can lead to jnks activation [ ] , the relationship between jnks and ire α during viral infection deserves further investigation. in the current study, we found that the ire α expression was up-regulated during hsv- infection, which suggests that the ire α branch may be involved in the regulation of hsv- replication. further study showed that when ire α rnase activity was increased by either pretreating cells with chemical compounds or xbp s over-expression, the hsv- replication was repressed, suggesting that the rnase activity of ire α is unfavorable for the virus. we presented evidence that the jnk is activated by ire α kinase activity, and thus facilitates viral replication. in conclusion, our research indicates that hsv- replication is regulated by both the rnase and the kinase activities of ire α, and these two activities have distinct effects on viral replication. irdye goat-anti-rabbit and irdye goat-anti-mouse were obtained from li-cor (lincoln, ne, usa). antibodies specific for glycoprotein d (gd- ), glyceraldehyde -phosphate dehydrogenase (gapdh), jun n-terminal protein kinase (jnk ), and radio immunoprecipitation assay (ripa) lysis buffer were purchased from santa cruz biotechnology (santa cruz, ca, usa). antibodies specific for ire α and phophorylated jun n-terminal protein kinase and (p-jnk / ) were purchased from cell signaling technology (beverly, ma, usa). anti-ire α (phospho s ) was purchased from abcam (cambridge, ma, usa). draq was obtained from ebioscience (san diego, ca, usa). thapsigargin (tg) and phosphonoacetic acid (paa) were purchased from sigma-aldrich (st. louis, mo, usa). apy and stf- were obtained from medchemexpresss (mce) (princeton, nj, usa). sp was purchased from beyotime (haimen, jiangsu, china). plasmid encoding xbp s was cloned into p ×flag-cmv- . expression vector (st. louis, mo, usa). anti-flagm antibody was purchased from sigma-aldrich. ire alpha ka-pcdna .egfp and ire alpha-pcdna .egfp were gifts from fumihiko urano (addgene plasmid # , addgene plasmid # ). hela, vero, and hec- -a cells were obtained from american type culture collection (atcc, manassas, va, usa). the cells were cultured in dulbecco's modified eagle's medium (dmem) or mccoy's a, supplemented with % fetal bovine serum (life technologies, carlsbad, ca, usa). hsv- (strain hf), a attenuated strain of the virus, originated from atcc ® vr ™ [ ] , was widely used in the researches of either antiviral study or the relationship between host cells and virus [ ] [ ] [ ] . hsv- (hf) was propagated and titrated on vero cells as described previously [ ] . the in vitro antiviral activity of apy , tg, and stf- were determined by in-cell western assay, or by titrating the infectious virion in drugs-treated cells as described [ ] . briefly, × cells were dispersed into each well of -well plates and, after h culture, the cells were either mock-pretreated or pretreated with drug for min at • c, and then infected with hsv- (hf) (multiplicity of infection (moi) = ) by adding the virus directly into the culture medium. viral protein expression was determined by in-cell western analysis of gd- expression as described below. viral titration analysis was evaluated as follows: confluent hec- -a cells cultured in a -well plate were pretreated with serial concentrations of drug for min and then infected with hsv- (moi = ) for h, and the cultural medium was replaced with µl fresh medium. hsv- -infected cells were frozen and then thawed with three cycles to release the virion. the virion-containing medium was diluted and dispensed on confluent vero cell monolayers. after h of incubation, viral titration was performed by counting the numbers of plaque-forming units (pfu). cells were lysed using ripa lysis buffer on ice for min and then centrifuged at , × g for min at • c to remove insoluble debris. the supernatants were collected and total protein concentrations were determined using bicinchoninic acid assay (bca) protein assay kit (pierce, rockford, il, usa). after boiling at • c for min, the proteins were separated on % sds-page with reducing condition and transferred to polyvinylidene difluoride (pvdf) membranes (millipore, billerica, ma, usa). the membranes were blocked using odyssey blocking buffer (li-cor) and then stained with primary antibodies for h at room temperature, followed by washing with phosphate-buffered saline (pbs)- . % tween- (pbs-t buffer) min for times and staining with infrared dye (irdye) igg ( : , ) for h at rt. the proteins were visualized under li-cor odyssey infrared imager (li-cor), after washing with pbs- . % tween- (pbs-t buffer) min for times. in-cell western assay was performed in -well plate. the cells were fixed with % paraformaldehyde for min at room temperature, and permeabilized by washes of . % triton-x in pbs with min for each wash. cell monolayers were blocked using odyssey blocking buffer (li-cor) for min and then incubated with primary antibodies being diluted in blocking buffer ( : ) for h at rt. after being washed with pbs-t buffer, the monolayers were stained in irdye igg ( : ) for h. the plate was rinsed and scanned in odyssey infrared imager. relative protein expression was normalized against draq fluorescence and quantified. total rna was extracted using trizol reagent (life technologies), according to the manufacturer's protocol. equivalent amounts of rna ( µg) from each sample were subjected to reverse-transcription using revertra ace qpcr rt kit (toyobo, osaka, japan). real-time pcr was performed in triplicate on abi prism sequence detection system using sybr green pcr master mix (life technologies). we determined the mrna level of spliced xbp by real-time pcr using a primer set that selectively amplifies the spliced variant of xbp cdna. the forward primer used in this reaction is designed to span the -base pair (bp) intron, so only the spliced xbp cdna was amplified [ ] . primers of xbp s, ire α, edem, herp, and gd used in real-time pcr are: xbp s forward: -ggtctgctgagtccgcagcagg- , and reverse: -gggcttggtatatatgtgg- , ire α forward: -cggcctttgcagatagtctc- , and reverse: -acgtccccagattcactg- , edem forward: -gcacaggccgaaacctcat- , and reverse: -tgctctttaagggcagggag- , herp forward: -caccgcgacttggagctgagtgg- , and reverse: -tctgtggattcagccaccttgg- , gd forward: -agcaggggttagggagttg- , and reverse: -ccatcttgagagaggcatc- . messenger rna transcription levels were standardized against housekeeping gene gapdh (forward, -tgcaccaccaactgcttagc- , and reverse, -ggcatggactgtggtcatgag- ). to measure the degree of xbp mrna splicing, the total cellular rna was isolated as described above. xbp was pcr amplified using platinum tm hot start pcr master mix (life technologies), according to the manufacturer s instructions. in order to amplify both the spliced and the unspliced variants of xbp cdna, the primers were designed circumventing the spliced segment as follows: forward primer -ccttgtagttgagaaccagg- , and reverse primer, -gggcttggtatatatgtgg- . for the analysis of pcr products, µl of each reaction mixture was loaded on a . % agarose gel in tbe ( mm tris-borate, mm edta, ph . ), and subjected to electrophoresis to separate the products. the bands were visualized using gel imaging system (tianneng , shanghai, china). to determine the effect of ire α kinase activity on hsv- replication, the ire α expression was knocked-down using x-treme gene sirna transfection reagent (sigma-aldrich, st. louis, mo, usa), according to the manufacturer's instructions. hela cells in -well plates were transfected with the target sirna ( . µg/well) or control sirna. the ire α sirna sequence is: ggacgugagcgacagaauadtdt, for negative control (nc): uucuccgaacgugucacgutt. sirnas were synthesized by genepharma (shanghai, china). cells were mock-infected or infected with hsv- (hf) (moi = ) at h post transfection, harvested at h post-infection (p.i.), and samples were prepared for translation and transcription expression analysis using western blot or real-time pcr methods. in order to examine the effect of xbp s on viral infection, hela cells were transiently transfected with plasmid cloning vehicle pcdna . or xbp s plasmid using lipofectamine transfection reagent (life technologies). . µg/well of plasmid was used to transfect hela cells in -well plate, ng/well of plasmid was used to transfect cells in -well plate. after incubation for h, the cells were infected with hsv- /blue (moi = ) for h (cultured in -well plate) or h (cultured in -well plate), before being lysed with ripa lysis buffer or % np- in pbs. the cell lysates in -well plate were harvested and preparing for western blot analysis, and in -well plate were then transferred to a new costar -well flat plate and mixed with cprg (chlorophenol red-β-d-galactopyranoside; boehringer, ingelheim, germany), and β-gal activity was measured in a tecan infinit m microplate reader at nm after h. to further investigate the effect of the kinase activity of ire on viral replication, hela cells cultured in a -well plate were transiently transfected with plasmid cloning vehicle pcdna . , or ire alpha ka-pcdna .egfp, or ire alpha-pcdna .egfp plasmid ( . µg/well) using lipofectamine transfection reagent (life technologies), according to the manufacturer's instructions. the cells were further cultured for h and then mock-infected or infected with hsv- (hf) (moi = ). total cellular protein was extracted after h p.i. preparing for western blot analysis with antibodies against total ire α, p-ire α and gd, respectively. the in vitro cytotoxicity of tg, apy and stf- was measured using a commercial cck- kit (dojindo, kumamoto, japan) via colorimetric method according to the manufacturer's instructions (see supplementary section). statistical analysis was performed using the two-tailed student's t-test, by using spss . (spss for windows release . , spss inc., chicago, il, usa). statistical significance: * p < . , ** p < . . both protein and mrna levels of ire α were analyzed to illustrate the effect of viral infection on ire α stress pathway after cells were infected with hsv- (hf) (moi = ). cells pretreated with tg, an er stress inducer which induces strong ire rnase activity [ , ] , served as a positive control. the results showed that the ire α protein expression was markedly up-regulated by hsv- infection from h p.i. ( figure a ). the mrna level of ire α was also up-regulated correspondingly ( figure b ). we further investigated whether the up-regulations of ire α protein and mrna levels were due to viral dna replication. before being infected with hsv- (moi = ), hec- -a cells were pretreated with paa ( µg/ml), an inhibitor of viral dna polymerase, and viral replication [ ] . both the mrna and protein levels of gd and ire α were determined by either real-time pcr or western blot analysis. the results showed that paa could efficiently reduce the translation and replication of hsv- , as indicated by the reduced gd protein and mrna expressions. both the protein and the mrna levels of ire α were also significantly reduced ( figure c -e), indicating that the cellular ire α signaling pathway is likely to be affected by hsv- replication. the ire α rnase activation can induce the splicing of xbp mrna [ ] . the translation product (xbp s) of the spliced xbp mrna is an important transcriptional regulator of genes involved in protein folding and degradation [ , ] . to investigate if ire α rnase activity was also affected by hsv- infection, we determined the spliced xbp mrna level by real-time pcr by using a primer set that selectively amplifies the spliced variant of xbp cdna [ ] , and the degree of xbp splicing using primers that circumvent the spliced segment, which allows for both the spliced and the unspliced variants of xbp cdna to be amplified (figure a,b) . tg is an effective inhibitor of the ca + ion pump proteins of intracellular membranes, located in sarcoplasmic reticulum (sr) and endoplasmic reticulum (er) [ ] . tg is widely used as an inducer of er stress [ , ] . the results showed that the xbp s mrna level and the xbp splicing degree were constant during viral infection, but increased in tg-treated cells ( figure c ), which is consistent with previous reports [ , ] , and confirms that ire α rnase activity was not activated by hsv- infection. xbp s plays a vital role in the regulation of a large number of genes functional in erad which may negatively regulate viral replication [ , , ] . previous study showed that hcv suppressed the ire -xbp pathway to facilitate its replication [ ] . recent research found that the ire -xbp pathway was also inhibited by hsv- ul protein [ ] , but the effect of ire α rnase activity on hsv- remains uncertain. therefore, we investigated the effect of ire α rnase activity on hsv- the data are presented as mean ± standard deviation (sd) from three independent determinations (* p < . , ** p < . ); (c-e) both ire α and gd expression were inhibited by paa. hec- -a cells were pretreated or mock-pretreated with phosphonoacetic acid (paa, µg/ml) for min and then infected or mock-infected with hsv- (hf) at moi = . both protein and mrna transcription levels of ire α and gd- at h p.i. were determined. columns and bars represent the mean ± sd of three independent experiments (** p < . ). all experiments were performed three times. representative results are shown. the ire α rnase activation can induce the splicing of xbp mrna [ ] . the translation product (xbp s) of the spliced xbp mrna is an important transcriptional regulator of genes involved in protein folding and degradation [ , ] . to investigate if ire α rnase activity was also affected by hsv- infection, we determined the spliced xbp mrna level by real-time pcr by using a primer set that selectively amplifies the spliced variant of xbp cdna [ ] , and the degree of xbp splicing using primers that circumvent the spliced segment, which allows for both the spliced and the unspliced variants of xbp cdna to be amplified (figure a,b) . tg is an effective inhibitor of the ca + ion pump proteins of intracellular membranes, located in sarcoplasmic reticulum (sr) and endoplasmic reticulum (er) [ ] . tg is widely used as an inducer of er stress [ , ] . the results showed that the xbp s mrna level and the xbp splicing degree were constant during viral infection, but increased in tg-treated cells ( figure c ), which is consistent with previous reports [ , ] , and confirms that ire α rnase activity was not activated by hsv- infection. xbp s plays a vital role in the regulation of a large number of genes functional in erad which may negatively regulate viral replication [ , , ] . previous study showed that hcv suppressed the ire -xbp pathway to facilitate its replication [ ] . recent research found that the ire -xbp pathway was also inhibited by hsv- ul protein [ ] , but the effect of ire α rnase activity on hsv- viruses , , of remains uncertain. therefore, we investigated the effect of ire α rnase activity on hsv- replication. stf- , an inhibitor of ire α rnase activity [ ] that suppressed the tg-induced xbp splicing in a dose dependent manner ( figure d) , was used as a means for demonstrating the effect of reducing rnase activity of ire α on hsv- infection. tg and stf- , which have opposite effects on xbp splicing, were used to treat cells before viral infection, and their effects on the viral infection were evaluated by measuring the pfu h p.i. tg but not stf- inhibited the replication of hsv- , as shown in figure e ,f. the inhibitory effect of tg on viral replication was supported by gd reduction in the presence of the drug, as shown in figure g . consistently, stf- showed no effect on gd expression ( figure h ). since the cell viability was not affected by the drug at the experimental concentrations as determined by cck- colorimetric assay, we ruled out the possibility that the viral inhibitory activity was due to the cytotoxic effect of the drug (the data was shown in the supplementary material). to further illustrate the role of xbp s on viral replication, we overexpressed xbp s by plasmid transfection, followed by the infection of the cells with hsv- . gd protein level was measured by western blot, and the result showed that the gd expression was reduced by about % in the cells overexpressing xbp s, indicating that viral replication was suppressed by xbp s (upper panel of figure i ). we also employed an hsv- /blue recombinant virus assay to confirm the effect of spliced xbp on viral replication. this recombinant virus contains an hsv- icp promoter-driven lacz gene, inserted into the hsv- tk gene loci. we determined the effect of xbp s overexpression on viral replication by measuring the regulated expression of β-galactosidase of the recombinant virus as a marker for viral growth [ , ] . the result showed that the replication of hsv- /blue was reduced, indicating that icp promoter activity was inhibited in the hela cells overexpressing xbp s (lower panel of figure i ). during the er stress responses, terminally unfolded or misfolded proteins may be degraded by erad. we speculated that the viral proteins would be degraded by erad when a large amount of viral proteins accumulated in er during replication. hsv- may have evolved countermeasures to ire -xbp action to prevent detrimental effect induced by xbp splicing, and this speculation was supported by a previous study which showed that xbp splicing induced by tg was repressed by hsv- ul protein [ ] . to explore if the downstream of xbp was also inhibited by hsv- infection, we measured the transcriptions of both edem and herp genes, both of which are regulated by xbp s and directly participate in erad [ , ] . hec- -a cells were cultured in the presence of tg ( µm) for min and then infected with hsv- (moi = ) for h and the transcriptions of both edem and herp were determined by real-time pcr. the results showed that increased transcriptions of these two genes induced by tg were downregulated by hsv- infection ( figure j ), suggesting that hsv- suppressed erad by inhibiting the rnase activity of ire α to avoid degradation of the viral proteins. stf- ( µm), used as control, significantly suppressed tg-induced xbp splicing ( d), and the transcription of both edem and herp ( j). taken together, the results suggest that rnase activity of ire α is detrimental to the viral replication, and were not activated during viral infection. replication. stf- , an inhibitor of ire α rnase activity [ ] that suppressed the tg-induced xbp splicing in a dose dependent manner ( figure d) , was used as a means for demonstrating the effect of reducing rnase activity of ire α on hsv- infection. tg and stf- , which have opposite effects on xbp splicing, were used to treat cells before viral infection, and their effects on the viral infection were evaluated by measuring the pfu h p.i. tg but not stf- inhibited the replication of hsv- , as shown in figure e ,f. the inhibitory effect of tg on viral replication was supported by gd reduction in the presence of the drug, as shown in figure g . consistently, stf- showed no effect on gd expression ( figure h ). since the cell viability was not affected by the drug at the experimental concentrations as determined by cck- colorimetric assay, we ruled out the possibility that the viral inhibitory activity was due to the cytotoxic effect of the drug (the data was shown in the supplementary material). to further illustrate the role of xbp s on viral replication, we overexpressed xbp s by plasmid transfection, followed by the infection of the cells with hsv- . gd protein level was measured by western blot, and the result showed that the gd expression was reduced by about % in the cells overexpressing xbp s, indicating that viral replication was suppressed by xbp s (upper panel of figure i ). we also employed an hsv- /blue recombinant virus assay to confirm the effect of spliced xbp on viral replication. this recombinant virus contains an hsv- icp promoter-driven lacz gene, inserted into the hsv- tk gene loci. we determined the effect of xbp s overexpression on viral replication by measuring the regulated expression of β-galactosidase of the recombinant virus as a marker for viral growth [ , ] . the result showed that the replication of hsv- /blue was reduced, indicating that icp promoter activity was inhibited in the hela cells overexpressing xbp s (lower panel of figure i ). during the er stress responses, terminally unfolded or misfolded proteins may be degraded by erad. we speculated that the viral proteins would be degraded by erad when a large amount of viral proteins accumulated in er during replication. hsv- may have evolved countermeasures to ire -xbp action to prevent detrimental effect induced by xbp splicing, and this speculation was supported by a previous study which showed that xbp splicing induced by tg was repressed by hsv- ul protein [ ] . to explore if the downstream of xbp was also inhibited by hsv- infection, we measured the transcriptions of both edem and herp genes, both of which are regulated by xbp s and directly participate in erad [ , ] . hec- -a cells were cultured in the presence of tg ( μm) for min and then infected with hsv- (moi = ) for h and the transcriptions of both edem and herp were determined by real-time pcr. the results showed that increased transcriptions of these two genes induced by tg were downregulated by hsv- infection ( figure j ), suggesting that hsv- suppressed erad by inhibiting the rnase activity of ire α to avoid degradation of the viral proteins. stf- ( μm), used as control, significantly suppressed tg-induced xbp splicing ( d), and the transcription of both edem and herp ( j). taken together, the results suggest that rnase activity of ire α is detrimental to the viral replication, and were not activated during viral infection. apy , an inhibitor of kinase activity of ire α, and also an activator of rnase activity of ire α [ , ] , was used to illustrate the roles of kinase activity of ire α on hsv- replication. to exclude the impact of rnase activity of ire α on viral replication, we used stf- to inhibit the apy -induced rnase activity since xbp splicing induced by apy was inhibited by stf- ( figure b ). hsv- replication was inhibited by apy in a dose dependent manner, as shown by in-cell western analysis on gd- expression ( figure c ). when cells were pretreated with both apy and stf- and then infected with hsv- , apy showed an inhibitory effect on viral replication independent of the presence of stf- ( figure d) , suggests that the suppression of kinase activity of ire α by apy is sufficient to inhibit viral replication. this conclusion was supported by the result that down-regulation of phosphorylated ire α, induced by apy compared with that of the control group, gd expression was suppressed, as shown in figure e . to further determine the role of kinase activity of ire α on viral replication, hela cells were transfected with a kinase-inactive mutant k a human ire α (ire ka) [ ] , and its effect on hsv- replication was analyzed by western blot analysis. the result showed that gd expression was completely inhibited by ire ka, but not the wild-type of ire α ( figure f) , suggesting that the kinase activity of ire α is required for the viral replication. stress-induced oligomerization and activation of the ire α lead to clustering of traf , resulting in the activation of proximal components of the jnk kinase cascade [ ] . studies showed that jnk activation could facilitate hsv replication [ ] . consistent with early observations, we showed that hsv- replication stimulated the jnk signaling ( figure a) , and that the inhibition of jnk activation by an jnk inhibitor (sp ) resulted in the suppression of viral replication ( figure b ). and gd- protein were determined by western blot at h p.i. all experiments were performed three times. the representative results were shown. apy , an inhibitor of kinase activity of ire α, and also an activator of rnase activity of ire α [ , ] , was used to illustrate the roles of kinase activity of ire α on hsv- replication. to exclude the impact of rnase activity of ire α on viral replication, we used stf- to inhibit the apy induced rnase activity since xbp splicing induced by apy was inhibited by stf- ( figure b ). hsv- replication was inhibited by apy in a dose dependent manner, as shown by in-cell western analysis on gd- expression ( figure c ). when cells were pretreated with both apy and stf- and then infected with hsv- , apy showed an inhibitory effect on viral replication independent of the presence of stf- ( figure d) , suggests that the suppression of kinase activity of ire α by apy is sufficient to inhibit viral replication. this conclusion was supported by the result that down-regulation of phosphorylated ire α, induced by apy compared with that of the control group, gd expression was suppressed, as shown in figure e . to further determine the role of kinase activity of ire α on viral replication, hela cells were transfected with a kinase-inactive mutant k a human ire α (ire ka) [ ] , and its effect on hsv- replication was analyzed by western blot analysis. the result showed that gd expression was completely inhibited by ire ka, but not the wild-type of ire α ( figure f ), suggesting that the kinase activity of ire α is required for the viral replication. stress-induced oligomerization and activation of the ire α lead to clustering of traf , resulting in the activation of proximal components of the jnk kinase cascade [ ] . studies showed that jnk activation could facilitate hsv replication [ ] . consistent with early observations, we showed that hsv- replication stimulated the jnk signaling ( figure a ), and that the inhibition of jnk activation by an jnk inhibitor (sp ) resulted in the suppression of viral replication ( figure b ). . the total and phosphorylated jnk, and gd protein were analyzed by western blot at h p.i. the number under each band represents the relative density of the band in comparison to the corresponding control normalized to gapdh; (c) ire α knock-down reduced hsv- viral gene replication in hela cells. hela cells were transfected with scramble sirna as negative control (nc) or ire α sirna for h and then infected with hsv- (moi = ), total rna was extracted at h p.i., both viral gd rna level and ire α mrna level were determined by real-time pcr and normalized to gapdh. data are means of three independent wells, and the bars represent the mean values ± sd, ** p < . ; (d) ire α knock down reduced the kinase activity of ire α and viral replication in hela cells. hela cells were treated according to material and methods section , total cellular protein was harvested at h p.i. and then phosphorylated ire α level, total ire α and gd expression were measured by western blot using indicated antibodies; (e) ire α knock-down attenuated jnk activation stimulated by hsv- in hela cells. hela cells were treated according to material and methods section , total cellular protein was harvested at h p.i. preparing for western blot analysis against gd, total and phosphorylated jnk antibodies; (f) hsv- stimulated jnk activation was suppressed by inactivation of ire α kinase activity in hela cells. hela cells were treated as describe in material and methods . total cell lysates were prepared after h p.i. for western blot analysis using antibodies directed against gd, phosphorylated jnk and jnk; (g) ire α kinase activity inhibitor repressed jnk activation induced by viral infection in hec- -a cells. hec- -a cells were pretreated or mock-pretreated with apy ( . µm), only or both apy ( . µm), and stf- ( µm) for min and then infected or mock-infected with hsv- (moi = ). total cell lysates were prepared and western blot analysis was performed using specific antibodies at h p.i. all experiments were performed three times. the representative results were shown. since jnk can be activated by ire α during er stress, and the activation of jnk is important to hsv replication [ , ] , we investigated if hsv- -induced ire α up-regulation could activate jnk signaling pathway. in a previous study, ire −/− fibroblasts were reported to be impaired in jnk activation during er stress induced by tg [ ] . we, therefore, analyzed the effect of ire α down-regulation by rna interference (rnai) on jnk activation during viral infection. the results showed that compared with sinc, siire α significantly down-regulated ire α and reduced gd expression ( figure c,d) . consistent with the observations, the jnk activation (p-jnk) induced by hsv- infection was also reduced by ire α knockdown, as shown in figure e , suggesting that ire α pathway is one of the mechanisms mediating the jnk activation during hsv- infection. to further explore the mechanism of ire α-mediated jnk activation, we examined the effect of wild-type ire α and ire ka on jnk activation and showed that both viral replication, and jnk phosphorylation were suppressed in ire ka ( figure f ). we further examined the effect of the combination of apy and stf- on jnk signaling activation and showed that the treatment of cells with these two drugs significantly inhibited the virus-induced jnk activation, and the viral replication ( figure g ). taken together, the evidences suggest that the kinase activity of ire α is involved in the regulation of hsv- replication through jnk signaling pathway. we, therefore, postulate that hsv- replication is facilitated by the kinase activity of ire α while inhibited by the rnase activity of ire α. as one of the most conserved branch of upr signaling pathways from yeast to mammals, ire α was reported being involved in many viral replications. regulators of ire α rnase activity, tg, and stf- , were reported to modulate rnase activity of ire α in both ridd and xbp splicing processes, both of these two processes are activated by tg and inhibited by stf- [ , ] . although ridd was induced during er stress, the target genes modulated by ridd are quite complicated, and those published substrates of ridd still lack fully validation [ ] , so most studies select xbp splicing to represent the rnase activity of ire α. in order to facilitate viral replication, ire -xbp pathway that mediates ire α rnase activity was suppressed in infection by the hepatitis c virus, human cytomegalovirus, and rotavirus while activated in infections of influenza a virus, murine coronavirus mouse hepatitis virus, hepatitis b virus, japanese encephalitis virus, flavivirus, and epstein-barr virus [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , indicating that the roles of ire α-xbp on viral replication depend on viruses. ire -xbp pathway is required for, and regulates efficient protein folding, maturation, and degradation in response to er stress [ ] . we speculate that ire α rnase activity activates cellular protein degradation pathway (erad), and leads to the degradation of viral proteins, which is unfavourable to viral replication. this assumption was supported by our data that tg-induced ire α rnase activity resulted in the inhibition of hsv- replication ( figure e ,g). results from an earlier study showed that perk branch of upr were activated by tg through up-regulating phosphorylation of perk [ ] . perk signaling regulates the suppression of translation initiation through phosphorylation of eif α to shutoff the cellular protein translation, and this response is used as an inhibitory mechanism on viral replication by the host. to facilitate viral replication, hsv- has evolved a countermeasure to avoid the detrimental effect of perk through dephosphorylation of the eif α, mediated by a virus encoded protein γ . [ , ] . the atf arm of upr was also activated by tg [ ] . researchers suggested that the activation of atf and its transcriptional activation of chaperone-encoding genes might benefit the virus by assisting the folding of accumulated proteins and preventing protein aggregation. this idea was supported by observations showing that atf signaling was stimulated by virus infections, such as hcv and african swine fever virus (asfv), and beneficial to viral replication [ , ] . therefore, we speculate that the tg induced inhibition of viral replication was specifically mediated by its regulation of ire α branch, which was further supported by the observation that xbp s over-expression also led to the viral inhibition ( figure i ). the limited inhibitory effect of tg-induced rnase activity of ire α (about % inhibition of hsv- replication ( figure e ,g)) may reflect a viral countermeasure to suppress rnase activity of ire α, as an earlier report showed that ire -xbp signal was inhibited by hsv- ul protein [ ] . we also observed that the transcriptions of xbp s target genes were up-regulated by tg, but this up-regulation was suppressed in hsv- infected cells ( figure j) ; however, detailed mechanisms of this feedback regulation need further investigation. in agreement with earlier studies that ire -xbp arm of upr was inactivated or suppressed by hsv- infection [ , , ] , we further presented evidence that the activation of the rnase activity of ire α is detrimental to hsv- replication, and suppression of the rnase activity may serve as a mechanism for the virus to escape from host restriction. jnk pathway is activated under cellular stresses [ ] . the protein kinases mitogen-activated protein kinase, kinase (mkk) and mkk activate the jnk in response to pro-inflammatory cytokines or cellular stresses [ ] . ire signaling also modulates the jnk activation when upr occurred [ ] . both jnk and p mapk are activated by hsv infection [ ] . however, evidence on how proximal signals are coupled to the activation of jnk kinase during hsv- infection is limited. our data showed that hsv- -stimulated jnk activation was repressed when ire α expression was down-modulated by rna interference, indicating that ire α was one of the upstream regulators of the jnk cascade during hsv- infection ( figure e ). we showed that ire α kinase activity positively regulated the hsv- replication (figure ), while ire α rnase activity negatively regulated viral replication ( figure e -i), suggesting that hsv- -induced jnk cascade activation was mediated by ire α kinase activity. we further confirmed this result by over-expressing ire ka, or using the kinase inhibitor, both of which resulted in the reduced activation of jnk and viral replication. these data strongly suggest that ire α kinase activity is required for hsv- replication due to its stimulation of jnk kinase cascade in infected cells. previous studies showed that hsv- viral proteins, such as icp , icp , or vp , were correlated with jnk activation [ , , ] . since co-localizations of icp or gd with ire α were observed in our preliminary study (data not shown), we speculate that the viral protein(s) might participate in the regulation of the ire α kinase activity to facilitate hsv- replication. further investigation still need be done to unveil the detailed mechanisms. in conclusion, our current study found that the activation of rnase activity of ire α has an inhibitory effect on hsv- replication, which might act as a cellular restriction to hsv- replication through activation of a downstream er protein degradation pathway to limit viral protein production. but ire α kinase activity was helpful to hsv- replication. to counter the cellular restriction, hsv- inactivates the rnase activity, while activating the kinase activity of ire α through a yet unknown mechanism. this differential role of ire α activation on hsv- replication allows the virus to avoid detrimental consequence of upr while exploiting the activation of jnk signaling pathway to facilitate its replication. it warrants further investigations to delineate the molecular mechanisms that dictate the elevation of kinase activities and the related viral factors. supplementary materials: the following are available online at www.mdpi.com/ - / / / /s , figure s : the cytotoxic effect of tg, stf- and apy on hec- -a and hela cells. the herpes simplex virus type regulatory protein icp enhances virus replication during acute infection and reactivation from latency early expression of herpes simplex virus (hsv) proteins and reactivation of latent infection hsv antivirals-current and future treatment options stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls endoplasmic reticulum stress links obesity, insulin action, and type diabetes endoplasmic reticulum stress induces p cytoplasmic localization and prevents p -dependent apoptosis by a pathway involving glycogen synthase kinase- beta regulated ire -dependent decay pathway is activated during japanese encephalitis virus-induced unfolded protein response and benefits viral replication viruses, endoplasmic reticulum stress, and interferon responses ire signaling affects cell fate during the unfolded protein response cab s inhibits the er stress-induced ire -jnk pathway and apoptosis via grp /bip hepatitis c virus subgenomic replicons induce endoplasmic reticulum stress activating an intracellular signaling pathway japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response herpes simplex virus infection activates the endoplasmic reticulum resident kinase perk and mediates eif- alpha dephosphorylation by the gamma( ) . protein a stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (ire p) in mammalian cells cloning of mammalian ire reveals diversity in the er stress responses on the mechanism of sensing unfolded protein in the endoplasmic reticulum dynamic interaction of bip and er stress transducers in the unfolded-protein response block of hac mrna translation by long-range base pairing is released by cytoplasmic splicing upon induction of the unfolded protein response xbp- regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response decay of endoplasmic reticulum-localized mrnas during the unfolded protein response physiological roles of regulated ire dependent decay coupling of stress in the er to activation of jnk protein kinases by transmembrane protein kinase ire influenza a viral replication is blocked by inhibition of the inositol-requiring enzyme (ire ) stress pathway coronavirus infection modulates the unfolded protein response and mediates sustained translational repression hepatitis c virus suppresses the ire -xbp pathway of the unfolded protein response human cytomegalovirus infection activates and regulates the unfolded protein response maintenance of endoplasmic reticulum (er) homeostasis in herpes simplex virus type -infected cells through the association of a viral glycoprotein with perk, a cellular er stress sensor association of a m(r) , phosphoprotein with protein kinase pkr in cells exhibiting enhanced phosphorylation of translation initiation factor eif- alpha and premature shutoff of protein synthesis after infection with gamma ( ) . -mutants of herpes simplex virus herpes simplex virus- disarms the unfolded protein response in the early stages of infection herpes simplex virus ul protein suppresses the ire /xbp signal pathway of the unfolded protein response via its rnase activity activation of cjun n-terminal kinase by herpes simplex virus type enhances viral replication activation of c-jun n-terminal kinase (jnk) pathway by hsv- immediate early protein icp contributions to the pathology of experimental virus encephalitis. iv. recurring strains of herpes virus zinc ionophores pyrithione inhibits herpes simplex virus replication through interfering with proteasome function and nf-kappab activation pyrrolidine dithiocarbamate inhibits herpes simplex virus and replication, and its activity may be mediated through dysregulation of the ubiquitin-proteasome system downregulation of cellular c-jun n-terminal protein kinase and nf-kappab activation by berberine may result in inhibition of herpes simplex virus replication protective vaccination against primary and recurrent disease caused by herpes simplex virus (hsv) type using a genetically disabled hsv- quantitative measurement of spliced xbp mrna as an indicator of endoplasmic reticulum stress unconventional splicing of xbp- mrna in the unfolded protein response xbp is essential for survival under hypoxic conditions and is required for tumor growth cytoplasmic ire alpha-mediated xbp mrna splicing in the absence of nuclear processing and endoplasmic reticulum stress drug action of thapsigargin on the ca + pump protein of sarcoplasmic reticulum differential contributions of atf and xbp to the activation of endoplasmic reticulum stress-responsive cis-acting elements erse, upre and erse-ii role of the endoplasmic reticulum-associated degradation (erad) pathway in degradation of hepatitis c virus envelope proteins and production of virus particles identification of an ire alpha endonuclease specific inhibitor with cytotoxic activity against human multiple myeloma herpes simplex type : lacz recombinant viruses. i. characterization and application to defining the mechanisms of action of known antiherpes agents herpes simplex virus (hsv) immediate-early (ie) promoter-directed reporter system for the screening of antiherpetics targeting the early stage of hsv infection a novel er alpha-mannosidase-like protein accelerates er-associated degradation divergent allosteric control of the ire alpha endoribonuclease using kinase inhibitors the unfolded protein response signals through high-order assembly of ire the molecular basis for selective inhibition of unconventional mrna splicing by an ire -binding small molecule ire alpha kinase activation modes control alternate endoribonuclease outputs to determine divergent cell fates regulation of insulin biosynthesis in pancreatic beta cells by an endoplasmic reticulum-resident protein kinase ire harmine blocks herpes simplex virus infection through downregulating cellular nf-kappab and mapk pathways induced by oxidative stress ire has distinct catalytic mechanisms for xbp /hac splicing and ridd getting ridd of rna: ire in cell fate regulation reovirus induces and benefits from an integrated cellular stress response hepatitis b virus x protein (hbx) activates atf and ire -xbp pathways of unfolded protein response flavivirus infection activates the xbp pathway of the unfolded protein response to cope with endoplasmic reticulum stress the lmp oncogene of ebv activates perk and the unfolded protein response to drive its own synthesis resistance of mrna translation to acute endoplasmic reticulum stress-inducing agents in herpes simplex virus type -infected cells requires multiple virus-encoded functions amino acid substitutions in the effector domain of the gamma( ) . protein of herpes simplex virus have differential effects on viral response to interferon-α the atf branch of unfolded protein response and apoptosis are activated to promote african swine fever virus infection jnk) signaling: recent advances and challenges synergistic activation of stress-activated protein kinase /c-jun n-terminal kinase (sapk /jnk) isoforms by mitogen-activated protein kinase kinase (mkk ) and mkk herpes simplex virus type infection stimulates p /c-jun n-terminal mitogen-activated protein kinase pathways and activates transcription factor ap- herpes simplex virus icp activation of stress kinases jnk and p the authors declare no conflict of interest. key: cord- - ubs q p authors: paim, francine c.; bowman, andrew s.; miller, lauren; feehan, brandi j.; marthaler, douglas; saif, linda j.; vlasova, anastasia n. title: epidemiology of deltacoronaviruses (δ-cov) and gammacoronaviruses (γ-cov) in wild birds in the united states date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ubs q p porcine deltacoronavirus (δ-cov) is the object of extensive research in several countries including the united states. in contrast, the epidemiology of δ-covs in wild birds in the us is largely unknown. our aim was to comparatively assess the prevalence of δ- and γ-covs in wild migratory terrestrial and aquatic birds in arkansas, illinois, indiana, maryland, mississippi, missouri, ohio, tennessee and wisconsin. a total of cloacal/fecal swabs collected during the period – were tested for γ- and δ-covs using genus-specific reverse transcription-pcr assays. a total of ( . %) samples were γ-cov positive, with up to positive samples per state. in contrast, only samples were positive for δ-cov ( . %) with only – originating from the same state. thus, unlike previous reports from asia, γ-covs are more prevalent than δ-covs in the us, suggesting that δ-covs may spread in birds with lower efficiency. this may indicate δ-cov emerging status and incomplete adaptation to new host species limiting its spread. phylogenetic analysis of the partial n gene revealed that the newly identified δ-cov strains were most closely related to the hku (wigeon) strain. further studies are necessary to investigate the role of aquatic bird δ-covs in the epidemiology of δ-covs in swine and terrestrial birds. coronaviruses (covs) are positive-sense rna viruses that are widespread in humans alongside various mammalian and avian species. they cause enteric, respiratory, or systemic diseases of variable severity [ , ] . covs belong to the coronaviridae family that contains four genera: alphacoronavirus (α-cov), betacoronavirus (β-cov), gammacoronavirus (γ-cov) and deltacoronavirus (δ-cov) [ ] . the virus evolves through an accumulation of point mutations and both homologous and non-homologous recombination. it is hypothesized that this ability to recombine allows the virus to evolve and create new forms which can target different species [ ] . α-cov and ß-cov infect multiple species of mammals, but γ-cov is only known to infect birds. in contrast, δ-covs are found in both birds a subset of avian cloacal/fecal swabs were selected from a collection of , that were collected for influenza a virus surveillance in different species of wild terrestrial and aquatic birds during the period - . a total of avian cloacal swabs were collected for avian influenza virus (aiv) surveillance in different species of wild terrestrial and aquatic birds along the mississippi flyway, us during the period - . of avian cloacal swabs, were from aquatic bird samples collected in ohio, illinois, missouri, maryland, wisconsin and tennessee in the period - . a total of were from terrestrial and aquatic bird samples collected in ohio, mississippi, indiana and arkansas in the period - . were from known species: aquatic and terrestrials; the remainder were from environmental fecal samples. rna from the avian cloacal/fecal swabs was extracted using a modified commercial protocol (ambion ® magmax™, applied biosystems, foster city, ca, usa) with mg/ml of bovine serum albumin (bsa) and % sodium sulfite. a total of avian cloacal/fecal swab rna samples were tested for γand δ-cov using pancoronavirus (in deg/in deg) and deltocoronavirus-specific reverse transcription-pcr (rt-pcr) assays, respectively [ , ] . δ-cov primers were designed using δ-cov nucleocapsid (n) universal primers (udcovf: '-rywgayksntcntggttyca- ' and udcovr: '-hgtgccwgtrtartaraagg- ') targeting bp [ ] . subsequently, after evaluating the results of next-generation sequencing (ngs), we designed hku -specific primers targeting a bp fragment of the n gene (hku -n-f '-tccgcgcctcatggctctc- ' and hku -n-r '-tcatgagaaggattctag- '). all the rt-pcr assays were performed under the same conditions using qiagen one-step rt-pcr kit (qiagen inc., valencia, ca, usa) in a geneamp pcr system thermal cycler (applied biosystems, foster city, ca, usa). the reaction-mixture (total µl) included × qiagen onestep buffer ( µl), dntp ( µl), upstream and downstream primers ( µmol/l, each µl), rnasin ( u/µl, . µl), enzyme mix ( µl), rnase-free water ( . µl) and rna template ( µl). the thermocycler protocol consisted of an initial reverse transcription step at • c for min, followed by pcr activation at • c for min, cycles of amplification ( • c for s, • c for s, • c for s), and a final extension step at • c for min. pcr products were analyzed on % agarose gel. the pcr product was purified with an agarose gel qiaquick gel extraction kit (qiagen inc., valencia, ca, usa). amplicons derived using the hku -specific primers were sequenced at the genomics shared resource of the ohio state university (columbus, oh, usa) by the sanger method [ ] . for ngs, previously extracted rna underwent cdna synthesis according to a random primer protocol performed using revertaid h minus first strand cdna synthesis kit (thermo scientific, waltham, ma, usa). pcr was conducted using true-start dna polymerase with mm dntps mix and pmol specific primers per reaction (thermo scientific), according to the manufacturer's protocols. truseq stranded total rna library prep kit was used with µg total rna for the construction of libraries according to the manufacturer's protocol. for rrna-depleted library, rrna was removed from . µg total rna using ribo-zero rrna removal kit (mixture : human/mouse/rat probe and bacteria probe), according to the manufacturer's protocol (with probe concentration for epidemiology kit protocol). all cdna libraries were sequenced using an illumina hiseq (illumina, san diego, ca, usa), producing × × bp paired end reads with multiplexing. reads were trimmed using default parameters with clc genomics workbench . . (qiagen bioinformatics, redwood city, ca, usa). trimmed reads were de novo assembled using a word size of , bubble size of , and sequences were assembled using bioedit sequence alignment editor and aligned using clustalw. the n gene sequences of avian and porcine δ-covs obtained through blast search and from genbank were included in the phylogenetic analysis. a maximum likelihood phylogenetic tree was constructed using mega software [ ] . a total of out of birds screened were positive for γ-cov with an apparent prevalence of . % ( % confidence interval: . %- . %). γ-cov was identified in the states of missouri (n = ), wisconsin (n = ), illinois (n = ), tennessee (n = ), ohio (n = ), and maryland (n = ) ( figure a ). the states where γ-covs were identified are shown in figure a . γ-covs were detected in different bird species: blue winged teal (spatula discors) (n = ), mallard (anas platyrhynchos) (n = ), american green-winged teal (anas crecca) (n = ), northern pintail (anas acuta) (n = ), ring-necked duck (aythya collaris) (n = ), and american wigeon (mareca americana) (n = ), all of which are aquatic bird species, meaning a prevalence of . % in aquatic birds and % in terrestrial birds. of interest, γ-covs were detected most frequently in samples from american green-winged teal, blue-winged teal and mallard that were among most frequently sampled waterfowl species (table ) . viruses , , x for peer review of a total of out of birds screened were positive for γ-cov with an apparent prevalence of . % ( % confidence interval: . %- . %). γ-cov was identified in the states of missouri (n = ), wisconsin (n = ), illinois (n = ), tennessee (n = ), ohio (n = ), and maryland (n = ) ( figure a ). the states where γ-covs were identified are shown in figure a . γ-covs were detected in different bird species: blue winged teal (spatula discors) (n = ), mallard (anas platyrhynchos) (n = ), american green-winged teal (anas crecca) (n = ), northern pintail (anas acuta) (n = ), ring-necked duck (aythya collaris) (n = ), and american wigeon (mareca americana) (n = ), all of which are aquatic bird species, meaning a prevalence of . % in aquatic birds and % in terrestrial birds. of interest, γ-covs were detected most frequently in samples from american green-winged teal, blue-winged teal and mallard that were among most frequently sampled waterfowl species (table ). there were of birds that screened positive for δ-cov, corresponding with an apparent prevalence of . % ( % confidence interval: . %- . %). thus, unlike previous reports from different countries [ , , ] , our study showed that in the us, γ-covs are more prevalent than δ-cov. δ-covs were detected in the states of illinois (n = ), arkansas (n = ), ohio (n = ), missouri (n = ), maryland (n = ) and mississippi (n = ) ( figure b ). the positive sampling locations are shown in figure . the bird species identified were the blue winged teal (spatula discors) (n = ), snow goose (anser caerulescens) (n = ), mallard (anas platyrhynchos) (n = ), red-tailed hawk (anser caerulescens) (n = ) and northern shoveler (spatula clypeata) (n = ). of these positive samples, only up to originated from the same state ( figure b) , suggesting that δ-covs spread with low efficiency in the avian species tested. of note, the red-tailed hawk was sampled upon intake to a wildlife rehabilitation center. interestingly, the prevalence of δ-cov in aquatic birds was . % compared to only . % in terrestrial birds, suggesting that aquatic birds represent an important natural reservoir for covs. blue-winged teal and snow goose were the two species associated with the most frequent recovery of δ-covs (table ) . * n/a-not available: was not analyzed because there was not enough rna. in contrast to γ-covs that are widespread and have been circulating in the us for decades, the prevalence and genetic characteristics of δ-covs in the us are not known. to address this gap, we conducted sequence and phylogenetic analysis of the δ-covs identified in this study. all amplicons generated with degenerate udcov-specific primers were confirmed to contain δ-cov n-gene sequences sharing ~ %- % nucleotide identity with various avian δ-cov species. however, due to a high number of degenerate nucleotides incorporated in these primers to allow for broad reactivity and detection of highly diverse porcine and avian δ-covs, the amplicons were not of satisfactory quality, with up to % of ambiguous nucleotides (ns). thus, we selected δ-cov positive samples ( table ) that had sufficient amounts of rna and subjected them to ngs sequencing ( table ). for one of the samples, blue winged teal coronavirus/usa/illinois / , ngs recovered approximately % of eukaryotic (host genome; normal; incidental), % bacteria, % virus, and % other. bacterial reads identified fusobacterium nucleatum ( %), while further analyses of viral and "other" reads identified various bacteriophage ( %). eighty-five reads were identified as δ-cov including fragments of orf a/b, s, m, n and ns a genes. blast search and phylogenetic analysis identified that these genomic fragments shared the highest nucleotide identity ( %- %) with wigeon cov hku , without evidence of recent recombination events (figure a-c) . for the other three samples, there were variable amounts of eukaryotic ( %- %), bacterial ( %- %), viral ( %- %) and other/bacteriophage ( %- %) reads; but no δ-cov sequence was recovered. this suggests that δ-cov rna in these samples was present in insufficient amounts or was of low quality. in contrast to γ-covs that are widespread and have been circulating in the us for decades, the prevalence and genetic characteristics of δ-covs in the us are not known. to address this gap, we conducted sequence and phylogenetic analysis of the δ-covs identified in this study. all amplicons generated with degenerate udcov-specific primers were confirmed to contain δ-cov n-gene sequences sharing~ %- % nucleotide identity with various avian δ-cov species. however, due to a high number of degenerate nucleotides incorporated in these primers to allow for broad reactivity and detection of highly diverse porcine and avian δ-covs, the amplicons were not of satisfactory quality, with up to % of ambiguous nucleotides (ns). thus, we selected δ-cov positive samples ( table ) that had sufficient amounts of rna and subjected them to ngs sequencing ( of eukaryotic (host genome; normal; incidental), % bacteria, % virus, and % other. bacterial reads identified fusobacterium nucleatum ( %), while further analyses of viral and "other" reads identified various bacteriophage ( %). eighty-five reads were identified as δ-cov including fragments of orf a/b, s, m, n and ns a genes. blast search and phylogenetic analysis identified that these genomic fragments shared the highest nucleotide identity ( %- %) with wigeon cov hku , without evidence of recent recombination events (figure a-c) . for the other three samples, there were variable amounts of eukaryotic ( %- %), bacterial ( %- %), viral ( %- %) and other/bacteriophage ( %- %) reads; but no δ-cov sequence was recovered. this suggests that δ-cov rna in these samples was present in insufficient amounts or was of low quality. based on the ngs results, we designed hku -specific primers targeting a fragment of the n gene and re-screened the δ-cov-positive samples for which we had sufficient amounts of rna. six out of re-screened samples were positive in rt-pcr using hku -specific primers. three samples yielded pcr fragments of sufficient quantity/quality and were used for capillary sequencing. phylogenetic analysis of these fragments demonstrated that similar to blue-winged teal coronavirus/usa/illinois / , the three δ-covs were most closely related to wigeon coronavirus viruses , , of hku (figure ) sharing . %- . % nucleotide identity in the n gene. the two samples positive in rt-pcr with udcov primers, but negative with hku primers (table ) could contain insufficient amounts of δ-cov rna to generate a longer amplicon (targeted by the hku -specific primers) or could possess genetic characteristics distinct from hku δ-cov and other δ-covs identified in this study. based on the ngs results, we designed hku -specific primers targeting a fragment of the n gene and re-screened the δ-cov-positive samples for which we had sufficient amounts of rna. six out of re-screened samples were positive in rt-pcr using hku -specific primers. three samples yielded pcr fragments of sufficient quantity/quality and were used for capillary sequencing. phylogenetic analysis of these fragments demonstrated that similar to blue-winged teal coronavirus/usa/illinois / , the three δ-covs were most closely related to wigeon coronavirus hku (figure ) sharing . %- . % nucleotide identity in the n gene. the two samples positive in rt-pcr with udcov primers, but negative with hku primers (table ) could contain insufficient amounts of δ-cov rna to generate a longer amplicon (targeted by the hku specific primers) or could possess genetic characteristics distinct from hku δ-cov and other δ-covs identified in this study. while γ-covs are endemic and highly prevalent in wild and domestic birds [ ] [ ] [ ] [ ] [ ] [ ] [ ] , δ-covs have only been discovered in the us only recently. thus, the role of wild avian species in the ecology of δ-covs in the us is unknown. environmental sampling of high-risk and co-mingling sites in alberta and saskatchewan canada, identified δ-cov in sparrows, that are closer phylogenetically to porcine δ-covs than to those from waterfowl [ ] . to date, the presence of δ-cov is confirmed in sparrows in canada and the us [ ] , in quail in brazil [ ] and in various birds in australia [ ] , suggesting that δ-covs circulate in different avian species in the americas. since porcine δ-cov often results in severe clinical disease and mortality in piglets, which impacts the swine industry, it is important to while γ-covs are endemic and highly prevalent in wild and domestic birds [ ] [ ] [ ] [ ] [ ] [ ] [ ] , δ-covs have only been discovered in the us only recently. thus, the role of wild avian species in the ecology of δ-covs in the us is unknown. environmental sampling of high-risk and co-mingling sites in alberta and saskatchewan canada, identified δ-cov in sparrows, that are closer phylogenetically to porcine δ-covs than to those from waterfowl [ ] . to date, the presence of δ-cov is confirmed in sparrows in canada and the us [ ] , in quail in brazil [ ] and in various birds in australia [ ] , suggesting that δ-covs circulate in different avian species in the americas. since porcine δ-cov often results in severe clinical disease and mortality in piglets, which impacts the swine industry, it is important to understand the morbidity and interspecies transmission rates between birds and pigs [ , ] . due to their flocking behavior and abilities to fly long distances, birds can play a role in the dissemination of δ-covs among themselves and other animals [ ] . the apparent prevalence of δ-cov in our study was only . %. however, this is higher compared with that observed in our previous study ( . %) that analyzed ohio samples from the period - [ ] . also, of these positive samples, only up to originated from the same state, suggesting that δ-covs spread with low efficiency in the avian species tested. additionally, our inability to recover cov sequences by ngs suggests that they are present in the samples at lower frequency compared with other microorganisms. the latter is more suggestive of asymptomatic carrier status as opposed to acute infection associated with clinical disease. these findings suggest that avian species likely represent a natural reservoir of δ-covs, while pigs and other mammals serve as spillover hosts. the higher prevalence of γ-covs in the us of . % is consistent with findings from some previous studies. in a screening study from brazil, quail were susceptible to both δand γ-cov [ ] . the higher prevalence of γ-versus-δ-covs and variations in preferred avian host species have been also reported by other researchers in other countries, such as australia [ ] , asia (hong kong and cambodia) [ ] and finland [ ] . this may indicate δ-cov emerging status and its ongoing spread in the us. in this study, a higher prevalence of δand γ-covs in aquatic vs. terrestrial birds was evident, with δand γ-covs prevalence in aquatic birds being . % and . %, respectively, compared with only . % and % in terrestrial birds. this suggests that aquatic birds may represent a natural reservoir for covs of terrestrial birds and pigs, and their concentration or survival may be increased in water sources compared with other avian habitats. similarly, in a surveillance study in hong kong and cambodia, δ-cov was found in different wild aquatic bird species including gray herons, pond herons, great cormorants, black-faced spoonbills, and several duck species, whereas γ-covs were found in little whistling ducks, tufted ducks, common teals, northern shovelers, eurasian wigeons, and northern pintails [ ] . in wild australian birds, δ-cov was detected in pacific black ducks, but it was also detected in terrestrial birds such as curlew sandpiper, red-necked stints, ruddy turnstones, and pied herons [ ] . this suggests that wild birds are major reservoirs of a wide range of δand γ-covs, and the circulation of covs without association with clinical disease is more common than previously recognized [ ] . however, it is important to note that δ-covs identified in terrestrial birds and pigs are more similar each other phylogenetically [ , , ] , and those from aquatic/wading birds are genetically more distinct. it is of interest, that prevalence of both γ-( %) and δ-covs ( %, table ) was higher in colder (october-february) than in warmer (march-september) months. this indicates that, similar to previous findings on avian, animal and human covs, there are may be seasonal (and migration associated) fluctuations in the prevalence of avian coronaviruses in wild bird in the us [ ] [ ] [ ] [ ] . because phylogenetic analysis of the partial n gene of the newly identified δ-covs (table ) revealed that they were most closely related to hku (wigeon) δ-cov, we hypothesized that it may be the parental strain recently introduced into the us. in our study, these newly identified δ-covs were more closely related to other δ-cov from aquatic birds, but not terrestrial avian species and pigs, which can be explained by the fact that aquatic birds occupy a separate ecological niche, while terrestrial birds and pigs may share some co-mingle sites. although, we did not identify δ-cov strains genetically similar to sparrow and porcine δ-covs, it is important to note that porcine δ-cov outbreaks in the us were predominantly detected in the states with the highest density of pig population that in turn have a significant geographical overlap with the mississippi flyway ( figure ) [ ] . while a small pilot study failed to identify porcine δ-covs in wild waterfowl in the mississippi flyway [ ] , the overlap of the bird migratory pathways and high density swine farms creates favorable conditions for birds and pigs to exchange δ-cov pools directly or through some other intermediate hosts. orange circles mark states with the highest pig inventory [ ] . black duck icons identify the states where avian δ-covs were found (this study). in conclusion, this is the first report of the presence of hku -like δ-covs in different avian species in the us. the close relatedness of all the strains we were able to sequence to hku suggests a recent introduction of δ-covs in the us and identification of this strain as potentially parental. we also demonstrated that aquatic birds are infected with δ-covs and γ-covs more frequently than terrestrial avian species. this, together with the observation that porcine δ-covs are more closely related to δ-covs identified in terrestrial birds, suggests that waterfowl might represent a natural reservoir for δ-covs. although γ-covs were more prevalent than δ-covs, consistent with previous studies, both covs were detected more frequently during the cold season in our study. further studies are necessary to investigate the role of aquatic bird δ-covs in the epidemiology of δ-covs in swine and terrestrial birds. it remains to be established: (i) if/how long avian δ-covs were present in south and north america prior to the porcine δ-cov outbreaks in the period - ; (ii) which avian species represent significant natural δ-cov reservoirs; and (iii) what is the potential of avian δ-covs to cross the interspecies barrier and infect swine. orange circles mark states with the highest pig inventory [ ] . black duck icons identify the states where avian δ-covs were found (this study). in conclusion, this is the first report of the presence of hku -like δ-covs in different avian species in the us. the close relatedness of all the strains we were able to sequence to hku suggests a recent introduction of δ-covs in the us and identification of this strain as potentially parental. we also demonstrated that aquatic birds are infected with δ-covs and γ-covs more frequently than terrestrial avian species. this, together with the observation that porcine δ-covs are more closely related to δ-covs identified in terrestrial birds, suggests that waterfowl might represent a natural reservoir for δ-covs. although γ-covs were more prevalent than δ-covs, consistent with previous studies, both covs were detected more frequently during the cold season in our study. further studies are necessary to investigate the role of aquatic bird δ-covs in the epidemiology of δ-covs in swine and terrestrial birds. it remains to be established: (i) if/how long avian δ-covs were present in south and north america prior to the porcine δ-cov outbreaks in the period - ; (ii) which avian species represent significant natural δ-cov reservoirs; and (iii) what is the potential of avian δ-covs to cross the interspecies barrier and infect swine. an overview of their replication and pathogenesis coronavirus genome structure and replication international committee on taxonomy of viruses. virus taxonomy: classification and nomenclature of viruses porcine deltacoronavirus infection: etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis porcine deltacoronavirus: overview of infection dynamics, diagnostic methods, prevalence and genetic evolution detection and characterisation of coronaviruses in migratory and non-migratory australian wild birds molecular and cellular biology: gamma and deltacoronaviruses in quail and pheasants from northern italy coronaviruses detected in brazilian wild birds reveal close evolutionary relationships with betaand deltacoronaviruses isolated from mammals comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group c coronavirus detection of 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molecular evolutionary genetics analysis version . for bigger datasets sampling in saskatchewan detects deltacoronavirus of avian origin gammacoronavirus and deltacoronavirus in quail origin, evolution, and virulence of porcine deltacoronaviruses in the united states coronavirus infections in hospitalized pediatric patients with acute respiratory tract disease global seasonal occurrence of middle east respiratory syndrome coronavirus (mers-cov) infection seasonality of infectious diseases and severe acute respiratory syndrome-what we don't know can hurt us detection and molecular characterization of infectious bronchitis-like viruses in wild bird populations simulating the distribution of individual livestock farms and their populations in the united states: an example using domestic swine (susscrofa domesticus) farms porcine epidemic diarrhea virus and porcine deltacoronavirus not detected in waterfowl in the north american mississippi migratory bird flyway in state rankings by hog and pigs inventory this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -jcu pasy authors: thor, sharmi w.; hilt, deborah a.; kissinger, jessica c.; paterson, andrew h.; jackwood, mark w. title: recombination in avian gamma-coronavirus infectious bronchitis virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jcu pasy recombination in the family coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. however, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. in this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (ibv) isolates were sequenced and along with other full-length ibv genomes available from genbank were analyzed for recombination. evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins , and , and the structural spike glycoprotein. the extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within ibv. these data indicate that reticulate evolutionary change due to recombination in ibv, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus. avian infectious bronchitis virus (ibv) is a gamma-coronavirus in the family coronaviridae, the order nidovirales, and the genus coronavirus that causes a highly contagious upper-respiratory disease of domestic chickens. in layer type birds it can cause a drop in egg production and some strains are nephropathogenic. infectious bronchitis remains one of the most widely reported respiratory diseases of chickens worldwide despite the routine usage of attenuated live vaccines to control the disease. control of ibv is difficult because there is little to no cross-protection between the numerous different serotypes of the virus. infectious bronchitis virus is an enveloped, single-stranded, positive-sense rna virus with a genome length of approximately kb. the ' end of the genome encodes four structural proteins; spike (s), envelope (e), membrane (m) and nucleocapsid (n) as well as several non-structural proteins [ ] . the s glycoprotein of ibv forms projections on the surface of the virion. spike is post-translationally cleaved into s and s subunits with the s subunit forming the outermost portion and s forming a stalk-like structure that is embedded in the viral membrane. the s subunit contains hypervariable regions that play a role in attachment to host cell receptors, and it contains conformationally-dependent virus-neutralizing and serotype-specific epitopes [ , ] . spike is also involved in membrane fusion and viral entry into the host cell. the e and m proteins are integral membrane proteins involved in assembly of the virus. the n protein is closely associated with the viral genome and plays a role in replication. the ' two-thirds of the genome, approximately kb, encodes two polyproteins a and ab. a minus one frame-shift mechanism is used to translate the ab polyprotein. the polyproteins are post-translationally cleaved into non-structural proteins (nsps), nsp - (ibv does not have an nsp ) that make up the replication complex. key nsps encoded, include a papain-like protease (plp ) within nsp , a main protease (mpro) within nsp , and the rna-dependent rna-polymerase (rdrp) within nsps and . genetic diversity in coronaviruses is due to adaptive evolution driven by high mutation rates and genetic recombination [ ] . high mutation rates are attributed to minimal proof reading capabilities associated with the rdrp. recombination is thought to be due to a unique template switching "copy-choice" mechanism during rna replication [ ] . evidence of recombination among strains of ibv has been observed both experimentally and in the field [ ] [ ] [ ] [ ] [ ] [ ] . the emergence of several alpha-and beta-coronaviruses has been attributed to recombination [ , ] but only recently was recombination shown to be the mechanism behind the emergence of a novel gamma-coronavirus, turkey coronavirus (tcov) [ ] . although "hot spots" of recombination in the genome of ibv have been reported [ , ] , a thorough study of recombination using multiple different strains across the entire genome has not been conducted. in this study we sequenced and analyzed the entire genome of eight ibv strains that represent different serotypes that have not been previously sequenced, and we compared these sequences with other gamma-coronavirus full-length genome sequences available in genbank for evidence of recombination [ ] . different serotypes of field viruses and vaccine type viruses were selected to provide a wide variety of sequences potentially capable of contributing gene fragments to recombinants. the full-length genomes of eight isolates of ibv were sequenced at × to × coverage, and the consensus sequences were assembled. the genome size (see the end of the 'utr in table ), organization of the genome, and the location and size of the open reading frames (orfs) are listed in table for each of the viruses. the gene order is the same for all the viruses examined; 'utr- a/ab-spike- a- b-envelope-membrane- b- c- a- b-nucleocapsid- 'utr. in addition, the genomes for cav/cav b/ , de/de / , fl/fl / , mass/h , iowa/iowa / and jmk/jmk/ contain orf b between nucleocapsid and the 'utr. the full-length genomes were aligned and phylogenetic trees were constructed using the neighbor-joining, minimum evolution, maximum parsimony and upgma programs in mega [ ] . the trees all had similar topology and bootstrap support, and a representative tree is shown in figure . the feline coronavirus fcov/fipv/wsu- - and the beluga whale virus belugawhalecov/sw / were included as out-groups. the wild bird viruses isolated from a munia (muniacov/hkuy / ), thrush (thrushcov/hku / ) and bulbul (bulbulcov/hku / ) formed a unique clade, which is not surprising as this group might represent a new coronavirus genus provisionally designated deltacoronavirus [ ] . the remaining viruses separated into clades consisting of ibv isolates from the us and vaccine viruses, tcov isolates, an ibv isolate from west africa and ibv isolates from china and taiwan. vaccines for ibv used in commercial poultry include the serotypes mass, conn, de and ark. the peafowlccv/gd/kq / , ck/ch/lsd/ / and ck/ch/zj / strains from china grouped with mass type viruses indicating that they are closely related, which is not surprising since mass type vaccines are used in china. the overall percent similarities between the various strains are listed in supplemental table . all ibv genomes examined are greater than % similar at the nucleotide level. genomes available in the database for ck/ch/ep , ck/ch/p , and mass/beaudette were excluded from the analysis because they are viruses not found in the field. the recombination programs can be used to detect recombination without reference sequences, and our analysis was conducted without regard to date of isolation because that information was not available for some of the viruses. although the programs attempt to identify major and minor parent sequences contributing to each recombinant, the data reported herein only represents sequences in other viruses that are most closely related to the sequence surrounding the transferred fragment (major sequence) and the sequence closely related to the transferred fragment (minor sequences) and doesn't imply origin or source of the transferred fragment. in many cases, the transferred fragment has undergone mutations making it difficult to identify all the endpoints for the major and minor sequences. in addition, some of the transferred fragments overlap suggesting that recombinations have occurred between recombinant viruses. twenty-five ibv strains were examined and the viruses with the most transferred fragments in table are cav/ b/ and mass/h both with fragments, and ck/ch/lsd/ / and ga / / both with fragments. the strains with the fewest transferred fragments are iowa/iowa / and tw/ / with only transferred fragments and the ck/ch/bj/ , holte/holte/ , and nga/a e / strains with only transferred fragment. the ark/ark-dpi-p / and ark/ark-dpi-p / strains are the same virus that was passaged and times in embryonated eggs, respectively. both viruses share identical transferred fragments indicating that they have identical recombination history. in addition, conn/conn / and conn/conn / share the same recombination history ( identical transferred fragments). the conn/conn / field virus was used to produce an attenuated live vaccine, which is currently used in commercial poultry. viruses that share the same recombination history are likely derived from the same parent virus suggesting that conn/conn / is reisolated conn vaccine derived from the conn/conn / virus. the fl/fl / virus also shares all transferred fragments with the conn viruses, however; fl/fl / and conn viruses are different serotypes suggesting that fl/fl / is a field virus that emerged due to point mutations accumulating in spike over time rather than from recombination. all of the transferred fragments in ck/ch/zj / are identical to all of the transferred fragments in vaccine strain mass/h , providing compelling evidence that ck/ch/zj / is reisolated mass/h vaccine. that observation was also reported by zhang et al. [ ] . it is interesting that mass/h ( transferred fragments) and mass/h ( transferred fragments) share only identical transferred fragments. the mass/h and mass/h viruses were isolated circa in the netherlands and it is widely accepted that h stands for holland, but it actually stands for houben, the owner of the broiler farm where the viruses were isolated [ ] . it is thought that mass/h was derived from mass/h but the actual relationship between the viruses is not certain. our data indicates that they are not necessarily parent and progeny but they are closely related. the gray/gray/ and jmk/jmk/ viruses share . % nucleotide similarity across the entire genome and have identical transferred fragments with jmk/jmk/ having one additional fragment located in the 'utr, which is not found in gray/gray/ . it is well known that the gray/gray/ virus is nephropathogenic, whereas the jmk/jmk/ virus is strictly respirotropic. perhaps sequence differences in the 'utr, which is involved in replication of the viral genome, play a role in the different pathobiologies observed for these viruses. there is evidence that some transferred fragments in field viruses come from vaccines. as an example, ck/ch/lsd/ / has of and of transferred fragments in common with vaccine strains mass/h and mass/h , respectively. in addition, the only fragments that usa viruses have in common with the viruses from china and taiwan are fragments also associated with mass type vaccines, which are used in both regions, providing further evidence that some of the fragments in field viruses come from vaccines. that result and the observation in figure that the viruses separated into clades based on geographic location also supports the conclusion that usa viruses have not recombined with asian viruses. a difference in the order of taxa in phylogenetic trees constructed from different regions of the genome is further evidence of recombination [ ] . the ordering of taxa in sequential trees [ , ] was conducted and inconsistent phylogenetic relationships were observed for all of the examined virus strains across the entire genome, indicating a substantial amount of recombination (data not shown). there is a high number of breakpoints in the a region of the genome and immediately upstream of the s gene, which has been previously shown to be a 'hot spot' for recombination [ ] . a phylogenetic compatibility matrix constructed at the % bootstrap level for bp sequence fragments at bp intervals also showed that recombination breakpoints were distributed throughout the ibv genomes (data not shown). to determine recombination hot and cold spots, a recombination breakpoint distribution plot (figure ) was generated in rdp using a nt window and , permutations [ ] . no global hot-spot regions were observed in the % and % confidence thresholds (dotted lines at the top of the graph). the detectable recombination breakpoint positions are shown at the top of the figure and were distributed throughout the genome with a relatively high number clustered just upstream of the s gene. that region also had the highest breakpoint count within the % local hot/cold-spot confidence interval. a high number of breakpoints were also observed in the a region of the genome; nsp , nsp , and nsp , in the envelope and matrix protein genes and in a small area near the 'utr. table shows that nsp , nsp , nsp and spike genes were associated with the greatest number of transferred fragments, which is consistent with the location and number of breakpoints in figure . recombination in the ab orf area, which encodes the nonstructural proteins involved in the viral replication complex, has the potential to alter the pathogenicity of the virus [ ] . the nsp contains hydrophobic residues that likely anchor the replication complex to the golgi [ ] . the nsp encodes the protease plp which cleaves nsps , , and and an area with adp-ribose '-phosphatase (adrp) activity. the protease plp has been shown to have deubiquinating-like activity [ ] and also to be a type i interferon (ifn) antagonist [ ] . changes in the amino acid composition of this area could affect the ability of the virus to replicate in a variety of cell types. the adrp region of nsp is conserved among coronaviruses [ , ] , and a recent study suggested a biological role for the coronavirus adrp in modulating the expression of pro-inflammatory immune modulators such as tumor necrosis factor alpha and interleukin- [ ] . recombination in this area could alter the pathogenicity of the virus by modulating host cytokine expression. the nsp is reported to be an s-adenosyl-l-methionine (adomet)-dependent rna (nucleoside- 'o)-methyltransferase ( 'o-mtase) responsible for capping the viral mrna nascent transcripts [ ] . an alteration in the efficiency of this protein could profoundly decrease not only viral replication but also pathogenicity. the spike glycoprotein of ibv on the surface of the virus plays a role in attachment to host cell receptors, membrane fusion and entry in this study, evidence was obtained that recombination is occurring among avian coronavirus ibv isolates across their entire genome. every sequence included in the analysis was recognized as a potential recipient of horizontally acquired sequences at some point in its viral evolutionary past. the nsp , nsp , nsp were associated with the greatest number of transferred fragments. in addition, the area immediately upstream of the spike gene had the highest number of recombination breakpoints. breakpoints in the ab polyprotein gene have the potential to alter pathogenicity of the virus, and breakpoints near or in spike have the potential to lead to the emergence of new serotypes of ibv or new coronaviruses. although the spike region determines the serotype of the virus, the remainder of the genome may be a mosaic of sequence fragments from a variety of gamma-coronaviruses. the only evidence of a gamma-coronavirus possibly recombining with an alpha or beta-coronavirus was the the pcr reaction was run on the same machine as the rt step and included a one-time initial denaturation step of °c for min, followed by cycles of °c for s, °c for s and °c for min. the pcr products were agarose gel purified using the qiaquick gel extraction kit (qiagen, valencia, ca, usa) according to the manufacturer's protocol. the pcr products were cloned into the topoxl vector using the topoxl cloning kit (invitrogen, carlsbad, ca, usa) according to manufacturer's protocol to prepare cdna libraries for sequencing. plasmid dna from the libraries of the cloned cdna fragments for each virus was isolated using an alkaline lysis method modified for the -well format and incorporating both hydra and tomtek robots. sequencing reactions were performed using the bigdye™ terminator® cycle sequencing kit version . (applied biosystems, foster city, ca, usa) and mj research (watertown, ma, usa) thermocyclers. sephadex filter plates were used to filter each reaction into perkin-elmer microamp optical -well plates. a / -strength sequencing reaction on an abi was used to sequence each clone from both the ' and ' ends. primers for one-step rt-pcr were specifically designed for each virus (supplemental table ). viral rna was amplified using the titan one tube rt-pcr kit (roche diagnostics, indianapolis, in, usa) following manufacturer's instructions. a dna engine peltier thermocycler (bio-rad laboratories, inc., hercules, ca, usa) was used for the rt-pcr reaction, which had the following steps: one cycle of °c for min and °c for min, followed by cycles of °c for s, °c for s, and °c for min s, and then cycles of °c for s, °c for s, °c for min and s adding s with each cycle. the resulting pcr products were agarose gel purified using the qiaquick gel extraction kit (qiagen, valencia, ca, usa) according to the manufacturer's protocol. the resulting cdna was sequenced using abi prism bigdye terminator cycle sequencing ready reaction kit (applied biosystems, foster city, ca, usa) following the manufacturer's protocol. the reactions were prepared for sequencing by centrifugation through either a centri-sep column (applied biosystems, foster city, ca, usa) or using the edge system (edgebio, gaithersburg, md, usa) plate. the samples were sequenced at the georgia genomics facility (university of georgia, athens, ga, usa). chromatogram files and trace data were read and assembled using seqman pro, and genome annotation was conducted with seqbuilder (dnastar, inc., madison, wi, usa). each sequence was aligned to a representative genome; mass/mass / (genbank accession #ay ), or cal /cal / (genbank accession #ay ) as a backbone for genome assembly. whole genome analyses were generated and phylogenetic trees constructed with the neighbor-joining method with bootstrap replicates as well as with minimum evolution, maximum parsimony and upgma methods [ ] . ibv strains in phylogenetic trees generated from sequential genome fragments using treeorder scan (version . , simmonics, university of warwick, coventry, uk) [ , ] . changes in the tree position of taxa supported at the % or greater bootstrap level for a bp sequence window were examined at bp intervals. in addition, a phylogenetic compatibility matrix was constructed and used to examine the frequency and location of recombinations across the entire genome. potential recombination sites were identified using the rdp software [ ] and a breakpoint map was constructed. a breakpoint density plot was then created from this map by moving a nt window nt at a time along the length of the map. the number of breakpoints falling within a window was plotted at the central window position. a % (upper) and % (lower) confidence threshold for globally significant breakpoint clusters (defined as windows with more breakpoint positions than the maximum found in > % of the , permuted plots) was calculated. in addition, % and % confidence intervals were calculated for local breakpoint clusters (defined as windows with more breakpoint positions than the maximum found in > % of the windows at that location in , permuted plots). genbank accession numbers virus genome sequences generated in this study were submitted to genbank and assigned the following accession numbers: cav/cav b/ (gu gray/gray/ (gu ) mass/beaudette (nc_ ), nga/a e / (fn ), ita/ / (fn ) references and notes the viruses and their replication does ibv change slowly despite the capacity of the spike protein to vary greatly? the neutralization epitopes on the spike protein of infectious bronchitis virus and their antigenic variation the evolution and emergence of rna viruses rna recombination in animal and plant viruses sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains origin and evolution of georgia (ga ), a new serotype of avian infectious bronchitis virus evidence of genetic diversity generated by recombination among avian coronavirus ibv a recombination event, induced in ovo, between a low passage infectious bronchitis virus field isolate and a highly embryo adaptedvaccine strain naturally occurring recombination between distant strains of infectious bronchitis virus coronavirus diversity, phylogeny and interspecies jumping recombinant canine coronaviruses related to transmissible gastroenteritis virus of swine are circulating in dogs emergence of a group coronavirus through recombination identification and analysis of the georgia serotype, a new serotype of infectious bronchitis virus molecular evolutionary genetics analysis (mega) software version . coronavirus genomics and bioinformatics analysis evolutionary aspects of recombination in rna viruses a simple and robust statistical test for detecting the presence of recombination recombination patterns in aphthoviruses mirror those found in other picornaviruses recombination detection and analysis using rdp complete genome sequence and recombination analysis of infectious bronchitis virus attenuated vaccine strain h comparative analysis of coronavirus hku genomes reveals a novel genotype and evidence of natural recombination in coronavirus hku frequency and dynamics of recombination within different species of human enteroviruses recombination in the genesis and evolution of hepatitis b virus genotypes the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity dynamics of coronavirus replication-transcription complexes the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis putative papain-related thiol proteases of positive-strand rna viruses. identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha-and coronaviruses mouse hepatitis virus liver pathology is dependent on adp-ribose- ''-phosphatase, a viral function conserved in the alphalike supergroup testing the hypothesis of a recombinant origin of the sars-associated coronavirus analysis of recombination and natural selection in human enterovirus infectious bronchitis application of phylogenetic networks in evolutionary studies neighbor-net: an agglomerative method for the construction of phylogenetic networks a modified bootscan algorithm for automated identification of recombinant sequences and recombination breakpoints possible emergence of new geminiviruses by frequent recombination analyzing the mosaic structure of genes evaluation of methods for detecting recombination from dna sequences: computer simulations sister-scanning: a monte carlo procedure for assessing signals in recombinant sequences an exact nonparametric method for inferring mosaic structure in sequence triplets evaluation of methods for detecting recombination from dna sequences: empirical data detecting and characterising individual recombination events: practice this work was supported by usda, csrees award number - - . the authors would like to thank the technical help of jon s. robertson and cornelia lemke with sequencing. discovery of the mosaic nature of the sars-coronavirus genome [ ] . although this type of recombination is possible it appears to be rare in nature.in this study, we characterized recombination in the full-length genomes of avian gammacoronavirus ibv strains from around the world. our bioinformatic analysis was similar to a previous study on enteroviruses [ ] and we found that recombination in ibv is more extensive than formerly thought, involving regions across the entire genome. our data suggests that reticulate evolution due to a high frequency of recombination in ibv, likely plays a major role in the generation of new serotypes of the virus. the characterization, distribution and frequency of recombination breakpoints are important information that will further our understanding of the mechanisms behind the diversity and evolution of these viruses so that better control methods can be developed. all of the viruses sequenced in this study (table ), were propagated in - day-old specific-pathogen-free (spf) embryonated eggs as described [ ] . total rna was isolated from μl of allantoic fluid collected from the infected eggs using the high pure rna isolation kit (roche applied science, mannheim, germany) following the manufacturer's instructions. the amplification reactions were carried out using strand displacement rt-pcr or one step rt-pcr. strand displacement rt-pcr uses a random (at the ' end) primer and an amplification primer. the sequence of the random primer was (agcgggggttgtcgaatgtttgannnn) and the sequence of the amplification primer was (agcgggggttgtcgaatgtttga). the rt-pcr reaction was carried out using the takara rna la pcr kit (takara bio. inc., otsu, shiga, japan) according to the manufacturer's protocol. a dna engine peltier thermocycler (bio-rad laboratories inc., hercules, ca, usa) was used for the rt reaction, which included an rna denaturing step at °c for min followed by °c for min, °c for min, °c for min, and °c for min. we used neighbor-net analysis to examine the ibv genomes for evidence of networked relationships and the pairwise homoplasy index (phi) in splitstree (version , simmonics, university of warwick, coventry, uk) [ , , ] to statistically determine the likelihood of recombination. in addition, the ibv genomes were examined for recombination breakpoints using the recombination detection program (rdp , version , simmonics, university of warwick, coventry, uk) [ , ] . unless otherwise stated, default settings were used in all of the programs. the specific algorithms used were rdp [ ] , geneconv [ ] , bootscan/rescan [ ] , maximum chi square [ ] , chimaera [ ] , siscan [ ] , and seq [ ] . we used more than one method to analyze the data because evaluation of these recombination detection methods using both simulated and empirical data showed that the results from only a single method were not very reliable [ ] . automasking was used for optimal recombination detection. the rdp analysis was run without a reference and a window size of , bootscan window size was increased to , maxchi and chimaera number of variable sites per window was increased to , and the window size and step size for siscan was increased to and , respectively. the window sizes were increased from their default settings because ibv has a high mutation rate, which can mask recombination signals. increasing the window size was shown to increase the ratio of recombination signals relative to mutational "noise" [ ] . inconsistent phylogenetic relationships between different regions of the viral genome provide further evidence of genetic recombination. herein, we examined the order of avian gamma-coronavirus key: cord- -txdc c j authors: laing, eric d.; sterling, spencer l.; weir, dawn l.; beauregard, chelsi r.; smith, ina l.; larsen, sasha e.; wang, lin-fa; snow, andrew l.; schaefer, brian c.; broder, christopher c. title: enhanced autophagy contributes to reduced viral infection in black flying fox cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: txdc c j bats are increasingly implicated as hosts of highly pathogenic viruses. the underlying virus–host interactions and cellular mechanisms that promote co-existence remain ill-defined, but physiological traits such as flight and longevity are proposed to drive these adaptations. autophagy is a cellular homeostatic process that regulates ageing, metabolism, and intrinsic immune defense. we quantified basal and stimulated autophagic responses in black flying fox cells, and demonstrated that although black flying fox cells are susceptible to australian bat lyssavirus (ablv) infection, viral replication is dampened in these bat cells. black flying fox cells tolerated prolonged ablv infection with less cell death relative to comparable human cells, suggesting post-entry mechanisms interference with virus replication. an elevated basal autophagic level was observed and autophagy was induced in response to high virus doses. pharmacological stimulation of the autophagy pathway reduced virus replication, indicating autophagy acts as an anti-viral mechanism. enhancement of basal and virus-induced autophagy in bat cells connects related reports that long-lived species possess homeostatic processes that dampen oxidative stress and macromolecule damage. exemplifying the potential that evolved cellular homeostatic adaptations like autophagy may secondarily act as anti-viral mechanisms, enabling bats to serve as natural hosts to an assortment of pathogenic viruses. furthermore, our data suggest autophagy-inducing drugs may provide a novel therapeutic strategy for combating lyssavirus infection. bats, order chiroptera, host a significantly greater richness of virus species compared to other mammalian taxa [ ] . viruses hosted by bats include several that are highly pathogenic in human hosts such as henipaviruses (e.g., nipah virus (niv) and hendra virus (hev)) [ ] , filoviruses (e.g., marburg virus (marv)) [ , ] , coronaviruses (e.g., sars-like coronavirus) [ ] , and lyssaviruses (e.g., rabies virus (rabv)) [ ] . bats experimentally infected with niv and hev [ , , ] , ebola virus in a dose-dependent manner, in both black flying fox-and human-derived cell lines, which we confirmed in primary black flying fox brain cells. activation of autophagy through pharmacological methods decreased ablv replication in both black flying fox and human cells, which suggested ( ) that autophagy functions as an anti-viral defense during ablv infection, and ( ) that activation of autophagy might be an effective treatment against neurotropic viruses such as ablv or related lyssaviruses. lastly, we observed that in contrast to human cells, black flying fox brain-derived cells withstood a high dose of ablv over a long incubation period and experienced significantly less cell death. our findings provide an initial in vitro exploration for future studies that may illuminate the importance of autophagy as an enhanced post-transcriptional anti-viral pathway in bats. black flying fox brain (pabrh) and kidney (pakit) tissue-derived cell lines and primary brain (pabr) cells have been previously described [ ] . pabrh and pakit cells were maintained in dmem (dulbecco's modified eagle medium (gibco laboratories; gaithersburg, md, usa), % hyclone ™ cosmic calf ™ serum (ccs) (thermo fisher scientific; waltham, ma, usa), and % l-glutamine (thermo fisher scientific)) complete cell culture media (dmem- ). primary pabr cells were maintained in dmem/nutrient f- ham media (sigma-aldrich; st. louis, mo, usa) with % fetal bovine serum (gibco) and % antibiotic-antimycotic (gibco). a human neuroblastoma cell line (nbf-l) was obtained from dr. aviva symes (uniformed services university, bethesda, md, usa) and maintained in dmem supplemented with % cosmic calf ™ serum (thermo fisher scientific), % fetal bovine serum (gibco), and % glutamax ™ (thermo fisher scientific). human embryonic kidney (hek) t (atcc ® crl- ™ ) and mouse neuro- a (atcc ® ccl- ™ ) cells were obtained from the american type culture collection (atcc; manassas, va, usa) and maintained in dmem- complete media. a recombinant australian bat lyssavirus (rablv), human isolate [ ] , anti-genome plasmid was used to generate a reporter virus through reverse genetics and a wild-type ablv (wt-ablv), pteropus isolate [ ] , was also used for infection studies (ncbi genbank accession number: af ). the open reading frame of turbo green fluorescent protein (gfp; evrogen; moscow, russian federation) was cloned into the rablv anti-genome plasmid between the ablv glycoprotein (g) and large rna polymerase (l) genes. to rescue rablv-gfp, hek t cells were seeded × cells/well in -well plates and transfected with µg full-length rablv-gfp anti-genome plasmid and µg nucleoprotein (n), . µg phosphoprotein (p), and . µg l protein helper plasmids. the total recombinant plasmid dna ( µg) was mixed with µl lipofectamine ltx ® (thermo fischer scientific) in opti-mem (gibco), following standard transfection protocol. hek t cells were maintained in dmem- complete media. cell culture supernatants were replaced hours post-transfection with dmem, % ccs and % l-glutamine. three days post-transfection, fluorescent microscopy was used to examine gfp expression. wells that contained gfp-positive cells were passaged and co-cultured with mouse neuro- a cells. cells were continuously monitored for gfp expression and cell culture supernatant was passaged to fresh hek t cells. to purify rablv-gfp, cell culture supernatant containing rablv-gfp was collected and ultracentrifuged ( , rpm, h) with a % sucrose cushion. pabrh, pakit, hek t, nbf-l, or primary pabr cells ( . × cells/well, -well cell culture plates) were infected with rablv-gfp or wt-ablv at the indicated multiplicities of infection (moi) (e.g., , . , ). to compare replication kinetics between pabrh, pakit, hek t, and nbf-l, cell lines were incubated with ml dmem- containing rablv-gfp (moi ) for h. after h, virus/dmem- was removed and replaced with fresh dmem- . cell culture supernatants were collected and centrifuged to remove cell debris ( rpm, min) then serially diluted and incubated with hek t cells to determine ablv titers. gfp expression from rablv-gfp was used to count the number of fluorescent virus plaques, whereas fitc-conjugated anti-rabies monoclonal globulin (fujirebio diagnostics, inc.; malvern, pa, usa) was used to quantify wt-ablv titers. to quantify gfp expression by flow cytometry (lsr ii; bd biosciences; san jose, ca, usa), cell culture supernatants were collected and centrifuged to collect floating cells, then mixed with the collected adherent cells, and fixed with % paraformaldehyde. gfp median fluorescence intensity (mfi) and the percentage of gfp positive cells were then analyzed. bafilomycin a (bafa ; nm; invivogen; san diego, ca, usa) was used to inhibit fusion of autophagosomes and autolysosomes, i.e., autophagic flux. to block autophagy, cells were incubated for two hours at the end of the infection time course with bafa prior to whole cell lysate processing. whole cell protein lysates were collected at indicated hours post-infection (hpi) and processed following protocol guidelines for autophagic quantification by lc b western blots [ ] . cell culture protein lysates were normalized to µg, and loaded into - % bis-tris protein gel (invitrogen; carlsbad, ca, usa); protein gels were transferred to pvdf membranes (bio-rad; hercules, ca, usa; ma, h) for standard western blotting techniques. antibodies used for immunoblots included: anti-lc b polyclonal antibody (sigma-aldrich), anti-p polyclonal antibody (sigma-aldrich), anti-ndp polyclonal antibody (aviva systems biology; san diego, ca, usa), anti-β-actin polyclonal antibody (abcam; cambridge, uk), anti-gapdh polyclonal antibody (abcam), and anti-turbo gfp polyclonal antibody (evrogen). an anti-rabies virus n protein polyclonal rabbit serum was provided by dr. ina smith and anti-rabies virus p protein polyclonal antibody was purchased (mybiosource; san diego, ca, usa). protein levels were quantified using image j software (nih; bethesda, md, usa). pabrh and nbf-l cell lines at a density of . × cells/well ( -well plate) were infected with rablv-gfp. cell culture supernatant was not removed after the addition of rablv-gfp until the conclusion of the post-infection time course. cell culture supernatant and cells were collected, centrifuged for min, × g, resuspended in ml pbs, and dyed with live/dead™ fixable violet dead cell stain kit, for nm excitation (thermo fisher scientific) for min following the manufacturer's guidelines. cells were fixed with % paraformaldehyde and incubated at • c for min. cells were then pelted and washed with pbs, this process was repeated twice. resuspended stained cells were analyzed with an lsr ii flow cytometer (bd biosciences) for ablv gfp expression and live/dead violet signal. pabrh, pakit, and nbf-l cells at a density of . × cells/well ( -well plate) were infected with rablv-gfp (moi . ) for h. at hpi, rapamycin (rapa; sigma-aldrich), small molecule enhancer of autophagy- (smer; tocris; bristol, uk), or nvp bez (bez; selleck chemicals; houston, tx, usa) were added to the wells and incubated with the ablv-infected cell culture for h. at hpi, cell culture supernatants and whole cell lysates were collected. whole cell lysates were processed as described above. supernatants were centrifuged for min at rpm to remove cell debris, then serially diluted and incubated with hek t cells at × cells/well ( -well plate) to determine rablv-gfp titers; fitc anti-rabies monoclonal globulin (fujirebio diagnostics) was used to quantify wt-ablv titers after bez treatments. we attempted to monitor autophagy levels by flow cytometry after rapa and smer induction using a cyto-id ® autophagy detection kit (enzo life sciences, inc.; famingdale, ny, usa), which stains autophagosomes with a green fluorescent dye; a positive stain was confirmed using the pakit cell line. ablv, like all lyssaviruses, has a '- '(-)ssrna genome that encodes five proteins: nucleoprotein (n), phosphoprotein (p), matrix protein (m), envelope glycoprotein (g), and large rna polymerase (l). we used reverse genetic techniques to generate a recombinant ablv reporter virus expressing a green fluorescent protein (rablv-gfp) ( figure a ). the ablv anti-genome plasmid was modified with the insertion of a gfp open reading frame between the g and l genes. first, we compared ablv replication in black flying fox and human cell lines. to conduct these experiments, we utilized brain (pabrh) and kidney (pakit) tissue-derived black flying fox cell lines [ ] . the pabrh cell line is morphologically fibroblast-like in appearance so we chose to compare ablv replication with a human neuroblastoma cell line (nbf-l) that also had fibroblast-like morphology [ ] . a human embryonic kidney cell line, that was similarly immortalized with a sv t antigen (hek t), was included for comparative purposes with the pakit cell line. plasmid was modified with the insertion of a gfp open reading frame between the g and l genes. first, we compared ablv replication in black flying fox and human cell lines. to conduct these experiments, we utilized brain (pabrh) and kidney (pakit) tissue-derived black flying fox cell lines [ ] . the pabrh cell line is morphologically fibroblast-like in appearance so we chose to compare ablv replication with a human neuroblastoma cell line (nbf-l) that also had fibroblast-like morphology [ ] . a human embryonic kidney cell line, that was similarly immortalized with a sv t antigen (hek t), was included for comparative purposes with the pakit cell line. lower rablv-gfp titers were quantified from both infected black flying cell lines compared to human cell lines at equivalent hpi ( figure b ). nearly -fold higher titers were achieved in both human compared to black flying fox cell lines at and hpi. quantification of gfp positive cells after rablv-gfp infection revealed that nearly double the number of human cells were infected compared to black flying fox cell lines at and hpi ( figure c ). investigation of ablv protein levels-including nucleoprotein (n), phosphoprotein (p), and the virus-expressed gfp (vgfp) ( figure d )-revealed lower amounts of structural and non-structural ablv proteins in black flying fox cells. gfp expression in black flying fox cells and human cells indicated that all four cell lines were susceptible to rablv-gfp infection, although multiple mechanisms of cellular entry, replication, or post-entry might contribute to the reduced level of replication in black flying fox cells relative to human cells. to avoid issues with inefficient ablv entry into black flying fox cells, in the following experiment the conditions of rablv-gfp infection were modified to promote infection of close to % of the cells. infection, although multiple mechanisms of cellular entry, replication, or post-entry might contribute to the reduced level of replication in black flying fox cells relative to human cells. to avoid issues with inefficient ablv entry into black flying fox cells, in the following experiment the conditions of rablv-gfp infection were modified to promote infection of close to % of the cells. we next sought to further investigate the ablv replication differences between the cells and determine whether black flying fox cells tolerated high doses of ablv infection. notably, ablv infection increased cell death in human and black flying fox cells in a dose-dependent manner ( figure a ). black flying fox (pabrh) cells experienced an increase in cell death at hpi (moi ), although a relatively lesser extent compared to human nbf-l cells, which was significantly higher than pabrh cells at this hpi ( figure b ). the percentage of gfp positive black flying fox cells was also dose-dependent. unlike in figure , rablv-gfp was not washed out after two hpi, which would lessen the effects of inefficient virus entry of black flying fox cells. gfp positive percentages between human and black flying fox cells were comparable with moi at (~ vs. %) and hpi (~ vs. %), whereas the percentage of gfp positive black flying fox cells was nearly half compared to human cells (~ vs. %) hpi, moi . ( figure c ). more strikingly, a significantly lower vgfp median fluorescence intensity (mfi), which is the quantification of vgfp expression and dependent on post-entry virus replication, was quantified in black flying fox cells compared to a human cell line hpi at both moi and ( figure d ). thus, despite a similar percentage of infected cells at and hpi (moi ) in both nbf-l and pabrh cells ( figure c ), lower vgfp in pabrh cells suggested that an intrinsic mechanism, not inefficient entry, restricted production of ablv encoded proteins (e.g., gfp) or replication. viruses , , x for peer review of we next sought to further investigate the ablv replication differences between the cells and determine whether black flying fox cells tolerated high doses of ablv infection. notably, ablv infection increased cell death in human and black flying fox cells in a dose-dependent manner ( figure a ). black flying fox (pabrh) cells experienced an increase in cell death at hpi (moi ), although a relatively lesser extent compared to human nbf-l cells, which was significantly higher than pabrh cells at this hpi ( figure b ). the percentage of gfp positive black flying fox cells was also dosedependent. unlike in figure , rablv-gfp was not washed out after two hpi, which would lessen the effects of inefficient virus entry of black flying fox cells. gfp positive percentages between human and black flying fox cells were comparable with moi at (~ vs. %) and hpi (~ vs. %), whereas the percentage of gfp positive black flying fox cells was nearly half compared to human cells (~ vs. %) hpi, moi . ( figure c ). more strikingly, a significantly lower vgfp median fluorescence intensity (mfi), which is the quantification of vgfp expression and dependent on postentry virus replication, was quantified in black flying fox cells compared to a human cell line hpi at both moi and ( figure d ). thus, despite a similar percentage of infected cells at and hpi (moi ) in both nbf-l and pabrh cells ( figure c ), lower vgfp in pabrh cells suggested that an intrinsic mechanism, not inefficient entry, restricted production of ablv encoded proteins (e.g., gfp) or replication. to determine whether an intrinsic defense mechanism was constraining ablv replication, we next focused on the anti-viral potential of the autophagy pathway. autophagy functions as an antiviral mechanism during vesicular stomatitis virus (vsv) infection [ , ] . vsv is a member of family to determine whether an intrinsic defense mechanism was constraining ablv replication, we next focused on the anti-viral potential of the autophagy pathway. autophagy functions as an anti-viral mechanism during vesicular stomatitis virus (vsv) infection [ , ] . vsv is a member of family rhabdoviridae, which includes lyssaviruses such as rabies virus (rabv) and rabies virus-related viruses (e.g., ablv). wild-type rabv infection of a human neuroblastoma cell line was shown to activate the autophagy pathway [ ] . a hallmark feature of autophagy induction is the cleavage and lipidation of the cytosolic microtubule-associated protein a/ b light chain b (lc b-i) to autophagosomal-associated lc b-phosphatidylethanolamine (lc b-ii). lc b-ii is incorporated into the developing autophagophore and retained in the autophagosome double membrane vesicle such that increased cellular levels of lc b-ii are used as a marker for the induction of autophagy [ ] . functional human and black flying fox lc b proteins are identical in amino acid sequence after post-translational cleavage. therefore, we compared quantities of lc b-ii between black flying fox and human cell lines by immunoblotting, following inhibition and stimulation of autophagy. bafilomycin-a (bafa ) inhibits autophagic flux by blocking the fusion of autophagosomes and autolysosomes [ , ] , causing a buildup of autophagosomes and lc b-ii. we treated pabrh, pakit, and nbf-l cells with bafa for two hours to inhibit autophagic flux and create a snapshot of basal autophagy levels in all three cell lines. as expected, bafa treatment significantly increased levels of lc b-ii in all three cell lines ( figure a,b) . interestingly, in the bafa treated cells, levels of lc b-ii were significantly higher in both pabrh and pakit compared to nbf-l ( figure a,b) , suggesting that these black flying fox cell lines maintained a higher basal level of autophagic flux. to determine whether autophagy was activated in bat versus human cells after ablv infection, we again monitored levels of lc b-i and lc b-ii. pabrh cells showed a significant percentage of lc b-ii conversion when infected with a moi at and hpi ( figure c,d) . the nbf-l cells had a significant percentage of lc b-ii when infected for hpi (moi . and ) ( figure e ,f). we confirmed these results in primary black flying fox brain (pabr) cells. a significant increase in lc b-ii was again dependent on a high virus dose, moi ( figure a,b) . additionally, treatment with bafa increased lc b-ii, however the levels of lc b-ii remained statistically equivalent regardless of ablv moi. as there was not an additive effect of ablv infection plus bafa treatment, it is possible that the basal level of autophagic processes are so rapid that external stimulation (a) has little observable effect by comparison or (b) additional stimulation via ablv infection increased autophagic flux such that autophagosome and lc b-ii turnover occurs more rapidly. these results suggested that although ablv induced autophagy in both black flying fox and human cells, further enhancement of autophagic flux in black flying fox cells required a high virus dose. this observation highlights the need for additional studies of autophagic kinetics, both basal and induced, in black flying fox cells. viruses , , x for peer review of to further characterize the increase in lc b-ii after ablv incubation, we monitored cytosolic levels of autophagy cargo receptor proteins, p and ndp , which are associated with selective autophagy and used to monitor autophagic flux [ ] [ ] [ ] [ ] . virus proteins, such as hiv nef protein and influenza m , inhibit autophagic flux by blocking fusion of autophagosomes with autolysosomes [ , ] . inhibition of autophagic flux and the resulting accumulation of autophagosomes and their contents such as lc b-ii could be misinterpreted as virus activation of autophagy. if autophagic flux is unobstructed by ablv infection, p and ndp will be degraded, whereas inhibition of autophagic flux will lead to an increase in these cargo proteins. we examined levels of p and ndp in pabrh cells after ablv infection, and in combination with bafa treatment. infection with ablv alone resulted in less p ( figure a ,b) and ndp ( figure a ,c) detected in pabrh cells at hpi. in nbf-l cells, a mean fold decrease in p was observed at both and hpi ( figure d ,e). treatment with bafa alone or in combination with ablv infection at all time points resulted in a significant increase of p and ndp in pabrh cells ( figure a-c) and nbf-l cell ( figure d ,e), however p and ndp fold-changes between ablv alone and ablv plus bafa were insignificant. since additive effects of bafa inhibition of autophagic flux were not observed during ablv infection and we observed a fold decrease of p and ndp hpi, we concluded that ablv alone was not inhibiting autophagic flux. together, increased lc b-ii (figures and ) and decreased p /ndp protein levels ( figure ) in black flying fox cells further demonstrated that ablv infection induced the autophagy pathway. viruses , , x for peer review of (a). all data are representative of three independent experiments, mean ± sem and * p < . anova (two-way), and tukey's multiple comparison test ( hpi moi vs. moi ). to further characterize the increase in lc b-ii after ablv incubation, we monitored cytosolic levels of autophagy cargo receptor proteins, p and ndp , which are associated with selective autophagy and used to monitor autophagic flux [ ] [ ] [ ] [ ] . virus proteins, such as hiv nef protein and influenza m , inhibit autophagic flux by blocking fusion of autophagosomes with autolysosomes [ , ] . inhibition of autophagic flux and the resulting accumulation of autophagosomes and their contents such as lc b-ii could be misinterpreted as virus activation of autophagy. if autophagic flux is unobstructed by ablv infection, p and ndp will be degraded, whereas inhibition of autophagic flux will lead to an increase in these cargo proteins. we examined levels of p and ndp in pabrh cells after ablv infection, and in combination with bafa treatment. infection with ablv alone resulted in less p ( figure a ,b) and ndp ( figure a ,c) detected in pabrh cells at hpi. in nbf-l cells, a mean fold decrease in p was observed at both and hpi ( figure d ,e). treatment with bafa alone or in combination with ablv infection at all time points resulted in a significant increase of p and ndp in pabrh cells ( figure a-c) and nbf-l cell ( figure d,e) , however p and ndp fold-changes between ablv alone and ablv plus bafa were insignificant. since additive effects of bafa inhibition of autophagic flux were not observed during ablv infection and we observed a fold decrease of p and ndp hpi, we concluded that ablv alone was not inhibiting autophagic flux. together, increased lc b-ii (figures and ) and decreased p /ndp protein levels ( figure ) in black flying fox cells further demonstrated that ablv infection induced the autophagy pathway. to understand whether the autophagy pathway had an anti-viral role during ablv infection, we examined the effects of pharmacological activation of autophagy on ablv replication. the mammalian target of rapamycin (mtor) is a key modulator of autophagy [ ] . we therefore tested the effects of both mtor-dependent autophagy activation using the mtor inhibitor rapamycin (rapa), as well as mtor-independent autophagy activation with small molecule enhancer of autophagy- (smer) stimulation [ , ] . rablv-gfp infected black flying fox cell lines, pabrh and pakit, were treated with rapa or smer, respectively, during ablv infection to elucidate whether activation of autophagy had anti-viral effects; human nbf-l cells were treated the same in comparison. rapa and smer activation of autophagy was monitored by cyto-id ® (enzo) staining of autophagosomes in pakit cells. treatment with either rapa or smer induced autophagy in pakit cells, with rapa showing a stronger effect ( figure a ). chloroquine (chq), an inhibitor of autophagic flux, was included as a positive control for autophagosome staining [ ] . treatment of black flying fox and human cells with rapa after ablv infection resulted in significant reductions in ablv replication ( figure b) . a significant reduction in ablv titers after smer treatment was only observed in human nbf-l cells, and a decreasing titer trend was observed in smer treated black flying fox cells. as a potential mechanism underlying the reduction in ablv, we examined whether treatment with rapa or smer resulted in degradation of virus proteins. cell lysates collected after rapa and smer treatment of pabrh and pakit cells, followed by analysis via western blot ( figure c -f). neither rapa or smer treatment had any significant effects on n or p protein levels in pabr cells, however, n protein fold change had a decreasing trend in rapa treated cells ( figure c,d) . ablv protein levels in bat pakit cells, infected and treated with rapa and smer, were examined by western blot (figure e,f) . as seen in the pabrh cells, n protein fold change had a decreasing trend in pakit cells when treated with rapa, but not smer. p protein levels were quantified for two independent experiments, and also had a trending decease in both rapa-and smer-treated cells. although, western blot quantification of vgfp can be difficult because of low replication and vgfp expression levels in bat cells, we successfully quantified vgfp from two of the four independent experiments with pakit cells. the noticeable vgfp fold decrease caused by rapa and smer stands in contrast to fold changes in n and p proteins. human nbf-l cells had a significant fold decrease of vgfp levels in both rapa-or smer-treated cells, but neither n nor p levels had any significant reductions ( figure g,h) . these results indicated that autophagy acts as an anti-viral response in both black flying fox cells and human cells during ablv infection. given our limited on-hand reagents (e.g., ablv n and p reactive antisera), we were only able assess the effects of autophagy activation on n and p protein levels. it is possible that other ablv proteins such as m protein or l rna polymerase, to which we lack reagents, are degraded by autophagy, and that degradation of these proteins leads to decreased ablv replication as assessed by lower ablv titers ( figure b ) and ablv vgfp expression ( figure f,h) . while conducting this study we observed that autophagy activation through pharmacological modulators (e.g., rapa) reduced ablv replication and protein expression in a human neuroblastoma cell line (nbf-l). infected neurons cannot rely on the anti-viral effects of ifn-induced cell death to control viral infection and risk potential damage to the central nervous system [ ] . given the neurotropism of lyssaviruses such as ablv and rabv, we asked whether potent autophagy induction with dactolisib (a.k.a. nvp bez or bez), a dual inhibitor pan-class pi k and mtor inhibitor evaluated in clinical trials, could also restrict ablv replication in nbf-cells. treatment of nbf-l cells with bez increased the amount of autophagosomal lc b-ii indicative of autophagy activation ( figure a ) without causing significant cytotoxicity ( figure b ). to examine the anti-viral effect of bez treatment on ablv replication, nbf-l cells were infected with wt-ablv for h then treated with bez. treatment with bez resulted in significant dose-dependent decreases of wt-ablv titers ( figure c ), in addition to n and p protein levels ( figure d,e) . although bez advanced to phase i/ii clinical trials as a potential anti-cancer therapy [ ] [ ] [ ] , its use was discontinued due to adverse toxicities [ ] . nevertheless, our results suggest that with improved selectivity or dosing, potent inducers like bez may be promising additional therapeutics to prevent lethality following lyssavirus exposure. fold change in ablv n, p, and vgfp proteins (β-actin-normalized) collected from nbf-l cell lysates. (c-h) whole cell lysates were collected hpi (rablv-gfp moi . ). fold change is a representation of at least three independent experiments, mean ± sem and ** p < . , * p < . anova (one-way) analysis, dunnett's multiple comparison test with treatments compared to dmso mock treated cells. while conducting this study we observed that autophagy activation through pharmacological modulators (e.g., rapa) reduced ablv replication and protein expression in a human neuroblastoma cell line (nbf-l). infected neurons cannot rely on the anti-viral effects of ifn-induced cell death to control viral infection and risk potential damage to the central nervous system [ ] . given the neurotropism of lyssaviruses such as ablv and rabv, we asked whether potent autophagy induction with dactolisib (a.k.a. nvp bez or bez), a dual inhibitor pan-class pi k and mtor inhibitor evaluated in clinical trials, could also restrict ablv replication in nbf-cells. treatment of nbf-l cells with bez increased the amount of autophagosomal lc b-ii indicative of autophagy activation ( figure a ) without causing significant cytotoxicity ( figure b ). to examine the anti-viral effect of bez treatment on ablv replication, nbf-l cells were infected with wt-ablv for h then treated with bez. treatment with bez resulted in significant dose-dependent decreases of wt-ablv titers ( figure c ), in addition to n and p protein levels ( figure d,e) . although bez advanced to phase i/ii clinical trials as a potential anti-cancer therapy [ ] [ ] [ ] , its use was discontinued due to adverse toxicities [ ] . nevertheless, our results suggest that with improved selectivity or dosing, potent inducers like bez may be promising additional therapeutics to prevent lethality following lyssavirus exposure. although bats are reservoir hosts for a richness of viruses, a comprehensive understanding of how their immune response can sense, respond to, and control viral infection is needed. the type i ifn pathway is well-conserved among vertebrates as an intrinsic first defense during virus infection, with unique anti-viral expression patterns noted in bat cells [ ] . in black flying fox cells, ifn-α was constitutively expressed regardless of virus infection, whereas ifn-β was not induced [ ] , consistent with another report that ifn-β was antagonized during hev infection [ ] . regardless, the ifn pathway is a constant target of virus antagonism and evasion. here, we focused on another well-conserved, intrinsic immune response and present a first experimental attempt to study the autophagy pathway during a virus infection in cell lines derived from a bat species. collectively, our findings demonstrated that ablv infection increased autophagosome generation and autophagy in black flying fox and human cells, and that autophagy activation in black flying fox cells was dependent on a high ablv moi. yet, how ablv rna or proteins are sensed by host sensory machinery during ablv replication remain unanswered. a related rhabdovirus, vsv, induces autophagy through g protein engagement independent of virus replication [ ] and rabv m protein may also contribute to autophagy activation [ ] . autophagy has been implicated in either anti-and pro-viral processes for distinct viruses [ , ] . for example, a protective role for autophagy was identified during vsv infection [ , ] . in our study, pharmacological activation of autophagy decreased ablv replication in both black flying fox and human cell lines, suggesting autophagy antagonizes lyssavirus infection. host-virus adaptations that promote tolerance to virus infection may contribute to the ability of bats to act as primary animal hosts without developing pathogenic disease [ ] . we found that the black flying fox brain cell line did not undergo significant cell death after what could be considered a pathogenic ablv infection (moi ; hpi). in contrast, ablv induced substantial cell death in human neuroblastoma cells, similar to that observed previously with rabv infection [ ] . crosstalk and interplay between autophagic and apoptotic processes have long been recognized. dependent on the environment or stimulation, autophagy can promote survival under stressful conditions (e.g., nutrient deprivation) or itself act as a mechanism of programmed cell death if left unchecked [ , ] . recently, we rescued a recombinant cedar virus [ ] , a non-pathogenic henipavirus species also isolated from pteropus species [ ] . such tools will facilitate opportunities to ask if autophagic response to ablv can be generalized to other bat-borne viruses, and better define how the balance between autophagy and cell death is calibrated in bat cells. the interactions between natural reservoirs hosts and viruses require that (a) the host is readily infected and (b) the virus needs to persist in the host long enough to be transmitted to another susceptible host, before it is either cleared by the host immune response or kills the host [ , ] . epidemiology models suggest a possibility that latently infected individual bats contribute to episodic shedding and natural persistence of bat-borne viruses [ ] . although horizontal transmission within a population is still regarded as the primary source of transmission [ ] , hev and niv recrudescence in pteropus hosts have been reported [ ] [ ] [ ] , yet the precise mechanism of latency or persistence is unclear. autophagy functions as a pro-survival defense by removing toxic virus n protein aggregates during sindbis virus infection, without having an effect on virus replication [ , ] . indeed, a pro-survival autophagic response that removes toxic protein aggregates and dampens virus replication might be critical for promoting viral persistence at the cellular level in natural hosts, maintaining the potential to shed virus with minimal disease presentation. more investigation is needed to translate in vitro bat immunity studies into a models of virus persistence and shedding within individuals and populations. in conclusion, our working hypothesis is that black flying fox cells maintain a higher basal rate of autophagy that serves as both an anti-viral and pro-survival mechanism when further stimulated by virus infection. similarly, brook and dobson, , postulated that autophagic removal of damaged mitochondria (i.e., mitophagy), a result of enhanced oxidative stress from the demands of flight, evolved a secondary anti-viral role for autophagy in bats [ ] . new cell lines have recently been derived from additional bat species facilitating a greater comparative study across the bat taxa [ ] . furthermore, comparisons with rodent cell lines will strengthen whether in vitro intrinsic responses to virus infection are unique to bats or shared co-adaptations among other natural host species. experimental challenges have limited our efforts to genetically silence or pharmacologically inhibit the autophagy pathways in black flying fox cells. although we cannot yet definitively associate 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evidence for nipah virus recrudescence and serological patterns of captive pteropus vampyrus recrudescent infection supports hendra virus persistence in australian flying-fox populations autophagy protects against sindbis virus infection of the central nervous system selective autophagy and viruses bats as 'special' reservoirs for emerging zoonotic pathogens tools to study pathogen-host interactions in bats we thank k. lund, for assistance with flow cytometry and c. olsen for statistical consultation. additionally, we thank a.j. symes for kindly providing human neuroblastoma cell lines. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- - imlae authors: van der gucht, winke; stobbelaar, kim; govaerts, matthias; mangodt, thomas; barbezange, cyril; leemans, annelies; de winter, benedicte; van gucht, steven; caljon, guy; maes, louis; de dooy, jozef; jorens, philippe; smet, annemieke; cos, paul; verhulst, stijn; delputte, peter l. title: isolation and characterization of clinical rsv isolates in belgium during the winters of – date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: imlae respiratory syncytial virus (rsv) is a very important viral pathogen in children, immunocompromised and cardiopulmonary diseased patients and the elderly. most of the published research with rsv was performed on rsv long and rsv a , isolated in and , yet recent rsv isolates differ from these prototype strains. additionally, these viruses have been serially passaged in cell culture, which may result in adaptations that affect virus–host interactions. we have isolated rsv from mucosal secretions of patients in the winters – and – , of which eight rsv-a subtypes and four rsv-b subtypes. passage of the isolates was assessed for viral replication kinetics and infectious virus production in hep- , a and beas- b cells, thermal stability at °c, °c and °c, syncytia formation, neutralization by palivizumab and mucin mrna expression in infected a cells. we observed that viruses isolated in one rsv season show differences on the tested assays. furthermore, comparison with rsv a and rsv b reveals for some rsv isolates differences in viral replication kinetics, thermal stability and fusion capacity. major differences are, however, not observed and differences between the recent isolates and reference strains is, overall, similar to the observed variation in between the recent isolates. one clinical isolate (be/ant-a / ) replicated very efficiently in all cell lines, and remarkably, even better than rsv a in the hep- cell line. respiratory syncytial virus (rsv), recently renamed to human orthopneumovirus, is a very important viral respiratory pathogen in infants, children and adult patients with immunodeficiency or cardiopulmonary disease and it is recognized as a major threat for the elderly population [ ] [ ] [ ] [ ] [ ] [ ] . an rsv infection starts with typical common-cold like symptoms but may progress to serious lower respiratory tract infections associated with a high rate of hospitalization of infants, children and elderly [ ] . no vaccines or therapeutics are available except for synagis ®® , also known as the humanized antibody palivizumab. palivizumab, which is solely used for passive immunization of high-risk infants, targets a specific, highly conserved epitope on the fusion protein [ , ] , resulting in fusion inhibition. currently, treatment of severe lower respiratory tract infections as a result of rsv infection consists of supportive care only, such as oxygen administration and nutrition. rsv is classified in the family pneumoviridae, genus orthopneumovirus and can be divided into two subtypes, rsv-a and rsv-b. it has a non-segmented, negative, single-stranded rna genome that consists of ten genes, encoding proteins. the viral envelope contains three proteins: the attachment protein (g), the fusion protein (f) and the small hydrophobic protein (sh). the g protein interacts with cellular receptors on the host cell membrane to attach the virus particle to the cell surface. the protein consists of a central conserved domain, two glycosylated mucin-like regions and an n-terminal region containing a transmembrane domain and a cytoplasmic domain [ , ] . sequencing of the g gene indicated that the two mucin-like regions flanking the central domain only have a % similarity at the nucleotide level between rsv-a and rsv-b and only % similarity at the deduced amino acid levels [ ] . consequently, the two mucin-like regions serve as excellent targets for rsv evolution studies. both subtypes are further divided into genotypes based on those genetic variations. for rsv-a, the genotypes ga - , saa - , na - and on [ ] [ ] [ ] [ ] [ ] [ ] have been defined, while for rsv-b, the gb - , sab - , uru - , ba - and thb [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] genotypes are reported. the f protein is responsible for the fusion of the viral envelope with the host cell membrane. an important side effect is the fusion of the cell membranes of an infected cell with adjacent cells, resulting in a giant cell with multiple nuclei, better known as a syncytium [ ] . the formation of syncytia is recognized as a means to efficiently spread the infection along epithelial surfaces, while minimizing contact with the immune system [ ] . one of the hallmarks of the pathology caused by rsv infection is increased mucus production in the lungs of infected individuals. mucus is a gel-like substance that consists of different mucins (muc), which are high molecular mass, highly glycosylated glycoproteins [ ] . airway mucus protects the epithelial surface from injury through mucociliary clearance, facilitating the removal of foreign particles and chemicals that enter the lung. twenty-one muc proteins have been described in humans and are divided in two families: secreted mucins and cell-tethered mucins. the major mucins produced in the airways are muc ac and muc b as secreted mucins and muc , muc , muc and muc as membrane-bound mucins [ ] . most of the published research on rsv was performed using the laboratory strains rsv-long and rsv a , which were isolated from the population in and , respectively [ , ] . not only have these viruses not circulated in the public for many years, they have been serially passaged in cell culture, which may have resulted in an accumulation of mutations that benefit the virus to grow in cell culture but may also affect virus-host interactions. low-passage, characterized clinical strains are hard to come by and consequently less used in research. therefore, we have isolated virus from mucosal secretions of patients in the winter seasons of - and - in belgium, resulting in eight rsv-a subtypes and four rsv-b subtypes. we have grown these viruses to passage and used them to assess their viral replication kinetics and infectious virus production in hep- , a and beas- b cells, thermal stability at • c, • c and • c, syncytium formation and neutralization by palivizumab. we have also obtained g protein sequences to assign genotypes and evaluated production of mucin mrna expression in a cells upon infection. the hep- , a and vero cell lines were obtained from and cultured to the instructions of atcc. the beas- b cell line was a generous gift from dr. ultan f. power (queens university belfast, ireland). all cells were cultured in dulbecco's modified eagle medium containing % inactivated fetal bovine serum (dmem- ) (thermo fisher scientific, waltham, ma usa). rsv reference strains a and b were obtained from bei resources, rsv a was cultivated in hep- cells as described by van der gucht w. et al. [ ] and rsv b was cultivated on vero cells in medium containing % inactivated foetal bovine serum (ifbs) until cytopathic effects (cpe) were visible throughout the flask. virus was collected as described for rsv a and quantified in a conventional plaque assay on hep- cells as described by schepens et al. [ ] . briefly, hep- cells were seeded at a concentration of , cells/well in clear well plates (falcon) one day prior to infection. cells were washed with dmem without ifbs (dmem- ) and infected with µl of a / dilution series made in dmem- . cells were incubated for h at • c after which the inoculum was replaced by dmem- containing . % avicel ®® (fmc biopolymer) and incubated for three additional days at • c ( % co ). afterwards, cells were washed with pbs, fixed with % paraformaldehyde solution and stained with palivizumab (leftovers provided by the department of paediatrics, antwerp university hospital) and goat-anti human secondary igg conjugated with horseradish peroxidase (hrp) (thermo fisher scientific) and visualized using chloronaphtol solution (thermo fisher scientific). this study was approved by the ethical committee of the antwerp university hospital and the university of antwerp ( / / ). nasal secretions were collected from children showing symptoms of an rsv-related bronchiolitis during the winter seasons of - and - after written parental consent was given. the secretions were extracted by a nasal swab and/or a nasopharyngeal aspirate, which were stored at • c for less than h. one day prior to mucosal extraction, hep- cells were seeded at a concentration of , cells/well in a clear well plate (falcon, corning, ny, usa). samples were vortexed for one minute with glass beads (sigma-aldrich; st. louis, mo, usa) before inoculating hep- cells with µl of a dilution series of the sample, made in dmem- . after h of incubation with the inoculum at • c, µl of dmem containing ifbs, antibiotics (penicillin/streptomycin (thermo fischer scientific), moxifloxacin (sigma-aldrich)) and anti-fungals (fungizone)(sigma-aldrich) was added to obtain a final concentration of dmem with % fbs. plates were incubated for seven days at • c ( % co ). after seven days, the plates were checked for syncytium formation and µl of the well with the lowest concentration of original sample but still presenting cpe, was transferred to a newly seeded plate, following the previously described protocol. after an additional seven days, the wells were rechecked for syncytium formation. a total of µl from wells presenting with syncytia was transferred to a freshly seeded t , which was incubated until cpe was visible throughout the culture. supernatant was collected, centrifuged for ten minutes at × g, aliquoted, and snap frozen in liquid nitrogen (passage ). virus obtained from these clinical samples was propagated until passage on hep- cells to obtain a plaque forming unit (pfu) count high enough to perform the following experiments. one sample did not propagate efficiently on hep- cells and was propagated for three passages on vero cells until a high pfu was reached. passage cultures were tested for the following contaminants by qpcr: hmpv, rhinovirus and , piv , , and , adenovirus, coronavirus e, nl , oc , paraechovirus, enterovirus and bocavirus and found negative for all. several virus cultures tested negative on mycoplasma presence and no signs of bacterial or fungal presence was observed when the cultures were test grown in absence of antibiotics and anti-fungals. rna for subtyping was extracted from passage virus using the qiamp viral rna extraction mini kit (qiagen, hilden, germany) following the instructions of the manufacturer. a multiplex reaction mix was made with superscript iii platinum one-step quantitative kit (thermo fisher scientific) in a final volume of µl containing µl rna, . µl pcr master mix, µl superscript rt/platinum taq polymerase and . µl of a pre-mixed primer/probe solution. this solution contains a final concentration of µm of each primer and µm of each probe. the primers for rsv-a are located in the viral rna was extracted from infected cell culture supernatants using the qiamp viral rna mini kit (qiagen) according to the instructions provided by the manufacturer. viral rna of the g gene was transcribed to cdna and amplified using the one-step rt-pcr kit (qiagen) and the following primers as described by houspie et al. [ ] . for rsv-a, g fw: -atg caa caa gcc aga tca ag- and f rv: -gtt atc aca ctg gta tac caa cc - , for rsv-b, bgf: -gca gcc ata ata ttc atc atc tct- and bgr: -tgc ccc agr ttt aat ttc gtt c- were used. primers were added to the reaction mix consisting of µl × rt-pcr buffer, µl dntp, µl enzyme, µl h o to a final amount of pmol. ten microliters ( µl) of rna extract was added to the reaction mix. the pcr was performed in a thermocycler (unocycler, vwr; radnor, pa, usa) with the following program: min at • c for the rt step, min at • c for pcr activation, amplification cycles consisting of s at • c, min at • c and min at • c followed by a final extension step of min at • c. the length of the amplified cdna was verified with % agarose gel electrophoresis and cdna was visualized with gelgreen™ (vwr). amplified cdna was delivered to the vib neuromics support facility (university of antwerp) for pcr cleanup and dna sequencing with the following primers as described by houspie et al. [ ] : in addition to the pcr amplification primers, for rsv-a: g r sequences were annotated in snapgene and contigs were built in bioedit with the cap application. multiple sequence alignments from reference strains and contigs, and phylogenetic trees were constructed in mega x using the maximum likelihood method. hep- , a and beas- b cells were seeded at a concentration of , cells/well in black cellstar ®® well plates with a µclear ®® flat bottom suitable for fluorescence microscopy (greiner-bio one) one day prior to inoculation. briefly before inoculation, the cells were washed with dmem- . clinical rsv and rsv a were diluted to infect the cells at a multiplicity of infection (moi) of . . virus was left to adhere for h at • c, % co and replaced with dmem- . cells were fixed with % paraformaldehyde after h, h and h, permeabilized with triton x- and stained with polyclonal goat-anti-rsv antibody (virostat; specificity all viral antigens) followed by donkey-anti-goat secondary antibody conjugated with alexa fluor (af ) (thermo fisher scientific) and additional dapi nucleus staining (sigma-aldrich). hep- , a and beas- b cells were seeded at a concentration of , cells/well in -well plates h prior to infection. briefly, before infection, cells were washed with dmem- and afterwards infected with clinical isolates and rsv a and rsv b at a moi of . . the supernatant was collected after h, h and h, aliquoted, snap frozen and stored at - • c. the supernatant was quantified using a conventional plaque assay on hep- cells as described above. aliquots of clinical isolates and rsv a and rsv b were thawed, diluted in dmem- to obtain a starting concentration of × pfu/ml and aliquots were made of each sample. immediately afterwards, one aliquot of each sample was snap frozen in liquid nitrogen as t . the other aliquots were stored at • c, at • c or at • c for h, h and h, snap frozen in liquid nitrogen and stored at - • c until quantification was performed. a conventional plaque assay on hep- cells, as described above, was used to quantify the remaining pfu in each aliquot. one day prior to inoculation, hep- cells were seeded at a concentration of , cells/well in black cellstar ®® -well plates with a µclear ®® flat bottom suitable for fluorescence microscopy (greiner bio-one). cells were inoculated with clinical rsv and rsv a at a moi of . for h at • c ( % co ). after h, the inoculum was removed and replaced by dmem- containing . % avicel ®® (fmc biopolymer). after h cells were washed with pbs, fixed with % paraformaldehyde solution, permeabilized with triton x- and stained with polyclonal goat-anti-rsv (virostat; westbrook, me, usa) followed by donkey-anti-goat secondary antibody conjugated with af (thermo fisher scientific). dapi staining was performed to stain the nuclei (sigma-aldrich). the plaque reduction assay was performed as described by leemans a. et al. [ ] . briefly, hep- cells were seeded at a concentration of , cells/well in a clear -well plate (falcon) h prior to inoculation. palivizumab was diluted / and further in a dilution series, which was incubated with diluted virus for h at • c ( % co ). afterwards, the cells were washed briefly with dmem- , and inoculated with µl of the virus-antibody solution for h at • c ( % co ). then, the inoculum was replaced with dmem- containing . % avicel ®® (fmc biopolymer). the plates were incubated for three days at • c ( % co ), washed with pbs and fixed with % paraformaldehyde solution. the cells were permeabilized with triton x- , stained with palivizumab antibody followed by goat-anti-human igg conjugated with hrp and coloured using chloronaphtol solution (thermo fisher scientific). the neutralization titer was defined as the reciprocal of the highest antibody dilution producing % reduction in plaques (ed ), relative to virus-control wells without antibody. a cells were seeded at a concentration of , cells/well in -well plates, h prior to inoculation (greiner bio-one). cells were infected with a moi of . for h at • c ( % co ). after h, inoculum was replaced by dmem- and was incubated for an additional h. afterwards, cell supernatant was collected, spun down at × g for min and only the pellet was kept. the still adherent cells were lysed with lysis buffer from the nucleospin kit (macherey-nagel; düren, germany) and added to the pellet. the solution was thoroughly mixed and frozen at - • c until extraction was performed. rna isolation was done following manufacturer's instructions of the nucleospin rna kit (macherey-nagel). concentrations were evaluated using the nanodrop ®® (thermo fisher scientific) and µg of rna was used to convert to cdna using the sensifast™ cdna synthesis kit (bioline; london, uk). relative gene expression was determined with the gotaq qpcr master mix (promega; madison, wi, usa) with sybr green fluorescence detection on a quantstudio real-time pcr instrument (thermo fisher scientific). standard quantitect primers available from qiagen were used for gapdh (qt ), ß-actin (qt ), muc (qt ), muc (qt ), muc ac (qt ) and muc b (qt ). analysis and quality control were performed using qbase+ software (biogazelle; ghent, belgium), relative expression of the target genes was normalized to the expression of the housekeeping genes gapdh and ß-actin. fluorescence images were acquired using an axio observer inverted microscope and a compact light source hxp c with filter sets , and for blue, green and red fluorophores, respectively (zeiss). image analysis was done using zeiss zen . blue edition imaging software and imagej version . . -rc- / . e. calculations were made in excel for mac and graphpad prism . data for viral replication kinetics, infectious virus production and thermal stability are presented as means (± sem) of the indicated independent repeats. to determine the significance between the clinical isolates and the reference (rsv a or b ), data was analysed with a two-way anova. fusion data and muc expression represents means (± sem), significance was calculated between the clinical isolates and their references with a one-way anova. data for plaque reduction represents means (± sd), significance was calculated between clinical isolates and references with a one-way anova. calculations were done using graphpad prism . nasal swabs and nasopharyngeal aspirates were obtained from one patient in december of and from patients between october and january - . rsv-a was detected in one sample of december and in samples collected between october and january . rsv-b was detected in samples collected between october and january . of the remaining two rsv-negative samples, one tested positive for human metapneumovirus (hmpv), one remained negative for rsv, hmpv and rhinovirus . hep- cells were infected with the samples on the day of the aspiration of secretions or the day afterwards, without freezing the samples. after two weeks of incubation, samples did not result in syncytium formation or positive fluorescent staining in either the nasal swab culture or the aspirate culture and were, therefore, not used in any of the following assays. cultures that showed syncytium formation were used to grow the virus on hep- cells. one sample was further grown on vero cells since no significant titers could be reached growing the virus on hep- cells (table ) . sequences of the g gene of all samples were obtained and aligned with previously reported representative sequences from genbank. the phylogenetic trees of rsv-a and rsv-b sequences are shown in figure a ,b. all rsv-a sequences cluster within the on genotype that contains a nt duplication and all rsv-b sequences contain a nt duplication in the g-gene, assigning them to the ba genotype, further differentiated into the baix genotype. sequences of the g gene of all samples were obtained and aligned with previously reported representative sequences from genbank. the phylogenetic trees of rsv-a and rsv-b sequences are shown in figure a ,b. all rsv-a sequences cluster within the on genotype that contains a nt duplication and all rsv-b sequences contain a nt duplication in the g-gene, assigning them to the ba genotype, further differentiated into the baix genotype. to study the dynamics of virus infection, both viral replication kinetics and infectious virus production were assessed in hep- , a and beas- b cells. to study the dynamics of virus infection, both viral replication kinetics and infectious virus production were assessed in hep- , a and beas- b cells. be/ant-b / ) and one strain grown on vero cells (be/ant-b / ) that resulted in significantly more infected cells at h than the reference rsv b , whereas just one strain (be/ant-b / ) seemed to result in comparable infection as the rsv b . infectious virus production of rsv-b strains shows that even though the be/ant-b / and be/ant-b / reach a very high percentage of infected cells, less infectious particles are produced compared to be/ant-b / , suggesting either that the particles may not be efficiently released in the supernatant and remain more cell-associated or that the particles may efficiently attach but replicate at lower levels. the same experiment was repeated in the a (figure ) cell line in which for the rsv-a isolates ( figure a ), the rsv a shows the highest percentage of infected cells, followed closely by the be/ant-a / , reaching only slightly lower percentages of infected cells than in the hep- cells after h. this strain also produced the highest amounts of infectious virus in a cells ( figure c ). in hep- cells both the rsv-b subtype isolates be/ant-b / and be/ant-b / reach a higher percentage of infected cells than the rsv b , whereas results of a replication kinetics suggest that the be/ant-b / and be/ant-b / strains reach similar infection rates ( figure b ). the be/ant-b / reached about % infection after h but the infection then seemed to flatten out towards h, resulting in a significant difference with infection rates of the rsv b . interestingly, the isolate be/ant-b / , which did not efficiently infect hep- cells now reached a near % infected number in a cells in h. unsurprisingly, the be/ant-b / achieved again the lowest number of infected cells and levels of virus production in a cells ( figure d ). as the beas- b cell line is also a highly permissive cell line for rsv infection and widely used as well, we assessed viral growth and production kinetics in this cell line (figure ). for all rsv-a clinical isolates, no major differences were observed after h and h of infection in percentage of infected cells ( figure a ). after h of infection, the amount of infectious virus released by the cells was the highest for rsv a and clinical isolate be/ant-a / . larger differences were observed between the clinical isolates of the rsv-b subtype ( figure b ). be/ant-b / reached percentages and infectious virus production that were comparable to rsv b ( figure b,d) . isolates be/ant-b / and be/ant-b / had the lowest infection rates and infectious virus production in both this cell line as well as in the hep- cells (figure b,d) . differences in the f protein are shown to be involved in thermal stability of viral particles [ ] . aliquots of each virus containing × pfu/ml were incubated at three different temperatures: °c (in vitro incubator temperature and core body temperature) ( figure a,b) , °c (upper airway overall, clinical isolate be/ant-a / replicated very efficiently in all cell lines, and remarkably, achieving even higher infection rates in the hep- cell line than the rsv a . additionally, two clinical isolates of the rsv-b subtype (be/ant-b / and be/ant-b / ) replicated very well in hep- and a cell lines and quite well in beas- b. overall, differences in infection kinetics were observed also among the different clinical isolates. differences in the f protein are shown to be involved in thermal stability of viral particles [ ] . aliquots of each virus containing × pfu/ml were incubated at three different temperatures: • c (in vitro incubator temperature and core body temperature) ( figure a,b) , • c (upper airway temperature) (figure c ,d) and • c (storage temperature) ( figure e ,f) for h, h and h. aliquots were snap frozen in liquid nitrogen and used for conventional plaque assay to quantify the remaining infectious virus. for all rsv-a isolates and rsv a , higher temperatures were associated with a faster decay of infectious virus. curiously, be/ant-a / maintained higher pfu at • c than other rsv-a isolates although at the other temperatures, there was no difference. additionally, be/ant-a / was preserved slightly better at • c, however at h no viable virus was detected. rsv-b isolate be/ant-b / retained higher titers for the duration of the experiment compared to other rsv-b isolates but its overall stability was less than the reference rsv b . the only exception is at • c, where its viral titers remained higher than rsv b . isolate be/ant-b / seems to decay especially fast at any other temperature than • c. syncytium formation has long been considered a typical characteristic of rsv infection in immortal cell lines, and it has been used as a measure of activity of the fusion protein [ ] . hep- cells were infected at a moi of . and incubated for h with an overlay of dmem- containing . % avicel ®® to allow spreading of the infection to neighbouring cells only. afterwards, cells were fixed, fluorescently stained and analysed with fluorescence microscopy. mean syncytium size was determined by calculating the mean of the number of nuclei in the syncytia formed per sample, (figure a ,b) as well as mean syncytium frequency ( figure c ,d) by counting the number of nuclei belonging to syncytia relative to the total number of nuclei of infected cells. mean syncytium size of all rsv-a clinical isolates ( figure a ) lies between four and seven nuclei per cell, with be/ant-a / , be/ant-a / and be/ant-a / having the largest syncytia. the smallest syncytia were produced by be/ant-a / . mean syncytium frequencies lie between % and %, with the lowest frequency found for be/ant-a / , which suggested that it promotes the formation of larger syncytia rather than many small syncytia ( figure c ). clinical isolate be/ant-b / formed significantly larger syncytia with a mean size of compared to all clinical isolates ( figure b ). reference strain rsv b formed almost no syncytia, with the smallest size and lowest frequency of all viruses tested. viruses , , x for peer review of figure . thermal stability profiles at °c, °c and °c. clinical isolates, rsv a and rsv b were aliquoted and exposed to °c (a,b), °c (c,d) or °c (e,f). one aliquot of each was snap frozen at h, h, h and h. aliquots were used for quantification by conventional plaque assay and calculated to the amount at h. data represents mean values ± sem (n = ), significant differences compared to the reference strains are indicated by *p < . ;**p < . ; ***p < . (two-way anova). produced by be/ant-a / . mean syncytium frequencies lie between % and %, with the lowest frequency found for be/ant-a / , which suggested that it promotes the formation of larger syncytia rather than many small syncytia ( figure c ). clinical isolate be/ant-b / formed significantly larger syncytia with a mean size of compared to all clinical isolates ( figure b ). reference strain rsv b formed almost no syncytia, with the smallest size and lowest frequency of all viruses tested. viral neutralization by palivizumab was assessed with a conventional plaque reduction assay. virus was incubated with a two-fold dilution series of palivizumab for h at • c and then transferred to hep- cells for h at • c to allow infection of non-neutralized virus. afterwards, the supernatant was replaced by dmem- containing . % avicel ®® and incubated for three days until plaques were visible to the naked eye. plaques were counted to determine the concentration of palivizumab that neutralizes the virus by %. figure shows that rsv-a clinical isolates be/ant-a / and be/ant-a / are % neutralized at lower palivizumab concentrations than most of the other clinical isolates and rsv a , resulting in better neutralization than the other isolates. remarkably, rsv a and rsv b neutralization was significantly different, with rsv-b strains having a higher sensitivity to palivizumab than rsv-a strains. overall no significant differences were observed between rsv-b clinical isolates and the reference rsv b for palivizumab neutralization. figure shows that rsv-a clinical isolates be/ant-a / and be/ant-a / are % neutralized at lower palivizumab concentrations than most of the other clinical isolates and rsv a , resulting in better neutralization than the other isolates. remarkably, rsv a and rsv b neutralization was significantly different, with rsv-b strains having a higher sensitivity to palivizumab than rsv-a strains. overall no significant differences were observed between rsv-b clinical isolates and the reference rsv b for palivizumab neutralization. with clinical isolates and reference strains that were pre-incubated for h with a palivizumab dilution series. inoculum was replaced with dmem- containing . % avicel ® and incubated for three days at °c. afterwards, the cells were fixed, stained with palivizumab as primary antibody and goatanti-human conjugated with hrp, plaques were visualized with chloronapthol. individual values are plotted as log ed , data represents mean values ± sem (n = ). with clinical isolates and reference strains that were pre-incubated for h with a palivizumab dilution series. inoculum was replaced with dmem- containing . % avicel ®® and incubated for three days at • c. afterwards, the cells were fixed, stained with palivizumab as primary antibody and goat-anti-human conjugated with hrp, plaques were visualized with chloronapthol. individual values are plotted as log ed , data represents mean values ± sem (n = ). rsv infection is hallmarked by an increase of mucus production and impaired mucociliary clearance. as muc ac and muc b are important players in the secreted airway mucins and muc and muc in the cell-tethered mucins [ ] , their mrna expression levels upon rsv infection of a cells was tested. mrna expression levels of the mucins were assessed by infecting a cells for h with a moi of . , followed by qrt-pcr with primers for the different mucin-encoding genes. a cells were incubated with virus of each isolate for h, after which the inoculum was removed and replaced with dmem- . cells were incubated for h, collected for lysis followed by an rna extraction and qrt-pcr. for all clinical isolates and controls, the relative expression of cell-tethered muc ( figure a ) is increased compared to the non-infected control. no significant differences can, however, be observed in between rsv isolates and controls. we have isolated rsv from nasal samples from patients in the winter seasons of - and - and found that isolating infectious virus from nasal samples is most efficient from nasopharyngeal aspirates ( %) compared to nasopharyngeal swabs ( %). however, yield may also depend on timing of sampling compared to onset of symptoms and time between sampling and inoculation of cell culture. hep- cells were used for isolation since they are permissive for most rsv strains and isolates described. one well-known exception is rsv b , which infects hep- cells, but viral titers remain low compared to other strains. however, infection of rsv b in vero cells does yield a higher titer, which is also the case for the clinical isolate be/ant-b / from this study. the clinical isolates were genotyped and characterized on their ability to grow and produce infectious virus in cell lines. thermal stability was assessed at °c (normal body temperature, incubation temperature), °c (estimated nasal temperature) and °c (storage temperature), fusion capacity and neutralization by palivizumab as specific features of the fusion protein and mucin mrna expression of muc and muc as cell-tethered mucins, and muc ac and muc b as secreted mucins. even though all rsv-a and rsv-b clinical isolates belong to the same genotype in the phylogenetic trees (on and baix, respectively), the results show differences between the different expression profiles of the cell-tethered muc show a considerable relative increase compared to the negative control ( figure b ). infection of be/ant-a / and be/ant-a / resulted in the highest relative increases of muc mrna among all of the rsv-a clinical isolates, whereas be/ant-a / and be/ant-a / resulted in the lowest increase. for the rsv-b clinical isolates, significantly lower increases are observed when compared to the rsv-a clinical isolates, but an increase is still observed. infection of isolates be/ant-b / and be/ant-b / resulted in the highest increase of muc mrna expression among the rsv-b isolates. muc ac is mainly produced in the epithelial goblet cells and was previously reported to slightly decrease in a cells under the influence of an rsv-infection after h [ ] . here, expression of muc ac is significantly reduced upon infection with all clinical isolates and reference strains, however no significant differences can be observed between the clinical isolates ( figure c ). muc b is produced by surface secretory cells throughout the airways and submucosal glands. our results show that muc b expression is downregulated as a result of rsv infection, with the strongest downregulation of rsv-a clinical isolates be/ant-a / , be/ant-a / , be/ant-a / and be/ant-a / . overall downregulation of muc b by the rsv-b clinical isolates is limited, with almost none in infections with be/ant-b / ( figure d ). we have isolated rsv from nasal samples from patients in the winter seasons of - and - and found that isolating infectious virus from nasal samples is most efficient from nasopharyngeal aspirates ( %) compared to nasopharyngeal swabs ( %). however, yield may also depend on timing of sampling compared to onset of symptoms and time between sampling and inoculation of cell culture. hep- cells were used for isolation since they are permissive for most rsv strains and isolates described. one well-known exception is rsv b , which infects hep- cells, but viral titers remain low compared to other strains. however, infection of rsv b in vero cells does yield a higher titer, which is also the case for the clinical isolate be/ant-b / from this study. the clinical isolates were genotyped and characterized on their ability to grow and produce infectious virus in cell lines. thermal stability was assessed at • c (normal body temperature, incubation temperature), • c (estimated nasal temperature) and • c (storage temperature), fusion capacity and neutralization by palivizumab as specific features of the fusion protein and mucin mrna expression of muc and muc as cell-tethered mucins, and muc ac and muc b as secreted mucins. even though all rsv-a and rsv-b clinical isolates belong to the same genotype in the phylogenetic trees (on and baix, respectively), the results show differences between the different isolates. according to the phylogenetic trees, the clinical isolates can be divided into three groups for the rsv-a isolates and two groups for the rsv-b isolates. however, differences can even be observed between clinical isolates that belong to the same group within the genotype. for example be/ant-a / , which, in comparison with be/ant-a / and other clinical isolates from the same season, infects a higher number of cells in viral replication kinetics experiments, produced a higher amount of infectious virus and retained its stability in thermal stability assays. it is only slightly less neutralized by palivizumab compared to other rsv-a clinical isolates that have been isolated from the same season. secondly, be/ant-b / and be/ant-b / are generally poor infectors and also have a lower thermal stability than other rsv-b clinical isolates from the same season. generally, differences can be observed between the different clinical isolates in viral replication kinetics, but all clinical isolates have the ability to infect all cell lines tested. these differences could be due to underlying genetic differences that could affect antiviral responses in the cells, or due to differences that could be accounted to changes in the way the virus replicates in the cells, such as the presence of defective interfering virus particles. our results suggest that the thermal stability of the clinical isolates is in general lower than the reference strains. the reason for this is unknown but it may be that repeated infection of the reference strains has resulted in the selection of variants with replicative fitness advantage at these temperatures. remarkably, rsv b remains rather stable at • c compared to the rsv a and other clinical strains. the f protein is indispensable for virus infection [ ] and its activity can be linked to the formation of syncytia [ , ] . mean syncytium size and mean syncytium frequency were determined after h of infection with a . % avicel ®® overlay to minimize free particle movement. the presence of avicel ®® would promote the formation of syncytia rather than spread and the formation of new syncytia during the incubation period. even with minimal free particle movement, different sizes of syncytia containing - nuclei per cell are formed by the clinical isolates. for example, be/ant-b / clearly forms large syncytia without infected satellite cells, which suggests that the produced particles remain more cell associated, and virus spread in hep- cells may be facilitated through syncytia formation rather than particle release. this could also be observed from the replication kinetics and infectious virus production: the number of infected cells rapidly increases, and about % of the culture is infected after h, whereas the amount of infectious virus in the supernatant is lower compared to most other clinical isolates. this suggests that the production of infectious virus particles is either limited or faulty in hep- cells for this isolate. a full characterization of the production of defective interfering particles could provide an explanation. we tested the neutralization of the clinical isolates by palivizumab, the monoclonal antibody currently used to passively immunize infants with risk factors of severe rsv-related respiratory infections. curiously, all rsv-b isolates and rsv b are better neutralized than all rsv-a clinical isolates. increased mucus production in the airways is an important characteristic and defence mechanism of the body to protect the airways from respiratory infections [ , ] . it has been shown that expression profiles of mucin mrna can change under the influence of the rsv infection [ ] . relative expression of muc and muc was increased in the infected cells compared to a non-infected control whereas relative expression of muc ac and muc b 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respiratory illness of a virus related to chimpanzee coryza agent (cca). i. isolation, properties and characterization respiratory syncytial virus (rsv) entry is inhibited by serine protease inhibitor aebsf when present during an early stage of infection protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein virological surveillance of influenza in belgium season circulation of hrsv in belgium: from multiple genotype circulation to prolonged circulation of predominant genotypes removal of the n-glycosylation sequon at position n located in p of the respiratory syncytial virus fusion protein elicits enhanced antibody responses after dna immunization a live rsv vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation differential mucin expression by respiratory syncytial virus and human metapneumovirus infection in human epithelial cells respiratory syncytial virus (rsv) sh and g proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated rsv subgroup b mutant n-glycans of f protein differentially affect fusion activity of human respiratory syncytial virus we would like to thank ultan power for providing us with the beas- b cell line for experiments. we thank the virology unit at sciensano, cyril barbezange and ilham fdillate, for the subtyping of the samples. thanks to bei resources for providing rsv a and rsv b reference strains. our gratitude is expressed towards the staff at the paediatric ward at the antwerp university hospital to provide us with fresh material for this study. we would also like to thank the parents for consenting to include their children in this study. the authors declare no conflicts of interest key: cord- -g a xf authors: shin, hye jin; kim, chonsaeng; cho, sungchan title: gemcitabine and nucleos(t)ide synthesis inhibitors are broad-spectrum antiviral drugs that activate innate immunity date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: g a xf nucleoside analogs have been frequently identified as antiviral agents. in recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. nucleoside analogs generally interfere with cellular nucleos(t)ide synthesis pathways, resulting in the depletion or imbalance of (d)ntp pools. intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos(t)ide synthesis inhibition. the precise crosstalk between these two independent processes remains to be determined. nonetheless, we summarize the current knowledge of nucleos(t)ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular dna/rna polymerases [ ] . more recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. some nucleoside analog drugs targeting specific viral polymerases (acyclovir for herpesviruses, zidovudine for human immunodeficiency virus (hiv), and sofosbuvir for hepatitis c virus (hcv)) have been successful in clinical trials [ ] [ ] [ ] [ ] and are currently in use for the treatment of virus-infected patients. another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with ifn-α [ ] . importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. the primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( figure ). alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. the other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. there have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [ ] [ ] [ ] [ ] . by targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)ntp pools. as viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. a more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes (isgs). importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. however, the precise mechanism of this crosstalk remains to be elucidated. there is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. they have been well-summarized in a previous report [ ] . in the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. more importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( figure ). alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. the other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. there have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [ ] [ ] [ ] [ ] . by targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)ntp pools. as viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. a more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes (isgs). importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. however, the precise mechanism of this crosstalk remains to be elucidated. there is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. they have been well-summarized in a previous report [ ] . in the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. more importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. figure . the mechanism of antiviral effect of nucleos(t)ide analogs. nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [ , ] . however, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of rna viruses, including middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov), zika virus (zikv), hcv, poliovirus (pv), influenza a virus (iav), hiv, and enteroviruses (ev) [ ] [ ] [ ] [ ] [ ] [ ] . the antiviral activities of gemcitabine against the abovementioned viruses are summarized in table . mers-cov and sars-cov belong to the family of coronaviridae and are causative agents of severe viral respiratory illness in humans. to efficiently select appropriate antiviral drug figure . the mechanism of antiviral effect of nucleos(t)ide analogs. nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [ , ] . however, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of rna viruses, including middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov), zika virus (zikv), hcv, poliovirus (pv), influenza a virus (iav), hiv, and enteroviruses (ev) [ ] [ ] [ ] [ ] [ ] [ ] . the antiviral activities of gemcitabine against the abovementioned viruses are summarized in table . mers-cov and sars-cov belong to the family of coronaviridae and are causative agents of severe viral respiratory illness in humans. to efficiently select appropriate antiviral drug candidates, dyall et al. screened fda-approved drugs in virus-infected vero e cells and identified gemcitabine as one of drugs with antiviral activity against both mers-cov and sars-cov (ec of . µm and . µm, respectively) [ ] . more recently, gemcitabine was shown to effectively suppress zikv infection and replication in human retinal pigment epithelium (rpe) cells, particularly at non-cytotoxic concentrations (ec of . µm vs. cc of > µm) [ ] . zikv, a member of the flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. effective antiviral activities of gemcitabine were also found for the replication of hcv in huh- cells and the infection of hiv in u -magi-cxcr cem cells, with estimated ec s of nm and . nm, respectively [ , ] , which were lower concentrations than those used in cancer therapy [ ] . in the case of hiv, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced hiv infectivity by increasing the viral mutation frequency [ ] . in a follow up study, clouser et al. further reported the antiviral effect of gemcitabine against hiv-related retrovirus, murine leukemia virus (mulv), in vitro (ec of . nm) and even in murine aids model [ ] . a significant antiviral effect of gemcitabine on iavs was also reported for rpe cells by denisova et al. (ec of . µm) [ ] . they also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on sindbis virus and herpes simplex virus- (hsv- ) (> log reduction in virus titer) but relatively weak effects on semliki forest virus and human echovirus , and minimal effects on bunyamwera virus, measles virus (mev), and vaccinia virus [ ] . the antiviral effect of gemcitabine on evs, initially performed on coxsackievirus b (cvb ), was found from screening fda-approved drugs in cvb replicon-harboring vero cells by our group (ec of . µm) [ ] . its broad-spectrum antiviral activity on evs was further identified by observing a similar inhibitory effect on enterovirus (ev ) and human rhinoviruses (hrvs) (ec s of and - µm, respectively). in the case of hrv, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model [ ] . in this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including tnf-α and il- β, and the number of lung infiltrating lymphocytes. more recently, zhang et al. also identified gemcitabine as the best anti-pv inhibitor from a screen of fda-approved drugs in pv replicon-harboring hela cells (ec of . µm) [ ] . as previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of rna viruses and has a therapeutic potential for the treatment of various virus-associated diseases. moreover, it is possible that gemcitabine is effective for other untested rna viruses. because gemcitabine is a deoxycytidine analog that interferes with dna as well as rna synthesis, dna viruses may not be the exception. consistent with this possibility, there has been a report that the infection of hsv- , which is a representative dna virus classified into the herpesviridae family, was strongly affected by gemcitabine [ ] . most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. this is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as mers-cov, sars-cov, and zikv, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. in this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. importantly, it is noteworthy that zikv was the most strongly affected by gemcitabine, with a low nanomolar ec , which was lower than that used in cancer therapy [ , ] . even for other viruses with a relatively high ec , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. in this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. as an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few rna viruses, was reported against evs such as cvb and ev [ ] . as previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed hiv infectivity both in vitro and in vivo [ , ] . however, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models [ , ] . more extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. however, our group has recently reported that gemcitabine had an anti-ev effect by targeting the salvage pathway of pyrimidine biosynthesis [ ] . moreover, gemcitabine strongly induced the expression of several isgs including cxcl , irf , irf , ifit , and ddx , which were the major effectors in the innate immunity that defended the host against the virus infection. these results were consistent with a previous report that gemcitabine stimulated the production of ifn-β and ifn-γ in iav-infected rpe cells [ ] . importantly, the activation of isgs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. similar phenomena in terms of isg activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in table [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid (mpa) are inhibitors of inosine- -monophosphate (imp) dehydrogenase (impdh), which is a key enzyme of the purine biosynthesis pathway. these inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. both have antiviral activities against viruses such as hcv, hepatitis e virus (hev), mers-cov, dengue virus, yellow fever, hepatitis b virus, west nile virus (wnv), chikungunya virus (chikv), and iav [ ] [ ] [ ] [ ] [ ] [ ] [ ] , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against hcv and hev shown to involve the stimulation of isgs [ , ] . for the antiviral activity of ribavirin against hcv, ribavirin specifically induced the expression of irf , irf , and isg mrnas, which are known to be important for anti-hcv immune responses [ ] . isg activation occurred through an undefined mechanism that was different from the classical ifn signaling, intracellular dsrna sensing pathway, toll-like receptor and nuclear factor b pathways. more importantly, ribavirin-induced isg activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting impdh inhibition-mediated isg activation as an alternative innate immunity pathway. like ribavirin, mpa remarkably induced the expression of several isgs, including irf , irf , isg , ifi , irf , cxcl , ifit , and ifitm mrnas in naïve or hev-infected huh- cells, and the induction of isgs was at least partially abrogated by the use of supplemented guanosine [ ] . mechanistically, the induction of isgs by mpa was independent of the classical jak/stat system, which is similar to that observed with ribavirin [ ] . similar results were obtained with several impdh or impdh inhibitors, with various affinities, that were custom-designed and synthesized [ ] . as shown in table , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase (dhodh), an essential enzyme in de novo pyrimidine synthesis. lucas-hourani et al. identified dd as an interferon-sensitive response element (isre)-stimulating compound from high-throughput screening, and further analyses suggested that it was a dhodh inhibitor with a strong antiviral activity against various viruses including mev, chikv, and wnv [ ] . dd enhanced the expression of several isgs, which were almost completely suppressed by the addition of supplemented uridine, indicating dhodh inhibition-mediated isg activation. moreover, the antiviral activity of and isg activation by dd required the interferon regulatory factor (irf ) transcription factor, a master regulator of antiviral gene expression [ ] , which was consistent with the observation that the anti-hcv activity of mpa was partially mediated by irf [ ] . in this study, similar results were shown with brequinar, another well-known dhodh inhibitor. fa- is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting dhodh and inducing the expression of isgs such as ifnb , cxcl , isg , and ccl [ ] . however, whether isg activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. the mechanism of nucleotide synthesis inhibitor-induced isg activation is still presently unclear. nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced isg activation is independent of the classical jak/stat-mediated ifn signal [ , , ] . first, wang et al. clearly showed that isg activation and anti-hev activity induced by mpa or brequinar was not mediated by jak [ ] . second, irf induction by ribavirin was not affected by knockdown of stat , while that of ifn-α was strongly affected under the same conditions [ ] . third, our recent study with gemcitabine further confirmed ifn signal-independent isg activation by parallel studies comparing the effects of gemcitabine and ifn-α. in our study, the phosphorylation of stat at tyr , which was dramatically triggered by ifn-α, did not occur when treated with gemcitabine [ ] . moreover, the upregulation of ddx mrnas induced by gemcitabine was not affected by irf knockdown, which was contrary to the result that ifn-α-induced upregulation of ddx mrnas was significantly suppressed under the same conditions. consistent with above observations, there have been some reports that isgs was induced in the absence of jak or stat activation [ , ] . despite limited data, we speculate the scenario of isg activation that is independent of jak/stat-mediated ifn signal. purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as impdh and dhodh, leading to the depletion or imbalance of the (d)ntp pool. inactivation of metabolic enzyme(s) itself or consequently altered nucleos(t)ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of isgs, possibly through the relay of kinases and transcription factors. based on the previously mentioned reports, this signal is less likely to be dependent on stat / -irf (ifn-stimulated gene factor ; isgf ), at least for gemcitabine, which is the major transcriptional complex in the ifn-induced jak/stat pathway. it should also be considered that thomas et al. excluded the involvement of an intracellular double-stranded rna sensing pathway, toll-like receptor and nuclear factor κb pathways, as well as a classical ifn signal in the activation of isgs induced by ribavirin [ ] . despite the consensus of isg activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of isgs, at least with different patterns [ ] . targeting an enzyme in which pathways (purine or pyrimidine synthesis) or steps (early/late and de novo/salvage) produce different levels of intermediates and nucleos(t)ides will consequently result in diverse outcomes of isg activations. there might be more than one signaling pathway involved. the synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward isg activation. systematic analyses of signaling kinases, irfs, and stats using sirna knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos(t)ides should therefore clarify the underlying molecular mechanisms of isg activation by purine/pyrimidine biosynthesis inhibitors. as newly emerging or re-emerged viruses such as sars-cov, mers-cov, and zikv have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. in this regard, nucleoside analogs that directly target viral rna-dependent rna polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. nucleoside analogs probably induce different subsets of isgs, at least with a different pattern, leading to various combinations of isgs and resulting antiviral outcomes. moreover, according to schoggins et al., different viruses are affected by distinct subsets of isgs and some isgs such as irf , mb d , hpse, ddx , mda, and ifitm act broadly on various viruses [ ] . thus, more systematic analyses on the subsets of isgs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. given the clinical side effects of ifn treatment, nucleotide analogs that differ from ifn in the activation of subsets of isgs need to be considered as alternatives. nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway 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roles in viral pathogenesis author contributions: hye jin shin, chonsaeng kim, and sungchan cho wrote the manuscript. the authors declare no conflict of interest. key: cord- -w finxf authors: heaton, nicholas s.; randall, glenn title: dengue virus and autophagy date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: w finxf several independent groups have published that autophagy is required for optimal rna replication of dengue virus (denv). initially, it was postulated that autophagosomes might play a structural role in replication complex formation. however, cryo-em tomography of denv replication complexes showed that denv replicates on endoplasmic reticulum (er) cisternae invaginations and not on classical autophagosomes. recently, it was reported that autophagy plays an indirect role in denv replication by modulating cellular lipid metabolism. denv-induced autophagosomes deplete cellular triglycerides that are stored in lipid droplets, leading to increased β-oxidation and energy production. this is the first example of a virus triggering autophagy to modulate cellular physiology. in this review, we summarize these data and discuss new questions and implications for autophagy during denv replication. classical autophagy is a homeostatic process wherein cytoplasmic material is sequestered in double membrane vesicles and degraded [ , ] . the process of autophagy is initiated when a cell perceives a signal such as starvation or pathogen infection. these signals are integrated through mtor to initiate a pathway autophagosome formation, which requires numerous autophagy (atg) proteins, which are conserved from yeast to humans [ ] . although the process of autophagosome formation is not completely understood, many of the required components are known [ ] . a limited description of autophagosome formation follows. for more comprehensive reviews on autophagy as a process, please open access see [ , , ] . initially, an immature autophagosome, termed a phagophore, forms from a variety of cytoplasmic membrane compartments [ ] . the atg /ulk complex positively regulates phagophore formation, while atg /beclin- recruits a class iii phosphatidylinositol -kinase (vps ) and atg to generate phosphatidylinositol -phosphate and nucleate the phagophore [ ] . two ubiquitin conjugating systems are involved in the elongation of the phagophore into an isolation membrane. first, atg is conjugated to atg , which then associates with atg l to form the pre-autophagosomal structure. second, atg /lc is cleaved by atg to form lc -i and subsequently becomes conjugated to phosphatidylethanolamine to form lc -ii and specifically associate with autophagosomal membranes [ ] . once the isolation membrane recognizes its cargo, it then engulfs the cargo and fuses, generating an autophagosome. lc -ii plays critical roles in cargo identification and membrane fusion [ ] . after a mature autophagosome has enveloped its cargo, it fuses with a lysosome to form a degradative compartment, the autolysosome. once the contents of the autolysosome have been broken down, the components are released for use by the cell. the process of autophagy is critical for the maintenance of cellular homeostasis as well as providing a mechanism to avoid cell death during starvation conditions [ ] . outside of the context of viral infection, deregulation of autophagy can lead to various pathologies including heart disease, neurodegeneration and cancer [ ] . during viral infection, autophagy can play either a pro-or anti-viral role [ ] . autophagy can act as an anti-viral component of the innate immune system, presumably by sequestering and degrading viral structures in the cells to help reduce viral replication. autophagy can be induced by toll-like receptor (tlr) ligands, which further indicates that autophagosomes can have anti-viral functions [ ] [ ] [ ] . in addition, autophagy can also play a role in delivering viral antigens for presentation to tlrs as has been reported for sendai virus and vesicular stomatitis virus [ ] . autophagy can also function in the adaptive immune response. recent studies have found that autophagy in antigen presenting cells can facilitate loading of antigen onto mhc class ii molecules [ , ] ; in at least one case this occurs via the fusion of autophagosomes with multi-vesicular mhc class ii loading compartments [ ] . despite its antiviral functions, autophagy is subverted by some viruses to perform pro-viral roles [ ] . poliovirus, coxsackievirus b virus, coronaviruses, hepatitis c virus, and dengue virus are among some of the best characterized examples of viruses that activate and require some aspect of autophagy for robust viral replication [ ] [ ] [ ] [ ] [ ] [ ] [ ] . while the exact mechanisms of how autophagy can contribute to viral replication are for the most part unclear, progress has been made in characterizing the proviral roles of autophagy. some picornaviruses appear to use autophagosomal membranes as components of the viral replication complex [ ] [ ] [ ] . other viruses such as coronaviruses can utilize specific components of the autophagy machinery; in this case a non-lipidated form lc , to help reorganize cellular membranes [ ] . hepatitis c virus also has a requirement for autophagy [ ] , likely for an early viral rna translation step [ ] and/or suppressing innate antiviral immunity [ ] . dengue virus (denv) is a positive-stranded rna virus of the family flaviviridae. it is composed of a group of four serotypes (denv - ). denv is transmitted to vertebrate hosts via the mosquito vectors aedes aegypti or aedes albopictus. infection with denv can lead to a spectrum of clinical diseases ranging from subclinical infection to dengue fever to the most severe forms, dengue hemorrhagic fever and dengue shock syndrome [ ] . globally, there are an estimated - million infections annually, making denv the most important arbovirus to human disease [ ] . due in part to the large impact on human health, basic research on denv has expanded in recent years. denv initiates infection of a permissive cell via clathrin-mediated endocytosis and then releases its genomic rna into the cytosol after fusing with the late endosome [ , ] . the viral rna is translated as one open reading frame, and is subsequently cleaved by cellular and viral proteases to release three structural proteins and seven non-structural proteins. the non-structural proteins replicate the viral rna and the structural proteins assemble with the nascent viral rna to generate new virions [ ] . during viral infection, the virus manipulates many different cellular pathways, including autophagy. denv has been published to induce and require autophagy by four independent laboratories [ ] [ ] [ ] [ ] [ ] . lee et al. performed the initial characterization of autophagy during denv infection in [ ] . the authors showed that denv infection of a hepatocyte cell line induced autophagy and that inhibiting autophagy with the drug -methyladenine ( ma) or sirnas targeting autophagy gene expression compromised viral infection. they further showed that the denv induced autophagosomes co-localized with lamp , a marker of lysosomal fusion. this work was expanded upon the next year by panyasrivanit et al. [ ] . it was again shown that denv infection of hepatocytes induced and required autophagy via immunofluorescence assays and drug inhibition. it was also shown that a proportion of denv nonstructural protein (ns ) protein co-localized with autophagosomes as well as lamp and the ribosomal protein l . the authors also showed that an endosomal marker (m p-r) co-localized with autophagosomes, indicating that some autophagosomes may fuse with endosomes to form organelles called amphisomes. since denv replicates on virally induced characteristic double membrane vesicles (dmvs), and autophagosomes are dmvs, the authors hypothesized that denv might replicate on amphisomes and thus link virus entry and replication. the authors also showed that inhibiting lysosomal fusion with autophagosomes increased viral replication, indicating a role for immature autophagosomes during denv replication. soon after this publication, work from the same lab (khakpoor et al.) examined the role of autophagy in denv infection [ ] . similar to denv , denv infection also induced and required autophagy. lamp was observed to co-localize with autophagosomes, but in contrast to the previous denv study, treatment with a lysosomal fusion inhibitor decreased denv replication. this indicated a role for mature autolysosomes in denv infection. the mechanism for how autolysosomes could contribute to viral replication remained unclear. following these initial characterizations of denv-induced autophagy, an electron tomography study was performed by welsch et al. which showed the d structure of denv replication complexes in hepatocytes [ ] . while traditional thin section em appears to show virally induced replication complexes to be a cluster of independent double membrane vesicles [ ] , the d reconstruction clearly showed that these vesicles were actually contiguous invaginations of the er. complementary immuno-em studies demonstrated that the viral replicase proteins are present on the er invaginations as well as double-stranded rna, the viral replication intermediate [ ] . interestingly, the er invaginations that contain the replication complex were physically linked to er membrane compartments that contained capsids [ ] . a model was proposed wherein denv rna is transported through a neck that links the replication complex compartment to sites of capsid assembly. following assembly, the capsid would bud into the er to acquire its envelope. thus, this study clearly ruled out the hypothesis that denv was replicating on classical autophagosomes; however, there was still a very clear requirement for autophagy during viral replication. this indicated that autophagy might be playing an indirect role to enhance viral replication. in addition to bulk macroautophagy, which is relatively non-specific, different types of selective autophagy exist that target specific organelles (reviewed in [ ] ). the hypothesized role of selective autophagy is that the cell frequently needs to initiate a physiological response that appropriately addresses a specific stress. relevant to denv infection, a type of selective autophagy termed lipophagy was described, wherein autophagosomes can target cellular stores of lipids known as lipid droplets (lds) to generate energy for the cell [ ] . heaton et al. performed a limited sirna screen to identify cellular co-factors of denv replication in hepatocytes, which identified, among others, a gene involved in the induction of autophagy [ ] . in subsequent work, the authors reproduced the published results that denv induces and requires autophagy for robust viral replication [ ] . the initial observations were expanded upon by showing that denv induced autophagosomes not only acquire lamp , but complete their maturation and become autolysosomes [ ] . these autophagosomes did not co-localize with markers of the viral replication complex, suggesting that they may play an indirect, non-structural role in denv replication. the denv-induced autophagosomes did, however, significantly co-localize with lipid droplets. lipid droplet volume decreased during denv infection in an autophagy-dependent manner, as did cellular triglycerides, a major component of lipid droplets. denv-induced autophagy stimulated the delivery of lipids to lysosomal compartments, resulting in the release of free fatty acids, which undergo -oxidation in the mitochondria to generate atp. this produces a metabolically favorable environment for viral replication. importantly, the authors showed that the defect in viral replication caused by inhibition of autophagy could be completely complemented by adding exogenous free fatty acids. this complementation of defective autophagy by free fatty acids required -oxidation. thus, despite the many roles of autophagy in regulating cellular homeostasis, its regulation of lipid metabolism is a major contributor for robust denv replication [ ] . many viruses, including cytomegalovirus, hcv, and denv alter cellular metabolism to promote their replication (reviewed in [ ] ). the induction of lipophagy is a novel mechanism by which viruses can manipulate the metabolic state of the infected cell. it is also a very different interaction with autophagy than has been proposed with other viral infections. many viruses appear to induce a bulk autophagy and then inhibit its progression at various steps to prevent anti-viral functions (reviewed in [ ] ). alternatively, denv infection induces a selective autophagy that is preferentially targeted to lipid droplets, which leads to changes in cellular metabolism. in addition to modifying cellular metabolism, it is possible that this serves a secondary function in immune evasion. the targeting of autophagosomes to lipid droplets may also divert autophagosomes from processing viral antigens for antigen presentation as an immune evasion strategy. the mechanism by which denv induces autophagy is unclear. a recent report showed that ns a expression can induce autophagosome formation during denv infection and help infected cells avoid apoptosis in renal epithelial cells and thus, contribute to prolonged viral replication [ ] . the unfolded protein response (upr)/autophagy pathways have been shown to modulate the denv pathogenassociated molecular pattern (pamp) rna-induced innate immune response [ ] , suggesting that autophagy may promote denv replication through repressing innate immunity. more work, however, is required to show whether the proposed viral triggers of autophagy reproduce all cellular signals and phenotypes that accompany autophagy induction in denv-infected cells.  denv infected cells are resistant to apoptosis by exogenous stimuli.  denv induces autophagy  ma treatment inhibits viral replication  inhibition of autophagy prevents denv mediated resistance to apoptosis  over-expression of ns a alone prevents apoptosis  ns a mediated protection from apoptosis is dependent upon autophagy given the many functions of autophagy in the cell, it is perhaps not surprising that different studies have identified multiple possible roles for autophagy in denv infection (figure ). while it is clear that denv replication does not occur on discreet, classical autophagosomes but rather the er, there are possible explanations as to why denv proteins are sometimes observed on membranes positives for autophagosomal markers. perhaps the components of the autophagosomal machinery are involved in the er membrane reorganization. this has been shown to be the case for the coronavirus mouse hepatitis virus [ ] . additionally, it is possible that denv may have different interactions with autophagy, dependent on the cell type. the majority of studies with denv and autophagy have focused on non-phagocytic cells, including hepatocytes and epithelial kidney cells. the impact of autophagy on denv replication also needs to be characterized in phagocytic cells. while the liver is an in vivo target during denv infection, it will be important to repeat these experiments in monocytes, which are thought to be a primary target during infection. further, the majority of this work has been done with denv , with only one report examining denv . it will be important to determine which of the interactions between denv and autophagy are conserved between serotypes. autophagy responds to various stress stimuli to maintain cellular homeostasis. since viral infections frequently induce stress, it was initially assumed that autophagy induction might be a byproduct of the infection. in this model, autophagy would be generically triggered and then subsequently inhibited by a viral protein prior to maturation into its degradative form. the demonstration that denv can induce at least one form of selective autophagy that goes to completion to modify cellular metabolism suggests that it triggers either the induction or the marking of lipid droplets as cargo in a very specific way. an important question is how the stimulation of atp production by denv-induced autophagy benefits replication. the answer that -energy is good‖ is not wholly satisfying. atp production might impact cellular energetics, atp-dependent enzymatic processes required for replication such as the ns atpase activity, or cellular signaling pathways that are regulated by atp levels. the importance of viral modulation of cellular metabolism is an emerging field within virology with more questions than answers at this stage. the cellular pathways that are modified and the viral functions responsible for lipophagy induction are not yet known. indeed, the cellular pathway of lipophagy induction is also poorly characterized. future studies characterizing the mechanism by which denv induces lipophagy should enlighten our understanding of how selective autophagy is triggered. it may also produce a novel antiviral therapeutic strategy. a generic interference with autophagy is a dubious antiviral approach given the importance of autophagy to cellular survival. however, it is possible that a denv-specific interaction with the autophagy machinery may be targeted for therapeutic intervention. inhibition of the denv-autophagy interaction stimulating lipophagy would limit cellular metabolic changes and inhibit denv replication. it may also enhance immune recognition of denv antigens, since autophagosomes would no longer be diverted to lipid droplets. in sum, there have been many proposals for the role of autophagy during denv infection. one role is the modulation of the cellular metabolic state, however, this is not mutually exclusive with additional pro-viral roles (such as the inhibition of apoptosis). future work will help to further characterize the role(s) and relative contributions of autophagy to the various aspects of denv replication. regulation mechanisms and signaling pathways of autophagy autophagy: basic principles and relevance to disease autophagy: process and function a comprehensive glossary of autophagyrelated molecules and processes the origin of the autophagosomal membrane the beclin -vps complex-at the crossroads of autophagy and beyond lc , a mammalian homologue of yeast apg p, is localized in autophagosome membranes after processing physiological role of autophagy as an intracellular recycling system: with an emphasis on nutrient metabolism viruses and autophagy myd and trif target beclin to trigger autophagy in macrophages toll-like receptors in control of immunological autophagy multiple regulatory and effector roles of autophagy in immunity autophagy-dependent viral recognition by plasmacytoid dendritic cells major histocompatibility complex class ii-restricted presentation of a cytosolic antigen by autophagy autophagy promotes mhc class ii presentation of peptides from intracellular source proteins antigen-loading compartments for major histocompatibility complex class ii molecules continuously receive input from autophagosomes subversion of the cellular autophagy pathway by viruses modification of cellular autophagy protein lc by poliovirus potential subversion of autophagosomal pathway by picornaviruses autophagosome supports coxsackievirus b replication in host cells coronaviruses hijack the lc -i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response the autophagy machinery is required to initiate hepatitis c virus replication activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro dengue and dengue hemorrhagic fever dengue-clinical and public health ramifications dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells dengue virus ensures its fusion in late endosomes using compartment-specific lipids recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis dengue virus nonstructural protein redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis autophagic machinery activated by dengue virus enhances virus replication a role for autophagolysosomes in dengue virus production in hepg cells co-localization of constituents of the dengue virus translation and replication machinery with amphisomes flavivirus ns a-induced autophagy protects cells against death and enhances virus replication composition and three-dimensional architecture of the dengue virus replication and assembly sites modification of intracellular membrane structures for virus replication dikic, i. a role for ubiquitin in selective autophagy autophagy regulates lipid metabolism dengue virus-induced autophagy regulates lipid metabolism multifaceted roles for lipids in viral infection we would like to thank kristi berger for critical reading of the manuscript. g.r. acknowledges membership within and support from the region v great lakes rce (nih award -u -ai- ). n.s.h. is funded by nih training grant t ai - and the william rainey harper fellowship. the authors declare no conflict of interest. key: cord- - uidpr authors: doyle, nicole; neuman, benjamin w.; simpson, jennifer; hawes, philippa c.; mantell, judith; verkade, paul; alrashedi, hasan; maier, helena j. title: infectious bronchitis virus nonstructural protein alone induces membrane pairing date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: uidpr positive-strand rna viruses, such as coronaviruses, induce cellular membrane rearrangements during replication to form replication organelles allowing for efficient viral rna synthesis. infectious bronchitis virus (ibv), a pathogenic avian gammacoronavirus of significant importance to the global poultry industry, has been shown to induce the formation of double membrane vesicles (dmvs), zippered endoplasmic reticulum (zer) and tethered vesicles, known as spherules. these membrane rearrangements are virally induced; however, it remains unclear which viral proteins are responsible. in this study, membrane rearrangements induced when expressing viral non-structural proteins (nsps) from two different strains of ibv were compared. three non-structural transmembrane proteins, nsp , nsp , and nsp , were expressed in cells singularly or in combination and the effects on cellular membranes investigated using electron microscopy and electron tomography. in contrast to previously studied coronaviruses, ibv nsp alone is necessary and sufficient to induce membrane pairing; however, expression of the transmembrane proteins together was not sufficient to fully recapitulate dmvs. this indicates that although nsp is able to singularly induce membrane pairing, further viral or host factors are required in order to fully assemble ibv replicative structures. this study highlights further differences in the mechanism of membrane rearrangements between members of the coronavirus family. viruses rely on their host cell to provide most of what they need to replicate and in order to do this, they hijack many cellular processes. a well-studied example is the ability of positive-sense single-stranded rna viruses (+rna) to induce cellular membrane rearrangements upon expression of viral proteins [ , ] . this reorganization of cellular membranes is a critical step in the viral replication cycle since these areas of restructured membranes act as a site for assembly of all components required for viral rna synthesis as well as offer protection from detection by the host antiviral defenses [ , ] . although the structures of these membranes are relatively well-understood, the mechanisms behind their formation, and particularly the viral and host proteins involved, are often not. the precise structure of virally induced membrane rearrangements varies between viruses [ , ] , but viruses generally cause proliferation of membranes, forming structures, such as convoluted membranes (cm), as well as distinct types of vesicles. most common are double membrane vesicles (dmvs), which are discrete from the cytoplasm and are produced by viruses, such as poliovirus [ , ] , hepatitis c virus [ , ] , human norovirus [ ] , and recently the equine torovirus, berne virus [ ] . spherules, which are invaginated vesicles with a channel connecting them to the cytoplasm, have been found in semliki forest virus [ ] , some flaviviruses [ ] [ ] [ ] [ ] , as well as brome mosaic virus (bmv), which is able to induce their formation with the expression of just one viral protein [ ] . an important +rna virus family, the coronaviruses, include pathogens of both animal and human importance, such as severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), mouse hepatitis virus (mhv), porcine epidemic diarrhea virus (pedv), and infectious bronchitis virus (ibv). within this subfamily of viruses, we see variations in membrane rearrangements formed. dmvs and cm are found in cells infected with the alphaand betacoronaviruses, such as sars-cov, mers-cov, and mhv [ ] [ ] [ ] [ ] [ ] [ ] . in the case of the gammacoronavirus ibv, although dmvs are found, the virus induces little cm and instead induces membrane zippering to form zippered endoplasmic reticulum (zer) as well as double membrane spherules, which are found tethered to the zer [ ] , producing a much more defined structure when compared to cm. subsequent to this discovery, mers-cov infection has also been shown to produce small circular structures similar in appearance to the spherules seen in ibv infection but less distinct [ ] . the coronaviral proteins involved in the production of membrane rearrangements have been recently investigated with the three transmembrane non-structural proteins (nsps) nsp , , and , which are the focus of these studies. nsps , , and from different coronaviruses are accepted as functional homologues, although amino acid sequence conservation is low (ranging from . to . % amino acid homology for nsps , , and between ibv strain beaur and mhv strain a ). these proteins do, however, have conserved secondary structure and conserved domains, including enzymatic domains in nsp , transmembrane domains in all three proteins, and cytoplasmic endo-domains in nsps and . for a detailed review of the domain organization and known functions of nsps , , and , see [ ] . nsp of mhv has been shown to be important for the normal function and stability of dmvs, where mutations in nsp resulted in attenuated virus and impairment of dmv formation [ ] [ ] [ ] . in addition, nsp has been shown to localize to dmvs and cm in sars-cov-infected cells [ ] . in a related group of viruses, the arteriviruses, expression of two nsps (nsps and ) was able to produce dmvs [ ] [ ] [ ] . these nsps of the arterivirus are considered functional homologs to coronavirus nsp and [ ] . upon co-expression of nsp and from mhv, both proteins located to areas of curved membranes from where they were shown to be able to recruit nsp and ; however, nsp and alone were not able to induce the formation of dmvs [ , ] . following on from this, it was shown that co-expression of sars-cov nsp and induced membrane pairing and with the addition of nsp the formation of dmv-like structures [ ] . in a subsequent study by others, it was shown that expression of only nsp and from either mers-cov or sars-cov was able to induce dmv formation, and furthermore, addition of nsp made no difference to their shape or size, and did not induce the spherule-like structures seen following infection with whole virus [ ] . interestingly, however, a small molecule inhibitor, k , has been shown to inhibit the replication of several coronaviruses in vitro. in hcov- e, k impaired dmv formation, while k resistance was associated with mutations in nsp , emphasizing a role for nsp in dmv formation [ ] . ibv is a pathogen of poultry, causing significant economic losses to the poultry industry worldwide as well as animal welfare problems. various strains of ibv cause disease that varies in severity from mild respiratory problems to virulent strains that can cause nephropathology and reproductive organ pathology. in this study, we compared the membrane rearrangements induced by viral proteins from two different strains of ibv, the pathogenic m and the apathogenic beaur. these strains were chosen because beaur and other strains of ibv induce dmv, zer, and spherule formation; however, m produces a low spherule phenotype when compared with other strains of the virus [ ] . as the role in membrane rearrangements for nsp and is well-established for several nidoviruses and considering that nsp may also play some role, here we investigated the role that these three nsps play in the formation of ibv membrane rearrangements. avian df cells were maintained in dmem (sigma aldrich, gillingham, uk) supplemented with % fcs (sigma aldrich, gillingham, uk). ibv strains beaur and m -ck (here referred to as m ) have been described previously [ , ] . plasmids expressing tagged nsps derived from either the apathogenic strain beaur or the pathogenic strain m were generated to produce pegfp-n -m nsp , pmcherry-n -beaur nsp , pmcherry-n -m nsp , pcdna . (-)-beaur nsp - xflag, and pcdna . (-)-m nsp - xflag. rna was extracted from virus-infected cells using an rnaeasy kit (qiagen, hilden, germany) following the manufacturer's protocol. rna was reverse transcribed using superscript iii (fisher scientific, loughborough, uk) and a random primer following the manufacturer's protocol. pcr was carried out on cdna using primers specific for each gene, including flanking restriction sites. pcr products were digested and ligated into pegfp-n (takara bio europe, saint-germain-en-laye, france) or pmcherry-n (takara bio) using xhoi and bamhi restriction sites. plasmid pcdna . (-) was modified by insertion of a xflag motif between the kpni and hindiii sites to generate pcdna . (-)- xflag. the pcr products were then ligated into this backbone using the xhoi and bamhi restriction sites. plasmid sequences were verified using sanger sequencing. the er marker plasmid pyfp-er was kindly provided by dalan bailey. df cells seeded into six-well plates were transfected with pegfp-n -m nsp , pmcherry-n -beaur nsp , pmcherry-n -m nsp , pcdna . (-)-beaur nsp - xflag or pcdna . (-)-m nsp - xflag, pegfp-c , pmchery-n , or pcdna . (-)-beaur nsp - xflag using lipofectamine (fisher scientific). cells were transfected with a total of ng plasmid with a dna:lipofectamine ratio of : following the manufacturer's instructions. after h, cells were lysed in cell lysis buffer ( mm tris-hcl (ph . ), mm nacl, mm edta, % v/v triton-x , % v/v glycerol, × halt protease inhibitor complex (fisher scientific). cell lysates were heated with × sample buffer (bio-rad laboratories, watford, uk) and separated on - % tgx gels (bio-rad). proteins were transferred to a nitrocellulose membrane and blocked in % milk in pbs-t. membranes were incubated with primary antibodies to detect gfp (biolegend, london, uk), mcherry (abcam, cambridge, uk), or flag (m ; sigma aldrich, gillingham, uk). after h, membranes were washed with pbs-t and incubated with irdye conjugated secondary antibodies (li-cor, cambridge, uk). membranes were imaged using an odyssey clx infrared imaging system (li-cor). df cells seeded onto glass coverslips were transfected with pegfp-n -m nsp , pmcherry-n -beaur nsp , pmcherry-n -m nsp , pcdna . (-)-beaur nsp - xflag, and pcdna . (-)-m nsp - xflag alone or in combination using lipofectamine . cells were transfected with a total of ng plasmid with a dna:lipofectamine ratio of : following the manufacturer's instructions. after h, cells were fixed for min in % paraformaldehyde in pbs at room temperature. cells were then permeabilized in . % triton x- in pbs for min and blocked in . % bsa in pbs for h. primary anti-flag m antibody (sigma aldrich) and anti-pdi antibody (enzo life sciences, exeter, uk) were diluted in blocking buffer and cells incubated for h. after three washes in pbs, alexa fluor conjugated secondary antibodies (fisher scientific) were diluted / and cells incubated for h. after a further three washes in pbs, nuclei were strained using topro (fisher scientific) or dapi (sigma aldrich) and coverslips mounted with vectashield (vector laboratories, peterborough, uk). cells were visualized using a leica sp confocal microscope (leica microsystems, milton keynes, uk). quantitation of transfected cells was performed manually on three randomly selected fields of view. df cells in six-well plates were either infected with beaur and incubated for h at • c when fresh × bes medium (mem, . % tryptose phosphate broth, . % bovine serum albumin, mm n,n-bis( -hydroxyethyl)- -aminoethanesulfonic acid (bes), . % sodium bicarbonate, mm l-glutamine, u/ml nystatin, u/ml penicillin, and u/ml streptomycin) was added, or were transfected with plasmids as described above. at hpi, cells were washed once in . % saline and scraped into the saline buffer. cells were pelleted at × g for min at • c and µl % glutaraldehyde in . m sodium cacodylate was added to the pellet. df cells were then rinsed three times in . m sodium cacodylate and incubated in % osmium tetroxide for h. after three washes in water, cells were incubated in % uranium acetate aqueous for h at • c. cells were dehydrated in increasing concentrations of acetone and then embedded in agar resin (agar scientific, stansted, uk). sections approximately to nm in thickness were cut and stained with % uranyl acetate to enhance contrast. data was recorded at kv on a phillips cm (amsterdam, netherlands) with a charge-coupled device (ccd) camera. cell sections used here each contained a single visible nucleus, with intact nuclear and plasma membranes. alternatively, df cells were seeded onto thermanox coverslips (fisher scientific) and either infected with beaur and incubated for h at • c, after which time fresh × bes medium was added, or cells were transfected with plasmids as described above. after h, cells were fixed in % glutaraldehyde for h, incubated in % aqueous osmium tetroxide solution for h, then dehydrated in increasing concentrations of ethanol. cells were embedded into agar resin and sections of nm were cut, collected on hexagonal thin bar copper grids, and stained with % uranyl acetate and lead citrate. data was recorded on a fei tecnai tem (fei, cambridge, uk) used at kv with a tvips f digital camera. df cells seeded onto thermanox coverslips were transfected and processed as before. sections or nm thick were cut from the resin-embedded blocks and collected on mesh copper hexagonal grids coated in formvar or pioloform-coated copper slot grids. ten or nm gold particles were applied to the grids to serve as fiducial markers for subsequent alignments. data was recorded on a jeol f tem (jeol, welwyn garden city, uk) used at kv with a tvips f digital camera, or on a tecnai tem (fei) used at kv with a fei k × k eagle ccd camera. samples were mounted in a jeol high angle tilt holder or a fischione double tilt tomography holder, respectively. a single axis tilt series was collected using serial em or fei software. each single axis tilt series was collected over • to • in increments of between • and . • and subsequently aligned and reconstructed in imod [ ] . our previous studies have shown that ibv is able to induce diverse membrane rearrangements in vero cells, primary chicken kidney cells (ckcs) and tracheal organ cultures (tocs). these membrane rearrangements include dmvs, zer, and spherules [ , ] . in order to further characterize membrane rearrangements induced by ibv, we analyzed the membrane rearrangements induced by beaur in df s. unlike primary ckcs, df s are a continuous avian cell line that are more easily transfected and are therefore used throughout this study. although the spike protein of beaur has increased tropism to allow for virus entry into additional cell lines, including df cells, m is not adapted to infect these cells [ , ] . df cells were infected with beaur, fixed after h, processed for em, and imaged. consistent with previous work, dmvs, zer, and spherules were all seen in ibv-infected df cells ( figure ). beaur. df cells were infected with beaur for h, fixed, and processed for electron microscopy (em). viral particles are indicated with arrowheads, double membrane vesicles (dmvs) with asterisks, and zippered endoplasmic reticulum (zer) and associated spherules with arrows. scale bar represents nm. other viruses in the nidovirales order have been shown to require expression of only two or three nsps to induce membrane rearrangements similar to those seen under virus infection conditions [ , , ] . to begin to understand the roles of ibv nsps in rearranging cellular membranes, nsps and from apathogenic beaur and nsps , , and from pathogenic m were tagged with fluorescent or epitope tags. it was not possible to generate a plasmid expressing nsp from beaur due to presumed toxic sequences, as has been found for this region in other coronaviruses [ ] [ ] [ ] . df cells were transfected with these plasmids and after h cells were lysed and proteins separated by sds-page and detected by western blot. all fusions proteins were found to be intact with bands detectable at the predicted molecular weights (figure a) , although an additional kda band was present in nsp -gfp expressing cells, presumably due to a cleavage event within nsp . it was also noted that nsp - xflag from m migrated at a higher molecular weight than nsp - xflag from beaur, most likely due to differences in post-translational modification. subsequently, df cells were transfected with these plasmids and after h cells were fixed, labelled with an anti-flag antibody, and visualized by confocal microscopy. all three nsps showed reticular cytoplasmic labelling consistent with localization to the er (figure b ), as has been observed previously [ , , [ ] [ ] [ ] [ ] [ ] . in addition to er localization, nsp was found in both small and large puncta in cells where the level of nsp expression was higher (comparison shown in figure b ). nsp was also found in small cytoplasmic puncta when expressed alone (figure b ). to confirm er localization, df cells were transfected with either the plasmid expressing nsp -gfp alone or plasmids expressing nsp or together with pyfp-er, as indicated. after h, cells were fixed and labelled with anti-flag-and nsp -expressing cells with anti-protein disulphide isomerase (pdi), a resident er protein. colocalization between yfp-er or pdi and nsp , , and was observed, confirming that these proteins localize to the er (figure c ). cells were transfected with plasmids expressing tagged nsps, as indicated, or empty vectors or nsp - xflag as controls. cell lysates were separated by sds-page and proteins detected by western blot. from left to right, nsp -gfp detected using anti-gfp, nsp -mcherry detected using anti-mcherry, and nsp - xflag detected using anti-flag, as labelled. molecular weight markers are shown on the left and asterisks indicate the nsp bands on each blot. (b) df cells were transfected with plasmids expressing nsp -mcherry and nsp - xflag from beaur, and nsp -egfp, nsp -mcherry, and nsp - xflag from m . after h, cells were fixed with % paraformaldehyde and imaged. nsp (green), nsp (red), and nsp (blue) were imaged as labelled. nuclei were stained with topro (grey) and scale bars indicate µm. (c) df cells were transfected with plasmids expressing nsp -mcherry and nsp - xflag from beaur, and nsp -egfp, nsp -mcherry, and nsp - xflag from m together with yfp-er. after h, cells were fixed with % paraformaldehyde and imaged. nsp (green) and nsp and nsp (red) were imaged along with markers for the er; pdi (red) or yfp-er (yellow) as indicated. nuclei were stained with dapi (grey) and scale bar represents µm. next, to understand whether co-expression of these proteins results in changes in their localization, df cells were transfected with combinations of the plasmids. after h, cells were fixed and labelled with an anti-flag antibody. upon co-expression of some combinations of these viral proteins, this staining pattern changed. expression of nsp with nsp resulted in both proteins localizing to cytoplasmic puncta, although some signals for both proteins also remained in the er (figure ). co-expression of nsp with nsp , or nsp with nsp , did not result in relocalization of either protein, with nsp remaining er-associated, nsp remaining both er-associated and localized in cytoplasmic puncta, and nsp remaining both er-localized and in cytoplasmic puncta (figure ) . interestingly, co-expression of nsps , , and resulted in relocalization of all three proteins to cytoplasmic puncta, some containing nsp and , some nsp only, and some puncta containing nsp , , and ( figure ). nsps and derived from either beaur or m exhibited the same pattern of localization. this demonstrates that co-expression of ibv nsps in the absence of any other viral components can result in their relocalization within the cell, presumably as a result of protein-protein interactions and potentially associated with rearrangement of cellular membranes. figure . co-expression of ibv non-structural proteins results in their relocalization from the er to cytoplasmic foci. df cells were transfected with plasmids expressing nsp -mcherry and nsp - xflag from beaur, and nsps -egfp, nsp -mcherry, and nsp - xflag from m in pairs or in a combination of three, as indicated. solid arrows indicate areas of nsp and colocalization, open arrows indicate areas of nsp , , and colocalization. nuclei were strained with topro (grey) and scale bar represents µm. to further understand the ability of ibv nsps , , and to rearrange cellular membranes, proteins were expressed in cells and analyzed by electron microscopy (em). initially, to assist with subsequent analysis by em, the percentage of total cells in figures b and that were expressing the nsps of interest, as well as the percentage of cells expressing other combinations of nsps, was quantified (table s ). df cells were transfected with tagged nsp , , and derived from beaur or m alone and in combination. after h, cells were chemically fixed, embedded in resin, and visualized using an electron microscope. a phenotype common to all transfected cells was small, tight whorl-like structures which stained more strongly than other structures (figure a ). these were considered an artefact of transfection. transfection of cells with empty pegfp-n , pmcherry-n , or pcdna . (-)- xflag did not result in changes to cellular membranes (figure a ). different types of membrane structures were observed in the transfected cell samples that were absent from mock treated cells, including paired membranes, disordered or piled membranes, and dmv-like structures. nsp in other coronaviruses has been shown to be important in membrane modifications, particularly in the formation of conventional dmvs [ , ] . initially, the effect of expression of nsp in df cells was investigated. interestingly, it was observed that expression of beaur nsp alone was capable of forming paired membranes. this the first time this has been observed for any coronavirus nsp . these paired membranes were observed both as very large areas of extensive accumulations or as small regions of shorter sections of paired membranes. the paired membranes were tightly apposed, often connected to the er, were largely free of ribosomes, and strongly resembled ibv-induced zer (figure b) , although the electron density often surrounding ibv-induced zer was missing here and no spherules were present. transfection of m nsp also induced membrane pairing (figure b) with an appearance comparable to that of beaur nsp -induced paired membranes. for cells transfected with the beaur nsp expression vector, out of cell sections ( %, percentage of total cells not transfected cells) contained piled membranes, and out of ( %) sections for m , significant to p < . by a fisher's exact test. it has previously been shown for other coronaviruses that membrane pairing requires co-expression of nsps and or that co-expression of these proteins results in dmv accumulation [ ] . therefore, the effect of co-expression of nsp with nsp was investigated. firstly, the effect of expression of nsp alone on cellular membranes was determined. although over cells were examined from multiple experiments, expression of nsp was found to have no striking phenotype with cellular membranes appearing unchanged in the presence of nsp derived from m when compared with untransfected cells. furthermore, surprisingly, expression of nsp with nsp had no effect on the membrane pairing ability of nsp (figure c) , with membrane rearrangements appearing comparable to cells expressing nsp alone, i.e., paired membranes connected to the er and lacking ribosomes, found covering both large and smaller areas of the cytoplasm. specifically, the numerous dmv-like structures observed in cells expressing nsp and from either mers-cov or sars-cov were not observed here [ , ] . overall, this data confirms that ibv nsp alone is the main driving factor in membrane pairing and co-expression of nsp does not alter this function. coronavirus nsp has previously been linked to autophagy induction when expressed alone [ ] . nsp derived from sars-cov has also been shown to induce single membrane vesicle accumulation and microtubule organizing center vesiculation [ ] . therefore, the cellular membrane rearrangements induced by expression of ibv nsp were analyzed. in cells expressing nsp alone from either beaur or m , large areas of tangled single membranes, which appear to be derived from the er, were observed ( figure ). these piled, disordered membranes strongly resemble the disordered membrane bodies seen previously upon expression of sars-cov nsp [ ] . to determine the effect of expression of nsp with other nsps on the formation of disordered membranes or any other structures, samples transfected with plasmids expressing nsp and either nsp or nsp were analyzed. in cells co-expressing nsp and , it was observed that cells expressing beaur nsp formed disordered membranes while those expressing m nsp did not. co-expression of nsp and nsp produced the paired membranes associated with nsp expression (for both beaur and m nsp ). disordered membranes were only found in cells co-expressing beaur nsp but none when co-expressing m nsp . this indicates that while nsp from either beaur or m can induce the formation of disordered membranes when expressed singly, co-expression of nsp with either nsp or disrupts this mechanism and to a greater extent in m . finally, the membrane rearrangements induced by co-expression of ibv nsps , , and were investigated by electron microscopy to determine whether co-expression of all three transmembrane nsps could result in the formation of structures comparable to replication organelles in ibv-infected cells. the major phenotype observed following co-expression of all three nsps was the paired membranes induced by expression of nsp alone ( figure ). when nsp and nsp derived from beaur were expressed with m nsp , a very limited number of dmv-like structures was observed ( in cell sections). in cells co-expressing nsp , , and derived from m , no dmv-like vesicles were found in cell sections with only nsp -associated paired membranes being detected. in neither combination were the spherules usually found during virus infection observed. therefore, although co-expression of ibv nsps , , and may be sufficient for formation of dmvs, this does not seem to be a very efficient process compared with dmv formation by nsp and from the betacoronaviruses studied previously [ , ] and nsp is unlikely to be the additional nsp required for ibv dmv formation. in order to further understand the paired membranes induced by expression of ibv nsp , electron tomography (et) was used to visualize membrane rearrangements in three dimensions. in addition, et was used to confirm that, unlike for other coronaviruses [ , ] , co-expression of ibv nsp and does not result in the formation of dmvs. df cells were transfected with plasmids expressing either beaur nsp or beaur nsp with m nsp . after h, cells were fixed and processed for et. the paired membranes produced by nsp expression (indicated by arrows) were found to form sheet-like structures with sections of paired membranes dilating in several places (arrowheads) (figure a , video s ). a comparison with cells expressing nsp and showed there is no noticeable difference between the areas of paired membranes induced upon expression of these nsps (figure b, video s ) . therefore, expression of ibv nsp alone results in the formation of paired er membranes. addition of nsp does not alter the membrane structures induced with no formation of either dmvs, as seen for other covs or spherules. induction of host cell membrane rearrangements is a tool used by many +rna viruses, such as coronaviruses [ , ] . these membrane rearrangements vary between the different members of the family, with the alpha and betacoronaviruses inducing convoluted membranes and dmvs and the gammacoronavirus ibv inducing zippered er, spherules, and dmvs [ ] [ ] [ ] [ ] [ ] , ] . the formation of these membrane rearrangements is, however, a well-conserved mechanism used by these viruses in order to provide a site for viral rna synthesis. although the pool of knowledge about these structures has been growing, the mechanisms behind their formation remain largely unclear. some light has been shed in recent years on the specific viral proteins involved in the formation of these structures; however, these studies were lacking in ibv. in this study, we looked at the involvement of nsps , , and , which have all been implicated in the formation of membrane rearrangements. as transmembrane proteins, these are likely candidates in reordering the host cell membranes to the advantage of the virus. we showed firstly that df cells are a suitable cell type to use for studying ibv membrane rearrangements in addition to those already tested [ ] . in order to assess the involvement of nsps , , and in virus-induced membrane rearrangements, plasmids expressing gfp, mcherry, or xflag fusion proteins were generated. to confirm expression of full-length fusion proteins, western blots were performed using antibodies against the tags. for all the constructs, full-length nsp fusion proteins were detected. however, in cells expressing nsp -gfp, an additional kda band was seen indicating that as well as full-length protein, a cleavage product corresponding to the c-terminus of nsp plus gfp was also being produced. next, we expressed nsps alone or in combination in df cells to assess their ability to rearrange cellular membranes. when expressed alone, all three nsps had a reticular, cytoplasmic localization consistent with previous observations that these nsps localize to the er [ , , [ ] [ ] [ ] [ ] [ ] , although nsp and nsp in addition had a punctate localization with nsp in particular forming large foci in some cells. er localization was subsequently confirmed by colocalization of the three nsps with er markers. when nsps and were co-expressed, both proteins localized to large and small cytoplasmic puncta with some protein also remaining in the er. this suggests that nsp and are able to interact with one another, again consistent with previous findings for other coronaviruses [ , ] , resulting in nsp moving into the nsp -containing puncta. co-expression of nsp and or nsp and did not result in alteration of their cellular localization. however, when nsp , , and were co-expressed, nsp and colocalized as seen before but some puncta now also contained nsp , although some puncta contained only nsp and or nsp alone. this suggests that, as seen in other coronaviruses, nsp and together, but not alone, are able to direct nsp into the nsp / puncta [ , ] . subsequently, em was used to identify changes to the structure of cellular membranes upon expression of these three proteins. surprisingly, expression of nsp did not induce any notable phenotype. expression of nsp from either sars-cov or mers-cov results in the production of disordered membrane bodies likely derived from the er [ , ] . it is not clear why nsp derived from ibv behaves so markedly differently from nsp s expressed by other coronaviruses. however, the previously studied nsp s have all been derived from betacoronaviruses so nsp from gammacoronaviruses, including ibv, may function somewhat differently. indeed, an amino acid sequence comparison between nsp sequences from beaur and the betacoronavirus mhv a shows only . % homology and . % similarity. therefore, although these are accepted as functional homologs, there is scope for these proteins to behave differently from one another. furthermore, given that we have previously demonstrated that ibv-induced membrane rearrangements are distinct from those induced by alphaand betacoronaviruses [ ] , differences in the mechanism of their formation might reasonably be expected. interestingly, expression of nsp alone induced membrane proliferation and the formation of disordered membranes similar to the disordered membrane bodies (dmbs) induced by sars-cov and mers-cov nsp [ , ] . expression of nsp alone did not appear to induce microtubule organizing center vesiculation as seen upon expression of sars-cov nsp [ ] and the presence of autophagosomes was also not apparent [ , ] , although this is likely due to differences in experimental approaches, namely the use of em in this study compared to immunofluorescence of whole cells used previously [ ] . therefore, ibv nsp also appears to function somewhat differently to nsp from sars-cov in its ability to rearrange membranes. the most striking phenotype came upon expression of nsp ; expression of nsp alone was sufficient to induce areas of paired membranes. furthermore, et demonstrated that these are sheet-like areas of paired er membranes, highly similar to zer in ibv-infected cells. it was noted that the paired membranes, although resembling zer in infected cells, lacked the electron density often surrounding the membranes [ ] . this reflects the lack of the other viral proteins making up the replication complex, which, presumably, accumulate on the cytoplasmic surface of the zer. nsp -induced paired membranes were observed as both small regions throughout the cytoplasm and also in extensive areas of paired membranes. these two phenotypes potentially reflect the different localizations observed by confocal microscopy with some cells containing nsp localized only to the er and some cells containing large cytoplasmic puncta corresponding to the large areas of paired membranes. use of correlative light electron microscopy (clem) in the future would confirm this. attempts were made to confirm the nsp homotypic interaction by co-immunoprecipitation; however, this was not successful. it has previously been shown for mhv that nsp can self-associate [ ] , although earlier attempts to demonstrate the interaction in sars-cov failed [ , ] , highlighting that detection of this interaction can be challenging. however, it is likely that self-interaction between nsp proteins located in both membranes of the er zippers the two er membranes together to generate the paired membranes seen, although it cannot be ruled out that instead an interaction with one or more cellular proteins is required. significantly, this is the first time for any coronavirus that, regardless of mechanism, a membrane pairing function for nsp alone has been described. surprisingly, addition of nsp did not alter the membrane rearrangements induced by nsp alone. previous work by others has shown that for other related coronaviruses and arteriviruses, membrane pairing requires the expression of nsp and (or their homologs) [ ] [ ] [ ] ] . in addition to this, however, co-expression of nsp and for other coronaviruses resulted in the formation of numerous dmv-like structures [ , ] . despite extensive searching and the use of electron tomography to gain three-dimensional information, we were not able to detect any dmvs in cells expressing nsp and . the reason for this difference is not clear. here, we used separate plasmids to express nsp and but this strategy was also used in previous work and when compared with a cleavable nsp - precursor did not yield different results [ ] . therefore, the protein expression strategy is unlikely to be the reason that dmvs were not formed. it is possible that the presence of the shorter nsp fragment detected by western blot prevented the formation of dmvs. however, full-length nsp was also present and therefore should have been capable of inducing dmvs in combination with nsp . in addition, dmvs were not detectable in cells expressing either nsp from m and nsp from beaur or cells expressing nsp and from m , indicating that the use of proteins from different virus strains was not the reason for the lack of dmvs. indeed, nsp relocalized to both beaur and m nsp -containing foci suggesting that m nsp is capable of interacting with both nsp proteins. again, attempts were made to confirm interaction between nsp and nsp by co-immunoprecipitation, but this was not successful. interactions between full-length or the c-terminus of nsp and nsp from other coronaviruses have been shown previously [ , ] . interestingly, sakai et al. showed that just two amino acid residues in nsp are necessary for the interaction with nsp ; however, these residues are only conserved in betacoronaviruses, not in alphaor gammacoronaviruses [ ] , so it is likely that the mechanism of any nsp /nsp interaction is different in ibv. overall, the data indicates that dmv formation by ibv requires the presence of additional viral protein(s), either to direct an interaction between nsp and nsp if it cannot occur directly or because dmv formation is via another mechanism. co-expression of nsps , , and did appear to result in the formation of a very small number of dmv-like structures. however, these were significantly less numerous and less easily identifiable than those observed by oudshoorn et al. [ ] . therefore, nsp does not appear to be the ibv protein required, in addition to nsp and , to induce dmvs and other viral proteins must play a role. throughout this study, we were unable to detect spherules associated with ibv infection, although we did identify membranes highly similar to zer. in our previous work, we demonstrated that m virus has a low spherule phenotype and the region of the genome from the end to nsp was responsible for this [ ] . unfortunately, we were unable to clone nsp from beaur due to toxicity problems in escherichia coli. it was also not possible to clone nsp from two further strains of ibv. as the nsp used in this study was derived from m , it is possible that this is the reason that spherules were not detected under any conditions. nsp from beaur and m are highly related with . % amino acid homology and . % similarity with the majority of the differences occurring within the non-functional papain-like protease domain. despite that fact, it cannot be ruled out that these differences are sufficient to prevent spherule formation. in future, cloning the c-terminal part of nsp from beaur, as other groups have done for mhv [ ] , may provide further insight into the role of nsp in membrane modifications. an alternative explanation for the lack of spherules could be that the precise molar ratio of nsps to one another, as well as the presence of cleavage intermediates, generated as a result of expression via a polyprotein during virus infection is critical for spherule formation. in that case, the expression approach taken here of transfecting multiple plasmids into cells would not result in the correct ratio of proteins or presence of cleavage intermediates, thereby preventing spherule formation. however, oudshoorn et al. were also unable to identify cms and spherule-like structures when combinations of nsps were expressed either from separate plasmids or as a polyprotein [ ] . instead, it is more likely that additional viral proteins are required for spherule formation. this is not necessarily surprising. for alphaviruses, spherules are only formed in the presence of all nsps and although they are able to form in the absence of rna, the length of rna present directly affects the size of the spherule produced [ , ] . furthermore, in the case of flock house virus, spherules only form when rna synthesis is actively occurring [ ] . therefore, spherule formation by ibv may require expression of additional nsps, including those required for rna synthesis, as well as an rna template. alternatively, it may require expression of additional nsps that direct interaction with cellular proteins that facilitate changes to the membrane. the mechanisms behind the formation of virus-induced membrane rearrangements required for replication organelle formation are doubtlessly complex. although we have identified a clear role for ibv nsp in membrane pairing and the formation of zippered er, numerous questions remain and further differences between ibv and members of the betacoronavirus sub-family have been highlighted. the identity of the ibv proteins required for both spherule and dmv formation remain unknown and further study is required to complete our understanding of the critical stage of the virus replication cycle. supplementary materials: the following are available online at http://www.mdpi.com/ - 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- journal: viruses doi: . /v sha: doc_id: cord_uid: kc gi cj severe acute respiratory syndrome virus (sars-cov- ) is responsible for the current global coronavirus disease (covid- ) pandemic, infecting millions of people and causing hundreds of thousands of deaths. the viral entry of sars-cov- depends on an interaction between the receptor-binding domain of its trimeric spike glycoprotein and the human angiotensin-converting enzyme (ace ) receptor. a better understanding of the spike/ace interaction is still required to design anti-sars-cov- therapeutics. here, we investigated the degree of cooperativity of ace within both the sars-cov- and the closely related sars-cov- membrane-bound s glycoproteins. we show that there exist differential inter-protomer conformational transitions between both spike trimers. interestingly, the sars-cov- spike exhibits a positive cooperativity for monomeric soluble ace binding when compared to the sars-cov- spike, which might have more structural restraints. our findings can be of importance in the development of therapeutics that block the spike/ace interaction. severe acute respiratory syndrome virus (sars-cov- ) is the cause of the current and rapidly evolving coronavirus disease (covid- ) pandemic. one potential therapeutic target receiving significant attention is the interaction between the sars-cov- spike (s) glycoprotein and its receptor, human angiotensin-converting enzyme (ace ). the s glycoprotein is a heavily glycosylated type i membrane protein present as a trimer on mature virions and consists of three s /s heterodimers [ , ] . the s subunit contains a receptor-binding domain (rbd) that specifically binds to ace and can undergo hinge-like movements to switch between an "up" position for receptor binding and "down" position for potential immune evasion [ ] [ ] [ ] [ ] . the s glycoprotein can only bind to ace with the rbd in the "up" state and this results in the dissociation of the trimer [ , ] . ace , the primary receptor for the plasmids expressing the different human coronavirus spikes (sars-cov- and sars-cov- ) were previously reported [ ] . the d g mutation was introduced using the quikchange ii xl site-directed mutagenesis protocol (agilent technologies, santa clara, ca, usa). the presence of the desired mutations was determined by automated dna sequencing. the plasmid encoding for soluble human ace (residues - ) fused with an xhistag was reported elsewhere [ ] . to generate the recombinant ace -fc fusion plasmid, dna encoding ace ( - ) was linked to fc segment of human igg and the whole fusion fragment was cloned into pacp-tag(m)- vector using nhei/noti as restriction sites. the vesicular stomatitis virus g (vsv-g)-encoding plasmid (psvcmv-in-vsv-g) was previously described [ ] . t human embryonic kidney cells (obtained from atcc, manassas, va, usa) were maintained at • c under % co in dulbecco's modified eagle's medium (dmem) (wisent, st. bruno, qc, canada) containing % fetal bovine serum (vwr) and µg/ml of penicillin-streptomycin (wisent). the generation and maintenance of t-ace cell line were previously reported [ ] . freestyle f cells (invitrogen, rockford, il, usa) were grown in freestyle f medium (invitrogen) to a density of × cells/ml at • c with % co with regular agitation ( rpm). cells were transfected with a plasmid coding for soluble ace or ace -fc using expifectamine transfection reagent, as directed by the manufacturer (invitrogen). one week later, cells were pelleted and discarded. supernatants were filtered using a . µm filter (thermo fisher scientific, waltham, ma, usa). the recombinant sace protein was purified by nickel affinity columns (invitrogen) and ace -fc was purified using protein a affinity column (cytiva, marlborough, ma, usa), as directed by the manufacturers. the protein preparations were dialyzed against phosphate-buffered saline (pbs) and stored in aliquots at − • c until further use. to assess purity, recombinant proteins were loaded on sds-page gels and stained with coomassie blue. using the standard calcium phosphate method, µg of spike expressor and µg of a green fluorescent protein (gfp) expressor (pires-gfp) were transfected into × t cells. to determine the hill coefficients, cells were preincubated with increasing concentrations of soluble ace ( to , nm), ace -fc ( to nm), or the monoclonal antibody cr ( to nm) h post-transfection. sace binding was detected using a polyclonal goat anti-ace (rnd systems). alexafluor- -conjugated goat anti-human igg (h+l) ab (invitrogen) and alexafluor- -conjugated donkey anti-goat igg (h+l) ab (invitrogen) were used as secondary antibodies. the percentage of transfected cells (gfp+ cells) was determined by gating the living cell population based on viability dye staining (aqua vivid, invitrogen). samples were acquired on an lsrii cytometer (bd biosciences, mississauga, on, canada) and data analysis was performed using flowjo vx. . (tree star, ashland, or, usa). hill coefficient analyses were done using graphpad prism version . . (graphpad, san diego, ca, usa). target cells were infected with single-round luciferase-expressing lentiviral particles. briefly, t cells were transfected by the calcium phosphate method with the lentiviral vector pnl . r-e-luc (nih aids reagent program) and a plasmid encoding for sars-cov- spike (wt or d g), sars-cov- spike, or vsv-g at a ratio of : . two days after transfection, the cell supernatants were harvested. each virus preparation was frozen and stored in aliquots at − • c until use. t-ace target cells were seeded at a density of × cells/well in -well luminometer-compatible tissue culture plates (perkin elmer, waltham, ma, usa) h before infection. luciferase-expressing recombinant viruses in a final volume of µl were incubated with increasing concentrations of soluble ace ( to , nm), ace -fc ( to nm), or the monoclonal antibody cr ( to nm) for h at • c and were then added to the target cells for an additional hours followed by incubation for h at • c; the medium was then removed from each well, and the cells were lysed by the addition of µl of passive lysis buffer (promega, madison, wi, usa) followed by three freeze-thaw cycles. an lb tristar luminometer (berthold technologies, bad wildbad, germany) was used to measure the luciferase activity of each well after the addition of µl of luciferin buffer ( mm mgso , mm kpo [ph . ], mm atp, and mm dithiothreitol) and µl of mm d-luciferin potassium salt (prolume, pinetop, az, usa). the neutralization half-maximal inhibitory concentration (ic ) represents the ligand concentration required to inhibit % of the infection of t-ace cells by recombinant lentiviral viruses bearing the indicated surface glycoproteins. ic values were determined using a normalized non-linear regression using graphpad prism software. to better understand the interactions between membrane-bound sars-cov- and sars-cov- s glycoproteins with their receptor, human ace , we sought to determine the cooperativity of ace within the respective trimers. to assess this, we calculated the hill coefficient, which is the steepness of a concentration-response curve and reflects the degree of cooperativity between a ligand and its receptor [ , ] . briefly, hek t cells were transfected with plasmids expressing the full-length sars-cov- s and sars-cov- s glycoproteins. we also tested the sars-cov- s d g mutant that is associated with higher infectivity and is now the strain circulating worldwide [ ] [ ] [ ] . transfected cells were incubated with increasing concentrations of sace and bound sace was revealed with an anti-ace antibody. these results were used to calculate the hill coefficient as indicated in material and methods. both the sars-cov- s and its d g counterpart demonstrated a positive cooperativity of sace binding (hill coefficient > ) ( figure a) , thus, indicating that the d g mutation does not overly affect s conformation, at least regarding sace interaction, in line with recent findings [ , ] . interestingly, sars-cov- s presented negative cooperativity (hill coefficient < ), suggesting that the interaction of sace with one sars-cov- s protomer reduces the efficiency with which additional sace molecules can engage adjacent s protomers. to evaluate if the differential hill coefficients observed between sars-cov- and sars-cov- was conserved among different rbd ligands, we tested two additional rbd-binding ligands: ace -fc, a molecule presenting two ace domains (residues - ) fused to a fc fragment, and the cr monoclonal antibody, which is specific to the sars-cov- rbd and has been shown to cross-react strongly with the rbd of sars-cov- and does not compete with the binding of ace [ , ] . interestingly, no differential cooperativity between sars-cov- and sars-cov- was observed for ace -fc, suggesting that the enhanced avidity provided by ace -fc, which allows for multiple spike protomers to bind, is able to overcome potential structural restraints present in the sars-cov- s ( figure b ). of note, we observed negative cooperativity of cr for all tested s glycoproteins ( figure c ), in line with previous findings showing that the binding of cr leads to the destruction of the prefusion sars-cov- s trimer [ ] . thus, it is possible that the binding of cr to one rbd protomer distorts the trimer structure, preventing additional cr molecules from binding. next, we tested the abilities of sace and ace -fc to neutralize sars-cov- and sars-cov- spike-bearing pseudovirions. we observed a higher neutralization potency of sace against sars-cov- s (wt or d g) when compared to sars-cov- (table ) . however, whether the negative cooperativity observed for the sars-cov- s/sace interaction could explain the ~ -fold lower neutralization potency of monomeric ace when compared to sars-cov- (ic = nm for sars-cov- s; nm for sars-cov- s) ( figure a ; table ) remains to be determined. in agreement with previous reports [ ] , ace -fc neutralized both sars-cov- s and sars-cov- s with a higher efficiency compared to monomeric sace ( figure b ; table ), further supporting previous observations that ligand multimerization enhances potency by providing higher avidity [ , , , ] . interestingly, we observed that sace ic was only reached upon its half-maximal binding to the respective s proteins, suggesting a model where the occupancy of at least two or more protomers of the sars-cov- s or sars-cov- s by sace is needed for virus neutralization ( figure d,e) . next, we tested the abilities of sace and ace -fc to neutralize sars-cov- and sars-cov- spike-bearing pseudovirions. we observed a higher neutralization potency of sace against sars-cov- s (wt or d g) when compared to sars-cov- (table ) . however, whether the negative cooperativity observed for the sars-cov- s/sace interaction could explain the~ -fold lower neutralization potency of monomeric ace when compared to sars-cov- (ic = nm for sars-cov- s; nm for sars-cov- s) (figure a ; table ) remains to be determined. in agreement with previous reports [ ] , ace -fc neutralized both sars-cov- s and sars-cov- s with a higher efficiency compared to monomeric sace ( figure b ; table ), further supporting previous observations that ligand multimerization enhances potency by providing higher avidity [ , , , ] . interestingly, we observed that sace ic was only reached upon its half-maximal binding to the respective s proteins, suggesting a model where the occupancy of at least two or more protomers of the sars-cov- s or sars-cov- s by sace is needed for virus neutralization ( figure d ,e). cryo-em data collected on free (receptor-unbound) sars-cov- s indicate that it assumes three distinct states. preferential are the -rbd-down state ( %) and the -rbd-up state ( %) that exists at a near : ratio, whereas less preferential is the -rbd-up conformation (approximately %) [ , , ] . this indicates that there is a relatively low barrier for the open-closed transition in its energy landscape. consistent with this more easy-to-open propensity of the sars-cov- spike, zhou et al. have identified % one-ace -bound, % two-ace -bound, and % three ace -bound sars-cov- spike by single-particle cryo-em when mixing spike and ace in a : molar ratio [ ] . the higher percentage of the two/three-ace bound state over the single ace -bound state corroborates the positive cooperativity seen between monomeric receptor and sars-cov- trimer. interestingly, in all ace -bound spike trimers, rbds not bound to ace are in the "down" conformation, suggesting that the down-to-up rearrangement of the spike trimer is the rate-limiting step in receptor binding. in contrast, available data reveal differences in the conformational dynamics of the sars-cov- spike which seems to have less propensity to engage multiple ace monomers. with a ratio of : (spike:ace ), as described by zhou et al. for the sars-cov- spike, the prevailing complex fraction observed for sars-cov- was the one ace monomer bound spike [ ] . interestingly, the cryo-em structure of this complex revealed three distinct conformations of spike in which the loaded rbd adopted three different tilted orientations and the other two rbds remained in the down state [ ] . this is different from the multiple ace loading of the sars-cov- spike which displays one essentially identical rbd up conformation. based on the structural dynamic data described above we propose a model as shown in figure . according to our model, the sars-cov- spike has more structural restraints from the ntd, sd , sd and s domains and, therefore, needs to overcome a higher energy barrier to open. as a result, the sars-cov- spike has a smaller population in the open conformation in the absence of ace , which is in line with the unavailability of structures of multi-ace bound sars-cov- spikes and our finding that monomeric sace binding suppresses the opening of the other two rbds. interestingly, these differences can be detected only if monomeric ace is used to form the complex but not with ace -fc. there is still a question of debate if ace monomer or dimer is involved in the entry process of both sars-cov- and sars-cov- . if the latter, the differences we observe for monomeric ace resulting from the intrinsic structural features of the two coronavirus spikes which leads to different energy landscapes could be mitigated during the process of viral entry in vivo. however, whether these differences are important for antibody-mediated neutralization remains to be determined. altogether, our results show differential inter-protomer conformational transitions between sars-cov- and sars-cov- s glycoproteins upon sace binding. a better understanding of conformational differences between the s glycoproteins between these two beta-coronaviruses might prove useful for the development of new therapeutics and/or vaccine design. we assume conformational landscapes for both species of spikes (sars-cov- : black; sars-cov- : red) that permit equilibration among four distinct states: all-receptor-binding-domains (rbds) -down (" "), one-rbd-up (" "), two-rbds-up (" "), and three-rbds-up (" "). we hypothesize that the sars-cov- spike energetic barrier for transitioning from all-rbds-down to one-rbd-up ( * ) is lower for sars-cov- than for sars-cov- , while both experience the same barrier for the reverse transition, making the one-rbd-up state more stable for sars-cov- than for sars-cov- , as illustrated by the relative energy differences ∆ . however, sars-cov- spike likely needs to overcome substantially higher energy barriers to transit from the one-rbd-up state to the two-rbdsup state ( * ), as compared with sars-cov- spike. this underlying conformational selection mechanism results in a larger population of sars-cov- spike in two or three rbds up state and has higher chance to engage multiple ace , which eventually spurs the complete open and dissociation of s from s and induces membrane fusion. cartoon representation of closed, one-rbd-up, two-rbds-up states of spike were generated from deposited structures in protein data bank ( zwv, vsb, × b, respectively). the three-rbds-up model was generated by c symmetry superposition of three one-rbd-up protomer in vsb. we assume conformational landscapes for both species of spikes (sars-cov- : black; sars-cov- : red) that permit equilibration among four distinct states: all-receptor-binding-domains (rbds) -down (" "), one-rbd-up (" "), two-rbds-up (" "), and three-rbds-up (" "). we hypothesize that the sars-cov- spike energetic barrier for transitioning from all-rbds-down to one-rbd-up (e * ) is lower for sars-cov- than for sars-cov- , while both experience the same barrier for the reverse transition, making the one-rbd-up state more stable for sars-cov- than for sars-cov- , as illustrated by the relative energy differences ∆e . however, sars-cov- spike likely needs to overcome substantially higher energy barriers to transit from the one-rbd-up state to the two-rbds-up state (e * ), as compared with sars-cov- spike. this underlying conformational selection mechanism results in a larger population of sars-cov- spike in two or three rbds up state and has higher chance to engage multiple ace , which eventually spurs the complete open and dissociation of s from s and induces membrane fusion. cartoon representation of closed, one-rbd-up, two-rbds-up states of spike were generated from deposited structures in protein data bank ( zwv, vsb, × b, respectively). the three-rbds-up model was generated by c symmetry superposition of three one-rbd-up protomer in vsb. author contributions: s.p.a., j.p., and a.f. conceived the study; s.p.a., j.p., and a.f. designed experimental approaches; s.p.a. and r.g. performed the experiments; s.p.a., y.c., j.p., c.f.a., m.p., and a.f. analyzed and interpreted the experiments; g.b.-b., y.c., and m.p. contributed unique reagents; s.p.a., y.c., m.p., and a.f. wrote the paper. every author has read, edited, and approved the final manuscript. all authors have read and agreed to the published version of the manuscript. funding: this work was supported by "ministère de l'Économie et de l'innovation du québec, programme de soutien aux organismes de recherche et d'innovation", by the fondation du chum, by the canada's covid- immunity task force (citf), in collaboration with the canadian institutes of health research (cihr) and a cihr foundation grant # to a.f. this work was also supported by the national institute of health grants r ai to mp and r ai to m.p. and a.f. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the authors declare no competing interests. a.f. is the recipient of a canada research chair on retroviral entry #rchs - . s.p.a., j.p., and g.b.b. are supported by cihr fellowships. r.g. is supported by a mitacs accélération postdoctoral fellowship. structure, function, and antigenicity of the sars-cov- spike glycoprotein cryo-em structure of the -ncov spike in the prefusion conformation cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding distinct conformational states of sars-cov- spike protein structure of the sars-cov- spike receptor-binding domain bound to the ace receptor sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor angiotensin-converting enzyme is a functional receptor for the sars coronavirus angiotensin-converting enzyme is an essential regulator of heart function inhibition of sars-cov- infections in engineered human tissues using clinical-grade soluble human ace a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin furin cleavage site is key to sars-cov- pathogenesis structural basis of receptor recognition by sars-cov- a ph-dependent switch mediates conformational masking of sars-cov- spike structures and distributions of sars-cov- spike proteins on intact virions characterization of the receptor-binding domain (rbd) of novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine trimeric sars-cov- spike interacts with dimeric ace with limited intra-spike avidity sars-cov- and three related coronaviruses utilize multiple ace orthologs and are potently blocked by an improved ace -ig neutralization of sars-cov- spike pseudotyped virus by recombinant ace -ig the membrane-proximal intracytoplasmic tyrosine residue of hiv- envelope glycoprotein is critical for basolateral targeting of viral budding in mdck cells cross-sectional evaluation of humoral responses against sars-cov- spike the hill equation revisited: uses and misuses cooperativity in binding processes: new insights from phenomenological modeling tracking changes in sars-cov- spike: evidence that d g increases infectivity of the covid- virus decline of humoral responses against sars-cov- spike in convalescent individuals the d g mutation in the sars-cov- spike protein reduces s shedding and increases infectivity potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants neutralization of sars-cov- by destruction of the prefusion spike the sequence of human ace is suboptimal for binding the s spike protein of sars coronavirus engineering human ace to optimize binding to the spike protein of sars coronavirus controlling the sars-cov- spike glycoprotein conformation cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace the authors thank m. gordon joyce (u.s. mhrp) for the monoclonal antibody cr . the authors declare no conflict of interest. the views expressed in this presentation are those of the authors and do not reflect the official policy or position of the uniformed services university, us army, the department of defense, or the us government.viruses , , key: cord- -xd f ct authors: brownsword, matthew j.; doyle, nicole; brocard, michèle; locker, nicolas; maier, helena j. title: infectious bronchitis virus regulates cellular stress granule signaling date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: xd f ct viruses must hijack cellular translation machinery to express viral genes. in many cases, this is impeded by cellular stress responses. these stress responses result in the global inhibition of translation and the storage of stalled mrnas, into rna-protein aggregates called stress granules. this results in the translational silencing of the majority of mrnas excluding those beneficial for the cell to resolve the specific stress. for example, the expression of antiviral factors is maintained during viral infection. here we investigated stress granule regulation by gammacoronavirus infectious bronchitis virus (ibv), which causes the economically important poultry disease, infectious bronchitis. interestingly, we found that ibv is able to inhibit multiple cellular stress granule signaling pathways, whilst at the same time, ibv replication also results in the induction of seemingly canonical stress granules in a proportion of infected cells. moreover, ibv infection uncouples translational repression and stress granule formation and both processes are independent of eif α phosphorylation. these results provide novel insights into how ibv modulates cellular translation and antiviral stress signaling. during replication within a host cell, all viruses must regulate a variety of cellular processes to generate an environment that allows progeny virus to be produced to continue the infection cycle. this includes promoting pathways that are favorable to replication and overcoming intrinsic immune pathways. cellular stress granules (sg) play an important role in the regulation of gene expression by regulating mrna translation and location, as well as integrating intracellular signaling and antiviral responses, and are therefore often targeted by viruses [ , ] . sg are cytoplasmic, non-membrane bound aggregations of mrna associated with translation initiation factors, the s ribosome and rna binding proteins. they primarily form under stress conditions that trigger the phosphorylation of translation initiation factor eif α [ ] . there are four eif α kinases; protein kinase r (pkr), recognizing dsrna, pkr-like endoplasmic reticulum kinase (perk), sensing er stress, heme regulated eif α kinase (hri) and general control nonderepressible (gcn ), activated by oxidative stress and amino acid deprivation [ ] [ ] [ ] [ ] . despite pkr being the assumed major kinase to activate the integrated stress response (isr) during viral infection, perk [ ] and gcn [ ] have also been found to play an important role. phosphorylation of eif α prevents delivery of the initiator trna to initiating ribosomes, therefore inhibiting translation initiation and leading to the accumulation of stalled s mrnps. sg can also be vero cells were maintained in × eagle's modified essential medium (sigma) supplemented with × l-glutamine (gibco) and % fetal bovine serum (sigma). recombinant ibv strain beau-r has been described previously [ ] . inactivated ibv was generated by treatment with binary ethylenimine (bei). briefly, the virus was incubated in . m bei for h at • c, followed by inactivation of bei by addition of m sodium thiosulfate. inactivation of virus was confirmed by rt-qpcr following the infection of cells. sodium arsenite, cycloheximide, puromycin and emetine were purchased from sigma. vero cells seeded onto glass coverslips were mock infected or infected with undiluted ibv (moi . ) and incubated at • c. after h, × bes (mem, . % tryptose phosphate broth, . % bovine serum albumin, mm n,n-bis( -hydroxyethyl)- -aminoethanesulfonic acid (bes), . % sodium bicarbonate, mm l-glutamine, u/ml nystatin, u/ml penicillin, and u/ml streptomycin) were added and cells were incubated for the indicated time. where indicated, cells were treated for h prior to fixation with µm sodium arsenite or µm cycloheximide, or for h prior to fixation with µm hydrogen peroxide. cells were fixed in % paraformaldehyde in pbs, permeabilized in . % triton x- in pbs and blocked in . % bovine serum albumin (bsa) in pbs. primary and secondary antibodies were diluted in blocking buffer. nuclei were stained with , -diamidino- -phenylindole (dapi). anti-dsrna j (english and scientific consulting) was diluted : , anti-nsp [ ] was diluted : , anti-s ( . , wageningen university and research) was diluted : , anti-ibv (abcam) was diluted : , anti-g bp (bd biosciences) was diluted at : , anti-g bp (sigma) was diluted : , anti-eif η (santa-cruz) was diluted : and anti-eif g (santa-cruz) was diluted : . alexa fluor-conjugated secondary antibodies (invitrogen) were diluted : . cells were visualized using a leica sp or nikon ti eclipse confocal microscope. to determine the percentage of the cells which were positive for sg, cells were counted manually, with at least cells counted over three independent biological replicates. vero cells seeded onto glass coverslips were mock or ibv infected (moi . ). after h, cells were fixed and labelled using the stellaris rna fish simultaneous labelling protocol (biosearch technologies). briefly, cells were fixed in % formaldehyde in pbs and permeabilized in % ethanol at • c. cells were incubated overnight at • c in a humidified chamber, with hybridization buffer containing nm probe and primary antibody. cells were then washed and labelled with alexa fluor-conjugated secondary antibody and dapi. finally, cells were mounted onto glass coverslips using vectashield and sealed with nail varnish. cells were visualized using a leica sp confocal microscope. stellaris fish probes with a quasar label were designed specifically for the nsp and nsp region of the ibv beau-r genome. vero cells seeded in -well plates were mock infected or infected with undiluted ibv (moi . ). at the indicated time points, cells were washed once with cold pbs and lysed in × sample buffer (biorad) containing dtt. cell lysates were heated to • c for min and briefly sonicated. proteins were separated on a %- % bis-tris gel (biorad) and transferred onto nitrocellulose membranes. these were blocked in . % bsa or % milk in tbs-tween (tbs-t), then incubated with primary antibody diluted in blocking buffer. following three washes in tbs-t, membranes were incubated with hrp labelled secondary antibodies (dako), diluted in blocking buffer. after three further washes in tbs-t, blots are incubated chemiluminescence substrate using the clarity western ecl substrate (bio-rad). labelled protein bands were visualized, using a vilber imaging system. anti-ibv was viruses , , of diluted : , anti-eif α (cell signaling technologies) was diluted : , anti-eif α-p (cell signaling technologies) was diluted : and anti-gapdh (invitrogen) was diluted : . vero cells seeded onto glass coverslips were mock or ibv infected (moi . ), as before. rpm was performed as described by david et al. [ ] briefly, one hour prior to processing, control wells were treated with µm sodium arsenite. at the indicated times post infection, cells were incubated with . µm puromycin for s at room temperature and then incubated with . µm puromycin and µm emetine at room temperature for min. cells were washed three times with room temperature × bes media, fixed and processed for immunofluorescence, as described above. anti-puromycin (sigma) was diluted : . to quantify immunofluorescence images, the puromycin signal in cells was determined using imagej [ ] . initially, the ability of ibv to induce sg during replication was assessed. vero cells were infected with ibv and at the indicated time points, cells were fixed and labelled with anti-dsrna to detect virus infection and with anti-g bp to detect sg. at each time point, infected cells were present, with the number of infected cells increasing over time, as expected ( figure a ). in addition, at each of the time points tested, g bp puncta were detected in a proportion of, but not all, infected cells, with diffuse g bp found in the remaining infected and uninfected cells. subsequently, the number of infected cells with and without g bp puncta was determined. the percentage of infected cells containing g bp puncta was found to be between and % ( figure b ) and this percentage remained unchanged over the course of infection, with no statistical difference between the percentages of cells containing puncta at any time point. therefore, ibv replication triggers the formation of g bp puncta, but interestingly, only in %- % of infected cells. following identification of sg in ibv infected cells, the requirement for active virus replication in induction of granules was assessed. cells were infected with wild type ibv or a bei-inactivated virus. after hours, cells were fixed and labelled with anti-s and anti-g bp . while cells infected with wild type ibv contained sg as observed before, cells infected with the inactivated virus did not ( figure s ). in addition, none of the compounds used for virus inactivation had any effect on sg formation or inhibition. therefore, the induction of sg requires actively replicating virus and is not a response by the cell to the presence of the virus particle. some viruses inhibit the formation of sg by degradation of sg nucleating proteins, such as g bp . therefore, it was investigated whether g bp is degraded during ibv infection. cells were infected with ibv and after h, cells were lysed, proteins separated by sds-page and levels of g bp , ibv n and gapdh were measured by western blot ( figure c ). g bp levels were unaltered compared to mock treated cells upon either ibv infection or sodium arsenite treatment. therefore, ibv infection does not result in the degradation of g bp . as ibv replication did not induce sg in every infected cell, it was hypothesised that ibv may be able to inhibit the formation of canonical sg. to test this, cells were infected with ibv for h and prior to fixation, cells were treated with sodium arsenite for hour or hydrogen peroxide for h to induce sg formation. sodium arsenite induces eif α-dependent sg by activating the eif α kinase hri. hydrogen peroxide induces sg via hri and gcn [ ] , but also in an eif α independent process, by disrupting the eif f complex. following fixation, cells were labelled with anti-dsrna to detect virus infected cells and anti-g bp to visualize sg. in uninfected cells, treatment with either sodium arsenite or hydrogen peroxide resulted in the formation of sg (figure a ). however, in ibv infected cells, both sodium arsenite and h o induction of sg were blocked with g bp in infected viruses , , of cells, remaining largely diffuse ( figure a ). the percentage of cells containing g bp foci was then determined ( figure b ). in the absence of chemical treatment, % of ibv infected cells contained sg. when mock infected cells were treated with sodium arsenite or hydrogen peroxide, % and % of cells were positive for sg, respectively. however, when ibv infected cells were sodium arsenite or hydrogen peroxide treated, only % and % infected cells contained sg, respectively. therefore, ibv infection inhibits both eif α-dependent and independent sg induction. were detected using an anti-g bp antibody (red) and ibv infection was detected with an anti-dsrna antibody (green). nuclei were stained with dapi (blue). positive control cells were treated with sodium arsenite (naas) to induce eif α-dependent sg. scale bar indicates μm. (b) images in (a) were quantified by manual counting of sg positive cells, identified by counting infected or treated cells with g bp foci. a minimum of cells were counted from three independent replicates. the mean and standard deviation is shown. asterisks indicate statistical significance, as measured by oneway anova, *** represents p < . and **** represents p < . , respectively. (c) vero cells were mock infected or infected with ibv. at hpi, where indicated, cells were treated with naas. cells were lysed at hpi and processed and labelled using anti-g bp , anti-gapdh or anti-ibv antibodies. mean g bp band intensities for ibv and naas treatments were normalized relative to mock band. blot representative of independent replicates. as ibv replication did not induce sg in every infected cell, it was hypothesised that ibv may be able to inhibit the formation of canonical sg. to test this, cells were infected with ibv for h and prior to fixation, cells were treated with sodium arsenite for hour or hydrogen peroxide for h to were detected using an anti-g bp antibody (red) and ibv infection was detected with an anti-dsrna antibody (green). nuclei were stained with dapi (blue). positive control cells were treated with sodium arsenite (naas) to induce eif α-dependent sg. scale bar indicates µm. (b) images in (a) were quantified by manual counting of sg positive cells, identified by counting infected or treated cells with g bp foci. a minimum of cells were counted from three independent replicates. the mean and standard deviation is shown. asterisks indicate statistical significance, as measured by one-way anova, *** represents p < . and **** represents p < . , respectively. (c) vero cells were mock infected or infected with ibv. at hpi, where indicated, cells were treated with naas. cells were lysed at hpi and processed and labelled using anti-g bp , anti-gapdh or anti-ibv antibodies. mean g bp band intensities for ibv and naas treatments were normalized relative to mock band. blot representative of independent replicates. viruses , , of infected cells, both sodium arsenite and h o induction of sg were blocked with g bp in infected cells, remaining largely diffuse (figure a ). the percentage of cells containing g bp foci was then determined ( figure b ). in the absence of chemical treatment, % of ibv infected cells contained sg. when mock infected cells were treated with sodium arsenite or hydrogen peroxide, % and % of cells were positive for sg, respectively. however, when ibv infected cells were sodium arsenite or hydrogen peroxide treated, only % and % infected cells contained sg, respectively. therefore, ibv infection inhibits both eif α-dependent and independent sg induction. figure . ibv inhibits eif α-dependent and independent stress granule induction. (a) vero cells were mock infected or infected with ibv for hours. prior to fixation, cells were treated for hour with μm sodium arsenite (naas) to activate the eif α-dependent pathway or for h with µ m hydrogen peroxide (h o ) to activate the eif α-independent pathway. at hpi, cells were fixed and stress granules (sg) were labelled with an anti-g bp antibody (red). ibv infection was detected with an anti-dsrna antibody (green). nuclei were stained with dapi (blue) and scale bar indicates μm. (b) images from (a) were quantified by manual counting of sg positive cells, identified by counting infected or treated cells with g bp foci. a minimum of cells were counted. data from three independent replicates. asterisks indicate statistical significance as measured by one-way anova, **** p < . . several viruses have been shown to promote the formation of specific virus-induced cytoplasmic foci, by recruitment and relocalisation of many sg components, including g bp and g bp [ , , , ] . therefore, following the identification of g bp puncta in some ibv infected cells, the nature of these puncta was investigated, to determine whether they were canonical sg or virusspecific granules. canonical sg contain multiple sg markers such as g bp , translation initiation factors, ribosomal subunits and mrna. therefore, the presence of punctate translation initiation factors eif η and eif g in infected cells was investigated. cells were infected with ibv and after h, cells were fixed and labelled with anti-dsrna and either anti-eif η or anti-eif g. as expected, eif η and eif g were diffuse within the cytoplasm in mock infected cells ( figure ). similar to previous observations using g bp , in a proportion of virus infected cells, both eif η and eif g were figure . ibv inhibits eif α-dependent and independent stress granule induction. (a) vero cells were mock infected or infected with ibv for hours. prior to fixation, cells were treated for hour with µm sodium arsenite (naas) to activate the eif α-dependent pathway or for h with µm hydrogen peroxide (h o ) to activate the eif α-independent pathway. at hpi, cells were fixed and stress granules (sg) were labelled with an anti-g bp antibody (red). ibv infection was detected with an anti-dsrna antibody (green). nuclei were stained with dapi (blue) and scale bar indicates µm. (b) images from (a) were quantified by manual counting of sg positive cells, identified by counting infected or treated cells with g bp foci. a minimum of cells were counted. data from three independent replicates. asterisks indicate statistical significance as measured by one-way anova, **** p < . . several viruses have been shown to promote the formation of specific virus-induced cytoplasmic foci, by recruitment and relocalisation of many sg components, including g bp and g bp [ , , , ] . therefore, following the identification of g bp puncta in some ibv infected cells, the nature of these puncta was investigated, to determine whether they were canonical sg or virus-specific granules. canonical sg contain multiple sg markers such as g bp , translation initiation factors, ribosomal subunits and mrna. therefore, the presence of punctate translation initiation factors eif η and eif g in infected cells was investigated. cells were infected with ibv and after h, cells were fixed and labelled with anti-dsrna and either anti-eif η or anti-eif g. as expected, eif η and eif g were diffuse within the cytoplasm in mock infected cells ( figure ). similar to previous observations using g bp , in a proportion of virus infected cells, both eif η and eif g were found in cytoplasmic puncta, with the remaining infected cells containing diffuse eif η or eif g ( figure ). therefore, ibv infection induces the formation of sg that contains multiple sg marker proteins. viruses , , x for peer review of found in cytoplasmic puncta, with the remaining infected cells containing diffuse eif η or eif g ( figure ). therefore, ibv infection induces the formation of sg that contains multiple sg marker proteins. in addition to containing multiple sg marker proteins, canonical sg are dissolved in the presence of cycloheximide. as mrnas are constantly shuttled between sg and ribosomes, cycloheximide binding to the ribosome, preventing release of mrna, inhibits recycling to sg. as a result, sg are dissolved. to further understand the nature of ibv induced sg, their susceptibility to cycloheximide treatment was determined. cells were infected with ibv for h, and one hour prior to fixation, cells were treated with cycloheximide. cells were then labelled with anti-dsrna and anti-g bp . firstly, it was confirmed that sg induced with sodium arsenite in uninfected cells were dissolved by treatment with cycloheximide ( figure a ). when cycloheximide treatment was applied to ibv infected cells, a significant decrease in the number of cells containing sg was observed. the percentage of infected cells containing sg was quantified ( figure b ) and, interestingly, the number of ibv infected cycloheximide treated cells containing sg was reduced to a value not significantly different from mock cells. together, this shows that ibv infection induces sg that contain multiple sg markers and are susceptible to cycloheximide, indicating that they are likely to be canonical sg. in addition to containing multiple sg marker proteins, canonical sg are dissolved in the presence of cycloheximide. as mrnas are constantly shuttled between sg and ribosomes, cycloheximide binding to the ribosome, preventing release of mrna, inhibits recycling to sg. as a result, sg are dissolved. to further understand the nature of ibv induced sg, their susceptibility to cycloheximide treatment was determined. cells were infected with ibv for h, and one hour prior to fixation, cells were treated with cycloheximide. cells were then labelled with anti-dsrna and anti-g bp . firstly, it was confirmed that sg induced with sodium arsenite in uninfected cells were dissolved by treatment with cycloheximide ( figure a ). when cycloheximide treatment was applied to ibv infected cells, a significant decrease in the number of cells containing sg was observed. the percentage of infected cells containing sg was quantified ( figure b ) and, interestingly, the number of ibv infected cycloheximide treated cells containing sg was reduced to a value not significantly different from mock cells. together, this shows that ibv infection induces sg that contain multiple sg markers and are susceptible to cycloheximide, indicating that they are likely to be canonical sg. it was previously found that during the replication of alphacoronavirus tgev, viral rna was targeted to virus-induced sg, and this was thought to be important for their anti-viral function [ ] . therefore, to further understand ibv induced sg and to determine whether ibv rna is also targeted to sg, viral genomic rna was visualized using fish. cells were infected with ibv or mock infected. cells were then mock treated or treated with cycloheximide (chx) to dissolve sg. at hpi, cells were fixed and labelled with anti-g bp (red) to detect stress granules (sg) and ibv infected cells were detected with an anti-dsrna antibody (green). nuclei were stained with dapi (blue). scale bar indicates µm. (b) images from (a) were quantified to determine the percentage of cells containing sg. a minimum of cells were counted. mean and standard deviation of three independent replicates are shown. asterisks indicate statistical significance, as measured by one-way anova, **** p < . ; ns, not significant. it was previously found that during the replication of alphacoronavirus tgev, viral rna was targeted to virus-induced sg, and this was thought to be important for their anti-viral function [ ] . therefore, to further understand ibv induced sg and to determine whether ibv rna is also targeted to sg, viral genomic rna was visualized using fish. cells were infected with ibv or mock infected. after hours, cells were fixed and labelled with fish probes specific for ibv genomic rna and anti-g bp ( figure ). viral genomic rna was found to be located in foci within the cytoplasm. in addition, g bp puncta were detected in a percentage of infected cells, as seen before. however, no co-localization was observed between viral genomic rna and g bp containing sg. therefore, ibv genomic rna is not targeted to sg. after hours, cells were fixed and labelled with fish probes specific for ibv genomic rna and anti-g bp ( figure ). viral genomic rna was found to be located in foci within the cytoplasm. in addition, g bp puncta were detected in a percentage of infected cells, as seen before. however, no co-localization was observed between viral genomic rna and g bp containing sg. therefore, ibv genomic rna is not targeted to sg. figure . ibv genomic rna is not diverted to stress granules during infection. vero cells were mock infected or infected with ibv for hours. ibv genomic rna (red) was detected using fluorescent in situ hybridization (fish) probes and an anti-g bp antibody was used to detect sg (green). nuclei were stained using dapi (blue). scale bars indicate µm. images are representative of three independent replicates. during the replication of several other viruses including ebola virus, west nile virus, dengue virus and tick-borne encephalitis virus, sg markers are redirected to sites of virus replication [ ] [ ] [ ] . to investigate whether ibv induced sg co-localize with sites of viral rna synthesis or virion assembly, cells were infected with ibv and after h, fixed and labelled with anti-g bp , as well as antibodies specific for dsrna, thought to be an intermediate in viral rna synthesis, nsp , the viral rna-dependent rna polymerase or spike protein (anti-s ), to label sites of progeny virus assembly. consistent with earlier experiments, dsrna did not co-localize with g bp foci (figure ). in addition, g bp did not to co-localize with either nsp or spike. therefore, ibv does not direct sg markers to sites of virus replication. . ibv genomic rna is not diverted to stress granules during infection. vero cells were mock infected or infected with ibv for hours. ibv genomic rna (red) was detected using fluorescent in situ hybridization (fish) probes and an anti-g bp antibody was used to detect sg (green). nuclei were stained using dapi (blue). scale bars indicate µm. images are representative of three independent replicates. during the replication of several other viruses including ebola virus, west nile virus, dengue virus and tick-borne encephalitis virus, sg markers are redirected to sites of virus replication [ ] [ ] [ ] . to investigate whether ibv induced sg co-localize with sites of viral rna synthesis or virion assembly, cells were infected with ibv and after h, fixed and labelled with anti-g bp , as well as antibodies specific for dsrna, thought to be an intermediate in viral rna synthesis, nsp , the viral rna-dependent rna polymerase or spike protein (anti-s ), to label sites of progeny virus assembly. consistent with earlier experiments, dsrna did not co-localize with g bp foci (figure ). in addition, g bp did not to co-localize with either nsp or spike. therefore, ibv does not direct sg markers to sites of virus replication. after hours, cells were fixed and labelled with fish probes specific for ibv genomic rna and anti-g bp ( figure ). viral genomic rna was found to be located in foci within the cytoplasm. in addition, g bp puncta were detected in a percentage of infected cells, as seen before. however, no co-localization was observed between viral genomic rna and g bp containing sg. therefore, ibv genomic rna is not targeted to sg. figure . ibv genomic rna is not diverted to stress granules during infection. vero cells were mock infected or infected with ibv for hours. ibv genomic rna (red) was detected using fluorescent in situ hybridization (fish) probes and an anti-g bp antibody was used to detect sg (green). nuclei were stained using dapi (blue). scale bars indicate µm. images are representative of three independent replicates. during the replication of several other viruses including ebola virus, west nile virus, dengue virus and tick-borne encephalitis virus, sg markers are redirected to sites of virus replication [ ] [ ] [ ] . to investigate whether ibv induced sg co-localize with sites of viral rna synthesis or virion assembly, cells were infected with ibv and after h, fixed and labelled with anti-g bp , as well as antibodies specific for dsrna, thought to be an intermediate in viral rna synthesis, nsp , the viral rna-dependent rna polymerase or spike protein (anti-s ), to label sites of progeny virus assembly. consistent with earlier experiments, dsrna did not co-localize with g bp foci (figure ). in addition, g bp did not to co-localize with either nsp or spike. therefore, ibv does not direct sg markers to sites of virus replication. the formation of sg in cells is closely associated with an inhibition of translation. previous work has demonstrated that ibv replication is associated with a shut-off of host translation from around hpi, with the translation of viral proteins also ceasing by hpi [ ] . however, translational activity on a single cell level has not been characterized. therefore, rpm was used to visualize actively translating ribosomes over a time course of infection [ ] . cells were infected with ibv and nascent polypeptides labelled with puromycin at , and hpi, followed by the stalling of translating ribosomes with emetine. cells were then fixed and labelled with an anti-puromycin antibody to detect nascent polypeptides and an anti-ibv antibody to detect infected cells. in mock infected cells, active translation was detected, with a diffuse puromycin signal throughout the cytoplasm. this signal was absent without puromycin treatment or upon treatment of cells with sodium arsenite to inhibit translation ( figure a ). following ibv infection at all three time points studied, two phenotypes were observed. some cells contained the diffuse puromycin signal detected in mock infected cells. alternatively, a proportion of infected cells showed reduced puromycin signal ( figure b) . to enable the level of translational activity to be determined, puromycin signal was quantified in at least infected cells and surrounding non-infected cells ( figure c ). this indicated that there was a shut-off of translation at all time points. furthermore, the degree of translational inhibition increased as infection progressed with a more pronounced shut-off at and than at hpi. therefore, consistent with previous work [ ] , this single cell rpm indicates that translational shut-off in infected cells is seen from hpi and increases with the duration of infection. as previously demonstrated, ibv induces a translational shut-off that increases over the duration of infection, with two phenotypes showing variation in the degree of translational shut-off. in addition to this, ibv induces sg in % of infected cells. the translational status of infected cells containing g bp foci was therefore assessed to determine whether the two different translational profiles could be attributed to the presence or absence of sg. cells were infected with ibv and then treated as before, using the rpm method. at hpi cells were fixed and labelled using anti-puromycin, anti-g bp and anti-ibv antibodies ( figure a ) and the intensity of puromycin label quantified as before ( figure b ). active translation was detected in mock infected cells, with a diffuse puromycin signal throughout the cytoplasm. upon stimulation with sodium arsenite, the accumulation of g bp into sg correlated with a strong reduction in puromycin signal, confirming that sg formation is coupled to translational shut-off. the level of translational activity was then determined in infected cells, with and without sg. while infected cells containing sg exhibited a potent translational impairment with puromycin labelling levels similar to those detected in cells stimulated with sodium arsenite, infected cells that did not contain sg also displayed markedly reduced puromycin signal compared to uninfected cells. this demonstrates that the translational shut-off seen in ibv-infected cells cannot be solely attributed to sg formation and that translation inhibition and sg formation are uncoupled. viruses , , x for peer review of asterisks indicate statistical significance, as measured by one-way anova, * p = . ; **** p < . . both sg formation and translational shut-off are usually associated with phosphorylation of eif α. previous work by others has demonstrated that ibv infection results in eif α phosphorylation at early time points, but that the virus inhibits this as infection progresses [ ] . therefore, the phosphorylation status of eif α was investigated. vero cells were infected with ibv and lysed at , , and hpi. proteins were separated by sds-page and transferred to nitrocellulose. blots were labelled using anti-eif α to detect total eif α, anti-eif α-p to detect the phosphorylated eif α, anti-ibv to detect virus and anti-gapdh as a loading control ( figure a ). total levels of eif α remained unchanged throughout infection. although eif α phosphorylation was achieved using sodium arsenite treatment, no eif α phosphorylation was detected at any time point during ibv infection, with levels remaining comparable to that of mock infected cells. subsequently, to determine whether ibv infection actively inhibits phosphorylation of eif α, vero cells were infected with ibv and then treated with sodium arsenite prior to cell lysis ( figure b ). as before, the sodium arsenite treatment of mock infected cells resulted in a significant increase in the level of phosphorylated eif α. when ibv infected cells were treated with sodium arsenite, there was also a significant increase in the level of phosphorylated eif α when compared to ibv infected untreated cells. significantly, the level of phosphorylated eif α in these cells appeared comparable to that in mock infected sodium arsenite treated cells ( figure b ). together, this demonstrates that sg formation and translational shut-off observed during ibv replication both occur in the absence of detectable levels of eif α phosphorylation, but that ibv infection does not actively inhibit eif α phosphorylation. data presented is representative of three independent replicates. asterisks indicate statistical significance, as measured by one-way anova, *** p = . ; **** p < . . both sg formation and translational shut-off are usually associated with phosphorylation of eif α. previous work by others has demonstrated that ibv infection results in eif α phosphorylation at early time points, but that the virus inhibits this as infection progresses [ ] . therefore, the phosphorylation status of eif α was investigated. vero cells were infected with ibv and lysed at , , and hpi. proteins were separated by sds-page and transferred to nitrocellulose. blots were labelled using anti-eif α to detect total eif α, anti-eif α-p to detect the phosphorylated eif α, anti-ibv to detect virus and anti-gapdh as a loading control ( figure a ). total levels of eif α remained unchanged throughout infection. although eif α phosphorylation was achieved using sodium arsenite treatment, no eif α phosphorylation was detected at any time point during ibv infection, with levels remaining comparable to that of mock infected cells. subsequently, to determine whether ibv infection actively inhibits phosphorylation of eif α, vero cells were infected with ibv and then treated with sodium arsenite prior to cell lysis ( figure b ). as before, the sodium arsenite treatment of mock infected cells resulted in a significant increase in the level of phosphorylated eif α. when ibv infected cells were treated with sodium arsenite, there was also a significant increase in the level of phosphorylated eif α when compared to ibv infected untreated cells. significantly, the level of phosphorylated eif α in these cells appeared comparable to that in mock infected sodium arsenite treated cells ( figure b ). together, this demonstrates that sg formation and translational shut-off observed during ibv replication both occur in the absence of detectable levels of eif α phosphorylation, but that ibv infection does not actively inhibit eif α phosphorylation. hpi, cells were washed and lysed. samples were then separated by sds-page and transferred to nitrocellulose. total eif α (eif α-t) was detected with anti-eif α, phosphorylated eif α (eif α-p) was detected with anti-eif α-p. ibv proteins were detected using an anti-ibv antibody, with a band corresponding to the ibv nucleocapsid protein shown (ibv (n)) and an anti-gapdh antibody was used as a loading control. (b) vero cells were mock infected or infected with ibv. at hpi, where indicated, cells were treated with naas. cells were lysed at hpi and processed and labelled as in (a). blots are representative of three independent replicates. here, we present a study that furthers our understanding of how ibv regulates the important cellular pathways of the isr and translation. firstly, we have demonstrated that ibv infection inhibits both eif α-dependent and independent sg formation. several other viruses, including kaposi's sarcoma-associated herpesvirus, zika virus, west nile virus and junin virus have been shown to inhibit sg signaling via regulation of the eif α-dependent pathway [ ] [ ] [ ] [ ] . these viruses achieve this by inhibiting activation of pkr [ ] , thereby preventing eif α phosphorylation [ ] or by total eif α (eif α-t) was detected with anti-eif α, phosphorylated eif α (eif α-p) was detected with anti-eif α-p. ibv proteins were detected using an anti-ibv antibody, with a band corresponding to the ibv nucleocapsid protein shown (ibv (n)) and an anti-gapdh antibody was used as a loading control. (b) vero cells were mock infected or infected with ibv. at hpi, where indicated, cells were treated with naas. cells were lysed at hpi and processed and labelled as in (a). blots are representative of three independent replicates. here, we present a study that furthers our understanding of how ibv regulates the important cellular pathways of the isr and translation. firstly, we have demonstrated that ibv infection inhibits both eif α-dependent and independent sg formation. several other viruses, including kaposi's sarcoma-associated herpesvirus, zika virus, west nile virus and junin virus have been shown to inhibit sg signaling via regulation of the eif α-dependent pathway [ ] [ ] [ ] [ ] . these viruses achieve this by inhibiting activation of pkr [ ] , thereby preventing eif α phosphorylation [ ] or by dephosphorylating eif α [ ] . zika virus was found to upregulate growth arrest, and dna-damage-inducible (gadd ), a component of the protein phosphatase (pp ) complex, and subsequent dephosphorylation of eif α [ ] . interestingly, in the present study, ibv was also found to inhibit eif α-independent sg signaling. flaviviruses and ebola virus also inhibit both eif α-dependent and independent signaling. ebola virus achieves this via an interaction between vp and several sg components including; g bp , eif and eef [ , , ] . whether any ibv proteins interact directly with sg components to inhibit sg formation remains to be determined. interestingly, hydrogen peroxide has also been found to inhibit sgs via the oxidation of tia- . arimoto-matsuzaki et al. ( ) also concluded that when cells are exposed to both oxidative stress and er stress via protein misfolding, these cells cannot form sg [ ] . it is therefore possible that the inhibition of hydrogen peroxide induced sgs presented here is achieved via er stress caused during ibv infection prior to hydrogen peroxide treatment [ ] . despite ibv regulation of multiple sg signaling pathways, infection results in the formation of sg in approximately % of infected cells. numerous viruses such as poxviruses and reoviruses are known to divert sg components to sites of virus replication to benefit the virus [ , ] . however, although not exhaustive, our analysis suggests that the sg formed in % of infected cells are canonical sg produced in response to virus replication. ibv induced sg were found to contain multiple sg marker proteins and were susceptible to cycloheximide treatment, hallmarks of canonical sg. in addition, the sg markers did not co-localize with markers for viral rna synthesis or particle assembly. therefore, they do not appear to resemble the virus-specific granules produced during the replication of other viruses. this then raises the interesting question of how sg form in this subset of cells. it is possible that the viral control of sg signaling may alter over the course of infection. however, eif α was not phosphorylated, even at early time points post infection, and the sg positive subpopulation of cells remained constant at all time points tested. therefore, this would not appear to be the case. other viruses have also been shown previously to induce sg in only a proportion of infected cells. for example, semliki forest virus infection resulted in sg in % of infected cells at hpi, with a further decrease after this point [ ] . in chronic hepatitis c virus (hcv) infection, an oscillation of the stress response is seen, in which % of hcv infected cells treated with interferon-α had sg, but in a live time course, this was shown to oscillate in a cell-specific rhythm, with % of cells displaying sg at some point during infection. this appears to be a strategy by hcv to modulate the cellular stress response by pkr activated eif α phosphorylation or conversely, dephosphorylation of eif α by the upregulation of gadd in a balancing act to prolong cell survival with oscillating stalls in translation and cell division [ ] . therefore, it is possible that in the ibv infected cells containing sg, viral regulation of eif α phosphorylation or other sg signaling pathways is less efficient, or these cells perhaps represent a more complex balancing act. another possible explanation for this subset of sg displaying cells is a pre-priming of the cellular innate immunity via a paracrine signaling effect, as seen with interferon (ifn) signaling, in which paracrine ifn activates the jak/stat pathway and upregulates interferon stimulated genes (isgs). vero cells do not secrete ifn, and so are unlikely to use this specific paracrine signaling pathway, however it cannot be ruled out for other signaling molecules. analysis at the single cell level will likely be required to tease apart the mechanism of sg formation in the subset of ibv infected cells that contain them. other members of the cov family have been found to display markedly different relationships with sg and their regulation. transmissible gastroenteritis virus (tgev) infection was shown to induce specific antiviral sg. in contrast to observations seen here where ibv genomic rna did not co-localize with g bp , these tgev specific granules feature an interaction between polypyrimidine tract binding protein (ptb) and viral genomic and sub-genomic rna [ ] . during mouse hepatitis virus (mhv) infection, translational shut-off and sg formation was also observed and mhv replication was enhanced upon infection of eif αs a −/− cells or tia −/− cells in which translational inhibition and sg formation are impaired, indicating an inhibitory role for sg during mhv replication [ ] . similar to our observations here, mers-cov inhibits sg formation. this is achieved via an interaction between mers-cov accessory protein a and dsrna, preventing pkr activation [ ] . therefore, taken together, and in agreement with our findings here that ibv inhibits multiple sg induction pathways, this suggests an antiviral function for sg during cov replication. sg usually form following translation inhibition, as a result of the aggregation of stalled mrnps, translation initiation factors and rna binding proteins. therefore, the translational activity of ibv infected cells was investigated. in agreement with previous work [ ] , translation inhibition occurred during ibv replication from hpi and the degree of inhibition increased as infection progressed. however, only % of infected cells contain sg and this remained constant across all time points studied. it was considered possible that cells with reduced translational activity are the % observed to contain sg, even though this would not account for all the cells found to have reduced levels of translation, particularly at later time points. therefore, the relationship between sg and translational inhibition in ibv infected cells was assessed using rpm and sg staining simultaneously. this confirmed that de novo protein synthesis in ibv infected cells without sg is significantly reduced, although it is reduced to a greater extent when sg are present. therefore, the translational inhibition seen during ibv infection cannot be solely attributed to sg formation and, interestingly, there is an uncoupling of translational arrest and sg formation. indeed, we observe both sg formation and translational inhibition in the absence of eif α phosphorylation, indicating altered signaling for both pathways. this situation has also been observed during murine norovirus (mnv) replication, where cellular translation is inhibited and canonical sg assembly is blocked through the repurposing of g bp independently from eif α phosphorylation [ , ] . mnv translational control is achieved via the phosphorylation of eif e by mnk , which in turn, is activated by the p kinase [ , ] . in addition, several cov have been shown to activate the interferon-induced endoribonuclease rnase l [ , ] . rnase l cleaves cellular and viral rnas producing ligands of pattern recognition receptors to amplify ifn production and the antiviral response [ ] . interestingly, rnase l has recently been shown to reduce translation independently of pkr, phospho-eif -alpha, and sgs [ ] . moreover, rnase l activity has been linked with the assembly of specific membrane-less organelles [ , ] . in cells treated with the pkr ligand poly(i:c), rnase l activation also resulted in the inhibition of chemically-induced sgs, similar to our observations with ibv, and the assembly rnase l-bodies (rlb) that are small sg-like punctate, that do not require g bp for assembly and are uncoupled from translation inhibition via pkr [ ] . interestingly, another study using the specific rnase l ligand - a showed that rnase l stimulation resulted in the assembly of antiviral stress granules (avsgs) [ ] . these avsgs are different from the rlbs in that they require g bp for assembly, which interacts with pkr and rig-i in these granules, but also oas and rnase l. the assembly of avsgs further stimulates irf -mediated ifn production, rather than ifn signaling, and may provide a platform for the interaction of rna ligands, with pattern recognition receptors to amplify the ifn production. despite these discrepancies, further investigation into the role of rnase l in ibv-mediated regulation of translation and sgs is warranted. the mechanism of ibv translational control is currently unknown. other covs have been shown to inhibit translation through the action of a viral non-structural protein, nsp , which binds the s ribosomal subunit and cleaves host mrna [ ] . however, ibv does not express nsp . instead, ibv accessory protein b was found to be responsible for translational shut-off and the stability of some mrnas tested was actually increased upon ibv infection, suggesting a completely different mechanism for the control of cellular translation [ ] . during this work, ibv replication did not result in phosphorylation of eif α, at any of the time points tested. furthermore, infection was not able to limit sodium arsenite induction of eif α phosphorylation, showing that ibv cannot actively inhibit eif α phosphorylation. this is in contrast to previous findings that ibv nsp is a weak antagonist of pkr and that gadd is upregulated during ibv infection, resulting in decreased levels of phosphorylated eif α [ ] . however, recently gadd has been shown to promote sg disassembly and block further stress sensing and eif a phosphorylation, but also to contribute to impairing sg assembly independently from the eif a pathway [ , ] . therefore, we do not exclude that gadd induction may contribute to the absence of sgs in ibv-infected cells. a subsequent study by the same laboratory found that ibv infection also induced the phosphorylation of perk, and the subsequent activation of atf and the proapoptotic gadd , again resulting in dephosphorylation of eif α [ ] . the reason for the inconsistency between our current findings and the previous work is not clear, although one methodological difference in the current study is the use of sodium arsenite to induce eif α phosphorylation, which acts via hri, whilst the previous studies showed the activation if eif α phosphorylation in virus infected cells, via activation of either pkr or perk. notably however, in our work presented here ibv replication did not induce phosphorylation of eif α at any time point, and it is therefore not necessarily surprising that mechanisms to dephosphorylate eif α are also not activated. indeed, the signaling molecule required for activation of pkr, dsrna, is known to be concealed within virus induced vesicles during coronavirus replication [ ] . furthermore, interferon signaling, which also relies upon the sensing of dsrna is not activated in ibv infected cells until very late time points, consistent with the shielding of dsrna from cellular detection [ ] . therefore, the activation of these various cellular signaling pathways in response to ibv infection is likely to be prevented, consistent with our findings. in the present study, we have demonstrated that ibv replication effectively blocks both eif α-dependent and eif α-independent sg signaling pathways. in addition, ibv replication results in a shut-off of translation. however, interestingly, in a proportion of infected cells, canonical sg are formed that do not localize with sites of viral replication and do not contain viral rna. this raises the interesting future possibility of being able to study the composition and function of canonical cellular sg in virus infected cells. in addition to these findings, in ibv infected cells, both translational repression and sg formation were found to occur in the absence of eif α phosphorylation, although ibv replication was not able to actively inhibit eif α phosphorylation. therefore, the ibv infection of cells results in a dysregulation and uncoupling of several important cellular signaling pathways. the mechanism behind this dysregulation remains to be determined, but we have furthered our understanding of how ibv changes the cellular environment to make it favorable for virus replication. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s . active replication is required for ibv induced stress granules. translation inhibition and stress granules in the antiviral immune response tinkering with translation: protein synthesis in virus-infected cells stress granules: sites of mrna triage that regulate mrna stability and translatability the dsrna protein kinase pkr: virus and cell control protein translation and folding are coupled by an 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middle east respiratory syndrome coronavirus ns b protein inhibits host rnase l activation activation of rnase l by murine coronavirus in myeloid cells is dependent on basal oas gene expression and independent of virus-induced interferon a scientific journey through the - a/rnase l system rnase l promotes the formation of unique ribonucleoprotein granules distinct from stress granules rnase l amplifies interferon signaling by inducing pkr-mediated antiviral stress granules coronavirus nonstructural protein : common and distinct functions in the regulation of host and viral gene expression protein synthesis inhibition and gadd control ifn-β heterogeneous expression in response to dsrna sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum activation of the chicken type i interferon response by infectious bronchitis coronavirus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to thank ambi batra for providing bei-inactivated ibv. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- - me ugkg authors: wang, xiaona; li, fengsai; han, meijing; jia, shuo; wang, li; qiao, xinyuan; jiang, yanping; cui, wen; tang, lijie; li, yijing; xu, yi-gang title: cloning, prokaryotic soluble expression, and analysis of antiviral activity of two novel feline ifn-ω proteins date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: me ugkg cats are becoming more popular as household companions and pets, forming close relationships with humans. although feline viral diseases can pose serious health hazards to pet cats, commercialized preventative vaccines are lacking. interferons (ifns), especially type i ifns (ifn-α, ifn-β, and interferon omega (ifn-ω)), have been explored as effective therapeutic drugs against viral diseases in cats. nevertheless, there is limited knowledge regarding feline ifn-ω (feifn-ω), compared to ifn-α and ifn-β. in this study, we cloned the genes encoding feifn-ωa and feifn-ωb from cat spleen lymphocytes. homology and phylogenetic tree analysis revealed that these two genes belonged to new subtypes of feifn-ω. the recombinant feifn-ωa and feifn-ωb proteins were expressed in their soluble forms in escherichia coli, followed by purification. both proteins exhibited effective anti-vesicular stomatitis virus (vsv) activity in vero, f (feline kidney cell), madin–darby bovine kidney (mdbk), madin–darby canine kidney (mdck), and porcine kidney (pk- ) cells, showing broader cross-species antiviral activity than the intercat ifn antiviral drug. furthermore, the recombinant feifn-ωa and feifn-ωb proteins demonstrated antiviral activity against vsv, feline coronavirus (fcov), canine parvovirus (cpv), bovine viral diarrhea virus (bvdv), and porcine epidemic diarrhea virus (pedv), indicating better broad-spectrum antiviral activity than the intercat ifn. the two novel feifn-ω proteins (feifn-ωa and feifn-ωb) described in this study show promising potential to serve as effective therapeutic agents for treating viral infections in pet cats. as cats continue to become more popular as household companions and pets [ ] , a variety of viral infections pose a serious threat to felines, including a high incidence of feline leukemia virus (felv) [ ] , feline coronavirus (fcov) [ ] , feline immunodeficiency virus (fiv) [ ] , and feline panleukopenia virus (fpv) [ ] . currently, the only preventative vaccines and therapeutic drugs available for use in pet cats have limited effectiveness. nevertheless, interferons (ifns) play an increasingly complementary role in homology and phylogenetic tree analysis of the feline ifn-ω genes isolated in this study were analyzed using dnastar and mega software. in addition, the characteristics of the feifn-ω genes and proteins were analyzed by several online bioinformatics software programs: signal peptide cleavage sites were analyzed by the signalp . server at http://www.cbs.dtu.dk/services/signalp- . /; phosphorylation sites were analyzed by the netphos . server at http://www.cbs.dtu.dk/services/netphos/; n-glycosylation sites were analyzed by the netnglyc . server at http://www.cbs.dtu.dk/services/netnglyc/; o-glycosylation sites were analyzed by the yinoyang . server at http://www.cbs.dtu.dk/services/yinoyang/; subcellular localization was analyzed by the targetp . server at http://www.cbs.dtu.dk/services/targetp; transmembrane regions were analyzed by the tmhmm . server at http://www.cbs.dtu.dk/services/tmhmm/; antigen epitopes and hydrophobicity were analyzed by the bepipred . server at http://www.cbs.dtu. dk/services/bepipred- . /; and secondary and three-dimensional structures were predicted by sopma at https://npsa-prabi.ibcp.fr/cgi-bin/npsa. in this study, the genes encoding feifn-ω that were isolated by rt-pcr were subcloned as a kpni and bamhi-generated (new england biolabs, ma, usa) gene fragment into the prokaryotic soluble expression plasmid pcold-tf, giving rise to recombinant pcold-feifn-ω. after that, the recombinant plasmid was transformed into e. coli bl (de ) competent cells and validated by pcr and sequencing analyses, thus generating the recombinant e. coli strain pcold-feifn-ω/bl . we then used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) analysis to determine the optimal conditions for expression of the recombinant feifn-ω in pcold-feifn-ω/bl cells via induction by isopropyl β-d-thiogalactoside (iptg, sigma, st. louis, mo, usa). briefly, for optimization of the iptg concentration, the recombinant e. coli strain was grown in luria-bertani broth (sigma, st. louis, mo, usa) supplemented with µg/ml ampicillin at • c until the optical density at nm was approximately . . then, iptg was added at final concentrations of . mmol/l, . mmol/l, . mmol/l, . mmol/l, or . mmol/l, and the cultures were continually cultivated for viruses , , of another h. after centrifugation at , × g for min, the cell pellets were lysed and analyzed by % sds-page. to determine the optimal induction time, the recombinant strain was induced by the optimized final concentration of iptg for h, h, h, h, and h. following centrifugation and cells lysis, the proteins were again analyzed by % sds-page. the fusion protein expressed by the pcold-feifn-ω/bl bacteria cells was subjected to cleavage by the c protease (sigma, st. louis, mo, usa). the target recombinant feifn-ω protein was then purified using ni + affinity chromatography columns according to the manufacturer's instructions, followed by confirmation analysis using sds-page. the purified feifn-ω protein was stored at − • c until use. vsv was used as a virus model to evaluate the antiviral activity of the recombinant feifn-ω via the in vitro microdose cytopathic effect inhibition assay (mcia) according to the method described previously [ , ] with slight modifications. briefly, µl of the purified feifn-ω sample was serially diluted ten-fold in dmem containing % fbs and transferred to confluent f cell monolayers in -well cell culture plates, and then incubated at • c in % co for h. f cells without ifn treatment were used as a control group. after incubation, vsv (moi = ) was prepared as described above, added into the -well plate, and incubated for - h until the cpe of the cells in the viral control group reached %. next, the culture medium was removed, and the cells were stained with . % crystal violet in % ethanol at • c for min. the cells were then destained with . % acetic acid in % ethanol at • c for min before determining the absorbance of each well at nm. each sample was performed with eight biological replicates and three technical replicates. the antiviral activity of the ifn was calculated as the method previously described, which is expressed as units per milligram according to the ratio of ifn titer and protein concentration [ ] . in parallel, the intercat ifn antiviral drug (toray industries, tokyo, japan) was used as an ifn treatment control. in addition, we determined the species-specific antiviral activity of the recombinant feifn-ω by conducting mcias using vsv in f , vero, mdck, mdbk, and pk- cells. broad-spectrum antiviral activity of the recombinant feifn-ω was determined by conducting mcias using vsv, fcov, cpv, bvdv, and pedv. the results are shown as mean ± sem (n = ) for three independent experiments. statistical analyses were performed using graphpad prism v . software. tukey's multiple comparison tests and one-way analysis of variance (anova) tests were used to analyze the significance of the differences between groups: * p < . ; ** p < . ; and *** p < . . we first collected splenic lymphocytes from cats infected with vsv and treated with poly(i:c) simultaneously. we then performed rt-pcr assays using a pair of degenerate primers to identify two genes encoding feifn-ω, referred to as feifn-ωa and feifn-ωb ( figure a) . these two novel genes were deposited in the genbank with accession numbers mk and mk . following nucleotide sequence homology analysis, we found that the feifn-ωa gene (mk ), with a size of bp, shared a maximum nucleotide sequence homology of . % ( . % amino acid homology) with the known subtypes of feifn-ω (feifn-ω to feifn-ω ) genes published in the genbank (figure c ). the feifn-ωb gene (mk ), with a size of bp, shared a maximum sequence homology of . % ( . % amino acid homology) with the known subtypes of feifn-ω ( figure d ). the sequence homology shared between the feifn-ωa and feifn-ωb genes was only . % (figure b) , indicating that the two genes identified in this study were novel feifn-ω genes. we constructed a phylogenetic tree comparing the feifn-ωa/ωb genes identified in this study with other ifn gene sequences published in genbank from different animals using the software dnastar (megalign) and mega . the results showed that the feifn-ωa/ωb genes belonged in the type i ifn family, but that their evolutionary relationship to the known feline ifn-ω genes already published in the genbank (figure ) was distant, indicating that the feifn-ωa and feifn-ωb genes, identified for the first time here, are new subtypes of feline ifn-ω. viruses , , x for peer review of we constructed a phylogenetic tree comparing the feifn-ωa/ωb genes identified in this study with other ifn gene sequences published in genbank from different animals using the software dnastar (megalign) and mega . the results showed that the feifn-ωa/ωb genes belonged in the type i ifn family, but that their evolutionary relationship to the known feline ifn-ω genes already published in the genbank (figure ) was distant, indicating that the feifn-ωa and feifn-ωb genes, identified for the first time here, are new subtypes of feline ifn-ω. the characteristics of the novel feline ifn-ω proteins (feifn-ωa and feifn-ωb) were analyzed using several online bioinformatics software programs, including the identification of potential signal peptide cleavage sites, n-glycosylation sites, o-glycosylation sites, phosphorylation sites, subcellular localization, and transmembrane regions. the results from these detailed analyses are displayed in table . in addition, we also used online software algorithms to predict antigen epitopes, hydrophobicity, and the secondary and three-dimensional structures of the feifn-ωa and feifn-ωb proteins. as shown in figure figure b ). the maximum hydrophobicity of feifn-ωa was . , and the minimum hydrophobicity was − . . the maximum hydrophobicity of feifn-ωb was . , and the minimum hydrophobicity was − . . the secondary structure of the two feifn-ω proteins was predicted using sopma software and revealed that feifn-ωa contained . % alpha helix, . % beta sheet, and . % irregular curl structures (figure c ), whereas feifn-ωb contained . % alpha helix, . % beta sheet, and . % irregular curl structures ( figure d ). the three-dimensional structures of feifn-ωa and feifn-ωb were predicted with swiss-model software and are shown in figure e ,f, respectively. ( ) . % extracellular . % intracellular % mitochondrion intracellular a signal peptide cleavage sites were analyzed by signalp . server at http://www.cbs.dtu.dk/services/ signalp- . /; b phosphorylation sites were analyzed by netphos . server at http://www.cbs.dtu.dk/services/ netphos/; c n-glycosylation sites were analyzed by netnglyc . at http://www.cbs.dtu.dk/services/netnglyc/ and o-glycosylation sites were analyzed by yino yang . at http://www.cbs.dtu.dk/services/yinoyang/; d subcellular localization was analyzed by targetp . server at http://www.cbs.dtu.dk/services/targetp; e transmembrane region was analyzed by tmhmm server v. . at http://www.cbs.dtu.dk/services/tmhmm/. the genes encoding feifn-ωa and feifn-ωb were subcloned into the prokaryotic soluble expression vector pcold-tf and then transformed into e. coli bl competent cells, thus generating the recombinant protein expressing e. coli strains pcold-feifn-ωa/bl and pcold-feifn-ωb/bl . after inducing their expression with iptg, the rfeifn-ωa ( figure a ) and rfeifn-ωb (figure b ) proteins fused with the trigger factor (tf) of approximately kda and were primarily expressed in their soluble forms. subsequently, we optimized the expression conditions of the rfeifn-ω proteins from the pcold-feifn-ωa/bl and pcold-feifn-ωb/bl e. coli strains. our observations indicated that the optimal induction conditions for rfeifn-ωa protein expression were an iptg concentration of . mmol/l ( figure c ) and an induction time of h (figure d) , and the optimal induction conditions for rfeifn-ωb protein expression were an iptg concentration of . mmol/l ( figure e ) and an induction time of h (figure f ). after that, the fused proteins (rfeifn-ωa/ωb+tf) were purified by his-tag ni + affinity column chromatography and subjected to cleavage using the c protease. the cleaved proteins were then purified by his-tag ni + affinity column chromatography once again, after which we collected the purified recombinant rfeifn-ωa and rfeifn-ωb proteins with molecular weights of approximately kda and kda, respectively ( figure g ). viruses , , x for peer review of the genes encoding feifn-ωa and feifn-ωb were subcloned into the prokaryotic soluble expression vector pcold-tf and then transformed into e. coli bl competent cells, thus generating the recombinant protein expressing e. coli strains pcold-feifn-ωa/bl and pcold-feifn-ωb/bl . after inducing their expression with iptg, the rfeifn-ωa ( figure a ) and rfeifn-ωb (figure b ) proteins fused with the trigger factor (tf) of approximately kda and were primarily expressed in their soluble forms. subsequently, we optimized the expression conditions of the rfeifn-ω proteins from the pcold-feifn-ωa/bl and pcold-feifn-ωb/bl e. coli strains. our observations indicated that the optimal induction conditions for rfeifn-ωa protein expression were an iptg concentration of . mmol/l (figure c ) and an induction time of h (figure d) , and the optimal induction conditions for rfeifn-ωb protein expression were an iptg concentration of . mmol/l (figure e ) and an induction time of h (figure f ). after that, the fused proteins (rfeifn-ωa/ωb+tf) were purified by his-tag ni + affinity column chromatography and subjected to cleavage using the c protease. the cleaved proteins were then purified by his-tag ni + affinity column chromatography once again, after which we collected the purified recombinant rfeifn-ωa and rfeifn-ωb proteins with molecular weights of approximately kda and kda, respectively (figure g ). we determined the antiviral activity of rfeifn-ωa and rfeifn-ωb using microdose cytopathic effect inhibition assays (mcias) and vsv as the viral model that was propagated on f cells. as shown in figure , both rfeifn-ωa and rfeifn-ωb demonstrated good antiviral activity similar to the intercat ifn (feline ifn antiviral drug) positive control. however, no antiviral effect in negative control groups (cells untreated with the ifns) was detected. viruses , , x for peer review of the black arrow represents the target fusion protein and red arrowhead represents the target fusion protein expressed under the optimized condition. we determined the antiviral activity of rfeifn-ωa and rfeifn-ωb using microdose cytopathic effect inhibition assays (mcias) and vsv as the viral model that was propagated on f cells. as shown in figure , both rfeifn-ωa and rfeifn-ωb demonstrated good antiviral activity similar to the intercat ifn (feline ifn antiviral drug) positive control. however, no antiviral effect in negative control groups (cells untreated with the ifns) was detected. we then tested whether the antiviral activities of rfeifn-ωa and rfeifn-ωb were species-specific. to address this question, we performed mcias with vsv propagated in several different cell lines from different animal species, including f cells (cat), vero cells (monkey), mdck cells (dog), mdbk cells (cattle), and pk- cells (pig). as shown in figure , the purified recombinant feifn-ωa and feifn-ωb proteins showed antiviral activity in both homologous animal cells (f cells) and heterologous animal cells (vero, mdck, mdbk, and pk- cells) in vitro. the antiviral activity of the rfeifn-ω proteins in heterologous animal cells was significantly stronger than that of the intercat ifn control. although the intercat ifn displayed high antiviral activity in f and vero cells, it had significantly weak antiviral activity in mdck and mdbk cells, and no antiviral activity in pk- cells. furthermore, the antiviral activity of rfeifn-ωb was better than that of rfeifn-ωa in homologous and heterologous animal cells, but especially in homologous animal cells that originated from cats. however, no antiviral activity was detected in cells' control groups (cells untreated with the ifns). we then tested whether the antiviral activities of rfeifn-ωa and rfeifn-ωb were species-specific. to address this question, we performed mcias with vsv propagated in several different cell lines from different animal species, including f cells (cat), vero cells (monkey), mdck cells (dog), mdbk cells (cattle), and pk- cells (pig). as shown in figure , the purified recombinant feifn-ωa and feifn-ωb proteins showed antiviral activity in both homologous animal cells (f cells) and heterologous animal cells (vero, mdck, mdbk, and pk- cells) in vitro. the antiviral activity of the rfeifn-ω proteins in heterologous animal cells was significantly stronger than that of the intercat ifn control. although the intercat ifn displayed high antiviral activity in f and vero cells, it had significantly weak antiviral activity in mdck and mdbk cells, and no antiviral activity in pk- cells. furthermore, the antiviral activity of rfeifn-ωb was better than that of rfeifn-ωa in homologous and heterologous animal cells, but especially in homologous animal cells that originated from cats. however, no antiviral activity was detected in cells' control groups (cells untreated with the ifns). next, we wanted to investigate whether the antiviral activities of the recombinant feifn-ωa and feifn-ωb proteins were also broad-spectrum. we performed mcias using a wide range of different viruses, including vsv, fcov, cpv, bvdv, and pedv. as shown in figure , rfeifn-ωa and rfeifn-ωb both displayed in vitro antiviral activity against vsv, fcov, cpv, bvdv, and pedv. their antiviral activities were especially strong against fcov and vsv. in contrast, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of rfeifn-ωa. no notable differences were observed between rfeifn-ωa and rfeifn-ωb with cpv, bvdv, or pedv. in addition, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of intercat ifn, and the antiviral activities of both rfeifn-ωa and rfeifn-ωb against cpv, bvdv, and pedv were significantly higher than that of intercat ifn. in fact, we observed no antiviral activity against bvdv or pedv by intercat ifn. unsurprisingly, there was no antiviral activity detected in cells' control groups (cells untreated with the ifns). figure . the broad-spectrum antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < . ; ** p < . ; *** p < . ) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the figure . the species-specific antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < . ; *** p < . ) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the intercat ifn group. comparison of the rfeifn-ωb group and rfeifn-ωa group also indicated significant differences (# p < . ). next, we wanted to investigate whether the antiviral activities of the recombinant feifn-ωa and feifn-ωb proteins were also broad-spectrum. we performed mcias using a wide range of different viruses, including vsv, fcov, cpv, bvdv, and pedv. as shown in figure , rfeifn-ωa and rfeifn-ωb both displayed in vitro antiviral activity against vsv, fcov, cpv, bvdv, and pedv. their antiviral activities were especially strong against fcov and vsv. in contrast, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of rfeifn-ωa. no notable differences were observed between rfeifn-ωa and rfeifn-ωb with cpv, bvdv, or pedv. in addition, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of intercat ifn, and the antiviral activities of both rfeifn-ωa and rfeifn-ωb against cpv, bvdv, and pedv were significantly higher than that of intercat ifn. in fact, we observed no antiviral activity against bvdv or pedv by intercat ifn. unsurprisingly, there was no antiviral activity detected in cells' control groups (cells untreated with the ifns). viruses , , x for peer review of figure . the species-specific antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < . ; *** p < . ) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the intercat ifn group. comparison of the rfeifn-ωb group and rfeifn-ωa group also indicated significant differences (# p < . ). next, we wanted to investigate whether the antiviral activities of the recombinant feifn-ωa and feifn-ωb proteins were also broad-spectrum. we performed mcias using a wide range of different viruses, including vsv, fcov, cpv, bvdv, and pedv. as shown in figure , rfeifn-ωa and rfeifn-ωb both displayed in vitro antiviral activity against vsv, fcov, cpv, bvdv, and pedv. their antiviral activities were especially strong against fcov and vsv. in contrast, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of rfeifn-ωa. no notable differences were observed between rfeifn-ωa and rfeifn-ωb with cpv, bvdv, or pedv. in addition, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of intercat ifn, and the antiviral activities of both rfeifn-ωa and rfeifn-ωb against cpv, bvdv, and pedv were significantly higher than that of intercat ifn. in fact, we observed no antiviral activity against bvdv or pedv by intercat ifn. unsurprisingly, there was no antiviral activity detected in cells' control groups (cells untreated with the ifns). figure . the broad-spectrum antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < . ; ** p < . ; *** p < . ) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the figure . the broad-spectrum antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < . ; ** p < . ; *** p < . ) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the intercat ifn group. a comparison of the rfeifn-ωb group and rfeifn-ωa group also indicated significant differences (# p < . ). currently, cats are continuing to become more and more common as household pets. we know of a wide variety of viral infections that can seriously endanger the health of pet cats, such as felv, fiv, fipv, fcov, and feline calicivirus (fcv). interferon represents a promising potential therapeutic agent that can effectively treat pet cats infected with these viruses. in this study, we identified two new genes encoding feline ifn-ω (feifn-ωa and feifn-ωb) in the spleen lymphocytes of cats. following sequence homology analysis, we found that feifn-ωa and feifn-ωb shared maximum nucleotide sequence homologies of . % and . %, and maximum amino acid homologies of . % and . % with the previously published subtypes of feline ifn-ω, respectively. in addition, phylogenetic tree analysis of ifns in cats and other species constructed using neighbor-joining analysis revealed that feifn-ωa and feifn-ωb do belong to the type i ifn family, but their evolutionary relationship to known feline ifn-ω genes was distant, indicating that feifn-ωa and feifn-ωb were indeed new subtypes of feline ifn-ω. we deposited these two genes into the genbank with the accession numbers mk (feifn-ωa) and mk (feifn-ωb), thus enriching the ifn-ω data submitted to the genbank [ ] . in this study, the characteristics of the feifn-ωa and feifn-ωb proteins, such as signal peptide sequences, signal peptide cleavage sites, phosphorylation sites, glycosylation sites, antigen epitopes, hydrophobicity, and transmembrane regions were analyzed using bioinformatics to provide better theoretical guidance for the functional study of these proteins. the mature proteins, with normal biological activity, were formed only after the signal peptide sequence was removed from the precursor protein, thus allowing them to be secreted outside the cell membrane [ , ] . we used online software to predict that the signal peptide sequence of the feifn-ωa/ωb proteins consists of amino acid residues, and that the signal peptide cleavage site is located between residues gly and cys . the results indicated that the recombinant feifn-ωa and feifn-ωb proteins could be expressed in vitro in their soluble forms. glycosylation is an important post-translational modification process that can affect the antigenic determinants, charge properties, enzymatic properties, and thermal stability of proteins. similar to previous reports for the known feifn-ω subtypes [ ] , we observed no n-glycosylation sites present in feifn-ωa or feifn-ωb. however, our analyses predicted nine potential o-glycosylation sites in feifn-ωa and six potential o-glycosylation sites in feifn-ωb, which is different from previous reports on the other known ifn-ω subtypes [ ] . studies have shown that glycosylation sites can play an important role in determining the activity of ifns [ , ] . for example, glycosylated ifn-ω has been observed to be markedly more potent than non-glycosylated ifn-ω against hepatitis c virus, bvdv, yellow fever virus, and west nile virus, with even more superior effects than ifn-α, ifn-β, and ifn-γ [ , ] . insect/baculovirus expression systems are one of the most effective eukaryotic expression systems for preparing feline ifns. these expression systems are capable of making post-translational modifications, such as glycosylation, which allows proteins to fold correctly, thus producing highly active and stable ifns [ ] [ ] [ ] [ ] [ ] . however, the application of these systems is limited by drawbacks, such as low yield of ifn protein and complicated operation requirements [ ] . therefore, e. coli expression systems are still the most widely used prokaryotic expression system for protein production with their characteristics of simple procedures, large-scale production, and low cost [ ] [ ] [ ] . generally, recombinant ifn is produced by e. coli expression systems in an insoluble inclusion body form that has not been modified or folded correctly, resulting in the loss of its biological activity [ , ] . to obtain a soluble and biologically active ifn protein, the inclusion bodies need to be denatured and then renatured, which is a time-consuming, laborious process [ ] . in this study, we selected the prokaryotic soluble expression system pcold-tf to prepare recombinant feifn-ωa and feifn-ωb. the pcold-tf system is a highly efficient soluble expression system with a his-tagged tf that allows the expressed protein to be effectively modified, folded, and secreted into the cytoplasm [ , ] . following the construction of recombinant e. coli strains and induction by iptg, the rfeifn-ωa/ωb proteins were expressed in their soluble forms and analyzed via sds-page. we optimized expression conditions for the rfeifn-ωa and rfeifn-ωb proteins and purified them using his-tag ni + affinity column chromatography. both proteins showed antiviral activity against vsv in microdose cytopathic effect inhibition assays using f cells. our results provide a basis for further studies into the development of rfeifn-ωa and rfeifn-ωb as therapeutic agents for viral infections. meanwhile, our data provide evidence that the pcold-tf expression system can be used to produce ifn with full bioactivity. recombinant feline interferon-ω (rfeifn-ω) was the first licensed immunomodulator for use in treating viral infections in cats, especially fiv and felv infections [ , , ] . furthermore, rfeifn-ω also exhibits therapeutic effects against other feline viral infections, such as fcv, feline parvovirus, and feline herpesvirus- [ ] , as well as viruses that originate in other animals, such as foot-and-mouth disease virus, influenza virus, bvdv, vsv, prv, and rotavirus [ , , ] . in this study, our results demonstrated that both rfeifn-ωa and rfeifn-ωb had antiviral activity in homologous animal cells (f cells, cat) and heterologous animal cells (vero cells, monkey; mdbk cells, cattle; mdck cells, dog; pk- cells, pig), indicating that rfeifn-ωa and rfeifn-ωb have broad cross-species antiviral activity in vitro, similar to previously published findings in mdbk and mdck cells [ ] . in contrast, the antiviral activities of rfeifn-ωb in homologous and heterologous animal cells were better than that of rfeifn-ωa. intriguingly, the rfeifn-ωa and rfeifn-ωb proteins exhibited antiviral activity in mdck cells, despite the absence of ifn-ω in canines [ , ] . we speculate that different ifns with different physiological functions are likely responsible for the difference of results obtained in our study compared to other published studies. furthermore, analysis of the broad-spectrum antiviral activities of rfeifn-ωa and rfeifn-ωb revealed that they were effective against vsv, fcov, pedv, bvdv, and cpv. out of those five viruses, antiviral activity was strongest against vsv and fcov. no significant differences in antiviral activity against cpv, bvdv, or pedv were observed between rfeifn-ωa and rfeifn-ωb. however, the overall broad-spectrum antiviral activity of rfeifn-ωb was significantly stronger than that of rfeifn-ωa. recently, combination antiviral therapy has become a common practice in treating feline viral infections due to pharmacokinetics and the short half-life of ifn alone [ , ] . there are many published studies focused on the combination of ifn-ω with other therapeutic agents, such as chemotherapeutic agents [ ] , ifn-α [ ] , and ribavirin [ ] , suggesting this is an attractive strategy to use against viral infections. we further evaluated the antiviral effects of rfeifn-ωa and rfeifn-ωb combined with feline il- against vsv and fcov in f cells. our results revealed that the in vitro antiviral activity of combination therapy was significantly increased compared to that of rfeifn-ωa or rfeifn-ωb alone, indicating that rfeifn-ω combination therapy may represent a more potent option than monotherapy for treating viral infections in cats; however, this hypothesis requires further exploration in vivo. in summary, the rfeifn-ωa and rfeifn-ωb obtained in this study exhibit significant broad-spectrum antiviral activity in both homologous and heterologous cells in vitro, particularly rfeifn-ωb, suggesting a promising candidate for the development of an effective therapeutic agent against viral infections in cats and other animals. in this study, two new subtypes of feline ifn-ω (ωa and ωb) were identified and characterized. they shared a maximum nucleotide sequence homology of . % and . % and a maximum amino acid homology of . % and . % with the previously known subtypes of feifn-ω, respectively. we analyzed the characteristics of feifn-ωa and feifn-ωb in detail using bioinformatics followed by soluble expression and optimization of induction conditions in e. coli. our data showed that purified recombinant feifn-ωa and feifn-ωb had broad-spectrum antiviral activity in homologous and heterologous animal cells, suggesting they are candidates for the development of effective therapeutic agents to be used against viral infections in pet cats. and, our research is underway to systematically evaluate the effectiveness of the two novel feifns as therapeutics agent for cat viral infections in vivo. in addition, the reported feifn-ω sequences will enrich the ifn data submitted to genbank. epidemiology of naturally occurring feline urologic syndrome feline leukemia virus infection: importance and current situation in switzerland effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis the protective rate of the feline immunodeficiency virus vaccine: an australian field study cell cycle s phase markers are expressed in cerebral neuron nuclei of cats infected by the feline panleukopenia virus interferon-omega: current status in clinical applications cloning, expression and antiviral bioactivity of 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interferon-omega for treatment of cats with acute upper respiratory viral disease constitutive and trophoblast-specific expression of a class of bovine interferon genes characterization and antivirus activities of a novel bovine ifn-omega safety pharmacology, toxicology and pharmacokinetic assessment of recombinant human omega-interferon produced from cho-ss cells a comparison of the antiproliferative properties of recombinant human ifn-alpha and ifn-omega in human bone marrow culture effect of recombinant feline interferon-ω alone and in combination with chemotherapeutic agents on putative tumour-initiating cells and daughter cells derived from canine and feline mammary tumours liposomal plasmid dna encoding human thymosin α and interferon ω potently inhibits liver tumor growth in icr mice this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -qb vp gr authors: happel, anna-ursula; varsani, arvind; balle, christina; passmore, jo-ann; jaspan, heather title: the vaginal virome—balancing female genital tract bacteriome, mucosal immunity, and sexual and reproductive health outcomes? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qb vp gr besides bacteria, fungi, protists and archaea, the vaginal ecosystem also contains a range of prokaryote- and eukaryote-infecting viruses, which are collectively referred to as the “virome”. despite its well-described role in the gut and other environmental niches, the vaginal virome remains understudied. with a focus on sexual and reproductive health, we summarize the currently known components of the vaginal virome, its relationship with other constituents of the vaginal microbiota and its association with adverse health outcomes. while a range of eukaryote-infecting viruses has been described to be present in the female genital tract (fgt), few prokaryote-infecting viruses have been described. literature suggests that various vaginal viruses interact with vaginal bacterial microbiota and host immunity and that any imbalance thereof may contribute to the risk of adverse reproductive health outcomes, including infertility and adverse birth outcomes. current limitations of vaginal virome research include experimental and analytical constraints. considering the vaginal virome may represent the missing link in our understanding of the relationship between fgt bacteria, mucosal immunity, and adverse sexual and reproductive health outcomes, future studies evaluating the vaginal microbiome and its population dynamics holistically will be important for understanding the role of the vaginal virome in balancing health and disease. despite the considerable literature published on bacterial communities, only a few human microbiota studies have focused on viruses, fungi, protists and archaea communities [ ] [ ] [ ] , yet shifts in one community likely modulate community structure of others and vice versa. it is estimated that only about % of the human virome has been described at the sequence level [ ] , and even less has been characterized functionally and hardly any studies have evaluated the virome in relation to reproductive health and the female genital tract (fgt). from various biological niches, such as the human gut, we know that viruses interact with other components of the microbiota and the human host, consequently influencing human health [ ] [ ] [ ] [ ] . however, little is known about the interactions of prokaryote-and eukaryote-infecting dna and rna viruses present in the fgt microbiota, collectively making up the vaginal virome, with other components of the vaginal microbiota or the human host, and its consequent impact on health outcomes. the primary outcome of this review was to summarize our current understanding of the pro-and eukaryote-infecting viruses making up the vaginal virome. secondary outcomes included describing interactions between the vaginal virome and other constituents of the vaginal microbiota, and possible associated adverse health outcomes. the literature search for this non-systematic review was conducted on march , using two databases (pubmed and googlescholar), with the search terms ("vagina" or "female genital tract" or "female reproductive tract") and ("virome" or "virus" or "viral" or "microbiome") and was limited to studies that were in published in english. a range of vaginal dna viruses infecting eukaryote cells have been identified by shotgun metagenomics of vaginal samples from generally healthy, asymptomatic women of reproductive age participating in the human microbiome project (hmp) [ ] , including double-stranded (ds) dna (families adenoviridae, herpesviridae, papillomaviridae and polyomaviridae) and single-stranded (ss) dna viruses (families anelloviridae) ( table and figure ). the most common viruses detected in the lower reproductive tract were alphapapillomaviruses, with % of the participants being infected with at least one alphapapillomavirus [ ] . to date, more than human papillomavirus (hpv) types have been identified, including at least that preferentially infect the genital mucosa [ , ] (table and figure ). longitudinal sampling in the hmp suggests that up to % of these papillomaviruses establish productive infections and were replication competent, as many of the viruses were detected over multiple time points in the same women. additional dsdna viruses have been identified in vaginal samples from pregnant women and women with reproductive disorders [ ] [ ] [ ] that share sequence similarities to those in the families alloherpesviridae, iridoviridae, marseilleviridae, mimiviridae, phycodnaviridae and poxviridae (table and figure ). in a cohort of women undergoing in vitro fertilisation, herpesviridae, polyomaviridae, papillomaviridae and anelloviridae were present, with papillomaviridae being the most common virus family detected [ ] , while in a cohort of sixty pregnant women, anelloviruses were the most commonly detected viruses in vaginal samples, being present in % of women screened [ ] . in a cohort of women coinfected with human immunodeficiency virus (hiv) and hpv, four viral families were identified in the fgt: papillomaviridae, anelloviridae, genomoviridae and herpesviridae [ ] . papillomavirus reads were more abundant in women with premalignant cervical lesions, which were also strongly associated with carrying multiple high-risk hpvs, while anellovirus read abundance was negatively correlated with host cd + t-cell counts [ ] . similarly, known hpv types were detected in the fgt of women living with hiv, in addition to viruses belonging to the polyomaand anelloviridae families [ ] . whether a core vaginal virome exists or whether differences in vaginal viruses identified in these studies are due to variable demographic or clinical characteristics of the cohorts, to differences in laboratory or sequencing methods, or to the viral databases and viral annotation tools applied remains unclear. to our knowledge, only one sequencing study [ ] reported rna viruses in the reproductive tract, belonging to the partitiviridae family (dsrna), of which fungi is the natural host. this is likely to be due to the poor stability of stored rna and more complex laboratory assays being required to sequence rna viruses. hiv- , an ssrna reverse transcribing virus (ssrna-rt) in the family of retroviridae, is also detectable in fgt secretions of women living with hiv during viral shedding [ ] [ ] [ ] . viruses , , x for peer review of stored rna and more complex laboratory assays being required to sequence rna viruses. hiv- , an ssrna reverse transcribing virus (ssrna-rt) in the family of retroviridae, is also detectable in fgt secretions of women living with hiv during viral shedding [ ] [ ] [ ] . while none of the women participating in the hmp had any genital symptoms, the high prevalence of eukaryote-infecting vaginal viruses raises the question of whether these vaginal viruses play a role in reproductive health. as the majority of humans remain asymptomatic to some viral infections, it has been proposed that viruses have become part of the metagenome of "healthy" individuals, rarely causing disease and remaining dormant within the host [ ] . however, some welldescribed disease-causing viruses, such as high-risk hpv types, herpes simplex virus (hsv)- and polyomaviruses were also described to be part of the vaginal virome of some individuals despite being asymptomatic (table and figure ). furthermore, viruses that do not overtly cause disease yet establish chronic infections have been shown to influence immunity at other mucosal surfaces, such as the gut [ , ] , and this is also likely to occur in the lower reproductive tract and is thus discussed in more detail below. while none of the women participating in the hmp had any genital symptoms, the high prevalence of eukaryote-infecting vaginal viruses raises the question of whether these vaginal viruses play a role in reproductive health. as the majority of humans remain asymptomatic to some viral infections, it has been proposed that viruses have become part of the metagenome of "healthy" individuals, rarely causing disease and remaining dormant within the host [ ] . however, some well-described disease-causing viruses, such as high-risk hpv types, herpes simplex virus (hsv)- and polyomaviruses were also described to be part of the vaginal virome of some individuals despite being asymptomatic (table and figure ). furthermore, viruses that do not overtly cause disease yet establish chronic infections have been shown to influence immunity at other mucosal surfaces, such as the gut [ , ] , and this is also likely to occur in the lower reproductive tract and is thus discussed in more detail below. * including only subfamilies, genera, species and types that have been described to infect the genital mucosa. risk of hpv types is indicated using different font types: high-risk, possibly high-risk and low risk. few shotgun metagenomic studies have investigated the presence or function of prokaryotic viruses in the lower reproductive tract of women. although prokaryotic-infecting viruses, from now on referred to as bacteriophages, are estimated to be amongst the most abundant living entities on earth [ ] and are thought to play an important role in shaping the bacterial microbiota and associated health outcomes in the human gut [ , [ ] [ ] [ ] , oral cavity [ , ] , skin [ , ] and lungs [ ] , their role in the lower reproductive tract is understudied. functionally, bacteriophages are divided into lytic (virulent) and temperate types based on their differential ability to either lyse host cells (to release progeny bacteriophages) or to incorporate their genomes into the host cell genome as prophages and remain dormant, respectively [ ] . several groups have identified functional and nonfunctional prophages in the genomes of vaginal bacterial species [ ] [ ] [ ] [ ] [ ] . strong bioinformatic and in vitro evidence indicates that vaginal lactobacillus strains (including l. crispatus, l. gasseri, l. jensenii and l. plantarum isolates) carry inducible prophages [ ] [ ] [ ] ] . one third of all vaginal lactobacillus strains from south african women that have been examined to date were found to harbour at least one prophage, with l. crispatus more commonly harbouring prophages than l. jensenii [ ] . for most of these lactobacillus prophages, however, factors determining their induction and permissive bacterial hosts are yet to be determined. prophages have also been described in the genomes of vaginal and urinary bacterial species that have been associated with adverse reproductive health outcomes, including gardnerella vaginalis [ ] , group b streptococcus (gbs) [ ] and enterococcus spp. [ ] . among gardnerella strains, a species associated with bacterial vaginosis (bv), more than annotated prophage sequences have been identified and evidence of ongoing prophage acquisition within these gardnerella populations was present [ ] . another study found that almost % of the examined genomes of vaginal and urinary gardnerella strains contained at least one prophage sequence [ ] . similarly, almost % of gbs isolates had at least one prophage, which carried genes encoding factors previously associated with host adaptation and virulence [ ] . the high abundance of prophage sequences within the genomes of vaginal bacterial species suggests that bacteriophages might play a role in shaping the bacterial microbiota of the fgt and associated health outcomes, similar to other biological niches. recent metagenomic sequencing of vaginal samples revealed that the majority of identified vaginal dna viruses are dsdna bacteriophages, similar to those in the families myo-, podo-and siphoviridae [ ] . in addition, various unclassified viruses within the order caudovirales were found to be present. notably, only % of vaginal viruses identified by metagenomic sequencing by jakobsen et al. ( ) targeted eukaryotes [ ] , confirming the high abundance of prokaryote-infecting viruses within the fgt microbiota (table and figure ). considering that a shift from lysogeny to a lytic lifecycle has been correlated with disease within the gut microbiota [ ] , the role of bacteriophages for fgt health remains a crucially important yet underexplored area. limited studies have examined the interactions between all components of the vaginal microbiota and the human host. observational studies have shown that the acquisition and transmission of viral sexually transmitted infections (stis), including hsv- , hpv and hiv, are more common in women with high diversity, nonoptimal vaginal bacterial microbiota [ ] [ ] [ ] . however, many studies did not adjust for confounders to this relationship, such as the presence of other stis, sexual risk behaviour (condomless sex) and circumcision status of sexual partners. recent evidence from longitudinal studies suggests that changes in the bacterial microbiota and associated immunomodulatory metabolites precede incidental stis [ ] and have been associated with a higher rate of hpv persistence and genital hsv- shedding [ ] . data from the hmp further indicated that alpha-papillomaviruses were more common in women with a high vaginal bacterial diversity than in those who had a lactobacillus-dominant microbiota [ ] . strong co-abundances between bacteriophages and predicted bacterial hosts were observed in a metagenomic study [ ] , further suggesting that viral and bacterial communities interact within the lower reproductive tract. as such, links between viral community composition and the presence of l. crispatus, l. iners, g, vaginalis and a. vaginae in the fgt were found [ ] . diversity changes in the vaginal eukaryotic dna virome over the course of pregnancy appeared similar to concomitant changes in the vaginal bacteriome, and pregnant women with highly diverse vaginal viromes tended to also have highly diverse vaginal bacteriomes [ ] . while a relationship between bacterial and viral communities within the human lower reproductive tract appears to be supported by these studies, the directionality of this relationship is not clear and causality is yet to be established. it remains to be determined if changes in the bacterial microbiota are driven by changes in the viral community structure, or if susceptibility, persistence and clearance of viral infections is influenced by the vaginal bacterial microbiota. it is also important to acknowledge that underlying indirect mechanisms may regulate both, bacterial and viral communities, in addition to a more direct microbial mechanism implied by available studies. there is clinical and in vitro evidence that the vaginal bacterial microbiota influences colonisation with fungal strains [ , ] , albeit studies investigating yeast-viral interactions in the fgt are limited. the majority of available research of yeast-viral interactions focuses on the relationship between viral stis and candida coinfections, in which vaginal candida infection was investigated as a risk factor for hiv transmission [ , ] . however, this is likely to be the results of mucosal barrier disruption and/or inflammation associated with vulvovaginal candidiasis. seventy-five percent of women experience at least one episode of candidiasis during their life, and the risk for frequent, more invasive and resistant infections in persons living with hiv is high, likely due to t cell immune defects [ ] . other in vitro studies have shown that hsv- significantly enhances binding of candida albicans to hela cells [ ] , which the authors conclude suggests that hsv- increases candida persistence in the fgt. interestingly, in vitro studies have also shown that hsv- and coxsackievirus-b were retained in and released by c. albicans biofilms, in which the viruses remained viable and protected from antiviral agents as well host factors [ ] . pseudomonas phages have been described to inhibit c. albicans biofilms as well as their planktonic growth [ ] . whether yeast-infecting bacteriophages are commonly present in the vagina is unknown, although as mentioned previously, partitiviridae, typically a fungal phage, have been identified in the female reproductive tract [ ] . in turn, hsv- has been shown to protect c. albicans by downregulating monocyte-mediated anti-candida immune system responses [ ] , suggesting bilateral interactions between fungal and viral communities in the fgt. hiv- envelope and transactivating proteins have also been shown to bind to c. albicans, which may promote fungal virulence by inducing hyphae formation [ ] [ ] [ ] . although the clinical relevance of these in vitro results remains to be elucidated and research on a broader range of vaginal viruses and fungi is needed, these studies suggest that there may be clinically relevant interactions between viral and fungal communities in the fgt and that an imbalance between these may influence reproductive health. microbial communities also interact with the host and influence innate immunity, immune cell activation and cytokine secretion. a balanced interplay between the vaginal microbiome and host immunity is crucial to prevent infections on the one hand but to maintain an immunotolerant environment, particularly during pregnancy, on the other hand. while the interaction of the vaginal bacteriome with the host has been reviewed by others [ , , ] , there are less data available on the effect of eukaryote-infecting viruses other than viral stis, prokaryote-infecting viruses and the collective virome on host immunity. the innate immune system, including epithelial cells and mucus, toll-like receptors, antimicrobial peptides and defensins, cytokines and innate immune cells, is an important host immune defence mechanism in the fgt, collectively minimizing the risk of viral infection upon exposure [ , ] . similarly, cellular and humoral adaptive immune responses to viral stis have been described in detail. while hiv- infection leads to induction of cd + t-cell responses in the cervical mucosa [ ] , hsv- infection has been associated with cervical cd + t cell numbers and a distinct cytokine profile including various cytokines without significant alterations in local proinflammatory cytokines [ ] , and hpv infection has been associated with a t helper (th) cytokine immune response and elevated proinflammatory and regulatory cytokines [ ] [ ] [ ] . in germ-free mice, expansion of enteric bacteriophages has been linked to immune cell expansion and increased inflammation in the gut, and phage dna isolated from human faeces stimulated interferon (ifn)-γ production by dendritic cells in mice [ ] . further, certain viruses may be important for conditioning of immune responses. in the gut, infection by a persistent strain of murine norovirus compensated for the absence of bacteria in germ-free mice by restoring intestinal morphology and by promoting lymphocyte differentiation [ ] . the same virus protected mice from lung injury following infection with pseudomonas aeruginosa and restored serum immunoglobulins in germ-free mice to levels observed in conventional mice [ ] , yet the relevance of these findings for humans and specifically the fgt remains to be confirmed. these initial studies point towards an important role of the vaginal virome in innate and adaptive immunity of the fgt. while many of these initial findings are based primarily on disease-associated viruses, they may suggest that changes in the collective virome may similarly lead to altered mucosal immunity in the fgt and thus may impact sexual and reproductive health outcomes. as reviewed elsewhere, sexually transmitted viruses, like hsv- , hpv, cytomegalovirus (cmv), hepatitis b or hiv, have been associated with a range of adverse health outcomes [ ] [ ] [ ] [ ] [ ] [ ] , including cervical cancer (hpv) [ ] , genital ulcers (hsv- ), aseptic meningitis as well as vertical infections in infants, such as neonatal herpes (hsv- ) [ ] or cirrhosis and liver cancer (hepatitis b) [ , ] . bv is a risk factor for severe reproductive complications [ ] [ ] [ ] and stis [ , , ] , and it has been suggested by several authors [ , , ] that bacteriophages are a contributing underlying biological cause for the rapid change in composition of vaginal bacterial communities associated with onset of bv and its difficulty to treat. with advances in shotgun metagenomics, further evidence is emerging that eukaryote-and prokaryote-infecting dna viruses, including bacteriophage communities, may differ between women with and without bv [ ] , although others previously did not find any differences by vaginal bacterial community state type [ ] , possibly due to different laboratory and analysis methods used. jakobsen et al. ( ) found that, while no significant difference in overall viral alpha diversity was present between groups, bacteriophage operational taxonomic units (otus) and predicted bacterial host otus strongly correlated in women with bv. miller-ensminger et al. ( ) observed variations between the prophage populations of women with and without overreactive bladder symptoms, suggesting that bacteriophages may contribute to genitourinary health [ ] . infertility is defined by the failure to establish pregnancy after months of regular sexual intercourse. while the most predictive factor for female infertility is a woman's age, biological and environmental factors are believed to contribute as well [ ] . changes in the vaginal bacterial microbiota have been described as a risk factor for infertility [ ] [ ] [ ] , and it is likely that viruses, such as human herpesviruses (hhv), also play a role. in up to % of infertile couples, the male or female partner had urogenital bacterial infections [ ] , and in agreement with older women being more likely to be infertile, it has been described that seroprevalence of hhv- , which similarly to hhv- can be transmitted via saliva, is increasing with increasing age [ ] . hhv- dna was found in % of endometrial epithelial cells from infertile women in contrast to being completely absent from the endometrium of fertile women [ , ] . hhv- a-specific endometrial natural killer (nk) cell numbers and cytokine responses were elevated in women who had hhv- a present [ ] , suggesting that hhv- infections may modify endometrial immune cell and inflammatory profiles, resulting in the inability to sustain a successful pregnancy. further, endometrial nk cells had increased expression of several chemokine receptors and endometrial epithelial cells upmodulated the corresponding ligands, including monocyte chemotactic protein (mcp and ccl ), interferon gamma-induced protein (ip- and cxcl ) and eotaxin- (ccl ) [ ] . others assessed whether hhv- played a role in preventing embryo implantation and found that % of women with two or more failed embryo transfers had detectable hhv- late viral proteins in endometrial epithelial cells and that women who were hhv- positive had undergone significantly more failed transfers than those who were hhv- negative [ ] , further providing evidence that hhv- infection might influence fertility and pregnancy outcomes. in subfertile women undergoing in vitro fertilisation with a fresh embryo transfer, an association of the eukaryotic vaginal virome with prophylactic antibiotic exposure and reproductive outcomes has been described [ ] . while there was no association between viral diversity and clinical pregnancy overall, a higher diversity of herpesviruses and alphapapillomaviruses was present in women who received prophylactic azithromycin treatment compared to women not receiving azithromycin. further, in women receiving azithromycin, viral diversity was higher in women whose embryo transfer did not result in clinical pregnancy compared with those who achieved clinical pregnancy [ ] , suggesting that both bacterial and viral components of the vaginal microbiota may influence the ability to achieve clinical pregnancy. a number of clinically relevant viruses can be vertically transmitted from mothers to their foetus, including zika virus, hiv, hepatitis b virus, hepatitis c virus, hsv- and - , varicella zoster virus, rubella virus, parvovirus b and cmv, and can cause stillbirth or severe morbidity in infants [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the context of preterm birth (ptb) and small-for-gestational age (sga) births, biological mechanisms are still poorly understood. sequence-based studies of the vaginal bacterial microbiota have revealed that high-diversity bacterial communities [ ] [ ] [ ] and possibly increased concentrations of vaginal inflammatory markers [ , ] might contribute to increased risk for these adverse birth outcomes (abos). recently, a higher richness of the vaginal eukaryotic dna virome, including viral sequences similar to those in the families adenoviridae, anelloviridae, herpesviridae, papillomaviridae, polyomaviridae and poxviridae, has been linked to spontaneous and medically indicated ptb in north american women, but not one specific virus or group of viruses could be linked to ptb [ ] . having both high bacterial and viral diversity in the first trimester of pregnancy was linked to the highest risk for ptb, indicating that the interplay of bacterial and viral communities or an imbalance thereof may be a mechanism by which ptb is triggered. neither whether higher viral diversity would also be associated with ptb in other populations nor how demographic factors influence the vaginal virome during pregnancy, are clear. focusing on specific viral groups rather than the collective virome, vaginal hpv infections have been linked to sga and low birth weight in observational studies, independently of other risk factors [ , ] . in silico analyses have revealed significant associations between invasive neonatal-infecting gbs isolates and harbouring of a specific group of prophages within their genomes [ ] . various viruses were also identified in amniotic fluid samples obtained from pregnancies with adverse outcomes, with adenovirus, cmv and enterovirus being the most common, while few healthy pregnancy controls had any virus detected [ ] . more than half of women delivering infants with intrauterine growth restriction had viruses in their amniotic fluid during pregnancy, and adenovirus was detected in the amniotic fluid collected by amniocentesis in % of these women [ ] . culture-and sequencing-based studies have found evidence of bacterial colonisation in amniotic fluid from pregnancies with abos [ ] [ ] [ ] , suggesting that similarly viruses might be present in amniotic fluid in pregnancies with adverse outcomes, where anatomical, physiological or immunological barriers are compromised. it remains to be determined whether bacterial and viral communities are present in amniotic fluid from healthy pregnancies, as recent sequencing-based studies have drawn conflicting conclusions [ ] [ ] [ ] [ ] . it also has been shown in vitro that adenoviral infection of extravillous trophoblast cells in the presence of maternal decidual lymphocytes induces trophoblast apoptosis [ ] , indicating that the maternal inflammatory response to adenovirus might induce placental cell death and subsequent abo. in mouse models of pregnancy, however, a combination of bacteria and eukaryotic-infecting viruses induced ptb, but neither alone was capable of causing ptb [ , ] . similarly, viral infection of the murine cervix and placenta has been shown to alter the inflammatory responses to subsequent bacterial infection of the fgt [ ] [ ] [ ] , indicating that viral infection of the fgt during pregnancy may alter the capacity to control ascending bacterial infections, which subsequently may lead to abo. data relating to severe acute respiratory syndrome coronavirus (sars-cov- ) infection of the fgt and the consequent relationship with pregnancy outcomes is sparse but continually accruing. a recent systematic review and meta-analysis of pregnant women with coronavirus-related symptoms, of which half had confirmed sars-cov- infection, reported a significant prevalence of placenta-mediated disorders with high rates of miscarriage, ptb, preeclampsia and foetal growth restrictions [ ] . while there is currently still conflicting yet continuously accumulating evidence for the presence of sars-cov- in vaginal secretions, amniotic fluid, cord blood or breastmilk in infected women [ ] [ ] [ ] [ ] , infection and visualization of sars-cov- in placental tissue has been demonstrated thoroughly [ , ] . the first case of transplacental transmission of sars-cov- from a pregnant woman affected by covid- during late pregnancy to her neonate has been described [ ] , suggesting potential perinatal sars-cov- transmission. further investigations are warranted to confirm vertical transmission of sars-cov- and long-term consequences thereof. the growing interest in the field of vaginal virome requires standardisation of laboratory protocols and analysis pipelines, including identification of rna viruses, adequate use of negative controls to account for contamination with environmental viruses, continued development of high-throughput sequencing accessibility, and advancement in viral annotation databases and tools. adequate use of viral nucleic acid extraction methods or kits that have not been associated with contamination as well as removal of bacterial and human nucleic acids either during the extraction process or after sequencing are crucial. it is concerning that various sequencing-based studies have described the presence of viruses in the fgt that share similarities to nonhuman, nonbacterial and nonfungal virus families. for example, vaginal iridoviridae have been described, which were previously only thought to infect ectothermic vertebrates, insects and crustaceans [ ] . similarly, phycodnaviridae were also described to be present in vaginal samples, which were previously only thought to infect algae [ ] . while this suggests that these viral sequences might have been present due to contamination or might have been incorrectly taxonomically classified or that they share similarities to sequences of viruses in these families (since there are clearly protein homologues found across viral families, especially the rna-dependent rna polymerases, helicase domains of replication proteins and, in some cases, capsid proteins [ ] ), it might be possible that these findings are real due to vaginal insertional or hygiene practices. further, analysis of low biomass samples, such as breastmilk, placenta or amniotic fluid, requires rigorous use of controls, including nucleic acid extraction and pcr controls, as well as environmental swabs collected and processed as samples as additional negative controls. there is a paucity of research on the vaginal virome and the interplay of vaginal bacterial and viral communities and host immunity and its likely effect on sexual and reproductive health outcomes. once systematic laboratory and analysis pipelines have been established for both dna and particularly rna viruses, the vaginal virome should be characterized in broader populations, such as pre-and postmenopausal women and women from different demographics, and in connection with other components of the vaginal microbiota and the human host. functional characterization of viruses present in the vaginal virome and evaluation of their effect on reproductive and sexual health outcomes are crucial, and highly detailed longitudinal clinical cohorts with frequent sampling or animal models will be required to assess causality. funding: this review was funded in part by r hd - s . the authors declare no conflict of interest. a comprehensive non-redundant gene catalog reveals extensive within-community intraspecies diversity in the human vagina archaea as emerging organisms in complex human microbiomes methanogenic bacteria in human vaginal samples the virome in mammalian physiology and disease bacteriophages in gut samples from 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the pregnant cervix predisposes to ascending bacterial infection type i interferon regulates the placental inflammatory response to bacteria and is targeted by virus: mechanism of polymicrobial infection-induced preterm birth outcome of coronavirus spectrum infections (sars, mers, covid - ) during pregnancy: a systematic review and meta-analysis sars-cov- is not detectable in the vaginal fluid of women with severe covid- infection clinical characteristics and intrauterine vertical transmission potential of covid- infection in nine pregnant women: a retrospective review of medical records transplacental transmission of sars-cov- infection detection of sars-cov- in human breastmilk visualization of sars-cov- virus invading the human placenta using electron microscopy unveiling crucivirus diversity by mining metagenomic data key: cord- -zfjy m i authors: alsaadi, entedar a. j.; neuman, benjamin w.; jones, ian m. title: identification of a membrane binding peptide in the envelope protein of mhv coronavirus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: zfjy m i coronaviruses (covs) are enveloped, positive sense, single strand rna viruses that cause respiratory, intestinal and neurological diseases in mammals and birds. following replication, covs assemble on intracellular membranes including the endoplasmic reticulum golgi intermediate compartment (ergic) where the envelope protein (e) functions in virus assembly and release. in consequence, e potentially contains membrane-modifying peptides. to search for such peptides, the e coding sequence of mouse hepatitis virus (mhv) was inspected for its amino acid conservation, proximity to the membrane and/or predicted amphipathic helices. peptides identified in silico were synthesized and tested for membrane-modifying activity in the presence of giant unilamellar vesicles (guvs) consisting of , -dipalmitoyl-sn-glycero- -phosphocholine (dppc), sphingomyelin and cholesterol. to confirm the presence of membrane binding peptides identified in the context of a full-length e protein, the wild type and a number of mutants in the putative membrane binding peptide were expressed in lenti-x- t mammalian and insect cells, and the distribution of e antigen within the expressing cell was assessed. our data identify a role for the post-transmembrane region of mhv e in membrane binding. the coronavirus envelope protein (e) is a small hydrophobic protein ranging from - amino acids [ , ] . it has an n-terminal domain, a long alpha helical transmembrane domain and a c-terminal hydrophilic domain, and is found incorporated into the virus particles of all coronavirus groups at low levels [ ] [ ] [ ] [ ] [ ] . two membrane topologies have been suggested for the e protein: hairpin or transmembrane [ , ] . e also interacts with the m protein, and mutants of m that are unable to bud from cells can be complemented by forms of e [ , ] . the membrane curving properties of e are such that the coexpression of m and e is adequate for the efficient formation of virus-like particles (vlp) [ , ] , and these can also incorporate the s protein if it is coexpressed [ ] . for many coronaviruses, including mhv, e protein also functions as an ion channel, a viroporin [ , ] , affecting the trafficking of virions in the secretory pathways and membrane permeability, both of which are essential for virus growth [ , , ] . while e function is critical for virus assembly, its viroporin activity in mobilizing calcium ions and its interactions with host cell tight junction proteins have also been implicated in the pathologies of some coronavirus infections [ , ] . an additional role for e in viral pathogenesis is an anti-apoptotic effect on host cells during virus replication [ ] . the relative significance of the e encoded ion channel activity to virus morphogenesis, as opposed to its structural role in binding m, is uncertain. however, its role as a virulence factor has been demonstrated by many studies on sars-cov e protein which have shown altered pathogenesis in a mouse model, either by an effect of virus production and by effects on expressing cells [ , , ] . stimulated by the pandemic of sars-cov- [ ] , a recent protein interaction map of virus infected hek cells identified host proteins of the vesicular sorting pathway and bromodomain proteins as interacting with e, the latter of which may have therapeutic potential [ ] . notwithstanding its multifunctional nature, the interaction of e with membranes is central to its biological role, and peptides with direct membrane binding properties are present within the coding region. the mapping of such peptides may help to interpret e mechanisms of action and offer the potential for inhibitor development. here, we test e-derived peptides for membrane binding activity in vitro and confirm those identified as positive in the context of the full length protein expressed in two different cell types. all chemical reagents were purchased from thermofisher scientific unless otherwise stated, and were used following the vendor's recommendations. lipids were obtained from avanti polar lipids. peptides used for the in vitro analysis (table ) were derived from the e protein of mhv coronavirus (accession number aau ), synthesized and supplied as a lyophilized product with a stated mean purity of ≥ % by cambridge research biochemicals, uk. peptide stock solutions were prepared in a buffer consisting of . mm sucrose, . mm glucose, mm dtt and . % (v/v) dmso. a positive control peptide, the m -influenza peptide, a well characterized amphipathic helix sufficient for budding into guvs and the formation of large luminal vesicles (luvs) [ ] , was used to validate the guv assay. negative controls were provided by buffer only in order to match test samples in all but the test peptide. guvs were generated by electroformation using a vesicle prep pro (nanion technologies gmbh, munich, germany) with a mix of mm , -dipalmitoyl-sn-glycero- -phosphocholine (dppc), mm egg sphingomyelin and . mol% cholesterol, which were first dissolved in chloroform. to visualize the guvs, . mol% of naphtha [ -alpha] pyrene (tokyo chemical industry uk ltd, oxford, uk) was added. lipids were mixed in an amber vial to a final total lipid concentration of mm before preparation by electroformation. briefly, µl of lipid stock was spread on the conductive side of an indium tin oxide slide (ito-slide), air dried and put into a vacuum desiccator for h to remove all solvent. a -mm o-ring was coated on one side with silicon grease and placed around the dried lipid film, which was then hydrated with µl of . mm sucrose solution in deionized water. a second ito slide was placed on top of the o-ring with the conductive sides facing, and a voltage of v peak to peak at a frequency of hz was applied to the ito slides over a period of h at • c [ ] . guvs were imaged using an evos-fl digital fluorescence microscope (evos, usa). following electroformation, the chamber was disassembled and the sucrose solution removed to leave the guvs attached to one ito slide. diluted peptide was added immediately at the desired concentration and the field was imaged , , and min thereafter. imagej was used to measure the shape and the relative size of the guvs. experiments were performed in triplicate and the average and standard deviation were calculated. buffer only and m -influenza peptide controls were treated in the same way. guv-peptide incubations were at room temperature. statistical significance was calculated using spss version , using linear mixed model (lmm) (p• . ). results were expressed as mean ± sem. figures were generated using graphpad prism . b software. lenti-x t cells were cultured and maintained in dulbecco's modified eagle medium (dmem) (sigma aldrich) supplemented with % fetal bovine serum (fbs) (ge healthcare) and . % penicillin-streptomycin solution (gibco/invitrogen, paisley, uk) at • c with % co . spodoptera frugiperda (sf ) cells (invitrogen) were maintained in ex-cell medium (sigma, gillingham, uk) supplemented with % fetal bovine serum and % penicillin/streptomycin solution at • c with shaking. the coding sequence of mhv e (genbank accession: ay . ) was amplified by pcr from cdna kindly supplied by dr. volker thiel using the primers hsv-e-fw ( 'gcgccatggcaca gccagaactcgccccggaagaccccgaggattttaatttattccttacagacacagtatggtat gtgggg- ') and hsv-e-rv ( '-gcgctcgaggatatcatccacctctaatag ggg- ') and the amplicon cloned into ptriex . (merck millipore, darmstadt, germany) between the ncoi and xhoi restriction sites. to provide markers for the detection of expression by western blot, the wt e coding region was cloned in frame with an upstream hsv and downstream his tag, both provided by the vector. following initial expression experiments, detection of the hsv tag was found to be poor. as a result, mutant e sequences were synthesized de novo (integrated dna technologies, dresden, germany) and cloned similarly, except they were in frame only with the downstream his tag. all constructs were confirmed by dna sequencing prior to use. one day before transfection, . × lenti-x t cells (takara, saint-germain-en-laye, france) were seeded into well plates containing a glass coverslip in each well and incubated for h at • c/ % co . the following day, the cells were transfected using lipofectamine transfection reagent (invitrogen) with plasmids encoding wildtype or mutant mhv-e and incubated for h at • c/ % co . a control vector ptriex . -gfp expressing green fluorescent protein (gfp) gene was used to visualize the efficiency of transfection, typically - %. twenty four hours after transfection, the media was removed and the cells were washed twice with cold pbs for min, then incubated in fix and permeabilization buffers for h at room temperature (ebioscience, san diego, usa). fixed and permeabilized cells were incubated with anti-his-alexa fluor conjugate ab ( e d hh /e , thermofisher scientific) for h at room temperature diluted : in × permeabilization buffer, washed twice with tbs for min each at room temperature in the dark, and then counterstained with dapi. the fixed cells were mounted with a drop of slowfade™ gold reagent before being imaged using an evos-fl digital fluorescence microscope. typical images were captured at × magnification and further manipulated, if required, using imagej software [ ] . the flashbac™ gold (fbg) baculovirus expression system (oxford expression technologies, oxford, uk) was used to produce recombinant baculoviruses. small-scale protein expression was performed by infection in a -well plate seeded with × sf cells per well using µl of a high titre stock of the recombinant baculovirus, typically passage , and incubated for days at • c. after incubation, cells were harvested and used for western blot with an anti-his antibody. proteins were separated by sds-page using - % precast tris-glycine sds polyacrylamide gels (invitrogen) for min at v. after electrophoresis, gels were either stained by coomassie blue r or transferred to pvdf membranes for western blot analysis. following transfer to pvdf membranes (whatman, chalfont st. giles, uk) using a semidry western blotting apparatus, membranes were incubated in blocking buffer ( % skimmed milk powder in tbst ( . % tween- , × tbs)) for h. membranes were incubated with the primary antibody at : , in × tbst buffer for h, followed by three washes for min each in tbst buffer, and subsequently, with a secondary horseradish-peroxidase (hrp) antibody conjugate (dako, glostrup, denmark) diluted : , in × tbst for h followed by three washes for min each with tbst buffer. the membrane was finally washed with tbs and the bound hrp activity was detected using chemiluminescence imagery on a syngene g: box. to allow reprobing of a pvdf membrane with a different antibody, the membrane was stripped using a stripping buffer ( mm β-mercaptoethanol, % sds, . mm tris-hcl ph . ) for min at • c. the membrane was then washed three times with tbst buffer, min each, and then reblocked and probed as before. a total of . × sf cells were seeded in t flasks as monolayers, infected with recombinant baculoviruses at an moi = and incubated for h at • c. the infected cells were harvested by loosening the monolayer into the media and collected by centrifugation at × g at • c for min. cell pellets were resuspended in µl of cold pbs and lysed by sonication for m with on/off pulses at -s intervals (sonics, vibracell tm ). the cell lysates were briefly clarified ( × g for min) and then centrifuged at low speed, i.e., , × g, at • c for min to collect large membrane debris. the pelleted material was kept as a low-speed pellet (lsp). the collected supernatants were then centrifuged at high speed, i.e., , × g, for min at • c in a beckman tl- ultracentrifuge and the pellets were retained as high-speed pellets (hsp). low-and high-speed pellets were tested for the presence of mhv e by western blot using an anti-his antibody, as before. several viral proteins encode amphipathic helices that modulate membrane curvature, e.g., the m protein of the influenza virus and the nonstructural protein b of the hepatitis c virus [ , , ] . the program amphipaseek [ ] was used to assess local amphipathy and putative membrane-binding regions for a phylogenetically diverse set of e proteins representing covs, and the output was visualized using jalview v . . b [ ] . cov e proteins have related topologies but otherwise limited direct homology outside of the clearly predicted tm domain. however, amphipathicity occurred in a region immediately downstream of the tm domain, a region which contains the previously mapped cysteine region [ ] at the end of the predicted transmembrane domain ( figure ) and which is also palmitoylated intracellularly [ , ] to facilitate contact with the lipid bilayer [ ] . in addition, two conserved proline residues occur in this region, plausibly providing conformational flexibility that may be important for e-e interactions or between e and other proteins [ , ] . spanning the first conserved proline, a residue sequence, i.e., - in mhv a e, including a number of hydrophobic residues, was evidently relatively well conserved among most α and β covs, and was also shared with the more distantly related γ and δ covs, with the exception that insertions containing further multiple hydrophobic residues were also present ( figure ). based on these criteria this peptide, the e post tm peptide (eptm), was considered to have potential for direct membrane interaction. ; green represents polar amino acids (n, q, s, t); pink represents cysteines (c); orange represents glycines (g); yellow represents prolines (p); cyan represents aromatic amino acids (h, y); white represents any unconserved/gap. the sars-cov- e protein is % identical to sars e, so was not included as a distinct entry. to address its potential as a membrane active peptide, the residue wild type peptide was synthesised and reconstituted with giant unilamellar membranes (guvs), and the effect on size and morphology were measured. in addition, peptides etm, representing the e protein tm domain itself, and the m influenza peptide were tested (table ) . guvs, composed of mm dppc, mm egg sm and . mol% cholesterol were reconstituted with the peptides at a concentration of µm and the guvs were measured at , and min thereafter by fluorescence microscopy, as described elsewhere [ ] . shape was measured as the ratio between the longest and shortest radii, while relative guv size was estimated by ramanujan's first approximation, taking an average of guvs per experiment for three separate experiments. peptide eptm changed the size but not the shape of the guvs membrane, leading to membrane deformation that increased with time, while guvs treated with buffer only showed no deformation ( figure ) . interestingly, peptide etm showed no effect on either the shape or size of the guvs. interaction of mhv-eptm peptide with the guv membranes made the guvs smaller in size and more rugged in appearance, which was possibly indicative of fragmentation of the lipid bilayers after peptide insertion. the lack of effect by the etm peptide suggested that this peptide alone is not membrane active and may need to be presented in the context of the complete e protein for biological activity. the assay was validated by use of the m -influenza peptide which showed a statistically significant change in guv shape with the formation of intraluminal-vesicles (ilvs) as described [ ] , although no significant change in size was apparent ( figure ). to validate the eptm peptide sequence as membrane active in the context of the full length e protein, e was expressed in both mammalian and insect cells through the use of a dual promoter vector, ptriex . , which permits expression from the same vector in both systems. mhv e protein was expressed as the wild type protein and also as a series of alanine scanning mutants in which conserved and hydrophobic residues were exchanged for alanine ( table ). in addition, the entire region was deleted within the e coding sequence. mammalian expression was done by transfection of lenti-x t cells where expression is driven by the vector resident cag promoter, and the expressed e protein was detected by immunofluorescence imaging with an anti his tag antibody h post-transfection. the wt mhv e protein was found to be distributed evenly throughout the cytoplasm with a slight concentration near the nucleus, plausibly the golgi body. in all the alanine scanning mutants of e, expression levels were similar with little diminution of the overall signal, but in most cases, the pattern of staining was altered to a more granular punctate appearance (figure ). this was particularly notable for mutation l a, where almost all of the fluorescence signal was associated with a punctate distribution. this data suggests that the eptm sequence, identified as causing membrane association in an isolated peptide, also influences the behaviour of the complete protein in a physiologically relevant environment. del ---------- to provide a more quantifiable measure of membrane association, a more productive protein expression system, i.e., expression in insect cells via the construction of recombinant baculoviruses, was investigated with the same panel of e variants. following the generation of recombinant viruses, insect cells were infected at high moi and their ability to express e protein assessed by western blot at days postinfection when synthesis from the vector encoded p promoter was maximal. to facilitate detection, the wt e protein was expressed with both hsv and his tags, at the n-and c-termini respectively, a predicted molecular mass of~ kda. of these two tags, however, only probing with the his antibody detected the expressed product consistently. as a result, all mutant e proteins were expressed with only the c-terminal his tag, a predicted molecular mass of~ kda. all e proteins were expressed at the molecular weight expected, but the expression level varied with mutation ( figure a ). half of the mutants expressed at levels similar to the wild type, but mutations l a, v a, l a and y a were present at reduced levels, consistent with a potential role of these residues in protein folding and stability. to ensure the levels of infection were equivalent, the blot was stripped and reprobed with a monoclonal antibody to the major baculovirus glycoprotein, gp , a marker for infection level, which showed near equivalent infection in all cases ( figure b ). to investigate whether the mutations introduced into mhv-e resulted in an effect on cellular localization in more detail, plausibly by altered membrane association, infection of insect cells with each recombinant virus was repeated on a larger scale and the infected cells harvested at days postinfection. the cells were lysed by sonication in the absence of detergent, clarified and subjected to differential membrane fractionation to produce low-speed (lsp) and high-speed membrane pellet (hsp) fractions. to confirm the fractionation, lsp and hsp samples were probed with an antibody to calnexin, an er marker, which was largely found in the lsp fraction ( figure ). the expression of the wt mhv e was found in both fractions, but was more strongly associated with the hsp fraction ( figure a ), consistent with its known primary localization in mhv-cov infected cells [ ] . similarly, mutations l a and v a partitioned mainly into the hsp fraction, although expression levels overall were reduced. strikingly, the "all" mutant, in which all targeted residues were mutated to alanine, and the "del" mutant, in which the target peptide was deleted from mhv e, were found almost exclusively in the lsp fraction, despite high levels of expression. the remaining mutants carrying mutations l a, p a, y a and y a also associated preferentially with the lsp, although, as before, expression levels were lower in some cases, notably l a and y a. relative densitometry of the hsp and lsp bands revealed significant differences among the mutants with regard to their localisation to the different membrane fractions of e expressing insect cells ( figure b ) and confirmed a role for the amphipathic mhv cov e - peptide in membrane interaction. the cov e proteins consist from a short hydrophilic amino terminal region, a hydrophobic transmembrane region and a carboxy-terminal region that encompasses the majority of the protein [ ] . the e proteins are multifunctional proteins involved in virus assembly and release, and have been located in the ergic and golgi of infected cells but do not traffic to the infected cell surface [ ] . as studies of e have often involved different coronaviruses and different experimental systems, the unambiguous basis of this localisation has not been described. an amphipathic helix, eptm, detected in the post-tm region of e, was suggested by bioinformatics analysis and assessed for direct membrane interaction in vitro by binding to guvs. for comparison, the predicted e protein tm domain, etm, and an established membrane active peptide from the influenza m protein were also included. eptm, but not etm, caused a change in guv size and appearance, consistent with direct membrane binding. following expression of the complete e protein with mutations in the same identified peptide, altered membrane binding in two distinct cell types, mammalian and insect, was apparent. these data support the hypothesis that the membrane binding observed for the eptm peptide in vitro occurs also in the context of the complete e protein expressed in a physiologically relevant environment. cov e protein is a known integral membrane protein, and it has been proposed that the basis of membrane interaction is by one of a number of possible topologies, e.g., as a type iii membrane protein, as a membrane hairpin or as a glycosylated type ii membrane protein [ ] . the expression of e in insect cells resulted in a single band detected by western blot that did not have the appearance typical of a glycosylated protein. moreover, in the latter topology, the eptm region is predicted to be free on the luminal side of the membrane and not to interact with it. the data here are inconsistent with this prediction and are more explicable by assuming that either the type iii membrane protein or the membrane hairpin topology is more likely. in both cases, it is postulated that the post-tm region folds back against the membrane, where it may be additionally anchored by cysteine palmitoylation, although the precise mechanism of interaction remains unknown. assuming that the predominant punctate staining in mammalian cells represents misfolded e protein that induced stress granules, then mutants l a, y a and y a formed a group with a shared phenotype. the "all" mutant, which necessarily included these mutations, was also punctate in appearance. by contrast, l a and v a were more similar to the wt straining pattern. biochemical fractionation of membrane from insect cells following the construction and validation of recombinant baculoviruses broadly recapitulated this division. the relative level of e protein following differential centrifugation of the cell membranes showed that wt, l a and v a mutants were predominantly associated with the hsp fraction with a minority in the lsp fraction ( . %, . % and . % respectively), while the remaining mutants were predominantly associated with the lsp fraction. some mutants, notably l a and y a, were expressed at lower levels but were hardly present in the hsp faction. as the wt exhibited an hsp pattern, we interpret this to be authentic partitioning into membranes, whilst the lsp pattern is likely to be aggregated protein trapped in the er by virtue of its inability to associate with membranes correctly. despite its conservation, the role of the p a mutant was equivocal, i.e., some way between the two extremes but tending towards non-wt-like mutants. these data suggest a possible model for eptm interaction with the membrane in which the c-terminal hydrophobic residues within the residue region are the most relevant. interestingly, these fall broadly on one side of the helix prediction for eptm, while those broadly tolerated fall on the other ( figure ). plausibly, this distinction relates to membrane binding verses the requirement for e to also bind other ligands. further work will be required to address the level of membrane curvature afforded by this interaction and the extent to which it relates to the various functions reported for cov e, such as reorientation of the plasma membrane within the golgi [ ] , interaction with the m protein [ , , ] and viral particle scission [ , ] . in sars cov, e residues which reportedly bind to the membrane include valine- , tyrosine- and lys- [ ] . these residues are fully conserved in sars-cov- within an overall e similarity of %, and are also equivalent in the mhv e peptide identified here. membrane binding by these residues in sars e is thus supported by the observations made here for v a and y a, although lys was not examined. e has been considered a therapeutic target for sars in relation to its viroporin function which is linked to inflammation [ ] , and more recently, for sars-cov- as a result of its mapped interactions with bromodomain proteins, for which drugs already exist [ ] . a more detailed mapping of 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the terms and conditions of the creative commons attribution (cc by) license we thank fellow members of the virology laboratory for their help during the course of this study. the authors declare no conflict of interest. key: cord- -egpyvqrw authors: mo, mei-lan; li, meng; huang, bai-cheng; fan, wen-sheng; wei, ping; wei, tian-chao; cheng, qiu-ying; wei, zheng-ji; lang, ya-hui title: molecular characterization of major structural protein genes of avian coronavirus infectious bronchitis virus isolates in southern china date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: egpyvqrw to gain comprehensive genetic information of circulating avian coronavirus infectious bronchitis virus (ibv) isolates in china, analysis of the phylogenetic tree, entropy of the amino acid sequences, and the positive selection as well as computational recombinations of s , m and n genes of ibv isolates was conducted in the present study. the phylogenetic trees based on the s , m and n genes exhibited considerably different topology and the ck/ch/lsc/ i-type isolates were the predominant ibvs based on the phylogenetic analysis of s gene. results of entropy of amino acid sequences revealed that the s gene had the largest variation; the m gene had less variation than the n gene. positive selections were detected in not only s but also m and n gene proteins. in addition, five s gene recombinants between vaccine strain / and ck/ch/lsc/ i-type field isolate were confirmed. in conclusion, multiple ibv genotypes co-circulated; genetic diversity and positive selections existed in s , m and n genes; / vaccine recombinants emerged in china. our results show that field ibvs in china are continuing to evolve and vaccine strains may have an important role in the appearance of new ibv strains via recombination. in addition, the present study indicates that ibv evolution is driven by both generations of genetic diversity and selection. infectious bronchitis (ib) is an acute, highly infectious and contagious disease of domestic chickens worldwide caused by avian infectious bronchitis virus (ibv), a member of genus gammacoronavirus, subfamily coronavirinae, family coronaviridae [ ] . ib affects chickens of all ages and ibv replicates primarily in the respiratory tract, and also in some epithelial cells of the kidney, gut and oviduct, resulting in reduced performance, reduced egg quality and quantity, increased susceptibility to infections with other pathogens, and condemnations at processing [ ] . multiple ibv serotypes or genotypes have been identified worldwide and different serotypes of ibvs confer little or no cross-protection against the others. ibv genome consists of a linear, single-stranded, positive-sense rna of . kb, which encodes four major structural proteins, the spike (s) glycoprotein, the membrane (m) glycoprotein, the nucleocapsid (n) protein and the envelope or small membrane (e) protein [ ] . the s glycoprotein is post-translationally cleaved into s and s subunits and s is the most divergent region, which carries conformationally-dependent virus-neutralizing and serotype-specific epitopes [ , ] . the n protein located in the capsid of the virion is involved in rna replication, assembly and carries group-specific antigenic determinants [ ] and has high immunogenicity, readily inducing antibodies and cytotoxic t-lymphocyte immunity in chickens [ ] . s and n genes have been used most frequently to determine the relatedness of emerging strains of ibv [ , ] . the m protein is a structural membrane protein and plays an important role in the viral assembly process and particularly is indispensable for many biological functions including viral core stability. interactions of m and e proteins are important for virus budding and formation of virus-like particles, which are involved in mucosal immunity [ ] . the genetic diversity and viral evolution of ibv are mainly monitored by analysis of the s gene because of its high variability and close serotype correlation [ ] , but viruses within the same serotype can have a high degree of genetic variability outside of the spike gene [ ] . pathogenicity of ibv is associated with the spike gene as well as genes outside of the spike gene [ ] . the m protein is associated with virus assembly and change this protein will affect the efficiency of virus particles formation and subsequent transmission of the virus [ ] . the n protein plays an important role in viral replication, assembly, and immunity. in addition to s glycoprotein, the n protein could represent an important target in the prevention of ib outbreaks [ ] . recent evidence revealed that there are significant variations in the n and m genes between strains [ , ] . therefore, it is necessary to analyze multiple genes especially to analyze the genetic variation of s , m and n genes considering their importance as structural proteins. the major challenge for the prevention and control of ib is the increasing number of new serotypes or variants of ibv, which was caused by frequent gene mutation and recombination [ ] [ ] [ ] [ ] . recombination is thought to be a contributing factor in the emergence and evolution of ibv or even the emergence of new coronaviruses and new diseases [ ] . the studies of ibv recombination are very important for ibv control, because they will further our understanding of the diversity and evolution mechanisms of these viruses and thus enable the development of better control methods [ , ] . ibv strains within a geographic region are unique and distinct [ ] although many countries share some common antigenic types. therefore, it is extremely critical to identify the prevalence of ibvs and genetic characteristics of circulating strains in a region or a country in order to develop effective vaccines for the control of the disease. outbreaks of ib have been occurring frequently in china in spite of intensive vaccinations for many years [ , , [ ] [ ] [ ] . ib is still a major problem in guangxi province [ , ] , which is located in southern china and produces a total of million birds per year and ranks third in china [ ] . it is very important to know the genetic characteristics of prevalent strains of ibvs in this region. we previously reported the genotype diversity of guangxi ibv isolates based on the hypervariable region i (hvr i) of s gene [ ] , but the available comprehensive genetic information of circulating ibv strains in this region was limited. therefore, in the present study we performed the analysis of the phylogenetic tree, of the entropy of the amino acid sequences, of positive selection as well as of computational recombination based on the sequencing results of the viral structural protein genes s , m and n in order to provide molecular epidemiology information of ibv and to lay a good foundation for the control of ib in the field. the nucleotide and deduced amino acid (aa) sequence identities of the s , m and n genes among the isolates were . %- . % (aa: . %- . %), . %- . % (aa: . %- . %) and %- . % (aa: . %- . %), respectively. the identities of nucleotide and deduced amino acid sequences of s , m and n genes between the isolates and all the reference strains were . %- . % (aa: . %- . %), . %- . % (aa: . %- . %) and . %- . % (aa: . %- . %), respectively. compared with the most popularly used vaccine strain h , all the isolates had lower nucleotide sequence identities (s : . %- %; m: . %- . %; n: . %- . %) except for gx-nn and gx-nn ( . % and . ) in the s gene, gx-nn , gx-nn and gx-nn ( . %, . % and . %) in the m gene, gx-nn ( . %) in the n gene (supplementary table s ). both the phylogenetic trees constructed with the neighbor-joining and maximum-likelihood method had very similar topography, so only the neighbor-joining trees are shown ( figure ). the phylogenetic trees based on s gene amino acid sequences showed that all ibv isolates except gx-c were divided into five distinct groups (figure a ). eleven out of isolates were grouped into the ck/ch/lsc/ i-type with china ibv reference strains ck/ch/lsc/ i, saibk and a , which were isolated during - . isolates gx-nn , gx-nn , gx-nn , gx-nn , gx-nn , gx-yl and reference vaccine strain / were classified into the / -type, but the latter five isolates occupied another offshoot. gx-yl and reference strains ldt , partridge/gd/s / and korea strain km were grouped as the tl/ch/ldt / -type. amazingly, gx-g and gx-xd isolated in were grouped with taiwan reference strains tw / , tw / as taiwan-type. isolates gx-nn and gx-nn showed a close relationship with commonly used vaccine strains h , h , ma , m and other china vaccine strains w , h , d , ibn, hk and grouped as mass-type. gx-c, isolated in , showed considerable low homology with the above five genotypes and belonged to a separate group. (a) s the phylogenetic trees of m and n genes showed that the isolates were segregated into distinct groups, which exhibited considerably different topology than that of the s gene (figure b,c) . in the phylogenetic trees of the m gene, , , and isolates were designated as ck/ch/lsc/ i-type, lx -type, mass-type and bj-type respectively. in the phylogenetic trees of the n gene, , , and isolates were designated as lx -type, new-type, ck/ch/lsc/ i-type and mass-type respectively. the results of the codon-based tests of positive selection (z-test, mega ) for analyzing the numbers of non-synonymous and synonymous substitutions per site (dn/ds ratio) on the s , m and n proteins were displayed as supplementary material (supplementary figures s ). no significant evidence for positive selection of s protein of taiwan-type and mass-type groups was observed (p > . ). however the result of analysis of entropy of s , m and n genes on amino acid sequences was shown in figure . the higher the peak is, the greater the entropy is, indicating the higher variation frequency of amino acid sites. numerous high entropy amino acid sites were distributed throughout the entire s gene; only a few high entropy amino acid sites were scattered within the m gene. the number of high entropy amino acid sites within the n gene is less than that of the s gene but more than that of the m gene (supplementary material). an entropy value bigger than . indicated the corresponding amino acid site was not conserved. the percentages of entropy bigger than . in amino acids sequences of s , m and n gene were . % ( / ), . % ( / ) and . % ( / ), respectively. the descending average entropy order were s ( . ) > n ( . ) > m ( . ). therefore, the s gene amino acid sequences had the largest variation; the m gene had less variation than the n gene. recombinant events were detected in the s gene of isolates gx-nn , gx-nn , gx-nn , gx-nn and gx-yl by all recombination detection methods implemented in the rdp . software. these five isolates were found to be recombinants between the vaccine strain / and the ck/ch/lsc/ i-type field strain gx-yl (figure ) one of the major problems caused by ibv in the field is the frequent emergence of new variants. ibv strains within a geographic region are unique and distinct [ ] . outbreaks of ib still occurred in guangxi [ , ] although vaccines have been applied and the molecular epidemiology information available was limited. hence, we investigated the genetic characteristics of s , m and n genes of ibvs circulating in this region. this is the first report on the analysis of entropy of amino acid sequence and the positive selection of s , m and n genes of ibvs. some investigators reported the genetic typing based on hvr i of the s gene is representative of the grouping based on the whole s gene [ , ] , but another study disagreed with these findings [ ] . the present results and our previous report from hvr i [ ] also indicated that genotypes based on hvr i are not representative of that based on the whole s gene. the reason was that mutations in non-hvr i of the s gene were also detected [ ] . in addition, in addition, our results showed that genotypes based on s gene exhibited considerably difference from m and n genes. the discordance of topology in the s -based tree and other gene-based trees were also described by other investigators [ , ] . the co-circulation of multiple ibv types and the ongoing emergence of ibv variants are the epidemiological challenges in china. nine genotypes including lx -type, ck/ch/lsc/ i-type, tl/ch/ldt / -type, ck/ch/ldl/ i-type, bj-type, ck/ch/lhlj/ i-type, mass-type, / -type, and n / associated circulated in china and lx -type was the dominant genotype [ ] . recently, the taiwan ii-type was firstly reported in china [ ] . in our study, ck/ch/lsc/ i-type, tl/ch/ldt / -type, / -type, taiwan-type and mass-type were identified, which suggested that multiple genotypes of ibvs were co-circulating in guangxi province. eleven out of isolates sharing . - . % s gene amino acid sequence similarity with the vaccine strain h belonged to ck/ch/lsc/ i-type. however, the prevalent genotype in this region was ck/ch/lsc/ i-type not the lx -type, which was different from other reports that lx -type was the dominant genotype in china [ , ] , indicating ibv strains within this region are unique and distinct. ibv has been diagnosed in china since the early s [ ] . surprisingly, the gx-g and gx-xd isolated in showed closed relationship with taiwan ii-type strain tw / and no taiwan-type strain occurred in recent year in our study. recently other investigators reported that taiwan ii-type strains of ibv occurred in mainland china [ ] . whether the taiwan-type ibvs entered china by long distances migration of wild birds or importing of poultry products or improper use of vaccines？it is unclear. identification of tai-wan and china-like recombinant ibvs in taiwan was reported [ ] . so it is very important to monitor the vaccine, birds and poultry products in china. natural selection generally causes a reduction in deleterious mutations while promoting advantageous mutations. a gene which undergoes positive selection promoted by natural selection usually has highly important functions [ ] . some investigators reported positive selection wasn't detected in the spike protein of ibvs although they differed markedly in the sequence of the spike protein [ , ] . however, other investigators showed different results. positive selection was detected in the spike protein of ibv california-type viruses, for which no vaccine exists but was not detected in massachusetts-and connecticut-types where attenuated live vaccines are routinely used [ ] . another report showed that positive selection was found in the s protein of variants isolated from layer-type birds but was not found in variants isolated from broilers, even though a high number of mutations was significantly associated with broiler-type chickens [ ] . positively selected sites in the nucleocapsid protein of the taiwan ibv and their effects on rna-binding activity were reported recently [ ] . previous reports on sars-cov indicated that positive selection on s protein was changeable in different epidemic groups and positive selection on replicase of sars-cov was detected only in human patients, not in any proteins of bat sars-like-cov [ ] . we found positive selection was observed on s protein of / -type and ck/ch/lsc/ i-type strains, m protein of ck/ch/lsc/ i-type and bj-type strains, n protein of ck/ch/lsc/ i-type and lx -type strains. thus, not only s protein but also m and n proteins experienced positive selection during the ibv epidemics. the variation of positive selection of s, m and n proteins among different groups may explain why these field variants escape immune pressure and may provide valuable evidence that these three structural proteins may be critical for virus evolution. it is the first time to analyze the positive selection of s , m and n genes of ibvs. the entropy is one useful quantification of diversity in a single position of amino acid sequences [ ] . high scoring amino acid positions may correlate with structurally or functionally important residues [ ] . the greater the entropy is, the higher variation frequency of amino acid sites is. an entropy value bigger than . indicated the corresponding amino acid site was not conserved [ ] . a shannon entropy analysis of immunoglobulin and t cell receptor revealed that the t cell receptor is significantly more diverse than immunoglobulin-suggesting t cell receptor has new complementarity determining regions, which represent a larger antigen combining site, additional combining sites, or an evolutionary strategy to avoid inappropriate interaction with other molecules [ ] . a recent study used the shannon entropy and relative entropy to measure the diversity of amino acid site of h ha between the - season and the - season and showed that the rate of evolution increases with the virus diversity in the current season and the shannon entropy of the sequence in the current season predicts relative entropy between sequences in the current season and those in the next season [ ] . according to our results, the average entropy of amino acid sequences, the percentages of entropy bigger than . and the number of amino acid sites with high entropy of s gene are biggest, and those of n gene were bigger than m gene. thus, these observations revealed that amino acid sequences of s gene had the largest variation; the m gene had less variation than the n gene. to our knowledge, it is the first time to analyze the entropy of s , m and n gene amino acid sequences. the variation of amino acids will have an important effect on the biological function and evolution of viruses. hence, observing the biological function of the amino acid residues with higher entropy and identifying the positively selected sites among ibvs will be further studied. recombination is involved in the emergence and evolution of ibv or can even directly lead to the emergence of new coronaviruses and related diseases [ ] recombination can occur between field isolates or between field and vaccine viruses [ ] [ ] [ ] . in our study, convincing evidence showed five s -gene recombinants gx-nn , gx-nn , gx-nn , gx-nn and gx-yl , with their putative parental strains of vaccine strain / and ck/ch/lsc/ i-type field strain gx-yl , and their crossover regions were at nucleotide position - or - . a recently report showed a recombinant (ck/ch/lzj/ strain) came from a chinese field isolate (ck/ch/ldl/ strain, lx -type) and a / -like strain, with switches at sites, namely upstream of s, the n gene and the ' utr [ ] . besides the mass-type vaccine, / -type live vaccines are also commonly used in china including during the breeding period [ ] , even without official authorization. our finding provides another evidence that / vaccine strains are contributing to the emergence of variants in the field in china. therefore, it is necessary to strengthen the vaccine licensing system before introduction of exotic ibv strains. we should continued / -type recombinants surveillance in china. the pathogenicity of / -derived recombinants should be assessed in further studies. twenty-three ibv strains, isolated as previously described [ ] were analyzed in the present study. the ibv field isolates were propagated in to -day-old specific pathogen free embryonated chicken eggs via the allantoic cavity route. allantoic fluids were harvested at h post-inoculation, frozen, and stored at − °c until used. for each ibv strain, the entire s , m and n genes were amplified. the s primers were designed according to the previous report and the anticipated amplification segment is about bp encompassing the entire s gene including the protease cleavage motif [ ] . the m gene sense primer was: '-cgagtttcctaagaacggttggaa- ', and the anti-sense primer was: '-cccctctctacacgcacacatttat- '. the n gene sense primer was: '-ccatggcaagcggtaaagcar- ', and the anti-sense primer was: '-ccactcaaagttcattctctcc- '. the anticipated amplification segments for m and n genes are bp and bp respectively. viral rna was extracted from the infectious allantic fluid by the trizol reagents (invitrogen, usa) according to the manufacturer's instruction. the first cdna strand was synthesized in µl mixture consisting of µl of rna extract, µl of µm/µl random mers, µl of ×reverse transcriptase first strand buffer, µl of u/µl rnase inhibitor (takara, japan), µl of u/µl amv reverse transcriptase (takara, japan) and µl of . mmol/l dntpmix (takara, japan). the mixture was incubated at °c for h, and then inactivated at °c for min. for the following pcr assays, a total of µl reaction mixture consisted of µl of the cdna, . µl of × pcr buffer, µl of . mmol/l dntpmix (takara, japan), µl of µmol/l of each of the two primers and . µl of u/µl taq dna polymerase (takara, japan). the pcr conditions for the s gene amplification were °c for min, cycles of °c for s, °c for s, and °c for min, followed by °c for min; that for the m gene were °c for min, cycles of °c for min, °c for min, and °c for min, followed by °c for min; and that for the n gene were °c for min, cycles of °c for min, °c for min, and °c for min, followed by °c for min. the pcr products were analyzed on . % agarose-gel electrophoresis. the pcr products were purified, cloned and then sequenced by sangon bio-company (shanghai, china). for each gene, three independent clones were selected randomly and sequenced twice from both directions. the open reading frames of s , m and n gene were determined using the dnastar version (dnastar, madison, wi). the nucleotide sequences of s , m and n genes have been submitted to genbank database and assigned accession numbers (supplementary table s ). the nucleotide and the deduced amino acid sequences alignments were generated using the clustalw multiple alignment method of bioedit version . . . and compared with those of reference ibv strains retrieved from the genbank database with the accession numbers listed in in supplementary material (supplementary table s ). phylogenetic trees were constructed based on the amino acid sequences of s , m and n genes with the neighbor-joining method (jones-taylor-thornton (jtt) model) and maximum-likelihood method (jtt model) using mega . version. the bootstrap values were determined from replicates of the original data. the entropy is one useful quantification of diversity in a single position of amino acid sequences. a large entropy means the amino acid in the given position is prone to be substituted. in order to understand the variation degree of s , m and n genes, the entropy of aligned amino acid sequences within these genes of the isolates was calculated by bioedit version . . . . in addition, codon-based tests of positive selection (z-test, mega ) were used to estimate the numbers of non-synonymous and synonymous substitutions per site (dn/ds ratio) within the s , m and n proteins in order to understand whether these proteins are submit to positive selection. . . computational recombination analysis. aligned nucleotide sequences of s , m and n genes were analyzed with the recombination detection program (rdp , version . ) to detect potential within-gene recombination events. the window size was adjusted to bp from the default setting bp because ibv has a high mutation rate, which can mask recombination signals. the highest acceptable p value was . and the detection of recombination events was applied between sequences sharing and % identity. seven algorithms in rdp . , including rdp, geneconv, bootscan, maxchi, chimaera, siscan and seq were used to confirm the recombination events. two phylogenetic trees, which were constructed from the portion of the alignment between the inferred breakpoints and the remainder of the alignment were made and compared to assess recombination 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recent years in china molecular epidemiological investigation of marek's disease virus from guangxi relationship between serotypes and genotypes based on the hypervariable region of the s gene of infectious bronchitis virus genetic diversity of avian infectious bronchitis virus california variants isolated between and based on the s subunit of the spike glycoprotein recombinational histories of avian infectious bronchitis virus and turkey coronavirus complete nucleotide sequences of s and n genes of infectious bronchitis virus isolated in japan and taiwan identification of taiwan and china-like recombinant avian infectious bronchitis viruses in taiwan differential stepwise evolution of sars coronavirus functional proteins in different host species identification of intertypic recombinant infectious bronchitis viruses from slaughtered chickens evolution of infectious bronchitis virus in taiwan: positively selected sites in the nucleocapsid protein and their effects on rna-binding activity quantifying selection and diversity in viruses by entropy methods, with application to the haemagglutinin of h n influenza incorporating background frequency improves entropy-based residue conservation measures crystal structure of the polymerase pa(c)-pb (n) complex from an avian influenza h n virus a shannon entropy analysis of immunoglobulin and tcell receptor molecular characterization of infectious bronchitis virus isolates from russia and neighbouring countries: identification of intertypic recombination in the s gene complete genome sequence and recombination analysis of infectious bronchitis virus attenuated vaccine strain h characterization of a recombinant coronavirus infectious bronchitis virus with distinct s subunits of spike and nucleocapsid genes and a ' untranslated region isolation and genetic analysis revealed no predominant new strains of avian infectious bronchitis virus circulating in south china during this work was supported by grants from the national natural science foundation of china ( , ), guangxi natural science foundation ( gxnsfca ), and the guangxi provincial programs for science and technology development ( - ). and the manuscript was kindly reviewed by richard roberts, aurora, co , usa. the authors declare no conflict of interest. key: cord- -yo ojr s authors: mendenhall, ian h.; kerimbayev, aslan a.; strochkov, vitaliy m.; sultankulova, kulyaisan t.; kopeyev, syrym k.; su, yvonne c.f.; smith, gavin j.d.; orynbayev, mukhit b. title: discovery and characterization of novel bat coronavirus lineages from kazakhstan date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: yo ojr s coronaviruses are positive-stranded rna viruses that infect a variety of hosts, resulting in a range of symptoms from gastrointestinal illness to respiratory distress. bats are reservoirs for a high diversity of coronaviruses, and focused surveillance detected several strains genetically similar to mers-coronavirus, sars-coronavirus, and the human coronaviruses e and nl . the bat fauna of central asia, which link china to eastern europe, are relatively less studied than other regions of the world. kazakhstan is the world’s ninth largest country; however, little is understood about the prevalence and diversity of bat-borne viruses. in this study, bat guano was collected from bat caves in three different sites of southern kazakhstan that tested positive for coronaviruses. our phylogenetic reconstruction indicates these are novel bat coronaviruses that belong to the genus alphacoronavirus. in addition, two distinct lineages of kazakhstan bat coronaviruses were detected. both lineages are closely related to bat coronaviruses from china, france, spain, and south africa, suggesting that co-circulation of coronaviruses is common in multiple bat species with overlapping geographical distributions. our study highlights the need for collaborative efforts in understudied countries to increase integrated surveillance capabilities toward better monitoring and detection of infectious diseases. bats are mammals in the order chiroptera that possess a range of unique ecological, immunological, and behavioral attributes. bats are exceptionally speciose, comprising % of all mammalian species, and they are the only mammals that are capable of true flight [ ] . most bat species are gregarious and roost in large colonies, which can number over one million individuals [ ] . they are relatively long-lived for their body size, and temperate species often undergo torpor or hibernation [ ] . bats also act as rich reservoirs of virus diversity with at least families of viruses detected, including double-stranded dna viruses, single-stranded dna viruses, and positive-and negative-sense single-stranded rna viruses [ ] . bats are incriminated as the source of several medically important virus families, including filoviruses, coronaviruses, paramyxoviruses, and reoviruses [ , ] . several recent zoonotic spillover events and outbreaks directly or indirectly originated from bats [ ] . coronaviruses are positive-sense rna viruses in the order nidovirales and the family coronaviridae. these viruses have the largest genome of any single-stranded rna viruses that infect vertebrates, and they are capable of recombining to create new strains. they infect a wide variety of mammals and birds, including infectious bronchitis virus in birds and transmissible gastroenteritis virus in pigs [ ] . in humans, seasonal coronaviruses can cause both upper and lower respiratory tract infections, with increased disease severity in the elderly, children, and immunocompromised patients [ ] . the zoonotic sars-coronavirus (sars-cov) outbreak originated in southern china from horseshoe bats, where wet markets permitted atypical contact between species, including subsequent spillover to humans [ ] . recent work showed that all genetic components of sars-cov co-circulate among different bat species sharing the same cave, underlying the opportunity for its re-emergence [ ] . on the other hand, camels are the putative natural reservoir for mers-coronavirus, although recent phylogenetic analysis indicated that bats harbor coronaviruses that are ancestral to the mers-cov lineage [ ] . more recently, an hku -cov outbreak caused by transmission from bats to pigs in china killed nearly , piglets [ ] . central asia is one of the largest grassland and steppe habitats in the world, although little is known about its resident bat fauna. this habitat type is primarily located in russia, mongolia, and kazakhstan. kazakhstan is the largest land-locked country in the world; it is relatively arid (< mm rainfall), and comprises plains and hills, with forested areas primarily restricted to the mountains in the south (tien shan) and the east (alatul and altai). described to date, there are species of bats in kazakhstan, with of these resident in turkestan oblast, and the most common species are vespertilio murinus (linnaeus, ) and myotis mystacinus (kuhl, ) [ , ] . while there is substantive bat research in russia and mongolia, there is little work on the kazakhstan bats and less on associated virus communities [ , ] . in this study, we collected fresh bat guano from three caves at different locations in kazakhstan and conducted molecular screening for coronaviruses. our objective was to explore and understand the diversity of bat coronaviruses in of kazakhstan. here, we identified and sequenced novel bat coronaviruses and determined the evolutionary relatedness of the viruses. to the best of our knowledge, this study represents the first detection of bat coronaviruses from kazakhstan. bat guano was collected from three sites in turkestan oblast from april to may . these sites were the kepterkhan tunnel and qaraungir cave in tulkibas rayon district, with additional guano collected in the ungirli cave in altyntau, sozak rayon ( figure ). bat feces were collected from plastic sheets placed underneath bat roosts. multiple fecal pellets were placed into cryovials with viral transport media using polyester swabs, which were subsequently placed into a liquid-nitrogen dry shipper and then transferred back to the research institute for biological safety problems (ribsp) in gvardeiskiy, kazakhstan, where all samples were stored at − • c. bat guano was vortexed for s and rna was extracted using a qiaamp viral rna mini kit (qiagen, hilden, germany), performed following the manufacturer's instructions. coronavirus nucleic acid was amplified using pan-coronavirus primers that amplified a -bp region of the rna-dependent reverse polymerase (rdrp) (pancor in- : ggttgggactatcctaagtgtga and pancor in- : ccatcatcagatagaatcatcata). a pcr was run using a superscript iii one-step rt-pcr system with platinum taq dna polymerase (invitrogen, carlsbad, ca, usa) in a rotorgene thermocycler (qiagen, hilden, germany). the reaction consisted of µl of rna, . µl of × reaction buffer, µl of reverse transcriptase, µm of forward and reverse primer, and water to a total of µl. the thermocycler protocol followed a previously described protocol with the reverse transcription (rt) step held at • c for min, followed by a denaturation step at • c for min, followed by cycles of • c for s, • c for s, and • c for min, with a -min extension period at • c [ ] . pcr products were visualized on a % agarose gel stained with ethidium bromide. a positive control, an rdrp sequence from human coronavirus e in a p-gem t-easy plasmid (promega, madison, wi, usa), was run with each set of reactions. positive samples with the appropriate amplicon size were purified and sequenced at ribsp using the genetic analyzer xl (thermofisher, waltham, ma, usa) with a big dye terminator cycle sequencing kit, v. . (thermofisher). electropherograms were inspected in geneious v [ ] . a total of rdrp sequences were newly generated from this study. to further understand the evolutionary relationships of these viruses, we analyzed novel bat coronavirus sequences in combination with rdrp sequences of coronavirus from different host species worldwide, representing the three genera: alpha-, beta-, and gamma-coronaviruses. global rdrp sequences were downloaded from national center for biotechnology information sequence database (genbank) and aligned using transalign [ ] . this large dataset was manually aligned and further down-sampled to sequences to reduce redundant and similar sequences. maximum-likelihood phylogeny of the partial rdrp gene ( bp) was reconstructed by raxml; gtr + gamma was selected for the model of nucleotide substitution as it allows rate heterogeneity among sites, as implemented in geneious v [ ] . branch support was assessed using bootstrap replicates; bootstrap values greater than % were indicated at major nodes. a total of bat guano samples were collected from three sites: kepterkhan tunnel (n = ), qaraungir cave (n = ), and ungirli cave (n = ). each cave was occupied by two bat species, the dominant species being myotis blythii (lesser mouse-eared bat) and the more infrequent species being hypsugo savii (savi's pipistrelle). overall, ( . %) of all guano samples screened were positive for coronaviruses: qaraungir cave with the highest percent positive ( %) and kepterkham tunnel with the lowest ( . %) ( table ) . sequence data were successfully generated for of the pcr positive samples. our rdrp phylogeny demonstrates that all new cov sequences (genbank accession mk -mk ) from kazakhstan bats belong to the genus alpha-cov (figure ; figure s , supplementary materials). the alpha-cov genus comprises a large number of coronaviruses from diverse hosts, including bats, shrews, dogs, cats, ferrets, pigs, and humans. in kazakhstan bats, the new cov sequences were found to be segregated into two different groups. the majority ( sequences) of the kazakhstan covs formed a strongly monophyletic single clade (bootstrap (bs) = %), referred to here as "kz ", with the nucleotide sequence identities ranging from . % to %. the kz clade was a sister group to three bat cov sequences from spain (miniopterus schreibersii and myotis blythii) and china (myotis ricketti). within the kz clade, two smaller co-circulating lineages (kz- a and kz- b) were formed that are also strongly monophyletic (bs = % and %, respectively). the kz- b sub-lineage contained seven bat sequences (ribsp- , , , , , , and ) with % nucleotide similarity; however, the samples were collected from two different sites. bat kz- b sequences (ribsp- , , , and ) were collected from the qaraungir cave, whereas bat kz- b sequences (ribsp- , , and ) were collected from the altyntau cave. this suggests the kazakhstan bats residing in two different caves appear to harbor highly similar cov strains. the kz group contains only one kazakhstan sequence (ribsp- ) from the altyntau cave, which is markedly divergent (nucleotide identities: . - . %) from the kz sequences. the ribsp- sequence is most closely related with pipistrellus pipistrellus cov (nucleotide identity: . %) described from france in . these sequences in turn grouped with diverse bat species from broad geographical regions including spain (nyctalus lasiopterus and myotis myotis), china (pipistrellus pipistrellus), and south africa (neoromicia cf capensis). taken together, our results indicate that kazakhstan bats may harbor a wider diversity of alpha cov, possibly by means of the regional and intercontinental spread of the virus. although the pooled feces prevented attribution to a singular species, the two lineages of alpha cov may be derived from the different species of bats, myotis blythii and hypsugo savii. kz is similar to myotis coronavirus sequences, while the kz is most similar to a pipistrellus sequence and h. savii is a pipistrelle bat. our approach was opportunistic sampling in three sites, and the total sample size was , limiting the conclusions we can make about bat-borne coronaviruses in central asia; however, this study provides a baseline for future studies. intense coronavirus surveillance is ongoing in china since the sars-cov outbreak, and it was demonstrated that co-roosting can maintain all strains with the necessary components to make sars [ ] . there is a paucity of bat-borne virus surveillance efforts across central asia, even though this region is one of the largest grassland biomes in the world. this area is also of interest because it is where the eurasian and asian bat populations may be panmictic, as seen in other bat species with large distributions [ , ] . the range of m. blythii and h. savii is extensive, with both species found from china and northern india through to spain, and these populations may be co-roosting at multiple sites due to the paucity of roosting areas in the steppe [ , ] (figure ). myotis blythii is considered an occasional migrant, with movement of greater than km recorded. the migratory behavior of hypsugo savii is not known [ ] . the potential panmixia and co-roosting of these bat species may lead to a similar mixing of viruses. these results provide a foundation to study bat-borne coronaviruses in kazakhstan and highlight the need for collaborative efforts in understudied countries to increase integrated surveillance capabilities toward better monitoring and detection of infectious diseases. the following are available online at http://www.mdpi.com/ - / / / / s : figure s . phylogenetic relationships of the rdrp gene sequences of coronavirus, inferred using the maximum-likelihood method with the gtr + gamma model in raxml. representative virus isolates from alpha-, beta-, and gamma-coronavirus were included in the analysis. colored branches and symbols denote viruses collected from different hosts. new cov sequences generated from this study are marked by red branches. bootstrap support values greater than % are displayed at major nodes. the scale bar indicates the number of nucleotide substitutions per site. bats: important reservoir hosts of emerging viruses microchiropteran bats: global status survey and conservation action plan bat ecology bats as viral reservoirs bats and their virome: an important source of emerging viruses capable of infecting humans host and viral traits predict zoonotic spillover from mammals evolutionary insights into the ecology of coronaviruses human coronaviruses: what do they cause? bats are natural reservoirs of sars-like coronaviruses discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat fatal swine acute diarrhoea syndrome caused by an hku -related coronavirus of bat origin catalog of the mammals of ussr bat study in the kharaa region bats of the russian far east. dalnauka vladivostok bat coronaviruses and experimental infection of bats, the philippines geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data using amino acids to facilitate the multiple alignment of protein-coding dna sequences raxml version : a tool for phylogenetic analysis and post-analysis of large phylogenies the conservation genetics of three cave-dwelling bat species in southeastern europe and anatolia continent-wide panmixia of an african fruit bat facilitates transmission of potentially zoonotic viruses the iucn red list of threatened species the iucn red list of threatened species bat migrations in europe: a review of banding data and literature; federal agency for nature conservation we wish to thank kairat tabynov and sulushash zhumabayeva at jacobs. we also thank s. nurabayev, e. burashev, n. rametov, and b. burabayev for their participation in the fieldwork. we also wish to thank jayanthi jayakumar at duke nus for technical assistance. the authors declare no conflict of interest. key: cord- -knswbb d authors: chang, chia-wen; leu, yan-lii; horng, jim-tong title: daphne genkwa sieb. et zucc. water-soluble extracts act on enterovirus by inhibiting viral entry date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: knswbb d dried flowers of daphne genkwa sieb. et zucc. (thymelaeaceae) are a chinese herbal medicine used as an abortifacient with purgative, diuretic and anti-inflammatory activities. however, the activity of this medicine against enteroviral infections has not been investigated. the water-extract of dried buds of d. genkwa sieb. et zucc. (dgfw) was examined against various strains of enterovirus (ev ) by neutralization assay, and its initial mode of action was characterized by time-of-addition assay followed by attachment and penetration assays. pretreatment of dgfw with virus abolished viral replication, indicating that dgfw inhibits ev by targeting the virus. gfw exerts its anti-ev effects by inhibiting viral entry without producing cytotoxic side effects and thus provides a potential agent for antiviral chemotherapeutics. enterovirus (ev ) is a positive-strand rna virus and belongs to the enterovirus genus of the family picornaviridae. it is a member of the picornaviridae family and has a (+)rna genome . kb in length that encodes amino acids [ , ] . the polyprotein is cleaved proteolytically into distinct proteins by the viral-encoded proteases a pro and c pro . the structural proteins comprising vp to vp and the nonstructural proteins a to c and a to d are required for viral replication [ ] . ev is a common enterovirus that usually causes hand, foot and mouth disease in children but is often associated with brainstem encephalitis, neurogenic shock and polio-like acute flaccid paralysis. ev caused an outbreak with mortalities in , with manifestations of neurological involvement and cardiopulmonary failure. in a long-term study, survivors of ev -induced cns infection were found to have neurological sequelae and retarded mental development [ ] . several epidemics occurred and caused a total of fatalities in - [ ] . unfortunately, there is no therapy with proven efficacy to control ev . given these recurring outbreaks and annual epidemics, there is a clear need to develop inhibitors for ev . viruses must deliver their genome into the host cells to initiate replication. most of them enter cells by endocytosis during which they hijack cellular machinery for the attachment to receptors and subsequent penetration across the plasma membrane. several receptors to facilitate ev entry have been reported, including scavenger receptor b , human p-selectin glycoprotein ligand- and sialylated glycans [ ] [ ] [ ] . a strategy to disrupt the interaction between viral particles and their receptors would be ideal for antiviral development. we have been identifying traditional chinese medicines with antiviral or prophylactic activity against ev infections [ , ] . the flower buds of daphne genkwa sieb. et zucc. (thymelaeaceae), known as won-hwa or yuanhua in chinese, are collected in the spring before blossoming. as a traditional medicine, they have been used as an abortifacient [ ] , with purgative, diuretic and anti-inflammatory actions [ ] . previous phytochemical studies on d. genkwa have led to the isolation of pharmacologically active flavonoids, diterpene orthoesters and coumarins, showing inhibitory activity against xanthine oxidase and adenosine , -cyclic monophosphate phosphodiesterase, as well as exhibiting antileukemic activity [ ] . in the present study, we used chinese traditional medicine extracts for anti-ev activity. we found that daphne genkwa sieb. et zucc. extracts (dgfw) could neutralize the ev -induced cytopathic effect (cpe) in human embryonic rhabdomyosarcoma (rd) cells. the half maximal effective concentration (ec ) of dgfw on ev was approximately . ± . mg/ml. dgfw inhibited ev viral protein and viral rna synthesis. additionally, dgfw suppressed viral activity at the adsorption stage of uptake as shown by a time-of-addition assay. cells pretreated with dgfw could not block viral infection. therefore, it is possible that dgfw binds to the virus to affect its activity. we conclude that dgfw possesses antiviral activity and has the potential for development as an anti-ev agent. in an endeavor to identify anti-ev chinese medicines, we initially screened a panel of extracts of chinese herbs with antioxidant properties. an assay to measure the inhibition of virus-induced cell death in rd cells was first employed using mg/ml of herbal extracts followed by ec determination if the candidates possessed antiviral activity from the primary screening. we identified that the water fraction of d. genkwa was a potent agent against ev isolate with an ec of . ± . mg/ml and a concentration producing % cytotoxicity (cc ) of . ± . mg/ml using rd cells (table ) . however, its solvent (chcl ) layer did not demonstrate anti-ev activity ( figure ) . dgfw was also able to inhibit successfully other isolates of ev except for isolate (genotype b ; table ). interestingly, this extract was effective against influenza virus, although the selectivity index was not satisfactory. however, dgfw did not show activity against cvs or two dna viruses, adenovirus and hsv- , at a concentration of mg/ml (table ) . the preparation of dgfw is shown in figure and the final dried extract was weighed and dissolved in dmso for antiviral analysis. we explored the effects of dgfw in protecting against virus-induced cpe on rd cells by microscopic assay (figure ). the rd cells were infected with ev isolate in the presence or absence of . mg/ml, which was not toxic to the cells by mtt assay ( figure s ). rd cells infected with virus displayed a typical cpe in which the cells became rounded and eventually detached from the culture dish (panel d, figure ). this effect was not caused by dgfw alone (panel b, figure ) or . % vehicle dmso (panel c, figure ). dgfw but not dmso protected the cells from virus-induced cpe (panel e versus f, figure ), indicating that dgfw inhibited viral replication. we therefore performed qpcr and western blotting to confirm the antiviral effect of dgfw ( figure ). the cells were first infected with virus in the presence or absence of dgfw and the cells were harvested at indicated points ( figure a ). the amount of viral rna was quantified against a standard curve of known concentrations. the synthesis of viral protein was measured as viral proteins a and c. dgfw substantially reduced the synthesis of viral rna and viral proteins ( figure b ,c). we assessed the antiviral mode of action using a time-of-addition assay in which dgfw was added at different stages of one infection cycle ( figure ). dgfw was either added before viral preadsorption, during adsorption (- to h p.i.), or after viral adsorption. the viruses from cells and culture medium were then collected for plaque assay at h p.i. ( figure b ). dgfw was able to reduce viral titer considerably during the viral preadsorption step between − and h p.i. (lane , figure ). dgfw did not target the cells when it was added before viral preadsorption (lanes - , figure b ). the late stages, such as viral package and release, were not inhibited by dgfw because no reduction of viral titer was seen when the extract was added immediately after viral adsorption (lanes - , figure b ). altogether, these results demonstrate that dgfw might target cellular uptake of the virus. dgfw ( . mg/ml) was added at the designated times. (b) viral adsorption was from − to h p.i. on ice. dgfw ( . mg/ml in e ) was added to the cells at the indicated times. after each treatment, dgfw was washed and replaced with fresh e . rd cells were pretreated with dgfw from − to − , − to − , or − to − h p.i. the cells were washed and inoculated with ev for h, and the unbound virus was removed and replaced with e and incubated for an additional h. the supernatants were collected and the viral yields determined by plaque assay; "virus" is a virus-only control without dgfw treatment. the ratio of viral titers is presented as the mean ± sem of the results of two experiments, and this is normalized to the virus-only control, set to . this is one representative result from two independent experiments. to test the mechanism of inhibition of viral entry into host cells by dgfw, we performed attachment and penetration assays. viral uptake into cells occurs first by binding to receptors followed by clathrin-dependent endocytosis into early endosomes [ ] . the attachment assay was used to examine the binding of the virus to receptors, and the penetration assay was used to test the entry step following receptor binding. we used hff cells for these assays because rd cells were easily detached from the dishes when incubated on ice, and hff cells are susceptible to ev infection [ ] . for the attachment assay, the hff cells were incubated with tcid of ev for h in the presence of dgfw on ice. the cytoprotection of dgfw was then determined. dgfw was able to inhibit attachment of ev into hff cells with an ec of . ± . mg/ml ( figure a ). for the penetration assay, dgfw was added during viral entry into cells at °c following viral preadsorption. dgfw was also able to inhibit penetration with an ic of . ± . mg/ml ( figure b ). dgfw might bind to and thus inhibit the virus, because dgfw displayed a similar ic against ev based on attachment and penetration assays. we next examined whether dgfw acted at the cell surface, such as via receptor-virus binding, by pretreatment of rd cells with dgfw for min at °c (lane , figure ). unbound dgfw was removed, the cells were then infected with ev for h, and the progeny viruses in the cells and culture medium were harvested for plaque assay at h p.i. similarly, ev was pretreated with dgfw or dmso for min at °c, and the infectivity of the treated virus was then determined by plaque assay (figure ). pretreatment of cells with dgfw did not show any reduction of viral titer, suggesting that dgfw did not affect viral binding to the cells directly. in contrast, pretreatment of the virus with dgfw caused a substantial reduction of viral titer, but this was not seen after treatment with dmso alone, implying that dgfw might target the virus directly and specifically ( figure ; lane versus lane ). this result was consistent with the time-of-addition assay (figure ). this direct targeting effect was further demonstrated by a lower concentration of dgfw. this was done by pretreating the virus with dgfw for min followed by diluting the mixture -fold, whereby the dgfw did not have inhibitory activity. the infectivity of the treated virus was then determined by plaque assay (figure b ). pretreatment with dgfw produced a significant reduction of viral titer by about % compared with the dmso control, suggesting again that dgfw suppresses viral activity by targeting the virus directly ( figure b ). to h p.i.) . similarly, rd cells were pretreated with dgfw for min before viral adsorption for h (- to h p.i.). at h p.i., the cells and culture medium were pooled for the determination of viral titers by plaque assays. "all" indicates the presence of dgfw all through from − . to h p.i.; "virus" is a nontreated virus control, and the titer was set to . the fold change of viral titers is presented as the mean ± sem of the results of two independent experiments. ** p < . ; (b) ev was preincubated with dgfw ( mg/ml) or dmso for min at °c. the mixture was diluted -fold for the plaque assay. the viral titer from pretreatment with dmso was set to , and the fold change of viral titers is presented as the mean ± sem of the results of two independent experiments. ** p < . . in this study, we demonstrated that dgfw suppressed viral adsorption to rd cells by time-of-addition assay. cells pretreated with dgfw did not show inhibition of viral infection, indicating that dgfw does not inhibit cellular viral receptors. however, dgfw was able to inhibit viral endocytosis by reducing viral attachment to and penetration into host cells. furthermore, pretreatment of virus with dgfw abolished viral replication, indicating that dgfw inhibits ev by targeting the virus directly ( figure ) . currently, there is no effective vaccine or antiviral agent to combat ev . many antienteroviral agents against poliovirus or rhinovirus have been thus tested for their efficacy against ev . pleconaril, a win compound and in clinical development, has been reported to inhibit several types of enterovirus-induced meningitis and aseptic meningitis [ ] . the win series of compounds inhibit viral replication primarily by binding the hydrophobic canyon region of capsid protein vp to stabilize the viral capsid structure, so that after the virus enters the host cell it cannot undergo the uncoating process [ ] . however, pleconaril did not effectively inhibit the cpe induced in cell cultures by the ev strain isolated from patients who had contracted hand, foot and mouth disease in [ ] . we demonstrated here that dgfw might target the viral particle for inhibition. however, dgfw did not show strong binding activity to the virus in a filter assay, suggesting that dgfw acts through transient binding but leads on to potent inhibitory activity ( figure s ). phylogenetic analysis of complete vp sequences has identified three ev genotypes: a, b and c. genotypes b and c can be further divided into subgenotypes b to b and c to c [ ] . dgfw inhibited ev /brcr, ev /tw/ / and ev /tw/ / , which belong to subgenotypes a, c and c , respectively. however, the ec of dgfw against ev /tw/ / (subgenotype b ) was greater than mg/ml, but this dose was equivalent to the effective cytotoxic concentration and therefore cannot be considered as having an inhibitory effect. we demonstrated that dgfw targets the virus to inhibit the attachment and entry steps. however, dgfw did not inhibit the b subgenotype, and this can be explained by the demonstrated differences in vp sequences [ ] . the phylogenetic analysis showed distinct differences between cv-b and ev , and consequently dgfw did not inhibit cv-b to b as expected (table ) . additionally, as dgfw appears to inhibit different genotypes of ev to different degrees, this might explain why dgfw does not target cellular factor(s). flavonoids are common constituents of plants used in traditional chinese medicine to treat a variety of diseases. d. genkwa has been used for the isolation of flavonoids, and studies have reported the isolation of luteolin, -o-methylluteolin, tiliroside [ ] , apigenin, genkwanin, sitosterol, benzoic acid [ ] and genkwadaphnin [ , ] from the dried flower buds. apigenin showed a strong inhibitory activity against xanthine oxidase [ ] , as well as against severe acute respiratory syndrome coronavirus (sars-cov) and influenza virus [ ] [ ] [ ] . luteolin and its glycosides have been widely used for their antimicrobial properties, and hbv, sars-cov and the influenza virus can be inhibited as shown by in vitro studies [ ] [ ] [ ] . however, no anti-ev activity of flavonoids isolated from d. genkwa has been demonstrated. interestingly, similar to our findings, apigenin was able to inhibit the influenza virus but did not show antiviral activity against hsv (table ) [ , , ] . the examination of anti-ev activities of apigenin and other purified compounds from d. genkwa is ongoing. on a cautionary note, daphne bud extracts, whether from the ether layer or the water layer, can also induce early antigen expression of epstein-barr virus (ebv) [ ] . up to % of the world's human population has a latent ebv infection, and it has a high association with nasopharyngeal carcinomas in southeast asia and certain other countries [ ] . in addition, d. genkwa possesses the harmful side effect of abortifacient activity. thus, there is a need to isolate the effective anti-ev ingredients that do not lead to the induction of ebv early antigen production or act as abortifacients. (table ) [ ] . influenza virus a/wsn/ was obtained from the american type cell culture and was amplified accordingly [ ] . overnight cultures of monolayers of rd cells were washed with dulbecco's phosphate-buffered saline (dpbs) before being inoculated with ev or cv-b at a multiplicity of infection (m.o.i.) of . . the cells and culture supernatants containing viral progeny were collected when cells developed a cpe of ~ %. the progeny viruses inside the cells were released by three cycles of freezing-thawing, and the resulting viral suspension was pooled with the culture medium, aliquoted and stored at − °c until use. dry flower buds of daphne genkwa were supplied and authenticated by the department of pharmacy, chang gung memorial hospital at chiayi, taiwan. a voucher specimen (no. cgu-dg- ) was deposited in the herbarium of chang gung university, taoyuan, taiwan. dry flower buds ( . kg) were extracted with methanol seven times ( l each) under reflux for h and concentrated to give a brown syrup ( . g) (figure ). the syrup was suspended in h o and partitioned with chcl and n-butanol, successively. the water-soluble layer was concentrated to gain a red-brown syrup ( . g). monolayers of rd cells (  per well) were cultured in -well plates overnight before use. ev tw/ / was used hereafter if not mentioned otherwise. a virus suspension with threefold serial dilution in dmem containing % fbs (e ) was added to the cells and incubated at °c for h and subsequently fixed with l of % paraformaldehyde (pfa) for h. the attached cells were stained with . % crystal violet at room temperature, and the density of staining was measured in a microplate reader (dynex technologies, chantilly, va, usa). the dilution causing cytopathology in half the cultures (the median tissue culture infective dose, or tcid ) was then calculated using the reed-muench method. the tcid determinations for the attachment and penetration assays were performed similarly except that preadsorption was carried out for h on ice. a neutralization assay (ec ) was used to test the antiviral efficacy of extracts or compounds by measuring the inhibition of cpe induced by enterovirus on rd cells. rd cells (  cells/well) were seeded into -well plates and incubated for - h at °c in a % co /air incubator. after infection, cells were overlaid with l of different concentrations of extracts and incubated at °c in a % co /air incubator for h. at the end of incubation, cells were fixed with pfa, followed by . % crystal violet staining. the concentration required for extracts to reduce the virus-induced cpe by % relative to the virus control was defined as the ec . aliquots of  rd cells per well were seeded overnight in -well plates before use. twofold serial dilutions of dgfw in e were added and incubated at °c for h. the medium was carefully withdrawn without touching the cells. mtt ( -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide; . mg/ml, sigma-aldrich, st louis, mo, usa) was added at l/well and incubated for h. dimethylsulfoxide (dmso; l/well) was added to dissolve the crystals, and the absorbance at nm of each well was measured using a microtiter plate reader (mrx elisa reader, dynex technologies). the concentration of dgfw that caused the death of % of the cells was defined as the % cytotoxic concentration (cc ). monolayers of rd cells ( .  per well) were seeded in -well plates. after an h culture, the cells were inoculated with serially diluted viral suspensions for h. the cells were washed with dpbs and overlaid with warm . % agarose in e . after incubation for days, the cells were fixed with % pfa for h. the agarose was carefully removed and subsequently stained with . % crystal violet. the titer of the virus was expressed as plaque-forming units (pfu) per ml. after electrophoresis, proteins were electrotransferred onto polyvinylidene fluoride membranes (millipore, billerica, ma, usa). the proteins of interest were incubated with primary antibodies for h and then incubated with appropriate secondary antibodies. proteins were detected by using an enhanced chemiluminescence western blotting detection system (millipore). polyclonal antibodies against a and a monoclonal antibody against c were as described [ , ] . rd cells (  cells/ml) were seeded into -well plates and then challenged with viruses at an m.o.i. of . the cells were pretreated with viruses for h on ice and washed with dpbs, and then e was added. after h incubation at °c, the cells were stained with . % sulforhodamine b (srb) in % acetic acid, and cpe was observed using a microscope. for dgfw treatment, the cells were pretreated with . mg/ml extracts and virus simultaneously for h and washed with dpbs, and then e containing . mg/ml dgfw was added. the protective effect exerted by dgfw was also observed after incubation for h at °c. the in vitro transcripts of the ev / infectious clone plasmid, which was kindly provided by dr. meishan ho of academia sinica, taiwan, was serially diluted tenfold for spectrophotometric analysis. after determining the rna concentration, the copy number of standard rna molecules can be calculated using the following formula: (x g/l rna /[transcript length in nucleotides  ])  .  = y molecules/l. the viral rna was then mixed with nm forward and reverse primers and sybr green pcr master mix (applied biosystems, foster city, ca, usa) followed by qpcr analysis using an abi stepone plus sequence detector system (applied biosystems). the primers used were: ev vp forward, -ctg atg gct tcg aat tgc aa- , and reverse, -gcg ttt atg tac ggc act att att gt- ; gapdh forward, -ggt ggt ctc ctc tga ctt caa ca- , and reverse, -gcg tca aag gtg gag gag tg- . a standard curve (plot of the ct value/crossing point against the log of the amount of standard added) was thus generated using different dilutions of the standard. rd cells (  cells/well) were seeded in -well plates overnight. the cells were then preadsorbed with ev at an m.o.i. of for h in the presence of . mg/ml dgfw. the culture medium was then replaced with e containing the same concentration of dgfw, and the cells were incubated at °c and harvested at , , and h post infection (p.i.) for total rna extraction using trizol. cdna was prepared by reverse transcription with m-mlv rt kits (invitrogen, carlsbad, ca, usa) for qpcr analysis using specific viral primers: forward, -ctg atg gct tcg aat tgc aa- , and reverse, -gcg ttt atg tac ggc act att att gt- . amplification was achieved using the following universal thermal cycling protocol: one cycle at °c for min, cycles at °c for s and °c for min. the absolute amounts of viral rna were then determined using an external standard curve. a -well tissue culture plate was seeded with rd cells (  cells/well), and incubated at °c for - h under % co in air. the cells were infected with ev at an m.o.i. of on ice. after viral adsorption, the cells were washed with hank's buffered salt solution to remove any unbound virus. dgfw ( . mg/ml) in e was added at − to − h, − to − h and − to − h (preadsorption), − to h (adsorption), to h, to h and to h (postadsorption). the supernatants were harvested at h p.i., and the viral titers were determined by plaque-forming assays. a -well tissue culture plate was seeded with hff cells (  cells/well), which were then incubated overnight at °c under % co in air. the cells were chilled on ice for h, and the medium was removed. the cells were infected with tcid of ev in the presence of increasing concentrations of dgfw on ice for h. the medium containing the unadsorbed virus was removed, and the cells were then washed three times with dpbs. after incubation for h in e at °c, cells were fixed with % pfa and stained with crystal violet. cell viability was determined by measuring the cell density in a microplate reader (mrx elisa reader, dynex technologies). a pueraria lobata extract at . mg/ml served as a positive control [ ] . this attachment method has been described [ ] . a -well tissue culture plate was seeded with hff cells (  cells/well), which were then incubated overnight at °c under % co . the cells were chilled on ice for h, and the medium was removed. the cells were infected with tcid of ev on ice for h. the medium containing unbound virus was then removed; various concentrations of dgfw in e were added, and the cells were incubated at °c for h to trigger endocytosis of the virus. the infected cells were then treated with alkaline phosphate-buffered saline (pbs; ph ) for min to inactivate any viruses that had not penetrated the cells, and then acidic pbs (ph ) was immediately added to neutralize the mix. the neutralized medium was removed, e was then replaced, and the cells were incubated at °c for h. the cells were fixed with pfa and subjected to . % crystal violet staining. a pueraria lobata extract at . mg/ml served as a positive control [ ] . this penetration assay was modified from previous reports [ , ] . data are expressed as the mean ± standard error of the mean [ ] and analyzed using two-tailed student's t-tests with p < . taken as significant. gfw exerts its anti-ev effects by inhibiting viral entry without producing cytotoxic side effects and thus provides a potential agent for antiviral chemotherapeutics. viral and host proteins involved in picornavirus life cycle the viruses and their replication neurodevelopment and cognition in children after enterovirus infection the enterovirus outbreak in taiwan: pathogenesis and management human p-selectin glycoprotein ligand- is a functional receptor for enterovirus scavenger receptor b is a cellular receptor for enterovirus sialylated glycans as receptor and inhibitor of enterovirus infection to dld- intestinal cells antiviral effects of salvia miltiorrhiza (danshen) against enterovirus anti-enterovirus activity screening of chinese herbs with anti-infection and inflammation activities some progress on the chemistry of natural bioactive terpenoids from chinese medicinal plants anti-inflammatory activity of (−)-aptosimon isolated from daphne genkwa in raw . cells daphnane diterpene esters isolated from flower buds of daphne genkwa induce apoptosis in human myelocytic hl- cells and suppress tumor growth in lewis lung carcinoma (llc)-inoculated mouse model the essential role of clathrin-mediated endocytosis in the infectious entry of human enterovirus glucose- -phosphate dehydrogenase deficiency enhances enterovirus infection discovery and development of antipicornaviral agents a novel basis of capsid stabilization by antiviral compounds design, synthesis, and structure-activity relationship of pyridyl imidazolidinones: a novel class of potent and selective human enterovirus inhibitors molecular epidemiology and evolution of enterovirus strains isolated from to reemergence of enterovirus in in taiwan: dynamics of genetic and antigenic evolution from to inhibitors of adenosine ′, ′-cyclic monophosphate phosphodiesterase in daphne genkwa sieb. et zucc inhibitors of xanthine oxidase from the flowers and buds of daphne genkwa genkwadaphnin, a potent antileukemic diterpene from daphne genkwa antiviral flavonoids from mosla scabra anti-influenza virus activities of flavonoids from the medicinal plant elsholtzia rugulosa biflavonoids from torreya nucifera displaying sars-cov cl(pro) inhibition anti-hbv active flavone glucosides from euphorbia humifusa willd orhan, i. cytotoxicity, antiviral and antimicrobial activities of alkaloids, flavonoids, and phenolic acids screening of epstein-barr virus early antigen expression inducers from chinese medicinal herbs and plants epstein-barr virus infection in the pathogenesis of nasopharyngeal carcinoma identification of bpr p as an inhibitor of cap-snatching activities of influenza virus reticulon binds the c protein of enterovirus and is required for viral replication enterovirus c protease cleaves a novel target cstf- and inhibits cellular polyadenylation a water extract of pueraria lobata inhibited cytotoxicity of enterovirus in a human foreskin fibroblast cell line inactivation of hsv- and hsv- and prevention of cell-to-cell virus spread by santolina insularis essential oil sch and analogs: in vitro activity and in vivo efficacy of novel agents for herpesvirus type inhibition of herpes simplex virus type penetration by cytochalasins b and d regulation of picornavirus gene expression this article is an open access article distributed under the terms and conditions of the creative commons attribution license this study was supported by chang gung memorial hospital (grant numbers cmrpd and cmrpg ). we thank hung-yao ho for the hff cells. the authors declare no conflict of interest. key: cord- - gbktpt authors: van brussel, kate; carrai, maura; lin, carrie; kelman, mark; setyo, laura; aberdein, danielle; brailey, juliana; lawler, michelle; maher, simone; plaganyi, ildiko; lewis, emily; hawkswell, adele; allison, andrew b.; meers, joanne; martella, vito; beatty, julia a.; holmes, edward c.; decaro, nicola; barrs, vanessa r. title: distinct lineages of feline parvovirus associated with epizootic outbreaks in australia, new zealand and the united arab emirates date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gbktpt feline panleukopenia (fpl), a frequently fatal disease of cats, is caused by feline parvovirus (fpv) or canine parvovirus (cpv). we investigated simultaneous outbreaks of fpl between and in australia, new zealand and the united arab emirates (uae) where fpl outbreaks had not been reported for several decades. case data from cats and clinical samples from additional cats were obtained to determine the cause of the outbreaks and epidemiological factors involved. most cats with fpl were shelter-housed, to weeks old at diagnosis, unvaccinated, had not completed a primary vaccination series or had received vaccinations noncompliant with current guidelines. analysis of parvoviral vp sequence data confirmed that all fpl cases were caused by fpv and not cpv. phylogenetic analysis revealed that each of these outbreaks was caused by a distinct fpv, with two virus lineages present in eastern australia and virus movement between different geographical locations. viruses from the uae outbreak formed a lineage of unknown origin. fpv vaccine virus was detected in the new zealand cases, highlighting the difficulty of distinguishing the co-incidental shedding of vaccine virus in vaccinated cats. inadequate vaccination coverage in shelter-housed cats was a common factor in all outbreaks, likely precipitating the multiple re-emergence of infection events. feline panleukopenia (fpl) is a highly contagious and often fatal disease characterised by acute severe enteritis, severe dehydration and sepsis due to lymphoid depletion and pancytopenia [ ] . fpl is usually associated with infection by feline parvovirus (fpv), a member of the genus protoparvovirus (formerly feline panleukopenia virus). protoparovirus is one of eight genera of vertebrate viruses within the subfamily parvovirinae of the family parvoviridae. collectively, fpv and canine parvovirus (cpv), along with associated variants found in various carnivore species such as mink and raccoons, constitute the species carnivore protoparvovirus [ ] . until the s, fpv was the only reported viral cause of fpl in cats. fpv is able to infect cats by first binding to the feline transferrin receptor (ftfr) expressed on the surface of cells, followed by clathrin-mediated endocytosis to initiate infection [ ] . canine parvovirus cpv- emerged in the late s and was initially unable to infect cats, as it could not bind to the ftfr [ ] . however, infectivity for feline cells was acquired soon after by the genetic variant cpv- a, which emerged in and replaced cpv- [ ] . the ability to infect cats has also been retained by subsequent antigenic variants of cpv- a, termed cpv- b and cpv- c, which only differ from cpv- a at a single amino acid position (vp ). these and other antigenic variants of cpv can cause fpl in both naturally acquired and experimental infections of cats [ ] [ ] [ ] [ ] . in contrast to parvoviral enteritis in dogs, estimated to cause , cases per year in australia, clinical cases of fpl have rarely been diagnosed in australia since the mid- s, and there have been no reports of fpl outbreaks for over years [ ] . in , fpl re-emerged in eastern australia, and subsequent outbreaks occurred between and in several locations in this region. similarly, outbreaks of fpl occurred in new zealand (nz) between and , as well as in the united arab emirates (uae) in , with no outbreaks of fpl reported in either country in recent decades, likely due to the widespread use of fpl vaccines. the contemporaneous re-emergence of fpl in geographically distinct settings long considered fpl-free has raised questions as to whether virus-related or other unknown risk factors played a role in the observed fpl outbreaks. herein, case data and clinical samples from and cats, respectively, were analysed to identify the lineages of carnivore protoparvovirus responsible for the outbreaks of fpl in australia ( to ), the uae ( ) and nz ( - ) and evaluate epidemiological factors associated with these outbreaks, including vaccination status. inclusion criteria for cases of fpl were (i) clinical signs typical of fpl (lethargy, fever, hypothermia, anorexia, vomiting, diarrhoea and/or sudden death) and a positive confirmatory test (faecal cpv antigen test or pcr) or (ii) clinical signs typical of fpl in a cat from a shelter with a confirmed contemporaneous outbreak of fpl. australian case data were extracted from a national online companion animal disease surveillance-reporting database launched in january [ ] and from the medical records of animal shelters and/or veterinary hospitals in outbreak regions for the period january to august . data recorded included case occurrence date, shelter location, shelter post code, age at diagnosis, sex, post code of owner residence or where found as a stray before entry into shelter, clinical signs at presentation, date of last vaccination, vaccination type (inactivated or modified live virus (mlv) vaccine), time interval between last vaccination and onset of clinical signs (days), method of diagnosis and outcome. data obtained were searched to identify cases for which serial monitoring of faecal shedding of fpv or cpv using qpcr testing had been performed. information about animal movements, biosecurity and vaccination protocols was obtained from shelter veterinarians and/or managers. new zealand case data were obtained from one shelter, comprising summary data of case diagnoses for two fpl outbreaks occurring between and as well as individual data for cases in which clinical samples were available for pcr and sequencing. individual case data for uae cases were available during the period of the outbreak in . data were analysed using microsoft excel ® for mac version . . and statistix ® version . (analytical software, tallahassee, fl, usa). descriptive statistics were generated for fpl case numbers, age at disease diagnosis, interval between vaccination and disease and interval between admission and disease. frequency distributions were created for age at disease diagnosis. mapping and geospatial analysis was performed with arcgis ® version . (ersi, redlands, ca, usa). residual diagnostic faecal samples or tissue (intestine or mesenteric lymph node) obtained post-mortem were collected prospectively in australia from april until august and from the uae and new zealand during suspected outbreaks of fpl in . in addition, a stored faecal sample from a cat diagnosed with fpl in the first outbreak in australia in was obtained for study. dna was extracted for sequencing of the carnivore protoparvovirus vp gene from tissue using the qiagen dneasy blood and tissue kit (qiagen, hilden, germany) or from faecal samples using the qiaamp fast dna stool mini kit (qiagen, germany) or by homogenisation, boiling and centrifugation, as previously described [ ] . the extracted dna was amplified by conventional pcr using u/µl of my taq hs red dna polymerase (bioline, usa), - ng of dna, x mytaq red reaction buffer and a final primer concentration of . µm in a final reaction volume of µl. three sets of overlapping primers were used to amplify the entire vp region ( bp) as described previously, with minor modifications (table ) [ ] . an initial denaturation step at • c for min, followed by - cycles of denaturation at • c for s, annealing at or • c for s and extension at • c for s, ending with a final extension at • c for min, was used for dna amplification. the pcr products were separated by electrophoresis on a % agarose gel (bio-rad laboratories, hercules, ca, usa) in × tris-acetate edta and visualized using sybr safe dna (invitrogen, carlsbad, ca, usa). sanger sequencing of positive pcr products was performed commercially (macrogen, seoul, korea). vp sequences were edited, assembled and aligned using the clustalw algorithm in geneious prime (biomatters ltd., auckland, new zealand) (although alignment was uncontroversial with no gaps needed). sequences were translated in megax, and amino acid substitutions compared to the ictv reference fpv sequence (fvp- genbank accession no. eu ) were identified. to determine the evolutionary history of the outbreak sequences from australia, new zealand and dubai, we performed a phylogenetic analysis including these sequences and representative vp sequences of fpv and cpv taken from genbank. in addition, we included an fpv vp sequence obtained from tissues of a healthy feral cat from tasmania, australia, sampled in (genbank accession no. mn ), for comparison. this resulted in a total data set of sequences, nt in length. phylogenetic analysis of these was performed using the phyml program [ ] and employing the gtr+i+Γ model of nucleotide substitution and a combination of nearest-neighbor interchange (nni) and sub-tree pruning & re-grafting (spr) branch swapping. nodal support was assessed using sh-like branch support. finally, the five cpv sequences included were used as an outgroup clade to root the tree. an fpv strain identical to that used in a commercially available mlv fpv vaccine was detected in faecal samples from new zealand cases. subsequently, qpcr was performed to determine fpv viral loads in dna extracts of mesenteric lymph nodes and tissues from these cases using a taqman qpcr assay as described previously [ ] . faecal samples containing vaccine virus were also submitted to a commercial laboratory for batch testing on a multiplex qpcr panel to detect potential faecal co-pathogens including feline coronavirus, tritrichomonas foetus, clostridium perfringens, giardia spp, salmonella spp, campylobacter jejuni, campylobacter coli and toxoplasma gondii (idexx pty ltd.). tissues (duodenum, jejunum, colon, liver, spleen, kidney, mesenteric lymph node, heart, lung, pancreas, brain and/or bone marrow) from representative australian fpl cases that had died or been euthanised, ranging in age from weeks to months, including one from melbourne in and from sydney in and , were available for histological examination. tissues (intestine, +/− mesenteric lymph node) from six suspected cases of fpl from new zealand were also examined. all tissues were collected post-mortem and animals were not euthanised for the purposes of this study. data were received from cases of fpl diagnosed in australia between january and september from animal shelters, rescue societies and veterinary hospitals. of the cases, % were shelter-housed, % were privately owned cats presenting to veterinary hospitals, and % were from foster carers working with rescue societies without premises. the distribution of australian cases by geographic region and year is shown in table and figure . figure s ). the distribution of cases varied with month of presentation and is shown in figure . frequent transfer of cats in victoria between shelters in mildura (outbreak site) and melbourne (outbreak site) suggested direct spread of the infection between these regions. cats from two private shelters in mildura were transported for rehoming to a shelter in melbourne in and . two of five shelters in nsw did not vaccinate cats while under their care. the other three shelters vaccinated healthy cats on admission using an inactivated fpv vaccine, whereas sick cats with suspected viral respiratory tract infections were not vaccinated until their clinical signs resolved. none of the three shelters in mildura vaccinated cats. of the three shelters in melbourne, one did not vaccinate cats, while two vaccinated cats on admission, one using an inactivated vaccine and the other an mlv vaccine. all shelters that used vaccinations gave a primary course to kittens starting at a minimum age of weeks, then every to weeks, finishing at to weeks of age, while adult cats were given two vaccinations one month apart. both rescue societies, one in nsw and one in victoria, accepted unvaccinated cats and kittens for adoption from municipal pounds (shelters), that were fostered until adoption. for shelter-housed cats, the median time from admission to diagnosis of fpl was days (iqr - ; range - days). of the cats diagnosed with fpl, ( %) had never been vaccinated or had an unknown vaccination history. sixty-five of the ( %) cats that had received at least one vaccination were < weeks of age at the time of their last vaccination. for cats ≥ weeks of age at the time of last vaccination, the vaccination-to-disease interval was > days for / cats vaccinated with ≥ inactivated vaccines and > days for / cats vaccinated with at least one mlv vaccine; vaccine type was unknown for one cat. for all vaccinated cats, the median time from last vaccination to diagnosis was . days (iqr - ; range - days). age, sex and breed data were available for fpl cases. the median age at diagnosis was weeks (iqr - weeks; range weeks- months). the frequency distribution of age at diagnosis is shown in table males ( %) and females ( %) were equally represented among fpl cases. most cats ( %) were domestic crossbred (domestic shorthair or domestic longhair). data on specific clinical signs were available for cats. the most common clinical signs were diarrhoea ( %), lethargy ( %), vomiting ( %), anorexia ( %), weight loss or failure to gain weight ( %) and sudden death ( %). diarrhoea was reported to be haemorrhagic in of cats ( %) with diarrhoea. overall mortality for all cats was %, noting that cases diagnosed in shelters were routinely euthanised at diagnosis to contain the infection. two fpl outbreaks occurred in wellington, new zealand, in december and january . the seasonal distribution of cases in the second outbreak is shown in figure . in total, cases of fpl were diagnosed, including in the first outbreak and in the second. during both outbreaks, % of feline admissions to the shelter were diagnosed with fpl. the median age at diagnosis was weeks (iqr - weeks, range - weeks). at diagnosis of fpl, % of cats were unvaccinated, % had received one vaccine, % had been vaccinated twice, and % had been vaccinated three times. the median time since the last vaccination was days (iqr - . days, range - days). the median time from shelter admission to diagnosis was days (iqr - days, range - days). of the nine cases for which clinical samples were available for pcr and sequencing, all cats had a positive faecal cpv antigen test result (fastest parvo strip, megacor hoerbranz, austria) and were euthanised at diagnosis. all cats had been vaccinated on admission using an mlv fpv vaccine (felocell or felocell , zoetis pty. ltd.). clinical data are presented in table . fourteen cases of fpl from dubai, uae, were diagnosed in domestic crossbreds with positive faecal cpv antigen test results. all cats were strays or strays that had been recently adopted, and all were unvaccinated, except an -month-old cat that had received a primary course of kitten vaccinations and a booster vaccination at months. clinical signs included vomiting, diarrhoea and lethargy, and the median age at diagnosis was months (range - months). all cats were treated, and outcomes were known in cases, of which died or were euthanised, and survived. in sections of duodenum and jejunum from the representative cases, there was a mild to severe necrotizing lymphoplasmacytic-to-suppurative enteritis, characterized by variable blunting, fusion and loss of villi, collapse of the lamina propria, loss of crypts and replacement by abundant coccobacilli and necrotic debris. the remaining crypts were often ectatic, with variable numbers of crypt abscesses ( figure ). evidence of multifocal crypt regeneration was also variably present. the submucosa and tunica muscularis were variably expanded by congestion, fibrin, oedema, haemorrhage, perivascular lymphocytes and plasma cells, macrophages exhibiting erythrophagocytosis, occasional neutrophils and rare eosinophils. in sections of mesenteric lymph nodes, the most common finding was multifocal follicular and paracortical lymphoid depletion with accentuation of the reticuloendothelial architecture ( figure ). depleted lymphoid follicles were small, poorly populated and contained central accumulations of hyalinized eosinophilic material (follicular hyalinosis) with variable numbers of tangible body macrophages. the subcapsular and medullary sinuses were expanded by erythrocytes, oedema fluid and moderate to markedly increased numbers of macrophages, some exhibiting erythrophagocytosis. mild-to-moderate multifocal lymphoid depletion was also apparent in splenic sections. extramedullary haematopoiesis characterized by the presence of myeloid and erythroid precursors was variably present within spleen and lymph nodes. sections of bone marrow examined were either hypocellular, characterised by increased numbers of, predominantly, adipocytes and extravasated erythrocytes interspersed with very low numbers of erythroid and myeloid precursors and megakaryocytes, or hypercellular, with increased numbers of myeloid and erythroid precursors. some sections of the liver demonstrated hepatocellular atrophy and congestion, with minimal periportal mononuclear infiltrates. among the sections of myocardium examined, there were no significant lesions. histopathology of the small intestine was performed in six cases, including five in which samples were collected for pcr and sequencing (table ). in four of these, lesions of mild enteritis (n = ) or fibrosis of the lamina propria (n = ) were present. in one case ( , table ), histological lesions were typical of fpl ( figure ) , with multiple sections of jejunum and ileum showing mild multifocal loss and replacement of crypts by karyorrhectic and cellular debris (necrosis) and low-to-moderate numbers of neutrophils within the lamina propria. low numbers of the remaining crypts were variably lined by degenerate or necrotic epithelium with intraluminal degenerate neutrophils, debris and sloughed necrotic epithelial cells (crypt abscesses). neutrophils frequently invaded the remaining crypt epithelium. rare epithelial cells contained - µm-diameter polygonal-to-ovoid, eosinophilic-to-amphophilic intranuclear inclusion bodies that occasionally marginated the chromatin. there was moderate multifocal crypt regeneration, and crypts were frequently lined by large epithelial cells with vesicular nuclei that were piled up or in mitosis. intestinal lymphoid tissue appeared mildly depleted. the morphological diagnosis was moderate diffuse acute neutrophilic enteritis with crypt necrosis, epithelial regeneration and rare intranuclear viral inclusions. these findings were consistent with fpv/cpv infection. (table ). of the cases where sections of mesenteric lymph nodes (n = ) or thymus (n = ) were available, lesions of mild follicular lymphoid depletion were present in one case, which was the same animal with small intestinal lesions consistent with fpv (case ). affected cortical follicles were small and poorly populated. histopathology was also performed on intestine and mesenteric lymph nodes from one unvaccinated cat with clinical signs of fpl, but for which samples were not collected for pcr and sequencing. changes in the duodenum were similar to but more severe than those described above in case , including crypt necrosis and abscessation, neutrophilic infiltration of the lamina propria and the presence within epithelial cells of intranuclear inclusion bodies which variably marginated the chromatin. there was moderate lymphoid depletion in mesenteric lymph node tissue. these findings were also consistent with fpv/cpv infection. the entire vp region of carnivore protoparvovirus was amplified from suspected cases of fpl, comprising from australia, from dubai and from new zealand. amino acid substitutions compared to the fpv reference strain are listed in table . all vp sequences in this study were identified as fpv, with no cases attributed to cpv. phylogenetic analysis of these vp sequences in comparison to a background global data set of fpv sequences revealed the presence of four distinct clades of australian viruses ( figure ). the first, denoted clade a, comprises viruses ( of which were identical) sampled from a wide geographic area (melbourne, geelong and mildura) between and . in contrast, australia clade b comprised just two identical sequences sampled in nsw in . these viruses were detected in the faeces collected from two cats in the same shelter in sydney, nsw, in november . both cats had severe diarrhoea and tested positive for salmonella spp. and carnivore protoparvovirus on a multiplex faecal qpcr panel (idexx pty ltd.). the largest australian clade (c) comprised viruses from nsw and one from queensland, collected between december and february . viruses collected from cats in north western nsw (gunnedah) and south eastern queensland (nambour) in were identical to those sampled from cats from four shelters and from privately owned cats in sydney. viruses from cats from the nsw central coast also fell into this clade. finally, one sequence isolated from a cat from melbourne in (fpv_ ) was identical to viruses in clade c, with the exception of a mutation at position (a g) that resulted in an amino acid substitution i to v. this mutation was also present in viruses in clade a. this study - a fourth (although not strictly monophyletic) clade (d) of viral sequences from australian cats contained three vp sequences identical to that of an fpv vaccine strain (purevax; genbank accession no. eu ) and two sequences that are very closely related to the vaccine strain. in one of these, from a -week-old kitten with acute vomiting and failure to gain weight, the vaccine strain was unlikely to be the cause of the cat's clinical signs. fpl was suspected because a multiplex qpcr faecal panel was positive for giardia spp. and canine protoparvovirus . however, the cat, from canberra in the australian capital territory, had been vaccinated days before with the same mlv vaccine strain. no littermates were affected, the cat recovered, and no other cases of suspected fpl were encountered in this region. another sequence in this clade was from a -week-old stray kitten with vomiting, diarrhoea and failure to gain weight. the kitten, from a shelter in melbourne, had been vaccinated days before with the same mlv vaccine. it also tested positive on a multiplex qpcr faecal panel for giardia spp. and canine protoparvovirus . the third sequence was from an owned -week-old kitten in sydney with acute onset vomiting and diarrhoea, vaccinated days before (vaccine type unknown). of the two closely related vp sequences, one (fpv_ ) was from a -week-old kitten vaccinated days before with the same vaccine strain, which presented for diarrhoea, vomiting and lethargy. it also tested positive for giardia spp. on a faecal antigen test. the other (fpv_ ) was from an unvaccinated stray -month-old cat that presented with vomiting, diarrhoea and anorexia and tested positive on a faecal cpv antigen test. finally, two other australian sequences-fpv / and tasmania/ -did not fall into a distinct clade but were relatively closely related to each other and to the vaccine cluster (clade d). all vp sequences from cats from dubai fell into a single clade containing four unique sequence types and which was not clearly related to any other of the fpv sequences. in contrast, those viruses sampled from new zealand in were either identical (eight of nine animals) or clearly very closely related to a vaccine strain (felocell, zoetis pty ltd.) and taken from cats with a history of recent vaccination. the single sequence that was not identical to that of the vaccine strain exhibited just two nucleotide substitutions, one of which was non-synonymous (e k) ( table ). the results of the qpcr analyses to quantify fpv viral load in faeces of nz cats and to detect faecal co-pathogens are presented in table . medical records were obtained from two unvaccinated kittens with fpl from australia, where serial monitoring of fpv faecal shedding using qpcr was performed on pooled faecal samples after diagnosis. testing was done at a single commercial laboratory, and results were reported as positive or negative (table ). table . results of serial monitoring of faecal shedding of fpv in faecal samples from two cats diagnosed with fpl. an understanding of the drivers of the re-emergence of fpl, the oldest known viral disease of cats, is essential to contain this fatal infection. we provide strong evidence of multiple outbreaks of fpl in three countries. there are no previous published reports of fpl outbreaks in any of these regions for comparison, although there is anecdotal first-hand experience of fpl among veterinarians practicing in australia from the late s to the mid- s, before commercial vaccines were used routinely [ ] . despite the widespread circulation of cpv in australia, this virus was not detected in any samples tested in these fpl outbreaks. canine parvovirus causes approximately % of fpl cases globally, but these have been confined to sporadic individual cases, and there are no reports of fpl outbreaks caused by cpv in multi-cat environments [ , , ] . whether this is due to viral or to host factors is currently unknown and warrants further investigation. a single fpv lineage (clade a) was responsible for disease in both outbreak regions in victoria, australia, even though these are geographically distant ( km) from each other. phylogenetic analysis revealed the closest related virus was taken from a domestic cat in japan in (genbank accession no. ab ), although it is likely that a closer ancestral virus exists but has not been sampled here. the % sequence identity of vp sequences from cases in mildura and melbourne is consistent with the epidemiological investigation, which found evidence supporting direct viral spread through transport of unvaccinated cats for adoption in and . the nonenveloped virions of fpv, shed in high titres in all excretions of infected animals, are environmentally resilient and remain viable in infected premises such as shelters for over a year [ , ] . indirect transmission by fomites is a major mechanism of parvovirus transmission among cats and dogs and was likely a crucial contributing factor to the canine global cpv pandemic in the late s [ ] . unexpectedly, the fpl outbreak in sydney, nsw, was not caused by spread of the victorian strain, but rather by a separate clade (c) comprising a relatively distinct virus [ , ] . this finding, together with the detection of additional australian fpvs (clades b and d), is consistent with multiple independent virus entries into australia, including the virus from an unvaccinated cat (clade d), which was seemingly derived from an fpv vaccine strain. three recently vaccinated cats, in which the same vaccine virus was detected (clade d), likely did not have fpl, including two with giardia co-infections initially diagnosed with fpl from a positive commercial faecal qpcr assay. mlv vaccine virus shed in faeces can be detected in qpcr assays and cpv faecal antigen tests [ , ] . quantifying viral loads can assist in the discrimination of vaccine and field strains. however, quantification data were not provided by the commercial laboratory for these two cases. cpv vaccine viral loads in recently vaccinated dogs are -to -fold lower than those of dogs infected with field strains [ ] . similarly, a recent report found several recently vaccinated cats had low fpv viral loads (< . × copies/mg of faeces) over a -day period of surveillance [ ] . before this investigation, only one fpv vp sequence from australia had been deposited in the genbank database (fpv / , genbank accession no. x ). the evolutionary relationships with fpv / cannot be inferred reliably, since the isolate, obtained from a -month-old cat in melbourne with peracute fatal fpl, had been serially passaged in vitro before sequencing in . interestingly, fpv / falls notably close to a clade of vaccine viruses in our analysis. in addition, we generated the vp sequence of an fpv strain detected in a healthy feral cat from tasmania in , which was genetically distinct from outbreak strains in this study and also fell into clade d, suggesting a vaccine-related origin. these findings, together with a report of fpv detection in the bone marrow of a healthy feral cat from melbourne in [ ] , provide evidence that fpv has been circulating widely in australia, at least since the s, although the viruses are phylogenetically unrelated to the - outbreaks. one possible explanation for the multiple independent fpv introductions described here is that fpv viruses circulating in the feral cat population may occasionally spill over into domestic cats. in comparison to the owned cat population, estimated at . million [ ] , some . to . million feral cats (free-roaming, unowned, unsocialised cats) inhabit australia [ ] . an fpv seroprevalence of % among feral cats in victoria and nsw, documented in , provides evidence of widespread fpv exposure [ ] . viral introductions could also have been anthropogenic, for example through fomite spread during international travel. we were also able to analyse sequence data from a contemporaneous fpl outbreak in the uae in . this is the first study to characterise full-length vp sequences from the middle east. viruses most closely related to the uae viruses, which formed a phylogenetically distinct lineage, were from a range of locations in europe, asia and the americas, but the long lineage leading to the uae viruses suggests that we have not yet sampled their ultimate ancestor. the vp sequence data from cases analysed from a third country with contemporaneous fpl outbreaks showed that the nz fpv strains segregated with fpv vaccine viruses. the virological findings from these cases yet again highlight the difficulty of diagnosing fpl in cats that have recently received mlv vaccines, as well as raising important questions about the potential capacity of reversion to virulence of mlv vaccines. the diagnosis of fpl in cases from nz was initially based on consistent clinical signs and a positive faecal antigen test. point-of-care immunochromatographic assays designed to detect cpv faecal antigen also detect fpv, so are used in practice for the diagnostic investigation of cats with clinical signs consistent with fpl [ ] . however, faecal antigen tests detect both field and vaccine strains of fpv and cpv. in one study evaluating three commercial cpv faecal antigen kits, % of cats vaccinated against fpv in the preceding days returned positive test results [ ] . the sensitivity of faecal antigen tests for the detection of parvoviruses is influenced by viral load in faeces, as well as by the presence of gut antibodies that can sequestrate viral particles and prevent binding to the test antibody [ ] . cpv vaccine viral loads in recently vaccinated dogs are usually below the limit of detection of antigen tests, ranging from . × to . × copies/mg of faeces in one study [ ] and from ≈ to . × copies/mg of faeces in another [ ] . the lower limit of detection of faecal antigen tests for cpv in dogs is to viral dna copies per mg of faeces [ ] . low titres of gut fpv antibodies in the new zealand cats could explain the positive faecal antigen test results in those cats with low faecal viral loads. all but one of the vp sequences obtained from the new zealand cases were identical to the fpv vaccine strain that the cats had been inoculated with. co-infection with other pathogens including feline coronavirus or giardia spp. could account for the clinical signs in five of these cases, which, although consistent with fpl, were non-specific. however, in one cat, high loads of vaccine strain virus were detected concurrently in lymph node and faeces (fpv_ , table ), which is more consistent with loads attained by pathogenic virus strains than with those attained by vaccine virus strains. further, unequivocal support of the diagnosis of fpl in this case was obtained from histological examination of the intestine, where lesions pathognomonic for fpl were identified ( figure ). an fpv-vaccine strain was detected in this cat, and the sequencing chromatogram showed no evidence of a coinfecting fpv or cpv. however, deep sequencing of the sample with examination of the metatranscriptome or metagenome would be required to rule out co-infection with other pathogenic viruses not included in the qpcr test panel. an alternative explanation is that there was reversion to virulence of the vaccine strain, although this has not been described previously in cats. molecular analysis of faeces from dogs displaying signs of acute gastroenteritis shortly after cpv vaccination ruled out reversion to virulence of cpv mlv, since co-infection with field strains of cpv or other pathogens including canine coronavirus, canine distemper virus and isospora canis was detected [ ] . in three pups in which only the vaccine virus was detected, cpv vaccine loads were lower than those associated with enteritis from cpv field strains, ranging from . × to . × dna copies/mg of faeces [ ] . the re-emergence of fpl in two australian states in relatively quick succession over a three-year period, caused by two distinct fpv lineages, raises the question as to why this disease is re-emerging now, after decades of apparent quiescence. animal shelters are conducive environments for pathogen emergence or re-emergence because of a large number of susceptible hosts living in a confined area. this is exacerbated by factors including young age, immunological naivety or immunosuppression, close contact and co-morbidities such as heavy parasite burdens. similarly, suboptimal biosecurity protocols favour pathogen persistence and fomite transmission [ ] . failure to vaccinate or inadequate or inappropriate vaccination in shelter-housed cats were major contributors to the re-emergence of fpl in australia. before these outbreaks, over half of the shelters from which cases were derived did not administer fpv vaccinations. in general, fpv vaccines are a highly effective tool in parvovirus control. for example, protective fpv antibody titres were measured in % of vaccinated cats in a field study of adult cats after mlv vaccination, of which % had pre-existing protective titres [ ] . in addition, viral challenge studies of fpv-vaccinated cats usually show vaccine efficacy > % [ ] . although state legislation mandates vaccination of owned cats in commercial boarding facilities in nsw and victoria, vaccination is not enforced for cats admitted to private or municipal animal shelters, until they are sold for rehoming. most shelters that did vaccinate, completed the primary course of vaccinations in kittens by to weeks of age and used inactivated vaccines, in contravention of the current guidelines. the world small animal veterinary association (wsava) vaccination guidelines recommend the use of mlv vaccines in shelter-housed cats for rapid induction of long-lasting humoral immunity [ , ] . a minimum age of weeks for the final vaccination in the primary course is recommended, based on the failure of % of kittens to seroconvert after vaccinations at and weeks of age with the commercial fpv mlv vaccination [ , ] . this immunity gap relates to persistent maternally derived antibodies (mda) in kittens, which decline below protective titres yet effectively neutralize the vaccine virus and thereby prevent seroconversion [ ] . persistent mda would explain the presence of vaccination non-responders in our study that were < weeks of age at the time of diagnosis of fpl, highlighting that vaccination alone cannot prevent disease in environments where susceptible kittens are exposed to fpv. among the % of vaccination non-responders that were older than weeks, some were likely exposed to fpv prior to seroconversion, either before or after admission to the shelter, as indicated by the median vaccination-to-diagnosis interval of . days. the incubation period for fpl is between and days [ ] . other reasons for apparent vaccine failure include persistence of mda for > weeks in kittens born to queens with high anti-fpv antibody titres or failure to mount an immune response to vaccination because of immunodeficiency (acquired or genetic), incorrect vaccine administration or storage errors [ , ] . the median time from shelter admission to diagnosis of fpl was days. the minimum holding time for unidentified cats in municipal shelters in the states of victoria and nsw is days, after which cats can be euthanised or rehomed (domestic animals act , companion animals act ). changes in euthanasia practices resulting in longer length of stay (los) may have contributed to a cumulative increase in selection pressure for fpl re-emergence, especially in shelters where vaccination was not practiced. the likelihood of exposure to infectious diseases increases within a shelter environment with increasing los [ ] . national data from royal society of prevention to cruelty (rspca) shelters show a progressive decline in euthanasia rates from % in to % in [ , ] . similar declines in euthanasia have occurred in shelters in other countries such as canada [ ] . a seasonal pattern of fpl was identified in australia and new zealand, with an annual peak in case numbers from december to may. this pattern may reflect waning maternal immunity lagging two to three months behind the peak kitten births. similar seasonality of fpl outbreaks was reported in the northern hemisphere, where seasons are inverted; case numbers peaked from july to december and were correlated with an influx of kittens born annually in spring and waning of maternal immunity over the subsequent two to three months [ ] . the duration of faecal shedding of fpv, which is relevant to infection control, has been inadequately studied. this study demonstrates that virus can be shed in faeces for an extended period of up to weeks from diagnosis. faecal shedding of vaccine virus was detected by qpcr post vaccination for the duration of one four-week study [ ] . shedding of field cpv strains in dogs has been detected by qpcr for - days [ , ] , while cpv vaccine virus was shed for - days on average [ , ] . further studies are warranted in cats to further define the duration of faecal shedding of both field and vaccine strains of fpv detectable by qpcr. inadequate vaccination coverage was identified in the fpl outbreaks described here and was a major contributor to the re-emergence of this disease. these outbreaks highlight the importance of adherence to vaccination 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maternally-derived antibodies in pups and protection from canine parvovirus infection canine parvovirus post-vaccination shedding: interference with diagnostic assays and correlation with host immune status this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors are grateful to the many shelters, veterinarians and veterinary nurses who contributed to this study. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -v kpmk b authors: hagemeijer, marne c.; rottier, peter j.m.; de haan, cornelis a.m. title: biogenesis and dynamics of the coronavirus replicative structures date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: v kpmk b coronaviruses are positive-strand rna viruses that are important infectious agents of both animals and humans. a common feature among positive-strand rna viruses is their assembly of replication-transcription complexes in association with cytoplasmic membranes. upon infection, coronaviruses extensively rearrange cellular membranes into organelle-like replicative structures that consist of double-membrane vesicles and convoluted membranes to which the nonstructural proteins involved in rna synthesis localize. double-stranded rna, presumably functioning as replicative intermediate during viral rna synthesis, has been detected at the double-membrane vesicle interior. recent studies have provided new insights into the assembly and functioning of the coronavirus replicative structures. this review will summarize the current knowledge on the biogenesis of the replicative structures, the membrane anchoring of the replication-transcription complexes, and the location of viral rna synthesis, with particular focus on the dynamics of the coronavirus replicative structures and individual replication-associated proteins. positive-strand rna (+rna) viruses are the most abundant viruses in nature. many important pathogens belong to this category, including poliovirus (pv), hepatitis c virus (hcv) and the severe acute respiratory syndrome (sars)-coronavirus (cov). a distinctive common feature of +rna viruses is the replication of their genomes in the cytoplasm of the host cell in association with rearranged cellular membranes that are remodeled into organelle-like membranous structures to which the viral replication-transcription complexes (rtcs) localize. the various membrane rearrangements observed in +rna virus-infected cells range in size from to nm, contain lipids that are derived from various cellular compartments and demonstrate an impressively diverse plethora of morphologies that include, among others, clusters of vesicles for the picorna-and togaviridae, spherule-like invaginations for the bromoviridae and nodaviridae, and vesicle packets and membranous webs for the flaviviridae (reviewed in [ ] [ ] [ ] ). these membrane rearrangements seem to be beneficial for (i) sequestering and concentrating all viral and cellular components necessary for viral rna synthesis and (ii) to provide a protective microenvironment against virus-elicited host defense mechanisms. also coronaviruses (covs), enveloped +rna viruses that belong to the family coronaviridae, extensively rearrange cellular membranes into organelle-like replicative structures during infection. these replicative structures consist of double membrane vesicles (dmvs) and convoluted membranes (cms). the viral replicase proteins involved in rna synthesis localize to both these structures, while double-stranded rna (dsrna), presumably functioning as replicative intermediate during viral rna synthesis, has been detected at the dmv interior [ , ] . recent studies have provided new insights into the assembly and functioning of the cov replicative structures. this review will summarize the current knowledge on the biogenesis of the replicative structures, the membrane anchoring of the rtcs, and the location of viral rna synthesis, with particular focus on the dynamics of the coronavirus replicative structures and individual replication-associated proteins. the coronaviridae are a family of evolutionary related, enveloped +rna viruses that together with the arteriviridae and the roniviridae, belong to the order of the nidovirales. historically, covs have been recognized as important infectious agents for domestic livestock, poultry and companion animals. in contrast to the animal viruses, human covs (hcovs) have been associated with relatively mild upper and lower respiratory tract infections, including ordinary common colds. however, in - the outbreak of a novel hcov in china, causing severe fatal atypical pneumonia in infected individuals, demonstrated that hcovs are also able to induce severe life-threatening disease in humans. this virus was called the sars-cov [ , ] and emerged in the human population from an animal reservoir, probably originating from bats, with palm civet cats acting as intermediate hosts [ , ] . among the +rna viruses, covs clearly distinguish themselves by carrying the most complex and largest genomes, which range in size from ~ to kb [ ] . despite the variation in size, the overall genome organization of the various covs is quite conserved. the genome contains all the genetic information necessary to direct both the synthesis of full-length genomic rna (replication) and the (discontinuous) production of subgenomic mrnas (transcription) [ ] . the linear +rna genome of covs is ' polyadenylated and has a ' cap structure, thereby mimicking cellular mrnas. the ' and ' ends of the genome contain untranslated regions (utrs) with cis-acting elements that are important for replication and transcription. two-thirds of the genome consists of two large open reading frames (orfs), orf a and orf b. the remaining ' one-third part encodes the structural proteins interspersed with sequences encoding some accessory proteins. a schematic picture of the prototype mouse hepatitis virus (mhv) genome is shown in figure a . the structural and the accessory proteins are expressed from a nested set of ' coterminal subgenomic (sg) mrnas that are generated via discontinuous transcription during subgenome-length minus-strand rna synthesis [ , ] . the rna-dependent rna polymerase (rdrp) copies the genomic positive-sense rna into a negative-sense template until it reaches a transcription-regulation sequence (trs). at this point, rna synthesis may either continue or the rdrp may relocate to the ' end of the genome and complete the negative-sense sgrna. these negative-sense sgrnas serve as templates for the synthesis of the corresponding positive-sense sgrnas. as a result, the positive-sense sgrnas form a nested set of mrnas, which extend for different lengths from a common ' terminus while also having a common ' end, which is known as the leader sequence. generally, only the ' first unique gene of each sgrna is translated (reviewed in [ ] ). minus-and plus-strand rna synthesis is already detected at to min post infection (p.i.) [ ] . minus-strand synthesis, of which the resulting rna species are mainly present as double-stranded intermediates because of their association with plus-strand rna molecules, peaks at to h p.i. after which the synthesis declines but does not stop [ ] . the plus-stranded rnas are produced in a -to -fold excess over their minus-strand counterparts [ , ] . the viral replicase is encoded by the two most ' orfs of the genomic rna, orf a and orf b. translation of orf a and orf b of the genomic rna generates two very large replicase polyproteins (pp), pp a and pp ab. the latter is synthesized via a − ribosomal frameshift mechanism mediated by a pseudoknot structural element at the end of orf a [ , ] . these replicase polyproteins are extensively processed by viral proteinases (reviewed in [ ] ), resulting in the generation of sixteen nonstructural proteins (nsps). a schematic representation of the cov replicase polyprotein is shown in figure b . the cov nsps form together with the nucleocapsid (n) protein, and presumably several host proteins, the membrane-associated rtcs. to generate the functional cov replication complexes, the replicase polyproteins pp a and pp ab have to be proteolytically processed to liberate the sixteen individual protein products. cleavage of pp a and pp ab is performed by two viral proteinases that reside in nsp and nsp : the papain-like proteases (plpro and plpro ) located in nsp and the chymotrypsin-like cysteine proteinase ( clpro), or main protease (mpro), present in nsp (reviewed in [ ] ). besides the mature nsps, also the intermediate and precursor polyproteins are likely to be functionally important. hence, the cleavage of these proteins may somehow be involved in the temporal regulation of plus and/or minus sense viral rna synthesis [ ] [ ] [ ] . schematic representation of the +rna genome of mhv-a . the coronavirus genome contains a ' cap structure and a ' poly(a) tail, together with untranslated regions (utrs). the first two-thirds of the genome consist of two large open-reading frames (orfs), orf a and orf b, which are translated into two large replicase polyproteins (pp a and pp ab). pp ab is synthesized via a − ribosomal frameshift mechanism at the end of orf a (rfs). the final one-third of the genome contains the canonical cov structural proteins-encoding genes (s, e, m and n), interspaced by several accessory genes ( a, he, , a); (b) a schematic representation of pp ab is shown. the coronavirus polyproteins are processed by viral proteinases residing in nsp (plpro and plpro ; grey arrowheads indicate cleavage sites) and nsp (mpro; black arrowheads indicate cleavage sites), thereby generating mature nsps. hydrophobic domains (tm , tm and tm ) in nsp , nsp and nsp are indicated, together with predicted and identified rna(-modifying) enzymes: the rna-dependent rna polymerase (rdrp; nsp ), the helicase (hel; nsp ), the exonuclease (exon; nsp ), the uridylate-specific endoribonuclease (n; nsp ), and the methyl transferase (mt; nsp ); (c). schematic representation of the topology of the coronavirus polyprotein. only the part of the polyprotein is shown that contains the hydrophobic domains (indicated by boxes) residing in nsp , nsp and nsp . nsp and nsp contain hydrophobic domains that do not span the lipid bilayer. mpro indicates the viral protease residing in nsp , in between nsp and nsp . the key enzyme involved in genome replication is the rna-dependent rna polymerase (rdrp), which is present in nsp [ ] . the nsp protein is able to utilize both homo-and heteropolymeric rnas as template but its rdrp activity is dependent on primers to copy the viral rna [ ] . these primers might be produced by a non-canonical rdrp activity that has been described for the nsp -encoded 'rna primase', as this protein is able to produce short oligonucleotides complementary to the rna genome [ ] . nsp has been shown to associate together with nsp into a hexadecameric complex, consisting of eight copies of each protein, thereby forming a channel that can harbor rna and may serve as a processivity factor for nsp [ ] . nsp also interacts with nsp in a : ratio [ ] and is able to synthesize much longer transcripts [ , ] . the cov nsp protein contains a superfamily helicase domain with an amino-terminal zincbinding domain that is important for the unwinding activity of duplex rna (and dna) in a '-to- ' direction [ ] [ ] [ ] . the resulting single-strands probably serve as templates for rna synthesis. the multifunctional nsp protein additionally possesses nucleotide triphosphatase activity [ , , ] and is likely to be involved in removal of one of the terminal phosphate groups at the ' end of the positivesense rnas, which is the first step in the formation of the ' cap structure. although the enzyme that subsequently adds the guanine to the terminal phosphates (guanylyl transferase) has not been identified yet, nsp has been shown to exert s-adenosyl-l-methionine (adomet)-dependent (guanine-n )methyltransferase (n -mtase) activity [ ] . finally, the cap- structure is formed by the adometdependent (nucleoside- 'o)-methyltransferase ( 'o-mtase) activity that is present in nsp [ ] , and for which the latter needs to form a complex with nsp [ , ] . covs possess the largest genomes among the +rna viruses [ ] . they encode a large number of additional rna-modifying enzymes, which are often not present in other rna viruses. these additional enzymatic activities are probably required to ensure proper rna synthesis and might account for their large size. in addition to the cap n -mtase activity, nsp has metal ion-dependent '-to- ' exoribonuclease (exon) activity [ ] and contains a nidoviral uridylate-specific endoribonuclease (nendou; nsp ) [ ] , both able to degrade rna and dsrna [ , ] . while the function of the nendou activity in the cov infection cycle is not known, it appears that the exon activity is required to ensure high replication fidelity of the extremely large cov genome [ , ] and that nsp may function as a proofreading enzyme [ ] . covs encode three nsps that contain hydrophobic stretches that are predicted to function as transmembrane domains: nsp , nsp and nsp . consistently, membrane association has been demonstrated for the nsp , nsp and nsp proteins of sars-cov [ ] [ ] [ ] and mhv [ ] [ ] [ ] [ ] . both nsp and nsp become n-glycosylated upon insertion into the membranes of the endoplasmic reticulum (er) [ ] [ ] [ ] . interestingly, transmembrane domain predictions based on the multiple alignment of cov replicase polyprotein sequences revealed an uneven number of hydrophobic domains for both nsp and nsp [ ] . this prediction is peculiar as it would separate the viral proteinases residing in nsp and nsp from their target sequences, implying that some of these hydrophobic domains might actually not span the membrane. in agreement herewith, and in contrast to the predictions, all three nsps were shown to have both their amino terminus and their carboxy terminus exposed in the cytoplasm. while all four hydrophobic domains of nsp span the lipid bilayer, this is the case for only two of the three hydrophobic domains in nsp and for six of the seven in nsp [ , ] . this experimentally established topology model ( figure c ) makes more sense, as it positions all of the proteinase cleavage sites on the same side of the membrane as the viral proteinases themselves. proteolytic processing of the replicase polyproteins probably starts during translation and prior to membrane insertion. interestingly, the cleavage between nsp and nsp appears to be a rapid event [ , , ] . this cleavage at the amino-terminus of the first hydrophobic/transmembrane domain of nsp probably facilitates the membrane insertion of nsp as it may enable the first hydrophobic domain to function as a signal peptide. the occurrence of conserved non-membrane spanning hydrophobic domains in nsp and nsp , which are likely to be peripherally associated with the membrane, suggests an important function for these domains, possibly in the biogenesis of the cov replicative structures. in this respect it is of interest to mention that the seventh hydrophobic domain of nsp contains putative palmitoylation sites (our own predictions and [ ] ). the addition of palmitic acid to this hydrophobic domain may stabilize its peripheral membrane association. in addition to the nsps mentioned above, other cov nsps are involved in rna binding (nsp and nsp ; [ , ] ) or in evasion of the antiviral response of the host (nsp and nsp ; [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). the function of nsp is not yet known, although this protein was shown not to be essential for virus replication [ , ] . the reader is referred to several excellent reviews on this topic for more detailed insights [ , , ] . upon infection of host cells, covs induce a variety of membranous structures of which some have been associated with viral rna synthesis. the first detectable membrane rearrangements in cov-infected cells are to nm organelle-like structures that have been described for both mhv [ , ] and the sars-cov [ , ] and consist of spherical vesicles containing double lipid bilayers, termed dmvs ( figure ). in between the clusters of dmvs, reticular cms are characteristically present [ , , ] . later in infection large virion-containing vesicles (lvcvs) [ , , , ] , highly organized cubic membrane structures [ , ] and condensed tubular bodies [ , ] are formed. the latter two structures are likely a result of the overexpression of cov structural proteins during infection and do not seem to be involved in cov replication [ ] . electron tomography studies demonstrated that in sars-cov infected cells the dmvs and cms form an interconnected membranous network that is also continuous with the er [ ] . this latter observation is in agreement with previous reports describing dmvs in close proximity to the er or continuous with it [ , , ] . moreover, (partial) colocalization of replicase proteins together with the er resident protein disulfide isomerase (pdi) has been reported [ ] , while also the translocon subunit sec α was found to be redistributed to the replicative structures upon sars-cov infection [ ] . the combined data indicate that the er is the most likely membrane donor for the dmvs, despite the absence of most conventional er markers on these structures [ , , , ] . the cov replicative structures, i.e., dmvs and cms (figure ) , have been associated with viral rna synthesis, as the mhv and sars-cov nsps have been shown to localize to these structures [ , , , , , , , [ ] [ ] [ ] [ ] . in addition, antibodies recognizing dsrna, the presumed replicative intermediates, label the interior of the sars-cov-induced dmvs [ ] . newly synthesized viral rna, visualized by -bromouridine '-triphosphate (brutp) labeling, was observed in mhvinfected cells in close proximity to the replicative structures by immunoelectron microscopy [ , ] . remodeling of eukaryotic cellular membranes into the replicative structures is likely dependent on the combined effects of both viral and cellular proteins and probably also on the specific lipid composition of the membranes themselves. nevertheless, only few studies have been published addressing the involvement of cellular constituents in cov replication and the generation of the replicative structures. it is conceivable that covs hijack cellular pathways to meet the conditions that are required for their replication, consequently adopting intrinsic properties of the utilized pathways themselves. in agreement with the er being the most likely membrane donor of the dmvs, an intimate association between the early secretory pathway and cov replication has been demonstrated. rtc formation and replication in mhv-infected cells were inhibited when the secretory pathway was interfered with by blocking protein export at er exit sites by treatment with the kinase inhibitor h or by expression of a dominant active sar mutant [ ] . also treatment with brefeldin a (bfa), an inhibitor of er-to-golgi trafficking, or knockdown of its target gbf , inhibited mhv replication while reducing the number of dmvs [ ] . similar results were published for sars-cov infected cells treated with bfa and it was noticed that the inner and outer membranes of the dmvs were separated in bfa-treated cells [ ] , which may explain the observed inhibition of viral replication. by their main ultrastructural characteristic, the double lipid bilayer, cov dmvs very much resemble autophagosomes. this similarity prompted studies into the role of the autophagy machinery in cov replication. initial studies revealed a colocalization between the autophagosomal protein marker microtubule-associated protein light-chain (lc /atg ) with the replicative structures [ ] ; moreover, viral replication was impaired and dmvs were not detected in the absence of the essential autophagy protein atg [ ] . in other studies however, these colocalization data could not be reproduced [ ] , while the absence of atg did not affect cov replication [ , ] . yet, lc was found in association with the replicative structures by zhao and coworkers [ ] . furthermore, others showed that, while the endogenous lc protein was recruited to the replicative structures, this was not the case for a gfp-tagged form of lc [ , , ] that is often used as a marker for autophagosomes [ ] . in agreement herewith, cov replication was shown to be unaffected in autophagy-deficient cells lacking atg , although depletion of lc severely affected cov replication [ ] . unlike autophagosomes, which may also be induced by expression of cov nsp [ ] , cov replicative structures were shown to be decorated with the non-lipidated form of lc . similar findings were also reported for edemosomes [ , ] , er-derived vesicles that transport er chaperones to lysosomes. as the edemosome cargo proteins edem and os- were also detected in association with the cov replicative structures, it was proposed that covs hijack edemosomes for their replication [ , ] . in agreement herewith, the transmembrane sel l protein, which was recently shown to interact with lc and to have a critical function in edemosome biosynthesis, was also shown to colocalize with dsrna foci in cov-infected cells, while its depletion negatively affected cov replication [ ] . the cov nonstructural membrane proteins, nsp , and probably play an essential role in the membrane rearrangements required for the induction of the replicative structures and in the anchoring of the rtcs to these structures. these proteins were shown to be engaged in homo-and heterotypic interactions [ ] . interestingly, co-expression of nsp with the c-terminal one-third part of nsp (nsp c ) resulted in the relocalization of these proteins from the er into discrete foci mostly localizing to the perinuclear region of the cell [ ] . although nsp was not required for the observed relocalization, it was recruited to the perinuclear foci when co-expressed [ ] , in agreement with this protein interacting with nsp [ ] . ultrastructural analysis of cells co-expressing nsp and nsp c revealed that the membranes of the er exhibited more curvature, although these membranes did not resemble the dmvs observed in cov-infected cells [ ] , which may not be surprising as only the cterminal part of nsp was used in these co-expressions with nsp . similar membrane rearrangements were not observed when the nsp from mhv was co-expressed with nsp from sars-cov (or vice versa), while (deletion) mutagenesis studies indicated essential roles for the large luminal loops of nsp and nsp in the relocalization of these proteins [ ] . while co-immunoprecipitation and immunofluorescence assays indicate that nsp and nsp of mhv interact, this interaction could not be confirmed using the venus protein-fragment complementation assay [ ] . we hypothesize that the interactions between nsp and nsp mediate some kind of "zippering" of the lipid bilayers of the er, which ultimately leads to the formation of the dmvs. in this model nsp and nsp interact via their luminal loops in such a way that their interaction prevents reconstitution of a functional venus protein. several other studies also suggest an important role for nsp in the generation of the replicative structures. disruption of the nsp glycosylation sites present in the loop between the first and second hydrophobic region, leads to the formation of aberrant dmvs in which the inner and outer membranes are detached, while the number of cms is increased [ ] . in agreement herewith, although the fourth hydrophobic domain of nsp is dispensable, the other three transmembrane regions are required for cov replication [ ] . furthermore, co-expression of the counterparts of the cov nsp and nsp of the distantly-related equine arteritis virus (eav), resulted in the rearrangement of host cell membranes into dmvs, albeit with a morphology [ ] differing from that observed in eav-infected cells [ ] . mutations of cysteine residues present in the luminal loop of eav nsp (the eav counterpart of cov nsp ) resulted in altered morphologies of the dmvs, while the introduction of a n-glycosylation site in this loop also affected their morphology to some extent [ ] . like covs, other +rna viruses also somehow induce membrane rearrangements that are required for their replication and transcription. for several of these viruses similar membrane rearrangements can be induced by the (co-)expression of nsps (for reviews see [ ] [ ] [ ] ). these nsps are either integral transmembrane proteins, examples being the ns a proteins of dengue virus (denv) [ ] and kunjin virus [ ] and the ns b protein of hepatitis c virus (hcv) [ ] , or alternatively the proteins are only peripherally associated to the lipid bilayer, such as the a protein of brome mosaic virus (bmv) [ ] and the c protein of poliovirus (pv) [ ] . strikingly, however, also for the integral membrane proteins the occurrence of hydrophobic/amphipathic regions that do not span the lipid bilayer but are peripherally associated with membranes, which has also been demonstrated for cov nsp and nsp [ , , ] , appears to be a common feature [ , [ ] [ ] [ ] . another similarity among the nsps of different +rna viruses appears to be their ability to assemble into larger protein complexes. such interactions have been observed not only between nsp , nsp and nsp of covs, but also between the nsps of other +rna viruses, including the bmv a protein [ , ] and the flavivirus ns a and ns b proteins [ , ] . how are the nsps of covs able to induce the observed membrane rearrangements? we speculate that the similarities between the membrane-associated nsps of different +rna viruses relate to their common ability to remodel membranes. the induction of membrane curvature in lipid bilayers is critical when remodeling host cellular membranes. the membrane-associated viral proteins may act as multimeric scaffolds able to impose such curvature, for instance by acting as wedges by inserting their amphipatic helices partially into one side of the bilayer (reviewed in [ ] [ ] [ ] ). the viral proteins may function similar to host cellular proteins known to induce membrane bending via a scaffold mechanism, as exemplified by the copi and copii complexes, and/or via the insertion of amphipathic domains into the lipid bilayer, as has been proposed for the small gtpase sar (reviewed in [ , ] ). the last few decades have provided virologists with exciting new information regarding the functions and structures of individual nsps and the characterization and formation of the membranous structures induced by +rna viruses. these insights were obtained by classical biochemical, immunofluorescent and ultrastructural approaches. unfortunately, little information regarding the dynamics of the (viral) proteins present at the membranous replicative structures in living cells is known. such studies are important as classical approaches only provide static views of cellular processes and do not necessarily reflect the dynamics underlying virus replication in living cells. to date, only few studies on the dynamics of the replicative structures of plus-strand rna viruses and their associated proteins have been published [ ] [ ] [ ] [ ] [ ] [ ] [ ] . by using a gfp-tagged version of nsp , shown by immuno-electron microscopy (iem) to be recruited to the dmvs and cms, as a marker the dynamics of the cov replicative structures were studied using live cell imaging [ ] . these studies showed for the first time that the cov replicative structures are moving through the cell. however, it appeared that the cov replicative structures consist of two classes that demonstrate different mobilities: large structures lacking any displacement and smaller structures with relatively high saltatory mobility. the smaller replicative structures were hypothesized to correspond to individual dmvs, while the larger ones supposedly represent the dmv/cm assemblies that have been observed in ultrastructural studies of cov-infected cells [ , ] . in sars-cov-infected cells the dmvs are confined to a reticulovesicular network [ ] . we therefore speculate that the small structures have not yet been 'captured' into this network. however, correlative light-electron microscopy studies will be required to solve this issue. live cell imaging studies of hcv-and semliki forest virus (sfv)-infected cells also reveal the presence of replicative structures that could be discriminated on the basis of their size and mobility. hcv induces the formation of a so-called membranous web, in which dmvs can be observed [ ] . large hcv structures, probably representing membranous webs, exhibited limited movement, whereas smaller ones were mobile and could travel long distances throughout the cytoplasm [ ] . sfv-induced vacuoles are assembled at the plasma membrane after which they are transported to modified lysosomes [ , ] . sfv-infected cells also harbor large acidic immobile perinuclear vesicles and smaller acidic cytoplasmic vesicles that showed saltatory movements. in addition to these acidic vesicles, sfv-infected cells contain a class of non-acidic highly mobile vesicles that displayed multidirectional short-distance movements. moreover, fusion of the neutral mobile structures with the acidic mobile structures resulted in the formation of the large acidic structures [ ] . such events of fusion of smaller replicative structures into larger ones have not been observed (yet) for the cov and hcv replicative structures [ , ] . the calculated velocities of the saltatory movements of the smaller nsp -positive structures in cov-infected cells [ ] correspond to those measured for microtubule-mediated transport [ ] . the observed association of these structures with microtubules and the inhibition of trafficking in the presence of a microtubule network-disturbing drug confirmed the transport of the smaller structures on microtubular tracks [ ] . a role of the cytoskeleton in the movement of replicative structures has also been described for hcv, sfv, and pv [ , [ ] [ ] [ ] . disruption of a functional microtubular network inhibited the movement of the small hcv and nascent pv replicative structures [ , ] and the trafficking of sfv neutral vesicles to acidic organelles [ ] , concomitant with dispersal of these structures throughout the cytoplasm. strikingly, inhibition of microtubule-dependent trafficking did not or only modestly affect the replication of these viruses [ , , ] . disruption of the actin network also did not affect cov replication much, although the replicative structures again failed to accumulate in the perinuclear area [ ] . collectively, these results show that the cytoskeleton is required for the perinuclear accumulation of the replicative structure rather than for replication per se. additional studies are required to clarify the role of the perinuclear targeting of the cov replicative structures during infection. up till now, only few studies have addressed the dynamics of individual viral proteins when present at the replicative structures. recently, we analyzed the dynamic properties of three replication-associated proteins, i.e., the soluble nsp and n proteins and the integral membrane protein nsp , and demonstrated that these proteins display different diffusional mobilities when present at the replicative structures. nsp , when expressed in trans is recruited to the replicative structures [ ] . after recruitment, nsp was shown to be immobilized by using fluorescent recovery after photobleaching (frap) methodology. in other words, nsp already associated with the replicative structures was not exchanged by nsp present at other locations in the cell. similar results have been demonstrated for other +rna virus replication-associated proteins, although the published literature on this subject is limited. also in hcv-infected cells, ns a-positive structures showed a static internal architecture when (part of) the ns a fluorescent protein pool was bleached [ ] . when expressed individually, ns a is highly mobile [ ] , similar to nsp . apparently, in the context of a viral infection, when other viral proteins are present, mhv nsp and hcv ns a are immobilized at the replicative structures presumably due to protein-rna or protein-protein interactions. in agreement herewith, large-scale protein-protein interaction studies demonstrated that nsp of mhv and sars-cov is engaged in a multitude of interactions with itself, nsp , nsp , nsp , nsp , nsp , nsp , nsp and nsp [ , [ ] [ ] [ ] . these observations are remarkable in view of the dispensability of nsp during cov infection in vitro [ ] . yet, nsp has to be somehow important in vivo as its coding sequence is maintained during evolution. in addition to its structural role in the coronavirion, i.e., in packaging of the genomic rna into the rnp complex, the structural n protein is also important in coronavirus replication and has been detected in the perinuclear region of infected cells colocalizing with markers for the replicative structures [ , , , ] . the interaction of the n protein with nsp is presumably important for the initial recruitment of n to the replicative structures [ ] , while n-n protein interactions were sufficient for recruitment of (mutant) n proteins in the presence of wild type n proteins [ ] . in contrast to nsp , the n protein is not immobilized at the cov replicative structures but is associated with it rather dynamically [ ] . this may not be surprising, as the multifunctional n protein is both involved in viral replication [ ] [ ] [ ] and virion assembly [ ] . as the cov replicative structures and virion assembly sites appear to be spatially separated, the newly synthesized genomic viral rna needs to be transported from the replication sites to the assembly sites. the n protein presumably facilitates this transport, consistent with its dynamic behavior. when expressed in trans in infected cells, the nsp -gfp fusion protein was detected at the er and at the replicative structures [ , ] . by performing fluorescence loss in photobleaching (flip) experiments continuity was demonstrated between the membranes of the er and the replicative structures that harbor nsp [ ] , in agreement with the model that the dmvs and cms form an interconnected network that is continuous with the er [ ] . however, nsp displayed different diffusional mobilities at different subcellular locations [ ] . it was more mobile in the er than at the replicative structures. this reduced mobility may be (partly) due to its engagement in interactions with the other transmembrane-containing nsps as well as with itself [ ] . also the mobility of the hcv ns b protein, which is an integral transmembrane protein as well, depends on its intracellular location. when expressed in the absence of other viral proteins, this protein is present at the er and at so-called membrane-associated foci (mafs) that are induced upon expression of this protein [ ] . frap analysis showed that ns b present at the mafs had a reduced mobility compared to ns b at the er, which was suggested to result from ns b being engaged in different interactions when present on mafs or the er [ ] . currently, one of the most enigmatic issues regarding cov replication is the precise localization of the sites of active viral rna synthesis. although cov rna synthesis appears to be protected by membranes [ ] it is still unclear whether synthesis of nascent viral rna occurs at sites of dsrna accumulation, as pores connecting the interior of the coronavirus dmvs with the cytoplasm have not been detected [ ] , while the nsps localize to both dmvs and cms [ , , , , , , , [ ] [ ] [ ] [ ] . to identify sites of nascent viral rna synthesis, one has to define what actually constitutes the active viral replication complexes. although dsrna molecules function as intermediates of replication and transcription, their presence at certain sites per se does not imply (all) these structures to be actively involved in rna synthesis. likewise, the location of viral enzymes that are required for rna synthesis does not need to correlate with active rtcs. moreover, newly synthesized rnas are not necessarily located at their site of synthesis as they may diffuse or be transported away to other subcellular locations. in view of these considerations, sites active in rna synthesis are expected to contain at least three components: the rdrp, dsrna intermediates active in replication/transcription and nascent viral rna. newly synthesized viral rnas, visualized by -bromouridine '-triphosphate (brutp) labeling, were shown to colocalize with antibodies recognizing either nsp or the c-terminal part of pp a [ ] , and were observed in close proximity to the dmvs by immunoelectron microscopy in mhv-infected cells [ , ] . recently, a new method was used to detect and visualize newly synthesized coronaviral rna by incorporation of an alkyne-modified uridine analog, -ethynyl uridine (eu), onto which an azide-derivatized fluophore was coupled via a copper (i)-catalyzed cycloaddition reaction (click chemistry). with this method, it was shown that throughout mhv infection foci of nascent rnas could be detected, which colocalize with the rdrp-containing nsp , indicating that they correspond with sites of active coronaviral rna synthesis. the relationship between nascent rna and dsrna is, however, less clear. while early in infection nascent rnas colocalize at or adjacent to patches of dsrna dots, presumably corresponding to dmvs, this correlation is much less apparent at later times when the dsrna dots are spread throughout the cell. many dsrna dots are apparently not transcriptionally active as no eu labeling was associated with them, while many foci of eu labeling did not appear to colocalize with the dsrna dots [ ] . different models can be put forward to explain these observations. in one model, dmvs function as the sites of active rna synthesis. at the later times in infection, many dmvs are no longer active, while the ones that are active may contain only little dsrna, resulting in less apparent colocalization of dsrna and eu labeling. in another model, dmvs are non-functional end-stage products. they are not actively involved in rna synthesis, but rather harbor dsrnas that are not (longer) functioning as intermediates in rna synthesis. in this model, which is in agreement with the presumed absence of pores in these structures [ ] , the cms would be the only plausible alternative for the sites of active rna synthesis. in yet another model, dmvs may be the initial sites of active rna synthesis, particularly early in infection, while at later times the membranes become sealed, connections are lost and rna synthesis shifts to the cm assemblies. clearly, ultrastructural studies will be required, ideally (co)localizing nascent rnas as well as the rdrp, to definitely determine the precise localization of cov rna synthesis. for most other +rna viruses the identification of the sites of active rna synthesis appears less complicated. nascent rnas, as well as nsps, have been shown to label the spherules that are observed in bmv- [ ] , sfv- [ ] or flock house virus (fhv)- [ ] infected cells at the modified er, lysosomes and mitochondria, respectively, indicating that these structures correspond with the sites of active rna synthesis. nascent rnas were previously also shown to colocalize with dsrna in kunjin flavivirus-infected cells [ ] . electron tomography of the membrane rearrangements observed in flavirus-infected cells revealed that the inner content of the dmvs, which contains nsps and dsrna, is connected to the cytoplasm via a pore [ , ] , indicating that these dmvs are actually spherule-like invaginations (once) active in rna synthesis. cov replicative structures/rtcs are macromolecular assemblies, the components of which are engaged in a plethora of protein-protein, protein-rna, and protein-lipid interactions [ ] [ ] [ ] ] . currently, the exact composition of the replicative structures/rtcs is not known, let alone the full arsenal of interactions occurring within these structures. moreover, the replicative structures are likely to be subject to some form of maturation [ , , , ] , as their composition appears to change during the course of infection as determined by biochemical and immunofluorescence analyses. thus, it will be of interest to confirm, extend and refine the previously published protein-protein interactome studies that have been published for sars-cov [ ] [ ] [ ] , for example by investigating proteinprotein interactions of other covs using novel (large-scale) screening approaches. likewise, it will be of interest to get more insight into the involvement of host proteins in the formation of these structures, for example by screening for host proteins that interact with the cov nsps or by elucidating the protein content of purified replicative structures -preferably in time-using mass spectrometry. one of the difficulties associated with these studies is the complexity of discriminating whether host proteins are directly involved in the formation of the replicative structures themselves or in rna synthesis per se, as inhibition of either process will result in reduced rna synthesis, protein expression and dmv formation. therefore, assays are needed in which the formation of replicative structures can be studied independent of viral replication. such assays may be provided by co-expression of viral proteins [ , ] that induce the rearrangement of cellular membranes. recently, by using such a replication-independent assay, reticulons, which form a family of er membraneshaping proteins, were implicated in the formation of the bmv-induced, er-derived spherules that are associated with viral rna synthesis [ ] . reticulons are involved in the induction of er membrane curvature [ ] [ ] [ ] and seem to be required by bmv for determining the size of the spherules and for stabilizing the spherule necks [ ] . it will be of interest to study the putative role of these proteins in cov replication and in reshaping of the er membranes by cov nsps. other proteins playing a role in shaping and remodeling of the er are the atlastins and climp- [ , ] . also these proteins are putative candidates involved in cov-induced membrane remodeling and would deserve to be investigated. in addition to the role of virus and host proteins in formation of the replicative structures, lipids may also be important in this process. dynamic alteration of the lipid composition can induce curvature of membranes to some extent [ , , , ] , which is, however, unlikely to be sufficient to generate the organelle-like structures observed in +rna virus-infected cells. it is more likely that lipids, together with lipid-modifying enzymes, contribute to the formation of the replicative structures by providing a suitable microenvironment to which the viral (and cellular) membrane shaping proteins are recruited. even more, +rna viruses may specifically hijack lipid-modifying enzymes for their own advantage [ , ] . this is underscored by several studies, which show that inhibition of lipid synthesis by using either drugs or small interfering rnas dramatically affects the replication of +rna viruses [ , [ ] [ ] [ ] [ ] . it will be of interest to study how and to what extent cov infection affects the lipid homeostasis in infected cells, for example by analyzing the lipid repertoire of infected cells by lipidomics techniques. finally, as all processes occurring in living cells are inherently dynamic in nature, it is also desirable to get more insight into the biogenesis and functioning of the replicative structures using various live cell imaging approaches. for example, photoactivatable fluorescent proteins can be used to investigate the formation of the 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α-helical segment mechanisms of membrane deformation mechanisms determining the morphology of the peripheral er viral reorganization of the secretory pathway generates distinct organelles for rna replication recruitment and activation of a lipid kinase by hepatitis c virus ns a is essential for integrity of the membranous replication compartment phospholipid biosynthesis and poliovirus genome replication, two coupled phenomena dengue virus nonstructural protein redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis synthesis of semliki forest virus rna requires continuous lipid synthesis dengue virus infection perturbs lipid homeostasis in infected mosquito cells photobleaching and photoactivation: following protein dynamics in living cells development and use of fluorescent protein markers in living cells single mrna molecules demonstrate probabilistic movement in living mammalian cells lambdan-gfp: an rna reporter system for live-cell imaging molecular beacons: probes that fluoresce upon hybridization copper-free click chemistry for dynamic in vivo imaging imaging intracellular fluorescent proteins at nanometer resolution sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (storm) breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy correlative light-electron microscopy (clem) combining live-cell imaging and immunolabeling of ultrathin cryosections we would like to thank oliver wicht and christine burkhard from the virology division, faculty of veterinary medicine, utrecht university for stimulating discussion and fulvio reggiori the authors declare no conflict of interest. key: cord- -jh dyyz authors: alenquer, marta; amorim, maria joão title: exosome biogenesis, regulation, and function in viral infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jh dyyz exosomes are extracellular vesicles released upon fusion of multivesicular bodies (mvbs) with the cellular plasma membrane. they originate as intraluminal vesicles (ilvs) during the process of mvb formation. exosomes were shown to contain selectively sorted functional proteins, lipids, and rnas, mediating cell-to-cell communications and hence playing a role in the physiology of the healthy and diseased organism. challenges in the field include the identification of mechanisms sustaining packaging of membrane-bound and soluble material to these vesicles and the understanding of the underlying processes directing mvbs for degradation or fusion with the plasma membrane. the investigation into the formation and roles of exosomes in viral infection is in its early years. although still controversial, exosomes can, in principle, incorporate any functional factor, provided they have an appropriate sorting signal, and thus are prone to viral exploitation. this review initially focuses on the composition and biogenesis of exosomes. it then explores the regulatory mechanisms underlying their biogenesis. exosomes are part of the endocytic system, which is tightly regulated and able to respond to several stimuli that lead to alterations in the composition of its sub-compartments. we discuss the current knowledge of how these changes affect exosomal release. we then summarize how different viruses exploit specific proteins of endocytic sub-compartments and speculate that it could interfere with exosome function, although no direct link between viral usage of the endocytic system and exosome release has yet been reported. many recent reports have ascribed functions to exosomes released from cells infected with a variety of animal viruses, including viral spread, host immunity, and manipulation of the microenvironment, which are discussed. given the ever-growing roles and importance of exosomes in viral infections, understanding what regulates their composition and levels, and defining their functions will ultimately provide additional insights into the virulence and persistence of infections. multivesicular bodies (mvbs) or late endosomes are components of the endocytic pathway that range from to nm in diameter. within mvbs are vesicles called intraluminal vesicles (ilvs) that range from to nm in diameter. mvbs can either be degraded or can fuse with the plasma membrane, releasing the ilvs into the extracellular space. the ilvs are called exosomes following their release from the mvb [ ] . the formation of the ilvs within the mvb and the budding of enveloped virions share many features. both processes require induction of membrane curvature, inclusion of specific cargo, and membrane fission for release. what is most striking is that evolutionarily unrelated viruses, with dramatically different genomes, have converged in their use of the host machinery for ilv formation to promote their own budding. important human pathogens viruses , , - more detail below [ ] . secretion of exosomes requires maturation of early endosomes into mvbs (a process reviewed in [ ] ), with concomitant formation of ilvs, and fusion of mvbs with the cell surface to release exosomes. at any point, material can be further internalized to the tgn and integrated in canonical secretory pathways [ , ] . viruses , , -x figure . the endocytic and secretory pathways. cargo binds to the plasma membrane, is endocytosed by a plethora of processes and, independently of the entry route, is transported to early endosomes (ee). from this sub-compartment, cargo is sorted to one of three destinations: recycling, degradation, or secretion. these routes require maturation of the ee into recycling endosomes or multivesicular bodies (mvbs), which can either fuse with lysosomes (l) to generate endolysosomes (el) or with the plasma membrane to release intraluminal vesicles to the milieu as exosomes. the membranes of the sub-compartments of the endocytic pathway have different compositions. specific members of the rab gtpase family, for example, are differentially enriched in each sub-compartment: rab is enriched in ee; rab in mvbs; rab , rab , rab , and rab in the slow and rapid recycling routes; and rab a/b in mvbs. rab is present in vesicles destined for retrograde transport to the trans-golgi network (tgn). in uninfected cells, interfering with these rabs affects exosome release. many viruses use these rabs in diverse steps of the viral life cycle, although whether this usage impacts in exosomal release has not been investigated. for example, at late stages of infection, viruses such as iav, rsv, sendai virus (sev), and andes virus (andv) were shown to hijack rab vesicles to transport their progeny rna to the cell surface. hiv, hsv , and human cytomegalovirus (hcmv) were shown to require rab a/b vesicles for assembly. human herpes (hhv- ) virions were shown to be secreted upon fusion of mvb with the plasma membrane, together with exosomes. the endocytic pathway is a convoluted web of interconnected sub-compartments with distinct cell localization, lipid and protein composition, and ph, which operates as follows: cells internalize ligands by endocytosis concomitantly with membrane proteins and lipids [ , ] . irrespectively of figure . the endocytic and secretory pathways. cargo binds to the plasma membrane, is endocytosed by a plethora of processes and, independently of the entry route, is transported to early endosomes (ee). from this sub-compartment, cargo is sorted to one of three destinations: recycling, degradation, or secretion. these routes require maturation of the ee into recycling endosomes or multivesicular bodies (mvbs), which can either fuse with lysosomes (l) to generate endolysosomes (el) or with the plasma membrane to release intraluminal vesicles to the milieu as exosomes. the membranes of the sub-compartments of the endocytic pathway have different compositions. specific members of the rab gtpase family, for example, are differentially enriched in each sub-compartment: rab is enriched in ee; rab in mvbs; rab , rab , rab , and rab in the slow and rapid recycling routes; and rab a/b in mvbs. rab is present in vesicles destined for retrograde transport to the trans-golgi network (tgn). in uninfected cells, interfering with these rabs affects exosome release. many viruses use these rabs in diverse steps of the viral life cycle, although whether this usage impacts in exosomal release has not been investigated. for example, at late stages of infection, viruses such as iav, rsv, sendai virus (sev), and andes virus (andv) were shown to hijack rab vesicles to transport their progeny rna to the cell surface. hiv, hsv , and human cytomegalovirus (hcmv) were shown to require rab a/b vesicles for assembly. human herpes (hhv- ) virions were shown to be secreted upon fusion of mvb with the plasma membrane, together with exosomes. no underlying mechanism has yet been reported to differentiate mvb formation for degradation or fusion with the cell membrane. there are, however, reports suggesting the existence of subpopulations of mvbs, depending on their fate. using a biotinylated derivative of the cholesterol-binding toxin perfringolysin o from clostridium perfringens to localize cholesterol at the subcellular level, möbius et al. identified two types of mvbs of similar morphology, one cholesterol-rich that was destined for secretion, and the other cholesterol-poor, which was destined for degradation [ ] . conversely, two sub-populations of mvbs differing in the presence or absence of lysobisphospatidic acid have been described. for example, egf and its receptor (egfr) are only present in mvbs negative for lysobisphospatidic acid [ ] . this supports the previously described role of phosphatidyl inositol (pi) in the formation of mvbs containing egfr [ ] , but not lysobisphospatidic acid [ ] . another study showed that a full-length p receptor was only released from sympathetic neurons and pc cells upon kcl-mediated cell depolarization. under this stimulus, p -containing mvbs escaped endolysosomes and degradation [ ] . whether this reflects distinct steps in mvb trafficking or even distinct mechanisms in mvb formation that mark them for degradation or fusion with the membrane is still unclear. ilv formation is characterized by inward budding of membranes, a process that starts in early endosomes but greatly augments as endosomes mature [ ] . evidence indicates that exosomes correspond to secreted ilvs of mvbs. relative to the composition of the cytoplasm, exosomes are enriched in components such as lipids, rnas, and proteins. lipids include cholesterol, sphingomyelin, glycosphingolipids, and phosphatidylcholine with saturated fatty acids [ ] [ ] [ ] . enriched rnas are specific mirnas, non-coding rnas, trnas, rrnas, and mrnas [ ] [ ] [ ] [ ] . finally, proteins found in higher concentration than in the cytosol include specific factors of the immune system, those of the escrt apparatus, those involved in trafficking, and lipid-rafts residents. the latter are, for example, cytokines, tetraspanins, major histocompability complex (mhc) class i and ii molecules, glycosylphosphatidylinositol-anchored proteins, rabs, snares, and flotillin [ , , , [ ] [ ] [ ] . importantly for the context of this review, cells infected with viruses were shown to release exosomes containing viral proteins and rnas [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (reviewed in [ ] ). some viral proteins, including the hiv nef, seem to have exosomal localization signals [ , ] . however, in the majority of cases, it is unclear whether the inclusion of viral components in exosomes results from direct sorting or from hijacking the machinery for exosome biogenesis, trafficking, and/or release. nevertheless, the specific composition of exosomes derived from cells, regardless of their infectious state, suggests that soluble and membrane-bound cargo are selectively incorporated into ilvs [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the formation of ilvs is accomplished by several molecular mechanisms. proteins belonging to the escrt family are, by far, the best characterized and have been extensively reviewed [ , , , [ ] [ ] [ ] . there are, however, ubiquitin-and escrt-independent pathways [ ] , including the oligomerization of the tetraspanin complexes [ ] , the sphingomyelinase pathway that catalyzes ceramide synthesis [ ] , or phospholipase d and adp rybosylation factor- -mediated ilv budding [ ] . one way for viral modulation of exosome release is by directly interfering with the machinery involved in exosome biogenesis; this has been reviewed elsewhere for the escrt machinery [ ] . however, in principle, anything that hinders mvb formation might impact, even if indirectly, on exosome biogenesis. for a clearer exploration of the latter hypothesis, we will briefly summarize some regulatory mechanisms operating in the endocytic pathway, the system in charge of exosome formation. there has been much debate on whether vesicles that deliver material from a donor compartment to a specific acceptor sub-organelle contain information on where to go [ , ] . in the s, it was found that different compartments, and the vesicles they produced, were populated by distinct rab gtpases. the rab gtpases belong to a large family of highly conserved proteins with members, which were discovered to regulate vesicular trafficking in eukaryotes [ ] . the description of the endocytic pathway in figure can be explained with a set of rabs as follows: after endocytosis, sorting in rab -positive early endosomes [ ] delivers cargo to return to the plasma membrane along fast (rab , rab ) [ ] or slow (rab a, rab b, rab ) [ , ] recycling processes. alternatively, rab endosomes acquire rab and release rab by a process called endosome maturation [ , ] . rab -containing endosomes sort material to ilv, decrease ph, and acquire hydrolytic proteases able to degrade their internal contents [ ] , or instead acquire rab a/b and fuse with the plasma membrane-releasing exosomes [ ] . at any point, vesicles might acquire rab to enter a retrograde transport to the tgn [ ] . in recent years, it has been shown that interfering with the levels and activation of rab gtpases influences exosome release. depending on the cell type, rab , rab , rab , rab , and rab were all implicated in the release of vesicles. rab overexpression was shown to inhibit progression of endocytosed material from early endosomes, impacting negatively on exosomal release of markers such as syndecan, cd , and alix, and this reduction was rescued by the overexpression of rab [ ] . in agreement, rab depletion severely impaired exosomal release of the same factors [ ] . the lack of a functional active rab reduced the secretion of exosomes in the erythroleukemia cell line k , drosophila s cells, and retinal epithelial cells, as evaluated by the following exosomal proxies: transferrin and hsc- , wingless and evi, or flotillin and anthrax-toxin, respectively [ ] [ ] [ ] [ ] .rab was identified in a screen performed in oligodendroglial cancer and primary cells to analyze phospholipase d -containing exosomes [ ] . rab has been found to facilitate the release of the exosomal markers mhc ii, cd , and cd in many cancer types, including hela cells [ , , ] . interestingly, rab and rab did not influence the release of wingless in drosophila s cells [ ] and rab did not affect the extracellular levels of flotillin or of anthrax toxin in retinal epithelial cells [ ] . the roles of these rabs in the endocytic pathway allowed for speculation that there might be extracellular vesicles derived from different routes such as recycling and mvb, but this awaits confirmation [ ] . in conclusion, altering the levels of any of the referred rabs has the potential to interfere with the progression of cargo at specific endocytic locations. consistently, it has been shown that switching off rab and repopulating the endosome with other rabs is a prerequisite for the maturation of early endosomes into other types of endosomes [ , ] . conversion to rab forms mvbs and endolysosomes. mvb acquisition of rab is, in some cases, required for exosome release [ , ] . additionally, exchanging rab for rab or rab allows progression from early endosomes to the slow recycling route and exchanging rab for rab or rab allows progression from early endosomes to the fast recycling route [ ] . many viruses were shown to use the rabs mentioned above to assist several steps of their replication. there is no evidence yet relating the viral usage of these proteins with exosome biogenesis and function. the picture of the viral usage of endocytic proteins has been built during years of research, making of viruses excellent tools to understand the crosstalk between different endocytic sub-compartments. in this sense, we provide an overview of identified interactions between viruses and endocytic proteins that regulate exosome biogenesis. examples of viruses using these rabs are identified in red in figure and in table . the orthomyxovirus iav, the paramyxoviruses sev and rsv, and the bunyavirus andv all share negative strand rna genomes, infect the lung epithelia, and use rab a pathway in their infectious cycle [ , [ ] [ ] [ ] [ ] [ ] [ ] . in the case of sev, rsv, and iav, progeny rna (in the form of viral ribonucleoproteins, vrnps) attach, facing the cytoplasm, to rab vesicles as a way to facilitate their transport to the apical side of the plasma membrane. for these three viruses and conversely to andv, viral interaction with the recycling endosome occurs via activated (gtp-bound) rab a [ , [ ] [ ] [ ] [ ] [ ] [ ] . the impact of rab viral hijacking in the recruitment and activation of other rabs and exosome biogenesis has not been investigated. however, as mentioned above, interfering with rab levels can inhibit or promote the release of exosomes containing transferrin, hsc- , flotillin, and anthrax toxin [ ] [ ] [ ] [ ] . during iav infection, the total levels of rab remain fairly constant, but it is possible that the amount of the activated form suffers viral-induced fluctuations. mechanistically, in uninfected cells, vesicular transport is promoted by binding of molecular motors to activated rab . amongst many of the rab effectors reported [ ] , the members of the rab -interacting family proteins (fips) have been well characterized in facilitating vesicular movement [ ] . reduction in the levels of some fips was reported to interfere with sorting of recycling vesicles [ , ] . for iav, our unpublished results suggest that the vrnp hijacking of the rab pathway "slows down" recycling efficiency. this system could then be used to assess the effects of impairing rab in exosome biogenesis. the effects in recycling efficiency might differ for rsv, as utley et al. [ ] have shown that several members of the recycling machinery were required for rsv vrnp transport. in the case of andv, viral replication and assembly is thought to occur in the lumen of a membranous delimited sub-compartment, followed by budding to the cytoplasm and transport to the periphery. rab depletion was shown to reduce over -fold the levels of produced virions [ ] . in this case, the viral structural protein n was shown to co-localize mainly with the gdp-bound rab near the tgn, at a perinuclear location, although it has not yet been addressed whether andv inhibits rab activation or affects mvb formation and exosome release [ ] . such analysis has also not been performed for rsv and sev. rab a regulates secretion of lysosome-related organelles, including mvbs [ , ] . it was reported that rab a levels increased in hcmv-infected cells [ ] , a dna virus member of the betaherpesvirinae subfamily. this rab was found in association with viral envelopes of hcmv in an undefined compartment related to the tgn or in vesicles being transported between the tgn and endosomes [ ] . the molecular mechanisms leading to increased levels of rab a in hcmv infection are still unclear, as well as their interference with exosome release. however, given the positive role of rab a in extracellular mhc ii-containing exosomes and the regulation of immune responses in glial cells, this role of rab a in hcmv infection should be further evaluated [ ] . another virus that hijacks rab a to promote assembly is hiv (figure ) , by a mechanism that is described below [ ] . hiv also uses the escrt pathway to facilitate budding [ ] and additionally viruses , , - increases the transcription of genes involved in mvb formation and exosome release [ ] . this viral interference with many different steps of mvb/exosome formation suggests a high dependency on the host ilv machinery. in conclusion, much work has been done in understanding how different rabs regulate particular steps in the life cycle of specific viruses. how the usage of various rabs impacts the overall regulation of the endocytic pathway, including exosome release and their still controversial associated functions, remains to be evaluated. as mentioned above, it was shown by many independent groups and using several models that specific rabs interfere with exosome release. however, rabs are themselves subjected to tight regulation and are not the only factors controlling membrane identity [ , ] . figure depicts the complexity behind the composition of each sub-compartment membrane, with many factors required to create the correct microenvironment. of these, seven phosphoinositide phosphates (pip) are crucial to recruit specific rabs, albeit indirectly (for a comprehensive review on pip chemistry and biology please refer to [ , ] ). these phospholipid derivatives are enriched in specific cellular membranes. for example, the plasma membrane contains mostly phosphoinositol- , -bisphosphate (pi( , )p ) and phosphoinositol- , , -triphosphate (pi( , , )p ), whilst the membranes of the endocytic pathway are decorated with phosphoinositol- -phosphate (pi p). during maturation of endosomes, mvbs acquire phosphoinositol- , -bisphosphate (pi( , )p ), a form that becomes prevalent in lysosomes [ ] . the pip isoforms are recruited by kinases and phosphatases that also occupy precisely defined sub-compartments [ ] . viruses have been shown to alter this pip equilibrium and by doing so alter the content of rabs involved in exosome formation. for example, phosphatidylinositol -phosphate (pi p) was shown to be recruited by flavivirus and picornavirus to sites near the endoplasmic reticulum. mechanistically, such enrichment was shown to be mediated by specific phoshoinositide (pi) kinases [ , ] , one of them able to recruit rab to the golgi [ ] . another recent paper explored the mechanisms of hiv- particles' assembly at the plasma membrane, a process that occurs in micro-domains enriched in pi( , )p and the viral protein pr gag . it was found that in t cells, rab a (and some of its effectors) boosted pi( , )p production by delivering the mvb-associated kinase pi kiiα to the cell surface [ ] . rab a was also implicated in exosome release in t cells; therefore, these hiv and exosome release could, in principle, be intertwined, although such relation has not been investigated yet. other factors that control rab delivery to specific membranes include the proteins that directly activate/deactivate them. being gtpases, rabs suffer cycles of gtp/gdp binding, which are a function of the protein families' rab guanine exchange factors (gef) and guanine-activating proteins (gap), respectively ( figure ) (reviewed in [ , , , [ ] [ ] [ ] ). there is also a clear cross-talk between rab proteins and other regulators of vesicular trafficking called adp-ribosylation factors (arfs) [ , ] . arfs are also gtpases, recruited by gefs and gaps that reside in specific membranes according to their pip composition. the complete picture of the regulators controlling the location and levels of the rabs mentioned above is far from complete and these factors have not been explored in the context of viral infection. however, it is widely accepted that cells react to stimuli to adjust the distribution and levels of intracellular pips as well as their cycles of degradation/secretion/recycling [ ] . all the above considerations raise important questions concerning the interplay between regulators of the endocytic process, exosome release and viral usurpation of specific steps in the endocytic pathway. first, can viral modulation of any of these regulators alter the content and levels of released exosomes? and second, would control of the levels and composition of secreted exosomes have associated functions? these are interesting questions that deserve more attention in the near future, especially given the ever-growing functions of exosomes in viral infections. concerning the interplay between regulators of the endocytic process, exosome release and viral usurpation of specific steps in the endocytic pathway. first, can viral modulation of any of these regulators alter the content and levels of released exosomes? and second, would control of the levels and composition of secreted exosomes have associated functions? these are interesting questions that deserve more attention in the near future, especially given the ever-growing functions of exosomes in viral infections. regulators of membrane identity. membrane composition is important to maintain the integrity of endocytic process. phosphoinositide (pi) kinases (and phosphatases) ensure the levels of specific pips in distinct membranes. these operate as docking platforms for guanine exchange and activator factors (gefs and gaps) able to recruit and turn on/off gtpases. gtpases involved in membrane integrity and vesicular biogenesis are mainly of two kinds: adp ribosylation factors (arfs) and arf-like proteins (arls); and rabs. arfs and arls are involved in early steps of vesicular biogenesis such as recruiting coating proteins and cargo, membrane curvature, and neck formation. rabs operate at later stages by recruiting effectors such as molecular motors, which are able to generate pulling forces and move released vesicles. vesicle scission and release are mediated by highly specialized proteins that recognize, encircle, and cut the membrane neck, using gtp or atp hydrolysis to drive the reaction. in normal circumstances, cells show remarkable processes to communicate with the exterior, including the release of exosomes. as mentioned throughout this review, exosomes can transfer functional proteins, lipids, and distinctive sets of rnas from cell to cell in homeostatic conditions [ , , ] . in the last decades, exosomes were also shown to facilitate cell-to-cell transport of disease-related proteins involved in neurodegenerative disorders, such as prions [ ] and betaamyloid peptides [ ] . this machinery could, in principle, also contribute to viral spread. for this to happen, two prerequisites would be necessary. first, viral rna and proteins would need to access ilv. indeed, vesicular stomatitis virus (vsv), dengue virus (and other flavivirus members), and hepatitis c virus (hcv) components were found in these sub-compartments [ , ] . a relevant question is: how are soluble viral rnas sorted into ilv? rna incorporation into exosomes has been suggested to operate via a selective and conserved mechanism linked to the lipid content of vesicles. for a comprehensive review on specific mechanisms, readers are directed to [ ] . nevertheless, the contribution of rna-binding proteins has been recognized. the hnrnpa b rna binding protein, for example, was shown to bind to a four triplet motif on mirnas and transport them into exosomes [ ] . in the case of viral transmembrane proteins, the most common mechanism of targeting molecules to ilvs is via ubiquitination and recruitment of the escrt machinery [ , , ] . for hcv, it was shown that the escrt component hrs is critical for release of nucleocapsid [ ] , and for hepatitis a virus (hav) the escrt protein vps b and the accessory proteins alix [ ] were deemed crucial for ilv budding. second, exosomes would need to enter a recipient cell and release their infectious content into the cytoplasm. this mechanism was recently reported for hcv [ ] , where exosomes derived from infected human hepatoma cells containing full-length viral rna, along with core and envelope figure . regulators of membrane identity. membrane composition is important to maintain the integrity of endocytic process. phosphoinositide (pi) kinases (and phosphatases) ensure the levels of specific pips in distinct membranes. these operate as docking platforms for guanine exchange and activator factors (gefs and gaps) able to recruit and turn on/off gtpases. gtpases involved in membrane integrity and vesicular biogenesis are mainly of two kinds: adp ribosylation factors (arfs) and arf-like proteins (arls); and rabs. arfs and arls are involved in early steps of vesicular biogenesis such as recruiting coating proteins and cargo, membrane curvature, and neck formation. rabs operate at later stages by recruiting effectors such as molecular motors, which are able to generate pulling forces and move released vesicles. vesicle scission and release are mediated by highly specialized proteins that recognize, encircle, and cut the membrane neck, using gtp or atp hydrolysis to drive the reaction. in normal circumstances, cells show remarkable processes to communicate with the exterior, including the release of exosomes. as mentioned throughout this review, exosomes can transfer functional proteins, lipids, and distinctive sets of rnas from cell to cell in homeostatic conditions [ , , ] . in the last decades, exosomes were also shown to facilitate cell-to-cell transport of disease-related proteins involved in neurodegenerative disorders, such as prions [ ] and beta-amyloid peptides [ ] . this machinery could, in principle, also contribute to viral spread. for this to happen, two prerequisites would be necessary. first, viral rna and proteins would need to access ilv. indeed, vesicular stomatitis virus (vsv), dengue virus (and other flavivirus members), and hepatitis c virus (hcv) components were found in these sub-compartments [ , ] . a relevant question is: how are soluble viral rnas sorted into ilv? rna incorporation into exosomes has been suggested to operate via a selective and conserved mechanism linked to the lipid content of vesicles. for a comprehensive review on specific mechanisms, readers are directed to [ ] . nevertheless, the contribution of rna-binding proteins has been recognized. the hnrnpa b rna binding protein, for example, was shown to bind to a four triplet motif on mirnas and transport them into exosomes [ ] . in the case of viral transmembrane proteins, the most common mechanism of targeting molecules to ilvs is via ubiquitination and recruitment of the escrt machinery [ , , ] . for hcv, it was shown that the escrt component hrs is critical for release of nucleocapsid [ ] , and for hepatitis a virus (hav) the escrt protein vps b and the accessory proteins alix [ ] were deemed crucial for ilv budding. second, exosomes would need to enter a recipient cell and release their infectious content into the cytoplasm. this mechanism was recently reported for hcv [ ] , where exosomes derived from infected human hepatoma cells containing full-length viral rna, along with core and envelope proteins [ ] , were shown to be infectious and a major route of transmission. interestingly, the non-enveloped virus hav has been reported to acquire a host-derived membrane for cell-to-cell transmission and the virus is still able to replicate in the recipient cell. the exosomes containing hcv rna and exosome-like vesicles containing whole hav capsids were less susceptible to antibody neutralization, and consequently this transmission mechanism has been reported to operate as an immune evasion strategy [ , , ] . upon reaching their destinations, exosomes containing viral proteins and rna would only be infectious provided they could enter target cells and reach the cytoplasm. exosomes containing vsv and other flaviviruses seem to enter the cell by being taken up by the endocytic pathway [ , ] . these two viruses were shown to escape late endosomes and reach the cytosol by a "back-fusion" process, a phenomenon by which ilvs fuse with the external late endocytic membranes [ ] . alternatively, exosomes containing viral antigens can induce signaling cascades at the surface upon binding their receptors to control host immune responses [ ] . research on exosome-mediated viral spread is still very limited; however, exosome modulation of the immune responses has been explored in some detail and will be discussed next. one of the main functions assigned to exosomes is the mediation of intercellular communication during innate and adaptive immune responses. in fact, many different cells of the immune system, including dendritic cells and b and t lymphocytes, have been shown to release exosome vesicles with immune modulatory properties. these exosomes can be found in bodily fluids (reviewed in [ ] [ ] [ ] ). in , raposo et al. demonstrated that b lymphocytes infected with epstein-barr virus (ebv), a human gammaherpesvirus associated with a variety of lymphoblastoid and epithelial cancers, released exosomes containing mhc ii molecules, and that these vesicles were capable of activating specific cd + t cell clones in vitro [ ] . two years later, zitvogel et al. published a study showing that exosomes released by dendritic cells had the ability to suppress the growth of tumors in vivo. this led to the interpretation that exosomes could be used as therapeutic agents modulating immune responses [ ] . subsequent studies with ebv-infected lymphoblastoid and nasopharyngeal carcinoma cells have shown that the exosomes secreted by these cells also harbor ebv-encoded latent phase mrnas [ ] , proteins [ , , [ ] [ ] [ ] , and mature mirnas [ , ] . accumulating evidence suggests that these exosomes exert immune inhibitory effects on tumor-infiltrating lymphocytes. latent membrane protein (lmp ), the major viral oncogene expressed in most ebv-associated tumors, has been detected in exosomes and was shown to inhibit immune response, namely t lymphocyte activation and proliferation, nk cytotoxicity, and the ability of cells to produce interferon gamma [ , , ] . exosomes secreted by ebv-infected nasopharyngeal carcinoma cells also contained high amounts of the immunoregulator protein galectin- , which is able to induce apoptosis of ebv-specific cd + t cells [ , ] . ebv mirnas present in the exosomes are internalized by dc where they downregulate specific immunoregulatory genes [ ] . in contrast, exosomes released from ebv-infected b lymphocytes were found to exert a stimulatory effect on non-infected b lymphocytes, driving their proliferation, class-switch recombination, and differentiation into plasmablast-like cells [ ] . it was recently reported that exosomes also regulate innate immunity. this was illustrated with the identification of important innate immune effectors (ifi , caspase- , interleukin b (il- b), il- , and il- ) in exosomes released from ebv-infected cells. such a strategy removes these effectors from infected cells to reduce innate immunity activation [ ] . another interesting approach was proposed for hsv infection. in this case, cells export the innate immune sensor sting (stimulator of ifn genes), viral mirnas, and mrnas through exosomes that are delivered to uninfected cells [ ] . the functional significance of this strategy is still not clear, but the fact that some mirnas are able to suppress reactivation of latent virus suggests that, in specific circumstances, hsv has evolved mechanisms to restrict, rather than expand, the spread of infection. exosomes harboring hcv rna are transferred from infected cells to non-permissive plasmacytoid dcs, where viral rna can trigger a type i ifn response [ ] . it is clear that in viral infections, exosomes play a dual role in the modulation of the immune system, both serving as a host program to induce innate and adaptive immunity and as a viral strategy to evade those same responses. viral infection is thought to be responsible for % to % of all human cancers, which makes the understanding of how pathogens modulate host cell functions during their transformation program seminal, both from a scientific and a clinical perspective [ ] . seven human tumor viruses have been identified, human papillomavirus (hpv), merkel cell polyomavirus (mcv), hcv and hepatitis b virus (hbv), the members of the herpes family ebv and kaposi's sarcoma associated herpesvirus (kshv), and the retrovirus human t-lymphotropic virus- . hiv is also tumorigenic, although indirectly, since the decrease of host immunity it provokes allows cell transformation, mostly by other viruses such as kshv. it is now well established that tumor cells secrete exosomes [ , , ] , but the cancer types in which exosomes are quantitatively, qualitatively, and functionally different from healthy tissues are incompletely characterized. many studies reported differential composition of exosomes in healthy organisms versus those infected with several tumor viruses [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nasopharyngeal carcinoma cells infected with ebv produced exosomes containing lmp [ , ] . these exosomes were shown to be taken up by neighboring cells [ ] and the transcription profiles of the recipient cell were subsequently altered [ , ] . the mechanism underlying transcriptional alterations occurred via lmp -mediated increase of egfr release by exosomes that lead to activation of erk and pi k/akt pathways in epithelial, endothelial, and fibroblast cells [ ] . erk and pi k/akt are renowned factors able to promote cell growth and migration. a specific mirna composition has been found in tumor viruses' derived exosomes [ , , , , [ ] [ ] [ ] [ ] [ ] . for example, there are several types of hpv-some tumorigenic, each associated with a different mirna exosomal profile [ ] [ ] [ ] . in the case of exosomes isolated from cancer patients infected with tumorigenic hpv, the mirna content was enriched for species controlling cell proliferation, senescence, and apoptosis. the exosomal mirna compositions were dependent on the expression of the viral oncogene e /e , suggesting that this is one mechanism by which the oncogene contributes to the growth of hpv-positive cancer cells [ ] . regardless of the infection status, the significance of exosomes in tumor development was demonstrated in breast cancer, where normal cells became immortalized when incubated with exosomes derived from tumor cells [ ] . another example was the injection of rab a depleted breast carcinoma cell lines in immunocompetent mice. the lack of rab a was associated with reduced release of exosomes [ ] and poor tumor development when compared to cells containing rab a, where tumor progression was normal and formed metastasis [ ] . one of the major difficulties clinicians face is the lack of efficient methods to diagnose tumors, especially in early stages. in the case of tumors that result from viral infection, the identification of biomarkers for poor prognosis of infection would greatly benefit patients. research needs to be done to identify clear populations of exosomes involved in infection and tumors, and to clearly define functions associated to exosomes in both conditions. these topics warrant investigation, as exosomes show great promise as biomarkers for cancer and/or infection, as therapeutic agents, and have the additional advantage of being accessible without the use of invasive techniques. the mechanisms regulating the levels, content, and function of exosomes in viral infection remain poorly characterized. there are four areas that need detailed investigation. first, it is unclear how the endocytic system controls the percentage of mvbs that fuse with the plasma membrane. we hypothesize that signaling events regulate the levels of activated rabs at each step of the endocytic pathway, and that this fluctuation allows for an adjustment of the levels of biomolecules sent for degradation and/or secretion. viruses provide excellent tools to better answer these questions as many efforts have been made to understand how each virus modifies distinct endocytic compartments. the next challenge is to better understand how these changes affect the endocytic process, including exosome release. second, little is known about the biological processes sorting material to ilvs of mvbs. the inclusion of viral proteins and rnas in exosomes offers a unique system to identify signals and sorting mechanisms into ilvs. third, the functional relevance of exosomes in infection and disease remains incompletely characterized. although exosome modulation of adaptive immunity has been extensively researched, its role in innate immunity remains largely unexplored. the recent identification of rna transfer that might affect host gene expression has been an important discovery that has renewed interest in this field. the role of exosomes in viral spread is far less explored, namely its overall contribution to viral infection. interestingly, using exosomes could be a means to mitigate exposure of viral antigens and operate as an immune evasion strategy. clearly a lot has to be done in identifying viruses able to use this pathway and the mechanisms leading to the inclusion of viral proteins/rna/capsids in ilvs-this also feeds into question two. finally, exosomes and many viruses share size, shape, and molecular characteristics. technical improvements in methods to separate and obtain pure exosomal fractions will facilitate the understanding of their role in infection, namely in immune activation, viral spread, and persistence. exosomes-vesicular carriers for intercellular communication herpes simplex virus type production requires a functional escrt-iii complex but is independent of tsg and alix expression virus budding and the escrt pathway multivesicular endosome biogenesis in the absence of escrts identification of complementation groups required for post-translational events in the yeast secretory pathway reconstitution of the transport of protein between successive compartments of the golgi measured by the coupled incorporation of n-acetylglucosamine the mechanisms of vesicle budding and fusion apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics endocytosis and intracellular processing of transferrin and colloidal gold-transferrin in rat reticulocytes: demonstration of a pathway for receptor shedding exosomes as divine messengers: are they the hermes of modern molecular oncology? electron microscopic 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a and rab b control different steps of the exosome secretion pathway syndecan-syntenin-alix regulates the biogenesis of exosomes hijacking multivesicular bodies enables long-term and exosome-mediated long-distance action of anthrax toxin drosophila s cells secrete wingless on exosome-like vesicles but the wingless gradient forms independently of exosomes exosomes go with the wnt the exosome pathway in k cells is regulated by rab rab a supports exosome-dependent and -independent mechanisms that modify the tumor microenvironment and can promote tumor progression melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through met inhibition of rab gtpase activity stimulates membrane fusion in endocytosis the rab pathway is required for influenza a virus budding and filament formation a rab -and microtubule-dependent mechanism for cytoplasmic transport of influenza a virus viral rna rab a is essential for transport of the influenza virus genome to the plasma membrane apical transport of influenza a virus ribonucleoprotein requires rab -positive recycling endosome roles for the recycling endosome, rab , and rab in hantavirus release from epithelial cells respiratory syncytial virus uses a vps -independent budding mechanism controlled by rab -fip rab proteins in health and disease the dynamic rab -fips the rip /rab -fip and kinesin ii complex regulates endocytic protein recycling rab -family interacting protein and myosin vb are required for cxcr recycling and receptor-mediated chemotaxis how to get out: ssrna enveloped viruses and membrane fission replication-competent influenza a virus that encodes a split-green fluorescent protein-tagged pb polymerase subunit allows live-cell imaging of the virus life cycle trafficking of sendai virus nucleocapsids is mediated by intracellular vesicles rab a controls hiv- assembly by regulating plasma membrane levels of phosphatidylinositol , -bisphosphate hiv- nef protein is secreted into vesicles 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exosomes for intercellular communication functional delivery of viral mirnas via exosomes blood diffusion and th -suppressive effects of galectin- -containing exosomes released by epstein-barr virus-infected nasopharyngeal carcinoma cells exosomes derived from burkitt's lymphoma cell lines induce proliferation, differentiation, and class-switch recombination in b cells constitutive interferon-inducible protein -inflammasome activation during epstein-barr virus latency i, ii, and iii in b and epithelial cells modulation of dna damage and repair pathways by human tumour viruses cancer exosomes perform cell-independent microrna biogenesis and promote tumorigenesis exosomal communication goes viral exosomes: fit to deliver small rna extracellular mirnas: the mystery of their origin and function virus-encoded micrornas: an overview and a look to the future micrornas are biomarkers of oncogenic human papillomavirus infections differential expression of cellular micrornas in hpv , - , and - transfected cells human papillomaviruses modulate microrna expression to directly control genome amplification dependence of intracellular and exosomal micrornas on viral e /e oncogene expression in hpv-positive tumor cells exosomes derived from epstein-barr virus-infected cells are internalized via caveola-dependent endocytosis and promote phenotypic modulation in target cells the authors would like to acknowledge fundação para a ciência e a tecnologia, portugal, key: cord- -gmw gl r authors: saiz, juan-carlos; de oya, nereida jiménez; blázquez, ana-belén; escribano-romero, estela; martín-acebes, miguel a. title: host-directed antivirals: a realistic alternative to fight zika virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gmw gl r zika virus (zikv), a mosquito-borne flavivirus, was an almost neglected pathogen until its introduction in the americas in , where it has been responsible for a threat to global health, causing a great social and sanitary alarm due to its increased virulence, rapid spread, and an association with severe neurological and ophthalmological complications. currently, no specific antiviral therapy against zikv is available, and treatments are palliative and mainly directed toward the relief of symptoms, such as fever and rash, by administering antipyretics, anti-histamines, and fluids for dehydration. nevertheless, lately, search for antivirals has been a major aim in zikv investigations. to do so, screening of libraries from different sources, testing of natural compounds, and repurposing of drugs with known antiviral activity have allowed the identification of several antiviral candidates directed to both viral (structural proteins and enzymes) and cellular elements. here, we present an updated review of current knowledge about anti-zikv strategies, focusing on host-directed antivirals as a realistic alternative to combat zikv infection. since the beginning of the st century, a number of infectious disease threats have emerged that demand a global response. among them, severe acute respiratory syndrome virus, avian influenza in humans, pandemic influenza a (h n ), middle east respiratory syndrome coronavirus, chikungunya virus, and ebola virus have been the most threatening ones. nonetheless, the emergency of a vector-borne virus, zika virus (zikv), which is responsible for congenital malformations and other neurological and ophthalmological disorders, was hard to predict. zikv is a mosquito-borne virus belonging to the spondweni serocomplex in the genus flavivirus of the family flaviviridae [ ] . the virus has been isolated from various mosquito species, although it seems that the natural transmission vectors are mosquitoes of the genus aedes [ , ] . besides mosquito bites, viral direct human-to-human transmission can occur perinatally, sexually, and through breastfeeding and blood transfusion [ ] . the zikv genome is a single-stranded rna molecule (≈ . kb) of positive polarity encoding a single open reading frame (orf) flanked by two untranslated regions at the and ends [ ] . zikv was first isolated from the serum of a monkey in , and one year later from aedes africanus mosquitoes caught in the same area, the zika forest [ ] . until it was detected in asia in the s, the virus had been confined to africa. later on, human outbreaks were reported in the pacific islands, micronesia in and, then, in french polynesia in [ ] . the natural course of zikv infection was usually asymptomatic or produce a relatively mild illness and an uneventful recovery [ ] , hence, the virus was considered an almost neglected pathogen until its recent introduction into the americas in , when it became a threat to global health, showing increased virulence, rapid spread, since the recent outbreak in in the americas, a quite high number of possible antiviral candidates are being tested in vitro and in vivo. however, until now, no specific therapy has been approved against any flavivirus [ ] , including zikv [ ] , and, thus, current treatments are mainly directed toward the relief of symptoms, such as fever and rash, by administering antipyretics, anti-histamines, and fluids for dehydration [ ] . nevertheless, it should be noted that some commonly used drugs, such as acetylsalicylic acid, are contraindicated in zikv-infected patients, since they increase the risk of internal bleeding, and other arboviruses (dengue or chikungunya viruses) that can co-infect the patients may produce hemorrhages [ ] . due to the natural course of zikv infection, which is usually asymptomatic or produce a relatively mild illness and an uneventful recovery, when facing anti-zikv strategies, a very important point to take into account is the main target population that would benefit from it, namely immunocompromised patients and pregnant women and their fetuses [ ] . in this sense, only for some of the tested drugs their safety profiles are known [ ] . however, in cases of food and drug administration (fda) (https://www.drugs.com/) category b compounds (animal reproduction studies have failed to demonstrate a risk to the fetus and there are no adequate and well-controlled studies in pregnant women), or even in those of category c (animal reproduction studies have shown an adverse effect on the fetus and there are no adequate and well-controlled studies in humans, but potential benefits may warrant use of the drug in pregnant women despite potential risks), or d (there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans, but potential benefits may warrant use of the drug in pregnant women despite potential risks), their use in pregnancy can be contemplated if the potential benefit outweighs the risks. even more, some of the assayed compounds cross the placenta and, thus, can also benefit the fetus. nonetheless, if used, this should be done in an individualized way, conditioning dosage and timings, and always under a clinician's control where the patient is informed of the pros and cons. current search for zikv antivirals is being conducted with different approaches: by screening of compounds libraries; by the repurposing of drugs of known active efficacy against other diseases now in use in clinical practice, many of which display broad-spectrum activity; and by testing natural products. two different strategies can be applied when pursuing for antivirals, those searching for compounds directed to viral targets (direct-acting antivirals) and those aimed to target cellular components needed for the viral life cycle (host-directed antivirals). among the virus-directed drugs tested [ , ] are those acting against the viral rna-dependent rna polymerase (non-structural protein (ns )) catalytic domain, including nucleoside analogs and polymerase inhibitors; the methyltransferase catalytic domain of the ns responsible for transferring the mrna cap; the ns b-ns trypsin-like serine protease needed for proper processing of the viral polyprotein; and the ns helicase. the crystal structures of all these proteins have already been resolved and will certainly help to find new antivirals [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the same way, structures from other viral proteins are also available that could help to design zikv therapeutic alternatives, such as those of the capsid c protein [ ], whose destabilization may impair zikv multiplication, the ns [ , ], an immuno-modulator, or the envelope glycoprotein [ - ], which mediates cell binding and endosomal fusion, constitutes a major target for neutralizing antibodies, and could be also the target for virucidal compounds [ ] . on the other hand, it has also been reported that passive transfer of neutralizing antibodies to pregnant mice suppresses zikv multiplication, inhibits cell death, reduces the number of progenitor neuronal cells, and prevents microcephaly [ , ] . likewise, administration of monoclonal antibodies (mabs) recognizing the domain iii of the zikv-e protein protect mice of lethal zikv challenge [ , ] and other mabs are able to bind and neutralize zikv, including those directed against the e dimer epitope [ ] . human polyclonal antibodies produced in transchromosomal bovines also protect mice from zikv lethal infection, eliminated zikv induced tissue damage in the brain and testes, and protected against testicular atrophy [ ] . thus, administration of therapeutic antibodies seems to also be a potential strategy against zikv. nevertheless, it should be noted that, although still controvertial in the case of zikv infection [ ] , the well-known antibody dependent enhancement effect (ade) [ ] , of which dengue virus (denv) is the prototypic model, may potentiate the risk of disease exacerbation. flaviviruses have small rna genomes (around . kb in length) and thus require many host factors and co-option of cellular metabolic pathways to successfully infect host cells and propagate efficiently [ ] . this offers an opportunity to search for host targets as therapeutic tools that, in many instances, as they are shared by different members of the flaviviridae family, can be envisaged as pan-flaviviral antivirals [ ] [ ] [ ] . this strategy can be directed to host factors implicated in infection, pathogenesis, and in the immune response, as it has been shown for denv and the west nile virus (wnv) [ ] . in addition, their effect would be less prone to the emergence of mutants that will escape their action, as often occurs with drugs targeting viral components. consequently, this kind of approach could ideally lead to the discovery of broad spectrum antivirals that could provide low cost but effective tools for the control of flaviviral threats. different approaches are being used to identify potential host factors as therapeutic targets against flaviviruses including the analyses of transcript levels (e.g., next generation rna sequencing) for altered expression patterns during infection, proteome changes, kinases activities variations, and protein-rna interactions (e.g., two-hybrid screenings and affinity chromatography). likewise, functional analysis can be applied by overexpressing cdnas or by rnai-mediated loss of function screens using dsrna, sirna, or shrna libraries, although it should be noted that in some cases downregulation is inefficient and some genes have redundant functions [ ] . replicons may also be used to specifically assay replication activity [ , ] . theoretically, host-acting antivirals can be directed to any molecule or pathway implicated in the different steps of the viral life cycle, from early events (binding, entry, and fusion), to the formation of the replication complex, and the viral maturation and egress. the first step of zikv infection is its binding to the cellular receptor ( figure ). several molecules have been proposed as a zikv receptor (members of the tyro /axl/mer (tam) family of receptor tyrosine kinases, t-cell immunoglobulin and mucin domain (tim) and dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin (dc-sign)) that are expressed in different neuronal and non-neuronal permissive cell types. these molecules are also receptors for other viruses, including flaviviruses such as denv and wnv, regulate several cellular activities (adhesion, migration, proliferation, and survival, release of inflammatory cytokines, antigen uptake, and signaling), and play important roles in the host's response to infection [ ] . however, elimination of a known receptor does not necessarily result in complete protection from viral infection, since flaviviruses use different receptors and, thus, there is always redundancy and alternatives. for instances, inhibiting, downregulating, knocking-down, or ablating axl, although in some cases they reduce zikv infection, they do not completely abolish it, pointing to the use of different cell surface receptors on different cell types [ ] [ ] [ ] [ ] . different molecules have been shown to inhibit zikv infection at the entry step ( figure ). r (an axl kinase inhibitor) and myd (an axl decoy receptor) compromises, but do not completely abolish, zikv infection of glial cells [ ] . r , as well as cabozantinib, an inhibitor of axl phosphorylation, that are currently in clinical trials for anticancer activities, significantly impairs zikv infection of human endothelial cells in a dose-dependent manner by affecting a post-binding step [ ] . likewise, curcumin, a widely used food additive and herbal supplement, reduces zikv infection in cell culture inhibiting cell binding while maintaining viral rna integrity [ ] , as does suramin, an anti-parasitic that interferes with attachment to host cells and with virion biogenesis by affecting glycosylation and maturation [ , ] . once zikv binds to the cell receptor, like other flaviviruses, it is internalized through clathrin-mediated endocytosis and transported to the endosomes with the involvement of cellular actin and microtubules to establish a productive infection ( figure ) [ ] . after internalization, to start translation and replication, the viral genome is released inside the cytoplasm by fusing the viral envelope with the membranes of the cellular endosomes, a process triggered by acidic ph inside them [ , ] . nanchangmycin, an insecticide and antibacterial polyether, inhibits zikv multiplication and, although the exact mechanism of action has not been completely elucidated, it probably targets axl and blocks clathrin-mediated endocytosis [ ] . acid endosomal ph triggers rapid conformational changes on viral envelope protein that result in its fusion with endosomal membrane in a ph-dependent manner, thus allowing nucleocapsid release to the cytoplasm for genome uncoating ( figure ). the optimal ph for conformational rearrangements and viral fusion is . - . , and these processes are likely dependent on the presence of cholesterol and specific lipids in the target membrane [ ] . these processes can be potentially druggable, and in fact, arbidol, a broad-spectrum antiviral and immunomodulatory use for human influenza a and b infections, inhibits zikv multiplication in cell culture probably because it intercalates into membrane lipids leading to the inhibition of membrane fusion between virus particles and plasma membranes, and between virus particles and the membranes of endosomes [ ] . chlorpromazine, an antipsychotic drug that also inhibits clathrin-mediated endocytosis, reduced zikv infection, confirming the requirement for clathrin-mediated endocytosis of zikv [ ] . in addition, -hydroxycholesterol ( hc) is increased in zikv-infected human embryonic cells and brain organoids, and reduces viremia and viral loads without affecting viral binding, but blocking internalization and suppressing viral and cell membranes fusion [ ] . even more, hc reduces mortality and prevents microcephaly in zikv-infected mice, and also decreases viral loads in the urine and serum of treated non-human infected primates [ ] . daptomycin, a lipopeptide antibiotic that inserts into cell membranes rich in phosphatidylglycerol, which suggests an effect on late endosomal membranes enriched in this lipid, has also been described as a zikv inhibitor [ ] . the dependence on endosomal acidification for zikv infection also provides a host target suitable for antiviral intervention. for instance, obatoclax (or gx - ), an anti-neoplastic and pro-apoptotic inhibitor of the bcl- that targets cellular mcl- , impairs zikv endocytic uptake by reducing the ph of the endosomal vesicles in cell culture, and thereby most likely inhibits viral fusion [ , ] . however, obatoclax, which presents a low solubility, has not produced satisfactory results in clinical trials for hematological and myeloid diseases. saliphenylhalamide (saliphe), which targets vacuolar adenosine triphosphatase enzyme (atpase) and blocks the acidification of endosomes, inhibits zikv multiplication in human retinal pigment epithelial cells [ ] that are natural targets for zikv infection [ ] . similar results were found by adcock et al. ( ) with saliphe using a different screening [ ] ; however, they reported that, contrary to that described by others [ ] , other compounds that interfere with the endocytic pathway, such as dynasore, that blocks clathrin-mediated endocytosis, or monensin, a cation transporter, were either toxic for the cells used or did not show any anti-zikv activity, as neither did chloroquine (cq). these contradictory results are probably explained by the different methodologies, cell types, and, to a lower extent, viral strains used to analyze the antiviral activities of the compounds and suggest that compounds showing different activities should be carefully evaluated before going further with investigations. in this line, and contrary to above mentioned report [ ] , cq, an fda-approved anti-inflammatory -aminoquinoline and an autophagy inhibitor widely used as an anti-malaria drug that is administered to pregnant women at risk of exposure to plasmodium parasites, was shown to have anti-zikv activity in different cell types (vero cells, human brain microvascular endothelial cells (hbmecs), and human neural stem cells (nscs)), affecting early stages of the viral life cycle, possibly by raising the endosomal ph and inhibiting the fusion of the envelope protein to the endosomal membrane [ , ] . cq has been shown to reduce placental and fetal zikv infection [ ] , and also attenuate zikv-associated morbidity and mortality in mice and protect the fetus from microcephaly [ ] . even more, cq attenuated vertical transmission in zikv-infected pregnant interferon signaling-competent swiss jim lambert (sjl) mice, significantly reducing fetal brain viral loads [ ] . similarly, cq, and other lysosomotropic agents (ammonium chloride, bafilomycin a , quinacrine, mefloquine, and n-tert-butyl isoquine (gsk )) that neutralize the acidic ph of endosomal compartments, block infection of a human fibroblast cell line and vero cells [ , ] . additionally, by medicinal chemistry-driven approaches, a series of new , -bis(trifluoromethyl)quinoline and n-( -(arylmethylimino)ethyl)- -chloroquinolin- -amine derivatives have been proved to inhibit zikv replication in vitro with a higher potency than chloroquine or mefloquine [ , ] . more recently, by screening fda-approved drugs using a cell-based assay, it has been shown that amodiaquine, another antimalarial drug, also has anti-zikv activity in cell culture by targeting early events of the viral replication cycle [ ] . niclosamide, a category b antihelmintic drug approved by fda, was capable of inhibiting zikv infection, and although its antiflaviviral effect has been associated to its ability to neutralize endolysosomal ph and interfere with ph-dependent membrane fusion, in the case of zikv, it seems that it was affecting other post-entry steps [ ] . in addition, recently, it has been reported that niclosamide decreases zikv production, partially restores differentiation, and prevents apoptosis in human induced nscs; even more, it can partially rescue zikv-induced microcephaly and attenuate infection in a developed humanized zikv-infected embryo model in vivo [ ] . likewise, tenovin- , which represses cell growth and induces apoptosis in cells expressing p by inhibiting the protein-deacetylating activities of sirt and sirt and, thus, affects endosome functions, potently inhibits zikv infection in primary placental fibroblast cells [ ] . iron salt ferric ammonium citrate (fac) also inhibits zikv infection through inducing viral fusion and blocking endosomal viral release by promoting liposome aggregation and intracellular vesicle fusion [ ] . overall, these studies evidence the potential of targeting viral entry to combat zikv. once zikv-rna is released from the endosomes in the cytoplasm, it acts as mrna to synthesize the negative-strand viral rna that directs positive-strand rna synthesis (figure ) [ ] . silvestrol, a natural compound isolated from the plant aglaia foveolata that it is known to inhibit the asp-glu-ala-asp (dead)-box rna helicase eukaryotic initiation factor- a (eif a) required to unwind structured -untranslated regions and thus impairing rna translation, exerts a significant inhibition of zikv replication in a cells and primary human hepatocytes [ ] . n-( -hydroxyphenyl) retinamide (fenretinide or -hpr), an activator of retinoid receptors that inhibits the proliferation of cancer cells and can induce apoptosis, inhibits zikv in cell culture and significantly reduces both serum viremia and brain viral burden in mice by decreasing the rate of viral rna synthesis, though not via direct inhibition of the activity of the viral replicase [ ] . zikv relies on polyamines for both translation and transcription [ ] , so that, drugs targeting the polyamine biosynthetic pathway, such as difluoromethylornithine (dfmo or eflornithine), an fda-approved drug that is used to treat trypanosomiasis, hirsutism, and some cancers, as well as diethylnorspermine (denspm) limit viral replication in bhk- cells [ ] . zikv replication and particle morphogenesis take place associated with a virus-induced organelle-like structure derived from the membrane of the endoplasmic reticulum (er) (figure ) [ ] . de novo synthesized positive strand-rna, once packaged, form enveloped immature virions in the er, enter the secretory pathway and, then, in the trans-golgi network, the prm is cleaved before the virus is released from the infected cell ( figure ) [ , ] . er-membrane multiprotein complexes, such as the oligosaccharyltransferase (ost) complex, have been reported to be critical host factors for flavivirus multiplication. in this regard, it has been shown that the n-linked glycosylation inhibitor- (ngi- ) chemical modulator of the ost complex blocks zikv-rna replication in different cell types [ ] . similarly, the host er-associated signal peptidase (spase) is an essential, membrane-bound serine protease complex involved in cleavage of the signal peptides of newly synthesized secretory and membrane proteins at the er and also for processing of the flavivirus prm and e structural proteins [ ] . it has also been reported that cavinafungin, an alaninal-containing lipopeptide of fungal origin, potently inhibits growth of zikv-infected cells [ ] . nitazoxanide, a broad-spectrum antiviral agent approved by the fda as an antiprotozoan and with potential activity against several viruses in clinical trials (rotavirus and norovirus gastroenteritis, chronic hepatitis b, chronic hepatitis c, and influenza), also inhibits virus infection targeting a post-attachment step, most likely virus genome replication [ ] . likewise, brefeldin a, a penicillium sp. product that inhibits protein transport from the er to the golgi apparatus, inhibits zikv multiplication [ ] , as does emetine, an anti-protozoal agent that inhibits both zikv ns polymerase activity and disrupts lysosomal function [ ] . zikv infection leads to cell-death by inducing host caspase- and neuronal apoptosis during its propagation [ ] . thereby, bithionol, a caspase inhibitor, inhibits zikv strains of different geographical origin in vero cells and human astrocytes [ ] . similarly, by using a drug repurposing screening of over molecules, it was found that emricasan, a pan-caspase inhibitor that restrains zikv-induced increases in caspase- activity and is currently in phase clinical trials in chronic hepatitis c virus (hcv)-infected patients, protected human cortical neural progenitor cells (npc) in both monolayer and three-dimensional organoid cultures, showing neuroprotective activity without suppression of viral replication [ ] . additionally, bortezomib, a dipeptide boronate proteasome inhibitor approved for treatment of multiple myeloma and mantle cell non-hodgkin's lymphoma that regulates the bcl- family of proteins, has also been described as a zikv inhibitor [ ] . similarly, different cyclin-dependent kinase (cdk) inhibitors, such as (alphas)- -(acetylamino)-alpha-methyl-n-( -( -methylethyl)- -thiazolyl)benzeneacetamide (pha- ), reduced zikv-infection and propagation [ ] . however, cdk inhibitors should not be suitable for the treatment of pregnant women but could be useful for the treatment of other non-pregnant patients, preventing the complications associated with zikv infection. the need for specific host lipids for flavivirus replication and particle envelopment make lipid metabolism a potential target for an antiviral search [ , ] , and, even though manipulating a major metabolic pathway such as lipid biosynthesis can be envisaged as a dangerous antiviral approach due to the undesirable effects that could be detrimental for the host, current use of drugs such as ibuprofen and aspirin (cyclooxygenase- (cox- ) inhibitors) or statins ( -hidroxi- -metil-glutaril-coa (hmg-coa) reductase inhibitors) highlights the feasibility of lipid-based therapeutics [ , ] . accordingly, inhibition of key enzymes involved in fatty acid synthesis, such as acetyl-coa carboxylase (acc) [ ] , and fatty acid synthase (fasn) [ ] [ ] [ ] , are potential targets for anti-zikv therapy. in this line, we have reported that nordihydroguaiaretic acid (ndga) and its derivative tetra-o-methyl nordihydroguaiaretic (m n or terameprocol), two compounds that disturb the lipid metabolism probably by interfering with the sterol regulatory element-binding proteins (srebp) pathway, inhibit the infection of zikv and wnv, likely by impairing viral replication, as did other structurally unrelated inhibitors of the srebp pathway, such as -[(diethylamino)methyl]-n-[ -( -methoxyphenyl)ethyl]-n-( r)- -pyrrolidinyl-benzamide dihydrochloride (pf- ) and fatostatin [ ] . in the same way, the dependence on cholesterol for different processes during flavivirus infection also provides a suitable target for antiviral strategies. as mentioned above, hc reduces viremia and viral loads in vitro, and also reduces mortality and prevent microcephaly in mice, and decreases viral loads in the urine and serum in non-human infected primates [ ] . lovastatin and mevastatin are hypolipidemic agents (hmg-coa inhibitors) belonging to the family of statins that are widely used for lowering cholesterol in patients with hypercholesterolemia and have been previously shown to present antiviral activity against dengue and hepatitis c viruses. both agents have been proposed as therapeutic candidates against zikv [ ] . in fact, lovastatin attenuates nervous injury in animal models of gbs [ ] . likewise, imipramine, an fda-approved antidepressant, inhibits zikv-rna replication and virion production in human skin fibroblasts, probably by interfering with intracellular cholesterol transport [ ] . regarding sphingolipid metabolism, which has been involved in flavivirus infection [ ] , treatment with the neutral sphingomyelinase inhibitor gw reduced zikv production by affecting viral morphogenesis [ ] as described for other flaviviruses [ ] . finally, since adenosine monophosphate-activated protein kinase (ampk) is a master regulator of lipid metabolism, its activation by pf- or metformin reduced zikv infection by impairing viral replication [ , ] . thus, targeting lipid metabolism could provide therapeutic alternatives for the discovery of host-directed antivirals against zikv. the ns protein is the viral rna-dependent rna polymerase responsible for the rna synthesis that also inhibits interferon (ifn) signaling by acting over the signal transducer and activator of transcription (stat ) protein [ ] , being, thus, a major target for antiviral design. besides the proven antiviral activities of different nucleosides analogs and inhibitors of the zikv-ns [ ] , several inhibitors of the biosynthesis of nucleosides (purines and pyrimidines) also impair zikv replication (figure ). ribavirin is an inhibitor of the inosine monophosphate dehydrogenase (impdh) with antiviral activity to several rna viruses [ ] , but its mechanism of action is not entirely clear. it may act as a guanosine synthesis inhibitor, a viral cap synthesis inhibitor, a viral rna mutagen, and as an inducer of lethal mutagenesis [ ] [ ] [ ] . by using a cell-based assay, no antiviral activity of the drug was initially observed [ ] but, later on, it was reported that although no activity against zikv was detected in vero cells, the drug did inhibit virus multiplication in human cell lines, including liver huh- and rhabdomyosarcoma (rd) cells [ ] . further studies have confirmed an inhibitory activity of ribavirin against zikv strains of different geographical origin in various types of cells, such as human neural progenitor cells (hnpcs), human dermal fibroblasts (hdfs), human lung adenocarcinoma cells (a ), and even in vero cells [ ] [ ] [ ] . still more, the drug was shown to abrogate viremia in zikv-infected stat- -deficient mice [ ] , which lack type i ifn signaling, are highly sensitive to zikv infection, and exhibit a lethal outcome. two other inhibitors of impdh, merimepodib (mmpd or vx- ) [ ] and mycophenolic acid (mpa) [ , , ] also inhibit zikv-rna replication in different cell types, including huh- cells, human cervical placental cells, and neural stem and primary amnion cells. however, other authors [ ] have described that mpa have little effect on zikv replication and showed significant cell toxicity. likewise, azathioprine, another inhibitor of purine synthesis and immunosuppressant, impaired zikv replication in hela and jeg cells [ ] ; nonetheless, its use in pregnant women is not recommended. the above described contradictory results stress again the differences that drug treatments may have as a consequence of the different viral strains, cell types, and methodologies used to assess them. as with the inhibitors of purine biosynthesis, compounds inhibiting the synthesis of pyrimidines have also effect on zikv replication (figure ). so that, the virus was highly susceptible to brequinar and cid treatments in cell culture [ ] . however, it should be noted that it has been reported that brequinar, as well as dd , antiviral activity may not be due to pyrimidine deprivation, but rather to the induction of the cellular immune response [ , ] . similarly, other inhibitors of the pyrimidine synthesis, such as gemcitabine, an activator of cellular caspases [ , ] , and, although with a lower efficiency probably due to its lower solubility, -azauridine and finasteride, a -azasteroid analog of testosterone that inhibit type ii and type iii α-reductase and is being tested for benign prostatic hyperplasia and male pattern baldness, reduce zikv replication [ , ] . several other compounds have been shown to have anti-zikv activity by inhibiting viral entry and/or rna synthesis, although their mechanisms of action have not yet been fully elucidated. among them are antiparasitics such as ivermectin (used mainly against worms infections) and pyrimethamine (a folic acid antagonist that inhibits the dihydrofolate reductase and, thus, dna and rna synthesis, is classified as a pregnancy category c, and was initially used to treat malaria and now toxoplasmosis and cystoisosporiasis) [ ] ; antibiotics such as azithromycin that prevents infection, replication, and virus-mediated cell dead [ ] , and kitasamycin (a natural product from streptomyces narbonensis that inhibits protein biosynthesis) [ ] ; drugs used to prevent chemotherapy-induced nausea and vomiting as palonosetron (a fda-approved -ht antagonist) [ ] ; antidepressants like sertraline (a selective serotonin reuptake inhibitor) [ ] and cyclosporine (that is also use for rheumatoid arthritis, psoriasis, crohn's disease, nephrotic syndrome, and in organ transplants, is believed to lower the activity of t-cells, and is currently in clinical trials for tis possible use in ameliorate neuronal cellular damage) [ ] . similarly, after chemical screening, it was found that hippeastrine hydrobromide (hh), an active component of traditional chinese medicine, and amodiaquine dihydrochloride dihydrate (aq), an fda-approved drug for treatment of malaria, inhibit zikv infection of human pluripotent stem cell-derived cortical npcs and in adult mouse brain in vivo even when the infection was already ongoing but, again, their mechanisms of action are not known [ ] . besides drugs that act against host targets directly implicated in the viral cycle, there are compounds that can prevent undesirable effects of zikv infection. in this regard, zikv infection leads to massive neuronal damage, especially of neural progenitor cells, and neurodegeneration [ ] [ ] [ ] , via both direct replication in neuronal cells and possibly through increased excitotoxicity via over activation of n-methyl-d-aspartate receptor (nmdar)-dependent neuronal excitotoxicity in nearby cells. memantine, a pregnancy category b fda-approved drug widely used to treat patients with alzheimer's disease, as well as other nmdar blockers (dizocilpine, agmatine sulfate, or ifenprodil), prevents neuronal damage and death and intraocular pressure increase induced by zikv infection in infected mice, but it does not affect virus replication, pointing to its possible use to prevent or minimize zikv-related microcephaly during pregnancy [ ] . ebselen (ebs), an antioxidant that reduces oxidative stress and improves histopathological features in a testicular injury study model and is currently in clinical trials for various diseases, showed minor effects in reducing zikv progeny production and viral e protein expression and on overall survival and viremia level of challenged ag mice; however, it should be noted that ebs reduced some zikv-induced effects, such as testicular oxidative stress, leucocyte infiltration, and production of pro-inflammatory response, whereas, in a model of male-to-female mouse sperm transfer, the drug improved testicular pathology and prevented the sexual transmission of zikv [ ] . ifns play a key role in the elimination of pathogens and they are release upon the activation of the innate immune response by infecting viruses. in this way, zikv infection induces ifn signaling pathways and further activates cytoplasmic retinoic acid inducible gene protein (rig )-like receptors (rlrs) and several type i and iii ifn-stimulated genes, driving to the subsequent activation of the janus kinase (jak)/stat innate immune pathway that confer resistance to zikv infection [ ] . different studies showed that ifn-α, ifn-β, and ifn-γ inhibit zikv replication in cell culture [ , , ] , and that treatment of pregnant mice with ifn-λ reduced zikv infection [ ] . in addition, ifitm and ifitm , which are interferon-induced transmembrane proteins, impair early stages of zikv infection. even more, ifitm prevents zikv-induced cell death [ ] . likewise, it has been reported that an interferon-activating small molecule ( -( -fluorophenyl)- -( -isopropyl- , , -thiadiazol- -yl)- , -ihydrochromeno [ , -c] pyrrole- , -dione (avc) strongly inhibits replication of zikv in cell culture [ ] . however, it is also known that the virus is capable of evading type i ifn responses by acting over the jak/stat signaling pathway [ , [ ] [ ] [ ] , and that type i ifns might be mediators of pregnancy complications, including spontaneous abortions and growth restriction [ ] . in any case, use of ifn against zikv, alone or in combination with other antivirals, deserve further studies. by screening a library of known human micrornas (mirnas), small, noncoding rnas (sncrnas) that modulate gene expression post-transcriptionally and regulate a broad range of cellular processes, several mirnas were found to inhibit zikv by increasing the capability of infected cells to respond to infection through the interferon-based innate immune pathway [ ] . another alternative is intervening over epigenetic regulation by using epigenetics modulators. for instance, histone h k methyltransferases (ezh and ezh ) suppress gene transcription and it has been shown that inhibitors such as -[( s)-butan- -yl]-n-[( , -dimethyl- -oxo- h-pyridin- -yl)methyl]- -methyl- -( -piperazin- -ylpyridin- -yl)indole- -carboxamide (gsk- ) reduce zikv multiplication in cell culture through the activation of cellular antiviral and immune responses [ ] . in any case, further studies are needed to evaluate the potential therapeutic capability of these immunomodulators against zikv infection. a great effort is being lately made to find compounds to fight zikv infection by applying different approaches, from repurposing of drugs with known antiviral activity to the screening of bioactive molecules from different libraries, as well as natural products. however, most of the already tested drugs have been found to inhibit viral replication in vitro, and only a few have been tested in vivo. hence, since, in many instances, the results will be difficult to extrapolate to humans, it would be hard for most of the tested antivirals to complete the entire drug development pipeline. in addition, it should be remarked that many drugs could have untoward effects and, thus, careful evaluation should be conducted before using them in clinical practice, as the main target populations for anti-zikv therapy will be pregnant women and patients with other medical complications. many of the already tested drugs are directed against viral structural and enzymatic proteins, including, for instance, anticancer and anti-inflammatory molecules, antibiotics, and antiparasitics; however, it is well known that this approach can easily lead to the appearance of resistance. since flaviviruses require many host factors and co-option of cellular metabolic pathways to successfully infect host cells and propagate efficiently, this offers an opportunity to search for host targets as therapeutic tools that, in many instances, can be broad spectrum agents, and which effect would be less prone to the emergence of mutants that will escape their action. because of that, and even though 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infection inhibitors of the histone methyltransferases ezh / induce a potent antiviral state and suppress infection by diverse viral pathogens key: cord- - sl xfur authors: sinha, anirban; lutter, rené; dekker, tamara; dierdorp, barbara; j. sterk, peter; frey, urs; delgado-eckert, edgar title: can measurements of inflammatory biomarkers be used to spot respiratory viral infections? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: sl xfur accurate detection of human respiratory viral infections is highly topical. we investigated how strongly inflammatory biomarkers (feno, eosinophils, neutrophils, and cytokines in nasal lavage fluid) and lung function parameters change upon rhinovirus infection, in order to explore their potential use for infection detection. to this end, within a longitudinal cohort study, healthy and mildly asthmatic volunteers were experimentally inoculated with rhinovirus , and time series of these parameters/biomarkers were systematically recorded and compared between the pre- and post-infection phases of the study, which lasted two months and one month, respectively. we found that the parameters’/biomarkers’ ability to discriminate between the infected and the uninfected state varied over the observation time period. consistently over time, the concentration of cytokines, in nasal lavage fluid, showed moderate to very good discrimination performance, thereby qualifying for disease progression monitoring, whereas lung function and feno, while quickly and non-invasively measurable using cheap portable devices (e.g., at airports), performed poorly. infections of the respiratory tract in humans are a major cause for global morbidity and mortality. about % of such infections are caused by viruses, the most prevalent being influenza, parainfluenza, respiratory syncytial virus (rsv), coronavirus, adenovirus, and rhinovirus [ ] . early detection and accurate identification of the virus in potentially affected individuals are necessary for appropriate and timely remedies and management. however, the differential diagnosis can be difficult due to nonspecific clinical presentation and symptoms [ ] , particularly in premorbid patients with, for example, bronchial asthma. historically, examination of clinical symptoms in combination with virus isolation, serological tests, imaging, and chest radiography [ ] have served as mainstays of viral detection [ ] . later, chest computed tomography [ ] , antibody staining and detection methods complemented with immune assays paved the way for better diagnostics but still lacked the specificity and sensitivity required for accurate detection. in the last two decades, there has been a remarkable improvement in the diagnosis of respiratory pathogens with the availability of molecular assays. indeed, mainly due to the advent of polymerase chain reaction (pcr) techniques, highly sensitive and robust methods such as nucleic acid sequence-based amplification (nasba), loop-mediated isothermal amplification (lamp), and micrornas [ , [ ] [ ] [ ] have come into the picture. multiplex assays coupled to microfluidics and high-resolution imaging constitute further developments towards more accurate diagnostics [ ] . these latest diagnostic tools rely on either finding the virus or traces of it in the patient's body, or virus specific antibodies generated by the patient's immune system. a topical example is sars-cov- . currently, on the one hand, the most accurate diagnosis method to detect sars-cov- infections in humans is based on detecting amplified nucleic acid sequences of the virus genome [ ] . however, pcr technology may not be fully accurate [ , ] and often requires multiple testing for reliable results [ ] . on the other hand, clinically available parameters for airways assessment such as inflammatory and lung function indices have not been traditionally used for the diagnosis of viral respiratory infections. this is mainly due to the fact that pathological changes in such indices have been found to lack pathogen-specificity [ , ] . furthermore, to the best of our knowledge, the discrimination accuracy of such parameters for the detection of any viral respiratory infection in humans, regardless of the specific virus involved, has never been systematically tested. whether a physiological parameter or biomarker qualifies for the detection of a viral respiratory infection depends on how accurately it can discriminate between infected and uninfected states. this can only be the case if the change in the parameter/biomarker elicited by the infection is stronger than its natural fluctuations during uninfected phases. moreover, ideally, this should be independent of other underlying disease processes, such as chronic ailments. therefore, we aimed at investigating how reliable inflammatory biomarkers and lung function are for the detection of respiratory viral infections in humans, by systematically comparing the values of these parameters/biomarkers during the participants' pre-and post-infection state within a longitudinal study cohort. our approach consisted of using longitudinally measured inflammatory biomarkers and lung function parameters to assess whether post-infection values exceeded their natural variability during uninfected phases. because of ethical reasons, we focused on experimental infection of humans in vivo with rhinovirus (rv ), which is a not particularly virulent pathogen that causes the common cold. in a prospective, longitudinally designed study comprising healthy and mildly asthmatic subjects, we measured time series of a set of inflammatory/immune biomarkers and standard lung function [ ] . the cohort demographics have been previously published in [ ] and are also summarized in table below. the serum antibody titer of rv required had to be less than : for all participants before enrollment in order to exclude any participants whose immune system had already been exposed to the virus. according to our previous findings, a serum antibody titer less than : is sufficient to exclude such participants [ ] . however, to increase recruitment of participants and without compromising the validity of the study outcomes, a relaxed titer of : was chosen. the participants were within - years of age and without concomitant disease or pregnancy. asthmatic subjects were not on steroids, at least in the previous weeks before study screening. the inclusion/exclusion criteria for participants along with the study design have been published in detail before [ ] . a summary of inclusion criteria can be found in table below. the parameters screened were cellular immune markers including eosinophil and neutrophil cell numbers in nasal lavage fluid. molecular immune markers, also measured in nasal lavage fluid, were the following cytokines: ifn-gamma, il- β, il- , il- , il- a, il- , il- , il- , ip- , and tnf-alpha, which are biomarkers known to play a role in asthma, inflammation, and rv infection [ ] . the inflammatory biomarker, measured in the exhaled air, was nitric oxide (feno). the lung function parameters measured were peak expiratory flow (pef), forced expiratory volume in st second (fev ), forced vital capacity (fvc), and the ratio fev /fvc. these parameters/biomarkers were repeatedly captured during two months prior to and one month following an experimental rv infection induced by controlled and deliberate inoculation with rv of healthy and mildly asthmatic volunteers [ ] . the sampling frequencies of the various parameters/biomarkers are detailed below in table . in order to minimize the influence of diurnal variations of the cytokines measured in nasal lavage fluid, for any given participant, the sampling visits were scheduled in such a way that samples were collected at approximately the same time of the day. the study protocol was approved by the hospital medical ethical committee. all study participants provided written informed consent of participation. the cohort study has been registered in the netherlands trial register, nl (ntr ). the efficacy of the inoculation with rv was established using serum antibody tests (rv seroconversion), clinical symptoms, and rhinovirus polymerase chain reaction (pcr) conducted on nasal lavage fluid taken from every participant after the inoculation. the viral load just after a few days from rhinovirus challenge, along with the seroconversion for antibodies against rhinovirus (measured at the end of the study for each participant), are summarized in table below. see also supplementary materials for symptoms data. in addition, the development of a host response was carefully assessed for each cohort participant, as described previously [ ] . all these tests provided strong evidence supporting the fact that all participants were effectively infected with the rv after inoculation. nevertheless, we excluded participants a and h from the analysis due to their negative outcomes of the viral load test during the first days after inoculation and negative seroconversion tests at the end of the study (see table ). thus, only those participants were included in our analysis who, either had a positive viral load in nasal lavage fluid when measured during the first days after inoculation, or a positive seroconversion test at the end of the study, or both. all participants were continuously monitored throughout the study. clinical symptoms and occurrence of any concomitant infection due to any exposures to pathogens apart from the experimental challenge were carefully recorded using a detailed questionnaire (see supplement materials), which was filled out at every visit. indeed, during the pre-challenge phase, none of the participants reported symptoms of any concomitant infection that could possibly confound the results during this phase of the study. apart from that, pcr tests using primers from a panel of common respiratory viruses were performed for every individual before experimental challenge to rule out the occurrence of concomitant infections. table . overview of the viral load (copies/ml) measured in nasal lavage fluid from each participant. samples of nasal lavage fluid were collected on days , , and after challenge. na means no sample was collected. the rightmost column displays, for each participant, the results of the antibody test against rhinovirus (rv) (from seroconversion) performed at the last visit of the study (approximately days after challenge). = no response and = response. for every inflammatory/immune/lung functional biomarker separately, we calculated the average value over the first days after inoculation and compared it to the average calculated over every possible time interval of consecutive days prior to the inoculation. previously published work has suggested that all important pathophysiological and immunological changes elicited by a rhinovirus infection in humans were most likely to happen within a time interval of days after exposure. this constituted the rationale behind our choice of time interval or "window" length [ ] . in order to assess the time dependency of the above-mentioned comparison of averages, the -day windows prior to the inoculation were glided, one day at a time. the start position of the gliding window is expressed relative to the day of the viral inoculation, which is marked as day . chronologically speaking, the first pre-inoculation window considered in our analysis started on day - relative to the day of the inoculation, and the last one at position - (the last window contains the day of the inoculation). see figure below for more details. viruses , , x for peer review of for every inflammatory/immune/lung functional biomarker separately, we calculated the average value over the first days after inoculation and compared it to the average calculated over every possible time interval of consecutive days prior to the inoculation. previously published work has suggested that all important pathophysiological and immunological changes elicited by a rhinovirus infection in humans were most likely to happen within a time interval of days after exposure. this constituted the rationale behind our choice of time interval or "window" length [ ] . in order to assess the time dependency of the above-mentioned comparison of averages, the day windows prior to the inoculation were glided, one day at a time. the start position of the gliding window is expressed relative to the day of the viral inoculation, which is marked as day . chronologically speaking, the first pre-inoculation window considered in our analysis started on day - relative to the day of the inoculation, and the last one at position - (the last window contains the day of the inoculation). see figure below for more details. figure . graphical representation of a participant's biomarker time series xi. the chronological order of measurements of the biomarker is expressed in days from the day of inoculation, which is marked as "day ". negative indexes represent measurements taken before the viral challenge, whereas positive indexes belong to measurements conducted after the challenge. note that for those biomarkers not measured on a daily basis, some of the values in the time series are missing. a moving figure . graphical representation of a participant's biomarker time series x i . the chronological order of measurements of the biomarker is expressed in days from the day of inoculation, which is marked as "day ". negative indexes represent measurements taken before the viral challenge, whereas positive indexes belong to measurements conducted after the challenge. note that for those biomarkers not measured on a daily basis, some of the values in the time series are missing. a moving window (depicted in green), covering ten days, glides, one day at a time, during the pre-challenge phase, starting from day − down to day − . for each window, the average over the ten days the window covers is calculated. for example, the window starting on day − yields the average µ - . after the challenge, only one window is contemplated, namely the window covering the first ten days immediately after inoculation. this window yields the average µ . for every participant, there is such an average µ j and µ , respectively, where j = − , . for a given day j between - and - during the pre-challenge phase, and for a given subgroup of the cohort (healthy, asthmatics, and pooled), the empirical distribution of averages µ j were compared to the empirical distribution of averages µ using a two-tailed paired mann-whitney u test. the results (p-values) of these comparisons are depicted for the biomarkers il- and feno in the top panels of figures and below. summary statistics for the pooled group can be found in tables "summary statistics of the averages of il- and feno concentration within each window" in supplementary material data. furthermore, roc curves were constructed assuming that the collection of averages µ j is characteristic of uninfected individuals, and the collection of averages µ is characteristic of infected individuals. the areas under these roc curves (auc) are depicted for the biomarkers il- and feno in the bottom panels of figures and below. the above-mentioned comparison of within-window averages was not done individually, but rather for all cohort participants using a two-tailed paired mann-whitney u test. thus, the test aims at establishing whether the distributions of values of a given parameter/biomarker prior to and during the infection are significantly different. for a given pre-inoculation window, the p-value resulting from this test is indicative of the ability of a given inflammatory/immune or lung function biomarker to discriminate between healthy and infected states as a function of the starting position of the pre-inoculation window at hand (see top panels in figures and ) . thereby, we looked at how the range observed during the healthy state varied in time and whether it remained, over time, statistically distinct from the range observed during the infected state. furthermore, this discriminatory ability was also quantitatively assessed by constructing a receiver operating characteristic (roc) curve [ ] and calculating the area under the same (see bottom panels in figures and ) . the area under a roc curve (auc) is an effective way to summarize the overall discrimination accuracy of a given parameter/biomarker at a given time point during the pre-challenge phase. on the one hand, if there is no apparent distributional difference between the values of the parameter/biomarker for a given pre-challenge window and the values for the post-challenge window, the auc will have a value of about . . on the other hand, if there is a perfect separation of the values for a given pre-challenge window and the values for the post-challenge window, the auc will be equal to . to relate the auc to its discriminatory performance, we followed [ ] , and used the following denominations of auc ranges: . - . excellent, . - . very good, . - . good, . - . sufficient, and . - . bad. this was how we quantitatively compared the discrimination accuracy of a given parameter/biomarker at a given time point during the pre-challenge phase to its discrimination accuracy at another time point during the pre-challenge phase (bottom panels in figures and ) . moreover, we used the auc to compare two different parameters/biomarkers in terms of their discrimination accuracy. for this comparison, however, it was also important to consider the stability over time of the discrimination accuracy of each of the two biomarkers. in order to explore whether the discrimination performance of the parameters/biomarkers at hand was independent of other underlying disease processes, such as chronic ailments, the above described analysis was conducted on the groups of asthmatics and healthy individuals separately. however, we also carried out the analysis on the data resulting from pooling these two groups. the cytokines il- ( for cellular immune markers measured in nasal lavage fluid, the emerging picture is more complex (see supplementary materials data). the eosinophil cell numbers were found to be a sufficient discriminator (auc values from . to . ), although bad during limited time intervals, whereas for neutrophil cell numbers, this appeared to be the other way around, with some episodes of good discrimination performance ( figures s a,b and s a,b in supplementary materials data) . surprisingly, the overall cell numbers in nasal lavage fluid oscillates between being a sufficient and a good to very good discriminator ( figure s a ,b in supplementary materials data). feno is, in general, a bad discriminator (auc . - . ), except for non-asthmatic patients, for which it is sufficient, and good only during a limited time interval (figure , see also "summary statistics of the averages of feno concentration within each window" in supplementary materials data for summary statistics of the pooled group). viruses , , x for peer review of to the best of our knowledge, this is the first study in which the utility of inflammatory/immune biomarkers and lung function has been tested for detecting respiratory viral infections, while the lung function parameters are throughout bad discriminators, whether measured in the morning or in the evening (see figures s a,b-s a ,b in supplementary materials data for the occasional exceptions to this rule). a general pattern, observed in our results, was how the ability to discriminate between healthy and rv -infected states varied over time. the amplitude of these temporal fluctuations was found to be relatively highest among the cellular immune markers and some of the cytokines. when comparing asthmatics and non-asthmatics in terms of the ability of the various parameters/biomarkers to discriminate between healthy and rv -infected, it appeared that for feno and the lung function parameters, with the exception of pef and the ratio fev /fvc, the discriminatory performance was higher in the non-asthmatics group. this difference between the two groups could be observed over time intervals of weeks (see figure and figures s a,b-s a ,b in supplementary materials data). for the cellular immune-markers and the cytokines, however, discriminatory performance differences were less pronounced or less consistent over time (see figure and figures s a,b-s a ,b in supplementary materials data). to the best of our knowledge, this is the first study in which the utility of inflammatory/immune biomarkers and lung function has been tested for detecting respiratory viral infections, while considering the natural temporal fluctuations of the parameters/biomarkers studied. our findings indicate that the concentrations of the various cytokines and the overall cell count in nasal lavage fluid are consistently at least sufficient discriminators of rv infection, the cytokines at times even excellent discriminators. furthermore, eosinophil and neutrophil cell numbers in nasal lavage fluid were found to reach, at times, the level of a sufficient discriminator. however, this was not consistently the case throughout the observation period. finally, feno and the lung function parameters were, in general, bad discriminators, except within the group of non-asthmatics, where sufficient discrimination power was reached during certain time intervals of the observation period. within a period of time of seven weeks prior to experimental and controlled inoculation with rv , each cohort participant was closely monitored, and no symptoms of any respiratory disease were reported during this time interval. nevertheless, our analysis has revealed a time dependency of the discriminative power of all the parameters/biomarkers studied herein. this can be interpreted in two ways as follows: ( ) during the phase prior to inoculation, the parameters/biomarkers fluctuate with an amplitude large enough to occasionally reach or nearly reach values characteristic of an infection. ( ) the effect elicited by the rv infection on these parameters/biomarkers is not strong enough, and thus the values remain within a "normal" range that can be observed during infection-free phases. in the context of discriminating between the non-infected and the infected state, the first interpretation hints at the possibility of finding false positives, whereas the second interpretation indicates that false negatives may result. whilst feno and lung function parameters can be quickly and non-invasively measured using cheap portable devices (e.g., at airports), our results suggest, on the one hand, that these parameters do not qualify as accurate discriminators of rv infection. on the other hand, we found that the concentrations of the various cytokines in nasal lavage fluid may indeed be successfully used for the detection of rv infection. in particular, il- ( figure ) and ifn-gamma ( figure s a ,b in supplementary materials data) are the best discriminating cytokines, despite the natural temporal fluctuations in their concentrations. this may not be surprising, as these cytokines are known to play critical roles in local anti-viral responses [ ] . furthermore, the influence of the asthma condition on ifn-gamma's discrimination performance is almost negligible. what is the clinical implication of these data? a nasal lavage is a relatively bothering procedure that needs to be carried out by trained health care workers. moreover, the laboratory assays to measure the concentration of these cytokines are expensive and require proficient staff and laboratory facilities. consequently, we conclude that measuring these cytokines as biomarkers in nasal lavage fluid will be most suitable in a hospital setting. in fact, some cytokines have been shown to be good predictors of disease progression in flu patients [ ] . our data analysis strategy of averaging biomarkers over a sliding -days window has, for our purposes, an undesirable effect of smoothing the time series data, thereby artificially reducing the true variability of the parameters/biomarkers. in fact, moving average filters are a widely used time series smoothing technique [ ] . however, on which days after rhinovirus infection a given patient will display the strongest changes in the parameters/biomarkers measured in our study was highly variable from patient to patient. previous research work has indicated that the strongest changes typically occurred within the first days after infection [ ] . therefore, we used the average of a given parameter/biomarker over the first ten days after inoculation as the magnitude that would be representative of the changes occurring upon infection, without having to choose a particular day after infection that may not be suitable for all individuals. consequently, and for the sake of consistency, we also used averages of the parameters/biomarkers over -day windows prior to inoculation. in previous work [ ] , we explored the potential of the whole parameter/biomarker time series to characterize each participant's state upon inoculation. those findings suggest that a "personalized time-series analysis" would most likely have more power and lead to better discriminatory performance. however, such an approach is challenging from a practical point of view, as patients would need to be constantly monitored, such as weekly, in order to detect a potential infection that no one knows in advance when it will hit. this would constitute a significant burden for most patients or healthy individuals, which discouraged us from pursuing such an approach in the context of rhinovirus infection detection. nevertheless, it is conceivable that, in the future, there could be portable patient telemonitoring technologies that allow for passive patient monitoring, i.e., longitudinal measuring of relevant parameters/biomarkers without the patient's active participation. with such technologies, a personalized time-series analysis approach would indeed become feasible. however, we believe that the development of such technologies is still in early stages. furthermore, and more importantly, we think that the potential ethical issues arising from constantly telemonitoring patients or healthy individuals need to be traded-off against the presumptive health benefits of such technologies. the choice of the age group for our study volunteers ( - years) was determined by the fact that, in this study, the participants were inoculated with an active virus. indeed, ethical approval by the hospital board for the inclusion of pediatric or older patients would have been very difficult to obtain. our results may have been influenced by the presence of atopy/allergy in our cohort. however, our study was designed to mimic a real-life setting in which the majority of the asthma patients have atopy as opposed to non-atopic asthma [ ] . moreover, some of the cytokines we decided to measure in this study (e.g., ifn-g) are known for their increased secretion during rhinovirus infections and less for their appearance during allergic reactions. furthermore, we selected mediators that would be released at sufficient abundancy in the nasal compartment to enable detection, even in the absence of any respiratory infection, and that have been consistently reported in the field [ , ] . although our study has an important clinical and epidemiological message, rhinovirus infections represent a milder form of respiratory infection as compared with other more virulent pathogens. hence, the conclusions derived from this study can only serve as potential reference for other viruses infecting the human respiratory system. moreover, the sample size of our study is relatively small owing to the number of sampling visits every volunteer was subjected to. however, this shortcoming was compensated for by the unprecedented exceptionally high sampling frequency of parameters/biomarkers. the following are available online at http://www.mdpi.com/ - / / / /s . figures s to s : discrimination performance (panel a: p-value plot; panel b: auc plot) of biomarker/parameter (respectively) ifn-gamma, il- , il- , il- , tnf-alpha, ip- , il- β, il- a, il- , percentage of eosinophils, percentage of neutrophils, cell density, normalized morning fev , normalized morning fev /fvc, normalized morning fvc, morning pef (% predicted), normalized evening fev , normalized evening fev /fvc, normalized evening fvc, and evening pef (% predicted). table s : summary of windowed averages for ifn-gamma. table s : summary of windowed averages for il- . table s : summary of windowed averages for il- . table s : summary of windowed averages for il- . table s : summary of windowed averages for tnf-alpha. table s : summary of windowed averages for ip- . table s : summary of windowed averages for il- β. table s : summary of windowed averages for il- a. table s : summary of windowed averages for il- . table s : summary of windowed averages for percentage of eosinophils. table s : summary of windowed averages for percentage of neutrophils. table s : summary of windowed averages for cell density. table s : summary of windowed averages for normalized morning fev . table s : summary of windowed averages for normalized morning fev /fvc. table s : summary of windowed averages for normalized morning fvc. table s : summary of windowed averages for morning pef (% predicted). table s : summary of windowed averages for normalized evening fev . table s : summary of windowed averages for normalized evening fev /fvc. table s : summary of windowed averages for normalized evening fvc. table s : summary of windowed averages for evening pef (% predicted). table s : summary of windowed averages for il- . table s : summary of windowed averages for feno. table s : symptoms from viral challenge and antibody detection. furthermore, a copy of the study questionnaire filled out for each participant at each visit. for every parameter/biomarker, each cohort participant's pre-challenge time series of windowed averages can be used by researchers as reference values to compare to values measured on their own patients infected with similar/different viruses known to affect the respiratory system. these time series can be made available upon request. detection of respiratory viruses by molecular methods clinical and financial benefits of rapid detection of respiratory viruses: an outcomes study chest radiological findings of influenza a h n pneumonia laboratory diagnosis of viral infection molecular imaging of influenza and other emerging respiratory viral infections identification of micrornas in throat swab as the biomarkers for diagnosis of influenza reliable detection of respiratory syncytial virus infection in children for adequate hospital infection control management improved detection of rhinoviruses in clinical samples by using a newly developed nested reverse transcription-pcr assay false negative tests for sars-cov- infection-challenges and implications reverse transcriptase pcr diagnostic assay for the coronavirus associated with severe acute respiratory syndrome evaluation of reverse transcription-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus laboratory diagnosis of respiratory tract infections in children-the state of the art cytokines in the respiratory airway as biomarkers of severity and prognosis for respiratory syncytial virus infection: an update loss of adaptive capacity in asthmatic patients revealed by biomarker fluctuation dynamics after rhinovirus challenge systemic tryptophan and kynurenine catabolite levels relate to severity of rhinovirus-induced asthma exacerbation: a prospective study with a parallel-group design new possible biomarkers for diagnosis of infections and diagnostic distinction between bacterial and viral infections in children lower airway rhinovirus burden and the seasonal risk of asthma exacerbation receiver-operating characteristic (roc) plots: a fundamental evaluation tool in clinical medicine measures of diagnostic accuracy: basic definitions host immune responses to rhinovirus: mechanisms in asthma the association between serum biomarkers and disease outcome in influenza a(h n )pdm virus infection: results of two international observational cohort studies digital signal processing: a practical guide for engineers and scientists upper airway· : allergic rhinitis and asthma: united disease through epithelial cells cxc chemokines and antimicrobial peptides in rhinovirus-induced experimental asthma exacerbations nasal cytokine responses to natural colds in asthmatic children we would like to acknowledge all the participants who volunteered to participate in our study along with all master students, research technicians, and study nurses who helped us with parts of the project. the authors do not have any commercial interests or associations to disclose that could result in a conflict of interest. key: cord- -ycjzitlk authors: simons, robin r. l.; gale, paul; horigan, verity; snary, emma l.; breed, andrew c. title: potential for introduction of bat-borne zoonotic viruses into the eu: a review date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ycjzitlk bat-borne viruses can pose a serious threat to human health, with examples including nipah virus (niv) in bangladesh and malaysia, and marburg virus (marv) in africa. to date, significant human outbreaks of such viruses have not been reported in the european union (eu). however, eu countries have strong historical links with many of the countries where niv and marv are present and a corresponding high volume of commercial trade and human travel, which poses a potential risk of introduction of these viruses into the eu. in assessing the risks of introduction of these bat-borne zoonotic viruses to the eu, it is important to consider the location and range of bat species known to be susceptible to infection, together with the virus prevalence, seasonality of viral pulses, duration of infection and titre of virus in different bat tissues. in this paper, we review the current scientific knowledge of all these factors, in relation to the introduction of niv and marv into the eu. bat-borne viruses with pandemic potential have been identified as the origin of a number of recent human disease outbreaks. examples include the paramyxoviruses nipah virus (niv) in malaysia and [ ] and the filoviruses ebola (ebov) and marburg viruses (marv) in africa [ ] . bats have also been linked with the more recent middle east respiratory syndrome coronavirus (mers-cov) [ , ] . nipah virus, in particular, has been suggested to have pandemic potential as it is capable of limited human-human transmission and rna viruses in general have particularly high mutation rates. a human-adapted strain of niv, were it to emerge in asia, could spread rapidly due to high population densities and global interconnectedness [ ] . a large, and still increasing, number of different viruses have been isolated from bats, many of which are asymptomatic in the host and are closely related to human pathogens. these viruses have the potential for cross-species transmission (i.e., -spillover‖) to other mammalian species, for example, marv in monkeys [ ] and niv in pigs [ ] , and to directly or indirectly infect humans [ ] . a recent paper describes the infection of a wildlife biologist with a novel paramyxovirus during a field trip to south sudan and uganda [ ] . a recent study found that bats have, on average, significantly more zoonotic viruses per species than rodents, which are also known to host a large range of viruses [ ] . additionally, the authors estimated that viruses had a broader host range in bats, averaging . host species per virus. bat-borne paramyxoviruses have been identified in various bat species across africa, australia, south america and asia and recently the detection and characterization of paramyxoviruses in free-ranging european bats has also been reported [ ] . further to this, recent evidence places bats as tentative hosts at ancestral nodes to paramyxoviridae and pneumoviridae [ ] . bat species can have very broad geographic ranges [ , ] and multiple species can share the same habitats and even roost sites [ ] . studies of pteropus bats in australia and asia found they could travel hundreds of kilometers between roosting sites with their home ranges extending across national boundaries and over sea [ , ] . factors that affect the degree and rate of contact between animals and humans are important for spillover of any zoonotic emerging infectious disease. most human outbreaks of bat-borne zoonotic diseases have been suggested to be as a consequence of human activities. for example, outbreaks of marv in africa have been linked to human contact with bat caves, for reasons such as mining operations [ ] or tourism [ , ] . deforestation has also led to bat colonies moving closer to areas inhabited by humans in search of food and roosting sites [ ] . bats are known to have varying degrees of contact with domestic animals and commercial food crops [ , ] , in particular contact of pteropus giganteus bats with date palm sap producing trees in bangladesh is considered a risk factor for human niv infection [ ] . livestock can act as an intermediate host between bats and humans. the outbreak of niv in malaysia in was linked to infection of pigs via fruit bats and resulted in the culling of over one million pigs and the deaths of over people [ ] . similarly, in australia hendra virus is transmitted to humans via horses; to date horses and seven people have been infected (four people have died). bats themselves are a known food source for humans in some areas of africa [ ] and asia [ , ] . while bats in the european union (eu) are known to harbor zoonotic viruses that can be transmitted via close contact, such as the european bat lyssaviruses and (eblv and ), there is currently no confirmation of the presence of viruses with pandemic potential in bats in the eu (i.e., at least capable of sustained human-human transmission). however, it is important to note that this should not be taken as proof of absence of such viruses, but rather that they have not been detected during surveillance of bat populations in the eu to date. historically, the first reports of human marv cases were in laboratory workers in germany and yugoslavia in , through direct contact with blood from african green monkeys (cercopithecus aethiops) imported from uganda [ , ] . however, more recent cases of bat-borne viruses affecting humans in the eu have been isolated incidents, notably the case of a dutch tourist returning from uganda after visiting a bat cave in which marv-infected bats roost [ , ] . similarly, as of november , cases of mers-cov have been detected in europe [ ] , several clusters of which represent chains of transmission in which the primary case had been infected in the middle east. previous studies have demonstrated the presence of similar viruses in eu bat populations, suggesting there is a risk of spillover of related viruses in the future. the first filovirus discovered in europe that was not directly imported from an endemic area of africa was lloviu virus (llov), detected in dead insectivorous bats in massive bat die-offs in caves in spain in [ ] . simultaneous bat die-offs were observed in portugal and france, although a causal relationship between llov and mortality was not shown. countries in the eu have strong historical links with many of the countries where zoonotic bat-borne viruses such as niv and marv are present and, consequently, commercial trade and human travel pose a potential route of introduction of such viruses into the eu. many patients in the ebov outbreak in uganda presented with mild clinical symptoms raising concerns that travel is possible whilst infected, [ ] . other factors, such as the importation of bushmeat, including bats and body parts of primates [ ] , could be potential routes of virus introduction. a qualitative risk assessment for the introduction of henipaviruses to the uk concluded that there was a low level of risk from southern asia, south-east asia and australia, through import of fruit and bushmeat and a very low level of risk through import of bat meat, horses and companion animals and through human travel [ ] . however, the assessment highlighted the high levels of uncertainty, reflecting the limited data and specific details of the routes involved. a risk assessment for niv establishing in australia also identified a lack of relevant data in various areas, reflected in the high levels of uncertainty in the risk estimates [ ] . in this paper we review the scientific literature relating to the risk of introduction of niv and marv into the eu, but do not formally assess the risk. we begin by reviewing the current evidence for the geographical location of the viruses and thus where a potential risk of introduction to eu member states (mss) may exist. next, we review the evidence for factors which may affect the probability of an eu introduction via the various routes, such as prevalence and transmission dynamics in bat species and survival and transmission potential of the viruses. finally we review the evidence directly relating to potential routes for introduction into the eu. the main routes we consider in detail are human travel, trade of fruit and pig products and illegal importation of bushmeat. other routes such as bat migration, the unintentional introduction of living or dead bats by aircraft and the effect of climate change are also given consideration. identification of countries which have had human cases of niv or marv is important for assessing the risk of introduction to the eu, as it identifies the areas from which human travel may be a particular risk. knowledge of the risk factors regarding human infection in these countries is also of relevance as they may suggest other potential routes of introduction, highlight similar factors which are present in the eu and may facilitate spread of the viruses, or indicate potential control measures. niv: studies suggest that human outbreaks are linked to one of two distinct nipah virus strains; niv-malaysia or niv-bangladesh [ ] [ ] [ ] . the malaysian strain emerged in with an outbreak in commercially farmed pigs, resulting in > human cases reported in malaysia and singapore with a case-fatality rate approaching % [ ] . pteropus vampyrus and pteropus hypomelanus were subsequently identified as likely natural reservoir hosts for the virus [ , ] . in this instance, pigs were implicated as amplifier hosts with viral isolates from both sick pigs and humans showing identical nucleotide sequences [ ] . the presence of large commercial pig farms with fruit trees in the vicinity meant that foraging bats could drop partially eaten fruit contaminated with niv into pig farms. pigs could then have access to this fruit and become infected with niv [ ] . transmission was attributed to direct contact with infective excretions and secretions and viral spread among pig farms was due to movement of pigs [ ] . there were no reported incidences of human-human transmission and there have been no further acute human cases attributed to niv-malaysia since , although relapsed and late onset cases have been reported [ ] . laws in malaysia preventing fruit farming in pig farming areas may have prevented further niv outbreaks. in contrast, there have been regular seasonal outbreaks of niv-bangladesh since its apparent emergence in , predominantly in bangladesh, although two outbreaks have also been reported in west bengal, india, close to the border of bangladesh [ ] . up to january , there have been reported human cases linked to niv-bangladesh with deaths, giving a reported case fatality rate of % [ , ].this appears much higher than the case-fatality rate of niv-malaysia, although direct comparison may be complicated by various biases such as method of surveillance and reporting. while human-human transmission is considered a major pathway for human infection with this strain [ ] , studies in india and bangladesh suggest the main risk factor is consumption of raw date palm sap [ , , ] . date palm sap is harvested from december through to march by cutting into the tree trunk and allowing the sap to flow overnight into an open clay pot [ ] . infrared camera studies have demonstrated that p. giganteus bats frequently visit date palm sap trees and lick the sap during collection [ ] , potentially contaminating it with niv from saliva and/or urine. other reported risk factors for human infection include consumption of alcoholic beverages made from date palm sap [ , ] , climbing trees and contact with other niv infected patients [ ] or animals [ ] . a recent study investigating the role of landscape factors in niv spillover risk in bangladesh [ ] found a significant correlation between niv spillover and villages with higher human population density, more fragmented forest cover and p. giganteus roost sites containing the tree species polyalithia longifolia or bombax ceiba. the geographical distribution of cases within bangladesh is shown in figure . cases generally occur in areas near inland water, predominantly the ganges, which provides a suitable habitat for date palm trees. in , there were cases reported from districts, more than in any other year, but generally only a few cases per district; the largest number was five cases reported from manikganj [ ] . the pattern of cases suggests multiple small outbreaks in different regions, rather than large outbreaks caused by one source. [ , ] . initial laboratory investigations at the time of the niv outbreak in siliguri (india) in did not identify an infectious agent. retrospective analysis, however, identified the presence of niv antibodies in serum samples by enzyme-linked immunosorbent assay (elisa) and rna by real-time pcr (rt-pcr) in (stored) urine samples [ , ] . sequence analysis confirmed that the pcr products were more closely related to the bangladesh strain ( . % identity) than the malaysia strain. to date, there is no reported evidence of niv outbreaks in humans emerging in other parts of india or in any other countries. however, given the delay in identification of the siliguri outbreak and large distribution of bats that can carry niv, it is possible that more human cases have occurred where niv has not been detected or reported. additionally, surveillance for niv in bangladesh may be more sensitive due to the previous cases reported to the authorities each year. as such, wider geographical distribution of human cases of niv should not be ruled out. marv: since the outbreak of marv in laboratory workers in europe [ ] , outbreaks have been confined to sub-saharan africa, although there have been reported cases of individuals acquiring infection in uganda and then travelling to the netherlands [ ] and the usa [ ] . human cases of filovirus infection in africa have been associated with hunting fruit bats for meat and with entry to mines or caves where large populations of fruit bat species, such as rousettus aegyptiacus, are present [ , , ] . it has been suggested that human infection could be through exposure to the excretions from bats roosting in the caves [ ] , although an experimental study on r. aegyptiacus bats inoculated with the hogan marv strain (originally from the south africa outbreak [ ] ) did not detect virus in the faeces of infected bats [ ] . one study reported -working as a miner‖ as a significant risk factor for a positive antibody result to marv, with % of the population who tested positive for marburg antibodies working in the local gold mines [ ] . there has not been a direct food consumption transmission route reported for marv, although index cases of filovirus infection have often had suspected contact with dead primates found in the forest, with exposure thought to occur during the butchering process [ , ] and the hunting process, which may involve the use of shotguns, potentially causing spray of body tissue and fluids [ ] . identification of countries where niv and marv have been detected in bats is important to understand the potential for infected bats from these areas to directly enter the eu, contaminated trade products destined for the eu, or infected humans who may travel to the eu. knowledge of the species of bats that are susceptible to niv and marv is also a relevant factor for assessing the susceptibility of bat species present in the eu. there is a growing body of literature on the geographical distribution and range of niv and marv in animal species with particular reference to bats. a complicating factor in defining the range is that studies in bats typically report serological or rna detection results, rather than detection of infectious virus. while a seropositive result is strong evidence of historical exposure to a virus, there may be cross reactivity with related viruses, precluding exact identification of the virus to which exposure has occurred, as seen for niv and hev in australasia [ ] . detection of rna demonstrates the presence of genetic material, but does not prove current presence of infectious virus. additionally, there can be difficulties in using serological assays outside their original, validated scope, for example when an existing assay is used with samples from an alternative species [ ] . here, the absence of positive and negative control samples and -gold standard‖ diagnostic assays makes it hard to determine an appropriate cutoff point to distinguish between seropositive and seronegative individuals [ ] . as such, positive identifications do not confirm active virus infection at the current time and, in some cases, could only be an indication of historical exposure to a related virus. in the absence of virus isolation or full genomic characterization, it cannot, therefore, be definitely confirmed whether the virus is currently present. niv: table shows a summary of reported testing for niv in bat species. niv is predominately associated with asian fruit bats of the genus pteropus, which have been suggested as the natural reservoir for henipaviruses [ ] . only a few studies have successfully isolated niv virus from bats. isolation has been reported from the urine of p. vampyrus [ ] and p. hypomelanus [ ] in malaysia and p. lylei in cambodia [ ] , but at very low prevalence, with only / samples yielding a virus isolate in the cambodia study. such low prevalence could be a factor for the inability to isolate niv in test samples of bats in other studies. rna positive pcr results have been obtained for p. vampyrus in indonesia [ ] and p. lylei in thailand [ ] , which identified both niv-malaysia and niv-bangladesh rna sequences. niv rna has also been detected in p. giganteus in india [ ] and p. vampyrus and rousettus amplexicaudatus in east timor [ ] . of particular interest to the eu is the identification of henipavirus antibodies in myotis daubentonii in china [ ] , as this species is also found across much of europe, although it should be noted that niv specific rna was not detected in this study and virus isolation was not attempted. while niv is predominantly associated with asia there is increasing evidence for the presence of related viruses in africa. paramyxovirus rna related to hev and niv has been detected in eidolon helvum bushmeat in the republic of congo [ ] and in the faeces and urine from roosting e. helvum bats in ghana [ , ] . viral concentrations were estimated to be low using rt-pcr assays [ ] . other studies have identified henipavirus antibodies in eidolon dupreanum and pteropus rufus in madagascar [ ] . a recent study combined genetic and serological analyses to determine the extent of connectivity among e. helvum populations across central africa. antibodies to henipaviruses were present in bats from all locations with henipavirus seroprevalences reported to be between %- . %, with an overall average of . % [ ] . however, the presence of infection on isolated islands suggested that large population size and connectivity may not be responsible for viral persistence. these studies do not confirm the specific presence of infectious niv virus in bats in continental africa and madagascar, but they now constitute a reasonably substantial body of work, from a number of independent sources, which suggest increasingly strong evidence for the presence of henipaviruses in bats that have a geographical range outside of asia and oceania. marv: table shows a summary of reported testing for marv in bat species. there are several studies reporting the prevalence of marv in bats in caves in africa including the countries of gabon, uganda and the democratic republic of congo (drc) [ , [ ] [ ] [ ] [ ] [ ] . marv is now considered endemic in r. aegyptiacus bats in gabon [ ] and, in general, those bat species which serve as potential reservoirs for marv are endemic to regions of central africa. there is little evidence for the potential of marv to occur outside africa at this point, although there are few published reports of testing for this virus on other continents; a study in india showed that none of bats tested, including p. giganteus (n = ), cynopterus sphinx (n = ) and megaderma lyra (n = ), were positive by pcr for marv rna [ ] . within africa, there are also reports of antibody or rna evidence of marv infection in bat species other than r. aegyptiacus, such as rhinolophus eloquens, miniopterus inflatus [ ] and hypsignatus monstrosus [ ] , but reports are less frequent. this highlights the importance of knowledge on the exact species of bats for the purpose of risk assessment, suggesting the main zoonotic risk is likely from r. aegyptiacus. in an experimental study, marv was found to be present in the blood and saliva of viraemic r. aegyptiacus bats but not in their faeces or urine [ ] , suggesting that close contact between adjacent bats of the same species within the roost may be important for marv transmission. marv rna has also been reported in a pooled liver, spleen, lung extract from a female r. aegyptiacus fruit bat in kenya in , although tissues from other bats including r. aegyptiacus from two locations were negative [ ] . from an eu perspective, r. aegyptiacus are known to be present in cyprus [ ] and turkey [ ] and populations were found in the wild in tenerife in the early s, as a result of escaped captive animals [ ] , before being effectively eradicated by . there is no reported evidence to suggest presence (or absence) of marv in these populations. bat host heterogeneity of virus prevalence is important both in terms of further spread of infection within the roost and spill-over to humans, e.g., through being hunted for bushmeat. some fruit bat colonies in trees in ghana have up to million bats, so the prevalence may vary spatially within the colony [ ] . fruit bat colonies in caves with more than , r. aegyptiacus bats are structured with juveniles more likely to be exposed to bat droppings due to their peripheral positions within the colony [ ] . a study on active infection of marv in a bat cave in uganda found a higher prevalence in older juvenile bats ( . %) than younger juveniles ( . %) or adults ( . %), the older juveniles were six months old at the time of capture and younger juveniles three months old [ ] . thus, an important consideration is whether juveniles and adult bats have different behaviors that would affect the onward transmission of marv. for example, are older juvenile bats and non-breeding adult bats more likely to range further in migration (and hence spread disease to other hosts) than younger juveniles or the breeding adults, or to be caught by bushmeat hunters (as they are less experienced in survival)? based on data gathered in tables - , those countries of the world where there is evidence of recent niv or marv infection in humans or bats are highlighted in figure . we define that a country is positive for human infection only if it has had a reported human case in the last years (i.e., since ). such a period of time without a reported case suggests that while there may still be potential for a human case in the country itself, the risk of import to the eu is extremely low. thus, malaysia and singapore are not considered positive for niv and south africa and kenya are not considered positive for marv. given the issues regarding use of serological positive results as an indicator of current virus presence, we do not consider serological positive results alone to be an indication of current viral presence in bats for this analysis. information from the iucn red list website is used to determine the geographical range of those bat species known to have been naturally infected [ ] , as there is a potential for undetected viral presence in these countries. it can be seen that while recent human infections of both niv and marv appear to be limited in geographical range (the red areas in figure ), there are a number of countries where bats have been identified as having the virus, but no human infection has been reported. it is also noted that the full geographical range of these bat species is extensive and in the case of r. aegyptiacus encroaches on the south-east boundary of europe, although the range of pteropus bats is much further east. however, if species serologically positive for henipaviruses are considered then m. daubentonii would be included and the geographical range would be much wider, encompassing europe and australia. viral load is a measure of the number of viral particles present in an organism or bodily fluid, e.g., the mass/volume of bat faeces, urine, saliva or bushmeat. the virus may be quantified in a number of ways including plaque-forming units (pfu), tissue culture infectious dose % units (tcid ) or number of genome copies. currently there are no published dose-response curves that convert pfu or tcid units in to risk of infection in humans or livestock animals. furthermore, the genomic copies may not all be equally infectious (due to the mutant spectrum) and some may be defective. it is not clear whether dispersion of the virions lowers the risk of transmission. however, the viral load is an important factor in a release assessment for any virus because it directly affects the risk of transmission. niv: while the studies mentioned previously demonstrate the likelihood of a continual reservoir of niv in many countries, the actual prevalence of bats currently shedding virus may be very low. as such, data on viral load is limited. however, with the application of real-time pcr, henipavirus-related sequences ranging from to , genome copies per . cm and . × per ml of bat urine have been reported [ ] . experimental studies have also been conducted in other animals. titres of up to pfu/ml from brain and basal turbinates and pfu/ml from trachea swabs were obtained from niv infected piglets [ ] , with lower levels found in lung and spleen and shedding peaks during the first week post inoculation. titre data are also available for niv strains from bangladesh and malaysia in experimentally infected rodents [ , ] . marv: one study reported that no viraemia or presence of marv rna could be detected in various tissues collected from r. aegyptiacus bats experimentally inoculated through oral or nasal routes [ ] , but subcutaneous and intraperitoneal inoculation resulted in high levels detected in plasma ( to tcid /ml) for five to nine days post inoculation, with titres up to . and . tcid /g in the liver and spleen respectively. virus was also occasionally detected in lung, heart, kidney and salivary glands with loads up to . tcid /g. ranges for tcid /ml of marv in tissues of naturally-infected r. aegyptiacus in uganda have also been derived from a standard curve of diluted stock virus using q-rt-pcr [ ] . high values of , - , , tcid /ml were obtained from liver, spleen and lung whilst values of - tcid /ml were obtained from multiple tissues including blood and intestines. a potential factor affecting the prevalence of viruses, regarding the risk of zoonotic transmission, is seasonal pulsing, or oscillations of prevalence, with peaks in prevalence at specific times of the year. periods of higher risk are relevant to eu incursion as they will affect factors such as the probability of eu tourists contacting an infected bat and thus impact on routes such as human travel to and from niv and marv areas. indeed, seasonal pulses of marv circulation in juvenile r. aegyptiacus bats coincide with periods of increased risk of human infection [ ] . the influx of susceptible young is a crucial driver of infection dynamics and bat reproduction and survival are thought to be major drivers of bat disease dynamics [ ] . many bat species exhibit highly synchronised parturition which can dramatically alter population contact rates and susceptibilities. sex differences in behaviour and distribution of bats during times of the year when the potential for disease transmission is greatest may also have important implications for disease dynamics [ , ] . the role of bat torpor in infection dynamics is largely unstudied [ ] . torpor typically reduces pathogen replication rates and hence lengthens the incubation periods. a study found a clear indication for torpor being a key factor in allowing perpetuation of rabies virus through the hibernation period, through prolonged incubation period and reduced mortality [ ] . this enabled the virus to persist in the population until susceptible individuals from the annual birth pulse could become infected and continue the cycle. migration and coloniality may also be important drivers of disease dynamics [ ] , altered migration behaviour may result in declining immunity within specific colonies which could lead to more explosive hev epidemics [ ] . niv: there is evidence of a seasonal pattern for spillover of niv to humans; a review of all human outbreaks of niv between and found that, except for the initial event in malaysia, they all occurred in the first five months of the year [ ] . a longitudinal study in thailand found the bangladesh strain of niv was dominant in the urine of p. lylei bats, with highest recovery of rna in may [ ] . in two sites, the bangladesh strain was almost exclusively detected between april and june while the malaysian strain was found dispersed during december to june. breeding of the bats (including mating and birthing) occurs in december to april, and may not be the only factor involved in bat transmission. there is some evidence that pregnant and lactating pteropus scapulatus and p. conspicillatus females had a significantly higher risk of hev infection [ , ] resulting in a seasonal pattern due to seasonality of reproduction of these bats. a study on an orchard in new south wales investigated the legal shooting of pteropus poliocephalus [ ] , found that the majority of bats shot were female (ratio : . ) and that % of these females were lactating. this suggests that pregnant and/or lactating females are more likely to be foraging for food and coming into contact with crops/orchards, which could not only be eaten by horses, but also could contribute to seasonality of human spillover for viruses such as niv bangladesh, for which oral transmission to humans through date palm sap is a route. the wild date palm produces sap seasonally from mid-october to mid-march and winter (december to early february) is the traditional date palm sap gathering season in bangladesh. outbreaks of niv generally coincide with this season, appearing between december and may. marv: a study of marv in r. aegyptiacus in the python cave in uganda predicted an oscillating biannual pattern of bat prevalence in the cave, with peaks in february and march. these peaks in prevalence coincided with the birthing seasons of the bats in the cave and the temporal clustering of previous reported spillover events of marv into humans [ ] . pcr data showed distinct oscillating pulses of marv infection in older juvenile bats (~six months of age) peaking in february and august that temporarily coincided with the peak twice-yearly birthing seasons. the authors speculate that the marv pulses reflect the pulses of newly weaned bats which populate the -low-lying‖ roosting areas where they are infected and may pass infection amongst themselves [ ] . as they age, and are recruited into the adult population, their colony positions are taken by the next generation of juvenile bats. it is not clear whether the oscillation peaks in juvenile bats coincide with other environmental/ecological factors affecting the bats such as local shortage of fruit or migration. knowledge of survival of virus in different media and under different environmental conditions is important for assessing the concentrations of virus on contaminated fruit and infected bushmeat over time and ultimately the risk to humans. this can be used to predict the concentrations of virus on the surface of fruit after export by taking into account the duration of transport to the eu. duration of infection in both humans and bats is also important when considering the probability of shedding infectious virus on arrival in the eu. niv: the incubation period for niv in humans has been reported to be as much as days [ ] . surveillance in bangladesh in found that among secondary cases, who had a single exposure to niv, the delay between exposure to onset of illness ranged from - days, with a median incubation period of nine days [ ] . the incubation period following a single intake of raw date palm sap to onset of illness varied between - days, with a median of seven days. a laboratory study on persistence of henipaviruses under various environmental conditions found that they were sensitive to ph, temperature and desiccation [ ] . the study showed a - log inactivation of henipaviruses in fruit juice (lychee, pawpaw and mango) over three to four days, although titres were still detectable after three days. there were also large variations in the half-life of the virus at different temperatures and ph values; e.g., in mango flesh, the half-life of niv was . hours for ph . at °c but . hours for ph at °c. for the purpose of risk assessment it is the rate of inactivation which is important, rather than the limit of survival, which depends on the starting titre. marv: investigation of the outbreak of marv in germany suggested that the incubation period could be as much as nine days [ ] . an experimental study on the effects of marv on the common marmoset (callithrix jacchus) found that animals became febrile after - days [ ] . an experimental study looking at marv, zaire ebolavirus (zebov) and reston ebolavirus (rebov), demonstrated survival for long periods in liquid media at both room temperature and °c, with virus recoverable from glass and plastic surfaces over three weeks after the start of the experiment [ ] . similar decay rates were found for marv and zebov, while rebov had significantly better survival within an aerosol. although data for survival of filoviruses on fruit are not available, a study looking at survival of poliovirus, simian rotavirus and feline calicivirus in the uk found prolonged periods of survival on fresh fruit and vegetable produce at refrigeration temperatures ( - °c), extending beyond the shelf life of the product [ ] . survival at °c was poorer, but some viruses remained viable for over a week. removal of viruses using conventional chlorine washing could give more than log reduction, but was only < log for poliovirus. however, it should be noted that these are non-enveloped viruses, and may, therefore, have different survival properties to the enveloped filoviruses. human-human transmission has been identified for both niv and marv. this, combined with incubation periods that could be over a week [ , ] , suggest that human travel could be an important route for transmission of bat-borne zoonotic viruses into the eu. the recent mers-cov cases in the eu highlight the risk of virus introduction from human travel [ ] . there has been one high profile case of tourism leading to an introduction of marv into the eu [ , ] . a similar, but non-fatal, incident from a person who visited the same bat cave in uganda also occurred in the usa [ ] . neither incident resulted in identified infection in other individuals. data from eurostat show that there are large numbers of people travelling between the eu and areas where niv and marv have been reported, both by air and by sea [ ] . the number of immigrants from these areas settling in eu countries is generally increasing and they will naturally have strong ties to their homeland. for example, the uk censuses of and show an increase in both number and percentage of the population of england reporting to be of indian, pakistani, bangladeshi and african ethnicity [ ] . combined, the indian, pakistani and bangladeshi ethnicity groups make up . % of the england population and % of the population of london in and similar data show that there has been an increase in the number of people reporting to be born in these countries [ ] . one could generally expect the individuals and their friends and families to have frequent trips to and from their native countries. data from the uk in suggested that out of , trips to the uk made by individuals using a bangladeshi passport, , were made by people returning after a temporary leave of absence [ ] . figure shows the migration into the eu from the niv and marv countries identified in figure . it is apparent that, in terms of migration from niv countries, the uk has the highest influx of migrants of all eu countries, while from marv countries it is france. further analysis showed that the majority of bangladeshi migrants go to the uk. this might suggest that when considering the risk from humans entering the eu from bangladesh, the uk is more likely to be at risk (before considering the impact of border control measures). previous research suggests that historically the bangladeshis that travelled to the eu were predominantly from sylhet [ ] , an area in the north east with very few reports of human niv cases, although more recently this may no longer be the case. it should be noted that some airports, such as heathrow, london, act as hubs for passengers going on to other destinations, which may lead to an overestimate for individual mss. however, an infected individual may be a risk even if only passing through the airport, as they will still likely have contact with airport staff and other passengers. a study looking at the risk of human-human transmission of viral haemorrhagic fevers (vhf), including marv, on airplanes found only a few events of vhf cases in the literature and no documented infection in follow up contacts [ ] . the study suggested that contact trace back should be undertaken for passengers and crew with direct contact with an infected individual, passengers seated within one seat of the case and cleaning staff responsible for cleaning the section occupied by the case. however, trace back of passengers seated more than one seat away from the infected individual was not considered necessary. this suggests that close contact is thought necessary for human-human transmission and so not everyone on an aircraft with an infected individual is likely to be at risk. as such, this would mean that spread of the virus to multiple locations in an eu ms, due to the dispersion of multiple individuals infected during the flight, is unlikely. however, the lack of data regarding vhf on flights and subsequent reliance on expert opinion in this study suggests that there is fairly high uncertainty surrounding the conclusions and further evidence should be sought, particular with regard to other viruses; factors such as stronger capability for airborne transmission could lead to different conclusions. tourism may have specific risks independent of other human travel. people who travel to foreign countries on holiday are likely to be there for only short periods of time, e.g., - weeks and some, particularly ecotourists, may visit bat caves or colonies, returning home soon afterwards. entering such areas carries the potential risk of direct contact with infected bats and contamination of shoes and clothing with potentially contaminated bat guano/faeces. there is a documented incident, in the python cave in uganda, of this leading to marv infection [ , ] , but there are numerous unofficial reports of similar such events. tourists are perhaps also more likely to be unaware of the risks of virus transmission, and therefore unaware of the appropriate precautionary measures. an author of this paper recently returned from west africa where they witnessed tourists entering an occupied bat cave and having contact with fresh bat guano. the tourists were not aware of the potential risks of virus transmission. however, the recent case of infection of a wildlife biologist with a novel paramyxovirus highlights that there is still a risk for people who are aware of and carrying out appropriate safety precautions [ ] . the short duration that tourists generally spend away means that, if infected, it is likely that they will return to the eu before clinical symptoms have developed, and there is little time for decay of pathogens in guano or indeed loss of guano from the clothing or shoes. it is well established that foodborne zoonoses can pose a threat to human health. pathogens may be present in products destined for human consumption either through infection of the source product in the natural environment (e.g., contamination of growing crops by infected animals or infection of animal tissues to be consumed while the animal was alive) or cross contamination of the product during processing, typically with urine or faeces. for viruses such as niv and marv, while cross-contamination during transportation could result in the presence of virus in other products, the most likely products to be contaminated are those that are associated with outbreaks, i.e., fruit and pig meat. while pig meat has not been directly associated with human infection, live pigs were identified as the source of human infection in the niv-malaysia outbreak, although pigs in malaysia are now considered free of niv [ ] . marv has not been associated with infection in any livestock animals to our knowledge (marv is known to infect primates which have been found in bushmeat seizures [ ] , but in this section we only consider animal products that would be traded legally for food). drinking raw data palm sap, or alcoholic beverages made from it, have been identified as risk factors for human niv infection in bangladesh [ , , ] , primarily due to the risk of direct contamination of the sap by the local pteropus bats. we have found no evidence of official trade of either of these products to the eu, although it is possible that individuals may bring alcoholic beverages with them in their personal belongings (the raw date palm sap ferments very quickly so is less likely to be brought over to the eu). while there are a number of products that involve its use, such as palm sugar, there are no reports of human infection as a result of consumption of such products. this suggests that the processing that takes place during the preparation of such products, in the case of palm sugar the sap is generally boiled, mitigates the risk. fruit bats are known to feed on a wide range of crops and they are often considered pests due to feeding in commercial orchards, although their importance in pollination is recognized [ , ] . a study on a vineyard in india found the old world fruit bat c. sphinx was responsible for > % damage to crops at the periphery of the vineyard [ ] . as such, it is common practice to protect commercial crops through the use of measures such as netting or shooting; one study on a stone fruit orchard in sydney, australia, consisting of four hectares of nectarine trees, where shooting was known to occur in order to protect the orchard, found a total of dead or injured flying foxes over days at the time when the nectarine crop was ripening and being harvested [ ] . niv-malaysia was isolated from fruit on tioman island, and contamination of fruit by bats is thought to be a potential route for the infection of pigs during the malaysian niv outbreak [ ] . a number of outbreaks in bangladesh have been linked with consumption of date palm sap [ ] , with the sap likely being contaminated with bat urine or saliva. while the date palm sap is the only identified foodborne source of human niv infection in bangladesh, bats could potentially have contact with, and contaminate with saliva or urine, any unprotected fruit grown in the region. while unlikely, if these crops are exported, there could be a risk of virus introduction into the eu. transport times can be less than hours by air travel, not long enough to allow significant decay of the virus. this route is less likely for marv as, to date, it has not been detected in the faeces or urine of either experimentally or naturally infected r. aegyptiacus bats [ , ] . faostat databases contain details of volumes of trade between eu mss and extra eu countries [ ] . the eu has strong trade links with the niv and marv identified areas in figure . while these databases show that there is little trade of pig products from niv and marv regions to the eu there is trade of fruit products (e.g., dates, apples, fruit juice). figure shows the relative volume of trade of fruit products from these areas and eu mss. the biggest importers from these areas are the netherlands and the uk, with germany having a relatively high volume from niv areas and france from marv areas. as with the human travel, it should be noted that some countries may act as hubs for trade products, with subsequent further distribution to other destinations. figure . trade data from faostat [ ] . trade of infected animals for non-food purposes could also pose a risk of viral introduction. marv has been identified in the african green monkey (cercopithecus aethiops), which have historically been traded for research purposes. there is considerable movement of horses around the world, primarily for sporting events. horses are known to be susceptible to hev with infections in australia [ ] , but to date there have been no reported cases of niv in horses. pet travel could also be a risk as the pets could potentially be infected with bat-borne zoonotic viruses in endemic countries. recently a kitten, infected with rabies virus, entered france from morocco demonstrating that such events can occur, even though the accompanying certificate of good health did not meet the regulatory provisions for the import of domestic carnivores from morocco [ ] . data from traces suggests that movement of live animals such as pets between niv or marv countries (as defined in figure ) and the eu are primarily animals not considered a risk of carrying the viruses (e.g., tropical fish) [ ] . however, there are a number of movements of cats and dogs. an experimental infection study of two cats with the malaysian strain of niv found that they started to develop clinical symptoms after five days [ ] . one cat developed acute clinical disease while the other recovered. virus was recovered from the tonsils and urine up to eight days post inoculation. while a very small sample size, and being aware that the experimental challenge dose is likely much higher than would be received in nature, this demonstrates that there is a potential risk of pets bringing the virus back into the eu. there is currently no quarantine regulation for third country pets, although the risk of bringing in pets from niv and hev areas is recognized by at least some mss [ ] . bat guano is also a potential trade product; it is sold for use as a fertilizer in several countries including thailand, indonesia, mexico, cuba and jamaica [ ] , and in theory could be imported into the eu. one study reported four of bat guano samples from a bat cave in ratchaburi province, thailand, were positive for group c betacoronavirus rna, although none contained niv rna [ ] . the legal importation of bats could also be a risk. the emergence of wild r. aegyptiacus bats in tenerife was believed to be a result of the escape of captive bats [ ] . there are no instances of live bat imports into the eu from niv countries on traces, but there are many instances of bats used in scientific research and zoos. r. aegyptiacus bats, known to be susceptible to marv, have been kept in zoo's in the eu; in two such bats died of rabies after being imported from a dutch zoo to a danish zoo [ ] . additionally there are reports of r. aegyptiacus being kept as pets. however, given the low numbers and the likely increased testing/surveillance of animals destined for these purposes the risk of importation from this route is likely very low. bushmeat is a term used to capture a variety of raw, smoked or partially processed meat that originates from the hunting of a variety of wild animals, including bats. it is well documented that bushmeat is illegally imported into both europe and the usa [ , , ] and, as such, it could act as a conduit for pathogen spread. in a recent study, illegal bushmeat imported into the united states was found to contain retroviruses and/or herpesviruses [ ] and henipavirus-like rna has been detected in internal organs of bat bushmeat sampled in the republic of congo [ ] . the perception of bushmeat as having zoonotic potential is not well recognized among bushmeat hunters, traders and consumers; one study reported that only % of bushmeat hunters in sierra leone are aware of the zoonotic disease risk [ ] and in a survey on bushmeat in the usa, participants in a focus group considered bushmeat to be a wholesome healthy and safe alternative to commercially produced meat from a shop [ ] . in an experimental study of r. aegyptiacus bats, marv was not detected in muscle, brain or skin tissues collected after cardiac exsanguination [ ] . this suggests that these tissues (including muscle) are not heavily infected, and that positive results in liver, spleen and kidney were not due to the presence of blood. if confirmed to be the case in naturally occurring infections, it could mitigate the risk of marv infection from the consumption of bushmeat if internal organs are not eaten. hunting of wildlife for food is a widely distributed practice in many parts of the world and constitutes an important source of animal protein for some rural communities. one paper reported that . % of households in brazzaville, congo consumed bushmeat [ ] and a survey of municipal markets identified different animals species, nine of which it is prohibited to hunt [ ] . economic recession over the past years has driven the commercialisation of bushmeat as a trade item; bushmeat now reaches the international markets as part of the $ billion annual global wildlife trade. the commercial trade in bushmeat occurs across almost all of tropical africa, asia and the neotropics, notably in the densely forested regions of west africa [ ] . estimates of bush meat harvests in ghana are around , tons annually [ ] . the bushmeat markets across west africa are nowadays dominated by small bodied, fast reproducing species such as rodents like the grasscutter (thryonomys swinderianus) [ ] . there is little officially reported information on the use of bats as bushmeat, a review of survey papers on bushmeat did not report anything on bats [ ] , but unofficial reports and eye witness accounts suggest it is not uncommon to see bats for sale in african markets. it is possible that bats do not follow a typical bushmeat commodity chain and amounts are therefore underestimated in standard bushmeat surveys [ ] . bats are often hunted for pre-arranged orders and regular customers rather than sale through wholesalers who may prefer to concentrate on larger animals with a higher value-to-weight ratio. one study estimates that , e. helvum are sold each year in southern ghana [ ] . this involves a commodity chain stretching up to km and involving multiple vendors. no official data regarding the size of the bushmeat trade exist as much of the trade is informal or illegal. while much trade is intra-country, trans-border trade does occur through known trade routes throughout the region and there is a limited amount of inter-continental trade from africa to europe [ ] . recently a quantity of bushmeat thought to be from the central african republic was seized by french police, and was reported to include bats, although the species were not named [ ] . imports of bushmeat into the uk do occur and mostly take place from those parts of africa with which the uk has close historical connections, in particular west africa [ ] . residents of the uk who have their ethnic and cultural origins in central and west africa and who are returning from a visit there often bring bushmeat into the uk for their own consumption. in comparison with the domestic market in bushmeat in central and west africa the amount of bushmeat coming into the uk represents only a very tiny fraction of the total turnover [ ] . a wildlife policy briefing report, which sets out bushmeat preferences in urban liberia provides a good indication of the sort of bushmeat likely to be imported into the uk, since returnees and visitors to the uk are most likely to buy their bushmeat in urban markets and are likely to reflect current local preferences [ ] . the list comprises ungulates, rodents, primates and pangolins. bats do not feature in the most preferred animals for taste from urban communities in west africa or in the more generic list of animal involved in the bushmeat trade in west africa. chaber et al. sampled passengers from flights from central and west africa to france over days in june [ ] . fifty-five passengers were found to be carrying fish or domestic meat and nine were carrying bushmeat. average individual consignments of bushmeat were over kg, compared with and kg for livestock and fish. most illegal imports detected by uk border agency are small amounts and continue to be typically gifts by travellers visiting family (or returning from visiting family abroad), or seizures from tourists, business people and students travelling to the uk for the first time. most do not involve deliberately smuggled goods but are made from passengers who are not aware of the current rules and prohibitions in place for products of animal origin (poao) imports [ ] . as well as personal carriage, bushmeat may be imported either by postal carriage or commercial freight to the eu. hm revenue and customs found bushmeat to constitute % of poao customs seizures for the period - . some bushmeat samples entering eu states from africa do so from european transit flights, as under the single market goods can travel freely from one member state to another without checks. thus the situation in any specific member state depends on the effectiveness of border controls in other member states. the bushmeat from animals hunted in tropical forests destined to be carried to the eu is likely to be preserved in some form for the duration of the journey. the bushmeat consumed in the uk imported from west africa is most often either smoked, dried or salted [ ] . because of this processing the initial load of viable organisms on the bushmeat would be expected to be reduced significantly. the average duration of smoking of bushmeat was found to be about hours minutes per day at a maximum temperature of . °c [ ] . to preserve the bushmeat it may be frozen on arrival in the uk. freezing in general promotes virus survival and a laboratory study suggested long survival times of marv at °c [ ] . throughout africa and asia, bats have been used in zootherapy, which is the treatment of human ailments with remedies made from animals and their products. around % of the population in africa uses traditional medicine and there is also a growing interest in many developed nations [ ] . there is evidence of bats being used for specific ailments in zootherapy and it is possible that they may still be used by migrants in european countries. treatment of ailments with bats include disorientation in patients with mental illness [ ] , fertility medicines and post birthing remedies, [ ] , the use of bat droppings of p. giganteus to treat patients with alcohol and drug addiction, [ ] , and night blindness [ ] . in asia, asthma is the most frequently cited disease for which bats are used as a remedy [ ] [ ] [ ] . these therapies are frequently practiced in countries where there is evidence of niv infection in bats. kanda tribal healers in bangladesh use p. giganteus in formulations for the treatment of fever [ ] , one pharmaceutical company in vietnam reportedly imported tonnes of faeces of rhinolophus bats [ ] . in a survey of asthma patients in singapore primary care clinics on the use of complimentary therapies, patients ( . %) had used complimentary medicine out of which ( . %) used animal products, ( . %) of which had used fruit bats [ , ] . whilst there is evidence that bats, as bushmeat, are eaten extensively in africa and asia there is little evidence of them being internationally traded or brought to the eu in personal possessions; a number of studies have investigated illegal imports of bushmeat, but rarely have bats been among the samples seized. however, these are relatively small studies and do not confirm the extent to which bats are exported as bushmeat. additionally, other animals, such as monkeys, were identified in the seized samples and are known to be susceptible to viruses such as marv. a review of possible microbiological hazards associated with the illegal importation of bushmeat concluded that although there was a lack of quantitative data relating to the microbiological risks, the risk of foodborne illness from consumption of bushmeat appeared to be very low and the risk of foodborne illness from cross contamination was also minimal [ ] . normal cooking would probably destroy any viruses and bacteria present although there were no data presented to verify this. the risk from use of bats in zootherapy is not as well understood. however, while the risk of contaminated bushmeat may be low, the consequence could be very high. migration is a seasonal, usually two-way movement from one place or habitat to another, to avoid unfavourable climatic conditions and/or to seek more favourable energetic conditions [ ] . some bat species are known to migrate large distances and cross national borders [ ] . such behaviour will connect seemingly distant bat populations, and an infected individual could therefore act as a vector to introduce a new virus into a naï ve population. bat flights are generally short distances for the purpose of foraging, hunting, changing roost sites or social behaviour. indeed, the majority of bat species in the world are sedentary. some bats, however, particularly those in the temperate regions of the world, perform annual long distance flights [ ] . bat migration typically occurs along rivers, as shown for bats in poland and central slovakia [ , ] and tends to avoid mountainous areas [ ] . with regards to bat species and geographical areas relevant to niv and marv, in congo a massive annual fruit bat migration takes place up the lulua river with hunting of the bats by villagers. direct exposure to the fruit bats may have led to an outbreak of ebov in [ ] . regular mass long-distance migrations have not been reported for r. aegyptiacus [ ] and a sedentary life history for r. aegyptiacus is also supported by the morphological record [ ] . in contrast, some e. helvum individuals migrate more than km [ ] , in some cases following the seasonal fluctuation in fruit abundance [ ] . thus, one study reported that that out of ( %) e. helvum ( %) were migratory, although % ( of ) were non-migratory [ ] . the median travel distance of the non-migratory bats was km (compared to km for the migratory bats) and similar to the observed daily commuting distances of r. aegyptiacus [ ] . based on available data and their own capture information, it was assumed that e. helvum has a core distribution in equatorial africa, with migrations in the northern direction, e.g., mauritania and niger from may to september and towards the south e.g., tanzania, zimbabwe and zambia during the months of october and december [ ] . thus, e. helvum from regions of africa north of the equator will generally migrate south in the autumn, away from europe. there is no evidence to suggest that the return migration routes in the spring would take the bats north of the sahara desert or that bats that might accidentally fly north (instead of south) in the autumn and reach europe. a review of data collected over years from banding of some one million bats within europe, provides information on which bats cross national borders [ ] . these data suggest there are a number of european bat species which migrate seasonally in the range of a few hundred kilometers and four species that are considered long distance migrants (regularly to km in one return flight). the migration routes are generally limited to europe, with the general trend from north-east to south-west europe. however, there are data showing movements of nyctalus noctula from russia into bulgaria [ ] and it is reported that pipistrellus nathusii killed in summer and autumn at german wind turbines originated from estonia or russia [ ] . an occurrence of vespertilio murinus on a north sea drilling rig confirmed that bats can fly across large bodies of sea [ ] . this raises the question of whether migration of bats from africa to europe can occur, for example, across the strait of gibraltar. there have been studies in relation to the genetic diversity in ibero-moroccan bats, but this does not address the frequency of vagrant african bats flying from morocco into southern europe. colonies of r. aegyptiacus, known hosts of marv, occur in cyprus and southern turkey. no banding studies have been done and existing knowledge is based on field observations in europe [ ] . in cyprus, no long distance flights are known, but seasonal altitudinal shifts have been observed [ ] , which could alter contact rates with other bat species. thus, despite the growing evidence on migration of bat species within europe, there are no data to suggest whether migration of bats into europe from niv or marv endemic areas (as outlined in figure ) could occur. a longer term risk factor is the gradual spatial creep of viruses due to transmission to previously uninfected species whose habitat spatially overlaps that of known infected species. for example p. vampyrus are known hosts of niv. they are not found outside of asia, according to iucn red list (see figure ), but have been reported in the shaanxi region of china, close to where m. daubentonii have also been recorded [ ] . m. daubentonii are also known to be present across europe and there is a report of henipavirus antibodies in three of four myotis bat species at a location in yunnan province, southern china in and . this included nine of m. daubentonii bats [ ] . although pteropid bats are not widespread in china, henipaviruses could be introduced to china by other susceptible bat species whose habitats and ranges overlap those of pteropid bats in neighbouring countries. this raises the question of whether henipaviruses could eventually emerge in european bats. however, there are a number of additional factors that may delay and/or prevent this from occurring, such as mountainous areas providing geographical barriers to interaction of neighbouring bat populations. indeed according to the iucn redlist the populations of m. daubentonii in china and europe are not contiguous. it would be interesting to know if bats in south-east asia migrate in a north-westerly direction to the same regions as those migrant european bat species to give a -virus cross-roads‖. the risk of eu bat infection with marv due to overlapping species populations is potentially higher than niv, due to shorter geographical distances, r. aegyptiacus are already present in some european countries where their range may overlap with some migratory european bat species, and the fact that some african fruit bat species (e.g., e. helvum) migrate large distances, although generally within the sub-saharan african continent [ ] . however, marv has not been isolated from any bats in cyprus or indeed northern africa, although there have been few published reports of attempts to find marv outside its normal range. additionally, marv has not been isolated from as many different bat species as henipaviruses, so the risk of virus transfer between species may be more limited. this may reflect the ubiquity of molecular receptors for henipaviruses among mammal species. there are a number of less obvious routes by which bat carcasses or products could enter europe. for example a bat strike on a long haul aircraft may result in the carcass of the bat being carried long distances across international boundaries. the remains of a bat were found in the wing flap of a boeing that had flown from heathrow (uk) to ben-gurion airport in israel [ ] . the plane had previously flown from ghana to london and pcr was used to identify the bat as having highest similarity with e. helvum. flying foxes and other bats were the animal species most often involved in aircraft strikes in australia between and with the majority of air strikes occurring at locations on the east coast of australia [ ] . for the year period ( - ) , strikes were reported to the united states federal aviation authority of which bats were involved in . % [ ] . this raises the question of what happens to the bat carcass remains and in particular how it is disposed of. in theory it could drop off the plane on coming into land at the destination airport as the carcass thaws or the wing flaps change position. this raises the possibility of the carcass being eaten by scavenging animals or even pet dogs or cats. accidental translocations of bats between land masses by ships or aircraft have also been known to occur, almost certainly with a far greater frequency than is actually reported [ ] . as some viruses such as coronaviruses can survive for long periods in water [ ] , bat guano or even dead bats transported in bilge waters of ships could, in theory, serve as route of transport of bat viruses around the world. another route, again involving aircraft, is where the bat is a stowaway either in the aircraft hold, or even the cabin itself. for example, in , a bat flew through the cabin of a commercial airliner minutes after takeoff during an early morning flight from wisconsin to georgia [ ] . the emergence of new viruses typically reflect change and combinations of events [ , ] . in this respect, anthropogenic changes, and in particularly globalization, are drivers. other changes including farming practice, environmental and climate change not only affect land use but also influence zoological and ecological factors including habitat and food supply. thus, over time, there may be changes in both the range and distribution of species and intensity and nature of species' interactions. climate change is associated with extreme weather events such as drought and flood. it is most likely to be linked to the geographical distribution of fruit bats through availability of food sources; the species p. nathusii has been observed to be adapting its range in response to recent climate changes [ , ] . this raises the question of how the range and population of fruit bats will change; ultimately, warming could convert forests to grassland savannas which are unsuitable habitats. a shift in the range of pteropid bats due to climate change could have an impact on the circulation of henipaviruses, by putting bats under stress [ ] . pteropus spp. may excrete viruses more often than usual in stressful situations such as when their food is destroyed by climatic events and extreme stress can result in immune suppression which can facilitate increased shedding of the virus [ ] . bats may also spread the virus between regions if they search for food in areas unaffected by flooding. additionally one study found a significant association with the dry season for spillover events [ ] . in this paper we have discussed factors that should be considered when assessing the risk of introduction of two bat-borne viruses, nipah virus and marburg virus, into the eu. the routes considered to pose a significant risk of introduction into europe include human travel, legal trade and illegal importation of bushmeat. a number of other potential routes should also be considered, including, bat strikes on aircraft and bat migration, although migration may not be significant as currently there is little evidence of significant migration pathways into europe. however, it is unclear whether the absence of knowledge of migration routes into the eu from the countries identified as having infection in bats from figure is because they do not exist or because their existence has not been comprehensively investigated. additionally, if niv or marv were to spread to areas on a european migration route, such as russia, then bat migration could become a greater risk. another, more long term risk for introduction to the eu could be transmission between bat species with overlapping distributions; r. aegyptiacus are hosts of marv and present in cyprus (although marv is not known to be present in bats in cyprus), where the range of this species may overlap with some migratory european bat species. it should also be noted that migration could pose a risk for other bat viruses which may be present on these migration routes. the two viruses discussed in this paper were chosen as they are not known to be present in eu bat populations, but published literature indicates their potential for causing large scale human outbreaks. there are many other bat-borne viruses of similar potential that we do not cover in detail here, but also require in depth consideration, such as ebola virus, hendra virus and mers-cov. at the time of writing there was limited and not conclusive evidence that mers-cov was a bat-borne virus [ , ] . while the risks of introduction of other bat borne zoonotic viruses should be considered on a case by case basis, there will likely be a degree of commonality with the factors and routes discussed in this paper, especially for viruses within the same family as marv or niv, namely filoviruses such as ebov and paramyxoviruses such as hev. while there is serological evidence of henipaviruses and filoviruses on multiple continents, the isolation of infectious virus in either bats or humans is currently limited to more confined geographical areas; niv in asia and marv in central africa. human infection of niv in particular is currently limited to bangladesh and west bengal in india. given the more widespread identification of niv amongst bat species and countries in asia, it is not clear why human outbreaks appear to be confined to this region. this could reflect the route of transmission, sensitivity of surveillance and also perhaps the greater titre of niv-bangladesh in bat saliva or urine compared to niv-malaysia [ ] . further knowledge of why these viruses do not currently seem to be spreading further, could help in assessing the risk of further spread, including the risk of reaching the eu. while a number of studies report high serological prevalence, actual virus infection in bats is rarely detected. this could explain why human spillover events of niv-bangladesh are fairly localised. p. giganteus roosts have been identified within km of villages in bangladesh and can consist of around individual bats [ ] , so even a low prevalence of infection within the roost can mean that there are still sufficient numbers of infected individuals able to contaminate local food sources such as date palm sap. in this paper we have discussed risks posed by bats, regarding entry of zoonotic viruses to eu, but the ecological importance of bats should also be recognized. insectivorous bats are responsible for controlling populations of other species considered to be pests such as mosquitoes and other insects, while fruit bats feed on nectar and pollen and so provide an important function as pollinators and/or seed dispersers [ ] . while the mass culling of pigs in malaysia undoubtedly helped to control the niv outbreak there, culling, or relocation, of wild bats could potentially increase levels of infection [ ] . for example, research in peru found that culling campaigns failed to reduce the seroprevalence of rabies among the studied vampire bat colonies [ ] . additionally culling of bats is considered by many to be unethical and methods are unavailable that comply with current standards of animal welfare. there are many alternative methods to help control virus disease, such as the use of bamboo skirts to prevent niv contamination of data palm sap in bangladesh [ , ] , limiting potential for indirect contact between livestock and bats at a local level, use of personal protective equipment by investigators dealing with suspect cases and a vaccine against hev in horses in australia [ , ] . this review identifies those routes which could provide a potential for introduction of niv and marv into the eu, but does not formally assess the risk associated with each route. for niv we have shown that, of the eu mss, the uk has the highest volume of relevant human travel (figure ), but the netherlands has the highest volume of relevant trade (figure ) , suggesting that the most probable route for introduction may vary between eu mss. however, to formally assess this it will be important to also take into account virus specific factors such as prevalence, titre and survival and ms specific factors such as border inspections or controls. therefore, it would be preferential to develop a quantitative risk assessment (qra), which would require large amounts of data. this review suggests that while data may be lacking to fully assess the risk for routes such as bushmeat, or indeed any other illegal activity, there are sufficient data available to assess legal routes such as volume of trade and human travel. in general, we found no evidence to suggest that the risk of niv release to the uk has changed from that reported in a previous qualitative risk assessment [ ] . reported human cases of niv continue to be limited to bangladesh and an increase in the number of those cases may be due to enhanced awareness and surveillance. a number of human cases of marv have been reported in uganda recently, but again this could be attributed to better surveillance. while there is evidence to suggest henipavirus infection of m. daubentonii in china and the presence of r. aegyptiacus in the eu country of cyprus, these are not sufficient factors on their own to warrant undue concern. however, it should be noted that there is a lack of research and surveillance in this area and the evidence for absence of niv or marv in bats present in the eu is limited. human migration patterns continue to change across some areas of the eu, suggesting the frequency of human travel to niv or marv areas and corresponding illegal imports of products such as bushmeat may change. this could increase the probability of a -rare event‖ occurring, such as importation of a bushmeat sample contaminated with virus and, as has been observed in the past, a single introduction event can be enough to cause an outbreak of disease in humans. a better understanding of surveillance sensitivity and biases in reporting, and further investigations of the presence and prevalence of these viruses in both bats and humans should be carried out, as high uncertainty remains about the risks associated with these diseases and how best to prevent or limit the risk of an introduction event. this work was funded by the european union fp project antigone (anticipating global onset of novel epidemics ) and the uk department for environment, food and rural affairs (defra) project se . the authors would also like to thank trevor drew and 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disease emergence: the past, present, and future drivers of nipah virus emergence the effect of bat (rousettus aegyptiacus) dispersal on seed-germination in eastern mediterranean habitats flying foxes carrying hendra virus in queensland pose a potential problem for other states ecological and anthropogenic drivers of rabies exposure in vampire bats: implications for transmission and control piloting the use of indigenous methods to prevent nipah virus infection by interrupting bats' access to date palm sap in bangladesh guidelines for veterinarians handling potential hendra virus infection in horses the authors declare no conflict of interest. key: cord- - rsk g l authors: kinast, volker; burkard, thomas l; todt, daniel; steinmann, eike title: hepatitis e virus drug development date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: rsk g l hepatitis e virus (hev) is an underestimated disease, leading to estimated million infections and up to , deaths annually. infections are mostly asymptomatic but can reach mortality rates up to % in pregnant women or become chronic in immunocompromised patients. the current therapy options are limited to the unspecific antivirals ribavirin (rbv) and pegylated interferon-α (pegifn-α). rbv leads to viral clearance in only % of patients treated, and is, similar to pegifn-α, contraindicated in the major risk group of pregnant women, emphasizing the importance of new therapy options. in this review, we focus on the urgent need and current efforts in hev drug development. we provide an overview of the current status of hev antiviral research. furthermore, we discuss strategies for drug development and the limitations of the approaches with respect to hev. with approximately million infected people per year, hepatitis e virus (hev) leads to more cases of acute hepatitis than any other human hepatotropic virus, such as hepatitis a virus (hav), hepatitis b virus (hbv), hepatitis c virus (hcv) and hepatitis d virus (hdv). hev is a quasi-enveloped positive strand rna virus ( figure a ) that is classified as a member of the genus orthohepevirus within the family of hepeviridae [ ] . the genotypes (gt) and of hev are obligate human pathogens and are primarily transmitted via contaminated drinking water. recent outbreaks of acute hepatitis linked to hev have, amongst others, been reported in nigeria [ ] , chad [ ] , and bangladesh [ ] . by contrast, the zoonotic gt and , which are endemic especially in europe and the americas, can cause in addition to acute infections also chronic infections in immunocompromised individuals. the most common infection routes are thought to be the consumption of undercooked meat of or contact with infected animals, such as pigs, wild boars, and deer, which constitute the virus reservoir. furthermore, the transfer of contaminated blood products is a man-made safety hazard, especially for risk groups such as immunocompromised patients [ ] . recent cases of human infections with gt [ ] and rat hev [ , ] extended the spectrum of hev gts, which are capable of jumping over the species barrier and are able to infect humans. acute hev infections are self-limiting in most cases, but especially for gt , they are linked to high mortality rates up to % in pregnant women [ ] . it is hypothesized that immunological and hormonal changes are responsible for the high mortality [ ] . hev has also been reported to cause a variety of extrahepatic manifestations, for instance, guillain-barré syndrome or pancreatitis (reviewed in [ ] , see figure b ). in total, . million estimated cases of acute illness and , - , deaths per year make this pathogen a non-negligible health burden. however, the current therapeutic options against hev are limited to the off-label use of the unspecific antivirals ribavirin (rbv) and pegylated interferon-α (pegifn-α). the treatment algorithm for figure . schematic representation of hepatitis e virus (hev) particle and the major clinical manifestations. (a) hev particle and genomic organization. the hev genome is composed of a single-stranded rna genome of~ . kb and is encapsulated in an icosahedral capsid. hev virions can occur in both a non-enveloped and in an enveloped form. the viral rna, which is capped with -methylguanosine ( mg) at the ´noncoding region and polyadenylated at the noncoding region, comprises three open reading frames (orf). furthermore, gt is believed to contain an additional orf (orf ). orf encodes the replicase proteins, including a methyltransferase (mt), cysteine protease (pro), helicase (hel), and rna polymerase (pol), as well as three regions without a reported enzymatic function (y, hypervariable region (hvr), and x). orf encodes the capsid protein, whereas orf encodes a viroporin. (b) major clinical manifestations. the majority of hev infections are asymptomatic. gt and gt infections can become chronic in immunosuppressed individuals, with high risk for developing severe complications, such as liver cirrhosis. hev has also been reported to cause a variety of extrahepatic manifestations, like guillain-barré syndrome. infections with hev gt cause acute hepatitis, with high mortality rates up to % in pregnant women. identification of novel therapy options can encompass several strategies. while some rely on de novo identification of compounds, another approach is to reuse already existing compounds, a process which is termed drug repurposing. drug repurposing can generally be described as the idea of using drugs or drug candidates for another than their primary indication. the pharmacokinetic and pharmacodynamic profile, including undesirable effects, is often already elucidated in vivo in animals and humans. the current first choice of anti-hev treatment, rbv, is an example for a repurposed drug that was originally developed for treatment of respiratory syncytial virus (rsv) in infants [ ] . rbv has not only been administered for antiviral therapy against rsv but also against hcv, influenza, viral hemorrhagic fevers [ ] and hev [ ] . recently, drug repurposing has gained increased attention in the scientific community with reviews covering either specific viruses [ ] or viral diseases in general [ , ] . surprisingly, there are only a few publications, in which this approach is used as a strategy to find an antiviral against hev. however, given the availability of public fda-approved compound libraries and the anticipated benefits of this approach over de novo development, publications screening for antiviral activity of already approved drugs are anticipated. de novo drug development can rely on screens, where compound libraries are tested for their capacity to interfere with the viral life cycle. both the target and the mode of action of the substance do not need to be identified. for structure-guided development, the target and ideally its crystal structure is already identified, which enables to specifically design antivirals. independent from the approach used to identify a prospect compound, the candidates have to be validated in vitro and in vivo. there are several in vitro models available, including different cell culture models as well as primary human hepatocytes and induced pluripotent stem cells. for a detailed overview, please see a recent review by meister et al. [ ] . similarly, there are several small animal models as rabbits, rats, ferrets, and birds, which are used for the different hev strains (reviewed in [ ] ). this review aims to give an overview over the current state of efforts to establish additional treatment options against hev. antiviral candidates are grouped according to the strategy that was used to identify them. before the concluding remarks, vaccination is also briefly discussed in terms of its potential to replace antiviral therapy options. '-c-methylcytidine ( -cmc), also known as nm , is a nucleoside analogue (na) originally developed against hcv. when first applied against hev in vitro, the compound demonstrated an inhibitory effect against hev replication (ic of µm/l) [ ] . furthermore, it inhibited the replication of both a luciferase replicon based on kernow c as well as the full-length virus without showing signs of resistance upon prolonged incubation [ ] . the effect could be reverted by addition of cytidine triphosphate, but not guanosine triphosphate to the cells. applying it together with rbv yielded in a moderate antagonistic effect, suggesting a convergent mechanism of inhibition. when assessed for treatment of hcv, insufficient oral bioavailability was reported. therefore, a prodrug termed nm was developed [ ] . however, the development was discontinued due to adverse toxic effects, which has been a problem for several 'methyl nucleosides [ ] . the toxicity correlates with the property to serve as a substrate for the mitochondrial dna polymerase and thus leading to the termination of mitochondrial rnas [ ] . the example of nm emphasizes that -cmc, although efficacious against hev in vitro, will probably need modifications to rule out undesired effects, in particular against the mitochondrial dna polymerase. recently, netzler and colleagues tested a collection of compounds with a reported inhibitory effect on the rna-dependent rna polymerase (rdrp) of different classes of viruses [ ] . both the class of nas as well as non-nucleoside inhibitors (nni) were considered in their study, covering compounds from late-preclinical stage to fda-approved drugs. they identified two compounds, gpc-n and nitd , inhibiting a gt derived subgenomic replicon. gpc-n had a half maximal effective concentration (ec ) of . µm and a therapeutic index (ti) of > (gpc-n ) whereas nitd had an ec . µm and a ti > . gpc-n has been described as an nni of the rdrp of multiple genera within the picornaviridae family [ ] . nitd has been reported to act as an antiviral on numerous viruses, amongst others hcv [ ] , west nile virus [ ] or zika [ ] . nitd has shown toxicity in vivo [ ] . so far, no clinical trials have been registered for both of these compounds. nishiyama and colleagues used an fda approved drug library for an in vitro screen against a gt strain containing a gaussia luciferase reporter [ ] . ciprofloxacin (cpfx) and ifn-λ - showed the best activity against the reporter genome. they also used an infection model, where cells infected with a full-length gt strain at a plateau phase were co-cultivated with naïve cells. all ifn-λ subtypes, but not cpfx, showed robust reduction of hev rna. sofosbuvir is a nucleotide prodrug, which can be incorporated into hcv rna by the ns b rdrp acting as a chain terminator [ ] . its antiviral potential against hev was demonstrated using gt hev replicons in huh and hepg cells. the ic values were in a low micromolar range, to orders of magnitude less potent than against hcv replicons [ ] . sofosbuvir showed an additive effect together with rbv. the inhibitory effect was confirmed by the same group in induced pluripotent stem-cell derived hepatocyte-like cells (ipsc-hlcs) for all four common gts [ ] and in cell culture for gt [ ] as well as gt [ ] by other groups. however, it was also described that both gt and gt replicons as well as full-length gt virus were not inhibited in hek t, u- mg, and huh cells [ ] . very recently, li et al. reported that a gt strain was moderately inhibited at µm by an isopropyl ester of sofosbuvir, psi [ ] . similar to the situation in vitro, the data on sofosbuvir's efficacy in vivo have been inconclusive. three case studies stated that sofosbuvir failed to clear hev in patients when administered together with rbv [ ] [ ] [ ] , while three reported successful treatment together with rbv [ ] [ ] [ ] . a multicenter phase ii study addressed whether sofosbuvir monotherapy is efficacious against hev. a total of mg sofosbuvir was administered once daily over a course of weeks to nine patients who had previously failed rbv therapy [ ] . none of the patients cleared the virus, although it reduced hev rna levels by at least one order of magnitude in five out of nine patients during the study period and significantly reduced alanine aminotransferase (alt) levels. with its antiviral efficacy being only moderate, it is probably not suited for use as monotherapy. however, it could further be investigated in combination with rbv. compound screening allows for the rapid testing of a large number of chemical substances and extracts with the aim of identifying new compounds. in the context of viral infections, important drugs identified with this method include maraviroc and etravirine [ ] , both of which target hiv as well as daclatasvir, which targets hcv [ ] . prerequisite for compound screening is a suitable assay, reflecting the physiological conditions of infection. importantly, the screening method has the benefit of not necessarily requiring profound knowledge about the viral lifecycle or specific targets. assays using purified enzymes, subgenomic replicons or full virus already have successfully been used in hcv research. there have been two reports about the antiviral activity of ethanol extracts of plants against hev. one extract was prepared from the plant lysimachia mauritiana and showed activity against a pshev -luc replicon in huh . cells, as well as while a full-length gt virus on rna and protein level in a cells [ ] . however, identification of the compound(s) responsible for the antiviral effect was not performed. the same group reported the antiviral effect of an ethanol extract of another plant, liriope platyphylla, against hev [ ] . both a pshev -luc-replicon as well as the c full-length genome were inhibited by the extract in huh . cells. by performing activity-guided fractionation and multicolumn chromatography, spicatoside a could be identified as the active compound, and its activity as a pure compound was demonstrated. no testing for toxicity, resistance induction or in vivo efficacy was conducted. zinc is an essential micronutrient that has been reported to reduce replication of, amongst others, hiv [ ] and coronavirus [ ] in high concentrations. in a study investigating the influence of different salts on hev replication, it was demonstrated that zinc salts inhibited replication of both gt and replicons as well as a gt clinical isolate in porf -huh cells [ ] . the effect was likely due to an inhibition of the rdrp, as an inhibitory effect on the protein was observed in vitro. a mild effect could be observed at µm, being strongest at µm. it will be of interest to investigate if zinc supplementation will prove an effective strategy against hev in patients. zinc levels in plasma have been reported to be usually around - µm depending on the study, as described in a meta-analysis [ ] . the authors also found that zinc supplementation increased plasma levels, with every doubling of the dose leading to a % increase [ ] . these data imply that zinc levels are highly regulated, suggesting that effective plasma levels in patients might be at best challenging to reach. there has been no evaluation of zinc monotherapy against hev in vivo so far. however, a case study [ ] investigated the influence of intra-erythrocyte zinc levels on the outcome of rbv treatment. they showed that treatment successes could not be attributed to increased zinc levels. although only comprising four patients in total, this suggests that it might not lead to viral clearance in immunocompromised individuals when combined with rbv. e is part of a small molecule compound library belonging to the diversity set ii of national cancer institute developmental therapeutic program. it was initially identified as an inhibitor of the rdrp of hcv, inhibiting hcv a replicon with an ec of . µm [ ] . the compound also inhibited hev, both the replicon p -luc as well as full-length p in huh or huh s - cells. interestingly, it did not show inhibitory effect on purified rdrp for both hcv and hev, suggesting that it does inhibit replication either by a affecting a host factor or that it does undergo modification within the host. no testing resistance induction or in vivo efficacy was performed. different from screening approaches, target-or structure-guided development aims at identification of suitable targets for an intervention first. prerequisite is that the function of a target is characterized. both the virus itself and the host factors necessary for the viral life cycle can be targeted ( figure ). only one protein of hev has been crystallized so far, which is a truncated version of the capsid protein porf [ , ] . although some conclusions regarding the involvement of certain domains in cell binding could be drawn, and heparan sulfates seem to be important for hev attachment to the host cell surface [ ] , this knowledge has not led to the establishment of a therapy concept yet. the scarcity of antiviral candidates directly acting on the virus can be explained by the lack of knowledge about hevs molecular virology. due to their small genomes, viruses depend on host factors for completion of their life cycle. this dependence on the host is a potential point to intervene. host-acting antivirals have the potential to interfere with the distinct steps of the viral life cycle, from blocking the entry receptor over preventing the formation of the replication complex to hamper the viral maturation by inhibiting cellular proteins. a detailed functional analysis of the host factor and its role in the context of hev infection is desirable and would help to optimize chemical intervention. additionally, direct acting antiviral specifically can target viral enzymes (e.g., helicase, polymerase, protease) without affecting host components. notably, the nucleoside analog ribavrin is reported to exert antiviral effects by targeting both the virus and the host [ ] . a hammerhead ribozyme is a small catalytically active rna molecule, capable of cleavage or self-cleavage [ ] . hammerhead ribozymes have been designed to target, amongst others, severe acute respiratory syndrome coronavirus (sars-cov) [ ] or hcv [ ] . however, there has only been one report so far describing the use of this technique in vivo [ ] , which dates back already more than a decade. in , it was reported that a hammerhead ribozyme could be designed to specifically cleave rna of hev gt in the 'cis-acting element [ ] . when expressed from a vector in hepg cells, they could decrease replication of a psgi-hev- 'luc construct. questions about delivery of the agent to target site in vivo in patients, as well as potential off-target effects, might remain unsolved, considering that there has been no follow-up study so far. peptide conjugated phosphorodiamidate morpholino oligomer (ppmo) are uncharged nucleic acid analogs containing a morpholine backbone connected by phosphorodiamidate linkage, rendering them resistant to many enzymes like nucleases, esterases, and proteases [ ] . ppmos bind rna via watson-crick base pairing and interfere with viral translation by steric blockage. ppmos complementary to sequences in sar have been shown to inhibit replication of a psk-e replicon in huh s - cells. they also reduced porf levels in hepg /c cells infected with kernow c and huh s - cells infected with psk-e [ ] . so far, there has been no follow-up on this study. mg , an inhibitor of the proteasome with low nanomolar inhibitory constant [ ] has been suggested as an antiviral against hev. it reduced rna and protein levels of a hev gt replicon in huh s - cells [ ] . however, when others reproduced the experiment in huh cells, they also found reduced expression of several housekeeping genes as well as overall rna levels [ ] . it is therefore assumed that mg inhibitory effect on hev is unspecific. some viruses can bind the cellular protein tumor susceptibility gene (tsg ) to hijack the escrt machinery for egress, for instance, hiv- [ ] . it was demonstrated that porf of hev can bind tsg via a psap motif in the viral protein and does colocalize with it in hev-transfected cells [ ] . consequently, porf is essential for viral egress, while having a neglectable effect on cell binding and replication [ ] . the interaction with tsg is mediated via the psap motif in the porf [ ] , the same motif that p gag of hiv- uses to engage with the cellular protein [ ] . cyclic peptides (cp) that had been developed to abrogate interaction of p gag and tsg and inhibited viral release of hiv virus like particles (vlps) [ ] were tested for their activity against hev [ ] . one of the inhibitors, cp , inhibited interaction of tsg and porf both in a yeast-three hybrid screen as well as in a pulldown assay. viral release of both gt and gt hev from huh porf or huh cells, respectively, was inhibited without showing significant toxicity. inhibition of viral release by targeting porf /tsg interaction is an interesting mechanism for antiviral development, with cp providing a starting point for further compound development and characterization. in general, targeting production of nucleotide synthesis to deplete or imbalance nucleotide pools is a strategy employed against several viruses [ , ] . the compounds rbv and mycophenolic acid (mpa), both of which target enzymes involved in nucleotide synthesis, are either already used as treatment against hev or have been reported for their potential to inhibit the virus. as its ester mmf, mpa inhibits the inosine- -monophosphate dehydrogenase (impdh), which is a pivotal enzyme in the purine nucleotide synthesis and is part of immunosuppressive regimens in organ transplant recipients. in a german study, a tendency was identified for its use in heart-transplant recipients being linked to clearance of hev infection without development of chronicity [ ] . in huh cells, mpa inhibited replication of a p -luc replicon, an effect that could be reverted by supplementation of guanosine, suggesting inhibition of impdh as inhibitory mechanism [ ] . in the same study, an additive effect with rbv was found. these results could not be confirmed in patients. a french study assessed the effect of immunosuppression by mmf on rbvs' potential to lead to a sustained virological response (svr) [ ] . they did not find evidence for an additive effect of mmf on rbv treatment. because of its immunosuppressive effect, mmf monotherapy to treat hev infections seems unlikely. given the lack of evidence for an additive effect with rbv, it will probably also not be used to treat hev infections together with rbv. although mmf seems unsuited as a drug against hev, targeting the impdh or enzymes involved in the purine synthesis pathway might still be a starting point for further studies. in a proof-of-concept study, wang and colleagues [ ] tested custom designed inhibitors of the impdh. all inhibitors decreased replication of a p -luc replicon, emphasizing the potential of this target. furthermore, inhibitors of pyrimidine synthase like brequinar and leflunomide inhibited the replicon. this is especially interesting, since both drugs have been tested in clinical trials and are therefore better characterized than newly developed inhibitors. targeting nucleotide synthesis, especially of pyrimidines, might therefore be an interesting approach to tackle hev, with brequinar and leflunomide already providing starting points for testing in vivo. it would be of interest to characterize these compounds better to evaluate their potential as hev antiviral. silvestrol is a natural compound belonging to the class of cyclopenta[b]benzofuran compounds, which are exclusively found in plants of the aglaia genus [ ] . silvestrol has been shown to target the translation initiation factor a (eif a) to rna, thereby preventing ribosome loading onto mrna and blocking translation [ ] . originally described in the context of cancer treatment, silvestrol has been reported to inhibit several viruses in vitro [ ] [ ] [ ] . silvestrol reduced viral titers, the number of infected a cells, as well as viral protein levels in infected cells in vitro [ ] . another study confirmed the inhibitory effect of silvestrol on gt replicons in hepg cells with an ic of around nm for gt hev [ ] . the inhibitory effects were consistent for different patient isolates covering the hev gt - , which were evaluated in ipsc-hlcs. silvestrol reduced viral titers in feces in xenograft mice infected with hev gt , therefore demonstrating its effectiveness in vivo. importantly, silvestrol was effectively inhibiting a p -luc replicon harboring the g r mutant, which confers rbv resistance [ , ] . these data demonstrate that silvestrol might provide a therapy option for otherwise untreatable rbv resistant cases. characterization of potential resistance barriers and a structure-activity relationship of the compound will be important next steps for further development of a promising candidate. vaccination may be an important strategy to reduce the global burden of the virus. in china, there is a vaccine licensed under the name hecolin ® . it consists of the amino acids - of the capsid e protein of hev gt , produced in e. coli [ ] . three doses at , , and months were tested in healthy patients - years of age [ ] . long-term studies following the same cohort up to months after vaccination showed an efficacy of . % in the intention-to-treat-analysis as well cross-protection against hev gt [ ] . there are currently several open questions about the vaccine, thereunder its efficacy and safety in risk groups, the cross-reactivity against other hev gts, and long-term protection after more than months. it is unknown whether hecolin ® is safe for pregnant women and their fetuses, which are a population especially at risk of fatal outcomes of hev gt infection. results obtained from pregnant women that had unintendedly been enrolled by mistake in the phase iii study suggest that the vaccine might be safe for them [ ] . however, because the study was not designed for that purpose, this finding is not a definitive conclusion. a study that does not exclude pregnant women is currently running in bangladesh and expected to finish in (clinicaltrials.gov identifier: nct ). it is also unknown if the vaccine is protective in the second risk group of immunosuppressed or immunocompromised patients. a french study investigated re-infections with hev in organ transplant recipients seropositive for anti-hev igg before transplantation [ ] . they showed that an igg titer up to . who units/ml does not guarantee protection from a re-infection up to one year after transplantation. the data from the long-term study on hecolin ® [ ] indicate that the geometric mean of igg titers months after the first dose is already below that value in individuals seronegative at baseline. notably, the igg titers of individuals, which were anti-hev igg positive before vaccination, fell below the threshold of . who units/ml after months. this raises the question whether chronic hev infections in immunocompromised patients can be attacked with the current vaccine. there are also no data on safety and efficacy in the risk group of patients with chronic liver disease. it is unknown if the vaccine confers cross-protection against hev gts , , or rat hev, although the cross-reactivity of the vaccine against gt hev is promising. the vaccine schedule requires six months; it would be to clarify if the vaccine can be used short-term to combat an outbreak. these limitations and uncertainties make research on antiviral therapy an important issue, regardless of the vaccine's benefits. the recently evolving attention on hev also led to an increased focus on finding a satisfactory therapy. however, sofosbuvir is the only candidate in clinical trials to date (figure ) . preliminary results indicate that it will not be a breakthrough in hev therapy options. apart from that, the nature of repurposed drugs might make them appealing to use for clinical trials. however, a -cmc derivative was discontinued due to toxic effects, while gpc-n or nitd never moved to clinical trials, which might indicate that they have adverse effects that could hinder development to an antiviral. the hits identified from other screens are either not well characterized yet, including toxicity, or are difficult to dose in vivo. poor characterization is also an issue for several of the target-based compounds, while one is an immunosuppressant. inhibition of porf and tsg might be an interesting target for drug development, as demonstrated using cp . silvestrol is a promising candidate, since it is comparably well characterized and shows efficacy in vivo and an additive effect with rbv. despite some challenges like scaling up its production, silvestrol is the most promising candidate at the moment. although the vaccine can be of great use in reducing hev burden, there are too many open questions, especially about their efficacy in risk groups and outbreak scenarios to solely rely on it to combat hev. an ultimate goal would be to discover specific agents only targeting the viral enzymes as, for instance, the hev protease or polymerase. these so-called direct acting antivirals (daas) are highly specific and were a breakthrough in hcv therapy with high cure rates and enhanced efficacy. being virus-specific, they might also be much more suited for the use in immunocompromised patients and especially pregnant patients, for whom there is no therapy yet. of note, none of the drug candidates presented in this paper has an approval for use in pregnant women. integral for the discovery of daa was in the case of hcv and will be in the case of hev the crystallization of the respective enzyme structures. this will enable structure-guided design of potent inhibitors by fitting the compound and the viral enzyme to complementary surfaces. as mentioned above, the structure of porf has been determined, but without further implications for drug development yet. regarding the polyprotein from orf , it is still debated whether it is cleaved into fragments upon translation or not. some studies argue that it is not cleaved [ ] [ ] [ ] [ ] , while others report cleavage into several fragments [ , ] . recently, a study was published suggesting that the proteases factor x and thrombin cleave the polyprotein and that silencing of these reduced psk-hev -luc replication [ ] . the assignment of functional domains to the porf polyprotein was done in , according to similarity to proteins in related viruses that had known functions [ ] . of these seven domains, the functionality could be confirmed with biochemical assays for four: methyltransferase [ ] , papain-like cysteine protease [ ] , helicase [ ] , and rdrp [ ] . the exact function of the other domains as well as the exact borders of each functional domain are not known to date. until it is not completely understood how the polyprotein is processed and how this does affect the structure and enzymatic activity of the functional domains, studies to identify antivirals based on structural insights seem at best unlikely. to date, the de novo development of candidate structures into a potential licensed drug is only a future perspective, underlined by the fact that none of the anti-hev candidates has been designed based on a structure of an hev protein. there are also only a few host factors known; therefore, the identification of novel host factors will be another cornerstone in combating hev. all -omics approaches to decipher the altered cellular environment during infection, as well as functional studies using cdna, shrna and sirna libraries to overexpress or silence host proteins, are powerful and successful tools to discover novel host factors. this will both give new starting point for drug discovery as well as potentially refine in vitro and in vivo models. overview of molecules/extracts with antiviral activity against hev. the depicted molecules/extracts are classified according to the strategy that was used to identify them. so far, the antiviral activity against hev of only four drugs (sofosbuvir, pegifn-α, ribavirin and silvestrol) was approved in experimental settings beyond in vitro cell culture systems. both drugs in the clinics are used off-label and therefore have not been tested in clinical trials against hev. funding: e.s. was supported by the german ministry of education and research (bmbf) through a ginaico grant gw and a silvir grant ggnatm . proposed reference sequences for hepatitis e virus subtypes a new hepatitis e virus genotype strain identified from an outbreak in nigeria a large outbreak of hepatitis e virus genotype infection in an urban setting in chad likely linked to household level transmission factors an outbreak of hepatitis e in an urban area of bangladesh abravanel, f. hev and 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processing for hepatitis e virus replication protein porf the in vitro-synthesized rna from a cdna clone of hepatitis e virus is infectious expression and processing of the hepatitis e virus orf nonstructural polyprotein activities of thrombin and factor xa are essential for replication of hepatitis e virus and are possibly implicated in orf polyprotein processing computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis e virus: delineation of an additional group of positive-strand rna plant and animal viruses virus-specific mrna capping enzyme encoded by hepatitis e virus hepatitis e virus (hev) protease: a chymotrypsin-like enzyme that processes both non-structural (porf ) and capsid (porf ) protein ntpase and ' to ' rna duplex-unwinding activities of the hepatitis e virus helicase domain the ' end of hepatitis e virus (hev) genome binds specifically to the viral rna-dependent rna polymerase (rdrp) this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to thank yannick brüggemann and patrick behrendt for insightful comments. the authors declare no conflict of interest. key: cord- - nyquokt authors: nemoto, manabu; schofield, warren; cullinane, ann title: the first detection of equine coronavirus in adult horses and foals in ireland date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: nyquokt the objective of this study was to investigate the presence of equine coronavirus (ecov) in clinical samples submitted to a diagnostic laboratory in ireland. a total of clinical samples were examined from equids with enteric disease in irish counties between and . a real-time reverse transcription polymerase chain reaction was used to detect ecov rna. nucleocapsid, spike and the region from the p . to p . genes of positive samples were sequenced, and sequence and phylogenetic analyses were conducted. five samples ( . %) collected in and tested positive for ecov. positive samples were collected from adult horses, thoroughbred foals and a donkey foal. sequence and/or phylogenetic analysis showed that nucleocapsid, spike and p . genes were highly conserved and were closely related to ecovs identified in other countries. in contrast, the region from p . and the non-coding region following the p . gene had deletions or insertions. the differences in the p . region between the irish ecovs and other ecovs indicated that the irish viruses were distinguishable from those circulating in other countries. this is the first report of ecov detected in both foals and adult horses in ireland. equine coronavirus (ecov) is a positive-stranded rna virus and belongs to the species betacoronavirus in the genus betacoronavirus [ , ] . the clinical signs associated with ecov infection during outbreaks in the usa [ ] and japan [ ] [ ] [ ] were fever, anorexia, lethargy and diarrhoea. the same clinical signs were also recorded in an experimental challenge study using japanese draft horses [ ] . the main transmission route is considered to be faecal-oral [ ] and ecov is usually detected in faecal samples. however, the molecular detection of ecov in faeces from horses with diarrhoea, does not prove causation. coronaviruses can cause both enteric and respiratory disease in many avian and mammalian species but ecov is less likely to be found in respiratory secretions than in faeces [ , ] . both molecular and seroepidemiology studies suggest that ecov may be more prevalent in the usa than in other countries [ ] . ecov was detected in samples collected from equids in states of the usa [ ] . in central kentucky, approximately % of both healthy and diarrheic thoroughbred foals were infected with ecov [ ] . all of the qpcr positive foals with diarrhoea were co-infected with other pathogens such as rotavirus or clostridium perfringens, suggesting that there was potential for ecov to be over-diagnosed as a causative agent in complex diseases. in contrast in japan, although an outbreak of diarrhoea occurred among ecov-infected draft horses at one racecourse [ ] [ ] [ ] , there have been no similar outbreaks subsequently, and all rectal swabs collected from diarrheic thoroughbred foals were negative. furthermore, only . % of the rectal swabs collected from healthy foals in the largest thoroughbred horse breeding region in japan were positive for ecov [ ] . in france, . % of faecal samples and . % of respiratory samples collected in counties tested positive for ecov [ ] . similar to the reports from japan and france, a low prevalence of ecov was also observed in the uk [ ] , saudi arabia and oman [ ] . the objective of this study was to investigate the presence of ecov in clinical samples submitted to a diagnostic laboratory in ireland. the samples were tested by real-time reverse transcription polymerase chain reaction (rrt-pcr) as it has been shown to be the most sensitive diagnostic method for ecov [ ] and is routinely employed as an alternative to virus isolation in diagnostic laboratories worldwide, both for timely diagnosis and in epidemiological studies [ , ] . virus isolation and biological characterisation were beyond the capacity of this study, which was similar in scope to that of the studies in horse populations in the usa, europe and asia [ , , , ] . the rrt-pcr assay was performed as previously described using a primer set targeting the nucleocapsid (n) gene (ecov- f, ecov- r and ecov- p) [ ] (table ) and agpath-id one-step rt-pcr kit (thermo fisher scientific, ma, usa) according to the manufacturer's instructions. to prove that the extraction was successful and that there was no inhibition during rrt-pcr amplification, an internal positive control primer/probe (primerdesign, southampton, uk) was added to the master mix. thermal cycling conditions were; • c for min and • c for min, followed by cycles at • c for s and • c for s. the superscript iii one-step rt-pcr system with platinum taq high fidelity (thermo fisher scientific, ma, usa) was used for sequencing analysis of two of the five ecov samples identified. there was inadequate viral nucleic acid in the other three samples for sequencing. the primer sets used to amplify the nucleocapsid (n) gene [ ] , the partial spike (s) gene [ ] , and the region from the p . to p . genes of non-structural proteins (oue, personal communication) are shown in table . the rt-pcr products were sequenced commercially by gatc biotech (cologne, germany). sequence analysis was performed using the blast and clustalw programs, and vector nti advance . software (thermo fisher scientific, ma, usa). phylogenetic analysis of nucleotide sequences was conducted with mega software version . [ ] . a phylogenetic tree was constructed based on nucleotide sequences of the k +g (n gene) and tn (s gene) using the maximum likelihood method. mega software was used to select the optimal substitution models. statistical analysis of the tree was performed with the bootstrap test ( replicates) for multiple alignments. the complete genome sequences of nc (ef ) [ ] , tokachi (lc ), obihiro - (lc ) and obihiro - (lc ) [ ] , the n (ab ) and s (ab ) genes of obihiro , the n gene of hidaka-no. / (lc ) and hidaka-no. / (lc ) [ ] , the s gene of ecov_fra_ / (kc ), ecov_fra_ / (kc ), ecov_fra_ / (kc ), ecov_fra_ / (kc ) and ecov_fra_ / (kc ) [ ] were used in sequence and/or phylogenetic analysis. the accession numbers registered in genbank/embl/ddbj are as follows: the complete sequences of the n gene; v /irl (lc ) and v /irl (lc ), the partial sequences of the s gene; v /irl (lc ) and v /irl (lc ) and the complete sequences from the p . to p . genes; v /irl (lc ) and v /irl (lc ). one six-week-old foal was the only clinical case on a public thoroughbred stud farm with approximately mares when it presented with diarrhoea. recovery took over three weeks during which it received fluid therapy, probiotics, antiulcer medication and antibiotics. the second foal was a -day-old filly, which had been hospitalised with diarrhoea two days prior to sample collection. the foal responded well to supportive treatment and at the time of sample collection, the diarrhoea had resolved. the five ecov positive samples tested negative for equine rotavirus. the nucleotide sequences of the complete n gene, the partial s gene and the region from the p . to p . genes of two positive samples ( v /irl/ and v /irl/ ) were determined. the nucleotide identities of the n and s genes of the two irish ecovs were . % ( / nucleotides) and . % ( / nucleotides), respectively. the nucleotide identities of the n gene of the two irish ecovs and the ecovs from other continents are summarised in table . phylogenetic analysis was performed for the nucleotide sequences of the complete n and partial s genes (figure ). the analysis for the n gene showed that irish ecovs were independently clustered although they were closely related to japanese viruses identified after . in the phylogenetic tree of the s gene, irish ecovs were closely related to all other ecovs analysed. the length of the region from the p . to p . genes in the two viruses was base pairs. compared with nc , irish ecovs, had a total of nucleotide deletions within p . and the non-coding region following the p . gene. compared with obihiro - and - , irish ecovs had a three-nucleotide insertion. when compared with tokachi , the irish ecovs had a -nucleotide insertion (see figure s ). the p . gene of the two irish ecovs did not have deletions or insertions, and the nucleotide identities were . - . % between these viruses and the other ecovs (nc , tokachi , obihiro - and obihiro - ). this study provides the first report of ecov circulating in ireland, the third european country with a significant horse industry where the virus has been detected in horses with enteric disease. however, detection of ecov in faeces samples from horses with enteric disease does not prove this study provides the first report of ecov circulating in ireland, the third european country with a significant horse industry where the virus has been detected in horses with enteric disease. however, detection of ecov in faeces samples from horses with enteric disease does not prove causation. in this study, samples collected between and from equids with enteric disease were tested, and only five samples ( . %) were positive for ecov. the inclusion of an internal positive control in the rrt-pcr eliminated the possibility of false negative results due to the presence of pcr inhibitors but the high content of nucleases associated with faeces samples may have caused some rna degradation. however, this low prevalence of ecov is similar to that identified in france [ ] and among thoroughbred foals in japan [ ] . although ecov has been identified on three continents, little is known about the genetic and pathogenic diversity in field viruses. in this study, sequence and phylogenetic analysis (figure ) demonstrated a high level of homology between viruses detected in a donkey and a horse in two provinces in ireland in different years. this suggests that irish ecovs may have low genetic diversity. compared with the ecovs of other countries, the n, s and p . genes of the two irish viruses were highly conserved. in contrast, the region from p . and the non-coding region following the p . gene had deletions or insertions ( figure s ). because of polymorphism in this region, this region could be useful for epidemiological investigation [ ] . the differences in the p . region between the irish ecovs and other ecovs indicated that the viruses in ireland may be distinguishable from those circulating in other countries. the positive samples were collected in november ( ), march ( ) and april ( ) in this study. higher case numbers are identified in the usa during the colder months (october to april) [ ] , and our results were consistent with the circulation period in usa. it has been reported that outbreaks mainly occurred among adult riding, racing and show horses in usa [ ] . the choice of cases to include in the current study may not have been optimal for detection of ecov as the majority of samples were from foals. however, two positive samples were collected from adult horses in a combined riding school/show jumping yard in the west of ireland. at the time of sample collection in april , the monthly mean temperatures were below long-term average and in parts of the west, were the coldest in years [ ] . cold weather may have been a predisposing factor to the ecov infection on the farm. two positive samples were collected from thoroughbred foals. a faeces sample collected from one foal with severe watery diarrhoea and inappetance was positive for ecov but a sample collected three days later tested negative. a potential difficulty in detecting ecov from naturally infected horses has been noted previously as serial samples from seven sick horses in the usa suggested that ecov only persisted for three to nine days in faeces [ ] . in both cases, the diarrhoea may have been caused by other unidentified coinfecting pathogens as has been suggested by investigators in the usa [ ] . this is the first report of ecov detection in faeces samples from both foals and adult horses in ireland. the viruses identified in ireland are genetically closely related to the japanese viruses and the results of this study give no indication of significant genetic or phenotypic diversity. in recent years, there has been an increase in awareness and testing for ecov in the usa and elsewhere [ ] . horse breeding and racing activities in ireland are the most prominent and important of any country on a per capita basis. there are over thoroughbred horses per , of population in ireland, compared to between three and five for great britain, france and the usa [ ] . thus, an investigation of ecov in ireland is pertinent not only to increase awareness nationally of the epidemiology of the virus and promote discussion on its clinical importance, but also to inform the industry globally of the health status of irish horses. ireland exports horses all over the world. by illustration, in the country was the second biggest seller of bloodstock at public auctions second only to the usa [ ] . many questions remain with regard to the clinical significance of ecov. the outbreak at a draft-horse racetrack in japan in affected of approximately horses and resulted in non-starters and the implementation of movement restrictions [ ] . however, draft horses appear to have a higher infection rate than other breeds and an outbreak of similar severity has not been reported in thoroughbred racehorses [ , ] . the much higher incidence of ecov positive thoroughbred foals identified in kentucky compared to similar populations internationally suggests an increased susceptibility to ecov infection in that population. in the past, specific environmental factors were associated with extensive reproductive loss in the kentucky area and to a lesser extent in other states [ ] , but predisposing regional factors such as differences in management, environment or husbandry have not been identified for ecov. it has been suggested that ecov is a coinfecting agent in foals with diarrhoea and clinical infections have predominantly been reported in adult horses with a mono-infection with ecov [ ] . there was no indication from the results of this study that coronavirus is a major cause of diarrhoea in irish horses but the introduction of rrt-pcr as a routine diagnostic test will assist in elucidating the significance of this virus to the irish breeding, racing and sports industries. the primary focus in future will 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attribution (cc by) license the authors are grateful to the horse owners and their veterinary surgeons for their support and discussions relating to the positive cases and also to dr marie garvey for assistance with manuscript preparation. the authors declare no conflict of interest. key: cord- -o i j b authors: li, yanpeng; gordon, emilia; idle, amanda; altan, eda; seguin, m. alexis; estrada, marko; deng, xutao; delwart, eric title: virome of a feline outbreak of diarrhea and vomiting includes bocaviruses and a novel chapparvovirus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: o i j b an unexplained outbreak of feline diarrhea and vomiting, negative for common enteric viral and bacterial pathogens, was subjected to viral metagenomics and pcr. we characterized from fecal samples the genome of a novel chapparvovirus we named fechavirus that was shed by / affected cats and identified three different feline bocaviruses shed by / cats. also detected were nucleic acids from attenuated vaccine viruses, members of the normal feline virome, viruses found in only one or two cases, and viruses likely derived from ingested food products. epidemiological investigation of disease signs, time of onset, and transfers of affected cats between three facilities support a possible role for this new chapparvovirus in a highly contagious feline diarrhea and vomiting disease. cats have an estimated world-wide population of over half a billion. members of at least viral families have been found to infect cats, including rabies virus, feline rotavirus (frv), feline panleukopenia virus (fpv), feline bocaviruses (fbov), feline bufavirus (fbuv) [ ] , feline astroviruses (feastv), feline picornaviruses (fepv), feline enteric coronavirus (fecv), feline calicivirus (fcv), feline herpesvirus (fhv- ), feline immunodeficiency virus (fiv) and feline leukemia virus (felv) [ ] [ ] [ ] . diarrhea in cats is common and possible infectious causes include bacteria, parasites, and/or viruses. some of the most prevalent feline enteric viruses include fbov, feastv, frv, and fpv [ , , ] . conditions in animal shelters contribute to pathogen emergence due to factors including intensive housing, rapid population turnover, animal stress, and the presence of many direct and indirect routes of possible exposure [ ] . the parvoviridae family consists of non-enveloped, icosahedral viruses with single-stranded dna genomes of to kb [ , ] . eight ictv-approved genera are currently included in the parvoviridae family [ ] . a recent reorganization of the parvoviridae has proposed the creation of a third subfamily named hamaparvovirinae that includes both invertebrate and vertebrate viruses as well as endogenized genomes in the germ lines of fish and invertebrates, suggesting an ancient origin [ , ] . vertebrate infecting members of this subfamily are classified within the chaphamaparvovirus genus (previously labeled as unclassified or chapparvoviruses) and have been identified in rats and mice [ , ] , bats [ , ] , rhesus macaques [ ] , cynomologus macaques [ ] , dogs [ ] , pigs [ ] , tasmanian devils [ ] , birds (red-crowned crane [ ] , chicken [ ] , and turkey [ ] ) and fish (tilapia [ ] ). a chapparvovirus has also been reported in a gulf pipefish and has been proposed to belong to a second genus named ichthamaparvovirus [ ] . a recent study showed that a murine chaphamaparvovirus named murine kidney parvovirus (mkpv) was the cause of nephropathy in laboratory mice [ ] and was widely distributed world-wide [ ] . here, we analyzed a multi-facility outbreak of vomiting and diarrhea in cats using the following approaches: a commercial feline diarrhea panel of pcr tests for known enteric pathogens; viral metagenomics; and follow-up pcrs. multiple mammalian viruses of varied origins were detected. diverse feline bocaviruses and a novel chaphamaparvovirus we named fechavirus were each shed by approximately half of the affected animals tested. the epidemiology of the outbreak points to a possible role for this newly characterized parvovirus in feline vomiting and diarrhea, pending larger studies. from november to january , a multi-facility outbreak of feline vomiting and diarrhea occurred in an animal shelter system in british columbia, canada. a case definition was created to identify all animals affected by the outbreak while excluding unaffected animals: cats housed in three affected facilities with one or more episodes of vomiting or diarrhea (with no apparent other cause) and direct or indirect exposure to cats from the case, between november and january (supplementary table s ). direct exposure was defined as being housed communally with possible direct contact with feces, vomit or a body surface of another animal meeting the case definition (based on time and animal location). indirect exposure was defined as exposure via fomites, personnel, or an environment potentially contaminated by another animal meeting the case definition. a total of cats met the case definition. an outbreak investigation was performed. stool samples from sick cats ( individuals, two pooled litters of co-housed kittens each) were collected and submitted for a variety of diagnostic tests (see supplementary table s ). as animal shelters are resource-limited, testing was focused on clinically sick cats and no samples were collected from healthy cats. initial diagnostic testing included fecal flotation (for parasites) ( cats), fecal antigen testing (helminth ( cats), giardia ( cats), and fpv ( cats)). these tests did not yield a causative agent that could explain the outbreak. testing was then expanded and fecal samples representing a subset of cats ( individuals, one pooled sample from a litter of kittens) were submitted to idexx laboratories, inc (sacramento, ca, usa) and subjected to a comprehensive feline multi-pathogen diarrhea screening panel [ ] . this panel tested for clostridium perfringens alpha toxin, clostridium perfringens enterotoxin, campylobacter coli and campylobacter jejuni, cryptosporidium spp., giardia spp., salmonella spp., tritrichomonas foetus, toxoplasma gondii, fpv, fecv, and frv using pcr and rt-pcr. this screening also did not yield a causative agent that could explain the outbreak. most samples were collected within five days (only were within days) after onset of vomiting or diarrhea with treatments generally initiated after sample collection. four feline vomit samples were also collected from a room of cats in the third affected shelter between january and , , but it was not possible to identify which cats had produced these samples. once the unusual nature of the outbreak became evident (indirect transmission pattern and large number of animals involved), any feces that was not discarded during the initial round of diagnostics and a small number of serially collected samples were frozen for possible further analysis. only initial samples from sick cats ( individuals and a pooled litter of kittens) were available for analysis. serial samples (collected on more than one day) were available from of these individual cats. one gram of feces from each of the cats was vortexed in ml phosphate buffer saline (pbs) with zirconia beads. viral particles were then enriched according to previously described methods [ ] by filtration through a . µm filter (merck millipore, massachusetts, usa) and digestion of the filtrate was achieved with a mixture of nuclease enzymes prior to nucleic acid extraction. nucleic acids (both rna and dna) were then amplified using random rt-pcr as described [ ] . briefly, reverse transcription was performed with primer with a random nonamer at the ' end ( 'gccgactaatgcgtagtcnnnnnnnnn), followed by second strand synthesis using klenow fragment dna polymerase (new england biolabs, massachusetts, usa). both cdna and dna were then amplified by amplitaq gold dna polymerase (thermo fisher scientific, massachusetts, usa) using a primer consisting of the fixed portion of the random nonamer containing primer ( 'gccgactaatgcgtagtc). ssdna genomes are converted to dsdna during the klenow dna polymerase primer extension step. an illumina library was then generated using the transposon-based nextera xt sample preparation kit and sequenced on the miseq platform ( × bases, dual barcoding) (illumina, ca, usa). adaptor and primer sequences are trimmed using the default parameters of vecscreen which is part of blast package v . . [ ] . reads are considered duplicates if base positions to are identical. one random copy of duplicates is kept. low sequencing quality tails are trimmed using phred quality score as the threshold. human and bacterial reads were subtracted by mapping to human reference genome hg and bacterial nucleotide sequences from blast nt database using bowtie v . . [ ] . the ensemble assembler program (v . ) [ ] was used for de novo assembly. both contigs and singlets were then analyzed using the blastx (v. . . ) to in-house viral proteome database, then the significant hits to virus are aligned to blast nr universal proteome database using diamond v . . . [ ] . the short reads sequencing data are available at ncbi sequence read archive (sra) under the bioproject number prjna (biosample accession samn - , and samn ). geneious r program was used to align reads and contigs to reference viral genomes and generate partial genome sequence. the genome gaps from the initial assembly were then filled by pcr whose products were sanger sequenced. dna was extracted from each individual fecal sample (and one pool of , cat# - ) shown in table plus vomit samples using the qiaamp minelute virus spin kit (qiagen, hilden, germany), and pcr assays were used for the detection of different viral nucleic acids in each sample. for fechavirus, the first round pcr primers fechaf ( '-ggtgcgacgacggaagatat- ') and fechar ( '-caacaccaccatctcctgct- ') amplified a bp region. the second round of primers: fechaf ( '-gctgcagttcaggtagctca- ') and fechar amplified a bp region. the pcr conditions for both rounds are as follows: × pcr gold buffer i, . mm dntps, . µm of each forward and reverse primers, u of amplitaq gold dna polymerase (applied biosystems, ma, usa) and µl dna template in a final µl reaction. the pcr programs for both rounds: • c min, cycles for • c s, • c s and • c s, followed by an extension at • c for min. pcr products were verified by gel electrophoresis and sanger sequencing. the pcr primers amplifying all three feline bocaviruses are as follows: fbov-f '-agaaccrccratcacartccact-' and fbov-r '-tggcraccgcyagcattt ca-' , the pcr conditions are the same as described before [ ] . the primers for fcv are: calici-f '-gcaaaggtggcgtcaaacat-' and calici-r '-gcaaaggtggcgtcaaacat-' . the pcr programs: • c min, cycles for • c s, • c s and • c s, followed by an extension at • c for min. the ns and vp protein sequences were aligned using the muscle program in mega . ; phylogenetic trees were inferenced using maximum-likelihood method. model test module imbedded in mega was used to determine the best substitution model. phylogenetic trees of both ns and vp protein sequences were generated using the bootstrap method under gtr+i+g model. the authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines, have been adhered to. no ethical approval was required as samples were collected as part of routine outbreak investigation. the data that supports the findings of this study are available in the main body of this article. a vomiting and diarrhea outbreak was identified across three animal shelters in british columbia, canada, lasting from november to january . a total of cats met the case definition (see methods). seventeen samples from cats (including a pool of samples from three cats), plus four vomit samples, were available for further study. the outbreak was first identified on november , in shelter when / cats housed in the main adoption room became sick with vomiting and diarrhea. diet, environmental, and toxic causes of the outbreak were ruled out clinically and an outbreak investigation initiated. the first cases in shelter (# , # ) had arrived from shelter via the organization's animal transfer program on november , and were sick during or shortly after transport. upon further investigation, the originating shelter, shelter , had more sick cats. on november , shelter became involved in the outbreak when a cat (# ) transferred from shelter became sick, several days after arrival and before the outbreak had been identified. ultimately, a total of cats were affected in shelter in november, cats were affected in shelter (november-january), and cats were affected in shelter (november-january) ( figure and supplementary table s ). nearly all transmissions were indirect ( figure ). because of this, it was not possible to definitively determine which animals had been exposed, except in specific rooms where housing was communal or exposure was known to be widespread prior to the introduction of control measures. attack rates (number of sick animals/total number of animals in a defined population) fr thesoe rooms were . % (shelter ) and . % (shelter ) (table ) . overall, diarrhea and vomiting were observed in . % and . % of the cases, respectively. there were likely more cats vomiting because vomitus that could not be attributed to a particular cat was found multiple times in the final wave of illness. of affected cats, . % and . %, respectively, also showed inappetence and lethargy, and . % required veterinary care ( table ). the minimum incubation period was h, and the maximum was estimated at - days based on estimated exposure dates. vomiting tended to start - days before diarrhea and last only a couple of days, but in some animals, the diarrhea lasted up to a week (longer in a few animals). the mean duration of illness was . days, with a median of . days (range to days) (supplementary table s ). no recovered cats relapsed and there were no cases where transmission was traced to a clinically recovered animal. the sheltering organization initiated control measures on november including cessation of all cat movement, use of personal protective equipment (gowns, gloves, caps, shoe covers) in all cat housing areas, and enhanced sanitation measures using accelerated hydrogen peroxide, which has good efficacy against bacteria and viruses (including non-enveloped viruses) [ ] . after control measures were initiated, the outbreak slowed and cases became sporadic, except for at shelter in january (due to communal housing). each time control measures failed, the failure was traced to a contaminated environment or fomite. seventeen fecal samples from cats in shelters and (collected after the outbreak was recognized) were available for further analysis. twelve samples representing cats were subjected to a comprehensive feline diarrhea panel pathogen screen (see materials and methods). fecv was detected in one sample, giardia dna in three samples, and clostridium perfringens alpha toxin dna in out of samples, while clostridium perfringens enterotoxin results were negative for all animals. clostridium perfringens is part of the normal feline intestinal flora; diarrheic and clinically healthy cats shed the organism at similar rates [ ] . type a, the most commonly isolated biotype, produces alpha-toxin and is commonly detected in feces of both diarrheic and healthy dogs [ ] . it is not considered a primary cause of diarrhea in cats, but may contribute to diarrhea in cases where a disturbance in the intestinal microenvironment has occurred, such as due to concurrent pathogen infection [ ] . giardia is a protozoal parasite that can infect multiple mammalian species and can be associated with diarrhea (and rarely, vomiting) in cats [ ] . it has a prepatent period of - days and while it can cause shelter outbreaks, it would not be capable of causing disease only h after exposure [ ] ; a shelter outbreak of giardia would have a more indolent course. frv was negative for all samples tested, only one sample (from a pooled litter) was positive for fpv. these tests ruled out the primary bacterial differentials for a shelter outbreak with a short incubation period. none of the bacteria/viruses/parasites testing positive were considered the main cause of the vomiting and diarrhea outbreak. in order to identify pathogens that could have caused the outbreak, all available fecal samples were analyzed using viral metagenomics. viral sequences assigned to five main viral families (anelloviridae, parvoviridae, papillomaviridae, polyomaviridae and caliciviridae) were detected in these fecal samples (table ) . feline anellovirus reads were detected in three cats, and unclassified anellovirus reads were detected in another four cats. anelloviruses are widely prevalent in humans and other mammals, and no clear pathologic role of anelloviruses has been identified [ , ] . lyon-iarc polyomavirus and feline papillomavirus (dyothetapappillomavirus ) were shed by one and two cats, respectively, with both genomes showing nucleotide identity of~ % with those genomes previously reported in cats [ , ] . lyon-iarc dna was previously shown to be shed by / cat feces in a hoarding situation diarrhea outbreak (one of which was co-infected with fpv) [ , ] . chicken anemia virus (cav), and gyrovirus and dna from family anelloviridae were each detected in one cat. cav is a highly prevalent chicken pathogen [ ] and gyroviruses have been frequently detected in human feces presumably originating from consumed infected chicken. [ , ] . also detected were two sequence reads with a perfect match to porcine parvovirus (protoparvovirus genus) also likely from consumed food [ ] . all cats were vaccinated subcutaneously upon entry into the three facilities with felocell vaccine (zoetis inc., parsippany, nj, usa) containing live attenuated fpv, fcv (mainly associated with the respiratory system in cats [ ] ), and feline herpesvirus- . the vaccine was also sequenced using metagenomics, yielding the near complete fcv genome (deposited in genbank mn ), approximately . % of the fpv genome, and more than , reads of the herpes virus. felocell vaccine-derived fpv and fcv sequences were compared to those detected by metagenomics in two and four of the diarrheic cats respectively. the sequence reads in the two cats shedding fpv showed %- % identity to the vaccine sequences. the few reads of fcv ( to sequence reads per sample) also showed a high level of similarity ( - %) to the sequenced vaccine fcv genome. given the extensive sequence diversity of fpv and feline fcv [ ] [ ] [ ] [ ] , we conclude that the fpv and fcv sequence reads detected in feces by metagenomics were vaccine derived. viral sequences belonging to the bocaparvovirus and chaphamaparvovirus genera were the most abundant and detected in / samples (table ) . three different feline bocaparvoviruses (febov - ) were found in , and samples, respectively. we then used a pan-febov pcr to test these samples. one cat, febov negative by metagenomics, was pcr positive (febov by sanger sequencing of pcr amplicon). two other cats positive for bocaviruses with only sequence reads were negative by pcr. all other pcr results were consistent with metagenomics results (table ) . all together, febov dna was detected by metagenomics and/or pcr in / samples. six cats also yielded sequence reads related to multiple parvoviruses in the chaphamaparvovirus genus. using de novo assembly and specific pcr to fill gaps, two near full-length genomes of and bases ( figure a) were generated from cats # and # (genbank accession numbers mn and mn ). the two genomes share . % identity of ns and . % identity of vp at protein level, and . % and . % at nucleotide level. these genomes encoded the two main orfs shared by all parvoviruses, a aa orf encoding non-structural replication protein (ns ), and a aa orf encoding viral capsid (vp ). the sequence of the inverted terminal repeats was not completed. the closest relative to these genomes was a parvovirus recently reported in canine feces named cachavirus (mk ) [ ] , with a nucleotide identity of . % and ns and vp protein identity of . % and . %, respectively. other major orfs consisted of a aa orf within the ns labeled np and a potential aa orf partially overlapping the ' of the ns orf. the np orf viruses , , of is widely conserved in chaphamaparvoviruses [ , ] . no significant similarity was found for the aa orf. at protein level, and . % and . % at nucleotide level. these genomes encoded the two main orfs shared by all parvoviruses, a aa orf encoding non-structural replication protein (ns ), and a aa orf encoding viral capsid (vp ). the sequence of the inverted terminal repeats was not completed. the closest relative to these genomes was a parvovirus recently reported in canine feces named cachavirus (mk ) [ ] , with a nucleotide identity of . % and ns and vp protein identity of . % and . %, respectively. other major orfs consisted of a aa orf within the ns labeled np and a potential aa orf partially overlapping the ' of the ns orf. the np orf is widely conserved in chaphamaparvoviruses [ , ] . no significant similarity was found for the aa orf. under an april ictv proposal updating the latest published classification [ ] , members of the same parvovirus species should show > % identity for ns . the fechavirus genome, therefore, qualifies as a member of a new species in the proposed chaphamaparvovirus genus. we named this novel virus feline chaphamaparvovirus or fechavirus. phylogenetic analysis confirmed the closest relative to be the canine cachavirus in both ns and vp orfs ( figure b,c) . specific pcr primers were designed based on the fechavirus genomes and used to test all fecal samples. besides the six fechavirus-positive fecal samples detected by ngs, two more samples were positive by pcr. all four vomit samples tested (from unknown cats in shelter ) were also pcr positive. a total of eight animals therefore shed fechavirus, of which one was co-infected with febov (cat # ). of the samples available for analysis, only one was negative for both febov and fechavirus dna (cat # ). under an april ictv proposal updating the latest published classification [ ] , members of the same parvovirus species should show > % identity for ns . the fechavirus genome, therefore, qualifies as a member of a new species in the proposed chaphamaparvovirus genus. we named this novel virus feline chaphamaparvovirus or fechavirus. phylogenetic analysis confirmed the closest relative to be the canine cachavirus in both ns and vp orfs ( figure b,c) . specific pcr primers were designed based on the fechavirus genomes and used to test all fecal samples. besides the six fechavirus-positive fecal samples detected by ngs, two more samples were positive by pcr. all four vomit samples tested (from unknown cats in shelter ) were also pcr positive. a total of eight animals therefore shed fechavirus, of which one was co-infected with febov (cat # ). of the samples available for analysis, only one was negative for both febov and fechavirus dna (cat # ). longitudinally collected fecal samples were available from four cats and pcr tested for fechavirus dna. cat # was fechavirus-positive on the last day of his illness (first available sample). cats # and # were each positive at only a single time point but manifested disease signs for and more days, respectively. cat # shed fechavirus over days while symptomatic plus a further days after recovery (table ). days (d -d ) reflect day samples were collected. days of illness are shaded starting at first sample collection. "+" and "−" means pcr positive or negative for the fechavirus. illness onset was , , , days before day for cats # , , and , respectively. dependoparvovirus sequence reads were also found in two cats. a near full-length genome of bases could be assembled from cat # (also infected with fechavirus) whose phylogenetic analysis showed it to be related to a recently reported dependoparvovirus genome from bats (desmodus rotundus) sharing ns and vp protein with . % and . % identities (figure ). longitudinally collected fecal samples were available from four cats and pcr tested for fechavirus dna. cat # was fechavirus-positive on the last day of his illness (first available sample). cats # and # were each positive at only a single time point but manifested disease signs for and more days, respectively. cat # shed fechavirus over days while symptomatic plus a further days after recovery (table ) . dependoparvovirus sequence reads were also found in two cats. a near full-length genome of bases could be assembled from cat # (also infected with fechavirus) whose phylogenetic analysis showed it to be related to a recently reported dependoparvovirus genome from bats (desmodus rotundus) sharing ns and vp protein with . % and . % identities (figure ). the timeline of disease presentation and fecal shedding status of key animals in the transmission chain between the three facilities was determined. there were no samples from cats in shelter available for testing, but cat # became sick and was fechavirus-positive shortly after being exposed to cats # and # , who originated in shelter and became sick during transfer to shelter . cat # entered shelter on november , . cats # and # were transferred to shelter on november , and cat # became sick on november . of the other affected cats in shelter a total of five individual cats and a sample pool (mixed feces from three cats) were febov -, -, or -positive. a second cat in shelter was shedding fechavirus (cat # ). this cat was transferred to shelter , where he was in direct contact with subsequently sick cat # , who was then in contact with cats # and # , initiating an illness transmission chain in shelter . despite clean breaks imposed by cleaning and emptying the facility in shelter , illness continued to recur and spread. the most likely cause of this was fomites that had been touched by humans caring for the cats, but were not in the cat housing area and had not been thoroughly disinfected; the outbreak resolved once this was addressed. of the seven cat fecal samples available for study from shelter , fechavirus was detected in five and bocaparvovirus in two animals. only a single case yielded both fechavirus and bocaparvovirus dna (# ). four additional vomit samples from unknown cat(s) in shelter were all fechavirus pcr-positive. last, an initially healthy cat in shelter developed disease signs immediately after cats from shelter were returned to shelter during the last wave of the outbreak and was found to be fechavirus-positive (cat # ). currently identified feline parvoviruses belong to two genera of the parvoviridae family, namely three bocaparvovirus species (carnivore bocaparvovirus / / , which include febov - [ , , ] ), and two protoparvovirus species (carnivore protoparvovirus which includes fpv [ ] and the still unclassified fbuv which is closely related to canine bufavirus [ ] ). febov was first discovered in multiple tissues of cats in hong kong [ ] and subsequently reported in cat feces in the us, japan, europe and china [ , , , ] . similar to human bocaparvovirus dna commonly found in the feces of healthy humans [ ] [ ] [ ] , frequent feline bocaparvovirus dna detection in healthy cats raises questions regarding its pathogenicity in cats [ , , , , ] . a recent study showed a possible association between fbov- infection, potentially aggravated by fpv, and hemorrhagic enteritis [ , , , , ] . fpv is an extensively studied pathogen that can lead to reduction in circulating white blood cells and enteritis [ , ] . the pathogenicity of febuv in cats is currently unknown [ , ] but the virus shares very high sequence identity with a canine bufavirus [ , , ] . using metagenomics, we found febov , , and and a novel chaphamaparvovirus we named fechavirus in a large fraction of fecal samples and fechavirus in all vomit samples from sick cats in a multi-facility outbreak. subsequent pcr testing confirmed the presence of either a bocavirus or fechavirus or both (n = cat # ) of these viruses in all but one of the samples tested (cat # ). the outbreak in shelter predominantly tested positive for febov ( / febov +ve and / fechavirus +ve), while the sick cats in shelter were mainly shedding fechavirus ( / fechavirus +ve, / febov +ve). another cat housed in shelter who developed vomiting after sick cats were transferred back from shelter was also shedding fechavirus as well as a novel dependovirus. many although not all dependoviruses are replication-defective and require a helper virus [ ] . the co-detection of fechavirus may provide the required help for replication of this new feline dependovirus. fpv and feline fcv reads were determined to originate from recently inoculated attenuated vaccine strains, while other viruses were deemed to be asymptomatic infections, derived from chicken and pork viruses in consumed food, or detected only in sporadic cases. fcv sequences were negative by pcr. this could be the result of low viral loads in those cats. the close genetic relationship of fechavirus to cachavirus found in both diarrheic and healthy dog feces [ ] may reflect a cross-carnivore virus transmission as occurred with fpv mutating into the highly pathogenic canine parvovirus [ , ] . serially collected fecal samples from symptomatic cats revealed transient shedding for / animals with fechavirus dna only detected at a single time point. one animal shed fechavirus dna for days both while exhibiting disease signs and days after disease resolution. the short duration of fechavirus shedding for / animals, also typical of fpv shedding [ ] , may account for its non-detection in some of the affected animals in shelter and . all but one of the fechavirus-negative samples were positive for one of three different feline bocaviruses indicating that both bocaviruses and fechavirus were circulating in these shelters. because of the high diversity of bocaviruses found here, belonging to three distinct species, and the typically asymptomatic nature of febov infections, a role for bocaviruses in the multi-facility transmission of this disease outbreak seems unlikely. a pathogenic role for fechavirus seems more likely as viral dna was detected in key animals with contacts across the three facilities experiencing outbreaks with similar disease signs. a key clinical attribute of this unusual outbreak was the prominence of indirect routes of transmission, including ongoing fomite transmission after robust control measures were implemented. another notable characteristic of this case was the very short minimum incubation period. combined with the absence of any known pathogen identified on routine screening, this points to a novel non-enveloped enteric virus as the most likely causative agent. animal shelters and veterinary hospitals should maintain good routine infection control procedures and consider novel viruses, including fechavirus, in the diagnosis of feline gastrointestinal disease. in this study, we characterized the virome from a cat diarrhea and vomiting outbreak, and showed that besides different febov, a new chaphamaparvovirus was associated with gastrointestinal disease. future studies on its prevalence, genetic diversity, and tissue distribution in healthy and diarrheic cats are needed to further investigate a possible etiologic role in feline disease. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : clinical signs and case definition ratings for all cats 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of dogs with diarrhoea parvovirus replication phylogenetic analysis reveals the emergence, evolution and dispersal of carnivore parvoviruses frequent cross-species transmission of parvoviruses among diverse carnivore hosts faecal shedding of parvovirus deoxyribonucleic acid following modified live feline panleucopenia virus vaccination in healthy cats this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to acknowledge melissa beall, roxanne chan, and phyllis tyrrel from idexx for helpful comments and assistance with clinical data. the other authors declare no conflict of interest. key: cord- -zpyz krq authors: barry, michele; van buuren, nicholas; burles, kristin; mottet, kelly; wang, qian; teale, alastair title: poxvirus exploitation of the ubiquitin-proteasome system date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: zpyz krq ubiquitination plays a critical role in many cellular processes. a growing number of viruses have evolved strategies to exploit the ubiquitin-proteasome system, including members of the poxviridae family. members of the poxvirus family have recently been shown to encode btb/kelch and ankyrin/f-box proteins that interact with cullin- and cullin- based ubiquitin ligases, respectively. multiple members of the poxvirus family also encode ubiquitin ligases with intrinsic activity. this review describes the numerous mechanisms that poxviruses employ to manipulate the ubiquitin-proteasome system. ubiquitin is a amino acid protein that is best known for its role in protein degradation [ ] , however, the addition of ubiquitin can also serve roles not associated with protein degradation [ ] . the post-translational addition of ubiquitin onto proteins occurs through a three step enzymatic cascade [ ] . ubiquitin is initially activated by one of two ubiquitin activating enzymes. activated ubiquitin is subsequently transferred to a ubiquitin conjugating enzyme. in the final step, a ubiquitin ligase is responsible for the transfer of ubiquitin to the target protein. proteins can be modified by open access mono-ubiquitin or poly-ubiquitin [ ] . any one of seven lysine residues present in ubiquitin allows for the formation of ubiquitin chains. lysine and are the most commonly used. polyubiquitin chains formed on lysine typically results in degradation through the s proteasome [ ] . conversely, polyubiquitin chains formed on lysine tend to alter protein function [ ] . more recently, linear ubiquitin has been associated with the regulation of nuclear factor b, and the ubiquitination of non-lysine residues has also been described [ , ] . the poxviridae are a large family of viruses that infect a wide range of vertebrates and invertebrates [ ] . the best known member of the family is variola virus, the causative agent of smallpox. global eradication of smallpox was achieved in through a vaccination program initiated by the world health organization (who) [ ] . smallpox eradication used vaccinia virus, a close relative of variola virus, as a live vaccine [ ] . by virus standards, poxvirus genomes are large ranging in size from - kbp; encoding upwards of or more open reading frames [ ] . much interest in poxvirus biology stems from the observation that poxviruses employ a vast array of effective immune evasion strategies [ , ] . additionally, the ease with which recombinant poxviruses are generated has made them attractive viruses for dissecting cellular signaling pathways [ ] . recently, protein ubiquitination has emerged as an important mechanism for the control of protein degradation and function, especially during virus infection [ ] [ ] [ ] [ ] [ ] . in this review we focus on the strategies that poxviruses have developed to exploit the ubiquitin-proteasome system. ubiquitination is a post-translational modification that plays an essential role in many cellular processes [ ] . ubiquitin is a small amino acid protein that is highly conserved in eukaryotes. in fact, only four amino acids differ between yeast, plants and mammalian ubiquitin sequences [ ] . two classes of ubiquitin-encoding genes are present in eukaryotic genomes. these including polyubiquitin genes that encode back-to-back ubiquitin sequences that are cleaved to produce ubiquitin monomers, and ubiquitin-carboxyl extension protein (cep) fusion genes that encode a single ubiquitin sequence fused to a ribosomal sequence at the c-terminus that is incorporated into the ribosome [ ] . ubiquitin contains seven lysine residues that can be used to build ubiquitin chains [ , , ] . traditionally, ubiquitination is associated with protein degradation; however, current evidence indicates that ubiquitination has additional regulatory functions [ , , ] . ubiquitin homologs have recently been identified in the genomes of poxviruses [ ] [ ] [ ] . two insect poxviruses: melanoplus sanguinipes (msev) ( table ) [ ] and amsacta moorei (amev) [ ] , as well as canarypox virus (cnpv), contain virus-encoded ubiquitin homologs [ ] . genomic analysis of msev, a poxvirus that infects locusts, identified the open reading frame msev ; encoding an amino acid protein that is % identical to human ubiquitin ( figure ) [ ] . however, the role that msev plays during viral infection has not been characterized. blast analysis also identified an additional poxvirus-encoded ubiquitin gene amev , in amev, a poxvirus that infects moths. amev is amino acids in length and % identical to human ubiquitin ( figure ) [ ] . sequencing of canarypox, a poxvirus that infects song birds, identified another ubiquitin homolog, cnpv , in canarypox virus ( figure ) [ ] . at amino acids in length, cnpv contains all of the residues required for protein ubiquitination and is % identical to human ubiquitin [ ] . interestingly, fowlpox virus (fwpv), a close relative of canarypox virus, contains fragmented remains of a functional ubiquitin gene [ ] . msev , cnpv , and amev are not part of the polyubiquitin gene class, and do not encode ribosomal peptides at the c-termini. in contrast to eukaryotes, which have multiple copies of ubiquitin-encoding genes, only one copy of each ubiquitin gene is present in the genomes of these viruses [ ] [ ] [ ] . virus-encoded ubiquitin genes have also been identified in baculoviridae, a family of dsdna viruses that infect insects [ ] . disruption of the ubiquitin gene in autographa californica nuclear polyhedrosis virus (acnpv) has no effect on virus viability, however, a decrease in virion budding and total infectious particles was observed [ ] . whether the ubiquitin-encoding genes in msev, amev, and cnpv are required for productive infection or virion budding remains to be determined. [ , ] . poxvirus amino acid sequences were obtained from the poxvirus bioinformatics resource center [ ] . residues representing % conservation are shaded. although most poxviruses do not encode their own ubiquitin genes, ubiquitin is associated with the virion. proteomic analysis of vaccinia virus indicates that ubiquitin accounts for approximately % of total virion protein [ ] . additionally, a lipid-modified form of ubiquitin is associated with several viruses [ , , ] . for example, baculovirus, african swine fever virus, herpes simplex virus and vaccinia virus incorporate lipid-modified ubiquitin in their envelopes [ , [ ] [ ] [ ] . previous analysis of baculovirus acnpv demonstrated that lipid-modified ubiquitin was present and that ubiquitin was host derived [ ] . scavenging ubiquitin from the host may represent another strategy used by poxviruses to increase the levels of ubiquitin available during infection. alternatively, lipid-modified ubiquitin may exist in cell membranes for a cellular function, such as autophagosome formation, and the virus simply acquires it passively during envelope acquisition. whether other poxviruses have lipid-modified ubiquitin incorporated into their envelopes has not been studied. it seems unlikely that poxviruses would maintain an open reading frame that has no role during infection. to date, the function of the poxvirus encoded ubiquitin sequences has not been determined. encoding additional pools of ubiquitin could be a mechanism used by entomopoxiruses and canarypox virus to increase efficiency of host cell modulation during infection. it is also possible that these viruses rely heavily on the ubiquitin-proteasome system. for example, it has been shown that members of the orthopoxvirus family require a functional ubiquitin-proteasome system for productive infection [ , ] . alternatively, viral-encoded ubiquitin homologs may function to inhibit the ubiquitin proteasome system. the acnpv-encoded ubiquitin functions as a chain terminator for k linked polyubiquitination, the linkage that targets proteins for degradation by the s proteasome [ ] . as such, it is possible that poxvirus-encoded ubiquitin may also act as chain terminators to inhibit degradation of certain substrates. at present, the reason that only a few members of the poxvirus family encode ubiquitin homologs remains unclear. poxviruses encode two families of proteins with intrinsic ubiquitin ligase activity; a membrane-associated ring-ch (march) ubiquitin ligase, and a really interesting new gene (ring) finger protein (figure a and b) [ ] [ ] [ ] [ ] . the march family of proteins contains a modified ring domain (ring-ch) at the n-terminus as well as transmembrane domains that promote localization to membranes (figure a ) [ ] . cellular march proteins play an important role in the down-regulation of membrane receptors including mhc class i, mhc class ii, and cd [ ] . in addition to a family of cellular march proteins, march ubiquitin ligases also exist in the genomes of herpesviruses and poxviruses (table ) [ ] . infection with myxoma virus (myxv), a rabbit specific poxvirus that causes myxomatosis, results in reduction of cell surface mhc class i [ , [ ] [ ] [ ] . the loss of mhc class i upon myxoma virus infection was later associated with ubiquitin ligase activity of the myxoma virus encoded march homolog, m r [ , ] . in addition to the down-regulation of mhc class i, m r also reduces cell surface expression of cd , alcam (cd ) and cd [ , , ] . the loss of cd by m r has been well characterized. upon infection, m r ubiquitinates the cytoplasmic tail of cd , leading to its internalization via endocytosis and subsequent lysosomal degradation [ ] . through the action of m r, myxoma virus induced ubiquitination and degradation of cell surface immune molecules provides an important mechanism for dampening the immune response. p is a virus-encoded ring finger ubiquitin ligase that plays an important role in virulence ( figure b ) [ , , ] . p is highly conserved among pathogenic poxviruses and is expressed at both early and late times during virus infection (table ) [ , ] . the p ubiquitin ligase contains two functional domains; an n-terminal dna binding domain, and a c-terminal ring domain ( figure b ). the dna binding domain of p , referred to as kila-n, remains largely uncharacterized [ ] . this domain is found in a number of large dna viruses as well as bacteria and bacteriophage [ ] . the kila-n domain plays an important role in the localization of p cytoplasmic viral factories [ , ] . in addition to being found in combination with a ring domain, the kila-n domain is also found independently in some poxviruses. for example, eight kila-n proteins are encoded in fowlpox virus (fwpv) and kila-n proteins are encoded in canarypox virus [ ] . however, only two proteins in fowlpox virus and canarypox virus combine a kila-n domain with a ring domain, likely encoding functional ubiquitin ligases [ , , ] . the c-terminal ring domain of p is responsible for ubiquitin ligase activity [ , ] . p displays sequence homology to a family of cellular proteins termed makorin (mkrn), however, this homology is restricted to the ring domain [ ] . it has been suggested that the p family of poxvirus proteins were acquired through a fusion event of an existing kila-n domain and a cellular mkrn [ ] . point mutations in the critical conserved residues of the ring domain disrupt ubiquitination [ , ] . using in vitro ubiquitination assays, p homologs in ectromelia virus (ectv), vaccinia virus (vv)-strain ihdw and variola virus (varv) were shown to function as ubiquitin ligases [ , ] . the p ortholog in variola virus, d r, functions in vitro with the ubiquitin conjugating enzymes, ubc and ubch c [ ] . work in our laboratory has demonstrated that expression of p targets conjugated ubiquitin to viral factories [ ] . k linked ubiquitin, which is associated with protein degradation, also co-localized at the virus factory with p [ ] . given that k linked ubiquitin is associated with proteasomal degradation it is likely that p plays a role in targeting substrates for degradation. interestingly, variola virus d r functions in vitro with ubc , the only known ubiquitin conjugating enzyme that promotes k linkages [ ] . in contrast to k linkages, k linkages are associated with non-proteolytic functions, suggesting that p may form k linkages during virus infection [ ] . to date, no p substrates have been identified. however, p has been implicated in the inhibition of apoptosis [ , ] . it is therefore tempting to speculate that p may be targeting pro-apoptotic proteins for degradation. since p localizes to viral factories, it is likely that potential substrates are located at the viral factory. additionally, since p is expressed early during infection, prior to virus factory formation, p may also be responsible for ubiquitinating cytoplasmic substrates. in vivo studies have shown that p is a critical virulence factor during ectromelia virus (ectv) infection [ ] . in susceptible strains of mice, ectromelia virus devoid of p was extremely attenuated and all mice recovered; this is in sharp contrast to mice infected with wild-type virus, which succumb to infection [ ] . both wild type ectromelia virus and ectromelia virus devoid of p replicated equally well in all cell lines tested except for primary peritoneal macrophages [ , ] . macrophages are thought to be critical for the transport of the virus, suggesting that the ubiquitin ligase activity of p plays an important role in peritoneal marcrophages [ ] . the role of p in virulence and its ability to function as a bona fide ubiquitin ligase suggests p is ubiquitinating substrates, however these substrates have yet to be identified. identification of p substrates will undoubtedly provide important clues into the role of p in virus virulence. the btb domain, also known as the poz, bric-a-brac, tramtrack, or broad-complex, is a highly conserved protein-protein interaction motif that is involved in many cellular functions, including transcriptional and cytoskeletal regulation [ ] [ ] [ ] . recently, cellular btb domain-containing proteins have been shown to function as substrate-specific adaptors of cullin- based ubiquitin ligase to target proteins for ubiquitination [ ] [ ] [ ] [ ] . unlike the well-characterized scf (skp /cul /f-box) and ecs (elonginc/cul /socs) e complexes, in which skp /f-box or elonginc/socs combine to bridge substrates to cullins, btb proteins fulfill this function through a single polypeptide containing the btb domain as a linker to cullin- and a substrate-recruiting domain, such as kelch, math or zinc fingers ( figure c ) [ ] [ ] [ ] [ ] . supporting this, the skp and elonginc proteins display similar three-dimensional structure as the btb domain [ ] [ ] [ ] . the kelch domain consists of multiple repeated kelch motifs, and is thought to mediate protein-protein interactions ( figure c ) [ ] . a large group of btb/kelch proteins have been identified in most members of the poxvirus family (table ) [ ] . for example, vaccinia virus encodes three btb/kelch proteins [ ] ; cowpox virus (cpxv) encodes six btb/kelch proteins [ ] ; ectromelia virus strain moscow (evm) encodes four such proteins [ ] ; while monkeypox virus (mpxv) encodes only one btb/kelch gene [ ] (table ) . although the specific roles of the poxvirus btb/kelch proteins are still unclear, it has been speculated that they may function as cullin- substrate-specific adaptors, similar to their cellular counterparts. in agreement with this idea, the btb domains of ectromelia virus encoded btb/kelch proteins evm and evm are essential and sufficient for interaction with cullin- [ ] . consistently, evm and evm associate with conjugated ubiquitin and roc , the ring-finger protein required for an active cullin- ubiquitin ligase complex [ ] . the other two ectromelia virus encoded btb/kelch proteins, evm and evm , also interact with cullin- [ ] . interestingly, evm , an ectromelia virus encoded protein containing only a btb domain, does not interact with cullin- , roc , or conjugated ubiquitin, suggesting that, unlike the other ectromelia virus encoded btb/kelch proteins, evm may function independently of the ubiquitin-proteasome pathway [ ] . the failure of evm to interact with cullin- is currently unknown. together, these findings suggest that poxviruses may employ btb/kelch-cullin- ubiquitin ligase complex as another strategy to manipulate the cellular environment. alternatively, the poxvirus btb/kelch proteins may function by simply sequestering cullin- to inhibit the cullin- -based cellular ubiquitin pathway. given that poxviruses encode multiple btb/kelch proteins with different kelch regions, it is probable that these viral btb/kelch proteins function to specifically target different substrates to the cullin- ubiquitin ligase for ubiquitination. the importance of the poxvirus btb/kelch proteins during virus infection has been studied. vaccinia virus devoid of the btb/kelch proteins c l, f l or a r, the orthologs of evm , evm and evm , respectively, displays an altered viral pathogenesis in the murine intradermal model [ ] [ ] [ ] . deletion of four btb/kelch genes, d l, c l, g l and a r, from cowpox virus strain gri- also results in altered host range and attenuated virulence [ ] . additionally, sheeppox virus (sppv) btb/kelch gene sppv- has been shown to modulate cellular adhesion and affect virus virulence using a sppv- knock-out virus model [ ] . these observations suggest that btb/kelch proteins function to manipulate the cellular host environment. to date, however, no definite substrates for the poxvirus btb/kelch proteins have been identified, although several targets for cellular btb/kelch proteins have been characterized. for example, nrf , a critical nuclear transcription factor regulating oxidative stress, is degraded by keap /cullin- ubiquitin ligase [ , ] . keap also functions as an ikk ubiquitin ligase [ ] . aurora b, a chromosomal passenger protein responsible for the proper progression of mitosis and cytokinesis, is targeted by klhl /cul e for ubiquitination [ ] . interestingly, the vaccina virus encoded btb/kelch protein, wr (cop-c l), was recently shown through yeast- -hybrid screening to interact with cellular crystallin alpha b (cryab), a small heat-shock protein [ ] . whether crystalline alpha b can be regulated by wr for cullin- -mediated ubiquitination needs to be investigated. although the role of poxvirus btb/kelch proteins is still undefined, many other viruses have evolved mechanisms to specifically recruit cellular proteins to cullin-based ubiquitin ligases [ , ] . future identification of the substrates targeted by the poxvirus btb/kelch proteins will provide new insight into the understanding of cellular anti-viral responses. ankyrin repeat proteins represent one of the largest families of proteins encoded by poxviruses. the ankyrin repeat consists of a amino acid helix-loop-helix motif with a highly conserved amino acid sequence [ ] [ ] [ ] . ankyrin repeats were first identified in the cytoskeletal structural protein called ankyrin, which contains ankyrin repeats [ ] . since its discovery, the ankyrin repeat has been characterized in a wide variety of cellular proteins, and generally mediates unique protein-protein interactions [ ] [ ] [ ] . with the exception of molluscipoxviruses, all other poxvirus families encode a large repertoire of ankyrin proteins ( table ). the largest family is encoded by canarypox virus and is comprised of ankyrin repeat proteins, representing % of the canarypox virus genome [ , ] . poxviral ankyrin repeat proteins are large proteins, ranging from - amino acids in length, containing between to ankyrin repeats located at their n-termini. although the poxviral ankyrin repeat proteins contain no obvious structural domains at their c-termini, many of the proteins display a conserved sequence, which upon closer inspection was shown to resemble the f-box domain that functions in the recruitment of substrates to the cellular scf (skp- , cullin, f-box) ubiquitin ligase complex [ ] [ ] [ ] . the poxviral f-box-like domain was later named pranc (pox protein repeat of ankyrin c-terminus) (pfam.janelia.org/family/pf ). the scf complex is a highly conserved ubiquitin ligase involved in regulation of the cell cycle, dna repair, and innate immunity [ , , ] . the complex consists of cullin- , which serves as the molecular scaffold, roc , a ring finger ubiquitin ligase, skp , the linker protein, and one of over known cellular f-box proteins which function in substrate recruitment ( figure d ) [ ] [ ] [ ] . cellular f-box proteins consist of n-terminal f-box domains in conjunction with c-terminal protein binding domains such as wd repeats or leucine-rich repeats (lrr) [ ] [ ] [ ] . the f-box domain consists of a highly conserved amino acid sequence, folding into three alpha-helices, which function to bind the linker protein, skp , while wd repeats or lrrs function to bind substrates which are subsequently ubiquitinated through the ubiquitin ligase activity of roc ( figure d ) [ ] . substrates of the scf ubiquitin ligase complex typically require a phosphorylation event prior to recognition by the substrate adaptor [ ] [ ] [ ] [ ] . until the recent identification of ank/pranc proteins in the parasitoid wasp, nasonia, ank/pranc proteins were thought to be unique to poxviruses [ ] . the poxvirus ank/pranc proteins differ from cellular f-box proteins in two important aspects. firstly, the c-terminal location of the poxvirus f-box-like domain is unique to this set of proteins, and secondly, the poxvirus ank/pranc proteins contain truncated f-boxes ( figure ) [ , , ] . the cowpox virus encoded ank/pranc protein cp contains a pranc domain that is only amino acids in length and may represent the minimum requirement for interaction with skp [ ] . a related family of proteins, the suppressor of cytokine signaling (socs)-box family appear in conjunction with ankyrin repeats, and function as substrate adaptor molecules for the ecs ubiquitin ligases [ ] . since the socs-box and the f-box share sequence similarity it has been proposed that the poxviral ankyrin/f-box proteins were acquired as socs-box proteins that have evolved to regulate the cullin- based ligase [ , ] . in addition to poxvirus encoded ank/pranc proteins, poxviruses also encode ankyrin only proteins [ , , ] . these ankyrin-only proteins do not contain pranc domains, and have been proposed to have arisen from full length ank/pranc proteins [ ] . figure . sequence alignment of ectromelia virus encoded ank/pranc proteins with cellular skp : alignx was used to align the c-termini of evm , evm , evm , and evm with the n-terminal f-box domain of skp , a cellular f-box protein [ ] . red dots indicate known contact points between skp and skp [ ] . h , h , and h represent alpha-helical secondary structures from skp . ank/pranc proteins have been identified in a wide range of poxviruses including vaccina virus, ectromelia virus, cowpox virus and orf virus. studies on myxoma virus identified the first interaction between a poxviral ank/pranc protein, m-t , and the scf complex [ ] . mt- , one of four ank/pranc proteins in myxoma virus, co-localizes with cullin- in the nucleus and regulates the cell cycle and interacts with akt [ , ] . myxoma virus encodes four ank/ pranc proteins (m-t , m , m , m ) all of which play a role in myxoma virus virulence [ ] [ ] [ ] . interestingly, m co-localized to the nucleus with the p subunit of nuclear factor kappa b (nf-b), suggesting that m is involved in inhibition of nf-b [ ] . each of the five orf virus encoded ank/pranc proteins have been shown to associate with a functional scf ubiquitin ligase complex, as demonstrated through in vitro ubiquitination assays [ ] . in the case of the orf virus proteins, the f-box-like domain was both necessary and sufficient to mediate the interaction with skp and cullin- [ ] . similarly, the f-box domains of proteins from ectromelia virus, cowpox virus, and vaccinia virus are also essential for interaction with the scf complex [ , , ] . the cowpox virus encoded ank/pranc protein, cp , functions as a host range protein that interacts with the nf-b transcription factor, p , to inhibit the transcription of inflammatory cytokines [ , ] . regulation of the nf-b signaling pathway by poxviral ank/pranc proteins appears to be a common trend. using a yeast two-hybrid screen, the variola virus encoded g r ank/pranc protein was shown to interact with the nf-b regulatory protein nfb /p as well as skp [ ] . g r, and its orthologs in cowpox virus, monkeypox virus, and ectromelia virus (cpxv , mpxv , evm ), bind p , and inhibit g r degradation following tnf stimulus [ ] . additionally, a cpxv deletion virus displayed increased release of proinflammatory cytokines in culture, and was slightly attenuated in c bl/ mice infected [ ] . although substrates have not been identified for the poxviral ank/pranc proteins, it has been hypothesized that the poxvirus ank/pranc proteins function as substrate adaptor proteins for the scf complex. although it is possible that the poxvirus ank/pranc proteins may simply bind and inhibit the scf complex, this seems unlikely due to the large number of unique ank/pranc proteins encoded by poxviruses. for example, fowlpox virus encodes ank/pranc proteins, each potentially targeting unique protein(s) for ubiquitination by the scf complex (table ) [ ] . additionally, the ectromelia virus and orf virus ank/pranc proteins have both been shown to associate with functional scf complexes, suggesting that these proteins do not simply function as inhibitors [ , ] . the identification of substrates recruited to the scf complex by poxviral ank/pranc proteins will be an essential step towards understanding this interesting family. the anaphase promoting complex/cyclosome (apc/c) is the largest known cellular ubiquitin ligase complex, composed of at least subunits ( figure e ) [ ] . since its discovery almost years ago its structure and regulation have proven to be increasingly complex. it is thought that the apc/c complex has evolved from an ancestral scf-type ubiquitin ligase since the subunits apc and apc resemble a cullin-family member and ring-type e ligase, respectively. apc functions as the molecular scaffold, and contains cullin homology and binds to the ring-finger protein apc [ ] . apc has been shown to recruit ubiquitin-conjugating enzymes to the apc/c in order to catalyze the in vitro transfer of ubiquitin onto target substrates [ ] . substrates for the apc/c are recognized through the presence of d-box or ken-box domains, which are recognized by a variety of apc/c components including cdh , cdc and doc [ , ] . the apc/c plays a major role in regulation of the cell cycle at several points, and most well known for its ability to degrade securin, a protein that regulates the separation of sister chromatids during anaphase [ ] . a family of poxvirus ring-finger proteins was recently identified that contain sequence similarity with the ring domain of the apc/c subunit apc (table ) [ ] . these apc homologs were identified in the parapoxviruses, molluscipoxviruses, as well as the crocodilepox and squirrelpox viruses. the poxvirus encoded apc homolog from orf virus, a member of the parapoxvirus family, is the only homolog studied to date and has been named pacr (poxvirus apc/cyclosome regulator) [ ] . pacr was shown to co-precipitate with apc/c subunits apc , apc and apc , and is shown to associate with the apc/c complex in a similar manner to apc [ ] . however, upon sequence analysis, pacr and the other poxvirus orthologs contain mutations within the ring domain that inhibit the binding of e ubiquitin-conjugating enzymes to the complex, and therefore inhibit substrate ubiquitination [ ] . it is thought that inhibition of apc/c may prompt cells into s-phase, a stage within the cell cycle where additional cellular factors may be present and contribute to virus replication. additionally, two of the targets of the apc/c are cellular ribonucleotide reductase and thymidine kinase proteins, proteins that contribute to the free nucleotide pools required for dna synthesis. typically poxviruses encode their own thymidine kinase and ribonucleotide reductase genes, however, the viral thymidine kinase and ribonucleotide reductases genes are absent from orf virus as well as other virus that encode homologs of parc. in contrast, many viruses that encode their own thymidine kinase genes, lack pacr orthologs. it has been hypothesized that one of the main reasons for encoding apc/c inhibitors is to upregulate cellular thymidine kinase and ribonucleotide reductase genes to enhance free nucleotide pools in poxviruses that lack the ability to promote this themselves. poxviruses are renowned for creating an optimal environment for viral replication and propagation [ , , ] . the ubiquitin-proteasome system, which plays a crucial role in protein degradation and cellular homoeostasis, is an attractive target for virus-encoded effector proteins. the ubiquitin-proteasome system is involved in regulating many important host pathways including antigen presentation, cell cycle progression, signal transduction, and dna repair [ , ] . individual interactions between poxviral proteins and the ubiquitin-proteasome system have been characterized [ , ] . the study of the ubiquitin-proteasome system has been aided greatly by the use of chemical proteasome inhibitors. these inhibitors block the catalytic action of the proteasome by preventing the degradation of ubiquitinated proteins and reducing the amount of free ubiquitin available within the cell [ ] . proteasome inhibitors, including mg , act to reversibly inhibit proteasome action while others, including mg , lactacystin, and epoxomycin irreversibly inhibit the proteasome [ , ] . notably, the bortezomib, sold under the trade name velcade®, and licensed for the treatment of multiple myeloma, is a potent inhibitor of the proteasome [ ] . the overall importance of a functioning ubiquitin-proteasome system during poxvirus infection has only recently been investigated [ , ] . it has now been demonstrated that a functioning ubiquitin-proteasome system is vital to a successful infection by members of the orthopoxvirus family [ , ] . in the presence of proteasome inhibitors, poxvirus replication is dramatically impaired [ , ] . early poxviral gene expression is unaffected while intermediate and late gene expression is greatly reduced through the action of chemically distinct proteasome inhibitors. viral factories, which normally appear as dna rich areas in the cytoplasm of infected cells, are unable to form in the presence of proteasome inhibitors. in addition, it has been shown that plasmid replication, which can normally occur during poxvirus replication at viral factories [ ] , is blocked by the use of proteasome inhibitors [ ] . the addition of proteasome inhibitors post-infection indicates that the block affects an early step during poxviral infection but does not affect the entry of poxvirus particles into the cell. intriguingly, inhibition of the ubiquitin activating enzyme results in a similar phenotype during infection. since overexpression of ubiquitin is unable to rescue late protein expression, dna production and the generation of progeny virus, this data suggests that a functional ubiquitin-proteasome system as a whole is required for successful poxvirus infection [ ] . together, these observations indicate that viral dna replication does not occur upon proteasome inhibition. the lack of viral dna replication along with the pattern of gene expression seen upon treatement with proteasome inhibitors, points to viral uncoating and dna replication as the likely candidates for the stage in the poxviral lifecycle actively blocked by proteasome inhibitors [ , ] . further studies will undoubtedly lead to a greater understanding of the interactions between poxviruses and the ubiquitin-proteasome system and specifically the role of the proteasome during infection. the dramatic effect of proteasome inhibitors on poxvirus infection, suggests the proteasome may be an attractive target for the development of antivirals. interestingly, proteasome inhibitors demonstrate an antiviral effect on a wide range of viruses including human immunodeficiency virus [ ] , influenza virus [ ] , vesicular stomatitis virus [ ] , coronavirus [ ] , human cytomegalovirus [ ] , respiratory syncytial virus [ ] , herpes simplex virus [ ] and hepatitis b virus [ ] . as such, proteasome inhibitors seem to demonstrate antiviral activity though distinct mechanisms among viral species. for example, proteasome inhibitors have been shown to impair entry and rna synthesis during coronavirus infection [ ] , inhibit the entry of herpes simplex virus into the nucleus [ ] , and inhibit influenza and vesicular stomatitis virus replication [ ] . however, in vivo studies recently conducted have produced mixed results. treatment with bortezomib results in a decrease of circulating rna in mice chronically infected with hepatitis b [ ] , but proteasome inhibition enhances the disease and mortality in mouse hepatitis coronavirus [ ] , as well, increasing inflammation and mortality was observed in human respiratory syncytial virus [ ] . a possible explanation for the seemingly conflicting results between the in vitro and in vivo experiments is through modulation of the immune system by proteasome inhibitors. it has been demonstrated that proteasome inhibitors affect antigen processing in vivo [ ] . while proteasome inhibition may be antiviral, the effects on the immune system caused by proteasome inhibitors may increase susceptibility and mortality in some viral infections. still, the proteasome remains a possible target for antiviral development against poxviruses and it would be interesting to determine whether proteasome inhibitors are able to inhibit poxvirus disease and mortality in vivo. since the first realization that poxviruses encode proteins with intrinsic ubiquitin ligase activity, the field has grown at a fast and exciting pace. it is clear from the current research that poxviruses encode multiple proteins that manipulate the ubiquitin-proteasome system. as discussed here, these strategies include the expression of poxvirus-encoded ubiquitin, ubiquitin ligases, btb/kelch proteins, ank/pranc proteins, as well as inhibitors of the apc/c complex. the presence of multiple poxvirusencoded proteins suggests that poxviruses exploit the ubiquitin-proteasome in order to regulate cellular processes. in support of this, our recent observations indicate that upon infection with vaccinia virus the ubiquitin proteasome system is fully 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to suppress human cytomegalovirus replication and virus-induced immune modulation decreased replication of human respiratory syncytial virus treated with the proteasome inhibitor mg- cellular proteasome activity facilitates herpes simplex virus entry at a postpenetration step bortezomib inhibits hepatitis b virus replication in transgenic mice the proteasome inhibitor bortezomib enhances the susceptibility to viral infection mass spectrometry for proteomics weighing in on ubiquitin: the expanding role of massspectrometry-based proteomics proteome-wide quantitation by silac proteomics by mass spectrometry: approaches, advances, and applications key: cord- -s n ldol authors: martin, javier; klapsa, dimitra; wilton, thomas; zambon, maria; bentley, emma; bujaki, erika; fritzsche, martin; mate, ryan; majumdar, manasi title: tracking sars-cov- in sewage: evidence of changes in virus variant predominance during covid- pandemic date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: s n ldol severe acute respiratory syndrome coronavirus (sars-cov- ), responsible for the ongoing coronavirus disease (covid- ) pandemic, is frequently shed in faeces during infection, and viral rna has recently been detected in sewage in some countries. we have investigated the presence of sars-cov- rna in wastewater samples from south-east england between th january and th may . a novel nested rt-pcr approach targeting five different regions of the viral genome improved the sensitivity of rt-qpcr assays and generated nucleotide sequences at sites with known sequence polymorphisms among sars-cov- isolates. we were able to detect co-circulating virus variants, some specifically prevalent in england, and to identify changes in viral rna sequences with time consistent with the recently reported increasing global dominance of spike protein g pandemic variant. low levels of viral rna were detected in a sample from th february, days before the first case was reported in the sewage plant catchment area. sars-cov- rna concentration increased in march and april, and a sharp reduction was observed in may, showing the effects of lockdown measures. we conclude that viral rna sequences found in sewage closely resemble those from clinical samples and that environmental surveillance can be used to monitor sars-cov- transmission, tracing virus variants and detecting virus importations. a global pandemic of coronavirus disease (covid- ) caused by a new betacoronavirus named severe acute respiratory syndrome coronavirus (sars-cov- ) is currently ongoing [ ] . the outbreak was first detected in wuhan (china) in december and spread rapidly to countries/territories with . million confirmed cases and , , deaths as of th september [ ] . while the majority of infections result in no apparent symptoms or mild ones, some progress to acute respiratory disease, multi-organ failure, and death [ ] . respiratory transmission is the primary route for sars-cov- infection although faecal-oral transmission is possible as high levels of viral rna have been detected in stool samples of a proportion of infected individuals [ ] . studies have shown that viral rna of titres up to . log genome copies (gc)/gram of faeces can be detected in stools of infected people one litre of inlet wastewater composite samples was collected during a h period at a sewage plant in south east england with a catchment area of approximately . × people. the samples were transported to the laboratory in chilled packages on the day of sampling and were stored at − • c until use. each sample was processed using a filtration-centrifugation method described before and previously validated for the detection of polio and non-polio enteroviruses during routine es for poliovirus as part of our role as a who global specialized polio network laboratory [ , ] . briefly, following the removal of solids by centrifugation at × g, wastewater was filtered through a ml nalgene rapid-flow™ . µm filter (thermo fisher scientific, waltham, ma, usa) and concentrated using centriprep centrifugal filter units with a kda molecular weight cutoff (merck life science uk limited, gillingham, uk) following manufacturer's instructions. starting volumes of raw sewage between and ml yielded - ml of concentrate. these were further concentrated in a second step, when necessary, with final volumes of concentrates reaching between and µl. a total of five sewage samples was processed, one from each month. at least two aliquots from each raw sewage sample were processed and analysed independently. as shown in the literature, and from our own experience, we know that human viruses in sewage often show a non-homogeneous distribution, particularly when virus concentrations are low [ , ] . aliquot samples from the same wastewater concentrate preparation almost always yield a different spectrum of polio and non-polio enterovirus serotypes when analysed by infection in cell cultures or direct molecular assays [ , ] . for this reason, following concentration of raw sewage, we tested a minimum of five replicate rna samples from at least two independent wastewater concentration processes for each sample. the sars-cov- rna content in wastewater concentrates was estimated by real-time rt-qpcr using a qscript xlt qpcr toughmix system (quantabio, beverly, ma, usa) in a rotor-gene q instrument (qiagen) and following a one-rt-pcr protocol. viral rna was purified from wastewater concentrates using the high pure viral rna kit (roche life science, mannheim, germany). previously reported primer reactions rdrp and e-sarbeco [ ] were used with the following amplification conditions: rt reaction was conducted at • c for min followed by pcr amplification cycles of • c for s, • c for s, • c for s, and • c for s. a standard curve for sars-cov- rna quantification was generated using serial dilutions of rna extracted from the national institute for biological standards and control (nibsc) virus reagent / containing noninfectious synthetic sars-cov- rna packaged within a lentiviral vector that had been calibrated with plasmid dna constructs to contain a concentration of . log sars-cov- genome copies (gc)/ml (https://www.nibsc.org/products/brm_product_catalogue/detail_page.aspx?catid= / , accessed on july ). the results were expressed in log sars-cov- gc/l of sewage. replicate assays were performed for each sample to improve quantification estimates. good laboratory practices were observed in all assays to reduce the possibility of cross-contamination: i.e., using different laboratory locations for sample processing, preparation of reaction mixtures, template addition, and post-processing analysis. two different operators tested each sample at least once. rna extraction and no template controls were included in every assay and were always found to be negative. an additional rt-pcr reaction using enterovirus primers was used as process control to rule out the presence of pcr inhibitors. the presence of live human enteroviruses in wastewater concentrates was also assessed using standard cell culture procedures as part of our routine process for poliovirus surveillance [ ] . whole-genome sars-cov- sequences collected up to st may were downloaded from the global initiative on sharing all influenza data (gisaid) database [ ] on th july . only sequences > , nt in length were used in our analysis. remarkably, . % of the whole-genome sars-cov- sequences analysed from the gisaid database ( , of , ) were from england. tables s -s show acknowledgments for authors who submitted the sequences analysed. whole-genome sars-cov- viral rna sequences were downloaded from the gisaid database [ ] to identify suitable genetic markers to be used in our sequence analyses, specifically we looked at sequence variations observed between viral rna sequences from england. geneious r software (biomatters, auckland, new zealand) was used for all nucleotide sequence analyses. whole-genome sequences were aligned to a reference sequence (wuhan-hu- strain) with national center for biotechnology information (ncbi) accession no. mn and the frequency of sequence variation at each nucleotide position was determined by standard single nucleotide polymorphism (snp) analysis using geneious r software default settings. rt-pcr fragments corresponding to different regions across the sars-cov- genome were amplified from purified viral rnas by one-step rt-pcr using a invitrogen superscript iii one-step rt-pcr system with platinum taq high-fidelity dna polymerase. genome location and nucleotide sequences of primer sets used for the pcr reactions are shown in figure s , npcr reactions were named npcr to npcr . amplification conditions were: • c for min followed by • c for min plus cycles of • c for s, • c for s and • c for min with a final extension step of • c for min. following the first pcr reaction, µl of amplified product was used for the second pcr reaction using the dreamtaq™ hot start pcr master mix with the same amplification conditions used for the first pcr step. final amplified products were purified using qiaquick ® pcr purification kit (qiagen, manchester, uk) ready for sanger and next-generation sequencing (ngs) analysis. primers were tested using serial dilutions of purified rna from nibsc's virus reagent / referred above. rna extraction and no template controls were included in every assay and were always found to be negative. primers used in this study did not closely match viral rna sequences from seasonal coronavirus that had been circulating worldwide the last several years. besides this, published nucleotide sequences of seasonal coronavirus serotypes in the pcr regions amplified, are at least % different to those from sars-cov- isolates, which means that full-sequence analysis can unequivocally demonstrate that the sequenced npcr products from this study were from sars-cov- and not seasonal coronavirus. all nucleotide sequences of npcr products in this study were identical or nearly identical to sequences from covid- isolates from england and none resembled those from seasonal coronaviruses. purified dna products were sequenced using an applied biosystems prism genetic analyser. sars-cov- sequences obtained in this study were compared to those available in the gisaid database [ ] . geneious r software was used for these analyses. sanger nucleotide sequences generated for this paper are available from the gisaid database [ ] with accession ids epi_isl_ and epi_isl_ -epi_isl_ . ngs libraries were constructed using the dna prep kit (formerly known as nextera dna flex) and dual-indexed using nextera dna cd indexes (both illumina, san diego, ca, usa). these libraries were pooled in equimolar concentrations and sequenced with bp paired-end reads on miseq v ( cycles) kits (illumina). initial demultiplexing was performed on-board by the miseq reporter software. fastq sequencing data was adapter and quality trimmed by cutadapt v . [ ] viruses , , of for a minimum phred score of q , minimal read length of bp, and ambiguous nucleotides. relevant fastq files used in this study are available from the ncbi short read archive under bioproject id: prjna . further processing and analysis of ngs data was performed with geneious r software using methods described before [ , ] . filtered reads were imported into geneious r , paired-end reads combined and sequence contigs built by reference-guided assembly. reads were mapped to references with a minimum base overlap, minimum overlap identity of %, maximum % mismatches per read, allowing up to % gaps, and index word length of . snps were identified using geneious r default settings. variants with strand bias > %, coverage < , average quality < , variant frequency < %, and the number of total variant reads < were excluded. rna extracted from nibsc's virus reagent / was used as control to measure background sequencing error. following concentration of raw sewage as described in section . , we tested a minimum of five replicate rna samples from at least two independent wastewater concentration processes for each sample. further replicate rnas were tested for positive samples to obtain more accurate viral rna quantification. sars-cov- rna in wastewater samples was quantified using a real-time quantitative polymerase chain reaction (rtqpcr) assay targeting the rna-dependent rna polymerase (rdrp) gene. a second rtqpcr assay targeting the envelope protein (e) gene was used for confirmation. the e-gene rtqpcr assay was less sensitive and accurate as the limit of quantitation (loq) was higher. the loq was genome copies of sars-cov- rna per reaction for the rdrp-gene assay and genome copies per reaction for the e-gene assay as found using rna extracted from the nibsc virus reagent / . these loq values correspond to . and . log gc/l of sewage, respectively, when maximum concentration is achieved. as shown in table , positive rtqpcr signals were obtained for the samples from march, april, and may. the sample from may was only positive in out of replicate assays with the rdrp-gene reaction and in none of the reactions with e-gene primers, so accurate quantification of viral rna in this sample was not possible. however, it was clear that there was a large reduction of sars-cov- rna concentration in sewage between th april and th may. positive and negative results were independently confirmed using a second real-time pcr platform (stratagene p) in a different nibsc laboratory. integrity of process was confirmed through use of previous experience with enteroviruses. this was demonstrated both by detection of enteroviral rna and recovery of infectious virus in cell cultures from all wastewater concentrates following who-recommended protocols as described in section . - -may- < . (n = ) - wastewater samples were concentrated using a standard filtration-centrifugation method (concentration factor: - ×). mean values of log sars-cov- genome copy (sc gc)/l wastewater with standard deviations are shown. dark grey indicates positive in at least / replicate npcr reactions. light grey indicates positive only after additional concentration (up to ×). positive pcr results were obtained for feb-may samples in at least two independent concentration processes for at least two different gene targets. the january sample remained negative even after a second concentration step. only / replicates gave positive rtqpcr signals with rdrp target, so viral rna quantification was not possible. details of the number of sequences analysed by date and country are given in table s . the frequency of sequence variation at each genomic nucleotide position was determined with respect to the reference wuhan-hu- strain (ncbi accession no. mn ) for each dataset. figure shows nucleotide positions at which sequence variation in > % of viral rna sequences from england and the rest of the world were observed. this sample was not possible. however, it was clear that there was a large reduction of sars-cov- rna concentration in sewage between th april and th may. positive and negative results were independently confirmed using a second real-time pcr platform (stratagene p) in a different nibsc laboratory (data not shown). integrity of process was confirmed through use of previous experience with enteroviruses. this was demonstrated both by detection of enteroviral rna and recovery of infectious virus in cell cultures from all wastewater concentrates following whorecommended protocols as described in materials and methods. details of the number of sequences analysed by date and country are given in table s . the frequency of sequence variation at each genomic nucleotide position was determined with respect to the reference wuhan-hu- strain (ncbi accession no. mn ) for each dataset. figure shows nucleotide positions at which sequence variation in > % of viral rna sequences from england and the rest of the world were observed. whole-genome sars-cov- sequences used in this analysis were downloaded from the gisaid database [ ] . the wuhan-hu- strain (ncbi accession no. mn ) was used as reference. whole-genome sars-cov- sequences used in this analysis were downloaded from the gisaid database [ ] . differences in nucleotide sequence frequencies at some of these common positions were noticeable indicating a different prevalence of some sequence variants between viral sequences in england and the rest of the world. we used this information to select genomic regions for our sequence analysis. key nucleotide positions , , , , , and , were targeted in two npcr products, npcr and npcr . npcr spans nucleotides - and npcr covers nucleotides , - , ( figure s ). the npcr product includes nucleotide variants a g and c t, which result in amino acid changes i v and p s in nsp protein and which are often associated between them. the npcr product also includes nucleotide c t, which is almost always associated with nucleotide sequence variations c t, c t, and a g; mapping in the leader sequence; rna polymerase (p l amino acid change); and spike protein (d g amino acid change), respectively. this virus variant containing these four nucleotide variations, named g , has become the dominant pandemic virus around the world [ ] . the npcr product includes nucleotide variant c t, also part of the dominant g pandemic strain, and synonymous change t c often associated with variation g t, which results in amino acid change g v in orf a protein. the frequency of t c is . % in england versus . % in the rest of the world. table s shows how nucleotide sequences at these selected five nucleotide positions most commonly combine in sars-cov- isolates. we also show in figure how the frequency of sequence variants at these five positions has changed during the pandemic in different countries/regions of the world. nucleotide variant c t, also part of the dominant g pandemic strain, and synonymous change t c often associated with variation g t, which results in amino acid change g v in orf a protein. the frequency of t c is . % in england versus . % in the rest of the world. table s shows how nucleotide sequences at these selected five nucleotide positions most commonly combine in sars-cov- isolates. we also show in figure how the frequency of sequence variants at these five positions has changed during the pandemic in different countries/regions of the world. as can be noted, differences in sequence composition at these positions were notable between clinical samples from different countries/regions, likely reflecting differences in the circulation of different virus variants. variants a g and c t were present in very low proportion in spain, asia, and usa as compared to the proportion in england. variant c t was particularly prevalent in spain and increase in the proportion of nucleotide variations characteristic of g pandemic variant was delayed in asia with respect to the other regions analysed. three additional npcr assays were designed as described in sections and . below. five different rna replicates from each wastewater concentrate were initially used to generate npcr products with the different primer combinations shown in figure s . as shown in table , positive rt-pcr products were obtained for all five npcr reactions using rna extracted from march, april, and may wastewater concentrates. the february wastewater concentrate only produced positive results with npcr and npcr reactions, and only after an additional concentration step to the standard - × concentration procedure was performed ( table ). the results obtained with npcr reactions were in good agreement with those from rtqpcr assays as the proportion of positive npcr reactions closely matched that of the viral rna concentration values. npcr assays allowed confirmation by sanger sequencing and ngs analysis. for all positive samples, positive npcr results were obtained for at least two different gene targets and from rna extracted from at least two different independent wastewater concentration processes. npcr positive reactions produced clean and clear bands following electrophoresis on agarose gels. none of the npcr reactions with rna from the wastewater sample viruses , , of collected on th january and none of the multiple rna extraction and pcr reaction negative controls produced sars-cov- npcr products. the rtqpcr and npcr results are summarized in figure in the context of epidemiological data. concentration step to the standard - × concentration procedure was performed ( table ) . the results obtained with npcr reactions were in good agreement with those from rtqpcr assays as the proportion of positive npcr reactions closely matched that of the viral rna concentration values. npcr assays allowed confirmation by sanger sequencing and ngs analysis. for all positive samples, positive npcr results were obtained for at least two different gene targets and from rna extracted from at least two different independent wastewater concentration processes. npcr positive reactions produced clean and clear bands following electrophoresis on agarose gels. none of the npcr reactions with rna from the wastewater sample collected on th january and none of the multiple rna extraction and pcr reaction negative controls produced sars-cov- npcr products. the rtqpcr and npcr results are summarized in figure in the context of epidemiological data. the amount of sars-cov- nucleotide sequences obtained by sanger analysis for each sample is shown in table and ranged between and nucleotides per wastewater concentrate. sequences from several npcr replicates were generated from each concentrate. nucleotide sequences for npcr , npcr , and npcr products for all samples were identical to those of the consensus sequence from clinical samples from england except for few nucleotide changes found in a few npcr replicates. nucleotide differences and mixed bases in sequence electropherograms were observed for npcr and npcr products from march and april at nucleotide positions where sequence variations had been observed between clinical samples in england as discussed above. the npcr products were analysed by ngs with an aim to quantify the proportion of different nucleotides at mixed base positions. between , and , filtered reads were sequenced per npcr product with > % of reads typically mapping to sars-cov- reference sequences. an example of the results for both sanger and ngs analyses of npcr products obtained with different rna replicates from each sample are shown in figure . ngs quantification results were in excellent agreement with those observed in sanger sequence electropherograms although no mixed peaks were detected in sanger sequences when the minor nucleotide component was below %, showing the inferior sensitivity of the sanger sequence analysis. differences in sequence composition were found between rna replicates from samples from march and april reflecting the presence of virus mixtures in both samples, replicate npcr and npcr products were sequenced from each sewage concentrate. mean sequence frequency values at the five selected nucleotide positions for each month are shown in figure . sequences when the minor nucleotide component was below %, showing the inferior sensitivity of the sanger sequence analysis. differences in sequence composition were found between rna replicates from samples from march and april reflecting the presence of virus mixtures in both samples, replicate npcr and npcr products were sequenced from each sewage concentrate. mean sequence frequency values at the five selected nucleotide positions for each month are shown in figure . error bars indicate standard error of the mean. whole-genome sars-cov- sequences used in this analysis were downloaded from the gisaid database [ ] . overall, the nucleotide sequence composition at all five selected nucleotide positions changed between march and april. viral rna samples containing a g and c t nucleotide variations decreased between march and april. the proportion of t at positions and , , genetic markers of the g dominant strain, increased between march and april. finally, the predominant sequence at position , also switched from t in march to c in april. the same trend in sequence composition continued in may although a similar in-depth analysis was not possible since fewer replicate npcrs were sequenced successfully. no mixed bases were identified by sanger or ngs analysis in any of the npcr products from the rna samples from may. sequence results from four npcr replicates from the may sewage found an a at nucleotide and a c at residue in all four replicates and a t at position in / of the replicates in agreement with their predominance observed in april. a single npcr product sequenced from may also contained the nucleotide sequences of g dominant strain at positions , and , . few additional sequence variations were identified in few pcr products, but none were present in more than one replicate. we detected sars-cov- rna in wastewater samples collected between february and may . a sample from th january was negative and only low levels of viral rna were detected in the sample from th february, days after the first two covid- cases had been confirmed in york, northern england, and days before the first case was reported in the population sampled in our study. the sars-cov- rna concentration estimated in wastewater samples was consistent with the number of cases reported at the time of sample collection. limitations in testing capacity early during the pandemic meant that there was an underestimation of cases in the community, and the extent of community transmission. it is therefore likely that the number of cases in early march was higher, which agrees with the viral rna levels we found in sewage. our results showed a large reduction of viral rna concentration in sewage between april and may, most likely due to the lockdown measures introduced in the country from rd march (figure ). this is in very good agreement with the observed reduction in covid- confirmed cases and infection estimates [ , ] . however, more frequent sampling would be required to estimate the rate of virus decay and establish firm conclusions about the relationship between cases and what is detected by es. previous studies have detected sars-cov- rna in wastewater samples worldwide [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] highlighting es as a potential tool to help establish early warning systems for the detection of peaks in virus circulation to be able to direct timely public health interventions. the turnaround of laboratory results could take as little as h using our current workflow. however, efforts to improve laboratory methods for sample processing, virus concentration, and viral rna quantification might be needed to increase the sensitivity for sars-cov- detection to ensure our ability to detect asymptomatic virus transmission, particularly in areas with low background transmission rates. the type of samples analysed, e.g., raw sewage versus primary sludge or different sample processing e.g., analysing aqueous versus solid phases may have an impact on viral rna recovery from sewage [ , , ] . the use of reference standards and collaborative studies between different laboratories using common samples would help in this process, allowing comparability between laboratories and methods. in addition, more detailed mathematical modelling studies similar to those conducted for poliovirus es [ ] will be required to understand the representativeness of replicate sampling, develop sampling strategies around high-risk communities, and establish how es can best complement clinical diagnosis to hopefully help prevent future lockdowns. early efforts conducted in australia [ ] to estimate the proportion of individuals infected with sars-cov- in a catchment area using es data should be expanded. although some relevant data are available, more detailed data on the dynamics of sars-cov- virus excretion in stools are necessary to conduct these analyses, such us knowing the proportion of infected individuals excreting virus in stools, the duration of virus shedding and the virus titres excreted during that period. estimating the total amount of stool shed per person per day during sars-cov- infection as well as the virus recovery rate from sewage in the laboratory would be additional factors to increase the accuracy of the modelling. a novel nested rt-pcr approach targeting five different regions of the viral genome improved the sensitivity of rt-qpcr results. next generation sequencing analysis of rt-pcr products revealed single nucleotide polymorphisms at five selected nucleotide positions, where sequence variation between viral rna in clinical samples from england had been observed confirming the co-circulation of virus variants and changes in virus variant predominance with time. the target nucleotide sites for our study were selected following analysis of whole-genome sars-cov- sequences from the gisaid sequence database [ ] . differences in sequence composition at these positions were notable between different countries/regions during the first few months of the pandemic, but in all cases, sars-cov- strains containing common nucleotide sequences at these five positions survived, largely reflecting the global dominance of sars-cov- variant g ( figure ). the g variant, containing a glycine (g) at residue in the sars-cov- spike protein, has become the dominant pandemic form globally showing a consistent increase at all national, regional, and local levels, which suggests a possible fitness advantage [ ] . however, no evidence exists yet that any observed changes among sars-cov- isolates, including g , have resulted in adaptation to the human host, increased transmissibility, or worsening disease severity [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . other possible explanations for g dominance exist such as being caused by purely neutral sampling processes as described for other viruses during previous pandemics [ ] . nucleotide sequences characteristic of variant g were present in % of the viral rna sequences reported from england in february but increased to around % in march, % in april, and % in may. variants a g and c t were specifically prevalent in clinical samples from england. a combination gt or at at these two nucleotide positions was present in (figure ). our sequencing results of sars-cov- rna from wastewater samples are consistent with these nucleotide sequence evolution patterns ( figure ). the nucleotide sequence composition changed at all five selected positions between the samples collected on th march and th april. variants a g and c t were only detected in low proportion in april and t, t, and c became the dominant sequences at these sites consistent with g global dominance [ ] . in line with our sequence variation results, a study that analysed sequence variation among u.k. sars-cov- isolates, found that the epidemic comprises a very large number of importations due to inbound international travel [ ] . the rate and source of introduction of sars-cov- lineages into the u.k. changed rapidly through time, peaking in mid-march, with most introductions occurring during march . many u.k. transmission lineages appeared to be very rare or extinct at the time of reporting ( th june). this would be consistent with notable changes expected in virus population dynamics with a likely decrease in sequence heterogeneity from mid-march onwards as seen in our results from sewage. overall, our study shows that es can be used to detect sars-cov- transmission with viruses identified in sewage resembling those found in clinical samples. we were able to detect virus variants specifically circulating in england and also to identify changes in viral rna sequences consistent with the increasing global dominance of g pandemic variant [ ] . our results are encouraging and suggest a potentially wider applicability of es to monitor sars-cov- transmission, tracing virus variants and detecting virus importations. we have shown that environmental surveillance can be used to monitor sars-cov- transmission detecting virus variants specifically circulating in england and identifying changes in virus variant predominance known to have occurred during the covid- pandemic. environmental surveillance could be used for the early detection of peaks in virus transmission for public health interventions to be timely implemented. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : nested-pcr strategy. table s : number of whole-genome sars-cov- sequences by region/country and date of collection analysed in this study. table s : most common nucleotide 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from environmental monitoring no evidence for distinct types in the evolution of sars-cov- on the origin and continuing evolution of sars-cov- emergence of genomic diversity and recurrent mutations in sars-cov- genotyping coronavirus sars-cov- : methods and implications emerging sars-cov- mutation hot spots include a novel rna-dependent-rna polymerase variant international expansion of a novel sars-cov- mutant discovery of a -nt deletion during the early evolution of sars-cov- no evidence for increased transmissibility from recurrent mutations in sars-cov- evaluating the effects of sars-cov- spike mutation d g on transmissibility and pathogenicity tracking changes in sars-cov- spike: evidence that d g increases infectivity of the covid- virus isolation of vaccine-like poliovirus strains in sewage samples from the uk detection by direct next generation sequencing analysis of emerging enterovirus d and c strains in an environmental sample from scotland world health organization. guidelines for environmental surveillance of poliovirus circulation; world health organization data, disease and diplomacy: gisaid's innovative contribution to global health detection of sars-cov- in raw and treated wastewater in germany-suitability for covid- surveillance and potential transmission risks atropos: specific, sensitive, and speedy trimming of sequencing reads high resolution identity testing of inactivated poliovirus vaccines potential spreading risks and disinfection challenges of medical wastewater by the presence of severe acute respiratory syndrome coronavirus (sars-cov- ) viral rna in septic tanks of fangcang hospital surveillance optimisation to detect poliovirus in the pre-eradication era: a modelling study of england and wales apparent founder effect during the early years of the san francisco hiv type epidemic ( - ) preliminary analysis of sars-cov- importation & establishment of uk transmission lineages this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we are kindly grateful to all authors submitting sequencing data to the gisaid database that were used for analyses shown in this manuscript. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.viruses , , key: cord- -oilurica authors: cui, tingting; theuns, sebastiaan; xie, jiexiong; den broeck, wim van; nauwynck, hans j. title: role of porcine aminopeptidase n and sialic acids in porcine coronavirus infections in primary porcine enterocytes date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: oilurica porcine epidemic diarrhea virus (pedv) and transmissible gastroenteritis virus (tgev) have been reported to use aminopeptidase n (apn) as a cellular receptor. recently, the role of apn as a receptor for pedv has been questioned. in our study, the role of apn in pedv and tgev infections was studied in primary porcine enterocytes. after seven days of cultivation, % of enterocytes presented microvilli and showed a two- to five-fold higher susceptibility to pedv and tgev. a significant increase of pedv and tgev infection was correlated with a higher expression of apn, which was indicative that apn plays an important role in porcine coronavirus infections. however, pedv and tgev infected both apn positive and negative enterocytes. pedv and tgev miller showed a higher infectivity in apn positive cells than in apn negative cells. in contrast, tgev purdue replicated better in apn negative cells. these results show that an additional receptor exists, different from apn for porcine coronaviruses. subsequently, treatment of enterocytes with neuraminidase (na) had no effect on infection efficiency of tgev, implying that terminal cellular sialic acids (sas) are no receptor determinants for tgev. treatment of tgev with na significantly enhanced the infection which shows that tgev is masked by sas. coronaviruses are known as human and animal pathogens that mainly infect the epithelium of the respiratory or intestinal tract. porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), and its variant porcine respiratory coronavirus (prcv) are classified as alphacoronavirus. they are enveloped viruses containing a single-stranded, positive-sense rna genome of approximately . kb. the positive ssrna serves as mrna for the generation of viral replicative proteins by translation of open reading frame (orf) a and orf b. the genome contains a untranslated region (utr), a utr, and at least seven orfs. orf a and orf b make up two-thirds of the viral genome and encode the non-structural replicase polyproteins (replicases a and b), which further guide the viral replication and translation, regulate cellular processes, and potentially fulfill other unknown functions. the remaining proximal third of the genome encodes four structural proteins (spike (s), envelope (e), membrane (m), and nucleocapsid (n) by orf , orf , orf , and orf , respectively). the s protein is a type i glycoprotein that projects from the virions surface forming in the present study, the co-culture system of primary porcine enterocytes that was established in our laboratory was used to study the role of apn in pedv and tgev infection in their target cells [ ] . further, it was investigated whether sialic acids are cellular receptors for tgev in primary enterocytes. primary porcine enterocytes were isolated from the ileum of three-day-old piglets and co-cultured with porcine myofibroblasts [ ] . euthanizing piglets was done in agreement with the european legislation on animal experiments. all experimental procedures were approved by the local ethical committee of the faculty of veterinary medicine, ghent university (ec / ), and all methods were carried out in accordance with the approved guidelines. the enterocytes were maintained with dulbecco's modified eagle's-f ham medium (dmem-f ). tgev purdue and miller grown on swine testicle (st) cells and pedv cv strain grown on vero cells were used in this study. pedv cv fecal suspension was collected from a three-day-old infected suckling piglet. a twenty percent fecal suspension was prepared in phosphate buffered saline (pbs) containing u/ml penicillin (continental pharma, puurs, belgium), mg/ml streptomycin (certa, braine l'alleud, belgium), mg/ml gentamicin (gibco brl, merelbeke, belgium), and . % v/v fungizone (bristol-myers squibb, braine l'alleud, belgium). hydrocortisone, spermidine, and wnt agonist were purchased from sigma-aldrich (sigma, st-louis, mo, usa). porcine insulin was purchased from protein specialists (prospec, rehovot, israel). the small intestinal contents (ic) were collected from the duodenum of a three-day-old suckling piglet. after euthanasia, a cm long segment of duodenum was closed by two surgical clamps and was removed from a piglet. then, one clamp was removed and the intestinal contents were released from the lumen into a ml centrifugation tube. in order to collect all the intestinal contents, the lumen was washed once by filling it with ml dulbecco's modified eagle's medium (dmem) containing u/ml penicillin, mg/ml streptomycin, mg/ml gentamicin, and . % v/v fungizone. then, the dmem was released from the lumen into the ml tube that already contained the intestinal contents. after centrifugation ( rpm, min at • c), the supernatant of the intestinal contents was collected and stored at − • c until use. three-and seven-day-old primary porcine enterocytes were fixed in hepes-buffered glutaric aldehyde (sigma, st-louis, mo, usa) for scanning electron microscopy as described previously [ ] . after h fixation, the samples were treated with % osmium tetroxide for h at room temperature (rt), followed by ascending grades of alcohol dehydration. in order to avoid the water vaporization obstructing the electron beam and interfering with image clarity, the dehydrated samples were transferred to a critical point drier (cpd, bal-tec, balzers, liechtenstein) for complete drying. finally, the dried samples were mounted on a metal stub and were sputter-coated with platinum. the microvilli of all the samples were acquired with a jeol jsm lv scanning electron microscope (jeol ltd., tokyo, japan). twenty-four h post co-cultivation, monolayers of enterocytes were cultured with medium containing hydrocortisone ( and µg/ml), spermidine ( and µm), insulin ( and µg/ml), wnt agonist ( . µm), or intestinal contents ( %) for h. the cells were fixed with % paraformaldehyde for min and immunofluorescence staining was conducted for apn expression analysis. cells were incubated with mouse monoclonal anti-porcine apn antibodies (imm ; kindly provided by prof. eric cox, ghent university) containing % normal goat serum for h at • c, followed by goat anti-mouse-igg fitc labeled antibodies for h at • c. nuclei were stained with hoechst for min at rt. the percentage of apn positive cells were analyzed by fluorescence microscopy (leica microsystems gmbh). at twenty-four h of co-cultivation, monolayers of enterocytes were treated with µg/ml hydrocortisone, µm spermidine, µg/ml insulin, . µm wnt agonist, or % intestinal contents for another h. then, the susceptibility of treated enterocytes to pedv and tgev was tested. cells were inoculated with µl of tgev miller at a multiplicity of infection (m.o.i.) of and µl of the pedv cv vero adapted strain at . tcid /ml or viral rna copies/ml of fecal suspension with µg/ml trypsin. after min of incubation at • c, unbound viral particles were removed by three washing steps with dmem. cells were further incubated in medium containing µg/ml hydrocortisone, µm spermidine, µg/ml insulin, . µm wnt agonist, or % intestinal contents for h ( • c, % co ) and fixed with % paraformaldehyde for immunofluorescence staining. to determine the co-localization of viral antigens and apn, co-cultured enterocytes were infected with tgev miller and purdue and pedv vero adapted and non-adapted strains. after h incubation, cells were fixed with % paraformaldehyde for min and immunofluorescence staining was performed. cells were incubated with mouse monoclonal anti-porcine apn antibodies containing % normal goat serum for h at • c, followed by goat anti-mouse-igg fitc labeled antibodies for h at • c. then, cells were permeabilized with . % triton x- for min at rt and incubated with mouse monoclonal anti-porcine pedv antibodies (kindly provided by prof. luis enjuanes, national center for biotechnology) or swine polyclonal anti-tgev antibodies [ ] containing % normal goat serum for h at • c, followed by goat anti-mouse igg a af labeled antibodies or goat anti-swine texas red labeled antibodies (molecular probes). nuclei were stained with hoechst for min at rt and the results were analyzed by a leica tcs spe laser scanning spectral confocal system linked to a dm b fluorescence microscope (leica microsystems). to remove sas from enterocytes, monolayers of co-cultured enterocytes were washed three times with warm dmem. then, cells were incubated with mu/ml na from vibrio cholerae (roche diagnostics, risch-rotkreuz, switzerland) at • c for h. cells that were mock-treated were incubated with dmem and underwent the same manipulations as na-treated cells. to remove sas from the virus, virus suspensions were incubated with mu/ml na from vibrio cholerae at • c for h. the mock-treated virus was incubated in dmem and underwent the same manipulations as the na-treated virus. afterwards, cells were inoculated with either na-treated or mock-treated virus (m.o.i of for tgev purdue and miller). after min incubation at • c, cells were washed three times with medium and further incubated in medium for h ( • c, % co ). then, cells were fixed with % paraformaldehyde for min at rt. immunofluorescence was performed to measure the percentage of infected cells. the cells were permeabilized with . % triton x- for min at rt. then, cells were incubated with swine polyclonal anti-tgev antibodies containing % normal goat serum for h at • c, followed by goat anti-swine-igg fitc labelled antibodies for h at • c. nuclei were stained with hoechst for min at rt. the percentages of infected cells were determined by fluorescence microscopy. to determine the co-localization of viral antigens and sas, co-cultured enterocytes were infected with tgev miller and purdue. after h incubation, cells were fixed with % paraformaldehyde for min and a double immunofluorescence staining was performed. cells were incubated with biotinylated maackia amurensis lectin ii (vector laboratories) for h at • c. the lectin was subsequently stained with streptavidin-fitc (invitrogen) for h at • c. then, cells were permeabilized with . % triton x- for min at rt and incubated with swine polyclonal anti-tgev antibodies containing % normal goat serum for h at • c, followed by goat anti-swine-igg texas red labeled antibodies. nuclei were stained with hoechst for min at rt and results were analyzed by a leica tcs spe laser scanning spectral confocal system linked to a dm b fluorescence microscope. to characterize the attachment of tgev to primary enterocytes, direct virus-binding studies were carried out with tgev particles. cells were chilled on ice for min and washed three times with dmem. then, cells were inoculated with tgev miller and purdue particles at a m.o.i. of for h at • c. unbound virus particles were removed by three washings with dmem. cells were fixed with % paraformaldehyde for min and a double immunofluorescence staining was performed. cells were incubated with biotinylated maackia amurensis lectin ii or mouse monoclonal anti-porcine apn antibodies for h at • c, followed by streptavidin-fitc or goat anti-mouse-igg fitc labeled antibodies for h at • c. then, cells were permeabilized with . % triton x- for min at rt and incubated with swine polyclonal anti tgev antibodies containing % goat serum for h at • c, followed by goat anti-swine-igg texas red labeled antibodies. nuclei were stained with hoechst for min at rt and the results were analyzed by a leica tcs spe laser scanning spectral confocal system linked to a dm b fluorescence microscope. data were statistically processed by spss (t-test). the data are represented as means with standard deviation (sd) of three independent experiments. results with p-values of < . were considered significant. the percentages of microvilli positive enterocytes were analyzed by scanning electron microscopy. a higher percentage of microvilli positive cells ( %) was observed at seven days cultivation compared to three days cultivation ( %). the expression of apn at seven days cultivation ( . ± . %) was significantly higher than at three days cultivation ( . ± . %; figure a ,b). the data suggest that primary enterocytes underwent a differentiation process in vitro. they terminally differentiate into mature enterocytes during the long cultivation time. next, enterocytes were inoculated with tgev and pedv at three and seven days of cultivation. the results showed that a significantly higher infection was detected in enterocytes at seven days cultivation (miller: . ± . %; purdue: . ± . %) than at three days cultivation (miller: . ± . %; purdue: . ± . %) for tgev. an increased trend of infection was detected in enterocytes at seven days cultivation ( infected cells per well) than at three days cultivation ( infected cells per well) for pedv but without significance (p = . ; figure c ). data are expressed as mean ± standard deviation (sd) of the results of three separate experiments. statistically significant differences in comparison with data from three days cultivation are presented as *p < . or **p < . . cells were treated with hydrocortisone, spermidine, porcine insulin, wnt agonist, or small intestinal contents to analyse their effects on enterocyte differentiation. cells were treated with the aforementioned products for h. afterwards, the differentiation marker apn was stained by immunofluorescence and the percentage of apn positive cells was counted ( figure a ). the results showed that without treatment, . ± . % of cells were apn positive. the treatment with and µg/ml hydrocortisone significantly increased the apn expression to ± . % and ± . %, respectively. the treatment with mm and mm spermidine significantly enhanced the apn expression to ± . % and ± . %, respectively. similarly, and µg/ml insulin treatment significantly increased the apn expression to ± . % and ± . %, respectively. since there was no dose-dependent enhancement, the lower concentration of each product ( µg/ml of hydrocortisone, µm of spermidine and µg/ml of porcine insulin) was used for the next cells were treated with hydrocortisone, spermidine, porcine insulin, wnt agonist, or small intestinal contents to analyse their effects on enterocyte differentiation. cells were treated with the aforementioned products for h. afterwards, the differentiation marker apn was stained by immunofluorescence and the percentage of apn positive cells was counted ( figure a ). the results showed that without treatment, . ± . % of cells were apn positive. the treatment with and µg/ml hydrocortisone significantly increased the apn expression to ± . % and ± . %, respectively. the treatment with mm and mm spermidine significantly enhanced the apn expression to ± . % and ± . %, respectively. similarly, and µg/ml insulin treatment significantly increased the apn expression to ± . % and ± . %, respectively. since there was no dose-dependent enhancement, the lower concentration of each product ( µg/ml of hydrocortisone, µm of spermidine and µg/ml of porcine insulin) was used for the next experiment. the treatment of wnt agonist and intestinal contents showed a trend of increased apn expression up to ± % (p = . ) and ± . % (p = . ), respectively ( figure b ). to determine the effect of apn on coronavirus replication, the enterocytes were precultured with µg/ml hydrocortisone, µm spermidine, µg/ml insulin, . µm wnt agonist, or % intestinal contents for h prior to inoculation with pedv cv vero adapted strain, cv fecal suspension, and tgev miller. for pedv cv vero adapted strain, only ± cells were infected per well without pretreatment. the highest infection ( ± infected cells per well) was observed in cells that were pretreated with intestinal contents. because of the variation between the three replicates, the treatment with intestinal contents was not significantly different from the mock treatment (p = . ). when cells were pretreated with wnt agonist, a significant increase of infection ( ± infected cells per well) was observed (p = . ). an increased trend of infection (but not significantly different) was observed after pretreatment with hydrocortisone ( ± infected cells per well, (p = . ), spermidine ( ± infected cells per well, p = . ), and porcine insulin ( ± infected cells per well, p = . ). for cv fecal suspension, the highest infection was observed when to determine the effect of apn on coronavirus replication, the enterocytes were precultured with µg/ml hydrocortisone, µm spermidine, µg/ml insulin, . µm wnt agonist, or % intestinal contents for h prior to inoculation with pedv cv vero adapted strain, cv fecal suspension, and tgev miller. for pedv cv vero adapted strain, only ± cells were infected per well without pretreatment. the highest infection ( ± infected cells per well) was observed in cells that were pretreated with intestinal contents. because of the variation between the three replicates, the treatment with intestinal contents was not significantly different from the mock treatment (p = . ). when cells were pretreated with wnt agonist, a significant increase of infection ( ± infected cells per well) was observed (p = . ). an increased trend of infection (but not significantly different) was observed after pretreatment with hydrocortisone ( ± infected cells per well, (p = . ), spermidine ( ± infected cells per well, p = . ), and porcine insulin ( ± infected cells per well, p = . ). for cv fecal suspension, the highest infection was observed when cells were pretreated with the wnt agonist ( ± infected cells per well), showing a four-fold higher infection compared to mock-treated cells ( ± infected cells per well). when cells were pretreated with intestinal contents, ± cells were infected. for tgev miller, incubation with intestinal contents, porcine insulin, and wnt agonist significantly increased the virus infection from . ± . % to . ± . %, . ± . % and . ± . %, respectively. hydrocortisone and spermidine increased the virus infection to . ± . % and . ± . % (figure ) . the results show that pretreatment of primary enterocytes with hydrocortisone, spermidine, porcine insulin, wnt agonist, and intestinal contents could stimulate the expression of apn and enhance the infection of pedv cv vero adapted and non-adapted strains and the tgev miller in the enterocytes. statistically significant differences in comparison with data from mock treatment are presented as *p < . or **p < . . to assess the role of apn in the replication of pedv and tgev, a double immunofluorescence staining of apn and virus was performed. for cv vero adapted strain, . ± . % of apn positive cells and . ± . % of apn negative cells were infected. for cv fecal suspension, . ± . % of apn positive cells and . ± . % of apn negative cells were infected. for tgev miller, . ± . % of apn positive cells and . ± % of apn negative cells were infected. for tgev purdue, more infection was found in apn negative cells ( . ± . %) than in apn positive cells ( . ± . %; figure ). the results suggest that for pedv and tgev miller, apn may be the predominant receptor, while tgev purdue mainly uses an additional receptor for virus infection. to assess the role of apn in the replication of pedv and tgev, a double immunofluorescence staining of apn and virus was performed. for cv vero adapted strain, . ± . % of apn positive cells and . ± . % of apn negative cells were infected. for cv fecal suspension, . ± . % of apn positive cells and . ± . % of apn negative cells were infected. for tgev miller, . ± . % of apn positive cells and . ± % of apn negative cells were infected. for tgev purdue, more infection was found in apn negative cells ( . ± . %) than in apn positive cells ( . ± . %; figure ). the results suggest that for pedv and tgev miller, apn may be the predominant receptor, while tgev purdue mainly uses an additional receptor for virus infection. to assess the role of sas as receptor determinants, enterocytes were pretreated with mu/ml na prior to inoculation with tgev miller and purdue. miller infected . ± . % of the na-treated enterocytes and . ± . % of mock-treated cells. for purdue, the percentage of infection was . ± . % and . ± . % for na-treated and mock-treated cells, respectively. this implies that tgev does not depend on terminal sa residues on the enterocytes surface for infection. since it has been reported that removal of sas on the surface of coronaviruses improves binding and infection [ ] , the replication of mock-treated, and na-treated viruses was compared in untreated epithelial cells. na pretreatment of virus significantly enhanced infection from . ± . % to . ± . % for miller. for purdue, na pretreatment of virus significantly increased infection from . ± . % to . ± . % ( figure ). these data show that removal of sa from tgev promotes binding and replication of tgev in enterocytes. viruses , , of to assess the role of sas as receptor determinants, enterocytes were pretreated with mu/ml na prior to inoculation with tgev miller and purdue. miller infected . ± . % of the na-treated enterocytes and . ± . % of mock-treated cells. for purdue, the percentage of infection was . ± . % and . ± . % for na-treated and mock-treated cells, respectively. this implies that tgev does not depend on terminal sa residues on the enterocytes surface for infection. since it has been reported that removal of sas on the surface of coronaviruses improves binding and infection [ ] , the replication of mock-treated, and na-treated viruses was compared in untreated epithelial cells. na pretreatment of virus significantly enhanced infection from . ± . % to . ± . % for miller. for purdue, na pretreatment of virus significantly increased infection from . ± . % to . ± . % ( figure ). these data show that removal of sa from tgev promotes binding and replication of tgev in enterocytes. data are expressed as mean ± sd of the results of three separate experiments. statistically significant differences in comparison with data from mock treatment are presented as *p < . . double immunofluorescence staining was further performed to assess the role of sa on tgev replication. tgev miller could infect both sa positive cells ( . ± . %) and sa negative cells ( . ± . %). the tgev purdue replicated slightly better in sas negative cells ( . ± . %) compared to sas positive cells ( . ± . %; figure ). double immunofluorescence staining was further performed to assess the role of sa on tgev replication. tgev miller could infect both sa positive cells ( . ± . %) and sa negative cells ( . ± . %). the tgev purdue replicated slightly better in sas negative cells ( . ± . %) compared to sas positive cells ( . ± . %; figure ). primary enterocytes were inoculated with tgev particles (m.o.i. = ) at °c. the binding of tgev to apn positive/negative and sas positive/negative cells was examined by double immunofluorescence staining. no significant differences were observed between the percentage of apn positive cells with bound tgev miller ( . ± . %) and the percentage of apn negative cells with bound tgev miller ( . ± . %; p = . ). the percentage of apn positive cells with bound tgev purdue ( . ± . %) was lower than the percentage of apn negative cells with bound tgev purdue ( . ± . %), but was not significantly different (p = . ). the percentage of miller particles that colocalized with apn ( ± %) was significantly higher than non-colocalized particles ( ± %). the percentage of purdue particles that colocalized with apn ( ± %) was significantly lower than non-colocalized particles ( ± %; figure ) . the percentage of sa negative cells with bound tgev miller ( . ± . %) and with bound tgev purdue ( . ± . %) was significantly higher than the percentage of sa positive cells with bound tgev miller ( . ± . %) and with bound tgev purdue ( . ± . %). the percentage of tgev miller particles ( ± %) and tgev purdue ( ± %) that colocalized with sa was significantly lower than the particles that did not colocalize, with miller at ± % and purdue at ± % (figure ). primary enterocytes were inoculated with tgev particles (m.o.i. = ) at • c. the binding of tgev to apn positive/negative and sas positive/negative cells was examined by double immunofluorescence staining. no significant differences were observed between the percentage of apn positive cells with bound tgev miller ( . ± . %) and the percentage of apn negative cells with bound tgev miller ( . ± . %; p = . ). the percentage of apn positive cells with bound tgev purdue ( . ± . %) was lower than the percentage of apn negative cells with bound tgev purdue ( . ± . %), but was not significantly different (p = . ). the percentage of miller particles that colocalized with apn ( ± %) was significantly higher than non-colocalized particles ( ± %). the percentage of purdue particles that colocalized with apn ( ± %) was significantly lower than non-colocalized particles ( ± %; figure ). the percentage of sa negative cells with bound tgev miller ( . ± . %) and with bound tgev purdue ( . ± . %) was significantly higher than the percentage of sa positive cells with bound tgev miller ( . ± . %) and with bound tgev purdue ( . ± . %). the percentage of tgev miller particles ( ± %) and tgev purdue ( ± %) that colocalized with sa was significantly lower than the particles that did not colocalize, with miller at ± % and purdue at ± % (figure ). porcine epithelial cells of the small intestines are the target cells for pedv and tgev in vivo. these cells show a high surface expression of apn, and apn has been reported to be the cellular receptor for pedv and tgev [ , ] . however, vero cells with undetectable apn expression were historically used for pedv propagation questioning the role of apn as a cellular receptor for pedv [ ] . in addition, overexpression of porcine apn in non-susceptible cells did not robustly support pedv infection, and knock-out of apn in susceptible cells did not abrogate pedv infection [ ] . the present study was performed to determine the role of apn in pedv and tgev infection in their target primary porcine enterocytes. we found that a higher infection of pedv and tgev was correlated with a higher apn expression. however, both pedv and tgev did not only infect apn porcine epithelial cells of the small intestines are the target cells for pedv and tgev in vivo. these cells show a high surface expression of apn, and apn has been reported to be the cellular receptor for pedv and tgev [ , ] . however, vero cells with undetectable apn expression were historically used for pedv propagation questioning the role of apn as a cellular receptor for pedv [ ] . in addition, overexpression of porcine apn in non-susceptible cells did not robustly support pedv infection, and knock-out of apn in susceptible cells did not abrogate pedv infection [ ] . the present study was performed to determine the role of apn in pedv and tgev infection in their target primary porcine enterocytes. we found that a higher infection of pedv and tgev was correlated with a higher apn expression. however, both pedv and tgev did not only infect apn positive enterocytes, but also apn negative cells. our results demonstrated that pedv and tgev may use another additional unknown receptor for entry in primary enterocytes. the epithelium of the small intestines is continuously and rapidly renewed in a process involving cell generation and migration from the multi-potent stem cells in the crypts to the differentiated cells at the tips of the villi within - days. in our study, after three days cultivation, the primary enterocytes were positive for sucrase and iso-maltase, which are considered as differentiation markers for epithelial cells [ ] . however, the expression of aminopeptidase n was only around %, indicating that the primary enterocytes are not fully mature at three days cultivation. therefore, we analyzed apn expression in enterocytes at a later time point (seven days cultivation). a significantly higher expression of apn was detected at seven days cultivation compared to three days cultivation. interestingly, the seven-day-cultured enterocytes with a higher apn expression showed significantly higher infection to tgev than that of enterocytes cultured for three days. this agrees with the fact that the virus mainly infects and destroys mature enterocytes lining the villi of small intestines [ ] . however, the infection efficiency of pedv in intestinal epithelial cells was still low, indicating that other factors than apn need to be considered for pedv infection. therefore, several positive enterocyte differentiation factors were tested. hydrocortisone plays an important role in the metabolism of proteins, lipids, and carbohydrates, and is a known promoter for differentiation of cultured cells. hydrocortisone was found to be a critical factor for the differentiation of skeletal muscle, osteoblasts, and endothelial cells [ ] . sorrell and colleagues demonstrated that hydrocortisone significantly upregulated the expression of apn in human dermal fibroblasts [ ] . besides, wnt signaling is required for the formation of normal crypt-villus units of intestines, and stimulates the differentiation of intestinal secretory epithelial cells [ ] . activated wnt signaling has also been shown to promote mesenchymal differentiation [ ] . the original rationale for including intestinal contents in our primary cell cultures was trying to mimic the in vivo situation in the intestinal tract. intestinal contents contain a large number of enzymes, such as: amylase, which digests carbohydrates to monosaccharides; pancreatic enzymes, which digest proteins into amino acids; and lipase which digests fats. it has been demonstrated that intestinal contents play an important role in virus infection. the proteases (trypsin) in the intestinal contents activate rotavirus infection by cleaving the outer capsid protein vp [ ] . the propagation of porcine enteric calicivirus (pec) on a cell line critically relies on the presence of intestinal contents in the culture medium [ ] . chang et al. demonstrated that the bile salts in intestinal contents are essential for growth of pec by inducing the protein kinase a (pka) signaling pathway [ ] . in our study, the intestinal contents collected from the upper duodenum promoted the expression of apn and enhanced the infection of both tgev and pedv, especially the cv vero adapted strain. further investigation is needed to determine which growth-promoting factor in the intestinal contents is responsible for the increase of coronavirus infection. to date, coronaviruses use four different proteins as cellular receptors. mouse hepatitis virus (mhv) initiates the infection by binding to the carcinoembryonic cell adhesion molecule on hepatocyte membranes and intestinal brush border membranes [ ] . next, apn was found to act as a receptor for porcine, feline, canine, and human coronaviruses [ ] . the receptors for the highly pathogenic human respiratory viruses sars-cov type and type as well as middle east respiratory syndrome coronavirus are angiotensin-converting enzyme (ace ) and dipeptidyl peptidase (dpp ) [ , ] . cong and colleagues proved that the porcine small intestine epithelial cell line was more susceptible to pedv when a high expression level of apn was present, and that interference with apn expression in epithelial cells inhibited pedv infection, demonstrating that apn serves as an essential receptor for pedv [ ] . in addition, a transgenic mouse model expressing porcine apn was proven to be susceptible to pedv, which confirmed that apn plays a role as the cellular receptor for pedv [ ] . in our study, we found that the higher infection of pedv in enterocytes is correlated with higher apn expression. both aged enterocytes and enterocytes treated with differentiation factors expressed more apn and were more susceptible to pedv, which indicates that apn may play role in pedv infection in enterocytes. moreover, we found that the apn was expressed at the apical surface of enterocytes and pedv infected times more enterocytes at the apical surface than at the basolateral surface (supplementary figure s ). these results were in agreement with the previous finding that pedv enters polarized cells via the apical membrane [ ] . our results indicate that apn facilitates the entry of pedv into primary enterocytes. shirato and colleagues indicated that apn may promote pedv replication in porcine kidney cell line, cpk cells via its protease activity [ ] . how apn facilitates pedv infection in enterocytes should be further investigated. however, due to the fact that other molecules besides apn will also be expressed at the apical plasma membrane during differentiation, they may also contribute to the higher susceptibility of differentiated enterocytes. recently, increasingly more data has been published that show that apn is not a cellular receptor for pedv. overexpression of apn in non-susceptible cells did not confer susceptibility of the cells to pedv and knocking out apn in susceptible cells did not abrogate pedv infection, which indicates that apn is not required as a cellular receptor for pedv in vitro [ , , ] . furthermore, apn knockout pigs retained their susceptibility to pedv confirming that pedv may use another additional receptor in pigs [ ] . in agreement with these findings, we found that pedv can infect apn negative primary enterocytes, which further confirmed that a cellular receptor different from apn exists in enterocytes for pedv replication. the primary enterocytes isolated and cultured in our study are not % positive for apn, as we not only get the villi epithelial cells, but also the crypt epithelial cells during our isolation procedure. by immunofluorescence staining of ileum tissue of a three-day-old piglet, we observed that the villi epithelial cells are apn positive, while the crypt epithelial cells are apn negative (data not shown). as pedv has been shown to infect goblet cells and crypt stem cells in addition to villous mature enterocytes [ ] , we believe that pedv may also use another cellular receptor besides apn to infect intestinal cells. taken together, our results indicate that although apn could significantly promote pedv infection in enterocytes, an additional cellular receptor exists in enterocytes for pedv replication. since ace is expressed in the gut epithelial cells, it will be tested in the near future if it can function as a pedv receptor. in addition to pedv, apn has been identified as a major cellular receptor for tgev. in the present study, it was shown that a higher expression of apn significantly increased the replication of tgev in enterocytes, indicating that apn plays an important role as a cellular receptor for tgev. whitworth and colleagues demonstrated that apn knockout pigs were resistant to tgev infection, indicating that apn is the sole functional receptor for tgev [ ] . however, we found that except apn positive enterocytes, tgev could also infect apn negative enterocytes, suggesting that an additional receptor also exists in enterocytes for tgev. the result obtained in our in vitro experiment may not fully reflect the in vivo experiments, as the in vivo situation is composed of a complex set of cells and tissues. the purdue strain used in our study has been passaged times in primary kidney cells and a nucleotide mutation (t to g at nucleotide position ), which causes a serine (s) to alanine (a) at aa [ , ] . this mutation may be correlated with the cell adaptation and also it may result in a broader cell tropism of the adapted strain, which may explain that the virus is able to grow more efficiently in apn negative cells. purdue has been proven to infect primary colon epithelial cells and porcine myofibroblasts, which are both negative for apn [ ] . in vivo, tgev shows a higher cell tropism to villous enterocytes of newborn piglets compared to older pigs and causes only high mortality in the early life of piglets. since apn is present on enterocytes from both newborn and older pigs, the age-dependent susceptibility to tgev infection may be caused by an additional receptor that is specifically present in newborn piglets. taken together, we hypothesize that apn is the determinant cellular receptor for tgev, but an additional receptor exists in young piglets. apart from apn, tgev also uses sa as an attachment mediator on the cells. shahwan and colleagues have shown that na treatment of jejunum epithelial cryosections did not reduce the tgev spike protein binding in vitro and were doubting on the role of sa during infection [ ] . in our study, removal of sa from intestinal cells had no effect on tgev infection, showing that terminal sa residues are not receptor determinants for tgev. removal of sa from tgev virions significantly enhanced the viral infectivity in vitro. this indicates that sa on the virion surface masks the binding site of the viral protein to cellular receptors. removal of sa from virions facilitates tgev to bind to its functional receptor on the enterocyte membrane. to confirm the role of apn and sa in tgev infection in primary enterocytes, a binding assay was performed in this study. the results demonstrated that both tgev miller and purdue could bind to apn positive and sa positive cells. meanwhile, miller and purdue could also bind to apn negative and sa negative enterocytes. furthermore, our study showed that apn and sa double-negative enterocytes could be infected by tgev miller and purdue (supplementary figure s ) , which suggests that besides apn and sa, tgev can use another cellular receptor for the replication in enterocytes. further investigation will focus on identifying this unknow receptor. a binding assay for pedv could not be conducted due to the low titer of the virus stock. based on our previously established primary enterocyte co-culture system, which is very relevant for the in vivo situation, it was shown that a higher expression of apn on the enterocytes resulted in a higher infectivity of tgev and pedv. however, tgev and pedv could also infect apn negative enterocytes, indicating that an additional receptor exists in enterocytes besides apn. tgev did not show binding activity on sas on the surface of enterocytes. these new insights stimulate the search for unknown receptors for pedv and tgev, which can assist further research on antiviral intervention. coronavirus spike proteins in viral entry and pathogenesis the coronavirus membrane glycoprotein mapping of the coronavirus membrane protein domains involved in interaction with the spike protein absence of e protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev identification of a putative cellular receptor kda polypeptide for porcine epidemic diarrhea virus in porcine enterocytes porcine aminopeptidase n is a functional receptor for the pedv coronavirus human aminopeptidase n is a receptor for human coronavirus e determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-n that is distinct from the enzymatic site porcine aminopeptidase n mediated polarized infection by porcine epidemic diarrhea virus in target cells aminopeptidase n is not required for porcine epidemic diarrhea virus cell entry porcine aminopeptidase n is not a cellular receptor of porcine epidemic diarrhea virus, but promotes its infectivity via aminopeptidase activity point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus receptor usage and cell entry of porcine epidemic diarrhea coronavirus sialic acids as receptor determinants for coronaviruses identification and comparison of receptor binding characteristics of the spike protein 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profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells trypsin cleavage stabilizes the rotavirus vp spike cell-culture propagation of porcine enteric calicivirus mediated by intestinal contents is dependent on the cyclic amp signaling pathway bile acids are essential for porcine enteric calicivirus replication in association with down-regulation of signal transducer and activator of transcription receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase structure of sars coronavirus spike receptor-binding domain complexed with receptor development of transgenic mouse model expressing porcine aminopeptidase n and its susceptibility to porcine epidemic diarrhea virus resistance to coronavirus infection in amino peptidase n-deficient pigs goblet cell depletion in small intestinal villous and crypt epithelium of conventional nursing and weaned pigs infected with porcine epidemic diarrhea virus a competitive inhibition elisa for the differentiation of serum antibodies from pigs infected with transmissible gastroenteritis virus (tgev) or with the tgev-related porcine respiratory coronavirus complete genomic sequences, a key residue in the spike protein and deletions in nonstructural protein b of us strains of the virulent and attenuated coronaviruses, transmissible gastroenteritis virus and porcine respiratory coronavirus sialic acid binding properties of soluble coronavirus spike (s ) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus we are grateful to e. cox for supplying the antibody against apn. we also thanks l. enjuanes for supplying the antibody against pedv. special thanks go to marthe pauwels for the excellent technical assistance. the authors declare no conflict of interest. key: cord- -jhiimglg authors: hayakawa, jun; masuko, tomomi; takehana, tae; suzuki, tohru title: genetic and antigenic characterization and retrospective surveillance of bovine influenza d viruses identified in hokkaido, japan from to date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jhiimglg influenza d virus (idv), which is a new member of the orthomyxoviridae family, is potentially involved in bovine respiratory diseases (brds). bovine idvs (bidvs) from japan have been distributed nationwide since and are genetically distinct from foreign idvs. we isolated bidvs from three brd outbreaks, in hokkaido during – , to understand their genetic and antigenic characteristics. retrospective surveillance was performed using sera collected throughout the last decade in hokkaido to investigate bidv existence. three bidvs were isolated using cell culture. comparative and phylogenetic analyses using sequence data of the three bidvs and idvs from japan and other countries available in genbank demonstrated that japanese bidvs, including the three bidv isolates, were genetically distinct from other idvs. genotype classifications based on the rotavirus genotype classification revealed multiple genotypes of rna segments – . two bidvs were of a new genotype, different from those of other japanese bidvs. neutralization assays against two bidvs with different genotypes using sera collected in acute and recovery phases of brd revealed differences in cross-reactivity to heterogenous bidvs. retrospective surveillance suggested that bidv existed in hokkaido, in . our findings suggest that bidvs of different genotypes and antigenicity are distributed and maintained in hokkaido and provide new insights into molecular characteristics and the evolution of idvs. influenza viruses are enveloped, segmented, single-stranded, negative-sense rna viruses, which belong to the family orthomyxoviridae, and are currently classified into the following four species: influenza a, b, c, and d (iav-idv). the genomes of iav and ibv consist of eight rna segments, whereas icv and idv have seven segments. both iav and ibv contain two major surface glycoproteins, i.e., hemagglutinin (ha) which binds to host cell receptors and mediates membrane fusion, and neuraminidase (na) which cleaves receptor sialic acids resulting in the release of newly assembled virus particles [ ] . in contrast, icv and idv have only one major glycoprotein, the hemagglutinin-esterase-fusion (hef) protein, which possesses "all-in-one" activities of receptor binding, receptor cleavage, and membrane fusion [ ] [ ] [ ] [ ] . the idv hef glycoprotein is structurally and functionally similar to the icv hef glycoprotein and is closely associated with the antigenicity and pathogenicity of the virus [ , ] . idv was first isolated from swine exhibiting influenza-like symptoms in oklahoma, united states of america (usa), in [ ] . subsequent observations have demonstrated that cattle, rather than pigs, were the natural reservoirs of the virus [ , ] . experimental infection indicated that the bovine idv (bidv) caused mild respiratory symptoms in cattle [ , ] . however, metagenomic analyses of feedlot cattle suffering from bovine respiratory disease (brd) suggested that this virus was a causative pathogen of brd [ , ] . moreover, a serological survey revealed that, apart from cattle and pigs, sheep, goats, horses, and camels were also susceptible to infection with idv, suggestive of its broad cell tropism [ ] [ ] [ ] . furthermore, other serological studies have reported the detection of virus-specific antibody titers in human sera [ , , ] . thus, these findings suggest that this virus has zoonotic potential. idvs have been mostly identified in cattle from north american, european, east asian, and african countries [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . to date, the phylogenetic analysis of hef genes from multiple swine and bovine idvs has revealed the existence of at least three lineages [ , ] . in particular, japanese bidvs have been identified as genetically distinct from those detected in other countries, and have been circulating nationwide, since , as described previously [ ] . in this study, we found that bidvs were associated with brd outbreaks the occurred in hokkaido, in recent years. we also demonstrated their genetic and antigenic characteristics via phylogenetic and antigenic analyses of the bidvs obtained in this study and other idvs reported in previous studies. furthermore, we performed a retrospective analysis of the existence of bidv in hokkaido using sera collected and stored in the last years and observed that bidv existed as early as . to investigate whether bidv was associated with brd, we diagnosed cattle from three brd outbreaks in herds occurring in hokkaido, japan from to , as follows: in outbreak , seven of calves (< months of age) developed fever and respiratory distress at cattle farm a on november . thereafter, brd slowly spread across most cattle in herds, between november and january . in outbreak , of calves ( - months of age) developed fever, severe cough, and nasal discharge at cattle farm b, in january . furthermore, some cattle developed pneumonia and became debilitated. in outbreak , weaned calves ( - months of age) developed nasal discharge at cattle farm c, in october . subsequently, of the cattle died due to pneumonia, by early january . viral rna was extracted from the nasal swab samples of five or six individuals from each outbreak using the high pure viral rna kit (roche, basel, switzerland), according to the manufacturer's instructions. these samples were subjected to conventional reverse transcription-pcr (rt-pcr) analysis specific for bovine viral diarrhea virus (bvdv), bovine respiratory syncytial virus (brsv), and bovine coronavirus (bcov) based on the recent prevalence of viruses associating with brd, in japan, reported in previous studies using the primesscript one step rt-pcr kit ver. (takara, shiga, japan), according to previously reported methodologies [ ] [ ] [ ] [ ] [ ] . in addition, all samples were subjected to real-time (rt)-pcr (qrt-pcr) specific for bidvs, according to a previous report [ ] . obtained samples were also tested for the presence of three bacterial species (mycoplasma bovis, pasteurella multocida, and mannheimia haemolytica) based on the recent prevalence of bacteria associating with brd in japan in previous studies, according to a routine methodology [ ] [ ] [ ] [ ] . all samples were collected as a part of routine diagnostic procedures, hence, permission with regard to animal ethics was not required. human rectal tumor cells (hrt- g, atcc crl- ) were maintained in dulbecco's modified eagle's medium (dmem; nissui, tokyo, japan) supplemented with % fetal bovine serum, u/ml penicillin, µg/ml streptomycin, and µg/ml gentamicin (nacalai tesque, kyoto, japan). to isolate viruses, nasal swab samples of five or six individuals from each outbreak were inoculated into hrt- g cells. the inoculum was removed after incubation for h, at • c, in a humidified atmosphere with % co . cells were washed two times with dmem, and then were incubated with dmem containing u/ml penicillin, µg/ml streptomycin, µg/ml gentamicin, and µg/ml pancreatin (sigma-aldrich, st louis, mo, usa), at • c, for a week. thereafter, the supernatants were harvested and passaged more times on hrt- g cells until cytopathic effects (cpes) were observed by microscopy. the isolated viruses were confirmed by the pathogen-specific rt-pcr or qrt-pcr. in addition, two remaining isolated bidvs, except one bivd isolated from outbreak (hkd ), were validated by a hemagglutination (ha) assay and transmission electron microscopy (tem) ( table s ). ha assay was performed according to the world health organization (who) manual on animal influenza diagnosis and surveillances (https://apps.who.int/iris/bitstream/handle/ / /who_cds_csr_ncs_ . .pdf) using . % turkey red blood cells (rbcs) in u-bottom -well plates. tem observation was performed according to the following procedure: the supernatants of infected cell cultures were partially concentrated and purified using an ultrafiltration device vivaspin - k (sartorius, gottingen, germany), negatively stained with % sodium phosphotungstic acid (ph = . ), and observed using an electron microscope (jem- ; jeol, ltd., tokyo, japan). titers (tcid , % tissue culture infectious dose) of isolated bidvs were determined according to the method reported by reed and muench [ ] . viral rna was extracted from the supernatant of each bidv isolate originating from the three brd outbreaks, according to the methodology described above. genomic sequences of individual rna segments were amplified via rt-pcr, using a set of primers originally designed by reference to the sequences of other idvs available in genbank (table s ). the primer sets were confirmed to not amplify pcr products from other species of ivs. rt-pcr was carried out using the primescript ii high fidelity one step rt-pcr kit (takara, shiga, japan) with the following cycling conditions: • c for min and • c for min; cycles of • c for s, • c for s, and • c for s; final extension step at • c for min. pcr products were sequenced using the bigdye terminator v . cycles sequencing kit on an automated abi prism genetic analyzer (thermo fisher scientifics, carlsbad, ca, usa). each genomic sequence from the three bidvs determined herein has been submitted to the dna data bank of japan and is retrievable via genbank (table s , genbank accession number lc -lc ). the sequence data were aligned using the clustal w method in the mega x software [ ] . genetic distances for the seven rna segments were calculated using the kimura two-parameter correction at the nucleotide level. phylogenetic analyses for all rna genomes, including the three bidvs and other previously reported idvs, were performed using the maximum-likelihood method with the general time reversible nucleotide substitution model and bootstrap replicates implemented in the mega x program [ ] . genotype classification of individual idv genes was conducted using cut-off values calculated based on the definition that was used in the full genome-based genotype classification of rotavirus, a segmented rna virus like idv [ , ] . briefly, the cut-off values for all genes were estimated as the percentage separating intra-genotype identity (nucleotide identity among strains belonging to the same genotypes), and inter-genotype identity (nucleotide identity among strains belonging to different genotypes). however, in cases when inter-and intra-genotype identity partially overlapped, the most appropriate cut-off value was chosen as the percentage at which the ratio of inter-genotype identity and intra-genotype identity dropped below . we collected acute (pre) and recovery (post) phase serum samples from and cattle from farms a and b, respectively. to compare antigenicity of bidvs (hkd , d/bovine/hokkaido/hkd / and hkd , d/bovine/hokkaido/hkd / ) isolated from the two farms, we performed cross-reactive neutralization tests using these serum samples. heat-inactivated sera ( µl) were serially two-fold diluted with dmem and mixed with an equal volume of tcid of hkd or hkd , at • c, for h. then, the mixture was added to hrt- g cells ( . × cells/ µl per well in -well plates), and cells were incubated, at • c, for days. on the basis of microscopic observation, the highest dilution of sera completely protecting the cells from cpes was recorded as the viral neutralizing (vn) antibody titer. antigenic cross-reactivity using pre-and post-brd outbreak serum samples from both farms against two different bidv isolates were compared and analyzed with the wilcoxon rank sum test. a p-value < . indicated a significant difference. we carried out neutralization assays against hkd and hkd using a total number of serum samples collected from cattle (over months of age) selected randomly at different farms in hokkaido, each year during to , in order to investigate the existence of bidv in hokkaido in the past (table s ). we diagnosed cattle from each brd outbreak occurring at three cattle farms in hokkaido between and . we tested five or six nasal samples from each outbreak through pathogen-specific rt-pcr and bidv-specific qrt-pcr, and virus and bacteria isolation (table ). in outbreak , three viruses (bcov, brsv, and bidv) were detected by rt-pcr and qrt-pcr. furthermore, three viruses (bpiv , bcov, and bidv) and two bacterial species (p. multocida and myc. bovis) were isolated in cell culture and agar, respectively. in outbreak , brsv and bidv were identified by rt-pcr and qrt-pcr, respectively. in addition, bidv and m. haemolytica were isolated using hrt- g cells and agar, respectively. in outbreak , three viruses (brsv, bcov, and bidv) and three bacteria (p. multocida, myc. bovis, and m. haemolytica) were detected in the six used nasal samples tested. moreover, bcov and bidv were also detected in hrt- g cell culture. in summary, we isolated five bidvs from all brd outbreaks, which were caused by multiple viruses and bacteria. the two bidv isolates (hkd and hkd ) were confirmed by a ha assay using turkey rbcs and tem observation (table s and figure s ). in addition, viral titers of hkd , hkd , and hkd determined according to the reed and muench's method were . , . , and . tcid / ml , respectively (table s ). amplification by rt-pcr, using a set of primers originally designed by reference to the complete genomes of other idvs available in genbank, successfully determined the nearly full-length nucleotide sequences of all rna segments, excluding several nucleotides at the ' and ' termini, of the five bidvs isolated in this study (table s ). we defined one of them as a representative strain, hkd , because the nucleotide sequences of seven rna segments of three bidvs isolated from outbreak were identical (data not shown). the lengths of the open reading frames (orfs) of all genes of the three bidvs (hkd , hkd , and hkd ) were almost identical to those of reference bidvs without insertions and deletions. comparative sequence analyses among the three bidvs identified in this study, as well as among these and other bidvs from japan detected in previous studies, demonstrated japanese bidvs had high genetic diversity, especially in hef gene (table ) . a comparison of the nucleotide sequences of the seven rna segments of japanese bidvs with those of idvs from other countries revealed that japanese bidvs are genetically distinct from idvs from other countries. phylogenetic analyses using orfs of individual genes were performed by adding data from the three bidvs to other idv data available in genbank (figure ). in addition, we also carried out the phylogenetic analyses using orf nucleotide sequences of ns and ns , because ns gene encodes two proteins (ns and ns ). however, these dendrograms revealed similarity with regard to that of ns gene (data not shown). according to the definition for genotype classification of rotavirus, cut-off values for the genotype classification of the pb , pb , p , hef, np, m, and ns genes were calculated from the frequency distribution of pairwise sequence identities and were set to . %, . %, . %, . %, . %, . %, and . %, at the nucleotide level, respectively (table and table s ). on the basis of these cut-off values, we revealed the existence of , , , , , , and genotypes for the pb , pb , p , hef, np, m, and ns genes, respectively ( figure and table ). in the analysis of all genes, bidvs from japan were clearly distinct from idvs from other countries and were classified into one or two genotypes. in addition, the hkd and hkd were grouped into a new genotype, different from the genotype of other japanese bidvs, including hkd , based on analyses of the pb , p , hef, np, and ns genes. moreover, hkd was classified into a pb genotype different from other idvs. there were significant differences (serum samples from farm a, p = . and serum samples from farm b, p = . ) in cross-reactivity to heterogenous bidv isolates in the neutralization assay using serum samples from farms a and b (table ) . briefly, the neutralization assay using serum samples collected from pre-and post-brd outbreak at farm a showed a clear increase of vn antibody titers against hkd isolated from farm a, but not against hkd isolated from farm b. in contrast, the assay using five serum samples from pre-and post-brd outbreak at farm b exhibited high increases of vn antibody titers against hkd but smaller increases of titers against hkd . retrospective surveillance was performed through a neutralization assay for two bidv isolates, hkd and hkd , using serum samples collected in hokkaido over the past decade. when considering vn antibody titers of more than as positive, the detection rates of antibodies against bidv hkd and hkd were % and %, respectively, almost identical between both assays (coincidence rate = %, and samples were positive and negative against both hkd and hkd , respectively; samples were positive against hkd , not hkd ; and samples were positive against hkd , not hkd ). our analysis revealed that antibodies against bidv in cattle sera have been continuously (in a range from % to %) detected in hokkaido from to (table ). in addition, our analysis also suggested that the type of dominant virus tended to fluctuate between and . however, we could not confirm the trend, because the vn antibody titers were affected by timing of sample collection and individual differences. table . viral neutralizing antibody titers of serum samples collected in the acute (pre) and recovery (post) phases of brd outbreaks that occurred at farms a and b against bovine influenza d viruses (hkd and hkd ) isolated from the two farms, as measured using a neutralization assay. and . interestingly, we also isolated bidvs from samples by using an hrt- g cell culture. these findings strongly support the notion that brd is caused by interactions between viruses and bacteria and that bidv is one of the causative pathogens of brd, as reported in previous studies [ , ] . sets of primers originally designed by reference to sequences at the ' and ' termini of the segments of other idvs available in genbank successfully amplified the pcr products of seven nearly full-length rna segments from the three bidvs obtained in this study. this suggests that the nucleotide sequences of the ' and ' termini are conserved between different idvs, as commonly observed for segmented rna viruses [ ] . comparative sequence analyses among the three bidvs isolated in this study and idvs from japan and other countries reported in previous studies suggested that these bidvs had large genetic variation. furthermore, hkd and hkd of the three bidvs are genetically distinct from other idvs. genotype classifications following the phylogenetic analyses for all rna genomic sequences of the three bidvs and other idvs revealed the existence of multiple genotypes for individual genes based on cut-off values calculated according to the definition of rotavirus genotype classification [ , ] . in addition, the analyses of all genes revealed that the genotypes to which six japanese bidvs belonged, including the three bidvs from the current study, were clearly different from the genotypes of other idvs, as reported in previous studies [ , ] . moreover, the data presented in this study classified hkd and hkd into a new genotype, which was distinct from genotypes that other idvs belonged to, for the five or six remaining genes, except for the pb or m genes. taken together, we successfully found a novel bidv with a unique genotype via a series of genetic analyses of bidvs identified, in hokkaido, in recent years. in the three brd outbreaks that occurred in hokkaido between and , cattle with brd from outbreaks and had more severe symptoms (a rapid spread in cattle herds, debilitation, and death due to pneumonia) than cattle from outbreak . in addition, cross-reactive neutralization assays against two bidvs (hkd and hkd ) using serum samples collected from infected cattle in farms a and b revealed significant differences in cross-reactivity to heterogenous bidvs, suggestive of the antigenic heterogeneity between the two bidvs. moreover, the phylogenetic analyses and genetic classifications of the seven rna segments revealed that hkd had a different genetic background from the two other bidvs (hkd and hkd ) . especially, the three bidvs were clearly classified into two different genotypes of the hef gene, which have been closely associated with the antigenicity and pathogenicity of the virus [ , ] . these findings suggest that several kinds of bidvs with different pathogenicity, antigenicity, and genotypes have been distributed and maintained in hokkaido, japan. retrospective surveillance using sera collected in hokkaido since revealed that antibodies against bidv in sera had been detected every year for the last decade. this observation suggests that bidv has existed in hokkaido since , which is earlier than previously reported [ ] . in conclusion, we isolated three bidvs from infected cattle with mild to severe respiratory diseases during brd outbreaks, in hokkaido, japan in recent years. two bidvs isolated from cattle with severe symptoms were classified into a new genotype, different from the genotype of previously described japanese bidvs, especially with regard to the idv hef gene. in addition, the neutralization tests using bidvs of two different genotypes suggested a significant difference in antigenicity between the two bidvs. moreover, our data demonstrated that the bidv had been distributed in hokkaido at least since . the genetic classification of idvs performed in this study will provide useful information for the monitoring and identification of a new idv, which could emerge in the future. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : image of bidv virus, which isolated from hrt- g cell culture, by transmission electron microscopy observation. the nasal swab sample collected from cattle with respiratory disorder was inoculated into hrt- g cells, and cells were kept, at • c, for a week. after a week, the supernatants were harvested from the cell culture which exhibited cytopathic effects, and then observed by transmission electron microscopy. bar indicates nm. table s : summary of ha and virus titers, and length of open reading frame determined by a sequence analysis and genbank accession number of three bovine influenza d viruses isolated from this study, table s : a set of primers for genomic sequence determination of seven rna segments from bovine influenza d virus, which were originally designed by reference to other influenza d viruses available in genbank, table s : summary of serum sample collected between and used in retrospective surveillance, table s : open reding frame (orf) nucleotide sequence identities among genotypes on individual rna segments of influenza d viruses. funding: this study was partially supported by a grant from national agriculture and food research organization (naro). the biology of influenza viruses the glycoprotein of influenza c virus is the haemagglutinin, esterase and fusion factor structure and function of the hef glycoprotein of influenza c virus hemagglutinin-esterase-fusion (hef) protein of influenza c virus novel influenza d virus: epidemiology, pathology, evolution and biological characteristics an open receptor-binding cavity of hemagglutinin-esterase-fusion glycoprotein from newly-identified influenza d virus: basis for its broad cell tropism isolation of a novel swine influenza virus from oklahoma in which is distantly related to human influenza c viruses characterization of a novel influenza virus in cattle and swine: proposal for a new genus in the orthomyxoviridae family cocirculation 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by) license we thank h. takahashi, k. uegaki, and k. kobayashi for sample collection and technical assistance. the authors have no other conflicts of interest to declare. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. (negative) in this study, we detected multiple viruses (brsv, bcov, and bpiv ) or bacteria (p. multocida, myc. bovis, and m. haemolytica) in samples from cattle from three brd outbreaks in hokkaido, between key: cord- -jtvpfsiu authors: abrams, anna; akahata, yoshimi; jacobson, steven title: the prevalence and significance of htlv-i/ii seroindeterminate western blot patterns date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jtvpfsiu human t-lymphotropic virus type i (htlv-i) infects an estimated – million persons worldwide. a number of diseases have been associated with the virus including adult t-cell leukemia (atl), htlv-associated myelopathy/tropical spastic paraparesis (ham/tsp), htlv-i uveitis, and htlv-i-associated infective dermatitis. once it was shown that there is an increased risk for developing ham/tsp associated with blood transfusion, screening for htlv- among blood banks was implemented in japan, united states, france, and the netherlands. this process includes detection by an enzyme immunoassay (eia) followed by a confirmatory western blot (wb) in which recombinant proteins specific for htlv-i env glycoproteins are incorporated into wb strips. htlv-i seropositive results are defined by the presence of antibodies against either gp or gp / (both env protein bands) and either p , p , or p (one of the gag bands). htlv-ii seropositivity is confirmed by the presence of rgp -ii. however, numerous cases have been documented in which serum samples are reactive by eia, but an incomplete banding pattern is displayed by subsequent confirmatory wb. although the significance of these htlv-i/ii seroindeterminates is unclear, it may suggest a much higher incidence of exposure to htlv-i/ii than previously estimated. the wb used to confirm seropositivity in an eia positive patient sample utilizes recombinant proteins specific for htlv-i/ii env glycoproteins incorporated into the wb strips. these recombinant proteins increase the sensitivity of the blot, as well as differentiate between htlv-i and htlv-ii. the specific sensitivity of an eia assay is approximately % and the eia specificity averages % while the sensitivity of the wb is . % with a specificity of . % [ , ] . a sample is classified as htlv-i positive if it meets the established criteria (see figure ). according to the world health organization, an htlv-i infected individual must have an antibody response to either gp or gp / (both env protein bands) and either p , p , or p (one of the gag bands). even more rigid criteria has been recommended by the htlv european research network, suggesting bands for both gag proteins p and p as well as the env proteins rgp and rgp -i must be present to ensure seropositivity [ ] . generally, htlv-i infected individuals such as asymptomatic carriers, ham/tsp patients, and atl patients exhibit the clear seropositivity illustrated in figure on the htlv blot . (mp diagnostics, solon, oh, usa). however, there have been reports around the world of htlv-i/ii seroindeterminate wb banding patterns that do not meet the strict criteria of htlv-i seropositivity [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these are referred to as htlv-i/ii seroindeterminates (see figure , blots ind- , ind- , and ind- ). htlv-i/ii seroindeterminate samples have been identified in otherwise healthy and normal individuals from jamaica, japan, brazil, and other htlv-i endemic areas and showed no other sign of disease or viral infection. however, of particular interest are the reports of htlv-i/ii seroindeterminate blotting patterns in patients with various neurological diseases including multiple sclerosis [ , ] . while the significance of htlv-i/ii seroindeterminates is still under investigation, it is clear from this finding that these samples may relate to disease (specifically neurodegenerative diseases) in at least a portion of cases. the reported prevalence of seroindeterminates has varied greatly from . % in taiwan, to . % in argentina, to as high as % of a high-risk population in brazil [ , , ] . interestingly, when dna isolated from peripheral blood mononuclear cells (pbmc) of these htlv-i/ii seroindeterminate individuals is amplified using polymerase chain reaction (pcr) assays, typically no htlv-i or htlv-ii virus is detected (however, recent reports from iran, argentina, and brazil have challenged this finding) [ ] . a seroindeterminate sample is defined by a positive eia result but an incomplete banding pattern by wb, as depicted in figure [ ] . incomplete banding patterns have manifested in various ways. a number of samples yielded all core protein bands, but lacked one or both of the recombinant bands (gd and rgp -i) necessary for seropositivity (for example, ind- in figure ). other samples seemed to exhibit a negative blotting strip except for a single protein band (as shown on ind- in figure ). classifying a blot as seroindeterminate can vary greatly between studies and cohorts. as research progresses, reports of large numbers of htlv-i/ii seroindeterminates have come from iran, brazil, japan, argentina, taiwan, the caribbean, central africa, and the united states, indicating a higher prevalence than originally thought [ ] [ ] [ ] [ ] [ ] [ ] [ ] . importantly, the reasons for these blotting patterns remain unclear. several possible explanations have been proposed for the occurrence of htlv-i/ii seroindeterminates that include cross-reactivities to other known retroviruses or a novel virus, antibody responses to a malaria parasite with epitope homology to htlv-i, a defective htlv-i or htlv-ii, and low copy numbers of prototypic htlv-i in the affected patient yielding the indeterminate antibody response. it has been reported that anti-htlv-i reactivity of sera can result from antibodies produced in response to plasmodium falciparum (p. falciparum). p. falciparum is a lethal malaria parasite and is the most prevalent of malaria parasites infecting humans [ ] . studies regarding this cross-reactivity concluded that p. falciparum and htlv-i must contain regions of immunogenic epitope homology. it was hypothesized that this homology may be a result of mimicry of host tissue by the two organisms [ ] . this suggests that in geographic regions known to be endemic for malaria, such as the philippines, and in which high levels of htlv-i antibody reactivity were reported, htlv-i/ii seroindeterminates are difficult to interpret, as it is difficult to rule out the possibility of cross-reactivity between htlv-i/ii and p. falciparum [ ] . it was later reported that this cross-reactivity might not be limited to p. falciparum. examination of data from a separate cohort showed cross-reactivity with htlv-i could be blocked by erythrocyte lysates (from areas where malaria is endemic) infected with p. falciparum, however htlv-i seroindeterminate samples from the united states failed to block antibody reactivities. this led to the assertion that the molecular mimicry may extend to epitopes other than the p. falciparum and plasmodial antigens [ ] . however, htlv-i/ii seroindeterminate banding patterns are being reported in areas where exposure to p. falciparum is extremely unlikely, such as the united states. furthermore, htlv-i/ii seroindeterminate patterns are observed in normal, healthy blood donors, showing no sign of malaria or similar parasite infection [ ] . while a subset of htlv-i/ii seroindeterminate samples may exhibit an antibody cross-reaction between htlv-i and p. falciparum, this cannot explain the wb banding patterns found in areas where these organisms are extremely rare or nonexistent. it should also be noted that cross-reactivity between antibodies against severe acute respiratory syndrome (sars) coronavirus and htlv has been observed on occasion [ ] . the htlv antibody response was exhibited when numerous serology tests were performed on samples from patients admitted to a hospital in taiwan for sars. however, this was immediately following the sars outbreak in , and has not been observed in other regions. various other hypotheses have been presented regarding the cross-reactivity of anti-htlv-i antibodies. in , it was reported that htlv i/ii indeterminate serologies were observed as responses to two types of simian t-lymphotropic virus (stlv) [ ] . an htlv-i/ii seroindeterminate sample that was also htlv-i pcr positive was sequenced and showed a divergent form of htlv-i [ ] . further investigations led to the suggestion that indeterminate blots may denote an antibody response to immunogenic regions of an endogenous retrovirus [ ] . also reported was the demonstration of elevated antibody levels to synthetic htlv-i epitopes in samples previously testing negative for htlv-i antibodies. the reactivity in this study reinforced a previous suggestion that increased levels of htlv-i antibodies may be an autoantibody response to the endogenous retrovirus hres-i [ , ] . the specific homologous regions between htlv-i and these endogenous retroviruses occur in the ltr, gag, and pol regions of the virus. however, a later report demonstrated the tax region of prototype htlv-i virus was amplified by nested pcr from one patient with an htlv-i/ii seroindeterminate wb that could not have been derived from the dna sequence of an endogenous virus [ ] . htlv-i/ii seroindeterminate banding patterns have also been reported in samples which were pcr positive for htlv-i, supporting the exciting possibility that an htlv-i/ii seroindeterminate pattern may result from cross-reactivity with a novel virus such as htlv-iii or htlv-iv [ ] . these newly discovered human retroviruses were found in cameroonese hunters showing no signs of htlv-related diseases, and all four htlv types show - % sequence homology with each other [ ] . due to the typically negative pcr results and lack of antibody response to some of the htlv-i antigens but reactivity to others, the most plausible suggestions seems to be that htlv-i/ii seroindeterminate blots may result from a low copy number of prototypic htlv-i [ ] . this explanation is supported by studies showing the ability to amplify the htlv-i tax region from pbmcs of some htlv-i/ii seroindeterminates, but not other regions of the virus [ , , ] . the same study reported the successful generation of an epstein-barr virus transformed b-cell line from a relapsing remitting multiple sclerosis patient with a seroindeterminate wb. the pbmcs from this patient had tested negative for regions of htlv-i by pcr, while an in vitro long-term generated b-cell line tested positive by primary pcr. the virus infecting the seroindeterminate b cell line was then sequenced in an attempt to identify any mutations or other factors that may be associated with an htlv-i/ii wb seroindeterminate status. the results indicated that the virus was globally > % homologous to prototypic htlv-i on the nucleotide level. fine analysis of the ′ ltr indicated that the htlv-i strain infecting the patient was of the cosmopolitan subtype [ ] . this was the first reported verification of a pcr negative seroindeterminate sample resulting from infection of a full length htlv-i virus [ ] . further support for the suggestion that these seroindeterminates may be the result of a low copy number of prototype htlv-i comes from a study of patients transfused with htlv- infected blood [ ] . eight seronegative individuals developed seroindeterimnate wb patterns after receiving a blood transfusion with the infected blood, further implicating the role of exposure to htlv-i in seroindeteriminates [ ] . the hypothesis that htlv-i/ii wb seroindeterminates (or subsets of these individuals) may be related to prototype htlv-i is further supported by the recent observations that demonstrated htlv-i pcr positive results in . % of htlv-i/ii seroindeterminates from iran [ ] (table ) . consistent with this observation are reports from brazil and argentina, which also demonstrated comparable rates of pcr reactivity in htlv-i/ii seroindeterminates ( . % and . %, respectively) [ ] (table ) . the most prevalent seroindeterminate banding pattern observed in iran was the appearance of gd alone, which is similar to the patterns seen in taiwan [ , ] . the data in table also demonstrates a relatively high frequency of htlv-i/ii wb seroindeterminates from htlv-i eia reactive samples that are observed in multiple cohorts throughout the world. these range from % in the united states to % in argentina. reports of pcr amplification of prototype htlv-i sequences from htlv-i/ii seroindeterminate individuals, especially in endemic regions such as iran and brazil, suggest a potentially far greater exposure to htlv-i at least in some sub-populations of htlv-i/ii wb seroindeterminates. while the significance of these prevalent seroindeterminate htlv-i/ii blotting patterns remains uncertain, it is clear that further investigation is necessary. the sequencing of a virus with homology to prototype htlv-i from a seroindeterminate cell line implicates exposure to the virus in at least some htlv-i/ii seroindeterminates. this finding may suggest that exposure to htlv-i is greater than previously estimated. of concern are reports that have demonstrated serum samples with negative htlv-i/ii results by eia, which may also have seroindeterminate banding patterns by wb [ ] . since these sera were initially screened as htlv-i/ii eia negative (see figure ), these samples (that also have an htlv-i/ii seroindeterminate wb pattern) would enter the blood supply and be available for transfusion [ ] . there have also been instances of serum samples testing eia positive and wb indeterminate at one point in time, and later testing eia negative, while remaining seroindeterminate by wb [ ] . this implies testing at one time point by eia cannot ensure the exclusion of seroindeterminate samples. if seroindeterminate blotting patterns are a result of infection or exposure to htlv-i (a virus associated with neurodegenerative disease, among other illnesses), and blood samples with these patterns can be negative by eia screening and enter the blood supply, this poses a possible risk to those transfused with htlv-i/ii seroindeterminate blood although the clinical consequence of obtaining blood from an htlv-i/ii wb seroindeterminate donor is not known. based on these observations there is a need for more stringent screening techniques for blood banks to ensure blood from seroindeterminate donors is not transfused. increasingly sensitive and specific assays are currently being optimized to better detect anti-htlv-i antibody responses. one such technique is lips assay. the lips assay yields high throughput results while also generating quantitative values of anti-htlv-i antibody responses [ ] . this creates data for quantitative cohort comparison as well as allows for differentiation between htlv-i infected disease patients and asymptomatic htlv-i carriers and the possible generation of a risk assessment for disease development. using antibody responses to the immunogenic epitopes of htlv-i, gag, env and tax, lips produces clear quantitative values for asymptomatic carriers, ham/tsp patients, atl patients, and may yield similar results for seroindeterminate individuals. the plasmids used in the lips assay are inserted with a gene including immunogenic epitope of a virus (for example, the gag, env, or tax region of htlv-i). renilla luciferase-fusion proteins (ruc-antigen) are obtained from cell lysates transfected with each plasmid. a preincubated serum/antigen mixture is added to a filter plate coated with immobilized protein a/g beads. this mixture is then washed to remove any fusion proteins that did not bind to the antibody. luciferase activity is then measured in a microplate luminometer. the lips assay provides a promising high throughput method of anti-htlv-i antibody detection. further examination of immunogenic epitopes, such as htlv-i rex and hbz, may enhance the specificity and sensitivity of the assay. currently, a whole gag protein is used for detection of antibody response; however, antibody responses for p , p , or other portion of the gag protein may yield even more specific results. a panel of these quantitative antibody responses may be able to detect seroindeterminate samples in a fast, inexpensive, and efficient manner. lips may also detect seroindeterminates that tested negative by eia, proving to be a more sensitive assay in terms of seroindeterminate detection. while the lips assay is under further development, other methods to improve detection and classification of seroindeterminates have also been proposed including: real-time pcr, molecular diagnostic analysis, detection of amino acid changes in the env region of htlv-i, inclusion of other synthetic peptides in serological assays, and optimization of an enzyme oligonucleotide assay (eoa) [ , [ ] [ ] [ ] . with further in depth research involving these newer methodologies, the significance of these htlv-i/ii seroindeterminate banding patterns will become clearer. as it becomes apparent that the prevalence of seroindeterminates is more widespread than originally thought, it becomes increasingly important to clarify the meaning of these results. if htlv-i/ii seroindeterminates (or subsets of these subjects) represent exposure to prototype htlv-i or htlv-ii, the frequency of infection in the general population will be greater than previously believed. it remains to be seen whether the incidence of exposure is also associated with any clinical disease outcome. the discovery of the first human retrovirus: htlv- and htlv- global epidemiology of htlv-i infection and associated diseases discovery of adult t-cell leukemia human t-cell leukemia virus specific antigens efficient transformation of previously activated and dividing t lymphocytes by human t cell leukemia-lymphoma virus clinical and 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characterization and sequencing of prototypic human t-lymphotropic virus type (htlv- ) from an htlv- / seroindeterminate patient caterino-de-araujo, a. serological patterns and temporal trends of htlv- / infection in high-risk populations attending public health units in sao paulo human t lymphotropic virus types i and ii proviral sequences in argentinian blood donors with indeterminate western blot patterns antibodies to human t lymphotropic virus type i in a population from the philippines: evidence for cross-reactivity with plasmodium falciparum immunologic crossreactivity between structural proteins of human t-cell lymphotropic virus type i and the blood stage of plasmodium falciparum false positive antibody results against human t-cell lymphotropic virus in patients with severe acute respiratory syndrome use of a generic polymerase chain reaction assay detecting human t-lymphotropic virus (htlv) types i, ii and divergent simian strains in the evaluation of individuals with indeterminate htlv serology demographic, ethnic, and geographic differences between human t cell lymphotropic virus (htlv) type i-seropositive carriers and persons with htlv-i gag-indeterminate western blots in central africa human t-cell lymphotropic virus (htlv)-related endogenous sequence, hres- , encodes a -kda protein: a possible autoantigen for htlv-i gag-reactive autoantibodies discovery and significance of new human tlymphotropic viruses: htlv- and htlv- the human htlv- and htlv- retroviruses: new members of the htlv family human t lymphotropic virus types i and ii western blot seroindeterminate status and its association with exposure to prototype htlv-i prevalence of human t-lymphotropic virus type among blood donors from mashhad, iran seroprevalence and demographic characteristics of htlv-i among blood donors in taiwan: - failure to detect evidence of human t-lymphotropic virus (htlv) type i and type ii in blood donors with isolated gag antibodies to htlv-i/ii long-term serological follow-up of blood donors with an htlvindeterminate western blot: antibody profile of seroconverters and individuals with false reactions sensitivity of two enzyme-linked immunosorbent assay tests in relation to western blot in detecting human t-cell lymphotropic virus types i and ii infection among hiv- infected patients from sao paulo, brazil sensitivity and specificity of a dna polymerase chain reaction nonisotopic-based detection method for the confirmation of infection with human t-lymphotropic virus types i and ii we would like to thank matthew mccormick and jussi virtanen for their assistance in preparation of this review. key: cord- -eva soja authors: hutson, christina l.; damon, inger k. title: monkeypox virus infections in small animal models for evaluation of anti-poxvirus agents date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: eva soja an ideal animal model for the study of a human disease is one which utilizes a route of infection that mimics the natural transmission of the pathogen; the ability to obtain disease with an infectious dose equivalent to that causing disease in humans; as well having a disease course, morbidity and mortality similar to that seen with human disease. additionally, the animal model should have a mode(s) of transmission that mimics human cases. the development of small animal models for the study of monkeypox virus (mpxv) has been quite extensive for the relatively short period of time this pathogen has been known, although only a few of these models have been used to study anti-poxvirus agents. we will review those mpxv small animal models that have been developed thus far for the study of therapeutic agents. an ideal animal model for the study of a human disease is one which utilizes a route of infection that mimics the natural transmission of the pathogen; the ability to obtain disease with an infectious dose equivalent to that causing disease in humans; as well having a disease course, morbidity and mortality similar to that seen with human disease. additionally, the animal model should have a mode(s) of transmission that mimics human cases. factors which subsequently allow more detailed open access inferences about disease pathogenesis include the availability of reagents to evaluate host innate and adaptive immune responses to the pathogen, and histopathological changes in the host which result from infection or the host response to infection. these findings can then be compared to what is known of human disease. the utility of a small animal model of human disease for study of therapeutic efficacy is augmented when large numbers of animals are available for use in appropriately, well-powered studies. even if all aspects of an animal model of disease are not completely faithful to what is known of human disease, important information regarding therapeutic efficacy can be gleaned from their use in -pre-clinical‖ studies. the published literature on clinical manifestations of systemic human orthopoxvirus disease is derived from historic literature descriptions of human smallpox and more recent descriptions of human monkeypox disease. the clinical-descriptive literature on human monkeypox is expected to grow in the next five years, as data acquisition and analysis from an ongoing study in the democratic republic of congo is finalized. currently available literature is largely derived from who-sponsored surveillance efforts in west africa and the congo basin in the s, after the first recognition of human disease in these areas, and subsequent analyses of public health response data and human research studies following the introduction of west african clade virus into the u.s. in . human monkeypox, as described through the active surveillance and case ascertainment studies sponsored by who in the s, was depicted as resembling discrete ordinary smallpox. in natural human infection, exposure leading to infection is believed to occur via a respiratory route, with subsequent progressive viremias/lymphemia, ultimately leading to seeding of the skin to generate a generalized rash. percutaneous exposure, also leading to generalized rash formation, has also been described for both viral infections. the disease pathogenesis has been conjectured and modeled largely from animal studies; initial models were using ectromelia infection of mice; some kinetic observations of virus shedding and viremia have been made in human studies of smallpox and monkeypox. the time course of disease is generally thought to include an asymptomatic phase of - days, during which time the virus initially enters the host, replicates, seeds reticuloendothelial organs, replicates, then spreads via the bloodstream (inducing a febrile response) which is the first symptomatic hallmark of disease. the fever is usually described as occurring - days post initial exposure/infection. the range has been - days. fever is accompanied by other symptoms, including headache, backache, myalgias, and or abdominal pain. two to three days following the fever, rash develops-initially presenting as a macular, then papular, then vesicular and pustular eruption. scabbing then begins. each stage of rash lasts - days. approximately - weeks post initial symptoms, scabs begin to separate from the skin. death and disease severity have had some correlation with rash burden in epidemiologic studies of hospitalized smallpox patients. severe outcomes are more frequent in unvaccinated, younger age groups; death occurs within the first week of illness in cases with hemorrhagic manifestations, and during the second or third week of illness in -ordinary‖ cases. in the human monkeypox cases studied in zaire/drc between - , of the deaths among patients, all occurred in unvaccinated children less than eight years of age. death occurred during the first week of illness in %, the second week in %, and the third week of illness in the remaining % [ ] . the development of small animal models for the study of monkeypox virus (mpxv) has been quite extensive for the relatively short period of time this pathogen has been known. initial animal models were designed to address natural history in potential or surrogate reservoir host species, as well as studies of disease in primates. routes of exposure were designed to evaluate disease if respiratory or percutaneous exposures occurred, or in some cases to simply address whether virus would replicate in the animal model system. factors that influence the outcome of a challenge study include the age of the animal at time of infection, inoculation route used, and the viral dosage given. additionally, the strain of mpxv (currently delineated as belonging to congo basin clade or west african clade) used in the study may influence the disease severity. (table ) guinea pigs and golden hamsters were found to be relatively resistant to mpxv (west african clade copenhagen strain) infection by multiple routes. guinea pigs were challenged via an intracardial, intranasal (in), oral or foot pad (fp) inoculation with no observable symptoms of disease except for edema at the fp inoculation site. golden hamsters were also resistant to mpxv infection via several routes of infection with no observable signs of disease, even with large dosages of virus ( . - . × ) [ ] . rabbits have also been considered as a possible animal model for the study of mpxv [ ] [ ] ; susceptibility depended greatly on the method of inoculation and the age of the animals. in adult rabbits challenged with mpxv (west african clade copenhagen strain) via an oral inoculation, no signs of disease were seen. however, if virus was delivered by intravenous route, acute illness was observed with generalized rash. young rabbits ( days old) inoculated via in or oral route developed severe illness; two day old rabbits were highly susceptible to infection by intracutaneous inoculation or skin scarification [ ] . the intracutaneous route led to the development of discrete white translucent lesions. another study found that intracerebral inoculation was % fatal and that intratesticular or intracorneal inoculation with mpxv was also pathogenic in rabbits [ ] . several rat species including white rats, cotton rats and multimammate rats have been challenged with mpxv. adult white rats inoculated with to plaque forming units (pfu) of west african mpxv were not susceptible to infection with intravenous, in, or cutaneous routes of infection. however, newborn white rats ( - days old) developed adynamia leading to death in - days when challenged with mpxv intranasally [ ] . cotton rats and multimammate rats were both found to be highly susceptible to mpxv infection. when cotton rats were challenged with pfu via an intravenous route of infection, % mortality was seen - days post infection (p.i.). the infection was characterized by difficulty breathing, cough, sneezing, cyanosis, rhinitis, purulent conjunctivitis, and progressive emaciation. an in mpxv challenge in cotton rats caused % mortality with a clinical picture such as that seen with the intravenous route [ , ] . multimammate rats were highly sensitive to both in and intraperitoneal (ip) inoculation [ , ] . marennikova et al. challenged adult common squirrels (sciurus vulgaris) with pfu of mpxv z- (congo basin clade) via in, oral or scarification routes of infection [ ] . disease progression occurred earlier in animals infected in or orally than those animals infected via a scarification route. skin lesions did not develop on any animals; symptoms of disease included fever, inactivity, inappetence, rhinitis, cough and difficulty breathing. infection was % lethal by day or p.i. regardless of inoculation route. shelukhina et al. challenged six african squirrel species (including members of the genera funisciurus, protexerus and heliosciurus) with congo basin mpxv via an in infection ( or pfu/ . ml) [ ] . all squirrel species were highly susceptible to congo basin mpxv challenge and developed an acute, generalized infection that was % lethal. however, some varying degree of susceptibility in the different squirrel species was seen with lesser dosages of virus. cutaneous inoculation of squirrels resulted in a thick, red papule at the inoculation site. skin lesions (restricted to non-fur-bearing areas of the skin or at the borders of the skin and mucous membranes of the nose and lip) occurred in only a few squirrels that had been infected by the oral or in route with small (nonlethal) doses of virus. most often the rash appeared in the later stages of disease ( - days p.i.). transmission studies were also conducted with squirrels and authors found that infected animals were able to transmit the disease to naïve animals via airborne and direct contact [ ] . ground squirrels (spermophilus tridecemlineatus) are very susceptible to mpxv infection. tesh et al. challenged adult ground squirrels with the west african mpxv clade either ip or in with . pfu [ ] . in both groups, symptoms of disease included anorexia and lethargy within - days of infection, with no other observable symptoms. weight loss was not measured for these animals. animals in the ip group died within - days p.i.; those in challenged all died within - days. a follow-up study compared the pathogenesis of the two mpvx clades in the ground squirrel model [ ] . inoculation of pfu by a subcutaneous route of infection was % lethal for both mpxv clades. however, the authors noted that the onset of severe respiratory distress was more rapid and uniform for the congo basin mpxv challenged animals. additionally, animals challenged with the congo basin mpxv began to die earlier than west african challenged animals. however, ld values were similar for the two strains using the ground squirrel mpxv model [ ] . animals were asymptomatic until days - when a generalized rash was observed on challenged animals. signs of disease included lethargy, inappetence, nasal discharge, respiratory distress, and diarrhea; morbidity was noticeably more for the congo basin mpxv challenged animals as was mortality. a follow-up study found the ld for the prairie dog mpxv model is approximately a hundred-times lower for the congo basin clade compared to the west african clade ( . × and . × , respectively) [ ] , utilizing an in route of infection. weight loss occurred in / west african mpxv challenged dosage groups and / congo basin mpxv challenged dosage groups; for both viral strains the highest percent weight loss calculated occurred in the highest viral inoculum group. a trend of increasing viral titers in oropharyngeal swabs with increasing viral inoculums dose was apparent for both mpxv strains, and when all mean values were combined, congo basin challenged animals had statistically higher levels of virus. furthermore, the duration of mpxv dna and viral shedding tended to occur earlier, attain higher levels, and persist longer for congo basin challenged animals. symptoms were also more numerous and severe for congo basin mpxv infected prairie dogs. schultz et al. challenged african dormice, graphiuris kelleni, with . × pfu of a congo basin clade of mpxv via fp route and observed % mortality [ ] . the authors further developed the model by infecting dormice with various dosages of congo basin mpxv by the in route and calculated the ld as pfu. animals became symptomatic at day (conjunctivitis and dehydration), and animals that succumbed to disease had a mean time to death of . ± to . ± days (depending on dose). morbidity for those animals that succumbed to disease included decreased activity, hunched posture, unkempt hair coat, dehydration, and conjunctivitis; lesions did not develop on any animals. weight loss was highest with , pfu (the highest dosage given), but also was seen in animals given or pfu. weight loss was not observed in the lowest dosage groups ( and . pfu). disease pathogenesis was described as localized inflammation, viral replication and hemorrhage in the nasal mucosa, followed by dissemination around day with subsequent necrosis of liver, spleen, lung and gastrointestinal tract tissues. when the west african strain of mpxv was used to challenge dormice by an in infection, similar days until death and mortality rates were seen as the congo basin mpxv challenged animals. as is the case for many pathogens, mice have been utilized numerous times for the study of mpxv. results have varied greatly depending on the type of mice used (i.e., wild strains or laboratory strains). in early studies [ , ] white mice were challenged via intracerebral, in, ip, fp, oral or intradermal (id) inoculation with a west african strain of mpxv and found to be highly sensitive to most inoculation routes. intracerebral inoculation was % fatal in adult white mice as was in inoculation of suckling mice [ ] . inoculation in eight day old mice via the fp, ip, or in route resulted in % mortality; id or oral inoculation caused % and % mortality, respectively. oral inoculation of day old white mice only caused % mortality; however in inoculation in day old animals led to % mortality [ ] . inbred laboratory mouse strains have also been studied by several groups. hutson et al. type i and type ii ifn signaling [ ] . mice were challenged with dosages between . to , pfu via an in route of infection. weight loss was seen with all dosages given except for the lowest ( . pfu). mortality occurred at - % at the pfu dose ( - days p.i.); % mortality occurred by day p.i. with the highest dose given ( , pfu). the calculated ld for the congo basin clade in the c bl/ stat-/-mice was and pfu for females and males, respectively. americo et al. screened inbred mouse strains and identified three that are highly susceptible to mpxv [ ] . of these three strains, the cast/eij was developed as a model. signs of morbidity in moribund animals included ruffled fur, hunched posture, and lethargy; no animals developed lesions. animals challenged with the highest dosages by an in route ( or pfu congo basin mpxv) lost up to % of the starting body weight and % died between days - . animals challenged with pfu all died between days - . animals challenged with pfu had an even longer delay in weight loss and death and % of the animals recovered. no animals perished in the pfu challenge group. the calculated ld for the congo basin clade in the cast/eij mice given an in challenge was pfu. animals were found to be even more sensitive with an ip congo basin mpxv infection and the calculated ld was pfu. challenging mice with or pfu of a west african mpxv strain resulted in rapid weight loss and % mortality by day p.i. lower dosages of west african mpxv resulted in less weight loss and lower amounts of death than what was observed for the congo basin mpxv; the calculated ld was , pfu, more than a log higher than for the congo basin clade. to date, four mpxv small animal models have been used for the testing of antiviral drugs cidofovir, cmx and st (tecovirimat). herein we will summarize those studies, efficacy data, and discuss the advantages, and limitations, of the animal models used. sbrana et al. utilized ground squirrels to test the efficacy of st- against a mpxv challenge [ ] . the authors used pfu of mpx-zai- ( × ld ) via a subcutaneous route of inoculation. squirrels ( - per group) were divided into five treatment groups; drug was given either at hours of infection, hours, hours, hours or hours p.i. mg/kg of drug was given once a day for days. two animals in each group were sacrificed at day to measure objective morbidity; the remainder of the animals were used to calculate survival rates. animals in the placebo group, that were not given st- , showed signs of illness beginning on day and all died between days - . signs of disease included lethargy, anorexia, nosebleeds, and terminal respiratory distress. at day , a sampling of placebo-treated animals exhibited significant leukocytosis, transaminitis, and coagulopathy; almost pfu/ml of infectious monkeypox was found in blood; at this time, between and pfu /ml of infectious mpxv was observed in % organ homogenates of liver, spleen and lung. animals treated on days , , or hours, before symptomatic disease onset, all survived infection and showed no signs of disease. at day , in a sampling of animals treated at hour , , or p.i., no virus was found in the liver, spleen, lung, or blood; although some abnormal values were apparently recorded, no clear trends in leukocytosis, transaminitis or coagulopathy were noted with delay in treatment onset. in animals initiating treatment at hours p.i., concurrent with symptomatic disease onset, % of animals survived infection. / survivors showed signs of disease. in those animals that succumbed to infection, st- prolonged the time to death; the mean time to death was day for animals receiving placebo and day for those receiving st- in the hour p.i. treatment group. the sampling of animals at day , initiating st- at hour p.i., demonstrated lower levels of viremia (~ log decrease) and ~ logs less virus in liver, spleen and lungs than that seen on the placebo treated animals at day . although some evidence of transaminitis was present, leukocytosis and coagulopathy were not observed in this treatment group. pathologic examination of tissues in general showed greater tissue necrosis in animals treated at later times p.i. this study was able to demonstrate a survival benefit in animals treated prior to, or at the onset of disease symptoms, in a disease model that has a time course attenuated with respect to what is seen in human disease. [ ] . four hours post intranasal infection with , × , or × pfu of mpxv, animals were intraperitoneally administered mg/kg cidofovir (the calculated ld for the dormouse mpxv model was pfu). aggregate data from all challenges showed animals treated with cidofovir had a mortality rate of % ( / ), whereas vehicle treated animals all ( / ) succumbed to disease. treatment initiation at later times p.i. was not evaluated; effects on viral load or histopathologic changes were not reported. as inbred mice have historically shown little disease symptomatology or pathogenesis post monkeypox infection, stabenow et al. utilized a laboratory mouse strain lacking stat (c bl/ stat-/-), which has been found to be sensitive to a range of viruses including sars, murine norovirus , respiratory viruses, dengue virus and mpxv [ , [ ] [ ] [ ] [ ] . these animals are deficient in their ability to transcribe many of the type i and type ii receptor interferon response genes. the authors used the congo basin clade virus mpx-zai- , evaluated disease and the protective efficacy of cmx and st . in untreated mice, % mortality was observed with . pfu challenge, % mortality with pfu of virus and % mortality with , pfu. over % total body weight loss, and mortality was observed on or prior to day p.i. in untreated animals. animals in the treatment studies were subsequently challenged with , pfu via an in infection. animals were then treated with mg/kg of cmx by gastric gavage on the day of challenge followed by every other day with . mg/kg until day p.i. all c bl/ stat-/-mice that were treated with drug survived infection, demonstrated < % body weight loss between days and , and developed a serologic response to monkeypox. similarly, mice treated daily, starting at the day of virus challenge, with mg/kg of st for days also survived infection and manifest < % body weight loss between days and . in this system, antiviral treated animals rechallenged with monkeypox at day post initial infection (at least days post reinitiation of steady weight gain), manifest % mortality. the model-again one with a short disease course-is useful for demonstrating immediate post exposure efficacy of antiviral treatment in the absence of a functioning interferon response system. additionally, in this animal model system, perhaps due to the immune defect, a monkeypox protective immune response was not elicited in all animals receiving antiviral treatment. this observation merits further observation in other animal model systems. smith et al. tested the efficacy of st in a prairie dog mpxv model [ ] . mpxv challenged prairie dogs have previously been shown to have an asymptomatic period followed by symptoms of disease including lethargy, nasal discharge, inappetence, weight loss and systemic lesion development most commonly between days - . in the current study, animals were inoculated via an in challenge with the congo basin clade virus roc- - . this is a different strain of mpxv than that used in the previous described studies, but is also a strain belonging to the congo basin clade. the challenge dose was . × , equal to × ld for the prairie dog model. animals were divided into three treatment groups; prophylactic (day ), post exposure (day ) and therapeutic (varying day based on rash onset), and a control vehicle treated group. st was formulated at mg/ml and administered daily, by oral gavage, for days. animals initiating treatment at day or were protected from death and apparent signs of illness. animals treated at rash onset had symptoms similar to the placebo control group; however symptoms were less severe in the treated animals. although all animals treated at rash onset survived infection, animals lost - % of body weight and did develop generalized rash (however, lesions resolved more quickly when compared to untreated prairie dogs in previous studies [ , ] . although asymptomatic, viable virus was shed sporadically from animals in the prophylaxis and post exposure groups (from two oropharyngeal samples in the day prophylaxis group, and five samples from the day post exposure group). more, sustained virus was detected in the oropharyngeal samplings of the animals in the therapeutic treatment group, but levels were less than the virus levels in the untreated group. / sham-treated animals survived infection. signs of disease and viral titers were all increased in this group of animals compared to the animals treated with st- . this is the first small animal study where a treatment and survival benefit has been demonstrated when animals are treated at later stages of illness. initiation of treatment at rash onset is similar to expectations of a human treatment regimen. the observation of virus shedding after treatment cessation in the prophylactically or post exposure treated animals merits further study to assess whether this reflects viral resistance or a blunted and delayed immune recognition and ultimate clearance of virus. animal models permit an advance beyond what can be gleaned from tissue culture evaluation of an antiviral effect. the evaluation of an antiviral, in the context of a host with a functioning immune system, enables better understanding of therapeutics' potential efficacy. the evaluation of an antiviral in the context of an impaired immune system enables better understanding of therapeutic use in a particular immunosuppressed population. pathogen host range, especially if not a simple issue of receptor utilization, can confound the ability to interpret, and extrapolate to the human, some of the nuances of the host pathogen interaction and prediction of potential human therapeutic benefit. of the small animal models used to evaluate antiviral efficacy, all have used stringent virus challenges (all greater than × ld ) and shown survival benefit. routes of infection have used methods that attempt to simulate potential human routes of infection and resultant human illness courses. given the uncertainties of what a human infectious or lethal monkeypox dose is, it is difficult to extrapolate the potential -best fit‖ of any of these models for human disease. the clinical time course of disease in the prairie dog model, however, has a temporal relationship that is close to what has been described with human systemic orthopoxvirus (variola or monkeypox) disease. however, a limitation of the prairie dog and some of the other described animal systems, with the exception of the mouse model, is a paucity of immune reagents. there are a handful of antiviral compounds which show promise in these small animal models using monkeypox virus as the challenge. additional studies evaluating treatment benefit when used in later stages of disease, their effect on elicitation of a protective immune response, evaluation of antiviral resistance, and their effect on viral shedding will improve our understanding of how they may be used in treatment of human disease, or in response to epidemic disease. the findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention. human monkeypox susceptibility of some rodent species to monkeypox virus, and course of the infection generalized monkeypox in orally infected rabbits and white mice a pox-like disease in cynomolgus monkeys histopathological and virological studies on monkeypox possible mechanism of orthopoxvirus preservation in nature laboratory diagnostics of human orthopoxvirus infections high sensitivity of multimammate rats to intranasal and intraperitoneal inoculation of monkeypox virus experimental infection of squirrels sciurus vulgaris by monkeypox virus experimental infection of tropical squirrels with monkeypox virus experimental infection of ground squirrels (spermophilus tridecemlineatus) with monkeypox virus comparative pathology of north american and central african strains of monkeypox virus in a ground squirrel model of the disease experimental infection of prairie dogs with monkeypox virus a prairie dog animal model of systemic orthopoxvirus disease using west african and congo basin strains of monkeypox virus dosage comparison of congo basin and west african strains of monkeypox virus using a prairie dog animal model of systemic orthopoxvirus disease experimental infection of an african dormouse (graphiurus kelleni) with monkeypox virus comparison of west african and congo basin monkeypox viruses in balb/c and c bl/ mice comparison of monkeypox viruses pathogenesis in mice by in vivo imaging a mouse model of lethal infection for evaluating prophylactics and therapeutics against monkeypox virus identification of wild-derived inbred mouse strains highly susceptible to monkeypox virus infection for use as small animal models efficacy of the antipoxvirus compound st- for treatment of severe orthopoxvirus infection resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat murine norovirus infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by stat -dependent interferon responses airway epithelial versus immune cell stat function for innate defense against respiratory viral infection critical roles for both stat -dependent and stat -independent pathways in the control of primary dengue virus infection in mice effective antiviral treatment of systemic orthopoxvirus disease: st- treatment of prairie dogs infected with monkeypox key: cord- -axkdf vu authors: kim, shin-hee; samal, siba k. title: newcastle disease virus as a vaccine vector for development of human and veterinary vaccines date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: axkdf vu viral vaccine vectors have shown to be effective in inducing a robust immune response against the vaccine antigen. newcastle disease virus (ndv), an avian paramyxovirus, is a promising vaccine vector against human and veterinary pathogens. avirulent ndv strains lasota and b have long track records of safety and efficacy. therefore, use of these strains as vaccine vectors is highly safe in avian and non-avian species. ndv replicates efficiently in the respiratory track of the host and induces strong local and systemic immune responses against the foreign antigen. as a vaccine vector, ndv can accommodate foreign sequences with a good degree of stability and as a rna virus, there is limited possibility for recombination with host cell dna. using ndv as a vaccine vector in humans offers several advantages over other viral vaccine vectors. ndv is safe in humans due to host range restriction and there is no pre-existing antibody to ndv in the human population. ndv is antigenically distinct from common human pathogens. ndv replicates to high titer in a cell line acceptable for human vaccine development. therefore, ndv is an attractive vaccine vector for human pathogens for which vaccines are currently not available. ndv is also an attractive vaccine vector for animal pathogens. infectious diseases have been emerging and reemerging over millennia [ ] . human immunodeficiency virus (hiv), severe acute respiratory syndrome coronavirus (sars-cov), and the most recent pandemic h n influenza virus are only a few of many examples of emerging infectious pathogens in the modern world [ ] . each of these diseases has global societal and economic impact related to unexpected illnesses and deaths, as well as interference with travel, business, and daily activities. to overcome emerging, reemerging, as well as stable infectious diseases, the demand for development of efficient vaccines has greatly increased. historically, live attenuated vaccines have provided the most effective protection against viral infection and disease [ ] . however, there have been safety concerns with the risk of reversion to the wild-type pathogen phenotype as shown with some traditional live attenuated vaccines such as the polio vaccine. furthermore, development of live attenuated vaccines has not been successful for many important pathogens. on the other hand, inactivated vaccines are generally not very effective and require a high containment laboratory for cultivation of highly virulent pathogens. also, there is a risk of incomplete inactivation for inactivated vaccines. therefore, there is a need for an alternative approach for development of vaccines. replicating viral vector vaccines offer a live vaccine approach without requiring involvement of the complete pathogen or cultivation of the pathogen [ ] . replicating viral vectors have the ability to synthesize the foreign antigen intracellularly and induce humoral, cellular, and mucosal immune responses. specifically, vectored vaccines can have advantages for (i) viruses for which a live attenuated vaccine might not be feasible (i.e., hiv); (ii) viruses that do not grow well in vitro (i.e., human papillomavirus, hepatitis c virus, and norovirus); (iii) highly pathogenic viruses that present safety challenges during vaccine development (i.e., sars-cov and ebola virus); (iv) viruses that lose infectivity due to physical instability (i.e., respiratory syncytial virus (rsv)); and (v) viruses that can exchange genes with circulating viruses (i.e., coronaviruses, influenza viruses, and enteroviruses) [ ] . a vectored vaccine can be rapidly engineered against a newly emerging pathogenic virus by inserting the gene of the protective antigen of the virus into the genome of the viral vector. in general, the magnitude of the immune response to live viral vector vaccines is substantially greater and broader than that induced by vaccines based on subunit proteins or inactivated viruses. furthermore, manufacturing of vectored vaccines against highly pathogenic viruses do not require a high level of biosafety containment laboratories. newcastle disease virus (ndv) is a fast-replicating avian virus that is prevalent in all species of birds [ ] . in most avian species, ndv infections do not result in disease. in chickens, ndv causes a highly contagious respiratory and neurologic disease, leading to severe economic losses in the poultry industry worldwide [ ] . ndv strains vary widely in virulence. based on the severity of the disease in chickens, ndv strains are classified into three pathotypes: lentogenic strains which cause mild or asymptomatic infections that are restricted to the respiratory tract; mesogenic strains which are of intermediate virulence; and velogenic strains which cause systemic infections with high mortality [ ] . naturally occurring low-virulent ndv strains, such as lasota and b , are widely used as live attenuated vaccines to control newcastle disease in poultry. although ndv primarily infects avian species, many non-avian species have also been shown to be naturally or experimentally susceptible to infection. the advent of a reverse genetics system to manipulate the genome of ndv not only allowed us to study the molecular biology and pathogenesis of ndv but also to develop ndv as a vaccine vector against diseases of humans and animals. ndv vector has several advantages over other replicating viral vectors. avirulent ndv strains are highly safe in avian and non-avian species. ndv replicates well in vivo and induces a robust immune response. in contrast to adeno, herpes, and pox virus vectors whose genome encodes a large number of proteins, ndv encodes only seven proteins and is thus less competition for immune responses between vector proteins and the expressed foreign antigen. ndv replicates in the cytoplasm, does not integrate into the host cell dna, and does not establish persistent infection. recombination involving ndv is extremely rare. ndv has a modular genome that facilitates genetic manipulation. ndv infects via the intranasal route and therefore induces both mucosal and systemic immune responses. a wide range of ndv strains exists that can be used as vaccine vectors. ndv-vectored vaccine can also be used as a "differentiating infected from vaccinated animals" (diva) vaccine. in this review article, we have reviewed the biology of ndv, development of reverse genetic systems for generation of ndv-vectored vaccines, and use of ndv vector for development of human and veterinary vaccines. ndv is a member of the genus avulavirus in the family paramyxoviridae [ ] . ndv virions are pleomorphic, but mostly spherical with a diameter of nm. the virion is enveloped with a bilayer lipid membrane. the genome of ndv is a non-segmented, negative-sense, single-stranded rna of , to , nucleotides containing six transcriptional units ( -n-p-m-f-hn-l- ) (figure ) . the genome encodes a nucleocapsid protein (n), a phosphoprotein (p), a matrix protein (m), a fusion protein (f), a hemagglutinin-neuraminidase protein (hn), and a large polymerase protein (l). an additional protein called the v protein is produced by rna editing of the p gene. the beginning and end of each gene contain control sequences, known as gene-start (gs) and gene-end (ge), respectively. the viral rna-dependent rna polymerase begins transcription at the end of the genomic rna, in a sequential manner by a stop-start mechanism [ ] . the re-initiation of transcription at the gs is not perfect, thus leading to a gradient of mrna abundance with high levels of mrna transcription viruses , , of located at the end. the genome length of ndv must be an even multiple of six for efficient virus replication following the "rule of six" [ ] . in ndv, the hn and f proteins are the two integral membrane proteins. the hn protein is responsible for attachment of the virion to sialic acid containing cell surface receptors. the f protein mediates entry of the virus into the host cell by fusion of the viral envelope to the plasma membrane. the f protein is synthesized as a precursor (f ) that is cleaved by host cell protease into two biologically active f and f subunits. cleavage of the f protein is a pre-requisite for virus entry and cell-to-cell fusion. the amino acid sequence at the f protein cleavage site has been identified as the primary determinant of virulence [ , ] . virulent ndv strains have multibasic residues that conform to the preferred cleavage site of the intracellular protease furin present in most cell types. in contrast, avirulent ndv strains typically contain one or two basic residues at the f protein cleavage site and are delivered to the plasma membrane in an uncleaved form for cleavage by extracellular proteases, thus restricting viral replication to the respiratory and enteric tracts where secreted proteases for cleavage are available. infectious ndv can be recovered entirely from cloned cdna by transfecting cultured cells with plasmids encoding the viral components of a functional nucleocapsid, full-length antigenomic rna, and the major proteins involved in replication and transcription, i.e. the n, p, and l proteins under the control of bacteriophage t rna polymerase promoter [ ] (figure ). this method, which is also known as reverse genetics technique, is now available for all three pathotypes of ndv strains [ ] [ ] [ ] [ ] . in general, a foreign gene flanked by ndv gs and ge sequences is inserted into a ′ non-coding region of an ndv genome as an additional transcription unit. due to a polar gradient transcription, foreign genes are expressed more efficiently when placed closer to ′ end of the genome. although a foreign gene can be placed between any two genes of ndv, the insertion site between the p and m genes has been found optimal for efficient expression of the foreign protein and replication of ndv [ ] [ ] [ ] . the insertion of a foreign gene into ndv genome increases its genome length and gene number and often has a growth retardation effect on virus replication in vitro and in vivo [ ] . ndv accommodates foreign genes (at least . kb in length) with a good degree of stability [ ] . a single ndv vector can also express at least three different foreign genes. in ndv, the hn and f proteins are the two integral membrane proteins. the hn protein is responsible for attachment of the virion to sialic acid containing cell surface receptors. the f protein mediates entry of the virus into the host cell by fusion of the viral envelope to the plasma membrane. the f protein is synthesized as a precursor (f ) that is cleaved by host cell protease into two biologically active f and f subunits. cleavage of the f protein is a pre-requisite for virus entry and cell-to-cell fusion. the amino acid sequence at the f protein cleavage site has been identified as the primary determinant of virulence [ , ] . virulent ndv strains have multibasic residues that conform to the preferred cleavage site of the intracellular protease furin present in most cell types. in contrast, avirulent ndv strains typically contain one or two basic residues at the f protein cleavage site and are delivered to the plasma membrane in an uncleaved form for cleavage by extracellular proteases, thus restricting viral replication to the respiratory and enteric tracts where secreted proteases for cleavage are available. infectious ndv can be recovered entirely from cloned cdna by transfecting cultured cells with plasmids encoding the viral components of a functional nucleocapsid, full-length antigenomic rna, and the major proteins involved in replication and transcription, i.e., the n, p, and l proteins under the control of bacteriophage t rna polymerase promoter [ ] (figure ). this method, which is also known as reverse genetics technique, is now available for all three pathotypes of ndv strains [ ] [ ] [ ] [ ] . in general, a foreign gene flanked by ndv gs and ge sequences is inserted into a non-coding region of an ndv genome as an additional transcription unit. due to a polar gradient transcription, foreign genes are expressed more efficiently when placed closer to end of the genome. although a foreign gene can be placed between any two genes of ndv, the insertion site between the p and m genes has been found optimal for efficient expression of the foreign protein and replication of ndv [ ] [ ] [ ] . the insertion of a foreign gene into ndv genome increases its genome length and gene number and often has a growth retardation effect on virus replication in vitro and in vivo [ ] . ndv accommodates foreign genes (at least . kb in length) with a good degree of stability [ ] . a single ndv vector can also express at least three different foreign genes. [ ] [ ] [ ] . the insertion of a foreign gene into ndv genome increases its genome length and gene number and often has a growth retardation effect on virus replication in vitro and in vivo [ ] . ndv accommodates foreign genes (at least . kb in length) with a good degree of stability [ ] . a single ndv vector can also express at least three different foreign genes. avirulent ndv strains lasota and b are commonly used as vaccine vectors because of their proven track records of safety. mesogenic and velogenic ndv strains are not used as vaccine vectors because they are virulent in chickens. in an experimental study, the mesogenic strain beaudette c (bc) was evaluated as a vaccine vector in nonhuman primates [ ] . strain bc replicated to a higher titer and induced a substantially higher level of antibody response compared to strain lasota, indicating it would be an effective vaccine vector. ndv has several advantages for use as a vaccine vector in humans. ndv is safe in humans, due to a natural host range restriction. in nonhuman primates, the intranasal and intratracheal inoculation of african green and rhesus monkeys with . plaque-forming units (pfu) per site of ndv did not cause any disease symptoms and its replication was restricted to the respiratory tract [ ] . in humans, infection by ndv appears to be limited and benign based on both anecdotal observations with bird handlers and in clinical studies using ndv as an oncolytic agent [ ] . according to a clinical study for ndv as an oncolytic agent in humans, intravenous administration of pfu of ndv to humans was safe without causing adverse effects [ ] . ndv shares only a low level of amino acid sequence identity with known human paramyxoviruses and are antigenically distinct from common human and animal pathogens, and thus would not be affected by preexisting immunity in humans. ndv infects via the intranasal route and has been shown to induce humoral and cellular immune responses both at the mucosal and systemic levels in murine and nonhuman primate models [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . ndv is a strong stimulator of the host immune response, thus providing an adjuvant effect. the use of avirulent pathotypes of ndv in humans prevents the possibility of accidental spread of a virulent virus strain from treated patients to birds. ndv grows to high titers not only in embryonated eggs ( pfu/ml) but also in vero cells ( pfu/ml), which is acceptable for human vaccine development. in fact, ndv has been used to express protective antigens of various human pathogens and has shown promising results in nonhuman primates. ndv-vectored vaccines for several human pathogens are discussed as follows (table ) . the potential of recombinant ndv strain b as an effective vaccine vector for humans was first evaluated by expressing an influenza virus (a/wsn/ ) hemagglutinin (ha) protein [ ] . the expressed ha protein was incorporated into virions and appeared to be cleaved, indicating that the ha protein was accessible to proteolytic enzymes. in vitro growth kinetics and pathogenicity test in embryonated chicken eggs indicated attenuation of the recombinant ndv. intravenous administration of mice induced higher titers of antibody to influenza virus ha than intraperitoneal administration. further, immunized mice by the intravenous route were completely protected against a lethal dose of influenza virus, suggesting that ndv can be a safe and effective vaccine vector for possible use in mammalian and avian species. the potential of ndv as a vaccine vector for use in humans was first determined in nonhuman primates. two ndv strains lasota and bc were evaluated as vaccine vectors in nonhuman primates by inserting the hn protein of human parainfluenza virus type (hpiv ) as a protective antigen [ ] . two doses of immunization with ndv strains confirmed their restricted replication in african green monkeys (ndv-bc and ndv-ls) and in rhesus monkeys (ndv-bc only). however, the serum antibody response following the second dose exceeded that observed with hpiv infection, even though hpiv replicated much more efficiently than ndv in these animals. this is the first study to demonstrate efficacy of ndv-vectored vaccine in nonhuman primates. ebola virus (ebov) causes severe hemorrhagic fever in humans with a fatality rate of up to % (species zaire ebolavirus) of infected individuals [ ] . due to the limitation of inactivated vaccines, viral vectors based on common human pathogens have been used for ebov vaccine. to overcome the high seroprevalence against vectors based on common human pathogens in the adult human population, recombinant ndv strain lasota expressing the ebov gp envelope protein was generated to evaluate its potential as a vaccine for ebov [ ] . following one intranasal and intratracheal inoculation of rhesus monkeys with ndv/gp, titers of ebov-specific antibodies and serum ebov-neutralizing antibodies, were undetectable or low compared to those induced by hpiv /gp. however, a second immunization resulted in a substantial boost in serum immunoglobulin (ig) g enzyme-linked immunosorbent assay (elisa) titers, yet the titers remained lower than those induced by a second dose of hpiv /gp. in contrast, the elisa iga titers in respiratory tract secretions and the serum ebov-neutralizing antibody titers were equal to those induced after the second dose of hpiv /gp, showing that the efficacy of ndv vector can be comparable to that of hpiv vector by prime-boosting vaccination [ ] . ndv was evaluated as a vaccine vector for another important emerging pathogen, the severe acute respiratory syndrome-associated coronavirus (sars-cov) [ ] . two ndv vectors were constructed: mesogenic strain bc (ndv-bc) and lentogenic strain lasota in which the f protein cleavage sequence was modified to that of strain bc (ndv-vf) [ ] . these ndv vectors were engineered to express the sars-cov spike s glycoprotein, the major protective antigen. two dose immunizations of african green monkeys induced a robust neutralizing antibody response, resulting in reduction of virus shedding after challenge with sars-cov ( % tissue culture infective dose (tcid )). specifically, immunization with ndv-vf vector resulted in sars-cov titers of a -fold, -fold, and -fold reduction in nasal turbinate, trachea, and lung, respectively, compared with the control animals. the ndv-bc vector was even more effective, with average reductions in viral titer of -fold, -fold, and -fold in the nasal turbinate, trachea, and lung, respectively. this study demonstrated the safety and protective efficacy of ndv as a topical respiratory vaccine vector for sars-cov. the use of viral vectors expressing selected hiv antigens has been a promising vaccine strategy. the potential of ndv-vectored vaccine against hiv infection was first evaluated by generating recombinant ndv expressing simian immunodeficiency virus (siv) gag protein (rndv/sivgag) [ ] . the vaccine virus induced gag-specific cellular immune responses in mice. among intravenous, intraperitoneal, and intranasal immunization routes, intranasal administration induced the strongest protective immune response against a surrogate challenge virus (rvac/sivgag) following a booster immunization with recombinant influenza viruses expressing immunogenic portions of siv gag. specifically, this heterologous vaccination approach resulted in approximately, a -fold reduction in rvac/sivgag titers at day after challenge compared to titers of control mice injected with phosphate-buffered saline (pbs). the magnitude of the protective immune response also correlated with the levels of cellular immune responses to gag. these results suggest that ndv vector can be a suitable candidate vaccine against hiv. the hiv gag and env proteins have been expressed by ndv vector [ , [ ] [ ] [ ] . the expression level of gag protein was optimized using different insertion sites in the ndv genome. it was found that the codon-optimized gag inserted between the p and m genes of ndv induced the highest level of protein expression and an enhanced immune response against hiv gag in mice [ ] . in another study, expression of gp env protein by ndv vector lasota also induced systemic and mucosal antibody responses in guinea pigs [ ] . priming/boosting by the intranasal route was more immunogenic than by the intramuscular route. further, coexpression of gp env and p gag by vector lasota enhanced both env-specific and gag-specific immune responses in guinea pigs [ ] . this approach was efficient in inducing cellular and protective immune responses to challenge with vaccinia viruses expressing hiv- env and gag in mice. these results suggest that vaccination with a single ndv vector coexpressing env and gag represents a promising strategy to enhance immunogenicity and protective efficacy against hiv. in addition, heterologous prime (ndv expressing gp ) and boosting (purified gp protein) approach induced high neutralizing antibody titer in guinea pigs [ ] . these findings suggest that vaccination with multiple hiv antigens in combination can broaden antiviral immune responses. respiratory syncytial virus (rsv) is a major cause of severe lower respiratory tract disease in infants and elderly [ ] . the development of an effective vaccine against rsv is a high priority. in order to develop a vector vaccine against rsv, ndv strain b was used to express the fusion glycoprotein of rsv [ ] . ndv was chosen as a viral vector because of its ability to induce a strong interferon (ifn)-α/β response. the rsv f protein was more immunogenic when presented by ndv-f than by live rsv, and this correlated with an increased ability of ndv to activate antigen-presenting cells in vitro and to induce high levels of ifn-α/β in vivo. rsv f-specific, cd + memory t cells were present in greater numbers in ndv-f-primed balb/c mice than in animals previously infected with rsv. consequently, ndv vaccine virus provided protection from rsv challenge. this study also highlights the adjuvant effect of ndv vector mediated by the potent ifn induction. ndv has also been used as a vector to express the immunogens of a bacterial pathogen. lyme borreliosis is a prevalent vector-borne disease in the united states, europe and parts of asia. ndv was used to express the basic membrane protein a (bmpa) and the outer surface protein c (ospc) of the lyme disease pathogen borrelia burgdorferi [ ] . c h or balb/c mice that were immunized intranasally with the ndv vectors mounted vigorous serum antibody responses against the ndv vector, but failed to mount a robust response against either the intracellular or extracellular forms of bmpa or ospc. in contrast, a single immunization of hamsters with the ndv vectors via the intranasal, intramuscular, or intraperitoneal route resulted in rapid and rigorous antibody responses against the bmpa and ospc. challenged with b. burgdorferi ( cells/animal), immunization with vector-expressing bmpa provided a reduction of the pathogen load in the joints. this study showed the potential of ndv as a vaccine vector against bacterial pathogens. nipah virus (niv) is a deadly emerging zoonotic pathogen that causes fatal encephalitis in humans and pigs [ ] . the glycoprotein (g) and fusion protein (f) are two major niv surface glycoproteins that stimulate protective immune responses. ndv strain lasota expressing the niv g and f proteins (rla-nivg and rla-nivf, respectively) were evaluated for their immunogenicity in mice and in pigs [ ] . following the second dose of immunization, rla-nivg and rla-nivf induced niv-specific neutralizing antibodies in mice and long-lasting neutralizing antibodies in pigs (at least for weeks). this study also showed that rla-nivg induced higher levels of neutralizing antibodies than rla-nivf. although the protective efficacy of the vaccines was not evaluated in this study, the vaccine viruses showed the potential to be used for protecting humans and animals against niv infection. norovirus (nov) is the most frequent cause of viral gastroenteritis in people of all ages [ ] . the inability of nov to grow in the cell culture system has greatly hindered development of effective vaccines. to circumvent this obstacle, virus-like particles (vlps) produced by the baculovirus expression system have been commonly used as nov vaccine candidates. as a live vaccine vector, lasota and modified bc strains were used to express the capsid protein (vp ) of nov strain va (gii. ) and norwalk virus (gi. ) [ , ] . for the modified bc vector, the multibasic cleavage site sequence of the f gene was changed to that of strain lasota. the nov-expressed vp protein formed vlps in cell culture and in allantoic fluid of embryonated chicken eggs. the modified bc-vectored vaccine induced higher levels of serum, cellular, and mucosal immune responses than the baculovirus-expressed vlps in mice. these results suggested that ndv has great potential for developing a live nov vaccine. alternatively, vlps produced in large quantities in embryonated eggs or in cell culture by ndv can be a cost-effective method for producing a vlp-based vaccine for humans. this study also has implications for development of ndv-vectored vaccines for other non-cultivable pathogens of humans. ndv-vectored vaccines have been evaluated in several animal species (i.e., chicken, cattle, sheep, cat, mouse, pig, and dog) for veterinary use [ ] (table ) . ndv is a natural vaccine vector for poultry pathogens. live attenuated ndv vaccines are widely used all over the world. therefore, an ndv vector carrying the protective antigen of another avian pathogen can be used as a bivalent vaccine. such a vaccine will be economical for poultry farmers. as a bivalent vaccine, the ndv strain lasota was first used to express the host-protective immunogen vp of infectious bursal disease virus (ibdv), a birnavirus, which causes a highly immunosuppressive disease in chickens [ ] . the protective efficacy of lasota-expressing vp protein was evaluated by challenging vaccinated chickens with a highly virulent ndv strain texas gb or a virulent ibdv variant strain. vaccination with rlasota/vp provided % protection against ndv and ibdv. booster immunization induced higher levels of antibody responses against both ndv and ibdv and conferred complete protection against both viruses. these results indicate that the recombinant ndv can be used as a vaccine vector for other avian pathogens. infectious laryngotracheitis is a major respiratory disease in chickens and caused by infectious laryngotracheitis virus (iltv), a herpes virus [ ] . bivalent ndv-vectored vaccines against iltv have been developed to improve the safety of current live attenuated iltv vaccines [ , ] . the protective efficacy of ndvs expressing the three major iltv surface glycoproteins, namely, gb, gc, and gd was evaluated against iltv infection in chickens [ ] . particularly, rndv-expressing gd induced the highest level of neutralizing antibodies among the tested vaccine candidates and completely protected chickens against the challenge of virulent iltv and ndv, showing its potential as a bivalent vaccine. this protective efficacy of rndv gd vaccine was attributed to high levels of envelope incorporation and cell surface expression of gd compared to gb and gc. in another study, lasota viruses expressing gb and gd of iltv were generated and vaccination of chickens with the two viruses conferred protection against virulent iltv and ndv challenges [ ] . in addition, lasota with gb showed the protection of commercial broilers against clinical disease. discrepancy of vaccine efficacy between these two studies could be due to the different levels of gb and gd expressions by ndv vectors and experimental conditions. infectious bronchitis virus (ibv), a coronavirus, is an important avian pathogen, causing respiratory disease in broilers and poor egg production in breeders and layers worldwide [ ] . the spike polypeptide s gene was expressed by lasota (rls/ibv.s ) [ ] . the vaccine virus effectively elicited hemagglutination inhibition antibodies against ndv and protected chickens against lethal challenge with virulent strain ndv/ca . ibv heterotypic protection was assessed using a prime-boost approach with a commercially available attenuated ibv massachusetts (mass)-type vaccine. chickens primed ocularly with rls/ibv.s and boosted with mass were completely protected against challenge with a virulent ark-type strain. the protective efficacy of this heterologous vaccination was similar to that of priming and boosting with mass (mass + mass). based on clinical signs, both vaccinated groups appeared equally protected against challenge compared to unvaccinated challenged chickens. in shedding of challenge virus in the trachea, viral rna was detected in % of rls/ibv.s + mass-vaccinated chickens while chickens vaccinated with mass + mass and unvaccinated challenged controls showed % and % incidence of ibv rna detection, respectively. these results demonstrate the potential of ndv-vectored vaccine for ibv infection. ndv vector has also been used for the prevention of economically important livestock diseases. rift valley fever virus (rvfv), a bunyavirus, causes recurrent large outbreaks in humans and in livestock [ ] . ndv expressing the rvfv structural glycoproteins gn was generated (ndfl-gn) [ ] . immunization of calves via the intranasal route elicited no detectable antibody responses, whereas intramuscular immunization elicited antibodies against both ndv and the gn protein. in general, the titers of rvfv-neutralizing antibodies were modest, varying from to . to improve the efficacy of ndv-vectored vaccine, gn was coexpressed with another glycoprotein gc [ ] , which resulted in the formation of vlps and subsequent release from the producing cells. a homologous prime-boost vaccination of mice with this vaccine virus induced neutralizing antibodies and provided complete protection from a lethal rvfv challenge. the immunogenicity of the vaccine virus was further evaluated in lamb, the main target species of rvfv. a single intramuscular vaccination induced neutralizing antibodies, and this response was significantly boosted by a second vaccination. although coexpression of the gn and gc induced a good immune response, protective efficacy of this vaccine needs to be further evaluated. bovine herpesvirus- (bhv- ) is a major cause of respiratory tract diseases in cattle. since modified live bhv- vaccines can cause latent infection in immunized animals, ndv expressing the glycoprotein d (gd) of bhv- was generated as a vectored vaccine [ ] . a single intranasal and intratracheal inoculation of calves with ndv elicited mucosal and systemic antibodies specific to bhv- . challenge with bhv- showed reduced virus shedding and clinical signs in immunized calves compared to unimmunized claves. in addition, the titers of serum antibodies specific to bhv- were higher in immunized animals compared to unimmunized animals, indicating that the vaccines primed for secondary responses. this indicates that ndv can be used as a vaccine vector in bovines, and bhv- gd may be useful as a mucosal vaccine against bhv- infection. however, vaccination might require augmentation by a second dose or the inclusion of additional bhv- antigens. rabies virus (rv), a rhabdovirus, causes a fatal neurologic disease in humans and in animals [ ] . to generate an effective, safe, and affordable rabies vaccine, ndv strain lasota expressing the rabies virus glycoprotein g (rl-rvg) was evaluated. the safety of rl-rvg vaccine virus was confirmed in cats and dogs. intramuscular vaccination with rl-rvg induced strong and long-lasting protective neutralization antibody responses against rabies virus in dogs and cats. although three doses of vaccination were conducted, the second dose induced the highest levels of immune responses in both cats and dogs. vaccination dose of % embryo infective dose (eid) completely protected dogs from challenge after one year. this study demonstrated protective efficacy of ndv-vectored vaccine against rabies in dogs. this vaccine may also have potential use in high-risk human individuals to control rabies virus infections. canine distemper virus (cdv), a morbillivirus, infects many carnivores and cause several high-mortality disease outbreaks [ ] . the current cdv live vaccine cannot be safely used in some exotic species, such as mink and ferret. ndv strain lasota expressing envelope glycoproteins, hemagglutinin (h, rla-cdvh) and fusion protein (f, rla-cdvf), were generated as vaccine candidates. in immunized minks, rla-cdvh induced higher titers of neutralization antibodies against cdv than rla-cdvf neutralizing antibodies. further, rla-cdvh provided complete protection against virulent cdv challenge during the four weeks of observation. in contrast, all animals immunized with rla-cdvf developed clinical signs of distemper and virus shedding. this study suggested that recombinant ndv expressing the h protein of cdv is a safe and efficient candidate vaccine against cdv in mink. the efficacy of rla-cdvh virus also needs to be evaluated in other host carnivore species. highly pathogenic avian influenza virus (hpaiv) is an economically important pathogen of poultry worldwide. the outbreaks involving h n or h n influenza viruses resulted in lethal infections in poultry and the death of a limited number of people [ ] . therefore, vaccination of poultry against hpaiv could play an important role in reducing virus shedding and raising the threshold for infection and transmission [ ] . however, development of vaccines against hpaiv has been hampered due to poor immunogenicity of the virus [ ] . furthermore, inactivated vaccines are not commonly used because of the high cost due to the requirement of enhanced biosafety level containment and the difficulty in "differentiating infected from vaccinated animals" (diva). the use of live attenuated influenza viruses as vaccines in avian or mammalian species can also raise a major biosafety concern, because the vaccine viruses may become virulent through mutation or genetic reassortment with circulating strains. alternatively, ndv can be an ideal vaccine vector for development of an avian influenza vaccine. ndv infects via the intranasal route and therefore induces both local and systemic immune responses at the respiratory tract [ ] . therefore, it provides a convenient platform for rapid, efficient, and economical immunization. in fact, ndv has been most commonly used as a vaccine vector against aiv. protective efficacy of ndv-vectored vaccines has been evaluated and verified by many different vaccination studies [ , , [ ] [ ] [ ] , ] . for the generation of vaccines, a major protective antigen, hemagglutinin (ha) of hpaiv has been placed between the p and m genes or between the f and hn genes in lentogenic ndv strains lasota or b . to address a safety concern, an ndv-vectored vaccine was further generated by replacing the polybasic cleavage site in hpaiv ha with that from a low-pathogenicity strain of influenza virus [ ] . in addition, the ha gene has been modified to enhance its expression levels by ndv. specifically, elimination of an ndv transcription termination signal-like sequence located within the ha open reading frame of h enhanced expression levels of ha protein by ndv and completely protected chickens after challenge with a lethal dose of velogenic ndv or highly pathogenic aiv, respectively [ ] . in addition, the ectodomain of an h n or h n avian influenza virus ha was fused with the transmembrane and cytoplasmic domains derived from the f protein of ndv [ , ] . this approach resulted in enhanced incorporation of the foreign protein into virus particles and the protection of chickens against both hpaiv and a highly virulent ndv. these studies also demonstrated that ndv can be used to generate a bivalent vaccine. although use of avirulent ndv vectors has been effective in protecting chickens against clinical disease and mortality, some studies also found virus shedding in chickens after challenge with hpaiv [ ] . to enhance the replication of vaccine virus, attenuated mesogenic ndv strain bc has been generated by changing the multibasic cleavage site sequence of the f protein to the dibasic sequence of strain lasota [ ] . additionally, the bc, f, and hn proteins were modified in several ways to enhance virus replication. the modified bc-based vectors replicated better than lasota vector, and expressed higher levels of ha protein and provided complete protection against challenge virus shedding, suggesting its potential to be safely used as a vaccine vector. for effective human vaccines against hpaiv, the immunogenicity of ndv expressing the ha of h n was evaluated in african green monkeys by the intranasal route of administration [ ] . two doses of ndv-vectored vaccine ( ˆ pfu) induced a high titer of h n hpaiv-neutralizing serum antibodies in all of the immunized monkeys. moreover, a substantial mucosal iga response was induced in the respiratory tract, which can potentially reduce or prevent transmission of the virus during an outbreak or a pandemic. the intranasal route of administration is also advantageous for needle-free immunization and is thus suitable for mass immunization. the protective efficacy of vaccine viruses was evaluated in african green monkeys by the intranasal/intratracheal route or by the aerosol route of administration [ ] . each of the vaccine constructs was highly restricted for replication, with only low levels of virus shedding detected in respiratory secretions. all groups developed high levels of neutralizing antibodies against homologous (a/vietnam/ / ) and heterologous (a/egret/egypt/ -namru / ) strains of hpaiv and were protected against challenge with ˆ pfu of homologous hpaiv. this study demonstrated that needle-free, highly attenuated ndv-vectored vaccines were immunogenic and protective in a nonhuman primate model of hpaiv infection. newcastle disease virus (ndv) is an attractive vaccine vector for both human and animal pathogens. the live attenuated vaccine strains used as vaccine vectors have a proven track record of safety and efficacy. ndv vectors not only induce robust humoral and cellular immune responses but also induce mucosal immune response. therefore, ndv can be a vector of choice for mucosal immunization. the ability of ndv to infect a wide variety of non-avian species makes it a potential vector for other animals. ndv is also a promising vaccine vector for use in humans. one advantage is that most humans do not have pre-existing immunity to ndv. ndv-vectored vaccines have also become available commercially (i.e., h n hpaiv vaccine for poultry). emerging infectious diseases: threats to human health and global stability the perpetual challenge of infectious diseases caiv-t comparative efficacy study group. live attenuated versus inactivated influenza vaccine in infants and young children newcastle disease virus as a vaccine vector for humans newcastle disease and related avian paramyxoviruses newcastle disease and other avian paramyxoviruses rescue of newcastle disease virus from cloned cdna: evidence that cleavability of the fusion protein is a major determinant for virulence role of fusion protein cleavage site in the virulence of newcastle disease virus recovery of a virulent strain of newcastle disease virus from cloned cdna: expression of a foreign gene results in growth retardation and attenuation high-level expression of a foreign gene from the proximal first locus of a recombinant newcastle disease virus evaluation of the contributions of the individual viral genes to newcastle disease virulence and pathogenesis generation by reverse genetics of an effective, stable, live-attenuated newcastle disease virus vaccine based on a currently circulating, highly virulent indonesian strain recombinant newcastle disease virus as a vaccine vector optimization of human immunodeficiency virus gag expression by newcastle disease virus vectors for the induction of potent immune responses recombinant newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication nonsegmented negative-strand viruses as vaccine vectors recombinant newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates phase i/ii trial of intravenous ndv-huj oncolytic virus in recurrent glioblastoma multiforme successful topical respiratory tract immunization of primates against ebola virus newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens immunization of primates with a newcastle disease virus-vectored vaccine via the respiratory tract induces a high titer of serum neutralizing antibodies against highly pathogenic avian influenza virus newcastle disease virus-vectored vaccines expressing the hemagglutinin or neuraminidase protein of h n highly pathogenic avian influenza virus protect against virus challenge in monkeys respiratory tract immunization of non-human primates with a newcastle disease virus-vectored vaccine candidate against ebola virus elicits a neutralizing antibody response newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous h n avian influenza viruses newcastle disease virus vector producing human norovirus-like particles induces serum, cellular, and mucosal immune responses in mice immunogenicity of newcastle disease virus vectors expressing norwalk virus capsid protein in the presence or absence of vp protein induction of cellular immune responses to simian immunodeficiency virus gag by two recombinant negative-strand rna virus vectors newcastle disease virus expressing human immunodeficiency virus type envelope glycoprotein induces strong mucosal and serum antibody responses in guinea pigs mucosal immunization with newcastle disease virus vector coexpressing hiv- env and gag proteins elicits potent serum, mucosal, and cellular immune responses that protect against vaccinia virus env and gag challenges enhanced immune responses to hiv- envelope elicited by a vaccine regimen consisting of priming with newcastle disease virus expressing hiv gp and boosting with gp and sosip gp proteins protection against respiratory syncytial virus by a recombinant newcastle disease virus vector newcastle disease virus-vectored nipah encephalitis vaccines induce b and t cell responses in mice and long-lasting neutralizing antibodies in pigs a host-restricted viral vector for antigen-specific immunization against lyme disease pathogen norovirus disease in the united states recombinant newcastle disease virus-vectored vaccines against human and animal infectious diseases a recombinant newcastle disease virus expressing vp protein of infectious bursal disease virus protects against ndv and ibdv recombinant newcastle disease viral vector expressing hemagglutinin or fusion of canine distemper virus is safe and immunogenic in minks a recombinant newcastle disease virus (ndv) expressing infectious laryngotracheitis virus (iltv) surface glycoprotein d protects against highly virulent iltv and ndv challenges in chickens infectious bronchitis virus s expressed from recombinant virus confers broad protection against challenge intramuscular inoculation of calves with an experimental newcastle disease virus-based vector vaccine elicits neutralizing antibodies against rift valley fever virus rift valley fever virus immunity provided by a paramyxovirus vaccine vector immunization of cattle with recombinant newcastle disease virus expressing bovine herpesvirus- (bhv- ) glycoprotein d induces mucosal and serum antibody responses and provides partial protection against bhv- . vaccine newcastle disease virus-vectored rabies vaccine is safe, highly immunogenic, and provides long-lasting protection in dogs and cats newcastle disease virus expressing h hemagglutinin gene protects chickens against newcastle disease and avian influenza immunization of chickens with newcastle disease virus expressing h hemagglutinin protects against highly pathogenic h n avian influenza viruses engineered viral vaccine constructs with dual specificity: avian influenza and newcastle disease toward a comprehensive phylogeny for mammalian and avian herpesviruses newcastle disease virus (ndv) recombinants expressing infectious laryngotracheitis virus (iltv) glycoproteins gb and gd protect chickens against iltv and ndv challenges the avian coronavirus infectious bronchitis virus undergoes direct low-ph-dependent fusion activation during entry into host cells rift valley fever virus (bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention oie (world organisation for animal health) avian influenza safety and immunogenicity of an inactivated subvirion influenza a (h n ) vaccine contributions of the avian influenza virus ha, na, and m surface proteins to the induction of neutralizing antibodies and protective immunity modified newcastle disease virus vectors expressing the h hemagglutinin induce enhanced protection against highly pathogenic h n avian influenza virus in chickens author contributions: s.h.k. and s.k.s. wrote the manuscript. the authors declare no conflict of interest. key: cord- -jsm o pq authors: chidlow, glenys r.; harnett, gerry b.; shellam, geoffrey r.; smith, david w. title: an economical tandem multiplex real-time pcr technique for the detection of a comprehensive range of respiratory pathogens date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jsm o pq this study used real-time pcr assays to screen small sample volumes for a comprehensive range of respiratory pathogens. initial thermocycling was limited to cycles to avoid competition for reagents, followed by a secondary real-time multiplex pcr. supplementary semi-nested human metapneumovirus and picornavirus pcr assays were required to complete the acute respiratory pathogen profile. potential pathogens were detected in ( %) of pernasal aspirates collected from children with acute respiratory symptoms. multiple pathogens were detected in ( %) of those samples. the tandem multiplex real-time pcr was an efficient method for the rapid detection of multiple pathogens. in recent years, improved nucleic acid amplification and detection technology has facilitated the identification of pathogens that had previously proved difficult or impossible to detect using traditional culture or immunofluorescent techniques. the increased use of molecular methods has also resulted in the discovery of novel respiratory pathogens including the human coronaviruses nl [ ] , hku [ ] and the sars coronavirus [ ] , human metapneumovirus [ ] , human bocavirus [ ] and two human polyomaviruses, ki [ ] and wu [ ] . despite these advances, there remain a significant proportion of respiratory disease episodes for which a pathogenic agent can not be identified [ , ] . the aim of this study was to modify real-time pcr assays to facilitate the rapid screening of respiratory samples for a comprehensive range of viral and bacterial pathogens. standard pcr assays require extraction of rna and dna with simultaneous removal of inhibitors of the pcr process from the sample. multiple extracts from each sample have been required to set up a comprehensive range of pcr assays for respiratory pathogens, and the sample volume available may be insufficient for all of the required tests. also, previous attempts at multiplex pcr have been limited by competition between different amplification reactions and by the limited number of fluorescent probes able to be monitored in the same tube. a multiplex tandem pcr assay which utilized a two step pcr system consisting of a limited multiplex pcr followed by a single target pcr has been reported [ ] . our study further modified that procedure to include real-time pcr with specific probes in the second step of the assay. it comprised two first round enrichment pcr assays (a and b) containing and primer pairs respectively, and the second step real-time pcr assays containing - primer pairs and specific taqman probes. the assay detects several adenovirus genotypes, bordetella species, human bocavirus, chlamydophila pneumoniae and psittaci, coronaviruses oc , e, hku and nl , haemophilus influenzae, influenza viruses a, b and c, legionella longbeachae and pneumophila, moraxella catarrhalis, mycoplasma pneumoniae, parainfluenzaviruses - , pneumocystis jirovecii, ki and wu polyomaviruses, respiratory syncytial virus types a and b, streptococcus pyogenes and pneumoniae. supplementary semi-nested human metapneumovirus and picornavirus pcr assays are required to complete the acute respiratory pathogen profile. this technique facilitates the investigation of a comprehensive range of respiratory pathogens from a single nucleic acid extract and a single reverse transcription reaction. in addition, the ability to amplify multiple targets efficiently aids in the detection of mixed infections. the sensitivity of the tandem multiplex real-time pcr assay was greater than or equal to that of singleplex real-time or traditional nested pcr assays for the majority of the agents investigated (table ) . exceptions included the detection of adenovirus and influenza virus c in the tandem multiplex realtime assay, which were -fold and -fold less sensitive respectively than the established nested pcr assay. while the adenovirus real-time assay performed similarly in the multiplex and singleplex assays, both had lower sensitivity than the traditional nested assay, suggesting the need to redesign the primers and probes used in the real-time assays. this differed from the influenza virus c assay, where the lower sensitivity appeared to be related to the multiplex format, and may therefore necessitate further fine-tuning of the assay. pernasal aspirates from hospitalized children aged between days and years (mean = months old, median = months old) collected from july to september were tested in the tandem multiplex real-time pcr assay and the results are presented in table . respiratory pathogens were detected in samples ( %) and multiple pathogens were detected in of those samples ( %). this detection rate excludes samples where moraxella catarrhalis and/or streptococcus pneumoniae were detected, as the significance of the detection of these agents in pernasal aspirates using pcr amplification techniques remains doubtful [ ] [ ] [ ] . further studies are under way to gather more information about asymptomatic nasopharyngeal carriage rates for viral and bacterial pathogens. respiratory syncytial virus (rsv) was the most common pathogen and was detected in samples. for of those samples, at least one, and up to three other respiratory agents, including human rhinovirus, human bocavirus, ki and wu polyomaviruses, human coronaviruses, parainfluenzavirus and group a streptococcus, were also detected. rhinoviruses were the next most commonly detected ( samples) and again, multiple agents were commonly detected ( samples) and included rsv, human coronaviruses, human bocavirus, parainfluenzavirus, adenovirus, ki and wu polyomaviruses and group a streptococcus. other pathogens detected included influenza virus type b, human metapneumovirus and pneumocystis jirovecii. the tandem multiplex real-time assay failed to detect pathogens that had previously been detected on initial routine testing in six samples. the agents were rhinovirus ( ), parainfluenza type , parainfluenza type , human metapneumovirus and influenza b. repeat testing of samples indicated that for the two parainfluenzavirus failures there had been a slight reduction in nucleic acid extraction sensitivity due to the deterioration of carrier rna used in the extraction process, and once that was overcome the samples performed as expected in the multiplex tandem assay. there was insufficient sample from the remaining four patients to complete similar further investigations. other samples with discrepant results were due to the absence of specific primers and probes to those agents in this tandem multiplex real-time assay. they included non-group b adenovirus ( ), cmv ( ) and non-serogroup b haemophilus influenzae ( ) . the tandem multiplex real-time assay detected respiratory pathogens in specimens for which no agent had previously been detected. the agents included rsv ( ), rhinovirus ( ), human bocavirus ( ), coronaviruses ( ), and parainfluenzavirus. a further specimens had multiple agents detected in the tandem multiplex real-time assay compared to the original test result. these additional detections were mostly due to the comprehensive range of respiratory pathogens detected by the tandem multiplex real-time assay compared to the selective range of tests originally performed as requested by the clinician. positive results were confirmed by repeat testing, and no-template controls were included after every fifth sample during the extraction and pcr process to detect contamination events. the samples in this study were collected between june and september , which represents midlate winter and early spring months, which have typically been the peak respiratory virus detection months in the temperate zones of western australia. despite the comprehensive range of agents detected by the assay described in this study, ( %) samples still failed to yield a positive result. some of these may be due to suboptimal specimen types for some viruses; poor collection, transport or storage conditions; samples collected too late in the time course of infection; or non-infectious causes of respiratory symptoms. however, it may also indicate possible infection with as yet unknown pathogens. the significance of the recently described ki and wu polyomaviruses as pathogens remains to be established since, although there has been an association with the respiratory tract [ ] , a recent report found no association between polyomavirus infection and respiratory disease [ ] . in our study ki and wu polyoma viruses were each detected in different samples ( %), but always in combination with another virus, including rsv, adenovirus, rhinovirus, bocavirus, coronavirus and parainfluenzavirus. the widespread and increasing use of molecular detection techniques has led to reports of multiple pathogenic agents detected in single samples [ ] [ ] [ ] . this study supports those findings with two agents detected in ( %) samples, and three or more agents detected in ( %) samples ( table ) . no data was available to determine whether this may have altered the severity of illness. some studies have reported more severe clinical presentation in the presence of mixed infection [ , , ] while others have reported no significant greater disease severity in dual respiratory infection [ ] . it has been reported that the detection of multiple agents in a multiplex pcr assay was severely compromised when the ratio of target materials in the sample exceeded : [ ] . we also found similar inhibition in a traditional multiplex which was less evident in the tandem multiplex real-time assay (data not shown). in practice, the tandem multiplex real-time assay detected multiple pathogens in / ( %) samples. traditionally, testing for a comprehensive range of respiratory pathogens has necessitated multiple pcr assays, immunofluorescence or multiple cell culture assays to be performed on each sample. considerable specimen volume is required to produce sufficient nucleic acid extracts for multiple pcr assays and, since many respiratory viruses have an rna genome, multiple reverse transcription reactions are required. multiple extraction and reverse transcription reactions significantly increase the cost of the assays. there have been recent reports of limited multiplex pcr assays [ , [ ] [ ] [ ] [ ] and realtime multiplex pcr assays for the detection of respiratory viruses and bacteria [ ] [ ] [ ] . immunofluorescent antigen detection tests may be used but require a substantial volume of sample, as well as needing an adequate cellular content. cell culture techniques require considerable sample volume, a range of different cell lines, and the means for detecting and identifying those agents that are successfully cultivated. many of the recently described novel viruses including human metapneumovirus, coronaviruses, human bocavirus and some rhinoviruses are difficult or impossible to cultivate in cell cultures [ ] [ ] [ ] , ] . a multiplex tandem pcr was recently described for gene expression profiling [ ] . the first enrichment reverse transcription pcr contained up to primer pairs, and the individual second pcr assays utilized sybr green detection of products. we have modified that technique to include specific taqman probes in the second pcr assay to permit specific real-time detection of the pcr product. the two enrichment pcr mixes contain and primer pairs, and the real-time pcr mixes contain - primer pairs with their corresponding fluorophore-labelled probes ( table ). the enrichment pcr was limited to amplification cycles to reduce amplification competition between targets, and the dilution of that enrichment pcr product prior to addition to the real-time pcr mix, limited non-specific product formation and carry-over primer competition in the secondary pcr assay [ ] . the use of nucleic acid extraction and liquid handling robots streamlined this tandem multiplex real-time assay. the nucleic acid extracted from the specimens and controls was eluted into tubes in a -well format. the enrichment pcr and dilutions were all performed in -well plates, and robots used to transfer diluted enrichment pcr products to the -tube discs preloaded with real-time pcr reaction mixes (genedisc- , corbett life science, australia). since the initial cycling step was limited to cycles the transfer of those products had a reduced the risk of contamination compared with a traditional nested assay. the addition of standardized amounts of equine herpesvirus and ms rna coliphage to the digest buffer supplied in the nucleic acid extraction kit, served as a control for the dna and rna extraction process, the removal of inhibitors to the cdna production and pcr amplification processes, and the successful completion of the cdna procedure. ct values in the specific real-time pcr assays for these internal controls were monitored to avoid false-negative reports. this tandem multiplex real-time pcr assay, in combination with the semi-nested picornavirus and human metapneumovirus pcr assays, tests for respiratory agents from a sample volume of µl compared to µl required for the individual assays. a single sample extraction and three reverse transcription reactions are required for the tandem multiplex real-time assay, compared to four extractions and reverse transcription reactions for the individual assays. in our laboratory the consumable costs are approximately aud for the individual assays compared to aud for the tandem multiplex real-time assay. further work is planned to incorporate the picornavirus and human metapneumovirus assays into the tandem multiplex real-time pcr assay, but so far we have been unable to design or use published real-time pcr primers and probes that detect all types of these viruses with the same sensitivity as our nested assays. the multiplex tandem pcr assay described has the potential to test for a much larger number of infectious agents, or to look for multiple targets in single agents. in this study, enrichment pcr-b contained only seven primer pairs, so it will be possible to add extra primers to detect a broader range of known or newly described agents. for example, it is planned to include primers directed against adenoviruses other than group b, and a broader range of haemophilus influenzae serogroups. similarly, continued experience with the assay and further assessment of the role of novel agents in respiratory disease, may lead to the removal of some primer pairs from the pcr mixes. this test also allows the flexibility to determine which of the organisms included in the enrichment pcr are to be included in the second round, usually with the aim of restricting the initial testing to the most common pathogens. if this is negative, tests for other organisms included in the enrichment pcr can then be performed without the need for more sample or further cdna synthesis. bacterial and viral rna and dna was extracted from respiratory specimens including nose swabs, throat swabs, pernasal aspirates and sputum samples, using a modified liquid sample protocol with the x-tractor gene instrument (corbett life science, australia). standardized doses of equine herpesvirus type (ehv ) and ms rna coliphage (ms ) were added to the lysis buffer supplied with the kit to monitor the efficiency of sample extraction, removal of reverse transcription and pcr inhibitors, and cdna production [ , ] . the ct values expected for the ehv and ms assays were between and , and samples with ct values greater than were retested. taqman primers and probes listed in table , were designed in-house using primer express software (applied biosystems, usa), with the exception of those for the influenza virus a matrix gene [ ] . the letter in the final column of the table indicates which of the enrichment pcr mixes contained the primers, and the number indicates which of the real-time pcr mixes contained the primers and probes. at present, not all of the real-time pcr mixes contain uniquely specific fluorophore-labelled probes, so further confirmation of some positive results is required. for example, in the parainfluenza mixes the types and probes both have fam labels and the type a and b probes both have vic labels, so individual real-time pcrs may be performed from the original enrichment pcr material if further typing is required. the mixes were prepared in large volumes, stored in single use aliquots at - °c, and batches were quality tested prior to use. enrichment pcr assays a & b, were set up in -well plates (axygen scientific, usa) using a liquid handling robot (cas corbett life science, australia) to add pcr reaction mix and the sample or control nucleic acid extracts. the first enrichment pcr used the superscript iii platinum one-step rt-pcr system (invitrogen, usa). the µl reaction mix a contained primers each at . - . µm, the initial enrichment pcr products were diluted : in pcr grade water, and a . µl sample was added to a µl reaction mix in tube rings (gendisc- , corbett life science, australia) containing - oligonucleotide primers at . µm, . µm taqman probes, mm mgcl , . mm dntp, and . unit of i-startaq dna polymerase. the dilution and product transfer procedures were performed by a liquid handling robot (cas corbett life science, australia). the reaction was cycled on a rotorgene real time thermocycler (corbett life science, australia). the cycling conditions were °c for min, followed by cycles of °c for sec, °c for sec and °c for sec. probe emission signals were acquired during the sec extension step of the cycling programme. the relatively long second annealing step was found to improve the sensitivity of the multiple primer and probe real-time pcr assay (data not shown). the singleplex real-time pcr assays used in the sensitivity comparison with the tandem multiplex real-time assays were performed as for the enrichment pcr assays described above, except that the only a single primer pair and the appropriate fluorophore-labelled oligonucleotide probe ( . µm) were included in the pcr reaction mix. the number of cycles was increased to , the denaturation, annealing and extension steps were reduced to , and secs, respectively, and performed on the corbett rg thermocycler (corbett life science, australia). the nested pcr assays used in the sensitivity comparison assays are mostly "in-house-modified" assays and have been used in our diagnostic laboratory for many years. since they are slowly being superseded by real-time amplification assays, the primer sequences have not been listed in this article but are available on application to the authors. briefly, the nested assays used µl of nucleic acid sample in a µl reaction volume in the superscript iii platinum one-step rt-pcr (invitrogen, usa) or the amplitaq gold dna polymerase system (applied biosystems, usa). appropriate enzyme activation was followed by cycles of °c for sec, °c for sec and °c for sec. similar conditions were used for the second pcr assay with . µl of the initial pcr product as the target dna. amplicons were detected by ethidium bromide agarose gel electrophoresis. the picornavirus and human metapneumoviruses were detected using traditional semi-nested pcr assays as we have not yet been able to design real-time pcr assays to reliably detect all strains of those viruses. the picornavirus pcr utilized published primer sets targeting the ' untranslated region of the virus genome [ , ] and enterovirus or rhinovirus identification was confirmed by dna sequencing. the in-house human metapneumovirus assay targets the matrix protein gene of the virus. well characterized dna and rna control materials were tested in traditional nested pcr assays, individual real-time assays and in the multiplex tandem real-time pcr assay. a collection of respiratory samples from hospitalized children was tested in the multiplex tandem real-time assay. the samples were received at the pathwest laboratory medicine wa facility for the routine investigation of respiratory infection and had previously been tested for a narrow range of respiratory pathogens. samples had been stored at - °c. no-template controls were included in the extraction process between every five samples and treated as samples for the completion of the assays. since all pcr mixes were quality checked for all agents prior to use, and the internal controls cover the extraction, inhibitor removal and cdna production processes, specific positive control materials were only included in the real-time taqman pcr assays. those control materials were well-characterized enrichment pcr products stored in aliquots at - °c. a tandem multiplex real-time assay is presented which detects a comprehensive range of respiratory pathogens from a specimen sample of µl. the initial enrichment pcr eliminates the requirement for individual reverse transcription steps for each of the rna viruses, resulting in considerable cost and specimen volume benefits. the secondary multiplex real-time pcr utilizes probes labeled with different fluorophores to facilitate a rapid result. the technique may be used in the routine diagnostic setting or in an outbreak or pandemic situation, to facilitate testing for a range of common, exotic and emerging pathogens. extra primers and probes for novel agents may be easily accommodated in this technique. identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia identification of a novel coronavirus in patients with severe acute respiratory syndrome a newly discovered human pneumovirus isolated from young children with respiratory tract disease cloning of a human parvovirus by molecular screening of respiratory tract samples identification of a third human polyomavirus identification of a novel polyomavirus from patients with acute respiratory tract infections frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections improved diagnosis of the etiology of community-acquired pneumonia with real-time polymerase chain 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multiplex pcr assay for detecting herpesvirus dna in clinical samples enhanced reverse transcription-pcr assay for detection of norovirus genogroup i a '-nuclease real-time reverse transcriptase-polymerase chain reaction assay for the detection of a broad range of influenza a subtypes, including h n improved detection of rhinoviruses in nasal and throat swabs by seminested rt-pcr polymerase chain reaction for human picornaviruses key: cord- -v dr hm authors: albert, manuel; bécares, martina; falqui, michela; fernández-lozano, carlos; guerra, susana title: isg , a small molecule with huge implications: regulation of mitochondrial homeostasis date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: v dr hm viruses are responsible for the majority of infectious diseases, from the common cold to hiv/aids or hemorrhagic fevers, the latter with devastating effects on the human population. accordingly, the development of efficient antiviral therapies is a major goal and a challenge for the scientific community, as we are still far from understanding the molecular mechanisms that operate after virus infection. interferon-stimulated gene (isg ) plays an important antiviral role during viral infection. isg catalyzes a ubiquitin-like post-translational modification termed isgylation, involving the conjugation of isg molecules to de novo synthesized viral or cellular proteins, which regulates their stability and function. numerous biomedically relevant viruses are targets of isg , as well as proteins involved in antiviral immunity. beyond their role as cellular powerhouses, mitochondria are multifunctional organelles that act as signaling hubs in antiviral responses. in this review, we give an overview of the biological consequences of isgylation for virus infection and host defense. we also compare several published proteomic studies to identify and classify potential mitochondrial isgylation targets. finally, based on our recent observations, we discuss the essential functions of mitochondria in the antiviral response and examine the role of isg in the regulation of mitochondrial processes, specifically oxphos and mitophagy. the innate immune response is the first line of defense against microbial and viral infections. invading microorganisms produce danger-and pathogen-associated molecular patterns that interact with host pattern-recognition receptors, triggering several intracellular signaling cascades that activate nuclear factor kappa-b (nf-κb), mitogen-activated protein kinases (mapks) and interferon (ifn) regulatory factors (irfs), resulting in the expression of a broad array of proteins involved in host defense such as type-i ifns and proinflammatory cytokines [ , ] . the release of type-i ifns has both autocrine and paracrine effects via ifnα/β receptors (ifnars) on the cell surface. binding to ifnars leads to the activation of the janus kinase-signal transducer and activator of transcription proteins (jak-stat) signaling pathway and the formation of the interferon-stimulated gene factor (isgf ) complex, with the subsequent expression of ifn-stimulated genes [ ] that establish an antiviral state and play important roles in determining the host innate and adaptive immune responses [ ] . one of the most highly induced genes in the type-i ifn signaling cascade is isg (interferon-stimulated gene ), which encodes a small ubiquitin-like protein involved in a post-translational modification ( ) . intracellular isg can be processed into its mature form and conjugated to de novo synthesized proteins in a process termed isgylation. isg processing exposes its carboxy-terminal lrlrgg motif, allowing its conjugation to lysine residues in target proteins to modulate their function. in addition, isgylation is reversible due to the action of the protease usp , which also regulates ifnar-mediated signaling ( ) . isg can remain unconjugated within the cell, regulating protein activity ( ), or be secreted as a cytokine, acting as a chemotactic and stimulating factor for immune cells ( ) . binding of isg to lfa- integrin receptor on the surface of nk cells promotes the activation, production and release of ifn-γ il- after il- priming. moreover, extracellular isg is able to form dimers/multimers through cysteine residues, to modulate cytokine levels. isg conjugation to target proteins is a covalent and reversible process through the action of a -kda deisgylase enzyme, ubiquitin-specific protease (usp ) [ , ] . interestingly, both isg and its conjugating and deconjugating enzymes are upregulated by type-i ifn [ ] , as well as by other stimuli such as type-ii and type-iii ifns [ ] [ ] [ ] , lipopolysaccharide [ ] , retinoic acid [ ] , dna damage or genotoxic reagents [ ] . usp not only acts as a deconjugating enzyme, but also as a negative regulator of the type-i ifn pathway (figure ) , with important implications in antiviral and antibacterial responses, immune cell development, autoimmune diseases and cancer [ ] . in humans, isg binds to usp , increasing its stability and leading to a decrease in ifn-α/β signaling. consequently, isg deficiency results in low usp levels, and therefore a sustained elevation in figure . intracellular and extracellular activities of isg . different stimuli trigger the expression of isg , which is produced as a precursor of kda with two ubiquitin-like domains linked by a hinge region ( ) . intracellular isg can be processed into its mature form and conjugated to de novo synthesized proteins in a process termed isgylation. isg processing exposes its carboxy-terminal lrlrgg motif, allowing its conjugation to lysine residues in target proteins to modulate their function. in addition, isgylation is reversible due to the action of the protease usp , which also regulates ifnar-mediated signaling ( ) . isg can remain unconjugated within the cell, regulating protein activity ( ), or be secreted as a cytokine, acting as a chemotactic and stimulating factor for immune cells ( ) . binding of isg to lfa- integrin receptor on the surface of nk cells promotes the activation, production and release of ifn-γ il- after il- priming. moreover, extracellular isg is able to form dimers/multimers through cysteine residues, to modulate cytokine levels. isg conjugation to target proteins is a covalent and reversible process through the action of a -kda deisgylase enzyme, ubiquitin-specific protease (usp ) [ , ] . interestingly, both isg and its conjugating and deconjugating enzymes are upregulated by type-i ifn [ ] , as well as by other stimuli such as type-ii and type-iii ifns [ ] [ ] [ ] , lipopolysaccharide [ ] , retinoic acid [ ] , dna damage or genotoxic reagents [ ] . usp not only acts as a deconjugating enzyme, but also as a negative regulator of the type-i ifn pathway (figure ) , with important implications in antiviral and antibacterial responses, immune cell development, autoimmune diseases and cancer [ ] . in humans, isg binds to usp , increasing its stability and leading to a decrease in ifn-α/β signaling. consequently, isg deficiency results in low usp levels, and therefore a sustained elevation in isg expression. this role for isg , which is absent in mice, seems to be predominant in humans, since patients appear not to be more susceptible to viral infections [ , ] . beyond the above-mentioned forms of isg -conjugated to target proteins or unconjugated within the cell-isg is also secreted into the serum, mainly by granulocytes via their secretory pathway [ ] . lymphocyte function-associated antigen receptor (lfa ) has recently been identified as the cellular receptor for isg ( figure ). isg binding to lfa triggers the activation of src family kinases, promoting ifn-γ and interleukin- (il- ) secretion in natural killer (nk) cells and, likely, also t-lymphocytes [ ] . the role of isg as an inductor of ifn-γ secretion seems to be the basis for the increased susceptibility to mycobacterial diseases in patients lacking a functional form of isg [ ] . secreted isg has also been described to promote nk [ ] and dendritic cell [ ] maturation, and to act as a chemotactic factor for neutrophils [ ] . along this line, a recent study highlighted the presence of dimeric and multimeric forms of extracellular isg important for its cytokine activity during parasite infection, and speculated on the existence of an unknown isg receptor on dendritic cells that mediates chemotaxis of these cells to the site of infection and il- β production [ ] . although there are several features of isg that are shared with ubiquitin, specially its structure, conjugation and deconjugation mechanisms [ ] , isgylation has not been shown to stimulate proteasomal degradation of its substrates [ ] . furthermore, some of the isgylation consequences are exerted by restricting the ubiquitin system, what might be mediated through the conjugation of isg to different e and e ubiquitin-conjugating enzymes [ ] , or even through the formation of mixed ubiquitin-isg chains [ ] . as a result, isgylation can decrease the polyubiquitylated proteins levels and downregulate protein turnover by the proteasome system [ ] . additionally, unlike ubiquitin, no poly-isg chains or specific isg -interacting motifs have been identified yet. in the following sections, we discuss the antiviral mechanisms mediated by isgylation of both viral and cellular proteins, with a focus on mitochondrial proteins, as we recently showed that isg modulates essential mitochondrial metabolic processes such as respiration and mitophagy in macrophages, with important implications for innate immune responses [ ] . the antiviral activity associated with isg and/or isgylation has been widely described since the first observation that isg -/mice were more susceptible to viral infections than their wild-type counterparts, albeit the role of isg and isgylation in viral life cycles is specific to the virus involved [ ] . early studies using isg -/mice demonstrated that isg has a protective effect against lethal infection by influenza virus, herpes simplex virus (hsv- ) and sindbis virus (sinv) [ ] . similarly, mice deficient in ube l-the e enzyme of isg -were also more susceptible to lethal infection by sinv [ ] . moreover, exogenous expression of wild-type isg by recombinant chimeric sinv protected ifnar-/-mice against systemic and lethal infections, whereas expression of isg mutants unable to conjugate to proteins did not show this protective effect [ ] , indicating an intrinsic antiviral role for isgylation. it should be noted that such an antiviral effect could be due to the conjugation of isg to viral and/or cellular proteins. by contrast, free isg , but not isgylation, has been described to promote antiviral responses against chikungunya virus (chikv) infection [ ] . to date, an antiviral effect mediated by isg or isgylation has been described using in vitro and/or in vivo systems for many other dna and rna viruses, including hepatitis b virus [ ] , vesicular stomatitis virus [ , ] , respiratory syncytial virus [ , ] , human immunodeficiency virus type (hiv- ) [ ] , and ebola virus [ ] . the antiviral effect of isg and isgylation has also been described against viruses of the genera novirhabdovirus, birnavirus and iridovirus in zebrafish, an example of the evolutionary conservation of the antiviral role of isg among vertebrates [ ] . given the importance of the antiviral response governed by isg , it is not surprising that viruses have evolved strategies to counteract its antiviral effects. for example, influenza b virus (ibv) ns protein [ ] , vaccinia virus e protein [ ] , and human cytomegalovirus (hcmv) ie and pul proteins obstruct isg antiviral action by preventing isgylation [ ] . similar mechanisms are also described for orthonairovirus and arterivirus otu-domain-containing proteases [ ] and for coronavirus papain-like proteases (plpro), which cleave isg from target proteins. remarkably, a plpro inhibitor was shown to protect mice from lethal infection in vivo [ ] . surprisingly, it has been reported that isgylation is necessary for robust production of hepatitis c virus (hcv), conferring a novel role for isg as a proviral factor that promotes virus production. indeed, in human hepatocytes, sirna silencing of isg was sufficient to both inhibit hcv replication and increase ifn expression [ ] . several reports have now highlighted a role for isg in the monitoring of hcv replication in cell cultures, as well as in the maintenance of hcv in liver, and pinpoint isg as among the predictor genes for non-response to ifn therapy [ ] . regarding the direct antiviral effect of isgylation via conjugation to viral proteins, perhaps the best-known example is the influenza a virus (iav) ns protein. this non-structural protein is abundantly expressed in infected cells and acts in multiple stages of the viral cycle, with important roles in ifn antagonism including sequestering double-stranded rna (dsrna), inhibiting dsrna-activated protein kinase (pkr) and contributing to the nuclear export of viral mrnas while blocking the splicing and export of cellular mrnas [ ] . seven lysine (k) residues in the ns protein were identified as potential target sites of isgylation [ ] . specifically, isg binding to k , which is part of the ns nuclear-localization signal, prevents its interaction with importin-α, inhibiting the translocation of ns to the nucleus and therefore repressing iav replication and viral rna processing [ ] . moreover, isgylation of the iav ns protein blocks its ability to counteract the innate immune response, prevents its interaction with pkr and, therefore, restores ifn-induced antiviral activities against iav [ ] . beyond ns , influenza virus nucleoprotein (np) and matrix protein (m ) have also been reported as targets of isg conjugation. isgylated np hinders the oligomerization of the more abundant unconjugated np, acting as a dominant-negative inhibitor of np oligomerization, impeding the formation of viral ribonucleoproteins and causing decreased viral protein synthesis and virus replication [ ] . interestingly, this study also identified a new role for influenza b virus ns in the sequestration of isgylated viral proteins, especially isgylated nps, which is perhaps an evolutionary mechanism to block the antiviral effect of isgylation. another example of isgylation of a viral protein with antiviral effects is the a protease ( apro) of coxsackievirus b (cvb ). isg conjugation to apro inhibits its ability to cleave the eukaryotic initiation factor eif g in cardiomyocytes, hindering the translational shutoff induced by cvb infection [ ] . consequently, isg conjugation to cvb leads to a reduction in virus titers and limits inflammatory cardiomyopathy, heart failure and lethality [ ] . similarly, isgylation of the hcmv scaffold protein pul interferes with the viral modulation of the innate immune response. specifically, isgylation of pul at k and k inactivates its function in the downregulation of tnfα-mediated nf-κb activation, suppressing hcmv growth [ ] . finally, another example of an isgylated viral protein is the human papillomavirus (hpv) l capsid protein. isgylated l proteins were shown to be incorporated into hpv pseudoviruses, resulting in a reduced infectivity; the precise mechanism that mediates this inhibitory effect remains elusive [ ] . knowledge about the impact of host protein isgylation in virus replication and cell homeostasis is still scant. in contrast to ubiquitylation, the molecular effect of isg conjugation on target proteins is not always clear. protein isgylation has been reported to increase protein degradation by selective autophagy [ ] , but there are also many examples where isgylation inhibits ubiquitylation, frustrating proteasome-mediated degradation of target proteins [ ] [ ] [ ] . with regard to proteins involved in antiviral response, many effectors of ifn signaling such as pkr [ ] , retinoic acid-inducible gene-i (rig-i) [ ] and myxoma resistance protein (mxa) [ ] have been reported to be targets of isgylation. pkr isgylation at k and k , both located in the dsrna-binding motif, triggers its activation. this modification occurs in the absence of viral rna and leads to the phosphorylation of eif α, preventing protein translation [ ] and suggesting that isgylation might mediate the activation of pkr in response to stressful stimuli beyond viral infection. further, isg conjugation to rig-i decreases rig-i cellular levels and downregulates rig-i-mediated signaling. accordingly, isgylation of rig-i represents a negative feedback loop that might control the strength of the antiviral response [ ] . interestingly, free isg also regulates rig-i levels by promoting the interaction between rig-i and the autophagic cargo receptor p , mediating rig-i degradation via selective autophagy [ ] . the interferon-induced mxa protein is also a target of isgylation, though the effect of this modification is not clear. other proteins involved type-i ifn signaling and regulation, such as components of the jak-stat pathway or regulators of signal transduction (e.g., jak and extracellular signal-regulated kinase [erk ]), are also bound by isg , although the functional consequences of isgylation remain unknown [ , ] . moreover, interferon regulatory factor (irf ), stat and the actin-binding protein filamin b are also targets for isg conjugation, with implications in the development of the innate immune response. irf is isgylated at k , k and k , which attenuates its interaction with the peptidyl-prolyl isomerase pin , preventing irf ubiquitylation. thus, isgylation of irf sustains its activation and enhances irf -mediated antiviral responses by inhibiting its degradation [ ] . in a similar manner, isgylation of phosphorylated stat (pstat ) inhibits its polyubiquitylation and further proteasomal degradation, supporting sustained stat activation [ ] . isgylation of filamin b, which acts as a scaffold of ifn signaling mediators, negatively regulates ifnα-induced c-jun n-terminal kinases (jnk) signaling, preventing apoptosis induction [ ] . beyond antiviral response, isgylation has been described to block the process of virus budding by interfering with the endosomal sorting complexes required for transport (escrt) machinery. for example, isgylation of chmp triggers its aggregation and the sequestration of the vps cofactor lip , impairing the membrane recruitment of vps and its interaction with the gag budding complex of avian sarcoma leukosis virus and hiv- , leading to the inhibition of virus release from the cell [ ] . similarly, isgylation of tumor susceptibility gene protein (tsg ), another component of the escrt sorting complex, inhibits the trafficking of viral hemagglutinin to the cell surface during iav infection [ ] , blocking virus release. isg has also been described to inhibit the interaction of hiv- gag protein with tsg , underscoring a critical role of isg in the ifn-mediated inhibition of hiv- budding and release [ ] . this sorting mechanism is also used in the generation of exosomes, which are small vesicles secreted to the extracellular environment by most cell types. interestingly, isgylation of tsg has been recently reported to inhibit exosome secretion [ ] . the above examples serve to illustrate the relevance of isgylation in the induction and regulation of the antiviral response (for a more complete review of isgylated cellular proteins see reference [ ] ), and highlight the complexity of fully understanding the consequences of isgylation in the regulation of biochemical processes where it is involved. although the significance of isgylation of host proteins has been elucidated for only a small set of cellular proteins, isgylation has a broad target specificity, and there is increasing evidence for its role in regulating many cellular functions. to address this concept, several proteomic studies have been performed to determine isgylated host proteins. zhao et al. [ ] transfected a tagged isg protein into ifn-stimulated hela cells, and used affinity selection to identify isgylated proteins. in a similar approach, giannakopoulos et al. [ ] used ifn-stimulated usp -/-mouse embryonic fibroblasts and human u cells to detect up to proteins conjugated to endogenously-expressed isg . a third proteomic study [ ] identified isgylated cellular proteins in ifn-stimulated a human lung adenocarcinoma cells stably expressing flag-isg . more recently, peng et al. [ ] examined isgylated proteins in influenza virus-infected a cells, identifying a total of cellular proteins in addition to viral ns protein. we have surveyed the proteins identified by these four studies, which rendered up to cellular proteins. identified proteins include, as previously outlined [ ] , abundant constitutively expressed proteins as well as diverse interferon-induced proteins. interestingly, there is only a low degree of overlap between the studies, and only four proteins are common to all four analyses (the glycolytic enzymes aldo and eno , the peroxiredoxin prdx , and stat ). these discrepancies may reflect the different transcriptional/translational patterns of the different cell lines included in each study, as it is believed that the biological effects of isgylation are dynamic and cell type/tissue-specific [ ] . we used david bioinformatics resources [ , ] to determine the subcellular localization of the proteins identified as isgylation targets in the aforementioned studies, with the aim to obtain a comprehensive picture of the broad range of actions of isg . in agreement with a previous report [ ] , our analysis ( figure ) shows that isg -targeted proteins are found almost throughout the cell, including nucleus, perinuclear space, cytosol, mitochondria, rough endoplasmic reticulum and cell membranes [ ] . moreover, a similar percentage of isgylation targets were predicted to be located in the nucleus, cytoplasm, extracellular space or as secreted proteins (figure ) . interestingly, proteins associated with cytoskeleton and cell junctions represent a significant percentage of the isg target proteins. other cell structures such as the melanosome or myelin sheath were also represented in the study, perhaps accounting for a specific role of isgylation in these organelles. the potential role of isg in mitochondria seems to be relevant, as a recent study predicted that % of free isg was localized to mitochondria [ ] . in our own analysis of the above proteomic studies, fifty-two isgylated proteins were predicted to localize to mitochondria, representing about % of the total isg target proteins ( figure ). further examination of these potentially isgylated proteins indicate that different mitochondrial processes could be affected by isg conjugation (table ) . remarkably, several subunits of the atp synthase (complex v of the respiratory chain) appear to be isg targets, which may be of relevance as mitochondrial atp production is the main source of energy for the cell. in line with these observations, our recent work linked isg to the control of the mitochondrial oxidative metabolism in macrophages in the context of viral infection [ ] . based on the evident association between isg and mitochondria, we will briefly review the role of mitochondria as antiviral mediators and targets of ubiquitin-like modifiers, focusing on the current knowledge about isg -and isgylation-mediated regulation of these multifunctional organelles. included in each study, as it is believed that the biological effects of isgylation are dynamic and cell type/tissue-specific [ ] . we used david bioinformatics resources [ , ] to determine the subcellular localization of the proteins identified as isgylation targets in the aforementioned studies, with the aim to obtain a comprehensive picture of the broad range of actions of isg . in agreement with a previous report [ ] , our analysis ( figure ) shows that isg -targeted proteins are found almost throughout the cell, including nucleus, perinuclear space, cytosol, mitochondria, rough endoplasmic reticulum and cell membranes [ ] . moreover, a similar percentage of isgylation targets were predicted to be located in the nucleus, cytoplasm, extracellular space or as secreted proteins (figure ) . interestingly, proteins associated with cytoskeleton and cell junctions represent a significant percentage of the isg target proteins. other cell structures such as the melanosome or myelin sheath were also represented in the study, perhaps accounting for a specific role of isgylation in these organelles. the potential role of isg in mitochondria seems to be relevant, as a recent study predicted that % of free isg was localized to mitochondria [ ] . in our own analysis of the above proteomic studies, fifty-two isgylated proteins were predicted to localize to mitochondria, representing about % of the total isg target proteins ( figure ). further examination of these potentially isgylated proteins indicate that different mitochondrial processes could be affected by isg conjugation (table ) . remarkably, several subunits of the atp synthase (complex v of the respiratory chain) appear to be isg targets, which may be of relevance as mitochondrial atp production is the main source of energy for the cell. in line with these observations, our recent work linked isg to the control of the mitochondrial oxidative metabolism in macrophages in the context of viral infection [ ] . based on the evident association between isg and mitochondria, we will briefly review the role of mitochondria as antiviral mediators and targets of ubiquitin-like modifiers, focusing on the current knowledge about isg -and isgylation-mediated regulation of these multifunctional organelles. [ , [ ] [ ] [ ] predicted to locate to mitochondria. proteins are grouped according to biological functions. acyl-coa thioesterase (acot ) [ ] complement c q binding protein (c qbp) [ ] receptor for activated c kinase (rack ) [ ] solute carrier family member (slc a ) [ ] solute carrier family member (slc a ) [ ] staphylococcal nuclease and tudor domain containing (snd ) [ ] negative regulation of apoptotic process nme/nm nucleoside diphosphate kinase (nme ) [ ] annexin a (anxa ) [ , ] glutathione s-transferase pi (gstp ) [ ] heat shock protein family a (hsp ) member (hspa ) [ ] interferon-induced protein with tetratricopeptide repeats (ifit ) [ ] positive regulation of protein insertion into mitochondrial membrane involved in apoptotic signaling pathway stratifin (sfn) [ ] tyrosine -monooxygenase/tryptophan -monooxygenase activation protein beta (ywhab) [ ] tyrosine -monooxygenase/tryptophan -monooxygenase activation protein épsilon (ywhae) [ ] tyrosine -monooxygenase/tryptophan -monooxygenase activation protein gamma (ywhag) [ ] tyrosine -monooxygenase/tryptophan -monooxygenase activation protein theta (ywhaq) [ ] tyrosine -monooxygenase/tryptophan -monooxygenase activation protein zeta (ywhaz) [ ] atp biosynthetic process atp synthase, h+ transporting, mitochondrial f complex, alpha subunit , cardiac muscle (atp a ) [ , ] atp synthase, h+ transporting, mitochondrial f complex, beta polypeptide (atp b) [ , ] atp synthase, h+ transporting, mitochondrial fo complex subunit g (atp l) [ ] oxidation-reduction process aldehyde dehydrogenase family member a (aldh a ) [ ] fatty acid synthase (fasn) [ , ] glutathione-disulfide reductase (gsr) [ ] lactate dehydrogenase b (ldhb) [ ] malic enzyme (me ) [ ] peroxiredoxin (prdx ) [ , , ] peroxiredoxin (prdx ) [ ] sorbitol dehydrogenase (sord) [ ] superoxide dismutase , soluble(sod ) [ ] thioredoxin reductase (txnrd ) [ , ] thioredoxin (txn) [ ] aminoacyl-trna synthetase alanyl-trna synthetase (aars) [ ] glycyl-trna synthetase (gars) [ ] phenylalanyl-trna synthetase , mitocondrial (fars ) [ ] tricarboxylic acid cycle malate dehydrogenase (mdh ) [ ] malate dehydrogenase (mdh ) [ ] glycolisis oxoglutarate dehydrogenase (ogdh) [ ] pyruvate kinase, muscle (pkm) [ , , ] chaperone chaperonin containing tcp subunit (cct ) [ ] heat shock protein alpha family class b member (hsp ab ) [ , , ] heat shock protein family a (hsp ) member a (hspa a) [ , ] heat shock protein family d (hsp ) member (hspd ) [ , ] ion channel chloride intracellular channel (clic ) [ , ] annexin a (anxa ) [ ] other functions creatine kinase, mitochondrial b (ckmt b) [ ] ubiquitin-like modifier activating enzyme (uba ) [ ] leucine aminopeptidase (lap ) [ ] -aminoimidazole- -carboxamide ribonucleotide formyltransferase/imp cyclohydrolase (atic) [ , ] clathrin heavy chain (cltc) [ , ] queuine trna-ribosyltransferase accessory subunit (qtrt ) [ ] enoyl-coa hydratase and -hydroxyacyl coa dehydrogenase (ehhadh) [ ] atp binding cassette subfamily f member (abcf ) [ ] mitochondria have myriad functions in the cell although they are best known for providing energy in the form of atp and for controlling metabolism to maintain energy homeostasis. owing to their endosymbiotic origin, mitochondria have their own genome, a single -kb circular dna which codes for mitochondrial proteins, ribosomal rnas and transfer rnas [ ] . the remainder of mitochondrial proteins are encoded by nuclear dna and are then transported to the mitochondria through the recognition of amino acid sequences known as mitochondrial targeting signals [ ] . as double-membrane organelles, mitochondria have an outer mitochondrial membrane (omm), where proteins responsible for transport of different molecules are embedded [ ] ; an intermembrane space (ims) and an inner mitochondrial membrane (imm), where electron transport chain (etc) proteins are localized and oxidative phosphorylation (oxphos) and atp production takes place [ ] , and a mitochondrial matrix (mm), compartment, where many metabolic pathways occur, such as the tricarboxylic acid cycle, fatty-acid oxidation, synthesis of biomolecules and regulation of apoptosis [ ] . the proper development of mitochondrial processes is critical for immune response, as the susceptibility to microbial infections and the risk of systemic inflammatory responses increases considerably when these organelles malfunction [ , ] . mitochondria are important for antiviral signaling. during rna-virus infection, viral rnas are initially recognized by cytoplasmic sensors, mainly rig-i-like receptors (rlrs) [ ] , whose interaction with mitochondria is essential for the coordination and development of an adequate antiviral response. the common structure of rlrs consists of a carboxy-terminal regulatory domain, a central rna helicase domain and amino-terminal caspase recruitment domains (cards) [ ] . after binding to viral rna, rlrs trigger ifn-mediated antiviral responses through their interaction with mitochondrial antiviral-signaling protein (mavs), a card-containing omm protein [ ] . the card-card interaction between rlrs and mavs causes mavs polymerization and consequent recruitment of a variety of downstream effectors, including tumor necrosis factor receptor-associated factor family proteins, ikb kinase epsilon (ikkε) and tank binding kinase , among others [ ] . this "mavs signalosome" activates nf-κb, irf and irf , promoting the expression of type-i ifn and antiviral molecules [ ] . given the central role of mavs in mitochondrial antiviral signaling, mavs and both upstream and downstream molecules are under tight regulation to ensure an adequate response [ , ] . mitochondria are dynamic organelles that undergo constant fusion and fission to regulate their morphology, activity and turnover according to the metabolic needs of the cell [ ] , and these mitochondrial dynamics are involved in the regulation of mitochondrial immune functions. mitofusins and optic atrophy protein are responsible for mitochondrial fusion, whereas the cytosolic gtp-ase dynamin-related protein (drp ) mediates mitochondrial fission through its interaction with adaptor proteins in the omm [ , ] . interestingly, these proteins have been shown to be implicated in the regulation of various mitochondrial immune-relevant processes, such as rlr signaling [ , , ] , apoptosis [ , ] , autophagy and mitochondrial bioenergetic conditions [ ] , which are important mechanisms to combat viral infections. mitophagy is a selective autophagic process in which defective mitochondria are engulfed in autophagosomes and eliminated by fusion with lysosomes [ ] . damaged mitochondria constitute a signal for the recruitment of pten-induced putative kinase protein (pink ), which surrounds the mitochondrial surface. the accumulation of pink and its kinase activity promote the translocation of the e ubiquitin ligase parkin from the cytosol to dysfunctional mitochondria, triggering the ubiquitylation of omm proteins. the formation of ubiquitin chains by parkin favors the binding of adaptor proteins (e.g., p and optineurin), which mediate the interaction with autophagosomes and the further degradation of dysfunctional mitochondria [ , ] . mitophagy is closely related to mitochondrial dynamics, as mitochondrial fragmentation promotes mitophagy whereas mitochondrial fusion hinders this process [ , ] . interestingly, defective mitochondria can play either positive or negative roles against viruses. for example, alterations in mitochondrial respiration trigger the production of reactive oxygen species (ros) which, in addition to being harmful for the cell in high levels, play an important role as second messengers in diverse intracellular signaling pathways [ ] . in the context of antiviral signaling, ros are involved in the regulation of the rlr pathway, potentiating rlr-mavs signaling and the production of type-i ifn [ ] . because healthy mitochondria are required for an adequate metabolic state and the activation of apoptotic processes [ , , ] , mitophagy must be finely regulated to modulate the mitochondria-mediated antiviral response. the implications of mitochondria in innate immunity are enormous. we have briefly discussed the interplay between different mitochondrial pathways, such as rlr signaling, mitochondrial dynamics, mitophagy, ros production and apoptosis, in the protection against viruses. however, mitochondria perform a plethora of functions in the establishment of a defensive state of the cell, which have been thoroughly reviewed by others [ , [ ] [ ] [ ] [ ] [ ] . moreover, the regulation of mitochondrial function is not only carried out by the host, but also by viruses with the aim to shut down defense mechanisms, complete their life cycle and spread [ ] , underscoring the relevance of mitochondria in antiviral response. such critical roles of mitochondria in the control of pathogen invasion and maintenance of cellular homeostasis must be strictly coordinated. as we previously discussed, ubiquitin and ubiquitin-like ptms are key regulatory processes of the innate and adaptive immune response against viruses, and both processes are finely regulated by mitochondria. in this regard, there is a broad spectrum of ptms [ ] , some of which occur within mitochondria, which are responsible for modifying their internal state and function [ , ] . thus, mitochondria are targets of ubiquitin and ubiquitin-like proteins, such as small ubiquitin-like modifiers (sumos) and isg . ubiquitin is a highly conserved . -kda protein known as a master regulator of cellular processes. its covalent conjugation to target proteins has proteolytic and non-proteolytic or regulatory outcomes, which fine-tune protein function and recycling [ ] . indeed, ubiquitylation is essential for the regulation of many mitochondrial processes, such as mitophagy [ , [ ] [ ] [ ] , mitochondrial dynamics [ , ] , and mitochondria-related immune signaling [ , , ] , establishing the importance of this modifier in the homeostasis of these organelles. sumos are a family of highly conserved -kda proteins that are essential in eukaryotic cells. similar to ubiquitin, sumo conjugation to specific lysine residues of target proteins (sumoylation) alters their function, their interaction with other proteins, and their stability [ , ] . although the major role of sumoylation is in the regulation of nuclear processes [ ] , it also targets mitochondria. sumos have been proven to be involved in the regulation of mitochondrial dynamics by binding to drp [ ] , with an important implication in programmed cell death [ ] [ ] [ ] . furthermore, sumoylation of the mitochondrial oxidative stress sensor dj- results in its stabilization and full activation, reinforcing its protective role against parkinson's disease [ ] , where mitochondrial dysfunction has great significance. although isg has been associated with mitochondria, its functions, both free or conjugated to mitochondrial proteins, are still being examined. one exception is the protein parkin. while not strictly a mitochondrial protein, its translocation to the omm from the cytoplasm is essential for parkin-mediated mitophagy [ ] . isg conjugation to parkin enhances its e ubiquitin ligase activity and its cytoprotective effect in parkinson's disease [ ] , an example of how isgylation affects mitochondrial processes. isg and isgylation for the regulation of mitochondrial metabolism [ ] . we undertook a comprehensive analysis of bone marrow-derived macrophages (bmdms) from wild-type and isg -/mice to interrogate how isg and isgylation could modulate the regulation of mitochondria in the context of stressful stimuli. monomeric isg and isgylated proteins were observed in mitochondrial fractions from wild-type bmdms after type-i ifn pre-treatment, and these proteins were preferentially located to the ims and imm (figure ) . given their localization, we hypothesized that isg and isgylation could impact mitochondrial respiratory metabolism, and we focused our study on oxphos and atp production. this analysis revealed that oxygen consumption and atp production were lower in isg -/-bmdms than in equivalent wild-type cells, indicative of defective oxphos. in accord with these observations, a clear difference in the distribution of etc supercomplexes was observed between the two groups, pointing to a possible role for isg in the correct assembly of etc proteins ( figure ). as recently reported by yoshizumi et al. [ ] oxphos activity is required for rlr-mediated antiviral signaling, and mice with oxphos defects showed increased susceptibility to viral infections. similarly, knockout mice for isg or the isg -activating e enzyme (ube l) were more susceptible to infection with many viruses than wild-type mice [ ] . since the lack of isg seems to cause alterations in oxphos, such increase in the sensitivity to viral infections in isg -/and ube l-/-mice might be explained as a result of defects in rlr-mediated antiviral responses, supporting the role of isg as a regulator of mitochondrial functions. regarding mitochondrial respiration byproducts, isg -/-bmdm also produced lower levels of ros. because ros production is tightly controlled by mitochondrial membrane potential [ ] , low levels of ros in isg -/-bmdm might be the result of abnormalities in the transmembrane proton gradient due to the absence of isg , and could affect the immune response against viral infections, as discussed earlier. mitochondrial ros also participate in the regulation of macrophage polarization [ ] and, interestingly, isg -/-bmdms displayed mixed features of m and m phenotypes, suggesting that alterations in mitochondrial oxphos could drive changes to immune cell function. finally, bmdms lacking isg accumulated non-functional mitochondria with an absence of parkin, suggesting that isg is also implicated in the regulation of mitophagy, perhaps through the control of parkin translocation from the cytosol (figure ) . taken together, these findings establish a relevant role for isg and isgylation in the control of mitochondrial oxphos and recycling, at least in murine bmdm, expanding the range of functions of this ptm and underscoring its importance in the regulation of essential cellular processes. viruses , , x for peer review of figure . impact of isg on mitochondrial activities. mitochondria are targets of isg and isgylation in murine bone marrow-derived macrophages (bmdms). isgylated proteins can be found in all mitochondrial localizations, mainly in the mitochondrial intermembrane space (ims) and inner mitochondrial membrane (imm), where free isg is also present. isg and isgylation are involved in the regulation of mitochondrial metabolism. absence of isg leads to alterations in oxphos, with lower oxygen consumption rates and atp production levels, in addition to aberrant etc supercomplexes assembly. such disruption of oxphos mechanisms decreases ros production, with repercussions for macrophage polarization. mitophagy is also altered in cells lacking isg . finally, isg -/-bmdm accumulate defective mitochondria and parkin cannot be found in mitochondrial extracts, suggesting that isg is important during the translocation of parkin from the cytoplasm to mitochondria. the functional significance of ptms in disease etiology, and the pathologic response to their disruption, is the subject of intense investigation. many of these reversible modifications act as regulatory mechanisms in mitochondria and show promise for mitochondria-targeted therapeutic strategies. with the advent of mass spectrometry-based screening techniques, there has been a vast increase in our current state of knowledge on mitochondrial ptms and their protein targets. detecting isgylated proteins in different organelles remains challenging, as it typically occurs in only a small portion of the total protein pool of the cell, albeit with essential roles in regulating protein fate and function. understanding the consequences of isgylation of mitochondrial proteins will require much work, but should be rewarding not only for developing new strategies to combat viral infections, but also for future applications in other biomedically relevant processes/diseases, for example inflammation, cancer and neurodegeneration. . impact of isg on mitochondrial activities. mitochondria are targets of isg and isgylation in murine bone marrow-derived macrophages (bmdms). isgylated proteins can be found in all mitochondrial localizations, mainly in the mitochondrial intermembrane space (ims) and inner mitochondrial membrane (imm), where free isg is also present. isg and isgylation are involved in the regulation of mitochondrial metabolism. absence of isg leads to alterations in oxphos, with lower oxygen consumption rates and atp production levels, in addition to aberrant etc supercomplexes assembly. such disruption of oxphos mechanisms decreases ros production, with repercussions for macrophage polarization. mitophagy is also altered in cells lacking isg . finally, isg -/-bmdm accumulate defective mitochondria and parkin cannot be found in mitochondrial extracts, suggesting that isg is important during the translocation of parkin from the cytoplasm to mitochondria. the functional significance of ptms in disease etiology, and the pathologic response to their disruption, is the subject of intense investigation. many of these reversible modifications act as regulatory mechanisms in mitochondria 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redox regulation of macrophage polarization we thank diego sanz for his technical assistance and kenneth mccreath for reviewing and correcting the manuscript. the authors declare no conflicts of interest. acknowledgments: we thank diego sanz for his technical assistance and kenneth mccreath for reviewing and correcting the manuscript. the authors declare no conflicts of interest.viruses , , key: cord- - ljxy authors: al-kassmy, jawad; pedersen, jannie; kobinger, gary title: vaccine candidates against coronavirus infections. where does covid- stand? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ljxy seven years after the middle east respiratory syndrome (mers) outbreak, a new severe acute respiratory syndrome coronavirus (sars-cov- ) made its first appearance in a food market in wuhan, china, drawing an entirely new course to our lives. as the virus belongs to the same genus of mers and sars, researchers have been trying to draw lessons from previous outbreaks to find a potential cure. although there were five phase i human vaccine trials against sars and mers, the lack of data in humans provided us with limited benchmarks that could help us design a new vaccine for coronavirus disease (covid- ). in this review, we showcase the similarities in structures of virus components between sars-cov, mers-cov, and sars-cov- in areas relevant to vaccine design. using the clinicaltrials.gov and world health organization (who) databases, we shed light on the current approved clinical trials worldwide in search for a covid- vaccine. the different vaccine platforms being tested are bacillus calmette–guérin (bcg) vaccines, dna and rna-based vaccines, inactivated vaccines, protein subunits, and viral vectors. by thoroughly analyzing different trials and platforms, we also discuss the advantages and disadvantages of using each type of vaccine and how they can contribute to the design of an adequate vaccine for covid- . studying past efforts invested in conducting vaccine trials for mers and sars will provide vital insights regarding the best approach to designing an effective vaccine against covid- . history always holds valuable lessons for us. as the influential chinese philosopher confucius once said, "study the past if you want to define the future". now, we certainly know that the novel severe acute respiratory syndrome coronavirus (sars-cov- ) is not entirely "novel" itself. multiple coronaviruses have been discovered in the past years [ ] . today, out of seven types of coronaviruses known to infect humans, only three are considered to be highly pathogenic: the sars-cov discovered in to be the cause of severe acute respiratory syndrome (sars), the mers-cov discovered in as the responsible virus for the middle east respiratory syndrome (mers), and the current sars-cov- causing coronavirus disease . the good news is that we find many similarities between the three coronaviruses. a review by wang et al. demonstrated the structural and immunological specific monoclonal antibodies (mabs) have previously been isolated [ ] [ ] [ ] [ ] . while many fail to cross-neutralize the sars-cov- , some mabs targeting the rbd were able to neutralize both viruses, making it a potentially interesting choice for vaccine design with a dual target. nevertheless, the non-rbd spike epitopes might be more immunogenic and have greater surface accessibility compared with rbd epitopes with bioinformatic analysis showing that the majority of high-score epitopes are located outside this domain [ ] . ten selected high-score epitopes in sars-cov- showed epitopes in the n-terminal region (ntd), the c-terminal of s , the n-terminal of s , the fusion protein (fp), and heptad repeat domain (hr ). only two were located in the rbd, and only one was considered conserved (located in the hr ). the hr region has previously been proposed as a pan-cov antiviral inhibitory target, and sars-cov studies have shown antibodies targeting this region have efficiently neutralized the virus in vitro, suggesting this domain might be interesting for a vaccine [ , ] . a recent cryogenic electron microscopy (cryo-em) analysis of the sars-cov spike protein after fusion also emphasizes the potential of the more conserved s , including the hr region, for vaccine developments for a wide range of sars-like cov [ ] . the s region of the sars-cov- spike protein, including the ntd, only shares % sequence identity with sars-cov compared with % within the s region; however, several predicted high-score epitopes were located in the ntd [ , ] . this region's potential for vaccine designs has been further established by a research team reporting neutralizing antibodies (nab) from recovered covid- patients targeting the ntd [ ] . even though these regions in s are less likely to be conserved, the antigenic potential might be superior to other conserved domains in s . while the s protein is by far the most studied region for vaccine designs, in silico analyses of potential immunogenic epitopes in the sars-cov- have suggested several domains within the n, m, and e protein as well as the non-structural proteins primarily focusing on t cell responses [ ] [ ] [ ] [ ] [ ] . using previously generated data from sars-cov with experimentally validated epitopes, seven areas were found within sars-cov- with % identity to sars epitopes and an % world population coverage [ ] . of these, two were found in the m protein and one in the n protein. lee et al., using a similar approach, also identified sars-cov-identical epitopes, with the majority being located in the n protein [ ] . kiyotani et al. compared human leucocyte antigens (hla) i and ii sars-cov- derived epitopes to both mers-cov and sars-cov and found a substantial number of hla-i epitopes shared by sars-cov and sars-cov- in both structural and non-structural proteins, whereas only epitopes located in orf ab were shared between all three viruses [ ] . the lack of shared t cell epitopes within all human cov structural proteins has previously been illustrated by liu et al., who found no conserved t cell epitopes between mers-cov, sars-cov, and two other human coronaviruses hcov-oc and hku [ ] . nevertheless, an informatics approach comparing the more conserved e protein of sars-cov- with taxonomically related cov including non-human cov suggests that some major antigenic epitopes in the envelope might play an important role and could be significant in potential partial protection arising from human-animal interaction [ ] . considering the risk of a future novel cov appearance, an approach looking at conserved areas within the same cluster of cov might be preferable for a more broadly protecting vaccine; nonetheless, considering the current pandemic, specific sars-cov- highly immunogenic targets might be preferable. since the first outbreak in , substantial work has been done in order to find protective vaccines against emergent coronaviruses, with several early predictions that another novel viral agent would emerge [ , , ] . various platforms have been evaluated and, while the majority were able to induce some level of antibodies and t cell responses, sterilizing immunity was not reported in several different animal models [ ] [ ] [ ] [ ] . the spike protein was the most extensively evaluated protein against both sars-cov and mers owing to its immunogenic properties. using various platforms such as viral vectors, nucleic acids, and subunits, the protein was shown to be able to induce potent humoral and cellular immune responses, which, in many cases, lead to some degree of protection or diminished viral shedding [ ] [ ] [ ] [ ] . the advantage of utilizing the whole spike compared with only s , rbd, or other subunits has been debated [ ] [ ] [ ] [ ] [ ] [ ] . looking at sars, several vaccines using different viral vectors or dna were able to induce high levels of neutralizing antibodies using the full-length s protein, which, in some models, provided protection against challenge [ ] [ ] [ ] [ ] [ ] . however, increased liver pathology was also reported in vaccinated animals after challenge, pointing to the risk of antibody disease enhancement (ade) when utilizing the full-length spike [ ] . this has led to several studies looking at protection following vaccination with various subunits of spike, including the s and rdb with promising results [ , , ] . similar considerations have been made in the quest of a mers-vaccine. the use of viral vectors, nanoparticles, proteins, dna with the full-length spike, or subunits like rbd and s protections has been observed to various degrees [ , , [ ] [ ] [ ] [ ] . in one study, increased lung hemorrhage was observed in animals vaccinated with s ; however, other groups reported no increase in lung pathology in vaccinated groups. even with the full-length spike underlining the risk of ade, using full-length or subunits of the spike protein would need further evaluation [ , ] . a few groups have examined the effect of non-spike proteins in challenge models against sars-cov [ , [ ] [ ] [ ] . while the protective efficacy was only slightly increased by the addition of m and e to the s vaccine using a parainfluenza virus type vector, no protection was observed without the s protein, underlining the importance of this protein [ ] . immunogenicity of mers-cov specific n vaccine has been shown, but the protective role has yet to be evaluated [ ] . however, a study using venezuelan equine encephalitis replicons encoding a sars-cov cd + t cell epitope conserved between sars-cov and mers-cov showed protection from a lethal challenge dose through interferon (ifn)-g production, implying the possible role for other antigens that are more conserved between the different coronaviruses in a vaccine strategy [ ] . when comparing different vaccine platforms, one study looked at the inactivated vaccines and adenovirus vectors expressing the s protein, or the n protein of sars reported increased protection when utilizing the whole inactivated virus. they argued that, perhaps, the exposure of several proteins would aid the immunogenic response [ , ] . however, historically, the inactivated vaccine produces a weaker immune response in humans, necessitating several prime-boost vaccinations for a sufficient response, making this a potentially less attractive platform [ ] . besides, using a whole inactivated mers-virus produced a hypersensitive reaction in the lungs, highlighting the possible adverse events with inactivated vaccines [ ] . the other vaccine platform tested in the study from see et al. was the adenoviral vector. for the whole inactivated vaccine, the immunogenic response was subsidiary, but a significant reduction in viral titers was reported in mice for both vaccines [ ] . moreover, van doremalen et al. tested the adenovirus-vectored vaccine chadox ncov- , which encodes the s protein of sars-cov- , in mice and in rhesus macaques. in mice, profiling of igg subclasses demonstrated a predominantly th response post vaccination and neutralizing antibodies were detected in all mice vaccinated with chadox ncov- . as for rhesus macaques, they found that, compared with control animals, a single vaccination significantly reduced the viral load in bronchoalveolar lavage fluid and respiratory tract tissue [ ] . the same group also previously demonstrated that a single dose of chadox mers, a chimpanzee adenovirus vector encoding the s protein of mers, protected non-human primates against mers-cov [ ] . in general, there are several advantages to utilizing viral vectors, such as the possibility of long-term gene expression, the high specificity of gene delivery to target cells, and the high immunogenicity after just one vaccination. that being said, the risk of pre-existing immunity against adenoviruses decreasing the desired immunogenic response against the protein of interest is a concern [ ] . the dna platform received increased attention over the years owing to its overall safety profile and high adaptability, followed by a fast-large-scale production [ ] . concerns remain regarding the lower generated immunogenicity and the need for several prime-boost regimes; however, several improved administration techniques and vector optimization have made this a valuable vaccine platform for emergent pathogens [ ] . several studies have looked at the dna vaccines as a platform for cov with encouraging results [ , , ] . wang and colleagues showed that dna expressing the full spike combined with s protein was superior to protein alone, reducing lung damage in a non-human primate (nhp) model [ ] . moreover, they showed higher immunogenicity using a dna-protein regime compared with dna or protein alone and underlined the advantage of including a different platform for the optimal response. finally, live-attenuated sars-cov and mers-cov viruses have been studied in mice models [ , ] . a group of researchers attenuated the e protein in sars-cov and tested the effects in mice. interestingly, they found that mice vaccinated with the attenuated virus had a significant reduction in the number of proinflammatory cytokines associated with lung injury, as well as increased cd + and cd + t cell counts [ ] . genetically modified viruses (lacking the envelope gene or the nsp gene, respectively) were shown to be able to protect mice from infection using both heterologous and homologous viruses. given the lack of sterilizing immunity from other platforms, some groups suggest this to be a possible way forward [ ] . that being said, the risk for clinical disease in immunocompromised people, combined with the risk of the vaccine reconverting back into its pathogenic forms, has made this option less prioritized [ ] . nonetheless, scientists and researchers worldwide must find the right balance between inducing an adequate immune response and assuring the safety of the vaccine. the optimal antigen with the right platform is crucial; however, another interesting point shown by previous studies is the potential improved protection using mucosal vaccine administration [ , , , , ] . one study comparing intramuscular (im) with intranasal administration (in) showed a better local iga response along with improved systemic t cell response in a mice model [ ] . in the study, both administration methods protected from infection. in another study, no virus was found in the lungs of in-vaccinated mice, while the same vaccine administered im and controls did not prevent viral amplification in lung tissue [ ] . other groups have looked at lung damage and found that, although protection in both in and im was observed, the changes in the lungs were less in the in-administered group [ ] . they also pointed out that, in the in group, serum nab titers were lower, indicating that the level of protection might not be correlated with circulating igg, but rather peripheral located antibodies. these findings suggest that increased protection might be achieved with mucosal administration rather than traditional parenteral administration. t cells, especially cd + and cd + , are crucial in eliciting a specific and adequate immune response and producing a long-term immunological memory. a concise review by grifoni et al. shed light on the importance of t cell response in covid- and its potential in developing a new vaccine. essentially, they looked up the antigens that the virus-specific t cells reacted to in exposed covid- patients and compared them to healthy individuals. the team found that % of the exposed patients had cd + t cells that responded to the s protein, n protein, and m protein. this is an interesting finding as adding the m protein and the n protein to the vaccines with the s protein increases the chance of mimicking a natural covid- infection, thereby eliciting a better immune response in the case of a potential future infection. as for the cd + response, the team found that, although the s protein and m protein were strongly recognized by cd + t cells, they were not a dominant target. they also found that the n protein and two other viral proteins, orf a and nsp , comprised an average of % of the total cd + t cell response [ ] . this indicates that we may be restricted in the types of antigens that can elicit a cd + t cell response. another study by peng et al. tested the immune memory of the t cell response of recovered patients compared with healthy controls. the team found that the t cell response was significantly higher in severe compared with mild covid- patients, with a significant response to the viral proteins s protein, m protein, and orf a. moreover, they identified separate peptides containing cd + and cd + epitopes [ ] . these findings showcase the importance of considering other viral proteins, such as the n protein and the m protein, to design a vaccine for covid- . despite the numerous pre-clinical studies testing various vaccine candidates in animal models, only a few have reached clinical trials in humans (summarized in table ). two sars-cov vaccine phase i trials have been completed, evaluating the safety and immunogenicity of an inactivated sars-cov vaccine and a dna vaccine expressing the spike protein [ , ] . both vaccines were safe and well-tolerated in the study groups and showed some degree of immunogenicity. the inactivated vaccine induced specific antibodies in % of participants after two doses; however, the genometric mean titer of nab decreased after only four weeks. the second study, conducted in the united states in , tested the safety and immunogenicity of a recombinant dna plasmid vaccine (vrc-srsdna - -vp), which expresses the s protein of sars-cov (enrollment: participants). the results were published in , showing that the vaccine generated a cd + t cell response in all recipients, along with a neutralizing antibodies response in % of the recipients. unfortunately, the vaccine was only able to provide a cd + t cell response in % of the recipients [ ] . the first human trial evaluating vaccine safety and immunogenicity of a mers vaccine was conducted in the united states. it was a phase i, open-label, dose-ranging trial (nct ) that tested a dna plasmid vaccine expressing the s protein of mers-cov (gls- ) [ ] . the published results indicated that the gls- was well tolerated. seroconversion, as measured by s -elisa, occurred in % of participants after two vaccinations, which then increased to % after three vaccinations. t cell responses were detected in % and % of participants, evaluated by elispot after two and three vaccinations, respectively, and neutralizing antibodies were seen in % of the participants. the response persisted when re-tested one year later [ ] . a recently published phase i trial (nct ) in the united kingdom concluded a chimpanzee adeno vector expressing mers spike (chadox mers) vaccine was safe and well-tolerated in different doses [ ] . they saw a significant increase in both t cells and igg after vaccination, and % of participants had neutralizing antibodies in the group receiving the highest dose. a study investigating the effect of a two-dose vaccine regiment will clarify whether or not this will enhance the nab response. importantly, pre-existing t cell or antibody response did not affect the response. still, the authors underline that the results must be evaluated with caution because of its small sample size and design. the same vaccine is being tested in a phase i clinical trial in saudi arabia, enrolling participants in an open-labeled, non-randomized clinical study (nct ). in russia, an ongoing phase i/ii open-dose, prospective randomized clinical trial is testing the safety and immunogenicity of an adenoviral-based vaccine, mers-bvrs-gamvac, in participants (nct ). the first in-human phase i trial (nct ) was conducted in germany in . the researchers tested the safety, efficacy, and immunogenicity of the vaccine candidate mva-mers-s, which is a modified vaccinia virus ankara (mva) vector expressing the s protein of mers-cov [ ] . in a homologous prime-boost immunization schedule in participants, no severe adverse events were reported. following the second immunization, % of low dose and % of high dose participants showed seroconversion, as measured by s elisa. neutralizing antibodies were detected after the second immunization; however, they decreased to pre-study levels after six months. t cell responses were detected in % and % of the subjects depending on dose and showed that mers-cov spike specific secretion of ifn-γ predominantly came from cd + t cells. the response also decreased over time. pre-existing immunity against the vector was observed; however, it did not correlate with antibody response. the covid- pandemic's significant global impact has advanced vaccine development at an unprecedented speed. a tremendous scientific effort supported by various regulatory and financial agencies has made it possible to accelerate the development, and the results from the first clinical vaccine trial have been reported within only five months [ ] . according to who, on may , vaccine candidates are now in different clinical phases, and vaccines are being evaluated in pre-clinical models [ ] . besides, other previously approved vaccines are being tested for their possible pan-protective effect thanks to their ability to induce non-specific interferon responses. the following active trials were taken from clinicaltrials.gov and who database. only trials testing active vaccines are included (summarized in table ). two different vaccines utilizing viral vectors are currently in clinical trial. they are phase i (nct ) and phase ii (nct ), randomized, double-blinded, and placebo-controlled clinical trials in wuhan, china. they are evaluating the safety and immunogenicity of an adenovirus type vector (ad -ncov) encoding the full-length s protein of sars-cov- in healthy adults years and older were evaluated [ ] . while most participants reported mild to moderate adverse events, including fever, muscle, or joint pain, most were transient and self-limiting. the vaccine was able to induce both humoral and t cell responses with - % showing nab, at day , and - % positive responders using elispot, an assay that quantitively quantitatively measures the frequency of cytokine secretion for a cell. the t cell response was further evaluated, and both cd + and cd + responses were noted, with polyfunctional phenotypes increasing with a dose of vaccine. however, diminished responses were observed owing to the presence of pre-existing anti-ad immunity. a phase i/ii single-blinded, randomized multicenter study in the united kingdom (nct ), not yet recruiting, wants to test the safety, immunogenicity, and efficacy of the chadox ncov- , similar to the previously evaluated chadox -mers vaccine. they intend to measure the number of virologically confirmed symptomatic cases in healthy adult volunteers aged to years and record the incidence of a serious adverse event (estimated enrollment: participants). a phase i, open-label, dose-ranging trial (nct ) in the united states is testing the safety and reactogenicity of a novel lipid nanoparticle (lnp)-encapsulated mrna-based vaccine (mrna- ) expressing the full length s protein of sars-cov- . one of the main primary outcomes of the trial is to test the frequency of solicited local reactogenicity adverse events in healthy adults years and older. the participants received two vaccinations, days apart. the results of the trial are promising as the mrna- vaccine elicited an anti-sars-cov- immune response in all participants, with no trial-limiting safety concerns [ ] . a phase i, open-label trial in the united states (nct ) is evaluating the safety, tolerability, and immunogenicity of ino- on healthy volunteers between and years old. the vaccine will be injected intra-dermally, followed by electroporation. using double-stranded dna plasmids allows researchers to synthesize and code for the spike protein, which will initiate an immune response. the primary objective of this study is to note the percentage of participants with adverse reactions post-vaccination (estimated enrollment: participants). a phase i/ii, randomized, placebo-controlled, observer-blind, dose-finding trial in the united states (nct /nct ) is evaluating the safety, tolerability, immunogenicity, and efficacy of four sars-cov- rna vaccine candidates (bnt a , bnt b , bnt b , and bnt c ) in healthy adults between and years old. the trial is evaluating multiple primary outcome measures, such as the percentage of participants reporting local reactions, systemic events, and adverse events (estimated enrollment: participants). two phase i/ii randomized, double-blinded, placebo-controlled trial in jiangsu, china (nct /nct ) are evaluating the safety and immunogenicity of inactivated sars-cov- vaccine in healthy adults > or to years old. the trials use a formalin-inactivated and alum-adjuvanted vaccine candidate. the primary objective is to note the occurrence of adverse reactions post-vaccination and evaluate immunogenicity (estimated enrollment: and participants). a phase i/ii, randomized, double-blind, placebo parallel-controlled phase i/ii clinical trial in shangqiu, china (chictr ) is evaluating the safety and immunogenicity of an inactivated sars-cov- vaccine in healthy individuals years and older. the primary outcome measure is the incidence of adverse events. a phase i randomized, placebo-controlled / phase trial by novavax in australia (nct ) is evaluating the safety and immunogenicity of a sars-cov- recombinant spike nanoparticle vaccine with and without matrix-m adjuvant in healthy participants between and years old. a phase i clinical trial (nct ), currently recruiting in china, is using inactivated artificial antigen-presenting cells expressing conserved structural and protease epitopes of sars-cov- . the primary objective of the trial is to evaluate the safety of injecting covid- /artificial antigen-presenting cells (apc) vaccine in healthy and covid- positive volunteers, including all age groups from months to years old (estimated enrollment: participants). the same institute is conducting another phase i/ii multicenter trial (nct ) testing the safety and efficacy of a lentiviral minigene vaccine (lv-smenp) of sars-cov- using modified dendritic cells and antigen-specific cytotoxic t-lymphocytes (ctls) (estimated enrollment: participants). currently, four trials are testing whether bacillus calmette-guérin (bcg) vaccine for tuberculosis is an efficient way to reduce the incidence of covid- . the first trial is a phase iii, two-group, multicenter, open-label randomized controlled trial (rct) (nct ) in australia, testing the covid- incidence measured over the six months post-randomization (estimated enrollment: participants). the second trial is a phase iii, placebo-controlled, adaptive multicenter rct (nct ) in the netherlands. the primary outcome measure is the number of days of healthcare workers' absence (estimated enrollment: participants). the vaccine is also being tested in south africa in a phase iii, randomized, double-blinded trial (nct ). the primary outcome measure is the incidence of healthcare workers' hospitalizations owing to covid- (estimated enrollment: participants). finally, there is a phase iv, randomized, double-blind trial in the united states (nct ) testing the incidence of covid- infection (estimated enrollment: participants). several other countries have planned similar studies with bcg vaccines, modified bcg vaccines, oral polio vaccine (opv), and the measles-mumps-rubella (mmr) vaccine, as listed on clinicaltrials.gov. there are several things to consider when choosing a vaccine platform for the ideal vaccine candidate that will be produced and distributed around the globe. the few clinical trials for sars and mers might give us a hint on what to expect. combining the results from the numerous animal studies with the few clinical trials suggests that the high immunogenicity seen in some animal models might not apply to humans. despite the correlates of protection for sars-cov- , it is believed that both a strong cellular and humeral response is necessary for full protection. several studies have underlined the importance of nabs [ , ] . using an inactivated vaccine in mice models, protection from challenge was reported against both sars and mers; however, the protection was only partial in ferrets [ , , ] . in humans, the response was short-lived, and if protection correlated to nab levels, several boosts would be required for continuing protection. this follows in line with previous vaccine trials with inactivated viruses and might prove to be an inferior choice compared with other strategies. there are four inactivated vaccine trials in evaluation in china, and with different optimization, including the use of an adjuvant, they might show novel insight. the dna platforms were able to protect against both sars and mers in animal models; however, a superior response was noted combining dna with protein in an nhp model [ ] . in humans, both the sars-dna (vrc-srsdna - -vp) and the mers-dna (gls- ) vaccine produced both t cell responses and nab. yet, against sars, the authors speculate it might not be sufficient for protection as only a minority produced cd + t cell responses [ , ] . furthermore, if nab are the most important players, a response of % would be suboptimal. a different prime-boost regime, including other platforms, might be beneficial if drawing from experience learned from animal models [ , ] . currently, there is only one clinical trial evaluating the protective effect of a covid- -dna vaccine (ino- ) with similar approaches as seen before. while small improvements or adjustments might add to the immunogenicity, major differences compared with previous trials are probably unlikely. nonetheless, the protective value of the generated response might be sufficient to slow the pandemic. considering viral vectors, adenovirus-based vaccines have been a frequently used platform and showed promise in mice with protection using both chadox and ad [ , , ] . however, in ferrets, incomplete protection was noted against sars [ ] . in humans, the chadox -mers-s was able to generate a long-lived t cell and nab response with just one injection. however, the nab response was not reported in more than %, raising concern about the protective value for the broad population. two ad-based vaccines are currently being evaluated against covid- (ad -ncov and chadox -ncov), and with the first results already published, nab were seen in up to % of participants and polyfunctional cd + and cd + phenotypes, making it a promising candidate [ ] . even so, the diminished response reported owing to pre-existing immunity is a concern. the use of chadox -vector for a covid- vaccine might overcome this issue; however, cross-reacting pre-existing cellular immunity should not be ignored. as different ad serotypes circulate globally, the vaccine-generated response might vary significantly depending on geographical location [ ] . the mva vector also had promising pre-clinical data, with different groups showing a protective response against both sars and mers in mice models [ , ] . still, concerns were raised about ade in ferrets as increased liver damage was observed [ ] . in humans, mva-mers vaccine generated both t cell responses and nab in the majority, but the response decreased over the six months, suggesting that a vaccine formulation and regime could be optimized [ ] . currently, there are no active mva-covid- vaccine trials. several other approaches not previously studied for sars and mers are currently being evaluated in clinical trials. mrna vaccines are advantageous for their capacity to initiate a potent immune response and minimize the risk of infection and insertion-induced mutagenesis, as well as the potential of large-scale production. despite that, one of the common downsides of mrna is its instability compared with dna and its high production costs. two different groups are evaluating mrna-vaccines in clinical trials (mrna- and bnt ). the moderna mrna- has been reported to be able to generate nab-titers at similar levels to those observed in convalescent sera according to the company [ ] . however, no combined results have been published, and whether or not this platform proves superior will be evaluated soon. vaccines using in vitro prepared specific apc have been studied extensively in cancer immunotherapy. while it has generally been proving safe, conflicting results remain about its benefits [ , ] . with the high cost and extensive preparation, this platform might be hard to distribute to the global population within a reasonable timeframe. yet, the two trials from china might add crucial new knowledge by trying a different approach and including several epitopes in structural and non-structural genes of sars-cov- . several countries are evaluating another alternative methodology by looking at the non-specific effect of live-virus vaccination, including the bcg vaccination. it is increasingly acknowledged that the innate immune system can display adaptive characteristics after certain infections or vaccinations somewhat comparable to the adaptive properties seen in the adaptive immune response [ ] . the bcg vaccine has previously been linked to several non-specific benefits by mounting a more efficient cytokine response [ ] . an encouraging study in found that upper respiratory infections were significantly decreased in an elderly population following bcg vaccination. as we see the same kind of infection in the upper respiratory tract in covid- , the use of bcg vaccine against sars-cov- might have some promising results [ ] . similar non-specific effects of opv and mmr vaccinations have been reported and high mmr vaccination coverage has been linked to few covid- deaths [ ] . a major advantage of this approach is that the protective effect of the bcg vaccine is currently being evaluated in several phase iii/iv trials. if a beneficial tendency is seen, no further evaluations will be necessary before implementation. whether or not this non-specific effect will be sufficient remains to be seen. interestingly, the different vaccine platforms in the clinical trials against sars and mers primarily use either the whole virion or s gene. while we can see more diversity for covid- , it remains dominated by the same antigens ( figure ). one concern from animal models is the possible ade using the full-length spike or s ; however, this challenge was far from reported in all studies, as less pathology in vaccinated groups was frequently reported [ , , , ] . despite that, if this adverse event appears, a few other non-spike candidates are in the pipeline, as listed in the pre-clinical landscape overview by who. besides, another antigen selected might help guide a broader immune response. as other structural proteins appear to be more conserved between cov, it might be an interesting approach for a pan-cov vaccine design. furthermore, no studies currently in clinical trial seem to evaluate the effect of mucosal administration, despite some pre-clinical studies showing clear evidence for improved impact ( figure ) [ , , , , ] . nevertheless, not all trials show clear information, and some might be evaluating these administration methods. no results have been published for a phase ii trial so far, and the protective value of these vaccines in humans remains unknown. this leaves a lot of questions unanswered, even after years since the sars outbreak. the last time coronavirus made its appearance was in during the mers outbreak. years later, and after completing only five phase i human vaccine trials against sars and mers, we still have many questions unanswered. the previous outbreaks were contained primarily with non-pharmacological methods (e.g., quarantines, social distancing, masks), leaving specific prophylactic and therapeutic options as a scientific question rather than a political priority [ ] . the current situation, with more than , confirmed deaths and a world in lockdown, has changed the tune, and all possible resources are now aimed at finding sustainable solutions. since , preclinical and clinical vaccine studies against these cov have shown some degree of immunogenicity and protection; however, no clear-cut success story has been written. many of the same approaches taken for sars and mers are being re-tested again, even though we probably can expect the same result and, for now, a lot of the suggested improvements are not being evaluated. nevertheless, much the last time coronavirus made its appearance was in during the mers outbreak. years later, and after completing only five phase i human vaccine trials against sars and mers, we still have many questions unanswered. the previous outbreaks were contained primarily with non-pharmacological methods (e.g., quarantines, social distancing, masks), leaving specific prophylactic and therapeutic options as a scientific question rather than a political priority [ ] . the current situation, with more than , confirmed deaths and a world in lockdown, has changed the tune, and all possible resources are now aimed at finding sustainable solutions. since , pre-clinical and clinical vaccine studies against these cov have shown some degree of immunogenicity and protection; however, no clear-cut success story has been written. many of the same approaches taken for sars and mers are being re-tested again, even though we probably can expect the same result and, for now, a lot of the suggested improvements are not being evaluated. nevertheless, much is still under development, and information is being kept close, so hopefully some lessons have been learned. finding potential vaccine candidates for covid- is naturally the first step taken, but subsequent vaccine production and distribution is an important subject that has previously been the bottleneck. in the h n influenza outbreak, the majority of vaccine supplies were bought by wealthy nations, leaving limited stocks for low-or middle-income countries [ ] . moreover, even though the united states had the vaccine, it was distributed to other countries only after six months, which was too late, as the second wave of the virus had already begun [ ] . today, world leaders are trying to find common ground to produce and distribute the best candidates to all countries based on needs and not economic power. still, national and financial interests might make this battle more difficult. most of the current clinical trials estimate to publish their results in , and this means that we have at least seven months of uncertainties regarding the fate of this virus. only time will tell whether we are on the right track to discovering the 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report. immunity funding: this research received no external funding. the authors declare no conflict of interest. key: cord- -x isowrt authors: ackerman, emily e.; shoemaker, jason e. title: network controllability-based prioritization of candidates for sars-cov- drug repositioning date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: x isowrt in a short time, the covid- pandemic has left the world with over million cases and staggering death tolls that are still rising. treatments for sars-cov- infection are desperately needed as there are currently no approved drug therapies. with limited knowledge of viral mechanisms, a network controllability method of prioritizing existing drugs for repurposing efforts is optimal for quickly moving through the drug approval pipeline using limited, available, virus-specific data. based on network topology and controllability, proteins involved in translation, cellular transport, cellular stress, and host immune response are predicted as regulators of the sars-cov- infected cell. of the , eight are prioritized as possible drug targets where two, pvr and scarb , are previously unexplored. known compounds targeting these genes are suggested for viral inhibition study. prioritized proteins in agreement with previous analysis and viral inhibition studies verify the ability of network controllability to predict biologically relevant candidates. as covid- spreads worldwide with million cases and , deaths occurring between january and august [ ] , there is an urgent need for novel treatment options. there are currently no known pharmaceutical treatments for sars-cov- infection. one strategy to accelerate the identification of possible leads is to reposition drugs with known targets and mechanisms that may have been through parts of the fda approval process [ ] . avoiding this development pipeline known for its low success rate [ ] advantageously saves invaluable time and monetary cost. while this is ultimately the fastest way to get treatments to patients in need, the most efficient way to discover drugs with the potential for repurposing is unclear. in the short time since the beginning of the pandemic, many attempts to predict candidate drugs for repositioning have been made. given the novel nature of the virus, methods of target prediction have been forced to utilize the limited data that is available or creatively repurpose data from related coronaviruses. in vitro screenings of chemical libraries have been used to identify inhibitors of sars-cov- replication [ , ] and cellular toxicity [ ] . screenings of experimentally verified sars-cov- interacting host proteins [ ] have elucidated key infection mechanisms which, when compared to drug databases, have predicted a range of possible targets for repurposing. network analyses using protein interaction data from up to related human coronaviruses [ , ] combined with in vitro screenings have identified additional sets of cellular pathways to consider for drug repurposing. topology of methods for robust controllability are sourced from liu et al.'s work [ ] . for any network with n total nodes, a subset, n d , of driver nodes is found using a maximum matching algorithm such as hopcroft-karp on the bipartite representation of the total network [ ] . this process is repeated iteratively after removing each node from the network (n = n − ) to identify a new maximum matching, n d . removed nodes are classified as indispensable (n d > n d ), neutral (n d = n d ), or dispensable (n d < n d ). calculations for jia classification were adopted from jia et al. [ ] . for any network with n total nodes, a subset, n d , of driver nodes is found using a maximum matching algorithm such as hopcroft-karp on the bipartite representation of the total network [ ] . control adjacent nodes of all n d are identified iteratively and used to create an input graph as described in zhang et al. [ ] . nodes are classified as critical (in all minimum input sets), neutral (in some minimum input sets), or redundant (in no minimum input sets) based on the input graph. after construction (see section ), the host interaction network (hin) contains , proteins and , interactions. median log degree and betweenness of the hin is . and . , respectively. in the construction of the virus integrated network (vin), of the sars-cov- proteins tested in gordon et al. [ ] are added to the network along with interactions with existing host proteins. four sars-cov- proteins, spike, nsp , orf b, and orf a, had no known interactions with host proteins of the hin and were omitted from the analysis. in total, the vin contains proteins and , interactions. median log degree and betweenness of the vin is . and . , respectively. there is no statistical difference between the degree or betweenness of the hin and vin (wilcoxon rank sum test p-values: . and . , respectively). however, all host proteins have higher betweenness in the vin compared to the hin. while viral proteins only interact with proteins, the topological effects are seen across the entire network. median log degree and betweenness of host proteins directly interacting with at least one sars-cov- protein, or "virus interacting proteins", in the hin are . and . , respectively. the same values in the vin are . and . , respectively. there is a significant difference in mean degree and betweenness distributions of virus interacting proteins compared to the total protein population of the vin (two sample t-test p-value: . × − and . × − , respectively). all described degree and betweenness distributions are found in figure . driver proteins are a subset of the network's proteins that must be directly controlled to manipulate total system behavior. this subset, size n d , is identified through maximum matching algorithms [ ] and serves as the first step in both methods of controllability. calculations identified n d = in the hin and n d = in the vin, implying that there is little change to the control structure of the network during infection. all sars-cov- proteins are driver proteins of the vin. the host proteins displaced by sars-cov- proteins as drivers are deemed "displaced proteins". their identities are listed in table . only five displaced proteins are not virus interacting proteins (hpr, cnnm , trim , dip a, mica). the removal of these five proteins as drivers of infected cell behavior in the vin suggests that they have fallen under the control of viral proteins or are part of a host cascade that has been activated in the response. two proteins are of note: first, trim is a member of the tripartite interaction motif family of innate immunity regulators [ ] . it is previously shown to be highly upregulated in the presence of tlr and tlr ligands [ ] of the viral rna sensing pathway [ ] . second, mica is an mhc class i cell surface protein which regulates the activation of both t cells and natural killer cells during a stress response along with other nkg d ligands such as rae [ , ] . the displaced protein set was analyzed with interferome v . [ ] to determine their status as interferon regulated genes (irgs) known to exhibit a fold change in expression greater than two in interferon knockdown studies. all displaced proteins are irgs with the exception of sigmar , eif e, and mica. the altered role of these immune proteins as drivers of network behavior is representative of the activation of immune response pathways. driver proteins are a subset of the network's proteins that must be directly controlled to manipulate total system behavior. this subset, size , is identified through maximum matching algorithms [ ] and serves as the first step in both methods of controllability. calculations identified = , in the hin and = , in the vin, implying that there is little change to the control structure of the network during infection. all sars-cov- proteins are driver proteins of the vin. the host proteins displaced by sars-cov- proteins as drivers are deemed "displaced proteins". their identities are listed in table . only five displaced proteins are not virus interacting proteins (hpr, cnnm , trim , dip a, mica). the removal of these five proteins as drivers of infected cell behavior in the vin suggests that they have fallen under the control of viral proteins or are part of a host cascade that has been activated in the response. two proteins are of note: first, trim is a member of the tripartite interaction motif family of innate immunity regulators [ ] . it is previously shown to be highly upregulated in the presence of tlr and tlr ligands [ ] of the viral rna sensing pathway [ ] . second, mica is an mhc class i cell surface protein which regulates the activation of both t cells and natural killer cells during a stress response along with other nkg d ligands such as rae [ , ] . the displaced protein set was analyzed with interferome v . [ ] to determine their status as interferon regulated genes (irgs) known to exhibit a fold change in expression greater than two in interferon knockdown studies. all displaced proteins are irgs with a robust controllability analysis was performed on the hin and vin as described in section to determine the effect of singular protein components on total system behavior. the classification results are shown in table . aside from the addition of viral nodes, there is very little change to the robust controllability of the vin as compared to the hin. the majority of all proteins are classified as neutral (vin: . %, hin: . %) and dispensable (vin: . %, hin: . %), suggesting that most proteins are regulated by neighboring protein pathways (neutral) or make the network easier to control in their absence (dispensable). conversely, the loss of a small proportion of indispensable proteins (vin and hin: . %) would make the network increasingly difficult to regulate. the driver protein population is skewed toward those with dispensable classifications (vin: . %, hin: . %) as compared to all proteins. classifications of virus interacting proteins are similar to those of the total network, eliminating the possibility that viral interactions target proteins that are advantageous to robust controllability. viral proteins e, nsp , and nsp are classified as dispensable in the vin. all other viral proteins are classified as neutral. the nine host proteins that change robust classification after the addition of viral interactions are listed in table . four of the "robust proteins" are also virus interacting proteins (impdh , rae , sigmar , nup ) and three belong to the displaced protein set (impdh , sigmar , nup ). while only the four virus interacting proteins exhibit an increase in degree from a singular viral interaction, all nine robust proteins demonstrate an increase in betweenness in the infected network, some by orders of magnitude. of note, the betweenness of impdh increases from to and sigmar reaches where it has a betweenness of in the hin. this trend demonstrates the importance of the robust protein set to network information flow and regulation in the infected cell. there is no trend in the changes to classification type for the robust group. only two robust proteins were identified as irgs with a fold change greater than two by interferome (impdh , dynlt ). to assess whether the robust controllability classifications of the driver and virus interacting proteins are a result of the network's connectivity structure, a randomization analysis was performed as developed in previous work [ ] . a random set of host proteins representing a "pseudo-virus interacting" protein set was pulled from the network and assessed for robust controllability. the resulting distributions from , iterations of this process are reported in figure a against the true values for all proteins, driver proteins, and virus interacting proteins. distributions are reflective of the true values of all proteins for all three robust controllability classifications. true values for virus interacting proteins also fall within the distributions for robust classifications implying that there is no regulatory advantage for the particular set of host proteins interacting with sars-cov- within the robust controllability framework. however, true values for driver proteins fall outside the distributions generated by the pseudo-sets, implying that the groups are distinctly different in regulatory function. a topological analysis including the median and mean values of the same distributions against the true values for virus interacting proteins is shown in figure . the mean log degree and betweenness is significantly higher than the corresponding distribution mean (one-sided t-test p-values: . × − , . × − ) implying that the virus prefers to interact with proteins that hold significance to network structure. similarly, a global controllability analysis was performed on the hin and vin as described in section . results are shown in table . as in the robust controllability analysis, global controllability classifications of the vin's proteins are almost identical to those of the hin. over half (vin: . %, hin: . %) of all proteins are classified as intermittent, suggesting that the majority of proteins are able to play a role in cellular regulation. only a small percentage (vin: . %, hin: . %) of all proteins are classified as critical, meaning they are involved in all combinations of network regulators. by definition, driver nodes cannot be redundant, therefore, they are predominately classified as intermittent (vin: . %, hin: . %). unlike the robust analysis, classifications of virus interacting proteins differ slightly from those of the total protein population. the eight critical virus interacting proteins of the hin become intermittent in the vin, losing some control over infected network regulation. there is a higher proportion of redundant virus interacting proteins in the vin similarly, a global controllability analysis was performed on the hin and vin as described in section . results are shown in table similarly, a global controllability analysis was performed on the hin and vin as described in section . results are shown in table . as in the robust controllability analysis, global controllability classifications of the vin's proteins are almost identical to those of the hin. over half (vin: . %, hin: . %) of all proteins are classified as intermittent, suggesting that the majority of proteins are able to play a role in cellular regulation. only a small percentage (vin: . %, hin: . %) of all proteins are classified as critical, meaning they are involved in all combinations of network regulators. by definition, driver nodes cannot be redundant, therefore, they are predominately classified as intermittent (vin: . %, hin: . %). unlike the robust analysis, classifications of virus interacting proteins differ slightly from those of the total protein population. the eight critical virus interacting proteins of the hin become intermittent in the vin, losing some control over infected network regulation. there is a higher proportion of redundant virus interacting proteins in the vin ( . %) compared to both the virus interacting proteins in the hin and all proteins of both the vin and hin (hin virus interacting proteins: . %, vin all proteins: . %, hin all proteins: . %), suggesting that proteins that directly interact with the virus are transitioning into deferential roles after the onset of infection. all viral proteins are classified as critical in the vin, always holding control of network regulation. eleven host proteins change classification after the addition of viral interactions ( table ). all eleven "global proteins" interact with sars-cov- proteins with six belonging to the displaced protein set (scarb , impdh , pvr, eif e , sigmar , and nup ). four global proteins are also identified as robust proteins (impdh , rae , sigmar , and nup ). with the exception of eif e , sigmar , and saal , all members of the set were identified as irgs with a fold change greater than two by interferome. a randomization analysis was performed as developed in the previous work [ ] using the same "pseudo-virus interacting" protein sets assessed for robust controllability classifications. the resulting distributions from the , iterations are found in figure b against the true values for all proteins, driver proteins, and virus interacting proteins. again, random distributions are reflective of the true values from the global controllability of all proteins. while the true value for intermittent virus interacting proteins reflects the random distributions, true values for critical and redundant proteins fall at the tails of the distributions suggesting a regulatory advantage in sars-cov- interacting with redundant host proteins. true values for driver proteins fall outside the distributions generated by the pseudo-sets, supporting the conclusion that the groups are distinct. assuming the identified robust and global proteins are acting as regulators of the infected state, it follows that they have potential as drug targets for sars-cov- infection treatments. the drugbank database [ ] was used to prioritize the predicted proteins for drug repositioning efforts by assessing which proteins act as targets for existing drugs. results were compared with the results of drug repurposing and viral inhibition studies performed by gordon et al. [ ] . of the combined robust and global proteins, six are drug targets registered in drugbank (pvr, scarb , nup , sigmar , impdh , and eif e ). nup , sigmar , impdh , and eif e were identified by gordon et al., though drugbank identified compounds for each target that were not included in the viral inhibition studies. the compounds associated with two additional targets that were identified by the controllability methods but are unregistered in drugbank (larp and rae ) were also previously identified. the targets and associated compounds for all eight genes are found in supplementary table s . a summary of compounds known to target pvr and scarb , i.e., the prioritized proteins that have not been recommended in previous repurposing studies are found in table . pvr is a known regulator of natural killer cell adhesion to host cells and lytic granule secretion after binding to dnam- , a receptor expressed by natural killer cells, t cells, and monocytes [ ] . first identified in the context of polio virus, it has also been identified for its role in motility during tumor cell invasion [ ] . it is a member of the displaced driver set and the global set and acts as the target for two experimental compounds: myristic acid and sphingosine. a previous study of cytokine storms resulting from influenza virus infection asserts that the use of sphingosine- -phosphate successfully blunts the overactive inflammatory response, limiting morbidity and mortality [ ] . given the similarly aggressive inflammatory response seen clinically in sars-cov- infected individuals [ ] , the prioritization of pvr is noteworthy. a randomization analysis was performed as developed in the previous work [ ] using the same "pseudo-virus interacting" protein sets assessed for robust controllability classifications. the resulting distributions from the , iterations are found in figure b against the true values for all proteins, driver proteins, and virus interacting proteins. again, random distributions are reflective of the true values from the global controllability of all proteins. while the true value for intermittent virus interacting proteins reflects the random distributions, true values for critical and redundant proteins fall at the tails of the distributions suggesting a regulatory advantage in sars-cov- interacting with redundant host proteins. true values for driver proteins fall outside the distributions generated by the pseudo-sets, supporting the conclusion that the groups are distinct. assuming the identified robust and global proteins are acting as regulators of the infected state, it follows that they have potential as drug targets for sars-cov- infection treatments. the drugbank database [ ] was used to prioritize the predicted proteins for drug repositioning efforts by assessing which proteins act as targets for existing drugs. results were compared with the results of drug repurposing and viral inhibition studies performed by gordon et al. [ ] . of the combined robust and global proteins, six are drug targets registered in drugbank (pvr, scarb , nup , sigmar , impdh , and eif e ). nup , sigmar , impdh , and eif e were identified by gordon et al., though drugbank identified compounds for each target that were not included in the viral inhibition studies. the compounds associated with two additional targets that were identified by the controllability methods but are unregistered in drugbank (larp and rae ) were also previously identified. the targets and associated compounds for all eight genes are found in supplementary table s . a summary of compounds known to target pvr and scarb , i.e., the prioritized proteins that have not been recommended in previous repurposing studies are found in table . pvr is a known regulator of natural killer cell adhesion to host cells and lytic granule secretion after binding to dnam- , a receptor expressed by natural killer cells, t cells, and monocytes [ ] . first identified in the context of polio virus, it has also been identified for its role in motility during tumor cell invasion [ ] . it is a member of the displaced driver set and the global set and acts as the target for two experimental compounds: myristic acid and sphingosine. a previous study of cytokine storms resulting from influenza virus infection asserts that the use of sphingosine- -phosphate successfully blunts the overactive inflammatory response, limiting morbidity and mortality [ ] . given the similarly aggressive inflammatory response seen clinically in sars-cov- infected individuals [ ] , the prioritization of pvr is noteworthy. table . status and structure of drugs known to target controllability predicted proteins. each target's sars-cov- protein interactor is given along with its known target function. viruses , , x for peer review of transport between nucleus and cytoplasm [ ] scarb is a cellular membrane protein involved in high-density lipid transport [ ] that mediates cell entry of hepatitis c virus as the receptor for the e protein [ ] . it was identified as both a displaced driver and global protein, and functions as the target for three compounds: phosphatidylserine, tocopherol/vitamin e, and pha- . while not specific to viral infection, scarb acts as a phosphatidylserine receptor on testicular sertoli cells which induce phagocytosis of spermatogenic cells [ ] . scarb also acts as one of the most important transport vehicles for vitamin e in the lung's alveolar cells, the presence of which largely regulates the receptor's expression [ ] . vitamin e is also known to have a positive effect on influenza a viral clearance in the lungs of mice [ ] . in addition to scarb , pha- targets nup , a nucleopore protein identified in all three protein sets of interest from controllability. knockout experiments reveal that nup has wide effects on t cell differentiation and response [ ] . while not studied in the context of viral infection, pha- is known to induce apoptosis in both tumor and vascular endothelial cells resulting from non-small cell lung cancer [ ] . sigmar and impdh were also identified in all controllability predicted groups. sigma receptors and (sigmar and sigmar ) have been discussed as regulators of cellular stress and the apoptotic response [ ] . several known targeting compounds show evidence of inhibition at the viral replication stage including haloperidol, pb , and widely discussed hydroxychloroquine. many drugbank predicted compounds targeting sigmar have not been tested for viral inhibition. impdh has long been a goal for targeting with immunosuppressive treatments, though inhibition with small molecules is notoriously difficult [ ] . while many drugbank-predicted compounds for impdh have not been tested in this context, mycophenolic acid and ribavirin were assessed in the viral inhibition screen with only the latter displaying active inhibition. transport between nucleus and cytoplasm [ ] scarb is a cellular membrane protein involved in high-density lipid transport [ ] that mediates cell entry of hepatitis c virus as the receptor for the e protein [ ] . it was identified as both a displaced driver and global protein, and functions as the target for three compounds: phosphatidylserine, tocopherol/vitamin e, and pha- . while not specific to viral infection, scarb acts as a phosphatidylserine receptor on testicular sertoli cells which induce phagocytosis of spermatogenic cells [ ] . scarb also acts as one of the most important transport vehicles for vitamin e in the lung's alveolar cells, the presence of which largely regulates the receptor's expression [ ] . vitamin e is also known to have a positive effect on influenza a viral clearance in the lungs of mice [ ] . in addition to scarb , pha- targets nup , a nucleopore protein identified in all three protein sets of interest from controllability. knockout experiments reveal that nup has wide effects on t cell differentiation and response [ ] . while not studied in the context of viral infection, pha- is known to induce apoptosis in both tumor and vascular endothelial cells resulting from non-small cell lung cancer [ ] . sigmar and impdh were also identified in all controllability predicted groups. sigma receptors and (sigmar and sigmar ) have been discussed as regulators of cellular stress and the apoptotic response [ ] . several known targeting compounds show evidence of inhibition at the viral replication stage including haloperidol, pb , and widely discussed hydroxychloroquine. many drugbank predicted compounds targeting sigmar have not been tested for viral inhibition. impdh has long been a goal for targeting with immunosuppressive treatments, though inhibition with small molecules is notoriously difficult [ ] . while many drugbank-predicted compounds for impdh have not been tested in this context, mycophenolic acid and ribavirin were assessed in the viral inhibition screen with only the latter displaying active inhibition. transport between nucleus and cytoplasm [ ] scarb is a cellular membrane protein involved in high-density lipid transport [ ] that mediates cell entry of hepatitis c virus as the receptor for the e protein [ ] . it was identified as both a displaced driver and global protein, and functions as the target for three compounds: phosphatidylserine, tocopherol/vitamin e, and pha- . while not specific to viral infection, scarb acts as a phosphatidylserine receptor on testicular sertoli cells which induce phagocytosis of spermatogenic cells [ ] . scarb also acts as one of the most important transport vehicles for vitamin e in the lung's alveolar cells, the presence of which largely regulates the receptor's expression [ ] . vitamin e is also known to have a positive effect on influenza a viral clearance in the lungs of mice [ ] . in addition to scarb , pha- targets nup , a nucleopore protein identified in all three protein sets of interest from controllability. knockout experiments reveal that nup has wide effects on t cell differentiation and response [ ] . while not studied in the context of viral infection, pha- is known to induce apoptosis in both tumor and vascular endothelial cells resulting from non-small cell lung cancer [ ] . sigmar and impdh were also identified in all controllability predicted groups. sigma receptors and (sigmar and sigmar ) have been discussed as regulators of cellular stress and the apoptotic response [ ] . several known targeting compounds show evidence of inhibition at the viral replication stage including haloperidol, pb , and widely discussed hydroxychloroquine. many drugbank predicted compounds targeting sigmar have not been tested for viral inhibition. impdh has long been a goal for targeting with immunosuppressive treatments, though inhibition with small molecules is notoriously difficult [ ] . while many drugbank-predicted compounds for impdh have not been tested in this context, mycophenolic acid and ribavirin were assessed in the viral inhibition screen with only the latter displaying active inhibition. facilitate cell entry, hepatitis c [ ] tocopherol/vitamin e (approved) viruses , , x for peer review of table . status and structure of drugs known to target controllability predicted proteins. each target's sars-cov- protein interactor is given along with its known target function. transport between nucleus and cytoplasm [ ] scarb is a cellular membrane protein involved in high-density lipid transport [ ] that mediates cell entry of hepatitis c virus as the receptor for the e protein [ ] . it was identified as both a displaced driver and global protein, and functions as the target for three compounds: phosphatidylserine, tocopherol/vitamin e, and pha- . while not specific to viral infection, scarb acts as a phosphatidylserine receptor on testicular sertoli cells which induce phagocytosis of spermatogenic cells [ ] . scarb also acts as one of the most important transport vehicles for vitamin e in the lung's alveolar cells, the presence of which largely regulates the receptor's expression [ ] . vitamin e is also known to have a positive effect on influenza a viral clearance in the lungs of mice [ ] . in addition to scarb , pha- targets nup , a nucleopore protein identified in all three protein sets of interest from controllability. knockout experiments reveal that nup has wide effects on t cell differentiation and response [ ] . while not studied in the context of viral infection, pha- is known to induce apoptosis in both tumor and vascular endothelial cells resulting from non-small cell lung cancer [ ] . sigmar and impdh were also identified in all controllability predicted groups. sigma receptors and (sigmar and sigmar ) have been discussed as regulators of cellular stress and the apoptotic response [ ] . several known targeting compounds show evidence of inhibition at the viral replication stage including haloperidol, pb , and widely discussed hydroxychloroquine. many drugbank predicted compounds targeting sigmar have not been tested for viral inhibition. impdh has long been a goal for targeting with immunosuppressive treatments, though inhibition with small molecules is notoriously difficult [ ] . while many drugbank-predicted compounds for impdh have not been tested in this context, mycophenolic acid and ribavirin were assessed in the viral inhibition screen with only the latter displaying active inhibition. transport between nucleus and cytoplasm [ ] scarb is a cellular membrane protein involved in high-density lipid transport [ ] that mediates cell entry of hepatitis c virus as the receptor for the e protein [ ] . it was identified as both a displaced driver and global protein, and functions as the target for three compounds: phosphatidylserine, tocopherol/vitamin e, and pha- . while not specific to viral infection, scarb acts as a phosphatidylserine receptor on testicular sertoli cells which induce phagocytosis of spermatogenic cells [ ] . scarb also acts as one of the most important transport vehicles for vitamin e in the lung's alveolar cells, the presence of which largely regulates the receptor's expression [ ] . vitamin e is also known to have a positive effect on influenza a viral clearance in the lungs of mice [ ] . in addition to scarb , pha- targets nup , a nucleopore protein identified in all three protein sets of interest from controllability. knockout experiments reveal that nup has wide effects on t cell differentiation and response [ ] . while not studied in the context of viral infection, pha- is known to induce apoptosis in both tumor and vascular endothelial cells resulting from non-small cell lung cancer [ ] . sigmar and impdh were also identified in all controllability predicted groups. sigma receptors and (sigmar and sigmar ) have been discussed as regulators of cellular stress and the apoptotic response [ ] . several known targeting compounds show evidence of inhibition at the viral replication stage including haloperidol, pb , and widely discussed hydroxychloroquine. many drugbank predicted compounds targeting sigmar have not been tested for viral inhibition. impdh has long been a goal for targeting with immunosuppressive treatments, though inhibition with small molecules is notoriously difficult [ ] . while many drugbank-predicted compounds for impdh have not been tested in this context, mycophenolic acid and ribavirin were assessed in the viral inhibition screen with only the latter displaying active inhibition. facilitate cell entry, hepatitis c [ ] nup /nsp transport between nucleus and cytoplasm [ ] scarb is a cellular membrane protein involved in high-density lipid transport [ ] that mediates cell entry of hepatitis c virus as the receptor for the e protein [ ] . it was identified as both a displaced driver and global protein, and functions as the target for three compounds: phosphatidylserine, tocopherol/vitamin e, and pha- . while not specific to viral infection, scarb acts as a phosphatidylserine receptor on testicular sertoli cells which induce phagocytosis of spermatogenic cells [ ] . scarb also acts as one of the most important transport vehicles for vitamin e in the lung's alveolar cells, the presence of which largely regulates the receptor's expression [ ] . vitamin e is also known to have a positive effect on influenza a viral clearance in the lungs of mice [ ] . in addition to scarb , pha- targets nup , a nucleopore protein identified in all three protein sets of interest from controllability. knockout experiments reveal that nup has wide effects on t cell differentiation and response [ ] . while not studied in the context of viral infection, pha- is known to induce apoptosis in both tumor and vascular endothelial cells resulting from non-small cell lung cancer [ ] . sigmar and impdh were also identified in all controllability predicted groups. sigma receptors and (sigmar and sigmar ) have been discussed as regulators of cellular stress and the apoptotic response [ ] . several known targeting compounds show evidence of inhibition at the viral replication stage including haloperidol, pb , and widely discussed hydroxychloroquine. many drugbank predicted compounds targeting sigmar have not been tested for viral inhibition. impdh has long been a goal for targeting with immunosuppressive treatments, though inhibition with small molecules is notoriously difficult [ ] . while many drugbank-predicted compounds for impdh have not been tested in this context, mycophenolic acid and ribavirin were assessed in the viral inhibition screen with only the latter displaying active inhibition. eif ae belongs to the e family of translation initiation factor proteins which bind to mrna ' cap structures to recruit ribosomal recruitment within the cytosol [ ] . the e family controls the rate of the early steps of the protein translation process. eif ae is a member of both the displaced driver set and the global protein set. a different translation regulator, larp , was identified as a druggable target for sars-cov- by gordon et al., given previous evidence that the downstream effects of a common kinase inhibitor, rapamycin, inhibited mers-cov infection by over % [ ] . studies show that rapamycin promotes the phosphorylation of eif ae, achieving similar inhibition of the mtor pathway [ ] ; however, evidence of viral inhibition after rapamycin treatment was inconclusive. still, several other mrna translation inhibitor compounds such as ternatin and zotatifin have tested as active inhibitors of sars-cov- , making eif ae an interesting prospect for future study. here, a set of drug targets is prioritized for drug repurposing efforts in the global fight against covid- . network controllability methods, with only disease-specific virus-host and host-host protein interaction data, create a large-scale representation of regulatory changes occuring during infection. with no additional biological information, the connectivity of the network is sufficient to predict the most biologically relevant components of the disease system, as evidenced by the high level of overlap between the presented results and the extensive biological analysis performed in gordon et al.'s study for sars-cov- [ ] . in total, this study demonstrates a simple computational approach to prioritizing drug target predictions with minimal biological context, an advantage in present times where viral understanding and data is even more sparse than usual. as seen in the previous study of influenza a virus [ ] , the magnitude of control needed to manipulate the total cell system (number of driver proteins) is comparable in the healthy and infected cellular networks. the small changes in driver proteins between the networks are seen in immunoregulatory proteins that are typically upregulated during viral infection (such as trim and mica) and many of the proteins identified in the controllability analyses. this is reflective of the activation of the immune response pathways and their effect on the cell as a whole. with respect to the ratio of resultant classifications in both controllability methods, outcomes are again similar to those achieved with the influenza a virus-host network [ ] . this is unsurprising due to the use of the same host network in the analyses. however, the low overlap between the controllability predicted proteins for the two diseases ( / proteins, pvr, rab , and saal ) demonstrates that while the method is easily applied to other viruses, the result is unique. one limitation of this method is the requirement of high-confidence virus-host protein interaction data where the host proteins exist in the hin. as experimentally-validated, directed networks are typically smaller than the available undirected networks, the method was unfortunately unable to use over half of the known sars-cov- -host protein interactions (in comparison, the influenza a virus network contains virus-host interactions). even so, the controllability analysis was able to predict biologically relevant proteins involved in functions like the cellular stress response, host translation, and cellular transport, proving the robustness of the method. of the eight prioritized targets, all but nup exhibit large increases in betweenness after the addition of virus-host protein interactions, placing particular importance on their role in infected cell behavior based on topology. further, most of the global protein set (including the novel predictions, pvr, and scarb ) have a betweenness of zero in the hin, implying that their individual significance to cellular network flow is truly unique to the infected cell. with the majority of the identified proteins being regulated by the interferon response (with a fold change in expression greater than two), this network result translates to immunological significance. the alterations in classification for all global proteins indicate a step down in network control where critical proteins have the most control and redundant the least. biologically, this could represent viral interruption of normal host function or activation of a new pathway, both being interesting prospects for drug development. given the biological relevance of the topologically predicted/controllability target proteins, there is a good reason to pursue these recommendations, either in drug repurposing or in novel drug development. the extended list of untested compounds found in supplementary table s will be considered for further viral inhibition studies, particularly tocopherol/vitamin e which is already approved and has documented positive effects on viral clearance for influenza a [ ] . predictions indicate opportunity to both interfere with the viral replication cycle or to modulate the immune response to infection. therefore, to most efficiently translate these findings to bedside, knockdown studies or sirna screens should be used to validate drug predictions for each target. cell culture studies that track interferon and cytokine activity may further establish a possible mechanism between the proposed targets and immune regulation. by narrowing the pool of drug target candidates with controllability methods, experimental validation will be efficient and timely. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : compounds targeting controllability-identified targets. author contributions: e.e.a. assisted in the conceptualization of the study, designed the study, performed all computational experiments, and wrote the manuscript. j.e.s. conceptualized and funded the study. all authors have read and agreed to the published version of the manuscript. funding: this research received no external funding. world health organization. coronavirus disease (covid- ) weekly epidemiological update drug repurposing: progress, challenges and recommendations diagnosing the decline in pharmaceutical r&d efficiency in vitro screening of a fda approved chemical library reveals potential inhibitors of sars-cov- replication a large-scale drug repositioning survey for sars-cov- antivirals identification of inhibitors of sars-cov- in-vitro cellular toxicity in human (caco- ) cells using a large scale drug repurposing collection a sars-cov- protein interaction map reveals targets for drug repurposing a sars-cov- protein interaction map reveals targets for drug repurposing network-based drug repurposing for novel coronavirus -ncov/sars-cov- the current landscape of coronavirus-host protein-protein interactions a protein interaction map identifies existing drugs targeting sars-cov- a dual controllability analysis of influenza virus-host protein-protein interaction networks for antiviral drug target discovery mathematical control theory: deterministic finite dimensional systems control capacity and a random sampling method in exploring controllability of complex networks controllability of complex networks emergence of bimodality in controlling complex networks a directed protein interaction network for investigating intracellular signal transduction combining multiple positive training sets to generate confidence scores for protein-protein interactions an $nˆ{ / } $ algorithm for maximum matchings in bipartite graphs input graph: the hidden geometry in controlling complex networks protein-mediated regulation of inflammatory and innate immune signaling and its association with antiretroviral activity expression profiling of trim protein family in thp -derived macrophages following tlr stimulation toll-like receptor signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection activation of nk cells and t cells by nkg d, a receptor for stress-inducible mica rae- ligands for the nkg d receptor are regulated by e f transcription factors, which controlcell cycle entry the database of interferon regulated genes identification of pvr (cd ) and nectin- (cd ) as cell surface ligands for the human dnam- (cd ) activating molecule cd /pvr plays a key role in cell motility during tumor cell invasion and migration cytokine storm plays a direct role in the morbidity and mortality from influenza virus infection and is chemically treatable with a single sphingosine- -phosphate agonist molecule understanding sars-cov- -mediated inflammatory responses: from mechanisms to potential therapeutic tools the human scavenger receptor class b type i is a novel candidate receptor for the hepatitis c virus mice deficient in nucleoporin nup develop peripheral t cell alterations cell entry of hepatitis c virus requires a set of co-receptors that include the cd tetraspanin and the sr-b scavenger receptor phosphatidylserine binding of class b scavenger receptor type i, a phagocytosis receptor of testicular sertoli cells regulation by vitamin e of the scavenger receptor bi in rat liver and hepg cells vitamin e supplementation decreases lung virus titers in mice infected with influenza a selective small molecule inhibitor of c-met, pha- , reverses lung premalignancy induced by mutant k-ras sigma- receptor chaperones at the er-mitochondrion interface regulate ca + signaling and cell survival highly selective inhibition of impdh provides the basis of antineuroinflammation therapy a novel ehp-gigyf translational repressor complex is essential for mammalian development antiviral potential of erk/mapk and pi k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis rapamycin enhances eif e phosphorylation by activating map kinase-interacting kinase a (mnk a) acknowledgments: thank you to the howard hughes medical institute for supporting this project through the james h. gilliam fellowships for advanced study program. the authors declare no conflict of interests. key: cord- - n ej f authors: masse, shirley; capai, lisandru; villechenaud, natacha; blanchon, thierry; charrel, rémi; falchi, alessandra title: epidemiology and clinical symptoms related to seasonal coronavirus identified in patients with acute respiratory infections consulting in primary care over six influenza seasons ( – ) in france date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: n ej f there is currently debate about human coronavirus (hcov) seasonality and pathogenicity, as epidemiological data are scarce. here, we provide epidemiological and clinical features of hcov patients with acute respiratory infection (ari) examined in primary care general practice. we also describe hcov seasonality over six influenza surveillance seasons (week to of each season) from the period / to / in corsica (france). a sample of patients of all ages presenting for consultation for influenza-like illness (ili) or ari was included by physicians of the french sentinelles network during this period. nasopharyngeal samples were tested for the presence of respiratory pathogens by real-time rt-pcr. among the ili/ari patients, were positive for at least one hcov ( . %). on an annual basis, hcovs circulated from week (november) to weeks – (may) and peaked in week (february). overall, among the hcov-positive patients detected in this study, hcov-oc was the most commonly detected virus, followed by hcov-nl , hcov-hku , and hcov- e. the hcov detection rates varied significantly with age (p = . ), with the age group – years accounting for . % (n = ) of hcov-positive patients. fever and malaise were less frequent in hcov patients than in influenza patients, while sore throat, dyspnoea, rhinorrhoea, and conjunctivitis were more associated with hcov positivity. in conclusion, this study demonstrates that hcov subtypes appear in ari/ili patients seen in general practice, with characteristic outbreak patterns primarily in winter. this study also identified symptoms associated with hcovs in patients with ari/ili. further studies with representative samples should be conducted to provide additional insights into the epidemiology and clinical features of hcovs. coronaviruses (covs) are an enveloped, single positive-strand rna species of viruses belonging to the coronaviridae family, which infect birds and mammals. in animals, covs cause respiratory, enteric, cardio-vascular, and neurological disorders [ ] . in humans, these viruses result in respiratory and gastro-intestinal symptoms, ranging from cold symptoms to severe diseases [ , ] . covs recognized to infect humans belong to the genera alphacoronavirus and betacoronavirus [ ] . seven cov species are known to cause human infection, of which four hcovs (namely hcov e, nl , oc , and hku ) are known as non-severe acute respiratory syndrome (sars)-like covs. hcov- e and hcov-nl belong to the genus alphacoronavirus. the genus betacoronavirus includes hcov-hku , hcov-oc , sars-cov- [ ] , the middle east respiratory syndrome (mers) coronavirus (mers-cov) [ ] , and the sars-cov- , which is currently associated with a global outbreak [ ] . the four non-sars/mers species circulate widely in humans and infect individuals of all ages [ , ] . hcov- e and hcov-oc were identified in and were primarily associated with mild upper respiratory tract infections [ , ] . hcov-nl was identified in from a -month-old child suffering from bronchiolitis and conjunctivitis [ ] and hcov-hku was discovered in in hong kong and isolated from patients with pneumonia [ ] . in general, the four common circulating hcovs mostly infect humans during the winter season (december-april) [ ] , whereas circulation of hcov-hku has been observed during the spring-summer period [ ] . the world health organization has highlighted the need to improve epidemiological surveillance and knowledge of the health burden imposed by non-influenza respiratory viruses [ ] . hcovs are generally associated with mild upper respiratory tract infections [ ] , but severe infections with hcov- e, hcov-nl , and hcov-oc have been reported [ ] [ ] [ ] . in the context of the spread of sars-cov- in the community, a better understanding of the seasonality and clinical features of patients with confirmed hcovs could be useful for mathematical modelling and clinical diagnosis. there is currently a debate about hcov seasonality and pathogenicity, as epidemiological data are scarce and mostly from hospitalized populations. here, we document the epidemiological and clinical features of hcov patients with acute respiratory infection (ari) observed in general practice. we also describe hcov seasonality over six influenza surveillance seasons ( / to / ) in corsica, france. nasopharyngeal samples were collected: i) as part of the community influenza surveillance conducted in collaboration with the french sentinelles network from patients seen in general practice, consulting for influenza-like illness (ili) or ari (for patients aged > years old) during six influenza seasons (week to of each season) from to in corsica, france; and ii) from ari patients enrolled throughout mainland france by general practitioners (gps) of the french sentinelles network, during an epidemiological study of the risk factors for seasonal influenza (iriis study; - influenza seasons (week to )) [ ] . notably, to ensure that the selection of ili/ari patients remained random, each gp was required to include, each week, the first two patients unrelated to one another, consulting within < h since symptom onset and consenting to provide a nasopharyngeal specimen. each patient could be included only once a year (table and figure ). the surveillance uses a specific definition of ili for patient recruitment: sudden onset of fever > • c with myalgia and respiratory signs, diagnosed by the physician. the case definition of ari was "any person with a sudden onset of symptoms and at least one of the following four systemic symptoms: fever or feverishness, malaise, headache, myalgia, and at least one of the following three respiratory symptoms: cough, sore throat, or shortness of breath." . this rrt-pcr did not provide details for hcovs and hadv, hbov, and hpiv were not analysed. thus, samples still available were retrospectively analysed for cov- e, cov-oc , and cov-nl by ftd. all nasopharyngeal samples collected by the french sentinelles network during the / season were analysed for sars-cov- . rdrp-ip and rdrp quantitative rtrt-pcr was used for detection of sars-cov- . the rdrp rtrt-pcr corresponds to the charité protocol [ ] . when a sample was positive for rdrp-ip , quantification of the number of rna copies was performed according to a scale ranging from to copies per µl [ ] . all analyses were conducted by the laboratory of virology, university of corsica. the hcov seasonality in corsica from / to / was studied using samples collected by gps of the corsican sentinelles network, as constant and homogeneous monitoring of this population of ili/ari patients has been conducted throughout six influenza seasons (table and figure ). to study the weekly number of hcovs detected among ili/ari patients seen in general practice during the six influenza seasons, we gathered all samples collected by gps for influenza surveillance and for the iriis study (table and figure ). the samples were obtained as part of influenza surveillance. the protocol was conducted in agreement with the helsinki declaration. we obtained authorization from the french data protection agency (cnil# ). the iriis study was approved by the ethics committee (cpp sud mediterranee v, / / ; ref. number . ). differences according to viral infection and epidemiological data (sex, age, clinical symptoms, and risk factors) were analysed and tested using fisher's exact test or chi-square test. results were considered statistically significant when the p value was lower than . . all statistical analyses were performed using r software version . . (r foundation, vienna, austria). during the six-year study, among the ili/ari patients enrolled by the corsican sentinelles network, . % (n = ) were positive for at least one of the respiratory viruses analysed ( table ) . hcovs were the third most frequently detected viruses ( . %; n = ) after influenza viruses subtypes a ( . %; n = ) and b ( . %; n = ) ( table ). the number of hcov infections ranged from . % the seasonal distribution per week of hcov, influenza viruses (a and b), rsv, and hrv was studied in ili/ari patients (table and figure ). among the patients, . % (n = ) were positive for at least one respiratory virus, of which . % (n = ) were positive for influenza a virus, . % (n = ) for influenza b virus, and . % (n = ) for hcovs. hcovs were detected during the entire winter season (weeks - ), with the highest number of hcovs detected in week ( figure ) . notably, week had a decrease in influenza a virus detection (figure ). since the number of hcovs detected by species per season was low, the seasonality of hcovs was established from all viruses detected per week over the six influenza seasons. the hcov peak trend detection among week to was clearly observed during three influenza seasons ( / ; / ; / ). among the hcov cases, . % (n = ) were typed. of these . % (n = ) of cases were infected with hcov-oc , . % (n = ) with hcov-nl , . % (n = ) with hcov-hku , and . % (n = ) with hcov- e ( table ). the weekly number of hcov types detected among the ili/ari patients is illustrated in figure . a degree of synchrony was observed during all winter seasons and in the timing of the peak (week ) for hcov-hku , nl , and e, whereas hcov-oc infections occurred earlier with a peak in weeks - ( figure ). among the ili/ari patients, ( . %) were positive for at least one hcov. among them, ( . %) were infected with one hcov, while ( . %) were infected with at least two different respiratory viruses (including two co-infections with three viruses) ( table ) . of these, the most frequently observed co-pathogens were influenza viruses (flu a: n = / ; . %, and flu b: n = / ; . %) ( table ) . two co-infection combinations were detected: hcov- e/nl and hcov-hku /oc . the demographic data and clinical characteristics of the confirmed hcov-positive patients are summarized in table . the age of patients infected with hcov varied from month to years with mean and median ages of years. the male to female ratio of hcov-infected patients was . ( / ). hcov infections were observed in all age groups (table and figure ). however, the detection rates in these different age groups varied significantly (p = . ) with the age group of - years accounting for . % (n = ) of hcov-positive patients. in the remaining groups, detection rates were observed of . % (n = ) in the - age group, % (n = ) in the - age group, . % (n = ) in the - age group, . % (n = ) in the - age group, and . % (n = ) in the group aged ≥ years old. * overall hcov infections also include strains without species determination (n = ). hcov-oc and hcov-hku were detected in patients of all ages (table and figure ). the oldest patients (≥ years old) were infected by hcov-oc and hcov-hku only ( figure a) , and hcov-hku was the most prevalent among patients aged ≥ years old ( figure b ), but a significant difference in the age-group distribution of hcov species was not observed (table ) . the epidemiological and clinical features of patients were compared according to their viral infection: influenza a cases, influenza b cases, and hcov cases (single or co-infection). fever and malaise were less frequent in hcov patients than in influenza patients, while sore throat, dyspnoea, rhinorrhoea, and conjunctivitis were more frequently associated with hcov positivity (table ) . sore throat and dyspnoea were more frequently detected in patients with hcov single infection ( . %, / ) than in patients with hcov co-infection ( . %, / ; p = . ) (table ) . notably, oseltamivir was prescribed more frequently to influenza a patients (table ) . lastly, when comparing epidemiological and clinical features of patients according to the hcov strain detected, there was a significant effect on the prevalence of sex (p = . ), with the hcov- e strain detected more frequently in male patients ( . %) ( table ). a significant difference in the prevalence of malaise (p = . ) among hcov strain patients was also identified, as this symptom was exclusively related to hcov-hku patients ( . %, / ) (table ). among the ari patient samples collected during the / influenza season and tested retrospectively for sars-cov- from week to week , four ( . %) were positive for sars-cov- . these four infections were detected during weeks (n = ), (n = ), and (n = ). fever, cough, and headache were the most commonly declared symptoms, while anosmia was reported for one patient. the mean age of the four patients was . years and the sex ratio was : (two women and two men). data from continuous surveillance are very important for identifying the pattern of hcov epidemiology. this report is the first to describe patterns of circulation of the four common hcov strains in french ili/ari patients seen in general practice over six influenza seasons. hcov was the third most detected respiratory virus in ari/ili patients after the influenza a and b viruses. similar to their susceptibility to other respiratory viruses, young children aged < years old had the highest risk of hcov infection. sore throat, dyspnoea, and conjunctivitis were more frequently described among hcov cases than among influenza cases. similar to other countries in the northern hemisphere, we reported a winter seasonal prevalence pattern for the four hcov strains [ ] , with the highest number of cases presenting in february [ , ] . in our study, the peak in february was attributable to the e, nl , and hku strains, whereas the oc strain had peaks in december. overall, among the hcov-positive patients in this study, the oc strain was the most commonly detected virus, followed by the nl , hku , and e strains, in agreement with previous studies [ , , ] . hcov-oc is routinely associated with~ % of acute respiratory infections and was the most commonly detected virus in hcov patients [ ] [ ] [ ] . a seroconversion study in hospitalized children reported that hcov-oc and hcov-nl might induce cross-immunity, and therefore reduce the subsequent number of clinically identified hcov-hku and hcov- e infections [ ] . we confirmed that, similar to other respiratory pathogens, hcovs were widespread in all age groups [ ] . a significant difference in the age distribution of hcov patients was noted, with the youngest ( - years old) patients displaying a threefold higher level of infection than those aged - years old and a sevenfold higher infection level than patients aged ≥ years old. among hcov patients aged < years old, the most frequently detected viruses were hcov-oc and hcov-nl , while among patients aged ≥ years old, hcov-hku was the most frequently detected. this trend of hcovs infection has already been reported in other studies [ , , , ] . a recent surveillance study on the occurrence and hospitalization rates in children with respiratory infections over years reported that hcovs were involved in . % of episodes [ ] . to date, in contrast to that observed for hcovs, few sars-cov- have been observed in children. official national statistics in italy reported that % of total cases diagnosed country-wide were below years [ ] . it could be possible that pre-existing cross-immunity provides protection and/or reduces the severity of covid- , thereby reducing the number of children who are tested/hospitalized [ ] . the fewer number of hcov infections in older than younger patients may result from higher antibody levels or other immune mechanisms of protection [ ] . the lower number of hcov infections in older than younger patients may result from higher antibody levels or other immune mechanisms of protection [ ] . it is well known that hcovs co-circulate endemically with other common respiratory viruses and co-infections are frequently observed. in our study, six other common respiratory viruses were detected simultaneously and . % of the hcov-positive patients were co-infected by at least one of the other respiratory viruses. the occurrence of co-infection of hcov, including other hcovs, rsv, influenza a and b viruses, hadv, and hmpv has been reported [ , ] . similar to other studies, we observed that influenza and rsv were the most common respiratory viruses co-infected with hcov [ , ] . we did not observe co-infection of sars-cov- and another respiratory virus among the four covid- patients detected. a recent co-infection of sars-cov- and hcov-hku has been reported [ ] . in this study, we compared the demographic data and clinical characteristics of hcov patients with those of influenza a and b patients, respectively (table ). sore throat, dyspnoea, rhinorrhoea, and conjunctivitis were more often observed in hcov cases than in influenza cases. while dyspnoea was reported as not common in seasonal influenza viruses [ ] , studies of hospitalized patients reported the role of hcov in causing lower respiratory illness [ , ] . fever was more often observed in influenza cases, as reported [ ] . these observations may be of importance, especially when hcovs and influenza virus co-circulate. the clinical impact of hcovs in co-detection is not fully understood, with previous studies observing unchanged morbidity or increased illness severity [ ] . when we compared the clinical presentations of patients with a single hcov infection with those with co-infection, sore throat and dyspnoea were more commonly reported in cases with a single infection. a growing number of studies are currently describing the clinical features due to sars-cov- infection [ ] [ ] [ ] [ ] [ ] . although most of the covid- studies have been conducted on hospitalized patients some comparisons with data of the present study might be useful. fever and cough were the most common symptoms reported for sars-cov- and hcov patients [ ] . compared to symptoms observed in hcov patients included in the present study, sars-cov- patients seem to show less headache ( . %- . % vs. . %), rhinorrhea ( % vs. . %) and myalgia ( . - . % vs. . %) [ ] . sore throat, which was one of the most detected symptoms among hcovs patients, was less common among covid- patients [ ] . about half of covid- patients had an underlying disease [ ] , whereas in our study less than a quarter of the hcov patients suffered from an underlying disease. this study has some limitations. first, we used two ili/ari patient samples, which can introduce bias, even if the methodology of enrolment was very similar. second, the number of hcov patients included did not allow the identification of meaningful associations by subanalyses. third, we studied hcov circulation during influenza season and not on annual basis. as previously described, hcovs co-circulate and showed marked winter seasonality, and are not detected in the summer period [ ] . lastly, we did not identify the species of hcov cases because relevant samples were not available. in conclusion, this study demonstrates that hcov subtypes appear in ari/ili patients seen in general practice, with characteristic outbreak patterns primarily in winter. hcovs were detected at a significantly higher rate in children aged < years 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characteristics of hospitalized patients with novel coronavirus-infected pneumonia in clinical findings in a group of patients infected with the novel coronavirus (sars-cov- ) outside of wuhan, china: retrospective case series clinical characteristics of coronavirus disease in china the epidemiology and clinical information about covid- we thank all general practitioners and paediatricians participating in the french sentinelles network. the authors declare no conflict of interest. the funder had no role in study design, data collection, data analysis, data interpretation, writing of the report, or in the decision to submit this article for publication. all researchers' decisions have been entirely independent from funders. key: cord- - gpyi ii authors: raev, sergei; yuzhakov, anton; bulgakov, alexandr; kostina, ludmila; gerasianinov, alexei; verkhovsky, oleg; zaberezhny, alexei; aliper, taras title: an outbreak of a respiratory disorder at a russian swine farm associated with the co-circulation of prrsv and prrsv date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gpyi ii we conducted a cross-sectional study to identify the major respiratory pathogen responsible for an outbreak of respiratory disease at a swine farm in west siberia in . we discovered that the peak of morbidity and mortality coincided with a high level of porcine reproductive and respiratory syndrome virus (prrsv) and -related viremia. based on longer prrsv viremia, the dominant role of prrsv over prrsv in the outbreak was assumed. phylogenetic analysis revealed that the prrsv strain belonged to sub-genotype —one of the predominant groups of genotype prrsvs in russia. a partial open reading frame sequence of the prrsv isolate demonstrated a high identity with modified live vaccine-related strains from denmark ( %) and wild-type vr ( %). we identified the first instance of prrsv /prrsv mixed infection in russia. this finding indicates that further field investigations are needed to access prrsv epidemiology in eastern europe. porcine reproductive and respiratory syndrome (prrs) has been simultaneously described in the usa and europe and remains a major problem for the pig industry. economic losses associated with this disease are estimated at us$ million per year globally. in sows, reproductive failure is characterized by abortions, fetus mummification, stillbirths, and birth of weak offspring. growing piglets mainly demonstrate respiratory symptoms such as dyspnea, coughing, and fever [ ] . further investigations have revealed that the causative agent of this disease is porcine reproductive and respiratory syndrome virus (prrsv), for which two genotypes have been described: the european (prrsv ) and north american (prrsv ) genotypes. significant genetic ( - % identity) and antigenic differences eventually led to their divergence into two separate species: betaarterivirus suid (the european type, or prrsv ) and betaarterivirus suid (the north american type, or prrsv ) [ ] . both viruses belong to genus betaarterivirus in the variartevirinae subfamily and arteriviridae family. prrsv's genome comprises about , nucleotides and contains ten open reading frames (orfs) including orf , which encodes the nucleocapsid protein [ ] . shortly after prrsv's discovery, both prrsv and prrsv were isolated beyond their original places of detection, namely europe and north america, respectively. prrsv strains are currently present in europe, north america, and asia, and prrsv strains are predominant in north america the blood samples were taken from piglets ages , , , , , , and weeks ( to animals per age group). serum samples were collected on the same day and stored frozen at − • c before analysis. all serum samples were tested for the presence of antibodies against prrsv capsid protein using two commercial elisa test kits. the first kit, ingezim prrs universal (ingenasa, madrid spain), is prepared on the basis of both prrsv and prrsv recombinant nucleoproteins. this kit detects antibodies against both prrsv and prrsv designated as "pan-prrsv antibodies". the second assay, rrss-serotest (vetbiochem, moscow, russia), is capable of detecting antibodies against prrsv only. both elisa test kits were used in accordance with the manufacturers' instructions. prrsv, pcv , swine influenza virus (siv), porcine parvovirus virus (ppv), and porcine respiratory coronavirus (prcv) in serum samples were detected using commercial polymerase chain reaction (pcr) kits (vetbiochem, russia) according to the manufacturer's instructions. in all serum samples, detection was performed using commercial real-time polymerase chain reaction (pcr) kits (vetbiochem, russia) in accordance with the manufacturer's instructions. according to previous data, both orf and orf sequences might be chosen for prrsv classification [ ] . we used the following primers for the amplification and sequencing of prrsv orf , kindly provided by ivan trus (ghe university, ghent, belgium): -tggcccctgcccaicacgt- (prrsv -orf -f) and viruses , , of -tgatcgccctaattgaataggtgact- (prrsv -orf -r). for the amplification and sequencing of prrsv orf , we used two pairs of nested primers. for the first round of pcr, we used the following primers: -ttctggcccctgcccayc- (f ) and -cgccctaattgaataggtgac- (r ). for the second round of pcr as well as for sequencing, we used '-ccaaataacaacggcaag- ' (amerf _ ) and '-tcatgctgagggtgatg- ' (amerr _ ). the expected size of pcr products of prrsv orf and prrsv orf was around and nucleotides, respectively. a one-tube, real-time rt-pcr kit (alpha ferment, moscow, russia) was used to perform reverse-transcription polymerase chain reaction (rt-pcr). the pcr amplification program consisted of min at • c, min at • c, cycles at • c for s, • c for s, and • c for s, as well as cycle of min at • c. the amplification products were visualized in % agarose gel in a buffer containing a mixture of tris base, acetic acid and edta (x tae) after which purification was performed using monarch dna gel extraction kit (new england biolabs, ma, usa) in accordance with the manufacturer's instructions. sanger sequencing was performed using the big dye®terminator v. . cycle sequencing kit (applied biosystems, ca, usa) in accordance with the manufacturer's instructions, and the primers specified above. the nucleotide sequences of the genome fragments were determined using an ab genomic automated analyzer (applied biosystems, usa). the nucleotide sequences were analyzed using lasergene . . . (dnastar, wi, usa). multiple alignment was performed using muscle (mega . . ). phylogenetic dendrograms were plotted using the maximum likelihood method, gtr model (mega . . ). the topology of the trees was confirmed following bootstrapping replication steps. viral rna was not detected in sera from either newborn or -week-old piglets, whereas in -week-old piglets, prrsv and prrsv , and viral rna were present in % and % of serum samples, respectively ( figure ). the mixed rna belonging to both viruses was detected in only one -week-old piglet. in sharp contrast to prrsv , where the rna was not present in samples from -week-old animals, prrsv viremia was detected in % of -week-old piglets. simultaneously, pcv dna was detected in % of -week-old piglets, and viremia rates increased with age until the age of weeks, with % of the piglets tested being viremic. all serum samples were negative for siv, ppv, and prcv. whereas the total concentration of prrsv -specific antibodies showed a clear tendency to rise from weeks (when all samples were seronegative) to weeks of age, a minor numerical decrease in the total level of antibodies against both strains from to weeks of age was detected ( figure ). comprehensive analysis of total prrsv antibodies (to both genotypes) and prrsv -specific antibodies (table ) did not reveal any certain tendency. prrsv rna was found in an only numerically higher (three out of nine) proportion of prrsv igg negative/total prrsv ig-positive samples compared to prrsv (one out of nine). serum samples from -week-old piglets were used for prrsv (sample # ) and for prrsv (sample # ) sequencing. the orf sequences were deposited into the genbank sequence database under the accession numbers orf _kem eu-mt and orf _kem na-mt . the orf _kem eu-mt (prrsv ) has nucleotides, and further phylogenetic analysis revealed that this isolate belongs to subtype of prrsv ( figure ). this strain is most closely related to strains eig иaus from lithuania and strain gk from the european part of russia [ ] . the partial nucleotide sequence of orf _kem na-mt (prrsv ) comprises nucleotides ( figure ). the comparative analysis of partial sequence orf _kem na-mt against those of reference strains demonstrated a high nucleotide identity with wild-type vr ( %) and mlv-related strains from denmark: kc . _dk- - - and af . _strain_ a ( %) [ ] . the level of identity between the hungarian non-mlv-related strain km . : - _hungary_ _ and orf _kem na-mt was only %. nucleotide identity with gu . , the only one prrsv strain detected in russia, was %. jq . _strain_qyyz kc _strain_gxlsf - ....-....................................-....................................................................... ef _strain_vr ..n.-....................................-....................................................................... af _strain_sp ...k-....................................-.............y......................................................... ef _strain_mn ...k-...n................................-.....................................................................a. ef _strain_mn ...k-................r................s..-.s...................................................a................. kc _isolate_dk- - - -....-....................................-...................................................?................... kc _dk- - - ....-....................................-....................................................................... kf _dk- - - - ....-....................................-....................................................................... af _md- _tw ..nk-......................s...v....r....-...........................................i........................... ay _strain_fj- ..r.-.....................h........e..i..-...........................................i........................... ay _strain_ch- a .r.k-...n................................-.s..................................................................... af _strain_ a ....-....................................-....................................................................... km _isolate_hungary_ _ ...k-....................................-..l........................................i........................... ef _strain_jxa ...k-...n..............................r.-...........................................a.................q.......a. gu _isolate_irkutsk/rus/ ...k-...n..............................r.-...........................................a.................q.......a. jq _strain_qyyz ...k-..........................a.......n.-......................v.........r...................................... jn _strain_nadc ..nk-......................s............n-r...............................r...................................... dq _isolate_s ....-....................................-....................................................................... af _isolate_nadc- _(e) . ...-....................................-....................................................................... mn _isolate_lung -s -....-....................................-....................................................................... af _isolate_ja- _(e) . ..k-.r.n.............................i.n-....................................................................... ay _isolate_ - .l..-..............p.........p...........-.............................................................?......... mk _isolate_ - ..nk-...g..................s............n-g...............................r............................r.......a. af _strain_respprrs_mlv ....-....................................-....................................................................... dq _isolate_ingelvac_atp ...k-.r.n.............................i.n-....................................................................... af _strain_primepac prrs ...k-....................................-.............y......................................................... kp _isolate_bacu_unam ....i...n................................-.....................................................................a. ay _isolate_ d ...kk.q.n................................-.....................................................a................. ef _isolate_ k . kc _isolate_dk- - - ..............................................-............................................................... dq _strain_porcilis_prrs ..............................................-............................................................... dq _strain_pyrsvac- ............n.................................-..r............................................................ mt _strain_tyu ......r....en..................k.m......skr..t-.r.r.......................................a....p.............. jf _strain_lena ......r.fr..n..................r...t..........-.................v.....g........................p.............. kp _strain_su -bel ......r.vr..n..................k...t..........-...r.............v............................................. strain_bor * ...r..r..rn.n......d...........k.m............n.r...............v.....a........................p.......... n.n......d...........k.m..........rvn.r...............v.....s....................................... kc _isolate_westsib ...n..r...n.na.....d...........k.......a....r.n...r.........t...v.....s........................a...i.......... af _strain_sid ...r..r...nnn......d........m..r.m.....r......n.r.r.............v.....la..................a................... af __strain_aus ...r..r..rn.n......d...........k.............vn.................v.....s........................p.............. kc _strain_eig ...r.pr...n.ni.....d...........k..............s.r...............v.....s....................................... eu _strain_gk . the results of this study indicate that the respiratory disease outbreak correlated with increased prrsv-related viremia rates, while it did not correlate with the prevalence of another major respiratory pathogen, pcv . remarkably, prrsv -related viremia peaked in a more pronounced manner and lasted longer compared to prrsv -related viremia. this might be an indication of the predominant role of prrsv in respiratory outbreak development. we assume that whereas total prrsv antibodies from piglets aged weeks might be considered as post-infectious, at weeks of age, these antibodies are of maternal origin. due to differences in sensitivity and specificity between the test kits used in this study and simultaneous prrsv and prrsv viremia detection, our effort to distinguish between prrsv and prrsv antibodies by using universal (prrsv /prrsv ) and prrsv -specific elisa test kits cannot describe the real situation. after the initial introduction of prrsv into europe, differentiation between antibodies against prrsv of different genotypes became crucial to prrsv diagnosis. an elisa assay for simultaneous detection and differentiation between antibodies to prrsv and prrsv was developed [ ] , but no such elisa test kit is commercially available in russia. hence, the use of universal pcr test kits for simultaneous detection of both viruses is the only option to prevent prrsv from remaining undetected. data on the comparative pathogenicity of prrsv , prrsv , and mixed infection are scarce. in one study, the predominant role of prrsv over prrsv in the course of prrsv - /prrsv (lineage ) mixed infection was demonstrated on the basis of replication and pathogenicity levels [ ] . significant differences in the pathogenicity of different genotypes/lineages of prrsv and prrsv should be considered in the case of mixed infection. further experimental studies will be required to establish the impact of at least prrsv - and prrsv (lineage ) presence. despite recombination never being demonstrated between prrsv and prrsv strains, the simultaneous presence of prrsv and prrsv might also be considered a possible risk for the emergence of recombinant strains. the use of mlv vaccines under such circumstances may only increase this risk [ , ] . the idea that prrsv is a homogenous group of viruses was revised after the discovery of genetically distinct isolates from eastern europe, especially from belarus and russia [ ] . thus, the detection of a new member of prrsv - in the present study offers additional proof of the wide distribution of this genotype in russia. isolates of prrsv - (including the so-called russian group of viruses), prrsv - , and prrsv - differ significantly in pathogenicity [ , , , ] . marked differences in the nucleotide sequence between prrsv - isolates and prrsv vaccine strains, which all belong to prrsv - , including differences in the orf sequence (which encodes a major epitope: the envelope glycoprotein), could potentially hamper vaccine efficacy [ ] . the benefits of using currently available vaccines against prrsv - , be they live, inactivated, or subunit, remain unknown. the circulation of prrsv further complicates the search for working control strategies, as no vaccines against prrsv are currently available in russia. phylogenetic analysis of a partial orf sequence of the prrsv strain isolated in the course of this study revealed that this strain was very dissimilar ( % identity) to the only known prrsv strain (jxa -related, sublineage . ) detected in east siberia, russia, in . this suggests that a different instance of prrsv introduction into russia occurred at a certain point in addition to the one resulting in the outbreak of . further genetic analysis (including an orf sequence) should be performed to classify this isolate more precisely. high homology within lineage of prrsv makes the distinction between the ingelvac prrs mlv vaccine strain, its parental strain vr- , vr- -related wild-type isolates, and vaccine-derived isolates very complicated and requires at least orf sequence analysis [ ] . the boehringer mlv vaccine has not been licensed in russia. however, there are known cases of the detection of an mlv-like prrsv strain in vietnam, regardless of the corresponding type of vaccine not being licensed in that country [ ] . notably, one locally-produced mlv vaccine against prrsv has been licensed but is currently commercially unavailable in russia, and the sequence of the vaccine virus has not yet been published. it is also conceivable that the virus originated from breeder animals infected with an mlv-related prrsv strain. it is well known that mlv-related strains (including strains that have a high identity level with orf _kem na-mt ) are widely spread in denmark, one of the major exporters of breeding pigs [ ] . for example, the average number of pigs imported from denmark to russia has been more than animals a year over the past three years. finally, an independent introduction of a wild-type vr -related strain could result in the emergence of the orf _kem na-mt virus. in the course of infection, nls interacts with nuclear transport proteins, leading to the penetration of the n protein into the nucleus and nucleolus of infected cells [ ] . a critical role of this element of the n protein in pathogenesis has been shown [ ] . a similar insertion is known to be found viruses , , of in a mexican field isolate dmzc / (genbank id mf . ) [ ] . this might indicate that both kem na and dmzc / have a nucleoprotein consisting of amino acids in length. complete orf sequencing should be performed to confirm this assumption. interestingly, kem na and dmzc / are genetically distant from each other, their nucleotide identity being only %, suggesting that this insertion may not be lineage-specific. to the best of our knowledge, this is the first report of prrsv /prrsv co-circulation in russia. considered alongside data from field veterinary laboratories, our observations indicate that prrsv circulates in both the european and asian parts of the country. the prevalence and distribution of prrsv in russia therefore warrants further investigation. porcine reproductive and respiratory syndrome: clinical disease, pathology and immunosuppression the molecular biology of arteriviruses genetic diversity of porcine reproductive and respiratory syndrome virus strains circulating in hungarian swine herds lelystad-like strain of porcine reproductive and respiratory syndrome virus (prrsv) identified in canadian swine characterization of emerging european-like porcine reproductive and respiratory syndrome virus isolates in the united states phylogeny-based evolutionary, demographical, and geographical dissection of north american type porcine reproductive and respiratory syndrome viruses definition of subtypes in the european genotype of porcine 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virus appearance of acute prrs-like symptoms in sow herds after vaccination with a modified live prrs vaccine comparison of molecular and biological characteristics of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine (ingelvac prrs mlv), the parent strain of the vaccine (atcc vr ), atcc vr , and two recent field isolates of prrsv pathogenesis and antigenic characterization of a new east european subtype porcine reproductive and respiratory syndrome virus isolate pathogenicity of three genetically diverse strains of prrsv type in specific pathogen free pigs genetic analysis of orf porcine reproductive and respiratory syndrome virus (prrsv) isolated in vietnam the localization of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication phylogenetic analysis of orf and orf of porcine reproductive and respiratory syndrome (prrs) virus and the frequency of wild-type prrs virus in méxico the authors would like to thank alexander mishin and alexander skrylev for their administrative support and yana streltsova for her excellent technical assistance. we thank tomasz stadejek and sara botti for providing the sequences of the strains bor- and ili- . the authors declare no conflict of interest. key: cord- -aesiff f authors: romero-brey, inés; bartenschlager, ralf title: membranous replication factories induced by plus-strand rna viruses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: aesiff f in this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) rna viruses. we discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. a general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (er) and double membrane vesicles, representing extrusions also originating from the er, respectively. we hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments. members of the family flaviviridae are enveloped viruses with a single stranded rna genome of positive polarity. this family contains four different genera: hepacivirus (from the greek hepar, hepatos, -liver‖), flavivirus (from the latin flavus, -yellow‖), pestivirus (from the latin pestis, -plague‖) and the recently included genus pegivirus [ , ] (figure ). hepatitis c virus (hcv) is the prototype species of the genus hepacivirus. it was first discovered by choo et al. [ ] in the serum and tissues of a chimpanzee experimentally inoculated with serum from an individual with chronic, non-a, non-b hepatitis. this virus was associated with a mild form of chronic hepatitis frequently observed in recipients of blood transfusions [ ] and was called hcv. a second species within the genus hepacivirus is gbv-b which was first identified in tamarins that developed hepatitis following inoculation with the serum from a surgeon (initials g.b.) with acute hepatitis. additional gb-like viruses were discovered later on and have been assigned to the new genus pegivirus (an acronym derived from pe, persistent; g, gb or g) within the family flaviviridae [ ] . and cms may represent the site of denv replication and rna translation/polyprotein processing, respectively [ ] . the most complete characterization of denv-induced intracellular membrane rearrangements elucidated their d architecture as well as their spatial connection with viral assembly sites [ ] . tem of resin-embedded infected cells revealed a complex collection of convoluted and vesicular structures, including cms that were usually surrounded by multiple vesicles, often appearing as longitudinal vesicle arrays. by using electron tomography (et), the latter were found to correspond to er tubules containing - nm single-membrane vesicles (ve) that result from the invagination of the er membrane into the er lumen. by conventional em, these vesicles appeared as double membrane vesicles, likely corresponding to the vps described earlier [ ] . immuno-em confirmed that the vesicles visible in resin-embedded cells were induced by denv infection and contained all ns proteins. however, only ns was detectable within the cms, which could be due to lower affinity of the antibodies or poor accessibility of the other ns proteins in the cms. double-stranded rna (dsrna) detected by immunostaining appeared as discrete electron-dense structures inside or on the cytosolic surface of a subset of vesicles, suggesting that dsrna might be present only in some of the vesicles at a given time point. furthermore, the vesicles contain rather uniform pores of ~ nm diameter towards the cytosol ( figure a ). thus, both the topology of the vesicles and the immunolabeling results support the idea that the vesicles might be the site of rna replication. moreover, these results showed that replication factories are a continuous membrane network that provides a platform for the transport of viral proteins and genomes between sites of rna replication, ribosome-containing compartments (rna translation) and virus assembly sites. in fact, virus budding sites were found in close proximity to the pores of the replication vesicles. this topological link may ensure efficient production and delivery of viral rna for the assembly of infectious virus progeny. consistent with these findings, a very recent publication using et showed that these virally modified structures were also observed in denv-infected mosquito cells, with one exception: cms were absent from denv-infected c / mosquito cells [ ] . in addition, after multiple rounds of virus replication, tubular structures were also observed in the vicinity of vps. these structures might represent a hallmark of chronically infected insect cells, since these structures are also induced by tbev in tick cells (see below). the first reports on wnv-infected cells described the visualization of virions [ ] . an extensive characterization of kunjin virus-the australian variant of wnv (wnv kun )-infected cells has been carried out more recently [ ] [ ] [ ] . three well-defined structures were found, corresponding to large cms, paracrystalline arrays (pcs) and vps that appeared as membrane sacs containing small vesicles (ve) [ , ] . based on immunolocalization studies, a distinct redistribution of the trans-golgi network (tgn) and colocalization of tgn markers with dsrna has been observed, suggesting that the replication factories of wnv kun were derived from the tgn [ ] . three-dimensional reconstructions of the wnv kun replication sites revealed an intimate association of the rough er (rer) with the bounding membrane of the vps [ ] (figure b ), resembling the vesicles observed in denv-infected cells. these results argue for an additional role of the rer in the formation of the wnv kun replication factories. similar to denv, individual necks were observed in the vesicles as well as the majority of the viral rna, as detected by immunolabeling with a dsrna-specific antibody, resided within these vesicles [ , , [ ] [ ] [ ] . in most cases, viral rna spanned the breadth of the vesicles and was juxtaposed to the necks open to the cytoplasm [ ] . . slices through tomograms of infected cells (on the left) and d top and lateral ( ° rotation) views of the same tomograms (on the right) are depicted, showing the characteristic virus-induced structures. the replication vesicles (ve) of denv, wnv and tbev (genus flavivirus) correspond to invaginations of er membranes that remain connected to the cytosol via nm-pores (highlighted with white arrows in the d lateral views), forming vesicle packets (vps). the replication factory of hcv (genus hepacivirus) is primarily composed of double membrane vesicles (dmvs) that seem to be formed aser protusions connected to er membranes via neck-like structures (highlighted with white arrows in the d lateral view). the er is shown in yellow (denv, tbev and hcv) or in red (wnv) and the replication organelles in brown (denv, tbev and hcv) or in white (wnv). the outer and inner membranes of dmvs are depicted in different shades of brown (outer membrane in dark brown and inner membrane in light brown). figure b is reproduced with permission from [ ] . in cells infected with tbev, one of the most important tick-transmitted viruses in europe and asia, virus particles and membrane-connected vesicles were also observed inside the er [ ] , similar to what was described for denv and wnv kun . the viral dsrna was only detected inside the vesicular structures within rer, suggesting that tbev rearranges internal cell membranes to generate a compartment that protects viral rna from detection by cytoplasmic pathogen recognition receptors (prrs) [ ] [ ] [ ] . this localization of dsrna might suffice to delay the onset of the ifn response [ ] . for tbev [ ] and wnv kun [ ] it was shown that treatment with brefeldin a (bfa), a drug which disrupts the golgi apparatus, did not interfere with viral replication. however, this treatment rendered wnv kun sensitive to the antiviral action of the ifn-induced protein mxa, indicating that bfa might have disrupted the membranous wnv kun replication compartments, thus leading to exposure of dsrna and its detection by prrs. in contrast, treatment of tbev-infected cells with bfa neither affected viral replication, nor the level of ifn production. these findings indicate that tbev dsrna might be stored inside bfa-resistant membrane vesicles that robustly protect the viral rna from recognition by cellular sensors. vector-borne flaviviruses like denv and tbev must replicate in both mammalian and arthropod cells. a few comparative studies have been published describing virus-induced structures such as cytoplasmic membrane proliferations and vesicle formation, also in insect cells [ , , , , ,] . a detailed comparative ultrastructural analysis of tbev-induced modifications revealed that the extent of membrane expansion and the abundance of vesicles were lower in insect cells [ ] . single-membrane vesicles, ranging in diameter from - nm were frequently found within proliferated er areas, often occurring in large groups contained within er cisternae. pore-like openings connected these vesicles to the cytoplasm and to other vesicles. apart from these vesicles, in tick-infected cells elongated vesicles or tubules were found that were much more prevalent in persistently than in acutely infected cells. these tubules were only occasionally noted in infected mammalian cells, similar to what was found with denv-infected cells [ ] . the tubular structures had a cross-sectional diameter of - nm, similar to the one of vesicles, reached up to nm in length, were closed at the ends and often arranged in fascicle-like bundles, shrouded with the er membrane. however, no pores between the tubules or towards the cytoplasm were observed [ ] . the function of these tubules is unclear and it is not known whether they represent bona fide features of replication factories, aberrant structures as a result of incorrect membrane remodeling, or the result of a cellular process to restrict infection [ ] . in any case, the tubules might be a feature of persistent infection, eventually linked to the high number of defective virus particles, because the lack of pores could prevent proper replication or packaging of the viral genome [ ] [ ] [ ] . further studies are required to shed light on the biogenesis and biological significance of these membranous tubular structures. a recent study identified nm-diameter vesicles within the er lumen of tbev-infected bhk- cells and in cells transfected with a tbev replicon [ ] . et revealed that these vesicles are invaginations of the er within a highly organized network of interconnected membranes with half of vesicles containing pore-like connections to the cytoplasm ( figure c ). however, no pore-like openings were observed between adjacent/neighboring vesicles, in contrast to what has been described for cells infected with langat virus (lgtv), a naturally attenuated tick-borne flavivirus [ ] or in wnv kun -infected cells [ ] . interestingly, in tbev replicon cells, the number of pore-containing vesicles was slightly larger (~ %) and they were found in much more fragmented er tubules as compared to tbev-infected cells. however, despite more extensive er rearrangements in replicon cells, they contained fewer vesicles, consistent with the lower level of viral replication [ ] [ ] [ ] . conventional em analysis of neurons infected with murray valley encephalitis virus (mvev) revealed several ultrastructural features, including proliferation of er and golgi complex membranes as well as the appearance of membrane-bound spherical vesicles ( - nm diameter) [ ] , similar to those observed for the related flaviviruses japanese encephalitis virus (jev) [ , ] and st. louis encephalitis virus (slev) [ ] . in the latter case, cylindrical membranous structures (or tubules) were also observed [ ] . the presence of vesicles was also detected in monkey liver cells infected with yellow fever virus (yfv) [ ] . these findings indicate that all members of the genus flavivirus utilize the er as a source of membranes for the formation of their replication factories, whereas assembly of new virions seems to occur at er sacs in close proximity to the replication sites [ , ] , thus creating an optimized membranous environment to support efficient viral replication and assembly. maturation of the newly synthesized virions takes place in the golgi apparatus, where flavirirus virions are often observed [ ] . in stark contrast to flaviviruses, hcv, the prototype of the genus hepacivirus, provokes an alternative rearrangement of intracellular membranes, originally designated -membranous web‖ (mw). this term referred to compact vesicle accumulations embedded into a membranous matrix [ ] as detected in cells inducibly expressing the hcv polyprotein. by using different em methods, we and others have recently found that the mw is primarily composed of double membrane vesicles (dmvs) [ ] [ ] [ ] . the fact that the kinetics of their appearance correlates with hcv replication suggests that these structures play an important role for viral rna amplification [ ] . indeed, immunolabeling of purified dmvs revealed an enrichment for viral proteins as well as dsrna [ , ] . importantly, dmvs contain enzymatically active viral replicase [ ] and they originate from er membranes, similar to what has been found for other members of the family flaviviridae. et analysis showed that most of the dmvs remain connected to the er via their outer membrane [ ] ( figure d ). although dmvs are primarily closed structures, ~ % of them have an opening towards the cytosol. late in infection, multi-membrane vesicles (mmvs) with an average diameter of nm are generated, likely originating from dmvs by secondary enwrapping events [ ] . by using huh . cells infected with the highly replicative hcv strain jfh- , ferraris and coworkers observed three different types of membrane alterations: vesicles in clusters (vics), contiguous vesicles (cvs) and dmvs [ ] . the vics were small single-membrane vesicles of variable size ( - nm) , grouped together in well-delimited areas. most of them had an internal invagination. the cvs were small single-membrane vesicles, present in large numbers and widely distributed throughout the cytoplasm, with a more homogeneous size (around nm). they were tightly associated to each other and tended to form a collar around lipid droplets (lds). dmvs were heterogeneous in size ( - nm) and had a thick, electron-dense membrane consisting of two closely apposed membranes. the increase of cvs' number correlated with an increase of intracellular hcv rna levels, arguing for a possible role of cvs in the early stages of viral replication. the presence of ns a in cvs, as demonstrated by immunogold staining, is consistent with this hypothesis. alternatively, cvs might constitute the membranous platform for viral assembly. in fact, the core protein is present in these structures ( %) as well as on the ld surface ( %). however, so far visualization of virus particles in infected cells has not been possible, making this hypothesis difficult to prove. while most of the dsrna signal was located within dmvs or at dmv membranes, vics were free of viral components and rna and these structures as well as cvs were very rarely observed in cells with a subgenomic jfh- replicon [ ] or absent in cells infected with a jfh- variant designated jc [ ] . the first d reconstruction of a complete hcv-infected cell revealed that all these three membrane structures were tightly connected and closely associated with ld clusters [ ] . taken together, these findings indicate a fundamental role of dmvs in hcv replication. an in-depth comparison of the study by ferraris and coworkers [ ] and our publication [ ] suggests that cvs might be also dmvs for several reasons: first, cvs have electron dense tightly apposed membranes; second, by using correlative light and electron microscopy, we also detected dmv accumulations around lds, reminiscent of the cvs described by ferraris and coworkers [ ] ; third, taking into consideration the density of content and morphology, some of the structures described as dmvs by ferraris and colleagues might correspond to mmvs according to our nomenclature. this might account for the differences in size between the dmvs reported in both studies (up to nm versus nm, respectively). alternatively, the difference might be due to the use of distinct virus strains (jfh- and jc ) that differ in their capacity to produce infectious virus particles by ~ orders of magnitude [ ] , which might also explain the presence of vics only in jfh- infected cells. much less about membranous replication factories is known for pestiviruses. tem-based studies from the times in which the genus pestivirus was still belonging to the family togaviridae reported that pestivirus-infected cells exhibited ultrastructural modifications of rer and contained small numbers of virus-like particles (vlps) [ , ] . gray and nettleton ( ) reported that border disease virus (bdv)-infected cells contained several profiles of er and many dense lamellar bodies, which when transversely sectioned appeared as multiple rows of tubules, nm in diameter [ ] . these lamellae were often found in association with rer and in one occasion vlps appeared to be budding within them. bovine viral diarrhea virus (bvdv)-infected cells contained rer modified into tubules, in which electron-dense vlps were present. more recent studies on bvdv-infected cells revealed cytoplasmic vacuolization and vlps in dilated er cisternae [ , ] . in addition, membrane structures consisting of vesicles of various sizes enclosed in much larger vesicles have been reported [ ] . these structures that morphologically resemble multivesicular bodies (mvbs) are distinct from the hcv-induced membranous web and more reminiscent of the flavivirus-induced vps. studies on the morphogenesis of pestiviral particles were hampered by a low rate of virion production. in a recent study, schmeiser and colleagues have overcome this problem by using high multiplicity of infection in mdbk cells with a distinct virus strain, the giraffe- strain [ ] . obtained results define the er as the site of pestivirus particle assembly, where budding of virions was observed. virus particles were also found inside the lumen of the golgi and in vesicles associated with the golgi compartment, suggesting that virus egress occurs via the conventional secretory pathway. interestingly, replication kinetics of pestiviral rna did not correlate with distinct membrane rearrangements and only slight dilatation of the er lumen was noticed. the absence of significant membrane rearrangements argues for a major difference between pestiviruses and other members of the flaviviridae family. interestingly, the authors detected the capsid protein and dsrna, the marker for viral replication intermediates, mainly in mvbs, indicating that pestiviruses are either using this compartment for replication or that viral rna and proteins are transferred to this compartment for degradation. similar assumptions have been made for hiv [ ] and marburg virus, a member of the filoviridae family [ , ] . alternatively, pestiviral rna and protein in mvbs might be intermediates of the entry process, prior to fusion of the envelope with the endosomal membrane. indeed, particles inside mvbs matching the morphological criteria of pestivirus virions were detected [ ] . however, mvbs of non-infected cells also contain vesicles for lysosomal degradation termed intraluminal vesicles (ilvs) that display a very similar morphology to pestiviral virions. thus, unambiguous discrimination between ilvs and pestivirus particles will require detailed immunolabeling approaches. the first visualization of the d architecture of a (+) strand rna virus replication factory was reported for flock house virus (fhv), a member of the family nodaviridae [ ] . this insect nodavirus induces the formation of invaginations at the outer mitochondrial membrane (omm) with an average diameter of ~ nm [ , ] ( figure a ). the interior of these vesicles (called spherules) is connected to the cytoplasm by a necked channel of ~ nm diameter, which is wide enough to allow import of ribonucleotides and export of synthesized rna (diameter < nm) [ ] . furthermore, metabolically labeled fhv rna localized between inner and outer mitochondrial membranes inside these spherules, thus validating the spherules as bona fide fhv-induced compartments for viral rna synthesis [ ] . conventional tem analyses of coronavirus-infected cells identified large numbers of isolated dmvs [ ] . at least in case of the severe acute respiratory syndrome (sars)-coronavirus, these dmvs are part of an elaborate reticulovesicular network (rvn) of modified er that consists of convoluted membranes, numerous dmvs (diameter - nm) ( figure b ) [ ] , and -vesicle packets‖ apparently arising from merging of dmvs. the cms were most intensively immunolabeled for viral replicase subunits whereas dmvs labeled abundantly for dsrna. while this result argues that dmvs might be the site of viral rna synthesis, et analyses failed to detect dmv connections to the cytoplasm to allow transport of nascent rna. instead, dmvs are connected to each other, to cms and to the er via their outer membranes. also, in case of another coronavirus, the mouse hepatitis virus (mhv), er membranes are thought to be the lipid donor of the membranous replication compartment [ , ] . qualitative and quantitative analyses by (immuno)-electron microscopy of mhv-induced membrane rearrangements revealed the appearance, in strict order, of dmvs (diameter - nm), cms, large virion-containing vacuoles, tubular bodies and cubic membrane structures [ ] . the recently identified coronavirus middle east respiratory syndrome coronavirus (mers-cov) induces extensive membrane rearrangements in the perinuclear region, including the formation of dmvs and cms [ ] . the diameter of mers-cov induced dmvs ranged from - nm, comparable to the sars-cov induced dmvs. in addition, cms were always surrounded by dmv clusters and were only observed in cells that appeared to be more advanced in infection. this observation strengthens the notion that dmv formation precedes the development of cms, as postulated previously for sars-cov [ ] . in addition to the betacoronaviruses (sars-cov, mhv and mers-cov), er-derived dmvs with a diameter of ~ nm have been observed in primary avian and mammalian cells infected with infectious bronchitis virus (ibv), an important poultry pathogen belonging to the genus gammacoronavirus [ ] . however, the most striking structures induced by ibv are zippered er membranes ( figure c ). the zippered er was associated to - nm diameter spherules, structures that are not present in cells infected with betacoronaviruses. et showed that these ibv-induced spherules are tethered to the zippered er and contain a . nm long channel connecting their interior to the cytoplasm of the cell, making them the ideal candidates for the site of ibv rna synthesis. er membranes are also targeted by nidovirales that belong to the family arteriviridae. cells infected with the prototypic arterivirus, equine arterivirus (eav), also contain dmvs associated to er tubules. these dmvs are ~ - times smaller (~ nm) as compared to coronaviruses [ ] . a recent in-depth ultrastructural analysis revealed that the outer membranes of eav-induced dmvs are interconnected with each other and with the er (figure d ), thus forming a reticulovesicular network (rvn) resembling the one previously described for the distantly related sars-cov [ ] . despite significant morphological differences, a striking parallel between the two virus groups, and possibly all members of the order nidovirales, is the accumulation of dsrna, the presumed intermediate of viral rna synthesis, in the dmv interior. along these lines, dmvs visualized by means of electron spectroscopy imaging contained phosphorus in amounts corresponding on average to a few dozen copies of the eav rna genome. like in sars-coronavirus infected cells, connections between dmv interior and cytosol could not be unambiguously identified, suggesting that dsrna is compartmentalized by the dmv membranes. in addition, et revealed a network of nucleocapsid protein-containing protein tubules, intertwined with the rvn. this potential intermediate in nucleocapsid formation, which was not observed in coronavirus-infected cells, suggests that arterivirus rna synthesis and assembly are spatially coordinated. membrane remodeling in picornavirus-infected cells has been studied for more than years. massive virus-induced membrane modifications have been reported already in [ ] , but the origin of these membranes is still a matter of controversy. several lines of evidence, including biochemical and structural data, suggest that the er must play a major role in the formation of those structures [ ] [ ] [ ] . early in infection membranous replication factories contain markers of the golgi [ , ] , whereas markers of the er, golgi and lysosomes were all found to be associated with poliovirus replication sites late in infection [ ] . initial reports identified membrane rearrangements as u-bodies because of their horseshoe-like shape [ ] . later, bienz et al. described rearranged membranes as clusters of single-membrane vesicles [ , ] , while other reports [ , ] noticed the double-membrane morphology of picornavirus-induced vesicles. the single-versus double-membrane morphology of the vesicles was first interpreted as two different models of their formation. however, several recent publications suggest that picornavirus-induced membrane rearrangements might occur in a consecutive manner. thus, early in poliovirus infection small clusters of single-membrane vesicles predominate that are transformed into bigger irregularly shaped single-membrane structures ( figure g ) and, late in infection, replaced by either round or irregularly shaped dmvs [ ] . interestingly, the small clusters of single-membrane vesicles of ~ - nm diameter contain gm , a cis-golgi marker, but did not stain positive for calnexin, an er marker. however, this does not exclude a role of the er for biogenesis of these vesicles, because er-resident proteins might be sorted out as these membranes are transformed. although dsrna and metabolically labeled viral rna were detected in single-membrane vesicles and dmvs, the exponential phase of viral rna synthesis correlates with the appearance of single-membrane and intermediate structures [ ] arguing that these structures are most relevant for high level poliovirus rna synthesis. similar results have been obtained with another member of the family picornaviridae, coxsackie b virus (cvb ) that also induces the formation of single-and double-membrane compartments, whose relative abundance correlates with the stage of the replication cycle [ ] (figure f ). based on the observation that the golgi apparatus disappears in cvb -infected cells, the membrane rearrangements might originate from this organelle (montserrat bá rcena, personal communication). similar to poliovirus, single-membrane tubular clusters occur predominantly early in infection, whereas the number of dmvs increases as infection progresses. a budding event could account for the formation of the tubules, depicting an average length of ± nm and an average diameter of ± nm. a subsequent enwrapping of these single-membrane tubules via an -autophagy-like‖ mechanism could then lead to the formation of dmvs that have an average diameter of nm ± nm. this transformation may require several steps: (i) membrane pairing; (ii) induction of curvature; and (iii) membrane fusion [ ] . this scenario would be consistent with the membrane surface of dmvs as an average-sized dmv with a diameter of nm would be equivalent to a tubule with a length of nm and a diameter of nm. er membranes were found near dmv clusters. however, in contrast to previous observations in nidovirus-infected cells, these dmvs were not connected to neighboring structures. in addition to these compartments, a third type of modification was detected in cvb -infected cells: multilamellar structures, which are typical for the late phase of infection and that correspond to enwrapped dmvs: despite their various shapes and degrees of complexity, in all instances they contained one dmv, surrounded by one or several layers of curved cisternae. in conclusion, these results, and similar observations made for foot and mouth disease virus (fmdv) (genus aphthovirus) [ ] suggest that members of the picornaviridae family induce singleand double-membrane vesicles. they appear in a time-dependent manner and seem to evolve from each other, possibly in coordination with the progression of the viral replication cycle [ ] . these membrane rearrangements occur independently from the used virus strain and cell line. rubivirus (family togaviridae) [ ] . rubv anchors its rna synthesis machinery to membranes of a cell organelle known as -cytopathic vacuoles‖ (cpvs) that is derived from modified endosomes or lysosomes and has an average diameter of - nm [ ] [ ] [ ] . freeze-fracture and et analysis of rubv-infected cells revealed a high complexity of cpvs that are composed of stacked membranes, rigid sheets, small vesicles and large vacuoles ( figure i ) [ ] . the cpvs are interconnected and linked to the endocytic pathway, as deduced from labeling experiments with endocytosed bsa-gold. furthermore, rer cisternae, mitochondria and golgi stacks are recruited around cpvs to build up rubv factories. cpvs have several contacts with cellular organelles: they are coupled to the rer through protein bridges of ~ - nm and closely apposed membranes and they are attached to golgi vesicles, whereas contacts with mitochondria were not detected [ ] . it has been proposed that rna synthesis occurs on vesicular membranes within the cpvs, which are linked to the cytosol and that the viral replicase molecules are associated with vesicles that transform with time into large vacuoles and straight elements [ ] . this is supported by immunogold labeling revealing replicase components and dsrna within the cpvs [ , , ] . the modification of late endosomes and lysosomes is a feature that rubv shares with alphaviruses, the other genus of the family togaviridae [ , ] . cells infected with alphaviruses like semliki forest virus (sfv), sindbis virus and western equine encephalitis virus (weev) contain large cpvs with a diameter ranging between and nm. the inner surface of these cpvs is covered with small invaginations or spherules that originate at the plasma membrane [ ] [ ] [ ] ( figure j ). these spherules are comprised of a single membrane forming a vesicle with a diameter of ~ nm. in addition, the spherules were shown to be the site of viral rna synthesis as deduced from metabolic labeling and detection by em [ , , ] . importantly, the inside of the spherule is connected to the cytoplasm by a pore with a diameter of - nm. the spherules are formed at the plasma membrane by the concerted action of the viral nonstructural proteins (nsp -nsp ) and genomic viral rna [ ] . furthermore, froshauer et al. [ ] showed that the cpvs that contain the spherules possess endosomal and lysosomal markers. time course studies revealed that the spherules of sfv undergo an unprecedented large-scale movement between cellular compartments [ ] . the spherules first form as blebs (exvaginations) at the plasma membrane. then, they are internalized by an endocytic process requiring a functional actin-myosin network. the spherules therefore represent an unusual type of endocytic cargo. after endocytosis, spherule-containing vesicles, namely cpvs-i fuse with acidic endosomes and move along microtubules. this leads to the formation of a very stable compartment, where the spherules accumulate as invaginations on the outer surface of unusually large, acidic vacuoles localized in the pericentriolar region [ ] . members of the genus norovirus (novs, family caliciviridae) are major agents of acute gastroenteritis [ ] . ultrastructural examination of murine norovirus (mnv- )-infected cells revealed a striking change in their overall morphology and intracellular organization [ ] . structures resembling virus particles were observed within or next to single-or double-membrane vesicles in the cytoplasm. the vesiculated areas increase in size with time and by hpi, large numbers of these vesicles and viral particles occupy most of the cytoplasm and displace the nucleus ( figure h ). in addition, a complete rearrangement of the er and loss of intact golgi apparatus was observed. both dsrna and mnv- nonstructural protein , the rna dependent rna polymerase, localize to the limiting membrane of individual vesicle clusters by immuno-em [ ] . immunofluorescence-based double-labeling showed that mnv- appears to recruit membranes derived from multiple cellular organelles and/or compartments: the er, trans-golgi apparatus and endosomes. however, despite extensive efforts, human norovirus cannot be grown in cultured cells [ ] . thus, detailed studies have not been possible, but it is assumed that replication structures are similar to those of mnv- . feline calicivirus (fcv), a member of the genus vesivirus within this family, is a major agent of respiratory disease in cats, which replication originates also membranous rearrangements and vesicles [ ] . brome mosaic virus (bmv, family bromoviridae) generates its replication factory by hijacking er membranes, similar to what has been described for other plant viruses like tobacco mosaic virus (tmv, family virgaviridae) [ ] , tobacco echt virus (tev, family potyviridae) [ ] and red clover necrosis mosaic virus (family tombusviridae) [ ] . however, other plant viruses such as alfalfa mosaic virus (amv) [ ] and cucumber mosaic virus (cmv), both belonging to the family bromoviridae, and turnip yellow mosaic virus (tymv, family tymoviridae) [ ] anchor their replication sites on chloroplasts. cucumber necrosis virus (cnv), family tombusviridae, utilizes peroxisomal membranes as replication platforms [ ] , while other plant viruses replicate on the surface of mitochondria [ ] . although the d architecture of these membranous replication sites remains largely unknown, their characteristics are strikingly similar to those for fhv (family nodaviridae). best studied is bmv that induces spherules, of similar size as the insect nodavirus fhv, in the er close to the nucleus, where viral rna synthesis and viral replication proteins are localized [ ] [ ] [ ] . in the case of beet yellows closterovirus (byv, family closteroviridae), tem of infected plant cells revealed the formation of ~ nm-diameter dmvs and multivesicular complexes (single-membrane vesicles surrounded by a common membrane) ( figure e ) [ ] . these multivesicular complexes often reside next to stacks of aligned filamentous byv particles [ , ] and resemble the dmvs and vps produced by nidoviruses and flaviviruses. several byv replication-associated proteins (l-pcp, mtr and hel) colocalize with dmv and vp membranes, supporting the role of these structures as replications platforms [ , ] . the membranes in closterovirus dmvs and vps are likely to be derived from er for members of the genus crinivirus [ ] or mitochondria in case of ampelovirus [ , ] . whether these structures are -closed‖ or -necked‖ remain unknown. based on amino acid sequence homologies of their rna-dependent rna polymerases, (+) rna viruses have been classified into three large supergroups [ , , ] : supergroup i (picornavirata or picorna-like group), including picorna-, corona-, arteri-and nodaviruses; supergroup ii (flavivirata or the flavi-like group), including tombus-, diantho-, pesti-, hepaci-and flaviviruses as well as single-strand rna bacteriophages; supergroup iii (rubivirata or the alpha-like group), including tobamo-, hordei-, alpha-and rubiviruses as well as hepatitis e virus (hev). these higher-order taxonomic units encompass diverse viruses infecting different hosts from almost all kingdoms of life. as discussed earlier [ ] , amongst these viruses two main architectures of remodeled membranes (morphotypes) can be found that may reflect two alternative strategies to induce the membranous microenvironments required to allow virus replication (summarized in table ). the first morphotype involves the formation of negatively curved membranes, initiated by invaginations of the pre-existing membrane bilayer and giving rise to spherules, vesicles or vacuoles towards the lumen of the targeted cell organelle. these structures have been identified in a broad range of mammalian, plant and insect cells infected with viruses belonging to supergroups ii and iii. the second strategy involves the formation of membranes with positive curvature, giving rise to double-membrane structures that are the predominant characteristic of the replication factories of the picorna-like virus supergroup. the conservation of these two sorts of morphotypes in distantly related viruses supports the assumption of an evolutionary conserved mechanism. a striking finding in this regard was the observation that hcv, despite belonging to the flavi-like supergroup, induces dmvs whereas flaviviruses induce the formation of negatively curved membranes. to our current knowledge, hcv is the only member of the family flaviviridae inducing the formation of membrane structures with positive curvature, suggesting that hcv might share common host cell pathways to induce membranous replication compartments with distantly related viruses such as corona-, arteri-, picorna-, calivi-or closteroviruses (table ) . however, it still remains to be elucidated whether other members of the family flaviviridae, belonging to the genera pestivirus and pegivirus, also utilize a picorna-like membrane remodeling strategy. a common feature associated with the spherule/vesicle/vacuole/-type of rearranged membranes is their size ( - nm diameter) and the presence of a pore connecting the interior of the vesicle with the cytoplasm [ , , , ] . since rna replication occurs in the vesicle interior, the pore allows exchange of nucleotides and rna products with the cytoplasm. the size of the pore is variable, ranging from . nm in case of ibv-induced spherules to ~ nm in case of membrane invaginations induced by flaviviruses. in contrast, in the majority of dmvs no such channel or pore has been detected. nevertheless, as exemplified with nidoviruses, the inner compartments enclosed by interconnected dmvs contain the bulk of dsrna [ ] , and in some cases they depict an electron-dense core assumed to correspond to viral rna (ibv and eav) [ , ] . although this represents a functional enigma in terms of rna synthesis and transport, the presence of dsrna in the dmv interior does not necessarily indicate active rna replication. assuming a temporal regulation, it is possible that dmvs might be sites of rna synthesis as long as they are linked to the cytoplasm, but replication would stop upon closure of the vesicles. yet, another strategy appears to be used by enteroviruses, where active rna replication has been detected on the cytosolic side of the membranous structures [ , ] , consistent with the membrane topology of the nonstructural proteins catalyzing rna replication [ ] [ ] [ ] . as described above, studies conducted with picornaviruses revealed that the exponential phase of viral rna synthesis coincides with the accumulation of single-membrane tubules [ , ] . importantly, pulse-radiolabeling experiments localized sites of active rna replication to the outer surface of single-membrane tubules [ ] and isolation of the membranous replication factories and their subsequent visualization by em revealed that they form rosette-like structures composed of virus-induced cytoplasmic vesicles [ ] . rna replication is thought to occur at sites where the vesicles cluster, whereas rna translation probably takes place on the exposed periphery of the vesicles. this raises the question, what the role of dmvs in the replication cycle of picornaviruses might be. it is possible that dmvs either support rna synthesis or serve as rna storage sites (especially in case of closed dmvs). in this manner, dmvs might be involved in regulating viral rna replication: by complete sealing of the viral replicase inside the vesicle, it would be inactive, thus regulating overall rna copy number in the infected cell. alternatively, dmvs might be an epiphenomenon, resulting from the over-expression of membrane-active proteins that accumulate especially during the late stages of infection. the mechanism responsible for dmv formation is not clear. in case of picornaviruses, it is thought that single-membrane structures are the precursors of dmvs [ , ] . nevertheless, dmv formation might also involve the autophagy machinery, or at least several components thereof, by a process analogous to the formation of autophagic vacuoles [ ] . this hypothesis is supported by the morphological resemblance of dmvs and autophagosomes. it has been shown that the inhibition or stimulation of autophagy results in a modest inhibition or stimulation of poliovirus and coxsackie b virus yield, respectively, and there are also data supporting the involvement of autophagy in the replication of rhinovirus and [ , ] . however, brabec-zaruba et al. [ ] reported that replication of rhinovirus was insensitive to pharmacological manipulation of autophagy and did not induce detectable modification of lc . this discrepancy might be due to the use of different cell types and experimental conditions. the mechanism of dmv formation in case of hcv and coronaviruses is also unclear. biochemical analysis of isolated host cell membranes associated with hcv rna and proteins identified markers of the autophagy machinery, including lc -ii, the lipidated form of lc (lc -ii) that is generated upon activation of the autophagy machinery [ ] . however, the role of autophagy in the hcv replication cycle is also a matter of controversy. for instance, immunolabeling did not identify lc -ii at those sites where nonstructural proteins accumulate [ ] . moreover, different roles of autophagy for the hcv replication cycle have been proposed. these include a role of autophagy in hcv rna translation [ ] , initiation of rna replication [ , ] , production of infectious virus particles [ ] or suppression of the innate antiviral defense [ , ] . to clarify these discrepancies, future studies should combine biochemical and cell biological approaches with ultrastructural analyses. the autophagy machinery might also be involved in the formation of virus-induced membrane invaginations/spherules. for instance, lee and coworkers provided evidence that denv infection enhances autophagolysosome formation and that inhibition of the autophagy machinery by -methyladenine ( -ma) reduces denv particle production [ ] . however, the effects were moderate, arguing that this pathway may contribute to denv replication to only a minor extent. moreover, autophagy includes membrane wrapping, leading to double-membrane compartments involved in lysosomal degradation whereas denv-induced vesicles are invaginations. finally, immunolabeling experiments failed to detect lamp- at these vesicles [ ] , arguing against the involvement of lysosomes in the formation of the denv replication vesicles. it remains to be determined whether autophagy is actively induced by these viruses to provide a compartment favoring replication or induced as a bystander defense against infection leading to degradation of the replicase proteins [ ] . another membrane compartment frequently induced by (+) rna viruses are convoluted membranes (cms) that were observed e.g., in sars-cov-, mhv-, wnv-or denv-infected cells [ , , , , , , ] . morphologically, cms resemble smooth er membranes, lack ribosomes and in case of denv are induced by the sole expression of ns a [ , ] . cms are often associated with late stages of infection, suggesting that dmv formation might precede the development of cms. in sars-cov-infected cells, dmvs appear to be connected with cms [ ] , while in mhv-infected cells no such connections have been observed [ ] . the role of cms for the viral replication cycle is not well understood. in case of wnv kun , cms are supposed to be the site of polyprotein processing [ , ] . this conclusion is based primarily on the strong immunolabeling for ns b and ns and the absence of ns and ns b. since polyprotein cleavage occurs co-translationally and thus, should happen at the rer, this model would require the formation of rather stable processing intermediates that are transferred from the rer to the cms where further cleavage would occur. alternatively, at least in case of denv, cms might represent a storage site for proteins and lipids involved in viral replication that can be recruited to vesicles upon demand. the fact that cms are physically linked with er-containing invaginations and contain ns would be consistent with this assumption [ ] . along these lines, the fact that insects are cholesterol auxotrophs and lack several enzymes in the cholesterol biosynthesis pathway [ ] , suggests that cholesterol might be a key component of cm structures, which would explain their absence in infected insect cells [ ] . viral replication complexes are targeted to the respective membranous organelle primarily by nonstructural (ns) proteins rather than viral rna [ ] . these ns proteins seem to have some specificity in recognizing organelle subpopulations and often contain multiple hydrophobic domains implicated in membrane targeting and rearrangement. the molecular mechanisms orchestrating the formation of these complex structures are still poorly understood, but it is clear that ns proteins, often working in a concerted action, are key players in replication factory biogenesis. one well-studied example among the single-membrane vesicle inducers is bmv where it was shown that the sole expression of the ns protein a is sufficient to induce the formation of single-membrane spherules resembling the ones observed in infected cells. these spherules had a diameter of - nm, resided in the er lumen and were shown to be the site of viral rna synthesis [ ] . in case of the insect nodavirus fhv, protein a and replication competent rna were required for induction of the spherules. expression of protein a alone induced only -zippering‖ of the surfaces of adjacent mitochondria, but did not induce spherules. thus, protein a is necessary, but not sufficient for spherule formation. moreover, spherules were not formed when replication-competent fhv rna templates were expressed with a protein a mutant lacking polymerase activity or when wild-type protein a was expressed with a replication-incompetent fhv rna template. thus, the membranous fhv replication compartment requires both a viral protein and active rna synthesis [ ] . feline calicivirus (fcv) infection results in rearrangement of intracellular membranes and production of numerous membrane-bound vesicular structures on which viral genome replication is thought to occur. expression of individual fcv nonstructural proteins revealed that p induces significant reorganization of the er into large, fenestrated membrane networks, resembling the structures found in infected cells [ ] . moreover, expression of p and p , two additional fcv ns proteins, induced extensive reorganization of the er and the nuclear envelope suggesting that the er is the primary source of the membranous replication factory [ ] . flavivirus membrane rearrangements are mainly induced by ns a, as suggested by recent studies with wnv kun and denv [ , ] , but it is unknown whether the same applies to ns a of tbev. in case of denv, ns a is thought to contain a central peripheral membrane domain that intercalates into the luminal leaflet of the er membrane [ ] . it is tempting to speculate that ns a oligomers [ ] might dilate the luminal leaflet, resulting in membrane invaginations towards the er lumen. however, the sole expression of ns a is not sufficient to induce er membrane invaginations that have been detected in infected cells. instead, expression of ns a lacking the c-terminal k fragment (corresponding to fully processed ns a) induced er membrane rearrangements reminiscent of cms, whereas unprocessed ns a/ k did not induce membrane alterations [ ] . these results provide strong evidence that processing at the ns a- k site is required for the induction of membrane alterations. the critical role of polyprotein cleavage for induction of membrane rearrangements is supported by studies conducted with wnv kun . there it was shown that a regulated cleavage of a ns a/ k/ b precursor by the viral ns b/ protease is needed for induction of membrane rearrangements [ ] . however, the same study reported that expression of full-length (uncleaved) wnv kun ns a/ k led to membrane alterations similar to those induced in infected cells whereas the k fragment impaired the ability of ns a to induce membrane rearrangements. whether this reflects a biological difference between wnv kun and denv ns a or is due to the use of alternative experimental approaches remains to be determined. one of the most fascinating mechanisms employed by (+) rna viruses to induce their replication factories is used by sfv. it was shown that the spherules of sfv arise by blebbing at the surface of the plasma membrane [ ] . these blebs are internalized and after fusion with lysosomes give rise to large cytoplasmic vacuoles. formation of these membrane alterations requires the viral protein nsp , which has several functions. it has guanine- -methyltransferase and guanylyltransferase activities and thus is critically involved in capping of the viral rnas [ ] [ ] [ ] , but at the same time has affinity to lipids [ ] . in fact, of the four ns proteins of sfv, only nsp has affinity for membranes, and when expressed alone, it is specifically targeted to the inner surface of the plasma membrane [ ] . nsp is a monotopic membrane protein and its affinity for membranes is dictated by an amphipathic α-helix, located in the central region of the protein [ , ] . nsp has a specific affinity for negatively charged phospholipids, which might account for its prevalent localization to the plasma membrane, where such lipids are enriched. membrane binding of nsp via its amphipathic α-helix is essential for alphavirus replication [ ] . however, nsp is not sufficient for cytoplasmic vacuole formation. for instance, it was found that nsp contributes to the transport of the replicase polyprotein from the plasma membrane to the surface of endosomes [ ] . these results indicate that nsp has to cooperate with other viral and cellular factors to allow formation of the cytoplasmic vacuoles. furthermore, in a recent study kallio et al. [ ] have shown that the size of the spherule is dependent on the length of the rna template, in contrast to what has been observed for fhv, another spherule-inducer [ ] . these results indicate that in addition to the ns proteins, the viral rna template itself critically determines the morphology of the membranous vesicles. in order to induce the variety of membrane alterations observed in cvb -infected cells ( figure f ), several membrane-remodeling mechanisms are required: induction of membrane curvature, membrane fusion and membrane-membrane interactions [ ] . these rearrangements require the enteroviral proteins bc and a; their coexpression generates er membrane-derived structures mimicking those observed during viral infection [ ] . importantly, b and c both contain an amphipathic α-helix [ ] [ ] [ ] , a well-known curvature-inducing motif [ ] . along the same lines, fmdv b and bc locate to the er when expressed on their own and cause a swelling of er cisternae [ ] . in case of hcv, we recently found that a concerted action of ns / a, ns b, ns a and ns b is required to generate the membranous web. furthermore, all these replicase components were capable of inducing membrane vesiculation with ns a having the highest potential to trigger membrane curvature. importantly, some of these ns a-induced structures corresponded to dmvs [ ] . in addition, ns b also plays an important role in triggering rearrangements of intracellular membranes [ ] . ns b is an integral membrane protein containing two n-terminal amphipathic α-helices, a highly hydrophobic central core domain composed of four putative transmembrane segments, and a highly conserved c-terminal domain that is thought to harbor two α-helices (reviewed in [ ] ). a recent study has demonstrated that ns b oligomerizes through multiple conserved determinants and that oligomerization appears to be required for membranous web induction [ ] . indeed, mutations affecting the highly conserved c-terminal domain impairing ns b self-interaction resulted in the formation of aberrant dmvs arguing for a central role of ns b in formation of functional replication compartments [ ] . studies on arteriviruses revealed that the sole expression of eav nsp and nsp is sufficient to induce membrane structures similar to those generated during eav infection [ ] . mutations within nsp , which is a tetra-spanning integral membrane protein, alter membrane rearrangements, highlighting the importance of this protein for the biogenesis of eav-induced dmvs [ ] . in case of sars-cov, nsp , nsp and nsp were found to be sufficient to induce the formation of dmvs that are similar to those observed in sars-cov-infected cells [ ] . these dmvs were, however, smaller in diameter, suggesting a role for other viral proteins or the presence of viral rna in determining the dmv morphology. importantly, em analysis of nsp mutants that are impaired in rna replication and virus growth, revealed an aberrant morphology of dmvs as well as an increased prevalence of cms [ ] . another important viral protein involved in inducing the sars-cov membranous replication factory is nsp , which is predicted to contain seven transmembrane segments and a hydrophilic cytoplasmic domain [ ] . nsp was shown to induce vesicles containing atg and lc -ii as well as phosphatidylinositol- -phosphate, thus sharing many features with omegasomes, which are omega-shaped membrane compartments that are formed during activation of autophagy [ ] . this result suggests that autophagy might contribute to the formation of the membranous replication site of sars-cov. in the last couple of years, our knowledge of the architecture of the replication factories induced by (+) rna viruses has increased substantially. this is primarily due to the more widespread use of et and other high-resolution imaging methods. nevertheless, our knowledge is still rather restricted to descriptions of the morphologies of these complex structures, whereas our understanding of their biogenesis in most cases is very rudimentary. more efforts are required to elucidate the role of the viral proteins in the formation of the replication vesicles, to identify the involved cellular components and the mechanisms used by these proteins to subvert and exploit cellular pathways to establish membranous replication factories. this includes determination of the d structure of involved (viral) proteins as well as evaluation of host cell factors and lipids contributing to biogenesis and activity of the replication compartment. in addition, further studies are needed to understand how viruses utilize these compartments to coordinate the different steps of their life cycle (replication, assembly and release) in space and time to achieve efficient replication. work in the authors' laboratory was supported by the deutsche forschungsgemeinschaft (sonder-forschungsbereich the gb viruses: a review and proposed classification of gbv-a, gbv-c (hgv), and gbv-d in 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nsp proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate the authors are very grateful to erik j. snijder (leiden university medical center, leiden, the netherlands) and kè vin knoops (european molecular biology laboratory, grenoble, france) for providing the unpublished pictures depicted in figure b and d and to montserrat bá rcena (leiden university medical center, leiden, the netherlands) and tero ahola (university of helsinki, helsinki, finland) for providing the unpublished pictures depicted in figure f and j, respectively. figures a, c , e, g, h and i are reproduced with permission from [ ] , [ ] , [ ] , [ ] , [ ] and [ ] , respectively.we also would like to thank jason m. mackenzie the authors declare no conflict of interest. key: cord- -olmein q authors: banerjee, arinjay; kulcsar, kirsten; misra, vikram; frieman, matthew; mossman, karen title: bats and coronaviruses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: olmein q bats are speculated to be reservoirs of several emerging viruses including coronaviruses (covs) that cause serious disease in humans and agricultural animals. these include covs that cause severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), porcine epidemic diarrhea (ped) and severe acute diarrhea syndrome (sads). bats that are naturally infected or experimentally infected do not demonstrate clinical signs of disease. these observations have allowed researchers to speculate that bats are the likely reservoirs or ancestral hosts for several covs. in this review, we follow the cov outbreaks that are speculated to have originated in bats. we review studies that have allowed researchers to identify unique adaptation in bats that may allow them to harbor covs without severe disease. we speculate about future studies that are critical to identify how bats can harbor multiple strains of covs and factors that enable these viruses to “jump” from bats to other mammals. we hope that this review will enable readers to identify gaps in knowledge that currently exist and initiate a dialogue amongst bat researchers to share resources to overcome present limitations. bats are an ancient and diverse group of ecologically important mammals, constituting almost a quarter of all mammalian diversity and inhabiting every continent except antarctica. more than species of bats belong to the order chiroptera and are further classified into two suborders, yinpterochiroptera and yangochiroptera [ ] [ ] [ ] . the yinpterochiroptera suborder includes the non-echolocating pteropodidae family and the echolocating rhinolophoidea superfamily. yangochiroptera contains the remaining echolocating microbat families. the two suborders diverged over million years ago [ ] [ ] [ ] . in addition to the important role that bats play in preservation of ecological balance, they have also been speculated to harbor a wide variety of viruses. many of the viruses in bats can cause disease in humans and agriculturally important animal species. these viruses include lyssaviruses, filoviruses, henipaviruses and coronaviruses [ , [ ] [ ] [ ] . in this mini-review, we focus on the role of bats as reservoir hosts for important human and animal coronaviruses. we discuss the evidence of coronavirus spillover from bats, how bat ecological niches may contribute to spillover and the need to further explore bat-coronavirus interactions using viruses that have been naturally detected in bats. sars-cov emerged in humans in and efficient human to human transmission resulted in a global sars epidemic which lasted months [ ] . initial studies investigating animal sources of the virus from "wet markets" in the guangdong province of china suggested that himalayan palm civets and raccoon dogs were the most likely hosts responsible for human transmission [ ] ; however, the role of bats as the original animal reservoir hosts of sars-cov was speculated as similar viruses were detected in them [ , ] . years later, during an ecological surveillance of bats in the same region, a sars-like cov that closely matched the human sars-cov was isolated from the chinese horseshoe bat. bat sars-like cov could replicate in hela cells expressing angiotensin-converting enzyme (ace ) receptor from human, civet and bat. the virus replicated in cells derived from human, bat and pig. no civet cells were tested [ ] . these data suggest that sars-cov could have spilled over into humans directly from the chinese horseshoe bat while the palm civets in the "wet market" were incidental hosts [ ] . however, the exact mechanism by which the zoonotic transmission event to humans occurred is still not clear. retrospective studies have found low levels of seroprevalence of sars-like cov in healthy individuals in hong kong dating back to . interestingly, in of of these seropositive patients, the antibodies responded more strongly against the sars-like-cov isolated from a himalayan palm civet compared to the human sars-cov isolate [ ] . these data suggest that low levels of human exposure to zoonotic sars-like covs occurred prior to the sars-cov epidemic that began in , but went unidentified. mers-cov emerged in saudi arabia in and continues to cause human disease with a case fatality rate of % [ ] . dromedary camels are a natural reservoir host for mers-cov. in the arabian peninsula and across northern africa, the seroprevalence rate for mers-cov in dromedary camels ranges from % to nearly % [ ] [ ] [ ] [ ] [ ] [ ] . live mers-cov has been successfully isolated and cultured from camel specimens [ ] . approximately % of primary mers-cov cases are a result of direct contact with dromedary camels or camel products [ ] ; however, the remainder of primary mers-cov cases have no history of contact with camels or infected individuals and thus, where they came into contact with the virus is unknown. a recent study found that out of camel workers surveyed in saudi arabia show evidence of prior mers-cov infection via seroconversion and/or virus-specific cd + t cell responses without any history of significant respiratory disease. this study suggests that camel workers with asymptomatic or mild disease may serve as another route of exposure [ ] . although camels are thought to be the primary zoonotic reservoir for human transmission, there is strong evidence that bats are the ancestral reservoir host for mers-cov [ , [ ] [ ] [ ] . mers-cov is a group c betacoronavirus and is phylogenetically related to batcovs identified in various bat species that belong to the vespertilionidae family. this includes batcov hku , batcov hku , neocov, and pdf- [ , , ] . furthermore, the spike protein from hku and mers-cov are highly similar and both use human dipeptidyl-peptidase (dpp ) for virus entry [ , , ] . it is not clear when mers-cov spread from bats to camels, but widespread exposure to the virus in the middle east and north and east africa dates back as early as the s, suggesting that camels have served as a zoonotic reservoir for mers-cov for at least years [ , , , , ] . in addition to emerging highly pathogenic coronaviruses, human coronaviruses that cause the common cold are also thought to have their origins in bats. hcov-nl was first identified in a pediatric patient with bronchiolitis in , but since then it has come to be appreciated that the virus causes approximately - % of the common colds each year and has most likely circulated in humans for centuries with worldwide distribution [ ] [ ] [ ] . a survey of bats in the u.s. found novel alphacoronaviruses, one of which was isolated from the north american tricoloured bat, perimyotis subflavus and was found to be a recent common ancestor of hcov-nl with an estimated divergence of~ years ago [ ] . hcov-nl -like sequences were also identified in bats in africa [ ] , further supporting a bat origin for hcov-nl . although hcov-nl -like viruses have been identified in bats, these viruses have sequences quite distant from the hcov-nl sequences, suggesting a possible intermediate host. hcov- e also appears to have its origins in bat species. hcov- e, another cause of the common cold, was first identified in and has been circulating in the human population for some time [ ] . hcov- e-related viruses have been found in hipposiderid bats during surveillance studies in kenya and ghana [ , ] . in , a novel alphacoronavirus was identified in an outbreak of respiratory disease in alpacas in the us, which is geographically separated from the bat species that harbor hcov- e-like viruses in africa [ ] . full genome sequencing and phylogenetic analysis of the alpaca cov placed it as an intermediate between the bat hcov- e-related viruses and hcov- e from humans [ ] . by analyzing more bat, alpaca and human hcov- e and hcov- e-related sequences, evidence of genomic changes that occurred between bat and alpaca hcov- e evolution and subsequently between alpaca and human evolution were identified [ ] . interestingly, during tests of dromedary camels for mers-cov, about % of the camels studied were positive for hcov- e [ ] . seropositive camels were more prevalent in the arabian peninsula compared to africa and the earliest seropositive sample was from in a study that looked at samples from to [ ] . these data all support the notion that hcov- e has its ancestral origins in bat species while camelids serve as a more recent zoonotic reservoir for human transmission. a recent study has shown that hcov- e (human strain) is incapable of infecting and replicating in cell lines from multiple bat species [ ] . thus it is critical to isolate bat and camel strains of hcov- e-related viruses to dissect the role of these mammals in the evolution of hcov- e. porcine epidemic diarrhea (ped) was recognized as an enteric disease in pigs in the united kingdom as early as . pedv was detected in belgium in [ ] . the full-length genomic sequence of the prototype belgian pedv cv strain was determined in [ ] . pedv cv is more closely related to a scotophilus bat coronavirus (btcov) / than to other known alphacoronaviruses, such as transmissible gastroenteritis coronavirus (tgev) and hcov- e and hcov-nl , in phylogeny as well as genome organization [ ] . this finding suggests that pedv and btcov/ / have a common evolutionary precursor and that cross-species transmission of coronavirus may have occurred between bats and pigs. pedv has since emerged in north america and continues to cause periodic outbreaks that significantly affect producers [ , ] . multiple pedv vaccine candidates have been shown to provide varying levels of protection in pigs [ , ] . an effective vaccine may enable control of future pedv outbreaks along with strict biosecurity practices. although pedv propagates in human embryonic kidney cells [ ] , no clinical cases of pedv have been reported in humans so far. we (banerjee and misra et al.) have also shown that pedv can infect kidney cells from big brown bats (eptesicus fuscus) [ ] . pedv replication in bat cells has not been extensively studied. efforts are focused on designing therapeutics and vaccines to prevent ped in pigs. more recently, a novel hku -related bat coronavirus, sads-cov has been shown to cause fatal enteric disease in pigs. zhou et al. identified sads-cov as the causative agent for a large-scale outbreak of fatal disease in pigs in china that caused the death of , piglets across four farms. sads-cov-like viruses with - % similarity to sads-cov were detected in . % of the bats that were sampled in this region [ ] . none of the human serum samples that were collected from farm workers were positive for antibodies against sads-cov [ ] . thus, sads-cov does not pose a risk for human transmission yet. further studies will be required to confirm the ability of sads-cov to infect and propagate in human cells. understanding how bats maintain a virus within a population is important for predicting spillover transmission events. for many viruses with known or suspected bat reservoirs, spillover transmission events typically occur within a defined time frame and location, which corresponds with higher than normal virus levels in the bat reservoir host. the reason for these "pulses" of virus within the reservoir host population are not clear, but proposed theories and evidence supporting these theories have been reviewed by plowright et al. [ ] . in the case of marburg virus (marv), for example, ecological surveillance data shows a clear biannual spike in the prevalence of marv positive bats within the kitaka cave population, which correlates with an increase in the number of juvenile rousettus aegyptiacus bats due to the biannual birthing cycle. this pulse of virus positive bats correlates with an increased incidence of human spillover events [ ] . horizontal transmission of marv between r. aegyptiacus bats was confirmed in a controlled experimental setting [ ] . furthermore, recent experimental data has shown that bats infected with marv clear infection and maintain long-term immunity. this finding suggests that susceptible naïve juvenile bats are critical for maintaining marv within the population [ ] . studies with hendra virus have shown that reproductive and nutritional stress can increase the levels of virus in little red flying foxes (pteropus scapulatus) [ ] . the increase in virus replication may enhance the chances of a virus spillover. similar ecological studies need to be undertaken for bats and covs. other stressors, such as secondary infections, may also affect the relationship between bats and their viruses. a recent study by one of our laboratories (misra et al.) suggest that infection of little brown bats (myotis lucifugus) with white-nose syndrome causing fungus (pseudogymnoascus destructans) leads to an increase in replication of a persistently infecting coronavirus in these bats [ ] . a recent study by anthony et al. evaluated the global diversity of coronaviruses in almost , animals and humans. during the course of this study, they found that the diversity of coronaviruses was highly associated with the diversity of bat species and this diversity separated into distinct geographical regions, which mirrored the distribution of different species of bats. the authors report particular associations between bat families and viral sub-clades that suggest co-evolution [ ] . a survey of coronaviruses isolated from bats in kenya found a high prevalence of coronaviruses in cardioderma cor, ediolon helvum, epomophorus labiatuc, hipposideros sp., miniopterus minor, otomops martiensseni, rhinolophus hildebrandtii, rhinolophus sp., and triaenops afer. the phylogenetic analysis of these novel covs found a number of cross-species transmission events, although the majority of these events appeared to be transient spillover events [ ] . the recombination frequency of coronaviruses, which can be as high as % for the entire genome [ ] , could lead to bats being an important reservoir for coronavirus recombination and virus evolution, much like birds and pigs are for influenza virus. indeed, there is strong evidence to suggest that a recombination event occurred between hcov- e-like viruses found in hipposideros bats and hcov-nl -like viruses found in triaenops afer bats, where the gene encoding for the spike protein is more closely related to the hcov- e virus [ ] . furthermore, the majority of recombination events identified in coronaviruses isolated from bats suggest recombination hotspots around the spike gene [ , ] . in theory, bats could serve as an important reservoir for coronaviruses and coronaviruses with altered host tropism may very well evolve in bats. although bats are known to harbor a wide variety of coronaviruses, the mechanisms for virus spillover into humans or livestock are widely unknown. there is evidence that there are seasonal fluctuations in virus replication [ , ] , however, the interconnectedness of virus replication rates and virus spillover have not been explored for bats. typically, coronaviruses found in bats have or require an intermediate host before spilling over into humans, like what is observed with mers-cov and camels. unlike the amount of information available from studies of other bat viruses such as nipah, hendra, ebola, and marburg viruses, we know very little, if anything about how coronaviruses are transmitted directly to humans or if direct human transmission does not occur and spillover via an intermediate host is required. bats are known to harbor a wide range of viruses including many that are highly pathogenic in humans. research to determine the mechanisms by which bats limit disease following virus infection is a relatively new field and can be difficult due to a lack of reagents and the need to develop appropriate in vitro and in vivo systems. even with these limitations, a variety of studies have been performed that evaluate the bat immune response to virus infection at the genomics level, in vitro using cell culture systems, and performing experimental infections in vivo. of note, very few of these studies are focused on coronavirus infections in bats and are rather centered around henipavirus and filovirus infections. future studies evaluating the virus-host interactions of bats and coronaviruses, particularly with bat cov isolates are important in determining why bats serve as important reservoirs for covs and how they control infection to limit severe pathological consequences. multiple studies have elucidated unique adaptations in the antiviral responses of bat cells. the primary bat species being used to study the bat immune response to virus infections in vitro and in vivo are pteropus alecto (black flying fox), rousettus aegyptiacus (egyptian rousette), and artibeus jamaicensis (jamaican fruit bat). papenfuss et al. were the first to sequence the p. alecto transcriptome and identified approximately genes ( . % of p. alecto transcribed genes) that encode immune-related proteins [ ] . a similar number of immune genes were also identified in the transcriptomes of r. aegyptiacus and a. jamaicensis [ , ] . this included the expression of canonical pattern recognition receptors including toll-like receptors (tlrs) - , retinoic acid-inducible gene i (rig-i), and melanoma differentiation associated protein (mda ) [ , ] . furthermore, genes for different immune cell subsets, t-cell receptors (tcrs), cytokines and chemokines, and interferon-related genes were detected, while genes encoding for natural killer (nk) cell receptors were largely absent. work has been done to characterize many of these genes in cell lines derived from various bat species including p. alecto [ ] [ ] [ ] . a large amount of interest in bat immune responses has focused specifically on the interferon response. genomic analysis of the interferon loci has shown species-specific evolution in which p. alecto has a contracted type i ifn locus [ ] whereas p. vampyrus, m. lucifugus, and r. aegyptiacus have expanded the number of type i ifn genes [ , ] . it has been observed that there may also be species specific differences in the baseline expression of type i ifns. p. alecto cells constitutively express three different ifnα genes [ ] whereas cells generated from r. aegyptiacus do not show constitutive expression of ifnα [ ] ; however, baseline expression of interferon alpha/beta receptors, ifnar and ifnar , as well as a variety of interferon-stimulated genes are upregulated in these bat cells compared to human cells [ ] . the molecular mechanisms that enable the differential expression pattern of ifns in bats are not known. thus, it is important to acknowledge that different species of bats may have evolved specific strategies to control viruses that they co-evolved with. although it appears that bats have many of the genes that are important for responding to virus infection, how this response compares between human and bat cells is just beginning to be examined. rna sensing and subsequent antiviral responses in bat cells have been studied using viruses known to induce an interferon response, such as sendai virus or newcastle disease virus or by transfecting a synthetic surrogate of viral double stranded rna (poly(i:c)) [ , [ ] [ ] [ ] [ ] [ ] . these studies show that bat cells respond to rna and induce an antiviral response. many viruses encode proteins that antagonize the host response to infection and dampen the innate antiviral response. it has previously been shown that the v and w proteins of nipah and hendra viruses can inhibit antiviral responses in bat cells, similar to what is observed in human cells [ ] . a more recent study showed that marv can inhibit the antiviral response in a r. aegyptiacus bat cell line and that this inhibition is dependent on the viral protein vp [ ] . coronavirus accessory proteins are dispensable for replication but they play an important role in pathogenesis and virus fitness under the natural environment of a host [ , ] . multiple studies with pedv, sars-and mers-covs have identified accessory proteins that can effectively inhibit an ifn response in mammalian cells [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, to date, there have been no published studies looking at the role of these accessory proteins in modulating antiviral responses in bat cells. in addition to studying the role of cov proteins in antagonizing the antiviral response in bat cells compared to other mammalian cell lines, it is also important to determine how covs isolated from bats compare to those isolated from humans. coronavirus accessory genes have co-evolved with their natural host for optimum functionality [ ] and thus it is important to identify the role of accessory proteins in both their natural and spillover hosts. many of the covs that have been reported in bats, with the exception of few, such as a sars-like cov (bat sl-cov-wiv ) [ ] , have been detected by molecular techniques that detect trace amounts of viral nucleic acids. to overcome this limitation, reverse genetics systems using the whole genome sequence from covs isolated from bats could be generated, propagated and evaluated in both bat and human cell lines [ ] . this would allow researchers to better understand the role of viral proteins in a species-specific context. the vast majority of studies evaluating the bat host response to virus infection has been performed in cell lines. however, there is a great need to understand what happens during a virus infection in bats in vivo. the ability to study these questions is a daunting task and requires specialized facilities and staff, appropriate species selection especially for covs, and generating the necessary reagents. because of these limitations, only a handful of studies have been performed looking at the in vivo response of bats to virus infection. in fact, there are only two published studies in which experimental infections in bats using covs was performed. the first study was performed in an attempt to rescue a bat cov isolate. watanabe et al. detected covs in . % of insectivorous bats and . % of frugivorous bats; however, they were unable to culture the virus in vitro. to propagate covs detected in a lesser dog-faced fruit bat (cynopterus brachyotis), they administered intestinal samples orally to leschenault rousette bats (rousettus leschenaulti). virus could be detected by quantitative real-time pcr (qpcr) on to days after infection and there was an increase in viral rna while no clinical disease was observed. based on these data, the authors reported that this bat cov replicates in leschenault rousette bats; however, they were not able to isolate live virus [ ] . this study emphasizes the importance of bat species selection for studying covs in bats. ideally, we would want to study a bat cov in the same species that it was detected in. the second study aimed to determine if bats could be infected with mers-cov and, if so, what the host response looks like. munster et al. infected ten jamaican fruit bats (artibeus jamaicensis) with mers-cov/emc . the authors detected virus shedding in the respiratory and intestinal tracts for days. although the bats showed evidence of virus replication, no overt signs of disease were observed. a moderate and transient induction of the innate immune response was seen, but there were no signs of inflammation. based on their observations, the authors reported that jamaican fruit bats support the replication of mers-cov and thus, bats could be potential ancestral hosts of mers-cov [ ] . this study has not been repeated in an insectivorous bat species. although several mers-like viruses have been detected in bats since the study in jamaican fruit bats, none have been successfully isolated [ , , , ] . another study focused on looking at species-specific tropism. in this study, the authors focused on the mers-cov receptor dipeptidyl peptidase (dpp ). widagdo et al. mapped the tissue distribution of dpp in multiple bat species to identify the differences in tissue tropism of mers-cov. in their study, the authors report that dpp in insectivorous bats is primarily detected in the gastro-intestinal (gi) tract and kidneys, whereas frugivorous bats express dpp in the respiratory and gi tracts [ ] . other studies determined that dpp expression in camels is primarily in the upper respiratory tract [ ] whereas dpp expression in humans is highest in the lower respiratory tract [ ] . these data suggest that the tissue tropism in bats may be different than that in other mammalian species and that this may dictate the course of disease and disease severity. the ability of bats to harbor several different coronaviruses may seem like a mystery, but the same is true for rodents. although bats harbor more zoonotic viruses per species, rodents harbor a larger total number of zoonotic viruses [ ] . after the sars outbreak, bats have been extensively sampled for coronaviruses and other viruses alike. we may be looking too hard and one may argue that we could find a similar diversity of viruses in other animals if we looked as robustly. metagenomics has enabled us to identify the broad range of viruses in bats and with time, we will expand this to other hosts of zoonotic viruses. for now, we know that bats are major evolutionary reservoirs and ecological drivers of cov diversity [ ] . we can leverage this knowledge to design studies that will allow us to identify factors that cause covs to spillover from bats to other hosts. a recent study demonstrated that secondary infection with the white-nose syndrome fungus (pseudogymnoascus destructans) increases cov replication in m. lucifugus [ ] . this study opens up a new avenue of investigation in infection dynamics. considering bats harbor multiple viruses, it is necessary to identify the impact these viruses have on each other. how do these viruses modulate the numerous host responses in bats and how does that affect virus replication? several such questions remain and studies are currently delayed due to the inability to isolate bat-covs similar to sars-cov, mers-cov, pedv and sads-cov. ecological and epidemiological studies to identify landscape changes and human practices that could enable a coronavirus to spillover from bats are also necessary [ ] . such studies enabled researchers to decipher 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middle east respiratory syndrome coronavirus bat coronaviruses and experimental infection of bats, the philippines replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) rapid detection of mers coronavirus-like viruses in bats: pote ntial for tracking mers coronavirus transmission and animal origin differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels dipeptidyl peptidase distribution in the human respiratory tract: implications for the middle east respiratory syndrome a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? ecological dynamics of emerging bat virus spillover community intervention to prevent nipah spillover key: cord- -oq cwka authors: tan, shaoyuan; dvorak, cheryl m. t.; murtaugh, michael p. title: characterization of emerging swine viral diseases through oxford nanopore sequencing using senecavirus a as a model date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: oq cwka emerging viral infectious diseases present a major threat to the global swine industry. since , senecavirus a (sva) has been identified as a cause of vesicular disease in different countries and is considered an emerging disease. despite the growing concern about sva, there is a lack of preventive and diagnostic strategies, which is also a problem for all emerging infectious diseases. using sva as a model, we demonstrated that oxford nanopore minion sequencing could be used as a robust tool for the investigation and surveillance of emerging viral diseases. our results identified that minion sequencing allowed for rapid, unbiased pathogen detection at the species and strain level for clinical cases. sva whole genome sequences were generated using both direct rna sequencing and pcr-cdna sequencing methods, with an optimized consensus accuracy of % and %, respectively. the advantages of direct rna sequencing lie in its shorter turnaround time, higher analytical sensitivity and its quantitative relationship between input rna and output sequencing reads, while pcr-cdna sequencing excelled at creating highly accurate sequences. this study developed whole genome sequencing methods to facilitate the control of sva and provide a reference for the timely detection and prevention of other emerging infectious diseases. emerging and re-emerging viral diseases have had a significant adverse impact on swine production and will be an ongoing challenge for the swine industry. emerging infections can be caused by previously unknown/undetected agents or known pathogens spreading to a new geographic location or host. the appearance of emerging diseases has usually been characterized by sudden unpredictable outbreaks, which then spread across regions and countries [ ] . this feature drives the need for effective emerging disease management via robust pathogen detection (including novel pathogens) and efficient epidemiological surveillance [ ] [ ] [ ] . over the last years, emerging pathogens that cause devastating swine diseases include porcine reproductive and respiratory syndrome virus (prrsv) first described in the late s [ ] , porcine circovirus type (pcv ) discovered in the late s [ ] and more recent porcine epidemic diarrhea virus (pedv) appearing in the us in the early s [ ] . the current introduction of african swine fever (asf) into china, affecting over half of china's swine herds, confirms the significant impact that viral diseases have on the swine industry. to date, asf has spread from china to neighboring countries and it is very likely to eventually enter other asf-free regions, such as the united states, despite all attempts to keep it out [ ] . the united states is now free of foot-and-mouth disease (fmd), an sva lab isolate (genbank: mn ) and clinical samples from swine sva-positive vesicular fluid were provided by dr. fabio a. vannucci at the university of minnesota veterinary diagnostic lab. the sva lab isolate was propagated in cell culture in nci-h non-small cell lung carcinoma cell line (atcc crl- ) as previously described [ ] . negative swine sera were spiked with the sva lab isolate to generate "spike-in" samples. we tested clinical samples in total, which were represented by vesicular fluid samples from sva-positive animals. sva rna was extracted from cell culture sva supernatants (cell culture samples), virus-free pig serum spiked with the sva lab isolate (spike-in samples), and clinical vesicular fluids (clinical samples) using the qiaamp viral rna mini kit (qiagen, germantown, md, usa) following the manufacturer's instructions without the addition of carrier rna and with a final elution in µl nuclease-free water. the concentration of the viral rna was performed using a speedvac lab concentrator (savant, ny, usa). a qubit . fluorometer (life technologies, carlsbad, ca, usa) and a nanodrop spectrophotometer (thermo scientific, waltham, ma, usa) were used for rna quantity and quality assessments. viral rna was sequenced using different kits, the direct rna sequencing kit or the pcr-cdna sequencing kit (ont, oxford, uk). the input rna for the direct rna sequencing (drs) library preparation was isolated sva rna with the addition of the rna calibration strand (rcs, bp), which was provided in the sequencing kit, to increase the amount of total input rna which is recommended for optimized results. the rcs is enolase ii mrna (yhr w, ncbi reference sequence: nc_ . ) provided at a concentration of approximately ng/µl. library preparation was performed according to the direct rna sequencing online protocol (drs_ _v , ont, oxford, uk), which includes the addition of a sequencing adaptor ligated at the end of the rna and is used for initiation of sequencing [ ] . the input rna for the pcr-cdna sequencing was the extracted sva rna only. pcr-cdna sequencing (pcs) libraries were generated according to the pcr-cdna sequencing online protocol (pcs_ _v , ont, oxford, uk). after library preparation, the drs and pcs libraries were loaded onto a r . . spoton flow cell and sequenced using a minion mk i sequencer (ont, oxford, uk) which was connected to a computer and remotely controlled by the minknow software (ont, oxford, uk). for the genome sequencing of the cell culture sva lab isolate, two sequencing replicates were performed for both drs and pcs, with the drs starting with ng sva rna plus ng rcs and ng sva rna alone for pcs. for the sequencing runs of clinical samples and negative pig serum spiked with the sva lab isolate, the same amount of sva rna was used for the drs and pcs library preparation, with the addition of ng rcs to each of the drs samples to increase the amount of total rna input amounts for optimized sequencing output. samples were sequenced for approximately h and the estimated sequence yield was monitored in real time. all samples were sequenced individually. flow cells were reused following nanopore guidelines until the number of total available pores was under . to minimize the potential contamination of previous runs and to protect the accuracy of the detection limit, samples with lower viral titers were sequenced first on a flow cell, followed by those with higher viral levels. basecalling was carried out using guppy (ont, oxford, uk). only raw sequencing reads that passed the quality filter of the phred quality score ≥ (pass reads) were used for downstream analysis. for the drs, raw reads of rna control strand (rcs, bp), which was used to enhance library preparation and sequencing performance, were filtered out by turning on the corresponding guppy parameter. the total yield, pass read yield, read quality, and the read length of raw reads from whole genome sequencing were analyzed using minionqc [ ] , a script written in r to provide quality control for oxford nanopore data. for the sequencing of cell culture samples, raw pass reads were mapped to the sva reference genome (genbank: mn ) using minimap [ ] , then analyzed using qualimap [ ] , generating raw error rates and coverage information which was then visualized using graphpad prism software (graphpad software, san diego, ca, usa). the reads that mapped the sva reference genome (sva reads) were extracted using samtools [ ] , and the sva yield, average read length, and quality were determined using nanoplot [ ] . for the spike-in and clinical samples, taxonomic analyses at the species level was performed to identify pathogens existing in the sample using what's in my pot (wimp), which is provided by ont's subsidiary metrichor [ ] . an sva custom database was created to analyze the sva sequencing reads by downloading all of the sva whole genomes available from genbank ( complete sva genomes as of march ). to detect sva at the strain level, pass reads were analyzed against this sva database using the basic local alignment search tool (blast) [ ] to identify the strain with the best match based on blast bit score. viral consensus sequences were generated using different assemblers and the results were compared to determine the optimal assembler for each sequencing method. four different assemblers, canu [ ] , miniasm [ ] , racon [ ] , and wtdbg [ ] , were used to examine the reads from direct rna sequencing and pcr-cdna sequencing. for drs, an optimal consensus sequence was generated, without need for reference or assembly, by extracting the longest read among all sequencing reads as a scaffold and mapping all pass reads to this longest read sequence using minimap [ ] followed by consensus generation using racon [ ] . for pcs, de novo assembly was performed using the canu assembler [ ] . after determining the consensus generation strategies for both sequencing methods, optimization was performed in terms of total input sequencing yield and the pre-treatment of raw reads using the cell culture virus in which the whole genome sequence was already known. groups containing different input sequencing yields, ranging from . to megabases (mb), were generated by random selection using fastq-tools- . (https://homes.cs.washington.edu/~dcjones/fastq-tools/) from the dataset of total pass reads. in the same yield group, three subgroups were formed using different raw reads filters; ( ) original pass reads (phred quality ≥ ) without further filters; ( ) pass reads with a read length > bp to remove short reads and the rcs; and ( ) pass reads that can be mapped to the sva database. the consensus length and accuracy were the two main parameters evaluated for comparison. consensus accuracy was determined by comparing the consensus genome to the reference genome and was analyzed using the clustalw pairwise alignment in geneious v . . software (https://www.geneious.com, san diego, ca, usa) [ ] . for the spike-in and clinical samples, a consensus sequence was able to be generated for most samples. sva reads were first extracted by mapping all of the reads against the custom sva database (generated above), followed by consensus generation using racon for drs and canu for pcs. the consensus length and accuracy were calculated to indicate the performance of consensus generation using varying amounts of input viral copies. sanger sequencing was performed for all clinical samples. primers were designed for pcr amplification of the end ( utr, d and partial c genomic regions) of the sva genome, similar to previous studies [ ] (forward primer_ gggtgacgacttacaaggga , reverse primer_ gagccagtgccgtgtgaagagt ; forward primer_ cgccaagtttcaatcccatc , reverse primer_ tcccttttctgttccgactg ). samples were sequenced as previously described [ ] . basically, sva rna was pcr-amplified using accustart pcr supermix (quantabio, beverly, ma, usa) and treated with exosap-it (thermo fisher scientific, waltham, ma, usa). sanger sequencing was performed at the university of minnesota genomics center. results and sequence quality were visualized using clc genomics workbench v . (https://www.qiagenbioinformatics.com/, redwood city, ca, usa). a consensus sequence for each clinical sample was generated using the geneious software version . (biomatters, auckland, new zealand) [ ] . the sanger sequence data were submitted to the genbank database and are available through accession numbers mn -mn , and mn . sanger sequencing references are also available at figshare (https://figshare.com/ articles/rapid_sva_detection_using_minion_sequencing/ ). analytical sensitivity was determined for both direct rna sequencing and pcr-cdna sequencing using spike-in and clinical samples containing a range of input virus amounts. for spike-in samples, an sva viral stock was -fold serially diluted from × to , × to generate decreasing amounts of virus which were then added into the sva-free pig serum. these spike-in samples ranged from to viral copies/ml (ct values ranging from to ). the ct value and viral copies for all samples were determined by rt-qpcr at the university of minnesota veterinary diagnostic lab. for the clinical samples, vesicular fluid clinical samples ranging from to viral copies/ml (ct values ranging from to ) were sequenced. for both sample sets, viral rna was extracted from ml of sample with half of the sample used for direct rna sequencing (drs) and half for pcr-cdna sequencing (pcs). for spike-in samples, the sva strain was determined by blasting raw sequencing reads to the custom sva database, and then the detected strain was compared to the known reference genome using clustalw pairwise alignment from the geneious software [ ] to identify the accuracy of the strain level detection. for the clinical samples, the consensus sequence generated from sanger sequencing was used as a partial reference genome. to obtain the whole genome reference (referred to as "reference sequence"), we performed blastn to get the whole genome of the strain in genbank with the best match. a comparison was made between the strain identified as the best blast match of the minion consensus sequence to the reference sequence. we then performed clustalw pairwise alignment to compare the "best match strain for the minion concensus" and the reference genome using geneious v . . software (https://www.geneious.com, san diego, ca, usa) to determine the detection accuracy [ ] . to minimize the effect of contamination from previous runs on the accuracy of analytical sensitivity, samples with lower viral titers were sequenced first when using the same flow cell. a correlation analysis was performed to test if drs and pcs were quantitative diagnostic methods. the total number of reads varied for each sequencing reaction, thus reads were normalized by calculating the ratio of sva reads/total reads in order to compare between samples. linear regression analysis was then performed to determine if there was any correlation using the sva reads/total reads ratio and the amount of input viral copies using graphpad prism software (graphpad software, san diego, ca, usa). the sequencing data were deposited at the ncbi sequence read archive (sra) and are available under accession numbers: srr to srr . detailed information of our pipeline used for analyzing raw sequencing reads can be found at https://github.com/shaoyuantan/svaproject. in order to evaluate and compare the general performance of drs and pcs for viral whole genome recovery, two whole genome sequencing runs using a cell culture-grown virus with a known reference sequence were carried out for each method (table ). all runs started with ng of sva rna for the library preparation and were sequenced for h. the available pores for each sequencing run were recorded to indicate the condition of the flow cell (table ). sva reads were extracted from total sequencing reads and analyzed. the pcs had a much better performance than drs in terms of higher sva yield (drs . mb, pcs . mb), longer average read length (drs bp, pcs bp), and lower raw error rates (drs . %, pcs . %) ( table ). these differences could be explained by the intrinsic features of oxford nanopore dna sequencing (pcs) and rna sequencing (drs), where the latter is a novel technology still under development and dna sequencing has been well optimized. the main reason for the higher sva yield from the pcs would be that although the two methods started with the same amount of sva rna, pcs involves a pcr amplification step which increases the number of sva dna strands available for sequencing. coverage analysis showed that pcs was able to generate a more even coverage distribution than drs ( figure ). for drs, significantly more coverage was seen at the end. this uneven distribution of drs has been observed previously and may be explained due to partially degraded rna and rna secondary structures hampering the movement of the rna through the nanopores, exhibiting higher coverage where sequencing is initiated, which is at the end of the genome [ ] [ ] [ ] . viruses , , x for peer review of reference sequence were carried out for each method (table ) . all runs started with ng of sva rna for the library preparation and were sequenced for h. the available pores for each sequencing run were recorded to indicate the condition of the flow cell (table ). sva reads were extracted from total sequencing reads and analyzed. the pcs had a much better performance than drs in terms of higher sva yield (drs . mb, pcs . mb), longer average read length (drs bp, pcs bp), and lower raw error rates (drs . %, pcs . %) ( table ). these differences could be explained by the intrinsic features of oxford nanopore dna sequencing (pcs) and rna sequencing (drs), where the latter is a novel technology still under development and dna sequencing has been well optimized. the main reason for the higher sva yield from the pcs would be that although the two methods started with the same amount of sva rna, pcs involves a pcr amplification step which increases the number of sva dna strands available for sequencing. . ± . . ± . * data shown as the mean ± sd of independent replicates. coverage analysis showed that pcs was able to generate a more even coverage distribution than drs ( figure ). for drs, significantly more coverage was seen at the ′ end. this uneven distribution of drs has been observed previously and may be explained due to partially degraded rna and rna secondary structures hampering the movement of the rna through the nanopores, exhibiting higher coverage where sequencing is initiated, which is at the ′ end of the genome [ ] [ ] [ ] . different assemblers were tested to determine the best fit assembler for the sequencing data from drs and pcs. in terms of consensus length and accuracy, drs datasets were assembled best using racon [ ] , and the pcs datasets were assembled best using canu [ ] . after choosing the assembler, pre-assembly read filters were examined to determine the optimal conditions for the generation of an optimized consensus sequence. different assemblers were tested to determine the best fit assembler for the sequencing data from drs and pcs. in terms of consensus length and accuracy, drs datasets were assembled best using racon [ ] , and the pcs datasets were assembled best using canu [ ] . after choosing the assembler, pre-assembly read filters were examined to determine the optimal conditions for the generation of an optimized consensus sequence. datasets containing different sequencing yields ( . , , and mb) were generated by randomly selecting reads from the total pass reads dataset. within the same yield dataset, three groups were generated based on different filters, with group containing all the pass reads (phred quality > ), group consisting of pass reads with a length filter > bp to remove short reads and all rcs reads (rcs was added to drs to increase the efficiency of library preparation), and group with pass reads that mapped to the sva database. the rationale behind the length filter was to test if a dataset with longer reads on average would help with consensus generation, and at the same time to delete all remaining rcs reads. although the rcs reads should all be removed during basecalling, in fact more than a third of the rcs reads remained in the pass reads dataset due to low filtering efficiency. the rationale for the use of the mappable filter was the assumption that a "less noisy" dataset would be beneficial for sva assembly and consensus generation, especially in some clinical samples where the desired viral rna reads would only account for < % of the total sequencing reads. using the " mb yield" datasets as an example, we evaluated the effect of the different filters on the read recovery, read length and read quality ( table ). the read recovery of the drs dataset after the length filter (group ) was % of the pass reads (group yield/group yield) and after the sva mappable filter (group ) it was % of the pass reads (group yield/group yield) ( table ). the low recovery was due to the large number of short reads present, mainly rcs reads. read recovery of the pcs dataset after the length filter (group ) and sva mappable filter (group ) was % and % of the pass reads (group ), respectively ( table ). the average read length and phred quality score was greater for pcs than for drs irrespective of the filters ( table ). the average read length of the unfiltered drs reads (group ) was especially low and was mainly due to the presence of short rcs reads, which account for > % of reads that are less than bp (table ). examination of each read filter at different sequence yields was performed to determine the optimal conditions for the generation of a consensus sequence. for the drs groups, as the starting yield increased, the length and accuracy of the generated consensus sequence increased ( table ). the highest consensus length and accuracy were observed at the mb yield (~ mb of sva reads, % sva mappable rate) (tables and ). at the same yield level, the consensus accuracy and length with different filters were similar, indicating that the sequencing yield is the leading factor for consensus accuracy and length and the raw read filters have minimal influence on results (table ) . a similar observation was observed for pcs sequencing, as within a sequencing yield, the different filters showed similar consensus length and accuracy (table ) . however, for pcs, an increase in yield did not always result in better consensus generation, as mb pass reads generated a lower accuracy and a shorter consensus than that of the mb read group (table ) . the most accurate consensus for pcs was generated using a total sequencing yield of mb (~ mb of sva reads, % sva mappable rate) (tables and ) . table . performance of consensus generation using different raw read filters at different yields *. sequencing method while both drs and pcs can generate a nearly full-length sva genome, the consensus from pcs achieved a % accuracy, much higher than that of drs which only reached a % accuracy (table ) . in this study, no obvious differences were observed when comparing the different filters using the cell culture samples. however, the filters may be useful in some situations not examined here such as in clinical samples from tissues that would contain a large amount of host rna. in order to make our pipeline applicable to all sample types, we used the sva mappable reads (group filter set) for the following spike-in and clinical sample analysis. the analytical sensitivity of oxford nanopore drs and pcs was evaluated by sequencing spike-in and clinical samples with a range of . × to . × viral copies. after sequencing, the number of total reads from each run was determined (table ). in order to detect sva at the species level in an unbiased and hypothesis-free manner, the taxonomic analysis was performed using wimp and the number of reads classified as sva were recorded (table ). results showed that sva was able to be easily detected using both sequencing methods in both spike-in and clinical samples containing more than × total viral copies. using drs to investigate spike-in samples, sva reads were detected in samples with as low as . × sva viral copies, while for pcs, sva reads were detected in samples with viral copies of . × or greater. in clinical samples, the detection limit was . × viral copies for drs and . × viral copies for pcs. the number of total reads indicated the overall performance of sequencing, while the ratio of sva reads to total reads suggested the presence and abundance of sva in a sample ( table ). as a reference for future experimental design, at least sva read should be obtained if sequencing a minimum of × viral copies from a clinical sample and generating around total reads (table ). of note, sva was not detected using pcs from a clinical sample with viral copies of . × , but was detected in other clinical samples with lower numbers of viral copies (table ). this could be explained by poor flowcell performance since few total reads were generated in the . × viral copy clinical sample. for example, even though the . × viral copy sample has five times greater viral copies than the . × viral copy sample, the total reads generated was times less, thus explaining why a sample with a higher viral copy number did not detect sva; poor sequencing performance and too few total reads generated for this sample. our observation of varying total and sva reads generated from samples with similar viral copies and sequencing time indicated inconsistent sequencing output for each run, mostly due to the condition of the flow cell used. for epidemiological and precise infection control purposes, it is necessary to know not only the infectious virus present, but the strain of the virus-causing disease. thus, to investigate whether drs or pcs can identify the strain of sva that is present in a sample, total reads were blastn analyzed against our sva whole-genome database and the sequence with the best match (top blast hit based on bit score) was considered as the sva strain present in the sample ( table ). the percent identity between the top blast hit and the known sequence of the sample was determined to identify the accuracy of strain level detection (table ). for the spike-in samples, a laboratory strain with a known whole genome reference sequence (mn ) was used and this sequence was also present in our sva whole genome database. the mn sequence was compared to the top blast hit to determine the percent identity which indicates the accuracy of strain level detection (table ). for each of the clinical samples, a partial genome reference sequence was obtained using sanger sequencing, which was then used to compare with the top blast hit, but since these reference sequences are partial sequences, they are not present in our sva whole-genome database, so we did not expect a % identity between the top blast hit and our reference sequence (table ). both sequencing methods were % accurate when detecting strains for the spike-in samples, in which the reference strain was present in the sva whole genome database and it was identified as the best match (table ) . for clinical samples, a comparison of the known partial genome to that of the top blast hit showed a sequence identity of . - . % for both sequencing methods (table ). some disagreements observed between the drs and pcs "best match" genome revealed a limitation of detection accuracy, which can be observed between highly similar strains (table ) . further examination of sequencing accuracy was determined by creating a consensus genome which was then compared to the known reference sequence to determine the sequencing accuracy. all raw reads that were mapped to the sva database were used to generate a consensus sequence. this consensus sequence (or longest read when no consensus could be generated) was then compared to the known viral reference sequence (table ) . a nearly complete sva consensus genome (breadth of coverage > %) was generated using both drs and pcs sequencing methods from samples containing . × viral copies or more, giving an accuracy greater than % for drs and greater than % for pcs (table ). in these experiments, a consensus genome coverage of % required a minimum level of sva reads for drs and sva reads for pcs. for samples containing less than viral copies, a shorter consensus genome was obtained with lower accuracy (table ) . then, a quantitative relationship between the output sva reads and the input sva viral copies was investigated. in order to minimize the inter-sequencing variations, sva reads were normalized for each sequencing run based on the total reads generated. a correlation analysis between the ratio of sva reads/total reads and input sva viral copies was performed. the results showed that drs had a strong linear regression with an r = . while pcs had a weak linear regression with an r = . , indicating that drs was a quantitative method while pcs was not. considering that pcs contains more steps than drs that can introduce bias, such as pcr amplification and amplicon selection, this was not surprising. the early and reliable detection of infectious agents as soon as clinical signs are observed is essential for efficient disease control. delays and misdiagnosis inevitably lead to the spread of disease and escalation of adverse impacts. prompt actions against an emerging pathogen are especially important because there is usually no existing immunity among the susceptible population, no vaccine, and no specific treatment against the pathogen. however, emerging infections are more difficult to identify since most diagnostics are based on previously known and expected infectious agents and miss unexpected pathogens. diagnostic methods that are rapid, available at the point-of-care, able to detect new pathogens, and robustly applicable across a wide range of pathogens are greatly needed to effectively fight against emerging eventualities [ , ] . among all pathogens, rna viruses have the highest mutation rates, and are anticipated to have the highest possibility to cause the next emerging event [ ] . they are also of special concern regarding zoonotic transmission due to their high adaptability to new hosts [ ] . in this study, we evaluated oxford nanopore minion sequencing for sva investigation, aiming to provide insights and tools for the investigation of emerging rna viral diseases through sequencing and bioinformatics. oxford nanopore provides two methodologies for rna sequencing: traditional amplicon sequencing (pcr-cdna sequencing, pcs), which has lower error rates and higher throughput, but involves reverse transcription and pcr amplification, which is time consuming and loses some rna genome structure information through the process; and direct rna sequencing (drs), which is an innovative technique under development that can sequence rna strands directly, thus eliminating the length limitations possibly coming from reverse transcription and allowing for the detection of nucleic acid base modifications. both sequencing methods used in this study can be used to detect unknown rna viral pathogens. however, a poly(a) tail, which is present in the sva genome, is needed for adapter ligation. thus, this approach lends itself readily to the sequencing of rna viruses with a poly(a) tail. many important swine rna viral pathogens have a poly(a) tail, such as coronaviruses (porcine epidemic diarrhea virus), picornaviruses (fmdv, sva) and arteriviruses (prrsv). the sequencing of rna pathogens which do not contain a poly(a) tail (such as rotaviruses) can be performed through the enzymatic addition of a poly(a) tail and this step can be added to any sequencing reaction without interfering with the sequencing of samples already containing a poly(a) tail [ ] . this study provided a thorough comparison between the pcs and drs methods, which are summarized in table , aiming to provide guidance on the selection of a sequencing method when in different clinical situations and for different purposes. we identified that pcs is more time consuming, but can generate a more accurate consensus, the advantage of which was especially obvious with higher viral copy number samples. although drs was observed to be less accurate, it was quicker to perform and just as sensitive and has unique and promising features such as the detection of nucleic acid modifications, as observed by other studies [ , ] . despite their differences, both sequencing methods were able to accurately detect sva at the strain level using raw reads and entry-level bioinformatics analysis (table ) . thus, a core sequencing laboratory with data analysis experts was not necessary for the detection of the strain of sva present, suggesting it could be run on a farm or at least more quickly than other more analysis-intense sequencing methods. the analytical sensitivity of a diagnostic method gives important information to help guide method selection based upon the situation. the evaluation of the analytical sensitivity of minion sequencing requires the definition of either sequencing time or the minimum number of total reads. in this study, the idea of a same day report was desired, so a rapid turnaround time frame using only h of sequencing was performed. the analytical sensitivity for both drs and pcs was shown to be similar with an input of × viral copies or more (in . ml starting material) always generating sva reads. drs was slightly more sensitive at approximately - viral copies (per . ml), while pcs needed approximately − viral copies (per . ml) to detect sva. previously, it had been shown that the minion sequencing of influenza virus had a detection limit of - genome copies/ml for h of sequencing, showing a similar sensitivity to our drs experiments [ ] . similar to other studies, we observed a great inconsistency between runs within the same sequencing time frame, which could be caused by factors such as varying flow cell conditions and sample quality [ ] . using the number of sequencing reads as well as sequencing time (monitored in real time using the minknow interface) can help minimize this sequencing run to run variation. in fact, we were able to determine that an input of more than × viral copies from a clinical sample and obtaining around total sequence reads were needed to generate a minimum of one sva read. if more sva reads were desired for other purposes, such as whole genome generation, or if a lower amount of sample was used, then more total reads should be set as a target. from this study, an advantage of direct rna sequencing over amplicon sequencing, such as pcr-cdna, was that drs showed a quantitative relationship between input viral titers and output sequencing reads. similarly, a strong relationship between influenza viral titers and influenza sequencing reads using oxford nanopore direct rna sequencing technology was observed by other research groups [ ] . however, in a hepatitis b virus (hbv) study using oxford nanopore amplicon sequencing (which includes a pcr step similar to our pcs protocol), considerable variability in total yields and the proportion of mapped hbv reads between sequencing runs was observed concluding that it was not quantitative [ ] . amplicon sequencing, such as the pcr-cdna protocol, includes more steps during library preparation, including the amplification and selection of pcr products which could possibly introduce bias, while the process of direct rna library preparation is simple and straightforward without additional amplification steps. while most sequencing is generally restricted to large laboratories, the portability of the minion sequencer makes it suitable for diagnosis in the field. on-site diagnosis can greatly improve emerging infectious disease management, especially considering that emerging disease outbreaks can happen anywhere and are more likely to occur in developing countries or remote areas where there is a lack of veterinary infrastructure, expertise, and diagnostic capacities [ , ] . in fact, several field studies have been conducted to confirm such advantages of a portable sequencer including a zika virus outbreak in brazil, a ebola outbreak, and a dengue virus field investigation [ , , ] , concluding that the use of the minion sequencer was advantageous for rapid in-field disease detection. there are a few limitations to using these sequencing methods for emerging disease detection. first, our method of species and strain detection largely depends on the genome database, genbank. while the examination of emerging viral diseases caused by known viruses expanding to new hosts or geographical regions will have viral genome information available in genbank, previously unknown or newly discovered pathogens will not. however, the sequencing information for these unknown or newly discovered pathogens can be determined following minion sequencing by carefully examining the unclassified sequencing reads. second, the analytical sensitivity of minion sequencing is lower than that of diagnostic pcr assays, but pcr assays are limited to the detection of known pathogens and during an outbreak, high levels of the pathogen should be present allowing for the ease of detection through minion sequencing. in addition, sequencing, even at the current sensitivity of detection, in the case of new pathogens can be used to support pcr by providing strain information for more effective disease control and for epidemiologic studies to track infection. third, while this study provided a benchmark and foundation for portable sequencer use in disease diagnostics, there is still more to do to achieve commercial diagnostics, such as the improvement of the accuracy, detection limit and consistency of flow cell performance. this study evaluated the ability of minion sequencing for use as a diagnostic tool for the detection of emerging viral diseases in swine by examining sva infection as a model of an emerging disease. we demonstrated that the portability, easy-operation, low-maintenance minion platform is an effective tool for the investigation of sva. we 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samples illumina and nanopore methods for whole genome sequencing of hepatitis b virus (hbv) one world, one health: the threat of emerging swine diseases. a north american perspective mobile real-time surveillance of zika virus in brazil serotyping dengue virus with isothermal amplification and a portable sequencer emerging infectious diseases: threats to human health and global stability this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to thank the university of minnesota veterinary diagnostic laboratory for providing samples for testing. the authors acknowledge the minnesota supercomputing institute (msi) at the university of minnesota for providing resources that contributed to the research results reported within this paper. url: http://www.msi.umn.edu. the authors declare no conflict of interest. key: cord- -gztmidn authors: sambri, vittorio; capobianchi, maria r.; cavrini, francesca; charrel, rémi; donoso-mantke, olivier; escadafal, camille; franco, leticia; gaibani, paolo; gould, ernest a.; niedrig, matthias; papa, anna; pierro, anna; rossini, giada; sanchini, andrea; tenorio, antonio; varani, stefania; vázquez, ana; vocale, caterina; zeller, herve title: diagnosis of west nile virus human infections: overview and proposal of diagnostic protocols considering the results of external quality assessment studies date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gztmidn west nile virus, genus flavivirus, is transmitted between birds and occasionally other animals by ornithophilic mosquitoes. this virus also infects humans causing asymptomatic infections in about % of cases and < % of clinical cases progress to severe neuroinvasive disease. the virus also presents a threat since most infections remain unapparent. however, the virus contained in blood and organs from asymptomatically infected donors can be transmitted to recipients of these infectious tissues. this paper reviews the presently available methods to achieve the laboratory diagnosis of west nile virus infections in humans, discussing the most prominent advantages and disadvantages of each in light of the results obtained during four different external quality assessment studies carried out by the european network for ‘imported’ viral diseases (enivd). west nile virus (wnv) is an enveloped spherical, single-stranded rna flavivirus that is transmitted to birds by ornithophilic mosquitoes, mainly belonging to the genus culex [ ] and is occasionally transmitted to mammalian hosts [ ] . the geographical area of wnv circulation encompasses most of africa, israel, north america and south america, australia and scattered areas in southern and central europe, including russia, czech republic, hungary, greece, romania, italy, southern france, portugal, turkey and spain [ ] [ ] [ ] . at least two distinct genetic lineages have been identified among the wnv isolates in diverse areas [ ]: lineage includes most of the american, european and some african strains, whereas lineage contains mainly sub-saharan african isolates, although some lineage strains have been detected in humans and mosquitoes outside africa [ ] [ ] [ ] [ ] . additional lineages have been proposed: lineages , and include viruses isolated from czech republic (rabensburg strain), caucasus and india, respectively [ ] [ ] [ ] . a sixth lineage was recently described in indonesia and a putative seventh lineage has been identified in spain [ ] .the strains belonging to lineages and have up to % nucleotide divergence [ ] . this wide diversity together with the elevated threat for human health posed by wnv infections resulted in the development of a variety of methods for laboratory diagnosis [ ] . it is important to emphasise that about % of the human infections caused by wnv remain asymptomatic and therefore approximately % of cases become clinically evident [ ] . the clinical syndromes associated with wnv human infections are predominantly mild flu-like fevers (wnv fever) of which less than % develops severe neuroinvasive disease [ ] . for detail about the case classification of the wnv infection and for proposals concerning laboratory diagnosis of this virus please refer to the conclusion of this paper. this paper reviews the currently available techniques for the identification of wnv infection in humans. four external quality assessment (eqa) studies have been performed: two for the molecular diagnostics of wnv infections, and two for the serological methods. the results of these studies are presented at the end of this review, focusing on the main diagnostic problems. currently, the laboratory methods for the diagnosis of infection by wnv belong to two main categories: serology and viral detection. a. virus isolation. wnv is not readily isolatable from tissues, plasma, serum and csf samples in cell culture using either mammalian or mosquito derived cell lines, such as vero e , rk- , ap or c / [ ] [ ] [ ] [ ] . moreover, these procedures must be performed under biosafety level conditions [ ] and it generally takes up to days before the appearance of cpe. virus isolation is most readily accomplished if the following procedures for collecting the samples are adhered to; whenever possible, the samples should be collected under sterile conditions, in collecting media containing antibiotics and immediately refrigerated at °c. under optimal conditions the samples should be inoculated onto susceptible cells in culture within minutes of collection but when this is not possible, the time interval should be kept to a minimum and always less than hours. in general these conditions can be met in reference laboratories and dedicated research facilities [ ] . the isolation of wnv strains provides the added value of allowing further studies and research on pathogenesis, genetic variation evolution, epidemiology, etc. b. molecular methods. wnv genomes in peripheral blood are usually detectable from - days to - days post-infection [ ] . during this time it is usually possible to identify the viral rna in serum and/or plasma samples. in addition the detection of viruria by molecular methods has been demonstrated to be a useful tool that enables the detection of wnv genomes, even after prolonged times post-infection [ , ] . for the routine detection of wnv rna using molecular techniques there are two distinct diagnostic settings: the first involves blood and organ donation screening from subjects living in an area where wnv circulation is known to be occurring, and the second involves the identification of viral genomes in serum, plasma and csf samples from patients presenting with a clinical picture typical of wnv infection [ ] . blood and organ safety screening. transmission of wnv between humans during blood transfusion and tissue and or organ transplantation has been recorded in the usa [ ] and recently in europe [ ] [ ] [ ] [ ] . in order to increase the biological safety of blood and organ donations, a screening policy has been implemented [ , ] in areas where human cases of wnv related diseases are known to be present, since it is believed that at least % of the wnv infected population remains asymptomatic during the phase in which viral rna is actually detectable in blood [ ] . in this diagnostic setting, the basic requirement for laboratories is to achieve very high sensitivity values, since these techniques must be applied in a healthy and totally asymptomatic population where the hypothetic wnv load could be as low as genome copies/ml. two commercial nucleic acid amplification tests (nats) are presently able to detect genomes at these low levels. the first is based on polymerase chain reaction (pcr) technology [ ] and is manufactured by roche diagnostics (mannheim, germany) and the second is a transcription mediated amplification (tma) test [ ] available from novartis diagnostics (emeryville, ca, usa). both assays are patented and the target sequence is not available to users. they are fully automated on high throughput instruments and allow the testing of hundreds of plasma samples per day, with an elevated numerical performance as required by the blood bank workflow. the analytical sensitivity of these tests (as stated by the manufacturers) is . [confidence interval (ci) %: . - . ] and . (ci %: . - . ) genome equivalents/ml, for the tma and pcr, evaluated on lineage wnv strains, respectively. these sensitivity values are within the standard for wnv blood screening products that have been set by the us food and drug administration within copies/ml [ ] . the specificity of these methods is considered to be quite high [ , ] . however, false positive reactions were reported due to the presence of usutu (usuv), a virus closely related to wnv [ , ] . depending on the local epidemiological prevalence of wnv infections, blood screening can be performed on individual samples (id-nat) or in pooled samples (usually or mini-pool specimens: mp-nat), the id-nat test being the more sensitive of the two [ - ] . as an alternative to these techniques, the use of manually performed real time rt-pcr (qiagen, hilden, germany) and nucleic acid sequence based amplification (nasba) have been recently reported [ ] in a population of tissue and organ donors, with good performance in terms of turnaround time and clinical performance. patients with suspected wnv neuroinvasive disease or wn fever. the viral load in biological specimens (blood, csf, urine) from patients with suspected wnv infection is hypothesized to be higher than in asymptomatic infected subjects and consequently the diagnostic methods do not need the same high sensitivity that is required for the screening tests used for asymptomatic individuals. reverse transcription pcr (rt-pcr). the sensitivity of conventional rt-pcr for wnv detection depends mainly on the target sequence. a test based on primers that target the c gene ( ') and the prm gene ( ') detected viral rna equivalent to approximately . plaque forming units (pfu) derived from a cell grown ny isolate [ ] . nested rt-pcr techniques can increase by up to fold the the rna detection threshold but these methods are more prone to contamination, require a longer time to perform and increased involvement of laboratory personnel [ ] . the use of pan-specific rt-pcr techniques, capable of identifying most of the flaviviruses and mainly based on nested assays [ , ] , can potentially differentiate wnv related disease from other infections caused by flaviviruses. the amplicons obtained using these labour intensive methods need to be sequenced and the obtained electropherograms analysed in order to identify the infecting virus. no precise indication can be provided presently about methods that can be used to differentiate the diverse wnv lineages one from another. most of the recently published papers in the field of wnv phylogeny are based on the analysis of the ns or e genes. real-time rt pcr. these methods are generally rapid and reliable and can be used for detection of wnv on a large variety of samples, including non-human specimens, such as animal tissues and mosquitoes [ ] . different technical modifications have been proposed in the last years. in general, the taqman probe-based assays are quite sensitive (approximate detection limit is . pfu of viral rna) [ ] but sometimes incapable of detecting emerging genomic variants of the virus [ ] . the sybr green based tests are as sensitive as the taqman tests, but less specific [ ] . the decreased specificity could be both a disadvantage, since false positive results can be generated by diverse viruses, and an advantage, given the broader spectrum of wnv variants that can be identified [ ] . another potential disadvantage of real-time rt-pcr is the represented by the generally short amplicons obtained, that are not suitable for sequencing and molecular characterization. given the strain variation of wnv from season to season and in different geographical locations, new diagnostic molecular approaches have been proposed. a multiplex pcr-ligase detection reaction assay (rt-pcr/ldr) based on the contemporary amplification of three different genomic regions (one in the coding sequence of ns and two in the nonstructural gene ns ) minimized the risk of false negative results due to wnv genetic variants [ ] . another method, based on genome-wide rt-pcr [ ] overcame the undetectability of wnv isolates with genomic differences. as mentioned, wnv isolates have been phylogenetically grouped into different lineages [ ] and lineages and overlap in some areas of europe [ ] . in order to distinguish between lineage and , a novel real-time quantitative rt-pcr test has been developed. this test showed an extremely high differentiation capacity on different cell derived wnv isolates at the level of - genome copies [ ] . wnv overlaps geographically with several other arboviruses including flaviviruses, alphaviruses and some bunyaviruses and as the number of recognised emerging viruses increases in both tropical and sub-tropical regions [ ] [ ] [ ] [ ] , new multiplex rt-pcr assays have been developed to facilitate simultaneous identification, for example, of chikungunya virus, denv [ ] , jev and wnv in patient samples [ ] . as these techniques improve the early detection of multiple arboviruses is becoming a practical reality with the potential for rapid and cost-effective differential diagnosis and epidemiological surveillance. c. immunohistochemistry. the detection of wnv antigen using histochemical protocols in tissues obtained from fatal encephalitis cases has been available for diagnostic purposes for many years. in today's laboratories this procedure is performed rarely and usually in order to improve the certainty of a clinical diagnosis in cases where laboratory data are minimal [ ] . data on the diagnostic performance of this assay have been mainly obtained in animals and the specificity of this method depends largely on the specificity and quality of the antiserum used. the sensitivity in many cases can be low and is often dependent on the amount of wnv present in the tissue and the quality of the tissue under investigation [ , ] . it is noteworthy to underline that most of the diagnostic approaches described in this paragraph could be hampered in the daily routine use for the laboratory diagnosis of wnv infection by the lack of standardized protocols and by the uncertainty about their specificity and sensitivity. specific antibody detection still remains the most widely used approach for the diagnosis of wnv infection in humans. in order to understand the application of serology for wnv diagnosis, it is useful to remember that the mean times from the detection of viral rna to igm and igg seroconversion are approximately and days respectively, as determined in a study performed amongst more than wnv viraemic blood donors in the usa [ ] . the main weakness that limits the clinical relevance of serological methods is the broad antigenic cross-reactivity that exists between all flaviviruses: the quite specific viral envelope (e) protein neutralising antibody response is often combined with less specific tests based on detection of antibodies against the membrane (m) and non-structural (ns) proteins of which the amino acid sequences are more conserved amongst the flaviviruses [ ] . based on this consideration, the principal serological methods can be subdivided into two main groups: the first includes the enzyme-linked immunosorbent assays (elisas) and immunofluorescence (if) based tests; the second includes the plaque reduction neutralization test which can be carried out using a highly sensitive % or less-sensitive % endpoint (prnt , and prnt respectively), both of which require the constant availability of standardised-validated infectious viruses and appropriate cell cultures. the hemagglutination-inhibition test (hia) is still used to detect pan-flavivirus immune response whereas the complement fixation test (cft) is rarely used in today's laboratories. the techniques included in the first group are widely used due to their relative applicability in routine laboratory and the ability to automate a part of the workflow but they are less specific as a consequence of their inability to distinguish between wnv-specific and cross-reactive antibody responses [ ] . thus, any positive result identified using these methods must be confirmed by the more specific tests, i.e., those that constitute the second group. for serosurveys involving individuals that appear healthy but may have been infected sub-clinically, the gold-standard for detecting immune responses is the prnt method which is able to detect, specifically, low titre, low avidity immune responses. the prnt assay, by definition, is less sensitive than the prnt assay and therefore is more appropriate for studies that involve the detection of immune responses in individuals that have presented with clinically apparent infection and have almost certainly developed detectable viraemia during their clinical infection. it is important to emphasise that in either of these analyses, the test should follow the guidelines of the world health organization [ ] and should include control standardised viruses that are known to be readily neutralisable and to be antigenically closely related but distinct species from wnv. this second group of techniques, particularly prnt assays are labour intensive and are generally limited to reference or dedicated research laboratories, where appropriate biosafety level could be attained [ ] . a. prnt. the flavivirus antibody response is directed against both family cross-reactive epitopes and virus specific epitopes [ ] . the e protein is one of the most prominent immunogenic polypeptides and elicits most of the neutralizing antibodies (nabs) [ ] . this test is generally performed following standard protocols [ ] . in detail, the method defined for dengue by the who could be adapted for wnv [ ] describes a standardised protocol for determining prnt titres of sera from patients. the same protocol could be used also to determine a prnt titre in sera obtained from sub-clinical infections. this last method is particularly useful to carry out a serosurvey on healthy individuals to determine whether or not they have previously experienced infection with wnv (or other viruses). given the broad antigenic cross-reactivity between different flaviviruses and the diverse neutralizing activity of the human igg subclasses [ ] , both prnt and prnt require evaluation of the nabs against a panel of related viruses [ ] . based on the present known epidemiology of flavivirus human infections in europe, the following viruses, grown either in mammalian cell lines e.g., vero e or mosquito-derived cells such as c / , should be included for differential diagnostic purposes and to act as the controls for cross-reactivity reactions [ ] . two strains of wnv lineage : one recently isolated in europe from human infections [ ] , preferably at a low passage number in cell culture, and one reference strain, for example, the italia or france ; while the use of old strains such as eg should be avoided. one strain of wnv lineage , since the presence of this lineage has recently been identified in the greek ongoing epidemic [ , ] . usuv which is now believed to be present in most of central europe [ ] and has recently been reported as causing human infections [ , ] . under selected clinical and epidemiological circumstances, such as when testing samples obtained from patients that travelled in diverse geographical areas prior to be tested, other flaviviruses should be included in this panel, as follows. japanese encephalitis virus (jev) which is widespread in asian countries [ ] from where a large human migration stream to europe is presently occurring and whose genome has recently been putatively identified in vectors in italy [ ] [ ] [ ] . tick-borne encephalitis virus (tbev) that is widespread in central and eastern european countries [ ] . in order to have a more complete panel, two additional viruses should also be included: one serotype of dengue virus (denv) in light of the large number of imported cases within the european union territory, [ ] [ ] [ ] [ ] in travellers returning from tropical endemic areas and of the recently reported autochthonous activity of denv in scattered areas in france [ ] and croatia [ ] . finally, yellow fever virus (yfv) should be included in this panel to ensure the absence of yfv-vaccine derived nabs [ ] . as a general rule, given the complicated pattern of antibody cross-reactivity among the family flaviviridae [ , ] , nab titres should be consistently higher against wnv than the titres detected against the control viruses in the panel. there are also technical variations in the testing protocol and this issue must be considered when comparing prnt results obtained in different laboratories. for example, variation to the techniques used for the detection of nabs could include the incubation time (usually from to hours), the method for cytopathic effect (cpe) detection (direct microscopy, staining with a vital dye, detection by if or by automated colorimetric detection [ ] ) and the diameter of the dish/well used to support the cell monolayer infected with the viruses (micro neutralisation titre assay: mnta [ ] . b. hia and cft. these tests, nowadays, are rarely used in the diagnosis of wnv infections in humans. the hia test requires treatment of each serum to remove non-specific inhibitors [ ] . it also requires the daily availability of fresh erythrocytes to maintain a high performance. moreover, hia is broadly cross-reactive. cft is very labour intensive both in the analytical stage and in the reading phase, and the test specificity and sensitivity suffer from the lack of standardization of the antigenic preparations obtained from different sources [ ] . all the tests reported above at points a and b are technically difficult, time consuming, relatively insensitive (with the possible exception of the prnt test) during early infection and require several days before a result can be delivered. consequently in the diagnostic laboratories of today, most serological diagnostic tests are performed by eia and if which are both technically simpler to perform. in addition, they can be highly specific when they incorporate monoclonal abs (mabs). c. eias. these methods can be subdivided into three main categories: ( ) the igm antibody-capture eia (mac-eia), ( ) the indirect igg eia and ( ) the epitope blocking eia. all of these methods have the advantage of rapidity and reproducibility when compared with those detecting nabs and with hia and cft. this test is suitable for routine detection of immune responses resulting from an acute viral infection. the igm response is usually detectable within the first week of wnv infection, sometimes during the pre-symptomatic stage, in plasma, serum or cerebrospinal fluid (csf) and generally persists for up to days in plasma/serum with prolonged persistence in the csf up to - days post infection [ ] and up to year in plasma/serum of viraemic blood donors [ ] . the standard commercially available mac-eias use plates coated with antibodies anti-human igm and wnv antigens obtained from the brain of experimentally infected mice. as the secondary immunoglobulin, monoclonal antibodies (mabs) directed to wnv conjugated with horse radish peroxidase can be used [ ] . the serum dilution for screening is generally at : . this test has a calculated sensitivity of . % with a specificity of . % [ ] . some technical modification of the igm detection has recently been proposed in order to increase the sensitivity, specificity and clinical performance of the tests. in particular, the introduction of a ratio method in the calculation of the igm eia results has been demonstrated to eliminate nonspecific reactivity [ ] . further, the use of recombinant highly specific antigens, such as the epitopes located in the domain iii of the e protein [ ] , the whole recombinant e protein produced in insect larvae [ ] , the virus-like particles (vlps) from prem and e proteins [ , ] , the ns , and proteins [ , ] have been proposed with promising results in order to increase the standardization of the antigen preparation and to reduce the costs of these assays. it is generally accepted that the identification of an igm response is a sign of probable wnv infection that needs to be further confirmed by other laboratory criteria, primarily by a fourfold increase of the serum antibody (ab) titres between different samples collected during the acute and convalescent stage of the disease. the presence of virus-specific igm in the csf is generally accepted as direct evidence of infection, thus prompting to identify the case as a confirmed one, according to the eu case definition [ , ] . although in general results of mac-eias correlate well with the prnt and the cross-reactivity with igm abs resulting from infection by jev serocomplex viruses is generally lower than for that of igg antibody responses [ , , ] , false positive results have been reported with the routine use of mac-eia [ ] [ ] [ ] . indirect igg eia. the detection of wnv igg by eias is now standardized and these methods are widely used for the determination of immune status to wnv either in suspected patients or in healthy asymptomatic populations [ , [ ] [ ] [ ] [ ] [ ] . a major limitation of these tests is, firstly, their restricted clinical specificity due to extensive cross-reactions with flaviviruses. for this reason it is recommended that the detection of igg should be performed in combination with the mac-eia in all cases of suspected infection, since persistence of igm may last for up to weeks after disease onset [ , , ] . all the cases of positive igg detected by this technique need to be further evaluated and confirmed either by a prnt test or by an increase of the igg response later on in the course of infection. the reported specificity and sensitivity values for the most commonly used commercially available igg eia techniques are about %- % and %- %, respectively [ , ] . in order to estimate the time course of infection, two commercially available eia methods (focus diagnostics, cypress, ca, usa and euroimmun, lübeck, germany) that evaluate the avidity of the igg immune response during wnv symptomatic disease have been used. in sera obtained within the first days after the onset of infection the igg avidity was lower than %, whereas an avidity index higher than %- % correlates with infections lasting at least days [ , ] . the determination of igg avidity is likely to provide additional useful data for clinical differentiation of recently and previously acquired wnv infections. a new test, based on the immune complex (ic) elisa method has recently been proposed for the detection of igg, showing an elevated capability of discriminating between antibody response directed against wnv and tbev [ ] . similar tests could be further developed in order to discriminate between wnv and usuv, jev, murray valley encephalitis virus and saint louis encephalitis virus infections. a technical variant of the eias for the identification of igg is the so called "epitope-blocking eia". this method is based on competition between the tested sera and wnv specific mabs for binding to wnv antigens and was originally developed for diagnosis and prevalence studies in animals since it is species-independent [ ] . this technique has also recently been evaluated for the diagnosis of human infection. the results obtained showed that this method is only applicable in populations not previously exposed to flaviviruses other than wnv [ ] which results in a lack of correlation with the prnt findings. previous vaccination against jev is another possible confounding factor for the interpretation of the results. d. if. the use of the if assay in routine diagnosis of wnv infection is mainly dependent on the number of samples that need to be evaluated and the quality of training of laboratory personnel in interpreting the test results. most of the tests used are developed "in-house" by individual laboratories usingwnv infected cell cultures. consequently this technique suffers from a lack of standardization when results are compared between laboratories and would greatly benefit from an external quality assurance (eqa) evaluation as shown later. a commercially available if test (euroimmun, germany) was shown to have about % specificity when compared with the prnt test, in a study performed on sera collected during a human wnv outbreak in south africa [ ] . in addition, new protocols for the if testing have been developed for the detection of igm and igg antibodies. these methods have been reported to exhibit higher specificity than mac eia [ , ] . to reduce the non-specific reactivity (against related vector transmitted viruses or against cellular antigens) defined quantities of uninfected cells should be mixed with wnv-infected cells: the uninfected cells are obviously expected to remain non-reactive with tested sera. in order to achieve practical recommendations about the possible routinely use of the serologic tests described above please refer to table . four external quality assurance (eqa) studies assessing the quality of wnv diagnostics worldwide were performed or supported by the european network for diagnostics of 'imported' viral diseases (enivd) [ ] . a first study assessing the quality of molecular detection methods of wnv infections was conducted in [ ] and a second one in [ ] . serological detection methods were assessed during a first eqa study in [ ] and a second in [ ] . for each eqa, participants were asked to analyse the provided eqa samples using the procedures routinely used by them in suspected human cases. assay details, such as the type of method used, suppliers of commercial kits, protocols and references were requested. the participants could have the choice of the diagnostic procedure employed. regarding the first eqa of molecular detection methods, the overall diagnostic performance for wnv ( ) was disappointing compared with earlier eqa studies on emerging agents such as ebola, lassa, pox, and severe acute respiratory syndrome viruses [ , ] . results showed a lower detection rate for lineage of wnv. the second eqa of molecular methods showed an improved proficiency of laboratories compared to the first eqa performed in . however, results suggest that detection of both wnv lineages is still problematic. therefore, further proceedings for the detection of both lineages are needed, particularly for in-house assays [ ] . for the first eqa of serological detection methods ( ), only % of participating laboratories passed the minimum requirements for successful performance. this result is mainly explained by the high rate of cross-reactivity with sera positive for related abs, particularly those specific for yfv. also the low sensitivity for igm detection results in a risk of overlooking wnv acute infections. in agreement with a previous eqa study for the serological detection of dengue virus infection [ , ] , there was no significant difference between the use of commercial or in-house assays. the second eqa of serological detection methods was conducted in and involved participants compared to in . compared to the previous study, results still showed low specificity for the igg detection methods demonstrating a high level of cross-reactivity with other flaviviruses. likewise, low sensitivity of igm detection was also observed indicating that there is still a need to improve wnv serological diagnostic tests. these studies demonstrate the importance of quality control measures in the detection of wnv infection. the results clearly indicate a need for laboratories to improve their tests: in particular by avoiding cross-reactivity with sera containing antibodies specific for heterologous flaviviruses and also by achieving lower detection limits in rt-pcr tests for all wnv lineages. by organising such comparative testing of well-characterised samples, laboratories are given the opportunity to identify their weaknesses 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proficiency study on west nile virus molecular detection second external quality assessment of the molecular diagnostic of west nile virus: are there improvements towards the detection of wnv? second international diagnostic accuracy study for the serological detection of west nile virus infection first international quality assurance study on the rapid detection of viral agents of bioterrorism sars molecular detection external quality assurance quality assurance for the diagnostics of viral diseases to enhance the emergency preparedness in europe quality control assessment for the serological diagnosis of dengue virus infections this article is an open access article distributed under the terms and conditions of the creative commons attribution license this work was supported in part by grant "fondi finalizzati lab p " from regione emilia romagna and by grants "ricerca corrente" from the italian ministry of health. parts of this study were also supported under the project "predemics" ( th the authors declare no conflict of interest. key: cord- -et rz authors: lauber, chris; gorbalenya, alexander e. title: genetics-based classification of filoviruses calls for expanded sampling of genomic sequences date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: et rz we have recently developed a computational approach for hierarchical, genome-based classification of viruses of a family (demarc). in demarc, virus clusters are delimited objectively by devising a universal family-wide threshold on intra-cluster genetic divergence of viruses that is specific for each level of the classification. here, we apply demarc to a set of filoviruses with complete genome sequences and compare the resulting classification to the ictv taxonomy of the family filoviridae. we find in total six candidate taxon levels two of which correspond to the species and genus ranks of the family. at these two levels, the six filovirus species and two genera officially recognized by ictv, as well as a seventh tentative species for lloviu virus and prototyping a third genus, are reproduced. demarc lends the highest possible support for these two as well as the four other levels, implying that the actual number of valid taxon levels remains uncertain and the choice of levels for filovirus species and genera is arbitrary. based on our experience with other virus families, we conclude that the current sampling of filovirus genomic sequences needs to be considerably expanded in order to resolve these uncertainties in the framework of genetics-based classification. for a steadily growing number of viruses the genome sequence is the first and only information available. because experimental characterization lags behind and is unlikely to be pursued for many viruses, comparative sequence analysis plays a central role in identifying commonalities and specifics between viruses. in this framework, researchers increasingly explore the usability of genetic sequences for virus classification. we have recently introduced a computational approach to hierarchically classify viruses of a family by relying solely on genetic data, coined demarc. briefly, in demarc virus clusters are delimited by devising a threshold on the maximum intra-cluster (intra-taxon) divergence of viruses. this is done separately for each level of the hierarchical classification, all of which are selected using a cost function that measures the quality of virus clustering. the approach was extensively evaluated in a case study of picornaviruses [ ] . strikingly, the demarc-based picornavirus classification showed only few, but biologically notable, deviations from the ictv taxonomy of the family picornaviridae [ ] , the latter being developed by extensive efforts of expert picornavirologists who rely on various virus characteristics [ ] . this analysis revealed important key parameters of demarc that distinguishes it from distance-based classification approaches and their applications of similar studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the demarc specifics include (i) the use of pairwise evolutionary distances (peds) instead of uncorrected p-distances, and (ii) a quantitative method to devise taxon levels and associated ped thresholds for virus clustering in a systematic and family-wide manner. we also reasoned that, in order to avoid a biased selection of genes/protein domains and to represent a virus genome as fully as possible, all family-wide conserved proteins must be used in a demarc-mediated analysis. as result, most incomplete genome sequences are excluded from the analysis. since some of these differences are evolutionary-based, the demarc-based picornavirus classification enabled biological implications that are not (yet) available in taxonomy or through other approaches to virus classification. they include the prediction of known and currently unknown genetic diversity in the family and the proposed genetic separation of members of virus species [ ] . so far, demarc was used to extensively revise the taxonomy of coronaviruses [ ] , and to propose the classification of two recently discovered insect nidoviruses [ , ] into the tentative new family "mesoniviridae" [ ] . in order to validate a general applicability of demarc in rna virus taxonomy a systematic analysis of viruses from diverse families is most wanted. in this study we sought to apply demarc to filoviruses, making it the first analysis of viruses with rna genomes of negative polarity (ssrna−). filoviruses form the family filoviridae of the order mononegavirales, the latter combining all known ssrna− viruses with non-segmented genomes [ ] . currently the filovirus genera marburgvirus and ebolavirus, which comprise one and five species, respectively, are recognized [ ] [ ] [ ] . additionally, a tentative genus "cuevavirus" with a single species formed by lloviu virus has been proposed [ ] . filovirus species are delimited using both phenotypic and genetic demarcation criteria including thresholds on percentage identity of full-length genomic sequences [ ] [ ] [ ] [ ] . the filovirus genome of about kb encodes seven structural proteins in separate open reading frames (orfs). they include a nucleoprotein (np), a spike glycoprotein (gp , ), two matrix proteins (vp and vp ), a multi-domain protein (l) with rna-dependent rna polymerase (rdrp) and methyltransferase function [ ] , an rdrp cofactor (vp ), and a transcriptional activator (vp ) [ ] . additionally, some filoviruses may encode lineage-specific proteins [ ] [ ] [ ] [ ] . marburg-and ebolaviruses are endemic to central africa and the philippines [ , , , ] while lloviu virus was discovered in southern europe [ ] . some filoviruses have been isolated from bats and domesticated pigs and can cause hemorrhagic fevers in primates with often fatal outcome. since they comprise some of the most dangerous pathogens in the world, the taxonomy of filoviruses, especially at the species level, may have considerable practical implications. the dataset of this study was formed by complete filovirus genome sequences that we downloaded into the viralis platform [ ] in february . for these filoviruses, a concatenated multiple alignment of the seven proteins that are conserved family-wide was constructed and submitted to ped calculation. the resulting ped values are distributed non-uniformly along the range of to . substitutions per amino acid position ( figure a ). using demarc we identified six distance threshold candidates, each associated with the optimal clustering cost of zero ( figure b ) (see experimental section). with this highest possible support all intra-cluster ped values would fall below the respective distance threshold, indicating that all clusters in a respective level may not be improved further (see [ ] for technical details). this result suggests the use of all six thresholds for building a classification. however, the number of ranks below the family level is limited to three in virus taxonomy (subfamily, genus, and species). to satisfy this limitation, three thresholds must be selected by using additional criteria, e.g., biological properties. within the demarc framework we used those thresholds that are associated with the highest threshold support measure (tsm) values. this use of tsm values is non-canonical: in demarc they are commonly used for the selection of ped ranges in which thresholds are further identified by local cost optimization if that is attainable (in situations where the optimal clustering cost of zero cannot be achieved; see [ ] for technical details). in the current analysis of filoviruses, however, using each of the ped threshold candidates within the six ped ranges would result in a clustering cost of zero ( figure b ). thus, for each range we arbitrarily selected the smallest observed ped value within the range as the threshold value (colored arrows in figure b ). we note that the produced classification accommodates laboratory-introduced genetic variation in some sequences due to virus propagation in tissue culture before sequencing. the observed continuous ranges with zero ped frequency imply that the scale of this variation is small compared to the natural genetic variation even at the lowest level of the derived classification. the first selected threshold (ped of . ) results in seven clusters ( figure b ) that match the official or tentative ictv species of the family filoviridae. these are species marburg marburgvirus (comprising virus sequences), species zaire ebolavirus ( ), species reston ebolavirus ( ), species sudan ebolavirus ( ), species taï forest ebolavirus ( ), species bundibugyo ebolavirus ( ), and tentative species "lloviu cuevavirus" ( ). according to the second threshold (ped of . ), three clusters that match the official or tentative genera-marburgvirus, ebolavirus, and "cuevavirus"-of the family are recognized ( figure b ). the third threshold (ped of . ) joins viruses of the genus ebolavirus and the tentative genus "cuevavirus" into a single cluster while viruses of the genus marburgvirus form the second cluster; these two clusters could be provisionally treated as tentative subfamilies. hierarchical relationships of the clusters according to the three applied distance thresholds are shown in figure a . all delineated clusters form monophyletic lineages in the phylogeny of the filoviruses (reciprocal monophyly) ( figure ) when assuming the root to be closest to the branch leading to marburgviruses (which would correspond to midpoint rooting). the three threshold candidates not considered for classification (ped of . , . , and . ) would result in eight, six, and five clusters, respectively ( figure b) . the clustering in eight clusters would split viruses of the species marburg marburgvirus into two clusters formed by the ravn and marv lineage, respectively ( figure b ). the clustering in six clusters would join viruses of the species taï forest ebolavirus and bundibugyo ebolavirus into a single cluster ( figure c ). the clustering in five clusters would join viruses of the species zaire ebolavirus, taï forest ebolavirus, and bundibugyo ebolavirus into a single cluster ( figure d ). it would be reasonable to consider these eight-, six-, and five-cluster scenarios as alternative species groupings. on the other hand, it could indicate that the three taxonomic ranks below the family level may be insufficient to accurately classify the genetic diversity of filoviruses. in the demarc-mediated classification of coronaviruses we also observe numerous ped thresholds with zero clustering cost [ ] . the ped threshold delineating the seven ictv species is controlled by a single cluster, species marburg marburgvirus, which shows by far the highest sampling ( % of all sequences) among all clusters of that level (figures a and ). this cluster contributes the largest intracluster ped value ( . ) of that level compared to other clusters for which values of at most . (between viruses of the species reston ebolavirus) are observed ( figure ). these considerable differences in the divergence of viruses from different filovirus species can be rationalized through two contrasting explanations that both exploit possible effects of biased virus sampling on the classification. first, the observed differences could be a result of the relatively poor sampling of virus sequences from the five ebolavirus and the "cuevavirus" species. once sampling is improved, viruses from these species may show a genetic divergence comparable to marburgviruses. alternatively, future improved virus sampling might show that the five ebolavirus species recognized by ictv actually form a single species. the full ped range from to around . ( figure a ) might then be populated which would merge viruses of the five ebolavirus species into a single cluster. this would result in three species clusters in total corresponding to the three ictv genera of the family filoviridae. consequently, ebolaviruses and "cuevaviruses" would form a single genus (in addition to the genus marburgvirus) as suggested by the ped threshold of . (figures and ) . the above scenarios are just few from many possible (e.g., see the alternative species groupings in figure b -d) and illustrate the current uncertainty about filovirus classification. the three-species scenario seems to be conceivable when comparing the filovirus ped distribution with that of the well-sampled family picornaviridae with up to available sequences per species (more than , sequences in total distributed among species clusters) [ ] . in the high-sampling case of picornaviruses, no ped values with zero frequency are observed which suggests that the current sampling of filovirus genome sequences may strongly underestimate the natural genetic diversity in the family. expanded virus sampling might also lead to a better differentiation of filovirus proteins by evolutionary criteria. currently, all seven proteins are conserved family-wide among known filoviruses, which may change for this family in the future when most diverged viruses are separated by (much) larger genetic distances, as we already observe for picornaviruses and many other families. this development would also affect the choice of proteins by demarc and, consequently, the resulting classification. furthermore, the ped threshold values are likely to change in the future, even if the underlying virus clusters will be stable, given the large ped ranges they currently represent ( figure b) . thus, a definite decision about number and virus composition of filovirus species and genera as well as stable demarcation thresholds will only be possible if the sampling of filovirus genome sequences is expanded in the future. we note that the use of pairwise sequence similarities is becoming increasingly popular for assisting decision-making in virus taxonomy (see the introduction for literature). on the other hand, there is the current paradigm that the use of a single (e.g., genetic) criterion is insufficient for the demarcation of viral taxa [ ] . in our opinion, a fully genetics-based classification provides a promising and meaningful foundation for virus taxonomy. indeed, the genome is the principal carrier of genetic information and heredity; and it was already acknowledged that virus taxonomy should reflect the evolutionary history of viruses [ ] which can be reconstructed from genetic data. however, analyzing the evolutionary record in genomes remains a challenging task with many parameters to define and is dependent on the amount of available genetic information (see above). consequently, the technical implementation and associated choices made during an analysis (e.g., using a relatively simplistic measure of pairwise distances) may affect the quality of genetics-based classification. different directions of future research efforts are conceivable, ranging from the development of improved evolutionary models for the calculation of genetic distances [ ] to entirely different techniques of utilizing the genetic information for virus classification [ , ] . as the development and application of demarc illustrates, this line of research offers a possibility of tackling the virus classification problem in a systematic manner. in the demarc framework, an upper limit on intra-species genetic divergence is imposed on loci encoding the family-wide conserved proteins. consequently, when there is a strong support for species, viruses of the same species are genetically separated from viruses outside the species in these loci. for picornaviruses with their rna genomes of positive polarity (ssrna+), this separation may be promoted by mutation and limited through homologous recombination [ , , ] , as we argued [ ] . recombination among ssrna− viruses was estimated to be generally rare [ ] and this was explained by major differences in the replication cycle (due to the negative polarity of genomes) compared to ssrna+ viruses, which limits the template-switching ability of the rdrp through rapid packaging of the genomic rna with ribonucleoproteins [ ] . however, homologous (intrasegmental) recombination can occur as was found for different ssrna− viruses [ ] [ ] [ ] including the prototype ebolavirus, ebola virus [ ] . the frequency with which both ssrna+ and ssrna− viruses recombine in nature remains to be shown but it may not be uncommon [ ] , and currently available recombination detection tools were shown to underestimate this frequency in certain situations [ ] . ultimately, both the rate of homologous recombination among rna viruses in nature and the model of genetic separation of virus speciation should be probed experimentally. ped values, which estimate the average number of amino acid substitutions per site between each pair of viruses. a mixture model (red curve) was fitted to the ped distribution and used to calculate a threshold support measure (tsm, green). for details on threshold delineation see [ ] . a threshold candidate (peak in the tsm measure) may be used to group the viruses into clusters in which virus pairs with a ped not exceeding the threshold join the same cluster. the three candidates with highest tsm scores were used to hierarchically group the filoviruses at three levels comprising seven, three, and two clusters, respectively. each of the thresholds is located within a continuous ped range for which no ped values are observed with the current filovirus sampling (striped background shading). ped values in these ranges (if sampled in the future) could be associated with either the classification level delimited by the respective threshold (intra-cluster distances) or with the next higher level (inter-cluster distances). the actual ped values of the thresholds are thus uncertain (bright-colored horizontal bars and question marks); for simplicity we selected the values ( . , . , and . ) that correspond to the smallest value within the respective ped range. (b) the change in the number of derived clusters with respect to the value of the ped threshold candidate is shown. for each threshold candidate a continuous ped range with optimal clustering cost of zero (no intra-cluster ped values exceed the threshold) is highlighted in green. the six threshold candidates considered in this study show the following ped ranges (from left to right): . - . , . - . , . - . , . - . , . - . , and . - . . the three threshold candidates used in (a) are indicated by colored arrows; three alternative ped thresholds for the species level are shown by black arrows. note that another clustering ( clusters) with zero cost was not considered in this study because of the marginal ped range and low tsm support of the associated threshold (ped of . ). genetics-based classification of filoviruses and alternative species groupings. a quadrant is used to visualize the classification of the filoviruses with three hierarchical levels. the axes indicate intervirus genetic divergence (as ped) which increases linearly from the perimeter of the quadrant (zero ped) to its origin (maximum ped of . ). the three classification levels are highlighted using three basic colors (orange, blue, and purple). each color exists in two shadings that highlight the limit on expected intragroup genetic divergence according to a distance threshold (soft shading) and the maximum observed intragroup genetic divergence (bright shading) of a cluster. (a) the genetics-based filovirus classification using the three top-ranked ped thresholds. it comprises seven, three, and two clusters, respectively, at the three hierarchical levels. the seven orange clusters of the lowest level correspond to the official or tentative ictv species of the family filoviridae [ ] and are indicated by names. the three blue clusters correspond to the official or tentative ictv genera of the family filoviridae (from left to right: "cuevavirus", ebolavirus, and marburgvirus); the two purple clusters correspond to supra-generic taxa currently not recognized in the ictv filovirus classification. outside the quadrant, the relative density of virus sampling per ictv species is shown as gray shadings from low (light) to high (dark) sampling, which is in the range of to . (b) an alternative classification with eight instead of seven clusters at the lowest level. it was derived by using the alternative species threshold at ped = . . (c) an alternative classification with six instead of seven clusters at the lowest level. it was derived by using the alternative species threshold at ped = . . (d) an alternative classification with five instead of seven clusters at the lowest level. it was derived by using the alternative species threshold at ped = . . figure . intragroup genetic divergence of filoviruses. box-and-whisker graphs are used to plot level-specific ped distributions for the seven clusters of the lowest classification level (bright-shaded orange, blue, and purple). the seven orange clusters correspond to the official or tentative ictv species of the family filoviridae [ ] and are indicated by names; virus sampling per ictv species is shown in brackets. the combined ped distributions of clusters of the second and third level are shown as gray box-and-whisker graphs. the expected range of level-specific ped values, bordered by two distance thresholds, is indicated by light-shaded background colors. the respective part of the ped distribution covering the full range of intragroup ped values of the three levels is show at the bottom. the ictv species are grouped vertically according to a maximum likelihood phylogeny shown at the left. internal nodes of the tree that correspond to ictv species are collapsed (triangles); all of them have a bootstrap support value of . the seven filovirus proteins that are conserved in all known members of the family (np, vp , vp , gp , , vp , vp , and l) were aligned separately at the amino acid level using the program muscle version . [ ] followed by manual correction. the seven protein alignments were concatenated to form a single alignment of , positions with a gap content of . %. to estimate the genetic similarity between virus pairs, ped values were calculated on this concatenated alignment using the program tree-puzzle version . [ ] ; the wag amino acid substitution matrix was applied [ ] . proteins that are not conserved family-wide, like sgp and ssgp of ebolaviruses and "cuevaviruses" or certain hypothetical proteins encoded by the anti-sense genomic rna, were not included in the calculation of ped values. for these proteins, an accurate estimation of genetic divergence may be approached only for the selected filoviruses that encode these proteins. the distribution of all ped values was partitioned into intra-rank and inter-rank ranges using a systematic approach implemented in demarc [ ] . this partitioning is achieved through the inference of ped thresholds below which two viruses are grouped together. we refer to the resulting virus groups as "clusters" in the context of genetic classification by demarc. a cluster might correspond to a viral taxon officially recognized by ictv. a derivative of the multiple alignment used for ped calculation, from which strongly conserved blocks [ ] (in total , alignment positions, . %) have been extracted by bagg [ ] , formed the dataset for unrooted tree reconstruction by phyml version . [ ] ; the wag amino acid substitution matrix was applied [ ] ; support for internal nodes was obtained through a non-parametric bootstrap analysis with replicates. demarc offers a systematic and quantitative framework for virus classification that utilizes genome sequences, the only information available for a growing number of viruses. the striking agreement on species and genus taxa between the demarc-mediated filovirus classification of this study and the taxonomy of the family filoviridae could be considered a cross-validation for both. however, we note that this classification is one of many equally strongly supported classifications, as demarc identified in total six potential taxon levels. each of these taxon levels is associated with a large continuous range of ped values that are not sampled (yet). consequently, we conclude that the current coverage of the natural genetic diversity of filoviruses is limited and needs to be considerably expanded, also concerning hosts not 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accepting gaps generator) a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood we thank igor sidorov, alexander kravchenko, and dmitry samborskiy for expert administration of the viralis platform. this research was partially supported through the european union seventh the authors declare no conflict of interest. key: cord- -fvirvpyl authors: srinivasan, suhas; cui, hongzhu; gao, ziyang; liu, ming; lu, senbao; mkandawire, winnie; narykov, oleksandr; sun, mo; korkin, dmitry title: structural genomics of sars-cov- indicates evolutionary conserved functional regions of viral proteins date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: fvirvpyl during its first two and a half months, the recently emerged novel coronavirus, sars-cov- , has already infected over one-hundred thousand people worldwide and has taken more than four thousand lives. however, the swiftly spreading virus also caused an unprecedentedly rapid response from the research community facing the unknown health challenge of potentially enormous proportions. unfortunately, the experimental research to understand the molecular mechanisms behind the viral infection and to design a vaccine or antivirals is costly and takes months to develop. to expedite the advancement of our knowledge, we leveraged data about the related coronaviruses that is readily available in public databases and integrated these data into a single computational pipeline. as a result, we provide comprehensive structural genomics and interactomics roadmaps of sars-cov- and use this information to infer the possible functional differences and similarities with the related sars coronavirus. all data are made publicly available to the research community. that of the severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) outbreaks combined [ ] [ ] [ ] . in spite of the instantaneous reaction by the scientific community and extensive worldwide efforts to address this health crisis, vaccines may be months and even years away [ , ] . for instance, a phase i trial for a vaccine that treats sars was announced in december , two years after the disease outbreak [ ] . additionally, a vaccine against mers, another infectious outbreak of the related coronavirus that emerged in , was patented in , with phase i trials introduced in the same year [ , ] . nevertheless, in the past two decades, a massive amount of work has been done to understand the molecular basis of the coronavirus evolution and infection, develop effective treatment in forms of both vaccines and antiviral drugs, and propose efficient measures for viral detection and prevention [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . structures of many individual proteins of sars-cov, mers-cov, and related coronaviruses, as well as their biological interactions with other viral and host proteins have been explored along with the experimental testing of the anti-viral properties of small-molecule inhibitors [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, experimental investigation of the same scale for sars-cov- may take the research community years to obtain. can it be facilitated? the answer lies in the use of the modern bioinformatics methods that can drastically streamline knowledge discovery by quickly providing important insights about possible molecular mechanisms behind infection, pinpointing likely protein targets for the anti-viral treatments, and predicting the efficacy of the existing antivirals developed for other coronaviruses. by leveraging previously known information on genome sequences as well as protein structures and functions, bioinformaticians have been successfully assisting virologists by structurally characterizing proteins of novel viruses, determining the evolutionary trajectories, identifying interactions with host proteins, and providing other important biological insights. in particular, a plethora of results has been achieved through comparative, or homology, modeling principles [ , ] . in addition to the global structural genomics, an initiative that focuses on determining the d structures of individual proteins on a genome scale [ ] , as well as to the specific efforts aimed at rapid structural characterization of proteins in emerging viruses [ ] [ ] [ ] [ ] , multiple works have used comparative modeling to predict the structures of protein-protein interaction complexes [ ] [ ] [ ] , facilitate structure-based drug discovery [ , , ] , infer protein functions [ ] , determine the macromolecular interaction network [ ] [ ] [ ] [ ] , and provide molecular insights into the viral evolution [ ] [ ] [ ] . here, using an integrated bioinformatics approach, we provide the first comprehensive structural genomics and interactomics analysis of the sars-cov- . the structural information on the individual sars-cov- proteins and their interactions with each other and with human proteins allows us to accurately determine the putative functional sites. these functional sites, combined with an evolutionary sequence analysis of sars-cov- and the closely related human sars-cov and bat coronavirus proteomes, provide us with a structure-based perspective of the evolutionary diversity of sars-cov- , and allow estimation of how similar the function of sars-cov- virus is when compared with sars-cov. consequently, we can forecast how likely the antibodies and candidate ligands that are efficient in inhibiting the sars-cov functions will be efficient in doing the same for sars-cov- . the goal of this work is to identify the evolutionary differences between sars-cov- and the closest coronavirus species, human sars-cov and bat sars-like coronavirus, and to predict their possible functional implications. to do so, we structurally characterized individual proteins as well as intra-viral and human-virus protein complexes, extracted the information on their interaction interfaces and ligand binding, and superposed the evolutionary difference and conservation information with the binding information. specifically, our integrative computational pipeline included the following five steps. first, for each of the candidate sars-cov- proteins a set of sequentially similar coronavirus proteins was determined and aligned. second, structural models of sars-cov- proteins were obtained using template-based, or homology, modeling. third, the protein-protein interaction complex structures of sars-cov- proteins interacting with each other and/or with the human proteins were determined using a multi-chain comparative modeling protocol. fourth, the protein-binding sites were extracted from the obtained models of protein-protein interaction complexes, and protein-ligand binding sites were extracted from the evolutionarily close coronavirus protein-ligand templates and mapped to the relevant structural models of sars-cov- through structural alignment. fifth, the information on the evolutionary differences and conservations between the protein sequences of sars-cov- and the related coronaviruses were extracted from the above protein sequence alignments and mapped onto the structural models of the sars-cov- proteins to determine if the proteinand ligand-binding sites were functionally conserved. lastly, a joint human-virus and virus-virus interactome was constructed through homology and further analyzed. all the obtained models and sequence alignments were made publicly available to the research community. available sequences for protein candidates ws, worf a, we, wm, worf , worf a, worf b, worf , wn, and worf were extracted from the ncbi virus repository [ ] (collected on january ) and then used in the sequence analysis and structural modeling (see supplementary materials containing sequence alignment files of each protein and the functional mappings of protein and ligand binding sites). the uniprot blast-based search was performed for each of the proteins using default parameters. from the results of each search, the final selection was done based on the pairwise sequence identity (> %) as well as the evolutionary relationship (coronaviridae family). each of sars-cov- proteins was then aligned with the corresponding coronavirus proteins using a multiple sequence alignment method clustal omega (embl-ebi, cambridge, uk) [ ] . the structure of each protein was determined using a single-template comparative modeling protocols with the modeller software (ucsf, ca, usa) [ ] . first, the template for each protein sequence was identified using a psi-blast search in the protein data bank (pdb) (research collaboratory for structural bioinformatics) [ ] . in general, a structural template with the highest sequence identity was selected out of those that covered at least residues of the target sequence with at least % sequence identity. the polyprotein worf ab was first split into putative proteins based on its alignment with the human sars-cov polyprotein, with each protein then independently searched against pdb. in total, structural templates for proteins were chosen ( table , figures - ) . in some cases, several independent templates, each covering an individual protein domain of a target sars-cov- protein, were selected. the obtained template was used in the comparative modeling protocol, generating five models. each model was assessed using the dope statistical potential [ ] ; the best-scoring model was selected as a final prediction. to model a protein-protein interaction complex, a multi-chain modeling protocol was used [ ] . specifically, we aligned the corresponding pairs of homologous proteins and combined them into a single alignment where the individual chains were separated by "/" symbol. the alignment was used as an input together with the multi-chain structural template of a homologous complex. in the case of a viral-human interaction (these were the only virus-host structural templates found), the human protein remained the same in the alignment. in total, structural templates of protein complexes involving homologs of sars-cov- proteins were retrieved ( figure ). similar to the single-protein protocol, five candidate models were generated for each complex conformation, and each model was assessed using the dope statistical potential, following by selection of the best-scoring model as a final prediction. we next extracted protein-and ligand-binding sites and mapped them onto the models of sars-cov- proteins. for protein binding sites, the obtained modeled structures of protein complexes that involved sars-cov- proteins were considered. for each sars-cov- protein in a protein-protein interaction complex, we identified all binding residues that constituted its protein-binding site. given an interaction between two proteins, a residue on one protein was defined as a protein binding site residue if there was at least one pair of atoms, one from this residue and another from a residue in the second protein, with van der waals (vdw) surfaces not farther than . Å from each other. using this definition, the binding sites were identified with ucsf chimera (ucsf, ca, usa) [ ] . the ligand binding sites were identified and mapped using a different protocol-we relied on the default definition of protein-ligand binding site residues from pdb d ligand view, since this standard was widely accepted. once all ligand binding site residues were identified for a protein from a related coronavirus, the residues were mapped onto the surface of the corresponding sars-cov- protein, guided by a structural alignment between the two proteins, which put residues from both proteins into a one-to-one correspondence. next, we leveraged the obtained protein homology information to predict and map all possible intra-viral and virus-host protein interactions at the systems level. since the sars-cov- genome exhibited substantial similarity to the sars-cov genome [ ] and proteome [ ] , we hypothesized that many of the interactions observed in the sars-cov proteome would be preserved in the sars-cov- proteome too, unless the corresponding binding sites were affected. the information in the interactome would help one understand the global mechanistic processes of the viral molecular machinery during the viral infection, survival within the host, and replication. with this knowledge, one could discern the protein interactions that were crucial for transmission and replication. these interactions could then be potential candidates for inhibitory drugs [ ] . furthermore, using the network biology approach, one could identify hubs and bottlenecks, new to sars-cov- , that could again be targeted by the antiviral drugs. for this purpose, we created a comprehensive integrated sars-cov interactome that consisted of both intra-viral and virus-host interactions. the sars-cov intra-viral interactome was created using the published data where the sars-cov orfeome was cloned, and a genome-wide analysis of viral protein interactions was performed through yeast-two-hybrid (y h) matrix screens [ ] . the y h matrix screen was summarized by combining interactions in one direction, both directions and self-interactions. we also included intra-viral interactions gathered from the literature review [ ] . the aggregated intra-viral interaction network consisted of proteins and unique interactions. we then constructed the sars-cov-host interactome through the published y h interaction data [ ] . we also included virus-host interactions mined from a literature survey of abstracts [ ] . the curated virus-host interaction network consisted of proteins, including host proteins, and unique virus-host interactions. next, to create an integrated network, the two individual networks were imported in cytoscape (cytoscape consortium) [ ] and merged to form a unified interactome, representing both intra-viral and virus-host interactions. finally, we included our predictions from structural modeling of sars-cov- intra-viral and virus-host interactions, surveyed recent literature on predicted sars-cov- interactions and annotated them accordingly in the unified interactome. the unified interactome consisted of proteins ( host proteins) and unique interactions. in addition, the networks were pruned for duplicate edges, and a network topology analysis was performed to compute a set of summary statistics (degree distribution, clustering coefficient, and other important characteristics) that characterized the three networks. finally, the putative interactions were annotated based on the extent to which the protein binding sites of the sars-cov- proteins were altered, compared to their sars-cov homologs. based on the evolutionary conservation analysis of the putative protein binding sites extracted from the modeled complexes, some interactions were annotated as potentially disrupted. the recently sequenced genomes of sars-cov- strains combined with the comparative analysis of the sars-cov genome organization and transcription allowed us to construct a tentative list of gene products [ ] . it was suggested that sars-cov- had predicted non-structural proteins (referred to as wnsp -wnsp here) constituting a polyprotein (worf ab), followed by (at least) downstream open reading frames (orfs): surface glycoprotein (or spike), orf a, orf b, envelope, membrane, orf , orf a, orf b, orf , nucleocapsid, orf a, orf b, and orf , which we refer in this work to as ws, worf a, worf b, we, wm, worf , worf a, worf b, worf , wn, worf a, worf b, and worf , respectively. the three viral species whose proteins shared the highest similarity were consistently the same: human sars coronavirus (sars-cov), bat coronavirus (btcov), as well as another bat betacoronavirus (btrf-betacov). searching against uniprot database (uniprot consortium) [ ] resulted in matches for the polyprotein (worf ab), all four structural proteins (ws, we, wm, and wn), and six orfs (worf a, worf , worf a, worf b, worf , and worf ), also referred to as the accessory proteins. the closest protein matches from uniprot shared sequence identity with the related sars-cov- proteins as high as % (with worf ab and wn) and as low as % (with worf ) (supplementary table s ). the majority of differences were single-residue substitutions spread across the protein sequence (see multiple sequence alignment files for all proteins in supplementary materials). perhaps the most profound differences lie in the sequences of the multi-domain protein wnsp and surface protein ws: our analysis revealed that, compared to related coronavirus proteins, the two proteins had large sequence inserts (multiple sequence alignments for both proteins can be found in supplementary materials, files worf ab_matches.aln and ws_matches.aln). in particular, wnsp had a novel large ( - res., region - of worf ab, depending on the alignment method) insert between its two putative functional domains, which are homologous to the n-terminal domain and adenosine diphosphate ribose " phosphatase (adrp, recently renamed to macrodomain- or mac- ) of sars-cov [ , ] (supplementary materials, file worf ab_matches.aln). interestingly, the closest matching peptide was found in c-jun-amino-terminal kinase-interacting protein (seq. identity is %) of labrus bergylta, a species of marine ray finned fish. being significantly more diverse than the other three structural proteins, ws was found to have inserts ( - res.) that seemed unique to sars-cov- and two additional inserts shared with human sars-cov proteins (supplementary materials, file ws_matches.aln). the unusually low conservation of orf , orf , and surface proteins between sars-cov- and human sars-cov, bat coronavirus btcov, as well as another bat betacoronavirus, btrf-betacov, prompted us to perform an expanded search for orf homologs using the blatp tool of ncbi blast against a large non-redundant protein sequence repository (nr) [ ] . our search resulted in three new homologs of orf not reported in uniprot that were originated from three different isolates of bat sars-like coronavirus: bat-sl-covzc (genbank id: mg , collected in ), bat-sl-covzxc (genbank id: mg , collected in ), and ratg (genbank id: mn , collected in ). the protein sequences from these isolates shared a striking similarity with worf , unseen in other strains before: the sequence identities between each of these three homologs and worf ranged between % and %. further analysis showed that the proteomes of these isolates shared even higher sequence identity with the other proteins of sars-cov- : from . % to % for and isolates, and even higher, . %- % for the isolate (supplementary table s ). in spite of the significant similarity of the three isolates to sars-cov- , important differences were observed. first, similar to other viruses, the and isolates did not have the four sequence inserts that were found in ws. second, neither of the two isolates had the large insert between the two domains of wnsp from worf ab described above. on the contrary, the isolate had both, the four sequence inserts in its surface protein, matching those in ws, and the large insert in nsp , although the sequence of the large insert is different from that one in wnsp . next, as a result of a comprehensive comparative modeling effort, we were able to structurally characterize individual proteins, including non-structural proteins of worf ab (wnsp , wnsp , wnsp , wnsp , wnsp , wnsp , wnsp , wnsp , wnsp , wnsp , wnsp , wnsp ), three structural proteins (we, wn, and ws), as well as one orf (worf a). for two proteins, wnsp and wn, multiple individual domains were modeled (figure , figure ). the templates for the majority of the models were the homologous protein structures from other coronaviruses, with a high target-to-template sequence similarity (seq. ids: - %, except for orf ). n-terminal and c-terminal domains of wn corresponded to n-terminal rna-binding domain and c-terminal dimerization domain of sars-cov, respectively. the modeled domains - of wnsp corresponded to ( ) (figure ) . a previously identified transmembrane domain of sars nsp was mapped to the sequence of wnsp , but could not be modeled due to the lack of a template structure. the structural analysis of the modeled proteins combined with the sequence conservation analysis revealed several findings. first, we found that the mutated residues tended to locate on the protein's surface, supporting previous observations in other families of rna viruses that the core residues of viral proteins were more conserved than the surface residues [ , , ] . furthermore, in a substantial number of proteins, distributions of mutated positions exhibited spatial patterns, with groups of mutations found to form clusters on the protein surfaces ( figure ). these obtained models were then used as the reference structures to map and analyze the protein-binding and ligand-binding sites. next, using comparative modeling, we structurally characterized protein interaction complexes for both intra-viral interactions (homo-and hetero-oligomers) and host-viral interactions, where the host proteins were exclusively human. in total, we obtained structural models for homo-oligomeric complexes, three hetero-oligomeric complexes, and eight human-virus interaction complexes including multiple conformations (figures and ) . the intra-viral hetero-oligomeric complexes included exclusively the interactions between the non-structural proteins (wnsp , wnsp , wnsp , wnsp , wnsp , and wnsp ). the modeled host-viral interaction complexes included three types of interactions: non-structural protein wnsp (papain-like protease, plpro, domain) interacting with human ubiquitin-aldehyde, surface protein ws (in its trimeric form) interacting with human receptor angiotensin-converting enzyme (ace ) in different conformations, as well as the same protein ws interacting with several neutralizing antibodies. based on the obtained models, the protein interaction binding sites were extracted and analyzed with respect to their evolutionary conservation. the analysis of the evolutionary conservation of protein binding sites revealed several patterns. first, we found that all protein binding sites of non-structural proteins involving in the intra-viral heteromeric complexes, wnsp -wnsp -wnsp , wnsp -wnsp , and wnsp -wnsp , were either fully conserved or allowed at most one mutation on the periphery of the binding region, in spite of the fact that each protein had multiple mutations on its surface ( figure a , and file worf ab_nsp _nsp _nsp _nsp _nsp _nsp _pbs_mapped.pdf in supplementary materials). furthermore, we observed the same behavior when analyzing the interaction between the papain-like protease plpro domain of wnsp and human ubiquitin-aldehyde ( figure b , and file worf ab_wnsp _domain _human_pbs_mapped.pdf in supplementary materials): the only two mutated residues were located on the border of the binding region and thus were unlikely to disrupt the protein-protein interaction. protein ws presented a striking contrast to the majority of sars-cov- proteins due to its heavily mutated surface ( figure , supplementary materials: ws_matches.aln and ws_human_pbs_mapped.pdf in supplementary materials) . first, the four novel sequence inserts and two inserts shared with the closest strains were expected to affect the protein's function. interestingly, three of the novel inserts were located in the first ntd domain, while the fourth one was located immediately before the s cleavage site and inside the homo-trimerization interaction interface. while the rbd domain of ws was not affected by those inserts, it was the most heavily mutated region of ws with potentially altering functional effects on the interactions with the human ace receptor and monoclonal antibodies mab [ ] and mab r [ ] ( figure b ). the ntd domain had been considered a target of another antibody, ab g , previously shown to work in mers-cov [ ] . however, based on the structural superposition of the ntd domain, it seemed likely that the potential interaction would be disrupted by the novel sequence inserts to ws. finally, the analysis of seven ligand binding sites (lbs) for multiple candidate inhibitors previously identified, with their interactions structurally resolved, for four proteins in sars-cov and mers-cov, showed that many of the lbs were intact in the corresponding sars-cov- proteins, such as wn, wnsp , wnsp , and wnsp ( figure and supplementary materials: wn_lbs_mapped.pdf, wnsp -domain _lbs_mapped.pdf, wnsp -nonsars_lbs_mapped.pdf, wnsp -sars_lbs _mapped.pdf, wnsp -sars_lbs _mapped.pdf, wnsp _lbs_mapped.pdf, and wnsp _lbs_mapped.pdf). for wnsp , the lbs for several inhibitors were mutated, while a co-localized binding site for another ligand was intact. after constructing the individual networks of intra-viral interactions and virus-host interactions, they were merged to form a unified network of sars-cov interactions, with the hypothesis that most interactions would be conserved in sars-cov- ( figure ). the network analysis of all three networks showed that unifying the intra-viral and virus-host networks reduced the number of disconnected components (islands) in the sars-cov-host interactome ( table ). the clustering coefficient and average node degree were both higher in the unified interactome compared to the virus-host interactome. table . network parameters of the sars-cov intra-viral, virus-host and unified networks. the table shows topological statistics for the three networks. among the many computed statistics, the shown parameters include the number of nodes and edges in the networks, the average degree, number of components (independent networks), diameter (maximum shortest path), and clustering coefficient. the network topology of the unified interactome indicated the presence of several viral hubs for the structural, non-structural, and accessory proteins ( figure ). the viral proteins exclusively formed all the hubs, with a few of specific interest. orf b was one of the largest hubs in the network thought to play a secondary role only in intra-viral interactions and not necessary for replication [ ] . however, a recent study showed that orf b hindered immunity by targeting the mitochondria and limited the host cell responses [ ] . nsp was the other major hub, which interacted with other replicase proteins, including two other hubs, nsp and nsp , which together played a crucial role in replication [ ] . another crucial non-structural protein was nsp , also present in the sars-cov- , which was known to be an inhibitor of the innate immune response. the host interaction partners of nsp modulated the calcineurin/nfat pathway that played an important role in immune cell activation [ ] . overexpression of nsp was associated with immunopathogenecity and long-term cytokine dysregulation as observed in severe sars cases. additionally, inhibition of cyclophilins/immunopilins (host interaction partners) by cyclosporine a (cspa) blocked the replication of covs of all genera. there was an important interaction between the sars-cov spike protein (s) and the host angiotensin-converting enzyme (ace ), as it was associated with cross-species and human-to-human transmissions. the same interaction was also inferred from structural modeling between sars-cov- ws and ace . surprisingly, it was recently established that the interaction was preserved [ ] , in spite of a number of mutations in the receptor binding site of ws. similar to sars-cov [ ] , ws protein in sars-cov- was expected to interact with type ii transmembrane protease (tmprss ) [ ] and was likely to be involved in the inhibition of antibody-mediated neutralization. thus, ws remained an important target for vaccines and drugs previously evaluated against sars and mers, while a neutralizing antibody targeting the ws protein could provide passive immunity [ ] . in addition, there were seven interactions from sars-cov determined by structural characterization of the protein complexes that were predicted to be either conserved or potentially disrupted in sars-cov- (green edges in figure ). an important target for vaccines was the surface (s) protein which was evaluated in sars-cov and mers-cov, with the idea that a neutralizing antibody targeting ws protein could provide passive immunity for sars-cov- [ ] . we also structurally modeled interactions between the sars-cov- ws protein and three human monoclonal antibodies that were previously studied in sars-cov for immunotherapy and mapped the information about the evolutionary conserved and diverse surface residues. we found that two interactions of ws, with mab [ ] and mab r [ ] , were likely to be disrupted due to the heavily mutated binding sites while another interaction, with ab g [ ] , was likely to be disrupted due to a novel insert into the ws sequence. together, these findings may provide structure-informed guidelines in the search for potential antibody treatment. this work provides an initial large-scale structural genomics and interactomics effort towards understanding the structure, function, and evolution of the sars-cov- virus. the goal of this computational work is two-fold. first, by making the structural road map and the related findings fully available to the research community, we aim to facilitate the process of structure-guided research where accurate structural models of proteins and their interaction complexes already exist. second, by providing a comparative analysis between the new virus and its closest relatives from the perspective of protein-and ligand-binding, we hope to help experimental scientists in their deciphering of the molecular mechanisms implicated in infection by the new coronavirus as well as in vaccine development and antiviral drug discovery. through integrating the information on structure, function, and evolution in a comparative study of sars-cov- and the closely related coronaviruses, one can make several preliminary conclusions. first, the extended peptide sequence newly introduced to wnsp between two structurally and possibly functionally independent domains of this protein, might act as a long inter-domain linker, thus extending the conformational flexibility of this multi-domain protein. second, the presence of the four novel inserts and one highly variable region of the surface protein ws and the analysis of this large-scale sequence change with respect to intra-viral and viral-host interactions lead us to conclude that these inserts might have structural impact on the homo-trimeric form of the protein as well as impact the functions carried out by the ntd domain. third, the structurally modellable repertoire of the sars-cov- proteome also points to the interesting targets for structural biology and hybrid methods. for instance, the whole structures of the multi-domain proteins wn and wnsp could be resolved by integrating comparative models of the individual domains with the lower-resolution techniques that cover the entire protein, such as cryogenic electron microscopy (cryoem). the structure of wnsp is especially interesting because of the presence of the novel peptide introduced between the two structural domains of the protein. the evolutionary analysis of the protein-and ligand-binding sites mapped on the surfaces of sars-cov- may provide new insights into the virus functioning and its future treatment. the % or near % evolutionary conservation of the protein binding sites on the surfaces of non-structural proteins wnsp , wnsp , wnsp , wnsp , wnsp , and wnsp that correspond to the intra-viral interactions for three complexes is consistent with our previous observations that the intra-viral interactions are significantly more conserved than viral-host interactions [ , , ] . however, the near-perfect conservation of the human ubiquitin-aldehyde protein binding site on the surface of wnsp is rather intriguing, suggesting the critical role of this 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(http://korkinlab.org/wuhan). key: cord- -t s s wo authors: gralinski, lisa e.; menachery, vineet d. title: return of the coronavirus: -ncov date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: t s s wo the emergence of a novel coronavirus ( -ncov) has awakened the echoes of sars-cov from nearly two decades ago. yet, with technological advances and important lessons gained from previous outbreaks, perhaps the world is better equipped to deal with the most recent emergent group b coronavirus. the third zoonotic human coronavirus (cov) of the century emerged in december , with a cluster of patients with connections to huanan south china seafood market in wuhan, hubei province, china. similar to severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) infections, patients exhibited symptoms of viral pneumonia including fever, difficulty breathing, and bilateral lung infiltration in the most severe cases [ ] . news reports of patients with an unknown pneumonia were first identified on st december with the wuhan municipal health commission saying they were monitoring the situation closely ( figure ). on st january , the seafood market was closed and decontaminated while countries with travel links to wuhan went on high alert for potential travelers with unexplained respiratory disease. after extensive speculation about a causative agent, the chinese center for disease control and prevention (cdc) confirmed a report by the wall street journal and announced identification of a novel cov on th january [ ] . the novel cov ( -ncov) was isolated from a single patient and subsequently verified in additional patients [ ] . while not yet confirmed to induce the viral pneumonia, -ncov was quickly predicted as the likely causative agent. the first sequence of -ncov was posted online one day after its confirmation on behalf of dr. yong-zhen zhang and scientists at fudan university, shanghai [ ] . subsequently, five additional -ncov sequences were deposited on the gsaid database on th january from institutes across china (chinese cdc, wuhan institute of virology and chinese academy of medical sciences & peking union medical college) and allowed researchers around the world to begin analyzing the new cov [ ] . by th january, there were confirmed cases in china and importantly, three exported cases of infected travelers who were diagnosed in thailand ( ) and japan ( ) [ ] . the sequences of these exported cases and several additional -ncov isolated in china have also been deposited on the gsaid database [ ] . diagnostic tests have subsequently been developed and some are being used on suspect cases identified in other locations including vietnam, singapore, and hong kong [ ] . to date there have been twenty-six fatalities associated with -ncov infection, many of these cases had significant co-morbidities and were older in age (> ). a range of disease has been observed highlighted by fever, dry cough, shortness of breath, and leukopenia; patients have included mild cases needing supportive care to severe cases requiring extracorporeal membrane oxygenation; however, compared to sars-cov ( % mortality) and mers-cov ( % mortality), the -ncov appears to be less virulent at this point with the exception of the elderly and those with underlying health conditions. initial monitoring of case close contacts had not revealed any further -ncov cases. however, modeling analysis based on official case numbers and international spread suggested that there may be cases going undetected [ ] . on th january, these fears were seemingly confirmed as an additional cases were added from further surveys raising the total in wuhan to infected patients [ ] . among the total cases in wuhan, remained in hospitals, mostly with mild symptoms, in serious condition, and in critical condition. the expanded numbers and extended range of onset dates ( december - january ) suggested likely human to human transmission or ongoing transmission from a market or other primary sources. on th january, the outbreak was further expanded to other parts of china (beijing, shanghai, & shenzhen) as well as another exported cases to south korea. as of january , the total case number has expanded to at least total cases with deaths across provinces in china and exported cases in countries [ ] . public health authorities have quarantined travel from wuhan to limit the spread of the virus and reports indicate other chinese cities have also been isolated [ ] . with the heavy travel season for lunar new year underway in asia, major concerns exist for the -ncov outbreak to continue and spread. the source of the -ncov is still unknown, although the initial cases have been associated with the huanan south china seafood market. while many of the early patients worked in or visited the market, none of the exported cases had contact with the market, suggesting either human to human transmission or a more widespread animal source [ ] . in addition to seafood, it is reported on social media that snakes, birds and other small mammals including marmots and bats were sold at the huanan south china seafood market. the who reported that environmental samples taken from the marketplace have come back positive for the novel coronavirus, but no specific animal association has been identified [ ] . an initial report suggested that snakes might be the possible source based on codon usage [ ] , but the assertion has been disputed by others [ ] . researchers are currently working to identify the source of -ncov including possible intermediate animal vectors. a zoonotic reservoir harkens back to the emergence of both sars-and mers-cov. sars-cov, the first highly pathogenic human cov, emerged in with transmission from animals to humans occurring in wet markets. surveillance efforts found sars-cov viral rna in both palm civets and raccoon dogs sold in these wet markets [ ] ; however, sars-cov was not found in the wild, suggesting that those species served as intermediary reservoir as the virus adapted to more efficiently infect humans. further surveillance efforts identified highly related covs in bat species [ ] . more recent work has demonstrated that several bat covs are capable of infecting human cells without a need for intermediate adaptation [ , ] . additionally, human serology data shows recognition of bat cov proteins and indicates that low-level zoonotic transmission of sars-like bat coronaviruses occurs outside of recognized outbreaks [ ] . mers-cov is also a zoonotic virus with possible origins in bats [ , ] , although camels are endemically infected and camel contact is frequently reported during primary mers-cov cases [ ] . for sars-cov, strict quarantine and the culling of live markets in se asia played a major role in ending the outbreak. with the cultural importance of camels, a similar approach for mers-cov was not an option and periodic outbreaks continue in the middle east. these lessons from sars and mers highlight the importance of rapidly finding the source for -ncov in order to stem the ongoing outbreak. with limited patient data, it is difficult to make robust declarations about populations that may be most susceptible to -ncov. however, disease severity following sars-and mers-cov corresponded strongly to underlying host conditions including age, biological sex, and overall health [ ] . early patient reports from -ncov find similar trends. severe illness with -ncov has been associated with elderly patients (> years old), including twenty-six lethal cases. these findings correspond to increased severity and death in people over the age of following both sars and mers-cov infection [ , ] . similarly, the underlying health of the patient likely plays a critical role in overall susceptibility. for the -ncov, limited comorbidity data is available; however, the twenty-six patients that have succumbed to the novel cov had significant health conditions including hypertension, diabetes, heart and/or kidney function issues that may have made them more susceptible. for the mers-cov outbreak, smoking, hypertension, diabetes, cardiovascular disease, and/or other chronic illnesses have been present in the majority of deaths and correspond to findings in animal models [ ] . the results indicate vigilance is necessary for these vulnerable patients following -ncov infection. the rapid sequencing of the nearly , nucleotide -ncov genome by dr. zhang's group at fudan university and several other groups in china illustrate the dedication and increased capacity of the scientific infrastructure in china [ , ] . for sars-cov, the causative agent was unknown for months and subsequently took over four weeks until a full genome was released [ ] . similarly, mers-cov was only identified after several months of testing and a full-length genome available about a month later [ ] . in contrast, time from the first date of patient onset ( december ) to the report of several -ncov full-length genomes took less than one month. combined with the immense pressure of an ongoing outbreak with an unknown agent, the effort of these scientists should be considered nothing less than remarkable. building from the sequence, the nucleotide alignment quickly distinguished the novel virus as a group b cov, distinct from the sars-cov strains [ , ] . examining the whole genome, -ncov maintains~ % nucleotide identity to the original sars epidemic viruses. its closest whole genome relatives are two bat sars-like covs (zc and zxc ) that shared~ % sequence identity with -ncov; these cov sequences were deposited in early from zhejiang province in r. sinicus bats in china. comparing across the deposited -ncov strains finds > . % conservation; the lack of diversity suggests a common lineage and source with emergence not likely having occurred that long ago [ , ] . a recent report has subsequently identified a bat cov sequence, ratg , with % sequence identity with the novel virus which argues for bat origins for the -ncov [ ] . we next shifted analysis to the nucleocapsid (n) protein, the most abundant protein produced in covs. generally, the n protein is well conserved across cov families including group b [ ] . the n protein for -ncov is no exception with~ % amino acid identity to the sars-cov n protein. while less conserved than other group b covs like hku -cov and shc -cov, -ncov antibodies against the n protein would likely recognize and bind the sars-cov n protein as well. n antibodies do not provide immunity to -ncov infection, but the cross reactivity with sars-cov n protein would allow a serum based assay to determine exposure to the novel cov in asymptomatic cases. while previous studies have found serum reactivity to group b virus n proteins in chinese populations [ ] , exposure to -ncov should increase the dilution factor substantially if exposure/infection had occurred. importantly, this information may provide insights about susceptibly and potential routes of spread through asymptomatic carriers. examining further, we next compared the spike proteins, the critical glycoprotein responsible for virus binding and entry. overall, the -ncov spike protein has roughly % amino acid identity with sars-cov, which is less conserved than other group b covs including hku -cov [ ] . however, narrowing analysis to the spike receptor binding domain (rbd) of sars-cov (amino acids - ), the -ncov rbd is % conserved relative to the epidemic rbd. this conservation level places the -ncov rbd between hku - ( . % conservation), a bat virus that cannot use human ace , and rshc ( . %), the most divergent bat cov spike known to use human ace for entry [ , ] . importantly, the key binding residues for sars-cov have been identified [ ] ; among these fourteen residues predicted to interact directly with human ace , the receptor for sars-cov, eight amino acids are conserved in -ncov. notably, several of these residues are also conserved relative to wiv -and wiv -cov, two bat strains closely related to sars-cov and known to use human ace [ , ] . initial structural modeling suggest that the -ncov may be able to use human ace as a receptor, although its affinity m be reduced relative to the epidemic sars-cov strains [ ] . a subsequent report demonstrated that the receptor binding domain of -ncov was capable of binding ace in the context of the sars-cov spike protein [ ] . in addition, another rapid report links demonstrates -ncov uses ace receptors from human, bat, civets, and swine [ ] . together, the modeling, pseudotyping, and infection data provide strong evidence for human ace being the receptor for -ncov. traditional identification of a microbe as the causative agent of disease requires fulfillment of koch's postulates, modified by rivers for viral diseases [ ] . at the present time, the -ncov has been isolated from patients, detected by specific assays in patients, and cultured in host cells (one available sequence is identified as a passage isolate), starting to fulfill these criteria. given the recentness of the -ncov outbreak, at this point there is no animal model available to fulfill the remaining criteria: ) testing the capability of -ncov to cause respiratory disease in a related species, ) re-isolating the virus from the experimentally infected animal and ) detection of a specific immune response. these efforts will surely be an area of intense research in the coming months both in china and in cov research laboratories around the world. notably, generating small animal models of coronavirus disease can be difficult. while sars-cov readily infected laboratory mice, it does not cause significant disease unless the virus is passaged to adapt to the mouse host [ ] . infection of primates produces a more mild disease than that observed in humans, although fever and pulmonary inflammation were noted [ , ] . mers-cov is incapable of infecting rodent cells without engineering changes in critical residues of the receptor protein, dpp [ , ] . however, mers-cov does infect non-human primates [ ] . as such, mers mouse models of disease required a great deal of time to develop and are limited in the types of manipulations that can be performed [ ] . at this point, the infectious capability of the -ncov for different species and different cell types is unknown. early reports suggest that the virus can utilize human, bat, swine, and civet ace [ ] ; notably, the group found mouse ace was not permissive for -ncov infection dissemination of virus stocks and/or de novo generation of the virus through reverse genetics systems will enable this research allowing for animal testing and subsequent completion of koch's postulates for the new virus. while the huanan seafood market in wuhan has been associated with the majority of cases, many of the recent cases do not have a direct connection [ ] . this fact suggests a secondary source of infection, either human to human transmission or possibly infected animals in another market in wuhan. both possibilities represent major concerns and indicate the outbreak has the potential to expand rapidly. for human to human transmission, there was limited data in the initial set of cases; one family cluster is of three men who all work in the market. similarly, a husband and wife are among the patients, with the wife claiming no contact with the market. in these cases, direct human to human infection may have been possible; alternatively, a contaminated fomite from the market may also be responsible as surfaces all around the market were found to test positive -ncov. however, the major increase in the number of cases, the lack of direct connection to the wuhan market for many cases, and the infection of health care works all suggest human to human spread is likely [ , ] . importantly, until the source of the virus is found, it will be difficult to distinguish zoonotic versus human to human spread. in the early part of the outbreak, the absence of infection in health care workers argued for inefficient human to human spread and distinguished -ncov from both sars-cov and mers-cov. in the two prior cov epidemics, health care settings served as a major transmission point fueling both outbreaks. based on who data, in mers-cov cases have been found to be health care workers; these patients generally have reduced disease and death likely due to younger age and absence of existing health conditions. the recent reports of numerous infected health care workers in wuhan indicate human to human infection can occur with -ncov and may be the product of a super spreading patient [ ] . however, while large swaths of healthcare workers are not getting sick as seen with sars and mers-cov, it may be too early to rule out their potential exposure to the novel cov as their disease may be asymptomatic. while not described during the sars-cov outbreak, asymptomatic cases ranged from . % to % in some mers-cov studies [ ] . a similar phenomenon may be occurring with -ncov and would make stopping the outbreak even more difficult to contain. another parameter to consider is the possibility of super spreading in the context of -ncov. super spreading is the amplified transmission of a virus by individuals in a population and has been suggested by at least one news report [ ] . both sars-and mers-cov outbreaks had documented evidence of super spreading patients [ ] . in general, both epidemic covs maintain a low r , the rate spread from an individual infected patient. however, roughly % of sars-and mers-cov patients have been associated with super spreading and an r > . these cases seeded a significant portion of the epidemic around the world. notably, neither mutations in the viruses nor severity of disease were found to be associated with super spreading, implying that host factors contribute to the phenotype [ ] . for -ncov, contact tracing to date suggest limited human to human spread and a low r . however, the recent increase in cases, both in and outside wuhan could signal the existence of super-spreading individuals fueling the outbreak. alternatively, super spreading could occur from the zoonotic source which has been seen in other disease outbreaks [ ] . in any event, the possibility of super spreading may continue to play a role in this ongoing -ncov outbreak. news of the -ncov came to widespread attention through the internet. over the years, websites like flutrackers.com, promed (promedmail.org), and others have permitted the collection of disease information from around the world and facilitated dissemination to interested parties. in , mers-cov first drew attention as a "novel coronavirus" entioned on promed mail and subsequently through conversation on twitter between science journalists, virologists, and public health experts. eight years later, a more connected network quickly dissected statements from the wuhan municipal health commission and speculated about possible causes. early during an outbreak, it can be difficult to distinguish between rumors with elements of truth versus baseless fear mongering. this fact can be exacerbated by language barriers and off the record sources. however, in this case, speculation of a novel coronavirus was fed by carefully worded statements that specifically excluding some virus families (influenza, adenovirus), but only excluded sars-cov and mers-cov for coronaviruses. coupled with memories of the sars outbreak, many worried that the truth may be held back. when the agent was finally confirmed as a cov, the world acted with both worry and relief: the outbreak would not be hidden. while far from perfect, the government response to -ncov provides a stark contrast to the sars outbreak at the beginning of the century. the rapid release of -ncov sequences permitted the research community to quickly become engaged, providing analysis and developing diagnostic tests. both the chinese cdc and the wuhan municipal health commission have posted regular updates of confirmed case numbers and patient statuses enabling public health authorities to monitor the situation in real time. researchers from around the world have connected on social media to compare updated sequence information and highlight key unknowns about the outbreak. while not always provided in a timely manner, the ability to share news updates and data in real time with researchers and public health officials around the world signals a major change in the response to outbreaks. this connectivity has facilitated awareness as well as new collaborations and a rapid response by the global research community. while there are many unknowns with -ncov, the world is engaged and prepared to battle the newest emergent virus strain. perhaps this means the lessons from the sars outbreak have truly been learned. wuhan municipal health commision. wuhan municipal health and health commission's briefing on the current pneumonia epidemic 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disease analysis of intrapatient heterogeneity uncovers the microevolution of middle east respiratory syndrome coronavirus key: cord- -vtgdqart authors: aston, emily j.; jordan, brian j.; williams, susan m.; garcía, maricarmen; jackwood, mark w. title: effect of pullet vaccination on development and longevity of immunity date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: vtgdqart avian respiratory disease causes significant economic losses in commercial poultry. because of the need to protect long-lived poultry against respiratory tract pathogens from an early age, vaccination programs for pullets typically involve serial administration of a variety of vaccines, including infectious bronchitis virus (ibv), newcastle disease virus (ndv), and infectious laryngotracheitis virus (iltv). often the interval between vaccinations is only a matter of weeks, yet it is unknown whether the development of immunity and protection against challenge when vaccines are given in short succession occurs in these birds, something known as viral interference. our objective was to determine whether serially administered, live attenuated vaccines against ibv, ndv, and iltv influence the development and longevity of immunity and protection against challenge in long-lived birds. based on a typical pullet vaccination program, specific-pathogen-free white leghorns were administered multiple live attenuated vaccines against ibv, ndv, and iltv until weeks of age (woa), after which certain groups were challenged with ibv, ndv, or iltv at , , , , and woa. five days post-challenge, viral load, clinical signs, ciliostasis, tracheal histopathology, and antibody titers in serum and tears were evaluated. we demonstrate that pullets serially administered live attenuated vaccines against ibv, ndv, and iltv were protected against homologous challenge with ibv, ndv, or iltv for at least weeks, and conclude that the interval between vaccinations used in this study (at least weeks) did not interfere with protection. this information is important because it shows that a typical pullet vaccination program consisting of serially administered live attenuated vaccines against multiple respiratory pathogens can result in the development of protective immunity against each disease agent. vaccination against respiratory viral disease is standard practice in commercial poultry operations. both live and killed vaccines are administered to poultry, and live vaccines are commonly used for a variety of pathogens because they are effective when mass applied and are relatively economical [ ] . in general, live vaccines induce local and cell-mediated immunity and provide a broader protective response than killed vaccines, whereas killed vaccines primarily induce humoral immunity and tend to be antigen-specific. the duration of immunity achieved following live vaccine administration depends on the age and type of bird, levels of maternal immunity, disease targeted by the vaccine, immunogenicity of the vaccine, method of vaccine application, number of and interval between boosters, virulence and similarity of the field challenge virus, interval between vaccination and challenge, and immunocompetency of the host [ ] [ ] [ ] . avian coronavirus infectious bronchitis virus (ibv) is an upper respiratory tract viral pathogen of poultry and leads to reduced weight gain and feed efficiency, drops in egg production and egg quality, stunted growth, and secondary bacterial infection resulting in airsacculitis [ ] . the virus initially replicates in the upper respiratory tract, followed by systemic replication in the reproductive tract and some strains can cause lesions in the kidney [ ] . infected birds may exhibit nasal discharge, coughing, sneezing, and tracheal rales [ ] . the disease is prevented by vaccination, and live vaccines are commonly used to induce local immunity and protection. live vaccines are generally administered to young birds to achieve early protection, and layers and breeders are also boosted with either live or inactivated vaccines, which vary based on their similarity to the circulating field viruses [ ] . newcastle disease (nd) is caused by virulent strains of avian paramyxovirus type , which has recently been reclassified as avian avulavirus (aavv- ) [ ] . depending on the strain of the virus, clinical signs of nd infection may be absent or may involve depression, inappetence, respiratory signs (nasal discharge, sneezing, coughing), reduced egg production and egg quality, and neurological signs (torticollis, circling, paralysis) [ ] . strains of newcastle disease virus (ndv) are characterized as lentogenic, mesogenic, and velogenic, according to their mean death time in embryos [ ] . lentogenic strains are of low pathogenicity causing mild respiratory or enteric infections, followed by mesogenic strains, while velogenic isolates are highly pathogenic often causing neurological signs and mortality [ ] . vaccination regimes against ndv vary and may utilize a combination of live, inactivated, and virus-vectored vaccines [ ] . in the united states, the most widely used traditional vaccine strains comprise lentogenic b (or virus clones of the b strain) and lasota strains [ ] . infectious laryngotracheitis (ilt) is a respiratory disease of poultry caused by gallid alphaherpesvirus i, and is economically important worldwide [ ] . clinical manifestations of ilt include increased mortality, reduced egg production, decreased body weight gain, conjunctivitis, tracheitis with expectoration of bloody mucus in severe cases, depression, severe dyspnea and susceptibility to other respiratory pathogens. live vaccines against ilt virus (iltv) may be of chicken embryo origin (ceo) or tissue culture origin (tco), in which they are passaged multiple times in eggs or tissue culture, respectively. although recombinant vaccines for ilt are commercially available, the ceo vaccine is the most widely used vaccine against iltv worldwide. because of the need to protect chickens against different viral pathogens from an early age, vaccination programs typically include multiple vaccines against a variety of pathogens. sample vaccination regimes in different poultry sectors are reviewed in the merck veterinary manual (www. merckvetmanual.com), in which the interval between vaccinations is often only a matter of weeks. however, there is little information showing that the intervals between vaccinations are sufficient for the birds to develop adequate immune protection against challenge for each virus. the literature shows that sequential viral infections may result in viral interference, in which one virus blocks the subsequent infection and/or replication of another virus in the host [ , ] , but until now it is unknown whether this phenomenon results in reduced protection from serially administered attenuated live vaccines in chickens. interestingly, it has been reported that simultaneous administration of viruses to chickens or turkeys does not result in viral interference [ ] . in this study, we investigate how a typical commercial vaccination schedule consisting of a combination of serially administered, live attenuated viral respiratory disease vaccines affects the development and longevity of immunity and protection against homologous challenge. a commercial ibv vaccine mildvac-ga- ® (merck animal health, summit, nj, usa) was used in this study. the vaccine was diluted according to the manufacturer's recommendations. the challenge virus used was ibv ga /cwl / and was prepared at a median embryo infectious dose (eid ) of . . virus titers were calculated by the reed and muench method [ ] . a commercial ndv vaccine b (newhatch-c , merck animal health, summit, nj, usa) was used for both vaccination and challenge and was reconstituted following the manufacturer's instructions. since mesogenic and velogenic strains of ndv require a biosafety level above bsl , which is not available in our laboratory, our experimental model for protection against ndv involves significantly reduced virus titers or sterile immunity against a second exposure (challenge) with the vaccine virus. an iltv commercial ceo vaccine (trachivax ® merck animal health, summit, nj, usa) and pathogenic iltv georgia broiler strain [ ] were used for vaccination and challenge, respectively. strain was propagated in chicken kidney cells obtained from -to -week-old specific pathogen-free (spf) chickens [ ] . the ceo vaccine was prepared following the manufacturer's recommendations. after inoculation, the median tissue culture infective dose (tcid ) was confirmed by titration of both viruses in chicken kidney cells as previously described [ ] . specific-pathogen-free eggs were obtained at days of incubation and hatched at the poultry diagnostic and research center, athens, ga. chicks were placed on fresh pine shavings in colony houses and pens. chicks were vaccinated with the manufacturers recommended dose in µl via the oculonasal route according to the following schedule; ibv at doa, ndv at weeks of age (woa), ibv at woa, iltv at woa, ndv at woa, and iltv at woa. in addition, a control group was not vaccinated. homologous challenges were conducted at , , , , and woa, and necropsies were performed five days post-challenge (dpc). at challenge, birds received one of four treatments: ibv ga , ndv b , iltv , or no challenge. the treatment groups for each challenge virus per time point were as follows: non-vaccinated, non-challenged (n = - ); vaccinated, non-challenged (n = - ); vaccinated, challenged (n = - ); non-vaccinated, challenged (n = - ). all ibv-challenged birds received an eid of × . per bird in µl intranasally. all ndv-challenged birds received the ndv b vaccine in µl intranasally, reconstituted according to the manufacturer's protocol. all iltv-challenged birds received the pathogenic strain at a dose of × . tcid per bird in µl split equally between eyedrop and intranasal routes. for ibv and ndv challenges, birds were observed at dpc for respiratory signs, as previously described [ ] : = absent; = mild; = moderate; = severe. for iltv challenges, birds were observed at and dpc for dyspnea, conjunctivitis, depression, and mortality, as described previously [ ] . the choanal cleft (ibv-and ndv-challenged and control birds at dpc) or trachea (iltv-challenged and control birds at and dpc) was swabbed for virus detection, and swabs were stored in pbs at − • c. at , , and woa, µl of tears was collected by adding granulated nacl to the eye. blood was collected by wing or cardiac puncture and added to a microcentrifuge tube to collect serum for antibody detection. birds were humanely euthanized, and the eyelid, harderian gland (hg), thymus, liver, spleen, cecal tonsils, and bursa of fabricius were collected and stored at − • c for virus detection and in % neutral buffered formalin. the trachea was removed, and one section was placed in % neutral buffered formalin, and the remaining portion of the trachea was submerged in tissue culture media for the ciliostasis test described below. the procedures were approved by the university of georgia institutional animal care and use committee (aup #: a - -r ). the ciliostasis test was performed on harvested tracheas collected in cell culture media (dulbecco's modified eagle's medium) at • c. for each trachea, five tracheal rings measuring approximately mm thick were cut and represented the proximal, middle, and distal portions [ , ] . cilia activity was observed using an inverted microscope (olympus, center valley, pa, usa). the scoring system follows: = all cilia beating; = % of cilia beating; = % of cilia beating; = % of cilia beating; = no cilia beating as previously described [ ] . the maximum possible score for each trachea is . each tracheal ring was scored by three individuals, and the average total score for each trachea was calculated. the ciliostasis protection score for each group was determined by the following formula: − [(total of the individual scores for the group)/(the number of individuals in the group × ) × ], and a score ≥ was considered protected. a section of each trachea was fixed in % neutral buffered formalin, processed, embedded in paraffin, and -µm sections were cut for hematoxylin and eosin staining. for ibv lesions, epithelial hyperplasia, lymphocyte infiltration, and epithelial deciliation were scored for each trachea. scores were determined as follows: = normal, = focal, = multifocal, and = diffuse, as described previously [ ] . for ndv lesions, a descriptive analysis was performed. for iltv lesions, microscopic lesions were scored on a scale of - (normal to very severe), as described previously [ ] . for ibv and ndv detection, viral rna extraction from µl of the pbs from each swab was conducted using a × magmax- viral isolation kit (thermo fisher, waltham, ma, usa) on a magmax™ express- deep well magnetic particle processor (thermo scientific, waltham, ma, usa), according to the manufacturer's instructions. the quantitative reverse transcription polymerase chain reaction (qrt-pcr) was performed with the agpath-id tm one-step rt-pcr kit (thermo fisher, waltham, ma, usa), following the manufacturer's protocol. each -µl reaction mixture contained . µl of × rt-pcr buffer, µm of each primer, µm of probe, µl of × rt-pcr enzyme mix, and µl of viral rna. the qrt-pcr reactions were run on the applied biosystems ® fast realtime pcr system (life technologies ltd., carlsbad, ca, usa) under the following conditions: one cycle of • c for min and • c for min, followed by cycles of • c for s and • c for s. the primers and probe for the ibv qrt-pcr were previously published [ ] , and are comprised of a forward primer ibv gu ( -gct ttt gag cct agc gtt- ), a reverse primer ibv gl ( -gcc atg ttg tca ctg tct att g- ), and a taqman ® dual-labeled probe ibv g probe ( -fam-cac cac cag aac ctg tca cct c-bhq - ). primers and probe for the ndv qrt-pcr were previously described [ ] and are comprised of a forward primer ndv m+ ( '-agt gat gtg ctc gga cct tc- '), a reverse primer ndv m- ( '-cct gag gag agg cat ttg cta- '), and a taqman ® dual-labeled probe ndv m+ ( '-fam-ttc tct agc agt ggg aca gcc tgc-bhq - '). the primers were obtained from integrated dna technologies (coralville, ia, usa), and the taqman probe was synthesized by biosearch technologies (novato, ca, usa). real-time rt-pcr components and thermocycler parameters were previously described [ ] . the data are expressed as the average cycle threshold (ct) value for all samples in each group, with positive ct values based on the limit of detection for this test associated with virus detection in eggs [ ] . each qrt-pcr reaction plate included a standard curve as an rna extraction control and as a positive control. ga ibv isolated from allantoic fluid was used as the template for the standard curve. negative controls were also included in each plate and consisted of pcr reagents with no rna. for iltv, total dna was extracted from the tracheal swabs using the megazorb ® dna extraction miniprep -well kit (promega, madison, wi, usa), as described previously [ ] . duplex real-time pcr assay that amplifies a fragment of the ul viral gene in iltv and a fragment of the chicken alpha -collagen gene was performed, as previously described [ ] . ibv-specific igg titers were detected using a commercial igg enzyme-linked immunosorbent assay (elisa) ibv antibody test kit (idexx, westbrook, me, usa). briefly, serum samples (stored at − • c) were diluted : , and the procedure was performed according to the manufacturer's protocol. tear ibv-specific iga was detected using a commercial igg elisa ibv antibody test kit (idexx, westbrook, me, usa). briefly, tears were serially diluted two-fold in pbs and incubated in duplicate in wells overnight at • c. all wash steps were performed using pbs-tween ( . % tween ). plates were incubated at • c for h in monoclonal mouse anti-chicken iga-biot ( : , clone a- , southern biotech, birmingham, al, usa), followed by hr in streptavidin-hrp ( : , southern biotech, birmingham, al, usa). final antibody detection steps were completed according to the manufacturer's instructions. endpoint titers were determined by reporting the lowest dilution at which the optical density (od), recorded at nm wavelength, was at least three standard deviations above the mean of control wells incubated with no tear samples. data from wells with a pinpoint color change due to residual substrate or air bubbles were excluded from analysis, and results were reported as log of the endpoint titer. the data were analyzed using prism v. . software (graphpad software, inc., la jolla, ca, usa; www.graphpad.com). for viral load data, a one-way analysis of variance (anova) with dunnet's posttest was used to compare treatment groups within each collection period. all other data were analyzed using a kruskal-wallis test with dunn's posttest to compare treatment groups within each collection period. significant differences were determined at p < . . at days following challenge with ibv ga , vaccinated/challenged birds had significantly lower rna loads compared to positive controls at all collection times and in all tissue samples, with the exception of cecal tonsil at woa (table ). in vaccinated controls, no ibv rna was detected at all collection times except in the cecal tonsils at and woa and in the choanal cleft at woa. ibv loads in negative controls in all tissues were below the limit of detection using the ct value of ≥ . as previously reported [ ] , at all collection times except the woa hg negative controls. clinical signs of ibv infection measured at days post-challenge were significantly reduced in vaccinated/challenged birds when compared to positive controls in all weeks except woa, but trends in clinical sign scores were numerically lower among vaccinated/challenged birds ( table ) . clinical signs were absent in non-challenged negative and vaccinated control birds, and signs in vaccinated/challenged birds were not significantly different from signs in non-challenged controls. notes: sem = standard error of the mean. table . clinical signs and microscopic lesions measured days following ibv ga challenge. letters (a-b) indicate significant differences among vaccine and challenge groups for each week (p < . ). histopathological examination of tracheas from all groups ranged from within normal limits to focal to multifocal minimal to moderate lymphocytic tracheitis; however, moderate lymphocytic infiltration was more frequently seen in positive controls. in all weeks, the proportion of vaccinated/challenged birds with deciliation or acute tracheal necrosis was significantly reduced compared to positive controls and was not different from negative and vaccinated controls (table ) . ciliostasis, defined as the cessation of tracheal ciliary movement, was measured at dpc, and the number of birds positive for ciliostasis and ciliostasis protection scores were calculated for each group (figure ). at all collection times, vaccinated/challenged birds were protected from ciliostasis (scores were > ), and positive controls were not protected (scores were < ). the non-challenged negative controls and vaccinated controls were protected (scores were > ) at all collection times. ibv-specific igg titers were measured in serum collected at dpc. at all times except at woa, vaccinated birds from both non-challenged and challenged groups exhibited significantly higher titers compared to non-vaccinated birds from both non-challenged and challenged groups (figure ). at woa, titers in vaccinated birds, regardless of challenge status, were significantly higher compared to positive controls. titers in vaccinated/challenged birds did not significantly differ from titers in vaccinated controls until woa, when vaccinated/challenged birds had significantly higher titers. compared to negative controls, vaccinated/challenged birds had significantly higher titers at all times. ibv-specific igg titers were measured in serum collected at dpc. at all times except at woa, vaccinated birds from both non-challenged and challenged groups exhibited significantly higher titers compared to non-vaccinated birds from both non-challenged and challenged groups (figure ). at woa, titers in vaccinated birds, regardless of challenge status, were significantly higher compared to positive controls. titers in vaccinated/challenged birds did not significantly differ from titers in vaccinated controls until woa, when vaccinated/challenged birds had significantly higher titers. compared to negative controls, vaccinated/challenged birds had significantly higher titers at all times. ibv-specific iga titers were measured in tears collected at dpc at , , and woa. at woa, titers in vaccinated/challenged birds were significantly higher compared to titers in non-challenged negative controls (figure ). at woa, vaccinated/challenged birds showed significantly lower titers compared to positive controls. in addition, the vaccinated/challenged birds had significantly ibv-specific iga titers were measured in tears collected at dpc at , , and woa. at woa, titers in vaccinated/challenged birds were significantly higher compared to titers in nonchallenged negative controls (figure ). at woa, vaccinated/challenged birds showed significantly lower titers compared to positive controls. in addition, the vaccinated/challenged birds had significantly higher titers than the vaccinated/unchallenged group at woa. no other significant differences were detected. at dpc with ndv b , vaccinated/challenged birds either had undetectable rna loads or significantly lower loads compared to positive control titers at all collection times and in all tissues sampled (table ) . non-challenged negative controls and vaccinated controls were negative for rna virus using the ct value of ≥ . as previously reported [ ] . at dpc with ndv b , vaccinated/challenged birds either had undetectable rna loads or significantly lower loads compared to positive control titers at all collection times and in all tissues sampled (table ) . non-challenged negative controls and vaccinated controls were negative for rna virus using the ct value of ≥ . as previously reported [ ] . table . average qrt-pcr ct values for ndv rna collected from choanal cleft, harderian gland, and conjunctiva days post-challenge with ndv b vaccine. letters (a-c) indicate significant differences among vaccine and challenge groups for each week (p < . ). clinical signs measured at dpc were significant at woa in positive controls, after which clinical signs in positive controls were no different from any of the other treatment groups (table ). no significant differences in clinical signs existed between vaccinated/challenged birds and non-challenged controls at any time. in all weeks, histopathological examination of tracheas was within normal limits or revealed focal to multifocal minimal to mild lymphocytic tracheitis, and there were no group-related differences. tracheas were also evaluated for ciliostasis, and no groups exhibited ciliostasis. ndv-specific serum igg titers in vaccinated/challenged birds and vaccinated controls were significantly higher than titers in both positive and negative controls at all collection times ( figure ). there was no significant difference in titers between vaccinated/challenged birds and vaccinated controls at any time. similarly, no significant difference in titers was detected between non-vaccinated/challenged and negative controls, except at woa in which titers from non-vaccinated/challenged controls were significantly higher. table . clinical signs measured days following ndv b challenge. letters (a-b) indicate significant differences among vaccine and challenge groups for each week (p < . ). clinical signs at dpc with iltv strain , vaccinated/challenged birds had significantly lower viral dna loads than positive controls at all collection times and in all tissues ( table ). the dna loads in the trachea and hg were undetectable or low and did not differ significantly from dna loads in nonchallenged negative and vaccinated controls, which were negative using the ct value of ≥ . as at dpc with iltv strain , vaccinated/challenged birds had significantly lower viral dna loads than positive controls at all collection times and in all tissues ( table ). the dna loads in the trachea and hg were undetectable or low and did not differ significantly from dna loads in non-challenged negative and vaccinated controls, which were negative using the ct value of ≥ . as previously reported [ ] . in the conjunctiva, dna loads in vaccinated/challenged birds were significantly higher than dna loads from both non-challenged negative and vaccinated controls, except at woa in which no significant difference was detected between vaccinated/challenged birds and vaccinated controls. clinical signs and viral dna were detected in challenged birds at dpc, but signs in positive controls had become more severe by dpc as tracheal viral load increased (table ). clinical sign scores measured at dpc were significantly reduced in vaccinated/challenged birds compared to positive controls, at all collection points except woa when clinical sign scores were numerically reduced. all negative controls and vaccinated controls had no clinical signs, and clinical signs in vaccinated/challenged birds were not significantly different compared to these controls, except at woa when clinical sign scores were significantly higher. table . clinical signs measured days following iltv challenge. letters (a-b) indicate significant differences among vaccine and challenge groups for each week (p < . ). clinical signs tracheal histological examination was within normal limits or revealed focal to diffuse minimal to mild lymphocytic tracheitis in tracheas from all groups. for the positive controls, / and / in weeks and , respectively, had acute focal necrotizing tracheitis with syncytia and intranuclear inclusions ( figure ). table . clinical signs measured days following iltv challenge. letters (a-b) indicate significant differences among vaccine and challenge groups for each week (p < . ). tracheal histological examination was within normal limits or revealed focal to diffuse minimal to mild lymphocytic tracheitis in tracheas from all groups. for the positive controls, / and / in weeks and , respectively, had acute focal necrotizing tracheitis with syncytia and intranuclear inclusions ( figure ). no ciliostasis was observed in the tracheas of birds from any of the groups. no ciliostasis was observed in the tracheas of birds from any of the groups. iltv-specific igg titers in serum collected days post-challenge were significantly higher in vaccinated birds from both challenged and non-challenged groups, compared to the positive and negative controls ( figure ). iltv-specific igg titers in serum collected days post-challenge were significantly higher in vaccinated birds from both challenged and non-challenged groups, compared to the positive and negative controls ( figure ). in the present study, we demonstrated that pullets serially administered live attenuated vaccines against ibv, ndv, and iltv were protected against homologous challenge with ibv, ndv, or iltv for at least weeks, as determined by challenge virus detection, clinical signs, histopathology and ciliostasis at days after challenge. additionally, our study showed that the age at vaccination and intervals between each vaccination did not interfere with the development of immunity to each virus and consequently protection against homologous challenge. we designed our vaccination protocol to represent a typical vaccination program for ibv, ndv, and iltv in commercial pullets. with the knowledge that live vaccine viruses can persist in flocks, it has been unclear, until now whether the immunity induced by a live vaccines could be compromised because of viral interference, a phenomenon in which one replicating virus blocks the infection and/or replication of another virus [ , ] . although the vaccines in the present study were administered at intervals of or weeks, it is feasible that virus from a previous immunization was still present at the time of the subsequent vaccination. ibv vaccines have been detected in the respiratory tract up to days post-vaccination [ ] , and ibv was isolated from tracheal and cloacal swabs collected at the point of lay and weeks of age in hens that had been virus-negative for several weeks following recovery from inoculation at one day of age [ ] . fentie, et al. [ ] reported that chickens vaccinated with ndv b shed vaccine virus days post-inoculation. in the present study, we demonstrated that pullets serially administered live attenuated vaccines against ibv, ndv, and iltv were protected against homologous challenge with ibv, ndv, or iltv for at least weeks, as determined by challenge virus detection, clinical signs, histopathology and ciliostasis at days after challenge. additionally, our study showed that the age at vaccination and intervals between each vaccination did not interfere with the development of immunity to each virus and consequently protection against homologous challenge. we designed our vaccination protocol to represent a typical vaccination program for ibv, ndv, and iltv in commercial pullets. with the knowledge that live vaccine viruses can persist in flocks, it has been unclear, until now whether the immunity induced by a live vaccines could be compromised because of viral interference, a phenomenon in which one replicating virus blocks the infection and/or replication of another virus [ , ] . although the vaccines in the present study were administered at intervals of or weeks, it is feasible that virus from a previous immunization was still present at the time of the subsequent vaccination. ibv vaccines have been detected in the respiratory tract up to days post-vaccination [ ] , and ibv was isolated from tracheal and cloacal swabs collected at the point of lay and weeks of age in hens that had been virus-negative for several weeks following recovery from inoculation at one day of age [ ] . fentie, et al. [ ] reported that chickens vaccinated with ndv b shed vaccine virus days post-inoculation. in addition, a study by hughes, et al. [ ] demonstrated intermittent shedding in the trachea from iltv-immunized chickens between and weeks post-vaccination. few experimental studies of sequential virus infections have been published, and fewer yet have been considered in the context of poultry viral respiratory pathogens. costa-hurtado, et al. [ ] demonstrated that chickens and turkeys serially infected with a mesogenic strain of ndv and highly pathogenic avian influenza virus (hpaiv) days apart resulted in an initial decrease followed by a subsequent increase in replication of the second virus. in a subsequent study [ ] , the same group found that low pathogenic avian influenza virus given days after a lentogenic strain of ndv did show viral interference whereas viral interference was not observed when the viruses were given simultaneously. we did not measure vaccine virus replication in the present study and, therefore, could not determine whether one vaccine virus compromised the infection and replication of a subsequent vaccine virus. however, our goal was to determine if chickens sequentially vaccinated with all three viruses were protected from viral replication and clinical signs following homologous challenge. thus, regardless of viral interference, our data shows that immunity to individual vaccine viruses was not compromised following sequential administration of multiple live attenuated vaccines targeting different viral respiratory tract pathogens. the detection of ibv rna in the cecal tonsils of vaccinated/non-challenged birds at and woa but not at subsequent times indicates that residual vaccine virus rna remained in the cecal tonsils until at least woa, following the second ibv vaccination. ibv has been isolated from the cecal tonsils at weeks post-infection and is known to persist for several months in various internal organs [ , ] . ibv rna was also detected in non-challenged negative control hg at woa but was absent from all other tissues collected from negative controls, which displayed no clinical signs of ibv infection. it is not clear why we detected ibv rna in samples from the hg in negative control birds but likely represents cross-contamination during processing of the samples since ibv was not detected in any other tissue. the tracheal histopathological lesions of deciliation in positive controls were consistent with previous reports of ibv-induced histopathology [ , ] , and were further confirmed by the presence of ciliostasis among positive controls. as expected, ibv-vaccinated/challenged birds were protected from ciliostasis. the european pharmacopoeia states that ciliostasis can be used to evaluate ibv vaccine efficacy, in which a lack of ciliostasis would indicate that the vaccine was efficacious [ ] . therefore, these observations further confirm that ibv-vaccinated birds were protected from homologous challenge. the observation of robust ibv-specific serum igg titers in vaccinated birds is consistent with previous studies showing that ibv infection stimulates a humoral response in chickens [ ] , but that circulating antibody titers do not correlate with resistance to infection [ ] . therefore, the presence of ibv-specific serum igg titers indicates only that the bird has been exposed to vaccine or challenge virus and should not be correlated with other measures of protection. the lack of significant titers in non-vaccinated/challenged birds can be explained by the early time of collection post-challenge. orr-burks, et al. [ ] found that significant changes in igg serum titer were not detected until days post-inoculation. orr-burks, et al. [ ] also demonstrated a lack of significance in ibv-specific iga titer in tears days after both primary and secondary exposure to ibv, but iga titer was significantly higher between and days after primary exposure to ibv. in this study, ibv-specific iga titers measured in tears at dpc did not reveal consistent trends and may be explained by the early time of collection post-infection. it was beyond the scope of this study to collect samples after dpc, but the production of ibv-specific iga in tears at later time points was demonstrated in a different experiment in which tears were collected between and dpc. in that experiment, naïve chickens challenged with ibv showed a higher trend of specific iga titer compared to non-challenged controls [ ] . the decision to use the b vaccine for ndv challenge was based on biosecurity regulations and the lack of appropriate biosafety level facilities needed for challenge with mesogenic or velogenic strains of ndv. since we used a lentogenic strain of ndv as challenge virus in our experimental design, protection was primarily based on significantly reduced or no virus detection at days after challenge, which is a measure of local immunity preventing virus infection and/or replication. presumably a bird protected from infection with a lentogenic strain of ndv would also show some level of protection from exposure to mesogenic or velogenic strains. vaccinated birds at each sampling time had significantly lower or no challenge virus rna compared to positive control groups, indicating that the vaccinated birds developed a local immune response and were indeed protected whereas the non-vaccinated positive controls were not. because lentogenic ndv only causes a mild respiratory or enteric infection [ ] , it was not surprising that respiratory signs and histological changes were mild or absent despite the presence of viral rna. we observed clinical signs of ndv infection only at woa in positive controls, while only a few birds showed mild clinical signs at and woa, and no clinical signs of disease were observed at later challenge times. this observation is consistent with existing knowledge that nd tends to be more severe in younger birds [ ] . the lack of ciliostasis observed in ndv b -infected positive controls contrasts with previous reports demonstrating that ndv caused ciliostasis in tracheal explants. butler, et al. [ ] demonstrated that ndv caused ciliostasis within to days after infection of tracheal explants, and malo, et al. [ ] reported that following vaccination of one-day-old chicks with lentogenic ndv, the peak of ciliostasis occurred at and dpv and waned by dpv. the discrepancy between our results and the previous studies may be explained by the use of a different lentogenic ndv strain; b in our studies and lasota in previous reports [ ] . it is well established that the lasota vaccine strain is more virulent than b [ ] , which may explain the ciliostasis observed in lasota-vaccinated chicks or lasota-infected tracheal explants, whereas ciliostasis was not observed in our study using b vaccine. robust ndv-specific circulating igg antibody responses developed following ndv vaccination and stayed elevated, which was consistent with prior research indicating that antibodies may be detected for up to one year in birds immunized multiple times against ndv [ ] . with the exception of woa, the igg titers in non-vaccinated/challenged birds were not significantly increased compared to titers in negative controls. this observation is not surprising given that ndv-specific antibodies are not detected in the serum until - days after exposure [ ] . for the iltv-challenged birds, vaccination prevented challenge virus replication in trachea and hg but did not completely block virus replication in the conjunctiva, especially at and woa. this may suggest a waning of the local immunity against iltv after the second vaccination at woa. in addition, the proximity of the conjunctiva to the inoculation site (eyedrop and intranasal) might explain why the local immune response was not able to completely block viral replication in the conjunctiva but successfully cleared the virus before it could replicate in hg and trachea. very few positive controls (non-vaccinated/challenged) demonstrated histological evidence of ilt infection in the trachea, though all positive controls tested positive for ilt dna by pcr. this finding is not surprising in light of a report by guy, et al. [ ] , which illustrated that histological detection of ilt is highly specific ( %) but poorly sensitive ( %). notably, intranuclear inclusion bodies are present only during the initial infection ( - days) and disappear following epithelial cell necrosis and desquamation [ ] , which may explain the observation that by dpc only / and / positive controls at and woa, respectively, had histological evidence of ilt infection despite the presence of ilt dna in the trachea. the absence of ciliostasis observed in both vaccinated and non-vaccinated, iltv-challenged birds at dpc was not surprising given that butler, et al. [ ] found that only some strains of iltv caused ciliostasis and did not correlate with virulence. moreover, the authors showed that ciliostasis rarely occurred before days, and sometimes even days, after inoculation. in addition, gerganov and surtmadzhiev [ ] also demonstrated ciliostasis in iltv-infected tracheal organ cultures at - days post-infection. therefore, our results combined with previous studies indicate that measuring ciliostasis may not be a reliable marker of protection from iltv infection and that ciliostasis in iltv infection studies may need to be evaluated at later times post-inoculation. the lack of significant iltv-specific serum igg titers in non-vaccinated positive controls may be explained by the early time of collection post-challenge, as iltv-specific antibodies do not become detectable until - dpi and peak at dpi [ ] . however, it is worth noting that antibody titers are not correlated to protection from iltv infection [ ] . igg titers were robust in vaccinated birds throughout the study and support previous data that antibodies may be detectable for at least a year [ ] . taken together, our data indicate that a typical pullet vaccination program consisting of serially administered live attenuated vaccines against ibv, ndv, and iltv does not interfere with immune responses to the individual vaccines, and the birds are adequately protected against homologous challenge until at least woa. this information is important because it shows that consecutively administered live attenuated vaccines against multiple respiratory pathogens can be an effective vaccination strategy for the development of protective immunity against each disease agent in long-lived birds. disease prevention and diagnosis infectious bronchitis virus variants: a review of the history, current situation and control measures the role of vaccination in risk mitigation and control of newcastle disease in poultry infectious bronchitis identification of vaccine strains of newcastle disease virus newcastle disease vaccines-a solved problem or a continuous challenge? current and future vaccines and vaccination strategies against infectious laryngotracheitis (ilt) respiratory disease of poultry review: a systematic approach to virus-virus interactions coinfections of the respiratory tract: viral competition for resources virus interference between h n low pathogenic avian influenza virus and lentogenic newcastle disease virus in experimental co-infections in chickens and turkeys in avian virus diseases: laboratory manual; 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prescott, joseph title: hantavirus immunology of rodent reservoirs: current status and future directions date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: me ajoyb hantaviruses are hosted by rodents, insectivores and bats. several rodent-borne hantaviruses cause two diseases that share many features in humans, hemorrhagic fever with renal syndrome in eurasia or hantavirus cardiopulmonary syndrome in the americas. it is thought that the immune response plays a significant contributory role in these diseases. however, in reservoir hosts that have been closely examined, little or no pathology occurs and infection is persistent despite evidence of adaptive immune responses. because most hantavirus reservoirs are not model organisms, it is difficult to conduct meaningful experiments that might shed light on how the viruses evade sterilizing immune responses and why immunopathology does not occur. despite these limitations, recent advances in instrumentation and bioinformatics will have a dramatic impact on understanding reservoir host responses to hantaviruses by employing a systems biology approach to identify important pathways that mediate virus/reservoir relationships. hantaviruses (family bunyaviridae, genus hantavirus) are negative-stranded, trisegmented viruses that cause about , disease cases annually, with case fatality rates of . %- %, depending on the virus [ ] . the viral gene segments encode four or five polypeptides. the large (l) segment encodes the rna-dependent rna polymerase (rdrp), the medium (m) segment encodes a precursor that is posttranslationally cleaved into gn and gc glycoproteins, and the small (s) segment encodes the nucleocapsid (n) protein. some hantaviruses encode a second nonstructural polypeptide (nss) downstream from the n start site in frame [ , ] . little is known about the immunomodulatory abilities of these proteins; however, there is evidence the n, gn and nss may alter host cellular responses during infection. more than hantavirus species have been classified, and many more unclassified hantaviruses have been identified. they are hosted by several species of rodents (rodentia), insectivores (insectivora), and bats (chiroptera) [ ] . little work has been conducted to understand hantavirus infections of insectivores or bats; however, much is known about the ecology of rodent-borne hantaviruses because of their impact on human health (table ) . a central problem of hantavirus/reservoir host research is the lack of reagents and methods for experimentally examining the immune response. recent experimental work on the immunology of rodent reservoirs, summarized in an exceptional review by easterbrook and klein [ ] , has begun to clarify these issues. the immune response is energetically expensive for wild animals, thus the findings of experimental studies will be critical for understanding the ecoimmunology of reservoir hosts of hantaviruses [ , ] , and experiments using wild rodents in natural or semi-natural environments [ , ] will be required to validate laboratory findings. rodentia is the largest mammalian order and is comprised of about , species [ ] . only a few dozen species have been identified as susceptible hosts, and most hantaviruses are hosted by a single rodent species [ ] . in each hantavirus/rodent reservoir relationship, infection results in two prominent features: no conspicuous pathology and persistent infection [ ] [ ] [ ] . the earliest report of experimental infection of a reservoir host with its hantavirus was by lee et al. [ ] that described infection of striped field mice (apodemus agrarius) with hantaan virus. inoculated mice developed chronic infection with transient viremia, and shed virus principally in urine, saliva and, to a lesser extent, feces, despite the production of neutralizing antibodies. currently, three laboratory infection systems have been developed to study hantavirus infections of reservoir hosts: seoul virus (seov) infection of the norway rat (rattus norvegicus), puumala virus (puuv) infection of the bank vole (myodes glareolus), and sin nombre virus (snv) infection of the deer mouse (peromyscus maniculatus) [ , , ] . in humans, these viruses are etiologic agents of hemorrhagic fever with renal syndrome (hfrs; seov, puuv) and hantavirus cardiopulmonary syndrome (hcps; snv) [ ]. these diseases share many pathologic similarities and because little damage to the endothelium occurs during infection, it is thought that the inflammatory immune response contributes to pathogenesis [ , ] . because the norway rat is a model organism with many specific reagents, including monoclonal antibodies to immune markers, significant progress has been made toward understanding the reservoir host immune response to seov [ ] [ ] [ ] . fewer methods and reagents are available for bank voles and deer mice. despite these limitations, emerging technologies will be useful for understanding why rodent reservoirs are infected without disease and why they are unable to clear infection. a significant limitation of hfrs research is that none of the hfrs-causing hantaviruses cause disease in animal models. however, two new world hantaviruses cause an hcps-like disease in syrian golden hamsters (mesocricetus auratus): andes virus (andv) and maporal virus (mapv) [ , ] . andv causes most hcps cases in south america; however, no human cases of disease have been associated with mapv. andv is hosted by the long-tailed pygmy rice rat (oligoryzomys longicaudatus) and mapv is hosted by the delicate pygmy rice rat (oligoryzomys delicatus) [ , ] . andv is considered an animal biosafety level- pathogen in most nations, whereas mapv is considered an absl- virus [ ] . the natural route of transmission among reservoir rodents is thought to be principally through aerosols and/or biting [ ] . however, experiments have been equivocal in clarifying transmission mechanisms. weanling bank voles caged with infected individuals lead to transmission as early as days post exposure [ ] . similar experiments examining snv transmission between deer mice have been less informative; however, in artificial enclosure experiments, transmission appeared to be facilitated by deer mice with higher amounts of viremia and wounding [ ] and males likely play a dominant role in transmission in natural populations [ , , ] . in bank voles, offspring of puuv-infected dams were less likely to be infected after exiting the nest because of protective maternal antibody [ ] . although it is thought horizontal transmission is most important, work by hutchinson et al. [ ] showed that vertical transmission occurred among cotton rats (sigmodon hispidus) infected with black creek canal virus, so it is possible that both routes may influence transmission at the population level. the route of transmission has important ramifications in terms of the host immune response where, presumably, a mucosal response occurs with aerosol transmission and a localized response at a bite site. experimental data have also shown that patterns of the expression of genes related to the immune response are different in infected males and females [ ] , and it is likely these differences have important roles in hantavirus ecology. spillover to other rodent species also occurs [ ] [ ] [ ] [ ] , but it is unknown if the rodents remain infected. recent work has shown that deer mice are experimentally susceptible to andv; however, virus is cleared several weeks after infection [ ] . a pronounced th /tfh gene expression profile occurs, including il- pathway activation, that does not appear to be substantially activated in snv-infected deer mice [ , ] . this system provides an opportunity to identify viral and reservoir host factors that are important for sterilizing immunity that clears infection. the principal target cells of infection in rodents (and humans) are the microvasculature endothelial cells of many tissues [ ] . experimental intramuscular infection of deer mice with snv resulted in detectable virus in the lungs as few as two days later [ ] . many organs appeared infected, although infection was limited to the vasculature within those tissues. two infectious outcomes occur in experimentally infected deer mice; a disseminated infection of three or more organs, or a restricted infection of the lungs and heart [ ] . the relevance of these two patterns to transmission efficiency is unknown. the levels of viral rna vary dramatically in infected deer mice, with most having modest to moderate levels of rna at the peak of infection. however, some deer mice have significantly greater amounts of viral rna, suggesting these deer mice may produce substantially more virus than others, and it is possible they transmit virus more efficiently (e.g., "supershedders") [ ] . this also occurs in semi-natural transmission experiments [ ] and suggests certain individuals may have a prominent role in population-level transmission of hantaviruses. most serological assays for detecting antibody responses in hantavirus reservoirs use virus neutralization, elisa or strip immunoblotting [ , , [ ] [ ] [ ] . while some of these assays are igg-specific, others use antiserum to whole igg, including the light chains. since light chains are shared by all immunoglobulins, these detection antibodies are not igg-specific. moreover, no assays are in place for detecting iga, which should be prominent in mucosal infections. igm assays have been problematic despite the availability of anti-igm capture antisera that are cross-reactive with igm from at least one hantavirus reservoir species [ ] . some immunoglobulins have isotypes with specific effector activities, such as complement fixation or antibody-dependent cell cytotoxicity. laboratory house mice have four igg isotypes; igg , igg a, igg b and igg . it is likely that reservoirs also have immunoglobulin isotypes with distinct effector functions and which might predominate during hantavirus infections. these reagent deficiencies are a current obstacle for assessing antibody responses in rodent reservoir hosts. despite these limitations, many field studies have been conducted examining antibody responses in natural and semi-natural hantavirus infections of rodent reservoirs [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in experimentally-infected deer mice, snv nucleocapsid-specific antibodies can be detected in serum as early as days post infection, and neutralizing antibody can be detected after three weeks post infection [ ] . similarly, experimentally-infected bank voles produce puuv-specific antibodies two to three weeks after inoculation [ ] and rats experimentally infected with seov also produce igg within two weeks post inoculation [ ] . the presence of igg in these naturally and experimentally infected reservoirs is an indicator of class switching and affinity maturation, events that are mediated by t cells [ ] . thus, rodents mount adaptive t cell/b cell immune responses to their reservoir hantaviruses; however, it appears to be inadequate for virus clearance. while inflammatory signatures are present [ , , ] , the magnitude of these signals appears to be modest relative to expression levels found in a syrian hamster pathology model of hcps [ ] . it is noteworthy that immunization of rodent reservoirs with homologous nucleocapsid antigen or plasmids encoding the antigen protects from subsequent challenge [ , ] , suggesting infection can be prevented in reservoir hosts. several hantavirus proteins have been implicated in modulation of the host cell's antiviral defenses ( table ). the gn glycoproteins of pathogenic new world hantaviruses and seov possess an immunoreceptor tyrosine activation motif (itam) in the cytoplasmic tail that binds to fyn tyrosine kinase, and the itam may also interact with lyn, syk, and zap- kinases found in lymphocytes [ , ] , although there is no evidence that lymphocytes are susceptible to hantaviruses. the itam may also promote polyubiquitination of the gn polypeptide to facilitate its degradation [ ] ; however, it is unclear how it impacts the host response to infection. presumably, the itam interferes with the antiviral response of an infected cell since the motif is cytoplasmic. the gn protein may also alter the rig-i pathway that leads to irf phosphorylation and subsequent ifnβ expression [ ] . the nucleocapsid may also antagonize the expression of ifnβ by binding to importin-α and interfering with nf-κb nuclear transport, which is required for ifnβ expression [ ] [ ] [ ] . additionally, both caspase and granzyme b are targets of the nucleocapsid of some hantaviruses [ ] and both are essential components of ctl-mediated apoptosis. the lack of damage to the endothelium of infected rodent reservoirs suggests this may be an important mechanism of preventing viral clearance. the nucleocapsid of andv, but not other hantaviruses, also inhibits autophosphorylation and activation of tbk , an enzyme that activates irf and nf-κb and induction of type i interferon gene expression [ ] . recently, putative nonstructural nss sequences have been identified in some hantaviruses [ , ] . this sequence is in an alternative reading frame of the nucleocapsid transcript. in other bunyaviruses, nss has anti-interferon activity [ ] [ ] [ ] ; however, its role in hantavirus infections is less well characterized. importantly, these studies have been conducted with cells from nonreservoir hosts where they, presumably, are not optimized for manipulating the immune response in a manner that benefits the virus but without host pathology. future studies should examine the roles of these proteins in cells from reservoir hosts. the presence of high-titer igg antibodies during hantavirus infections of reservoir hosts indicates both t cell and b cell responses occur because t cells induce class switching and affinity maturation of antibodies produced by antigen-specific b cells. in experimental infections of rats with seov and deer mice with snv, early infection results in subtle inflammatory signatures, but a regulatory t cell (treg) response predominates at persistence (figure ) [ , ] . treg responses are critical for suppressing inflammation [ , ] ; however, inflammation is a prominent feature of hantavirus disease in humans [ ] and hamsters [ , ] . in other viral diseases, the occurrence of a treg response is associated with persistent infection because these cells, while suppressing inflammation, also prevent virus clearance [ , ] . for reservoirs of hantaviruses, the treg response may limit inflammatory immunopathology to an otherwise innocuous infection, but it may also impair virus clearance. how this relationship evolved is unknown, but considering the presence of hantavirus proteins with immunomodulatory activities, it suggests the viruses may manipulate the host response to favor persistence; a treg response may prevent sterilizing immunity, thus allow virus to remain in a population. it may also explain the ecoimmunology and ecology of hantavirus infections of reservoir hosts, thus studies assessing the energetics of inflammatory and anti-inflammatory immune responses should be performed. much of the work examining reservoir responses to hantaviruses has been conducted in rats infected with seov and deer mice infected with snv or andv. using cdna arrays, klein et al. [ ] identified nearly genes that were differentially expressed in male and female rats infected with seov. many immune-associated transcription factors, proinflammatory, antiviral, t cell and ig family member genes were significantly higher in females, which may reduce transmission from females. in deer mice infected with snv, early expression signatures of a mixed th /th /treg response were present in virus-specific cd + t cells, including ifnγ, il , il and tgfβ, before transitioning to a treg-like response at persistence [ ] . the expression of immune response genes differed in the spleen, where signatures of inflammation occurred within two days, peaking by days to before subsiding, and lungs, where little immune gene expression occurred [ ] . some deer mice produced nucleocapsid-specific igg by day while others had igg around day or later, and neutralizing antibody was not detected until after weeks. . during acute infection, snv elicits a modest inflammatory response that initially limits, but does not clear, virus. within a few weeks, the response transitions to a regulatory response that may allow episodic recrudescence of virus that can be shed. in deer mice infected with snv, early expression signatures of a mixed th /th /treg response were present in virus-specific cd + t cells, including ifnγ, il , il and tgfβ, before transitioning to a treg-like response at persistence [ ] . the expression of immune response genes differed in the spleen, where signatures of inflammation occurred within two days, peaking by days to before subsiding, and lungs, where little immune gene expression occurred [ ] . some deer mice produced nucleocapsid-specific igg by day while others had igg around day or later, and neutralizing antibody was not detected until after weeks. assessment of cytotoxic t cell responses of reservoir hosts has not been reported. most assays that assess ctl functions require susceptible syngeneic target cells, which have been difficult to obtain with reservoir hosts. susceptible primary cell lines from reservoir hosts have been produced [ , ] , but these are typically obtained from embryonic fibroblasts, thus matching of mhc alleles for use in ctl assays is difficult. many zoonotic viruses antagonize the innate immune response in human cells, and their pathogenic potential often correlates with their abilities to inhibit the innate response in vitro ( [ ] [ ] [ ] [ ] for review). pathogenic hantaviruses inhibit antiviral responses despite high levels of replication, whereas nonpathogenic viruses are efficiently recognized and elicit innate responses that limit replication [ , ] . this antagonistic capacity must have evolved in the reservoir hosts of these hantaviruses because humans are dead-end hosts. to date, few studies have addressed the interactions between hantaviruses and their rodent hosts in vitro. this is partially due to the unavailability of cell lines, and the reagents and techniques to generate primary cell cultures from the various reservoirs of hantaviruses. the norway rat/seov system is the most tractable for studying virus/reservoir interactions. inoculation of rat lung-derived endothelial cells with seov resulted in virus replication, but little or no induction of cytokines or chemokines, suggesting seov can efficiently antagonize antiviral responses [ ] . despite this, endothelial cells increased their expression of the protein pd-l , which correlated with the ability of these cells to induce treg cell activity. in addition, antigen presenting cells isolated from norway rats and infected with seov in vitro were resistant to stimulation, suggesting that virus infection inhibits the normal signaling activities of these cells [ ] . thus antagonism of the innate immune response likely allows for viral replication, while at the same time promotes an anti-inflammatory response that limits immunopathology. similar studies have been performed using bank vole cells infected with puuv [ ] . embryonic fibroblasts inoculated with puuv did not express increased amounts of ifnβ or mx , although non-related viruses were able to induce up-regulation of these genes. this suggests, as with seov, puuv efficiently antagonizes host innate responses in its natural reservoir. the syrian golden hamster develops an hcps-like disease when infected with andv or mapv [ , ] . hamsters inoculated with andv mount an inflammatory response, as measured by elevated mrna encoding pro-inflammatory mediators, prior to succumbing to the disease [ ] . infection also results in the activation of the adaptive immune response, characterized by antigen-specific proliferation of t cells and the generation of virus-specific antibodies [ , ] . in contrast, snv, which is highly pathogenic in humans, replicates in hamsters, but does not cause disease and is cleared by the immune response [ ] . passaging of snv in hamsters results in a virus that is able to replicate efficiently and cause a persistent infection similar to what is seen in the rodent reservoir, yet still does not cause disease [ ] . examination of immune responses elicited by andv (pathogenic in hamsters) and snv (non-pathogenic in hamsters) showed that passaged snv evoked a stronger adaptive immune response than did andv; however, andv infection induced a much stronger innate immune response at late time points, despite both viruses replicating to similar levels. depletion of t cells did not alter the outcome of infection [ , ] ; thus, these data suggest that, at least in the hamster model, the activation of the t cell-mediated immune response is not responsible for immunopathogenesis, and perhaps the innate immune response, either elicited from infected endothelial cells, macrophages and/or neutrophils, might contribute to disease. infectious diseases and the immune response are complex processes that are challenging to study, even with substantial reagent resources and mature methodologies. many difficulties exist for studying hantavirus infections of their reservoir hosts, including the lack of molecular and immunological reagents, methods for experimental investigation, and that few reservoir species have been colonized for laboratory use. in addition, pathogenic hantaviruses require bsl- and/or absl- containment, which presents logistical hurdles for examining the reservoir host/hantavirus relationship. despite these limitations, novel instrumentation, particularly those for transcriptome profiling (e.g., rna-seq) and metabolomics, and development of molecular and cellular methods, provide an opportunity to rapidly develop the tools necessary for examining reservoir host responses using a systems biology approach. the key feature of these new technologies is that they are species-independent in the data they generate, but they require significant computational and bioinformatics resources. infection triggers a cascading host response that is highly orchestrated by the vertebrate immune system. many genes are modulated (expressed or repressed) during the course of infection and identification of mrna and noncoding rnas can be used to identify the mechanisms that control, or fail to control, disease. moreover, some infectious diseases, including hantavirus disease, have substantial immunopathologic components. the quantitative assessment of the transcriptional landscape (patterns of gene expression) can be used to profile the host responses in infected and uninfected animals of the same species, or a disease model species to reservoir host species to identify mechanisms of susceptibility or resistance. rna-seq is one such method for profiling transcriptional landscapes [ ] . the depth of coverage and costs of rna-seq have improved dramatically in the last few years, and it is likely to become less expensive. however, the computational resources necessary for using rna-seq for studying host responses is substantial, often requiring hundreds of gigabytes of ram and multicore, multiprocessor systems typically found in servers running a linux operating system [ ] . this depth is often necessary to detect rnas that occur in extremely low abundance because their proteins are highly potent (e.g., cytokines). even then, it is possible that differentially expressed genes may not be detected and other, more sensitive methods, such as real-time pcr, may be required to validate pathway signatures. despite these requirements, bioinformatics tools for rna-seq are now widely available, many of which are free. a typical first step of differential gene expression profiling is the de novo assembly of all rna-seq samples from an experiment, which represents the totality of expressed genes from the experiment. there are several de novo assemblers available, including the trinity suite [ ] and oases [ ] . each of these packages has advantages and disadvantages, thus it is important to understand how each performs assemblies, particularly isoforms that may have specific activities. included in the trinity package is rsem [ ] that estimates transcript abundances, including isoforms, in experimental samples by counting reads from replicates against the de novo assembly. an important feature of rsem is that it does not require an annotated genome; it determines the abundance of transcripts from the unannotated assembly, identifies differentially expressed transcripts, and provides a % credibility interval for each gene. additional tools, such as deseq and edger [ , ] provide statistical evaluation of differential gene expression between samples and provide quantitative (higher/lower) and qualitative (on/off) data. the differentially expressed transcripts are subsequently identified by other means (e.g., blast) and can then be mapped to pathways (such as reactome or kegg) [ , ] to visualize [ ] where viruses may influence the host response and identify mechanistic targets of hantaviruses. in recent years, micrornas (mirna) have been identified that are important regulators of antiviral responses. activation of tlr/rig-i pathways leads to the expression of several mirnas [ ] that likely are important in lymphocyte functions [ ] . because hantavirus gn targets rig-i [ ] , it is possible that these mirnas are dysregulated, which could provide the virus an advantage over the host cell response. while mirna expression has been evaluated in hantavirus-infected human endothelial cells, epithelial cells and macrophages [ , ] , no work has been conducted to examine the role of mirnas in reservoir host cells infected with hantaviruses. considering the importance of mirna in host responses, it is likely they play an instrumental role in the immunological events leading to persistent infection of the reservoir hosts. the use of rna-seq can identify global mirna expression [ ] and clarify their regulatory roles in infected reservoirs. metabolic products can provide substantial information about the interactions of viruses and infected host cells, and the how immune system responds to infection [ , ] . the field of metabolomics is young but potentially informative for understanding hantavirus/reservoir host interactions. many viruses metabolically remodel the host cell to optimize infection. because metabolic products (e.g., carbohydrates, lipids, prostaglandins, etc.) are identical or highly similar between vertebrate species, this approach may help identify which enzymes, by virtue of their metabolic products, may be targeted by hantaviruses. while there are no reports of metabolic assessment of hantavirus infections, rift valley fever virus (rvfv) modulates the activity of adenosine ' monophosphate-activated protein kinase (ampk) in infected cells. this enzyme is a regulator of several metabolic pathways, including enhancement of catabolic pathways such as autophagy and atp production, but it represses anabolic pathways, such as lipid biosynthesis. infection of cells by several viruses, including rvfv, results in activation of ampk and restriction of viral replication, suggesting an antiviral role for this enzyme [ ] . other studies have revealed metabolic pathway targeting by viruses [ ] [ ] [ ] , thus efforts to examine how hantaviruses may manipulate the metabolomes of infected cells could lead to the identification of therapeutic targets for treating hantavirus disease. the use of monoclonal antibodies has been particularly challenging for hantavirus/reservoir research. identification of cell surface markers could shed light on what cells respond during hantavirus infection of reservoir hosts. while cell surface antigens tend to be more divergent, intracellular proteins, such as antiviral proteins, tend to be more conserved, particularly phosphoepitopes. thus, it is likely that many available antibodies to house mouse (mus musculus) or norway rat antiviral proteins will be cross-reactive with orthologs from hantavirus rodent reservoir species. without information as to which proteins may be of interest, screening of antibodies is a daunting and expensive task since most likely will not be informative. however, combined with rna-seq and metabolomic data, it is likely that target proteins and pathways can be identified so that investigators can focus their efforts and resources. in addition, software tools can help predict whether antibodies may be cross-reactive to proteins from reservoir hosts [ ] . the use of cytokines or neutralizing cytokine antibodies to perturb the host response during infection has identified many mechanisms that contribute to susceptibility or resistance. for example, administration of ifnγ to laboratory mice facilitates clearance of lcmv, whereas antibody that neutralizes ifnγ impairs ctl responses and clearance, thus leading to persistent infection [ , ] . depletion of immune cell subsets with antibodies can also determine the roles of those cells during infection. this approach revealed a critical role for cd + t cells for sustaining ctl responses to lcmv [ ] . other than the norway rat, an array of cytokines and antibodies for experimental manipulation of the host response of hantavirus reservoirs is substantially limited. thus, it is difficult to determine the mechanisms controlling the host response of reservoirs. some cytokines are broadly cross-reactive and can be used for studying reservoir responses. recombinant house mouse gm-csf and human il- stimulate deer mouse cells [ ] , and it is likely that other commercially-available cytokines can also be used. identification of cross-reactive cytokines should be a high priority of the hantavirus community. with genome and transcriptome sequencing, many cytokine genes can be rapidly cloned into expression vectors that are codon-optimized for the expression system of choice using de novo synthesis services (e.g., geneart, life technologies). moreover, some antibodies may be cross-reactive with reservoir species' orthologs [ ] . anti-mouse cd (clone gk . ) and anti-rat cd β (clone ) antibodies can be used to deplete cd + and cd + t cells from hamsters, respectively [ , ] , and they may also be useful for reservoir host studies to examine the roles of these cells. finally, it is difficult to assess ctl activity in reservoir hosts. most colonies are established with wild rodents that are highly polymorphic. this limits use of traditional ctl assays that require mhc class i-matched target cells. the generation of highly inbred strains is challenging and may result in alleles that do not represent the natural biology of hantavirus infection, thus it may not be desirable to generate highly inbred rodents. it is possible to establish mhc homozygotes with controlled breeding and screening of littermates for the same haplotypes. even then, the generation of susceptible cell lines can be problematic. while endothelial cell lines can be generated with retroviral transformation, it is possible the cells may have activated antiviral pathways that could alter in vitro ctl responses. the use of growth factors for expanding endothelial cells in culture may be more attractive. until methods are established for generating syngeneic, susceptible target cells, assessment of ctl responses in reservoir hosts will be difficult. the immunological relationships between hantaviruses and their rodent reservoir hosts are complex. infection typically leads to disseminated infection within a few days but without conspicuous signs of disease. expression of immune genes can be detected in as little as a few days that suggests innate and adaptive immune activation, but the magnitude of expression is substantially less than in hantavirus pathology models. the lack of reagents and methodologies for studying hantavirus reservoirs, most of which are not model organisms, presents a significant challenge. however, new technologies have recently emerged that are cost effective and species-independent, but which generate large amounts of data that require substantial computational and bioinformatics support for data reduction. with these tools, it should be possible to accelerate research to understand the relationships of hantaviruses and their reservoir hosts. work was provided by the department of microbiology, immunology and pathology, colorado state university (ts), nih grant ai (ts), and intramural program of niaid, nih (jp). t.s. and j.p. reviewed the literature and wrote the manuscript. the authors declare no conflict of interest. hantaviruses: a global disease problem tula and puumala hantavirus nss orfs are functional and the products inhibit activation of the interferon-beta promoter the andes hantavirus nss protein is expressed from the viral small mrna by a leaky scanning mechanism phylogeny and origins of hantaviruses harbored by bats, insectivores, and rodents immunological mechanisms mediating hantavirus persistence in rodent reservoirs long-term patterns of immune investment by wild deer mice infected with sin nombre virus refining 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computational analysis of noncoding rnas lipids at the interface of virus-host interactions multifaceted roles for lipids in viral infection amp-activated kinase restricts rift valley fever virus infection by inhibiting fatty acid synthesis dengue virus infection perturbs lipid homeostasis in infected mosquito cells metabolic effects of influenza virus infection in cultured animal cells: intra-and extracellular metabolite profiling viral effects on metabolism: changes in glucose and glutamine utilization during human cytomegalovirus infection modulation by gamma interferon of antiviral cell-mediated immune responses in vivo mechanism of recovery from acute virus infection. viii. treatment of lymphocytic choriomeningitis virus-infected mice with anti-interferon-gamma monoclonal antibody blocks generation of virus-specific cytotoxic t lymphocytes and virus elimination mechanism of recovery from acute virus infection: treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that lyt- + t lymphocytes mediate clearance of virus and regulate the antiviral antibody response generation of competent bone marrow-derived antigen presenting cells from the deer mouse (peromyscus maniculatus) discrimination of peromyscus maniculatus leukocytes by flow cytometry the authors thank brian hjelle, charles h. calisher, rushika perera and heinz feldmann for helpful comments, support and advice leading to the preparation of this manuscript. support for this key: cord- - lso w o authors: adney, danielle r.; brown, vienna r.; porter, stephanie m.; bielefeldt-ohmann, helle; hartwig, airn e.; bowen, richard a. title: inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: lso w o the middle east respiratory syndrome coronavirus (mers-cov) was first recognized in and can cause severe disease in infected humans. dromedary camels are the reservoir for the virus, although, other than nasal discharge, these animals do not display any overt clinical disease. data from in vitro experiments suggest that other livestock such as sheep, goats, and horses might also contribute to viral transmission, although field data has not identified any seropositive animals. in order to understand if these animals could be infected, we challenged young goats and horses and adult sheep with mers-cov by intranasal inoculation. minimal or no virus shedding was detected in all of the animals. during the four weeks following inoculation, neutralizing antibodies were detected in the young goats, but not in sheep or horses. the middle east respiratory syndrome coronavirus (mers-cov) is an emerging pathogen first described from saudi arabia in [ ] that can cause severe respiratory disease and death in roughly % of infected humans [ ] . there is considerable field and experimental evidence that dromedary camels serve as an important reservoir host involved in transmission to humans [ ] [ ] [ ] [ ] [ ] [ ] , but whether other livestock such as goats, sheep, and horses play a role in transmission has only been assessed indirectly. the virus is phylogenetically similar to betacoronaviruses previously detected in bats and there has been speculation that this disease originated through a cross-species transmission from bats to camels or humans [ ] [ ] [ ] . serologic testing of sheep, goats, and cattle from jordan [ ] and saudi arabia [ ] failed to identify animals with neutralizing antibodies to mers-cov. similarly, horses tested in the united arab emirates lacked antibodies to mers-cov [ ] . direct contact with dromedaries in saudi arabia was found to be independently associated with mers-cov illness, while contact with goats, sheep, or horses was not associated with human illness [ ] . in vitro studies in which replication of mers-cov in cultured cells was assessed have yielded mixed results with respect to species susceptibility. cells from goats, but not sheep or cattle, supported replication of mers-cov [ ] , and primary equine kidney cells supported virus replication, albeit at lower levels than observed with vero cells [ ] . transfection of the dpp receptor from goats, sheep, and horses into non-permissive mouse or hamster cells allowed replication of mers-cov [ , ] . collectively, these in vitro studies suggest the possibility that some livestock are susceptible to infection, but demonstration of infection in live animals is required to better assess their potential as reservoir hosts. the objective of this study was to determine if goats, sheep, and horses can be infected with mers-cov and assess their potential importance in viral transmission. goats (n = ) were evaluated for viral shedding, organ burden, and seroconversion and transmission to co-housed goats (n = ). limited viral shedding was observed without demonstration of viral transmission. due to the lack of transmission, only viral shedding and serology were evaluated in horses (n = ) and sheep (n = ). these animals did not become productively infected or seroconvert, indicating that such livestock are unlikely to serve as reservoirs for mers-cov and are unimportant in viral transmission. these studies were approved by the animal care and use committee of colorado state university (approval number - a) and were conducted in an association for the assessment and accreditation of laboratory animal care, international (aaalac) approved facility. two goats, three sheep, and four horses were purchased in colorado, usa. both of the goats were bred on site and gave birth to either two (doe ) or three kids (doe ). all animals were fed a complete pelleted feed supplemented with hay, and were observed at least once daily for nasal discharge, demeanor, food consumption, and clinical status. sheep, goat kids and horses were each inoculated intranasally with . × to . × plaque-forming units (pfu) of a low passage human isolate of mers-cov (strain hcov-emc/ ) propagated in vero e cells as described previously [ ] . the goat kids were maintained at all times in a room with their mothers, who served as in-contact controls to test for virus transmission. rectal temperature and nasal swabs were taken daily for seven days. one goat kid from each doe was euthanized days post-inoculation (dpi) and the remaining kids and mother goats were euthanized on day post-inoculation. the horses and sheep were monitored for viral shedding and seroconversion, and were euthanized on day post-inoculation, with the exception of horse , which was euthanized on day due to an injury. samples of nasal secretions were collected by inserting and rotating a swab into each nare and were immediately placed in viral transport medium and frozen until plaque assay was performed. plaques originating from all animals having low titers of virus were confirmed to be mers-cov by immunofluorescence using a rabbit polyclonal antiserum against hcov-emc- antigen as a primary antibody. nasal turbinates, trachea, larynx, and lung samples were collected from two kids (goat c and a) on day post-infection and frozen for virus titration or fixed in % neutral-buffered formalin for greater than days prior to being embedded in paraffin. tissue sections (hematoxylin/eosin and immunohistochemistry) were prepared and evaluated by a veterinary pathologist as previously described [ ] . in order to detect mers-cov antigen immunohistochemical analysis was performed with a previously described rabbit polyclonal antiserum against hcov-emc- antigen [ , ] . serum was collected immediately prior to inoculation and weekly thereafter until necropsy. neutralizing antibodies in sera were assayed using a plaque reduction neutralization test (prnt) with a % neutralization cutoff as described previously [ ] . goats were assessed for clinical disease, viral shedding, seroconversion, and viral transmission to their mothers. fevers were not detected in any of the goats, and no nasal discharge was observed. low levels of infectious virus were detected in two of the inoculated goat kids from doe (figure ), but not from either of the adult goats that had intimate contact or the kids from doe . serum was collected immediately prior to inoculation and weekly thereafter until necropsy. neutralizing antibodies in sera were assayed using a plaque reduction neutralization test (prnt) with a % neutralization cutoff as described previously [ ] . goats were assessed for clinical disease, viral shedding, seroconversion, and viral transmission to their mothers. fevers were not detected in any of the goats, and no nasal discharge was observed. low levels of infectious virus were detected in two of the inoculated goat kids from doe (figure ), but not from either of the adult goats that had intimate contact or the kids from doe . in order to study acute pathology and determine organ burden, two goats were euthanized on day -post infection and nasal turbinates, trachea, and lung were collected. very small but confirmed quantities of virus were isolated from the turbinates of both goats euthanized days post-infection (dpi) (figure ), which may reflect input virus or very low level virus replication. goat kid c was histologically unremarkable, however, the turbinates of goat kid a had multifocal areas of loss of goblet cells, epithelial necrosis or squamous metaplasia and attenuation and/or erosion of the epithelium, accompanied by mild to moderate neutrophil and monocyte/macrophage infiltration and occasional minimal hemorrhage. small amounts of cellular debris, leukocytes and mucus (exudate) were present in the nasal cavity, mainly associated with the aforementioned affected areas. these tissues were negative for viral antigen by immunohistochemistry (ihc) and the histopathologic lesions were very likely the result of trauma from daily swabbing rather than due to virus replication. in order to study acute pathology and determine organ burden, two goats were euthanized on day -post infection and nasal turbinates, trachea, and lung were collected. very small but confirmed quantities of virus were isolated from the turbinates of both goats euthanized days post-infection (dpi) (figure ) , which may reflect input virus or very low level virus replication. goat kid c was histologically unremarkable, however, the turbinates of goat kid a had multifocal areas of loss of goblet cells, epithelial necrosis or squamous metaplasia and attenuation and/or erosion of the epithelium, accompanied by mild to moderate neutrophil and monocyte/macrophage infiltration and occasional minimal hemorrhage. small amounts of cellular debris, leukocytes and mucus (exudate) were present in the nasal cavity, mainly associated with the aforementioned affected areas. these tissues were negative for viral antigen by immunohistochemistry (ihc) and the histopathologic lesions were very likely the result of trauma from daily swabbing rather than due to virus replication. the remaining goats were euthanized on day post-infection and the serological status of the experimentally infected kids and their cohoused dams were assessed. each of three kid goats held past day seroconverted, however, neutralizing antibodies were not detected in either of the dams (table ) . table . neutralizing antibody titers in goats experimentally infected or exposed by contact to mers-cov. mother goats are indicated as or , and their corresponded kids are indicated as a, b, c (doe ), a, or b (doe ). titers represent dilutions of serum which neutralized ≥ % of input virus. nd = not done. three sheep were experimentally infected and evaluated for clinical disease, viral shedding, and seroconversion. no nasal discharge or clinical disease was observed and all three sheep maintained consistent food intake and activity levels. a small quantity of virus was detected in nasal swabs from sheep on days , , , , and and from sheep on day ( figure ). the remaining goats were euthanized on day post-infection and the serological status of the experimentally infected kids and their cohoused dams were assessed. each of three kid goats held past day seroconverted, however, neutralizing antibodies were not detected in either of the dams (table ) . table . neutralizing antibody titers in goats experimentally infected or exposed by contact to mers-cov. mother goats are indicated as or , and their corresponded kids are indicated as a, b, c (doe ), a, or b (doe ). titers represent dilutions of serum which neutralized ≥ % of input virus. nd = not done. three sheep were experimentally infected and evaluated for clinical disease, viral shedding, and seroconversion. no nasal discharge or clinical disease was observed and all three sheep maintained consistent food intake and activity levels. a small quantity of virus was detected in nasal swabs from sheep on days , , , , and and from sheep on day ( figure ) . unlike the goats, acute pathology in sheep was not evaluated in this study. the sheep were euthanized on day post-infection, and serum samples from each week were assessed for the presence of mers-cov neutralizing antibodies. sheep developed a low titer of neutralizing antibody on day ( ), but neutralizing antibodies were not detected in either of the other two sheep at any time-point tested ( table ). unlike the goats, acute pathology in sheep was not evaluated in this study. the sheep were euthanized on day post-infection, and serum samples from each week were assessed for the presence of mers-cov neutralizing antibodies. sheep developed a low titer of neutralizing antibody on day ( ), but neutralizing antibodies were not detected in either of the other two sheep at any time-point tested ( table ) . table . neutralizing antibody titers in sheep experimentally infected with mers-cov. titers were determined by plaque reduction neutralization test (prnt) using a % cutoff. < despite no detectable rise in rectal temperature or change in appetite and activity, horses and showed mild intermittent nasal discharge prior to inoculation and throughout the days experiment. low levels of virus were detected in nasal swab samples from three of the four inoculated horses. virus was detected on day in horse , day in horse , and day in horse ( figure ) . virus was not detected in any of the nasal swab specimens tested from horse . virus isolation was performed by plaque assay from nasal swab specimens obtained from sheep experimentally infected with mers-cov. the limit of detection for this assay was . log pfu/ml, indicated by the dashed line. table . neutralizing antibody titers in sheep experimentally infected with mers-cov. titers were determined by plaque reduction neutralization test (prnt) using a % cutoff. despite no detectable rise in rectal temperature or change in appetite and activity, horses and showed mild intermittent nasal discharge prior to inoculation and throughout the days experiment. low levels of virus were detected in nasal swab samples from three of the four inoculated horses. virus was detected on day in horse , day in horse , and day in horse ( figure ). virus was not detected in any of the nasal swab specimens tested from horse . serum was collected weekly until day (with the exception of horse , which was euthanized early due to an injury unrelated to the experiment), and evaluated for the presence of mers-cov neutralizing antibodies. unlike the inoculated goat kids, none of the infected horses seroconverted (table ). serum was collected weekly until day (with the exception of horse , which was euthanized early due to an injury unrelated to the experiment), and evaluated for the presence of mers-cov neutralizing antibodies. unlike the inoculated goat kids, none of the infected horses seroconverted (table ) . the objective of this study was to determine if goats, sheep, or horses could be experimentally infected with mers-cov. very limited amounts of infectious virus were detected in nasal swab specimens of some of the experimentally infected animals, but not in uninfected, co-housed goats. it is possible that the infectious virus detected was residual from the input virus, at least on day post-infection. however, a previous study with alpacas re-challenged days after an initial infection was not able to detect infectious virus upon re-challenge indicating that input challenge virus is not detected one day after infection, although the role of secretory antibodies was not addressed in that study [ ] . similarly, another study with alpacas re-challenged on day post-infection was unable to detect viral rna upon re-challenge [ ] . in comparison to experimentally infected dromedaries, significantly less virus was isolated from the livestock in this study. since the main objective of this study was to determine the role these animals might play in transmission of the virus, we chose to test these samples by plaque assay in order to determine the amount of infectious virus present, rather than by rt-pcr which only reveals the presence of viral rna regardless of infectivity. previous studies of naturally or experimentally infected camels and experimentally infected alpacas showed variable levels of nasal discharge. studies in camels demonstrated that infected camels have nasal discharge while infected alpacas did not have any observable discharge [ , , , ] . the goats and sheep in this study did not have any observable discharge; in contrast horses and had discharge throughout the entire study. all animals were examined and healthy prior to the study, but due to the dust associated with the housing of horses we believe this discharge was unrelated to infection and instead an effect of the environment. current evidence suggests that dromedary camels are the primary reservoir of mers-cov. however, elucidating the role that other livestock such as goats, sheep, or horses could play in transmission is important for designing field studies and biosecurity strategies, and in assessing individuals at risk for viral transmission. while in vitro studies suggested that these animals could be naturally or experimental infected, the lack of support from field data coupled with the experimental data presented here suggest that these animals are unlikely to be infected and are not important in viral transmission of mers-cov. the authors declare no conflict of interest. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia evidence for camel-to-human transmission of mers coronavirus an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia serologic assessment of possibility for mers-cov infection in equids risk factors for primary middle east respiratory syndrome coronavirus illness in humans replicative capacity of mers coronavirus in livestock cell lines host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas infection with mers-cov causes lethal pneumonia in the common marmoset experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus key: cord- -wfla l authors: popov, vsevolod l.; tesh, robert b.; weaver, scott c.; vasilakis, nikos title: electron microscopy in discovery of novel and emerging viruses from the collection of the world reference center for emerging viruses and arboviruses (wrceva) date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: wfla l since the beginning of modern virology in the s, transmission electron microscopy (tem) has been an important and widely used technique for discovery, identification and characterization of new viruses. using tem, viruses can be differentiated by their ultrastructure: shape, size, intracellular location and for some viruses, by the ultrastructural cytopathic effects and/or specific structures forming in the host cell during their replication. ultrastructural characteristics are usually sufficient for the identification of a virus to the family level. in this review, we summarize years of experience in identification of novel viruses from the collection of the world reference center for emerging viruses and arboviruses (wrceva). since the beginning of modern virology in the s, transmission electron microscopy (tem) has been one of the most important and widely used techniques for identification and characterization of new viruses. two tem techniques are generally used for this purpose: negative staining on an electron microscopic grid coated with a support film, or (ultra) thin-section tem of infected cells, fixed, pelleted, dehydrated and embedded in epoxy plastic. negative staining can be conducted on highly concentrated suspensions of purified virus or on cell culture supernatants. for some viruses, tem can be conducted on contents of skin lesions (e.g., poxviruses and herpesviruses) or on concentrated stool material (rotaviruses and noroviruses). for successful detection of viruses in ultrathin sections of infected cells, at least % of cells must be infected, so either high multiplicity of infection (moi) or rapid virus multiplication is required. viruses can be differentiated by their specific morphology (ultrastructure): shape, size, intracellular location, or from the ultrastructural cytopathic effects and specific structures forming in the host cell upolu, aransas bay [ ] , sinu [ ] , and trinity [ ] orthobunyaviruses [ ] [ ] [ ] , nyamiviruses [ ] , a new reovirus from cameroon (fako virus) [ ] and colombia [ ] , a new paramyxovirus [ ] , an insect-specific (capable of replication in insects but not in vertebrates) alphavirus [ ] , a new flavivirus genus [ ] and other novel flaviviruses [ ] [ ] [ ] [ ] and rhabdoviruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . lastly, wrceva scientists discovered the new family of mesoniviruses [ , ] and the new taxon of the negeviruses [ , ] . in the past decade, the wrceva has also been critical in contributing to the surveillance of dengue, chikungunya and zika viruses during their spread through various parts of the world and identifying their sources and mechanisms of emergence. in many of these discoveries, transmission electron microscopy (tem) was essential to determine the taxonomic position (family) of the virus, which will be demonstrated by the following examples. paramyxoviruses form at the cell surface using plasmalemma as their envelope. usually they are pleomorphic or spherical in shape, varying in size from nm to several hundred nanometers, but they can also be finger-like or filamentous ( figure a ). filamentous virions are nm to nm in diameter and can reach up to µm in length. virions have spikes at the surface~ - nm long, which make their surface look "fuzzy" in ultrathin sections. they contain a helically packed nucleocapsid which in cross-sections looks like tubules~ nm in diameter with~ nm periodicity. at earlier stages of infection, cells can contain intracytosolic inclusions consisting of nucleocapsid strands nm to nm in diameter ( figure b ). viruses , , x for peer review of paramyxovirus [ ] , an insect-specific (capable of replication in insects but not in vertebrates) alphavirus [ ] , a new flavivirus genus [ ] and other novel flaviviruses [ ] [ ] [ ] [ ] and rhabdoviruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . lastly, wrceva scientists discovered the new family of mesoniviruses [ , ] and the new taxon of the negeviruses [ , ] . in the past decade, the wrceva has also been critical in contributing to the surveillance of dengue, chikungunya and zika viruses during their spread through various parts of the world and identifying their sources and mechanisms of emergence. in many of these discoveries, transmission electron microscopy (tem) was essential to determine the taxonomic position (family) of the virus, which will be demonstrated by the following examples. paramyxoviruses form at the cell surface using plasmalemma as their envelope. usually they are pleomorphic or spherical in shape, varying in size from nm to several hundred nanometers, but they can also be finger-like or filamentous ( figure a ). filamentous virions are nm to nm in diameter and can reach up to μm in length. virions have spikes at the surface ~ - nm long, which make their surface look "fuzzy" in ultrathin sections. they contain a helically packed nucleocapsid which in cross-sections looks like tubules ~ nm in diameter with ~ nm periodicity. at earlier stages of infection, cells can contain intracytosolic inclusions consisting of nucleocapsid strands nm to nm in diameter ( figure b ). rhabdoviruses have characteristic bullet or finger-like morphology with cross-striations - nm in periodicity, reflecting helical packaging of the nucleocapsid. in cross-sections, they appear ring-like in structure ( figure c ). the size of virions varies depending on virus, with diameters ranging from - nm and lengths of - nm; some virions may be much longer, reaching nm ( figure ). defective-interfering particles are shorter and wider, giving them a conical shape. virions bud from host cell membranes either from the plasmalemma into extracellular space (figure a ,b) or into intracellular vacuoles of varying sizes ( figure c,d) . inside vacuoles, virions can be packed in stacks of up to µm ( figure d ). some rhabdoviruses, especially in animal models, form intracytoplasmic inclusions consisting of nucleoprotein, sometimes with intravacuolar virions [ ] . these inclusions correspond to negri bodies seen in rabies virus infection. rhabdoviruses have characteristic bullet or finger-like morphology with cross-striations - nm in periodicity, reflecting helical packaging of the nucleocapsid. in cross-sections, they appear ringlike in structure ( figure c ). the size of virions varies depending on virus, with diameters ranging from - nm and lengths of - nm; some virions may be much longer, reaching nm ( figure ). defective-interfering particles are shorter and wider, giving them a conical shape. virions bud from host cell membranes either from the plasmalemma into extracellular space (figure a ,b) or into intracellular vacuoles of varying sizes ( figure c,d) . inside vacuoles, virions can be packed in stacks of up to μm ( figure d ). some rhabdoviruses, especially in animal models, form intracytoplasmic inclusions consisting of nucleoprotein, sometimes with intravacuolar virions [ ] . these inclusions correspond to negri bodies seen in rabies virus infection. the family nyamiviridae includes six genera. nyamanini virus (nymv), midway virus, and sierra nevada virus (snvv) were recently assigned to the genus nyavirus [ ] [ ] [ ] . they are mostly spherical in shape and bud from the cell surface. nymv virions are - nm in diameter ( figure c ), snvv is larger and more variable in size, its virions measuring - nm in cross-sections ( figure d ). the family nyamiviridae includes six genera. nyamanini virus (nymv), midway virus, and sierra nevada virus (snvv) were recently assigned to the genus nyavirus [ ] [ ] [ ] . they are mostly spherical in shape and bud from the cell surface. nymv virions are - nm in diameter ( figure c ), snvv is larger and more variable in size, its virions measuring - nm in cross-sections ( figure d ). arboviruses assigned to the family orthomyxoviridae are predominantly tick-borne and recently classified into the genera thogotovirus and quaranjavirus [ ] . the genus thogotovirus currently comprises viruses: thogoto virus (thov) and dhori virus (dhov). probable members also include batken virus and its variants, araguari virus (arav) [ ] , jos virus (josv) [ ] , upolu virus (upov), aransas bay virus (abv) [ ] and the recently isolated bourbon virus [ ] . as for other orthomyxoviruses, virions form at the cell surface. thov and dhov exhibit significant variations in morphology: they can be pleomorphic, spherical or filamentous, nm in diameter and up to nm long. other viruses that may be assigned to this genus are mostly spherical but display some polymorphism in sizes: virions of abv can be - nm in diameter ( figure a ), upov − - nm ( figure b ), josv − - nm, arav − - nm, having spikes at their surface~ nm long. virions of bourbon virus are mostly pleomorphic, but can be spherical or filamentous [ ] . comprises viruses: thogoto virus (thov) and dhori virus (dhov). probable members also include batken virus and its variants, araguari virus (arav) [ ] , jos virus (josv) [ ] , upolu virus (upov), aransas bay virus (abv) [ ] and the recently isolated bourbon virus [ ] . as for other orthomyxoviruses, virions form at the cell surface. thov and dhov exhibit significant variations in morphology: they can be pleomorphic, spherical or filamentous, nm in diameter and up to nm long. other viruses that may be assigned to this genus are mostly spherical but display some polymorphism in sizes: virions of abv can be - nm in diameter ( figure a ), upov - - nm ( figure b ), josv - - nm, arav - - nm, having spikes at their surface ~ nm long. virions of bourbon virus are mostly pleomorphic, but can be spherical or filamentous [ ] . the genus quaranjavirus currently includes two named viruses: quaranfil virus (qrfv) and johnston atoll virus (jav). lake chad virus (lkcv) [ ] , wellfleet bay virus (wfbv), cygnet river virus [ ] and tjulok (tyulek or Тюлёк) virus (tlkv) [ ] are also probable members of the genus. these viruses are mostly spherical but also display size polymorphism: jav virions are - nm in diameter, lkcv - - nm, wfbv - - nm ( figure c ). most virions of tlkv are ~ nm in diameter but their size can vary from - nm ( figure d ). in ultrathin sections of some virions, up to seven dense granules can be observed representing ribonucleoprotein complexes [ ] . the morphology and size of the virions initially led to their misidentification as bunyaviruses or arenaviruses [ , , ] . virions mostly form at the cell surface, but with lkcv and tlkv, virions have been observed inside intracytosolic vacuoles with single or two to four virions in a tight vacuole. the genus quaranjavirus currently includes two named viruses: quaranfil virus (qrfv) and johnston atoll virus (jav). lake chad virus (lkcv) [ ] , wellfleet bay virus (wfbv), cygnet river virus [ ] and tjulok (tyulek or Тюлёк) virus (tlkv) [ ] are also probable members of the genus. these viruses are mostly spherical but also display size polymorphism: jav virions are - nm in diameter, lkcv - - nm, wfbv - - nm ( figure c ). most virions of tlkv are~ nm in viruses , , of diameter but their size can vary from - nm ( figure d ). in ultrathin sections of some virions, up to seven dense granules can be observed representing ribonucleoprotein complexes [ ] . the morphology and size of the virions initially led to their misidentification as bunyaviruses or arenaviruses [ , , ] . virions mostly form at the cell surface, but with lkcv and tlkv, virions have been observed inside intracytosolic vacuoles with single or two to four virions in a tight vacuole. from the largest family of rna viruses (bunyaviridae), we illustrate the morphology of new and emerging tick-borne viruses that are likely to be assigned to the genus phlebovirus. phleboviruses are spherical, enveloped viruses - nm in diameter ( figure ) sometimes with a dense nucleocapsid core ( figure d ). virion envelopes are formed either from the host cell plasmalemma ( figure a ,d) or from the membrane of small intracytosolic vesicles ( figure b ,c), usually originating in the golgi. envelope glycoproteins are organized into small hollow cylindrical units~ nm in diameter arranged in an icosahedral surface lattice revealed in negatively stained virions [ ] . from the largest family of rna viruses (bunyaviridae), we illustrate the morphology of new and emerging tick-borne viruses that are likely to be assigned to the genus phlebovirus. phleboviruses are spherical, enveloped viruses - nm in diameter ( figure ) sometimes with a dense nucleocapsid core ( figure d ). virion envelopes are formed either from the host cell plasmalemma ( figure a ,d) or from the membrane of small intracytosolic vesicles ( figure b ,c), usually originating in the golgi. envelope glycoproteins are organized into small hollow cylindrical units ~ nm in diameter arranged in an icosahedral surface lattice revealed in negatively stained virions [ ] . flaviviruses are spherical enveloped viruses ~ nm in diameter in ultrathin sections, usually with a dense nucleocapsid core. immature virions are larger in size (~ nm) and are located inside the cisterns of granular endoplasmic reticulum within infected cells ( figure ). replicating flaviviruses induce formation of characteristic intracytoplasmic membraneous structures: convoluted membranes ( figure c ), paracrystalline arrays and smooth membrane structures (sms) within usually expanded cisterns of granular endoplasmic reticulum ( figure a ,b), visible in conventional (epoxy plastic-embedded) ultrathin sections, or vesicle packets in ultrathin cryo-sections [ ] . these structures apparently are connected with each other and the membrane system of the cell flaviviruses are spherical enveloped viruses~ nm in diameter in ultrathin sections, usually with a dense nucleocapsid core. immature virions are larger in size (~ nm) and are located inside the cisterns of granular endoplasmic reticulum within infected cells ( figure ). replicating flaviviruses induce formation of characteristic intracytoplasmic membraneous structures: convoluted membranes ( figure c ), paracrystalline arrays and smooth membrane structures (sms) within usually figure a,b) , visible in conventional (epoxy plastic-embedded) ultrathin sections, or vesicle packets in ultrathin cryo-sections [ ] . these structures apparently are connected with each other and the membrane system of the cell (endoplasmic reticulum and golgi) and serve as sites of virus rna replication and processing thus representing flavivirus replication complexes [ , ] . the presence of even one of these structures can serve as an identifier of an infection of the cell with a flavivirus. sms can be spherical, - nm in diameter, or slightly elongated ( figure a ,b) but in some viruses they can be extremely long, up to µm ( figure d ). representing flavivirus replication complexes [ , ] . the presence of even one of these structures can serve as an identifier of an infection of the cell with a flavivirus. sms can be spherical, - nm in diameter, or slightly elongated ( figure a ,b) but in some viruses they can be extremely long, up to μm ( figure d ). in ultrathin sections of infected cells virions of arthropod-borne reoviruses appear as spherical particles - nm in diameter with central dense cores (figures c,d and a ). family reoviridae includes two subfamilies, spinareovirinae and sedoreovirinae, according to morphology of virion surface which can be visualized in negatively stained preparations of purified viruses. virions of viruses assigned to the spinareovirinae have short flat spikes or turrets. in fako virus (unassigned; probable genus dinovernavirus), recently isolated from mosquitoes in cameroon [ ] , these turrets are ~ nm tall and ~ nm wide ( figure a ). in the sedoreovirinae, virions are devoid of spikes but can display round surface capsomere subunits ~ nm in diameter ( figure b ). reoviruses reproduce in cytoplasmic inclusions -virus factories, or viroplasms which appear in ultrathin sections as medium density masses composed of fine fibrils and granules and containing virus particles of different stages of maturity and empty shells ( figures c,d and a ). in coltiviruses (genus coltivirus) and orbiviruses (genus orbivirus), virus factories are often associated with randomly distributed fibrils and/or microtubules as demonstrated by f.a. murphy [ , ] . in ultrathin sections of infected cells virions of arthropod-borne reoviruses appear as spherical particles - nm in diameter with central dense cores ( figure c ,d and figure a ). family reoviridae includes two subfamilies, spinareovirinae and sedoreovirinae, according to morphology of virion surface which can be visualized in negatively stained preparations of purified viruses. virions of viruses assigned to the spinareovirinae have short flat spikes or turrets. in fako virus (unassigned; probable genus dinovernavirus), recently isolated from mosquitoes in cameroon [ ] , these turrets are~ nm tall and~ nm wide ( figure a ). in the sedoreovirinae, virions are devoid of spikes but can display round surface capsomere subunits~ nm in diameter ( figure b ). reoviruses reproduce in cytoplasmic inclusions -virus factories, or viroplasms which appear in ultrathin sections as medium density masses composed of fine fibrils and granules and containing virus particles of different stages of maturity and figure a ). in coltiviruses (genus coltivirus) and orbiviruses (genus orbivirus), virus factories are often associated with randomly distributed fibrils and/or microtubules as demonstrated by f.a. murphy [ , ] . arenavirus virions are spherical and pleomorphic with variable sizes, from nm to nm in diameter ( figure b ). they are surrounded by an envelope ~ nm thick covered with spikes and typically contain several ribosomes nm to nm in diameter. virions bud from the cell surface ( figure b ). alphavirus virions are icosahedral particles which appear spherical in ultrathin sections, - nm in diameter with dense nucleocapsid core ( figure c ). alphavirus replication complexes are associated with specific vesicles -spherules formed as invaginations of either plasma membrane (in vertebrate cells), [ ] or limiting membranes of endosomes (occasionally in vertebrate but mostly in mosquito cells) [ , ] . these spherule-loaded endosomes (and/or lysosomes) are termed "type cytopathic vacuoles" [ ] and are very characteristic for alphavirus infection ( figure d ) although recently they have been encountered in negevirus-infected c / mosquito cells ( figure d ). alphavirus dense nucleocapsid cores ~ nm in diameter can accumulate around cytopathic vacuoles [ ] and can be found in the cytoplasm close to plasma membrane ( figure c , arrows). alphavirus virions form by budding from the plasma membrane and can form paracrystalline arrays at the cell surface ( figure c ). electron microscopy was instrumental in the recent discovery of the insectspecific alphavirus, eilat virus ( figure b,c) , which cannot replicate in mammalian cells [ ] , allowing it to serve as a safe platform for vaccine development [ , ] . mesoniviruses were recently classified as a family [ , ] in the order nidovirales, being distantly related to coronaviruses (family coronaviridae, order nidovirales). they appear to be very common and widespread in mosquito populations from different geographical and ecological locations [ ] . mature virions are spherical ~ nm in diameter with a dense nucleocapsid core ~ nm in diameter arenavirus virions are spherical and pleomorphic with variable sizes, from nm to nm in diameter ( figure b ). they are surrounded by an envelope~ nm thick covered with spikes and typically contain several ribosomes nm to nm in diameter. virions bud from the cell surface ( figure b ). alphavirus virions are icosahedral particles which appear spherical in ultrathin sections, - nm in diameter with dense nucleocapsid core ( figure c ). alphavirus replication complexes are associated with specific vesicles -spherules formed as invaginations of either plasma membrane (in vertebrate cells), [ ] or limiting membranes of endosomes (occasionally in vertebrate but mostly in mosquito cells) [ , ] . these spherule-loaded endosomes (and/or lysosomes) are termed "type cytopathic vacuoles" [ ] and are very characteristic for alphavirus infection ( figure d ) although recently they have been encountered in negevirus-infected c / mosquito cells ( figure d ). alphavirus dense nucleocapsid cores~ nm in diameter can accumulate around cytopathic vacuoles [ ] and can be found in the cytoplasm close to plasma membrane ( figure c , arrows). alphavirus virions form by budding from the plasma membrane and can form paracrystalline arrays at the cell surface ( figure c ). electron microscopy was instrumental in the recent discovery of the insect-specific alphavirus, eilat virus ( figure b,c) , which cannot replicate in mammalian cells [ ] , allowing it to serve as a safe platform for vaccine development [ , ] . mesoniviruses were recently classified as a family [ , ] in the order nidovirales, being distantly related to coronaviruses (family coronaviridae, order nidovirales). they appear to be very common and widespread in mosquito populations from different geographical and ecological locations [ ] . mature virions are spherical~ nm in diameter with a dense nucleocapsid core~ nm in diameter and surrounding envelope ( figure a ). usually they are found within large vacuoles often filling their entire space ( figure a ) but can be localized in individual small vacuoles and at the cell surface ( figure b ). some infected cells had intravacuolar paracrystalline arrays consisting of empty and full virus particles but with less electron density than mature virions. at the periphery of these arrays, mature virions could be observed either free in cytosol or inside vacuoles [ ] . viruses , , x for peer review of and surrounding envelope ( figure a ). usually they are found within large vacuoles often filling their entire space ( figure a ) but can be localized in individual small vacuoles and at the cell surface ( figure b ). some infected cells had intravacuolar paracrystalline arrays consisting of empty and full virus particles but with less electron density than mature virions. at the periphery of these arrays, mature virions could be observed either free in cytosol or inside vacuoles [ ] . insect-specific viruses isolated recently from mosquitoes and phlebotomine sandflies have been characterized and proposed to represent a new genus (negevirus) related to genera of mite-infecting plant viruses (blunervirus, cilevirus, and higrevirus) in the new family kitaviridae [ , ] , or novel members of entomobirnavirus, family birnaviridae ( figure d ). they appear to be very common and widespread in insect populations on different continents as isolates have been obtained from pools of mosquitoes and sandflies collected in israel, north and south america, africa, and indonesia. in negatively stained preparations of piura virus, isolated from culex sp. mosquitoes in peru, spherical particles with diameters of ~ nm and ~ nm were found. the striking feature of negevirus infection insect-specific viruses isolated recently from mosquitoes and phlebotomine sandflies have been characterized and proposed to represent a new genus (negevirus) related to genera of mite-infecting plant viruses (blunervirus, cilevirus, and higrevirus) in the new family kitaviridae [ , ] , or novel members of entomobirnavirus, family birnaviridae ( figure d ). they appear to be very common and widespread in insect populations on different continents as isolates have been obtained from pools of mosquitoes and sandflies collected in israel, north and south america, africa, and indonesia. in negatively stained preparations of piura virus, isolated from culex sp. mosquitoes in peru, spherical particles with diameters of~ nm and~ nm were found. the striking feature of negevirus infection of c / mosquito cells are the cytopathic effects -enormous expansions of perinuclear space -a granular endoplasmic reticulum system which becomes filled with vesicles and/or microtubules ( figure c , figure a ,b andfigure a). they have a diameter of - nm but can be of different lengths. the shortest can resemble a rice grain in morphology and can be arranged in rosettes in cross-sections of er. others can be very long, reaching up to several micrometers ( figures c and a,b) . expanded perinuclear space can occupy almost the whole cytoplasm of the cell. some negeviruses cause long protrusions of the nucleus and deep invaginations of perinuclear membranes leading to the fragmentation of the nucleus ( figure b ). another peculiarity of negevirus infection of c / cells is the formation of cytopathic vacuoles with spherule-like structures - nm in diameter at the inner periphery of their limiting membrane ( figure c,d) . these vacuoles can reach . µm in diameter and are morphologically similar to type cytopathic vacuoles in alphaviruses. they have been found in all described negeviruses [ ] and later in many others that have not yet been fully characterized. their role in negevirus replication is not known. the wrceva collection at the university of texas medical branch in galveston, texas, plays an important role in virology research as a depository of natural viral biodiversity, and as a valuable resource for knowledge of viruses that are not only pathogenic for animals and humans but also are naturally occurring in arthropods. propagation in cell cultures and subsequent examination of virus morphology by electron microscopy have been useful initial steps in the identification and characterization of novel viruses, giving indications for their further genetic characterization. author contributions: all authors contributed sections for the completion of this manuscript. sometimes, after inoculation of c / mosquito cells with homogenates of mosquito pools, several viruses can be observed in the culture, or even in the same cell. figure c illustrates mixed infection with karang sari virus (genus alphamesonivirus) (virions nm in diameter inside er cisterns) and an unknown flavivirus (smaller virions, nm in diameter in different er cisterns). figure d demonstrates a co-infection with a kamphaeng phet virus (unclassified; probable genus alphamesonivirus; virions nm in diameter) and an unknown reovirus (virions nm in diameter with a characteristic dark nucleocapsid core). we have also observed co-infection with bontag baru virus (unclassified; probable genus alphamesonivirus) and an unknown flavivirus, ngewontan (unclassified; proposed genus negevirus) and nam dinh virus (genus alphamesonivirus); eilat virus (genus alphavirus) ( figure b ,c) and negev virus (unclassified; proposed genus negevirus) ( figure a ). three viruses have also been observed to infect the same culture: bontag baru virus (unclassified; probable genus alphamesonivirus), an unknown flavivirus and an unknown rhabdovirus, the latter budding into an er cistern occupied by a flavivirus virion. it is most likely that these viruses originate from different individual mosquitoes in the pool, but the possibility of a co-infection of one mosquito with several different viruses cannot be excluded. the wrceva collection at the university of texas medical branch in galveston, texas, plays an important role in virology research as a depository of natural viral biodiversity, and as a valuable resource for knowledge of viruses that are not only pathogenic for animals and humans but also are naturally occurring in arthropods. propagation in cell cultures and subsequent examination of virus morphology by electron microscopy have been useful initial steps in the identification and characterization of novel viruses, giving indications for their further genetic characterization. author contributions: all authors contributed sections for the completion of this manuscript. funding: this work was supported in part by nih grant r ai and contract hhsn /d . the funding agencies had no involvement in the writing of the report or in the decision to submit this article for publication. the foundations of virology -discoverers and discoveries, inventors and inventions, developers and technologies the foundations of virology -discoverers and discoveries, inventors and inventions, developers and technologies 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mosquito-specific viruses with extensive geographic distribution and host range negevirus: a proposed new taxon of insect-specific viruses with wide geographic distribution genetic characterization, molecular epidemiology, and phylogenetic relationships of insect-specific viruses in the taxon negevirus quaranfil, johnston atoll, and lake chad viruses are novel members of the family orthomyxoviridae araguari virus, a new member of the family orthomyxoviridae: serologic, ultrastructural, and molecular characterization genomic and antigenic characterization of jos virus novel thogotovirus associated with febrile illness and death, united states cyclic avian mass mortality in the northeastern united states is associated with a novel orthomyxovirus taxonomic status of the tyulek virus (tlkv) (orthomyxoviridae, quaranjavirus, quaranfil group) isolated from the ticks argas vulgaris filippova, (argasidae) from the birds burrow nest biotopes in the kyrgyzstan tick-borne viruses structurally similar to orthomyxoviruses electron microscopy and antigenic studies of uncharacterized viruses. i. evidence suggesting the placement of viruses in families arenaviridae, paramyxoviridae, or poxviridae generation and analysis of infectious virus-like particles of uukuniemi virus (bunyaviridae): a useful system for studying bunyaviral packaging and budding ultrastructure of kunjin virus-infected cells: colocalization of ns and ns with double-stranded rna, and of ns b with ns , in virus-induced membrane structures wrapping things up about virus rna replication modification of intracellular membrane structures for virus replication genus coltivirus (family reoviridae): genomic and morphologic characterization of old world and new world viruses functional sindbis virus replicative complexes are formed at the plasma membrane alphavirus rna replicase is located on the cytoplasmic surface of endosomes and lysosomes the host range phenotype displayed by a sindbis virus glycoprotein variant results from virion aggregation and retention on the surface of mosquito cells cytoplasmic structures associated with an arbovirus infection: loci of viral ribonucleic acid synthesis eilat virus host range restriction is present at multiple levels of the virus life cycle novel insect-specific eilat virus-based chimeric vaccine candidates provide durable, mono-and multivalent, single-dose protection against lethal alphavirus challenge a chikungunya fever vaccine utilizing an insect-specific virus platform an insect nidovirus emerging from a primary tropical rainforest identification and characterization of genetically divergent members of the newly established family mesoniviridae the authors thank julie wen and zhixia ding for expert assistance in electron microscopy and dora salinas for excellent help in preparation of the manuscript.funding: this work was supported in part by nih grant r ai and contract hhsn /d . the funding agencies had no involvement in the writing of the report or in the decision to submit this article for publication. the authors declare no conflicts of interest. the authors thank julie wen and zhixia ding for expert assistance in electron microscopy and dora salinas for excellent help in preparation of the manuscript. the authors declare no conflict of interest. key: cord- -amv los authors: widagdo, w.; sooksawasdi na ayudhya, syriam; hundie, gadissa b.; haagmans, bart l. title: host determinants of mers-cov transmission and pathogenesis date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: amv los middle east respiratory syndrome coronavirus (mers-cov) is a zoonotic pathogen that causes respiratory infection in humans, ranging from asymptomatic to severe pneumonia. in dromedary camels, the virus only causes a mild infection but it spreads efficiently between animals. differences in the behavior of the virus observed between individuals, as well as between humans and dromedary camels, highlight the role of host factors in mers-cov pathogenesis and transmission. one of these host factors, the mers-cov receptor dipeptidyl peptidase- (dpp ), may be a critical determinant because it is variably expressed in mers-cov-susceptible species as well as in humans. this could partially explain inter- and intraspecies differences in the tropism, pathogenesis, and transmissibility of mers-cov. in this review, we explore the role of dpp and other host factors in mers-cov transmission and pathogenesis—such as sialic acids, host proteases, and interferons. further characterization of these host determinants may potentially offer novel insights to develop intervention strategies to tackle ongoing outbreaks. middle east respiratory syndrome coronavirus (mers-cov) is a novel pathogen that was isolated in late [ ] . since then, the virus has caused multiple outbreaks and infected more than individuals, [ ] who then develop a respiratory infection ranging in severity from asymptomatic to fatal [ , ] . severe-to-fatal mers-cov patients have a higher chance of transmitting this virus since they shed a higher amount of virus progeny in comparison to the asymptomatic-to-mild ones [ ] [ ] [ ] [ ] . identifying and quarantining these patients in healthcare facilities where outbreaks have occurred, together with implementing proper infection control, has been effective in reducing transmission and containing these outbreaks [ , ] . however, new mers-cov cases are still being reported, especially in the arabian peninsula [ , ] . this is partly due to the continuous zoonotic introduction of this virus to the human population in this region by dromedaries [ ] . the dromedary camel is the only animal species that has been reported to transmit this virus to humans [ ] [ ] [ ] [ ] . mers-cov infection in these animals merely causes mild upper respiratory tract infection [ , ] , but seroepidemiological studies showed that this virus has been circulating in dromedary camels for decades, suggesting the efficient transmission of mers-cov in this species [ ] [ ] [ ] [ ] . although the clinical manifestations, as well as transmission, are remarkably different in mers-cov-infected humans and dromedary camels, the viruses isolated from these two species are highly similar, if not indistinguishable [ , ] . this indicates that host factors play a significant role in mers-cov pathogenesis and transmission. however, the identity of these host factors and how they affect the pathogenesis and transmission of mers-cov are generally not well understood. dipeptidyl peptidase- (dpp )-the mers-cov receptor, sialic acids, proteases, and interferons are ; a cartoon representation of mers-cov s protein binding to dpp (pdb code l ) (b). the s protein consists of the s and s subunits. the α/β hydrolase domain of dpp is indicated in red, β-propeller domain in green, while part of the mers-cov s protein is shown in blue. other mers-cov-interacting host factors besides dpp are less extensively studied and have mostly been investigated in vitro. glycotopes of α , -sialic acids coupled with -n-acetylated neuraminic acid are recognized by the s protein of mers-cov during attachment [ ] . in the absence of these glycotopes, mers-cov entry is reduced but not abolished, indicating their function as an attachment factor rather than a receptor [ ] . besides α , -sialic acids, ceacam and grp have also been suggested to be attachment factors for mers-cov, but their roles in vivo during mers-cov infection are not clear at this moment [ , ] . post attachment, mers-cov uses the c-terminal part of its s protein-known as s ( figure a )-to interact with host proteases, such as furin, tmprss , and cathepsins [ ] [ ] [ ] [ ] . these proteases cleave the s protein and induce conformational changes, allowing fusion between viral and host cellular membranes, resulting in the release of viral rna into the cell cytoplasm [ ] . tmprss and dpp are held in one complex at the cell surface by a scaffolding protein, the tetraspanin cd , leading to a rapid and efficient entry of mers-cov into the susceptible cells [ ] . once fusion with host cell membranes has occurred, mers-cov subsequently replicates its genetic material and produces viral proteins in the cell cytoplasm to generate new virus progeny. during this stage, mers-cov uses its nsp - polyproteins to build its replication organelles as well as its accessory proteins such as the a and b proteins to inhibit host anti-viral defense mechanisms [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, the capacity of mers-cov accessory proteins to impede several pathways of host immune response in the lungs may be limited. mers-cov inoculation of macaques and genetically modified mice generally results in limited clinical manifestations; thus, adapting this virus through serial passaging or defecting the type i interferon pathway may be needed to enhance viral replication and pathogenesis in these animals [ , [ ] [ ] [ ] [ ] . these observations, together with studies showing type i interferon capacity to inhibit mers-cov infection in vitro [ , ] , highlight the importance of the innate immune response, especially type i interferon, as an inhibiting factor for mers-cov. so far mers-cov has been isolated from dromedary camels and humans [ , ] . both species are not only susceptible to mers-cov infection, but also capable of transmitting this virus [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] ]. however, current data indicate that virus spread is more efficient in dromedary camels than in humans [ , , [ ] [ ] [ ] ] . this difference in transmissibility could be partially due to the different tropism of mers-cov in these two species. in dromedaries, mers-cov has been shown to replicate in the nasal epithelium upon experimental in vivo infection [ ] , while in humans, mers-cov mainly replicates in the lower respiratory tract, particularly in the bronchiolar and alveolar epithelia [ , [ ] [ ] [ ] [ ] . higher viral rna levels in the sputum and lavage samples of mers-cov patients compared to nasal and throat swabs are consistent with the tropism of mers-cov in humans [ ] [ ] [ ] . this different mers-cov tropism in dromedary camels and humans is in line with the localization of dpp in the respiratory tract tissues of these two species. in humans, dpp is absent in the nasal epithelium but present in the lower respiratory tract epithelium, mainly in type ii pneumocytes [ , ] . in contrast, dpp is expressed in the nasal epithelium of dromedary camels [ ] . this difference in dpp localization between humans and dromedary camels therefore explains mers-cov tropism in these two species and highlights dpp as an essential determinant of mers-cov tropism. dpp localization has also been investigated in many other mers-cov-susceptible species. in gambian and egyptian fruit bats, dpp is expressed in the respiratory tract and intestinal epithelium, suggesting that mers-cov can target both tissues [ ] . in line with this finding, mers-cov inoculation via intranasal and intraperitoneal routes in the jamaican fruit bat led to viral rna shedding both in the respiratory tract and the intestinal tract [ ] . in contrast to frugivorous bats, dpp is limitedly expressed in the respiratory tract epithelium of two insectivorous bats, i.e., common pipistrelle and common serotine bats, but abundant in their intestinal epithelium [ ] . accordingly, sequences of mers-like-covs were mainly obtained from rectal swabs and fecal samples of insectivorous bats [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these findings not only support insectivorous bats as the origin host of mers-cov [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , but also indicate the importance of intestinal tropism and fecal-oral transmission of mers-like-cov in these insectivorous bats. besides bats, humans, and dromedary camels, other animal species have also been proposed as potential hosts of mers-cov. remarkably, dpp of horses, llamas, alpacas, pigs, bovines, goats, sheep, and rabbits has been demonstrated to recognize the s protein of mers-cov [ , ] . in most of these species, there is a preferential upper respiratory tract expression of dpp observed. rabbits express dpp in the upper and lower respiratory tract epithelium, and thus may allow mers-cov to replicate in both compartments [ , ] . horses, llamas, and pigs mainly express dpp in the upper respiratory tract-particularly the nasal epithelium [ ] . upon intranasal mers-cov inoculation, llamas, alpacas, and pigs developed upper respiratory tract infection, while horses did not seroconvert and only shed infectious virus in a limited amount [ ] [ ] [ ] [ ] [ ] . the reason why horses seem to be less permissive to mers-cov remains to be investigated, but a chronic co-infection in the guttural pouch, a common disease among horses, might be one of the explanations. this guttural pouch infection results in excessive mucus production that might hinder mers-cov from attaching and entering the nasal epithelium [ , , ] . sheep, on the other hand, did not seem to express significant levels of dpp in their respiratory tract, and thus did not seroconvert nor shed infectious virus upon experimental mers-cov inoculation [ , ] . comparable to sheep, goats limitedly shed infectious virus upon experimental infection and did not transmit this virus to other naïve goats upon direct contact [ ] . the results of experimental mers-cov infection in livestock animals are in line with data from epidemiological studies. mers-cov seropositive llamas and alpacas are present in the field, while horses, goats, and sheep are generally found to be seronegative [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . given the fact that experimental in vivo infection studies and dpp expression analysis in different animal species revealed that dromedary camels are not the only animals in which mers-cov has an upper respiratory tract tropism [ , , , ] , it is then relevant to question whether other animals can potentially spread mers-cov as well. new world camelids, i.e., alpacas and llamas, are able to transmit the virus to respective naïve animals upon contact [ ] . pigs and rabbits, on the other hand, hardly transmit the virus-neither by contact nor airborne routes [ , ] . most likely, this is caused by the fact that pigs and rabbits, unlike dromedary camels, shed low levels of infectious virus upon mers-cov inoculation ( figure ) . this difference indicates that other host factors besides dpp could cause interspecies variation in mers-cov infection. indeed, several glycotopes of α , -sialic acids that function as attachment factors of mers-cov are present in the nasal epithelium of dromedary camels but absent in that of rabbits and pigs ( figure ) [ , ] . the lack of these glycotopes in pigs and rabbits might limit the susceptibility and transmission of mers-cov in these animals. although the role of these glycotopes in mers-cov transmission still requires further investigation, it remains plausible that an efficient transmission of this virus might require the presence of both dpp and mers-cov-recognized glycotopes of α , -sialic acids (figure ). . it has also been reported that the lysosomal proteases from bat cells support coronavirus spike-mediated virus entry more efficiently than their counterparts from human cells [ ] . these observations suggest that host proteases from different host species may determine the species and tissue tropism of mers-cov. because mers-cov has been circulating in dromedary camels for decades before emerging in the human population [ ] [ ] [ ] [ ] , it is plausible that this virus inhibits the immune response of dromedary camels more efficiently than that of other species, including pigs and rabbits. the difference in immune response among mers-cov-susceptible species is therefore another factor that might yield interspecies variation in permissiveness to mers-cov. characterizing the difference in host proteases and immune responses among mers-cov-susceptible species, as performed for dpp and mers-cov-recognized α , -sialic acid glycotopes (figure ) , has not yet been investigated. these data, however, may further explain interspecies variation in mers-cov infection and transmission. mers-cov causes respiratory infection in humans ranging from asymptomatic to severe pneumonia [ , ] . however, it is currently unclear what causes this intraspecies variation. epidemiology data indicate that individuals with certain risk factors are at higher risk of developing severe mers-cov infection [ , ] . this implies that some host factors may dictate the outcome of mers-cov infection, thus rendering intraspecies variation. two of the risk factors, i.e., smoking and chronic obstructive pulmonary disease (copd), have been shown to upregulate dpp expression in the lungs [ , [ ] [ ] [ ] , suggesting dpp as a possible reason for intraspecies variation observed among mers-cov patients. in healthy human lungs, dpp is almost exclusively expressed in type ii pneumocytes [ , ] . type ii pneumocytes are small cuboidal cells that can regenerate alveolar epithelium upon injury, and roughly cover % of the alveolar surface area. meanwhile, around % of the surface area of the alveolus is occupied by type i pneumocytes that are morphologically flat and responsible for gas exchange [ , ] . in the lungs of smokers and copd patients, unlike in healthy human lungs, dpp is prominently expressed in both type i and ii pneumocytes, indicating upregulated expression on type i pneumocytes [ ] . autopsy reports from fatal mers-cov patients showed that both type i and ii pneumocytes expressed dpp and became infected by mers-cov, proposing a role of dpp -expressing type i pneumocytes in mers-cov pathogenesis [ , ] . damage to type i cells in the lung alveoli during viral infection may lead to diffuse alveolar damage [ ] . in line with observations made in human mers cases, common marmosets that express dpp in both type i and ii pneumocytes have been reported to produce more infectious virus upon experimental mers-cov infection, compared to rhesus and cynomolgus macaques that merely expressed dpp in type ii pneumocytes [ , [ ] [ ] [ ] [ ] . accordingly, these common marmosets developed moderate-to-severe infection, while macaques generally developed mild transient pneumonia [ , , [ ] [ ] [ ] [ ] . similarly, in genetically modified mice that displayed mers-cov tropism for type ii pneumocytes, only mild clinical manifestations were observed upon mers-cov infection [ , ] . adapting mers-cov through serial passaging or upregulating dpp expression throughout the airway epithelium in mice, however, will induce severe clinical disease [ , ] . these data altogether support the role of dpp -expressing type i pneumocytes in the pathogenesis of severe mers-cov infection. the differential expression of host factors that limits the infection should also be taken into account. dpp in soluble form has been demonstrated to protect against mers-cov infection in vitro and in a mouse model [ , ] ; however, its presence in the lungs and role in mers-cov pathogenesis remain to be investigated. the host immune response also has the capacity to inhibit mers-cov infection. mers-cov has been shown to replicate to higher levels in immunocompromised rhesus macaques [ ] , consistent with the observation that immunocompromised individuals have difficulties clearing mers-cov upon infection [ , , ] . the survivors of mers-cov infection have been shown to develop virus-specific cd + and cd + t cell responses, implying the role of t cells in virus clearance [ ] . however, the depletion of t cells in mice can either lead to failure in mers-cov clearance or improvement in clinical outcome, depending on the type of mouse model used [ , ] . therefore, the role of adaptive immune response in mers-cov pathogenesis is currently unclear. on the other hand, one of the main components of the host innate immune response, type i interferon, inhibits mers-cov replication in susceptible cells, partly by inhibiting double membrane vesicles (dmv) formation [ , , , , ] . the absence of type i interferon signaling in mice also resulted in more severe clinical manifestations and histopathological lesions upon mers-cov infection [ ] . advance age, which can cause delayed type i interferon response upon viral infection, is a well-known risk factor for fatal mers-cov infection [ , , [ ] [ ] [ ] . collectively, these data highlight the role of host innate immune response as a potent inhibitor for mers-cov infection. it is indubitable that severe mers-cov infection is not solely driven by the pathogen. additional underlying conditions increase mers-cov replication and induce severe-to-fatal clinical manifestations [ , , , , ] . it is plausible that more than one underlying condition is needed to yield a fatal outcome. dpp upregulation in type i pneumocytes and insufficient type i interferon response might be crucial determinants for severe mers-cov infection (figure ). further investigation of the host determinants of mers-cov pathogenesis may offer insights for developing novel therapeutic measures. although mers-cov has been reported to undergo some genotypic changes since it emerged in the human population [ , [ ] [ ] [ ] [ ] , this has not resulted in distinct phenotypic changes so far [ , ] . therefore, host factors remain the most significant determinant in explaining inter-and intraspecies variations observed in mers-cov pathogenesis and transmission. dpp and mers-cov-recognized α , -sialic acids might partially explain these variations, since their localization has been demonstrated to be variable between mers-cov-susceptible species [ , , , ] . dpp expression in human lungs has also been shown to vary due to certain comorbidities [ , , ] . nevertheless, it is undoubtable that the inter-and intraspecies variation in mers-cov pathogenesis and transmission is a complex phenomenon influenced by more than one host 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recombinant virus multihospital outbreak of a middle east respiratory syndrome coronavirus deletion variant deletion variants of middle east respiratory syndrome coronavirus from humans key: cord- - me p authors: lópez-roig, marc; bourhy, hervé; lavenir, rachel; serra-cobo, jordi title: seroprevalence dynamics of european bat lyssavirus type in a multispecies bat colony date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: me p we report an active surveillance study of the occurrence of specific antibodies to european bat lyssavirus type (eblv- ) in bat species, scarcely studied hitherto, that share the same refuge. from to , sera were obtained from nine bat species. blood samples were subjected to a modified fluorescent antibody virus neutralization test to determine the antibody titer. eblv- -neutralizing antibodies were detected in six of the nine species analyzed (pipistrellus pipistrellus, p. kuhlii, hypsugo savii, plecotus austriacus, eptesicus serotinus and tadarida teniotis). among all bats sampled, female seroprevalence ( . %, % ci: . %– . %) was not significantly higher than the seroprevalence in males ( . %, % ci: . %– . %). the results showed that the inter-annual variation in the number of seropositive bats in t. teniotis and p. austriacus showed a peak in (> % of eblv- prevalence). however, significant differences were observed in the temporal patterns of the seroprevalence modeling of t. teniotis and p. austriacus. the behavioral ecology of these species involved could explain the different annual fluctuations in eblv- seroprevalence. wildlife plays a key role in emerging infectious diseases by providing a -zoonotic pool‖ from which pathogens may emerge [ ] . zoonotic pathogens represent approximately % of all pathogens able to infect humans [ ] . in recent years, bats have been implicated in numerous emerging infectious disease events and have been recognized as important reservoir hosts for viruses that can cross the species barrier to infect humans and other domestic and wild mammals [ ] . the role of bats in viral diseases is well established, particularly their role as hosts for lyssaviruses, coronaviruses, flaviviruses, astroviruses and adenoviruses [ ] [ ] [ ] . bats have several unique features that may maximize their effectiveness as reservoir hosts for viruses. bats are the second largest order of mammals. currently, there are about recognized bat species worldwide, accounting for approximately % of all mammalian species. bats have the potential to rapidly and widely spread viruses (having a high mobility, they are the only mammals capable of flight). they have a long lifespan and a high survival rate, and many bat species have a gregarious behavior. bats can fly long distances between their summer and overwintering sites, permitting the exchange of viruses between conspecifics or bats of other species, i.e., in france, rabies virus infections have been associated with the migratory routes of nathusius' pipistrelle (pipistrellus nathusii) bats [ ] . persistent viral infections occurring among long-lived bats, coupled with their often gregarious roosting behavior, could greatly increase the potential for intra-and inter-species transmission of viruses [ ] , especially in summer and winter periods. seasonality in temperate zone bats includes birthing periods, migration, gregarious behavior and torpor. each of these strategies may affect population density, contact rates and immune response, thus leading to spatiotemporal variation in infection dynamics [ , ] . numerous bat species have been found to be infected by lyssaviruses [ ] . bats serve as reservoirs of of the lyssavirus species described (the only lyssavirus species that have not been isolated from bats, to date, are mokola virus and ikoma virus). furthermore, recently described lyssavirus species enlarged the genetic diversity of lyssaviruses found in bats [ ] [ ] [ ] , suggesting that the lyssaviruses originated in these mammals and progressively diverged from a common ancestor [ , ] . in europe, four of the lyssavirus species recognized, european bat lyssavirus types and (eblv- and eblv- , respectively), bokeloh bat lyssavirus (bblv), the west caucasian bat virus (wcbv) and one tentative species, lleida bat lyssavirus, circulate among several bat species [ , , ] . eblv- is widely distributed throughout europe, and two variants have distinct distributions and evolution histories: one is eblv- a, which has an east-west distribution from russia to france, with very little genetic variation; and the other is eblv- b, which exhibits a south-north distribution and far more genetic diversity [ ] . different studies showed that lyssavirus dynamics exhibits a strong seasonal pattern [ ] and that the breeding period could favor the infection of bats [ ] [ ] [ ] . many bat species roost in very large and dense maternity colonies. this dense clustering of individuals can provide large opportunities for viral exchange in bat colonies [ ] . previous studies have observed a higher seroprevalence in multispecies colonies compared to monospecific colonies, suggesting that interspecific virus transmission plays an important role in eblv- dynamics [ ] . however, in some cases, infection cycles may be maintained among specific host species and transmission may be minimal among sympatric bats [ ] . furthermore, differences in the ecological behavior of species (e.g., migration, torpor) can drive different bat infection dynamics. in this sense, a higher number of species might not only increase the rates of contact between bat groups, but could also facilitate virus entry or spread through the higher mobility of individuals among colonies, especially if there are migratory species involved [ ] . few studies have addressed the inter-annual dynamics of lyssavirus among bat multispecies that are roosting in the same refuge, despite these studies giving a better understanding of the dynamics of bat lyssaviruses. our previous investigations have analyzed the temporal dynamics of lyssavirus in one bat species (myotis myotis) roosting in two colonies [ , ] . the present report is based on a long-term (nine years) longitudinal study of the prevalence of eblv- neutralizing antibodies and provides the first report on the inter-annual dynamics of eblv- in p. austriacus and t. teniotis, both being bat species scarcely studied hitherto. we chose this locality, because we found three species (p. pipistrellus, p. austriacus and t. teniotis) that were eblv- rna-positive by nested reverse transcriptase-polymerase chain reaction in the first year of study [ ] . our specific goals were: (i) to provide information about eblv- seroprevalence in the wild bat community where several european bat species share the same refuge; and (ii) to compare the temporal patterns of seroprevalence mainly in two less-studied bat species that, moreover, exhibit different ecological strategies. this study was carried out at the san pedro de los griegos pothole ( ° ' n, ° ' e; elevation: m), situated km from oliete village (teruel province). the cavity is an enormous hole with an entrance of × m and a -m maximum depth. crevices in the walls are optimal roost sites for many birds and bat species. however, the pothole is totally illuminated and shows a large lagoon inside ( figure ). around the cavity, the vegetation is dominated by a mix of low growing stipa sp., brachypodium retusum, rosmarinus officinalis and thymus vulgaris. local weather is characterized by continental climate with a mean annual temperature of . °c and a mean annual precipitation of mm (mainly in spring). however, mean daily temperature is over °c between june and august (with . °c and . °c as the mean minimum and maximum temperatures, respectively). the permanent availability of water and nutrients, the dampening of hard external climatic conditions and the suitability of the habitat for the reproduction of various vertebrate species make the san pedro pothole a site of unprecedented high biodiversity in europe [ ] . bats were captured in summer (from june to july) over a -year period ( - ). mist nets were employed to capture bats at sunset when emerging from the pothole to forage. all bats were identified to species based on published identification keys of the bats of europe [ ] . individuals were sexed, and the reproductive status of adult females was classified as pregnant or lactating, based on palpation of the abdomen and nipple condition [ ] . blood samples were obtained by a small puncture made in the median artery. the amount of blood sampled varied from . ml to . ml, according to the size of the animal. pressure with a sterilized absorbent hemostatic sponge impregnated with gelatin was applied to prevent bleeding and facilitate healing. the bats were given % glucose water to drink to prevent dehydration and to provide rapidly assimilated compounds for energy. once bleeding ceased, the bat was released. vials containing blood were stored at °c for a few hours. samples were centrifuged for minutes at × g, and the serum was extracted with a micropipette. serum samples and blood pellets were stored at - °c before analysis. all animals were handled in strict accordance with good animal practices, as defined by current european legislation. bat capture and blood sampling were authorized by permit from the spanish regional committee for scientific capture. the technique used to detect eblv- neutralizing antibodies is an adaptation of the rapid fluorescent focus inhibition test (rffit) [ , ] . a constant dose of a previously titrated (calibrated to give % fluorescent foci-infected cells), cell culture-adapted eblv- challenge virus ( fra) was incubated with -fold dilutions of the sera to be labelled. after incubation of the serum-virus mixtures, a suspension of bsr cells (a clone of bhk cells) was added. after hours incubation, the cell monolayer was acetone-fixed and labelled with a fluoresceinated anti-nucleocapsid antibody (bio-rad, marnes-la-coquette, france). the optimal challenge dose (the dilution giving % infected cells for each virus production) was calculated. titers are presented as an arithmetic mean of two independent repetitions. serum samples with antibody titers < are considered negative for eblv- neutralizing antibodies. this cut-off value is similar to that applied in other studies [ , , , ] . to study the variation in eblv- -antibody prevalence, we conducted two analyses: first, three explanatory variables (sex, species and year) were first screened using a univariate analysis and a chi-square test to check for statistically significant associations with serological status ( : negative; : positive). in the second analysis, we used a generalized additive model (gam) to study the temporal patterns of eblv- -antibody prevalence in only two species (p. austriacus and t. teniotis). more specifically, we used a generalized additive model with the binomial error distribution, where the seroprevalence was the response variable and sex, species and year ( - ) were the explanatory variables. the -year‖ variable was modeled as a covariate fitted with penalized cubic regression splines and sex and species as a fixed categorical factor. to avoid over-fitting and to retain more easily interpretable relationships in the gam smoothing function, an upper limit of degrees of freedom was set for the year variable when fitting the models. we used an information-theoretic procedure and the akaike information criterion corrected for small sample sizes (aicc) to compare models [ ] . modeling was performed using the -lme ‖ and ‗‗mgcv'' packages in the r program v. . [ ] . we report the results of the prevalence of specific eblv- neutralizing antibody analysis from the - period in nine bat species roosting in the same refuge. five of these species (eptesicus serotinus, p. kuhlii, p. pygmaeus, myotis myotis and m. daubentonii) were captured sporadically (sample size < individuals during the whole study period), while the rest of the species sampled (p. pipistrellus, hypsugo savii, plecotus austriacus and tadarida teniotis) were captured often. the larger samples (> individuals) were obtained in p. austriacus and t. teniotis, because they form large colonies in this cavity. t. teniotis form a colony of several hundred individuals. the colony of p. austriacus is smaller and consists of individuals, approximately [ ] . we observed pregnant females in all bat species, except in e. serotinus, p. pygmaeus and m. myotis, where females were never captured, indicating that this cavity is a breeding roost for the rest of the species found. males were also captured during the breeding period, indicating that males, either as solitary individuals or forming part of the maternity colonies (e.g., p. austriacus), are present during the breeding period in the cave. among the sera obtained, ( . %) were positive for eblv- -neutralizing antibodies. eblv- antibodies were detected in ( . %) of the nine species analyzed (p. pipistrellus, p. kuhlii, h. savii, p. austriacus, e. serotinus and t. teniotis) ( table ) . no significant differences in eblv- seroprevalence were detected among seropositive bat species (χ = . , df = , p = . ). the highest seroprevalence was observed in h. savii. we did not find any difference in eblv- seroprevalence between females ( . %, % ci: . %- . %) and males ( . %, % ci: . %- . %) (χ = . , df = , p = . ) when all species were analyzed together and when only bat species with a large sample size-p. austriacus and t. teniotis-were considered (table ) . capture-mark-recapture of some bats during the study period allowed the tracking of temporal changes in eblv- seroneutralization titers. seven p. austriacus were captured and analyzed almost two times at intervals of one or several years. four of these seven bats showed positive antibody titers, becoming negative in the following recapture sessions after some years, indicating that these bats survive at least several years after their seroconversion (table ) . the models that incorporate sex and species variables were not significantly different from the model without these variables (Δaicc < ) ( table ). the best model showed a significant different nonlinear pattern in the eblv- seroprevalence along p. austriacus and t. teniotis. the effect of year fitted with the spline was highly significant for two species (p. austriacus: df = . , p < . and t. teniotis: df = . , p = . ), suggesting a different inter-annual pattern among these species (figure , table ). although no positive sera were detected in three bat species (m. myotis, m. daubentonii and p. pygmaeus), this result is probably due to the very low sample size. the high percentage ( %) of seropositive species found and the lack of significant differences in eblv- seroprevalence among seropositive species suggest that most of the bat species can be exposed to eblv- in this pothole although most of these species are not considered as lyssavirus reservoirs by previous studies [ , , , ] . previous studies have shown higher prevalence in females than in males [ , ] . this difference may be due to the gregarious behavior of female bats in summer (nursing colonies are composed almost exclusively of adult females). in these colonies, virus transmission may be favored by high contact rates during social grooming, nursing or olfactory or lingual contact with body fluids. reproductive activity may also play a role in virus transmission [ ] , because an increased susceptibility to infectious disease during pregnancy and lactation has been demonstrated in bats [ ] and other mammals [ ] . however, we report in this study no sex differences of eblv- seroprevalence. the presence of males in this cavity during summer could indicate that males also are present in maternity colonies, as observed in p. austriacus colonies, or roost near these colonies. significant fluctuations in the percentage of seropositive bats are indicative of several different episodes of eblv- infection occurring in p. austriacus and t. teniotis colonies during the period of study. a quick increase and a high seropositive percentage after a lyssavirus episode are not unusual in a gregarious behavior species and could explain the sudden increase in the percentage of seropositive bats in t. teniotis and p. austriacus colonies. a similar quick increase with seropositive peaks of %- % was observed in different colonies of m. myotis in mallorca [ , ] . however, in m. myotis colonies, the evolution of seroprevalence after infection peaks follows a more gradual decline over subsequent years, until a new episode takes place, very different from what is observed here. the delay between the waves is then dependent on the rate of inflow of susceptible bats into the colonies as a consequence of new births, bat immigration from neighboring colonies and the expiration of eblv- specific immunity in previously infected animals [ ] . when a sufficient fraction of susceptible bats in the colony is reached, the virus spreads again if infected individuals join the colony. in the t. teniotis and p. austriacus colonies, the increase of seroprevalence is followed by a rapid decline until seropositive bats are not detected. the difference in the seropositive percentage evolution can be due to a higher rate of inflow of individuals in colonies of t. teniotis and p. austriacus. no data of inflow are available on t. teniotis, but very few recaptures were obtained during the study, indicating probably a high inflow rate in this colony. however, recapture rates in the p. austriacus colony were higher, suggesting a lower inflow in this species. another hypothesis could be a different lifespan of immunity in these species. recent studies estimated the lifespan of the m. myotis immunity from eblv- to be around two years [ ] . in this respect, it is possible that the immunity lifespan would be shorter in p. austriacus and t. teniotis than in m. myotis. the best model obtained by gam analysis indicated that inter-annual patterns of seroprevalence evolution were significantly different for t. teniotis and p. austriacus. annual fluctuations could result from the behavioral ecology of the species involved [ ] . t. teniotis and p. austriacus are two species with a different social organization and behavior. while t. teniotis forms large maternity colonies and can make long seasonal movements, p. austriacus forms smaller maternity colonies constituted by both sexes and makes shorter seasonal movements [ ] . different host ecology, behavior and movement could explain the different temporal variations in seroprevalence in these two species. changes in density during migration or colony formation may affect contact rates and, thus, disease dynamics [ , ] . differences in eblv- exposure dynamics could also be related to host community composition and inter-species interaction. higher eblv- seroprevalence was observed in large and multispecies colonies compared to smaller and monospecific colonies, suggesting that interspecific virus transmission plays an important role in dynamics. a higher number of species might not only increase the rates of contact between bat groups, but could also facilitate virus entry or spread through the higher mobility of individuals among colonies, especially if there are migratory species [ ] . in this sense, m. schreibersii (a species that often shares roost with m. myotis) has been considered as a regional reservoir and an essential species for eblv- persistence in the balearic islands [ ] . other bat species present in the san pedro pothole, such as p. pipistrellus and p. kuhlii, showed lower eblv- seroprevalence than p. austriacus and t. teniotis. however, previous studies of bat rabies surveillance in europe did not find eblv- -neutralizing antibodies in both species of pipistrellus (for review see [ , ] ). these results could be indicative of a low public health risk associated with these synanthropic species. furthermore, the lack of a standardized serological test procedure, including arbitrary cut-off values, makes the comparison between previous european studies difficult. however, the higher values of eblv- seroprevalence in our study could be due to differences in virus circulation and dynamics resulting from regional differences or selection of different types of colony (large multispecies maternity colonies in this case) [ , ] . research programs that focus mainly on multi-host systems will help advance our understanding of the ecology of bat diseases. this research addresses the role of multiple hosts in the 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inference: a practical information-theoretic approach r: a language and environment for statistical computing. r foundation for statistical computing aná lisis demográ ficos y sanitarios en las colonias de plecotus austriacus y tadarida teniotis de la sima de san pedro (oliete, parque cultural del rí o martí n) (in spanish) active surveillance of bat rabies in france: a -year study reproduction and nutritional stress are risk factors for hendra virus infection in little red flying foxes (pteropus scapulatus) immunosuppression during pregnancy and lactation insights into persistence mechanisms of a zoonotic virus in bat colonies using a multispecies metapopulation model bat migrations in europe. a review of banding data and literature; federal agency for nature conservation animal migration and infectious disease risk bat rabies surveillance in europe twenty years of active bat rabies surveillance in germany: a detailed analysis and future perspectives the authors wish to thank sergi vives, departament d'estadística de la facultat de biologia, university of barcelona, for his mathematical support. we thank pepe royo of the centro de arte rupestre -antonio beltrán‖ del parque cultural del río martín of ariño (teruel, spain) for providing access to installations and for support during sample collection. we thank xavier bayer and cisco guasch for sharing his team's fieldwork.the research leading to these results has received funding from ministerio de sanidad y servicios sociales e igualdad, dirección general de salud pública y sanidad exterior. the authors declare no conflict of interest. key: cord- -eiobmxp authors: zhao, shan; li, wentao; schuurman, nancy; van kuppeveld, frank; bosch, berend-jan; egberink, herman title: serological screening for coronavirus infections in cats date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: eiobmxp coronaviruses (covs) are widespread among mammals and birds and known for their potential for cross-species transmission. in cats, infections with feline coronaviruses (fcovs) are common. several non-feline coronaviruses have been reported to infect feline cells as well as cats after experimental infection, supported by their ability to engage the feline receptor ortholog for cell entry. however, whether cats might become naturally infected with covs of other species is unknown. we analyzed coronavirus infections in cats by serological monitoring. in total cat serum samples and fcov type or type -specific antisera were screened for the presence of antibodies against the s receptor binding subunit of the cov spike protein, which is immunogenic and possesses low amino acid sequence identity among coronavirus species. seventy-eight sera were positive for antibodies that recognized one or more coronavirus s s whereas serum exclusively reacted with human coronavirus e (hcov- e) and two sera exclusively reacted with porcine delta coronavirus (pdcov). we observed antigenic cross-reactivity between s s of type and type fcovs, and between fcov type and porcine epidemic diarrhea virus (pedv). domain mapping of antibody epitopes indicated the presence of conserved epitope(s) particularly in the cd domains of s . the cross-reactivity of fcov type and pedv was also observed at the level of virus neutralization. to conclude, we provide the first evidence of antigenic cross-reactivity among s proteins of coronaviruses, which should be considered in the development of serological diagnoses. in addition, the potential role of cats in cross-species transmission of coronaviruses cannot be excluded. coronaviruses (covs) are enveloped viruses with a positive-stranded rna genome and classified into four genera (alpha-, beta-, gamma-and deltacoronavirus) within the subfamily orthocoronavirinae in the family coronaviridae of the order nidovirales. covs are found in a variety of mammals and birds, in which they can cause respiratory, enteric and systemic infections [ ] [ ] [ ] . additionally, covs have proven ability for cross-species transmission, exemplified by the emergence of severe acute respiratory syndrome (sars) coronavirus in / , and of the middle-east respiratory syndrome (mers) coronavirus in [ ] . both viruses belong to the betacoronavirus genus and have an animal origin. sars coronavirus crossed over from bats via intermediate hosts to humans, became human-adapted and quickly spread worldwide before its containment. mers coronavirus recurrently enters the human population via its dromedary camel reservoir host, with limited, non-sustained human-to-human transmission particularly in healthcare settings [ ] [ ] [ ] . apart from sars-and mers-cov, all four globally endemic human covs (hcov-oc , hcov-nl , hcov- e and hcov-hku ) originate viruses , , of from animals [ ] [ ] [ ] [ ] . in addition, cross-species transmission potential of covs is also illustrated by the occurrence of chimeric coronaviruses that resulted from recombination events between feline covs (fcov) and canine covs (ccov) [ , ] . in order to get insight into the frequency of interspecies transmission of coronaviruses within and between animal and human populations and the risk of subsequent development of a pandemic, it is useful to screen for coronavirus infections in animal species; especially those that are in close contact with humans. serological assays that can detect virus-specific antibody responses against infection play an important role in these epidemiological studies [ ] . cats live in close contact with humans and often roam around freely in the environment. hence cats are an interesting species to study for infections with coronaviruses. infections with feline coronaviruses (fcovs) are recognized and widespread [ , ] . fcovs are classified into two types, type and type , based on the genetic and antigenic difference of their spike (s) protein [ ] . in the field, the majority of fcov infections are caused by fcov type , while fcov type , derived from recombination events of type fcovs and ccovs obtaining the s gene and some flanking regions of ccovs, is less prevalent [ , ] . depending on the virulence of the fcov strain and the immune response of the cat, the clinical presentation can range from apparently asymptomatic, through diarrhea, to full-blown feline infectious peritonitis [ ] . fcovs are members of the genus alphacoronavirus, to which also hcov- e, porcine transmissible gastroenteritis virus (tgev), and ccov belong. the latter three viruses and fcov type have been proven to use feline aminopeptidase n (fapn) as a functional receptor in vitro [ ] . the receptor for type fcov has still not been identified [ ] . notably, previous studies have shown that hcov- e and ccov could infect cats after experimental inoculation, causing an asymptomatic infection [ , ] . thus, cats might potentially become naturally infected with covs of other species which may lead to virus-host adaptation e.g., mutation or recombination, resulting in emergence of novel coronaviruses and potentially new diseases [ , ] . the extent to which infections with covs of other species occur in the field, has not been explored in previous epidemiological studies of cov infections in cats [ , [ ] [ ] [ ] . being the main envelope protein of coronaviruses, the spike (s) protein mediates cell attachment and membrane fusion to allow viral entry. s functions as the main determinant of cell-, organ-and host-tropism. additionally, it is also the major target of neutralizing antibodies. spike comprises two functionally interdependent subunits, s and s , with s responsible for receptor binding and s for membrane fusion [ , ] . the s subunit is the least conserved and the most variable immunogenic antigen between coronavirus species [ ] . therefore, the s subunit is well suited as an antigen to screen for coronavirus type specific antibodies [ ] . in this study, covs infection in cats were detected through profiling antibody presence in serum samples from cats. recombinant cov spike s subunits of different animal and human covs were expressed in a mammalian expression system and used for screening of cat sera for the presence of antibodies against the respective proteins. positive samples were also tested by virus neutralization assays to support the specificity of the reaction [ ] [ ] [ ] . this investigation intends to extend our knowledge of cov epidemiology, potential reservoirs, and cross-species transmission. specific fcov type and fcov type sera were obtained from specific pathogen free (spf) cats previously infected with strain uu or rm and fipv- respectively [ , ] . in addition, for the serological survey, feline sera were retrieved from the serum bank in our lab. these had all been collected from cats in the netherlands. most of the samples (> %) were from a study on antibody titer testing for feline panleukopenia virus. the other samples were send to our lab for fip or felv-fiv diagnostics. sera of uninfected spf cats were included as negative controls. all samples were stored at − • c until analysis. african green monkey kidney cells (vero-ccl ), human hepatoma cells (huh ), pig kidney epithelial cells (llc-pk ), human embryonic kidney cells stably expressing the sv large t antigen (hek- t) were maintained in dulbecco modified eagle medium (dmem, lonza, basel, switzerland) supplemented with % fetal bovine serum (fbs, bodinco, alkmaar, the netherlands). virus strains used in this study have been described previously [ ] [ ] [ ] . briefly, recombinant porcine epidemic diarrhea virus (pedv) (rpedv-s dr -gfp) was propagated and titrated in vero cells, and hcov- e in huh cells. pdcov was propagated and titrated in llc-pk cells, but supplemented with µg/ml tpck-treated trypsin (sigma-aldrich, inc., st louis, mo, usa) in dmem. synthetic sequences of coronavirus spike s subunits (hcov-hku (gb: yp_ . ), mers-cov (gb:yp_ . ), sars-cov (gb: aax . ), hcov-oc (gb: aar . ), hcov- e (gb: np_ . ), hcov-nl (gb: yp_ . ), tgev (gb: abg . ), pedv (gb: aog . ), bcov (gb: p . ), pdcov (gb: aml . ), fcov type (gb: fj . ), fcov type (gb: ay . )) and different domains of pedv s subunit (s and s a-d , as identified and described in [ ] ) were cloned into pcaggs expression plasmids as described previously [ ] . similarly, the expression constructs encoding chimeric proteins in which s s were fused to the fc domain of mouse igg a. for protein production, hek- t cells were transfected with plasmid dna conjugated to polyethyleneimine (polysciences, inc., warrington, pa, usa). at - h post transfection, inoculum was removed and the transfection mixture was replaced by sfm ii expression medium (gibco®, life technologies inc., grand island, ny, usa). at - days post transfection, cell supernatants were harvested and proteins were collected by protein a sepharose beads (ge healthcare bio-sciences ab, uppsala, sweden). proteins were then eluted with . m citric acid, ph . and neutralized with m tris-hcl, ph . . concentrations of proteins were assessed by nanodrop spectrophotometry (thermofisher scientific inc., waltham, ma, usa) and confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page) with bovine serum albumin (bsa, bioivt, west sussex, uk) as standard. typical yields for proteins were . - . mg/ml. for long term storage, proteins were stored at − • c upon usage. to study the potential cross-reaction between fcov type and in more detail, models of fcov type (strain: uu ; genbank accession no.: fj . ) and fcov type (strain: - ; genbank accession no.: ay . ) s proteins were generated via the automated protein structure swiss-model homology modelling server (https://swissmodel.expasy.org/) [ ] using the elucidated hcov-nl cryo-em structure (pdb code: szs) as the input model. figures were made with pymol (the pymol molecular graphics system, version . schrödinger, llc.). fcov s domains of both type and , namely s -cd , were expressed as murine fc fusion proteins in hek- t cells as described above. high binding microtiter plates (greiner bio-one bv, alphen aan den rijn, the netherlands) were coated overnight at • c with equal molar amount of protein ( . pmol per well, diluted in phosphate buffered saline (pbs, ph . )). after three washes with washing buffer (pbs containing . % tween- ), the plates were blocked for h at • c with blocking buffer (pbs containing % milk powder (protifar, nutricia, zoetermeer, the netherlands), . % tween- ). protein coating efficiency was assessed by binding of anti-mouse igg antibodies in a direct elisa, and confirmed the equimolar coatings of all proteins. to detect antigenic reaction with serum samples, sera were tested in duplicate at a : dilution in blocking buffer, and then incubated in the plates at • c for h. after washing, plates were incubated with a : diluted horseradish peroxidase (hrp)-conjugated goat anti cat igg (rockland immunochemicals, inc., pottstown, pa, usa) at • c for h. the peroxidase reaction was then visualized via adding tmb super slow one component hrp microwell substrate (biofx®, surmodics ivd, inc., eden prairie, mn, usa) for min. reaction was stopped with . % sulfuric acid and optical densities (od) were measured at nm. negative sera (from uninfected spf cats) were included to determine the elisa cut-off values; sera with od values higher than -fold the od of negative sera were considered positive. all s proteins were coated on the same elisa plates making it easy to screen and compare the od values of individual sera in one assay. hereby we excluded the sera that give high background od values against all proteins being considered false positive. neutralization assays were performed with some of the covs to support the specificity of elisa results. cat sera were serially diluted -fold in dmem and mixed : with rpedv-s dr -gfp, hcov- e or pdcov ( % tissue culture infective doses [tcid ]/ml). these mixtures were then incubated at • c for h, and µl of each mixture was used for inoculation with vero, huh and llc-pk cell monolayers in -well plates, respectively. for pdcov infection, tpck-treated trypsin (sigma-aldrich, inc., st louis, mo, usa) was supplied to llc-pk tissue culture medium at a final concentration of µg/ml. at - days post infection, cytopathic effect (cpe) could be observed via microscopy. virus neutralization titers (vnt) were expressed as the highest serum dilution resulting in % reduction of cytopathic effect (hcov- e and pdcov) or virus-induced fluorescent cells (pedv). before virus neutralization, sera were inactivated through incubation at • c for min. experiments were performed in triplicate. feline sera (n = ) were screened by indirect elisa for antibody reactivity against cov s antigens. the od values against these antigens are shown in figure . in total, of the sera ( . %) contained anti-cov antibodies, while sera showed reactivity against more than one cov s antigen. none of the samples had to be discarded because of reactivity against all of the proteins indicating a potential false positive result. the frequency of different combinations of cov-s reactive samples is summarized in table . reactivity against eight out of cov s antigens could be observed, whereas none of the sera recognized the s protein of hcov-hku , mers-cov, sars-cov and hcov-oc . different s antigens were grouped by amino acid sequence phylogeny (left panel) using mega . each column of the heat map represents an individual sample, and columns were arranged in a descending order based on elisa-reactivity against feline coronavirus (fcov) type . table . numbers of positive cat samples and different combinations of reactivity found. the number of positive sera against each individual s is shown in the bottom row. positive elisa reactions are colored in orange. cut-off value was determined as the -fold over the od of negative sera. fcov type -s fcov type -s pdcov-s tgev-s e-s bcov-s different s antigens were grouped by amino acid sequence phylogeny (left panel) using mega . each column of the heat map represents an individual sample, and columns were arranged in a descending order based on elisa-reactivity against feline coronavirus (fcov) type . table . numbers of positive cat samples and different combinations of reactivity found. the number of positive sera against each individual s is shown in the bottom row. positive elisa reactions are colored in orange. cut-off value was determined as the -fold over the od of negative sera. number of cats (total = ) fcov type -s fcov type -s pedv-s pdcov-s tgev-s e-s nl -s bcov-s as expected, many sera were positive for fcov s , with sera ( . %) positive for fcov type and sera ( . %) for fcov type s ( figure ). all of the fcov type s positive sera also tested positive for fcov type s , while of fcov type s positive sera also reacted with tgev s . the fcov type s and tgev s elisa reactivities showed a strong nonparametric spearman correlation (spearman r = . , p < . ). with respect to this, we suggest that the tgev s positivity was due to cross-reactivity of fcov type s , as fcov type shows close antigenic and genetic relationship with tgev (s shares . % amino acid sequence identity). the remaining fcov type s positive but tgev s negative sera do react with fcov type s . an explanation might be the cross-reactivity between fcov type and type . remarkably, feline sera were reactive with s proteins from human, porcine and bovine covs (table ) , including hcov- e ( / ), hcov-nl ( / ), pedv ( / ), pdcov ( / ) and bcov ( / ). od values of feline sera positive for hcov-nl s and bcov s were relatively low ( figure a ). elisa reactivity towards non-feline cov s proteins might be explained by infection with the respective or related covs or by the presence of cross-reacting antibodies, although there was low sequence identity (< . %) between s proteins of fcov type and related non-feline coronaviruses (for the complete comparison of s sequence identities, see table ). yet, all of pedv-s positive sera were also positive for fcov type s ( figure b , table ). the elisa results of fcov type s and pedv s showed a strong nonparametric spearman correlation (spearman r = . , p < . ). thus, this might indicate the occurrence of antibody cross-reactivity against fcov type and pedv s antigens. many of the hcov- e and pdcov s positive sera also reacted with fcov type s , but no strong nonparametric spearman correlation was observed (hcov- e, r = . ; pdcov, r = . ). one feline serum only reacted with hcov- e viruses , , of s , and two feline sera only recognized pdcov s . ( figure b , table ). this observation led us to hypothesize that cross-reactivity may not play a role in elisa reactivity of these three sera, but that the three cats had been infected with these viruses or related viruses. fcov type -s (fj . ) fcov type -s (ay . ) . pedv-s (aog . ) . . pdcov-s (aml . in our screening, samples were shown to be positive for two or more s proteins including fcov type . the data prompted us to test different hypotheses which may explain this phenomenon: specific reaction through natural virus infection or reaction due to cross-reactivity with fcov-s antigens. to explore this further, we employed fcov type specific sera derived from specific pathogen free (spf) cats that had been experimentally infected with fcov type i strain rm (n = ) or strain uu (n = ). these sera were tested for their elisa reactivity against seven cov s proteins (excluding tgev s ) that showed positive reactivity in the previous serological screening. as expected, all sera were positive for fcov type s in our elisa; interestingly, four samples also reacted with fcov type s , and five samples with pedv s . no positive elisa-reactivity was detected with s of hcov- e, pdcov, hcov-nl or bcov (table s ). thus, fcov type infection could lead to the generation of antibodies that cross-react in the s -elisa with fcov type and pedv s proteins. the elisa cross-reactivity of fcov type specific sera with fcov type s antigens prompted us to map the domains responsible for cross-reaction within the s subunit. hence, to identify domain borders within s , we built homology-based models of both fcov type and type spike using the related elucidated hcov-nl cryo-em structure as the template model. as shown in figure a , continuous structural domains can be identified for the s subunit of both spikes, namely s , and s a through s d . amino acid sequence identities of these domains between fcov type and type differ, ranging from . % to . % ( figure b ). several s proteins for both type and type fcov-s comprising one or two domains were expressed and purified ( figure c ). fcov type specific sera (n = for strain rm and n = for strain uu ) and type (n = , strain - ) specific sera were then tested against these proteins in elisa format. the four fcov type specific sera that cross-reacted with s of fcov type again showed binding to fcov type s . but the fcov type specific sera showed little to no reactivity against fcov type s . as shown in figure , the type specific antisera reacted with all of the homologous s domains, with s and s b of both type and type displaying the strongest reaction. interestingly, the cd domain showed the highest level of cross-reactivity between fcov type and , in agreement with its highest sequence identity among s domains (figure ) . the other three domains showed little to no cross-reactivity. antisera reacted with all of the homologous s domains, with s and s b of both type and type displaying the strongest reaction. interestingly, the cd domain showed the highest level of crossreactivity between fcov type and , in agreement with its highest sequence identity among s domains ( figure ). the other three domains showed little to no cross-reactivity. the s subunit of one protomer are colored, with s shown in cyan, s a in blue, s b in green, and the domains s cd in red. the s part of the protomer is marked in light gray. (b) schematic presentation of the fcov type (strain uu ) and type (stain - ) s protein with the signal peptide (sp), the s subunit (the domains are colored as described in the legend of figure a ) and the s subunit (the c-terminal transmembrane domain is indicated by a black box). amino acid sequence identities between fcov type and type s domains are indicated. (c) diagram of the different s subdomains sequence. all s subdomains were c-terminally tagged with the fc part of mouse igg a (not shown in the figure) and expressed as fc fusion proteins. the s subunit of one protomer are colored, with s shown in cyan, s a in blue, s b in green, and the domains s cd in red. the s part of the protomer is marked in light gray. (b) schematic presentation of the fcov type (strain uu ) and type (stain - ) s protein with the signal peptide (sp), the s subunit (the domains are colored as described in the legend of figure a ) and the s subunit (the c-terminal transmembrane domain is indicated by a black box). amino acid sequence identities between fcov type and type s domains are indicated. (c) diagram of the different s subdomains sequence. all s subdomains were c-terminally tagged with the fc part of mouse igg a (not shown in the figure) and expressed as fc fusion proteins. viruses , , x for peer review of figure . elisa-reactivity of fcov specific antisera against different s subdomains of fcov type and . equimolar amount of purified s proteins and the four s subdomains were coated onto well plates and antibody binding was determined by elisa. the fcov type and specific antisera used in the screening were derived from experimentally infected specific pathogen free (spf) cats and are indicated at the right side of each panel, absorbance values and antigens in use are shown on the y-and x-axis, respectively. graphs represent the mean values from three independently performed experiments. standard deviations are indicated as error bars. because the fcov type specific cat sera also showed elisa reactivity with pedv-s (table s ), we analyzed the reaction of the five pedv-s positive cats in more detail. samples were analyzed via elisa using antigens comprising different pedv-s domains, as described in our previous study [ ] . cat sera taken pre-and post-fcov infection were collected and tested. as indicated in figure , all five cats had developed pedv-s reactivity to different extent after fcov type inoculation. noticeably, all sera showed the highest od values with the cd domain, while the other domains, including the s b containing the presumed receptor binding domain (rbd) [ ] , were non-reactive ( figure ). on the other hand, the swine pedv positive control serum exhibits strong reactivity against all pedv-s domains. the next question we asked was whether fcov type specific sera could neutralize pedv infection in tissue culture, as they showed no reactivity with the s b of pedv spike. as shown in figure , pedv neutralizing antibodies were detected in three out of five fcov type i specific cat sera. figure . elisa-reactivity of fcov specific antisera against different s subdomains of fcov type and . equimolar amount of purified s proteins and the four s subdomains were coated onto -well plates and antibody binding was determined by elisa. the fcov type and specific antisera used in the screening were derived from experimentally infected specific pathogen free (spf) cats and are indicated at the right side of each panel, absorbance values and antigens in use are shown on the y-and x-axis, respectively. graphs represent the mean values from three independently performed experiments. standard deviations are indicated as error bars. because the fcov type specific cat sera also showed elisa reactivity with pedv-s (table s ) , we analyzed the reaction of the five pedv-s positive cats in more detail. samples were analyzed via elisa using antigens comprising different pedv-s domains, as described in our previous study [ ] . cat sera taken pre-and post-fcov infection were collected and tested. as indicated in figure , all five cats had developed pedv-s reactivity to different extent after fcov type inoculation. noticeably, all sera showed the highest od values with the cd domain, while the other domains, including the s b containing the presumed receptor binding domain (rbd) [ ] , were non-reactive (figure ). on the other hand, the swine pedv positive control serum exhibits strong reactivity against all pedv-s domains. the next question we asked was whether fcov type specific sera could neutralize pedv infection in tissue culture, as they showed no reactivity with the s b of pedv spike. as shown in figure , pedv neutralizing antibodies were detected in three out of five fcov type i specific cat sera. the experiment was carried out in duplicate and repeated three times. error bars indicate standard deviations. sera were collected from spf cats prior (cat - p) and after (cat - ) experimentally inoculated with fcov type . positive serum: pedv positive swine serum collected from the field; negative serum: serum from fcov negative spf cat. several serum samples from field cats, but not virus-specific serum samples from fcov inoculated spf cats, were found to be elisa positive for hcov- e (n = ) and pdcov s (n = ) ( figure a) . also, a few feline sera displayed unique elisa positivity for s of hcov- e (n = ) or pdcov (n = ) ( figure b, table ). this could indicate that these antibodies were induced upon infection with these specific viruses. to corroborate the possibility of a natural infection in these cats with hcov- e or hcov- e-like viruses, we tested sera neutralization antibody titers. the results showed that one of the hcov- e s reactive feline sera was able to neutralize hcov- e infection (vnt = ); no neutralization of pdcov was detected for the all pdcov-s positive sera. coronavirus infections are endemic and ubiquitous in feline populations. two viral types, type and , are distinguished and both of them could well sustain themselves in the cat reservoir [ , ] . both have been shown to have worldwide distribution, with the seropositivity rate up to % among animal shelter populations and in multi-cat households [ , ] . the majority of natural infections are caused by type fcovs, while in the field type fcovs are less common and mainly occur in asia [ , , [ ] [ ] [ ] . covs are generally considered to be host-specific; however, cross-species transmission does occur which may lead to incidental infections like the spillover of mers-cov from dromedary camel to humans, where humans function as an incidental and ultimately dead-end host [ ] . but covs might also adapt to the new host exemplified by the animal origin of all four endemic human covs (hcov-oc , hcov-nl , hcov- e and hcov-hku ) [ ] [ ] [ ] [ ] . whereas in cats infections with fcov are well recognized, studies regarding possible natural infections with other animal and human coronaviruses are lacking to the best of our knowledge. knowing the genetic variability of coronaviruses and the use of orthologous receptors by non-feline covs, studies on cross-species transmission are desirable. this may provide insight regarding whether cross-species transmission does occur. in the present study we used the highly immunogenic s antigens to screen cat sera for the presence of antibodies against feline and non-feline coronaviruses, as a first indication of possible infections with these viruses. in our study, of the cat sera were shown to be seropositive for coronaviruses. the seropositive rate ( . %) against s of fcov type is consistent with previous studies [ , ] . all of the fcov type s positive sera of naturally infected cats were also positive for fcov type s , which might be the result of cross reaction between the two proteins, despite their low amino acid identity. elisa with specific antisera from experimentally fcov type and type infected cats showed that sera of several fcov type infected cats could cross-react with fcov type s . domain mapping elisa results showed that fcov type specific sera react to different levels with the s domains of fcov type s protein, and also reacts with fcov type s cd . vice versa, fcov type specific sera also reacted with s cd of fcov type . these observations pose a potential two-way cross-reactivity between s cd domains. interestingly, in parallel with our findings on feline coronaviruses, we identified a number of samples that were seropositive against the s of pedv, a viral pathogen that mainly replicates in the porcine intestinal epithelium. to study the possibility of cross-reaction, samples derived from preand post-fcov infected cats were screened against pedv s in elisa. the reactivity found against pedv s with fcov specific sera shows that cross-reaction can occur at the level of domain s cd ; the other pedv s domains showed no reaction with the fcov positive sera. judging from these observations, it seems that s cd plays an important role in cross-reaction between fcov type and , and also fcov and pedv. as s cd is the most conserved domain among fcov and also between fcov and other alphacoronaviruses (for a systematic assessment of sequence identities, see table ), it is reasonable to hypothesize that antibodies can develop against conserved epitopes within this region and subsequently cause cross-reaction. this should be taken into account when developing and interpreting serological assays. table . identities of amino acid sequences of fcov type (strain: uu ) s and s domains compared with the amino acid sequences of other alphacoronaviruses. (identities are shown in %; na: not available) the genbank accession numbers of these viruses are as follows: fcov type (uu ), fj . ; fcov type (rm), fj . ; fcov type , ay . ; tgev, abg . ; pedv, aog . ; hcov- e, np_ . ; hcov-nl , yp_ . . amino acid % identity to fcov type (uu ) s s s a s b s cd noticeably, elisa reactivity among cat sera towards the n-terminal fcov s domains and a was less consistent and generally lower compared to whole s , which seems to correlate with the higher antigenic variation in those domains found among fcov type strains [ ] (figure ). especially the sera from fcov-rm infected cats (cat , , and ) showed lower od values against s a . this phenomenon could be explained as the samples displaying higher reactivity were from cats inoculated with fcov-uu (cat , and ), the particular strain from which the s region was used as an antigen in the elisa studies. in the meantime, the possibility of the variable elisa reactivity might be due to the difference in individual antibody levels. in principle, the distinct antigenic reactivity of s and s a between the two fcov types might facilitate the development of a specific elisa method which allows the serological discrimination of fcov type and type infections in cats. in order to provide further insight regarding cross-reactivity between fcov type and pedv, we performed virus neutralization assays. cross-neutralization of pedv infection could be observed for some of the feline fcov type post-infection sera, in contrast to the pre-infection serum counterparts. since fcov specific pedv neutralizing sera did not react with pedv s , s a or s b , it is likely that the cross-neutralizing antibodies are targeting conserved epitopes in the s cd domain or the s subunit of the pedv spike protein [ ] . given the unknown tgev infection background of the pedv positive pigs, the cross-reaction of pedv specific sera against fcov type could not be explored in our study, as tgev positive pig samples would certainly influence the outcome [ , , ] . of note, our findings cannot exclude the possibility that field cats might incidentally get naturally infected with pedv or pedv-like viruses, as there had been one report showing the detection of pedv in one stray cat via pcr assay [ ] . it would be interesting to include more sera of cats from pig farms in future studies. considering the fact that cats play an important role in human society and have constant interaction with humans, it is of interest to conduct serological surveys for possible reverse zoonosis of human pathogens. in our study s antigens of several human coronaviruses were included and this led us to identify hcov- e seropositive feline samples in our elisa survey (table ) ; one serum in particular reacted solely with hcov- e s but not with any other coronavirus. of the hcov- e s reactive feline sera one showed low neutralizing activity against hcov- e infection. this might suggest that positive cats were indeed exposed to hcov- e or related viruses. mers seropositivity is also seen in other species besides the dromedary host [ ] . rare cases of seropositivity might be considered as spill-over infections from the dromedary camel reservoir. similar (perhaps dead-end) spill-over infections of e from the human reservoir to cats might also occur. a similar principle could also apply for pdcov, a porcine pathogen that emerged rather recently. both hcov- e and pdcov use apn as their receptor and have been reported to also be able to use feline apn for cellular entry [ , ] . although reports are lacking regarding the natural infection of these two viruses in cats, hcov- e was shown to cause a priming effect of fcov antibody in experimentally fcov infected cats suggesting that infection occurred [ ] . therefore, the detection of antibodies against s of hcov- e in a portion of the cats might be specific and due to the exposure to hcov- e through daily interaction with humans. eight cats were seropositive for pdcov of which two cats were seropositive only for pdcov and not for any other covs. this could be caused by infection with pdcov or pdcov-related viruses through avian sources, considering the fact that cats are natural avian predators and the presumed avian origin of pdcov [ , ] . our findings emphasize the potential role of cats as incidental hosts for non-feline coronaviruses and the need of in-depth study of naturally infected pathogens in cats. besides serological studies, efforts should also focus on isolation and identification of these viruses in cats. in conclusion, we presented a thorough serological survey in cats using s proteins of different animal and human coronaviruses. we demonstrated, despite the low amino acid identity, cross-reactivity between s proteins of fcov type and , and between that of fcov type and pedv. this should be considered when developing fcov serological assays as well as interpreting the results. our observation that some feline sera displayed antibody reactivity exclusively against non-feline cov s proteins warrant further research into the epidemiology and cross-species transmission of coronaviruses in cats and other animals that are in close contact with humans. further large scale serological studies regarding coronaviruses infection across animal species using arrays of cov s antigens can shed light into the hitherto unresolved host promiscuity of coronaviruses and the risk of cross-species transmission. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / / s , table s : elisa reactivity (od values) of fcov type specific antisera against s antigens of different coronaviruses. the authors declare no conflict of interest. pre-fusion structure of a human coronavirus spike protein discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the 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relationship between feline, porcine, and canine coronaviruses a review of feline infectious peritonitis virus infection: - seroprevalence study of feline coronavirus in owned and feral cats in sydney prevalence of korean cats with natural feline coronavirus infections persistence and transmission of natural type i feline coronavirus infection spike protein fusion peptide and feline coronavirus virulence the s glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes recombinant canine coronaviruses related to transmissible gastroenteritis virus of swine are circulating in dogs prevalence of swine viral and bacterial pathogens in rodents and stray cats captured around pig farms in korea middle east respiratory syndrome coronavirus infection in non-camelid domestic mammals this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -ko bdvo authors: vasilakis, nikos; tesh, robert b.; popov, vsevolod l.; widen, steve g.; wood, thomas g.; forrester, naomi l.; gonzalez, jean paul; saluzzo, jean francois; alkhovsky, sergey; lam, sai kit; mackenzie, john s.; walker, peter j. title: exploiting the legacy of the arbovirus hunters date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ko bdvo in recent years, it has become evident that a generational gap has developed in the community of arbovirus research. this apparent gap is due to the dis-investment of training for the next generation of arbovirologists, which threatens to derail the rich history of virus discovery, field epidemiology, and understanding of the richness of diversity that surrounds us. on the other hand, new technologies have resulted in an explosion of virus discovery that is constantly redefining the virosphere and the evolutionary relationships between viruses. this paradox presents new challenges that may have immediate and disastrous consequences for public health when yet to be discovered arboviruses emerge. in this review we endeavor to bridge this gap by providing a historical context for the work being conducted today and provide continuity between the generations. to this end, we will provide a narrative of the thrill of scientific discovery and excitement and the challenges lying ahead. almost years have passed since walter reed, james carroll, aristides agramonte, and jesse lazear established that yellow fever is caused by a filterable infectious agent which is transmitted by the bite of a mosquito, then known as stegomyia fasciata (aedes aegypti). lazear, who like his colleagues, had been stationed by the us army in cuba to study the disease, died of yellow fever in september after being exposed experimentally to mosquitos that had fed on sick patients. at about the same time in south africa, james spreull and sir arnold theiler demonstrated that bluetongue disease of sheep is caused by an "ultravisible" agent that could be transmitted by the injection of an infected serum. epidemiological evidence suggested that the agent was vector-borne, and it was subsequently shown by r.m. du toit that the disease occurred in sheep inoculated experimentally with suspensions of wild-caught biting midges (culicoides imicola). these and other seminal discoveries precipitated a century of research into vector-borne and zoonotic viral diseases, resulting in the discovery and isolation of many hundreds of novel viruses from insects or vertebrate hosts. some were identified as important human or veterinary pathogens. many other viruses were archived in reference collections, with only basic characterization of their biological or molecular properties. in recent years, the advent of next generation sequencing (ngs) has transformed this situation. complete genome sequences are now available for many of the archived isolates, allowing more accurate taxonomic assignments, analysis of their phylogenetic and evolutionary relationships with other viruses, and evaluation of the potential risks they may present to humans and wild or domestic animal populations. ngs has also opened the door to viral metagenomics, which has greatly increased the pace of new virus discovery from a wide range of hosts, usually with complete or near-complete viral coding sequences, but no virus isolate and minimal biological data. this has presented both opportunities and challenges for virologists and epidemiologists, as well as viral taxonomists, evolutionary biologists, and bioinfomaticians. sadly, this technological revolution has been accompanied by a period of progressive disinvestment in training in classical virology. in this review, we recall the rich history of the discovery of arboviruses and other zoonotic viruses in various settings around the world and the many outstanding scientists who have contributed to the endeavour. we also consider the impacts of ngs and metagenomic analysis, and the implications of these new technologies for the future of this important field of research. the rockefeller foundation (rf) was organized in for "the well-being of mankind throughout the world" [ ] . at the time, yellow fever was still epidemic in many tropical and subtropical regions of africa and the americas, so in , the rf established the yellow fever commission, with the lofty goal of eradicating yellow fever from the world. during the next years, the rf supported an international group of scientists working on yellow fever in new york city in the united states (u.s.); rio de janeiro and salvador in brazil; bogota, colombia; yabba, nigeria; and entebbe, uganda [ , ] . much of the work and accomplishments of rf-funded personnel during this period was described in strode's classic book, entitled "yellow fever" [ ] . the major accomplishments included: confirmation of earlier work by the reed commission in cuba, demonstrating that yellow fever was caused by a filterable agent, yellow fever virus (yfv), that was transmitted by the bite of infected mosquitoes, aedes aegypti; . discovery that the rhesus monkey and the white mouse are susceptible to infection with yfv, providing models for subsequent studies on the pathogenesis, transmission, epidemiology, and control of the disease; . demonstration that convalescent sera of humans and animals infected with yfv neutralize the virus. this discovery led to the development of the mouse neutralization test, which allowed investigators to map the geographic distribution of the virus; . discovery of the forest or sylvan cycle of yfv and the importance of mosquitoes other than ae. aegypti in the transmission and maintenance of the virus; . development of the d vaccine strain of yfv and its first human trials. max theiler, son of south african bluetongue researcher sir arnold theiler, received a nobel prize in for this work. as a by-product of the overseas yellow fever investigations, rf-funded researchers also isolated a number of other previously unknown arboviruses, including west nile, zika, semliki forest, bunyamwera, bwamba, uganda s, ilheus, and anopheles a and b viruses [ ] . the threat and onset of world war ii changed the priorities of the rf, and most of its overseas yf research activities ceased during this period. many of the former american rf staff became involved in studies of diseases of military importance, such as typhus, malaria, sandfly fever, and dengue, some as civilians and others as members of the u.s. armed forces. in , after the end of the war, the rf decided to develop a major new program to study arthropod-borne viruses and to discover "what might be out there", as well as their disease associations, life cycles, and vectors. this led to the development of the rockefeller foundation virus program, a world-wide virus discovery program that was funded from to [ ] . over the next several years, field laboratories staffed by both rf and local scientists were established with foreign governments in pune, india; port of spain, trinidad; belem, brazil; johannesburg, south africa; ibadan, nigeria; cali, colombia; and cairo, egypt [ ] . in egypt, the cairo laboratory was associated with the u.s. naval medical research unit no. . scientists in these field laboratories were involved in the detection and investigation of human diseases in their respective geographic regions, surveying human and animal populations for serologic evidence of past viral infection, and searching for viruses in a wide variety of arthropods, mammals, birds, reptiles, and amphibians [ ] . all virus isolations were done on site, using the classic technique of intracranial inoculation of newborn white mice, but later, as vertebrate cell cultures became available, inoculation of cell cultures was also used. in addition, sentinel animals, such as non-human primates, mice, and hamsters, were also used in the field to detect virus activity. viruses isolated in the overseas laboratories were initially characterized locally, lyophilized for preservation and storage, and then aliquots of each new virus were shipped back to the rf central virus laboratory in new york for further characterization and study. it was during this period that jordi casals, delphine clarke, loring whitman, and others developed the sucrose-acetone method for preparation of viral antigens and began to adapt the hemagglutination inhibition (hi) and complement fixation (cf) tests to identify and group arboviruses [ ] [ ] [ ] . the rf virus program was extremely productive and many novel arboviruses, as well as non-arthropod-borne viruses (i.e., hantaviruses and arenaviruses), were discovered by rf-funded investigators during this period. the productivity of this search strategy in detecting novel viruses affecting humans was recently reviewed by rosenberg et al. [ ] . the virus discovery rate was highest in the period - , which coincided with the rf virus program. a similar approach was also practiced by the institut pasteur and other international groups involved in virus discovery during this period, as described in this article and in other publications [ ] [ ] [ ] [ ] [ ] [ ] , resulting in the detection of a high proportion of pathogenic arboviruses. the second advantage of the strategy was that it yielded actual virus isolates, whose pathogenesis could be studied experimentally in vivo and in vitro in vertebrate and arthropod models. in contrast, current search methods for new viruses, which generally use metagenomics and other sophisticated genetic techniques to detect novel viral agents, do not usually yield live viruses, only their nucleotide sequences. a complete or partial genetic sequence alone rarely provides insight into the epidemiology and ecology, host range, pathogenesis and disease potential, or transmission modes of the new viruses. in , the rf made a decision to phase-out its virus program and to devote more of its efforts and resources to other projects, such as population control and increasing food production ("the green revolution"). over the next years, the rf-funded investigators working in overseas laboratories were withdrawn and the respective laboratories were turned over to local institutions or governments. in , the rf made arrangements with yale university to transfer its arbovirus group to new since the establishment of the institut pasteur in paris in , louis pasteur sent collaborators to various countries, mainly in the french colonies of indochina and africa. in that early time, pasteur wanted to set up rabies centers where the disease was highly and dramatically prevalent; naturally, the research potential of these centers was rapidly extended to tropical infectious diseases [ ] . currently, spread among countries on five continents, there are institutions, of which bear the name of "institut pasteur" (ip). altogether, these institutions constitute a structure long called the "institut pasteur d'outre-mer", which in became the international network of pasteur institutes (réseau international de l'institut pasteur, riip) and associated institutes. from this emerging network, the first african laboratory for microbiology was created in saint louis, senegal in , and then transferred to dakar to become the pasteur institute of dakar (institut pasteur de dakar, ipd yellow fever was to play a key role in the research at ipd. in the s, ipd had developed a vaccine against yellow fever (strain fnv) and produced it commercially. the re-emergence of yellow fever in africa in the s occurred mainly in the savannah zone and revealed the lack of knowledge about the natural maintenance of the virus during the inter-epidemic period. a large study to understand the emergence and re-emergence of sylvatic yellow fever was established between the ipd, institute pasteur abidjan (ipa) in côte d'ivoire, and institute pasteur bangui (ipb) in the central african republic, in order to detect the circulation of the yfv and map its emergence in the different ecozones. permanent research stations were developed in rain forests and savannas to detect virus circulation in vectors and hosts from the tree canopies to the savanna ground. year-round mosquito and monkey sampling over a period of more than years made possible the detection of yfv in various monkey and mosquito species (e.g., aedes africanus, aedes opok, aedes furcifer-taylori, and aedes luteocephalus), thus elucidating the mechanism of yfv maintenance in nature. epidemiological observations during this period allowed max germain to formulate the concept of a "yellow fever zone of emergence" in west and central africa [ ] . these ecological transition zones constitute ecotones adjacent to sylvatic environments, where prevailing ecological conditions, such as vector abundance, presence of non-human primates, and closeness for human contact, enable the cross-species transmission of viruses into humans-a "zone of emergence", which clearly appeared as the main source of epidemics in west africa [ ] . thus, this specific ecosystem constitutes an ideal transition ecotype, where vaccination campaigns for the containment of epizootics and ultimately eradication ought to concentrate [ ] . lastly, virus isolation in male mosquitoes (ae. furcifer-taylori) allowed the documentation of vertical transmission, allowing for virus maintenance in the inter-epidemic periods [ ] . in the early s, research laboratories focusing broadly on arboviruses were established under the umbrella of the collaborating center for reference and research on arboviruses (crora), led by paul brés. crora laboratories were created at various ips throughout africa, including ipd, ipa, and ipb, as well as ip yaoundé (cameroon), and ip tananarive (madagascar). one of the major activities was to establish an inventory of arboviruses circulating in various ecosystems. although virus isolations were made at various ips, virus characterization and identification were carried out at the crora reference center at ipd, followed by confirmation at yaru, with final registration in the international arbovirus catalogue [ ] . at the end of the s, following an outbreak of the ebola epidemic in zaire in , ipb set up a research program on viral hemorrhagic fevers that lasted until [ ] . this research program was the result of an important collaboration between the various pasteur institutes in africa and researchers of orstom. in , orstom extended its field of research and expertise to tropical medical virology, thus supporting teams from the riip. the partnership between riip and ostrom was instrumental in expanding the scope of field research in africa, with seminal studies on the vertical transmission of arboviruses in mosquitoes [ ] or the role of environmental factors, such as climate and latitude, in arbovirus transmission from arthropod to vertebrate hosts, using yfv [ , ] and dengue virus (denv) [ ] [ ] [ ] as models. research at ipb was also critical in elucidating the etiology of exanthematous fevers, coined "congolese red fevers", that have long been attributed to rickettsia. a total of arboviruses (chikungunya, igbo-ora, o'nyong nyong, sindbis, bouboui, yellow fever, wesselsbron, west nile, zika, ilesha, bwamba, dugbe, tataguine, nyando, bangui, and rift valley fever viruses) were associated with these syndromes. symptoms, consisting of fever, diffuse pain, and exanthem, were present in more than % of the cases, with etiology dominated by four viruses-chikungunya, ilesha, bwamba, and tataguine [ , ] . between and , two field research stations were established in the central african republic, one located adjacent to the forest (bouboui) and the other in the wooded savanna (bozo) (figure ). during this period, more than , mosquitoes of identified species in , pools were preserved for virus isolation. the most common anthropophilic mosquitoes caught were aedes (stegomiya) africanus and ae. (st.) opok, inside the forest gallery, aedes vittatus, in the savannah, and anopheles gambiae and an. funestus, in the houses of the village of bozo. a total of viruses were isolated and assigned to different species. these included chikungunya, bagaza, bouboui, bozo, bwamba, ilesha, kamese, kedougou, middleburg, mossuril, m'poko, nyando, orungo, pata, pongola, simbu, sindbis, tataguine, wesselsbron, west nile, yellow fever, and zika viruses. altogether, six arboviruses were found in the forest gallery, including bouboui, bozo, chikungunya, orungo, yellow fever, and zika viruses, vectored primarily by ae. africanus. research at ipd and ipb was also instrumental in demonstrating the expanded range of the crimean-congo hemorrhagic fever virus (cchv) in west and central africa [ ] [ ] [ ] , followed by repeated isolation of the virus. this allowed a clear understanding of its eco-epidemiology in the region, including north-south migration of the infected ticks through the cattle traffic migration patterns in the sahel [ , ] . likewise, active circulation of the rift valley fever virus (rvfv) was also demonstrated in senegal [ ] , mauritania [ ] , upper volta (present day burkina faso) [ , ] , and the central african republic [ ] . support for riip laboratories located in africa was provided by the ipp and ostrom teams, as well as through collaboration with various research centers in the u.s., such as harvard university, supporting the initial study on yellow fever and fnv vaccine; yaru, as a partner for new arbovirus classification; the centers for diseases control (cdc) at fort collins colorado, for the study of arboviruses; the cdc in atlanta and the u.s. army medical research institute of infectious diseases (usamriid), for the initiation of viral hemorrhagic fever research in central and west africa. the era of virus discovery in australia can be traced to the summer of - , when a major epidemic of encephalitis swept through southeastern australia. there were clinical cases reported in victoria, new south wales, and south australia, of which ( %) were fatal [ ] . a similar epidemic of unknown etiology (named australian x disease) had occurred in eastern australia from until , with almost reported cases and an average case/fatality rate of % [ , ] . surprisingly, no further cases were reported in the intervening years. amongst those investigating the - epidemic were john a.r. miles and colleagues at the institute of medical and veterinary science in adelaide, and eric l. french of the walter and elisa hall institute of medical research in melbourne who, almost simultaneously, reported the isolation of a virus from the brain tissue of clinical cases [ , ] . the virus, named murray valley encephalitis virus (mvev), was shown to be closely antigenically related to japanese encephalitis virus (jev), a flavivirus (then designated group b arbovirus) known to cause fatal encephalitis in east and southeast asia [ ] . the subsequent development of arbovirology and the pathway to virus discovery in australia were linked intimately with the establishment of the queensland institute of medical research (now the qimr-berghofer institute) at herston in brisbane ( figure ). ralph l. doherty joined the staff and, in , established a program of virus isolation from mosquitoes, based at a field station at innisfail in the far north queensland. in , doherty isolated the ross river virus (rrv) from aedes vigilax mosquitoes collected in townsville [ ] . he subsequently showed that rrv neutralizing antibodies occurred commonly in human sera in eastern australia [ ] . he also showed that individuals suffering from a severe debilitating syndrome, known as epidemic polyarthritis, had high antibody titres to rrv, suggesting a causal relationship [ , ] . in , doherty and his colleagues isolated the virus from a boy from the edward river mission aboriginal settlement in cape york [ ] . rrv and the related alphavirus, the barmah forest virus (see below), are now recognized as important public health problems in much of australia and the pacific islands, causing arthritis, myalgia, and fatigue for six months or longer. several thousand cases of the disease are notified annually [ ] . , normanton, and cairns in far north queensland. these included four novel flaviviruses (kunjin, kokobera, edge hill, and stratford), three orthobunyaviruses (koongol, wongal, and maputta) and one orbivirus (corriparta) [ ] . the study also identified two alphaviruses previously unknown in australia (sindbis and getah) and the first isolations of mvev from mosquitoes [ ] . with support from the rockefeller foundation, a field station was established at kowanyama and further expeditions were undertaken to collect arthropods and potential mammalian hosts throughout queensland. anopheline mosquitoes and a swamp pheasant (centropus phasianinus) collected at kowanyama from to yielded three novel viruses (kowanyama, trubanaman, and alfuy viruses) [ ] . in - , three novel viruses were isolated at kowanyama (wongorr and mitchell river viruses from mosquitoes and the ngaingan virus from biting midges), three novel viruses were isolated from mosquitoes collected near charleville in western queensland (charleville, warrego, and wallal viruses) and two viruses (belmont and d'aguilar viruses) were isolated from mosquitoes and biting midges, respectively, collected near brisbane [ , ] . further expeditions to charleville to collect mosquitoes resulted in the isolation of two novel viruses in (facey's paddock and murweh viruses) and two novel viruses in (parker's farm and little sussex viruses) [ ] . leanyer virus was also isolated in from mosquitoes collected near darwin in the northern territory [ ] . viruses were also isolated from wildlife hosts; the almpiwar virus was isolated from a skink (cryptoblepharus virgatus) at kowanyama in [ ] and the mossman virus was first isolated from a rodent (rattus leucopus) captured near mossman in [ ] . in collaboration with doherty and his qimr team, expeditions were also conducted to isolate viruses from ticks associated with sea birds. in , harald n. johnson from yaru collected soft ticks (ornithodoros capensis) from sooty tern (onychoprion fuscatus) colonies on the great barrier reef near cairns, from which two viruses (upolu and johnston atoll viruses) were isolated [ ] . the saumarez reef virus was subsequently isolated by toby d. st. george and colleagues from australia's commonwealth scientific and industrial research organization (csiro) in , from the same species of ticks associated with sooty terns on a coral cay in the southern coral sea [ ] . in , m. durno murray from csiro undertook an expedition to the australian territory of macquarie island in the southern ocean to collect hard ticks (ixodes uriae) associated with sea birds, resulting in the isolation of two novel viruses (nugget and taggert viruses) [ ] . a second csiro expedition to macquarie island in yielded two additional novel viruses (gadget's gully and precarious point viruses) from hard ticks collected in royal penguin (eudyptes chrysolophus schlegeli) rookeries [ ] . other research groups in australia also joined the hunt for arboviruses during the late s and early s, including ian d. marshall at the australian national university in canberra and neville f. stanley at the university of western australia. from to , marshall and his colleagues conducted surveys for arbovirus activity, particularly rrv and mvev, in coastal regions of new south wales and in the murray river valley. in addition to these and other known arboviruses, marshall and colleagues isolated several novel arboviruses from mosquitoes, including gan gan, yacaaba, tilligerry and termeil, paroo river, picola, and barmah forest viruses [ , ] and a novel reovirus, nelson bay virus, from a fruit bat (pteropus poliocephalus) [ ] . like the related alphavirus rrv, the barmah forest virus was subsequently shown to be a cause of epidemic polyarthritis in humans [ , ] , with infections occurring commonly throughout australia [ ] . the gan gan virus also infects humans and is suspected of an association with epidemic polyarthritis [ ] . marshall also conducted a number of collecting trips to papua new guinea from to funded by the rockefeller foundation. during an expedition to the sepik river district of papua new guinea in - , he isolated the joinjakaka virus from a mixed pool of culicine mosquitoes and the japanaut virus from both culicine mosquitoes and a fruit bat (syconycteris crassa). in western australia, stanley and colleagues surveyed for arbovirus activity in the ord river valley from to [ , ] . from , mosquitoes of species, virus isolates were recovered, including isolates of mvev and isolates of the kunjin virus from cx. annulirostris, suggesting the region may be an endemic focus in australia [ ] . the study also identified eight novel viruses, including kimberly, parry's creek, ord river, and kunnanurra viruses, as well as four unknown isolates (or , or , or , and or ), which have yet to be characterized [ , ] . continuing surveillance in western australia by others has continued to reveal novel arboviruses, including oak vale, stretch lagoon, parry's lagoon, and fitzroy river viruses [ ] [ ] [ ] [ ] . in , csiro established a new virology laboratory at long pocket in brisbane, headed by toby d. st. george, to investigate endemic diseases of livestock in northern australia. in , doherty and colleagues had isolated bovine ephemeral fever virus (befv) from cattle during a major epizootic in queensland [ ] but, despite epidemiological evidence suggesting vector-borne transmission, the virus had never been isolated from insects. this led st. george and colleagues to attempt virus isolations from a location in northern australia, where serological monitoring of a sentinel herd of cattle indicated that befv was likely to be enzootic. for a continuous period from october until may , insect collections for virus isolation were conducted at beatrice hill southeast of darwin. from the , mosquitoes and , biting midges processed, one isolate of befv was recovered (from a mosquito pool). however, the collection also yielded other virus isolates from different serological groups, including four novel viruses (csiro village, marrakai, beatrice hill, and humpty doo virus) [ ] . most significantly, the collection also yielded a single isolate of a novel serotype of bluetongue virus (btv serotype , btv- ), a major pathogen of sheep and goats that had previously been regarded as exotic to australia [ ] . the isolation of btv- and the consequences for international trade dramatically changed the landscape with respect to virus discovery and characterization in australia. an immediate consequence was the approval of government expenditure for the establishment of the $ million csiro australian animal health laboratory (aahl) in geelong, victoria, providing high-level biosecure containment for laboratory work and live animal studies. the discovery also led to the establishment of a permanent veterinary virology capability at the berrimah veterinary laboratory in darwin under geoff p. gard, and a national program for serological monitoring of sentinel cattle herds and the collection of insect vectors. efforts to isolate viruses from arthropods and livestock intensified. in the northern territory, a second novel serotype of bluetongue virus (btv- ) was isolated from a healthy sentinel cow at victoria river station in [ ] , and four other novel arboviruses (coastal plains, berrimah, adelaide river, and koolpinyah viruses) were isolated from healthy cattle between and [ ] [ ] [ ] [ ] . in queensland, eight novel arboviruses were first isolated between and from biting midges (tibrogargan, tinaroo, peaton, wongabel, and walkabout creek viruses), healthy sentinel cattle (the douglas virus), and soft ticks (argas robertsi) (vinegar hill and lake clarendon viruses) [ , [ ] [ ] [ ] [ ] [ ] . surveillance activities by the berrimah veterinary laboratory have continued to the present, with regular reports of the isolation of novel arboviruses. continuing surveillance by others in northern australia has also resulted in the isolation from mosquitoes of the bamaga virus and new mapoon virus from cape york [ , ] . the s also saw several significant disease emergence events in australia, which drew particular attention to bats as reservoir hosts of highly pathogenic viruses. in september , an outbreak of a severe respiratory disease occurred in horses at a stable in brisbane. of the affected horses were euthanized or died of the disease. one of two severely affected humans who had contact with the horses also died. cooperation between the queensland government, the newly established csiro australian animal health laboratory, and others resulted in rapid isolation of the hendra virus, a novel paramyxovirus [ ] , and the identification of fruit bats as reservoir hosts [ , ] . the hendra virus has since re-emerged regularly in australia, with more than confirmed cases in horses and seven infected humans, four of whom have died. in , an injured female fruit bat (pteropus alecto) was found at ballina in new south wales. tissue homogenates from the euthanized bat were injected into mice, resulting in the isolation of a novel lyssavirus, subsequently named australian bat lyssavirus (ablv) [ ] . three fatal human cases of ablv infection have subsequently been reported [ ] [ ] [ ] . the virus is now known to occur at low prevalence in five of six families of bats endemic to the northern territory, queensland, and western australia [ ] . in april , another novel paramyxovirus, the menangle virus, emerged at a commercial piggery in new south wales, causing stillbirths with abnormalities of the brain, spinal cord, and skeleton [ ] . two humans exposed to the pigs also developed an influenza-like illness [ ] . fruit bats were again implicated as reservoir hosts [ ] . the role of bats in the ecology and emergence of pathogenic viruses has been a major focus of study in australia since that time, primarily involving research teams led by hume e. filed and linfa wang. in all, more than novel rna viruses representing families and genera have been isolated from humans, livestock, wildlife, and arthropods in australia and papua new guinea, and reported in the literature (table s ). many others have been isolated but remain uncharacterized and/or unreported. more complete characterization of these viruses will be facilitated greatly by the use of ngs. south and southeast asia have also been a fertile area for virus discovery, particularly novel mosquito-borne and tick-borne flaviviruses and viruses with reservoirs in bat species. early studies in india, partly funded by the rockefeller foundation, by telford work and his indian colleagues from the virus research centre in pune, including d.p. murthy, p.n. bhatt, h. trapido and k. pavri, led to the discovery of the kyasanur forest disease (kfd) virus [ ] . the virus was isolated from sera and tissues collected from a moribund black-faced langur (presbytis entellus). this followed reports of an epizootic of unknown etiology causing large numbers of deaths in non-human primates and a number of cases of severe febrile illness in villages close to forested areas where dead monkeys had been found. the virus was shown to be closely related to the russian spring-summer encephalitis (rsse) serocomplex of flaviviruses, now known as the tick-borne encephalitis (tbe) serocomplex of flaviviruses. the virus was also isolated from some larvae and nymphs of hemaphysalis spinigera ticks [ ] . kfd in humans followed a biphasic course, not unlike tbe, but with some hemorrhagic manifestations not seen in tbe, and without either meningitis or encephalitis. another tick-borne virus related to the rsse serocomplex, langat virus, had been isolated two years earlier from a pool of hard ticks, ixodes granualtus, collected from forest rats caught near kuala lumpur, malaysia, by c.e. gordon smith, then working at the institute for medical research in kuala lumpur [ ] , but it is not known to be a human pathogen. a number of mosquito-borne flaviviruses were first isolated in southeast asia. the most important with respect to human disease are three of the four dengue serotypes. although dengue serotype had been first isolated independently by hotta in japan in [ ] , and shortly after by sabin in cincinnati in with material collected in hawaii [ ] , the other three serotypes were first isolated from material collected in southeast asia. dengue serotype was also isolated by sabin in from material obtained from new guinea [ ] and dengue serotypes and were first isolated in from human sera and aedes aegypti and culex tritaeniorhynchus mosquitoes collected during a major outbreak of epidemic hemorrhagic fever in manila, philippines, by w.m. hammon, a. rudnick, and colleagues at the university of pittsburgh [ ] . other novel flaviviruses have been isolated in malaysia, thailand, and papua new guinea [ ] . the tembusu (tmuv) virus was isolated in kuala lumpur in from various mosquito species [ ] and was the first of several closely related viruses, including the thcar virus, which was isolated from a pool of cx. tritaeniorhynchus mosquitoes collected in chiang mai, thailand, in [ ] ; the sitiawan virus, from sick broiler chicks in malaysia [ ] ; and the duck tembusu virus, an infection of ducks and geese causing an egg-drop syndrome in china and southeast asia [ , ] . neutralising antibodies were found in humans in malaysia [ ] but the virus has not been implicated in human disease. two other mosquito-borne flaviviruses have been described, jugra virus and sepik virus. little is known about the jugra virus, which was isolated from aedes spp. and uranotaenaia spp. mosquitoes and from the blood of a cynopterus brachyotis fruit bat [ ] . the sepik virus was isolated in by ian d. marshall and colleagues from a pool of mansonia septempunctata mosquitoes collected in the sepik district of papua new guinea [ ] . it was associated with a hospitalized case of febrile illness of unknown origin, with rising neutralising antibody to the virus. the sepik virus is particularly interesting, as its nucleotide sequence analysis shows it to be the closest known flavivirus to yellow fever virus [ ] . a novel lineage of the west nile virus was isolated in sarawak, east malaysia, by d.i.h. simpson, e.t.w. bowen, and colleagues, from cx. pseudovishnui group mosquitoes [ ] . initially called kunjin virus, it was shown to differ significantly in genomic sequence from the australian kunjin viruses, which have been shown to comprise west nile lineage b viruses, and have been described as west nile lineage virus [ ] . two flaviviruses with no known vector have been isolated in southeast asia, both from cy. brachyotis fruit bats: the carey island virus was isolated from a bat in the jugra forest, malaysia, in by a. rudnick and colleagues from the institute of medical research, kuala lumpur and the international center for medical research, university of california [ ] , and the phnom penh virus was isolated by j.j. salaun and colleagues in from the salivary glands and brown of bats [ ] . a closely related virus, the batu cave virus, is considered to be a variant of phnom penh virus. a considerable number of novel bunyaviruses have been isolated from south and southeast asia, particularly by scientists from the national institute of virology (formerly the virus research centre) in pune, including p.n. bhatt, k. pavri, k.r. singh, c.n. dandawate, f.m. rodrigues, d.t. mourya, p.d. yadev, a.c. mishra, and many others. recently reviewed in [ ] , these viruses include the orthobunyaviruses, umbre, kaikalur, thimiri, and sathuperi viruses; a nairovirus, ganjam virus; phleboviruses, bhanja, and malsoor viruses; a hantavirus, thottapalayam virus; and two uncharacterized viruses, kaisodi and wanowrie viruses. additionally, novel bunyaviruses, batai and oya viruses, have been isolated in malaysia, the former from cx. gelidus mosquitoes [ ] and the latter from pigs [ ] , and the kaeng khoi virus was isolated from tadarida plicata bats in thailand. novel orbiviruses from south and southeast asia include the sathuvachari virus, isolated from starlings (brahminy myna) collected in vellore, tamil nadu, india, and most closely related to the mosquito-borne orbiviruses [ ] ; and the japanaut virus, isolated from a mixed pool of culicine mosquitoes from papua new guinea [ ] . new rhabdoviruses first isolated in south asia include the chandipura virus and joinjakaka virus-the former, a major human pathogen in india, was isolated from a human infection in near nagpur city [ ] , whereas the latter, isolated in from a mixed culicine pool in the sepik district of papua new guinea, is not associated with disease in humans or animals. chandipura infection is characterized by fever, chills, arthralgia, myalgia, vomiting, and weakness. two novel alphaviruses were reported in kuala lumpur-bebaru and getah viruses. bebaru was first isolated from cx. (lophoceraomyia) spp. collected in , but although neutralising antibodies have been found in human sera, it has not been associated with human disease [ ] . getah virus was first isolated from cx. gelidus mosquitoes collected in near kuala lumpur [ ] . it causes a mild disease in horses, characterized by pyrexia, edema of the hind limbs, swelling of the submandibular lymph nodes, and urticarial rash. it also causes a mild disease in pigs, with occasional reproductive problems, including abortion and neonatal infections. neutralising antibodies have been found in a number of animals and in humans. the important role of bats as reservoirs of a wide range of viruses was underlined by a number of the viruses described above, and particularly by the discovery of their role as the reservoir of the nipah virus in malaysia in [ ] and subsequently their probable role as the origin of severe acute respiratory syndrome coronavirus (sars-cov) [ , ] . the nipah virus, a virus closely related to the hendra virus in australia, was first isolated k.b. chua and s.k. lam during an outbreak of severe disease of humans and pigs in - in peninsula malaysia [ ] , resulting in human cases with a mortality of %, and the culling of over million pigs. transmission to humans was from infected pigs. the disease in humans was a rapidly progressive encephalitic syndrome, with a significant pulmonary syndrome in some patients [ ] . in pigs, the disease was spread via the respiratory tract, and the symptoms were either neural or pulmonary, or both. subsequent epidemics in bangladesh and india have substantially expanded our knowledge of the nipah virus, and have demonstrated that direct transmission of the virus from bats to humans can occur through the consumption of date palm juice, and possibly by other routes, and that mortality rates may often be significantly greater than in malaysia [ ] . furthermore, evidence of nipah-like and hendra-like viruses have been detected either by isolation or serology from other pteropid bats across the geographic range of the genus, and related viruses may be carried by other bat species on other continents. sars-cov was first isolated by m. peiris and colleagues in hong kong [ ] , and contemporaneously in the u.s. [ ] and europe [ ] . it was shown to be unrelated to other coronaviruses. transmission to humans is believed to have been via an intermediate host, such as the himalayan palm civets (paguna larvata), through wet markets in southern china. continued studies of the nipah virus in malaysia, and subsequently in bangladesh and india, and investigations of sars-cov in bats have resulted in an enormous explosion of knowledge of viruses carried by bats, with many examples from most viral families, although in many cases the information is from genomic fragments [ ] . virus isolations have been made from bats, especially frugivorous bats, from india and malaysia. one of the earliest isolations was a paramyxovirus of the genus rubulavirus, which was isolated from a rousettus leschenaultia bat collected near pune in india [ ] . a novel adenovirus from the genus mastadenovirus was also isolated from the same fruit bat species caught in maharashtra state [ ] . a number of viruses have been isolated from fruit bats in malaysia by k.b. chua, s.k. lam, l.f. wang, and their colleagues-tioman virus, a paramyxovirus in the genus rubulavirusi, isolated from pteropus hypermelanus and related to the australian menangle virus, but not known to cause human disease [ ] ; pulau virus, an orthoreovirus related to the nelson bay virus of australia, and not associated with human or animal disease [ ] ; melaka virus, an orthoreovirus, causing acute respiratory disease in humans [ ] ; and kampar virus, an orthoreovirus related to the melaka virus, and also causing acute respiratory disease [ ] . the importance of orthoreoviruses originating in pteropid bats was assessed in an outpatient clinic, where it was found that pteropine orthoreoviruses are among one of the common causative agents of acute upper respiratory tract infection (urti), with a cough and sore throat as the most common presenting clinical features [ ] . the history of arbovirus research in the ussr began in , when an expedition under the leadership of lev a. zilber (at the time, the head of central virological laboratory of narkomzdrav ussr in moscow) went to the russian far east to study seasonal epidemic encephalitis. this disease with high mortality rates affected forest workers and soldiers stationed in the taiga, mainly those who came from other regions of the ussr. the disease had a pronounced seasonality-the cases started being recorded at the beginning of may, reached the peak in early june, and declined by august. local doctors designated the disease "spring-summer encephalitis" (sse) and assumed that it had been caused by some kind of virus. it has also been suggested that there were some similarities between sse and "summer encephalitis" (japanese b encephalitis and st. louis encephalitis) described at the time, it was also assumed to be a toxic form of influenza [ ] . however, the etiology and transmission routes of sse remained unclear. during summer of , zilber and colleagues isolated at least strains of a new virus from the blood and cerebrospinal fluid of sick people, and from brain tissues of dead patients. the isolated virus had a weak antigenic relationship (in complement fixation tests) with japanese b encephalitis virus [ ] . based on comparative analysis of epidemiologic data and the seasonal abundance of ixodes persulcatus ticks in the taiga, zilber assumed that sse was transmitted by ticks, in contrast to "summer encephalitis", which is transmitted by mosquitoes [ ] . several strains of the virus were isolated from the ix. persulcatus ticks, and their ability to transmit the virus by biting laboratory animals was shown experimentally [ ] . one of the first isolated strains (sofjin) was used for infecting rhesus macaques, which developed the clinical symptoms with signs of central nervous system (cns) impairment, similar to those in sick people [ , ] . so, the etiological agent of sse, which was subsequently given the name tick-borne encephalitis, was discovered and is now known by the name tick-borne encephalitis virus (tbev). in - , subsequent expeditions under the leadership of pavlovsky and smorodintsev studied in detail various aspects of the ecology, epidemiology, and pathogenesis of tbev, as well as the protective properties of the first anti-tbe vaccine, obtained from the brain tissue of mice infected by the tbev strain sofjin [ ] [ ] [ ] . further studies showed that tbev is also prevalent in other regions of the ussr, including the european part, where the main vector of the virus is ix. ricinus ticks. at the same time, it was found that tbev is also an etiological agent of some seasonal encephalitis or febrile illnesses, such as central european encephalitis or biphasic milk fever [ ] [ ] [ ] . the strains of tbev were initially divided into two geographical subtypes ("far eastern" and "central european"). these subtypes differed in severity of the illness and had antigenic differences in virus neutralization tests with serum of convalescents [ ] . a third subtype of tbev ("west siberian") was described by pogodina and her colleagues in [ ] . genetic data that has been accumulating since the late s confirms the existence of three main tbev subtypes (or genotypes). the nucleotide difference between genotypes reaches - % when comparing complete genomes [ ] [ ] [ ] . tbe is the most important arboviral infection in russia. despite significant progress in the development of anti-tbev vaccines, thousands of cases are recorded annually in russia, mainly in siberian and far eastern regions [ ] . in the modern classification, tbev belongs to the species tick-borne encephalitis virus of the genus flavivirus (flaviviridae) [ ] . tbev is widely distributed within the area of its main arthropod vector-ix. persulcatus and ix. ricinus ticks, including russia, eastern and central europe, baltic and scandinavian countries [ , ] . the discovery of tbev as a causative agent of sse gave an impetus to studies of similar diseases throughout the ussr. during subsequent years, the major virological centres were established as parts of the academy of medical science of the ussr (ams ussr), such as the department of neurovirology at the institute of neurology ( ), the institute of virology ( ), the institute of poliomyelitis and viral encephalitis ( ), as well as departments of virology at regional medical institutes in siberia and the far east. scientists from these centres were actively involved in the study of various zoonotic viral infections distributed in the ussr. many participants of the first expeditions subsequently became famous virologists. one of the most notable ones is michael p. chumakov, who later headed the institute of virology ams ussr ( ) ( ) ( ) ( ) ( ) and the institute of poliomyelitis and viral encephalitis ams ussr ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in moscow. chumakov organized numerous expeditions that aimed to study the etiology of zoonotic human infections. in the s, outbreaks of the disease, designated by local doctors as "atypical tularemia", "anicteric leptospirosis", and "omsk spring-summer fever", were recorded in several rural regions of the omsk district in western siberia. clinicians from the omsk medical institute, under the leadership of ahrem-akhremovich, described the disease in detail and named it omsk hemorrhagic fever (ohf), as the patients often developed hemorrhagic diathesis. they also suggested that ohf was transmitted by dermacentor reticulates ticks, which are highly prevalent in the region [ , ] . in , chumakov and colleagues investigated the blood of patients with ohf and isolated strains of a new virus, which was similar but different from tbev in serologic tests. the virus was named omsk hemorrhagic fever virus (ohfv). several strains of ohfv were also isolated from de. reticulates ticks collected in the natural foci of ohf [ , ] . in subsequent years, the ecology of ohfv was extensively studied by scientists from the omsk medical institute and the institute of poliomyelitis and viral encephalitis ams ussr. the de. reticulatus ticks and their host, a narrow-headed vole (microtus gregalis), are considered an original natural reservoir of ohfv. the european water vole (arvicola amphibius), the tundra vole (microtus oeconomus), and some species of shrews are also involved in the circulation of ohfv [ ] . however, the emergence of ohf outbreaks in the s was presumably a consequence of the introduction by humans of muskrats (ondatra zibethicus) to this region in - [ ] . muskrats are highly susceptible to ohfv and serve as an extremely effective amplifying host. the appearance and growth of the muskrat population in the natural foci of ohf led to an increase in infection rates in other animals and ticks [ , ] . in addition to transmission of ohfv by ticks, humans can get infected while hunting and skinning, by direct contact with blood and excretions of infected animals. such "muskrat outbreaks" among hunters and their family members have been registered in the region at different times of the year, including winter, which is the season of active hunting for muskrats [ , ] . based on antigenic relationships, ohfv was assigned to the tbe antigenic complex [ ] , and later was classified as a separate species, omsk hemorrhagic fever virus of the genus flavivirus (family flaviviridae) [ ] . genome sequence analysis confirmed the close evolutionary relationships of ohfv with tbev [ ] [ ] [ ] . in , virologists led by chumakov studied the etiology of an outbreak of a febrile illness which was accompanied with hemorrhagic manifestations ("acute infectious capillary toxicoses") in a rural area in the northwest part of the crimean peninsula. they designated the disease as crimean hemorrhagic fever (chf) and suggested that is transmitted by hyalomma (plumbeum) marginatum ticks. despite the absence of virus isolates from specimens from chf patients or from ticks, the viral etiology of chf and its zoonotic nature were proven experimentally by infecting volunteers with the blood of chf patients or a filtered suspension of ticks collected from a hare caught in the focus of the disease [ ] . sporadic cases and outbreaks of chf were subsequently recorded almost annually in southern regions of the european ussr and central asian soviet republics. the first strains of the chf virus were isolated by alexander m. butenko from chumakov's team in , from sera of chf patients and from hy. marginatum nymphs isolated in southern russia [ , ] . later, the chf virus was shown to be identical to the congo virus isolated from a patient with hemorrhagic fever in zaire (present day democratic republic of congo) and the virus received its present name, the crimean-congo hemorrhagic fever virus (cchfv) [ ] . cchfv is a one of the prototypic nairoviruses and today is assigned to the species crimean-congo hemorrhagic fever virus, of the genus orthonairovirus (nairoviridae: bunyavirales). during spring and summer , chumakov, together with libíková from the institute of virology in bratislava (former cžechoslovakia), investigated an outbreak of fibrile illness in the kemerovo district in western siberia. initially, it was assumed that the patients were affected by tbe, but the sera of the patients did not react with tbev-specific antigen in serological tests. on the contrary, a new virus, named the kemerovo virus (kemv), was isolated from the blood of patients. several strains of kemv were also isolated from ix. persulcatus ticks collected in the region where the outbreak occurred [ , ] . similar to kemv, the tribeč virus and lipovníc virus were isolated from ix. ricinus ticks in czechoslovakia in [ , ] . based on morphological studies, kemv was classified to the genus orbivirus (family reoviridae) [ ] . the ecology of kemv in russia has not been studied sufficiently, but recent research has shown that its prevalence in ix. persulcatus, ix. ricinus, ix. pavlovsky, and de. reticulatus ticks varies from zero to . % in different regions of the country [ , ] . from the above, it follows that in the period - , arboviruses in the ussr were studied mostly as causative agents of human disease. examinations of arthropods and vertebrates in the natural foci of important human disease often led to exploring some other arboviruses. for example, butenko isolated the west nile virus (wnv) and dhori virus (dhov) from hy. marginatum ticks for the first time in the ussr while studying the natural foci of cchfv in the southern region of russia in [ ] . in the late s, there was an ecological trend in virology developing in the ussr. the founder of the ecological approach to virology in the ussr was dmitry k. lvov, who established the department of the ecology of viruses at the d.i. ivanovsky institute of virology in moscow ( ) , and later headed the institute ( - ). under his leadership, an ecological and virological survey was organized, aimed to identify the arboviral diversity in hematophagous arthropods and wild animals of the entire ussr. the survey included collecting and examining mosquitoes, ticks, and vertebrate animals (mostly rodents and birds), as well as samples from humans, in different types of biocenoses located in different climatic zones of the ussr. lvov and colleagues isolated more than strains of different mosquito-borne viruses, including viruses of the california encephalitis antigenic group (species california encephalitis orthobunyavirus) and batai and batai-like viruses (species bunyamwera orthobunyavirus) in the genus orthobunyavirus, family peribunyaviridae [ ] [ ] [ ] [ ] . the other mosquito-borne viruses whose circulation was discovered and studied extensively, are the sindbis virus (sinv) and getah virus (getv) (genus alphavirus, family togaviridae) [ ] [ ] [ ] . one of the important subjects of d. lvov's research was ix. uriae ticks, which parasitize on colony-nesting sea birds. in - , more than virus strains were isolated from ix. uriae ticks collected in the nests of sea birds on the coasts and islands in the sea of okhotsk, the bering sea, and the barents sea [ ] . the isolated strains were mostly classified as novel bunyaviruses, flaviviruses, and orbiviruses, often based on morphological studies of the virion structure only, because their antigenic relationships with other viruses were not known at the time. among them, the sakhalin virus (sakhv) and paramushir virus (prv) were described as a novel bunyaviruses and later classified to the species sakhalin orthonairovirus (genus orthonairovirus, family nairoviridae) [ ] . several other new viruses (zaliv terpenia, comandory, and rucutama viruses) were discovered and now belong to the species uukuniemi phlebovirus (genus phlebovirus, family phenuiviridae) [ ] . the tyuleniy virus (tyuv) was isolated for the first time, which is one of the prototypic viruses of seabird tick-borne flaviviruses group (genus flavivirus, family flaviviridae) [ ] . the prevalence of the okhotsky virus (okhv) and aniva virus (aniv), two newly described viruses belonging to the species great island virus (genus orbivirus, family reoviridae), have been studied in detail [ , , ] . many new viruses were discovered by lvov and his colleagues while exploring the territories of central asia and transcaucasia. they isolated and studied the issyk-kul virus (iskv), which is associated with bats of the family vespertionidae and their argasid ticks [ , ] . new viruses, tamdy (tamv) and burana (burv), were isolated from hyalomma spp. ticks collected from sheep or cows in pasture lands [ ] . some novel viruses (artashat, chim, and geran viruses) were isolated from argasid ticks collected in rodent burrows [ ] . morphological studies of these viruses by electron microscopy identified them as bunyaviruses. recently, they were classified as different species of the genus orthonairovirus (family nairoviridae) [ ] . in total, during the ecological and virological surveys of the s to s, thousands of strains of different arboviruses were isolated, some of which remain to be classified. based on the studies conducted by soviet and russian virologists, we now know that at least zoonotic viruses, assigned to eight viral families, circulate on the territories of the former ussr [ ] . does this number reflect the true diversity of viruses circulating in the vast territory of northern eurasia? this question can only be answered by additional research aimed at finding new viruses, using new methods and approaches. the concept of viruses developed from the observations of ivanovsky and beijerinck of "filterable agents", with the discovery of the causative agent of tobacco mosaic in the s. yellow fever virus and dengue virus were the first two arboviruses to be isolated early in the th century [ , ] . the pioneering work of alexis carrel on the development of many cell and tissue culture methods in the s at the rockefeller institute, and later refinements by maitland, eagle, and enders, led to the widespread use of various culture systems as indispensable tools for virus studies [ ] [ ] [ ] . although these tools have since been used extensively for the in vitro characterization of viruses, they were inadequate for the identification and classification of a flood of novel viruses collected through the yfv surveillance program supported by the rockefeller foundation. jordi casals, among others, led the use of the complement fixation (cf) test in order to study viruses affecting the central nervous system. the cf test exploits the unique affinity of complement for antigen-antibody complexes. the original assay was developed in the s for the serologic study of yfv, was improved in the s [ , ] , and its sensitivity and specificity improved again in the s [ ] . using cf tests, casals and his colleagues were able to classify viruses into antigenic groups. however, the inherent complexity and labour consuming aspects of the assay (titrations of antigen, complement, and hemolysin for optimal outcomes), technical demands (accurate interpretation of outcomes) and the development of alternative assays (see below), have restricted its applicability in laboratories worldwide. hirst observed in that chicken erythrocytes agglutinated in the presence of the influenza a virus and that virus-specific antibodies inhibited agglutination, forming the foundation for the hemagglutination inhibition (hi) test [ ] . a decade later and on theiler's suggestion, casals showed that many arboviruses also agglutinated erythrocytes, establishing the hi test as a diagnostic tool for arbovirus infection and identification [ ] . the gold standard for arbovirus identification, the plaque reduction neutralization test (prnt), has its origins in the observations of stokes and colleagues, dating back in the s when monkeys could be protected against yfv by the inoculation of convalescent sera from patients who had recovered from the disease [ ] . by the early s, max theiler had adapted the assay for use in mice, in which mixed serum and virus was inoculated intracerebrally [ ] . the cell culture adaptation of the test was first demonstrated by itoh and melnick in for studying the seroconversion of chimpanzees infected with echoviruses [ ] , and a year later by henderson and taylor to detect antibodies to the eastern equine encephalitis virus [ ] . versions of this assay are now widely used, including the microprnt [ ] , the virus reduction neutralization test (vrnt) [ ] , the focus reduction neutralization test (frnt) [ ] , the rapid fluorescent inhibition test (rffit) [ ] , the flow-cytometry neutralization [ , ] , the colorimetric micro-neutralization assay (cmnt) [ ] , and the reporter virus particle-based neutralization assays [ ] [ ] [ ] [ ] . historically, these methods (virus isolation, hi, cf, and neutralization tests) served as the basis of arbovirus diagnosis for many years, augmenting electron microscopy (see section below), which allowed the visualization of viruses in infected tissues and cell cultures. however, an inherent limitation of the serology-based assays has been their inability to determine whether antibodies in the examined serum were the result of a recent or past infection. this conundrum was solved by determining whether antibodies were igm (recent infection) or igg (past infection), using the enzyme-linked immunosorbent assay (elisa) [ ] . the introduction of elisa revolutionized the field by also offering increased specificity and sensitivity for the accurate detection of many viruses. overall, although identification of pathologic agents through serologic assays is quite straightforward, there are instances where accurate identification may not be possible due to cross-reactivity. for example, cross-reactivity among flaviviruses poses a challenge in their identification, even when the "gold standard" of prnt for arbovirus detection is applied, especially in hyper-endemic settings of flavivirus circulation. several diagnostic labs faced this challenge during the recent emergence and explosive spread of the zika virus in the americas. a similar challenge is also common for the serologic diagnosis of bunyavirus infections, which is attributed to their ability to reassort. in this scenario, a novel bunyavirus may be misidentified as a known pathogen due to the presence of the m segment (contributed by the known pathogen), which encodes the immune-reactive envelope proteins (reviewed in [ ] ). since the beginning of modern virology in the s, transmission electron microscopy (tem) has been one of the most important and widely used techniques for the identification and characterization of new viruses. two tem techniques are usually used for this purpose: negative staining on an electron microscopic grid coated with a support film and (ultra) thin section tem of infected cells, fixed, pelleted, dehydrated, and embedded in epoxy plastic. negative staining can be conducted on highly concentrated suspensions of purified virus or cell culture supernatants. for some viruses, tem can be conducted on contents of skin lesions (e.g., poxviruses and herpesviruses) or concentrated stool material (rotaviruses and noroviruses). for successful detection of viruses in ultrathin sections of infected cells, at least % of cells must be infected, and so either high multiplicity of infection (moi) or rapid virus multiplication is required. viruses can be differentiated by their specific morphology (ultrastructure): shape, size, intracellular location or, for some viruses, from the ultrastructural cytopathology and specific structures forming in the host cell during virus replication. usually, ultrastructural characteristics are sufficient for the identification of a virus at the level of a family. in certain cases, confirmation can be obtained by immuno-em performed either on virus suspension before negative staining or on ultrathin sections. this requires virus-specific primary antibodies, which might be not available in the case of a novel virus. for on-section immuno-em, oso post-fixation must be omitted and the partially dehydrated sample must be embedded in a water-miscible acrylic plastic (usually lr white). the ultrastructure of most common viruses is well documented in good atlases and book chapters [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and many classical publications of the s, s, and s. several excellent reviews were recently published on the use of tem in the detection and identification of viruses [ ] [ ] [ ] . the advent of next generation sequencing (ngs) has expanded the tool kit of the virus hunter. for many years, sanger-based sequence analysis has been employed in the identification and characterization of viruses [ ] [ ] [ ] [ ] . however, with the completion of the human genome sequence, the necessity for a high throughput approach that provided a massively-parallel sequencing strategy was fully apparent [ , ] . the automated sanger method was considered a first-generation technology and newer methods are referred to as next generation sequencing (ngs). commercially available technologies from roche/ , illumina, life technologies/apg, oxford nanopore, and pacific biosciences offer unique ngs platforms, and all have been extensively reviewed [ ] [ ] [ ] [ ] [ ] . unlike sanger sequencing, ngs does not require prior knowledge of the viral sequence and thus can be used for viruses of unknown sequence. many viruses cannot be cultured in the cell culture systems currently in use. ngs has shifted the paradigm by removing the need for cell culture and so opening the door to the discovery of many new viruses. the first instruments for ngs were developed in the s and commercialized in the early s, and they were quickly adopted for the identification of novel viruses from a wide range of sources. the number of known viruses increased from about , in to approximately , viruses in [ ] , and that number has since increased dramatically. the technology has also been applied to sequence analysis of many previously known but poorly characterized viruses. this has significantly expanded the known virosphere and assisted in understanding the diversity and evolutionary relationships of viruses. the expansion of the known virosphere has also allowed the taxonomic assignment of an increasing number of viruses, with a total of orders, families, genera, and species of viruses currently approved by the international committee on taxonomy of viruses (ictv) [ ] [ ] [ ] [ ] [ ] [ ] . ngs has also allowed sequence analysis of hundreds or thousands of isolates known pathogenic viruses, facilitating epidemiological studies at scales extending from very local to global. this has generated a trove of new knowledge of significant public health importance. however, without the availability of isolates, ngs has not necessarily increased our understanding of the ecology of novel viruses, their host range, and the risks they may pose to public, veterinary, agricultural, or environmental health. the application of ngs to the unbiased mass sequencing and bioinformatic analysis of total nucleic acids extracted from biological samples obtained from a wide range of sources has led to an explosive increase in the number of complete or near-complete viral genomes. for example, a recent study using ngs to sequence the transcriptomes of arthropods representing species from four classes (insecta, arachnida, chilopoda, and malacostraca) identified novel viruses, many of which are represented by complete or near-complete genomes [ ] . the novel viruses encompass the entire taxonomic diversity of previously known families and/or genera of (-) ssrna viruses and include divergent viruses with entirely novel and unusual genome architecture. similarly, sequence analysis of the transcriptomes of animals of more than species sampled across nine metazoan phyla (arthropoda, annelida, sipuncula, mollusca, nematoda, platyhelminthes, cnidaria, and echinodermata), as well as chordates of the subphylum tunicata (salps and sea squirts), resulted in the discovery of rna viruses, mostly represented by complete or near-complete genomes [ ] . based on phylogenetic analysis of rna polymerase (rdrp) domain sequences, the novel viruses included clades representing many established families of plant and animal rna viruses, as well as at least five clades that are so divergent that they are considered as likely new virus families or orders. also, the sequencing of transcriptomes of gut, liver, and lung or gill tissue of fish, reptiles, amphibians, and birds identified novel vertebrate-associated viruses, representing every family or genus of rna virus associated with vertebrate infection, including those containing important human pathogens (orthomyxoviruses, arenaviruses, and filoviruses) [ ] . these and other similar studies have heralded a new era in virology, revealing new dimensions in viral biodiversity and providing largely unexpected insights into the deep evolutionary history of viruses. however, only a minor subset of newly discovered viruses has been subject to full phenotypic characterization which can provide critical and fundamental insights into their biology and virus-host interactions, ultimately transforming our understanding of the evolutionary forces that shape the virosphere and disease emergence. realistically, as important as these discoveries are to the advancement of science, very few of the viruses will ever cause human disease or influence the global economy. this mass sequencing approach, which has been called viral metagenomics, can also be applied in a more targeted way to identify viruses in clinical cases of diseases of unknown etiology or to survey for potentially novel zoonotic viruses that may represent a significant risk of transmission to humans. indeed, ngs is being used increasingly in conjunction with real-time pcr as a front-line tool in medical and veterinary settings for rapid detection and identification of exotic or unknown emerging viruses. for example, in , an outbreak of acute hemorrhagic fever occurred in mangala, democratic republic of congo (drc), involving three human cases, two of which were fatal. as no positive diagnosis was obtained using real-time pcr for known viral hemorrhagic fevers in africa, ngs was conducted on acute phase serum collected from the surviving patient, revealing the near-complete sequence of a novel rhabdovirus, bas-congo virus (basv; species bas congo tibrovirus) [ ] . although the disease was attributed by the investigators to the novel virus, no isolate was obtained and there was no evidence of neutralising antibodies in serum samples from undiagnosed hemorrhagic fever cases or random serum donors from the drc. subsequently, ngs of blood collected from healthy individuals from nigeria identified two related viruses, ekpoma virus (ekv- ; species ekpoma tibrovirus) and ekpoma virus (ekv- ; species ekpoma tibrovirus), and a serological survey indicated that antibodies to these or similar viruses occur commonly in healthy humans in nigeria [ ] . however, once again, neither virus was isolated. interestingly, several other tibroviruses had previously been isolated from healthy cattle or biting midges (culicoides spp.) in australia and florida [ , , , ] . none have been associated with either disease in livestock or the infection of humans [ , ] . these and other studies raise important issues regarding the significance of ngs data, even when providing complete or near-complete viral genome sequences, when investigating disease etiology. most viruses can cause asymptomatic infections and many newly discovered viruses may be benign in their natural host. therefore, in the absence of a virus isolate which can be used for experimental studies, establishing a causal association with disease based only on detection of the viral genome should be approached cautiously. the availability of a rapidly expanding number of novel viral genomes identified by metagenomic studies has also presented challenges for virus taxonomy-a system for classification of viruses that is administered by the international committee on taxonomy of viruses (ictv). should viruses detected only by their nucleotide sequence be classified and assigned to species and other higher taxa (genus, family order, etc.) alongside viruses for which we have a viable isolate? the ictv (then the international committee on nomenclature of viruses) was established in at the ninth international congress for microbiology in moscow, publishing its first report in [ ] . it operates under the auspices of the international union of microbiological societies (iums), with the authority to develop, refine, and maintain a universal virus taxonomy. historically, the description and classification of a new virus by the ictv required significant information, such as host range, serology, replication cycles, and structure, aspects that could be determined from the study of isolates. on the other hand, sequences alone provide a trove of information, including evolutionary relationships (e.g., phylogeny), genome organization (e.g., the number of genes and their order), presence or absence of distinctive motifs (e.g., protein cleavage sites, terminal sequences, internal ribosome entry sites), as well as genome composition (e.g., codon usage, gc content), which of course could be used to inform classification into species. these concerns framed the contents of a workshop of experts and members of the ictv, resulting in a seminal consensus statement in which viruses identified only from metagenomic data are considered to be bona fide viruses and thus candidates for taxonomic assignment [ ] . the expanding diversity of the known virosphere also presented challenges. a recent analysis of metagenomes of geographically and ecologically diverse samples led to the discovery of , new partial dsdna viral genomes encoding more than . million proteins, % of which had no sequence similarity to proteins from known virus isolates [ ] . other metagenomic studies have revealed similar diversity in ssdna and rna viruses, particularly in marine ecosystems [ ] . this has led ictv to consider a far broader framework for taxonomic assignment of viruses, recently approving the establishment of a taxonomic hierarchy that includes ranks (realm, subrealm, kingdom, subkingdom, phylum, subphylum, class, subclass, order, suborder, family, subfamily, genus, subgenus, and species), thus expanding the range even beyond those currently available for other organisms [ ] . the sheer volume of new virus genomes identified by metagenomic studies has also led to the development of new bioinformatic tools, that are increasingly being applied for automated virus classification of viruses, based almost exclusively on nucleotide sequence data [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . while the classic tools of virus discovery and characterization (e.g., em, serology, and tissue culture) are still widely used, ngs allowed for the rapid identification of an enormous repertoire of viruses, thus exponentially expanding the boundaries of the known virosphere. the resultant genomic sequences allowed for their accurate taxonomic assignments, analysis of their phylogenetic and evolutionary relationships with other viruses, and evaluation of the potential risks they may present to humans and wild or domestic animal populations. below are a few representative examples of how ngs transformed the known relationships of arboviruses within their respective families. rhabdoviruses contain negative-sense (-) single-stranded rna (ssrna) genomes. they are amongst the most numerous and diverse of rna viruses, naturally infecting mammals, birds, fish, reptiles, and amphibians, as well as insects, arachnids, crustaceans, nematodes, and a wide range of plants [ , ] . most (but not all) rhabdoviruses that infect vertebrates are transmitted by hematophagous arthropods. the rhabdoviridae currently comprises genera containing species and one unassigned species [ ] . of these, viruses assigned to seven of the genera (vesiculovirus, ephemerovirus, tibrovirus, hapavirus, ledantevirus, curiovirus, and sripuvirus) are considered to be arboviruses. viruses assigned to the newly characterized genus almendravirus appear to be insect-specific [ ] . viruses assigned to the genus tupavirus have been isolated only from vertebrates but may possibly have arthropod vectors. complete coding sequences are now available for more than other rhabdoviruses, many of which have been isolated from or detected in arthropods, but they have not yet been formally classified. while the classic tools of viral discovery (e.g., em, serology, and tissue culture) are still widely used, ngs has played a central role in recent genome sequencing efforts, revealing diversity, not only in the ecology of rhabdoviruses but also in the structural diversity of genome architecture [ , [ ] [ ] [ ] [ ] [ ] [ ] . in addition to the five canonical rhabdovirus structural protein genes (n, p, m, g, and l), it is now recognized that rhabdoviruses commonly contain multiple long open reading frames encoding putative accessory proteins, mostly of unknown function. ngs has also facilitated studies of the evolution of rhabdovirus genome organization [ ] and revealed the importance of arthropods in the evolutionary history of rhabdoviruses [ ] . according to the current international committee on taxonomy of viruses (ictv), classification of the order bunyavirales encompasses families of segmented (-) ssrna viruses-arenaviridae, hantaviridae, nairoviridae, peribunyaviridae and cruliviridae, mypoviridae, fimoviridae, wupedeviridae, phasmaviridae, and phenuiviridae-a classification based on structural, genetic, and antigenic characteristics [ ] . bunyavirales is a diverse order with a large number of viruses associated with human, veterinary, and plant disease, as well as being vectored by arthropods (mosquitoes, ticks, sandflies, and thrips) and infecting a wide range of other invertebrates. the recent discovery of gouléako (golv) [ ] and cumuto (cumv) [ ] viruses (the latter with ngs) which are evolutionarily related to but distinct from viruses in the genus phlebovirus, family phenuiviridae, suggesting they are to be assigned to the new genus goukovirus. in the past five years, ngs has revolutionized understanding of the vast diversity of this order, by allowing genetic identification of previously uncharacterized and unassigned bunyaviruses, which in turn provided a complimentary approach to the gold standard of classification (structural, genetic, and antigenic characteristics) for a more refined taxonomic classification. combined, these approaches will be instrumental for enhancing our understanding of their ecologic and geographic distribution, as well as public health impact. the family flaviviridae comprises positive-sense (+) ssrna viruses. the family contains several serious human pathogens, including dengue, yellow fever, zika, japanese encephalitis, west nile, and tick-borne encephalitis viruses (all arboviruses in the genus flavivirus) and the hepatitis c virus (a member of the genus hepacivirus). members of the genus flavivirus, like the alphaviruses (see section . ), are a diverse group of arthropod-borne viruses that are found on every continent except antarctica. several flaviviruses have been discovered and characterized recently, mainly through chance or increased surveillance efforts, and facilitated through ngs. newly characterized viruses include the mercadeo virus (mecdv), from pools of culex species mosquitoes in panama [ ] , sabethes flavivirus (sbfv), from sabethes belisarioi in brazil [ ] , the la tina virus (ltnv), from aedes scapularis in peru [ ] , the long pine key virus (lpkv), from anopheles crucians in the florida everglades, and the kampung karu virus (kpkv), from anopheles tesselatus in borneo [ ] , aedes flavivirus (aefv), from aedes albopictus laboratory colony that originated in thailand [ ] , xishuangbanna flavivirus (xfv), from aedes albopictus in china [ ] , and the cuacua virus (cucuv), from mansonia ssp. in mozambique [ ] . one unexpected finding was the discovery of segmented viruses that grouped in the family flaviviridae and were more closely related to flaviviruses than members of other established genera. the jingmen tick virus (jmtv) is unique in the family flaviviridae with a four segment positive-sense genome [ ] . traditional sanger sequencing generated the ns and ns gene sequences but could not connect these within a single genome segment. phylogenies based on either ns -like or ns -like sequences, which are encoded on two of the four segments, showed jmtv falling basal on the flavivirus tree and clearly distinct from the other viruses in this genus. the remaining two segments did not appear to have a flavivirus origin. jmtv has been isolated from cattle, monkeys, and ticks (rhipicephalus and haemaphysalis spp.) [ ] . this prototype virus has lent its name to the newly identified segmented flaviviruses, and other viruses have recently populated the jingmenvirus clade. similarly, the guaico culex virus (gcxv) consists of five segments, and initial studies suggested gcxv is insect-specific, based on its isolation from culex ssp. mosquitoes [ ] . additional potentially insect-specific four-segmented viruses include shuangao insect virus (saiv ), the wuhan flea virus (whfv), wuhan aphid virus (whav ), wuhan aphid virus (whav ), and the wuhan cricket virus (whcv) [ ] . interestingly, sequences that are similar to toxocara canis, a larva roundworm, and tentatively named t. canis larva agent virus (tclav), are distantly related to jmtv and appear to be the first evidence of a flavivirus-line organism in a member of the phylum nematoda [ ] . the discovery and characterization of a segmented genome flavivirus has significant implications, as it reveals that rna virus segmentation is an evolutionary process that has occurred in previously unanticipated circumstances. the alphaviruses are a diverse group of (+) ssrna arthropod-borne viruses that are found on every continent except antarctica [ ] . there were no known mosquito-only alphaviruses until , when analysis by ngs of a mosquito-pool from the negev desert in israel showed the presence of an alphavirus, the eilat virus (eilv) [ ] . further analysis showed that, although eilv could infect mosquito cells, it was unable to replicate in vertebrate cells [ ] . eilv is considered a vaccine candidate, as it can produce immunity without replication in the vertebrate host, illustrating the importance of such discoveries, which can lead to novel platforms for the prevention and/or treatment of disease [ ] . reoviruses are a diverse family of double-stranded rna (dsrna) viruses that infect a wide range of hosts and have a wide range of characteristics. of the genera, there are only three for which novel viruses have been described using ngs, and the majority of these are in the genus orbivirus. interestingly, because of the structure of the genomes of reoviruses and the conserved sequences at the ' and ' ends of the segments [ ] , it is relatively simple to sequence segments of reoviruses using more traditional techniques. this may be why there are so few novel reoviruses determined by ngs. novel viruses identified using ngs have been assigned to four other genera (seadornavirus, orthoreovirus, dinovernavirus, and cypovirus), all of which are shown in table . refers to year of the publication and not of the isolation of the virus. *not yet formally classified. newly recognized viruses containing a (+) ssrna genome have been proposed to form a new genus negevirus, related to genera of mite-infecting plant viruses (blunervirus, cilevirus, and higrevirus) in the new family kitaviridae [ , ] . originally, six viruses with restricted host range in insects (isvs), designated as negev (negv), ngewotan (nwtv), piura (piuv), loreto (lorv), dezidougou (dezv), and santana (sanv), were identified in and isolated from mosquitoes and phlebotomine sandflies, collected in brazil, the ivory coast, israel, indonesia, peru, and the usa, [ ] . their widespread geographic distribution was documented by several other groups, who reported the isolation and characterization of related viruses in the philippines (tanay virus; tanav) [ ] , trinidad and tobago (wallerfield virus; walv) [ ] , côte d'ivoire (goutanap virus; ganv) [ ] , portugal (ochlerotatus caspius negevirus; ocnv and culex univittatus negevirus; cunv) [ ] , brasil (brajeira and wallerfield viruses), colombia, and nepal. collectively, the close relationship of negeviruses with plant viruses of the genera cilevirus, higrevirus, and blunervirus, coupled with their heterogenous genome organization and architecture, provides support for the possibility that negeviruses are plant-like viruses that could eventually anchor a new virus family [ ] . members of the family mesoniviridae, a newly discovered family of (+) ssrna viruses assigned to the order nidovirales [ , ] , appear to have an extensive geographic distribution but a restricted host range within members of the family culicidae (flies) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the mesoniviridae comprise of a single genus alphamesonivirus with nine recognized species: alphamesonivirus , including nam dinh (ndiv) [ ] , cavally (cavv) [ ] , ndiv a . [ ] , ndiv ngewotan, and ndiv houston [ ] viruses; alphamesonivirus , including the single isolate of the karang sari (ksav) virus, and the four bontag baru (bbav) isolates sampled in the early s in indonesia, but only recently characterized [ ] ; alphamesonivirus , including the dak nong virus (dknv), the three isolates of kamphaeng phet (kphv), sampled in indonesia and thailand in the mid- s [ , ] ; alphamesonivirus , including the casuarina virus (casv), isolated in australia from coquillettidia xanthogaster mosquitoes [ ] ; alphamesonivirus , the african hana virus (hanav) [ ] ; alphamesonivirus and , including ofaie (ofav) and kadiweu (kadv) viruses, isolated in the pantanal of brasil from mansonia sp. mosquitoes, respectively [ ] ; and alphamesonivirus- and , including the african nse (nsev), and meno (menov) viruses [ ] ; and the distinct yet unassigned species, the yichang virus [ ] , isolated from culex sp. mosquitoes in china. across several generations, virus hunters have left a profound legacy, both to science and to the broader global community. in the field, their work has often been conducted in difficult, demanding, and sometimes dangerous circumstances. in the laboratory, they have developed and applied tools and methodologies that led to improved diagnosis, prevention, and treatment of viral disease, and still lie at the center of virology today. others working at more fundamental scientific levels have used their virus isolates as scientific models, opening the door to a revolution in molecular and cellular biology. yet, their work continues. viral disease emergence remains one of the most serious threats to humanity, with potentially devastating social and economic consequences. recent examples, such as sars, mers, henipa-, ebola, and zika viruses, illustrate the threat that unidentified or poorly characterized viruses can, and almost certainly will, continue to present. equally devastating emerging diseases of livestock, fisheries, and crops threaten food production and livelihoods. as history has repeatedly shown us, technological revolutions have been often accompanied by periods of progressive disinvestment in training for the eclipsed technologies of the past, and in our case, classical virology. the recent emergence of the zika virus in the americas has reinforced the notion that traditional methods of virus discovery and new technologies offer a complimentary toolkit, especially in resource-poor settings that can be seamlessly integrated in the service of public health. it is our hope and expectation that new generations of virus hunters will appreciate the legacy of their predecessors and complement the power of ngs, metagenomics, and bioinformatics, not only to advance viral evolutionary biology, but to understand and limit the potential impacts of emerging viral disease. funding: this work was supported in part by nih grants r ai and u ai . the funding agencies had no involvement in the writing of the report or in 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interferon-gamma (ifn-γ) is an important component of this response, and we show that sinv-infected differentiated neurons respond to ifn-γ in vitro by induction of antiviral genes and suppression of virus replication. to determine the in vivo effects of ifn-γ on sinv clearance and t cell responses, c bl/ mice lacking ifn-γ or ifn-γ receptor- were compared to wild-type (wt) mice after intracranial sinv infection. in wt mice, ifn-γ was first produced in the cns by natural killer cells and then by cd (+) and cd (+) t cells. mice with impaired ifn-γ signaling initiated clearance of viral rna earlier than wt mice associated with cns entry of more granzyme b-producing cd (+) t cells. however, these mice established fewer cd (+) tissue-resident memory t (t(rm)) cells and were more likely to experience reactivation of viral rna synthesis late after infection. therefore, ifn-γ suppresses the local development of granzyme b-expressing cd (+) t cells and slows viral rna clearance but promotes cd (+) t(rm) cell establishment. response to viral infections of the central nervous system (cns) poses a unique problem for the immune system. the restrictive nature of the blood-brain barrier limits the ability of proteins and immune cells to enter into the brain and spinal cord in response to a virus infection [ ] . resident cells of the cns, particularly neurons, have a limited capacity to express major histocompatibility complex (mhc) molecules [ , ] . because neurons are a valuable but finite and minimally renewable cell population, preservation of neuronal function requires that infected neurons be allowed to survive, necessitating noncytolytic immune mechanisms to control virus infection. sindbis virus (sinv) is the prototypic member of the alphaviruses, a genus of enveloped, positive-sense, single-stranded rna viruses belonging to the togaviridae family [ ] . sinv is neurotropic in mice, and when mice are infected with a strain of sinv that does not cause fatal encephalomyelitis (e.g., te), clearance of infection from the cns occurs in three phases [ ] . in phase , during the first to days post infection (dpi), both infectious virus and viral rna increase rapidly, followed by clearance of infectious virus that occurs primarily through cooperative effects of anti-sinv antibody and the cytokine interferon-gamma (ifn-γ) [ ] [ ] [ ] [ ] . in phase , from approximately to days, infectious four to six week-old male and female wild-type c bl/ (wt) mice, mice deficient in ifn-γ receptor (ifngr −/− , strain b . s -ifngr tm agt /j, jackson labs), and mice deficient in ifn-γ (ifng −/− , strain b . s -ifng tm ts /j, jackson labs) were intracranially (ic) inoculated with plaque forming units (pfu) of the te strain of sinv diluted in µl pbs or µl pbs vehicle while under light isoflurane anesthesia. for fresh tissue collection, mice were euthanized by an overdose of isoflurane anesthesia, perfused with ice-cold pbs, and cervical lymph nodes (clns), brains, and/or spinal cords were collected. the johns hopkins university institutional animal care and use committee approved protocols for all studies performed (mo h approved / / ; mo h approved / / ). for preparation of lysates, dap- cells pooled from three wells were incubated on ice for min in ripa buffer ( mm tris, mm nacl, % sds, % np- , . % na-deoxycholate, mm edta) and centrifuged at , rpm for min. total protein was quantified by dc protein assay (bio-rad, hercules, ca, usa) using a bsa standard curve, and µg was boiled in x sds loading buffer ( . m tris (ph . ), % glycerol, % sds, . % bromophenol blue, % β-mercaptoethanol) for min. samples were run on a % sds-polyacrylamide gel electrophoresis (page) gel and transferred to a nitrocellulose membrane (bio-rad). membranes were blocked in tbs- . % tween- (tbst) + % milk for h at room temperature on a rocker and incubated overnight at • c on a rocker with primary antibody diluted in tbst + % bsa ( : rabbit polyclonal anti-nsp ; : , rabbit polyclonal nsv anti-sera; : , mouse monoclonal anti-β-actin, (millipore, burlington, ma, usa) [ , ] . membranes were incubated with secondary antibody diluted in tbst + % nonfat milk ( : horseradish peroxidase (hrp)-conjugated donkey anti-rabbit igg for nsp and poly-nsv; : hrp-conjugated sheep anti-mouse igg for β-actin, ge healthcare) for h on a rocker and developed using amersham ecl western blotting detection reagent (ge healthcare) according to manufacturer's instructions. rna was isolated from dap- cells using the qiagen rneasy (germantown, md, usa) or rneasy plus mini kit following the manufacturer's directions. for mouse cns tissue, right brain halves or whole spinal cords were homogenized in one ml qiazol in lysing matrix d tubes (mp biomedicals, irvine, ca, usa) at . m/s for s using a fastprep- homogenizer (mp biomedicals). the qiagen rneasy lipid tissue mini kit was used to isolate rna, and cdna was synthesized using a high capacity cdna reverse transcription kit with random primers (life technologies, carlsbad, ca, usa), and quantitative real-time pcr (qrt-pcr) was performed using taqman universal pcr master mix (roche, indianapolis, in, usa) on a fast real-time pcr system. sinv rna copies were measured using taqman probe ( - -carboxyfluorescein (fam)-cgcatacagacttccgcccagt- -carboxytetra-methylrhodamine (tamra)- , applied biosystems, waltham, ma, usa) with primers to the sinv e gene (forward, -tgggacgaagcggacgataa- ; reverse, -ctgctccgctttggtcgtat- ). sinv e copies were quantified using a standard curve made of ten-fold dilutions of a plasmid containing the sinv subgenomic region genes and normalized to endogenous rodent gapdh. mrna was measured using commercially available taqman gene expression assays (applied biosystems or integrated dna technologies, coralville, ia, usa), and relative quantification was performed by the ∆∆ct method using endogenous rodent gapdh mrna for normalization. single cell suspensions were made from clns, brains, and spinal cords pooled from to mice per strain per time point as previously described [ ] . briefly, clns were dissociated using gentlemacs c tubes and dissociator (miltenyi biotech, auburn, ca, usa), and red blood cells were lysed with an ammonium chloride solution (sigma-aldrich, st. louis, mo, usa or ebioscience, san diego, ca, usa). brains and spinal cords were dissociated in a solution containing collagenase d (roche) or collagenase iv (worthington labs, worthington, oh, usa) and dnase i (roche), and mononuclear cells were separated on a supplemented percoll gradient. live mononuclear cells were quantified using trypan blue exclusion. for degranulation assessment, - × cells were stimulated for h at • c with ng/ml of phorbol- -myristate -acetate (pma, sigma), µg/ml ionomycin (sigma), and antibody against cd a (clone ebio d b, ebioscience) in rpmi + % fbs. after h, monensin (golgistop, : , bd pharmingen, franklin lakes, nj, usa) was added to block cellular protein transport. for intracytoplasmic cytokine staining (ics), - × cells were stimulated for h at • c with ng/ml pma and µg/ml ionomycin in the presence of brefeldin a (golgiplug, bd pharmingen) in rpmi + % fbs. following live/dead and surface antibody staining (see above), cells were fixed for min using fixation/permeabilization solution from the bd cytofix/cytoperm kit. cells were stained for min on ice with monoclonal antibodies against ifn-γ (clone xmg . ), il- (clone b ), il- a (clone ebio b ), granzyme b (clone ngzb), granzyme a (clone gza- g . ), gm-csf (clone mpi- e ), and tnf-α (clone mp -xt ) from ebioscience or bd pharmingen diluted in bd perm/wash buffer. cells were resuspended in µl facs buffer and run on a bd facscanto ii cytometer using bd facsdiva software, version , and analyses were carried out using flowjo software, version . cells were characterized as follows: cd t cells (cd hi cd + cd + ), cd t cells , and tissue resident memory t (t rm ) cells (cd hi cd l − cd + ). all flow cytometry data are presented as averages of to independent experiments. following euthanasia, mice were perfused with ice-cold % paraformaldehyde (pfa), and brains and spinal columns were collected. brains were cut into three coronal sections using an adult mouse brain slicer (zivic instruments, pittsburgh, pa, usa), fixed overnight in % pfa, and embedded in paraffin. spinal columns were fixed overnight in % pfa, decalcified for h in a % sodium citrate/ % formic acid solution, cut to isolate the l -l spinal cord regions, and embedded in paraffin. viruses , , of µm tissue sections from to mice per group were stained with hematoxylin and eosin (h&e). brain slides were coded and sections scored as previously described [ ] using a - ( ) scale: , no detectable inflammation; , one or two small inflammatory foci; , moderate inflammatory foci in up to % of × magnification fields per section; , moderate to large inflammatory foci in greater than % of × magnification fields. an additional point was given for excessive parenchymal cellularity, allowing for a maximum score of . spinal cord slides were coded and sections scored using a modified - ( ) scale as previously described [ ] : , no detectable inflammation; , one to two small inflammatory foci; , greater than two inflammatory foci per spinal cord or moderate to marked inflammatory foci. an additional point was given for excessive parenchymal cellularity, allowing for a maximum score of . statistical analyses were performed using graphpad prism software. time-course studies were analyzed by two-way anova with bonferroni's or tukey's multiple comparison post-test for two group and three group comparisons, respectively. comparisons between three groups at a single time point were made using one-way anova with tukey's multiple comparisons post-test. a p value of < . was considered significant for all analyses. to elucidate a potential role for ifn-γ in virus clearance from neurons, immature cycling cap- olfactory neuronal cells and mature bipolar differentiated dap- cells [ ] were infected with sinv (moi = ). both cap- and dap- cells supported virus replication, with cap- cells producing higher peak titers than dap- cells and dying by hpi ( figure a ). dap- cells continued to produce virus through hpi and were used for subsequent studies of the effect of ifn-γ on virus replication. dap- cells infected with sinv (moi = ) were treated with u/ml rat recombinant ifn-γ h before infection, hpi, or at hpi. virus replicated in all treatment groups, with titers peaking at about hpi ( figure b ). dap- cells treated with ifn-γ prior to infection and at hpi had significantly decreased virus production compared to untreated cells at , , and hpi, with the greatest effect seen in pretreated cells, demonstrating the ability of neurons to develop an ifn-γ-induced antiviral response. treatment with ifn-γ at hpi did not alter virus production. to assess the effect of ifn-γ on virus replication after infection was established, production of sinv proteins was examined by immunoblot in dap- cells infected with sinv alone or treated with ifn-γ at hpi. in untreated sinv-infected cells, production of the nonstructural nsp protein and structural capsid protein reached high levels by hpi ( figure c ). production of the e and e structural glycoproteins (along with precursor to e , pe ) was evident by hpi and diminished by treatment with ifn-γ. these results show that ifn-γ can decrease the production of sinv by neurons. we next sought to determine the effects of ifn-γ on production of viral rna. sinv-infected dap- cells were treated with ifn-γ at hpi, and cell pellets collected to quantify viral rna by qrt-pcr. viral rna copies were comparable at hpi. copy number in untreated cells continued to rise, peaking at hpi, while sinv rna levels plateaued in treated cells and by hpi had begun to decrease ( figure d ). overall, viral rna synthesis was significantly inhibited by ifn-γ treatment compared to untreated dap- cells from to hpi. these studies show that ifn-γ signaling affects production and clearance of both infectious virus and viral rna from neurons in vitro. because ifn-γ facilitates virus clearance from neurons, we examined induction of antiviral genes by ifn-γ. mrna expression of representative antiviral isgs was examined by qrt-pcr in dap- cells infected with sinv (moi = ) and treated with u/ml ifn-γ at hpi. gbp ( figure a ) and irgm ( figure b ), two genes associated with autophagy [ ] , were highly expressed by sinvinfected dap- cells treated with ifn-γ, as were oasl ( figure c ), a member of the '- 'oligoadenylate/rnasel system [ ] , and rsad ( figure d ), which encodes viperin, a protein that interferes with assembly and release of many viruses [ ] . zc hav ( figure e ), which encodes zap/parp , a protein involved in viral rna degradation and induction of the innate immune response that restricts alphavirus and flavivirus replication [ , ] , was less highly upregulated. all of these genes required ifn-γ for induction and generally were not induced by sinv infection alone. because ifn-γ facilitates virus clearance from neurons, we examined induction of antiviral genes by ifn-γ. mrna expression of representative antiviral isgs was examined by qrt-pcr in dap- cells infected with sinv (moi = ) and treated with u/ml ifn-γ at hpi. gbp ( figure a ) and irgm ( figure b ), two genes associated with autophagy [ ] , were highly expressed by sinv-infected dap- cells treated with ifn-γ, as were oasl ( figure c ), a member of the '- 'oligoadenylate/rnasel system [ ] , and rsad ( figure d ), which encodes viperin, a protein that interferes with assembly and release of many viruses [ ] . zc hav ( figure e ), which encodes zap/parp , a protein involved in viral rna degradation and induction of the innate immune response that restricts alphavirus and flavivirus replication [ , ] , was less highly upregulated. all of these genes required ifn-γ for induction and generally were not induced by sinv infection alone. to characterize the time course and source of ifn-γ throughout the course of sinv infection of the cns, flow cytometry was used to characterize cells from the clns, the draining lymph nodes of the brain, and the brains of wt mice ( figure ). cd + t cells, cd + t cells, and nk cells were examined for cytokine production during phase ( and dpi), phase ( and dpi), and phase ( dpi) of infection. in the clns, few cells produced ifn-γ at any time ( figure a ,c). in the brain, nk cells were the predominant source of ifn-γ at dpi, both as percentage of live cells and absolute numbers ( figure b ,d). numbers of ifn-γ-producing cells in the brain peaked at dpi and were predominantly cd + t cells. as cd + t cells decreased, cd + t cells became comparable contributors by dpi. in phase of infection, fewer t cells were present in the brains and cd + and cd + t cells produced the majority of ifn-γ. therefore, nk cells produce most of the local cns ifn-γ early in the course of infection, but at later times, cd + , and especially cd + , t cells become the predominant source. we next characterized the percentages of each cell population producing ifn-γ. little change was seen in the clns, with less than % of cd + t cells, less than % of cd + t cells, and - % of nk cells producing ifn-γ at any time after infection ( figure e ). in the brain, the percentage of cd + and cd + t cells producing ifn-γ increased over the course of infection, going from approximately to % at and dpi to over % at , , and dpi ( figure f ). in contrast, the percentage of brain nk cells producing ifn-γ remained between to %, similar to that in the clns. to compare the relative amounts of ifn-γ produced by each cell type, median fluorescence intensities (mfis) were determined. in the clns, the mfi for ifn-γ remained low and did not change for any cell population ( figure g ,i). however, in the brain, the ifn-γ mfis for cd + and cd + t to characterize the time course and source of ifn-γ throughout the course of sinv infection of the cns, flow cytometry was used to characterize cells from the clns, the draining lymph nodes of the brain, and the brains of wt mice ( figure ). cd + t cells, cd + t cells, and nk cells were examined for cytokine production during phase ( and dpi), phase ( and dpi), and phase ( dpi) of infection. in the clns, few cells produced ifn-γ at any time ( figure a ,c). in the brain, nk cells were the predominant source of ifn-γ at dpi, both as percentage of live cells and absolute numbers ( figure b ,d). numbers of ifn-γ-producing cells in the brain peaked at dpi and were predominantly cd + t cells. as cd + t cells decreased, cd + t cells became comparable contributors by dpi. in phase of infection, fewer t cells were present in the brains and cd + and cd + t cells produced the majority of ifn-γ. therefore, nk cells produce most of the local cns ifn-γ early in the course of infection, but at later times, cd + , and especially cd + , t cells become the predominant source. cells, but not nk cells, increased over time, with the greatest increase occurring between and dpi ( figure h ,j). . also evaluated were the percentage of each cell type producing ifn-γ (e, f) and the mfi of ifn-γ for each cell type presented in graph form (g,h) and as histograms (i,j) (n = - pooled mice per time point from three independent experiments, except for data from dpi clns, which were from two independent experiments; data are presented as the mean ± sem). to determine the in vivo role of ifn-γ during sinv encephalomyelitis, the responses of mice deficient in ifn-γ (ifng −/− ) or in the α-chain of the ifn-γ receptor (ifngr −/− ) were compared to those of wt mice. to assess expression of antiviral isg mrnas for the five antiviral isgs previously selected for in vitro analysis of neuronal responses plus oas a, a protein associated with the '- 'oligoadenylate/rnase l system that is more active than oasl in mice [ ] were examined by qrt-pcr ( figure ) . gbp , irgm , oasl , rsad , and zc hav were up regulated in the brains and spinal cords of all mice during sinv infection, likely reflecting the overlap with isgs induced by type i ifn. however, expression of gbp ( figure a ) and irgm ( figure b ) at - dpi in the brain and spinal . also evaluated were the percentage of each cell type producing ifn-γ (e, f) and the mfi of ifn-γ for each cell type presented in graph form (g,h) and as histograms (i,j) (n = - pooled mice per time point from three independent experiments, except for data from dpi clns, which were from two independent experiments; data are presented as the mean ± sem). we next characterized the percentages of each cell population producing ifn-γ. little change was seen in the clns, with less than % of cd + t cells, less than % of cd + t cells, and - % of nk cells producing ifn-γ at any time after infection ( figure e ). in the brain, the percentage of cd + and cd + t cells producing ifn-γ increased over the course of infection, going from approximately to % at and dpi to over % at , , and dpi ( figure f ). in contrast, the percentage of brain nk cells producing ifn-γ remained between to %, similar to that in the clns. to compare the relative amounts of ifn-γ produced by each cell type, median fluorescence intensities (mfis) were determined. in the clns, the mfi for ifn-γ remained low and did not change for any cell population ( figure g ,i). however, in the brain, the ifn-γ mfis for cd + and cd + t cells, but not nk cells, increased over time, with the greatest increase occurring between and dpi ( figure h ,j). to determine the in vivo role of ifn-γ during sinv encephalomyelitis, the responses of mice deficient in ifn-γ (ifng −/− ) or in the α-chain of the ifn-γ receptor (ifngr −/− ) were compared to those of wt mice. to assess expression of antiviral isg mrnas for the five antiviral isgs previously selected for in vitro analysis of neuronal responses plus oas a, a protein associated with the '- 'oligoadenylate/rnase l system that is more active than oasl in mice [ ] were examined by qrt-pcr ( figure ) . gbp , irgm , oasl , rsad , and zc hav were up regulated in the brains and spinal cords of all mice during sinv infection, likely reflecting the overlap with isgs induced by type i ifn. however, expression of gbp ( figure a ) and irgm ( figure b ) at - dpi in the brain and spinal cord were higher in wt mice than ifngr −/− and ifng −/− mice. smaller differences in expression levels in brain were seen at day and for oasl ( figure c ) and at dpi oas a ( figure d ) and at dpi for rsad ( figure e ) and zc hav ( figure f ). these results show that while isgs are induced during sinv infection in the cns of mice with impaired ifn-γ signaling, expression is diminished compared to that of mice with intact ifn-γ signaling. viruses , , x for peer review of cord were higher in wt mice than ifngr −/− and ifng −/− mice. smaller differences in expression levels in brain were seen at day and for oasl ( figure c ) and at dpi oas a ( figure d ) and at dpi for rsad ( figure e ) and zc hav ( figure f ). these results show that while isgs are induced during sinv infection in the cns of mice with impaired ifn-γ signaling, expression is diminished compared to that of mice with intact ifn-γ signaling. clearance of viral rna from the cns was examined by quantifying sinv rna in brains and spinal cords of sinv-infected wt, ifng −/− and ifnrg −/− mice by qrt-pcr using primers specific for the e gene ( figure ). viral rna peaked at to dpi in the brains ( figure a ) and spinal cords ( figure b ), with comparable amounts for all mice. however, at dpi, viral rna levels were higher in the brains and at and dpi in the spinal cords of wt mice compared to ifngr −/− and ifng −/− mice. viral rna levels were comparable throughout phase of infection; however, at occasional times during phase , when viral rna had reached a low-level steady state, viral rna increased, especially in the spinal cords of ifng −/− mice. the results indicate that while impaired ifn-γ signaling results in delayed infectious virus clearance [ , , ] , initiation of viral rna clearance was accelerated, but the likelihood of reactivation of viral rna synthesis late after infection was increased. clearance of viral rna from the cns was examined by quantifying sinv rna in brains and spinal cords of sinv-infected wt, ifng −/− and ifnrg −/− mice by qrt-pcr using primers specific for the e gene ( figure ). viral rna peaked at to dpi in the brains ( figure a ) and spinal cords ( figure b) , with comparable amounts for all mice. however, at dpi, viral rna levels were higher in the brains and at and dpi in the spinal cords of wt mice compared to ifngr −/− and ifng −/− mice. viral rna levels were comparable throughout phase of infection; however, at occasional times during phase , when viral rna had reached a low-level steady state, viral rna increased, especially in the spinal cords of ifng −/− mice. the results indicate that while impaired ifn-γ signaling results in delayed infectious virus clearance [ , , ] , initiation of viral rna clearance was accelerated, but the likelihood of reactivation of viral rna synthesis late after infection was increased. to examine the effect of ifn-γ signaling on the inflammatory response to sinv infection, we first examined brain and spinal cord pathology of sinv-infected wt, ifng −/− and ifngr −/− mice. most of the pathological changes seen with alphavirus encephalomyelitis are associated with infiltration of immune cells [ , , , [ ] [ ] [ ] . brains and spinal cords were examined for histopathological changes and inflammation at , , , and dpi ( figure ). sporadically at dpi and consistently at dpi, brains from wt mice had diffuse bilateral dilation of the lateral ventricles, extending along the dorsal aspects of the hippocampi. this change was rarely seen in brains from ifngr −/− or ifng −/− mice, and when present, was less severe. compared to mock-infected control mice ( figure a ), both perivascular cuffing and infiltration of mononuclear cells into the parenchyma in the brain were present at dpi in all sinv-infected mice ( figure b ). in the spinal cord, compared to mock-infected controls ( figure c ), sinv-infected wt and ifngr −/− mice had more inflammation than ifng −/− mice ( figure d ). for quantitative comparison of the inflammation in brain ( figure e ) and spinal cord ( figure f ) at , , and dpi, a scoring system was used to evaluate coded h&e-stained sections [ , ] . inflammation steadily increased during the first week of infection, peaking at dpi in wt and ifng −/− mice and at dpi in ifngr −/− mice. in both tissues, inflammation scores were lower in ifng −/− mice than in wt or ifngr −/− mice. minimal inflammation present in the brain but not spinal cord of mock-infected mice was likely due to trauma from the ic inoculation. to examine the effect of ifn-γ signaling on the inflammatory response to sinv infection, we first examined brain and spinal cord pathology of sinv-infected wt, ifng −/− and ifngr −/− mice. most of the pathological changes seen with alphavirus encephalomyelitis are associated with infiltration of immune cells [ , , , [ ] [ ] [ ] . brains and spinal cords were examined for histopathological changes and inflammation at , , , and dpi ( figure ). sporadically at dpi and consistently at dpi, brains from wt mice had diffuse bilateral dilation of the lateral ventricles, extending along the dorsal aspects of the hippocampi. this change was rarely seen in brains from ifngr −/− or ifng −/− mice, and when present, was less severe. compared to mock-infected control mice ( figure a ), both perivascular cuffing and infiltration of mononuclear cells into the parenchyma in the brain were present at dpi in all sinv-infected mice ( figure b ). in the spinal cord, compared to mock-infected controls ( figure c ), sinv-infected wt and ifngr −/− mice had more inflammation than ifng −/− mice ( figure d ). for quantitative comparison of the inflammation in brain ( figure e ) and spinal cord ( figure f ) at , , and dpi, a scoring system was used to evaluate coded h&e-stained sections [ , ] . inflammation steadily increased during the first week of infection, peaking at dpi in wt and ifng −/− mice and at dpi in ifngr −/− mice. in both tissues, inflammation scores were lower in ifng −/− mice than in wt or ifngr −/− mice. minimal inflammation present in the brain but not spinal cord of mock-infected mice was likely due to trauma from the ic inoculation. , and ifng −/− (white bars) mice either mock-infected or sinv-infected at , or dpi were scored for inflammation using a four-point (brain) or three-point (spinal cord) system (data are presented as the mean score ± sem for - mice per strain per group; * p < . and ** p < . , tukey's multiple comparisons test). to determine the effects of ifn-γ signaling on immune cell subsets after sinv infection, cells isolated from the clns and brains of wt, ifng −/− and ifngr −/− mice were examined by flow cytometry. in the clns at dpi, there were more total mononuclear cells in wt mice than ifng −/− mice ( figure a ), but similar numbers at and dpi. neither the percentage nor absolute number of cd + t cells ( figure c ) or cd + t cells ( figure d ) in clns were affected by impaired ifn-γ signaling, with a general overall decrease from dpi to dpi. , and ifng −/− (white bars) mice either mock-infected or sinv-infected at , or dpi were scored for inflammation using a four-point (brain) or three-point (spinal cord) system (data are presented as the mean score ± sem for - mice per strain per group; * p < . and ** p < . , tukey's multiple comparisons test). to determine the effects of ifn-γ signaling on immune cell subsets after sinv infection, cells isolated from the clns and brains of wt, ifng −/− and ifngr −/− mice were examined by flow cytometry. in the clns at dpi, there were more total mononuclear cells in wt mice than ifng −/− mice ( figure a ), but similar numbers at and dpi. neither the percentage nor absolute number of cd + t cells ( figure c ) or cd + t cells ( figure d ) in clns were affected by impaired ifn-γ signaling, with a general overall decrease from dpi to dpi. in contrast, wt mice had more total mononuclear cells in the brain at dpi than ifngr −/− and ifng −/− mice ( figure b ). numbers of infiltrating cd + t cells peaked at dpi, while infiltration of cd + t cells occurred later with peaks at dpi. wt mice had more cd + t cells than ifng −/− mice at dpi ( figure e) , while both the percentage of cells and absolute numbers of cd + t cells were lower in brains of wt mice than ifngr −/− and ifng −/− mice at dpi ( figure f ), despite the fact that wt mice had more overall brain mononuclear cells at this time ( figure b ). therefore, while the absence of ifn-γ signaling did not affect the proliferation of t cells in clns in response to sinv infection, it did affect recruitment of these cells to the site of infection in the brain. because wt mice have more inflammation ( figure ) and more mononuclear cells ( figure b ), but fewer cd + t cells ( figure f ) in brain compared to ifngr −/− and ifng −/− mice at dpi, non-t cell immune cell populations were assessed. macrophages ( figure g ) and nk cells ( figure i ), both as a percentage of the total live mononuclear cell population and absolute numbers, were higher in wt mouse brains compared to mice with impaired ifn-γ signaling, while neutrophils tended to be higher in ifngr −/− mouse brains ( figure h) . microglial cells as a percentage of the total brain mononuclear cell population were higher in ifng −/− compared to wt and ifngr −/− mice ( figure j ), but absolute numbers of microglia were comparable. therefore, the larger number of mononuclear cells in the brains of wt mice at dpi compared to mice with impaired ifn-γ signaling was due to infiltration of more macrophages and nk cells. because impaired ifn-γ signaling altered the numbers of cd + and cd + t cells infiltrating the brain during sinv infection, we sought to determine the effector function of these cells. brain cd + t cells at dpi were characterized further by measuring production of signature cytokines and transcription factors ( figure a -d) and expression of cytokine ( figure e -h) and transcription factor ( figure i -l) mrnas associated with different t helper (th) subsets. as expected, ifn-γ (th cells) was not produced by cd + t cells in ifng −/− mice, and the percentage of cd + t cells producing ifn-γ did not differ between wt and ifngr −/− mice ( figure a ). the percentage of cd + t cells producing il- (th cells) was lower in ifng −/− mice than wt or ifngr −/− mice ( figure b ). although not significant, more cd + t cells in ifngr −/− mice produced il- a (th cells) compared to wt and ifng −/− mice ( figure c) , and the percentage of cd + t cells expressing both cd and foxp (tregs) trended lower in ifng −/− mice ( figure d ). these results show that ifn-γ signaling affects cd + t cell function during sinv infection, but impaired signaling has only a modest effect on th subset profile. to further evaluate th subsets, mrnas for four cytokines associated with specific th profiles were examined: il for th cells ( figure e ), il for th cells ( figure f ), il a for th cells (figure g ), and il for tregs ( figure h ). at dpi, mrna expression of il and il was lower in ifng −/− mice, and expression of il a was higher in ifngr −/− mice. il expression did not significantly differ among strains. expression of mrnas for transcription factors tbx for th cells (figure i ), gata for th cells ( figure j) , rorc for th cells (figure k ), and foxp for tregs ( figure l ) was also measured. while significant differences were found between strains at various time points, no major trends were identified. therefore, cd + t cell differentiation mostly affected ifng −/− mice with fewer th and treg cells and ifngr −/− mice with more th cells. modulation of th profiles during sinv infection by ifn-γ signaling warrants further examination. to examine how ifn-γ signaling affects cd + t cell production of effector proteins at dpi, ifn-γ, tnf-α, gm-csf, and granzyme b in cells from the brains of wt, ifngr −/− , and ifng −/− mice were examined by flow cytometry. cd + t cells from ifng −/− mice did not produce ifn-γ, and the percentage of cd + t cells producing ifn-γ did not differ between wt and ifngr −/− mice ( figure a ). the percentage of cd + t cells producing tnf-α was not significantly different between strains ( figure b ), but more cd + t cells from wt mice than ifng −/− mice produced gm-csf ( figure c) , while more cd + t cells from both ifngr −/− and ifng −/− mice than wt mice produced granzyme b ( figure d) . furthermore, the granzyme b mfi of cd + t cells from ifngr −/− mice was also higher than wt mice ( figure e ), indicating that individual cd + t cells in the brains of mice with impaired ifn-γ signaling produced more granzyme b than cells with intact ifn-γ signaling. expression of granzyme a ( figure f ) and granzyme b ( figure g ) mrnas in brain was lower in ifng −/− mice compared to wt and ifngr −/− mice, but expression differences among strains for granzyme k (figure h ), granzyme m ( figure i ), and perforin ( figure j ) were less pronounced. effector function of cd + t cells in the spinal cords of infected mice were similar to the brain in that ifn-γ-producing cd + t cells were not detected in ifng −/− mice and were comparable between wt and ifngr −/− mice ( figure k ). tnf-α ( figure l ) and gm-csf ( figure m ) production by cd + to examine how ifn-γ signaling affects cd + t cell production of effector proteins at dpi, ifn-γ, tnf-α, gm-csf, and granzyme b in cells from the brains of wt, ifngr −/− , and ifng −/− mice were examined by flow cytometry. cd + t cells from ifng −/− mice did not produce ifn-γ, and the percentage of cd + t cells producing ifn-γ did not differ between wt and ifngr −/− mice ( figure a ). the percentage of cd + t cells producing tnf-α was not significantly different between strains ( figure b ), but more cd + t cells from wt mice than ifng −/− mice produced gm-csf ( figure c) , while more cd + t cells from both ifngr −/− and ifng −/− mice than wt mice produced granzyme b ( figure d) . furthermore, the granzyme b mfi of cd + t cells from ifngr −/− mice was also higher than wt mice ( figure e ), indicating that individual cd + t cells in the brains of mice with impaired ifn-γ signaling produced more granzyme b than cells with intact ifn-γ signaling. expression of granzyme a ( figure f ) and granzyme b ( figure g ) mrnas in brain was lower in ifng −/− mice compared to wt and ifngr −/− mice, but expression differences among strains for granzyme k (figure h ), granzyme m ( figure i ), and perforin ( figure j ) were less pronounced. t cells were not different, but the percentage of cd + t cells producing granzyme b ( figure n ) was lower in wt mice than mice with impaired ifn-γ signaling. these findings indicate that ifn-γ signaling not only inhibits infiltration of cd + t cells into the cns, but also inhibits cd + t cell synthesis of granzyme b. effector function of cd + t cells in the spinal cords of infected mice were similar to the brain in that ifn-γ-producing cd + t cells were not detected in ifng −/− mice and were comparable between wt and ifngr −/− mice ( figure k ). tnf-α ( figure l ) and gm-csf ( figure m ) production by cd + t cells were not different, but the percentage of cd + t cells producing granzyme b ( figure n ) was lower in wt mice than mice with impaired ifn-γ signaling. these findings indicate that ifn-γ signaling not only inhibits infiltration of cd + t cells into the cns, but also inhibits cd + t cell synthesis of granzyme b. . . effect of ifn-γ signaling on cd + t cell and nk cell degranulation and cytotoxic function during sinv infection cd + t cells and nk cells primarily exert their cytolytic effects through secretion of cytotoxic granzymes that kill target cells [ ] . because infiltration of cd + t cells and nk cells into the brain were differentially regulated by ifn-γ signaling, the extent of degranulation, as identified by cd a expression [ ] , and granzyme production were examined in cells from the clns and brains of wt, ifngr −/− , and ifng −/− mice at dpi. in clns, cd a expression by cd + t cells was minimally affected by ifn-γ signaling ( figure a ). in brain, the percentage of cd + t cells expressing cd a was higher in ifng −/− mice than wt mice, but absolute numbers were comparable ( figure c ). in contrast, wt mice had more degranulated nk cells in the clns ( figure b ) and the brain ( figure d ) than either ifngr −/− or ifng −/− mice. therefore, ifn-γ signaling affected the cytotoxic function of local cns nk cells and cd + t cells during sinv infection, but in opposite directions. because long-term control of viral rna is likely required to prevent reactivation of virus production and relapse of clinical disease, immune cells remain in the cns long term after sinv infection [ ] . tissue-resident memory (trm) cells are a subset of memory t cells that do not circulate, but instead permanently remain at sites of infection [ ] . cd + trm cells were assessed in clns and brains in wt, ifngr −/− , and ifng −/− mice at , , and dpi to determine a role for ifn-γ in their development, maintenance, and survival ( figure a ). very few cd + or cd + trm cells were present in clns of sinv-infected mice at any time after infection ( figure b ). higher percentages of cd + t cells in the brain were trm cells, but they did not change over time or differ among mouse strains granzymes a and b are the primary granzymes involved in cytotoxicity of cd + t cells and nk cells [ ] , so we next identified the granzymes produced in the brain at dpi during sinv infection and the effect of ifn-γ signaling using boolean gating ( figure e,f) . approximately to % of cd + t cells and to % of nk cells in the brain produced both granzyme a and b. however, if producing only one granzyme, cd + t cells preferentially produced granzyme b, while nk cells preferentially produced granzyme a ( figure f ). additionally, granzyme production was lower in cd + t cells but higher in nk cells of wt mice compared to mice with impaired ifn-γ signaling. taken together, these data suggest that ifn-γ promotes nk cell cytotoxicity but suppresses the cytotoxic potential of cd + t cells in the brain during sinv infection. because long-term control of viral rna is likely required to prevent reactivation of virus production and relapse of clinical disease, immune cells remain in the cns long term after sinv infection [ ] . tissue-resident memory (t rm ) cells are a subset of memory t cells that do not circulate, but instead permanently remain at sites of infection [ ] . cd + t rm cells were assessed in clns and brains in wt, ifngr −/− , and ifng −/− mice at , , and dpi to determine a role for ifn-γ in their development, maintenance, and survival ( figure a ). very few cd + or cd + t rm cells were present in clns of sinv-infected mice at any time after infection ( figure b ). higher percentages of cd + t cells in the brain were t rm cells, but they did not change over time or differ among mouse strains ( figure c , left panel). however, percentages of cd + t rm cells in the brain increased over time ( figure c , right panel) and were more abundant in the brains of wt mice than ifngr −/− mice at dpi and ifng −/− mice at and dpi. the results suggest that ifn-γ signaling promotes the development of cd + t rm cells in the brain during sinv infection and affects their presence following infectious virus clearance. ifn-γ is an important determinant of the outcome of virus infections of the cns, with both induction of antiviral genes and regulation of the immune response to infection. previous studies have shown that ifn-γ facilitates clearance of infectious virus from spinal cord neurons during sinv infection [ ] , and in vitro, ifn-γ treatment inhibits replication of sinv in neurons through the jak/stat signaling pathway [ , ] ; however, the antiviral genes induced were not identified. the current study showed that mrnas for gbp and irgm gtpase proteins associated with autophagy were figure . effect of ifn-γ signaling on t rm cell populations. flow cytometry was used to examine t rm cell populations by gating, denoted by blue frames, around cd + cells (a) at , , and dpi in the clns (b) and brains (c) of wt (black bars), ifngr −/− (gray bars), and ifng −/− (white bars) mice, and results are presented as a percentage of cd + (left graphs) and cd + t cells (right graphs) (n = - pooled mice per strain per time point from three to four independent experiments; data are presented as the mean ± sem; * p < . , *** p < . by tukey's multiple comparisons test). ifn-γ is an important determinant of the outcome of virus infections of the cns, with both induction of antiviral genes and regulation of the immune response to infection. previous studies have shown that ifn-γ facilitates clearance of infectious virus from spinal cord neurons during sinv infection [ ] , and in vitro, ifn-γ treatment inhibits replication of sinv in neurons through the jak/stat signaling pathway [ , ] ; however, the antiviral genes induced were not identified. the current study showed that mrnas for gbp and irgm gtpase proteins associated with autophagy were highly induced by ifn-γ signaling in sinv-infected neurons as well as in the cns of sinv-infected mice where multiple cells may be responding to secreted ifn-γ. autophagy can decrease virus replication by destroying virus or viral replication components, including alphaviruses, or by delivering them to endosomes for toll-like receptor (tlr) induction of the innate immune response [ ] [ ] [ ] . rna viruses, including the alphavirus chikungunya virus (chikv), target irgm to promote virus replication [ , ] . another antiviral protein system highly induced by ifn-γ signaling during sinv infection of neurons was the - -oligoadenylate synthetase (oas) family, a pathway that affects replication of several neurotropic viruses, including rabies virus, canine distemper virus, west nile virus (wnv), and jev [ , [ ] [ ] [ ] . two other antiviral genes usually more robustly induced by type i than type ii ifns, rsad and zc hav , were induced later and to lesser extents. rsad -encoded viperin can impair budding of enveloped viruses [ ] [ ] [ ] and alphavirus and flavivirus replication and assembly [ ] [ ] [ ] [ ] . zc hav encodes zinc-finger antiviral protein (zap) or poly(adp-ribose) polymerase- (parp ), an rna-binding protein that recruits rna decay factors to degrade viral rna and inhibit viral rna translation [ ] [ ] [ ] [ ] [ ] . however, the specific role for these isgs in ifn-γ-mediated virus clearance from neurons will require further study. the current studies show that the in vivo role of ifn-γ in pathogenesis of sinv-induced encephalomyelitis is complex. although ifn-γ is important for clearance of infectious virus, particularly from spinal cord neurons [ , ] , and was sufficient for viral rna clearance in neurons in vitro, ifn-γ delayed clearance initiation of viral rna from both the brain and spinal cord in mice ( figure ), suggesting an extra neuronal effect that secondarily affects viral rna clearance. ifn-γ signaling improved recruitment of nk cells but differentially affected the recruitment of t cells, with more cd + t cells and fewer cd + t cells infiltrating the brains of sinv-infected wt mice than ifngr −/− or ifng −/− mice. this modification of the local t cell response potentially explains the differential initiation of rna clearance among mice with intact and impaired ifn-γ signaling, leading us to further evaluate the composition and functionality of these t cells. cd + t cells primarily exert their effects via cytokine secretion, which identify th subsets that influence the differentiation and activation of other immune cells [ ] . treg cells, defined by expression of both foxp and cd , tended to be lower in ifng −/− mouse brains, and mrna expression of the regulatory cytokine il- was significantly lower compared to wt and ifngr −/− mice at dpi. th cells have been associated with fatal encephalomyelitis and virus persistence during infection with several viruses, such as sinv nsv, tmev, and jhmv, especially in the absence of ifn-γ [ , [ ] [ ] [ ] . ifngr −/− mice expressed more il a mrna and trended toward increased il- a production by cd + t cells compared to wt and ifng −/− mice. similar results were seen in ifngr −/− mice infected with sinv nsv and suggest preferential expansion of th cells [ ] . neutrophils, also associated with a th profile [ ] , were also increased in brains of ifngr −/− mice. these results show that ifn-γ signaling affects cd + t cell function, although major differences in th profile were not seen, and reiterates the differences between mice lacking ifn-γ production and mice with impaired receptor function in the immune response to sinv infection. in our mouse model of alphavirus encephalomyelitis, fewer cd + t cells infiltrated the cns in wt mice with intact ifn-γ signaling, an effect similar to the suppression of the cd + t cell response to friend virus infection observed in association with lactate dehydrogenase virus-induced ifn-γ [ ] . as with nk cells, cd + t cells primarily exert their effector function against virus infections through cytotoxic activity and produce granzymes and perforin for delivery through granule exocytosis to activate caspases and induce target cell apoptosis [ ] . a clear role for nk cells in pathogenesis of alphavirus encephalomyelitis has not been identified [ , ] and, in the current studies, greater numbers of nk cells and granzyme a expression in wt mice did not offset the diminished cd + t cell response. specific targeting of infected cells by cd + t cells is achieved through direct contact with an infected cell expressing mhc class i molecules. neurons have a limited capacity for mhc class i expression [ , [ ] [ ] [ ] [ ] , but cd + t cells can directly engage virus-infected neurons [ ] , and clearance of wnv, jev, and lcmv from the cns is dependent on the granzyme/perforin pathway [ ] [ ] [ ] . perforin production is decreased in the brains of wt mice compared to ifngr −/− and ifng −/− mice during sinv nsv infection [ ] , and perforin-mediated effector function is impaired during jhmv infection [ ] however, ifn-γ promotes granzyme b production during experimental coronavirus retinopathy [ ] . therefore, the effect of ifn-γ on the granule exocytosis pathway during cns infection appears to be virus-specific, and the mechanisms by which ifn-γ influences granzyme production during sinv infection remain to be understood. it is becoming increasingly understood that granzymes have non-cytotoxic as well as cytotoxic roles [ , [ ] [ ] [ ] . for instance, cd + t cells can inhibit reactivation of hsv- in neurons via a non-cytolytic mechanism [ ] [ ] [ ] . mouse granzyme k induces macrophages to release il- β during lcmv infection [ ] , and granzyme substrates with direct antiviral activity, either viral proteins or host cell proteins essential for virus replication, have also been identified. granzymes a, h, and m cleave viral proteins important for replication of moloney mouse leukemia virus, adenovirus, and human cytomegalovirus [ ] [ ] [ ] . granzymes b and h both cleave the rna-binding protein la, which is important in viral rna metabolism for several viruses [ ] [ ] [ ] [ ] . hnrnp k, which modulates viral rna replication of chikv, enterovirus , dengue virus, and hiv- [ ] [ ] [ ] [ ] and interacts with sinv nsp and subgenomic mrna [ , ] , is a substrate for granzyme b. silencing of hnrnp k or disruption of hnrnp-vrna binding decreases sinv rna replication in vitro [ , ] . therefore, we postulate that the increased granzyme b produced by cd + t cells of ifngr −/− and ifng −/− cleaves one or more cellular proteins important for sinv rna replication or stability to accelerate noncytotoxic viral rna clearance from the brain and spinal cord. further studies regarding viral rna clearance, the noncytotoxic roles of granzymes during sinv infection, and regulation by ifn-γ, are warranted. persistence of viral rna after cns infection is common [ , , [ ] [ ] [ ] [ ] [ ] and presents the potential for virus reactivation and relapse of disease. indeed, during phase of infection, transient increases in viral rna were seen in sinv-infected mice, particularly in ifng −/− mice. therefore, prevention of virus reactivation is likely achieved through continued presence of immune cells at the previous site of infection [ , ] . over the course of infection, the percentage of cd + , but not cd + , t cells expressing cd , a marker for t rm cells in the brain, increased. as permanent residents at sites of previous infection, t rm cells provide a rapid response to pathogen reactivation [ ] [ ] [ ] . ifn-γ produced by cd + t cells is required for generating cd + t rm cells in the lung after influenza virus infection [ ] , and rapid clearance of lcmv by brain t rm cells depends on ifn-γ signaling and cytotoxic granule release [ ] . brains of sinv-infected mice defective in ifn-γ signaling had both fewer cd + t cells and cd + t rm cells than wt mice, indicating a need for ifn-γ to promote the residence of t rm cells in the brain after infection. in conclusion, ifn-γ signaling has both positive and negative effects on sinv clearance and control. ifn-γ facilitates infectious virus clearance through direct antiviral effects on infected neurons and inducing production of b cell-attracting chemokines that fosters local production of anti-sinv antibody [ ] . however, ifn-γ impairs viral rna clearance, possibly by suppressing the cd + t cell response in the cns and granzyme b production. finally, ifn-γ promotes the development of cd + t rm cells in the brain, likely helping prevent reactivation of persistent virus. better understanding of the complicated interplay between the virus and host immune system in inducing pathology and promoting virus clearance is critical to developing effective therapies. the immune response in viral encephalitis induction of mhc class i genes in neurons the role of cd (+) t cells and major histocompatibility complex class i expression in the central nervous system of mice infected with neurovirulent sindbis virus fields virology alphavirus-induced encephalomyelitis: antibody-secreting cells and viral clearance from the nervous system antibody-mediated clearance of alphavirus infection from neurons interferon-gamma-mediated site-specific clearance of alphavirus from cns neurons synergistic roles of antibody and interferon in noncytolytic clearance of sindbis virus from different regions of the central nervous system interferon gamma modulation of disease manifestation and the local antibody response to alphavirus encephalomyelitis persistence of viral rna in mouse brains after recovery from acute alphavirus encephalitis virus specificity and isotype expression of intraparenchymal antibody-secreting cells during sindbis virus encephalitis in mice interleukin modulation of pathogenic th cells during fatal alphavirus encephalomyelitis contribution of t cells to mortality in neurovirulent sindbis virus encephalomyelitis interleukin- modulation of virus clearance and disease in mice with alphaviral encephalomyelitis biologic functions of the ifn-gamma receptors noncytolytic clearance of sindbis virus infection from neurons by gamma interferon is dependent on jak/stat signaling the molecular cell biology of interferon-gamma and its receptor antiviral actions of interferons an olfactory sensory neuron line, odora, properly targets olfactory proteins and responds to odorants molecular basis of sindbis virus neurovirulence in mice the nsp macrodomain is important for sindbis virus replication in neurons and neurovirulence in mice basis of neurovirulence in sindbis virus encephalomyelitis of mice the inflammatory response to nonfatal sindbis virus infection of the nervous system is more severe in sjl than in balb/c mice and is associated with low levels of il- mrna and high levels of il- -producing cd + t cells glutamine antagonist-mediated immune suppression decreases pathology but delays virus clearance in mice during nonfatal alphavirus encephalomyelitis immunity-related gtpase m (irgm) proteins influence the localization of guanylate-binding protein (gbp ) by modulating macroautophagy characterization of the "- -"oligoadenylate synthetase ubiquitin-like family the antiviral response. microbes infect multiple interferon stimulated genes synergize with the zinc finger antiviral protein to mediate anti-alphavirus activity inhibition of japanese encephalitis virus infection by the host zinc-finger antiviral protein mice deficient in interferon-gamma or interferon-gamma receptor have distinct inflammatory responses to acute viral encephalomyelitis distinct immune responses in resistant and susceptible strains of mice during neurovirulent alphavirus encephalomyelitis immunopathogenesis and immune modulation of venezuelan equine encephalitis virus-induced disease in the mouse protective effects of glutamine antagonist -diazo- -oxo-l-norleucine in mice with alphavirus encephalomyelitis cytotoxic and non-cytotoxic roles of the ctl/nk protease granzyme b sensitive and viable identification of antigen-specific cd + t cells by a flow cytometric assay for degranulation granzyme a activates another way to die tissue-resident memory t cells gamma interferon-dependent, noncytolytic clearance of sindbis virus infection from neurons in vitro autophagy protects against sindbis virus infection of the central nervous system eating oneself and uninvited guests: autophagy-related pathways in cellular defense autophagy-dependent viral recognition by plasmacytoid dendritic cells irgm is a common target of rna viruses that subvert the autophagy network capsid, membrane and ns are the major viral proteins involved in autophagy induced by japanese encephalitis virus common host genes are activated in mouse brain by japanese encephalitis and rabies viruses interferon-stimulated genes-mediators of the innate immune response during canine distemper virus infection oas b-dependent immune transcriptional profiles of west nile virus infection in the collaborative cross the interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts hiv- infection of human macrophages directly induces viperin which inhibits viral production in vivo and in vitro studies on the antiviral activities of viperin against influenza h n virus infection the antiviral protein viperin inhibits hepatitis c virus replication via interaction with nonstructural protein a viperin restricts chikungunya virus replication and pathology cell-type-and region-specific restriction of neurotropic flavivirus infection by viperin viperin inhibits classical swine fever virus replication by interacting with viral nonstructural a protein expression of the zinc-finger antiviral protein inhibits alphavirus replication inhibition of filovirus replication by the zinc finger antiviral protein zinc-finger antiviral protein inhibits hiv- infection by selectively targeting multiply spliced viral mrnas for degradation inhibition of hepatitis b virus replication by the host zinc finger antiviral protein zap's stress granule localization is correlated with its antiviral activity and induced by virus replication cd t cells: fates, functions, and faults th cells enhance viral persistence and inhibit t cell cytotoxicity in a model of chronic virus infection il- signal affects both protection and pathogenesis of virus-induced chronic cns demyelinating disease ifn-γ protects from lethal il- mediated viral encephalomyelitis independent of neutrophils interleukin- : a novel inflammatory cytokine that bridges innate and adaptive immunity negative impact of ifn-γ on early host immune responses to retroviral infection natural killer cells appear to play no role in the recovery of mice from sindbis virus infection nk cell-mediated immunopathology during an acute viral infection of the cns viral persistence in neurons explained by lack of major histocompatibility class i expression consequences of cytotoxic t lymphocyte interaction with major histocompatibility complex class i-expressing neurons in vivo regulation of class i mhc gene expression in the developing and mature cns by neural activity detailed in vivo analysis of interferon-gamma induced major histocompatibility complex expression in the central nervous system: astrocytes fail to express major histocompatibility complex class i and ii molecules rapid formation of extended processes and engagement of theiler's virus-infected neurons by cns-infiltrating cd t cells control of lymphocytic choriomeningitis virus infection in granzyme b deficient mice cd + t cells require perforin to clear west nile virus from infected neurons cytolytic effector pathways and ifn-γ help protect against japanese encephalitis perforin-mediated effector function within the central nervous system requires ifn-γ mediated mhc up-regulation the critical role of ifn-γ in experimental coronavirus retinopathy are proteinases functional molecules of t lymphocytes? human and mouse granzyme a induce a proinflammatory cytokine response granzyme b-dependent proteolysis acts as a switch to enhance the proinflammatory activity of il- α gamma interferon can block herpes simplex virus type reactivation from latency, even in the presence of late gene expression selective retention of herpes simplex virus-specific t cells in latently infected human trigeminal ganglia noncytotoxic lytic granule-mediated cd + t cell inhibition of hsv- reactivation from neuronal latency mouse granzyme k has pro-inflammatory potential a secretable serine proteinase with highly restricted specificity from cytolytic t lymphocytes inactivates retrovirus-associated reverse transcriptase granzyme h destroys the function of critical adenoviral proteins required for viral dna replication and granzyme b inhibition granzyme m targets host cell hnrnp k that is essential for human cytomegalovirus replication functional characterization of the interaction between human la and hepatitis b virus rna cleavage of la protein by granzyme h induces cytoplasmic translocation and interferes with la-mediated hcv-ires translational activity la protein binds the predicted loop structures in the ' non-coding region of japanese encephalitis virus genome: role in virus replication la protein can simultaneously bind to both -and -noncoding regions of japanese encephalitis virus genome heterogeneous nuclear ribonuclear protein k interacts with the enterovirus ' untranslated region and participates in virus replication hiv nef enhances tat-mediated viral transcription through a hnrnp-k-nucleated signaling complex vimentin interacts with heterogeneous nuclear ribonucleoproteins and dengue nonstructural protein and is important for viral replication and release mapping of chikungunya virus interactions with host proteins identified nsp as a highly connected viral component heterogeneous nuclear ribonuclear protein k interacts with sindbis virus nonstructural proteins and viral subgenomic mrna identification and characterization of sindbis virus rna-host protein interactions magnetic fractionation and proteomic dissection of cellular organelles occupied by the late replication complexes of semliki forest virus long term intraparenchymal ig secretion after acute viral encephalitis in mice persistence of japanese encephalitis virus in the human nervous system long-term effects of semliki forest virus infection in the mouse central nervous system persistence of west nile virus in the central nervous system and periphery of mice t cells facilitate recovery from venezuelan equine encephalitis virus-induced encephalomyelitis in the absence of antibody persistence of virus-specific immune responses in the central nervous system of mice after west nile virus infection recruitment and retention of b cells in the central nervous system in response to alphavirus encephalomyelitis dendritic cell-induced memory t cell activation in nonlymphoid tissues long-lived epithelial immunity by tissue-resident memory t (trm) cells in the absence of persisting local antigen presentation brain-resident memory t cells represent an autonomous cytotoxic barrier to viral infection cd + t cell help guides formation of cd + lung-resident memory cd + t cells during influenza viral infection this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank kimberly schultz, kirsten kulcsar, lisa mangus, and lauren peiffer for helpful discussions and elizabeth troisi and jane yeh for technical assistance. the funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. the authors declare no conflicts of interest. key: cord- - ngr l authors: han, xiaoxiao; tian, yiming; guan, ru; gao, wenqian; yang, xin; zhou, long; wang, hongning title: infectious bronchitis virus infection induces apoptosis during replication in chicken macrophage hd cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ngr l avian infectious bronchitis has caused huge economic losses in the poultry industry. previous studies have reported that infectious bronchitis virus (ibv) infection can produce cytopathic effects (cpe) and apoptosis in some mammalian cells and primary cells. however, there is little research on ibv-induced immune cell apoptosis. in this study, chicken macrophage hd cells were established as a cellular model that is permissive to ibv infection. then, ibv-induced apoptosis was observed through a cell viability assay, morphological changes, and flow cytometry. the activity of caspases, the inhibitory efficacy of caspase-inhibitors and the expression of apoptotic genes further suggested the activation of apoptosis through both intrinsic and extrinsic pathways in ibv-infected hd cells. additionally, ammonium chloride (nh( )cl) pretreated hd cells blocked ibv from entering cells and inhibited ibv-induced apoptosis. uv-inactivated ibv also lost the ability of apoptosis induction. ibv replication was increased by blocking caspase activation. this study presents a chicken macrophage cell line that will enable further analysis of ibv infection and offers novel insights into the mechanisms of ibv-induced apoptosis in immune cells. infectious bronchitis virus (ibv) can cause avian infectious bronchitis, an acute and highly infectious disease of chicken. ibv is a member of the family coronaviridae and genus coronavirus. it is a single stranded positive sense, enveloped rna virus - kb in length [ , ] . like some other members of the coronavirus family, ibv mainly causes upper-respiratory tract disease. ibv is characterized by nephritis, proventriculitis and reduction in both laying rate and egg quality in infected chickens [ ] . vaccination is an effective prevention measure, but ibv's ability to mutate has decreased vaccine protection [ ] [ ] [ ] . in order to develop better prevention and control measures, the interactions between host and ibv needs to be better studied. almost all wild-type ibv strains are only able to proliferate in embryonated chicken eggs or primary chicken embryo kidney cells. the beaudette strains were used previously to study the resistance of ibv to the antiviral state induced by type i interferon (ifn) [ ] , induction of apoptosis through endoplasmic reticulum stress in vero cells by ibv infection [ ] and activate autophagy by ibv nonstructural protein (nsp) [ ] . however, there have been limited studies of the interactions between ibv infection and immune cell apoptosis. here, the beaudette strain was used to study the mechanism of ibv infection. apoptosis is a form of programmed cell death that results from the activation of intracellular self-destruction biochemical programs [ ] . the activation of caspases (a family of cysteine protease) is a significant regulatory event in the apoptosis process [ ] . caspase cascades are triggered by both extrinsic and intrinsic signals to mediate the cell apoptosis [ ] . the relationship between cell apoptosis and virus infection is complex. cell apoptosis induced by virus may cause tissue damage, especially in the immune and nervous systems, suggesting that apoptosis is a pathogenic mechanism in virus-induced disease. at the same time, apoptosis of infected cells can directly interfere with viral replication, and immune cells can engulf apoptotic cells to prevent inflammation [ , ] . previous studies demonstrated that ibv induced apoptosis in cultured mammalian cells and primary cells [ , , ] . however, there is limited information about the apoptosis signaling pathways induced by ibv infection in immune cells. some studies indicated that ibv can transform certain elements of the innate immune system to promote secondary bacterial infections, and macrophages, as an important component of the innate immune system, may play a role in this process [ ] . a nephropathogenic ibv strain (b ) can productively replicate in blood monocytic cells, and the infected cells may act as carrier cells to play a crucial role in cell-associated viremia and the dissemination of virus to the internal organs [ ] . some viruses have been shown to induce apoptosis in macrophage, like human immunodeficiency virus (hiv)- [ ] , chikungunya virus (chikv) [ ] and influenza virus [ ] . therefore, additional study is required to investigate the functional roles of macrophages in ibv infection to help understand the mechanistic details of immune responses during virus infections [ ] . in this report, we used chicken macrophage hd cells considered an accurate representation of primary avian macrophages [ , ] . the hd cells were identified and characterized as a novel model that is permissive to ibv infection. the molecular and morphological variations in ibv-infected cells revealed that cell apoptosis was induced by ibv infection and appeared to activate caspase- by the fas/fas ligand (fasl)-mediated signaling pathway and to activate caspase- by the b-cell lymphoma (bcl- ) family-mediated signaling pathway. apoptosis required viral replication in ibv-infected cells. the chicken macrophage hd cells were kindly provided by prof. xin-an jiao (jiangsu key laboratory of zoonosis, yang zhou university, yang zhou, jiangsu province, china). hd cells were cultured in dulbecco's modified eagle's medium (dmem) (hyclone, logan, ut, usa) supplemented with % fetal bovine serum (fbs) (gemini bio-products, west sacramento, ca, usa), u/ml penicillin and µg/ml streptomycin (hyclone, logan, ut, usa) at ph . and were kept at • c with % co . the vero cell-adapted ibv beaudette strain (p ) [ ] used in the current study was kindly provided by prof. shi-qi sun (state key laboratory of veterinary etiological biology, lanzhou veterinary research institute, chinese academy of agricultural sciences, gansu province, china). traditional ibv strain m , vaccine ibv strain h and virulent ibv strain sabik [ ] were housed in our laboratory (viruses were propagated in specific pathogen free (spf) days old embryonated chicken eggs). susceptibility of hd cells to ibvs was measured by morphological changes, growth curves using % tissue culture infective doses (tcid ) and indirect immunofluorescence assay (ifa). here, tcid were applied to hd cells to quantitate virus titers as described previously [ ] . hd cells were cultured in -well plates, and ten-fold dilutions of the virus were prepared in dmem supplemented with % fbs. cultured cells were infected with the virus and then observed daily for cytopathic effects (cpe). in order to evaluate the virus growth kinetics in hd cells, the cells were infected with ibv at multiplicity of infection (moi). the infected cells were collected at the indicated time points and analyzed using tcid assay. a cell counting kit- (cck- ) assay (beyotime, haimen, jiangsu province, china) was utilized to identify the viability of cells as described previously [ ] . hd cells were cultured in -well plates and infected with ibv at different moi ( . , . , , , and moi) for specific lengths of times. in parallel, a negative control was set up. the cells were incubated with µl/well cck- solution (beyotime, haimen, jiangsu province, china) and allowed to react for h at • c. the absorbance was detected using a microplate reader (model , bio-rad, hercules, ca, usa) at nm. the negative control was set at %, and the treated samples were calculated according to the following formula: survival rate (%) = optical density (od) of the treated cells/od of the negative control × . hd cells were pre-incubated in -well plates and infected with ibv at moi. to assess apoptosis, the condensed and fragmented nuclei were observed using hoechst staining (keygen biotech, nanjing, jiangsu province, china). at the specified time points, the cells were immobilized with % paraformaldehyde (keygen biotech, nanjing, jiangsu province, china) for min and then incubated with hoechst (keygen biotech) in the dark for min. the typical apoptotic morphological changes were observed using a fluorescence microscope (olympus ix , olympus, tokyo, japan) with uv excitation at nm. hd cells were grown overnight to % density in -well plates and were then infected with ibv at an moi of . after the appearance of typical cpe, the cells were immobilized with % paraformaldehyde for min. a mouse polyclonal antibody against the ibv nucleocapsid (n) protein ( : dilution, prepared in our laboratory) was added, followed by incubation for h at • c. next, the cells were treated with a fluorescein isothiocyanate (fitc)-conjugated goat anti-mouse igg ( : , transgen biotech, beijing, china) for h at • c. the specimens were viewed with an olympus ix fluorescence microscope (olympus) with the appropriate excitation and emission wavelengths for fitc ( nm and nm, respectively). to identify the apoptotic rate, the percentage of cells undergoing apoptosis was determined by an annexin v-fitc apoptosis detection kit (absin, shanghai, china). hd cells were cultured in -well plates and infected with ibv at moi. cells were harvested and washed three times with phosphate-buffered saline (pbs) at the indicated times. the cells were centrifuged at g for min and then suspended in µl of binding buffer containing µl fitc-conjugated annexin v antibody and µl propidium iodide (pi). the mixture was incubated at room temperature for min in the dark. the cells were detected by flow cytometer (bd biosciences, san jose, ca, usa) within an hour. the activities of caspase- , - , and - were detected by colorimetric assay kit (keygen biotech). the cells were incubated with lysis buffer, and the concentrations of protein were detected by bicinchoninic acid (bca) protein assay reagent (vazyme biotech, nanjing, jiangsu province, china). the protein ( µg/sample) was treated with caspase- , - , and - substrate for each sample at • c for h. samples were read by a microplate reader (model , bio-rad) at nm. total rna was isolated using trizol agent (invitrogen, carlsbad, ca, usa), and each rna sample was reverse-transcribed to complementary dna (cdna) by primescript™ rt reagent kit (takara, dalian, liaoning province, china). cdna was used for quantitative real-time polymerase chain reaction (qrt-pcr) analysis. the sets of primer pairs of apoptotic regulating genes are listed in table [ ] . for qrt-pcr reactions, the µl reaction mixture included µl cdna, . µl sybr premix ex taqtm ii (takara), . µl of forward and . µl of reverse primer and . µl rnaase-free water (takara). reaction conditions were • c for min followed by cycles of • c for s, the specific melting temperature (tm) of a primer pair for s, and then • c for s, and • c for s, using a bio-rad iq thermal cycler (bio-rad). β-actin was selected as a reference gene. the expression fold changes were calculated using the −∆∆ct method [ ] . table . sequences of chicken primer pairs used for quantitative real-time polymerase chain reaction (qrt-pcr). fas all data are expressed as the mean ± standard error of the mean (sem) from three independent experiments performed in triplicate. the statistical analyses were conducted using student's t-test in graphpad prism version (graphpad software, san diego, ca, usa). a p-value < . was considered significant, and a p-value < . was considered highly significant. to determine whether ibvs replicate in the chicken macrophage cell line, four ibv strains (beaudette, m , h and sabik strains) were utilized in this study to test the infective processes in hd cells. for the m , h and sabik strains, the infected cells were blindly passaged five times, and cpe was not observed. the growth curve using tcid and ifa showed that replication of the three ibv strains in hd cells was severely restricted, and no significant replication was observed. however, the hd cells were highly permissive for the propagation of the attenuated ibv beaudette strain. the results showed that hd cells could be infected by ibv beaudette in passage one. first, morphological changes of ibv beaudette-infected hd cells were observed. after infection with ibv beaudette at moi, cpe appeared in hd cells at h post-infection (h.p.i.) and were evident at h.p.i. when compared with the mock infection ( figure a ). the normal hd cells could also be re-infected with the culture supernatant from the virally infected cells. the susceptibility of hd cells to ibv beaudette infection was evaluated by growth kinetics. the growth kinetics of the virus were observed upon infection at an moi of in hd cells. the virus titers increased until reaching the maximal level of . tcid /ml ( figure b ). ibv beaudette replication in hd cells was also studied by performing an immunofluorescence assay. the production of fitc-stained virus was observed by h.p.i. in contrast, mock-infected hd cells showed no fluorescence ( figure c ). infection of hd cells with ibv beaudette caused cell death in a time-and dose-dependent manner, as tested by cck- assay. (figure a ). the infected cells showed chromatin condensation and nuclear fragmentation. after h of infection, large amounts of apoptotic bodies were observed in hd cells ( figure b ). the rate of apoptotic cells was measured by flow cytometry. the rate of apoptosis significantly increased at h.p.i. in virus-infected cells when compared with the mock- infection of hd cells with ibv beaudette caused cell death in a time-and dose-dependent manner, as tested by cck- assay. (figure a ). the infected cells showed chromatin condensation and nuclear fragmentation. after h of infection, large amounts of apoptotic bodies were observed in hd cells ( figure b ). the rate of apoptotic cells was measured by flow cytometry. the rate of apoptosis significantly increased at h.p.i. in virus-infected cells when compared with the mock-infected cells ( figure c ). these findings indicated that apoptosis was induced by ibv beaudette infection in hd cells. viruses , , of infected cells ( figure c ). these findings indicated that apoptosis was induced by ibv beaudette infection in hd cells. activation of the caspase proteinases is a significant event in the occurrence of apoptosis. the activity of caspases that play important roles in the activation of the apoptosis pathway was activation of the caspase proteinases is a significant event in the occurrence of apoptosis. the activity of caspases that play important roles in the activation of the apoptosis pathway was investigated in this study. when hd cells were infected with ibv beaudette at moi, the levels of caspase- , - , and - were significantly increased from h.p.i. and then increased further over time ( figure a ). to further identify the function of caspase- , - , and - in the apoptotic pathway, we measured the viability of infected-cells incubated with specific inhibitors of caspase- , - , and - (z-devd-fmk, z-ietd-fmk, and z-lehd-fmk; keygen biotech, nanjing, jiangsu province, china). the data revealed that cell viability was significantly increased by the specific inhibition of caspase- , - , and - ( figure b ). to confirm the function of caspase- and caspase- to activate caspase- , the inhibitory efficacy of the caspase- or caspase- inhibitors on caspase- activity was also determined. when cells were pretreated with the caspase- or caspase- inhibitor, the activity of caspase- was significantly decreased in cells, and more significantly decreased when the two inhibitors were added together ( figure c ). these results revealed that caspase- activation and ibv beaudette-induced apoptosis may be triggered via both extrinsic and intrinsic pathways. viruses , , of investigated in this study. when hd cells were infected with ibv beaudette at moi, the levels of caspase- , - , and - were significantly increased from h.p.i. and then increased further over time ( figure a ). to further identify the function of caspase- , - , and - in the apoptotic pathway, we measured the viability of infected-cells incubated with specific inhibitors of caspase- , - , and - (z-devd-fmk, z-ietd-fmk, and z-lehd-fmk; keygen biotech, nanjing, jiangsu province, china). the data revealed that cell viability was significantly increased by the specific inhibition of caspase- , - , and - ( figure b ). to confirm the function of caspase- and caspase- to activate caspase- , the inhibitory efficacy of the caspase- or caspase- inhibitors on caspase- activity was also determined. when cells were pretreated with the caspase- or caspase- inhibitor, the activity of caspase- was significantly decreased in cells, and more significantly decreased when the two inhibitors were added together ( figure c ). these results revealed that caspase- activation and ibv beaudette-induced apoptosis may be triggered via both extrinsic and intrinsic pathways. caspase- has an important effect on apoptosis that is mediated by fas/fasl. the activity of caspase- was increased in the ibv beaudette-infected hd cells. this implied that apoptosis is induced by ibv beaudette infection through the fas/fasl pathway. to investigate this further, the expression levels of fas and fasl were detected in ibv beaudette-infected hd cells by qrt-pcr. the data revealed increased gene expression of fas and fasl over time ( figure a) . furthermore, the the data are shown as the mean ± sem, * p < . , ** p < . versus ibv infection alone. (c) the effect of initiator caspase- or - on the activation of caspase- : µm of each caspase inhibitor was utilized to pretreat cells for h. then, the treated and untreated cells were infected with ibv at moi for h. caspase- activity was detected using a colorimetric assay kit. data are shown as the mean ± sem, * p < . , ** p < . versus virus infection alone. caspase- has an important effect on apoptosis that is mediated by fas/fasl. the activity of caspase- was increased in the ibv beaudette-infected hd cells. this implied that apoptosis is induced by ibv beaudette infection through the fas/fasl pathway. to investigate this further, the expression levels of fas and fasl were detected in ibv beaudette-infected hd cells by qrt-pcr. the data revealed increased gene expression of fas and fasl over time ( figure a) . furthermore, the members of the bcl- family are generally distributed on the surface of mitochondria, and their activation may regulate the intrinsic apoptosis pathway. to test this, the expression levels of bcl- and bcl- -associated x protein (bax) were quantified by qrt-pcr in ibv beaudette-infected hd cells. the results showed the mrna levels of bcl- were obviously downregulated from h.p.i. and declined over time. conversely, the mrna levels of bax were upregulated from h.p.i. and continuously increased until h.p.i ( figure b) . moreover, the activation of caspase- was partly inhibited in ibv beaudette-infected cells pretreated with the inhibitor of caspase- ( figure c ). this suggested that caspase- activation was affected by the blocking of caspase- activity. taken together, these findings suggested that the fas/fasl-mediated signal contributes to the activation of caspase- . additionally, bcl- and bax might play important roles in regulating the activation of caspase- . the activation of caspase- can also affect the extrinsic apoptosis pathway in ibv beaudette-infected cells. viruses , , of members of the bcl- family are generally distributed on the surface of mitochondria, and their activation may regulate the intrinsic apoptosis pathway. to test this, the expression levels of bcl- and bcl- -associated x protein (bax) were quantified by qrt-pcr in ibv beaudette-infected hd cells. the results showed the mrna levels of bcl- were obviously downregulated from h.p.i. and declined over time. conversely, the mrna levels of bax were upregulated from h.p.i. and continuously increased until h.p.i ( figure b) . moreover, the activation of caspase- was partly inhibited in ibv beaudette-infected cells pretreated with the inhibitor of caspase- ( figure c ). this suggested that caspase- activation was affected by the blocking of caspase- activity. taken together, these findings suggested that the fas/fasl-mediated signal contributes to the activation of caspase- . additionally, bcl- and bax might play important roles in regulating the activation of caspase- . the activation of caspase- can also affect the extrinsic apoptosis pathway in ibv beaudette-infected cells. following incubation with z-ietd-fmk for h, the cells were infected with ibv beaudette for h. caspase- activity was detected using a colorimetric assay kit. data are shown as the mean ± sem. * p < . versus virus infection alone. to determine whether apoptosis plays a pivotal role in inhibition of virus replication, the virus titers of untreated cells or those treated with caspase inhibitors were detected by tcid . the results showed that the caspase- inhibitor could increase the titer of ibv beaudette, but did not show obvious effects on caspase- and - inhibitor-treated cells ( figure a ). to test whether the ability of virions to enter cells was crucial to apoptosis, endosomal acidification was blocked by nh cl to prevent the viruses from being released [ ] . the virus titer was significantly decreased with nh cl treatment ( figure b ). compare with non-treated cells, the rate of apoptotic cells was also decreased in nh cl-treated cells when infected with ibv beaudette ( figure c ). next, the uv-treated virus was used to test whether apoptosis induction required virus replication. when the virus was subjected to uv treatment, the virus titer could not be detected ( figure d ). consistently, the rate of apoptotic cells was dramatically decreased in cells infected with uv-inactivated virus, when compared with the uv-untreated virus ( figure e ). in conclusion, apoptosis induction required viral replication in ibv beaudette-infected cells. viruses , , of to determine whether apoptosis plays a pivotal role in inhibition of virus replication, the virus titers of untreated cells or those treated with caspase inhibitors were detected by tcid . the results showed that the caspase- inhibitor could increase the titer of ibv beaudette, but did not show obvious effects on caspase- and - inhibitor-treated cells ( figure a ). to test whether the ability of virions to enter cells was crucial to apoptosis, endosomal acidification was blocked by nh cl to prevent the viruses from being released [ ] . the virus titer was significantly decreased with nh cl treatment ( figure b ). compare with non-treated cells, the rate of apoptotic cells was also decreased in nh cl-treated cells when infected with ibv beaudette ( figure c ). next, the uv-treated virus was used to test whether apoptosis induction required virus replication. when the virus was subjected to uv treatment, the virus titer could not be detected ( figure d ). consistently, the rate of apoptotic cells was dramatically decreased in cells infected with uv-inactivated virus, when compared with the uv-untreated virus ( figure e ). in conclusion, apoptosis induction required viral replication in ibv beaudette-infected cells. compared with other coronaviruses, ibv is not easily adapted to cell culture. several mammalian cell lines and primary cells have been previously revealed to be permissive to ibv infection. some strains of ibv can replicate and produce cpe in primary chicken embryo kidney cells. ibv holte and beaudette- strains can proliferate in the bhk- cell line [ ] , and the beaudette strain of embryo-culture ibv has adapted to vero cells [ ] . however, previous studies of chicken immune cells and the pathogenesis of ibv focused on primary immune cells separated from the blood or spleen [ , ] . in this study, hd cells, a chicken macrophage cell line, were shown to be susceptible to ibv beaudette. additionally, the ibv beaudette-infected cells produced infectious virus progeny with a high virus titer. morphological assessment of the cells during ibv beaudette infection showed that cpe can be observed after h.p.i. the virus growth kinetics for hd cells also showed peak viral titers at h.p.i. immunofluorescence was used to identify and analyze virus infection, and strong fluorescence signals were observed in the ibv beaudette-infected cells. based on these results, the chicken macrophage hd cells will serve as an essential tool for future studies of ibv infection. apoptosis is an important part of the antiviral host response. however, some viruses actively trigger this process to facilitate their replication [ ] . infection with coronavirus induced apoptosis in various cell types. transmissible gastroenteritis virus (tgev)-induced apoptosis in pk- cells was dependent on viral replication [ ] . porcine hemagglutinating encephalomyelitis virus (phev) induced apoptosis through a caspase-dependent pathway in pk- cells [ ] . severe acute respiratory syndrome (sars) coronavirus membrane (m) and nucleocapsid (n) proteins can induce apoptosis in hpf cells [ ] . according to previous studies, apoptosis occurs in response to ibv infection in vero cells, df cells and chicken embryo kidney cells [ , ] . this study is the first demonstration that ibv induces apoptosis in chicken macrophage hd cells. the ibv beaudette-infected hd cells exhibited typical characteristics of apoptosis including the condensation of the cell nucleus, reduction of cell viability, and an increased rate of apoptotic cells. caspases are a family of cysteine-catalyzed proteases that cleave aspartic acids. the triggering of caspase cascades plays indispensable roles in apoptosis and can be induced by many viruses. there are two major signaling pathways that contribute to caspase activation: death receptor and mitochondrial pathways [ ] . some viruses have exhibited cell apoptosis that is mediated by fas/fasl signaling as a reaction to viral infection, such as hepatitis c virus (hcv) [ ] and dengue virus (denv) [ ] . cell apoptosis can be induced by some viruses by regulating the levels of bcl- family members, such as sars coronavirus [ ] and epstein-barr virus (ebv) [ ] . our results showed that the activation of caspase- in ibv beaudette-infected cells was regulated by fas and fasl. the results also showed that activation of caspase- in ibv beaudette-infected cells was regulated by decreased expression of bcl- and increased expression of bax. the caspase- activation and virus-induced apoptosis might be triggered through both extrinsic and intrinsic pathways. in most cases, cell apoptosis induced by virus is a process of interaction between extrinsic and intrinsic pathways. the activation of caspase- was inhibited by z-ietd-fmk, and the activation of caspase- was not completely eliminating by blocking caspase- activity, suggesting that the activation of caspase- is not the only pathway to activate caspase- , requiring further research. ibv are known to induce apoptosis through caspase-dependent pathway [ ] and intrinsic-dependent pathway regulated by bcl- family proteins [ ] in vero cells. in this study, these two pathways were demonstrated that play an important role in ibv beaudette-infected hd cells. additionally, this is the first report that the extrinsic pathway regulated by fas/fasl was activated in ibv-induced apoptosis. improved knowledge of the mechanisms by which ibv activates the extrinsic and intrinsic apoptotic pathways will help to better understand the pathogenic properties of epidemic ibv strains in the host. some viruses can induce cell apoptosis through viral replication. uv-inactivated bhv- and tgev could not induce apoptosis, or the nh cl-pretreated cells prevented the appearance of apoptosis [ , ] . some other viruses can induce apoptosis without viral replication, such as vaccinia virus and vesicular stomatitis virus [ , ] . here, infection with uv-inactivated ibv beaudette or treatment of hd cells with nh cl reduced virus apoptosis induction, indicating that ibv beaudette -induced apoptosis in hd cells depends on viral replication. this finding is similar to that of a previous study showing that uv-inactivated ibv lost the capacity to induce apoptosis in mammalian cells [ ] . we tested whether caspase activation is needed for ibv replication in cells. the finding revealed that treatment with the caspase- inhibitor can increase the virus titer in ibv beaudette-infected cells, suggesting that the caspase inhibitor might increase the survival time of cells to promote replication. from a therapeutic standpoint, available drugs controlling apoptosis could be used to limit ibv spreading [ ] . the innate immune response is the first line of defense against viruses, and macrophages are an important component of this system. some viruses have evolved strategies to induce apoptosis to enhance the production of virus progeny and promote dissemination to neighboring cells with limited host immune/inflammatory responses. the presence of apoptotic cells may also lead to the mobilization and initiation of innate immune defenses [ ] . previous studies have shown that virus-induced apoptosis of macrophage has an important impact on virus infection. porcine reproductive and respiratory syndrome virus (prrsv) stimulates anti-apoptotic pathways in macrophages early in infection, and these prrsv-infected macrophages die by apoptosis late in infection [ ] . chikv infection induces apoptosis and enhances expression of major histocompatibility complexes (mhcs) and co-stimulatory molecules and interleukin (il)- and monocyte chemoattractant protein (mcp)- production in macrophages [ ] . however, little is known about ibv-induced immune cell apoptosis. it has been reported that phagocytic cells may play a crucial role in dissemination of virus to the blood circulation and internal organs. therefore, establishment of this macrophage system of ibv beaudette infection and determination of the apoptotic mechanism might be proof of principle for ibv infection in the host. in conclusion, chicken macrophage hd cells were established for attenuated ibv strain beaudette infection. ibv beaudette induced cell apoptosis through caspase- activation mediated by fas/fasl and caspase- activation mediated by bcl- /bax. in addition, ibv beaudette replication was essential to apoptosis induction, and ibv beaudette replication increased when caspase activation was blocked. based on these findings, this study has shown the establishment of a chicken macrophage cell line that will facilitate the further analysis of ibv infection. additional studies are required to clarify the detailed molecular mechanisms underlying ibv-induced apoptosis. an efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus ibv coronavirus avian infectious bronchitis virus the long view: years of infectious bronchitis research recombinant infectious bronchitis coronavirus beaudette with the spike protein gene of the pathogenic m strain remains attenuated but induces protective immunity vaccination against infectious bronchitis virus: a continuous challenge recombinant infectious bronchitis virus (ibv) h vaccine strain expressing the hemagglutinin-neuraminidase (hn) protein of newcastle disease virus (ndv) protects chickens against ibv and ndv challenge infectious bronchitis coronavirus inhibits stat signaling and requires accessory proteins for 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vaccine porcine hemagglutinating encephalomyelitis virus induces apoptosis in a porcine kidney cell line via caspase-dependent pathways m and n proteins of sars coronavirus induce apoptosis in hpf cells pathogenicity and tissue tropism of infectious bronchitis virus is associated with elevated apoptosis and innate immune responses apoptosis initiated by bcl- -regulated caspase activation independently of the cytochrome c/apaf- /caspase- apoptosome hepatitis c virus protects human b lymphocytes from fas-mediated apoptosis via e -cd engagement fasl/fas pathway is involved in dengue virus induced apoptosis of the vascular endothelial cells induction of apoptosis by the severe acute respiratory syndrome coronavirus a protein is dependent on its interaction with the bcl-x l protein epstein-barr virus interactions with the bcl- protein family and apoptosis in human tumor cells bovine herpes virus type induces apoptosis through fas-dependent and mitochondria-controlled manner in madin-darby bovine kidney cells vesicular stomatitis virus induces apoptosis primarily through bak rather than bax by inactivating mcl- and bcl-x l dose-dependent lymphocyte apoptosis following respiratory infection with vaccinia virus caspase inhibitors: viral, cellular and chemical recent progress in studies of arterivirus-and coronavirus-host interactions porcine reproductive and respiratory syndrome virus modulates apoptosis during replication in alveolar macrophages the authors declare no conflict of interest. key: cord- - tgovkr authors: wu, nicholas c.; wilson, ian a. title: structural biology of influenza hemagglutinin: an amaranthine adventure date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: tgovkr hemagglutinin (ha) glycoprotein is an important focus of influenza research due to its role in antigenic drift and shift, as well as its receptor binding and membrane fusion functions, which are indispensable for viral entry. over the past four decades, x-ray crystallography has greatly facilitated our understanding of ha receptor binding, membrane fusion, and antigenicity. the recent advances in cryo-em have further deepened our comprehension of ha biology. since influenza ha constantly evolves in natural circulating strains, there are always new questions to be answered. the incessant accumulation of knowledge on the structural biology of ha over several decades has also facilitated the design and development of novel therapeutics and vaccines. this review describes the current status of the field of ha structural biology, how we got here, and what the next steps might be. four types of influenza virus, a, b, c, and d, are known. influenza a and b viruses can cause severe symptoms and mortality in the human population, whereas influenza c virus only manifests itself in mild disease and influenza d virus does not circulate in humans. a major difference between influenza a and b viruses is that influenza b virus is almost exclusively observed in humans, whereas influenza a virus has a diverse and extensive reservoir in aquatic birds that occasionally spills over to humans directly or via domestic animals, such as pigs, as new pandemics or emerging viruses [ ] . as a result, influenza a viruses receive much more attention than other influenza types even though influenza a and b both co-circulate in the human population as seasonal viruses. influenza a virus can be further divided into subtypes based on the antigenicity of the surface glycoproteins hemagglutinin (ha) and neuraminidase (na), with known subtypes of ha (h -h ) and subtypes of na (n -n ). similar to influenza a virus, influenza b virus also has two surface glycoproteins ha and na, which diverged into two lineages, victoria and yamagata, during the s [ ] . in contrast, influenza c and d viruses only have one surface glycoprotein hemagglutinin-esterase fusion (hef) [ ] that encompasses both ha and na activities. four known influenza a pandemics have been documented in human history, namely spanish flu (h n ), asian flu (h n ), hong kong flu (h n ), and swine flu (h n ), although others undoubtedly have occurred prior to these [ ] . occasionally, other influenza a subtypes, such as h n , h n , h n , h n , h n , h n , and h n , also infect back in the early s, george hirst reported the ability of influenza virus to agglutinate chicken red blood cells (rbcs) [ ] and attributed this to adsorption of the ha onto the rbcs [ ] . in the late s, an enzyme from vibrio cholerae was discovered with the ability to prevent influenza virus from agglutinating red blood cells [ , ] . subsequent identification of the enzymatic product revealed sialic acid as the receptor of influenza virus [ ] . however, the location and molecular characteristics of the rbs were unclear until the first ha structure was determined in [ ] . the rbs was identified partly due to its sequence conservation, structural resemblance to the wheat-germ agglutinin sialic acid-binding site [ ] , and from mutations that affect receptor specificity [ ] . the first structure of ha in complex with sialic acid in confirmed the location of the rbs and sialic acid as the host receptor of influenza virus [ ] . the rbs of influenza a ha is composed of four structural elements, -loop, -loop, -helix, and -loop, which are named after their positions on the primary amino acid sequence. similarly, rbs of influenza b ha is composed of the -loop, -helix, and -loop, which are structurally equivalent to the -loop, -loop, and -helix receptor specificity can also continue to evolve when seasonal viruses circulate in the human population, due to natural mutations that are likely a response to immune selection pressure. this phenomenon has recently been reported in human h n viruses, which have evolved a preference for long, branched sialylated glycans with extended poly-n-acetyl-lactosamine (poly-lacnac) [ . in fact, evolutionary contingency, which describes sequence variants that were previously fit but then become unfit and extinct, as well as evolutionary entrenchment, which describes sequence variants that were previously unfit and then become fit and emerge, are common in the ha rbs of human h n viruses [ ] . as seasonal influenza viruses continue to evolve in the human population, it will be fascinating to observe how the receptor-binding mode is able to change (or not) in the future, which would allow the h n virus to continue its over years of sustained circulation in the human population. interestingly, bat influenza a viruses h n and h n do not utilize sialylated glycans as receptors [ , ] . crystal structures of the ha from h n and h n viruses indicate that their rbs is highly acidic, which would electrostatically repulse sialic acid and hence would have substantially different biochemical properties from the other ha subtypes (i.e., h -h ) even although the overall architecture of the rbs is roughly similar [ , ] . recent studies have revealed that major histocompatibility complex class ii (mhc-ii) human leukocyte antigen dr isotype (hla-dr) can act as a receptor for bat influenza a viruses [ , ] . however, a structure of the complex between bat influenza ha and hla-dr has not been reported. therefore, the receptor-binding mechanism of bat influenza ha remains elusive. influenza viruses have also been discovered in species as diverse as eel, toad, and hagfish using a meta-transcriptomic approach [ ] . nonetheless, the has from these influenza viruses have not been functionally characterized and their receptors are currently unknown. additional influenza virus subtypes as well as types will likely be discovered in the future, and it will be to interesting to see whether other host receptors are employed. after attaching to the host receptor, endocytosis transports the influenza viral particle to the endosome, where the ph becomes acidic. the acidic ph triggers viral-host membrane fusion that is mediated by conformational rearrangements in the ha. the prerequisite for such conformational rearrangements is proteolytic processing of the ha. ha is translated as a single polypeptide chain ha , which is then cleaved by host proteases into the ha and ha subunits. the membrane fusion machinery is encoded mainly by ha , while ha is entirely responsible for receptor binding, as outlined in the previous section. the overall structure of uncleaved ha is almost identical to the cleaved ha [ ] . the cleavage site on ha is presented as a surface loop on the ha stem, which is proximal to the viral membrane compared to the ha head. the amino acid sequence at the cleavage site is a well-characterized pathogenic factor [ ] . while most influenza a strains carry a monobasic cleavage site, some highly pathogenic avian influenza a strains carry a polybasic cleavage site that can be processed by ubiquitously expressed furin. upon cleavage, the c-terminus of ha remains solvent exposed, whereas the n-terminal of ha , which represents the hydrophobic fusion peptide, inserts into a buried cavity that is composed of ionizable residues including ha his , as well as ha asp , asp , and lys [ ] . this metastable conformation is then poised for low ph-induced structural rearrangements to accomplish viral-host membrane fusion. in fact, the ability of ha to undergo ph-dependent structural rearrangement has been known since the early s based on circular dichroism, electron microscopy, and sedimentation analyses [ ] . subsequent analyses demonstrated that, after conformational changes, ha is susceptible to trypsin digestion, where ha residues to (globular domain) are released, while ha residues to remain covalently linked through a disulfide bond to the intact ha subunit [ , ] . a crystal structure of this trypsin-digested product containing the intact ha subunit, which represents the post-fusion conformation of ha, was reported in the mid- s [ ] . the post-fusion conformation of ha features substantial rearrangements of helices and connecting segments to form a Å-long α-helix in each protomer, which assemble as a three-stranded coiled coil at trimer interface ( figure ). in addition, the hydrophobic fusion peptide relocates to the top of the helix ready for membrane insertion. [ ] , and state (post-fusion conformation, pdb qu ) are shown [ ] . of note, after fusion peptide is released from state , the fusion peptide becomes disordered [ ] . in state , the membrane proximal region (yellow) is also disordered [ ] . different components in the ha that are involved in structural rearrangements between pre-and post-fusion structures are in different colors. structures of intermediates during the ha fusion process have been probed by low-resolution cryo-electron microscopy (cryo-em) [ , ] , as well as x-ray crystallography [ ] . nonetheless, a more complete picture of ha fusion intermediate structures was described only recently [ ] , by taking advantage of advances in high-resolution cryo-em [ ] . specifically, after incubation of ha at low ph for different times ( s, s, s, and min), ha conformational changes were examined by cryo-em [ ] (figure ). three-dimensional ( d) classification and reconstruction at different time points revealed three sequential intermediate forms of ha, including one with a Å-long three-stranded α-helix coiled coil [ ] . however, it is still unclear how the lipid bilayer membranes from the virus and host are fused together because most structural studies use the ha ectodomain and membranes are often excluded. the feasibility of structurally characterizing full-length ha, which includes the transmembrane region, has also recently been demonstrated by cryo-em [ ] . therefore, future studies should be able to explore the conformational changes during influenza virus-host membrane fusion in the context of full-length ha and in the presence of a membrane. based on analysis of the first ha structure [ ] with known natural antigenic variants and laboratory escape mutants at the time [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , four major antigenic sites (a-d) in the h ha were identified and reported in a back-to-back paper with the ha structure in [ , ] . in the s, a fifth antigenic site (e) was also identified [ , ] . similarly, five major antigenic sites, namely sa, sb, ca , ca , and cb, were identified in h ha during the early s [ , ] . all of the major antigenic sites in h and h has as well as influenza b ha [ , ] are located in the ha globular head domain and their immunodominance can change over natural evolution ( figure a,b) . however, the first structure of an antibody (hc ) in complex with ha was not reported until [ ] . antibody hc targets the rbs, which explains its neutralization activity. however, hc also recognizes rbs-proximal regions, which are highly variable across strains. as a result, escape mutants to hc could be readily identified. consistently, subsequent studies demonstrated that major antigenic drift in seasonal influenza viruses is mostly driven by mutations within or near the rbs [ , ] . it is therefore not surprising that some of the mutations that arise during natural evolution of human influenza virus can alter both ha antigenicity and receptor binding [ , , , ] . furthermore, egg-based seasonal influenza vaccines often carry egg-adaptive mutations in the ha rbs that allow the vaccine strain to bind to α - linkage sialylated glycans on the chorioallantoic membrane but can also alter the antigenicity of ha, thereby decreasing vaccine effectiveness [ , [ ] [ ] [ ] . for example, one of the egg-adaptive mutations t k would abolish an n-glycosylation site at n and appears to contribute to the poor seasonal influenza vaccine effectiveness in the - influenza season. in fact, accumulation of n-glycosylation in the ha globular head domain plays an important role in the antigenic drift of seasonal influenza virus [ , , ] . a recent study has shown that n-glycosylation sites are added to has of seasonal influenza virus at discrete -to- -year intervals, with an upper limit of~ and~ glycans in the ha globular head domains of h n and h n , respectively [ ] . the glycan form, occupancy, and heterogeneity at each n-glycosylation site on ha can be probed by mass spectrometry [ , ] . moreover, some of the n-glycans on ha can be observed by x-ray crystallography and cryo-em [ , ] . as compared to the variable globular head domain in ha , the stem domain in ha is much more conserved. it had long been thought that neutralizing antibodies (nabs) do not target the stem domain until the discovery of a mouse ha stem antibody c in [ ] . nevertheless, this observation was largely unappreciated and stem antibodies were not found in humans until the late s [ ] [ ] [ ] (figure c ). in the subsequent decade, many stem neutralizing and protective antibodies have been isolated and structurally characterized. unlike neutralizing antibodies to the ha head, which generally block receptor binding, stem antibodies typically protect by interfering with the fusion machinery [ ] [ ] [ ] . due to high sequence conservation of the stem domain, stem antibodies usually exhibit higher breadth (i.e., broadly neutralizing antibodies, bnabs) and interact with a greater range of influenza subtypes and strains compared to nabs to the ha head. recurring molecular features are observed in stem abs. for example, the ighv - antibody heavy-chain germline gene is commonly used by the immune system for generation of stem antibodies due to the presence of a germline-encoded ify motif, which can engage three highly conserved hydrophobic pockets in the ha stem region [ , [ ] [ ] [ ] [ ] [ ] [ ] . in addition, ighd - , one of the heavy-chain diversity genes that encodes for an important part of complementarity determining region of the heavy chain (cdr h ), is also utilized in many stem antibodies. the ighd - gene encodes an lxyfxwl motif that makes favorable interactions with four hydrophobic pockets in the ha stem. however, the breadth of some ha stem antibodies is often restricted to group has (h , h , h , h , h , h , h , h , h , and h ), since a conserved n-glycan at ha residue in most of the group has (h , h , h , h , h , and h ) can sterically hinder accessibility to the ha stem epitope [ , ] . a few select ighv - -encoded stem antibodies can manage to navigate around the n-glycan at ha residue to achieve cross-group neutralization [ , ] . some group -specific abs bind to an epitope that is in the lower part of the stem domain and closer to the viral membrane ( figure c ) and hence can avoid a clash with the n-glycan at ha residue [ , ] . more recently, ighv - was found to be a germline gene that is often utilized in cross-group stem abs [ ] [ ] [ ] [ ] . interestingly, the ancestral precursors of ighv - -encoded cross-group stem abs can be either group -or group -specific, depending on the cdr h sequences and conformations [ ] . there is an accumulation of glycosylation sites during human h n evolution. while many antigenic sites have now been masked by glycans (yellow), antigenic site b (blue) remains exposed due to its proximity to the rbs, making it immunodominant in recent human h n strains [ , ] . (c) broadly neutralizing epitopes that have been identified in the past decade are shown. (d) a recently identified trimeric interface epitope is illustrated. over the past decade, several cross-group bnabs that target ha rbs have also been discovered and characterized [ ] [ ] [ ] [ ] [ ] [ ] [ ] (figure c ). while the reactivity of some rbs bnabs is mostly limited to a particular subtype [ ] [ ] [ ] [ ] [ ] [ ] , they are still considered as broadly neutralizing in the sense of covering most if not all strains within a subtype (e.g., j [ ] and ch [ ] to h ha, as well as f - [ ] and - - c [ ] to h ha). such antibodies could be very useful in protecting against antigenic drift in seasonal viruses, for example. in addition, subtype-specific bnabs that target the vestigial esterase subdomain [ , ] , "lateral patch" epitope on ha [ ] , and the junction between the ectodomain and membrane anchor have also been identified [ ] . in , an h -specific bnab was shown to target an epitope that partly involves the ha protomer-protomer interface in ha [ ] . such a finding demonstrated that an antibody epitope does not need to be completely solvent exposed in the prefusion conformation. in the same year, three other papers have reported an epitope that is almost exclusively in the ha protomer-protomer interface in ha [ ] [ ] [ ] (figure d ). some interface-targeting antibodies can cross-react with all influenza a subtypes and confer in vivo protection despite the lack of neutralization activity [ , ] . therefore, it is now quite clear that "breathing" of the ha trimer [ ] can allow antibodies to access cryptic epitopes that are transiently exposed and were not originally thought to be accessible in the ha prefusion conformation. during the early s, structural-based computational screening of around , small molecules resulted in the identification of benzoquinones and hydroquinones as ha fusion inhibitors [ ] . one of the compounds, tert-butyl hydroquinone (tbhq), had its binding mode to ha reported in [ ] . tbhq binds to a hydrophobic pocket in an interface region between ha monomers, which in turn stabilizes the ha prefusion conformation and prevents the conformational changes required for membrane fusion. interestingly, arbidol, which was developed as a general antiviral medication in russia especially for respiratory diseases during the late s [ ] , was more recently shown to inhibit ha-mediated membrane fusion by stabilizing the prefusion conformation [ , ] . in , the structure of arbidol in complex with ha revealed that the arbidol binds to a similar location in the ha stem as tbhq, but its binding site is much larger and more complex [ ] . structure-based optimization of arbidol resulted in a compound with its affinity improved by two to three orders of magnitude, although it manifested low stability [ ] . thus, the stem region on ha that is targeted by both arbidol and tbhq represents a promising target for future influenza antiviral development. over the past decade, structural characterization of bnabs to the ha have stimulated antiviral design, ranging from small protein binders [ ] [ ] [ ] [ ] to peptides [ ] to small molecules [ , ] . in addition, the discovery and characterization of bnabs to ha have reignited aspirations and novel approaches towards a more universal influenza vaccine [ ] . while universal influenza vaccine design has largely been focused on the stem domain [ ] [ ] [ ] [ ] [ ] , our recent study demonstrated the need to consider the potential for escape mutations to stem bnabs, which can more rapidly emerge in the h subtype compared to other subtypes, such as h n [ ] . indeed, some escape mutations have already been observed in low frequency in naturally circulating strains. to escape stem bnabs, mutations can either decrease binding of stem bnabs or enhance ha fusion ability [ ] . similarly, escape mutants to rbs bnabs can be even more readily isolated [ , ] . notwithstanding, studies in zika virus, ebola virus, hbv, and sars-cov- have shown that use of a well-designed antibody cocktail can minimize the emergence of escape mutants [ ] [ ] [ ] [ ] . thus, a universal influenza vaccine may need to induce a polyclonal response that targets both the rbs and stem domain to prevent or mitigate against escape. the advantage of simultaneously targeting rbs and the stem domain has been demonstrated by a multidomain antibody composed of four physically linked camelid single-domain antibodies-three of which target the stem domain and one the rbs [ ] . this multidomain antibody is able confer "universal" in vivo protection against both influenza a and b viruses [ ] . furthermore, the recent development of a "mosaic" nanoparticle that co-displays has from multiple subtypes provides possibilities to induce such a polyclonal bnab response [ ] . our understanding of ha biology has advanced relentlessly every year since the first ha structure was reported in . however, new unknowns in the structural biology of ha emerge as influenza viruses continues to evolve, new subtypes are found, and new zoonotic viruses enter the human population. for example, accumulation of natural mutations in the ha rbs has revealed unexpected changes in the receptor-binding modes during h n evolution and motivated greater understanding of how the sialic acid receptor can continue to engage to an ever-changing binding site. in addition, discovery of the initial human bnabs to the ha stem has inspired the discovery of new epitopes in the ha targeted by different families of bnabs. the elucidation of how bnabs target neutralizing epitopes on the ha has further galvanized efforts to design a variety of different classes of therapeutic candidates against the ha [ ] [ ] [ ] , ] . such therapeutics could prevent influenza entry and infection compared to ameliorating infection as for drugs like tamiflu and relenza [ ] . recent advances in cryo-em have greatly complemented x-ray crystallography and enhanced the ability to investigate full-length ha embedded in micelles or membranes [ , ] . thus, many of the new as well as perennial unanswered questions can now begin to be addressed. in addition, the neuraminidase (na) is also undergoing its own reincarnation from the initial antibody work in the s [ ] and first structures in the s [ ] [ ] [ ] . na has been a neglected target on influenza virus [ ] but is now undergoing a renaissance for vaccine design [ ] . structural biology of ha, as well as na, will therefore remain a key component of influenza research until influenza virus ceases to be a global health concern, which is not yet on the horizon. we have experienced the wrath of the sars-cov- pandemic in and do not want to also experience an influenza pandemic like h n . thus, effective utilization of the available and emerging structural information on ha and na needs not only to continue to be developed but put into practice through licensed universal vaccines and therapeutics. host and viral determinants of influenza a virus species specificity reappearance and global 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hemagglutinin elicitation of broadly protective immunity to influenza by multivalent hemagglutinin nanoparticle vaccines neuraminidase inhibitors for preventing and treating influenza in healthy adults and children. cochrane database syst reactions of antibodies with surface antigens of influenza virus structure of the influenza virus glycoprotein antigen neuraminidase at . a resolution structure of the catalytic and antigenic sites in influenza virus neuraminidase three-dimensional structure of a complex of antibody with influenza virus neuraminidase broadly protective human antibodies that target the active site of influenza virus neuraminidase we thank the two anonymous reviewers for providing insightful comments. the authors declare no conflict of interest. key: cord- -f htekhz authors: yu, meiling; wang, li; ma, sunting; wang, xiaona; wang, yusai; xiao, ya; jiang, yanping; qiao, xinyuan; tang, lijie; xu, yigang; li, yijing title: immunogenicity of egfp-marked recombinant lactobacillus casei against transmissible gastroenteritis virus and porcine epidemic diarrhea virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: f htekhz porcine transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) are the causative agents of highly fatal acute diarrhea in pigs, resulting in enormous losses in the pig industry worldwide. to develop an effective bivalent oral vaccine against tgev and pedv infection, the d antigenic site of the tgev spike (s) protein and the major antigen site (core neutralizing epitope—coe) of the pedv s protein were used as immunogens, and the enhanced green fluorescent protein (egfp) gene was used as a reporter to construct genetically engineered lactobacillus casei rlppg(f)-t g -egfp- d-coe. the expression of proteins of interest by the recombinant l. casei was confirmed by confocal laser scanning microscopy and a western blot assay, and the immunogenicity of rlppg(f)-t g -egfp- d-coe in orally immunized mice was evaluated. the results showed that levels of anti-pedv and anti-tgev serum immunoglobulin g (igg) and mucosal secreted immunoglobulin a (siga) antibodies obtained from the mice immunized with rlppg(f)-t g -egfp- d-coe, as well as the proliferation levels of lymphocytes, were significantly higher than those in mice orally administered phosphate-buffered saline (pbs) or rlppg-t g . moreover, the serum igg antibodies showed neutralizing effects against pedv and tgev. our data suggest that the antibiotic resistance-free genetically engineered l. casei bivalent oral vaccine provides a safe and promising strategy for vaccine development against pedv and tgev. larger-scale outbreak in the united states in . herds vaccinated with the cv -inactivated vaccine were also infected, resulting in tremendous losses to the swine industry [ ] [ ] [ ] . accumulating evidence indicates that this large-scale recurrence of ped was caused by highly virulent pedv variants [ ] [ ] [ ] [ ] . in addition, co-infection of tgev and pedv often causes higher morbidity and mortality in newborn piglets. therefore, the development of a safe and highly efficient vaccine against tge and ped would be of great importance. it is now clear that the gastrointestinal mucosas are the primary sites of pedv and tgev infection [ , ] and that mucosal immunization is a promising way to prevent pedv and tgev infection. however, the existing live attenuated/inactivated vaccines administered parenterally cannot effectively induce mucosal immunity against pedv and tgev infection [ ] [ ] [ ] [ ] . although some vaccines for tge and ped developed in china can effectively stimulate intestinal mucosal immunity through houhai acupoint injection, this immunization procedure is more time-consuming and labor-intensive. therefore, vaccines that can induce a mucosal immune response against pedv and tgev would be of great significance, in particular those demonstrated to be safe, inexpensive, easy to use, and effective. the spike (s) glycoproteins of tgev and pedv possess antigen epitopes, and previous reports have demonstrated that the d antigen site (amino acids - ) in the s protein of tgev and the core neutralizing epitope (coe; amino acids - ) in the s protein of pedv can elicit neutralizing antibodies against tgev and pedv infection, respectively. this suggests that these are promising candidate antigens for the development of a genetically engineered vaccine [ , , ] . lactobacillus casei (l. casei) is a potential delivery vehicle for oral vaccines, because it is a probiotic bacterium characterized by its safety and resistance to gastric acid and bile [ , ] . previous reports have shown that recombinant l. casei live vaccine is able to colonize the murine intestines for five days [ ] and the swine intestines for longer [ ] . moreover, we have previously constructed a recombinant l. casei live vaccine expressing the d antigen site of the tgev s glycoprotein combined with muramyl dipeptide and tuftsin as adjuvants, suggesting the possibility of a promising oral vaccine against tgev challenge [ ] . however, plasmid-mediated antibiotic resistance is commonly used as a selective marker for genetically engineered bacteria [ ] [ ] [ ] . this could result in potential biosafety issues due to the transfer of antibiotic resistance from genetically engineered bacteria to environmental pathogens. enhanced green fluorescent protein (egfp) is a luminescent jellyfish protein with the amino acid substitutions necessary to generate a strong fluorescence signal when excited by ultraviolet or blue light. it is widely used in biological research, including in studies of cell differentiation, gene tracking, and protein localization and operation in vivo [ , ] , providing a potential candidate for a screening marker to replace antibiotic resistance. in this study, a genetically engineered l. casei strain, rlppg f -t g -egfp- d-coe, was constructed using the tgev s protein d antigen site and pedv s protein-neutralizing antigen epitope region coe as immunogens, l. casei as an antigen delivery vehicle, and egfp as a selective marker, combined with a constitutive expression plasmid, ppg-t g . the immunogenicity of this strain when orally administered in mice was evaluated, suggesting a potential approach for the prevention of tgev and pedv infection. all applicable international and national guidelines for the care and use of animals were followed. approval ( nefu- , april ) was obtained from the institutional committee of northeast agricultural university for the animal experiments. tgev strain th and pedv strain hlj- were isolated by our laboratory from pedv/tgev-positive samples collected from pig farms in which a severe outbreak of acute diarrhea had been reported in piglets [ , ] . l. casei atcc was kindly provided by jos seegers (nizo institute, netherlands). african green monkey kidney cells (vero cells; atcc ccl- ) and swine testicle (st) cells were purchased from the china center for culture collection (wuhan, china) and were cultured in dulbecco's modified eagle medium (dmem; gibco, gaithersburg, md, usa) supplemented with % fetal bovine serum (fbs; gibco) at • c with % co . the details of all plasmids used in this study are listed in table . the primers used for amplifying genes encoding d (a peptide of the the d antigenic site of the tgev spike (s) protein was repeated six times), coe, and egfp are listed in table . the linker sequence (ggggs) was added in primers fs and re. a schematic diagram of the dna plasmid construction is shown in figure . in brief, the gene encoding d was inserted into plasmid pmd t-coe at saci and mlui sites, generating plasmid pmd t- d-coe; then, the fusion dna fragment d-coe, obtained from pmd t- d-coe by saci and apai digestion, was inserted into the corresponding sites of plasmid ppg-t g , generating recombinant plasmid ppg-t g - d-coe. next, the gene encoding egfp was inserted into ppg-t g - d-coe at saci and kpni sites, generating recombinant plasmid ppg-t g -egfp- d-coe; finally, a chloramphenicol resistance (cm r ) gene selective marker in ppg-t g -egfp- d-coe was deleted using restriction enzymes stui and ncoi, followed by blunt-end treatment and ligation, giving rise to recombinant plasmid ppg f -t g -egfp- d-coe. all recombinant plasmids were identified by restriction enzyme digestion and sequencing. for construction of the recombinant lactobacillus strain, l. casei competent cells were prepared according to a method previously described [ ] , followed by electroporation. briefly, ng of recombinant plasmid ppg f -t g -egfp- d-coe was gently mixed with µl of l. casei competent cells at • c for min; then, the mixture was transferred into a pre-cooled gene pulser (bio-rad, hercules, ca, usa) disposable cuvette (inter-electrode distance of . cm) and subjected to a single electric pulse ( . v; Ω; µf) with a gene pulser (bio-rad). after growth at • c for h, the recombinant lactobacillus strain with green fluorescence signal was collected through flow cytometry using a facscalibur (bd biosciences, san diego, ca, usa) at nm and was grown on an de man-rogosa-sharpe (mrs) plate at • c for h. this was followed by pcr confirmation and a chloramphenicol sensitivity assay, giving rise to recombinant strain rlppg f -t g -egfp- d-coe. the hereditary stability of recombinant l. casei strains was detected, and rlppg f -t g -egfp- d-coe was analyzed for stability by serially transferring the cultures after h of incubation into mrs medium at • c ( % inoculum; generations). plasmids were extracted from the cells, and pcr was used to confirm the presence of the gene egfp- d-coe in the plasmid using the primers fe and rs . plasmids ppg-t g - d-coe and ppg f -t g -egfp- d-coe were generated according to the steps indicated by the arrows. ① the gene encoding d was inserted into plasmid pmd -t-coe at saci and mlui sites, generating plasmid pmd -t- d-coe; ② fusion dna fragment d-coe, obtained from pmd -t- d-coe by saci and apai digestion, was inserted into the corresponding sites of plasmid ppg-t g , generating recombinant plasmid ppg-t g - d-coe; ③ the gene encoding enhanced green fluorescent protein (egfp) was inserted into ppg-t g - d-coe at saci and kpni sites, generating recombinant plasmid ppg-t g -egfp- d-coe; ④ the chloramphenicol resistance (cm r ) gene as a selective marker in ppg-t g -egfp- d-coe was deleted using restriction enzymes stui and ncoi, followed by blunt-end treatment and ligation, giving rise to recombinant plasmid ppg f -t g -egfp- d-coe. for analysis of the expression of proteins of interest by recombinant strains rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe, the bacterial strains were grown in basal mrs broth at °c for h (optical density of sample measured at a wavelength of nm (od ) ≈ . ) without shaking and were harvested by centrifugation at , × g for min. cells were washed twice with sterile phosphate-buffered saline (pbs; ph . ) and lysed using a mini-beadbeater (biospec, bartlesville, ok, usa). after centrifugation, the same quantity of total protein in the supernatants of each sample was isolated by sodium dodecyl sulfate % polyacrylamide gel figure . schematic drawing of the construction of dna plasmids. plasmids ppg-t g - d-coe and ppg f -t g -egfp- d-coe were generated according to the steps indicated by the arrows. the gene encoding d was inserted into plasmid pmd -t-coe at saci and mlui sites, generating plasmid pmd -t- d-coe; fusion dna fragment d-coe, obtained from pmd -t- d-coe by saci and apai digestion, was inserted into the corresponding sites of plasmid ppg-t g , generating recombinant plasmid ppg-t g - d-coe; the gene encoding enhanced green fluorescent protein (egfp) was inserted into ppg-t g - d-coe at saci and kpni sites, generating recombinant plasmid ppg-t g -egfp- d-coe; the chloramphenicol resistance (cm r ) gene as a selective marker in ppg-t g -egfp- d-coe was deleted using restriction enzymes stui and ncoi, followed by blunt-end treatment and ligation, giving rise to recombinant plasmid ppg f -t g -egfp- d-coe. for analysis of the expression of proteins of interest by recombinant strains rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe, the bacterial strains were grown in basal mrs broth at • c for h (optical density of sample measured at a wavelength of nm (od ) ≈ . ) without shaking and were harvested by centrifugation at , × g for min. cells were washed twice with sterile phosphate-buffered saline (pbs; ph . ) and lysed using a mini-beadbeater (biospec, bartlesville, ok, usa). after centrifugation, the same quantity of total protein in the supernatants of each sample was isolated by sodium dodecyl sulfate % polyacrylamide gel electrophoresis (sds-page) and was transferred onto polyvinylidene fluoride membranes, followed by development with mouse anti- d monoclonal antibody/rabbit anti-coe polyclonal antibody (diluted at : ) prepared in our lab, or mouse anti-egfp monoclonal antibody (zsgb-bio, beijing, china; diluted at : ). horseradish peroxidase (hrp)-conjugated goat anti-mouse/rabbit igg antibody (sigma, st. louis, mo, usa) was utilized as a secondary antibody, diluted at : . immunoblots were visualized with chemiluminescent substrate reagent (pierce, rockford, il, usa) according to the manufacturer's instructions. laser confocal microscopy was used to confirm the expression of fusion protein egfp- d-coe on the surface of rlppg f -t g -egfp- d-coe using rlppg-t g - d-coe as a control. briefly, recombinant strains were cultured in mrs medium at • c for h; then ml of culture was collected by centrifugation at × g for min. the pellets were washed three times with pbs, re-suspended in ml of pbs, and smeared on a microscope slide. images were viewed by laser confocal microscopy (zeiss, oberkochen, germany). in order to evaluate the immunogenicity of recombinant strain rlppg f -t g -egfp- d-coe used as an oral vaccine, -week-old female specific pathogen-free (spf) balb/c mice (derived from mus musculus) (n = ) were obtained from liaoning changsheng biotechnology co., ltd. (liaoning, china) and kept under spf conditions for one week with free access to a standard chow diet and water, in accordance with institutional guidelines. prior to oral administration, the recombinant lactobacillus strains were cultured for h in mrs medium without shaking, washed with sterile pbs, and re-suspended in pbs at a concentration of cfu ml − . spf balb/c mice were randomly divided into four groups ( mice per group): pbs, rlppg-t g , rlppg-t g - d-coe, and rlppg f -t g -egfp- d-coe. the immunization dosages are shown in table . the mice were immunized once a day for consecutive days and boosted twice at week intervals. after immunization, serum samples were collected from the immunized mice on days , , , , and , and were stored at − • c until they were required for use. mucosal lavage samples were obtained from the vaginas of the mice by washing with µl of sterile pbs (ph . ) and were stored at − • c until analysis. in addition, fecal samples were collected and treated according to a method previously described [ ] . briefly, a . g fecal pellet was suspended in µl of pbs containing mmol l − phenylmethylsulfonyl fluoride (sigma) and % bovine serum albumin (bsa) and was then incubated at • c for h. after centrifugation, the supernatants were stored at − • c until use. to detect tgev-and pedv-specific antibodies in the collected samples, polystyrene microtiter plates were coated with purified tgev/pedv for h at • c, using cultured st/vero cells as a negative antigen control. after blocking with % skim milk at • c for h and washing three times with pbs- . % tween (pbst), serum and mucus samples serially diluted in pbs- % bsa were added to wells in triplicate, and then plates were incubated for h at • c. after washing with pbst, hrp-conjugated goat anti-mouse igg or iga antibody (invitrogen, carlsbad, ca, usa) was added to each well ( : ) and incubated for an additional h at • c. the substrate o-phenylenediamine dihydrochloride (sigma) was used for color development, and the absorbance was measured at nm. the neutralizing capacities of serum antibodies obtained from the mice immunized with rlppg f -t g -egfp- d-coe were determined. briefly, the % tissue culture infective dose (tcid ) values of tgev and pedv were detected by the reed-muench method. serum antibodies collected from the vaccinated mice on day post-immunization were diluted at : - : (in a total of µl), mixed with an equal volume of pedv or tgev ( tcid per µl) and incubated at • c for h. then, the treated viruses were added to a confluent monolayer of vero and st cells cultured in -well plates. the cells were overlaid with % methylcellulose, and the plates were incubated at • c in a % co atmosphere and examined daily for five days for tgev-and pedv-specific cytopathic effects (cpe). on day post-immunization, splenocytes from three mice from each group were prepared for a lymphocyte proliferation assay as previously described [ ] . briefly, µl of splenocytes ( × cells ml − ) was suspended in roswell park memorial institute (rpmi) medium containing % fetal calf serum and then transferred to a -well flat-bottom plate; the cells were re-stimulated for h with . , . or µg ml − tgev- d protein and pedv-coe protein (produced by escherichia coli prepared in our lab), using . µg ml − concanavalin a (cona) and culture medium as positive and negative controls, respectively. the plates were supplemented with µl of -( , -dimethylthylthiazol- -yl)- , -diphenyltetrazoliumbromide (mtt) per well and incubated for an additional h, and then proliferation was measured using od values. the experiment was carried out in triplicate, and the lymphocyte proliferation index in the spleen (si) was calculated as the mean reading of triplicate antigen stimulation wells divided by the mean reading of triplicate negative control wells. data are shown as the means ± standard errors of three replicates per test in a single experiment repeated three times. tukey's multiple comparison tests were used to analyze differences among the groups. a p-value of < . was considered statistically significant, and p < . was considered highly significant. following flow cytometry screening and growth on antibiotic-free mrs plates, egfp-marked recombinant lactobacillus strain rlppg f -t g -egfp- d-coe was obtained and identified with colony pcr (figure a) . as shown in figure b , the strain rlppg f -t g -egfp- d-coe could not grow on mrs agar medium in the presence of chloramphenicol. the result of the hereditary stability of recombinant l. casei strains showed that rlppg f -t g -egfp- d-coe is stable for more than generations. for confirming the expression of the proteins of interest by the recombinant lactobacillus strain constructed in this study, the cell lysates of recombinant strains rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe were analyzed by a western blot assay. as shown in figure , the expected immunoblot bands of fusion protein pgsa- d-coe of kda expressed by rlppg-t g - d-coe (figure a ,b) and the fusion protein pgsa-egfp- d-coe of kda expressed by rlppg f -t g -egfp- d-coe were observed (figure a-c) , but these proteins were not expressed in rlppg-t g or l. casei. moreover, green fluorescence could be observed by laser confocal microscopy on the surface of rlppg f -t g -egfp- d-coe but not on rlppg-t g - d-coe (figure ) , indicating that egfp could successfully be used as a selective marker. for confirming the expression of the proteins of interest by the recombinant lactobacillus strain constructed in this study, the cell lysates of recombinant strains rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe were analyzed by a western blot assay. as shown in figure , the expected immunoblot bands of fusion protein pgsa- d-coe of kda expressed by rlppg-t g - d-coe (figure a ,b) and the fusion protein pgsa-egfp- d-coe of kda expressed by rlppg f -t g -egfp- d-coe were observed (figure a-c) , but these proteins were not expressed in rlppg-t g or l. casei. moreover, green fluorescence could be observed by laser confocal microscopy on the surface of rlppg f -t g -egfp- d-coe but not on rlppg-t g - d-coe (figure ) , indicating that egfp could successfully be used as a selective marker. for confirming the expression of the proteins of interest by the recombinant lactobacillus strain constructed in this study, the cell lysates of recombinant strains rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe were analyzed by a western blot assay. as shown in figure , the expected immunoblot bands of fusion protein pgsa- d-coe of kda expressed by rlppg-t g - d-coe (figure a ,b) and the fusion protein pgsa-egfp- d-coe of kda expressed by rlppg f -t g -egfp- d-coe were observed (figure a-c) , but these proteins were not expressed in rlppg-t g or l. casei. moreover, green fluorescence could be observed by laser confocal microscopy on the surface of rlppg f -t g -egfp- d-coe but not on rlppg-t g - d-coe (figure ) , indicating that egfp could successfully be used as a selective marker. anti-tgev/pedv-specific secreted iga (siga) and igg antibodies were assessed to evaluate the ability of rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe to induce mucosal and systemic immune responses using balb/c mice as a model. as shown in figure , anti-tgev/pedv-specific antibodies were detected at high levels days post-immunization and were significantly increased after the booster immunization, peaking at days post-immunization. the levels of anti-tgev/pedv-specific mucosal siga in mouse feces and vaginas, as well as the levels of anti-tgev/pedv-specific serum igg antibody in mice orally immunized with rlppg-t g - d-coe/rlppg f -t g -egfp- d-coe, were significantly higher (p < . ) than those in the pbs or rlppg-t g groups. in addition, the levels of anti-tgev/pedv siga in the vaginas of mice orally immunized with rlppg f -t g -egfp- d-coe days post-immunization were significantly higher (p < . ) than those in the rlppg-t g - d-coe group. there was no statistical difference (p > . ) observed in the pbs or rlppg-t g groups before and after immunization. anti-tgev/pedv-specific secreted iga (siga) and igg antibodies were assessed to evaluate the ability of rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe to induce mucosal and systemic immune responses using balb/c mice as a model. as shown in figure , anti-tgev/pedv-specific antibodies were detected at high levels days post-immunization and were significantly increased after the booster immunization, peaking at days post-immunization. the levels of anti-tgev/pedv-specific mucosal siga in mouse feces and vaginas, as well as the levels of anti-tgev/pedv-specific serum igg antibody in mice orally immunized with rlppg-t g - d-coe/rlppg f -t g -egfp- d-coe, were significantly higher (p < . ) than those in the pbs or rlppg-t g groups. in addition, the levels of anti-tgev/pedv siga in the vaginas of mice orally immunized with rlppg f -t g -egfp- d-coe days post-immunization were significantly higher (p < . ) than those in the rlppg-t g - d-coe group. there was no statistical difference (p > . ) observed in the pbs or rlppg-t g groups before and after immunization. the neutralizing capacities of the serum antibodies induced in mice orally immunized with recombinant strains against tgev (figure a ) and pedv (figure b ) showed inhibitory activity against viral infection. the anti-tgev neutralizing antibody titers were − ( : ) and − . ( : . ) and the anti-pedv neutralizing antibody titers were − . ( : . ) and − . ( : . ) in mice immunized with rlppg f -t g -egfp- d-coe and rlppg-t g - d-coe, respectively. these were significantly different from those obtained in the rlppgf-t g and pbs groups (p < . ). bars represent the mean ± standard error of each group. * p < . , ** p < . vs. phosphate-buffered saline (pbs) and vector control groups; # p < . , ## p < . vs. rlppg f -t g -egfp- d-coe group. the neutralizing capacities of the serum antibodies induced in mice orally immunized with recombinant strains against tgev (figure a ) and pedv (figure b ) showed inhibitory activity against viral infection. the anti-tgev neutralizing antibody titers were − ( : ) and − . ( : . ) and the anti-pedv neutralizing antibody titers were − . ( : . ) and − . ( : . ) in mice immunized with rlppg f -t g -egfp- d-coe and rlppg-t g - d-coe, respectively. these were significantly different from those obtained in the rlppgf-t g and pbs groups (p < . ). the proliferation of spleen lymphocytes upon stimulation with purified coe or d proteins was analyzed by an mtt assay. the results showed that the proliferation levels of spleen lymphocytes from mice orally immunized with rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe were significantly higher than those of spleen lymphocytes from mice in the rlppgf-t g and pbs control groups (p < . ). there was no significant difference (p > . ) observed between the rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe groups (figure ). over the past decades, large-scale outbreaks of diarrhea in swine caused by pedv and tgev have occurred in america, europe, and asia, resulting in considerable economic losses to the pig industry; in particular, pedv infection has been responsible for many of these outbreaks [ , ] . the pedv strain hlj- used in this study was isolated from an outbreak of acute diarrhea in piglets in hegang, heilongjiang province, china [ ] , and phylogenetic analysis based on the s gene revealed that hlj- shares high homology with u.s. pedv strains. both belong to the giia subgroup, which represents epidemic and pandemic field strains [ , ] . occasionally, changes in the antigenicity of pedv due to amino acid mutations have resulted in vaccination failure [ , ] . the the proliferation of spleen lymphocytes upon stimulation with purified coe or d proteins was analyzed by an mtt assay. the results showed that the proliferation levels of spleen lymphocytes from mice orally immunized with rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe were significantly higher than those of spleen lymphocytes from mice in the rlppgf-t g and pbs control groups (p < . ). there was no significant difference (p > . ) observed between the rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe groups (figure ) . the proliferation of spleen lymphocytes upon stimulation with purified coe or d proteins was analyzed by an mtt assay. the results showed that the proliferation levels of spleen lymphocytes from mice orally immunized with rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe were significantly higher than those of spleen lymphocytes from mice in the rlppgf-t g and pbs control groups (p < . ). there was no significant difference (p > . ) observed between the rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe groups (figure ). over the past decades, large-scale outbreaks of diarrhea in swine caused by pedv and tgev have occurred in america, europe, and asia, resulting in considerable economic losses to the pig industry; in particular, pedv infection has been responsible for many of these outbreaks [ , ] . the pedv strain hlj- used in this study was isolated from an outbreak of acute diarrhea in piglets in hegang, heilongjiang province, china [ ] , and phylogenetic analysis based on the s gene revealed that hlj- shares high homology with u.s. pedv strains. both belong to the giia subgroup, which represents epidemic and pandemic field strains [ , ] . occasionally, changes in the antigenicity of pedv due to amino acid mutations have resulted in vaccination failure [ , ] . the over the past decades, large-scale outbreaks of diarrhea in swine caused by pedv and tgev have occurred in america, europe, and asia, resulting in considerable economic losses to the pig industry; in particular, pedv infection has been responsible for many of these outbreaks [ , ] . the pedv strain hlj- used in this study was isolated from an outbreak of acute diarrhea in piglets in hegang, heilongjiang province, china [ ] , and phylogenetic analysis based on the s gene revealed that hlj- shares high homology with u.s. pedv strains. both belong to the giia subgroup, which represents epidemic and pandemic field strains [ , ] . occasionally, changes in the antigenicity of pedv due to amino acid mutations have resulted in vaccination failure [ , ] . the s protein neutralizing antigenic epitopes of pedv have been reported to provide cross-protection against variant strains [ , ] . therefore, we selected the coe of the pedv s protein as an immunogen for developing an effective vaccine against pedv infection. pedv and tgev infections initially occur on mucosal surfaces, especially the intestinal mucosal epithelial surface. therefore, mucosal vaccination is an effective strategy for preventing viral diarrheal diseases [ ] . in this study, we used l. casei to deliver the tgev s protein protective d antigenic site (repeated six times) and the pedv s protein coe for developing an oral mucosal vaccine against pedv and tgev. our results suggested that the genetically engineered l. casei strain rlppg f -t g -egfp- d-coe can be used as a bivalent oral vaccine for pedv and tgev, eliciting mucosal and humoral immune responses against both tgev and pedv via oral immunization. this was evidenced by significantly higher levels of virus-neutralizing antibodies, anti-pedv/tgev serum igg, and mucosal siga in mice orally immunized with rlppg f -t g -egfp- d-coe, compared to the levels for the rlppg-t g or pbs groups. moreover, the genetically engineered l. casei vaccine is safe and easy to administer, making it practical and convenient. mucosal immunity plays an important role in preventing viral diarrheal diseases, and siga antibodies from durable lactogenic immunity are a good way for piglets to obtain passive immunoprotection, indicating the importance of the siga antibody in the control of viral infection. at the same time, the level of siga can reflect the status of viral infection and the protective efficacy of vaccines [ ] . in the present study, anti-tgev/pedv siga antibodies could be effectively induced at high levels in the feces and vaginas of mice orally treated with rlppg f -t g -egfp- d-coe. iga is reported to peak at weeks and decline at weeks in piglets [ ] ; thus, if durable immunity was present at days post-immunization, newborn pigs could obtain immune protection. therefore, we assessed the levels of the siga antibody for days post-immunization. our data showed that the levels of siga in the feces of mice orally immunized with rlppg f -t g -egfp- d-coe gradually increased and peaked at , and days post-immunization, indicating that a mucosal immune response can be effectively elicited by rlppg f -t g -egfp- d-coe after oral immunization. therefore, the developed vaccine provides a promising strategy for protecting piglets from pedv and tgev infection via oral immunization. moreover, the neutralizing activity of an antibody is an important index used for evaluating the immunoprotective efficacy of a vaccine. in this study, high levels of serum antibody were elicited following oral immunization with rlppg f -t g -egfp- d-coe. this could effectively neutralize the pedv/tgev infection, and higher antibody titers reflected higher neutralizing activities. as a comparison, the neutralizing antibody titer induced by the recombinant l. casei oral vaccine developed in this study was higher than that induced by a previously developed dna vaccine [ ] . therefore, oral immunization with genetically engineered l. casei strain rlppg f -t g -egfp- d-coe may provide effective protection for piglets against pedv and tgev infection. in addition, our results showed that the egfp-marked recombinant lactobacillus oral vaccine rlppg f -t g -egfp- d-coe (antibiotic-free selective marker vaccine) exhibited a similar immunogenicity to the antibiotic resistance marker vaccine rlppg-t g - d-coe, as the levels of antibodies induced by the rlppg f -t g -egfp- d-coe and rlppg-t g - d-coe vaccines were not significantly different (p > . ). notably, the use of egfp as a selective marker in rlppg f -t g -egfp- d-coe would avoid the main disadvantage of traditional plasmid expression systems by eliminating the use of antibiotic resistance genes as selective markers for genetically engineered bacteria [ , , ] . moreover, rlppg f -t g -egfp- d-coe was constructed with a constitutive expression plasmid developed by our lab, exhibiting a significant advantage to inducible gene expression systems that require the use of an inductive agent. additionally, a pgsa-derived anchoring matrix from bacillus subtilis [ , ] was used to express the fusion proteins, which were displayed on the bacterial surface, eliciting good immunogenicity. the improved plasmid expression system used in this study therefore provides a powerful tool for the development of recombinant lactobacillus oral vaccines. in conclusion, an egfp-marked recombinant lactobacillus oral vaccine, rlppg f -t g -egfp- d-coe, was constructed in this study and provides a promising strategy for the development of a bivalent oral vaccine against tgev and pedv infection. further investigations are underway to evaluate the immunogenicity of this vaccine following its oral 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isolation and identification of porcine epidemic diarrhea virus (pedv) strain nj using porcine intestinal epithelial cells physiological and biochemical characteristics of poly gamma-glutamate synthetase complex of bacillus subtilis ter beek, a. rodz and pgsa play intertwined roles in membrane homeostasis of bacillus subtilis and resistance to weak organic acid stress the authors declare no conflict of interest. key: cord- - v oru l authors: bolatti, elisa m.; zorec, tomaž m.; montani, maría e.; hošnjak, lea; chouhy, diego; viarengo, gastón; casal, pablo e.; barquez, rubén m.; poljak, mario; giri, adriana a. title: a preliminary study of the virome of the south american free-tailed bats (tadarida brasiliensis) and identification of two novel mammalian viruses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: v oru l bats provide important ecosystem services as pollinators, seed dispersers, and/or insect controllers, but they have also been found harboring different viruses with zoonotic potential. virome studies in bats distributed in asia, africa, europe, and north america have increased dramatically over the past decade, whereas information on viruses infecting south american species is scarce. we explored the virome of tadarida brasiliensis, an insectivorous new world bat species inhabiting a maternity colony in rosario (argentina), by a metagenomic approach. the analysis of five pooled oral/anal swab samples indicated the presence of different taxonomic viral families infecting a wide range of hosts. by conventional nucleic acid detection techniques and/or bioinformatics approaches, the genomes of two novel viruses were completely covered clustering into the papillomaviridae (tadarida brasiliensis papillomavirus type , tbrapv ) and genomoviridae (tadarida brasiliensis gemykibivirus , tbgkyv ) families. tbrapv is the first papillomavirus type identified in this host and the prototype of a novel genus. tbgkyv is the first genomovirus reported in new world bats and constitutes a new species within the genus gemykibivirus. our findings extend the knowledge about oral/anal viromes of a south american bat species and contribute to understand the evolution and genetic diversity of the novel characterized viruses. bats belong to the order chiroptera, which is the second-largest mammalian group, comprising families and species distributed globally, with the exception of polar areas [ , ] . approximately % of the world's bat species are endangered, causing concerns about the negative conservation impact and its influence on the ecosystem services these bats provide, such as arthropod regulation, seed dispersal, and pollination [ , ] . on the other hand, certain specific aspects of bats-including their relatively long lifespan in relation to their body size [ ] , the reliance of some species on prolonged torpor [ ] , and flight-may make them suitable for hosting a wide variety of viruses [ ] , including zoonotic viruses highly pathogenic to humans [ ] , such as severe acute respiratory syndrome (sars)-related coronavirus, ebola virus, nipah virus, and hendra virus [ ] [ ] [ ] [ ] . nevertheless, little is known about their own pathogens [ ] . in addition, the gregarious behavior of many bat species, such as free-tailed bats tadarida brasiliensis (i. geoffroy saint-hilaire, ), may facilitate rapid transmission of pathogens between bats and other species [ ] . t. brasiliensis is the most abundant migratory and cosmopolitan species of the new world bats, widespread throughout the americas [ ] [ ] [ ] and protected by international agreements [ ] . using next-generation sequencing (ngs) technologies, an enormous variety of viral species and genotypes [ , ] have been identified in the tissues and feces of bats mainly inhabiting asia [ , ] , africa [ , ] , europe [ , ] , and north america [ , ] . on the other hand, the viromes of bats from south america remain understudied [ , ] . for example, the identification of viruses infecting t. brasiliensis is principally limited to detection of specific viral families, such as rabies lyssavirus [ ] , alphacoronaviruses [ ] , polyomaviruses [ ] , circoviruses [ ] , and anelloviruses [ ] . in order to contribute to the preservation of t. brasiliensis and to evaluate its possible role as a pathogen reservoir, greater efforts directed at identifying the viruses present in this species are needed. in this study we report a detailed description of two novel complete genome sequences, one describing a new papillomavirus genus and the other representing a novel variant of an existing gemykibivirus species. in addition, we report a preliminary overview of the t. brasiliensis virome composition. altogether, our findings add to the knowledge of viral diversity in a south american bat species, providing insights for understanding their role as reservoirs, as well as their own pathogens, which may have consequences for the animals' health. the bat colony investigated occupies the attic of the law school building at the universidad nacional de rosario in downtown rosario, argentina ( • . s • . w) [ ] . in this place, t. brasiliensis (molossidae) establishes a maternity colony every year that can reach about , individuals during the maternity season (november to march), after which they migrate [ , ] . a total of swab samples ( oral and anal) were collected from adult female specimens inhabiting this colony from december to february . briefly, bats were manually captured from the walls and held in individual cotton bags for determination of their species based on anatomical and morphological characters, reproductive condition, and age [ ] . the oral cavity and anal regions of each individual were sampled using individual sterile cottontipped swabs (deltalab, barcelona, spain), rolled back and forth ( times), suspended in µl of saline solution (nacl . %), and stored at • c until further processing. the bats were rehydrated and released. during this study, every effort was made to minimize interference with and suffering of the animals; no breeding or pregnant females were captured, and no animals were sacrificed. sample collection was conducted by trained professionals as approved by the ministry of environment of the argentinian santa fe province (file - ) and facultad de ciencias bioquímicas y farmacéuticas (universidad nacional de rosario) animal ethics committee (file / , march ). samples were processed according to previously published protocols that have been successfully applied for identification of papillomavirus (pv) in human skin swab samples [ ] [ ] [ ] . briefly, the cells were centrifuged at , × g for min and the pellets were resuspended in µl te buffer (qiagen, hilden, germany) containing µg of proteinase k (qiagen), and incubated overnight at • c. following proteinase k inactivation ( • c for min), the lysates were stored at − • c. subsequently, the obtained samples were tested for the presence of pv dna using improved versions of fap [ , ] and cut pcrs [ ] , as described previously [ , ] . circular dna molecules in lysates of five selected pv-positive samples (four anal and one oral swab) were enriched using rolling-circle amplification (rca) with the illustra templiphi amplification kit (ge healthcare, chicago, il, usa) [ ] [ ] [ ] . the pool of rca-enriched samples was sequenced on an illumina hiseq instrument at the sequencing facility of gatc biotech (ebersberg, germany). sequencing libraries were prepared using the gatc automatic library preparation approach, and the sequencing reads were sequestered in the format of × bp. reads were subjected to quality trimming and filtering using the bbduk program (bbtools v . ). end trimming was performed on the first and last bases of each read, clipping bases with phred scores below . trimmed reads shorter than bp and with an average phred score below were discarded. the read pairs contained in the metagenomic sample, which shared k-mers, sliding-window subsequences of nt, with the sequencing datasets of samples (six in total) that were processed and analyzed in the same sequencing batch, were discarded using the bbduk program (referred to as laboratory-batch background screen in figure ). the primary purpose of this step was to conservatively limit the possibility of falsely identifying viral taxa that did not originate from the bat metagenomics sample and that could have been introduced by aerosol during sample processing or index hopping during sequencing. in order to limit the content of bacterial reads, the metagenomic dataset was mapped to the bacterial reference-index files (obtained november , from ftp://ftp.ccb.jhu.edu/pub/infphilo/ centrifuge/data/p_compressed.tar.gz) using the centrifuge sequence classification system (centrifuge version . . -beta) [ ] . reads not mapping to any bacterial taxon were used in further metagenomic analyses (unless stated otherwise). two types of metagenomic characterization workflows were used: ( ) taxonomic classification of ngs read pairs and ( ) taxonomic classification of contigs assembled de novo from ngs read pairs. in both cases, the centrifuge metagenomic classification system with the reference nucleic sequence index files, obtained from ftp://ftp.ccb.jhu.edu/pub/infphilo/centrifuge/data/p_compressed+h+v.tar.gz (version june , downloaded november ), was used to obtain the final taxonomic calls (default parameter settings). taxonomic classification of sequences was further summarized to the taxonomic level of family using pavian [ ] . de novo assembly was performed with two different de brujin graph assembly tools: spades (v . ) [ ] and unicycler (obtained from github: october ; github commit: d daebc d f e acb c a ff ), adapting various parameter settings. altogether, six different metagenomic de novo assemblies were constructed, using settings specified in table s . all contigs assembled de novo by any of the six approaches exhibiting a minimum length of nt were collected and subjected to taxonomic classification (workflow ). the circularity of the complete genome assemblies was determined by matching the sequence stretches (minimum match length nt) at their and ends. coverage statistics of the novel complete viral genome sequences were obtained by remapping the trimmed read dataset to the constructed genome assemblies using bowtie (v . . ) [ ] . ( ) and contigs assembled de novo ( ) . pair reads quality filtering and trimming were performed with the bbduk program (bbtools v . ). the centrifuge metagenomics classification system was used for the taxonomic classification of pair reads and contigs (centrifuge version . . -beta) [ ] . de novo assembly was performed using spades v . [ ] and unicycler. ( ) and contigs assembled de novo ( ) . pair reads quality filtering and trimming were performed with the bbduk program (bbtools v . ). the centrifuge metagenomics classification system was used for the taxonomic classification of pair reads and contigs (centrifuge version . . -beta) [ ] . de novo assembly was performed using spades v . [ ] and unicycler. sequences of the e , e , l , and l genes of reference pv genomes, downloaded from pave (http://pave.niaid.nih.gov/ on march ), and the corresponding genes from the novel pv, tadarida brasiliensis papillomavirus type (tbrapv ), were used in the phylogenetic analysis. additional information on nucleotide sequences (genbank accession number and virus name abbreviations) used in these analyses is summarized in table s . the e e l l concatenation was constructed by first obtaining the amino acid-guided multiple sequence alignments of each gene. multiple sequence alignments were obtained using muscle (v . . ) [ ] . as suggested by bernard et al. ( ) [ ] , the multiple sequence alignments used for phylogenetic analysis of tbrapv were guided by the amino acid alignments and the pv identity calculation was based on patristic distance measurements, as determined by seaview [ ] . phylogenetic clustering was conducted using iq-tree [ ] . the most appropriate substitution models were determined using modelfinder [ ] , according to the bayesian information criterion. branch support values were calculated using uf bootstrap ( replicates) [ ] , sh-alrt ( replicates), and abayes tests [ ] . phylogenetic analysis of genomoviridae was conducted using a reference dataset of complete genome nucleotide sequences and rep protein sequences downloaded from ncbi genbank ( march ). the complete genome sequences were rotated to all begin in the start codon of the rep gene using circulator (version: github commit a befb c dbbcd b ad a aa e d b ) [ ] , and the two subunits of the rep gene were concatenated into a single protein sequence in which the genbank record indicated them as parts of different genes/coding sequences. pairwise sequence identity values used for taxonomic classification of tadarida brasiliensis gemykibivirus (tbgkyv ) were obtained using sequence demarcation toolkit (sdt v . ) [ ] . in this scope, the pairwise sequence alignments were produced using muscle (v . . ) [ ] . phylogenetic trees were rendered using figtree (v . . ) (http://tree.bio.ed.ac.uk/software/figtree/), and sequence identity histograms were visualized using gnuplot (v . ). open reading frames (orfs) of novel viral genome sequences were marked using orffinder (ncbi); the manual annotation process of the orfs was guided by the use of ncbi blastp, and the identification of viral-family specific sequence motifs was performed using regular expressions with the linux grep utility (v . ). members of papillomaviridae and genomoviridae identified in bat species so far are summarized in table s . the complete genome sequence of the novel pv type (tbrapv ) was obtained by generating four overlapping amplicons in different pcr reactions, using x pcr buffer, . mm of mgcl , µm of each dntp (thermo fisher, walthem, ma, usa), . u of gotaq hotstart polymerase (promega, madison, wi, usa), and . µm of each of the primers (tbrapv - f -cagggtattcagggtgtttctcc- and tbrapv - r -aatgtttctaatctgcaacc- ; tbrapv - f -gtgcgcggcgacttctcatactta- and tbrapv - r tcagcctcattgtcctcatcattg- ; tbrapv - f -tgggcttgaaacctggacactaca- and tbrapv - r -atgcccgggaa tatggatgga- ; tbrapv - f -ggcctgcaagaccacctac- and tbrapv - r -gggggcatctgacctgtta- ). cycling conditions for the four reactions were the same and were performed as follows: initial denaturation at • c for min, followed by cycles of s at • c, s at • c, and min at • c, with a final extension at • c for min. the amplicons were resolved in a % agarose gel electrophoresis and the~ kb fragments were gel purified, ligated into the pgem-t easy vector (promega), and transformed into e. coli cells. sanger sequencing was performed using sequencing facilities at macrogen inc. (seoul, korea). in august , dna clones and the corresponding nucleotide sequences were subsequently submitted to the animal papillomavirus reference center (http://www.animalpv.org/) for its confirmation and official designation. the genbank/embl/ddbj accession numbers for the novel viruses reported in this paper are tbrapv (mn ) and tbgkyv (mn ). the relevant raw high throughput sequencing data obtained in this study was deposited at the ncbi sequence read archives (sra) with the following accession number: prjna . the contigs, obtained by de novo assembly as part of the metagenomic workflow ( ), have also been made available for download (supplementary data s ). ngs data analysis workflows and centrifuge-based taxonomic assignments of reads and contigs are depicted in figure and table , respectively. briefly, a total of , , read pairs were sequestered from the rca-enriched samples, and , , of them passed the quality filtering and trimming procedures. out of these, , , read pairs were removed during the laboratory-batch background screen and an additional , read pairs were identified as originating from bacteria. the final metagenomic characterization was carried out using the remaining , , read pairs ( figure ). metagenomic analysis revealed that only a small proportion of read pairs ( , read pairs; . %) and de novo assembled contigs longer than nt ( out of total , ; . %) mapped to viral taxa. overall, a large number of phage-related sequences were detected ( . % of viral read pairs and . % of viral contigs), likely representing the most abundant entities infecting bacteria present in the bat digestive system, which exhibited similarity mostly to the families inoviridae, siphoviridae, and myoviridae ( table ). the eukaryotic viral sequences (insect, invertebrate, plant, protist, and vertebrate viruses) could be summarized into a total of viral families, corresponding to viral families with dna genomes, and to families with rna genomes. sequences of different viral families infecting insects and crustaceans, mostly found related to the families baculoviridae, ascoviridae, iridoviridae, and nimaviridae, were detected ( . % of viral read pairs and . % of assembled viral contigs). on the other hand, sequences related to viruses infecting plants (five viral families, . % of viral read pairs, and . % of viral contigs) were mostly associated with phycodnaviridae or potyviridae, whereas those related to viruses infecting protists (five viral families, . % of viral read pairs, . % of viral contigs) clustered predominantly in the family mimiviridae. sequences classified as originating from vertebrates, predominantly mammalian viruses, were represented by . % of viral read pairs and . % of viral contigs. the principal viral families identified included retroviridae, genomoviridae, herpesviridae, papillomaviridae, and poxviridae. the analysis also identified (although in low counts) viral sequences related to the family alloherpesviridae, which infects fish and amphibians. of note, the metagenomic analysis indicated that out of read pairs were assigned to the family retroviridae ( . % of viral read pairs), and assembled contigs ( . % of viral contigs; table ) exhibited resemblance to the nucleotide sequence of desmodus rotundus endogenous retrovirus isolate (genbank accession number: nc_ ) [ ] . however, a more detailed analysis of this sequence revealed the presence of two flanking regions at the and ends of approximately nt, which probably derived from the host genome (desmodus rotundus). in fact, contigs and reads previously classified as retroviridae in our study aligned with these flanking regions. this finding explained the initial misclassification and indicated that great care should be taken when using genbank sequence data as reference material because a large portion of sequences and their respective annotations may not have been curated adequately. finally, a total of read pairs and assembled contigs were classified as similar to viruses unassigned to taxonomical families (table s ). the metagenomic analysis suggested that a total of read pairs (workflow ) and contigs (workflow ) could be attributed to pvs (table ). the longest contig obtained by assembly de novo covered the complete genome of tbrapv , which was subsequently confirmed by conventional molecular methods (pcr, cloning, and sanger sequencing; data not shown). remapping read pairs (quality-, background-, and bacteria-filtered read pairs) to the confirmed tbrapv genome sequence indicated a complete sequence length of nt, with a gc content of %. the complete novel genome sequence was covered on average . × by a total of read pairs ( , reads) . detailed analysis of the tbrapv viral genome (table and figure a) , showed a typical genomic organization of bat pvs, potentially encoding four early genes (e , e , e , and e ) and two late genes (l and l ) [ , ] . a putative e gene was found overlapping the e gene ( figure a) , with its own start and stop codons, and the presence of e -characteristic proline-rich stretches ( . %) with an important role in cell cycle arrest was found [ ] . typical domains were additionally identified in the putative viral proteins encoded by tbrapv . the e protein contained two characteristic zinc-binding domains, separated by amino acids [ ] , and four internal and likely not functional pdz-binding motifs (rtnv, isdl, ssil, lssl) [ ] . the e protein contained a prb-binding motif (lwcde) [ ] and a single zinc-binding domain ( table ) . analysis of the e protein, the largest protein encoded by tbrapv (table ) , showed the typical atpbinding site of the atp-dependent helicase (gpsnsgks) [ ] , and several cdk-phosphorylation and the upstream regulatory region (urr) of tbrapv contained two typical tata boxes, three putative e protein binding sites, an e protein binding site [ ] , and three putative polyadenilation sites for late gene transcripts (table ) . multiple potential binding sites for transcriptional regulatory factors, such as ap- , nf- , and sp- , were also present within the urr (data not shown). typical domains were additionally identified in the putative viral proteins encoded by tbrapv . the e protein contained two characteristic zinc-binding domains, separated by amino acids [ ] , and four internal and likely not functional pdz-binding motifs (rtnv, isdl, ssil, lssl) [ ] . the e protein contained a prb-binding motif (lwcde) [ ] and a single zinc-binding domain ( table ) . analysis of the e protein, the largest protein encoded by tbrapv (table ) , showed the typical atp-binding site of the atp-dependent helicase (gpsnsgks) [ ] , and several cdk-phosphorylation and cyclin-binding sites. a highly conserved bipartite-like nuclear localization signal (nls) and a leucine-rich crm -dependant nuclear export signal (nes) (lspvlekvti), which together allow shuttling of the e protein between the cell nucleus and the cytoplasm in most human pvs [ ] [ ] [ ] , were identified at the n-termini of the e protein. no conserved leucine zipper domain was present at the c-termini of the putative tbrapv e protein, in agreement with other bat pvs (espv and rfpv ) [ ] . at the n-termini of the l protein, a highly conserved furine cleavage motif (rrkr), as well as a transmembrane-like domain (gtggggrgvpigprvatgrpggpinsvg) [ ] , were identified. in addition, a canonical polyadenylation site, necessary for regulation of early viral transcripts [ ] , was also found in the tbrapv l gene (table ) . phylogenetic analysis, based on pv l gene nucleotide sequences, indicated peak sequence identities of tbrapv to hpv and rfpv , which amounted to . and . %, respectively (figure , charts a and a ). maximum likelihood phylogenetic clustering of l sequences (figure ) suggested common ancestry of tbrapv , edpv , and hpv , and that tbrapv branched away prior to the delineation of edpv and hpv , with high sh-alrt, abayes, and uf bootstrap support values. further phylogenetic analyses were conducted based on the concatenated alignments of e , e , l , and l gene nucleotide sequences, and they indicated a peak sequence identity of tbrapv to rfpv ( . %, figure , charts a and a ), whereas the maximum likelihood phylogenetic clustering indicated analogous common ancestry to the concatenated pv genes ( figure ) , with high sh-alrt, abayes, and uf bootstrap support values. the metagenomic analysis indicated the presence of several genomovirus-related read pairs and sequence contigs (table ) , and the complete genome sequence ( nt) of tbgkyv was recovered using de novo assembly. remapping to the tbgkyv genome sequence indicated a mean coverage of . × by a total of read pairs ( paired reads + unpaired reads) and did not reveal any abnormalities that would indicate misassembly. completeness of the circular genome sequence was determined by matching and ends, and the sequence was rotated to begin with the characteristic genomoviridae nonanucleotide motif [ ] . three non-overlapping orfs, with a minimum protein length of aa, were found, exhibiting peak amino acid similarities to the genomoviridae rep and cp/cap proteins. the rep protein of tbgkyv was encoded across two different rep-encoding orfs separated by an intron, representing a catalytic and a central protein domain ( figure b , table ), which is characteristic for replication-associated proteins encoded by single-stranded (cress) dna viruses [ ] . phylogenetic analysis and pairwise sequence comparison demonstrated that tbgkyv shares its highest identity in the rep protein ( . %) and the complete genome level (nucleotide sequence, . %) with the "mongoose associated gemykibivirus " (genbank accession number kp ) [ ] ( figures b and ) . nucleic sequence identity histograms of the pv nucleotide sequences (a and a ) and tbrapv (a and a ) based on the concatenated e , e , l , and l gene sequences (a and a ) and on the l gene sequence (a and a ). multiple sequence alignments were constructed using muscle (v . . ) [ ] , and the distance matrices were estimated using seaview v . [ ] , as suggested in bernard et al. ( ) . red arrows indicate the maximum sequence similarities of tbrapv for each of the sequence contexts (e , e , l , and l concatenation and the l gene sequence). the blue arrows (a and a ) indicate the overall maximum sequence identity in the depicted context (e , e , l , and l concatenation and the l gene sequence). the histograms were visualized using gnuplot (v ). (b) pairwise sequence identity histograms based on complete genome nucleotide (purple) and rep gene (green) amino acid sequences from the genomoviridae family, based on the entire pairwise identity distance matrices (b ) and on the matrix slices representing pairwise identities of tbgkyv to all other genomoviridae sequences (b ). the pairwise similarity matrices were obtained through pairwise sequence alignments (muscle v . . ) [ ] , using sequence demarcation toolkit (sdt v ) [ ] . for the arrows in the histogram: red = the species demarcation threshold/criteria (sdc) for genomoviridae [ ] ; green and blue = maximum pairwise identity values of tbgkyv for the complete genome (green) and the rep protein sequence contexts (blue). histograms were visualized using gnuplot (v ). **nt = nucleotide, aa = amino acid, scd = sequence demarcation threshold/criteria [ ] . cg: complete genome. nucleic sequence identity histograms of the pv nucleotide sequences (a and a ) and tbrapv (a and a ) based on the concatenated e , e , l , and l gene sequences (a and a ) and on the l gene sequence (a and a ). multiple sequence alignments were constructed using muscle (v . . ) [ ] , and the distance matrices were estimated using seaview v . [ ] , as suggested in bernard et al. ( ) . red arrows indicate the maximum sequence similarities of tbrapv for each of the sequence contexts (e , e , l , and l concatenation and the l gene sequence). the blue arrows (a and a ) indicate the overall maximum sequence identity in the depicted context (e , e , l , and l concatenation and the l gene sequence). the histograms were visualized using gnuplot (v ). (b) pairwise sequence identity histograms based on complete genome nucleotide (purple) and rep gene (green) amino acid sequences from the genomoviridae family, based on the entire pairwise identity distance matrices (b ) and on the matrix slices representing pairwise identities of tbgkyv to all other genomoviridae sequences (b ). the pairwise similarity matrices were obtained through pairwise sequence alignments (muscle v . . ) [ ] , using sequence demarcation toolkit (sdt v ) [ ] . for the arrows in the histogram: red = the species demarcation threshold/criteria (sdc) for genomoviridae [ ] ; green and blue = maximum pairwise identity values of tbgkyv for the complete genome (green) and the rep protein sequence contexts (blue). histograms were visualized using gnuplot (v ). nt = nucleotide, aa = amino acid, scd = sequence demarcation threshold/criteria [ ] . cg: complete genome. figure . phylogenetic tree of the genomovirus rep amino acid sequence. the tree was constructed using the lg+i+g substitution model, and branches are annotated with sh-alrt ( replicates), abayes, and uf bootstrap support ( replicates) values, respectively. maximum support values are shown with asterisks (*). novel tbgkyv is depicted in bold. genomoviridae genera were classified according to varsani and krupovic ( ) . unclassified sequences were not depicted. table and figure b . the large intergenic region of the novel virus contains the characteristic nonanucleotide (tataaatag) motif, which is likely to be important for rolling-circle replication initiation [ ] [ ] [ ] . the rep protein's catalytic domain of tbgkyv contained rolling circle replication motif i (lftysq), possibly involved in the recognition of iterative dna sequences associated with the origin of replication [ ] , motif ii (thlhv), which may regulate the nicking/joining endonuclease activity at the origin of dna replication [ , ] , motif iii (yatk), involved in the double-strand dna cleavage [ , ] , and a geminivirus rep protein sequence (grs) (rlfdvenfhpnivpsr), which allows appropriate spatial arrangements of motifs ii and iii [ ] . furthermore, rep helicase motifs walker-a (gpsrtgkt), walker-b (vfddi), and walker-c (wlmn) [ ] [ ] [ ] [ ] were identified in the central rep protein domain. walker motifs contribute to atp binding, which is used as an energy source to unwind the dsdna intermediate in the - direction by the rep helicase [ , ] . more than viruses from taxonomic families have been isolated from or detected in bats so far [ ] , with a few of them implicated in the etiology of several severe diseases in humans. except for rabies, no direct evidence of zoonotic diseases transmitted by new world bats has been found [ ] . new world, especially south american, and old world bat species had different evolutionary histories, leading to distinct immunological features [ , ] . viruses infecting south american bat species have been poorly studied [ , ] , and further research focused on evaluating their viromes is required. here, a first attempt to assess to the virome composition in pooled oral and anal swabs of t. brasiliensis was presented. during our study a total of , , read pairs were removed during the laboratory batchbackground screening, due to traces of sequences from co-processed samples. the power of ngs stems from non-specific sampling of nucleic acids and automated repetition, yielding vast numbers of sequencing reads, providing the opportunity to characterize populations of nucleic acids with unprecedented sensitivity, accuracy, and non-specificity. due to its super-sensitivity, even the slightest addition of environmental nucleic acids to a sample may be detected using ngs and can potentially further complicate the interpretation of the results. laboratory background and/or dna isolation kit-derived contamination has been addressed previously as a major factor that can severely impede the interpretation of high throughput sequencing data, and the use of negative controls has been proposed [ ] . moreover, positive/negative control samples have been recommended in metagenomic experiments aimed at detecting pathogens in clinical samples [ ] . in this study, the metagenomic sample was processed alongside six samples of molluscum contagiosum skin lesions, and laboratory background filtering was initially considered due to the detection of approximately molluscum contagiosum virus read pairs in the trimmed read dataset. in order to prevent the identification of human skin microflora in the pooled bat swab sample, a strict k-mer based negative filtering was used, effectively removing any read pair that contained at least one bp subsequence that could be identified in any of the read pairs from the six background datasets. because the background samples also originated from mammalian (human) skin, it could be that a large portion of mammalian reads were removed during this step, explaining the somewhat extreme number of read pairs classified as laboratory-batch background. moreover, it is also likely that the viral composition of human and bat skin could be shared to some degree, but due to the filtering scheme reads originating from bats' anal and oral microflora sharing nucleotide sequence similarity to that of human skin may have also been removed prior to metagenomic classification. as a consequence, taken strictly, only the subset of viruses that are present in t. brasiliensis, but not in human skin, was explored here. although dna spillovers could be suspected and controlled for to some degree, in this case it may be a greater challenge in studies where multiple samples from different species or anatomical sites of bats are processed. in light of the present results, it would likely be beneficial to process the samples in such studies as independently as possible and to include negative controls that would characterize the laboratory background, such as sequencing libraries of buffer solutions that underwent the same treatment as the samples, as suggested previously [ , ] . specifically, a total of , virus-related read pairs ( . % out of , , ) and virus-related contigs ( . % out of , ) were assembled de novo, mapped to viral taxa, and identified by ngs. although the proportion (and number) of virus-related sequences detected in this study (< %) is comparable to reports of previous studies of bat viromes based on illumina sequencing [ , , ] , it may be that additional physical viral dna/rna enrichment steps, such as centrifugation, filtration, and/or nuclease-treatment, could further augment the viral read yields, as suggested previously [ ] . initially, this study was focused on the identification of pvs in t. brasiliensis, in order to explore their diversity in different hosts. accordingly, swab samples included in this work were first processed using experimental protocols, designed previously to suit our aforementioned initial aim [ ] [ ] [ ] . viral dna was enriched using rca, as suggested previously by others [ ] [ ] [ ] . however, it should be noted that rca may have favorably facilitated the amplification of circular genomes and, as a consequence, hindered the detection of linear genomes. thus, more than % of the classified viral sequences ( , / , ) identified in our analysis corresponded to circular viral genomes. in this study about . % of viral reads and % of contigs corresponded to sequences from eukaryotic viral families, mostly with dna genomes. interestingly, virus-associated sequences from rna viruses belonging to families were also detected, most likely reflecting the presence of traces of viral rna. limited but highly accurate reverse transcriptase activity has indeed previously been reported for the phi dna polymerase, used in rca [ ] . most of the insect-infecting viral sequences detected belonged to viral families infecting lepidopteran adults or larvae, which may represent the diet of t. brasiliensis, as detected previously in feces and anal swabs from various insectivorous bat species (myotis sp., rhinolophus sp., molossus sp., neoromicia sp.), including t. brasiliensis [ , , , ] . in addition, the detection of various plant viral families in this study could reflect the plant diet of the insects ingested by the bats. a total of different mammalian viral families were identified in t. brasiliensis samples, representing approximately % ( / ) of the eukaryotic viral families interrogated herein. several mammalian viral families, supported by the contigs and sequencing reads, have been identified previously in new world [ , , ] and old world [ , , ] bat species. the mammalian viral families identified in t. brasiliensis included typical zoonotic viruses identified previously in bats, such as polyomaviridae [ ] , rhabdoviridae [ ] , coronaviridae [ , ] , poxviridae, flaviviridae, and adenoviridae [ ] . the identification of circoviridae and astroviridae in t. brasiliensis was also in line with the results of previous studies [ , ] . on the other hand, this study indicated the presence of genomoviridae, alloherpesviridae, papillomaviridae, herpesviridae, paramyxoviridae, and reoviridae in t. brasiliensis for the first time. notably, the presence of incorrect annotations in public databases, such as the sequences assigned to the retroviridae family in this study, highlight the need for the curation of data (whenever possible) to avoid the under-and/or overestimation of the classified sequences derived from metagenomics studies. pvs are a highly diverse family of non-enveloped and double-stranded circular dna viruses that are known to infect a wide variety of mammals, as well as birds, reptiles, and fish [ , ] . various human and non-human pvs, including bat pvs, have frequently been identified in healthy epithelia and may represent part of the native epithelial microflora [ , , ] . several studies have suggested the presence of pvs in old world bat species using conventional [ , , ] or ngs aproches [ , , , ] . the only pv type (mmopv ) identified in a new world bat species (molossus molossus) has recently been described [ ] , suggesting a crude sampling imbalance and a severe lack of information to elucidate the evolutionary mechanisms driving pv diversification on the global scale. in this study, tbrapv has been identified in pooled oral and anal swabs of t. brasiliensis by ngs, and its sequence has been completely characterized by conventional molecular techniques. tbrapv is the first reported pv type found in t. brasiliensis and the second pv type identified in new world bat species. according to the current ictv papillomaviridae classification guidelines (published in june ), based on the nucleotide sequence of the l gene [ ] , tbrapv is the founding member of a novel pv genus in the firstpapillomavirinae taxonomical subfamily, sharing more than % sequence identity to other pv types included in this subfamily. although nucleotide sequences analysis in the l gene indicated that tbrapv shares a . % identity with hpv (nu-pv) and should be included within the same genus (more than % of nucleotide identity in l gene) [ ] , the mentioned demarcation criteria suggests a visual inspection of phylogenetic trees derived from concatenated e , e , l , and l nucleotide sequences to delineate pv genera [ ] . in the present study, such analysis clustered tbrapv basal to the delineation of hpv (nu-pv) and edpv (sigma-pv), identified in a north american porcupine (erethizon dorsatum) and, therefore, may represent a novel genus within the papillomaviridae family. this phylogenetic clustering also indicated that tbrapv shares common ancestry with other bat pvs such as espv , espv , rfpv , ehpv , and mscpv . on the other hand, tbrapv is only distantly related to rapv , ehpv , ehpv , espv , mscpv , and mrpv , which have been identified from different tissues and bats species. the idea that different bat pvs evolved during a process of strict host coevolution is further refuted by the observation that different bat pvs appear scattered around the papillomaviridae phylogenetic tree in a highly polyphyletic manner [ , ] . in addition, under strict host coevolution it would be expected for tbrapv and mmopv , both molossid pvs, to be closely related; nevertheless, mmopv has a basal taxonomic position with respect to tbrapv . these observations support the idea of multiple evolutionary forces as drivers of pv evolution, including coevolution, adaptive radiation, broad host range, host switch, and recombination [ ] . genomoviruses are single-stranded circular dna viruses that belong to the recently proposed genomoviridae family [ ] . members of this family encode two genes-the cap/cp and the rolling-circle replication-associated protein (rep)-and an intergenic region. it has been proposed that a novel viral complete genome sequence of the same species exhibits more than % similarity to any other complete genomovirus genome [ ] . in addition, in previous studies, the authors aimed to establish nine genera within the family genomoviridae based on pairwise comparisons of complete genome sequences [ ] . cress dna viruses, including genomoviruses, have been found in association with a great variety of animal species, such as camels [ ] , bats [ ] , mongooses, badgers [ ] , wolves [ ] , pigs [ ] , and humans [ ] , as well as in environmental-associated [ ] and plant-associated [ ] samples. however, no direct implication with a disease has been demonstrated so far. in particular, bat-associated genomoviruses have been identified from feces [ , ] or pharyngeal and anal swab samples [ ] of asiatic [ , ] or european [ ] bat species and have been attributed to various taxonomic genera of the genomoviridae family [ ] . tbgkyv is a novel species within the gemykibivirus genus according to the classification criteria [ ] . it should be noted that the rep and cap proteins of tbgkyv exhibited different percentages of similarity, the cap being considerably more divergent than the rep, indicating differences in their evolutionary histories due to their respective molecular functions [ , ] . to the best of our knowledge, this is the first report demonstrating the presence of genomoviral sequences in mucosal swab samples of a new world bat species. finally, it is worth noting that the results of our metagenomic screening of the pooled t. brasiliensis oral and anal swab samples is effectively provided at three different levels of specificity/sensitivity. the two complete genome sequences (tbrapv and tbgkyv ), that have been described at the highest level of detail, also confer the highest level of confidence. in other words, we have complete confidence that these two viruses were present in the pooled nucleic acid samples. assigning sequences that did not assemble at the level of complete viral genomes to taxonomical families could therefore be a valid approach offering a higher level of sensitivity than only the complete genome assemblies, but at the cost of diminished specificity. in addition, identifying a sequence fragment that resembles a known viral genome in a given genomic region, may not always be sufficient to infer that that specific virus was present in the sample. viruses are highly promiscuous entities, which can easily exchange parts of their genomes with their hosts, be integrated or even naturalized into the host genomes [ ] . taxonomical viral families identified among the assembled contigs could be interpreted as viral families that were probably represented in our samples. lastly, and due to the low number of assembled contigs, that were found related to known viral sequences, we attempted to increase the sensitivity even further by obtaining taxonomical family mappings also for the source read pairs. these results, however, should be interpreted with utmost caution, because they likely confer a very low level of specificity due to the limited sequence length ( × bp). taxonomical viral families identified by read-pair taxonomy mapping only, merely suggest the possibility that these families were present and should be replicated in the future by taxonomical mappings conferring greater specificity, for example with longer sequences, such as those assembled from illumina or nanopore reads. this study presents an initial description of the oral/anal virome composition of t. brasiliensis, a widely-distributed new world bat species living in close contact with the human population, for the first time. although their biological significance is not clear, this work contributes to a better understanding of the evolution and genetic diversity of these viruses. using conventional nucleic acid detection techniques and/or bioinformatics approaches, the whole genomes of two novel viruses were completely covered, tbrapv and tbgkyv , clustering into the papillomaviridae and genomoviridae families. tbrapv is the first pv type identified in this host and the prototype of a novel genus in the firstpapillomavirinae taxonomic subfamily. tbgkyv is the first genomovirus reported in new world bats and constitutes a new species within the genus gemykibivirus. future studies are required to investigate the possible health impact of the viruses described on bat colonies and to identify the factors that contribute to their dispersal. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , supplementary data : details of the assembled nucleotide sequences obtained by de novo assembly and used in the taxonomic classification as a part of the metagenomic workflow ( ) (figure ). table s : de novo assembly settings in metagenomic workflow: taxonomic classification of contigs assembled de novo. post-assembly contig correction was applied in all cases. in the case of unicycler assembly, the post-assembly contig-correction program was pilon v . 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mathieu; talbot, pierre j. title: human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: lavcsqov respiratory viruses infect the human upper respiratory tract, mostly causing mild diseases. however, in vulnerable populations, such as newborns, infants, the elderly and immune-compromised individuals, these opportunistic pathogens can also affect the lower respiratory tract, causing a more severe disease (e.g., pneumonia). respiratory viruses can also exacerbate asthma and lead to various types of respiratory distress syndromes. furthermore, as they can adapt fast and cross the species barrier, some of these pathogens, like influenza a and sars-cov, have occasionally caused epidemics or pandemics, and were associated with more serious clinical diseases and even mortality. for a few decades now, data reported in the scientific literature has also demonstrated that several respiratory viruses have neuroinvasive capacities, since they can spread from the respiratory tract to the central nervous system (cns). viruses infecting human cns cells could then cause different types of encephalopathy, including encephalitis, and long-term neurological diseases. like other well-recognized neuroinvasive human viruses, respiratory viruses may damage the cns as a result of misdirected host immune responses that could be associated with autoimmunity in susceptible individuals (virus-induced neuro-immunopathology) and/or viral replication, which directly causes damage to cns cells (virus-induced neuropathology). the etiological agent of several neurological disorders remains unidentified. opportunistic human respiratory pathogens could be associated with the triggering or the exacerbation of these disorders whose etiology remains poorly understood. herein, we present a global portrait of some of the most prevalent or emerging human respiratory viruses that have been associated with possible pathogenic processes in cns infection, with a special emphasis on human coronaviruses. the central nervous system (cns), a marvel of intricate cellular and molecular interactions, maintains life and orchestrates homeostasis. unfortunately, the cns is not immune to alterations that lead to neurological disease, some resulting from acute, persistent or latent viral infections. several viruses have the ability to invade the cns, where they can infect resident cells, including the neurons [ ] . although rare, viral infections of the cns do occur [ ] . however, their incidence in clinical practice common virus, is associated with febrile illness, fever, cough and congestion [ , ] , as well as a characteristic rash and koplik's spots [ ] . in rare circumstances, significant long-term cns diseases, such as [ ] post-infectious encephalomyelitis (pie) or acute disseminated encephalomyelitis (adem), occur in children and adolescents. other examples of rare but devastating neurological disorders are measles inclusion body encephalitis (mibe), mostly observed in immune-compromised patients, and subacute sclerosing panencephalitis (sspe) that appears - years after infection [ ] . yet, with the exception of hiv, no specific virus has been constantly associated with specific human neurodegenerative disease. on the other hand, different human herpes viruses have been associated with alzheimer's disease (ad), multiple sclerosis (ms) and other types of long-term cns disorders [ ] [ ] [ ] . as accurately stated by majde [ ] , long-term neurodegenerative disorders may represent a "hit-and-run" type of pathology, since some symptoms are triggered by innate immunity associated with glial cell activation. different forms of long-term sequelae (cognitive deficits and behavior changes, decreased memory/learning, hearing loss, neuromuscular outcomes/muscular weakness) were also observed following arboviral infections [ , , [ ] [ ] [ ] . including the few examples listed above, more than one hundred infectious agents (much of them being viruses) have been described as potentially encephalitogenic and an increasing number of positive viral identifications are now made with the help of modern molecular diagnostic methods [ , , [ ] [ ] [ ] . however, even after almost two decades into the st century and despite tremendous advances in clinical microbiology, the precise cause of cns viral infections often remains unknown. indeed, even though very important technical improvements were made in the capacity to detect the etiological agent, identification is still not possible in at least half of the cases [ , ] . among all the reported cases of encephalitis and other encephalopathies and even neurodegenerative processes, respiratory viruses could represent an underestimated part of etiological agents [ ] . respiratory syncytial virus (rsv), a member of the orthopneumovirus genus [ ] , infects approximately % of infants before the age of and almost % by the age of years old [ ] , making it the most common pathogen to cause lower respiratory tract infection such as bronchiolitis and pneumonia in infants worldwide [ , ] . recent evidence also indicates that severe respiratory diseases related to rsv are also frequent in immunocompromised adult patients [ , ] and that the virus can also present neuroinvasive properties [ ] . over the last five decades, a number of clinical cases have potentially associated the virus with cns pathologies. rsv has been detected in the cerebrospinal fluid (csf) of patients (mainly infants) and was associated with convulsions, febrile seizures and different types of encephalopathy, including clinical signs of ataxia and hormonal problems [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, rsv is now known to be able to infect sensory neurons in the lungs and to spread from the airways to the cns in mice after intranasal inoculation, and to induce long-term sequelae such as behavioral and cognitive impairments [ ] . an additional highly prevalent human respiratory pathogen with neuroinvasive and neurovirulent potential is the human metapneumovirus (hmpv). discovered at the beginning of the st century in the netherlands [ ] , it mainly causes respiratory diseases in newborns, infants and immunocompromised individuals [ ] . during the last two decades, sporadic cases of febrile seizures, encephalitis and encephalopathies (associated with epileptic symptoms) have been described. viral material was detected within the cns in some clinical cases of encephalitis/encephalopathy [ ] [ ] [ ] [ ] [ ] but, at present, no experimental data from any animal model exist that would help to understand the underlying mechanism associated with hmpv neuroinvasion and potential neurovirulence. hendra virus (hev) and nipah virus (niv) are both highly pathogenic zoonotic members of the henipavirus genus and represent important emerging viruses discovered in the late s in australia and southern asia. they are the etiological agents of acute and severe respiratory disease in humans, including pneumonia, pulmonary edema and necrotizing alveolitis with hemorrhage [ ] [ ] [ ] [ ] . although very similar at the genomic level, both viruses infect different intermediate animal reservoirs: the horse for hev and the pig for niv as a first step before crossing the barrier species towards humans [ ] . in humans, it can lead to different types of encephalitis, as several types of cns resident cells (including neurons) can be infected [ , ] . the neurological signs can include confusion, motor deficits, seizures, febrile encephalitic syndrome and a reduced level of consciousness. even neuropsychiatric sequelae have been reported but it remains unclear whether a post-infectious encephalo-myelitis occurs following infection [ ] [ ] [ ] . the use of animal models showed that the main route of entry into the cns is the olfactory nerve [ ] and that the nipah virus may persist in different regions of the brain of grivets/green monkeys [ ] , reminiscent of relapsing and late-onset encephalitis observed in humans [ ] . influenza viruses are classified in four types: a, b, c and d. all are endemic viruses with types a and b being the most prevalent and causing the flu syndrome, characterized by chills, fever, headache, sore throat and muscle pain. they are responsible for seasonal epidemics that affect to million humans, among which , to million cases are lethal each year [ , ] . associated with all major pandemics since the beginning of the th century, circulating influenza a presents the greatest threat to human health. most influenza virus infections remain confined to the upper respiratory tract, although some can lead to severe cases and may result in pneumonia, acute respiratory distress syndrome (ards) [ , ] and complications involving the cns [ ] [ ] [ ] . several studies have shown that influenza a can be associated with encephalitis, reye's syndrome, febrile seizure, guillain-barré syndrome, acute necrotizing encephalopathy and possibly acute disseminated encephalomyelitis (adem) [ ] [ ] [ ] [ ] [ ] [ ] . animal models have shown that, using either the olfactory route or vagus nerve, influenza a virus may have access to the cns and alter the hippocampus and the regulation of neurotransmission, while affecting cognition and behavior as long-term sequelae [ , , [ ] [ ] [ ] [ ] . the influenza a virus has also been associated with the risk of developing parkinson's disease (pd) [ ] and has recently been shown to exacerbate experimental autoimmune encephalomyelitis (eae), which is reminiscent of the observation that multiple sclerosis (ms) relapses have been associated with viral infections (including influenza a) of the upper respiratory tract [ ] [ ] [ ] . another source of concern when considering human respiratory pathogens associated with potential neuroinvasion and neurovirulence is the enterovirus genus, which comprises hundreds of different serotypes, including polioviruses (pv), coxsackieviruses (cv), echoviruses, human rhinoviruses (hrv) and enteroviruses (ev). this genus constitutes one of the most common cause of respiratory infections (going from common cold to more severe illnesses) and some members (pv, ev-a and -d , and to a lesser extent hrv) can invade and infect the cns, with detrimental consequences [ ] [ ] [ ] [ ] . even though extremely rare, hrv-induced meningitis and cerebellitis have been described [ ] . although ev infections are mostly asymptomatic, outbreaks of ev-a and d have also been reported in different parts of the world during the last decade. ev-a is an etiological agent of the hand-foot-mouth disease (hfmd) and has occasionally been associated with upper respiratory tract infections. ev-d causes different types of upper and lower respiratory tract infections, including severe respiratory syndromes [ ] . both serotypes have been associated with neurological disorders like acute flaccid paralysis (afp), myelitis (afm), meningitis and encephalitis [ , [ ] [ ] [ ] [ ] . last but not least, human coronaviruses (hcov) are another group of respiratory viruses that can naturally reach the cns in humans and could potentially be associated with neurological symptoms. these ubiquitous human pathogens are molecularly related in structure and mode of replication with neuroinvasive animal coronaviruses [ ] like phev (porcine hemagglutinating encephalitis virus) [ ] , fcov (feline coronavirus) [ , ] and the mhv (mouse hepatitis virus) strains of mucov [ ] , which can all reach the cns and induce different types of neuropathologies. mhv represents the best described coronavirus involved in short-and long-term neurological disorders (a model for demyelinating ms-like diseases) [ ] [ ] [ ] . taken together, all these data bring us to consider a plausible involvement of hcov in neurological diseases. the first strains of hcov were isolated in the mid- s from patients presenting an upper respiratory tract disease [ ] [ ] [ ] [ ] . before the severe acute respiratory syndrome (sars) appeared in and was associated with sars-cov [ ] [ ] [ ] , only two groups of hcov, namely hcov- e (previous group , now classified as alphacoronavirus) and hcov-oc (previous group , now classified as betacoronavirus) were known. several new coronaviruses have now been identified, including three that infect humans: alphacoronavirus hcov-nl [ ] and betacoronaviruses hcov-hku and mers-cov [ , ] . the hcov- e, -oc , -nl and -hku strains are endemic worldwide [ , , [ ] [ ] [ ] [ ] [ ] [ ] and exist in different genotypes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in immunocompetent individuals they usually infect the upper respiratory tract, where they are mainly associated with - % of upper respiratory tract infections (uri): rhinitis, laryngitis/pharyngitis as well as otitis. being highly opportunistic pathogens [ ] , hcov can reach the lower respiratory tract and be associated with more severe illnesses, such as bronchitis, bronchiolitis, pneumonia, exacerbations of asthma and respiratory distress syndrome [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the - sars pandemic was caused by a coronavirus that emerged from bats (first reservoir) [ ] to infect palm civets (intermediary reservoir) and then humans [ ] . a total of probable cases were reported and almost % ( cases in more than countries) of these resulted in death [ ] [ ] [ ] . the clinical portrait was described as an initial flu-like syndrome, followed by a respiratory syndrome associated with cough and dyspnea, complicated with the "real" severe acute respiratory syndrome (sars) in about % of the patients [ , ]. in addition, multiple organ failure was observed in several sars-cov-infected patients [ ] . in the fall of , individuals travelling from the arabian peninsula to the united kingdom were affected by the middle-east respiratory syndrome (mers), a severe lower respiratory tract infection that resembled sars, leading also to gastrointestinal symptoms and renal failure among some patients [ ] . molecular sequencing rapidly showed that the new epidemic was caused by a new coronavirus: the mers-cov [ , , ] . mers-cov most probably originated from bats before infecting an intermediary reservoir (the dromedary camel), and also represented a zoonotic transmission to humans. phylogenetic analyses suggest that there have been multiple independent zoonotic introductions of the virus in the human population. moreover, nosocomial transmission was observed in multiple hospitals in saudi arabia [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although possible, human-to-human mers-cov transmission appears inefficient as it requires extended close contact with an infected individual. consequently, most transmission have occurred among patients' families and healthcare workers (clusters of transmission). a more efficient human-to-human transmission was observed in south korea, during the outbreak of mers-cov [ , ] . even though it has propagated to a few thousand people and possesses a high degree of virulence, mers-cov seems mostly restricted to the arabic peninsula and is not currently considered an important pandemic threat. however, virus surveillance and better characterization are warranted, in order to be prompt to respond to any change in that matter [ , [ ] [ ] [ ] . as of october , , the world health organization (who) reported that mers-cov had spread to at least different countries, where laboratory-confirmed human cases have been identified with being fatal (https://www.who.int/emergencies/mers-cov/en/). as observed for the four circulating strains of hcov [ , ] , both sars-cov and mers-cov usually induce more [ , ] severe illnesses, and strike stronger in vulnerable populations such as the elderly, infants, immune-compromised individuals or patients with comorbidities [ , ]. over the years, like sars-and mers-cov, the four endemic hcov have also been identified as possible etiological agents for pathologies outside the respiratory tract. indeed, myocarditis, meningitis, severe diarrhea (and other gastrointestinal problems) and multi-organ failure [ , , [ ] [ ] [ ] [ ] have been reported, especially in children. recent investigations on hcov as enteric pathogens demonstrated that all hcov strains can be found in stool samples of children with acute gastroenteritis; however, no evidence of association could yet be clearly demonstrated with disease etiology [ , ] . different reports also presented a possible link between the presence of hcov within the human central nervous system (cns) and some neurological disorders among patients examined [ ] [ ] [ ] [ ] [ ] [ ] . like all viruses, hcov may enter the cns through the hematogenous or neuronal retrograde route. in the human airways, hcov infection may lead to the disruption of the nasal epithelium [ ] and, although they bud and are released mostly on the apical side of the epithelial cells, a significant amount of viruses is also released from the basolateral side [ ] . thus, although hcov infections are, most of the time, restricted to the airways, they may under poorly understood conditions pass through the epithelium barrier and reach the bloodstream or lymph and propagate towards other tissues, including the cns [ , , , ] ; this was also suggested for other respiratory viruses that can reach the human cns, namely, rsv [ , ] [ , ] . moreover, persistently-infected leukocytes [ ] may serve as a reservoir and vector for neuroinvasive hcov [ ] . therefore, neuroinvasive hcov could use the hematogenous route to penetrate into the cns. the second form of any viral spread towards the cns is through neuronal dissemination, where a given virus infects neurons in the periphery and uses the machinery of active transport within those cells in order to gain access to the cns [ , ] . although the olfactory bulb is highly efficient at controlling neuroinvasion, several viruses have been shown to enter cns through the olfactory route [ , ] . after an intranasal infection, both hcov-oc and sars-cov were shown to infect the respiratory tract in mice and to be neuroinvasive [ ] [ ] [ ] [ ] [ ] . over the years, we and others have gathered data showing that hcov-oc is naturally neuroinvasive in both mice and humans [ , , , , ] . experimental intranasal infections of susceptible mice also indicate that, once it has invaded the cns, the virus disseminated to several regions of the brain and the brainstem before it eventually reaches the spinal cord [ ] [ ] [ ] . furthermore, based on more recent work [ ] , figure illustrates the olfactory route, which is clearly the main route of neuroinvasion used by hcov-oc , as well as the early steps of subsequent neuropropagation within the cns in susceptible mice and recapitulates the suggested equivalent pathway in humans. nevertheless, our data suggest that hcov-oc may also invade the cns from the external environment through other pathways involving other cranial peripheral nerves [ ] , reminiscent of what was shown for other human respiratory viruses such as rsv and influenza virus [ ] . therefore, on the one hand, an apparently innocuous human respiratory pathogen such as the hcov may reach the cns by different routes and induce short-term illnesses, such as encephalitis. on the other hand, it may persist in resident cells of the human cns and may become a factor or co-factor of neuropathogenesis associated with long-term neurological sequelae in genetically or otherwise predisposed individuals. because of their natural neuroinvasive potential in humans and animals, a possible association between the presence of ubiquitous human coronaviruses in the triggering or exacerbation of neurological human pathologies has often been suggested over the years. it is now accepted that hcov are not always confined to the upper respiratory tract and that they can invade the cns [ , , , ] . as other viruses listed herein, hcov are neurotropic and potentially neurovirulent. even though no clear cause and effect link has ever been made with the onset of human neurological diseases, their neuropathogenicity is being increasingly recognized in humans, as several recent reports associated cases of encephalitis [ ] , acute flaccid paralysis [ ] and other neurological symptoms, including possible complications of hcov infection such as guillain-barré syndrome or adem [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the presence and persistence of hcov in human brains was proposed to cause long-term sequelae related to the development or aggravation of chronic neurological diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . given their high prevalence [ , ] , long-term persistence and newly recognized neuropathogenesis, hcov disease burden could currently be underestimated. this suggest that better surveillance, diagnoses and deepened virus-host interactions studies are warranted in order to gather more knowledge that will make possible the development of therapeutic strategies to prevent or treat occurrences. potential short-term neuropathologies sars-cov, hcov-oc and - e are naturally neuroinvasive and neurotropic in humans and therefore potentially neurovirulent [ , , , , , ] . furthermore, animal models showed that sars-cov could invade the cns primarily through the olfactory route [ ] or even after an intra-peritoneal infection [ ] , and induce neuronal cell death [ , ] . to our knowledge, no reports on the presence of the three other coronaviruses that infect humans in the cns have been published. however, neurological symptoms have been described in patients infected by all three viruses [ , , ] . making use of our in vivo model of hcov neuropathogenesis, relying on the natural susceptibility of mice to hcov-oc -the most prevalent strain among endemic hcov [ , ] -encephalitis and transient flaccid paralysis associated with propagation towards the spinal cord and demyelination and long-term persistence in surviving mice were observed [ , [ ] [ ] [ ] [ ] [ ] [ ] ; thus, recapitulating the neurological afflictions reported in some patients infected by hcov [ , , , , [ ] [ ] [ ] ] . although we must interpret data obtained in rodents with all the caution dictated by the use of a non-human host, it is likely that the underlying mechanisms described will have relevance to the human situation or at least provide leads to investigate neurotropic hcov in humans. in susceptible mice, hcov-oc has a selective tropism for neurons in which it is able to use axonal transport as a way of neuron-to-neuron propagation [ ] . these results, together with data harvested with the use of microfluidic devices (xona microfluidic), helped to elaborate a putative model of propagation adapted from tomishima and enquist [ ] , in which infectious hcov-oc could either be assembled in the cell body or at different points along the axon using the anterograde axonal transport to propagate between neurons or from neurons to glial cells surrounding neurons in the cns (figure ). furthermore, based on previous data using different mutant recombinant viruses harboring mutation in the s protein [ , , ] and making use of a luciferase expressing recombinant hcov-oc [ ] [ ] [ ] , we are now showing that the rate and success of virus propagation towards the spinal cord, in part through the neuron-to-neuron pathway, correlates with the exacerbation of neurovirulence ( figure ). [ ] and adapted from tomishima and enquist [ ] . in this model, solid arrows represent fully assembled virus transport and dashed arrows represent subvirion assemblies [ ] . schematic representations were assembled with the motifolio neuroscience toolkit, . [ ] was injected intra-nasally (i.n.) into mice. virus spread was assessed by bioluminescence imaging (bli) with the xenogen vivo vision ivis imaging system (perkin-elmer) in infected anaesthetized mice placed in a light proof specimen chamber after intraperitoneal injection of d-luciferin. images were taken with a ccd camera mounted in a light-tight imaging chamber, using the acquisition software living image version . . (caliper-lifesciences). evaluation of associated clinical scores: (levels to : is asymptomatic; is mice with early hunched back; is mice presenting slight social isolation, weight loss and abnormal gait; is mice presenting total social isolation, ruffled fur, hunched back, weight loss and almost no movement; and is mice moribund or dead (presented elsewhere; [ ] ), indicate that only mice with a positive signal at both the level of the brain and spinal cord were evaluated to be at level to . hcov-oc structural and accessory proteins are important for infection and some clearly represent virulence factors [ , [ ] [ ] [ ] [ ] , , ] . using neuronal cell cultures and our murine model, we gathered data indicating that some of these proteins also play a significant role in viral dissemination [ , ] and now aim to exploit these promising leads to fully understand the course and determinants of propagation to and through the cns and complete the neurologic portrait of short term hcov neuropathogenesis. the presence of hcov rna in the human cns establishes the natural neuroinvasive properties of these respiratory viral agents. moreover, it also suggests that they persist in human cns [ ] as they do in human neural cells [ , ] and in the cns of mice that survive acute encephalitis. these surviving mice exhibited long-term sequelae associated with decreased activity in an open field test and a reduced hippocampus with neuronal loss in the ca and ca layers [ ] , reminiscent of what was observed after infection by the influenza a virus and rsv [ , ] and to the significant loss of synapses within the ca region after infection by wnv [ , ] . the precise and complete etiology of several long-term neurological pathologies still represent a conundrum. multiple sclerosis (ms) represents one such neurological disease for which an infectious agent or agents may play a triggering role, with viruses the most likely culprit in genetically predisposed individuals [ ] . it has been suggested that several neurotropic viruses could be involved in ms pathogenesis but that they may do so through similar direct and/or indirect mechanisms [ ] [ ] [ ] [ ] [ ] . however, although research has not yet led to a direct link to any specific virus, association of coronaviruses with ms has been suggested [ ] . even though hcov-oc and - e were detected in some control brains and in some brains coming from patients with different neurological diseases, there was a significantly higher prevalence of hcov-oc in brains of ms patients [ ] . moreover, autoreactive t cells were able to recognize both viral and myelin antigens in ms patients but not in controls during infection by hcov-oc and hcov- e [ , ] . thus, the immune response may participate in the induction or exacerbation of long-term neuropathologies such as ms in genetically or otherwise susceptible individuals. furthermore, it was shown that in recombination activation gene (rag) knock-out mice, hcov-oc -induced encephalitis could be partially mediated by the t-cell response to infection [ ] . this underlines the possibility that, like its murine counterpart mhv, long term infection of the cns by hcov [ ] may participate in the induction of demyelinating ms-like lesions. immune cell infiltration and cytokine production were observed in the mouse cns after infection by hcov-oc . this immune response was significantly increased after infection by viral variants, which harbor mutations in the viral glycoprotein (s) [ ] . these variants also induced glutamate excitotoxicity [ , ] , thus increasing damage to neurons [ ] and/or disturbing glutamate homeostasis [ ] and thereby contributing to neuronal degeneration and hind-limb paralysis and possible demyelination [ ] [ ] [ ] [ ] . the degeneration of neurons may eventually lead to death of these essential cells by directly generating a cytotoxic insult related to viral replication and/or to the induction of different regulated cell death (rcd) pathways [ ] [ ] [ ] . our results indicate that the underlying mechanisms appear to involve different cellular factors and pathways of rcd, described and reviewed elsewhere [ , ] . virus-cell interactions are always important in the regulation of cell response to infection. for hcov-oc , we clearly showed that the viral s and e proteins are important factors of neurovirulence, neuropropagation and neurodegeneration of infected cells [ ] [ ] [ ] , ] . we have also demonstrated that the he protein is important for the production of infectious hcov-oc and for efficient spreading between neuronal cells, suggesting an attenuation of the eventual spread into the cns of viruses made deficient in fully active he protein, potentially associated with a reduced neurovirulence [ , ] . coronavirus accessory proteins have been extensively studied and are now considered as important viral factors of virulence implicated in pathogenesis while counteracting innate immunity [ ] [ ] [ ] [ ] . two of these accessory proteins (ns and ns ) produced during infection by hcov-oc play a significant role in virulence and pathogenesis in the mouse cns [ ] . like for several other respiratory viruses, accumulating evidence now indicate that hcov are neuroinvasive in humans and we hypothesize that these recognized respiratory pathogens are potentially neurovirulent as well, as they could participate in short-and long-term neurological disorders either as a result of inadequate host immune responses and/or viral propagation in the cns, which directly induces damage to resident cells. with that in mind, one can envisage that, under the right circumstances, hcov may successfully reach and colonize the cns, an issue largely deserted and possibly underestimated by the scientific community that has impacted or will impact the life of several unknowing individuals. in acute encephalitis, viral replication occurs in the brain tissue itself, possibly causing destructive lesions of the nervous tissue with different outcomes depending on the infected regions [ ] . as previously mentioned, hcov may persist in the human cns as it does in mice [ , ] and potentially be associated with different types of long-term sequelae and chronic human neurological diseases. in their famous review on cns viral infection, published a few years ago, koyuncu et al. [ ] insisted that, under the right conditions, all viruses can have access to the cns. what "under the right conditions" means certainly represents a subject of debate among virologist and physicians. nevertheless, as stated in the introduction of this review, viral factors (mutations in specific virulence genes), host factors (immunodepression, age) or a mixture of both (underlining the importance of virus-host interactions), are all good candidates to refer to if one intends to find the beginning of an explanation. a fast and accurate diagnosis would certainly improve prognosis for patients with a suspected cns infection. identification of a specific virus provides relevant information on how to treat a patient; therefore, the development of modern technologies, such as high throughput sequencing (next generation sequencing) are warranted as it represents a potentially unbiased marvelous tool for rapid and robust diagnosis of unexplained encephalitis or other types of encephalopathies or neuronal manifestations, especially in the context where more traditional techniques have failed to identify the etiological agent [ , , , , , ] . therefore, although our attention is mainly on a few different viruses such as hsv, arboviruses and enteroviruses, it may now be the time to look at cns viral infection from another perspective. these viruses truly represent an important proportion of cns viral infection associated with encephalitis, meningitis, myelitis and long-term neurological disorders. nevertheless, accumulating evidence in the scientific literature strongly suggest that many other viral candidates could be underestimated in that matter. several human respiratory viruses are neuroinvasive and neurotropic, with potential neuropathological consequences in vulnerable populations. understanding the underpinning mechanisms of neuroinvasion and interaction of respiratory viruses (including hcov) with the nervous system is essential to evaluate potentially pathological short-and long-term consequences. however, viral infections related to diseases that are rare manifestations of an infection (like long term chronic neurological diseases), represent situations where koch's postulates [ ] need to be modified. a series of new criteria, adapted from sir austin bradford hill, for causation [ , ] was elaborated by giovannoni and collaborators concerning the plausible viral hypothesis in ms [ ] . these criteria certainly represent a pertinent tool to evaluate the involvement of human respiratory viruses as a factor that could influence long-term human neurological diseases. to continue the gathering of epidemiological data is justified to evaluate the clear cause and effect link between neuroinvasive respiratory viruses and short-and long-term human neurological diseases. understanding mechanisms of virus neuroinvasion and interactions with the central nervous system is essential for different reasons. first, to help better understand 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infectious agents clinical metagenomic sequencing for diagnosis of meningitis and encephalitis human virome in nasopharynx and tracheal secretion samples the aetiology of tuberculosis (translation of die aetiologie der tuberculose ( ) sequence-based identification of microbial pathogens: a reconsideration of koch's postulates the environment and disease: association or causation? infectious causes of multiple sclerosis this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank jessy tremblay for his excellent work in confocal microscopy images. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -a xu d authors: aboughdir, maryam; kirwin, thomas; abdul khader, ashiq; wang, brian title: prognostic value of cardiovascular biomarkers in covid- : a review date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: a xu d in early december , the coronavirus disease (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ) first emerged in wuhan, china. as of may th, , a total of over million covid- cases and , deaths have been reported globally, reflecting the raised infectivity and severity of this virus. amongst hospitalised covid- patients, there is a high prevalence of established cardiovascular disease (cvd). there is evidence showing that covid- may exacerbate cardiovascular risk factors and preexisting cvd or may lead to cardiovascular complications. with intensive care units operating at maximum capacity and such staggering mortality rates reported, it is imperative during this time-sensitive covid- outbreak to identify patients with an increased risk of adverse outcomes and/or myocardial injury. preliminary findings from covid- studies have shown the association of biomarkers of acute cardiac injury and coagulation with worse prognosis. while these biomarkers are recognised for cvd, there is emerging prospect that they may aid prognosis in covid- , especially in patients with cardiovascular comorbidities or risk factors that predispose to worse outcomes. consequently, the aim of this review is to identify cardiovascular prognostic factors associated with morbidity and mortality in covid- and to highlight considerations for incorporating laboratory testing of biomarkers of cardiovascular performance in covid- to optimise outcomes. in early december , the coronavirus disease (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ) first emerged in wuhan, china [ ] . on january st, , the world health organisation declared covid- a public health emergency of international concern, and on march th, , it was finally characterised as a pandemic [ ] . as of may th , , a total of over million covid- cases and , deaths have been reported globally, reflecting the raised infectivity and severity of this virus, yet the lack of widespread testing availability means these figures are likely even higher than reported [ ] . it is therefore important to predict the risk of morbidity and mortality, especially in vulnerable patients. sars-cov- is an enveloped, non-segmented, single-stranded, positive-sense rna virus belonging to the coronaviridae family [ ] . sars-cov- is a zoonotic virus not too dissimilar to the sars-cov outbreak of and the middle east respiratory syndrome coronavirus of [ ] [ ] [ ] [ ] . this novel coronavirus enters cells via binding of the viral surface spike protein to the angiotensin-converting enzyme (ace) protein [ ] . ace is highly expressed in lung alveolar cells, providing the route of entry for the virus [ ] . in addition, ace is also widely present in the myocardium, which has raised concerns due to the possibility of direct viral infection of the cardiovascular system [ ] . although covid- patients present primarily with symptoms of respiratory disease and therefore follow a pneumonia-like treatment plan, it is essential that the cardiovascular system is not ignored and to recognise those presenting with early signs of acute myocardial injury. of the patients hospitalised for covid- thus far, the prevalence of cardiovascular comorbidities has been staggering. based on early reports, patients with cardiovascular disease (cvd) may represent % of those in an intensive care unit (icu) plus those with hypertension accounting for % of patients [ ] . additionally, zhou et al. found that myocardial injury, defined by raised serum cardiac troponin i (ctni) levels, in covid- patients was associated with over % mortality rate [ ] . furthermore, heart failure was prevalent in % of patients presenting with covid- , which was also more prevalent amongst patients who died compared to those who survived ( . % vs. . %) [ ] . this demonstrates how essential it is to recognise those presenting with early signs of acute myocardial injury and to initiate a more intensive treatment plan. based on these observations, several theories surrounding the interplay between the pathophysiology of covid- and the cardiovascular system have been postulated [ , ] . namely, covid- may exacerbate cardiovascular risk factors and preexisting cvd or may increase susceptibility for the development of new cardiovascular complications. alternatively, cvd or myocardial injury may predispose to worse outcomes in covid- patients, which is reflected by a number of studies whereby established cvd is associated with more severe covid- , leading to higher morbidity and mortality. with such staggering mortality rates reported, it is fundamental during this time-sensitive covid- outbreak to identify patients with an increased risk of adverse cvd outcomes and/or myocardial injury. one may achieve this through laboratory investigations of biomarkers such as ctni, brain natriuretic peptide (bnp), d-dimers, and fibrinogen-all of which reflect cardiovascular function and are used as diagnostic tools in addition to assessing the risk of cvd in patients [ ] [ ] [ ] [ ] . while these biomarkers are recognised for cvd, there is an emerging prospect that they may aid prognosis in covid- , especially in patients with cardiovascular comorbidities or risk factors that predispose to worse outcomes. this is crucial as the speed of deterioration of many covid- patients means any early biomarkers indicative of severe morbidity or mortality may then help prevent this rapid deterioration. consequently, the aim of this review is to identify cardiovascular prognostic factors associated with morbidity and mortality in covid- and to highlight considerations by summarising the evidence for utilising laboratory testing of biomarkers of cardiovascular performance in covid- to optimise outcomes. covid- patients at risk of serious illness and icu admission tend to be older and to present with similar comorbidities, including heart failure, hypertension, and coronary artery disease [ , , ] . in a meta-analysis of studies ( , covid- patients in total), cvd was reported as the third most prevalent comorbidity in covid- patients ( %, % ci %- %), and patients with severe covid- symptoms had a higher risk of cvd (or . , % ci . - . ) [ ] . whilst these results were limited by significant heterogeneity due to variations in the severity of covid- patients and follow-up period, a similarly high prevalence of cvd in covid- patients ( %) was observed in the study by huang and colleagues [ , ] . notably, yang and jin state that covid- patients with established cvd are susceptible to more adverse complications-these patients are therefore also at a greater risk of myocardial injury, which mainly manifests as elevated serum ctni levels [ ] . ctni is a gold-standard necrotic biomarker for myocardial risk assessment worldwide [ ] . it is released virtually exclusively in the myocardium in the presence of myocardial injury irrespective of the mechanism of insult [ ] . other biomarkers of myocardial injury that are of diagnostic value include creatine kinase-myocardial band (ck-mb) and bnp, which may provide insight into the severity of symptoms in covid- . although they are already established for cvd, results from emerging studies, as discussed below, elucidate the potential role of these biomarkers, particularly ctni and cardiac troponin t (ctnt), as predictors of prognosis in covid- patients, as shown in table . the predictive potential of troponin proteins for severe morbidity in covid- patients has been demonstrated. for instance, huang et al. reported a substantial elevation of ctni (> ph/ml) in out of ( %) covid- patients [ ] . all then developed acute myocardial injury, and out of the were admitted into an icu-this allows the conceptualisation of ctni as a prognostic tool in other diseases such as covid- [ ] . in addition, a meta-analysis of studies with covid- patients reported a significantly higher ctni mean difference in patients with more severe covid- symptoms compared with patients with non-severe covid- presentation ( . ng/l, % ci . - . ng/l), although heterogeneity was relatively high, posing a limitation similar to the previously mentioned meta-analysis [ ] . nevertheless, shi et al. also identified that out of ( . %) covid- patients presented with myocardial injury, diagnosed by significantly raised serum ctni levels [ ] . amongst these patients, there was a significantly higher mortality rate of . % compared to a . % mortality rate in those with normal ctni levels and no myocardial injury, signifying the serious nature of myocardial injury in covid- patients [ ] . importantly, it demonstrates the potential value of ctni in foreshadowing the outcomes of covid- patients. the possible role of ctnt in covid- prognosis is also exemplified by guo et al., who reported the elevation of ctnt levels in out of ( . %) hospitalised covid- patients, all of whom developed myocardial injury [ ] . in those patients, mortality was a staggering . % compared to . % in those patients with normal serum ctnt levels [ ] . whilst covid- patients with raised ctnt levels and established cvd had an alarming mortality rate of . %, those with raised serum ctnt levels but no history of cvd still had a relatively high mortality rate of . % [ ] . this indicates the prognostic value of detecting elevated ctnt levels in all covid- patients, irrespective of the presence of underlying cvd. conversely, patients with normal serum ctnt levels and established cvd had a much lower mortality rate of . % compared to the . % rate in patients with elevated ctnt levels [ ] . interestingly, guo et al. also observed a significant positive linear correlation between serum ctnt and plasma c-reactive protein (p < . ), suggesting a link between the severity of inflammation observed in covid- and myocardial injury [ ] . indeed, several myocarditis autopsy findings of inflammatory mononuclear infiltrate in myocardial tissue have been reported in patients with high viral load-these studies also further explore the changes in cardiac inflammatory markers during covid- manifestation [ ] [ ] [ ] . it is therefore plausible that, through these inflammatory changes, there is an increased risk of myocardial injury, which manifests as elevated serum ctnt levels and consequently leads to more severe symptoms. whilst ctni and ctnt have demonstrated remarkable potential in predicting covid- outcomes, bnp too has shown some prospect in the prognosis of covid- . guo and colleagues found that raised ctnt levels were significantly associated with elevated serum bnp levels (p < . ) [ ] . they reported that, alongside the gradual elevation of serum ctnt levels, bnp levels likewise progressively increased in covid- patients whose health deteriorated, contrasting the low and stable serum bnp levels in successfully discharged patients [ ] . similarly, a case report presented the cardiac involvement in deterioration of a covid- patient without preexisting cvd, whereby serum levels of bnp ( pg/ml), ctnt ( . ng/ml), and ck-mb ( . ng/ml) were all elevated-this patient was then admitted to the icu with myocarditis [ ] . moreover, shi et al. report significantly raised bnp levels in covid- patients with myocardial injury compared to those without ( pg/ml vs. pg/ml, p < . )-these patients consequently also had a high mortality rate of . % [ ] . as such, the aforementioned findings in these studies are groundbreaking as they reflect the prospect of routinely measuring serum bnp levels in covid- patients at admission to reduce mortality and to prevent deterioration where possible. in addition to ctni and bnp, ck-mb may similarly hold prognostic value in covid- . in the study by wang et al., out of ( . %) covid- patients were admitted to the icu with severe symptoms, all of whom had significantly elevated serum ctni and ck-mb levels (p = . and p < . , respectively) compared to non-icu patients [ ] . perhaps this implies that patients with more severe covid- symptoms have adverse outcomes of acute myocardial injury-reflected by the elevation in ck-mb and ctni levels. likewise, this study provides insight into the value of ck-mb, along with ctni, in categorising covid- patients with an increased risk of adverse outcomes and admission to icu for health deterioration. the value of ck-mb and ctni in covid- is also exemplified in the study by zhou et al., whereby a significant association between elevated ck-mb and ctni levels and in-hospital death was illustrated (p = . and p < . , respectively) [ ] . similarly, wan et al. found that creatine kinase was significantly higher in covid- patients with severe symptoms compared to those with mild symptoms (p = . ) [ ] . these studies demonstrate the benefit of utilising ck-mb in determining the patients that require urgent intervention. although bnp and ck-mb have evidently demonstrated some prognostic value in covid- , it is important to highlight that, in all studies measuring bnp or ck-mb, ctni was also measured and it provided the same, if not a clearer, link between myocardial injury and covid- outcomes. additionally, contrasting findings are reported between studies regarding creatine kinase levels and severe covid- presentation. for instance, whilst wan et al. found creatine kinase to be significantly elevated in covid- patients with severe symptoms, huang et al. found no significant difference in serum creatine kinase levels between icu and non-icu patients (p = . ) [ , ] . therefore, more studies that clearly illustrate a conclusive link between ck-mb and bnp and covid- outcomes will provide a better understanding of their prognostic role. it is through the lack of evidence in the literature that one, therefore, postulates ctni may be a preferred option compared to ck-mb and bnp, mainly due to its high sensitivity in detecting worsening prognosis and myocardial injury in covid- patients. it is worth noting that raised serum ctni levels are similarly associated with a higher risk of mortality in other diseases such as pneumonia (odds ratio = . ), sepsis (odds ratio = . ), chronic obstructive pulmonary disease (hazard ratio = . ), and acute respiratory distress syndrome (hazard ratio = . ) [ ] [ ] [ ] [ ] . hence, one may logically also predict a correlation between elevated serum ctni levels and a higher risk of mortality in covid- patients. studies have clearly illustrated a significant difference in serum ctni levels between covid- patients who survived and those who died. ctni levels provide novel insight into a multi-faceted prognostic use of ctni in other diseases than cvd as it has proven to be a reliable marker of mortality in the previously discussed studies. during a crisis such as the current covid- pandemic, measuring serum ctni levels may enable healthcare professionals to predict prognosis and to therefore avoid worsening outcomes in vulnerable patients by identifying them at an earlier stage and by providing them with an intensive treatment plan which tackles both the myocardial injury and covid- . it has been clear from early reports in china that abnormal coagulation is associated with poor prognosis in covid- patients. tang et al. clearly illustrated this by retrospectively analysing the coagulation parameters of hospitalised covid- patients [ ] . strikingly, . % of non-survivors matched the diagnostic criteria for disseminated intravascular coagulation (dic) in later stages of the disease (according to the criteria described by the international society on thrombosis and haematosis) [ ] . this contrasts with only one ( . %) survivor meeting the criteria. hence, abnormal coagulation is a principal factor involved in the deterioration and high mortality seen in covid- . however, it is less clear if coagulation parameters, as seen in table , could be used to stratify patients on admission, thus highlighting those more likely to develop severe disease in order to prompt swift intensive treatment. thus far, d-dimer has demonstrated promise in its ability to hold prognostic value in covid- patients. wang et al. conducted a retrospective single-centre case series including patients with confirmed covid- [ ] . those who were eventually admitted to icu had significantly increased d-dimer levels (median d-dimer mg/l vs. mg/l, p < . ) on admission compared to those who avoided intensive treatment [ ] . this finding was substantiated by a smaller retrospective cohort study that found d-dimer levels were four times the upper limit of normal in patients subsequently admitted to the icu, a level much higher than non-icu patients (median d-dimer level . mg/l vs. . mg/l, p = . , reference range < . mg/l) [ ] . remarkably, a multi-centre retrospective cohort study of patients demonstrated that, even after multi-variant analysis, an increased d-dimer on admission was highly associated with in-hospital death (or . , p = . ) [ ] . furthermore, % of those who did not survive had a d-dimer > µg/ml on admission compared to just % of those who survived [ ] . this striking evidence greatly supports the prognostic ability of d-dimer. however, those still hospitalised at the end of the study were excluded; thus, only those who had died or been discharged during the study period were counted. therefore, this may have exaggerated the difference between the groups as only those with more severe disease at an earlier stage would be included in the analysis of those who died. nonetheless, in addition to d-dimer being raised on admission, numerous studies from china have demonstrated that, in non-survivors, d-dimer continues to rise throughout the clinical course of the disease [ , ] . this is compared to a low and stable d-dimer in those who survived. importantly, an increased d-dimer was highly associated with acute myocardial injury, diagnosed via a raised ctni, which as mentioned previously has been correlated with an increased risk of in-hospital death [ ] . therefore, it is reasonable to suggest that d-dimer has prognostic value when taken on admission and could also be used to highlight patients who are deteriorating. however, the practicalities of such an implementation would need to be considered. for example, whilst wan et al. found that a raised d-dimer was associated with a more severe disease, the median d-dimer level in the severe cases was still within the normal range on admission [ ] . this could present a barrier in confidently triaging patients on their d-dimer level if it is still below the cutoff. although, in this study of patients, there was only one fatality, a death rate much lower than previously reported. therefore, the raised yet normal d-dimer could be explained by a relatively well cohort. prothrombin time (pt) may also hold some predictive value in covid- patients. contrasting evidence has surfaced concerning the association of an extended pt with admission to icu. whilst a smaller retrospective cohort study found that those who were admitted to icu had a significantly longer pt on admission (median pt . s vs. . s, p = . ), wang et al reported no significant difference [ , ] . although, of the patients included in wang et al.'s analysis, a large proportion was still hospitalised and not discharged ( . %) [ ] . therefore, patients who were not admitted to icu may have deteriorated and subsequently required intensive care; thus, comparing patients by icu admission may be unreliable in this cohort. nevertheless, there is good evidence to support that a prolonged pt is associated with in-hospital death. a large multi-centre retrospective cohort study found that a pt over s was greatly associated with in-hospital death (or . , p = . ), whilst tang et al. found that pt time was significantly increased in non-survivors (median pt . s vs. . s, p < . ) [ ] . tang et al. also demonstrated that, from admission, pt continued to rise in those who did not survive, supporting its association with in-hospital death [ ] . like d-dimer, an increased pt was also associated with acute cardiac injury, implying that abnormal coagulation parameters on admission are associated with myocardial injury [ ] . however, as previously discussed, the pathology of this injury, whether infarction or myocarditis, is still unclear. similar trends have also been noted in platelet counts. there was no difference in the platelet counts on admission of those admitted to icu [ , ] . however, a reduced platelet count was associated with in-hospital death and cardiac injury. zhou et al. reported a much lower platelet count in those who died (median platelet count, . × /l vs. . × /l, p < . ) with % of non-survivors having a platelet count less than × /l on admission compared to just % of those who survived [ ] . in addition, those with raised ctni on admission had a significantly lower platelet count compared to those without cardiac injury (median platelet count, × /µl vs. × /µl, p < . ). this further illustrates that abnormal coagulation is associated with cardiac injury in hospitalised covid- patients [ ] . lastly, a study of patients found that fibrinogen degradation products (fdp) were also significantly raised on admission in patients that did not survive (median fdp . µg/ml vs. . µg/ml, p < . ) [ ] . whilst fibrinogen levels showed no significant difference on admission, it was significantly lower in non-survivors in late hospitalisation [ ] . this suggests that a decreasing fibrinogen level is associated with the progression of the disease; thus, it may aid in the identification of deteriorating patients. in light of the striking rate of dic in patients who did not survive, it has been suggested that the use of heparin in covid- may be beneficial. therefore, tang et al. conducted a retrospective analysis of covid- patients and found that the use of low molecular weight heparin was associated with improved prognosis in severe covid- cases with a markedly elevated d-dimer [ ] . this further supports the pivotal role that abnormal coagulation plays in the deterioration of covid- patients and how coagulation parameters may help in determining the prognosis of patients. furthermore, it demonstrates that a raised d-dimer may also aid treatment optimisation in severe cases of covid- . evidently, coagulation parameters have demonstrated their prognostic potential in covid- patients. however, in all studies that demonstrated an association between covid- and coagulation markers, d-dimer consistently provided the clearest link to icu admission and in-hospital death. additionally, as seen in table , it has been the most widely studied biomarker and thus may be the most reliable in predicting the outcome in covid- patients. as previously mentioned, sars-cov- uses the ace receptor for entry into target cells, found commonly in the lungs, heart, and vessels [ ] . ace converts angiotensin i to angiotensin ii, which can then activate the angiotensin ii receptor type [ ] . angiotensin ii has profound effects not limited to the cardiovascular system, including vasoconstriction; the release of pro-inflammatory cytokines, such as il- ; as well as pro-oxidative effects [ ] [ ] [ ] . numerous studies have demonstrated that the use of ace inhibitors (aceis) and angiotensin ii receptor i blockers (arbs) lead to an increase in expression of the ace receptor [ ] . this has sparked debate surrounding the use of acei/arbs due to potentially enhancing the risk of infection by increasing the entry way, ace . interestingly, it has been revealed recently that the plasma levels of angiotensin ii were raised in infected patients compared to that of healthy controls [ ] . this may be in part explained by the reduction of ace due to the binding and internalisation of the enzyme caused by the virus. moreover, the level of angiotensin ii in covid- patients was strongly associated with viral load and lung injury, suggesting that covid- was causing an imbalance in the renin-angiotensin system [ ] . this implies that angiotensin ii may be a mediator of the disease, leading to pulmonary vasoconstriction and inflammatory or oxidative organ damage. therefore, it would be not unreasonable to suggest that the use of an acei or arb may be beneficial in the treatment of covid- . whilst there have been no other studies to date to our knowledge, this suggests that angiotensin ii could be used as a biomarker to stratify patients as those with higher angiotensin ii would have increased risk of organ failure, thus requiring more intensive treatment. furthermore, this could provide an explanation for the increased risk of myocardial injury whilst hospitalised with covid- . the powerful vasoconstrictive effects of angiotensin ii may increase the demand on the heart whilst potentially inducing oxidative damage. this risk of myocardial injury may then be further increased by the coagulative state mentioned previously. however, further studies are essential to support these findings, especially when considering the small sample size of patients in the study. it is relatively nonspecific [ , ] . increased d-dimer is associated with • icu admission • in-hospital death • acute myocardial injury +++ [ , , , , , ] pt pt is used to evaluate the extrinsic and common pathways of coagulation. it is the time taken for plasma to clot after adding thromboplastin. pt is increased in dic and can be a sign of liver disease or vitamin k deficiency [ ] . increased pt is associated with • in-hospital death • acute myocardial injury • potentially associated with icu admission ++ [ , , , , ] platelet count number of platelets in a volume of blood: decreased in many conditions, namely dic, anaemia, and marrow failure [ ] . reduced platelet count is associated with • in-hospital death • acute myocardial injury ++ [ , , ] fibrinogen is an acute phase protein involved in platelet aggregation and is decreased acutely by consumption due to dic or chronically due to hepatic impairment [ ] . decreasing fibrinogen levels correlates with deteriorating clinical parameters in covid- patients. fdp fdp are fragments released following plasmin-mediated degradation of fibrinogen/fibrin and raised in inflammatory and thrombotic conditions [ ] . raised fdp is associated with in-hospital death in covid- patients. + [ ] angiotensin ii angiotensin ii is a circulating hormone involved in the renin-angiotensin system. it is a regulator of blood pressure through vasoconstriction and sympathetic nervous stimulation [ ] . angiotensin ii is raised in infected patients compared to control. raised plasma levels of angiotensin ii associated with • viral load • lung injury raised levels of d-dimers, prothrombin time (pt), fibrinogen degradation products (fdp), and angiotensin ii and reduced platelet count and fibrinogen levels are all associated with deteriorating clinical parameters in covid- patients. prognostic potential is judged by the authors on association with clinical findings and the quality of the literature in support of this. pt, prothrombin time; fdp, fibrinogen degradation products; dic, disseminated intravascular coagulation; icu, intensive care unit. biomarkers of acute myocardial injury have evidently revealed their potential in predicting worsening prognosis for covid- patients with and without myocardial injury. ctni provides remarkable prognostic value for patients at increased risk of worsening outcomes and in-hospital mortality, though studies have also shown the association of raised ck-mb and bnp levels with more severe symptoms of covid- . raised serum ctnt and ctni levels show a clear correlation with deteriorating health and increased mortality in patients with established cvd or cardiovascular risk factors and even in those presenting without a history of cvd. as a result, detecting elevated serum ctnt or ctni levels on admission as a routine procedure may be invaluable to reduce mortality and severe covid- patients during a time when icus are operating at maximum capacity. additionally, it may allow healthcare professionals to initiate intensive treatment in those vulnerable patients before covid- symptoms worsen. collectively, the evidence presented suggests a common coagulation activation in patients that die from covid- . d-dimer has demonstrated predictive value for both icu treatment and in-hospital death when taken on admission. furthermore, fdp, pt, and platelets when taken on admission may also highlight those more likely to die in hospital. therefore, the measurement of coagulation parameters on admission may help in the assignment of scarce icu beds. the continued activation of coagulation throughout the clinical course of non-survivors, evidenced by an increasing d-dimer level and pt plus a decreasing fibrinogen level, may help identify deteriorating patients that require extra support or palliative care. furthermore, plasma levels of angiotensin ii may offer a novel method of predicting disease severity. also, the pathogenic role of angiotensin ii in covid- and the potential use of ace/arbs needs to be more clearly elucidated. nonetheless, when considering the prognostic potential of these biomarkers, it is poignant to contemplate whether they are causative in the deterioration of covid- or simply a consequence of disease progression. additionally, the mechanism concerning the abnormal biomarker levels should be elucidated. for instance, many of the markers of coagulation are raised in inflammatory or hepatic diseases and, thus, are nonspecific (table ) . hence, whilst it is clear that the body is in a pro-coagulative phase, the cause of this is unclear. further investigation into the role of these biomarkers may permit insight into the pathogenesis of sars-cov- . similarly, the exact pathology of myocardial injury in covid- is unknown, although this review has highlighted possible mechanisms to be explored. firstly, the high association with abnormal coagulation may suggest a causative link. alternatively, a common trigger, such as angiotensin ii, might instigate both coagulation activation and myocardial injury. nevertheless, more studies are required to elucidate the specific mechanism of myocardial injury and its association with severe inflammation in covid- , along with the subsequent detrimental symptoms that often lead to mortality in vulnerable patients. when reviewing the literature published thus far on covid- , the requirement for multi-centre studies with larger cohorts and clinical power is abundantly clear. furthermore, due to the high demand for research to published, numerous papers included in the review comprise of patients still not discharged from hospital. consequently, the data has incomplete endpoints, thus reducing the potential clinical translation of their findings. moreover, the evidence presented only concerns patients presenting to hospital, and 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cardiovascular disease: focus on natriuretic peptides and cardiac ischemia. scand cardiac troponin i and creatine kinase-mb release after different cardiac surgeries methods of creatine kinase-mb analysis to predict mortality in patients with myocardial infarction treated with reperfusion therapy comparison of myoglobin, creatine kinase-mb, and cardiac troponin i for diagnosis of acute myocardial infarction abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia towards definition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation anticoagulant treatment is associated with decreased mortality in severe coronavirus disease patients with coagulopathy covid- ) infection and renin angiotensin system blockers role of angiotensin ii in blood pressure regulation and in the pathophysiology of cardiovascular disorders angiotensin ii and inflammation: the effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockade oxidative stress-mediated effects of angiotensin ii in the cardiovascular system evaluation of d-dimer in the diagnosis of suspected deep-vein thrombosis how to interpret and pursue an abnormal prothrombin time, activated partial thromboplastin time, and bleeding time in adults thrombocytopenia: an update key: cord- -b xjlqg authors: zapatero-belinchón, francisco j.; dietzel, erik; dolnik, olga; döhner, katinka; costa, rui; hertel, barbara; veselkova, barbora; kirui, jared; klintworth, anneke; manns, michael p.; pöhlmann, stefan; pietschmann, thomas; krey, thomas; ciesek, sandra; gerold, gisa; sodeik, beate; becker, stephan; von hahn, thomas title: characterization of the filovirus-resistant cell line sh-sy y reveals redundant role of cell surface entry factors date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: b xjlqg filoviruses infect a wide range of cell types with the exception of lymphocytes. the intracellular proteins cathepsin b and l, two-pore channel and , and bona fide receptor niemann–pick disease c (npc ) are essential for the endosomal phase of cell entry. however, earlier steps of filoviral infection remain poorly characterized. numerous plasma membrane proteins have been implicated in attachment but it is still unclear which ones are sufficient for productive entry. to define a minimal set of host factors required for filoviral glycoprotein-driven cell entry, we screened twelve cell lines and identified the nonlymphocytic cell line sh-sy y to be specifically resistant to filovirus infection. heterokaryons of sh-sy y cells fused to susceptible cells were susceptible to filoviruses, indicating that sh-sy y cells do not express a restriction factor but lack an enabling factor critical for filovirus entry. however, all tested cell lines expressed functional intracellular factors. global gene expression profiling of known cell surface entry factors and protein expression levels of analyzed attachment factors did not reveal any correlation between susceptibility and expression of a specific host factor. using binding assays with recombinant filovirus glycoprotein, we identified cell attachment as the step impaired in filovirus entry in sh-sy y cells. individual overexpression of attachment factors t-cell immunoglobulin and mucin domain (tim- ), axl, mer, or dendritic cell-specific intercellular adhesion molecule- -grabbing non-integrin (dc-sign) rendered sh-sy y cells susceptible to filovirus glycoprotein-driven transduction. our study reveals that a lack of attachment factors limits filovirus entry and provides direct experimental support for a model of filoviral cell attachment where host factor usage at the cell surface is highly promiscuous. ebola and marburgviruses are enveloped, negative single-strand rna viruses of the filoviridae family [ ] . since the discovery of marburg virus (marv) in [ ] and ebola virus (ebov) in [ ] , the us centre of disease control has reported several epidemic outbreaks in humans and nonhuman primates [ , ] . despite intense world-wide research efforts, no antiviral treatments or vaccines have yet been licensed. in addition to primates, filoviruses infect pigs, dogs, duikers, and fruit bats in nature, and rodents and ferrets can be infected experimentally [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the viral glycoprotein (gp), the only viral surface protein, exclusively mediates the entry and internalization of filoviruses into cells. the precursor protein gp is synthesized on the endoplasmic reticulum, and cleaved in the constitutive secretory pathway into the surface unit gp , which binds to host cell factors, and the transmembrane unit gp , which mediates fusion of viral envelopes with endosomal membranes. filoviruses display a broad cell tropism [ ] . almost any cell type with the notable exception of lymphocytes is susceptible to infection by authentic filoviruses in vitro [ , ] , or to transduction by retrovirus particles pseudotyped with gp [ , ] . moreover, immortalized cell lines cultured in suspension are resistant to filovirus entry, while cell adhesion enhances susceptibility to infection [ , ] . thus, the broad cell tropism observed in infected primates, where virus can be isolated from all organs but not from lymphocytes [ , , ] , is also recapitulated in vitro. the availability of host factors on the cell surface that interact with viral envelope gp or with envelope lipids such as phosphatidylserine (ptdser) often determines viral cell tropism. such virus-host interactions mediate virus attachment, and are a necessary prerequisite for virus internalization, viral fusion with host membranes, and viral genome release into the cytosol for transcription and replication [ , , ] . several plasma membrane proteins have been implicated in filovirus attachment: cellular lectins such as asialoglycoprotein receptor (asgr-r), dendritic cell-specific intercellular adhesion molecule- -grabbing non-integrin (dc-sign), liver/lymph node-specific intercellular adhesion molecule- -grabbing non-integrin (l-sign), human macrophage c-type lectin specific for galactose and n-acetylglucosamine (hmgl), or liver and lymph node sinusoidal endothelial cell c-type lectin (lsectin) [ ] [ ] [ ] [ ] [ ] , t-cell immunoglobulin and mucin domain and (tim- , tim- ) [ , ] , members of the tam family (tyro , axl, mer) of receptor tyrosine kinases [ ] , integrin αvβ [ , ] , and scavenger receptor a. however, none of these factors seems to be essential for filoviral infection across cell lines. rather, their role in cell entry is considered to be cell type dependent, and some of them may promote entry indirectly by regulating downstream processes such as macropinocytosis or gp proteolytic cleavage [ ] [ ] [ ] [ ] . in contrast, several intracellular proteins are essential for filovirus infection in all cell types studied thus far. the endosomal and lysosomal cysteine proteases cathepsin b and cathepsin l cleave gp and thereby expose its receptor binding domain [ ] , and the two-pore channel (tpc ) and two-pore channel (tpc ) mediate endolysosomal ca + efflux [ ] . finally, the endolysosomal cholesterol transporter niemann-pick c (npc ) [ , ] binds to processed gp [ ] . the remarkable diversity of plasma membrane proteins implicated in filovirus cell entry prompted us to analyze twelve cell lines for a potential correlation of host factor expression to filovirus susceptibility. we could show that the neuroblastoma sh-sy y cell line is specifically resistant to filovirus infection although all intracellular proteins known to be essential were expressed, and although its overall transcriptome was very similar to that of susceptible cell lines. heterokaryon assays revealed that sh-sy y cells did not express a dominant restriction factor that inhibited filovirus gp-driven cell entry, but recombinant gp could not bind to their plasma membrane. by individual overexpression of a wide range of different filovirus attachment-promoting proteins, sh-sy y cells became susceptible to filovirus gp-driven transduction. our findings demonstrate that filoviruses-in contrast to many other viruses-can highjack a diverse range of different plasma membrane proteins for the critical cell attachment step, which is a mandatory prerequisite for productive infection of any cell and thus any host. the cell lines huh- . /tet g and sh-sy y/tet g were engineered to stably express tet-on g transactivator protein, and the cell line hek t/zsgreen was developed to stably express zsgreen fluorescent reporter protein under the control of tet-on g-inducible p treg v promoter. genetically modified cell lines were generated by stable transduction of parental cell lines either with lentiviral particles of the plvx-tet g plasmid vector (clontech) for transactivator expression or plvx-tre g-zsgreen for reporter expression. . mg/ml g or µg/ml puromycin were added in the media of transduced cell lines to select for transactivator or reporter expressing cells, respectively. clone hek t-h /zsgreen was isolated by cell subcloning to obtain a permanent cell line with homogenous reporter expression. stable cell lines were maintained in culture as parental cell lines with % tet-free fbs (clontech, takara bio, mountain view, ca, usa) in instead of fcs. surrogate hiv-based pseudovirion packaging plasmid hiv gag-pol, transfer plasmids cslucw (firefly luciferase expression) or csgw (hrgfp), and envelope plasmid for vsv-g protein and its backbone plasmid pcdna . have been described previously [ ] . the pwpi-mcherry-bsd bicistronic expression vector was generated by introduction of the mcherry dna sequence into a pwpi-bsd empty vector [ ] . both plasmids were digested with restriction enzymes bamhi and spei. the mcherry encoding fragment was gel isolated, purified and ligated into the new lentivirus expressing vector. mcherry expression was confirmed by pseudovirion generation and fluorescence-activated cell sorting (facs) analysis. lenti-x tm tet-on ® inducible expression system plasmids plvx-tet g and plvx-tre g-zsgreen were purchased from takara bio. (mountain view, ca, usa). gp encoding plasmids pvr -gp(z) (ebov mayinga), pvr -gp(r) (restv pennsylvania), pvr -gp(s) (sudv boniface), and pvr -gp(ic) (tafv cote d´ivoire) were provided by drs. anthony sanchez and gary nabel (nhi, bethesda, usa). pcaggs-marv, pcaggs-bdbv or were commercially purchased (vectorbuilder inc, shenandoah, united states). mlv-based dc-sign overexpressing plasmid pqcxip_hdc-sign-au has been described [ ] . for construction of pwpi-havcr -bsd vector, the coding sequence of human tim- (htim- ) was synthesized (integrated dna technologies) and amplified by pcr using the forward primer 'cctgcaggcgcgccggatccgccacc atgcatcctc aagtggtcat cttaagcctc atcctacatc tggcagattc tgtagctggttctgtaaagg ttggtgg and the reverse primer gatatccggagccgcctcctccatccgtggcataaagactattc and cloned into the lentiviral expression vector pwpi-bsd [ ] by gibson assembly. the htim- insert was confirmed by sequencing. the drugs z-phe-tyr(tbu)-diazomethylketone (fydmk) ( , calbiochem, san diego, ca, usa), u a ( , calbiochem, san diego, ca, usa) and tetrandrine (sc- , santa cruz biotechnology, santa cruz, ca, usa) were dissolved in dmso according to supplier's instructions. working solutions were prepared by dilution of the stock solution with culture medium. doxycycline hyclate (dox) (d , sigma-aldrich, san luis, ms, usa) for cell-cell fusion reporter expression activation was prepared as mg/ml stock solution in dmso and stored at − • c. the stock solution was diluted in culture media supplemented with tet-free approved fbs (clontech, takara bio, mountain view, ca, usa) to ng/ml. recombinant conjugated protein egf-alexafluor for egf intravesicular accumulation analysis was purchased from invitrogen™ (e ) (carlsbad, ca, usa). filipin iii (f ) for unesterified cholesterol detection was obtained from sigma-aldrich (san luis, ms, usa). mouse monoclonal allophycocyanin(apc)-conjugated antibodies (mabs) against human surface entry factors axl (fab a), human integrin β (fab a), mer (fab a), or dc-sign (fab a) and their respective isotype controls, igg (ic a) and igg b (ic a) as well as alexafluor -conjugated mab against human integrin αv (fab g- ) and its igg isotype control (ic g) were purchased from r&d systems (minneapolis, mn, usa). mouse apc mab against human tim- ( ) or apc-migg κ isotype control ( ) were obtained from biolegend inc. (san diego, ca, usa). for immunoblot detection of intracellular factors rabbit polyclonal antibodies (pabs) directed against human procathepsin l + cathepsin l ab (ab ) or mouse antihuman niemann pick-c mab (ab , ab ) were purchased from abcam (cambridge, uk). antihuman procathepsin b and cathepsin b rabbit mab ( ) was acquired from cell signaling technology inc. (danvers, ma, usa). rabbit pabs directed against human two pore channel segment (tpc ) (ls-c ) was purchased from lifespan bioscience inc. (seattle, wa, usa) or against human two pore channel segment (tpc ) (acc- ) from alomone labs (jerusalem, israel). mouse mab against human β-tubulin (t ) or gapdh (g ) were purchased from sigma-aldrich (san luis, ms, usa). horseradish peroxidase-coupled (hrp) goat pabs directed against mouse (a ) or against rabbit (a ) iggs were purchased from sigma-aldrich (san luis, ms, usa). mouse pab conjugated with fluorescein isothiocyanate (fitc) and directed against human igg, fc γ fragment specific ab ( - - ) for fusion protein cell binding assays was purchased from jackson immunoresearch europe ltd. (newmarket, uk). hiv-based pseudotype viral particles were generated as described previously [ ] . in brief, % hek t cells were cotransfected in -well plates with µg total dna using polyethylenimine (pei) (sigma-aldrich, san luis, ms, usa) according to manufacturer's instructions. dna ratios were part transfer plasmid: part packaging plasmid: parts envelope plasmid for reporter pseudotype virus generation or : : for transgene delivery. cell supernatants were collected at and h post-transfection and passed through a . µm pore filter. viral stocks were stored at • c for short time (less than days) or aliquoted and frozen at − • c for long-term storage. for transduction assays, viral supernatants were first mixed with polybrene (h - g, sigma-aldrich, san luis, ms, usa) at a final concentration of µg/ml and µl or ml was added to - % confluent target cells ( - % for heterokaryon transductions) in or -well plate format, respectively, for h. seventy-two hours post transduction, cells were lysed for luciferase quantification or processed for fluorescence detection. for virus-cell specificity studies, gfp-based pseudoparticles were titrated by limiting serial dilution and subsequent transduction of the susceptible cell line huh- . . transducing units per ml (tu/ml) were calculated as [((% infected cells/ ) * # transduced cells) / virus volume (ml)] by facs analysis. for pharmacological inhibition studies, target cells were preincubated with drugs in dmso or dmso alone for h prior to addition of pseudoparticles and further incubation. luciferase-based gp-driven entry was measured as previously described [ ] . fully infectious filovirus work was performed under biosafety level (bsl ) conditions at the institute of virology, phillips-marburg university, germany. susceptible cell lines sk-n-be( )-c and hek t and resistant cell lines sk-n-mc and sh-sy y seeded on -well plates with coverslip per well were mock treated, or infected with ebov (mayinga isolate) (genbank accession number nc ) or marv (musoke isolate) (genbank accession number nc ) at a moi of . tcid /ml for h at • c. after inoculation, virus was removed, and cells were further incubated for h in dmem with % fcs. three days later, the cells were fixed with % pfa for h. for immunofluorescence analysis, free aldehyde groups were quenched with . mm glycine in pbs, and the cells were permeabilized with % triton-x (tx ). permeabilized cells were incubated with goat α-ebov or α-marv antisera (α-ebov serum was used for mock infection) against virus nucleoprotein (np) as primary ab. α-goat alexafluor was used as secondary ab with dapi for nuclear staining. np staining was visualized using a zeiss axiophot microscope with a x objective with an illumination of ms for fitc channel and ms for dapi. remnant cells were scraped from the wells into % sds/pbs for immunoblot analysis. % lysates were run through an sds/page gel and transferred onto a nitrocellulose membrane. np or tubulin protein detection was carried out by the same goat ebov and marv antisera as used for if and a tubulin specific mab, respectively, followed by antigoat irdye -conjugated secondary ab incubation. immuno-labelled proteins were detected with and odyssey infrared-imaging system (li-cor). briefly, hek t and sh-sy y cells were infected with µl/well of recombinant vsv bearing ebov mayinga strain gp (rvsv∆g-ebovgp) at a moi of ( . × tcid ) or mock infected for h at • c in dmem without fcs. at hpi, . ml dmem with % fcs was added to the virus inoculum and further incubated for h ( replicates) or h ( replicate). cell supernatant was collected each day, and viral titers were determined by tcid limiting-dilution assay - h after starting of assay [ ] . simultaneously, bright-field images were acquired at h ( replicates) and hpi ( replicate) with a nikon ts microscope using a × objective. for cell surface protein detection immunostaining and subsequent fluorescence quantification was performed. in brief, adherent cells were detached with versene ( , gibco, gaithersburg, md, usa), a nonenzymatic cell dissociation reagent. the cell suspensions were spun at rpm for min, and the cells were resuspended in µl % fcs in pbs (facs buffer) solution per staining. fc receptors were blocked with µl human fcr blocking reagent (miltenyi biotec gmbh, bergisch gladbach, germany) for min. subsequently, saturating concentrations of conjugated specific or isotype control abs were added and incubated for at least min. stained cells were washed with facs buffer and resuspended in µl facs buffer for fluorescence detection. cells were kept at • c and in the dark throughout the staining. protein expression was evaluated by apc or fitc channel intensity in a bd facscanto cytometer (bd, frankin lakes, nj, usa). protein expression was calculated as the difference of geometric mean fluorescence intensity (∆mfi) between specific ab and isotype control staining. cell surface detection of bound gp -fc fusion protein was detected as described before [ ] . briefly, adherent cells were detached with versene. × cells/well were washed with × dpbs containing ca + and mg + ( , gibco, gaithersburg, md, usa) and fc receptors blocked with human fcr blocking reagent. subsequently, cells were incubated with nm gp -fc or fc for . h, washed with facs buffer ++ ( % fcs in pbs with ca + /mg + ) and incubated with fitc-conjugated mouse abs directed against human fc fragment ab ( : ) for min. cells were washed twice with facs buffer ++ and fixed in part facs buffer ++ / parts % pfa for min. cells were maintained at • c throughout the processing. bound protein was quantified by fitc channel intensity in a bd facscanto cytometer (bd, frankin lakes, nj, usa) and subtracted from mfi of secondary ab control. to monitor gfp, cells were detached with trypsin, washed with × pbs and fixed with % pfa for min at • c and analyzed with a bd facscanto cytometer. cell-cell fusion/transduction fluorescence quantification analysis was performed at the hannover medical school cell sorting core facility with a bd facsaria fusion (bd, frankin lakes, nj, usa). cells were prepared as described for gfp quantification. the gating strategy for heterokaryon detection and transduction is depicted in figure s . , events were recorded for single cell line controls, , for the pbs treatment control, and , zsgreen positive events (heterokaryon cells) for peg treatment. data was analyzed with flowjo software (tree star, ashland, or, usa). plasmids encoding soluble ebov gp -fc or fc alone were transfected by polyethylenimine into hek tcells. cell supernatants with ebov gp -fc and fc, respectively, were collected and h post transfection, concentrated using a tangential flow cassette (vivaflow ) (sartorius, gottingen, germany) and diluted with an equal amount of mm phosphate buffer (ph ). proteins were purified by affinity chromatography using a hitrap protein g hp ml column (ge healthcare, braunschweig, germany) according to the manufacturer's instructions followed by concentration and size exclusion chromatography using a superose increase / gl column (ge healthcare, braunschweig, germany) equilibrated with mm tris ph , mm nacl. to detect intracellular filovirus entry factors immunoblotting was conducted. cells were lysed in cell lytic m (c , sigma-aldrich, san luis, ms, usa) plus protease inhibitors ( , roche, basel, switzerland). protein concentrations were determined by bradford assay ( , thermo fischer scientific, waltham, ma, usa) using a nanodrop™ spectrophotometer (nd- , thermo fischer scientific, waltham, ma, usa). . µg of protein was solubilized with × dtt sample buffer (tris-hcl, glycerin, sds, dtt, bromophenol), boiled for min at • c, cooled down and resolved by electrophoresis. proteins were blotted to a pvdf membrane (ge healthcare, braunschweig, germany) by mini trans-blot ® (bio rad, hercules, ca, usa) wet transfer system. membranes were blocked and incubated with ab in % milk powder with . % pbs-tween either for h at rt or o/n at • c with constant shaking. protein was detected using the hrp-based ecl chemiluminescence system (ge healthcare, braunschweig, germany) and blots were documented with a las- mini (fujifilm, düsseldorf, germany). cells were grown to about % confluency on cm dishes prior to extraction. total rna was extracted as instructed in the rneasy midi kit (qiagen, hilden, germany) guidelines. microarray analysis was carried out at the research core unit transcriptomics of hannover medical school. the whole human genome oligo microarray kit x k v (g a, design id , agilent technologies, santa clara, ca, usa), covering probes and about , transcripts, was used to analyze mrna levels. quick amp labeling kit, two-color ( - , agilent technologies, santa clara, ca, united states) was used for the generation of cy -or cy -labelled crna. raw data was acquired as relative light units (rlu). microarray data has been deposited in public genomic data repository gene expression omnibus (geo) under the accession number gse . microarray data was compiled for gene expression clustering analysis among studied cell lines. briefly, matrices were prepared by assigning attributing individual expression values of each gene (row) versus the corresponding cell line (column). gene probes that yielded no detectable signal in any of the studied cell lines were excluded from the analysis. an unbiased heatmap was generated from these matrixes through the r software heatmpap. gplot package [ ] by applying the "euclidean" distance method and the "average" (upgma) clustering method. color scales represent values between the maximum and minimum expression values of the individual cell lines. cysteine proteases cathepsin b and l proteolytic activities were measured using the fluorometric cathepsin l (ab ) and cathepsin b (ab ) activity assay kit (abcam, cambridge, uk) according to the manufacturer´s protocol with the modification of the starting material. × cells were spun down, washed and lysed in µl chilled cl lysis buffer. lysates were split into wells with equal volumes (≈ . × cells/well) for intra-assay variation evaluation. fluorometric values were recorded with a microplate reader using a bio-tek flx multifunction fluorescence luminescence microplate reader (bio-tek, winooski, usa) with a / emission filter. npc and tpc / functionality was evaluated by unesterified cholesterol and egf endolysosomal accumulation, respectively. × hek t or × sh-sy y cells were seeded onto poly-l-lysine coated coverslips in a -well plate. on the next day, cells were treated with vehicle (dmso) control or target-specific drugs for h. twenty-four hours post-treatment, cells for npc -mediated cholesterol shuttling analysis were washed and fixed with % pfa in pbs for min at rt. furthermore, cells for tpc / functionality assessment were incubated for min with µg/ml egf-alexafluor conjugated protein in serum-free dmem without or with µg/ml tetrandrine. subsequently, cells were washed with serum-free dmem and treatments applied for extra . h in normal culture medium. finally, cells were washed and fixed as for cholesterol accumulation. fluorescence microscopy was used to assess npc or tpc / functionality or heterokaryon formation and the susceptibility of heterokaryons to filovirus gp-mediated transduction. sample preparation was performed as described previously [ ] . for functionality assays, cells were fixed with % paraformaldehyde in pbs for min followed by quenching of the remaining fixative with mm nh cl for min and permeabilization with . % tx for exactly min. the cells were washed in pbs and blocked with . % bsa in pbs for min. to assess the functionality of npc or tpc / , samples were then stained with . % bsa/pbs solution containing µg/ml filipin iii (unesterified cholesterol) or . mg/ml dapi, respectively, for min followed by extensive washing. all incubations were done at rt. lastly, coverslips were washed with h o and mounted with mowiol containing . % (wt/vol) , -diazabicyclo-[ . . ]octane on glass slides. for cell-cell fusion/transduction assays, coverslips were processed identically as for tpc / samples. egf accumulation (tpc / ) and heterokaryon formation and transduction were analyzed at a confocal fluorescence microscope (tcs sp , leica microsystems, wetzlar, germany) with plan-apochromat ×/ . oil immersion objectives, and -, -, -, and -nm lasers. cholesterol accumulation (npc ) was analyzed at a leica dm b epifluorescence microscope (leica microsystems, wetzlar, germany) coupled to a uv light filter. pictures were acquired with a x magnification objective. documentation was performed with las af lite software (leica microsystems, wetzlar, germany) and image processing with imagej /fiji package [ ] or adobe photoshop cs (adobe systems inc., mountain view, ca, usa). cells expressing either dox-inducible tet-on g transactivator (huh- . , sh-sy y) or zsgreen under the control of the p treg v promoter (hek t-h ) were chemically fused. cell-cell fusion methodology has been previously described [ ] . × huh- . or × sh-sy y cells were coseeded with × hek t-h cells in -well plates. twenty-four hours after coculture, the cells had reached about - % confluency increasing the chance of cell-cell contacts and hence fusion efficiency. cocultured cells were either treated with µl pbs as fusion control or with pre-warmed % peg ( roche, sigma-aldrich, san luis, ms, usa) as fusogenic agent for min at • c. after incubation, any trace of peg was removed by extensive washing with pbs. finally, cells were allowed to recover for at least h at • c with fresh new media prior to further experimentation. unless stated otherwise, experiments were carried out as biological replicates with technical replicates for intra-assay control and quantified as the mean of the data points with error bars representing standard deviation (sd). viral transductions were performed with different viral stocks. microscopy experiments were performed twice for functionality assays. cell-cell fusion/filovirus and transduction was documented once by microscopy and quantified by flow cytometry in three independent experiments. data analysis and representation was done with graph pad prism statistical software (la jolla, ca, usa). multiple t tests was performed for statistical significance discovery correcting for multiple comparison with the holm-sidak method. p-value significance was represented as: n.s. p > . ; * p ≤ . ; ** p ≤ . ; *** p ≤ . ; **** p ≤ . to identify potential cell-type differences in filovirus susceptibility, we transduced a panel of twelve cell lines with hiv-based particles coding for the reporter luciferase and pseudotyped with the figure a,b) . particles bearing no gp did not result in any reporter expression, while vsv-g particles lead to strong luciferase activity in all cell lines. susceptibility to filovirus gp-driven transduction was heterogeneous with the endothelial cell lines hpmec and ea.hy being much more susceptible than the fibroblasts and epithelial cells, and the lymphocytic cell line jurkat being resistant, as reported before [ ] . remarkably, the adherent, neuroblastoma cell lines sk-n-mc and sh-sy y [ , ] were also not susceptible to filovirus gp-mediated transduction. due to this unexpected phenotype, we set out to investigate the determinants underlying the observed resistance. to identify potential cell-type differences in filovirus susceptibility, we transduced a panel of twelve cell lines with hiv-based particles coding for the reporter luciferase and pseudotyped with the ebov strain mayinga gp (ebovpp), the marv strain musoke gp (marvpp), the vsv -g indiana strain g (vsv-gpp), or lacking any viral gp (noenvpp). at h post transduction, we measured the luciferase activity and normalized it for each cell line to the one obtained upon vsv -gpp transduction ( figure a,b) . particles bearing no gp did not result in any reporter expression, while vsv-g particles lead to strong luciferase activity in all cell lines. susceptibility to filovirus gpdriven transduction was heterogeneous with the endothelial cell lines hpmec and ea.hy being much more susceptible than the fibroblasts and epithelial cells, and the lymphocytic cell line jurkat being resistant, as reported before [ ] . remarkably, the adherent, neuroblastoma cell lines sk-n-mc and sh-sy y [ , ] were also not susceptible to filovirus gp-mediated transduction. due to this unexpected phenotype, we set out to investigate the determinants underlying the observed resistance. (vsv-gpp) were used as negative and positive control, respectively. seventy-two hours post transduction, cells were lysed and luciferase activity was measured. data were normalized to vsv-gpp activity as indicated in material and methods. graphs plot the mean values of three independent experiments performed in triplicate (n = ) with error bars representing the standard deviation (sd). individual values are represented as dots, squares or triangles. to validate our results obtained with lentiviral pseudotypes, we infected the different cell lines with the replication-competent vsv virus bearing the ebov gp (rvsv∆g-ebov-gp) or with authentic filoviruses. we inoculated the susceptible hek t and sk-n-be ( ) (figure a,b) . of note, tubulin was also degraded in the susceptible sk-n-be( )-c cells but not in the resistant sh-sy y upon inoculation, a sign of extensive infection previously observed in our lab. the sk-n-mc did not express any ebov np protein, but displayed sporadic marv np labelling in few cells. therefore, we focused our further analyses on sh-sy y cells. next, we inoculated hek t or sh-sy y cells with rvsv∆g-ebovgp virus at a moi of . we evaluated cell rounding and detachment, an established phenotype of ebov gp-expressing cells [ ] , and measured the titers of released virus at hpi. in line with our other experiments, the hek t cells had initiated cell rounding at hpi ( figure s ), and completed it at hpi ( figure c ), while the morphology of the sh-sy y cells had not changed upon inoculation by h (figure s ) or h ( figure c ). furthermore, viral titers had increased by -fold in hek t but decreased in sh-sy y by hpi ( figure d) . altogether, these experiments demonstrated that sh-sy y cells were resistant to filovirus infection. the genus ebolavirus comprises five species: zaire ebolavirus (ebola virus (ebov)), sudan ebolavirus (sudan virus (sudv)), reston ebolavirus (reston virus (restv)), bundibugyo ebolavirus (bundibugyo virus (bdbv)), and taï forest ebolavirus (taï forest virus (tafv)) [ ] . moreover, a new filovirus species, lloviu cuevavirus (lloviu virus (llov)), was discovered in spain in [ ] . we asked whether the gps of these viruses could mediate productive entry in sh-sy y cells. to test this, we transduced either hek t or sh-sy y cells with pseudoparticles bearing no viral envelope protein (noenvpp) or gps from the respective ebola species or lloviu cuevavirus. for ebov, we used gps of mayinga strain, makona strain (wt), and the cell entry-enhancing makona variant a v [ , ] . vsv-gpp served as positive control ( figure a ). consistent with our other results, hek t cells but not the sh-sy y cells were susceptible to transduction mediated by all gps (figure a) . the genus ebolavirus comprises five species: zaire ebolavirus (ebola virus (ebov)), sudan ebolavirus (sudan virus (sudv)), reston ebolavirus (reston virus (restv)), bundibugyo ebolavirus (bundibugyo virus (bdbv)), and taï forest ebolavirus (taï forest virus (tafv)) [ ] . moreover, a new filovirus species, lloviu cuevavirus (lloviu virus (llov)), was discovered in spain in [ ] . we asked whether the gps of these viruses could mediate productive entry in sh-sy y cells. to test this, we transduced either hek t or sh-sy y cells with pseudoparticles bearing no viral envelope protein (noenvpp) or gps from the respective ebola species or lloviu cuevavirus. for ebov, we used gps of mayinga strain, makona strain (wt), and the cell entry-enhancing makona variant a v [ , ] . vsv-gpp served as positive control ( figure a ). consistent with our other results, hek t cells but not the sh-sy y cells were susceptible to transduction mediated by all gps ( figure a ). to determine whether sh-sy y cells were susceptible to transduction driven by other viral gps, we generated pseudoparticles encoding for gfp and bearing chikungunya virus envelope proteins to determine whether sh-sy y cells were susceptible to transduction driven by other viral gps, we generated pseudoparticles encoding for gfp and bearing chikungunya virus envelope proteins (chikv e -e ), severe acute respiratory syndrome virus s protein (sars-s), lassa virus gpc (lasv-gpc), rabies virus g protein (rabv-g), or vsv-g gps ( figure b ). filovirus susceptible huh- . and hek t cells ( figure a -b) as well as resistant sh-sy y and jurkat cells were transduced at a moi of . . while huh- . cells were susceptible to all pseudoparticles tested, jurkat cells were effectively transduced only with vsv-gpp. hek t cells were susceptible to all pseudoparticles with the exception of sarspp, which could only transduce huh- . cells. lasvpp efficiently transduced epithelial huh- . and hek t cells. sh-sy y cells were susceptible not only to vsv-gpp, but also to transduction driven by rabv-g and chikv e -e . in addition, marginal transduction was detected with lasv-gpc pseudoparticles. in summary, sh-sy y cells are resistant to transduction driven by filovirus gps but not by the majority of the other viral glycoproteins tested. continuous virus-host co-evolution has led to the emergence of host restriction factors that limit many infections, and of viral proteins that counteract these intrinsic defense mechanisms. for instance, hiv- has evolved proteins that target the restriction factors apobec g, trim , or serinc (reviewed in [ ] ). similarly, ebov gp antagonizes the antiviral protein tetherin [ ] . to investigate whether the resistance of sh-sy y cells to gp-driven transduction was due to the expression of a host restriction factor, we used doxycycline (dox)-controlled transcriptional activation [ ] , and combined it with experimental cell-cell fusion assays [ ] . this combination allowed us to differentiate between heterokaryons of two different cell types expressing either the transactivator or its inducible counterpart from homokaryons derived from only one cell type. in the latter case, the inducible gene would not be activated, and hence there would be no reporter gene expression even in the presence of dox ( figure s ). we engineered huh- . and sh-sy y cells to express transactivator proteins, and hek t-h cells to express the tet-inducible zsgreen reporter gene. we next assessed whether noenvpp, ebovpp, marvpp, or vsv-gpp lentiviral particles expressing the mcherry reporter were able to transduce hek t-h cells, huh- . cells, sh-sy y cells, hek t-h /huh- . heterokaryons, or hek t-h /sh-sy y heterokaryons. the engineered cell lines exhibited the same susceptibility to gp-driven transduction as the respective parental cell lines ( figure b and figure s a ). moreover, heterokaryons of susceptible hek t and huh- . cells or of hek t and sh-sy y cells were susceptible to gp-driven transduction ( figure a ,b and figure s b ). importantly, reporter expression depended on viral gps, since noenvpp did not transduce heterokaryons. taken together, heterokaryons from resistant and susceptible cells were transduced by pseudoparticles bearing filovirus gps, suggesting that sh-sy y cells do not express a specific restriction factor but rather lack an important enabling filovirus entry factor, which is delivered to resistant cells by fusion with susceptible cells. to further investigate the resistance phenotype of sh-sy y cells, we analyzed protein expression ( figure a ,c,e) and functionality ( figure b ,d,f) of the intracellular proteins operating during the endolysosomal phase of filovirus infection [ ] [ ] [ ] . cathepsin l was expressed to a similar extend in all cell lines, while expression of procathepsin b ( kda) and the active cathepsin b isoforms ( kda and kda) varied among cell lines but did not correlate with susceptibility to filovirus infection ( figure a ). for instance, sh-sy y cells expressed the kda active isoform at a level comparable to that of susceptible hpmec cells. in addition, we measured substrate-specific cleavage activity of cathepsin b and cathepsin l in the cell lysates ( figure b ), but the relative cleavage activities did not correlate with susceptibility to filovirus gp-driven transduction. notably, the resistant sh-sy y cells showed cathepsin b and cathepsin l substrate-specific cleavage activity, suggesting that gp-processing can occur on this cell line. finally, specific inhibition of cathepsin b/cathepsin l with fydmk [ ] confirmed that filovirus gp-driven transduction into susceptible cell lines required cathepsin b and cathepsin l activity as expected ( figure s a ). the bona fide receptor npc was ubiquitously expressed but its relative expression did not correlate with filovirus susceptibility ( figure c ). for instance, hpmec, the cell line with the highest susceptibility to ebov or marv gp-mediated transduction ( figure a,b) , displayed the lowest npc expression. we also assessed npc functionality in hek t or sh-sy y cells by inhibiting its cholesterol transport activity with u a [ ] (figure d ). in untreated cells, cholesterol was distributed throughout the entire cells with the highest signal at the plasma membrane. in contrast and as reported before [ , ] , cholesterol accumulated more in the cell center in u a treated cells, most likely in late endosomes and endolysosomes, indicating that these cells expressed functional npc . finally, u a treatment inhibited filovirus gp-driven transduction of susceptible cells ( figure s b ), confirming that entry into these cells is npc dependent. in sum, the data show that sh-sy y cells express functional npc . similarly, all cell lines expressed also the endosome traffic regulators tpc and tpc ( figure e ). as for the other intracellular host factors, tpc relative expression was heterogeneous and did not correlate with susceptibility to transduction driven by the gps of ebov or marv. on the other hand, tpc was expressed to a similar extent with sh-sy y cells displaying the lowest level of tpc . to test whether tpc accounted for the resistance of sh-sy y cells to gp-driven transduction, susceptible hek t and resistant sh-sy y cells were treated with the inhibitor tetrandrine, which blocks tpc / and results in epidermal growth factor (egf) accumulation in lamp- positive endosomes [ ] . both cell lines displayed larger and brighter organelles after drug treatment, indicating egf accumulation and suggesting that also tpc / were functional in sh-sy y cells. finally, tetrandrine inhibited filovirus gp-driven transduction of all susceptible cell lines tested as expected ( figure s c) . collectively, these results confirmed the essential functions of cathepsin b, cathepsin l, npc , tpc , and tpc for filovirus infection, and demonstrated that neither an absence nor a dysfunction of these intracellular proteins can explain the resistance of sh-sy y cells to gp-mediated transduction or infection. besides these intracellular proteins essential for filovirus infection, the resistance of sh-sy y could also result from a lack of plasma membrane proteins implicated in filovirus entry. since a long list of host factors seems to contribute to filovirus attachment (table s ) , we performed a global gene profiling analysis. we extracted total rna from our panel of cell lines as well as from primary human hepatocytes (phh) and quantified relative mrna levels by microarrays. we used hierarchical clustering analyses to compare the respective gene expression profiles. figure a represents an unbiased clustering analysis of all detected transcripts and figure b depicts clustering of all filovirus cell surface or soluble extracellular entry factors described so far. the cell lines were grouped in two major clusters by an unbiased analysis ( figure a ). this observation also hold true for the analysis considering only reported entry factors ( figure b) , except that the huh- . cells were outside the cluster closest to phh. whereas one cluster contained most of the susceptible cell lines, the other enclosed all resistant cell lines, including sh-sy y, together with the susceptible hek t, sw , and huh- . cell lines (unbiased analysis only). thus, the gene expression profiles of the susceptible cell lines hek t and sw were more similar to resistant cell lines than to other susceptible lines. moreover, neither the global gene expression profiles nor expression patterns of reported filovirus attachment factors correlated with susceptibility or resistance to filovirus gp-driven cell entry. gene expression of attachment factors implicated in filovirus entry using the heatmaps. of the r library "glplots" package. transcript probes that yielded no detectable signal were removed prior to analysis. heatmaps were generated by plotting cell lines as columns and genes as rows using the "complete" method for clustering and "euclidean" method for distance calculation. in the bar above the heatmaps dark blue represents susceptible cell lines and light blue resistant cell lines. we next analyzed the microarray data in more detail, as the clustering analysis did not reveal any correlation between susceptibility to filovirus infection and host gene expression. we considered gene expression values below relative light units (rlu) on a specific probe as likely negative, ˂ rlu ˂ as uncertain, and above as likely positive (table s ). mrna levels that scored consistently above (ex. tyro ) or below (ex. cd ) the threshold in all cell lines were not further considered. of the remaining, havcr (tim- ) and axl (axl) mrna levels were below the threshold in resistant sh-sy y and jurkat cells but above in some susceptible cell lines. therefore, we use facs to analyze their surface expression (figure ) , as well as of integrin αv and β (figue s ), which have been implicated in filovirus entry too [ , ] , and whose mrna were less abundant in resistant than in susceptible cell lines (table. s ). figure . hierarchical clustering analysis (hca) does not correlate gene expression to susceptibility to filovirus infection. microarray data of cell lines tested for susceptibility to filovirus infection as well as primary human hepatocytes (phh) were clustered based on (a) their global gene expression or (b) gene expression of attachment factors implicated in filovirus entry using the heatmaps. of the r library "glplots" package. transcript probes that yielded no detectable signal were removed prior to analysis. heatmaps were generated by plotting cell lines as columns and genes as rows using the "complete" method for clustering and "euclidean" method for distance calculation. in the bar above the heatmaps dark blue represents susceptible cell lines and light blue resistant cell lines. we next analyzed the microarray data in more detail, as the clustering analysis did not reveal any correlation between susceptibility to filovirus infection and host gene expression. we considered gene expression values below relative light units (rlu) on a specific probe as likely negative, < rlu < as uncertain, and above as likely positive (table s ). mrna levels that scored consistently above (ex. tyro ) or below (ex. cd ) the threshold in all cell lines were not further considered. of the remaining, havcr (tim- ) and axl (axl) mrna levels were below the threshold in resistant sh-sy y and jurkat cells but above in some susceptible cell lines. therefore, we use facs to analyze their surface expression (figure ) , as well as of integrin αv and β ( figure s ), which have been implicated in filovirus entry too [ , ] , and whose mrna were less abundant in resistant than in susceptible cell lines (table s ) . most cell lines had detectable axl expression at the cell surface ( figure a,b) . among the susceptible cell lines, we observed both high levels (hpmec or ea.hy ) and low levels (huh- . , hek t). conversely, tim- was expressed only in three susceptible cell lines (huh- . , a , and -o) ( figure c,d) . integrins αv and β were present in all cells with some susceptible cell lines expressing similar levels to resistant ones ( figure s a-d) . notably, resistant sh-sy y cells lacked or expressed only low levels of all tested plasma membrane proteins. in summary, the presence or absence of a specific cell attachment factor previously implicated in filovirus entry does not explain the resistance of the sh-sy y cells to filovirus infection. most cell lines had detectable axl expression at the cell surface ( figure a,b) . among the susceptible cell lines, we observed both high levels (hpmec or ea.hy ) and low levels (huh- . , hek t). conversely, tim- was expressed only in three susceptible cell lines (huh- . , a , and -o) ( figure c,d) . integrins αv and β were present in all cells with some susceptible cell lines expressing similar levels to resistant ones ( figure s a-d) . notably, resistant sh-sy y cells lacked or expressed only low levels of all tested plasma membrane proteins. in summary, the presence or absence of a specific cell attachment factor previously implicated in filovirus entry does not explain the resistance of the sh-sy y cells to filovirus infection. to further characterize the step of filovirus infection impaired in sh-sy y cells, we studied filovirus gp-specific attachment using recombinant soluble ebov gp fused to human fc or a human fc alone [ ] (figure a ). we chose hepg cells as a control, since they express the gp-binding ctype lectin asgp-r and bind to filovirus gp [ , ] , and compared them with susceptible hek t and resistant sh-sy y cells. ebov gp -fc bound most efficiently to hepg cells and to hek t cells with lower efficiency. however, it did not bind to sh-sy y cells, suggesting that the latter lacks any attachment factor for ebov gp. to further characterize the step of filovirus infection impaired in sh-sy y cells, we studied filovirus gp-specific attachment using recombinant soluble ebov gp fused to human fc or a human fc alone [ ] (figure a ). we chose hepg cells as a control, since they express the gp-binding c-type lectin asgp-r and bind to filovirus gp [ , ] , and compared them with susceptible hek t and resistant sh-sy y cells. ebov gp -fc bound most efficiently to hepg cells and to hek t cells with lower efficiency. however, it did not bind to sh-sy y cells, suggesting that the latter lacks any attachment factor for ebov gp. we next asked whether ectopic expression of surface factors that promote filovirus attachment could rescue gp-driven entry into sh-sy y cells ( figure b ). we chose to overexpress axl and tim- proteins because their expression was either very low or absent in sh-sy y cells, but substantial in a subset of susceptible cell lines ( figure a -d, table s ). furthermore, we overexpressed mer tyrosine kinase since it was expressed well in hek t and sw susceptible cell lines, which had clustered in the transcriptome analysis with the resistant cell lines ( figure b , table s ). finally, we overexpressed dc-sign as one ca + -dependent c-type lectin and npc as negative control. we could confirm expression and correct subcellular localization of these attachment proteins ( figure s a,b) in the respective sh-sy y derived cell lines. next, we transduced the novel overexpressing sh-sy y cell lines, sh-sy y/empty vector, sh-sy y wild type (wt), or hek t with mock, ebovpp, marvpp, vsv-gpp, or noenvpp lentiviral particles coding for firefly luciferase. strikingly, ectopic expression of any of the four attachment protein tested was sufficient for robust transduction of sh-sy y cells by both, ebovpp or marvpp ( figure b ), but not by noenvpp. as expected, npc overexpression did not increase filovirus susceptibility, supporting the notion that filovirus attachment proteins had been missing. among the ectopically expressed proteins, dc-sign increased ebov gp-driven transduction by -fold as compared to sh-sy y wt. tim- , axl, and mer had a less potent yet significant effect on ebov gp-mediated entry with fold change values of . , , or . , respectively. the transduction by marvpp was stronger than by ebovpp for both axl ( . ) and mer ( . ) tyrosine kinases, but not for tim- ( . ). moreover, dc-sign expression augmented marvpp transduction by -fold, but this was not statistically significant. finally, ptdser receptors mer and tim- enhanced vsv-gpp transduction by approx. -fold, as reported before [ , ] . however, axl failed to promote transduction. these results indicate that attachment was the limiting step of filovirus gp-driven entry into sh-sy y cells, and that filoviruses can utilize multiple alternative surface proteins for successful attachment to cells. the large number of host proteins reported to promote filoviral internalization and thus infection prompted us to analyze systematically the cellular requirements for filovirus susceptibility. we tested a panel of immortalized cell lines for ebov and marv gp-mediated transduction to correlate susceptibility to the presence of plasma membrane proteins or endosomal proteins required during the early phases of infection. as previously reported, fibroblast, epithelial, and endothelial cells were susceptible to filovirus gp-driven transduction, whereas the lymphocytic cell line jurkat was fully resistant to pseudoparticle mediated reporter expression [ , ] . however, we identified two neuroblastoma cell lines, sk-n-mc and sh-sy y, which were resistant to filovirus gp-driven transduction. this was unexpected, as most human cell lines previously described to be resistant to filovirus infection are nonadherent or lymphocytic cell lines [ , , ] . sk-n-mc was isolated from metastatic neuroblastoma tissue of a years old caucasian female [ ] . these cells were not susceptible to ebov-gp transduction or ebov infection but supported a low level of marv-gp transduction and marv infection. this disparity might be due to different receptor usage among filoviruses, to different internalization mechanisms, or even indicate a host factor specifically required for ebov but not for marv [ ] . on the other hand, sh-sy y cells, a thrice cloned subline of the neuroblastic sk-n-sh cell line [ , ] , were highly resistant to both ebov and marv by all assays (figures and ) . these cells are susceptible to chikungunya virus, dengue virus, varicella-zoster virus, and neurovirulent poliovirus [ ] [ ] [ ] [ ] . our study revealed that sh-sy y cells are also susceptible to transduction driven by the g protein of rabies virus (figue b) . together, these results suggest that the entry defect observed is largely filovirus specific. according to the current model on filovirus cell entry, one or several proteins on the cell surface facilitate infection in addition to the essential intracellular endosomal proteins. at the plasma membrane, ca + -dependent carbohydrate binding c-type lectins interact with n-glycans attached to filoviral gp [ ] and function as attachment factors [ ] . furthermore, members of the tam (tyro , axl, mer) and tim- ptdser binding protein families enhance filovirus cell entry, tams likely by enhancing macropinocytosis [ ] and tim- by a process depending on gp or ptdser [ , ] . finally, the intracellular proteins cathepsin b and cathepsin l, tpc and tpc , and the bona fide receptor npc are essential for productive filovirus infection in vitro and in vivo [ , , ] . our panel of cell lines was largely lacking expression of lectins at the mrna level with the exception of asgr and asgr , which were expressed on huh- . cells, as reported previously [ ] . the absence of expression of other entry promoting lectins is in line with the tissue distribution of dc-sign, hmgl (dendritic cells and macrophages), l-sign, and lsectin (endothelial cells of the liver and lymph nodes) [ , , ] . therefore, although lectins might contribute to the cell tropism of filoviruses [ ] , clearly they are not essential for filovirus infection. in line with previous studies [ , , , , ] , tam and tim- mrna expression was heterogeneous among the cell lines studied here, suggesting that they are not essential but may facilitate filovirus attachment and internalization. indeed, studies on the role of axl in filovirus cell entry have shown a cell-type-specific effect with antibodies against axl reducing axl-dependent filoviral gp-driven transduction in some axl-positive cell lines but not in others [ ] . similarly, overexpression of dc-sign but not of tim- renders jurkat cells susceptible to filovirus virus-like particles (vlp) [ ] and infection studies with tim- -/mice also suggest a dispensable role of tim- since ebov viral loads are similar in ko and wt mice [ ] . our data clearly demonstrate that tim- , axl, mer, and dc-sign have important yet redundant roles during filovirus entry. this is in line with published studies supporting the function of c-type lectins, tam tyrosine kinases, and tim proteins in cell entry [ , , , , , , [ ] [ ] [ ] . prospective further attachment factor protein expression profiling among susceptible and resistant cell lines could provide even a clearer picture on the cellular requirements for productive filovirus cell entry. in sum, expression of tim- , axl, mer, or dc-sign is an important determinant of susceptibility to filoviruses. finally, the required intracellular endosomal proteins were expressed and functional in all cell lines studied here including the resistant sh-sy y cells, irrespective of their susceptibility to ebov infection, suggesting that they are not the determinants of resistance in sh-sy y cells. a similar scenario has been observed in resistant lymphocytic cell lines, which clearly express npc yet they are resistant to infection [ ] . in fact, the jurkat cells analyzed here had higher npc mrna levels than primary human hepatocytes ( over rlu) (table s ) and expressed npc protein ( figure b ). thus, resistance to filoviral cell entry is more common than previously thought and not restricted to either nonadherent or lymphocytic cells or to the presence of intracellular host factors. the disparity between susceptibility to infection and surface protein expression could be attributed to different reasons. first, resistant cell lines might express a dominant restriction factor that blocks productive internalization or viral fusion (reviewed in [ ] ). however, this was not the case for the sh-sy y cells reported here, as heterokaryons of susceptible hek t-h and sh-sy y cells were also susceptible to filovirus gp-driven transduction. second, filoviruses might rely on different cofactors in different cell types. in jurkat cells, this might be conceivable. an overexpression of dc-sign but not of tim- renders otherwise resistant jurkat cells susceptible to ebov-gp virus-like particles [ ] . moreover, ebov virions bind to resistant jurkat and activated cd + t-cells in a tim- dependent-manner without productive infection [ ] . however, ectopic expression of axl, an inhibitor of the type i interferon signaling [ ] , renders jurkat cells susceptible. this suggests that jurkat cells might be able to resist filovirus infection because they have an intrinsic antiviral innate immune mechanism activated. this hypothesis could be tested in the future by determining whether heterokaryons of resistant jurkats with susceptible cells will be susceptible or resistant to filovirus infection. in sh-sy y cells, however, an ectopic expression of any potential attachment-promoting plasma membrane protein of filoviruses, such as dc-sign, axl, mer, or tim- , conferred susceptibility to filovirus gp-driven transduction or infection. interestingly, the enabling potency was different among attachment factors. these differences may be related to the mechanism by which the attachment factors promote susceptibility in sh-sy y cells. for instance, tam receptors may confer susceptibility by enhancing micropinocytosis, whereas tim- or dc-sign could bind to the virus gp [ , , ] . further mechanistic studies on the role of the studied attachment factors could shed light on the differences observed in gp-mediated filovirus susceptibility in sh-sy y cells. nevertheless, these data highlight the concept that virus-receptor interactions at the cell surface are required for productive entry but several alternative host cell factors can be utilized. to our knowledge, this is the first study which directly shows that several unrelated proteins can be utilized alternatively to initiate filoviral cell entry in one cellular context. these findings may help to explain the broad cell tropism of filoviruses. in summary, we have characterized sh-sy y cells as a novel adherent nonlymphocytic cell line exhibiting a specific resistance to filoviral cell entry, and show that gp-mediated filovirus attachment to cells is impaired. this lack of susceptibility could be circumvented by expression of any one of several unrelated cell surface proteins that had previously been implicated in filoviral cell entry using other cellular backgrounds and experimental settings. we thus provide the most direct experimental evidence to support the current model of filoviral cell entry with alternative unspecific interactions between filoviral particles and plasma membrane host proteins mediating attachment and subsequent infection of target cells. moreover, the filovirus-specific resistance of sh-sy y cells makes this cell line an interesting new tool for studying filoviral cell entry and for potentially identifying new cellular factors in the filoviral entry puzzle. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : rvsv∆g-ebov infection, figure s : gating strategy for detection of heterokaryons and transduced heterokaryons, figure s : susceptibility of single cell lines and heterokaryons of huh- . and hek t-h cells, figure s : intracellular factors inhibition assays, figure s : integrin αv and β cell surface expression, figure s : detection of ectopic expression of filovirus attachment factors, table s : reported filovirus attachment host factors, table s : filovirus entry 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lysosomal cholesterol export and ebola infection the transport of low density lipoprotein-derived cholesterol to the plasma membrane is defective in npc cells the intracellular transport of low density lipoprotein-derived cholesterol is inhibited in chinese hamster ovary cells cultured with -beta-[ -(diethylamino)ethoxy]androst- -en- -one analysis of ebola virus entry into macrophages enveloped viruses disable innate immune responses in dendritic cells by direct activation of tam receptors coordinate morphological and biochemical interconversion of human neuroblastoma cells differentiated neuroblastoma cells provide a highly efficient model for studies of productive varicella-zoster virus infection of neuronal cells differences in replication of attenuated and neurovirulent polioviruses in human neuroblastoma cell line sh-sy y dengue virus induces apoptosis in sh-sy y human neuroblastoma cells characterization of chikungunya virus infection in human neuroblastoma sh-sy y cells: role of apoptosis in neuronal cell death how c-type lectins detect pathogens interaction between tim- and npc is important for cellular entry of ebola virus tyrosine kinase receptor axl enhances entry of zaire ebolavirus without direct interactions with the viral glycoprotein ebola virus binding to tim- on t lymphocytes induces a cytokine storm gp mrna of ebola virus is edited by the ebola virus polymerase and by t and vaccinia virus polymerases dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein, of severe acute respiratory syndrome coronavirus role of phosphatidylserine receptors in enveloped virus infection the authors declare no conflict of interest. key: cord- -tnxokfwu authors: kim, sung-jae; nguyen, van-giap; huynh, thi-my-le; park, yong-ho; park, bong-kyun; chung, hee-chun title: molecular characterization of porcine epidemic diarrhea virus and its new genetic classification based on the nucleocapsid gene date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: tnxokfwu porcine epidemic diarrhea virus (pedv) causes continuous, significant damage to the swine industry worldwide. by rt-pcr-based methods, this study demonstrated the ongoing presence of pedv in pigs of all ages in korea at the average detection rate of . %. by the application of bayesian phylogenetic analysis, it was found that the nucleocapsid (n) gene of pedv could evolve at similar rates to the spike (s) gene at the order of (− ) substitutions/site/year. based on branching patterns of pedv strains, three main n gene-base genogroups (n , n , and n ) and two sub-genogroups (n a, n b) were proposed in this study. by analyzing the antigenic index, possible antigenic differences also emerged in both the spike and nucleocapsid proteins between the three genogroups. the antigenic indexes of genogroup n strains were significantly lower compared with those of genogroups n and n strains in the b-cell epitope of the nucleocapsid protein. similarly, significantly lower antigenic indexes in some parts of the b-cell epitope sequences of the spike protein (coe, s d, and c ) were also identified. pedv mutants derived from genetic mutations of the s and n genes may cause severe damage to swine farms by evading established host immunities. porcine epidemic diarrhea virus (pedv) is an enveloped, single-stranded rna virus belonging to the family coronaviridae, subfamily coronavirinae, genus alphacoronavirus, and subgenus pedacovirus. pedv is one of the major pathogens causing acute enteritis disease, which is characterized by vomiting and watery diarrhea and commonly leads to high rates of mortality and morbidity in suckling piglets [ ] . the disease was first reported in the uk in , and the prototype virus-designated as pedv cv -was subsequently identified in belgium [ ] . since the s, pedv has been widespread throughout asia, where it has been regarded as an endemic disease for many years [ , ] . in the late s, new and highly pathogenic strains were reported in china. these new strains were pathologically more critical than the classic strains, resulting in morbidities of - % and mortality rates of - % in infected suckling piglets [ ] . in may , these new highly pathogenic strains moved from china to the usa and rapidly spread across the country, massively impacting the swine industry; they affected more than farms, accounting for the death of more than million piglets within the year [ , ] . subsequently, these strains have become pandemic [ ] . pedv has an approximately kb long genome and consists of seven open reading frames (orf), encoding non-structural or structural proteins [ ] . orf ab and orf genes encode non-structural proteins. orf a codes for the large polyprotein pp a, while orf b is always expressed with pp a as the fusion protein pp a/b through ribosomal frameshifting. pp a and pp a/b are further processed into non-structural proteins (nsp to nsp ). orf codes an accessory protein that is likely to be an additional non-structural protein [ ] . the envelope (e), membrane (m), spike (s), and nucleocapsid (n) genes encode four major structural proteins [ ] . the n protein, which is the most abundant protein in the virus particle, provides the structural basis for the helical nucleocapsid surrounding the virus genome [ , ] . the m and e proteins form a viral envelope by assembly. the m protein is the most abundant component, and the e protein is less abundant in the viral envelope. [ ] . the s protein is very exposed and forms large petal-shaped spikes on the surface of the virus [ ] . among these structural proteins, the s protein plays important roles in virus infection and the induction of neutralizing antibodies [ ] and shows substantial genetic diversity [ ] . as a result of these features, genetic analyses based on the s gene have been commonly used to investigate pedv evolution [ , , ] . the pedv has diverged into several subgroups based on the genetic diversification of the s gene. so far, two genotypes (g and g ) have been identified by s gene phylogenetic analysis. each genogroup can be further divided into two subtypes, g a/b and g a/b, respectively [ , ] . recently, a third subtype (g c) was identified within the g genogroup [ ] . the n protein is involved in several biological and immunological activities of the virus, including viral nucleolar localization, host cycle er stress, s-phase prolongation, the inhibition of interferon-β production [ ] [ ] [ ] , and the induction of abundant antibodies [ , ] . despite the significant features of the n protein, there has been less investigation on the n protein compared to the s protein. in order to better understand the evolutionary aspects of pedv, this study applied multiple bioinformatic tools to investigate the genetic diversity of pedv. six hundred and seventy-two fecal samples were collected from farms in , and fecal samples were collected from farms in . rectal swabs were randomly collected from pigs of all stages (suckling, weaned, growing, finishing, gilt, sow) in swine farms dispersed throughout provinces of south korea. rna extraction from the samples was performed using an rna/dna extraction kit (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions, and the extracted rna samples were stored at − • c. the rna was converted into cdna with a commercial kit (rna to cdna ecodry premix, clontech, otsu, japan), following the manufacturer's protocol. pedv in the collected fecal samples was detected by pedv-pcr [ ] . commercial pcr kits (median diagnostics, chuncheon-si" korea) were used to detect porcine reproductive and respiratory syndrome virus (cat. ns-prr- ), porcine parvovirus (cat. ns-abo- ), and japanese encephalitis virus (cat. ns-abo- ). the other three enteric pathogens were detected by specific primers and pcr conditions reported previously, such as porcine deltacoronavirus [ ] , transmissible gastroenteritis virus, and porcine group a rotavirus [ ] . full-length genome sequencing was conducted with overlapping primer pairs using positive samples from swine farms that were severely damaged by porcine epidemic diarrhea [ ] , and strains (y , s , s , s , s , s and s ) were fully sequenced. information relating to the strains is provided in table . the full-length genome sequences of the strains were registered in genbank (accession numbers mh -mh ). for the genetic analyses, other previously registered complete genome sequences were retrieved from genbank, and the sequences originated from asia (china, korea, japan), america (usa), and europe (germany, belgium) from to . rdp v . program [ ] with default algorithms (rdp, geneconv, maxchi, siscan, and bootscan) was applied to identify recombinants in the alignment of the s and n genes. the general options were "sequences are linear", "highest acceptable p-value = . ", and "bonferroni correction" = true. upon detection, new recombination-free alignments were created by the option of "save alignment with recombinant regions removed". four datasets with identical number of sequences (n = , seven obtained in this study) were generated for subsequent analyses: complete s gene (d ), s gene without recombinant regions (d ), complete n gene (d ), and n gene without recombinant regions (d ). beast package v . . [ ] , which is available at the cipres science gateway [ ] , was used to infer the phylogenetic relationships between sequences and co-estimate the substitution rates from the above-mentioned d -d datasets. the genetic classification of pedv based on the s gene followed previous publication [ ] . for the model of nucleotide substitution, bmodeltest tool [ ] implemented in beast was selected, which helps infer the most appropriate substitution model. for the molecular clock model, four models were specified: strict clock, uncorrelated lognormal and exponential relaxed-clock [ ] , and random local clock [ ] . for tree prior, three coalescent models implemented in beast were tested, including coalescent constant population, coalescent exponential population, and coalescent bayesian skyline plot [ ] . each analysis was run for million chains, sampling every , generations. the output log files were analyzed in tracer v . . [ ] to assess the convergence (effective sample size > ). path sampling analyses [ ] were also performed to select the best fitting molecular clock and tree prior models for each dataset. for that analysis, the number of path steps was , and the length of each chain was one million iterations. the nucleotide substitution rates and phylogenetic tree of each d -d dataset were inferred from the data best-fit combining models (supplementary material tables s and s ). the phylogenic trees were summarized with treeannotator v . . to produce the maximum clade credibility tree, which was displayed using figtree v . . . pairwise genetic distances (p-distances) within each dataset were calculated using mega v. software [ ] . the option for gaps/missing data treatment was specified as "partial deletion". the obtained results were displayed in a frequency distribution histogram of p-distance. basically, a lower genetic relationship between two sequences indicated a higher p-distance, and well-bounded areas with peaks in the histogram indicated the presence of different genetic clusters [ ] . the baseml program implemented in package paml . j [ ] was used to reconstruct amino acid changes on the evolutionary path of pedv based on the n gene. the input tree topology for that analysis was the maximum clade credibility tree inferred by beast under the data best-fit combining models. nonsynonymous substitutions that occurred on the given branches of a phylogeny were annotated by the treesub program [ ] . amino acid sequences deduced from nucleotide sequences of the n gene were aligned and comparatively analyzed to investigate the nonsynonymous changes according to genetically distinct clusters. subsequently, antigenic index analysis of the complete amino acid sequences of the n gene was performed to evaluate the possible antigenic variation of n proteins. the antigenic index of each amino acid was calculated by the jameson-wolf method [ ] , and the calculated indexes of each strain were compared. antigenic index analyses were performed to evaluate the antigenic variation of s proteins within the korean pedv strains. the antigenic index of each amino acid was calculated by the jameson-wolf method for previously identified s protein-neutralizing epitopes, coe (within the s b region) [ ] , s d [ ] , and c [ ] . subsequently, the calculated indexes of the korean strains were compared. the detection rate of pedv from to was . % ( / ). specifically, the positive rates in and were . % ( / ) and . % ( / ), respectively. the positive rate in had somewhat increased compared with that in . from the detection rates of each growth stage, the highest rate was seen in the suckling stage (table ) . geographically, the province with a higher concentration of swine farms showed higher positive samples of pedv (figure ). in the phylogenetic trees inferred from the d -d datasets of the s gene ( figure , supplementary figure s -s ), the pedv strains were classified into five sub-genogroups (g a, g b, g a, g b, and g c), which were previously designated [ ] . however, the relationships between sub-genogroups differed. the d dataset contains the complete s gene supported for the clusters of (g a, g b) and (g c, (g a, g b)). the d dataset contains the s gene without recombinant regions supported for the different clusters of (g b, g c) and (g b, g a). in both datasets, histograms of pairwise p-distances exhibited a discrete distribution betwen sub-genogroups. that was in agreement with the tree topologies and posterior support values at the nodes to each sub-genogroup ( figure ). in both the d and d datasets, the seven korean strains identified in this study (s , s , s , s , s , s , and y ) were clustered within sub-genogroup g a. in the phylogenetic trees inferred from the d -d datasets of the s gene ( figure , supplementary figures s and s ) , the pedv strains were classified into five sub-genogroups (g a, g b, g a, g b, and g c), which were previously designated [ ] . however, the relationships between sub-genogroups differed. the d dataset contains the complete s gene supported for the clusters of (g a, g b) and (g c, (g a, g b) ). the d dataset contains the s gene without recombinant regions supported for the different clusters of (g b, g c) and (g b, g a). in both datasets, histograms of pairwise p-distances exhibited a discrete distribution betwen sub-genogroups. that was in agreement with the tree topologies and posterior support values at the nodes to each sub-genogroup ( figure ). in both the d and d datasets, the seven korean strains identified in this study (s , s , s , s , s , s , and y ) were clustered within sub-genogroup g a. regions (d ) and with the recombinant regions removed (d ). the sub-genogroups were designated as g a, g b, g a, g b, and g c. each sub-genogroup was colored consistently between the d and d datasets. the inserted histograms of pairwise p-distances were between sub-genogroups g a-g b, g a-g a, g a-g b, and g a-g c. the p-distance is calculated by dividing the number of nucleotide differences by the total number of nucleotides compared. after removing recombinant regions, some sequences might contain large deletions. in other words, the total number of nucleotides became smaller. thus, the p-distance on the right panel was larger than that on the left panel. the korean strains identified in this study are marked by arrows. supported by high posterior probability values ( . - ), the phylogenetic trees inferred from the d -d datasets of the n gene (figure , supplementary figures s and s ) suggested that the classification of pedv strains into four s gene-based sub-genogroups g a, g b, g b, g a/g c was more reliable. in both datasets, it was observed that the s gene-based g a and g c were not monophyletic (pink branches). differing from the s gene-based phylogenies (figure ) , the ngene-based trees with or without the elimination of recombinant regions had identical topologies of (g a, (g b, (g b, g a/g c))). as the result, this study proposed an n-based genotyping of pedv as n , n , n b and n a, which were equivalent to the s-based genotyping of g a, g b, g b, and g a/g c, respectively. that classification was also supported by a clear bimodal distribution of genetic distance between the proposed genogroups n -n and n -n a/n b (inserted histograms, figure ). according to that scheme, the seven korean strains identified in this study (s , s , s , s , s , s , and y ) were within sub-genogroup n a. the estimated mean nucleotide substitutions of the s and n genes were at the order of − nucleotide substitutions/site/year ( table ). the substitution rates of the complete s and n genes were not significantly different, as the % highest posterior density (hpd) overlapped (table ) . inferring from two datasets where the recombinant regions were removed, the s gene showed substantially higher substitution rates than the n gene because of non-overlapping % hpd ( . × − - . × − versus . × − - . × − , respectively). the estimated mean nucleotide substitutions of the s and n genes were at the order of − nucleotide substitutions/site/year ( table ). the substitution rates of the complete s and n genes were not significantly different, as the % highest posterior density (hpd) overlapped (table ) . inferring from two datasets where the recombinant regions were removed, the s gene showed substantially higher substitution rates than the n gene because of non-overlapping % hpd ( . × − - . × − versus . × − - . × − , respectively). * random local clock model (rlc). ** the geometric mean nucleotide substitution rate (substitutions/site/year) was inferred from the data that best fit the molecular clock and coalescent tree prior of constant population size, exponential population size, and bayesian skyline plot (bsp). *** highest posterior density (hpd). several consistent changes allowed the differentiation of the genogroups and sub-genogroups based on the n gene (figure , supplementary figure s ). the complete list of nonsynonymous changes was given in the supplementary figure s . genogroup n differed from genogroups n and n by nonsynonymous substitutions (a g, k n, m v, p l, q l, n h, and v a). unique changes leading to branches (n , (n a, n b)) were a t, h l, q l, and e d. genogroup n was further characterized by two nonsynonymous substitutions (a t, k i). finally, the main branch leading to sub-genogroup n a displayed five changes (k n, m v, r k, k r, and n s). in the antigenic index analysis of n gene sequences, significant reductions were found in amino acid positions - according to the genogroups ( figure ). genogroup n exhibited lower antigenic indexes, below . (the cut-off value), compared with genogroups n and n . these amino acid sequences were located in a b-cell epitope sequence of the n protein, which is one of the pedv-specific epitopes (amino acids - and - ) previously identified [ ] . in the antigenic index analysis of n gene sequences, significant reductions were found in amino acid positions - according to the genogroups ( figure ). genogroup n exhibited lower antigenic indexes, below . (the cut-off value), compared with genogroups n and n . these amino acid sequences were located in a b-cell epitope sequence of the n protein, which is one of the pedv-specific epitopes (amino acids - and - ) previously identified [ ] . nonsynonymous substitutions were mapped to the branches of the phylogeny. for clarity, posterior values were shown for main separating nodes. in the antigenic index analysis of n gene sequences, significant reductions were found in amino acid positions - according to the genogroups ( figure ). genogroup n exhibited lower antigenic indexes, below . (the cut-off value), compared with genogroups n and n . these amino acid sequences were located in a b-cell epitope sequence of the n protein, which is one of the pedv-specific epitopes (amino acids - and - ) previously identified [ ] . when analyzing the korean pedv strains-newly identified in this study-based on the three major b-cell epitope sequences of the s protein (coe, s d, and c ), the six strains exhibited significantly lower antigenic indexes in some parts of the b-cell epitope sequences ( figure ). the strains s , s , s , and s exhibited lower antigenic indexes, below the cut-off value of . , in amino acid positions - of the coe region, which contained the nonsynonymous change k n. the s strain exhibited lower antigenic indexes, below the cut-off value of . , in amino acid positions - in the coe region, and the nonsynonymous change e v was observed in this region. the y strain also showed antigenic indexes below . at amino acids - of the s d region, which contained the nonsynonymous change s f. it is likely that each of the mentioned nonsynonymous changes affected the antigenic indexes of the adjacent regions. viruses , , x for peer review of when analyzing the korean pedv strains-newly identified in this study-based on the three major b-cell epitope sequences of the s protein (coe, s d, and c ), the six strains exhibited significantly lower antigenic indexes in some parts of the b-cell epitope sequences ( figure ). the strains s , s , s , and s exhibited lower antigenic indexes, below the cut-off value of . , in amino acid positions - of the coe region, which contained the nonsynonymous change k n. the s strain exhibited lower antigenic indexes, below the cut-off value of . , in amino acid positions - in the coe region, and the nonsynonymous change e v was observed in this region. the y strain also showed antigenic indexes below . at amino acids - of the s d region, which contained the nonsynonymous change s f. it is likely that each of the mentioned nonsynonymous changes affected the antigenic indexes of the adjacent regions. figure . antigenic index analysis of s protein b-cell epitopes in korean pedv strains. strains s , s , s , and s exhibited lower antigenic indexes, below the cut-off value of . , at amino acids figure . antigenic index analysis of s protein b-cell epitopes in korean pedv strains. strains s , s , viruses , , of s , and s exhibited lower antigenic indexes, below the cut-off value of . , at amino acids - of the coe region. strain s exhibited reduced antigenic indexes, below . , at amino acids - of the coe region. strain y exhibited reduced antigenic indexes, below . , at amino acids - in the s d region. first of all, this study reflected the ongoing circulation of pedv in korea at about % (table ) and its wide distribution (figure ). at the same time, obtaining genomic sequences of pedv from field samples provided good opportunity for studying the genetic evolution of the virus. as a result, this study applied multiple bioinformatics tools to investigate the genetic diversity of pedv. based on phylogenetic analysis of the complete s gene or s gene excluding recombinant regions, global pedv strains ( figure ) can be classified into two genogroups, and each genogroup may be further subdivided into two (g a and g b) and three (g a, g b, and g c) sub-genogroups. this result was consistent with that of guo et al. [ ] . additionally, guo et al. reported that all these different subgroups existed in korea, with the most prevalent subgroup being g a, when they analyzed the korean pedv strains identified prior to . similar to the previous data, all the korean field strains identified from to in this study were included in subgroup g a, indicating that the most prevalent korean subgroup has not changed since . genogroups g and g are known to have different s protein neutralization activities [ ] . however, differences within the genogroups have not been investigated. the six korean strains (s , s , s , s , s , and y ) had significantly reduced antigenic indexes compared with other korean strains in some parts of the coe [ ] and s d [ ] , which encode the b-cell epitopes of the s protein. these antigenic index reductions may induce somewhat different s protein antigenicities, even within the same genogroup. in fact, strains s , s , s , and s originated from swine farms that had suckling piglet mortalities of almost %. these swine farms had been regularly using killed vaccines containing the new genogroup (g ) pedv strain but were seriously damaged by porcine epidemic diarrhea. in the literature, the n gene had been used to infer the phylogenetic relationships of pedv strains [ , ] . it was noteworthy that the n gene showed a similar evolutionary rate to the s gene in this study. this high evolutionary rate implies that the n gene, as well as the s gene, is likely to have high genetic diversity; accordingly, several sub-genogroups could have diverged. comparing to the s-based phylogenetic topology, the classical strains (g strains) presented the same topology in the n-based and s-based analysis. however, there were some differences in the classification of subgroups on the new genotype strains (g strains). the n genogroup consisting of the g strains was divided into only subgroups (n a and n b) not following the s-based subgroups (g a, g b and g c). specifically, the n b strains were consistent with the g b strains, but the g a and g c strains were grouped into the same subgroup, n a, in the n-based topology. this classification of the three genogroups (n , n , and n ) was also supported by the consistent variation in the antigenic indexes depending on the genogroups. the number of antigenic indexes of the n strains significantly decreased compared to those of the n and n strains within amino acids - , which code for the b-cell epitope of the n protein [ ] . the pedv n protein has an important immunological aspect. abundant antibodies against the n protein are induced at the early stages of pedv infection [ , ] . furthermore, the n protein is considered to play an important role in inducing cell-mediated immunity [ ] . as a result of these features, the n protein is commonly used as a target for diagnosis and vaccine development [ , ] . as mentioned above, several geno-and sub-genogroups based on the n gene were identified in this study. this genetic diversity may change their antigenicities according to their geno-or sub-genogroup ( figure ). in the immunological diagnosis of pedv, commercial pedv elisa kits have been showing poor performance, and the results of neutralizing assays using cell culture sometimes mismatch with those of the elisa assays. in fact, chang et al. recently reported that the antibodies induced by the g b pedv strain poorly reacted with a commercial n-based elisa kit that showed a sensitivity of % [ ] , which may be the result of antigenicity differences between the genogroups. however, further study is required to validate this hypothesis. indeed, if there are antigenic differences between the genogroups, a combination of n proteins derived from both genogroups would be required for the accurate immunological diagnosis of pedv. overall, this study revealed that pedv displayed genetic diversity in both the s and n genes, which resulted in the divergence into different sub-genogroups and altered antigenic indexes. such pedv mutants derived from genetic mutations of the s and n genes may cause severe damage to swine farms because of their ability to evade the unprepared host immune systems. table s : supplementary-material-table-s -model-comparison- . table s : supplementary-material- table- experimental infection of pigs with a new porcine enteric coronavirus, cv a new coronavirus-like particle associated with diarrhea in swine isolation of porcine epidemic diarrhea virus (pedv) in korea isolation of porcine epidemic diarrhea virus during outbreaks in south korea new variants of porcine epidemic diarrhea virus, china ped virus reinfecting u.s. herds. virus estimated to have killed million-plus pigs distinct characteristics and complex evolution of pedv strains evolutionary and genotypic analyses of global porcine epidemic diarrhea virus strains emerging and re-emerging coronaviruses in pigs porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis the molecular biology of coronaviruses coronavirus immunogens identification of the membrane protein of porcine epidemic diarrhea virus cellular entry of the porcine epidemic diarrhea virus neutralization of genotype porcine epidemic diarrhea virus strains by a novel monoclonal antibody molecular characterization of a korean porcine epidemic diarrhea virus strain nb molecular characterization of us-like and asian non-s indel strains of porcine epidemic diarrhea virus (pedv) that circulated in japan during - and pedvs collected from recurrent outbreaks detection and phylogenetic analysis of porcine epidemic diarrhea virus in central china based on the orf gene and the s gene porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf and tbk molecular characterizations of subcellular localization signals in the nucleocapsid protein of porcine epidemic diarrhea virus porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin- expression detection and phylogenetic analysis of porcine deltacoronavirus in korean swine farms multiplex reverse transcription-pcr for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group a rotavirus phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china rdp : detection and analysis of recombination patterns in virus genomes : an advanced software platform for bayesian evolutionary analysis creating the cipres science gateway for inference of large phylogenetic trees bayesian phylogenetic site model averaging and model comparison relaxed phylogenetics and dating with confidence bayesian random local clocks, or one rate to rule them all bayesian coalescent inference of past population dynamics from molecular sequences posterior summarization in bayesian phylogenetics using tracer . improving the accuracy of demographic and molecular clock model comparison while accommodating phylogenetic uncertainty mega : molecular evolutionary genetics analysis software new phylogenetic groups of torque teno virus identified in eastern taiwan indigenes phylogenetic analysis by maximum likelihood annotating ancestral substitution on a tree the antigenic index: a novel algorithm for predicting antigenic determinants heterogeneity in membrane protein genes of porcine epidemic diarrhea viruses isolated in china spike protein region (aa ) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies the gprlqpy motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus the identification and characterization of two novel epitopes on the nucleocapsid protein of the porcine epidemic diarrhea virus sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china genetic variation of nucleocapsid genes of porcine epidemic diarrhea virus field strains in china development and evaluation of enzyme-linked immunosorbent assay based on recombinant nucleocapsid protein for detection of porcine epidemic diarrhea (pedv) antibodies development and comparison of enzyme-linked immunosorbent assays based on recombinant trimeric full-length and truncated spike proteins for detecting antibodies against porcine epidemic diarrhea virus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to thank eun ok kim and jung ah kim for their excellent technical assistance. the authors declare that there is no conflict of interest. key: cord- -sfr x ob authors: röst, gergely; bartha, ferenc a.; bogya, norbert; boldog, péter; dénes, attila; ferenci, tamás; horváth, krisztina j.; juhász, attila; nagy, csilla; tekeli, tamás; vizi, zsolt; oroszi, beatrix title: early phase of the covid- outbreak in hungary and post-lockdown scenarios date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: sfr x ob covid- epidemic has been suppressed in hungary due to timely non-pharmaceutical interventions, prompting a considerable reduction in the number of contacts and transmission of the virus. this strategy was effective in preventing epidemic growth and reducing the incidence of covid- to low levels. in this report, we present the first epidemiological and statistical analysis of the early phase of the covid- outbreak in hungary. then, we establish an age-structured compartmental model to explore alternative post-lockdown scenarios. we incorporate various factors, such as age-specific measures, seasonal effects, and spatial heterogeneity to project the possible peak size and disease burden of a covid- epidemic wave after the current measures are relaxed. a cluster of pneumonia cases of unknown origin was detected in wuhan city, the capital of hubei province, china, with a population of million in december . on december china alerted the world health organization (who) china country office [ ] . on january the causative pathogen of the pneumonia outbreak was identified as a novel coronavirus, and, on february, the who officially named the novel coronavirus as sars-cov- and the disease it causes as covid- . sars-cov- infection quickly spread from china, where it emerged in december , to europe, where the first cases were confirmed on january in france (where, later in april, covid- was retrospectively confirmed for a patient hospitalized in late december ) [ , ] . around the same time, on january, the first infection in germany was confirmed in bavaria that led to a local outbreak. by february , subsequent cases have been confirmed and high-risk contacts have been identified via agile contact-tracing [ ] . the first epidemic in europe started in the lombardy region of italy with the first detection on february [ ] . the who director-general declared the covid- outbreak a public health emergency of international concern under international health regulations ( ) on january [ ] and then a pandemic on march [ ] . by that time, the number of daily new cases of covid- was over in several countries, including italy, france, and germany. manipulation and shiny version . . . [ ] for creating an interactive dashboard to carry out epidemiological analyses online (available in hungarian [ ] ). the full source code of this dashboard and related analysis is available at [ ] . effective reproduction number (r t ), the average number of secondary cases per primary case for those primary cases who turn infectious on day t, was tracked in real time based on the daily number of reported new cases using the methods of cori et al. [ ] and that of wallinga and teunis [ ] , among the several methods aimed to estimate r t [ , ] . in brief, the method of cori et al., is based on calculating the ratio of the actual number of infections on a day to the total infectiousness of all past cases on that day. thus, it measures r t by assuming that infected individuals will infect in the future as if conditions remain unchanged. in contrast, the method of wallinga and teunis makes no such assumption; it uses a likelihood-based inference on the possible infection networks underlying the epidemic curve. the fundamental difference is that the method of cori et al., solely uses past information ("backward looking approach"), due to which the result is sometimes called instantaneous reproduction number, while the wallinga-teunis method more closely corresponds to the concept of the usual definition of effective reproduction number; however, it requires future information in exchange ("forward looking approach"). for a discussion on the relative merits of these two approaches, see [ , ] . the wallinga-teunis method was used with the addition of cauchemez et al., who aimed to provide real-time estimation capability [ ] . both methods require-in addition to incidence data-information on the serial interval. depending on the used dataset, different estimations of the serial interval have been published: a mean of . days was found in [ ] , and . days in [ ] . here, we assume an intermediate value following [ ] , where the mean and standard deviation (sd) of the serial interval were estimated at . days ( % cri: . , . ) and . days ( % cri: . , . ), respectively. (the serial interval is assumed to follow gamma distribution.) they also concluded that the serial interval of covid- is close to or shorter than its median incubation period, which is coherent with our choice of parameters in the transmission dynamics model. the estimation was carried out using r packages r version . - [ , ] and epiestim version . - [ , ] . case fatality rate (cfr) is defined as the (conditional) probability of death from a disease for those contracting the disease (for diseases where asymptomatic state also exists, infection fatality rate (ifr) is defined analogously) and is estimated as the ratio of cumulative deaths and cumulative cases. this definition, i.e., where c t and d t are the daily, c t and d t are cumulative number of cases and deaths, respectively, on day t is however biased when used during the epidemic (thus the name crude/naive cfr or ncfr). the reason for this is that a proportion of cases counted in the denominator will die (in the future), thus they should have been counted in the numerator as well, but, as they're not, the ratio underestimates the true value [ , ] . fortunately, it is relatively easy to correct for this bias using information on the distribution of the diagnosis-to-death time [ , ] . the likelihood that the cumulative number of deaths on day t is d t is given by where f i denotes the (conditional) probability that death happens on day i after onset (for those who die) and π stands for the true value of the cfr. this observation allows both maximum likelihood and bayesian estimation for π using the observed series of c i and d i , the latter of which was employed in the present study, using a beta( , ) (i.e., uniform) prior. it was assumed that diagnosis-to-death time follows lognormal distribution with a mean of days and a standard deviation of . days, as found by linton et al. [ ] . the bayesian estimation was manually coded using the r package rstan version . . [ ] . a markov chain monte carlo approach was used to carry out the estimation with no-u-turn sampler, using chains, warmup iterations and iterations for each chain. the cfr mentioned in the previous subsection is still not the true value of the fraction of fatal outcomes of all infections, as there is another source of bias, but this time leading to overestimation: the underascertainment of cases. this is a substantial issue now as a-precisely not yet known, but epidemiologically significant-fraction of the covid- cases are asymptomatic or mildly symptomatic. since in many countries testing was extended to contacts (and in a few instances, even random sampling was carried out), the confirmed cases include some asymptomatic cases as well. however, the value of the estimated (corrected) ifr can also be used to estimate the ascertainment rate: by assuming that the ifr in reality takes a benchmark value (one derived from large-sample, well-designed studies accounting for underascertainment or sero-epidemiological surveys) and-crucially-assuming that the difference of the actual estimated ifr from that value is purely due to underascertainment. then, the ascertainment rate can be obtained by simply dividing the assumed true value of the ifr with the actual estimated cfr [ ] . note that this might be a strong assumption, as it rules out that there is a real difference in the country's ifr from the benchmark value; in particular, it rules out different virulence of the pathogen, different age-and comorbidity-composition in the country and different effect of the healthcare system on survival. mathematical models have been developed to better understand the global spread [ ] and the transmission dynamics of covid- for many countries, including australia [ ] , france [ ] , germany [ ] , uk [ ] , and the usa [ , ] . such models have been used to project the progress of the outbreak and to estimate the impact of control measures on reducing disease burden. the two most common approaches are compartmental models formulated as systems of ordinary differential equations, and agent based models used to generate an ensemble of stochastic simulations for possible outcomes. here, we establish a compartmental population model, adjusted to the specific characteristics of covid- , considering the following compartments. we denote by s the susceptibles, i.e., those who can be infected by the disease. latents (l) are those who have already contracted the disease but do not show symptoms and are not infectious yet. in accordance with studies indicating that viral shedding peaks before the onset of symptoms [ ] , in our model, we have introduced the presymptomatic infected compartment i p for those who do not have symptoms, but who already are capable of transmitting the disease to susceptibles. we divided the latent period into two compartments l and l , thus, together with i p , the incubation period follows a hypoexponential distribution, having a shape matching empirical observations [ , ] . since a large fraction of infected shows only mild or no symptoms, after the incubation period, we differentiate these individuals from those with symptoms. we assume a gamma-distributed infectious period with erlang parameter m = , similar to the sars study [ ] , hence, we have three classes for both asymptomatic and symptomatic infectious individuals (i a, , i a, , i a, and i s, , i s, , i s, , respectively). individuals from the i a, compartment will all recover and hence proceed to the recovered class r. immunity is assumed for those who have recovered from the disease, at least for the time scale of this modeling. individuals from i s, may either recover without requiring hospital treatment (and thus move to r) or become hospitalized. it is of crucial importance to project the number of hospital beds and intensive care unit (icu) beds needed; thus, in the model, we further differentiate symptomatically infected individuals who need hospital care and critical care, denoted by i h and i c , respectively. we operate with the assumption that the healthcare system will not be overwhelmed, and thus disease-induced death is only considered from critical care that fits with the data obtained from nphc. hence, individuals from i h will proceed to r after recovery. those from i c with fatal outcome transit to the d compartment. those who are out of icu and on the path to recovery are collected into the i cr , from where they eventually recover and move to the r class. to take into account the different characteristics of the disease in various age groups, we stratified the hungarian population into seven groups, corresponding to the available choices in the hungarian online questionnaire for the assessment of changes in the number of contacts following the lockdown [ ] . the compartments listed above corresponding to the different age groups are denoted by an upper index i ∈ , . . . , . accordingly, all of our parameters can be calibrated age-specifically. the transmission rates from age group k to age group i are denoted by β (k,i) j , with j ∈ {p, a, s}, where the three subscripts p, a, s stand for presymptomatic, asymptomatic, and symptomatic infected, respectively. the parameters described in the following all have an upper index i which stands for the corresponding age group. a fraction p i of exposed people will not show symptoms during his/her infection, while ( − p i ) will develop symptoms. the average length of the incubation period is (α i l, ) − + (α i l, ) − + (α i p ) − days, with the transition rates α i l, , α i l, , α i p , respectively. similarly, the average infectious period of asymptomatic and symptomatic infected individuals are with the corresponding transition rates, respectively. a fraction h i of the infectious compartment i i s, will be hospitalized, the remaining fraction − h i will recover without hospital care. out of those who need hospitalization, a fraction ξ i needs intensive care. for the hospitalized classes i i h , i i c , i i cr , the average time spent in these compartments is given as (γ i h ) − , (γ i c ) − and (γ i cr ) − , respectively. a fraction µ i of those leaving the i i c compartment will die due to the disease, while the remaining fraction will proceed to the i i cr class. the transmission dynamics of our model for one age group is illustrated in figure . . reproduction numbers are calculated using the next generation matrix method in section . . . we discuss the application and, then, some limitations of this model in sections . . and . . , respectively. the codes were implemented in wolfram mathematica and are available at [ ] . the governing equations of the transmission model described in section . take the form where the index i ∈ { , . . . , } represents the corresponding age group. next, we add the spatial locations of the population to the previous model. the population is divided into patches, where each patch represents a separate geographic region. within each region, we use the same compartmental model (but possibly with different parameters), and we also include spatial movement of individuals between the patches. the governing equations of such a metapopulation model, where p ∈ { , , . . . , #patches} are we have chosen our model parameters based on comprehensive literature review and present them here, except the transmission rates β (k,i) s,_ which are left for section . . . for the incubation period, we assume hypoexponential (generalized erlang) distribution with parameters ( . , . , ). this way, the average incubation period is . days: the same length and very similar shape of the probability distribution function was estimated in [ ] , and this distribution has the observed concavity properties as well (see [ ] ). in addition, this estimation is consistent with [ ] , and such values have been used in [ , , , ] . the first . days are the latent period [ ] and the past two days are the presymptomatic period [ ] , when transmission is already possible with similar rate as at symptom onset [ ] . therefore, we use the same transmission rates for the presymptomatic and symptomatic infectious periods. for the transmission rate of asymptomatic infected individuals, we use a reduction factor . [ , , ] . for the length of infectious periods (both symptomatic and asymptomatic), we assume a gamma distribution with erlang parameter (coherent with the sars study [ ] ), and an average length days of infectivity. although full recovery and viral shedding may take much longer, the infectiousness throughout the course of infection is mostly concentrated to this period [ , ] . the choice of days is also justified by [ , ] , who estimated that around % of transmissions occur during the presymptomatic period, and it is also within the range of infectious periods used by [ , ] . the average stay in hospital is assumed to be days, in accordance with the seven days median reported in [ ] using over , patients' data in the uk. similarly, the average duration of critical care is assumed to be days, in accordance with the intensive care national audit & research center (icnarc) report [ ] . very similar numbers were reported in the us [ ] , and were used in other modeling studies [ , , ] . for those who recover from intensive care, we assumed a -day hospitalized rehabilitation period. the periods above associated with the average time an individual spends in each compartment over the course of the infection are age-independent and summarized in table . table . age-independent epidemiological parameters of covid- . assumed to be valid for all age groups. references and explanations are in section . . . incubation period (α i l, ) − + (α i l, ) − + (α i p ) − . days latent period (α i l, ) − + (α i l, ) − . days presymptomatic (infectious) period (α i p ) − . days infectious period of i i a (γ i a, ) − + (γ i a, ) − + (γ i a, ) − . days infectious period of i i s (γ i p, ) − + (γ i p, ) − + (γ i p, ) − . days hospitalization (γ i h ) − presymptomatic vs symptomatic β next, we discuss the age-specific parameters, which are mostly related to the outcome of infections. we stratified the population into the following seven age groups: - , - , - , - , - , - , + years old. using the data from the hungarian central statistical office (ksh), we obtain the division shown in table . according to [ ] , a fraction . of infected children (under years old) are asymptomatic or mild cases. this value was used in [ ] as well. we set the probabilities of the infection following mild or asymptomatic course in an individual according to weitz et al. [ ] . the probabilities of hospitalization given infection h i and of requiring intensive care in addition ξ i are based on the work of moss et al. [ ] . the ratios of fatal outcomes µ i are derived from the icnarc report [ ] comprising icu case reports from uk. all these age-dependent parameters are listed in table . for creating our contact matrix m cont , we have utilized the work by prem, cook, and jit [ ] , where the estimated matrices are written for age groups, namely - , - ,. . . , - , +. as we have divided the hungarian population into seven age groups, see table , we aggregated the higher resolution data. first, we derived a symmetric matrix m total with elements where m = [m i,j ] is the original contact matrix and [p i ] is the age distribution of hungary for the same age groups as in [ ] . thus, m total contains the total number of contacts among age groups in its upper triangular part (with values relative to the contact pattern in m). the total number of contacts, w.r.t. the age distribution used in our work, is then obtained by summing up the corresponding elements of this matrix of size × resulting in m total cont of size × . finally, dividing element-wise each column of m total cont by the aforementioned population vector given in yields the following × contact matrix: for more insight, we include its heatmap in figure . additional technical details are to be found in our source code available at [ ] . recall that we have assumed presymptomatic patients, which are members of classes i i p , to be as infectious as symptomatic patients. in addition, patients with no or mild symptoms (those in i i a ) possess a transmission coefficient half of the baseline. thus, our task is to give reasonable estimates for the rates β (k,i) s,_ corresponding to the transmission rate of the symptomatic individuals from age group k to group i. to that end, we follow the terminology and techniques of [ ] to compute the next generation matrix (ngm) and the baseline transmission rate β . finally, the desired coefficients are obtained by taking into account the relative contact rates between age groups via the contact matrix presented in section . . . we note that the probabilities p i have a special role during ngm computations as their effect is what ultimately specializes the resulting transmission rate matrix for covid- . first, let us consider the infectious subsystem of ( ), namely, equations describing l i . . , }. linearizing this w.r.t. the disease free equilibrium yields the linearized infectious subsystem: where the matrices t and Σ are referred to as the transmission part and transitional part, respectively; the state is described by recall that the transmission matrix t has the form on the other hand, the transitional matrix Σ is block diagonal with blocks then, the ngm with large domain is given by follows with the, again, block diagonal e with e i = [ ]. the baseline transmission rate β may be factored out from k as β hence, k = β ·k, wherek may be readily constructed and we can compute its spectral radius ρ(k). then, we obtain the baseline transmission rate using the assumed basic reproduction number r as for other scenarios, the final steps are altered to align with the desired reproduction number r, resulting in an appropriate β and then the scaled transmission rates β (k,i) s . we omit presenting all transmission matrices but give the computed baseline transmission rates in table . we use the compartmental model described above to explore possible future scenarios, assuming widespread transmission in the population. in particular, we investigate the disease dynamics when different levels of general reductions of transmission, compared to the baseline, are in place. by manipulating the contact matrix, we investigate the effect of age-specific interventions, such as school closures and special measures aimed to protect the elderly. seasonality of respiratory viruses can be attributed to a combination of factors, including the survival of the virus in different environmental conditions, changes in contact patterns (such as school holidays), less time spent in closed spaces where the highest number of transmissive contacts are made, and potentially seasonal changes in the health conditions of the population as well. to express this behavior, we define a time-dependent parameter by which we scale the transmission rate β. parameter c denotes the magnitude of the effect of seasonality on the number of contacts. using such a time-dependent transmission rate, we compare possible disease dynamics generated by the interplay of control measures with different degrees of seasonal behavior. spatial heterogeneity is also considered using our patch model, where the country is divided into distinct geographic regions (patches). the transmission dynamics is described within each patch by our compartmental model (but potentially with different parameters and age group composition), and individuals may move between those patches. for obvious reasons, individuals in compartments i i h , i i c , i i cr and d i do not travel. let travel p,q denote the number of travels from patch p to patch q. to derive travel rates t p,q for each age group i, we divide the number of travels with the population of the appropriate patch numerical simulations for such situations show the differences in the transmission dynamics, healthcare demand, mortality, and overall disease burden. these scenarios are summarized in table . our work has several limitations. due to limited testing and the large number of asymptomatic and mild cases, there was a huge uncertainty in the number of true cases, especially in the early weeks. now, with the help of [ ] , we have a good estimation of the overall ascertainment rate over this period, but it is still unclear how this rate evolved in time. the transmission model has the same weaknesses that all compartmental models have: we assume a homogeneous population with random mixing, apart from the age structure. we added some further heterogeneity in space (patch model) and time (seasonality). in our scenarios, we assumed a constant reduction in transmission, while in reality the control measures and the behavior of the people were continuously changing. hence, such scenarios cannot be considered as predictions, as we cannot expect such unchanging circumstances for months. the role of children in this pandemic is still not clear, in our modeling, we assumed that they are equally susceptible, and equally infectious once they develop symptoms, but we used an age-specific probability for developing symptoms. since our transmission model is deterministic, it is suitable only when there is significant spread in the population. for very low case numbers, the development of the epidemics is largely influenced by random events. stochastic effects are important when considering extinction or resurgence of the disease, and possible case importations after travel restrictions are lifted. however, these issues are not in the scope of the present work. the model has a large number of parameters, many of those have uncertainty. the most important ones in regard to the burden on the healthcare system are hospitalization rates, probability of intensive care need, mortality, all of those depending on age. we do not have too much data for this from hungary, hence we used parameters taken from the literature. a full sensitivity analysis is beyond the scope of this study, but we present a sensitivity chart for a crucial output of an outbreak, see section . , which is of concern in many countries: the peak icu demand, including the need for mechanical ventilators, to assure that all patients receive the necessary care, and no additional excess mortality is caused by an overwhelmed healthcare system. this was one of the key questions in other modeling studies. the sensitivity analysis was conducted by running many simulations, sweeping through a two-parameter plane, and retrieving the icu peak from each individual run. the code can be found in [ ] . the first hungarian covid- cases were reported during the first week of march through the hungarian notifiable disease surveillance system operated by nphc which is the source of data described in this section (for the most recent information, see [ ] ). the first case, an iranian -year old man (studying and residing in hungary) who recently returned from tehran, was reported on march . by may , the cumulative number of reported confirmed covid- cases were ( . cases per , population), including deaths (crude cfr . %), see figure for the daily reported numbers. out of the cases, . % ( , cases) occurred in the + age group, . % ( cases) in the - age group, . % ( cases) in the - age group and . % ( cases) among people under -years old. age specific morbidity was highest in the + age group ( . cases per , population) and more than twice of the overall in the - age group ( . out of deaths, . % ( deaths) belonged to the + age group. as seen in figure , the highest crude cfr was observed in the + age group ( . %), followed by the - age group ( . %) and the - age group ( . %). no deaths were reported under years of age, see figure . additional details are provided in table . out of the cases, . % ( cases) were female and . % ( cases) male (gender is unknown for two cases). the morbidity among women was higher ( . vs. . cases per , population), so men were . ( % ci . - . ) less likely to become ill. however, men aged years and older had a . ( % ci . - . ) higher risk to die than women aged years and older ( . cases vs. . cases per , population). out of cases, at the stage of data consolidation as of may , , we have information about the symptoms of . % ( cases). out of cases, . % ( cases) had no symptoms, . % ( cases) had mild symptoms, and . % ( cases) had severe disease (including cases required intensive care and/or ventilation). most of the cases were reported from the central part of hungary, from the capital ( cases) and the surrounding pest county ( cases). see figure for a comparison of the capital region with the rest of hungary. the morbidity (per , population) was also the highest in budapest ( . ). the epidemic curve ( figure ) reflects a propagated source epidemic especially when we consider only those cases that cannot be connected to outbreaks in closed communities (like long-term care facilities or hospitals) or to health care associated infections. out of cases, . % ( cases) were associated with health care and/or outbreaks in hospitals, contributing to the daily reported new cases since mid-march. health care workers had . times ( % ci . - . ) higher risk to become a confirmed covid- case in comparison to the general population ( . cases vs. . cases per , population). out of cases, . % ( cases) were reported from long-term care facilities (nursing homes and other closed communities like homeless shelters) contributing to the daily reported new cases since early april. at the peak of the epidemic curve, . % ( cases) of cases on april were reported from the same retirement and assisted living facility. figure shows the results for the real-time estimation of the reproduction number. it showed a steady decline-apart from an outlying effect in early april-and became close to, or even below by mid-april, and remained at that level since then. this conclusion is robust to the chosen methodology. results for the real-time estimation of cfr are shown in figure . note that-as the outbreak is coming to its end-the naive method converges to the final value that was readily well estimated almost a month earlier by the corrected technique. (the naive estimator is increasing as deaths still occur, but case count is already low at the end of the epidemic.) the final cfr to characterize this phase in hungary is about %. various ifr estimations have been published, for example . % for china [ ] , . % for uk [ ] . recent serological studies found ifr values spanning from . % in a german town [ ] , to . % in milan [ ] . note that the testing intensity-and therefore the ascertainment rate-may very well change over time, e.g., with the increase of testing intensity. this analysis is based on the data from the early phase as a whole and, therefore, it is considered as an estimation of the average. the results for the estimation of the ascertainment rate are shown in table , where we explore a reasonable range of ifrs from . % to . %. note that earlier estimates based on [ , ] are consistent with the preliminary results of a large-scale hungarian sero-epidemiological study [ ] . most studies concerning the early growth-rate of the epidemic in wuhan estimated the value of the basic reproduction number to be around . - . (see e.g., [ , ] ), also later studies regarding the spread in other countries [ , ] used similar values. our estimations given in section . shows that in hungary the highest value of the effective reproduction number was . , by the wallinga-teunis method. hence, we choose r = . for the basic reproduction number (comparable with a similar reproduction number for germany in the early phase [ ] , . for italy [ ] ). modeling studies [ , , , ] highlighted that the worst case, i.e., "do nothing" scenarios lead to an outbreak when the healthcare demand substantially exceeds the capacities at the peak and the overall mortality reaches severe levels. given the current level of preparedness, we do not consider a "do nothing" scenario, and our most pessimistic case assumes that, even in the absence of any control measures, a % reduction in transmission is realized due to population awareness and behavior. on the other hand, the best case is the continuation of the current suppression scenario with r ≈ , resulting in very small case numbers. however, it is questionable whether it can be sustained until a vaccine is developed and deployed. below, we consider three scenarios illustrating the loss of control for suppressing the outbreak, and assuming a wide community spread of the disease. the efficacy of the mitigation efforts is expressed by a percentage in the reduction of transmission. the primary tool for this is the decrease of contact numbers, but other preventive measures such as hand hygiene or mask wearing may also have an effect in the reduction of transmission. first, let us consider a weak control of the epidemic assuming there is no centralized control measure introduced, but the number of transmissions is reduced by % following a level of behavioral response due to social awareness. such a reduction decreases the reproduction number to r = . . the first column of figure shows the hospitalization and icu demand on the top row and the daily incidences on the bottom row as a function of time with the application of this weak control. according to the simulations, in this case, there would be approximately . million infections with about , deaths by the end of the outbreak. this suggests that we can expect % of the population to gain immunity against the virus and this number is slightly larger than the threshold of herd immunity (that is ( − /r ) ∼ . % with r = . for the "do nothing" scenario). at the peak, there would be a need for more than icu beds and for , hospital beds with such a weak measure. we remark that there is a -days window when the daily incidences exceed , , and during this period more than . million people ( % of the population) get infected. in other words, % of all the infections occur during these three weeks. for further details, see table . we perform similar simulations for the case of a moderate control, assuming that the reproduction number is decreased to r = . as a result of the control measures. the simulations (second column of figure ) show that the number of hospital beds and icu beds needed is significantly reduced to and at the peak, respectively. meanwhile, the daily incidence at the peak is around , . we expect almost . % percent of the population to be infected throughout the epidemic and gain immunity upon recovery. this is less than required to reach herd immunity. for further information, we refer to table . finally, we consider a stronger control achieving a % reduction of transmission. this results a decrease of the reproduction number to r = . . the outcome of this strong control is shown in the third column of figure . a control of such strength significantly reduces the number of all infected and hospitalized cases and of those needing intensive care treatment. the number of required intensive care beds (around ) is far below the available capacity even at the peak of the epidemic and also the number of hospital beds needed is reduced to a rather low level-around at the peak. the total number of fatalities in this scenario is about . meanwhile, the epidemic would last for more than a year and the cumulative number of all infected remain far below the level of herd immunity threshold, so we can expect further outbreaks when the measures are relaxed. several key parameters of the model are highly dependent on age. intervention strategies and the relaxation of various measures have to take into account the fact that different age groups have different risks and different roles in the transmission. although the number of children infected with covid- has been reported worldwide relatively small in comparison with other age groups [ ], some evidence shows that children and adolescents may become infected and spread the disease as other age groups [ , ] . moreover, children and adolescents usually have a high number of contacts. thus, school closures can be expected to be an efficient tool to reduce the contacts and transmissions. besides school closures, it is important for younger individuals to avoid meeting older and other high risk people. elderly people have a higher chance of developing symptoms, and a higher percentage of them needs hospitalization and intensive care, hence these groups need more protection. age-specific interventions include avoiding contacts with elderly by providing special time slots for shopping, in post offices, etc., or closing/reopening schools. the introduction of various age groups in our model enables us to study such age-specific interventions and analyze their direct and indirect effects on all groups. on the stacked diagrams of figure , we present the contributions of the age groups to the mortality and the number of recovered individuals. columns of this figure show the effect of the weak, moderate, and strong control that we previously discussed in details in section . and table . here, we would like to emphasize that, in the case of each control measure, the most vulnerable age groups are the groups of elderly ( - , - , +) people as they suffer most of the fatalities; meanwhile, they are predicted to produce only a small fraction of the cases in the population. figure . age-specific mortality and recovery. the figure shows the effect of the weak, moderate, and strong control ( %, % and % general contact reduction, respectively). every age group covers at most one decade except the group of "middle aged" that represents three decades. according to our model, elderly people ( +) are predicted to produce most of the fatality cases in each scenario. the legend on the bottom applies for all figures. we consider two school closure scenarios: an optimistic and a pessimistic one (with respect to the outcome of the outbreak); both use the weak control scenario ( % general decrease in transmission, cf. section . ) as a starting point. the optimistic case is comprised of omitting the school component of the contact matrix and halving the other contacts [ ] of children and young adults (between age - ), which provides a new global contact matrix for this intervention. in the pessimistic scenario, we omit the school component of the contact matrix as well, but, instead of halving, it considers a % increase in the other contacts of children and young adults. arguably, the students might replace some school contacts by new other contacts, due to other activities. however, many of such contacts are lost as well: for example, they do not use public transportation to/from the school, and extracurricular activities also drop. since the exact balance is difficult to estimate, our two closure scenarios serve as a boundary to explore this regime of possibilities. note that, by school closure, we mean the closure of educational institutions from preschools to universities. as a reference, we also incorporate the weak control scenario to this analysis. figure shows that this measure decreases the peak hospital bed and icu needs to approximately % compared to the case when we only apply weak control in the optimistic scenario and by % in the pessimistic one. moreover, closing schools postpones the peak of the epidemic (by about one month in case of the above setting), suggesting that children may play a significant role in transmission due to their large number of contacts, even though they give negligible contribution to the overall mortality, cf. top row of figure ). note that this conclusion is based on the assumption that all age groups are equally susceptible, and symptomatic children are equally infectious to adults, and age specific difference appears only in the probability of developing symptoms, which is much smaller for children in our model (see parameters p i in table ). school closure in addition to the % contact reduction (pessimistic approach) school closure in addition to the % contact reduction (optimistic approach) figure . effect of school closure. simulations suggest that school closures-if maintained for a long period-effectively decrease peak hospital bed and icu needs and significantly postpone the peak of the epidemic. the effect of school closure combined with the % general reduction in transmission is comparable, in the optimistic case, with the effect of moderate control ( % reduction in transmission, cf. section . ) regarding the peak hospital bed and icu need, but not as significant in decreasing the mortality (figure middle column). however, to achieve this, schools need to be closed for an extended period of time, which may not be feasible. we also point out that a standalone closure of preschools and primary schools is not sustainable without a certain amount of home office of the parents, but this opens up sociological and economical questions that we do not address here. the elderly being the most vulnerable group of the population, when it comes to relaxation of measures introduced against the spread of covid- . most countries handle these age groups separately from the rest of the population, e.g., separate time slots for shopping continue to exist and elderly are encouraged to keep the same level of social distancing [ , ] . to include these effects in our model, we manipulate the entries of the contact matrix involving older age groups separately from the remaining parts. figure illustrates that, in addition to the weak control, if % and % reduction of the outside household connections of elderly people is applied, then we can expect about % and % reduction in the hospital, icu bed needs, and mortality. the epidemic curves only slightly shift to the right suggesting that elderly people do not play an important role in the transmission of the disease due to their low number of contacts. in addition, % reduction of contacts outside the household is again not feasible, as this would mean the complete isolation of a large sub-population. we plotted this scenario only to show the theoretical limits of this approach. general % contact reduction (weak control) % contact reduction for elders outside of households + weak control % contact reduction for elders outside of household + weak control figure . protection of the elderly. the figures show the effect of an additional contact reduction of elderly people in case of a weak control. the figures suggest that the selective protection of elderly people can successfully reduce the peak icu need and the overall mortality, yet it has a theoretical limit. in this section, we investigate the epidemic curves in case of the weak, moderate, and strong control with seasonality of various strengths expressed by parameter c ∈ { , . , . , . }, see ( ) . during the summer, these values of c eventuate a %, %, and % further decrease in transmission as that is when the seasonality curve attains its minimum. the case c = means that there are no seasonal effects at all, while c = . is a strong seasonality, which is similar to h n [ ] . see the top left image of figure for the seasonality functions ω(c, t) corresponding to the different c values. as we have seen in section . , decreasing the reproduction number decreases and postpones the peak of the epidemic curves. seasonality causes a similar delay in the peak of the epidemic due to decreased transmission rates in the summer months. counter-intuitively, it cannot be said in general that stronger seasonality leads to a smaller peak (cf. bottom left image of figure ). the reason for this is that the impact of seasonality is not only determined by the decrease in the transmission rate, but the temporal relation between the peak of the epidemic and the minimum of the seasonality function is also an important factor. this phenomenon is well illustrated in figure where three scenarios (weak, moderate, and strong control) are presented along with the assumed seasonality functions for the aforementioned values of c. in the upper right image of figure , corresponding to a weak control, one can observe that increasing the effect of seasonality first decreases the peak, but, after a certain value (c = . in our example), the epidemic is so much suppressed in the summer months that the peak shifts to the right and even slightly increases in winter months compared to the c = . scenario. for the case of moderate control, shown in the lower left figure, this effect is much more significant. note that the peak of the epidemic (without seasonality) is so far from summer (the minimum of the seasonality curves) that increasing the effect of seasonality results in a significantly higher peak. it can be seen that strong seasonality eventuates a long "plateau" phase when the epidemic curve does not increase in a period of six months. during this time, only a small fraction of the population goes through the infection and a massive number of susceptibles remain in the system, only to get infected a few months later. this phenomenon is responsible for the increased peak of c = . compared to the c = . case. the lower right figure shows that the reduction of transmission during the warm months together with a strong control can decrease the number of infected in such an extent that the peak, even if arriving in the winter months, is significantly smaller. a general observation is that seasonality has the largest impact on the epidemic curve if the peak time is close to the summer months. of course, this is highly dependent on the starting time of the outbreak. hungary is a relatively small country; however, significant differences were observed between regions in the reported case numbers. the capital, budapest, has . million inhabitants and a further . million people live in its surrounding pest county. budapest and pest county are highly connected by commuters with connections to other regions as well [ ] . the high connectivity of the capital with other countries contributed to the earlier appearance of the disease in budapest, and most of the cases were reported from this central region of the country. to address the role of spatial heterogeneity in the evolution of the epidemic curve, we considered a metapopulation model as in ( ) . hence, the population is distributed among patches, representing geographic regions of the country. for the sake of simplicity, here we only present results from a two-patch model, separating budapest and pest county (patch , population of approx. , , ) from the remaining parts of the country (patch , population , , ). we assumed different transmission parameter β for each patch. based on hungarian mobility data on commuters [ ] , we assumed , daily travels between the two patches in the case of normal circumstances and investigated the effect of the lockdown of budapest and the surrounding pest county by decreasing the number of daily travels to , . we considered the contact matrix for both patches to be the same as in the uniform model described in section . . . the biological and medical parameters are assumed to be the same in each patch, but the local reproduction number may differ, as well as the age structure of the population. the left-hand side of figure illustrates that the two-patch model reproduces the uniform model in case we use the same r = . for both patches as well as for the uniform model and we assume , daily travels between the patches. the middle figure shows that the uniform model slightly overestimates the size of the epidemic as the peak of the aggregated two-patch model is smaller than that of the uniform model in case r = . remains the same, and we reduce the daily travels to , corresponding to the separation of budapest and pest county from other regions. although the epidemic curves of the patches are shifted, the aggregated result shows that this setup does not provide significantly different dynamics. lastly, on the right-hand side of figure , we further investigate the scenario of , daily travels, and choose the local reproduction numbers of the patches to vary around r = . , namely, we take r budapest = . and r other regions = . . these values were selected to reflect the higher population density of the capital, proportionally to the population in the two patches. due to the difference in the local reproduction numbers, we may observe an increased number of cases in budapest with an earlier peak and fewer infections in other regions. figure . epidemic curves of the regions: sum of the infective compartments (i p , i a, , i a, , i a, , i s, , i s, , i s, ). first, we consider identical reproduction numbers r = . for both patches (budapest with pest county and other regions). without any travel reductions, the two-patch model gives identical results to the one-patch version, as seen in the left figure. next, if travel reductions are put in place, the one-patch model overestimates slightly the size of the epidemic for equal r values. finally, assuming different reproduction numbers and large reduction in travel, the peak occurs earlier in the patch with larger r (budapest and pest county); furthermore, the one-patch model and the aggregated two-patch model differ in both time and size of the peak. for an uncontrolled epidemic in the uk, ref. [ ] estimated a peak in icu bed demand more than times greater than the maximum capacity in these countries. in a study for the united states, ref. [ ] projected that, at the outbreak peak, three times more icu beds would be needed than the total number of icu beds in the us, and % isolation of cases reduces the demand for icu beds to the normal capacity. in the Île-de-france region, ref. [ ] estimated that the peak number of icu beds needed would exceed more than times the regional capacity if no strategy is implemented after lockdown, and only efficient case-finding and isolation applied parallel with social distancing could decrease icu demand below the maximum capacity throughout the epidemic. for australia, ref. [ ] studied three capacity expansion scenarios ( , and times expansion, respectively), and, even in mitigated scenarios, demand is estimated to be higher than the number of available beds. additional social distancing measures were shown to reduce the epidemic to a level where a reasonable expansion of icu capacity can be sufficient. the peak icu demand crucially depends on two factors: the probabilities of developing severe disease, and the shape (in particular the peak size) of the epidemic curve. we plotted a heatmap of the peak icu demand in figure , compiled from hundreds of numerical simulations. transmissibility (vertical axis) is expressed by the reproduction number r. disease severity, for simplicity, is expressed by the ifr. in fact, here we used a scaling factor for the probability of hospitalization, with the baseline corresponding to the parameters in table . in our weak control scenario (section . ), the ifr is . %, which is a bit lower than the finding of [ ] . however, during the first wave in hungary, the schools were closed and covid- disproportionately affected the vulnerable population. in our scenarios, we assume a widespread community spreading, hence younger generations appear in higher numbers, thus the ifr is expected to be smaller. in any case, by the scaling of the hospitalization rate (while leaving the probability of intensive care and fatal outcome given hospitalization intact), we explored a wider range of ifrs. we found that indeed the peak icu demand can vary across a large interval. from the shape of the level curves in the heatmap, we can conclude that the peak icu demand is more sensitive to r than to the ifr, hence flattening the curve is indeed of utmost importance to avoid exceeding healthcare capacities. [ ] . the white dot is our most pessimistic scenario (weak control). the most important implemented measures are summarized in table . to assess their impact, we compared the reported case numbers adjusted by the ascertainment rate : to the simulated outbreak curve with r = . ( figure on the left, logarithmic scale). here, we assumed that the ascertainment rate did not change in time, which may not be the case. one can see that the epidemic was on the r = . trajectory, which could have resulted in substantially more infections. the data shows a clear deviation from this scenario early april, two weeks after strict social distancing started. the slope of the epidemic curve further decreased mid-april, following the stay at home measures by two weeks. overall, due to the compliance of hungarian society with the social distancing measures, around half million infections were averted by the end of april, compared to the "do nothing" scenario, which could have reached - million in may if further doublings would have been allowed. the first covid- case was detected, the laboratory confirmed, and then reported through the hungarian notifiable disease surveillance system on march . well tailored, effective, combined non-pharmaceutical control measures have been introduced promptly in hungary in the very early phase of the outbreak (see table ), accompanied with a high level of compliance for social distancing. online surveys [ ] , polling, and indirect data (such as traffic data, passenger volumes on public transportation, etc.) all showed a drastic reduction in the number of contacts and mobility. in particular, the online questionnaire maszk [ ] showed a - % decrease (depending on the locality) in the daily number of physical contacts as well as in the number of closed contacts per capita, based on the replies of , respondents by may , constituting a non-representative, but rather large sample. accordingly, the hungarian epidemic curve was strongly suppressed. as of may , the cumulative number of reported confirmed covid- cases were ( . cases per , population), including deaths. the epidemic peaked on april with newly reported cases. sars-cov- was not able to sustain long transmission chains in the community; however, it was able to cause outbreaks mostly in healthcare institutions and long-term care facilities: nearly two thirds of the reported cases are connected to such institutions. the proportion of cases in health care workers gradually increased during the epidemic. they had tenfold risk to become confirmed covid- cases compared to the general population. due to effective measures, the virus could not spread significantly from closed communities and health care workers to the wider population. the age specific cfr showed a similar pattern to other countries: of the deaths reported by may, ( . %) belonged to the + age group. we tracked the temporal variation of the effective reproduction number in real time, which showed a steadily decreasing trend, interrupted by an outlying outbreak in a long-term care facility. we identified the time intervals when the effective reproduction number was below or around the critical threshold . the adjusted cfr was also estimated real-time, and predicted the eventual cfr one month in advance well. benchmarking the cfr to other countries, we estimated underascertainment rate to be - times, and the true cumulative number of covid- cases to be between , and , . these results are consistent with data from the preliminary results of a large scale seroepidemiological survey, carried out in hungary in may , where the seroprevalence of sars-cov- infection was estimated to be between , and , [ ] . based on these data and the number of reported cases, underascertainment is likely to be between . - . , and the true cfr may be lower than . %, and the ifr is roughly half of that. as control measures are being successively relaxed since may , we established an age-structured compartmental model to investigate several post-lockdown scenarios, and projected the epidemic curves and the demand for critical care beds assuming various levels of sustained reduction in transmission. special measures designed to reduce the contact number of the elderly population as well as school closures can reduce the peak hospital bed demand and the overall mortality; however, these measures also have their limitations. a metapopulation version of the transmission dynamics model has also been studied, and we reported some results for a two-patch case, where the budapest region is considered separately from the rest of the country. due to the high connectedness, the epidemic curves of the two-patch system are not much different from the spatially uniform case. to achieve a noticeable reduction in the overall peak size due to spatial heterogeneity (where the local peak times are shifted in the regions), a large reduction in the mobility rates is necessary. since the majority of the population is still susceptible (over %, according to [ ] ), a weak or even a moderate reduction in the transmission, compared to the baseline, could result in a large second outbreak with significant mortality and high peak icu demand. therefore, a high level of alertness needs to be maintained to avoid such scenarios. the seasonal behavior of sars-cov- is not completely understood yet [ , ] , thus we considered a range of possibilities from the absence of seasonality to a strong seasonality, which is similar to h n . the interplay of seasonal effects with the post-lockdown contact numbers can generate a variety of disease dynamics; thus, a confident forecast of the timing and the size of a potential second wave is not possible at the moment. the effectiveness of strict social distancing measures, such as school closures and stay at home measures with good compliance is likely to be very high; however, such interventions have negative consequences on the society and on the economy and are thus not sustainable in the long term. modeling results [ , ] suggest that combined multiple interventions, including moderate contact decrease, high covid- detection rate, effective contact tracing, and good compliance with personal protective instructions, may have substantial impact on transmission, and are able to keep the reproduction number around one. situation reports; world health organization france: surveillance, investigations and control measures cov- was already spreading in france in late investigation of a covid- outbreak in germany resulting from a single travel-associated primary case: a case series covid- epidemic in italy: evolution, projections and impact of government measures who. statement on the second meeting of the international health regulations ( ) emergency committee regarding the outbreak of novel coronavirus director-general's opening remarks at the media briefing on covid- ; world health organization covid- ) in the eu/eea and the uk-ninth update multiple sars-cov- introductions shaped the early outbreak in central eastern europe: comparing hungarian data to a worldwide sequence data-matrix covid- announcements of hungary r: a language and environment for statistical computing; r foundation for statistical computing elegant graphics for data analysis table: extension of 'data.frame'. r package version . . shiny: web application framework for r real-time epidemiology of covid- in hungary (a magyarországi koronavírus járvány valós idejű epidemiológiája-in hungarian) real-time epidemiology of covid- in hungary (a magyarországi koronavírus járvány valós idejű a new framework and software to estimate time-varying reproduction numbers during epidemics different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures how generation intervals shape the relationship between growth rates and reproductive numbers the effective reproduction number of pandemic influenza: prospective estimation association of public health interventions with the epidemiology of the covid- outbreak in wuhan effective reproduction number estimation estimating in real time the efficacy of measures to control emerging communicable diseases serial interval of covid- among publicly reported confirmed cases epidemiological characteristics of covid- cases in italy and estimates of the reproductive numbers one month into the epidemic serial interval of novel coronavirus (covid- ) infections the r package: a toolbox to estimate reproduction numbers for epidemic outbreaks r : estimation of r and real-time reproduction number from epidemics. r package version . - estimate time varying reproduction numbers from epidemic curves. r package version . - . methods for estimating the case fatality ratio for a novel, emerging infectious disease assessing the severity of the novel influenza a/h n pandemic early epidemiological assessment of the virulence of emerging infectious diseases: a case study of an influenza pandemic real-time estimation of the risk of death from novel coronavirus (covid- ) infection: inference using exported cases incubation period and other epidemiological characteristics of novel coronavirus infections with right truncation: a statistical analysis of publicly available case data rstan: the r interface to stan using a delay-adjusted case fatality ratio to estimate under-reporting ( ). cmmid risk assessment of novel coronavirus covid- outbreaks outside china modelling the impact of covid- in australia to inform transmission reducing measures and health system preparedness expected impact of lockdown in Île-de-france and possible exit strategies a first study on the impact of current and future control measures on the spread of covid- in germany report -impact of non-pharmaceutical interventions (npis) to reduce covid- mortality and healthcare demand projecting hospital utilization during the covid- outbreaks in the united states covid- epidemic risk assessment for georgia temporal dynamics in viral shedding and transmissibility of covid- early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia effects of latency and age structure on the dynamics and containment of covid- appropriate models for the management of infectious diseases hungarian data supply questionnaire (maszk-magyar adatszolgáltató kérdőív-in hungarian) code basis for covid modelling in hungary contact tracing assessment of covid- transmission dynamics in taiwan and risk at different exposure periods before and after symptom onset quantifying sars-cov- transmission suggests epidemic control with digital contact tracing features of , hospitalised uk patients with covid- using the isaric who clinical characterisation protocol report on covid- in critical care incidence, clinical outcomes, and transmission dynamics of severe coronavirus disease in california and washington: prospective cohort study children with covid- in pediatric emergency departments in italy projecting social contact matrices in countries using contact surveys and demographic data the construction of next-generation matrices for compartmental epidemic models preliminary results of the h-uncover study estimates of the severity of coronavirus disease : a model-based analysis infection fatality rate of sars-cov- infection in a german community with a super-spreading event sars-cov- seroprevalence trends in healthy blood donors during the covid- milan outbreak pattern of early human-to-human transmission of wuhan covid- outbreak in italy: estimation of reproduction numbers over two months toward the phase . medrxiv ministry of health, welfare and sport, netherlands. children and covid- an analysis of sars-cov- viral load by patient age covid- and the consequences of isolating the elderly seasonal transmission potential and activity peaks of the new influenza a (h n ): a monte carlo likelihood analysis based on human mobility the salient targets of commuters social distancing strategies for curbing the covid- epidemic temperature, humidity, and latitude analysis to estimate potential spread and seasonality of coronavirus disease (covid- ) cmmid covid- working group. effects of non-pharmaceutical interventions on covid- cases, deaths, and demand for hospital services in the uk: a modelling study cmmid covid- working group. effectiveness of isolation, testing, contact tracing, and physical distancing on reducing transmission of sars-cov- in different settings: a mathematical modelling study the authors declare no conflict of interest. key: cord- - uwiys a authors: hung, yu-fu; schwarten, melanie; hoffmann, silke; willbold, dieter; sklan, ella h.; koenig, bernd w. title: amino terminal region of dengue virus ns a cytosolic domain binds to highly curved liposomes date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: uwiys a dengue virus (denv) is an important human pathogen causing millions of disease cases and thousands of deaths worldwide. non-structural protein a (ns a) is a vital component of the viral replication complex (rc) and plays a major role in the formation of host cell membrane-derived structures that provide a scaffold for replication. the n-terminal cytoplasmic region of ns a( – ) is known to preferentially interact with highly curved membranes. here, we provide experimental evidence for the stable binding of ns a( – ) to small liposomes using a liposome floatation assay and identify the lipid binding sequence by nmr spectroscopy. mutations l e;m e were previously shown to inhibit denv replication and to interfere with the binding of ns a( – ) to small liposomes. our results provide new details on the interaction of the n-terminal region of ns a with membranes and will prompt studies of the functional relevance of the curvature sensitive membrane anchor at the n-terminus of ns a. dengue virus (denv), the causative agent of dengue fever, is a positive strand rna, enveloped virus belonging to the flaviviridae family. the viral rna is translated into a single polyprotein which is processed by cellular and viral proteases into three structural proteins (capsid, premembrane, and envelope) and the seven non-structural (ns) proteins ns , ns a, ns b, ns , ns a, ns b, and ns [ ] . the ns proteins are not found in the mature virion but are crucial for viral replication. synthesis of viral rna takes place in replication complexes (rcs) that contain essential ns proteins, viral rna and host cell factors [ ] . upon denv infection, a complex and continuous network of er membrane-derived vesicular structures and convoluted membranes is formed. these structures contain the viral replication complexes and the sites of virion assembly [ ] [ ] [ ] . ns a is small integral membrane protein containing four predicted transmembrane segments (ptmss) [ ] . although ptms , often referred to as the k fragment, is not part of the mature ns a, it serves as a signal peptide for the er localization of ns b and is cleaved from the mature ns a [ ] . experimental data verify that ptms and ptms span the membrane while ptms is embedded in the luminal leaflet of the er membrane and does not span it [ ] . ns a is crucial for the formation of the virus-induced membrane structures. expression of ns a lacking the k fragment alone is sufficient to induce membrane alterations that resemble the denv-induced highly curved membranes that harbor the rcs [ ] . clearly, to induce these structures ns a will have to closely interact with host membranes. however, the mechanism by which ns a induces the curved morphology of these newly formed membranes is still unknown. insertion of amphipathic helices into one leaflet of a membrane bilayer, as well as oligomerization of membrane proteins are among the mechanisms known to participate in the induction of membrane curvature [ ] . molecular dynamics (md) simulations suggest that ptms of ns a could support membrane undulations upon stable association with the membrane [ ] . curved vesicular structures might also be induced via homooligomerization of ns a [ , ] . while we have previously shown that the ns a n-terminal cytoplasmic region is implicated in its oligomerization [ ] , a recent study demonstrated that ptms is the major determinant in this process [ ] . introduction of two point mutations at the n-terminal of ns a (l e and m e) reduced both the amphipathic character of this region and ns a homooligomerization and abolished viral replication [ ] . ns a is an essential component of the viral rc [ ] . direct interaction of ns a with the cytoskeletal protein vimentin was reported to be necessary for correct localization of the rc at the perinuclear site [ ] . the vimentin binding site was found to be located at the n-terminal residues of ns a [ ] . ns a was also reported to bind ns b, another component of the rc, via ptms [ ] . mutational analysis suggests a functional relevance of this interaction for viral replication [ ] . it was speculated that the interaction between ns a and ns b in concert with ns a oligomerization and ns b dimerization may play a role in the spatial and temporal regulation of distinct molecular complexes involved in the viral infection cycle [ ] . our previous circular dichroism (cd) data demonstrated that ns a( - ) interacts with highly curved small unilamellar liposomes under α-helix formation, while mutated ns a( - , l e;m e) does not [ ] . surface plasmon resonance data indicated a seven fold-greater association of wild type ns a( - ) with immobilized liposomes compared to the mutant [ ] . the structure of ns a in presence of membrane mimicking sds micelles was characterized by nmr [ ] . to further extend these results we used liposome flotation for direct proof of ns a( - ) binding to free liposomes. the exact location of lipid binding sites in the amino acid sequence of ns a( - ) was addressed by nmr spectroscopy. our findings provide a basis for specific structure-function studies that will enhance our understanding of the role of ns a and might provide future targets for anti-viral intervention. the peptide ns a( - ) corresponds to amino acid residues - from the n-terminal of ns a of dengue virus serotype (ncbi protein database accession number: np ). a mutant peptide containing the mutations l e and m e was designated ns a( - , l e;m e). the two ns a peptides were recombinantly produced in e. coli bl cells and enzymatically cleaved from the affinity tag as described earlier [ ] . uniform isotope labeling with n or c, n was achieved by expression in m medium containing n ammonium chloride and c glucose (eurisotop, saarbrücken, germany) as the sole source of nitrogen and carbon, respectively. unlabeled peptides were expressed in lb media. alexa fluor succinimidyl ester (nhs ester) was purchased from life technologies, darmstadt, germany. the dye was dissolved in anhydrous dmso at a concentration of mm immediately prior to the labeling reaction. for the reaction µl from a µm ns a( - ) or ns a( - , l e;m e) stock in sample buffer ( mm sodium phosphate, ph . , mm nacl) were combined with µl of . m nahco and the ph was adjusted to . . this µl peptide solution was supplemented with µl of the alexa fluor nhs ester in dmso resulting in an approximately ten-fold excess of dye-over-peptide. the labeling reaction was wrapped in aluminum foil and incubated on a rocking platform shaker at ˝c for h. labeled protein and free dye were separated on a superdex / gl column (ge healthcare, freiburg, germany) operated on an Äktapurifier system (ge healthcare). the lipid -palmitoyl- -oleoyl-sn-glycero- -phosphocholine (popc) in chloroform solution was purchased from avanti polar lipids (alabaster, al, usa). small unilamellar lipid vesicles (suvs) were prepared from chloroform-free popc dispersions ( mg¨ml´ ) in sample buffer as described earlier [ ] . suvs were obtained by sequential extrusion through nm ( times) and nm ( times) nuclepore polycarbonate membranes (ge healthcare) with nominal pore diameter of either or nm, followed by sonication with a mm microtip of a branson sonifier ( cycles of sonication, s each, interrupted by cooling for min after each cycle). sonicated suvs were centrifuged for min at , ˆg and ˝c in a refrigerated eppendorf r tabletop centrifuge to remove any titanium abrasion of the microtip from the sample. the hydrodynamic radius of each liposome preparation was determined by dynamic light scattering (dls) using a dyna pro instrument (protein solutions, lakewood, nj, usa) equipped with a mm path length µl quartz cell. liposome solutions ( mg of popc per ml) were diluted -fold with buffer directly after extrusion or sonication and measured immediately. data were analyzed with dynamics v software distributed with the instrument. experimental data were fitted to the model of rayleigh spheres. equal volumes of µm suspensions of alexa fluor -labeled peptides or free dye and sonicated popc suv ( mg¨ml´ ) in sample buffer were combined and mixed at room temperature for min. µl of each of the three resulting samples were thoroughly mixed with µl of a % (w/v) sucrose solution to obtain homogeneous solutions containing µm of either alexa fluor -labeled peptide or free alexa fluor dye, popc liposomes ( mg¨ml´ ) and % (w/v) sucrose in sample buffer. sucrose solution ( µl of a % (w/v) solution in sample buffer) was transferred to the bottom of a polyallomer centrifuge tube ( mmˆ mm; beckman coulter) followed by a second layer formed by the µl of % (w/v) sucrose solution containing one of the labeled ns a peptides or the free dye and popc liposomes. finally, each sample was carefully overlaid with two cushions of decreasing sucrose concentration, i.e., . ml of % (w/v) followed by µl of % (w/v) sucrose in sample buffer (cf. scheme in figure , left). samples were centrifuged for h at , ˆg and ˝c in a beckman coulter optima max-xp ultracentrifuge using a tls swinging bucket rotor. fluorescence images were taken in front of a mini transilluminator (bio-rad, munich, germany) prior to and immediately after centrifugation. nmr experiments were conducted at ˝c on bruker avance iii hd nmr and varian vnmrs instruments, equipped with cryogenic z-axis pulse-field-gradient (pfg) triple resonance probes operating at proton frequencies of and mhz, respectively. samples for resonance assignment contained µm [u- n, c]-labeled ns a( - ) in sample buffer ( mm sodium phosphate, ph . , mm nacl) as used for the liposome flotation experiments but supplemented with % (v/v) deuterium oxide and . % (w/v) nan (referred to as nmr buffer). assignment of protein backbone resonances was accomplished using a combined set of heteronuclear multidimensional nmr experiments: d ( h- n)-hsqc [ , ] , d ( h- c)-hsqc [ ] , d hnca [ ] , d bt-hnco [ ] , and d hncaco [ ] . h and c chemical shifts were referenced directly to internal , -dimethyl- -silapentane- -sulfonic acid (dss) at ppm and n chemical shifts were referenced indirectly to dss using the absolute ratio of the n and h zero point frequencies [ ] . nmr data were processed using nmrpipe, v. . [ ] and evaluated with ccpnmr v. . [ ] . binding of fluorescently labeled proteins to liposomes can be visualized using a simple floatation assay [ ] . popc bilayers have a mass density very close to g· cm − [ ] at room temperature. thus, popc liposomes will migrate to the top layer containing the lowest sucrose concentration in a step gradient of decreasing sucrose concentrations upon centrifugation. in contrast, a small protein of ~ kda molecular weight is expected to have a mass density well above . g· cm − [ ] and thus will accumulate in a sucrose rich layer of high mass density. alexa- -labeled ns a( - ) apparently binds to small sonicated popc liposomes and migrates with the liposomes to the top of the gradient ( figure a ). in contrast, the mutated peptide ns a( - , l e;m e) remains in the high density layer with % (w/v) sucrose even after h of centrifugation ( figure b) , indicating that this peptide binding of fluorescently labeled proteins to liposomes can be visualized using a simple floatation assay [ ] . popc bilayers have a mass density very close to g¨cm´ [ ] at room temperature. thus, popc liposomes will migrate to the top layer containing the lowest sucrose concentration in a step gradient of decreasing sucrose concentrations upon centrifugation. in contrast, a small protein of " kda molecular weight is expected to have a mass density well above . g¨cm´ [ ] and thus will accumulate in a sucrose rich layer of high mass density. alexa- -labeled ns a( - ) apparently binds to small sonicated popc liposomes and migrates with the liposomes to the top of the gradient ( figure a ). in contrast, the mutated peptide ns a( - , l e;m e) remains in the high density layer with % (w/v) sucrose even after h of centrifugation ( figure b) , indicating that this peptide does not bind to liposomes. as a negative control, the alexa- free dye was loaded to the % (w/v) sucrose layer with popc liposomes. the free dye remained in the high-density region after centrifugation ( figure c ) indicating a lack of association with the popc suvs. the fluorescent band of the free dye has a larger vertical extension than that of the labeled ns a( - , l e;m e) after h of centrifugation ( figure b,c) . this probably reflects the larger diffusion coefficient of the low molecular weight dye molecule ( . g¨mol´ ) . in a second control experiment alexa- -labeled ns a( - ) was loaded to the % (w/v) sucrose layer without adding popc liposomes. as expected, the peptide did not significantly migrate during the h of centrifugation and remained almost completely in the % (w/v) sucrose layer ( figure d ). the floatation results confirm the binding of the wild type peptide to small liposomes. amide hn, n cross peaks in hsqc spectra of ns a( - ) were used to monitor peptide interaction with sonicated popc liposomes in an amino acid residue resolved manner. the hydrodynamic radius of the popc liposomes was " nm based on dls measurements. backbone cross peaks for most of the amino acid residues of ns a( - ) were identified in buffer without liposomes except for s , l , p , m , h , n and h . the observed cross peaks characterize the free peptide conformation. addition of increasing amounts of sonicated popc liposomes at constant peptide concentration caused gradual peak intensity reductions in a peptide region specific manner ( figure a) . interestingly, peak positions did not significantly change, except for r , which showed a small shift. such a behavior is typical for slow or intermediate exchange of the peptide between the free and liposome-bound state. strongest peak intensity reduction is observed in the n-terminal region extending up to k (figure a ). some peaks in this region completely disappear already at . mg¨ml´ popc (n , i , e , g , k ) while the others are reduced to less than %. at the highest liposome concentration studied ( mg¨ml´ ) only two cross peaks from this peptide region remain visible and show low intensity. it is likely, that a number of amino acid residues of the n-terminal region bind directly to the liposome. binding will change the chemical environment and strongly reduce the rotational correlation time of the amino acid residues in direct contact with lipids. nmr signals of bound residues are likely broadened beyond detection. exchange dynamics may differ somewhat among the amino acid residues in this region, explaining the variable intensity reduction of the free state cross peaks. cross peaks of the central region from a through l of ns a( - ) show rather uniform intensity reduction upon gradual liposome addition (figure a ). all free state peaks remain visible and retain about % of their original intensity even in the presence of mg¨ml´ popc. different scenarios might contribute to the reduction of the free state peak intensities. peptides that are anchored with their n-terminal region in the liposome might retain their free state conformation in the central domain, albeit with a reduced overall rotational correlation time and thus lower peak intensities. in addition, amino acid residues of the central region of some ns a peptides might bind directly to the liposome leading to the disappearance of the corresponding nmr signals. the uniform peak intensity reduction pattern in the central region may suggest a concerted binding of this amino acid stretch, e.g., as one secondary structure element, to the liposome. viruses , scenarios might contribute to the reduction of the free state peak intensities. peptides that are anchored with their n-terminal region in the liposome might retain their free state conformation in the central domain, albeit with a reduced overall rotational correlation time and thus lower peak intensities. in addition, amino acid residues of the central region of some ns a peptides might bind directly to the liposome leading to the disappearance of the corresponding nmr signals. the uniform peak intensity reduction pattern in the central region may suggest a concerted binding of this amino acid stretch, e.g., as one secondary structure element, to the liposome. finally, amino acid residues in the c-terminal region of ns a( - ) from t to l show the smallest reduction in the free state peak intensities upon liposome addition (figure a) . perhaps, these small intensity reductions might be entirely caused by anchoring of the peptides via amino acid residues in the n-terminal and perhaps the central regions. interaction of ns a( - , l e;m e) inspection of hsqc spectra of mutant ns a( - , l e;m e) in buffer and with increasing amounts of sonicated popc liposomes revealed no changes in cross peak positions and only a minor finally, amino acid residues in the c-terminal region of ns a( - ) from t to l show the smallest reduction in the free state peak intensities upon liposome addition (figure a) . perhaps, these small intensity reductions might be entirely caused by anchoring of the peptides via amino acid residues in the n-terminal and perhaps the central regions. influence of lipid addition on cross peak intensities of backbone amide signals. all cross peaks observed in buffer remain visible in presence of liposomes and retain at least % of their original intensity upon addition of mg¨ml´ of popc liposomes ( figure b ). almost no peak reduction is observed for the c-terminal region of ns a( - , l e;m e). peak intensity reductions in the n-terminal and central regions are quite moderate, in particular at low liposome concentration ( . and mg¨ml´ popc). even at mg¨ml´ popc liposomes cross peak intensities remain between % and % (n-terminal region) or between % and % (central region) with respect to intensities measured in buffer. apparently some interaction of the mutant peptide with liposomes is retained at the highest lipid concentration studied. amino acid residues in the central and perhaps in the n-terminal part of the mutant peptide are likely to make transient contact with the liposome. however, the observed peak intensity reductions are much weaker in case of ns a( - , l e;m e) than for wild type. the l e and m e mutations that disrupt the amphipathic character of the ns a n-terminal abolish viral replication, indicating that the n-terminal residues of ns a play a crucial role in replication. furthermore, these mutations had a similar effect when inserted as single mutations [ ] . we have previously shown that ns a( - ) interacts preferentially with highly curved liposomes. cd spectroscopy demonstrated that these two point mutations severely compromise this interaction [ ] . the interaction of wild type ns a( - ) with highly curved membranes has been demonstrated for three different lipid compositions, i.e., pure popc, a popc/dops mixture at a molar ratio of : , and a blend of synthetic lipids resembling the composition of membranes in the er (er lipid mix), but no dependence on lipid composition was detected [ ] . here, we confirm these results using a liposome floatation assay and nmr. initial floatation experiments were conducted with pure popc and with the er lipid mix. again, no influence of lipid composition on ns a( - ) binding was observed. the detailed analysis presented in the current manuscript was conducted with single component popc suvs. liposome floatation experiments ( figure ) clearly show that wild type ns a( - ) binds to highly curved popc liposomes, this interaction was not observed with the mutant peptide. the interaction between ns a( - ) and liposomes was further characterized using nmr. these experiments indicate that the main lipid binding sites of the peptide are located at the n-terminal amino acid residues of ns a( - ). residues a through l may also be involved in liposome binding while the remaining c-terminal residues are only weakly affected by liposome binding and do not seem to play a direct role in this process. the backbone resonance assignment of ns a( - ) and ns a( - , l e;m e) reported in this manuscript contains information on the secondary structure of the two peptides in lipid-free buffer. analysis with the talos-n software [ ] clearly shows the lack of secondary structure for both peptides in buffer. the nmr data on ns a( - ) recorded in presence of liposomes do not allow any straightforward conclusion on the structure of the liposome-bound peptide. however, interaction of ns a( - ) with sodium dodecyl sulfate (sds) micelles induces the formation of two amphipathic helices (ah) encompassing residues n to e (ah ) and t to l (ah ) of ns a( - ) [ ] . cd spectra of ns a( - ) recorded in presence of either sds micelles or small popc liposomes are very similar [ ] . therefore, it is conceivable that the two amphipathic helices ah and ah are also formed in the liposome-bound peptide. the n-terminal region of ns a( - ) that forms ah , an interhelical linker and the n-terminal half of ah in sds micelles seem to be crucial for peptide binding to liposomes. interestingly, this region also contains the two mutations l e and m e, which abolished liposome binding in the floatation experiment ( figure ) . moreover, mutagenesis of other residues in this region including p a [ ] , r a and k a [ ] was also shown to reduce or abolish denv replication. the central part of ns a( - ) encompassing residues a to l shows less pronounced nmr signal intensity reductions upon titration with popc liposomes than its n-terminal amino acid residues (figure a) . a comparatively weak nmr signal intensity reduction is observed for both the n-terminal and central regions of ns a( - , l e;m e) ( figure b ) indicating some residual interaction with liposomes. however, the liposome floatation assay clearly shows that this interaction of the mutant peptide with popc liposomes is too weak for stable anchoring of the peptide at the membrane. the amino acid sequence of a through l is identical in both peptides. we conclude that this amino acid stretch a to l is not sufficient for stable membrane anchoring of ns a( - ). denv ns a apparently contains two separate membrane anchors. the membrane spanning helices ptms and ptms stably integrate the protein into the membrane. the n-terminal region of the cytosolic domain specifically binds to the convex surface of highly curved membranes [ ] and may serve as a second membrane anchor. therefore, one hypothesis might be that ns a can bridge two adjacent membranes or connect separate patches of the same membrane that come into close proximity due to membrane convolution. we speculate that membrane bridging by ns a might play a crucial role in stabilizing the complex morphology of denv-induced er-derived membrane structures, which include stacks of convoluted membranes (cm), double membrane vesicles and tubes [ ] . the vesicles were described as invaginations of the er, which are connected to the cytosol via pore-like openings [ ] . ns a may play different roles in the reorganization of these er-derived membranes. asymmetric insertion of ptms into the luminal leaflet of the inner membrane of the vesicles as well as oligomerization of ns a may induce concave membrane curvature required for vesicle formation [ ] . binding of the n-terminal region of ns a to the saddle-shaped neck region connecting the vesicle and the pore may further stabilize the vesicular structures. all positive-strand rna viruses form their replication complexes on modified host membranes. however, the source of the membranes and the nature of the modifications vary (for review see [ ] ). in general, the role of these modifications is twofold; to provide a scaffold concentrating and correctly positioning the viral and host factors for efficient viral replication and to protect the replicating virus from detection by the host immune system. the mechanisms driving the formation of these structures are still incompletely understood. convoluted membranes are a form of membrane modification induced by several positive-strand rna viruses including, in addition to denv, severe acute respiratory syndrome coronavirus (sars-cov) and kunjin virus (kunv) [ , , ] , for example. in kunv convoluted membranes are thought to be the site of polyprotein processing [ ] . while in denv the role of these structures is still unclear, they are thought to be a depot for factors required for replication [ ] . in summary, the liposome floatation data provide direct proof for specific binding of ns a( - ) to highly curved free liposomes. the main lipid binding sites in ns a( - ) are located within the n-terminal amino acid residues. the exact role of this specific interaction in the viral life cycle is still under investigation. nevertheless, this important structural information may assist in further understanding of the role of ns a and the mechanism by which it induces the membrane alterations underlying the viral rc formation. the non-structural protein a of dengue virus is an integral membrane protein inducing membrane alterations in a k-regulated manner modification of intracellular membrane structures for virus replication viral rna replication in association with cellular membranes wrapping things up about virus rna replication composition and three-dimensional architecture of the dengue virus replication and assembly sites cleavage at a novel site in the ns a region by the yellow fever virus ns b- proteinase is a prerequisite for processing at the downstream a/ b signalase site membrane curvature and mechanisms of dynamic cell membrane remodelling membrane undulation induced by ns a of dengue virus: a molecular dynamics simulation study determinants of dengue virus ns a protein oligomerization an n-terminal amphipathic helix in dengue virus nonstructural protein a mediates oligomerization and is essential for replication cellular vimentin regulates construction of dengue virus replication complexes through interaction with ns a protein dengue virus ns a cytoplasmic domain binding to liposomes is sensitive to membrane curvature recombinant production of the amino terminal cytoplasmic region of dengue virus non-structural protein a for structural studies natural abundance nitrogen- nmr by enhanced heteronuclear spectroscopy amino acid type determination in the sequential assignment procedure of uniformly c/ n-enriched proteins pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity a novel approach for sequential assignment of h, c, and n spectra of proteins: heteronuclear triple-resonance three-dimensional nmr spectroscopy. application to calmodulin best-trosy experiments for time-efficient sequential resonance assignment of large disordered proteins a suite of triple resonance nmr experiments for the backbone assignment of n, c, h labeled proteins with high sensitivity h, c and n random coil nmr chemical shifts of the common amino acids. i. investigations of nearest-neighbor effects nmrpipe: a multidimensional spectral processing system based on unix pipes the ccpn data model for nmr spectroscopy: development of a software pipeline arfgap responds to membrane curvature through the folding of a lipid packing sensor motif specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase average protein density is a molecular-weight-dependent function protein backbone and sidechain torsion angles predicted from nmr chemical shifts using artificial neural networks proteins c and ns b of the flavivirus kunjin translocate independently into the nucleus sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum we would like to thank matthias stoldt for excellent nmr support. d.w. acknowledges funding from the sonderforschungsbereich sfb . bernd w. koenig, silke hoffmann, yu-fu hung and melanie schwarten conceived and designed the study; yu-fu hung and melanie schwarten performed the experiments and analyzed the data, ella h. sklan, silke hoffmann and dieter willbold discussed the results and revised the manuscript; bernd w. koenig wrote the manuscript. the authors declare no conflict of interest. key: cord- -aixq mhf authors: charlton, frank w.; pearson, hayley m.; hover, samantha; lippiat, jon d.; fontana, juan; barr, john n.; mankouri, jamel title: ion channels as therapeutic targets for viral infections: further discoveries and future perspectives date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: aixq mhf ion channels play key roles in almost all facets of cellular physiology and have emerged as key host cell factors for a multitude of viral infections. a catalogue of ion channel-blocking drugs have been shown to possess antiviral activity, some of which are in widespread human usage for ion channel-related diseases, highlighting new potential for drug repurposing. the emergence of ion channel–virus interactions has also revealed the intriguing possibility that channelopathies may explain some commonly observed virus induced pathologies. this field is rapidly evolving and an up-to-date summary of new discoveries can inform future perspectives. we herein discuss the role of ion channels during viral lifecycles, describe the recently identified ion channel drugs that can inhibit viral infections, and highlight the potential contribution of ion channels to virus-mediated disease. the human "channelome" contains over known channels [ ] that selectively and rapidly transport ions across biological membranes in response to specific stimuli. ion channels are present on the plasma membranes and organelles of all cells, where they regulate organelle ion homeostasis, mitochondrial function, inflammasome activation, action potential firing, membrane potential, cell volume, and autophagy [ ] [ ] [ ] [ ] . given their importance, it follows that their dysfunctions leads to human diseases, termed channelopathies [ ] . these include disease states of the nervous [ ] , musculoskeletal [ ] , cardiovascular [ ] , and immune systems [ ] . this has motivated research on compounds that can modulate ion channel activity;~ % of current fda-approved drugs are ion channel modulators, second only to drugs targeting g-protein coupled receptors [ , ] . many viruses encode their own ion channels [ ] [ ] [ ] termed "viroporins," highlighting the importance of ionic balance during viral infection. this field has spurred intense research and several drugs that target viroporins have emerged (reviewed in [ ] ). more recent evidence highlights how viruses can regulate and/or depend on the ion channels expressed by host cells, highlighting them as new host targets for therapeutic intervention (reviewed by hover et al., ) [ ] . given recent and important advances in this field, we herein provide an up-to-date review of the virus-ion channel literature and discuss the future prospects of ion channel drugs as anti-viral agents. firstly, we highlight recent evidence that suggests that viruses have adapted to take advantage of endolysosomal ionic balance as a cue for viral entry. we then discuss how intracellular ion channels contribute to the two-pore channels and (tpc / ) gunaratne et al., [ ] severe fever with thrombocytopenia syndrome virus (sftsv) unknown channel li et al., [ ] severe acute respiratory syndrome coronavirus (sars-cov- ) two-pore channel (tpc ) ou et al., [ ] bunyamwera orthobunyavirus (bunv) two-pore domain k + (k p ) hover et al., / [ , ] hazara orthonairovirus (hazv) unknown k + channel punch et al., [ ] charlton et al., [ ] human immunodeficiency virus (hiv) g-protein coupled inwardly rectifying k + (girk) atp-sensitive k + k atp dubey et al., [ ] merkel cell polyomavirus (mcpyv) the involvement of ca + channels during viral entry is now well-documented [ ] . fujioka et al. showed that influenza virus (iav) hemagglutinin (ha) triggers intracellular [ca + ] oscillations that are required for viral infection [ ] . the initial modulation of ca + by iav was demonstrated using förster resonance energy transfer (fret)-based imaging of the ca + sensor yellow cameleon (yc . ). these oscillations in ca + were mediated by a specific voltage-gated ca + channel (ca v . ) identified through sirna silencing approaches. assays subsequently revealed that iav directly binds to ca v . via the interaction of ha and a sialylated site on ca v . . accordingly, iav entry could be inhibited by diltiazem, a clinically available ca + blocker, highlighting the potential of these compounds for drug repurposing. ebola virus (ebov) also requires ca + channels for its entry into host cells. ebov enters cells through endolysosomes positive for both niemann-pick c (npc ) and two-pore ca + channel (tpc ) [ ] . to further characterise this pathway, penny et al. expanded the pharmacology of tpcs using a virtual screen of~ fda-approved drugs. all identified tpc modulators were cross-referenced with two recent anti-ebov screens, with four dopamine receptor antagonists and five oestrogen receptor modulators identified. as such, it was reasoned that these drugs exert their inhibitory effects on ebov through the blockade of tpcs ( figure e ), subsequently confirmed through ebov virus-like particle (vlp) assays [ ] . das et al. further characterised the role of ca + in ebov entry using single-molecule fret (smfret)-imaging. it was shown that ca + and ph synergistically induce a conformational change in the ebov glycoprotein gp (a key mediator of receptor binding and viral entry) to form a reversible intermediate state primed for npc binding. npc binding then further promotes the conformational transition into a fusion-ready "primed" state of invading ebov virions [ ] . of importance to the current pandemic, it has been shown that middle east respiratory syndrome coronavirus (mers) [ ] is dependent on tpcs to escape endosomes [ ] . as an enveloped virus, mers must fuse its envelope with host membranes to enter cells. following receptor attachment, mers particles can fuse at either the cell surface or intracellularly in the endosomal network. fusion is mediated by the proteolytic cleavage of the viral spike (s) protein at its s /s site. at the surface of the cell, this proteolytic event is facilitated by tmprss , which in turn precludes exposure of the fusion loop and coalescence of host and viral membranes [ ] . alternatively, fusion can occur in late endosomes following translocation through the endocytic network and proteolytic processing by proprotein convertases, including furin, in a process regulated by ca + [ , ] . in studies by gunaratne et al., genetic silencing of tpc and tpc prevented the entry of pseudotyped mers ( figure f ). the dependence of mers upon tpcs was further demonstrated through its inhibition by tetrandrine and fangchinoline (tpc inhibitors), which prevented a post-internalisation but pre-fusion entry event. the mechanism through which tpc blockade inhibited mers was multi-faceted: tpc and tpc silencing impaired furin activity, whilst pharmacological and genetic inhibition of tpc impaired endosomal motility. of note, the related sars-cov- , the causative agent of covid- [ , ] , was similarly inhibited by tpc blockade. specifically, treatment of cells with tetrandrine reduced the entry of a lentiviral vector pseudotyped with the sars-cov- s (spike) [ ] . the bunyavirus severe fever with thrombocytopenia syndrome virus (sftsv) is an emerging arbovirus with fatality rates of - % and the potential to cause future pandemics [ ] . using a library of fda-approved drugs, the ca + channel blockers benidipine hydrochloride and nifedipine were shown to inhibit sftsv infection ( figure c) , with in vivo activity confirmed using c bl/ and humanised mouse models. through a retrospective analysis of human sftsv cases, clinical evidence of the efficacy of nifedipine as an anti-sftsv therapeutic was also demonstrated. a cohort of patients receiving nifedipine prior to and during hospital admission showed enhanced viral clearance and reduced frequency of neurological syndromes, often associated with fatal outcomes [ , ] . the fatality rate of patients receiving nifedipine was reduced -fold compared to untreated patients, which corresponded to abnormal serum ca + levels at admission. the viral processes through which sftsv requires ca + channels were subsequently shown to be during virus internalisation and genome replication. the involvement of k + channels in viral entry has been extensively characterised for the model bunyaviruses bunyamwera orthobunyavirus (bunv); and hazara orthononairovirus (hazv), a model for crimean congo haemorrhagic fever virus, which causes severe viral haemorrhagic fever outbreaks, with a case fatality rate of up to %. initial work using known k + channel pharmacology suggested that the blockade of two-pore k + channels (k p ) inhibited the early stages of the bunv lifecycle ( figure d ) [ ] . subsequent work identified both acidic ph and k + in endosomes as crucial biochemical cues for the endosomal escape of bunv [ ] . similar studies in hazv highlighted a dependence on k + channels for infection, and that k + primarily accumulated in cholesterol-rich endosomes ( figure b) [ , , ] . the k + dependence of hazv involves the glycoprotein spikes; a change in k + concentration triggers conformational changes in the glycoproteins, as revealed through cryo-electron tomography of hazv virions incubated with k + that "primed" them for insertion into target membranes (figure a ). moreover, it was shown that both bunv and hazv could be "primed" in vitro in buffers containing high [k + ], which expedited entry and subsequent viral gene expression. this phenomenon was analogous to earlier studies for iav, in which acid bypass in the presence of k + revealed that the exposure of iav virions to low ph and high [k + ] weakened interactions between the m matrix protein and ribonucleoprotein (rnp) bundles, a pre-requisite for genome release ( figure b ). the exposure of iav virions to k + therefore drives viral uncoating and expedites iav infection [ ] . recent work also highlights a requirement for k + channels during human immunodeficiency virus (hiv) infection. using pharmacological approaches ( figure a ) [ ] hiv entry could be blocked with ifenprodil and the broad spectrum k + channel blocker tetraethylammonium (tea). khan et al. also showed that the pharmacological activation of the endolysosome-resident transient receptor potential mucolipin channel (trpml ) enhanced the degradation of hiv-tat (a multi-function viral protein involved in transcription, splicing, capping, and translation), which in turn reduced the transition from viral latency [ ] . trpml activates large conductance ca + -activated potassium (bk) channels in endosomes [ ] , implying that this channel is required for hiv infection. the reliance of viruses upon ion channels is not restricted to rna viruses. it was recently shown that both k + and ca + channels are important host factors for polyomavirus infection [ ] . using a panel of ion channel modulators, the entry of merkel cell polyomavirus (mcpyv), the causative agent of merkel cell carcinoma (mcc), was shown to be sensitive to -aminopyridine ( -ap), a blocker of voltage-gated k + (k v ) channels ( figure g ). moreover, both mcpyv and simian virus (sv ) ( figure h ) were sensitive to verapamil, a broad-spectrum ca + channel blocker. the identities of the ca + channels involved in polyomavirus entry were further explored, revealing a requirement for transient (t-type, low-voltage activated) channel family members in mcpyv infection but not sv . tetrandrine, a tpc blocker, restricted both viruses. the role of tpcs was found to be during endoplasmic reticulum (er) disassembly and/or er docking for sv [ ] , which may be explained by the recent demonstration that ca + ions mediate the stabilization of sv capsids and contribute to its disassembly [ ] . figure i ) [ ] . time of addition assays using the cftr inhibitors cftr and glibenclamide, combined with the assessment of exposure of vp /vp minor capsid proteins, indicated a role for cftr in the trafficking of bkpyv to the er. whilst the mechanism of cftr involvement in bkpyv er trafficking remains unclear, it is hypothesised that the channel may be important in the acidification and er docking of bkpyv-containing endosomes. cftr has been implicated in the fusion of endosomes [ ] , and co-localises with vacuolar atpase (vatpase) to provide the counter-charge during endosomal acidification [ ] . figure i ) [ ] . time of addition assays using the cftr inhibitors cftr and glibenclamide, combined with the assessment of exposure of vp /vp minor capsid proteins, indicated a role for cftr in the trafficking of bkpyv to the er. whilst the mechanism of cftr involvement in bkpyv er trafficking remains unclear, it is hypothesised that the channel may be important in the acidification and er docking of bkpyv-containing endosomes. once viral genomes are released inside the host cell, replication can commence. recent evidence suggests that this process is partially controlled by ion concentrations and can therefore be targeted by ion channel drugs. flaviviruses establish replication complexes in modified intracellular membranes, often derived from the er. the er stores the majority of intracellular ca + , and so it is perhaps unsurprising that an array of flaviviruses depend on intracellular ca + ion channels for their replication (table ) . japanese encephalitis virus (jev) is an arthropod-borne virus linked to acute encephalitis. wang et al. performed a screen of fda-approved drugs to assess in vitro activity against jev [ ] . within the screen, three of the five most potent inhibitors were blockers of voltage-gated ca + channels (vgccs), including manidipine, cilnidipine, and benidipine hydrochloride. time of addition assays suggested that these three drugs did not act during viral entry, nor were they virucidal, but specifically inhibited virus replication. the potential of these drugs as broad-acting anti-flavivirus treatments was further assessed and each led to a concentration-dependent inhibition of dengue virus denv), west nile virus, and zika virus (zikv) replication. yellow fever virus (yfv) was, however, insensitive to manidipine. interestingly, the in vitro selection of a manidipine-resistant jev identified a q r mutation in the non-structural protein ns b. sequence alignments confirmed that the q site was conserved in each of the flaviviruses susceptible to manidipine, but not yfv. the efficacy of manidipine against jev was further confirmed in in vivo mouse models, with manidipine-treated mice exhibiting significantly higher survival rates compared to untreated mice challenged with jev. a role for ca + channels during denv replication was further revealed by dionicio et al. [ ] . denv was shown to inhibit intracellular ca + release from the er, which in turn activated the store-operated ca + (soce) pathway. in addition, specialised ca + release activated ca + channels (cracs) were identified as a requirement for denv replication. small molecule blockers of these channels resulted in a % reduction in virus yield. additionally, yeast two-hybrid screens identified the ca + -permeable non-selective transient receptor potential vanilloid (trpv ) in complex with the dead-box helicase (ddx x) as a key regulator of viral mrna translation for zikv [ ] . the hepatitis b virus (hbv) x protein is an oncoprotein that regulates cytosolic [ca + ] [ ] . recently, yao et al. characterised this mechanism, revealing its interaction with orai , a critical component of the soce pathway [ ] . hbv replication was also dependent on k + in studies by chakraborty et al. [ ] . they reported that k + -dependent nucleolytic activity in the presence of hbv rna mediates the self-cleavage of a nt oligomer with ribozyme activity that is required for viral replication. chikungunya virus (chikv) is a re-emerging arbovirus associated with long-term complications and high morbidity. using sirna silencing of the cl − intracellular channels (clic) and , müller et al. demonstrated a requirement for both channels during the replication of a chikv sub-genomic replicon in mammalian and invertebrate cells [ ] . the voltage-dependent anion channel (vdac ) is also implicated in viral replication; it is upregulated by infectious bursal disease virus (ibdv). han et al. showed that knockdown of vdac inhibited ibdv replication through the reduction of viral polymerase activity, and that the overexpression of vdac promotes polymerase activity. immunoprecipitation (ip) experiments showed that vdac interacts with ibdv vp and vp , components of rnps, indicating a role for this channel in rnp formation [ ] . viral pathologies are becoming increasingly linked to the dysregulation of host ion channels (table ). this reveals an interesting and new avenue for ion channel drugs, as the pharmacological manipulation of virus-targeted channels may not only impair viral infection at the cellular level, but may circumvent virus-induced channelopathies. recent studies have linked viral infection to neuronal pathologies through the dysregulation of ca + signalling. the fda-approved alzheimer's drug memantine protected against neuronal cell death induced by zikv infection [ ] . memantine acts upon the n-methyl-d-aspartate receptor (nmdar), which mediates ca + signalling to govern synaptic plasticity [ ] . the overstimulation of nmdar is linked to neurodegeneration, a pathology commonly associated with zikv. upon challenge with memantine, zikv replication was unaffected, but antagonism of nmdar invoked a neuroprotective effect in vivo. whilst the exact mechanism(s) of zikv neuropathologies are unknown, it is predicted that the virus hyper-stimulates nmdar to upregulate ca + signalling to the point of ca + overload and postsynaptic neuronal death, a process termed "glutamate excitotoxicity." the reactivation of herpes simplex virus (hsv- ) can lead to cranial nerve disorders and severe pain. zhang et al. revealed that hsv- disrupts the expression of t-type ca + channels in differentiated sensory-like neurons, as a means to disrupt pain responses [ , ] . proteomics and transcriptomics showed that hsv- decreased the expression of the ca v . t-type ca + channel subunit at the protein level, despite increasing ca v . mrna synthesis. this upregulation of ca v . mrna synthesis was postulated to be a compensatory response to decreased expression of the channel subunit. the loss of ca v . initially led to reduced pain transmission in infected neurons; however, the release of interleukin- (il- ) in response to viral infection was subsequently shown to restore ca v . current density and pain responses. rotavirus (rv) dysregulates cellular ca + homeostasis through the depletion of er stores. using genetically-encoded ca + indicators in infected cells, cytosolic ca + increased in distinct peaks [ ] [ ] [ ] , which was mediated by the non-structural protein nsp . rv-nsp acts as a ca + -permeable viroporin to release ca + from the er, which in turn activates the er ca + sensor stim , subsequently leading to soce activation. rv-infected cells then secrete a cleavage product of nsp (ensp ), which elicits an inward inositol triphosphate (ip )-dependent ca + signal, which in turn stimulates cl − release through ca + -activated cl − channels (caccs) [ ] . the efflux of cl − from cells is a known causative factor of diarrhoea in vivo, thereby identifying nsp as the first viral enterotoxin. this multi-faceted control of ca + signalling suggests a crucial role for ca + channels in the pathophysiology of rv. in this regard, it was shown that the blockade of caccs, including tmem a, reduces intestinal motility and fluid loss in vivo with no direct effects on the levels of virus infection [ ] . coxsackie virus b (cvb ), amongst other enteroviruses, is associated with cardiomyopathies and sudden cardiac death [ ] . kcnq is a k v channel (k v . ) that mediates a delayed, slow rectifying k + current in ventricular tissue to regulate contractility. kv . trafficking and activity are regulated by the serine/threonine kinase sgk [ ] , which is upregulated by cvb . as such, kcnq currents are elevated to % in cvb infected cells, whilst herg (k v . ) and ca v . activity decrease by % and % respectively. these results corroborated with localisation studies of each channel and demonstrated that the surface expression of kcnq increased, whilst herg and ca v . expression decreased in infected cells. inherited mutations in each of these three ion channels are associated with heart rhythm disorders. importantly, decreased herg expression increases the risk of drug-induced arrhythmias; the additional inhibition or reduced trafficking of this channel in cells targeted by small therapeutic molecules reduces the number of redundant repolarisation currents, in turn depleting the overall repolarisation reserve. together, these data suggest that cvb re-programmes ion channel expression in cardiac tissue, leading to an increased risk of arrhythmia. this highlights these channels as therapeutic targets to prevent the sudden cardiac death that results from cvb infection. a number of viruses that primarily infect the airway system have been shown to dysregulate airway epithelial na + transport. human respiratory syncytial virus (hrsv) primarily infects airway epithelial cells, and dysregulates epithelial na + channels (enac) to disrupt na + flux in the airways [ ] . enacs are a critical mediator of osmotic fluid absorption across airway epithelia, through the selective transport of na + . electrochemical balance is maintained through apical cl − channels, which include cftr. clinical studies of infants diagnosed with hrsv showed a negative correlation between enac mrna expression and the severity of hrsv bronchiolitis [ ] . hrsv has been shown to manipulate airway ion flux through upregulation of channels involved in the cough reflex, namely, transient receptor cation channel, subfamily a, member (trpa ), and the acid sensing ion channel receptor (asic ), a member of the enac family of na + channels [ ] . hrsv induced a -fold increase in asic mrna in normal bronchial epithelial cells, compared to the -fold increase observed in cells challenged with measles virus (mev). interestingly, uv-inactivated hrsv and mev maintained their ability to upregulate asic , suggesting these effects were independent of genome replication. the virion-induced upregulation of il- and il- was subsequently shown to inhibit trp receptor activity, identifying these receptors as potential targets for virus-induced cough symptoms. enac and cftr channel expression are also modulated by iav. from single-cell recordings in intact lung epithelial cells, brand et al. described a reduction in enac and cftr activity upon iav-infection, through reduced surface expression. the mechanisms governing this downregulation were not characterised, but it is thought that iav promote er stress, known to cause deficits in enac abundance [ ] . the loss of enac and cftr surface expression was accompanied by a reduction in airway surface liquid (asl), a known contributor to cystic fibrosis. importantly, treatment with the fda-approved cftr corrector lumacaftor could restore iav-mediated asl perturbations, highlighting how virus-induced pathologies can be treated by therapies targeting host ion channels [ ] . the altered activity of na + channels was observed in cells latently infected hsv- dorsal root ganglion neurones. upon acute lytic infection, hsv- was found to reduce functional voltage-gated sodium channel (vgsc) expression within h and abolish vgsc activity within days, whilst latent hsv- infection led to a recovery of these currents and increased na v . channel expression. furthermore, hsv- reactivation from a dormant state decreased vgsc activity. it is known that vgscs play a role in the transmission of pain signals [ ] . similarly, post-herpetic neuralgia associated with varicella-zoster virus is associated with an increase in na + current amplitude through the activity of na v . and na v . [ ] . na v . dysregulation has also been associated with hereditary pain disorders. gain-of-function mutations in scn a, the gene encoding na v . , are causative of primary erythromelalgia (pe), a rare neuropathy characterised by recurring pain, warmth, and redness of the extremities. research into the management of pe identified two novel selective na v . blockers, pf- and tv- , which may hold promise in ameliorating pain symptoms associated with pe [ ] and viral-induced neuropathies. stakaitytė et al., used a proteomics approach to identify changes in the host channelome in response to the overexpression of mcpyv small tumour antigen (st). the analysis revealed a role for two clic proteins [ ] , clic and clic ( -fold and -fold up regulation in cells overexpressing st vs. control cells, respectively). pharmacological and genetic inhibition of these channels reduced st-induced motility and migration, implicating their function in mcpyv, st-induced metastatic processes [ ] . these data were reinforced by the finding that mcpyv-positive mcc tumours showed enhanced clic and clic expression ( . -fold and . -fold increase respectively), implicating their involvement in mcc tumorigenesis. these findings align with earlier evidence that clic and clic are involved in the metastatic progression of specific tumour types, through switching of cellular localisation and function to integral transmembrane proteins as active anion channels and signal transducers [ ] . it is now clear that host cell ion channels play an important role during viral infection at the cellular level, and as causative factors of disease states in infected tissues. ion channels have been linked to multiple stages of viral lifecycles, in which viruses are either passively dependent upon or actively modulate channel functionality. given this knowledge, evidence is emerging that ion channel inhibitors represent a new antiviral strategy. whilst toxicity profiles for ion channel inhibitors are only available in the context of those used to treat hereditary channelopathies, in vivo evidence is emerging that these drugs can be efficacious against viruses. moreover, overlapping mechanisms of acquired and hereditary channelopathies may underpin the efficacy of ion channel modulators in treating virally-induced pathophysiologies. the manipulation of host ion homeostasis presents an attractive target for the treatment of many clinically important viruses and their associated pathologies, whilst circumventing the risks of resistance associated with direct-acting antiviral drugs. as such, continued studies of host-virus interactions may guide future antiviral approaches. funding: j.m. and j.f. are supported by the academic fellow scheme at the university of leeds. the authors declare no conflict of interest. overview of molecular relationships in the voltage-gated ion channel superfamily genetic neurological channelopathies heptad repeat -based peptides inhibit avian sarcoma and leukosis virus 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ebolavirus glycoprotein directs fusion through npc + endolysosomes mining of ebola virus entry inhibitors identifies approved drugs as two-pore channel pore blockers naadp-dependent ca + signaling regulates middle east respiratory syndrome-coronavirus pseudovirus translocation through the endolysosomal system calcium channel blockers reduce severe fever with thrombocytopenia syndrome virus (sftsv) related fatality characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov modulation of potassium channels inhibits bunyavirus infection potassium is a trigger for conformational change in the fusion spike of an enveloped rna virus cellular cholesterol abundance regulates potassium accumulation within endosomes and is an important determinant in bunyavirus entry g protein-coupled and atp-sensitive inwardly rectifying potassium ion channels are essential for hiv entry identification of potassium and calcium channel inhibitors as 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extracellular tat-mediated hiv- ltr transactivation bk channels alleviate lysosomal storage diseases by providing positive feedback regulation of lysosomal ca + release two-pore channels control ebola virus host cell entry and are drug targets for disease treatment calcium bridge triggers capsid disassembly in the cell entry process of simian virus intracellular cftr: localization and function functional vacuolar atpase (v-atpase) proton pumps traffic to the enterocyte brush border membrane and require cftr screening of fda-approved drugs for inhibitors of japanese encephalitis virus infection dengue virus induced changes in ca + homeostasis in human hepatic cells that favor the viral replicative cycle the trpv channel links calcium influx to ddx x activity and viral infectivity calcium signaling by hbx protein in hepatitis b virus dna replication hepatitis b virus x protein upregulates intracellular calcium signaling by binding c-terminal of orail protein the epsilon motif of hepatitis b virus rna exhibits a potassium-dependent ribonucleolytic activity voltage-dependent anion channel interacts with ribonucleoprotein complexes to enhance infectious bursal disease virus polymerase activity chikungunya virus requires cellular chloride channels for efficient genome replication n-methyl-d-aspartate (nmda) receptor blockade prevents neuronal death induced by zika virus infection can an fda-approved alzheimer's drug be repurposed for alleviating neuronal symptoms of zika virus? mbio regulation of t-type ca + channel expression by interleukin- in sensory-like nd / cells post-herpes simplex virus (hsv- ) infection regulation of t-type ca + channel expression by herpes simplex virus- infection in sensory-like nd cells rotavirus calcium dysregulation manifests as dynamic calcium signaling in the cytoplasm and endoplasmic reticulum imaging intraorganellar ca + at subcellular resolution using cepia use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells a functional nsp enterotoxin peptide secreted from rotavirus-infected cells shikonin inhibits intestinal calcium-activated chloride channels and prevents rotaviral diarrhea modulation of voltage-gated sodium channel (vgsc) activity in human dorsal root ganglion (drg) neurons by herpesvirus quiescent infection a kidnapping story: how coxsackievirus b and its host cell interact respiratory virus infection up-regulates trpv , trpa and asics receptors on airway cells influenza-mediated reduction of lung epithelial ion channel activity leads to dysregulated pulmonary fluid homeostasis the cellular chloride channels clic and clic contribute to virus-mediated cell motility long qt syndrome-associated mutations in kcnq and kcne subunits disrupt normal endosomal recycling of i ks channels inhibition of na+ transport in lung epithelial cells by respiratory syncytial virus infection decreased airway epithelial ion transport was associated with the severity of the respiratory syncytial virus infection and complications in infants epithelial sodium channel abundance is decreased by an unfolded protein response induced by hyperosmolality varicella-zoster viruses associated with post-herpetic neuralgia induce sodium current density increases in the nav- . neuroblastoma cell line primary erythromelalgia: a review merkel cell polyomavirus small t antigen mediates microtubule destabilization to promote cell motility and migration chloride channels in cancer: focus on chloride intracellular channel and (clic and clic ) proteins in tumor development and as novel therapeutic targets this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - p scli authors: majzoub, karim; wrensch, florian; baumert, thomas f. title: the innate antiviral response in animals: an evolutionary perspective from flagellates to humans date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: p scli animal cells have evolved dedicated molecular systems for sensing and delivering a coordinated response to viral threats. our understanding of these pathways is almost entirely defined by studies in humans or model organisms like mice, fruit flies and worms. however, new genomic and functional data from organisms such as sponges, anemones and mollusks are helping redefine our understanding of these immune systems and their evolution. in this review, we will discuss our current knowledge of the innate immune pathways involved in sensing, signaling and inducing genes to counter viral infections in vertebrate animals. we will then focus on some central conserved players of this response including toll-like receptors (tlrs), rig-i-like receptors (rlrs) and cgas-sting, attempting to put their evolution into perspective. to conclude, we will reflect on the arms race that exists between viruses and their animal hosts, illustrated by the dynamic evolution and diversification of innate immune pathways. these concepts are not only important to understand virus-host interactions in general but may also be relevant for the development of novel curative approaches against human disease. the animal kingdom, including humans, has evolved while facing constant threats from viral elements. viruses can be, in some cases, beneficial for a given animal species and drive its evolution [ ] . however, their uncontrolled replication may cause disease and prove fatal to their hosts. consequently, animal cells have evolved devoted pathways which ( ) sense and recognize pathogen-associated molecular patterns (pamps) and, more particularly, virus-associated molecular signatures; ( ) initiate signaling cascades stemming from the site of detection, translocating the information to the nucleus; and ( ) induce a transcriptional program that confers an antiviral state to the host ( figure ). interestingly, a closer examination of individual factors constituting these pathways shows a different conservation status between different animal species. while genes encoding sensors and signaling platforms are generally well conserved amongst animals, virus-stimulated genes (vsgs) are clearly less so and are subject to faster evolution [ , ] . in vertebrates, one such vsg is the secreted interferon (ifn) cytokine, that signals in an autocrine and paracrine fashion. secreted ifn molecules bind to cell-surface receptors and initiate signal transduction involving the janus kinase/signal transducer and activator of transcription (jak-stat) pathway. this pathway induces the transcription of a major antiviral program composed of hundreds of so-called ifn-stimulated genes (isgs) that comprise effectors of the cell-autonomous antiviral defense [ ] . a lot of our understanding of the innate antiviral immune system in animals is a result of studies conducted in vertebrates and more particularly in mammalian species. therefore, the ifn system has been heavily studied over the last years. however, during the last two decades, we came to appreciate that the ifn system, as we know it, is a vertebrate particularity. indeed, while some animal species like insects or nematodes are devoid of ifns and rely on rna interference (rnai) as the major antiviral pathway, some others, like mollusks, have conserved all the components that lead to ifn production but have no obvious homologs of type i ifn cytokines. nevertheless, these are predicted to use an ifn-like antiviral cytokine [ ] . in fact, the ifn cytokine itself seems to be an evolutionary novelty, however, the pathways dictating its production existed early in metazoan evolution [ , ] (figure ). interestingly, ifn is not the only vsg induced upon viral detection in mammals. certain isgs that directly interfere with the viral life cycle like viperin are also immediately induced after viral infection in an ifn-independent fashion [ ] [ ] [ ] . in this review, we will focus on the conserved pathways that are responsible for sensing viral pamps, signaling and inducing antiviral genes upon infection in animals. we will start by describing our current view on the immediate activation of the ifn and nf-kb pathways in vertebrate species. we will then zoom in on two important viral nucleic acid receptor families, toll-like receptors (tlrs) and rig-i-like receptors (rlrs), describe their function in viral rna detection and their conservation across animal species. next, we will focus on a central hub in the signaling pathways induced by dna viruses, the stimulator of ifn genes (sting). we will then examine how the evolutionary conflicts between viruses and host immune factors are shaping antiviral immunity in animals. viruses and transposable elements are very powerful drivers of evolution [ , ] . however, their uncontrolled replication and spread can be catastrophic to host cells. it is therefore not surprising that every known living species on the planet has evolved measures to recognize and counteract parasitic genetic elements. it is suggested that anti-sense mediated targeting of viral nucleic acids was the most primordial strategy protocells used to fend off viral threats [ ] . this is illustrated by argonaute and crispr-based defenses in bacteria, archaea and rnai systems in plants, all of which rely on anti-sense nucleic acids that program a nuclease to target and degrade the complementary invading viral genome [ ] [ ] [ ] . because many of the core rnai machinery components can be found in all eukaryotic superkingdoms, it is thought that this antiviral defense mechanism predated the emergence of pattern-recognition receptor (prr)-based immunity ( figure ) [ ] . interestingly, antiviral defense in eukaryotes has diversified greatly during evolution, with some species maintaining rnai-based defenses [ ] and others innovating and adopting completely novel antiviral strategies. in chordates and more particularly in vertebrate animals, the emergence of ifn-i and a recombinational adaptive immune system seems to coincide with the loss of rnai as the main antiviral mechanism in somatic cells. there is strong evidence of an intrinsic incompatibility between an antiviral rnai and the prr-ifn system. for instance, while long dsrnas can produce functional small interfering rnas (sirnas) in stem cells in the absence of ifn, the same dsrna molecule is not processed onto sirna and is sensed as a pamp in vertebrate somatic cells [ , ] . interestingly, experimental evidence of antiviral rnai in mammals seems to be limited to specialized pluripotent cells in which the prr-ifn system is not fully deployed yet. the emergence of both the ifn-i (innate) and somatic dna recombination systems (adaptive) in vertebrates constituted a major evolutionary event that dispensed them from using rnai for antiviral purposes. interestingly, retrotransposition and selfish transposable elements were determinants in the acquisition of these two systems [ , [ ] [ ] [ ] . the emergence of somatic dna recombination in vertebrate animals was considered an "immunological big bang" [ , ] . indeed, somatic dna recombination in specialized b and t cell lineages provided jawed vertebrates with large repertoires of major histocompatibility complexes (mhc), t-cell receptors (tcrs) and immunoglobulins (igs). until relatively recently, adaptive immunity was believed to be exclusive to gnathostomes (jawed vertebrates). we now know that agnathans (jawless vertebrates), including lampreys and hagfish, have also evolved an equivalent adaptive immune system with specialized lymphocytes termed, vlra, vlrb and vlrc cells, in which specific variable lymphocyte receptors (vlrs) are produced through somatic leucine-rich repeat (lrr) rearrangements [ ] . in both gnathostomes and agnathans, somatic recombination events in specialized cells permitted a pathogen-tailored response and endowed vertebrate species with an immune memory. until the end of the last century, most vertebrate immunologists concentrated their efforts on studying the adaptive arm of the immune system. nearly years ago, charles janeway predicted the presence of an evolutionary ancient immune system that detects conserved microbial and danger signals, termed the pathogen-associated molecular patterns (pamps) and danger-associated molecular patterns (damps), respectively. janeway predicted that this innate immune system precedes and instructs the adaptive system [ , ] and posited that pamps and damps must be sensed by germ-line encoded prrs. at that time, the innate immune components were severely understudied and tlrs, rlrs and sting's respective functions in immunity were completely unknown. this illustrates the immense leap forward the innate immunity field has experienced in the last three decades. today, we know that vertebrates share with other invertebrate animals specialized phagocytic cells that are able to discriminate between self and non-self. by recognizing general pathogen molecular patterns (pamps, e.g., viral double-stranded rna) and danger signals (damps), these cells establish an immediate and general inflammatory response translated into an antimicrobial and/or antiviral state. although mainly studied in vertebrates, the machineries responsible for these responses transcend this group and are fairly conserved in all animals. we will describe a particular arm of the innate immune pathways, the innate antiviral system that is best studied in mammals. unlike bacteria, viruses represent a unique challenge for prrs because they possess few unique signatures that could serve as pamps [ ] . however, viral nucleic acids (dna or rna) could have peculiar biochemical features that differentiate them from endogenous host rna [ ] . in rna molecules, for example, the lack of a -methylguanosine cap structure, double strandedness or the trior bi-phosphorylation at their ends are often used by prrs for self/non-self-discrimination. prrs that detect viral infection can be classified into four families: tlrs, rlrs, aim -like receptors (alrs) and the cgas-sting sensors [ ] . after viral detection, prr-mediated signaling directly or indirectly induces transcription factors, including ifn-regulatory factors (irfs) and nuclear factor k-b (nf-kb) to upregulate expression of vsgs including pro-inflammatory cytokines. another class of prrs such as double-stranded rna (dsrna) activated protein kinase r (pkr; also known as eif ak ), adenosine deaminase acting on rna (adar ) and - -oligoadenylate synthetase (oas ) also contribute to innate immunity [ ] . these also recognize viral signatures; however, their main function is not necessarily to induce a transcriptional immune response, but rather to directly attack viral rna by degrading it or inhibiting its translation. for this reason, these are not usually considered receptors. viruses are obligatory intracellular parasites, therefore their detection by prrs most often occurs in the intracellular milieu. endosomal transmembrane tlrs, including tlr , tlr and tlr recognize dsrna in the endosome lumen [ ] [ ] [ ] . rlrs including rig-i [ ] , melanoma differentiation associated gene (mda ) [ ] , and laboratory of genetics and physiology (lgp ) [ , ] detect viral rnas in the cytosol, whereas cytosolic viral dna is mainly recognized by cyclic-gmp-amp (cgamp) synthase (cgas) [ ] . therefore, the nature of the viral particle (e.g., enveloped vs. non-enveloped) and the viral genome (e.g., dna vs. rna) dictates which of these receptors recognizes the infection first ( figure single-stranded rna (ssrna) is a potent tlr and tlr ligand, while tlr is specific for dsrna. tlr , for example, recognizes dsrna viruses from reoviruses [ ] , but can probably also recognize dsrna intermediates from (+) strand rna viruses like coxsackievirus and west nile virus (wnv) and (-) strand rna viruses like the human respiratory syncytial virus (hrsv) [ ] . indeed, all rna viruses are thought to produce dsrna intermediates as part of their replication cycle, so both ssrna and dsrna viruses have the potential to be sensed by tlr . tlr and , on the other hand, have been shown to prefer ssrna ligands from (-) strand rna viruses such as vesicular stomatitis virus (vsv) and influenza a virus (iav) [ , ] . when it comes to rlrs, most rna viruses have been shown to be detected by rig-i or mda , as these receptors have a high affinity to dsrnas. while rig-i prefers viral rnas bearing di-or tri-phosphate groups at their while the cytosolic recognition of viral rna is almost exclusively mediated by rlrs, several proteins have been proposed to play a role in dna sensing and triggering innate immune responses, such as the dna-dependent activator of ifn-regulatory factors (dai), ddx , rna polymerase iii, ifi and dna-pk [ ] [ ] [ ] [ ] [ ] [ ] . however, among all the proposed sensors, only cgas knock-outs can completely shut down ifn production in response to cytosolic dna [ ] . the cgas protein is now thought to be the major viral dna sensor and has been shown to detect adenovirus, human papillomavirus (hpv), herpes simplex virus- (hsv- ) and cytomegalovirus (cmv) [ , [ ] [ ] [ ] . aim has also been shown to activate the inflammasome upon dna stimulation [ ] but will not be discussed in this review. rna ligands cause the endosomal transmembrane tlr , and to dimerize and then to oligomerize through their cytoplasmic tir (toll/il- receptor) domains. this allows tlrs to recruit signaling adaptors via tir-tir interactions [ ] . tlr recruits the adaptor protein trif (tir-domain-containing adapter-inducing ifn-β) [ , ] . trif is able to play a dual role by inducing the ifn or the nf-kb pathways. when it comes to ifn, after activation, trif recruits the ubiquitin ligase traf (tumor necrosis factor receptor-associated factor ) through an ubiquitination mechanism which in turn recruits tank-binding kinase (tbk ) [ , ] . the trif/tbk complex is then able to phosphorylate the transcription factor irf , triggering its dimerization and nuclear translocation. phosphorylated irf dimers specifically bind to ifn-stimulated response elements (isres) present in the ifn-β gene promoter which leads to the transcription of this cytokine [ ] . trif can also recruit ripk (receptor-interacting serine/threonine-protein kinase ) that leads to the activation of the ikk complex, releasing the nf-kb transcription factor from its ikb inhibitory subunit and resulting in its translocation to the nucleus to induce the transcription of pro-inflammatory cytokines [ ] (figure ). unlike tlr , the activation of tlr and tlr recruits the adaptor protein myd (myeloid differentiation primary response ) through tir-tir domain interaction. myd death domains oligomerize which triggers the formation of the myddosome signaling complex consisting of myd and the irak family of kinases (il- receptor-associated kinases), irak , and . through a series of phosphorylations and the help of the e ubiquitin ligase traf , the myddosome is able to recruit and activate the transcription factors irf , irf and nf-kb that translocate to the nucleus to induce the transcription of ifn-α genes and other proinflammatory cytokines [ ] [ ] [ ] [ ] [ ] [ ] [ ] (figure ). rlrs (rig-i, mda and lgp ) are characterized by a central dead-box helicase/atpase domain and a c-terminal regulatory domain (ctd) essential for rna recognition and autorepression in the absence of rna ligands. with the exception of lgp , rlrs also possess two n-terminal caspase activation and recruitment domains (cards). upon rna binding rig-i is remodeled into an active conformation in which the ctd and helicase domains organize into a ring around the rna ligand and the card domains are exposed [ , ] which facilitate their interactions with other card domains resulting in rig-i tetramers. although rig-i and mda share similar domain architectures, mda seems to prefer longer dsrna, assembling along these molecules to form helical, filamentous oligomers [ , ] . a poly-ubiquitination reaction by ubiquitin ligases like riplet and trim (tripartite motif-containing ), is thought to enhance rig-i and mda oligomerization and activation [ ] [ ] [ ] [ ] . rig-i and mda oligomers then serve as a scaffold for binding to the adaptor protein mavs (mitochondrial antiviral signaling protein, also known as ips- , visa, and cardif) [ ] [ ] [ ] . mavs has been shown to be critical for mounting an efficient immune response to infection by several rna viruses [ ] . its c-terminal transmembrane domain is inserted into the outer mitochondrial membrane [ ] , whereas its n-terminal card domain mediates its aggregation on the mitochondrial surface by interacting with the tandem cards of rig-i or mda oligomers [ , ] . mavs aggregates then recruit several e ubiquitin ligases including traf , traf and traf . although traf-mediated ubiquitination is essential to activate mavs downstream signaling, the ubiquitination targets of traf remain unknown [ ] . subsequently, the ubiquitin sensor nemo (nf-κb essential modulator, also known as ikkγ) [ , ] is then recruited to the mavs/trafs complex, which in turn recruits ikk and tbk to the mavs complex leading to activation of nf-kb and irf and their translocation to the nucleus to induce the transcription of antiviral genes [ ] [ ] [ ] [ ] (figure ). after trif and mavs were discovered, sting was identified as a third adaptor protein that is also able to activate irf and ifn production [ , ] . sting is an endoplasmic reticulum (er) resident membrane protein with cytoplasmic c-and n-termini. sting has been shown to be essential for dna-mediated ifn production in different tissues, for example, it is crucial for host defense against the dna virus hsv- [ ] . sting has also been shown to sense cyclic dinucleotides (cdns), which are the second messengers known to be produced by bacteria such as listeria monocytogenes [ ] [ ] [ ] [ ] . although it can bind bacterial cdns, sting is unable to bind dna and relies on an upstream sensor, cgas [ ] . cgas is an enzyme that contains a nucleotidyltransferase (ntase) domain and can synthesize the second messenger -cyclic gmp-amp (cgamp) from atp and gtp upon dna recognition ( figure ). loss of cgas in various cell lines and also in vivo results in a complete loss of type i ifn induction upon dna delivery or viral infections [ , ] . cgas preferentially binds longer dna (> bp) as a dimer to form stable protein-dna ladder networks responsible for strong cgamp production [ , ] . a unique cgamp isomer termed -cgamp with particular phosphodiester linkages is produced by cgas [ , ] . -cgamp is a potent sting ligand and has a higher affinity to this protein than other cgamp molecules containing different phosphodiester linkages such as -cgamp, -cgamp or bacterial cdns [ , ] . apart from activating sting in the cell where cgas initially detects viral dna, cgamp second messengers can also travel to neighboring cells, through gap-junctions [ ] or after being packaged in newly formed virions [ , ] . this intercellular transfer of free or packaged cgamp permits uninfected cells to mount a preventive ifn response, protecting them from infection or providing a faster response to dna viruses that encode cgas antagonists. upon cgamp binding, sting undergoes a conformational change that results in the release of its c-terminal tail (ctt) from its autoinhibitory state and in the formation of sting homodimers that translocate to perinuclear regions to colocalize with tbk [ , , ] . tbk recruitment results in the phosphorylation of sting and the phosphorylated site serves as a platform for irf dimerization and activation which ultimately results in ifn-β induction [ ] (figure ). sting has also been shown to induce nf-kb, map kinase and stat activation, as well as the stimulation of lc puncta formation, a hallmark associated with autophagosome formation [ , [ ] [ ] [ ] . however, the molecular mechanisms by which sting induces these non-ifn responses remain poorly understood. tlrs comprise an ancient family of membrane-spanning receptors that recognize ligands through their extracellular domains and initiate an intracellular response upon stimulation (see above). the toll gene was first identified as a developmentally important gene in drosophila in [ ] . in the mid- s the discovery that this gene also plays an essential role in the ability of drosophila to resist fungal infections connected for the first time toll receptors to innate immunity [ , ] . although in flies toll functions as a cytokine receptor, a human toll receptor (tlr ) was rapidly identified [ , ] and shown to induce an immune response in mice after induction by lps [ ] . we now know that there are ten tlrs in humans that can respond to many bacterial and viral pamps [ ] . prototypical tlrs contain three structural elements, a hydrophobic ectodomain containing a variable number of lrrs, a transmembrane domain and a tir domain, which mediates downstream signaling through adaptor proteins [ ] . tlrs are likely very ancient immune sentinels since two of their characteristic building blocks (lrr and tir domains) are observed in placozoans (e.g., trichoplax animals) [ ] and porifera (e.g., sponges) [ ] . full tlrs were detected in cnidarian species, like the starlet sea anemone (nematostella vectensis; one single tlr) [ , ] and the acroporid corals (acropora digitifera; four tlrs) [ ] (figure ) . interestingly, both developmental and immunological roles of tlrs have been described in cnidarians. tlrs from both the sea anemone (nematostella vectensis) and the mountainous star coral (orbicella faveolata) have been shown to signal via myd leading to nf-kb activation [ , ] . in the bilateria phylum, tlrs can be found in most studied species, however, their numbers vary greatly among species, ranging from a single tlr in nematodes like caenorhabditis elegans, to over two hundred in echinoderms like the pacific purple sea urchin strongylocentrotus purpuratus (figure ). the expansion of the tlr repertoire in some animals like the sea urchin, reflects the adaptation of their immune arsenal to rapidly changing environmental stressors [ ] . amongst a multitude of other innate immune factors in this species, such as nacht domain-lrrs and scavenger receptors, sea urchin genomes encode for tlrs. among those, tlrs belong to a greatly expanded set of genes with vertebrate like features, many of which seem to have duplicated recently. the high prevalence of pseudogenes ( % to %) among those might reflect a history of strong positive selective pressures. another phylum where tlrs have undergone a significant expansion is in mollusca [ ] , like the pacific oyster crassotrea gigas [ ] (figure ). the pacific oyster encodes for tlrs in total, potentially reflecting a highly specialized response to environmental challenges and response to pathogens. the spread of pathogens in c. gigas natural habitats occurs very quickly, which is highlighted by the mass mortality events the ostreid herpesvirus (oshv ) has caused in many oyster nurseries. tlr sensing of oshv results in the differential regulation of more than a thousand genes, many of which are related to viral infection (e.g., cytosolic dna sensing and dna replication) [ , ] . in contrast to the very diverse set of tlr repertoires found in other bilateria species (e.g., nematodes, sea urchins and oysters), chordates and more particularly vertebrates contain roughly equal numbers of tlrs, reflecting the reduced need for highly diversified pattern recognition due to the acquisition of adaptive immune components (figure ). in general, vertebrate tlrs can be grouped into six major families [ ] . the families responsible for sensing of viral pamps are the tlr family, which recognizes dsrna, the tlr family (including tlrs , and ) which recognizes nucleic acid motifs and the large tlr family (tlr , , , , , , , and ) . the reduced number of tlrs in vertebrates does not necessarily mean that the tlr-response in those species cannot be tailored to a particular environment. a peculiar example is tlr , one of two virus sensing tlrs present in the pufferfish takifugu rubripes. tlr is widely conserved among teleosts and amphibians but does not seem to be present in avian or mammalian animals, which indicates that tlr might be required only in vertebrates living in water [ ] . in mammals, one last case of tlr adaptation and rapid evolution that is worth mentioning comes from bat species. analyses of tlr evolution in bats reveal adaptations acquired by tlrs , , and , with unique mutations fixed in ligand-binding sites [ , ] . these adaptations are thought to stem from the unique lifestyle of bat species, that are the only known flying mammals, and that represent important viral reservoirs [ ] . evolutionary studies paint a complex and dynamic picture of the emergence and functional diversification of rlrs across the animal kingdom. initially, several studies proposed that rig-i and mda /lgp evolved in animals independently through gene fusion and domain grafting events [ , ] . for instance, it has been proposed that the two card domains have been acquired by rig-i and mda in two separate events: the first domain being gained by the ancestor of rig-i and mda before their duplication and the second acquired after their divergence [ ] . these studies suggested that full-length rlrs are a vertebrate-specific evolutionary novelty, although their building blocks may have been present in closely related invertebrate animals [ , ] . a more recent study challenges this view and finds that the rlr-based immunity is not vertebrate-specific but originated in the earliest multicellular animals [ ] (figure ). in this study, the authors show that rlrs functionally diversified through a series of gene duplication events, followed by protein-coding changes that modulated their rna-binding properties. using homology-based gene prediction based on confirmed human rlrs the authors were able to identify full-length rlrs in early-branching animal genomes, including porifera (e.g., sponges) and cnidaria (e.g., jellyfish). however, they were unable to identify rlrs in non-metazoan eukaryotes, including fungi and choanoflagellates [ ] (figure ). it is therefore proposed that the ancestral rlr (rig-i/mda /lgp anc) duplicated in bilateria to give rise to rig-i and mda /lgp lineages, followed by a more recent duplication of the mda /lgp ancestor, giving rise to mda and lgp lineages in jawed vertebrates after their split from jawless vertebrates [ ] . the emergence of rlrs early in animal evolution is a very plausible scenario, since other components of the signaling pathways downstream of rlrs, like the irf genes, are also found in early metazoans [ ] ( figure ). another recent evidence suggesting that rlrs predated vertebrate evolution comes from studies performed in mollusks (pacific oyster; c. gigas). the invertebrate c. gigas not only encodes up to rlrs, but also mavs, traf , tbk and irf family proteins, which have been shown to have functional antiviral roles [ , , ] (figure ) . even though there is no consensus on the exact evolutionary history of rlrs, it is clear that these receptors (and/or their building blocks) existed very early in metazoan evolution and most importantly, they are subject to a very dynamic evolution. this is illustrated by the lineage-specific loss of rlr genes in many species. for example, although mda and lgp homologs were found in many teleost fish, rig-i homologs have only been identified in some fish species like salmon and carp [ ] . rig-i is absent in the chicken genome although mda and lgp are both present [ , ] . interestingly, chickens suffer severely from avian influenza virus (aiv) infection compared to ducks (that do possess the rig-i gene) which could be due to the loss of rig-i affecting their first line of defense in epithelial cells [ ] . most studied mammals possess rig-i, however, it has been lost in at least one mammalian species; the chinese tree shrew [ ] . interestingly, with the loss of rig-i, both mda and lgp have undergone strong positive selection in chinese tree shrews, and positively selected sites in mda endowed the substitute function for the lost rig-i [ ] . another eloquent example illustrating the dynamic evolution of these receptors is the loss of all rlr genes (rig-i, mda and lgp ) in insects (figure ). in drosophila, for example, although the nf-kb and jak/stat pathways are present and contribute to antiviral defenses [ , ] , all components of the rlr-mavs-irf-axis have been lost. instead, drosophila like other insects and relies on the rnai mechanism as the major antiviral system protecting it from viral infections [ , , ] . interestingly the rnase iii dicer- , a central player in insect antiviral immunity, responsible for generating small interfering rnas (sirnas), also contains an n-terminal dexd/h-box helicase domain that is highly homologous to the helicase domains of vertebrate rlrs [ , ] . moreover, dicer- has been shown to be responsible for the transcriptional upregulation of an antiviral gene (vago) that could function as a cytokine by activating the jak/stat pathway and triggering systemic antiviral immunity in various mosquito tissues [ , ] . although the pathway leading to the transcriptional activation of vago is still poorly understood in insects, these studies established that dexd/h-box helicase containing proteins, like dicer and rlrs, may represent an evolutionarily conserved set of viral nucleic acid sensors that direct antiviral responses in animals [ ] . one last observation exemplifying the dynamic and rapid evolution of these receptors comes from mammalian species. indeed, rlrs seem to be experiencing very recent adaptive changes in some mammals. for example, rig-i seems to have accumulated adaptive changes altering its rna-binding properties throughout mammalian evolution [ ] . moreover, in humans, for example, a number of protein-coding polymorphisms have been identified in rig-i which may contribute to differences in viral susceptibility and risk of autoimmune diseases [ , , ] . sting presence in animal genomes is probably more ancient than that of rlrs, since sting homologs can be found in most animal phyla including unicellular choanoflagellates ( figure ) [ , ] . furthermore, the ability of sting to bind cdns seems to be an ancient property. in an elegant study, kranzusch and colleagues show that a sting homolog in the starlet sea anemone n. vectensis (nvsting) is not only structurally very similar to that of human sting but is also able to bind cgamp with very high affinity [ ] . however, sting's ctt domain, which is crucial for tbk recruitment and downstream ifn induction, appeared only in vertebrate species [ ] . consequently, nvsting lacking the ctt is unable to induce ifn-β production in response to cdns when transfected in mammalian cells [ ] . the lack of a ctt domain in invertebrates does not mean that sting could not have an immune function in these animals. a first indication comes from invertebrate species like the lophotrochozoa phylum that includes the pacific oyster c. gigas and the annelid worm capitella teleta. in these animals, an unusual sting architecture can be found, where a sting domain is fused to a tir domain, known to be involved in innate immune signaling [ , ] . the second indication that sting lacking a ctt could function in immunity comes from arthropods. recent studies in drosophila, that lack an ifn system, clearly show that sting is important for antimicrobial and antiviral nf-kb activation in this model [ , ] (figure ) . interestingly, the emergence of the ctt domain of sting in vertebrate species seems to coincide with the development of the ifn system. nevertheless, sting ctt domain function, which dictates downstream signaling, seems to be plastic amongst vertebrate species. in a recent study, authors show that sting ctt-dependent activation of irf and nf-kb varies between vertebrate species [ ] . while sting ctt from mammalian species is able to induce a strong ifn-β and a weaker nf-kb response, an extension of this domain in ray-finned fish species elicits a dramatic enhancement of nf-kb activation and weaker irf -ifn signaling [ ] . another indication of sting ctt structure-function plasticity comes from bat species. a highly conserved and functionally important serine residue (s ) in sting's ctt domain is lost in bats [ ] . the replacement of this critical residue in this mammalian species significantly dampens sting-dependent ifn activation. the authors of this study suggest that the lifestyle of bat species (e.g., flight induced cytosolic dna, high viral titers) imposes a strong selective pressure on sting. this results in functionally dampened sensing and signaling mechanisms to avoid ifn overactivation and to cope with high cytosolic dna content. taken together, present studies suggest an evolutionarily ancient role of sting in antiviral immunity and modulation of its structure and function to accommodate species-specific pathogen burdens. the picture is less clear for the cgas enzyme when it comes to antiviral immunity. although cgas homologs have been identified in a variety of ancient metazoan lineages [ , ] , it is believed that the ability of cgas to bind and detect dsdna emerged in vertebrates. indeed, cgas' zinc-ribbon domain, required for dna binding and cgamp synthesis in response to dna in the cytosol, seems to be a vertebrate innovation [ ] [ ] [ ] . interestingly, primate cgas seems to have undergone rapid evolution in this lineage, as observed by the positive selection at its nucleic acid binding interfaces [ ] . these studies argue that although the cgas enzyme existed early in metazoans, its function has been repurposed for dna sensing only recently in vertebrates. clearly, cgas and sting seem to have acquired novel features throughout evolution. specifically in vertebrates cgas evolved the zinc ribbon motif to detect dna and sting evolved the ctt domain that expanded its signaling potential. as obligate intracellular parasites, viruses have evolved an array of evasion mechanisms to escape their elimination by the host's immune system. interestingly, viral antagonism is a general strategy and is not a peculiarity of animal viruses. many bacteriophages, for instance, encode crispr-cas inhibitors, termed anti-crisprs, to counter prokaryotic antiviral systems [ ] . plant viruses also encode viral suppressors of rnai (vsrs) the main antiviral system in plant cells [ ] . likewise, several evasion strategies and immune antagonisms by animal viruses have been described [ ] [ ] [ ] [ ] [ ] . these include hiding the viral genome from immune detection, shutting off host translation or transcription machineries, inhibiting host rna processing and trafficking and interfering directly with either proteins that sense viral presence, or factors that signal the information to the nucleus. since interfering with the innate immune system is less damaging for the host than targeting vital cellular machineries (e.g., translation), many studied viruses seem to have opted for this strategy. several studies describe viral evasion mechanisms at both the recognition and sensing step (tlrs, rlrs and cgas-sting) or at the downstream signaling steps through the targeting of proteins such as mavs, tbk , irf , irf and nf-κb. evasion strategies and immune antagonisms by animal viruses are a very active area of research, that have yielded a rich literature in the past few years. we will here just give some select examples of viral strategies that curb sensing and signaling by tlrs, rlrs and cgas-sting in animals, with an obvious bias towards viruses infecting humans. for a more complete picture on the subject, readers can refer to excellent reviews, published recently, describing those strategies [ ] [ ] [ ] [ ] [ ] . tlr signaling has been shown to be inhibited by the vaccinia virus (vacv) protein a r, that targets specific tir-domain-containing adaptor proteins. a r itself contains a tir domain which allows it to competitively interact with tir-domain-containing complexes such as myd , trif or tram, thereby inhibiting the activation of both nfkb and irfs [ , ] . human t-cell leukemia virus type- (htlv- ) is also able to interfere with tlr -dependent signaling. the htlv- encoded viral protein p binds and disables a transcription factor, pu. , required for tlr surface expression [ ] . trif, an important player in the tlr signaling cascade, is a target of choice of many viruses. the ns / a protease of hcv and the c proteases of several picornaviruses such as coxsackievirus b, ev and hepatitis a virus (hav), can all recognize and proteolytically cleave trif, producing trif fragments that are unable to signal [ ] [ ] [ ] [ ] [ ] . when it comes to rlrs, one basic strategy used by cytosolic viruses to escape surveillance is to simply prevent these receptors from accessing viral genomes. denv, for example, replicates in convoluted membranes of the er concealing its dsrna intermediates from the cytosol and thereby prevents the activation of rlrs [ ] . other viruses like ebola virus (ebov) and marburg viruses encode viral protein (vp ) that tightly binds and 'shields' the viral genome from detection by rig-i [ , ] . another strategy used by viruses to 'hide' from rlrs consists of modifying the very molecular features these receptors rely on to recognize viral genomes. for example, both, hantaan viruses from the bunyaviridae family and borna disease virus (bdv) from the bornaviridae family, encode phosphatases that process the triphosphate group at their genome termini, to a -monophosphate to escape rig-i surveillance [ , ] . lassa virus (lasv) from the arenaviridae family evolved a unique strategy in which its nucleoprotein (np) acquired a - exonuclease activity, that enables it to digest free dsrna, preventing the activation of rig-i [ ] . however, the most direct way of interfering with rlr function and their signaling partners is to either directly target them for cleavage and degradation or to manipulate their phosphorylation and ubiquitination statuses, which are crucial for their activation. indeed, many viruses encode proteases that directly cleave rlrs. while the cpro proteases of both poliovirus and ev cleave rig-i, the apro of ev cleaves mda [ , ] . mavs, a crucial hub for both rig-i and mda -mediated signaling is also frequently targeted and cleaved by numerous viral proteases, such as cpro from hav, apro from ev , ns -ns a from hcv, apro and cpro from rhinovirus and cpro from coxsackievirus b (cvb ) [ , , [ ] [ ] [ ] . mavs can also be indirectly degraded by particular viruses. for instance, measles virus (mev) can trigger a selective form of autophagy, called mitophagy, responsible for the degradation of mitochondria, which leads to a decrease of mavs abundance [ ] . another example of indirect mavs degradation comes from studies with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov). this virus has evolved a strategy in which its b protein localizes to mitochondria and subverts the cellular e ubiquitin ligase atrophin- -interacting protein (aip ) to degrade mavs [ ] . post-translational modifications of both mavs and rlrs have also been shown to be subverted by viruses to inhibit their downstream signaling. ns proteins from many influenza a virus strains (iav) interact with the host ubiquitin ligase trim and inhibit its oligomerization, a crucial step for its enzymatic activity of attaching lys -linked polyubiquitin to the card domains of rig-i [ , ] . other viruses encode deubiquitinating enzymes (dubs) to remove the lys -linked ubiquitination off rig-i. orf from kaposi's sarcoma herpesvirus (kshv), papain-like protease (plp) from sars-cov, leader proteinase (lpro) from foot-and-mouth disease virus (fmdv) and the ovarian tumor (otu)-type proteins of arteriviruses and nairoviruses have all been shown to possess a deubiquitination activity and interfere with rig-i mediated signaling [ , [ ] [ ] [ ] . rig-i and mda phosphorylation status can also be subverted by viruses. in normal conditions, phosphorylation of serine or threonine residues keeps rig-i and mda in an inactive state. upon viral infection, pp phosphatases are recruited to dephosphorylate specific marks on those receptors and activate them. v proteins from measles and nipah viruses (mev and niv) act as decoys and have been shown to bind pp -α and pp -γ, sequestering them away from mda and rig-i [ , ] . similar to evasion strategies that counter the rna sensing machinery described earlier, dna viruses use numerous strategies to escape cgas-sting-dependent detection and signaling. they could either hide their viral genomes or cleave, degrade, post-translationally modify or even relocalize dna sensing and signaling factors [ ] . hepatitis b virus (hbv), that causes chronic hepatitis and increases the risk of developing liver cirrhosis and hepatocellular carcinoma, has developed an array of mechanisms to inhibit the host's immune systems (reviewed in [ ] ). notably, the hbv polymerase can bind to sting to block its lys -linked ubiquitination, inhibiting the production of ifn-β [ ] . moreover, even though cgas is expressed in human hepatocytes and is able to sense and signal upon transfection of naked relaxed-circular hbv dna; during a natural infection, hbv dna seems to escape cgas detection, likely due to packaging of the genome into the viral capsid [ ] . kshv, another dna virus has been shown to act on both cgas and sting. several kshv proteins (e.g., orf and lana) can either sequestrate stimulatory dna or directly bind to cgas inhibiting its enzymatic activity [ , ] . kshv has been also shown to encode a viral interferon regulatory factor (virf ) that interacts with sting thereby preventing tbk binding and sting activation by tbk -dependent phosphorylation [ ] . the ns protease of denv, together with its ns b co-factor, has been shown to target the residues - (lrrg) of human sting, leading to its cleavage and degradation [ , ] . interestingly, mouse sting lacks these lrrg residues, and ns b/ns of denv is neither able to cleave the murine sting, nor to block murine ifn-β production. therefore, it has been proposed that the inability of denv to cleave mouse sting might explain its host tropism, as murine cells are not very susceptible to denv infection [ , ] . in animals, prrs and their associated signaling pathways are early and potent cellular sensors of viral elements, that mobilize the organism's defenses by inducing an antiviral state. major advances have been made in the last two decades in the understanding of their function in mammalian immunity. new genomics data and gene editing tools can now let us interrogate prr-like pathways in poorly studied animal species and define their evolutionary trajectories. studying the evolution of immune components and their interplay with viral pathogens is extremely important since our immune responses to contemporary viruses have been shaped by our evolutionary responses to previous infections. the modern innate immune system is generally not yet optimized against modern viruses but rather was selected for by previous rounds of co-evolution with ancient viruses [ ] . studying the biological arms race between host and virus, referred to as the "red queen hypothesis" [ ] , in which each entity maintains a relatively constant fitness cost, will be instrumental in the fight against future infections. such studies will help us understand many aspects of viral infections including viral zoonoses, tropism, global epidemics and disease progression. furthermore, exploring these pathways and mechanisms for therapeutic purposes may offer novel strategies to cure human disease. indeed, modulating the action of the aforementioned immune sensors is proving to be an effective strategy to develop vaccines and vaccine adjuvants [ ] [ ] [ ] [ ] [ ] or to treat viral infections [ ] [ ] [ ] [ ] [ ] [ ] . finally, the use of tlr, rlr and sting modulators, to treat inflammation, auto-immune disease [ , ] and also in cancer immunotherapy [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] provides an eloquent incentive to continue studying these pathways and to look ahead with great optimism. a virocentric perspective on the evolution of life evolution of innate immunity: clues from invertebrates via fish to mammals evolution of interferons and interferon receptors transcriptional regulation of antiviral interferon-stimulated genes antiviral defense and innate immune memory in the oyster dynamic evolution of immune system regulators: the history of the interferon regulatory factor family ifn regulatory factor- bypasses ifn-mediated antiviral effects through viperin gene induction constitutive expression of an isgf /irf transgene leads to interferon-independent activation of interferon-inducible genes and resistance to virus infection transcriptional profiling of interferon regulatory factor target genes: direct involvement in the regulation of interferon-stimulated genes phylogenetic analysis of the endoribonuclease dicer family early origins and evolution of micrornas and piwi-interacting rnas in animals molecular evolutionary and structural analysis of the cytosolic dna sensor cgas and sting ancient origin of cgas-sting reveals mechanism of universal , cgamp signaling ancient origins of vertebrate-specific innate antiviral immunity the evolution of vertebrate toll-like receptors evolution of mda- /rig-i-dependent innate immunity: independent evolution by domain grafting nf-kappab signaling pathways in mammalian and insect innate immunity the evolution of antiviral defense systems the frustrated gene: origins of eukaryotic gene expression antiviral immunity directed by small rnas crispr-cas immunity in prokaryotes the evolutionary journey of argonaute proteins on the origin and functions of rna-mediated silencing: from protists to man rna interference to treat virus infections specific interference with gene expression induced by long, double-stranded rna in mouse embryonal teratocarcinoma cell lines antiviral rna interference in mammalian cells transposition mediated by rag and rag and its implications for the evolution of the immune system primordial emergence of the recombination activating gene (rag ): sequence of the complete shark gene indicates homology to microbial integrases evolution of immune systems from viruses and transposable elements re-evaluation of the immunological big bang pattern recognition receptors and control of adaptive immunity approaching the asymptote? evolution and revolution in immunology crosstalk between cytoplasmic rig-i and sting sensing pathways discriminating self from non-self in nucleic acid sensing innate immune pattern recognition: a cell biological perspective recognition of double-stranded rna and activation of nf-kappab by toll-like receptor recognition of single-stranded rna viruses by toll-like receptor human tlr- -, - -, and - -mediated induction of ifn-alpha/beta and -lambda is irak- dependent and redundant for protective immunity to viruses the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses inhibition of the rna polymerase iii-mediated dsdna-sensing pathway of innate immunity by vaccinia virus protein e rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate dna-pk is a dna sensor for irf- -dependent innate immunity dlm- /zbp ) is a cytosolic dna sensor and an activator of innate immune response the interferon response to intracellular dna: why so many receptors? immunobiology ifi is an innate immune sensor for intracellular dna the helicase ddx senses intracellular dna mediated by the adaptor sting in dendritic cells adenovirus detection by the cgas/sting/tbk dna sensing cascade pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna aim recognizes cytosolic dsdna and forms a caspase- -activating inflammasome with asc ticam- , an adaptor molecule that participates in toll-like receptor -mediated interferon-beta induction role of adaptor trif in the myd -independent toll-like receptor signaling pathway specificity in toll-like receptor signalling through distinct effector functions of traf and traf critical role of traf in the toll-like receptor-dependent and -independent antiviral response type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors cutting edge: tnfr-associated factor (traf) is essential for myd -dependent pathway but not toll/il- receptor domain-containing adaptor-inducing ifn-beta (trif)-dependent pathway in tlr signaling interferon-alpha induction through toll-like receptors involves a direct interaction of irf with myd and traf helical assembly in the myd -irak -irak complex in tlr/il- r signalling protein kinase ikkbeta-catalyzed phosphorylation of irf at ser induces its dimerization and nuclear translocation in myeloid cells an oligomeric signaling platform formed by the toll-like receptor signal transducers myd and irak- ikkbeta is an irf kinase that instigates inflammation interleukin- receptor-associated kinase- plays an essential role for toll-like receptor (tlr) -and tlr -mediated interferon-{alpha} induction structural basis of rna recognition and activation by innate immune receptor rig-i structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna mda assembles into a polar helical filament on dsrna cooperative assembly and dynamic disassembly of mda filaments for viral dsrna recognition trim ring-finger e ubiquitin ligase is essential for rig-i-mediated antiviral activity ubiquitin-induced oligomerization of the rna sensors rig-i and mda activates antiviral innate immune response riplet/rnf , a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf essential role of ips- in innate immune responses against rna viruses an autoinhibitory mechanism modulates mavs activity in antiviral innate immune response mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response structural basis for the prion-like mavs filaments in antiviral innate immunity mavs recruits multiple ubiquitin e ligases to activate antiviral signaling cascades activation of ikk by tnfalpha requires site-specific ubiquitination of rip and polyubiquitin binding by nemo sensing of lys -linked polyubiquitination by nemo is a key event in nf-kappab activation key role of ubc and lysine- polyubiquitination in viral activation of irf sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling the adaptor protein mita links virus-sensing receptors to irf transcription factor activation sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity sting is a direct innate immune sensor of cyclic di-gmp coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for v. cholerae virulence mpys is required for ifn response factor activation and type i ifn production in the response of cultured phagocytes to bacterial second messengers cyclic-di-amp and cyclic-di-gmp the n-ethyl-n-nitrosourea-induced goldenticket mouse mutant reveals an essential function of sting in the in vivo interferon response to listeria monocytogenes and cyclic dinucleotides cyclic gmp-amp synthase is an innate immune sensor of hiv and other retroviruses pivotal roles of cgas-cgamp signaling in antiviral defense and immune adjuvant effects cgas senses long and hmgb/tfam-bound u-turn dna by forming protein-dna ladders cyclic gmp-amp synthase is activated by double-stranded dna-induced oligomerization cgas produces a - -linked cyclic dinucleotide second messenger that activates sting cyclic gmp-amp containing mixed phosphodiester linkages is an endogenous high-affinity ligand for sting viruses transfer the antiviral second messenger cgamp between cells transmission of innate immune signaling by packaging of cgamp in viral particles structure-function analysis of sting activation by c[g( , )pa( , )p] and targeting by antiviral dmxaa atg a controls dsdna-driven dynamic translocation of sting and the innate immune response phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf activation activation of stat by sting is critical for antiviral innate immunity cytosolic-dna-mediated, sting-dependent proinflammatory gene induction necessitates canonical nf-kappab activation through tbk activation of autophagy by alpha-herpesviruses in myeloid cells is mediated by cytoplasmic viral dna through a mechanism dependent on stimulator of ifn genes establishment of dorsal-ventral polarity in the drosophila embryo: the induction of polarity by the toll gene product signals from the il- receptor homolog, toll, can activate an immune response in a drosophila hemocyte cell line the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults chromosomal localization of til, a gene encoding a protein related to the drosophila transmembrane receptor toll, to human chromosome p prediction of the coding sequences of unidentified human genes. i. the coding sequences of new genes (kiaa -kiaa ) deduced by analysis of randomly sampled cdna clones from human immature myeloid cell line kg- defective lps signaling in c h/hej and c bl/ sccr mice: mutations in tlr gene the role of pattern-recognition receptors in innate immunity: update on toll-like receptors evolutionary origins of toll-like receptor signaling innate immunity in the simplest animals-placozoans sea anemone model has a single toll-like receptor that can function in pathogen detection, nf-kappab signal transduction, and development the innate immune repertoire in cnidaria-ancestral complexity and stochastic gene loss differential and convergent utilization of autophagy components by positive-strand rna viruses a conserved toll-like receptor-to-nf-kappab signaling pathway in the endangered coral orbicella faveolata genomic insights into the immune system of the sea urchin massively parallel rna sequencing identifies a complex immune gene repertoire in the lophotrochozoan mytilus edulis massive expansion and functional divergence of innate immune genes in a protostome teleost tlr recognizes rna duplex to induce ifn and protect cells from birnaviruses adaptive evolution of virus-sensing toll-like receptor in bats the evolution of bat nucleic acid-sensing toll-like receptors immune system modulation and viral persistence in bats: understanding viral spillover origin and evolution of the rig-i like rna helicase gene family characterization of the mollusc rig-i/mavs pathway reveals an archaic antiviral signalling framework in invertebrates retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) in fish: current knowledge and future perspectives chicken cells sense influenza a virus infection through mda and cardif signaling involving lgp association of rig-i with innate immunity of ducks to influenza genome of the chinese tree shrew loss of rig-i leads to a functional replacement with mda in the chinese tree shrew the kinase ikkbeta regulates a sting-and nf-kappab-dependent antiviral response pathway in drosophila the jak-stat signaling pathway is required but not sufficient for the antiviral response of drosophila the rna silencing endonuclease argonaute mediates specific antiviral immunity in drosophila melanogaster essential function in vivo for dicer- in host defense against rna viruses in drosophila sensing viral rnas by dicer/rig-i like atpases across species the dexd/h-box helicase dicer- mediates the induction of antiviral activity in drosophila secreted vago restricts west nile virus infection in culex mosquito cells by activating the jak-stat pathway dicer- -dependent activation of culex vago occurs via the traf-rel signaling pathway nucleic acid sensing in invertebrate antiviral immunity the rig-i atpase core has evolved a functional requirement for allosteric stabilization by the pincer domain the selective footprints of viral pressures at the human rig-i-like receptor family evolution and functional impact of rare coding variation from deep sequencing of human exomes cyclic di-nucleotide signaling enters the eukaryote domain evolutionary origins of cgas-sting signaling toll signaling: the tireless quest for specificity analysis of drosophila sting reveals an evolutionarily conserved antimicrobial function modular architecture of the sting c-terminal tail allows interferon and nf-kappab signaling adaptation dampened sting-dependent interferon activation in bats structure of human cgas reveals a conserved family of second-messenger enzymes in innate immunity ] is the metazoan second messenger produced by dna-activated cyclic gmp-amp synthase structural mechanism of cytosolic dna sensing by cgas overlapping patterns of rapid evolution in the nucleic acid sensors cgas and oas suggest a common mechanism of pathogen antagonism and escape the discovery, mechanisms, and evolutionary impact of anti-crisprs decoding type i and iii interferon signalling during viral infection viral evasion of dna-stimulated innate immune responses ten strategies of interferon evasion by viruses viral evasion of intracellular dna and rna sensing viral evasion and subversion of pattern-recognition receptor signalling a r and a r from vaccinia virus are antagonists of host il- and toll-like receptor signaling vaccinia virus protein a r targets multiple toll-like-interleukin- receptor adaptors and contributes to virulence the htlv-i p interferes with tlr signaling and modulates the release of pro-and anti-inflammatory cytokines from human macrophages innate immunity evasion by enteroviruses: insights into virus-host interaction toll-like receptors in antiviral innate immunity hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity the coxsackievirus b c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling disruption of tlr signaling due to cleavage of trif by the hepatitis a virus protease-polymerase processing intermediate the dengue virus conceals double-stranded rna in the intracellular membrane to escape from an interferon response ebola virus vp protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling structural basis for marburg virus vp -mediated immune evasion mechanisms sequestration by ifit impairs translation of o-unmethylated capped rna old world hantaviruses do not produce detectable amounts of dsrna in infected cells and the termini of their genomic rnas are monophosphorylated structure of the lassa virus nucleoprotein reveals a dsrna-specific to exonuclease activity essential for immune suppression rig-i is cleaved during picornavirus infection enterovirus apro targets mda and mavs in infected cells cleavage of ips- in cells infected with human rhinovirus disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor mitophagy enhances oncolytic measles virus replication by mitigating ddx /rig-i-like receptor signaling sars-coronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/traf /traf signalosome influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirus-encoded deubiquitinase orf deubiquitinase function of arterivirus papain-like protease suppresses the innate immune response in infected host cells antagonism of the phosphatase pp by the measles virus v protein is required for innate immune escape of mda measles virus suppresses rig-i-like receptor activation in dendritic cells via dc-sign-mediated inhibition of pp phosphatases the cgas-sting defense pathway and its counteraction by viruses immune evasion strategies during chronic hepatitis b and c virus infection. vaccines (basel) , , hepatitis b virus polymerase disrupts k -linked ubiquitination of sting to block innate cytosolic dna-sensing pathways hepatitis b virus evasion from cyclic guanosine monophosphate-adenosine monophosphate synthase sensing in human hepatocytes inhibition of cgas dna sensing by a herpesvirus virion protein cytoplasmic isoforms of kaposi sarcoma herpesvirus lana recruit and antagonize the innate immune dna sensor cgas modulation of the cgas-sting dna sensing pathway by gammaherpesviruses denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus targets the adaptor protein mita to subvert host innate immunity evolutionary conflicts between viruses and restriction factors shape immunity the evolutionary conundrum of pathogen mimicry enhanced influenza virus-like particle vaccination with a structurally optimized rig-i agonist as adjuvant pika as an adjuvant enhances specific humoral and cellular immune responses following the vaccination of mice with hbsag plus pika a tlr ligand that exhibits potent inhibition of influenza virus replication and has strong adjuvant activity has the potential for dual applications in an influenza pandemic as , an aluminum salt-and tlr agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity the tlr agonist, monophosphoryl lipid a, attenuates the cytokine storm associated with respiratory syncytial virus vaccine-enhanced disease sting agonists enable antiviral cross-talk between human cells and confer protection against genital herpes in mice the tlr antagonist eritoran protects mice from lethal influenza infection targeting innate immunity for antiviral therapy through small molecule agonists of the rlr pathway direct antiviral properties of tlr ligands against hbv replication in immune-competent hepatocytes safety, efficacy and pharmacodynamics of vesatolimod (gs- ) in virally suppressed patients with chronic hepatitis b antibody and tlr agonist delay viral rebound in shiv-infected monkeys therapeutic effects of the artemisinin analog sm on lupus-prone mrl/lpr mice via inhibition of tlr-triggered b-cell activation and plasma cell formation tak- (resatorvid), a small-molecule inhibitor of toll-like receptor (tlr) signaling, binds selectively to tlr and interferes with interactions between tlr and its adaptor molecules essential for the antitumor effect of immune checkpoint blockade sa- - bbl and monophosphoryl lipid a constitute an efficacious combination adjuvant for cancer vaccines magnitude of therapeutic sting activation determines cd (+) t cell-mediated anti-tumor immunity immunogene therapy using immunomodulating hvj-e vector augments anti-tumor effects in murine malignant glioma promising targets for cancer immunotherapy: tlrs, rlrs, and sting-mediated innate immune pathways immunogenic cell death of human ovarian cancer cells induced by cytosolic poly(i:c) leads to myeloid cell maturation and activates nk cells sting-mediated dna sensing promotes antitumor and autoimmune responses to dying cells sting agonist formulated cancer vaccines can cure established tumors resistant to pd- blockade this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this work has been supported by the h marie-curie actions msca-if- -hipshot (km). this work was also supported in part by the national institutes of health grants u -ai (tfb) by arc, paris and institut hospitalo-universitaire, strasbourg (therahcc and therahcc . ihuarc ihu and ihuarc to t. acknowledgments: the authors would like to thank jean-luc imler and joao t. marques for their critical reading of the manuscript. the authors apologize to colleagues whose work could not be cited due to space limitations. the authors declare no conflict of interest. key: cord- -g n cm authors: matczuk, anna karolina; chodaczek, grzegorz; ugorski, maciej title: production of recombinant eav with tagged structural protein gp to study artervirus minor protein localization in infected cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: g n cm equine arteritis virus (eav) is a prototype member of the arterivirus family, comprising important pathogens of domestic animals. minor glycoproteins of arteriviruses are responsible for virus entry and cellular tropism. the experimental methods for studying minor arterivirus proteins are limited because of the lack of antibodies and nested open reading frames (orfs). in this study, we generated recombinant eav with separated orfs and , and gp carrying ha-tag (gp -ha). the recombinant viruses were stable on passaging and replicated in titers similar to the wild-type eav. gp -ha was incorporated into the virion particles as monomers and as a gp /gp -ha/gp trimer. gp -ha localized in er and, to a lesser extent, in the golgi, it also co-localized with the e protein but not with the n protein. the co-localization of gp -ha and the e protein with ergic was reduced. moreover, eav with gp -ha could become a valuable research tool for identifying host cell factors during infection and the role of gp in virus attachment and entry. equine arteritis virus (eav) is a prototype member of arteriviridae, a family of enveloped positive-stranded rna viruses comprising porcine reproductive and respiratory syndrome virus (prrsv), a major pathogen in the swine industry [ ] . eav infects horses and donkeys and leads to abortions in pregnant mares and respiratory illness with flu-like symptoms, which can even lead to death in young animals. the virus is transmitted via the respiratory route and via the contaminated semen of previously infected stallions. despite available vaccines, eav remains an important pathogen in the horse industry [ ] . the infectious genomic rna has a length of~ kbp, is -polyadenylated, and presumably -capped. the large replicase open reading frames (orfs) a and b occupy most of the eav genome and are directly translated from the genomic rna [ , ] . however, the genes for the structural virion proteins, which are located at the '-end of the arterivirus genome, overlap with each other and are expressed from the ' co-terminal nested set of six leader-containing subgenomic rnas [ ] . the structural proteins of arteriviruses include the nucleocapsid n and several membrane proteins, such as the gp /m dimer, the gp / / complex, the small and hydrophobic e protein, and the orf a protein [ ] . all structural proteins, in addition to orf a, are essential for eav infectivity; however, only n along with viral rna and gp /m dimer is required for budding. note that gp , gp , gp , and e are minor viral components, and knocking them out individually from eav does not prevent the the cell line bhk- (baby hamster kidney cells; atcc c ) was maintained as an adherent culture in dmem mixed in a : ratio with the leibovitz l- medium (cytogen, lodz, poland), which was supplemented with % fetal calf serum (fcs) (biological industries, cromwell, ia, usa), u of penicillin per ml, and mg of streptomycin and l-glutamine per ml (biowest, lodz, poland). the cells were maintained at • c in an atmosphere of air with % co and % humidity. recombinant dna techniques were performed according to standard protocols. newly constructed plasmids were propagated in competent escherichia coli strain dh alpha (thermo scientific, warszawa, poland). cloning vector peav , which is a derivate of the peav (genbank y . ) and described in reference [ ] , was used to generate mutant eavs. we generated specific dna fragments by overlap extension pcr and purified them from the gel with the aid of the gel-out kit (a&a biotechnology, gdansk, poland). rgeavbamhi and rgeavecori were used as an external primers. first, the translation initiation codon of the eav orf was mutated (atg > acg) with primers gp kofor and gp korev, and the . bp fragment cloned back to peav with the restriction enzymes bamhi and ecori (thermo scientific, poland) to generate peav gp ko. in the second step, the asci site was added after the stop codon of the orf with primers eav ascifor and eav ascirev to generate the plasmid peav gp koasci. the separation of the orf and orf was achieved by the overlap extension pcr with primers reconascgp for and rgeavecori on the peav template and cloned to peav koasci with the asci and ecori restriction enzymes to generate peav s / . as the last step, the ha tag was added directly to orf 's '-end with the use of primers rgeavbamhiifor and eavgp harev and peav s / as a dna template. the ligation after bamhi and asci restriction enzyme digestion of the bp product produced the peav gp -ha vector. all of the generated plasmids were sequenced with the rgeavecorirev primer (genomed, warszawa, poland). table shows the list of used primers, while figure shows the cloning schematics. all the genes that were subjected to mutations in plasmids were sequenced before use in experiments (genomed, warsaw, poland). the aid of the gel-out kit (a&a biotechnology, gdansk, poland). rgeavbamhi and rgeavecori were used as an external primers. first, the translation initiation codon of the eav orf was mutated (atg > acg) with primers gp kofor and gp korev, and the . bp fragment cloned back to peav with the restriction enzymes bamhi and ecori (thermo scientific, poland) to generate peav gp ko. in the second step, the asci site was added after the stop codon of the orf with primers eav ascifor and eav ascirev to generate the plasmid peav gp koasci. the separation of the orf and orf was achieved by the overlap extension pcr with primers reconascgp for and rgeavecori on the peav template and cloned to peav koasci with the asci and ecori restriction enzymes to generate peav s / . as the last step, the ha tag was added directly to orf 's '-end with the use of primers rgeavbamhiifor and eavgp harev and peav s / as a dna template. the ligation after bamhi and asci restriction enzyme digestion of the bp product produced the peav gp -ha vector. all of the generated plasmids were sequenced with the rgeavecorirev primer (genomed, warszawa, poland). table shows the list of used primers, while figure shows the cloning schematics. all the genes that were subjected to mutations in plasmids were sequenced before use in experiments (genomed, warsaw, poland). full length clones peav , peav s / , and peav gp -ha were linearized using xhoi and in vitro-transcribed using amplicap-max t high yield message maker kit (cellscript, madison, wi, usa), and µg rna was then introduced into the bhk- cells suspended in pbs using the gene pulser xcell electroporation apparatus and electroporation cuvettes with a -mm electrode gap (bio-rad, warszawa, poland). the cells were pulsed twice at v, f; resuspended in dmem/l- % fcs; and seeded into two wells of the -well plate. the cells were then maintained at °c until the cpe was observed. the cells that adhered were detached using a plastic cell scraper and collected together in the supernatants. the cells were then centrifuged at a low speed. while half of the cells were subjected to rt-pcr and sequencing, the second half were subjected to western blotting with anti-n and anti-ha antibodies. the remaining supernatants were collected, aliquoted, and stored in − °c as a p stock. the monolayers of bhk- cells grown in -well plates were inoculated with each of the wild-type eav-wt (derived from peav ), eavs / , and eavgp -ha viruses at a multiplicity of infection (moi) of . and incubated at °c for h. the cells were then washed two times with pbs, with calcium and magnesium, and then overlaid with ml of dmem/l- % fcs and % l-glutamine culture medium. at , , , , and h post-infection, the supernatants were harvested and virus titers were determined on the bhk- cells using the plaque assay. virus aliquots were stored at − °c. this experiment was carried out in triplicate. full length clones peav , peav s / , and peav gp -ha were linearized using xhoi and in vitro-transcribed using amplicap-max t high yield message maker kit (cellscript, madison, wi, usa), and µg rna was then introduced into the bhk- cells suspended in pbs using the gene pulser xcell electroporation apparatus and electroporation cuvettes with a -mm electrode gap (bio-rad, warszawa, poland). the cells were pulsed twice at v, f; resuspended in dmem/l- % fcs; and seeded into two wells of the -well plate. the cells were then maintained at • c until the cpe was observed. the cells that adhered were detached using a plastic cell scraper and collected together in the supernatants. the cells were then centrifuged at a low speed. while half of the cells were subjected to rt-pcr and sequencing, the second half were subjected to western blotting with anti-n and anti-ha antibodies. the remaining supernatants were collected, aliquoted, and stored in − • c as a p stock. the monolayers of bhk- cells grown in -well plates were inoculated with each of the wild-type eav-wt (derived from peav ), eavs / , and eavgp -ha viruses at a multiplicity of infection (moi) of . and incubated at • c for h. the cells were then washed two times with pbs, with calcium and magnesium, and then overlaid with ml of dmem/l- % fcs and % l-glutamine culture medium. at , , , , and h post-infection, the supernatants were harvested and virus titers were determined on the bhk- cells using the plaque assay. virus aliquots were stored at − • c. this experiment was carried out in triplicate. plaque assay was performed on the bhk- cells grown on -well plates with gmem supplemented with % fcs, % l-glutamine, and . % carboxymethyl cellulose (cmc, sigma-aldrich, poznań, poland). the overlays were fixed with % formaldehyde and stained with crystal violet for three days p.i. to determine the stability of the ha-tag fluorescence, the recombinant eavgp -ha virus was subjected to sequential serial passages at an moi of in the bhk- cells. after the appearance of cpe, the supernatants were collected, centrifuged at a low speed, and stored in − • c. the remaining cells were washed with pbs, centrifuged, and stored in − • c for further rt-pcr and sequencing. to verify the stability of the ha-tag, the bhk- cells were grown on glass coverslips that were placed on a -well plate and infected with different passage recombinant viruses at an moi of . h p.i. the cells were subjected to immunofluorescence with anti-ha tag antibodies ( : , ab , abcam, uk) and anti-n antibody ( : , vmrd, usa), as has been described later in the materials and methods section. furthermore, the infected cells from passages p , p , p , and p were subjected for rt-pcr and sequencing. the total cellular rna was extracted from transfected or infected cells (from -well plates each) with rneasy mini kit (qiagen, wrocław, poland) according to manufacturer's instructions. the cdna was generated with maxima h minus first strand cdna synthesis kit (thermo, poland) according to the manufacturer's instructions with µg of rna and oligot primer. the obtained cdna was subjected to pcr reaction with eavfor and eavrev primers (each mm) listed in table and one-fusion high-speed-fidelity polymerase (geneon, abo, poland). the thermal profile was as follows: initial denaturation at • c for min, followed by cycles of denaturation at • c for s, annealing at • c for s and extension at • c for s, and a final extension at • c for min. the rt-pcr products were gel-purified using a qiaquick gel extraction kit (qiagen inc., valencia, ca, usa), and the sense and antisense strands were sequenced (eurofins mwg operon, huntsville, al). the sequence data were analyzed using the software finchtv . . . pcr products were subjected to agarose gel electrophoresis, purified (gel-out, a&a biotechnology, gdynia, poland), and the sense and antisense strands were sequenced (genomed, warsaw, poland). the bhk- cells seeded on -well plates were infected with p stock at an moi of or left mock infected. h p.i. cells were detached using a plastic cell scraper, pelleted, and lysed either directly in the sds sample buffer without dtt or resuspended in µl × glycoprotein denaturing buffer and boiled for min at • c. to analyze glycosylation, the samples were digested with peptide-n-glycosidase (pngase f; u/l, h at • c) or endo-beta-n-acetyl-glucosaminidase (endo h; u/l, h at • c) according to the manufacturer's instructions (new england biolabs, hitchin, uk). after the deglycosylation reaction, the samples were supplemented with reducing sds-page buffer and subjected to sds-page and western blotting. the bhk- cells were seeded in the complete medium onto glass coverslips in -well plates. after h, the cell culture medium was replaced with dmem/l- medium with the cells infected with eav wt, eav s / , eavgp -ha, or left uninfected. after h, the cells were washed two times with pbs and the cell culture medium was replaced with dmem/l- containing % fcs, % penicillin/streptomycin and, % l-glutamine. the cells were then fixed h p.i. with paraformaldehyde viruses , , of ( % in pbs) for min at room temperature (rt), washed twice with pbs, permeabilized with . % triton in pbs for min at room temperature, and washed again twice with pbs. after blocking (blocking solution contained % bovine serum albumin in pbst) for h at room temperature, the cells were incubated with a rabbit polyclonal anti-ha tag antibodies ( : , ab , abcam, cambridge, uk) and mouse monoclonal anti-n ( : , vmrd, pullman, wa, usa) antibody diluted in a blocking solution at room temperature for h. these cells were then washed three times with pbs and incubated with secondary antibodies ( : , goat anti-mouse igg h&l alexa fluor and : goat anti-rabbit igg h&l alexa fluor , abcam, uk). after immunostaining in all the cases, the cells were washed three times with pbs. finally, the stained cultures were mounted on glass slides in a fluoroshield mounting medium with dapi (abcam, cambridge, uk) and stored at • c. the images were recorded using a zeiss cell observer sd confocal microscope (zeiss, oberkochen, germany) equipped with an emccd qimaging rolera em-c camera and - × oil objectives ( . µm and . µm per pixel, respectively). the imaging was performed sequentially using nm, nm, and nm laser lines with a quadruple dichroic mirror + + + and / , / , and / emission filters. subsequently, the images were deconvoluted with huygens software (svi, hilversum, the netherlands). for localization with cellular markers, the bhk- cells were infected with eavgp -ha at an moi of or left uninfected. the cells at h p.i were subjected to immunofluorescence as described above with the following antibodies: rabbit polyclonal anti-ha tag antibodies ( : each co-localization experiment was conducted at least times, and at least cells were taken to quantify the co-localization in the jacop plugin using the fiji software. in this study, all the graphs presented were created using graphpad prism software. the significance was estimated using one-way anova. the images were obtained from the confocal microscope were deconvoluted using huygens essential x (scientific volume imaging, hilversum, the netherlands) and processed in imagej fiji [ ] . the co-localization analyses were performed in imagej using the jacop plugin [ ] in which pearson's and mander's coefficient were calculated. for each condition, at least cells were taken for measurements of co-localization. fluorescence intensities in the antibody accessibility experiment were measured as a mean gray value in imagej fiji for fields per condition. the detached and low-speed centrifuged, transfected, or infected cells were solubilized in the ripa buffer (sigma-aldrich, poland) with the complete protease inhibitor tablet (roth, sigma-aldrich, poland/merc, poland). samples in the sds-page loading buffer, with or without dtt, were subjected to sds-page using % or % polyacrylamide. then, the gels were blotted onto polyvinylidene difluoride (pvdf) membrane (ge healthcare, warszawa, poland). after blocking of the membranes (blocking solution; % skim milk powder in pbs with . % tween (pbst)) overnight at • c, the antibodies in the blocking solution were incubated for . h at room temperature. rabbit-anti-ha tag antibodies ( : ); ab ; abcam, cambridge, uk) were used to detect gp with the ha tag, mouse monoclonal anti-n antibody ( : , vmrd, usa), and rabbit anti-e ( : , described in [ ] , a gift from eric snijder, university of leiden, belgium). after washing ( times for min each with pbst), suitable horseradish peroxidase-coupled secondary antibodies ( : ; anti-rabbit or anti-mouse; dako, carpinteria, ca, usa) were applied for h at room temperature. after washing with pbst, the signals were detected by chemiluminescence using the ecl plus reagent (pierce/thermo, poland). the bhk- cells seeded in t bottles were infected with eavgp -ha or mock infected at an moi of . two hours after infection cells were washed with pbs, and the cell culture medium was replaced with dmem/l- containing % fcs, % penicillin/streptomycin and, % l-glutamine. h p.i. supernatants were collected and low-speed centrifuged to remove the cells. then, the supernatants were subjected to purification and concentration on amicon ultra- , ultracel- k filters (merck, warszawa, poland). briefly, ml of cell-free supernatants were transferred to filters and centrifuged at × g for min at room temperature, to obtain ul of concentrated supernatant. the sds sample buffer with or without dtt was added to % of the volume of the obtained concentrated virions, while the remaining % was lysed in the mnt buffer ( mm mes, mm tris, mm nacl, % tx- , ph . ) and subjected to ip with rabbit polyclonal anti-ha (ab ; abcam, cambridge, uk) overnight at • c. the antibody-protein complexes were pulled with a-sepharose (sigma-aldrich, poland), washed with mnt, boiled with reducing or non-reducing sds buffer, and subjected to sds-page and western blotting as described above. for recombinant eav with a tagged gp , we selected the junction between orf and orf because, in the peav vector, the overlap between the orf and orf consists of nt, while that between orf and orf is nt. the orf '-end overlaps with two orfs coding gp and orf a proteins. moreover, joining the ha-tag directly with the gp did not abolish the expression of gp [ ] , while the direct tagging of gp with flag-tag made its expression impossible [ ] . the cloning procedure is shown in figure a . first, the start codon of gp was mutated to produce the gp knock-out virus, and then the restriction site asci was generated to allow the separation of orf and orf by duplicating the missing orf nt long sequence. finally, the ha-tag sequence was directly inserted to the end of orf . all these mutations were generated using pcr methods. subsequently, the in vitro-transcribed full-length viral rnas from the xhoi-linearized plasmids peav , peav s / , and peav gp -ha were introduced into the bhk- cells using electroporation. after h, the cells were harvested and examined by western blotting analysis using antibodies that are specific to the n protein and ha-tag. the expression of gp -ha was only observed in cells transfected with in vitro-transcribed rna produced from peav gp -ha; however, it was not observed in peav (wt), peav s / , or mock-transfected cells. note that the size of the gp -ha was~ kda ( figure b ). as shown in figure b , which shows the successful replication of the eav, the expression of the n protein was observed in cells transfected with in vitro-transcribed rna produced from peav (wt), peav s / , and peav gp -ha, but not in the mock-transfected cells. when new cultures of the bhk- cells were infected using culture supernatants from bhk- cells previously transfected with in vitro-transcribed rna from peav (wt), peav gp -ha, or peav s / , i.e., passage (p ), gp -ha was detectable using only immunofluorescence (if) for the eavgp -ha infected cells ( h post-infection) ( figure c ). however, the viral n protein was detected in cells infected with wild-type eav (eav-wt), eavs / , and eavgp -ha, but not in mock-infected cells. to summarize, these data demonstrate that rnas derived from peav s / and peav gp -ha viruses are fully replication competent when introduced into the susceptible mammalian cells. in addition, the progeny viruses that have either separated orf and orf or express ha-tagged gp are infectious. the in vitro growth properties of eavs / and eavgp -ha viruses were compared to those of eav-wt using one-step growth curve experiments for bhk- cells (figure a ). all tested viruses replicated to titers that exceeded pfu/ml of cell culture fluid, although the eav-wt reached a slightly higher titer at h post-infection. this suggests that the separation of the orf and orf , as well as introduction of the ha-tag to the gp protein did not have a significant effect on the eav progeny's production. the in vitro growth properties of eavs / and eavgp -ha viruses were compared to those of eav-wt using one-step growth curve experiments for bhk- cells (figure a ). all tested viruses replicated to titers that exceeded pfu/ml of cell culture fluid, although the eav-wt reached a slightly higher titer at h post-infection. this suggests that the separation of the orf and orf , as well as introduction of the ha-tag to the gp protein did not have a significant effect on the eav progeny's production. the stability of gp -ha expression was investigated by a serial passage of eavgp -ha virus in the bhk- cells. the expression of gp -ha was analyzed by if using cells infected with p , p , p , and p ( figure b ). the levels of n and gp -ha protein expression was stable up to p ; however, in cells infected with p , the number of cells expressing gp -ha and fluorescence intensity were reduced. to determine whether these results were caused by the emergence of viruses containing mutations, the intracellular rna was isolated after infection with p , p , p , and p eavgp -ha virus and subjected to rt-pcr, followed by sequence analysis (figure c ). note that the consensus sequence was still present in p ; however, the chromatogram of the nt sequence of the ha-tag and the neighboring sequence showed the presence of mutated nucleotides, which may indicate the rise of the quasispecies viruses with accumulated mutations within the ha-tag sequence. such quasispecies of viruses, which are present in the supernatant along with the original recombinant virus, can lead to diminished fluorescence intensity and loss of fluorescence from the ha-tag in certain infected cells. to summarize, this data indicate that the separation of orf and orf and introduction of gp -tag can be stably accommodated within the eav genome and that the ha-tag added to c-terminus of gp is expressed at least up to p . to investigate the molecular weight of gp -ha, the bhk- cells were infected with the eavgp -ha virus. cell lysates were subjected to both non-reducing and reducing sds-page, followed by western blotting with anti-ha antibodies. in the non-reducing conditions, most of the gp -ha protein was present at the band of~ kda, and only a small fraction migrated to two faint bands of~ kda ( figure a ). this indicates that the gp -ha in infected cells is present mostly in the monomeric form; however, some of it forms a multimeric structure that might correspond to the gp -ha dimer or the gp -ha associated with other viral or cellular proteins. in reducing conditions, the gp -ha displays a characteristic double-band pattern, which is caused by the heterologous glycosylation of the overlapping nntt sequon, indicating that certain gp -ha molecules have and some have n-glycans [ ] . to confirm that gp -ha in infected cells is indeed n-glycosylated, the cell lysates were digested with peptide n-glycosidase f (pngase f) (cleaves of all types of n-linked carbohydrates) or endo-β-n-acetyl-glucosaminidase (endo h; cleaves only the high-mannose-type carbohydrates) prior to sds-page ( figure a ). the carbohydrate chains from the intracellular gp were completely cleaved by both these enzymes, confirming that gp -ha is retained in er or a cis-golgi compartment, as endo h cannot cleave the n-glycans with additional modifications occurring in the medial or trans-golgi. therefore, the gp -ha expressed in infected cells exhibits the same behavior as in previously published data for the wild-type gp [ , , ] . the stability of gp -ha expression was investigated by a serial passage of eavgp -ha virus in the bhk- cells. the expression of gp -ha was analyzed by if using cells infected with p , p , p , and p ( figure b ). the levels of n and gp -ha protein expression was stable up to p ; however, in cells infected with p , the number of cells expressing gp -ha and fluorescence intensity were reduced. to determine whether these results were caused by the emergence of viruses containing mutations, the intracellular rna was isolated after infection with p , p , p , and p eavgp -ha virus and subjected to rt-pcr, followed by sequence analysis ( figure c ). note that the consensus sequence was still present in p ; however, the chromatogram of the nt sequence of the ha-tag and the neighboring sequence showed the presence of mutated nucleotides, which may indicate the rise of the quasispecies viruses with accumulated mutations within the ha-tag sequence. such quasispecies of viruses, which are present in the supernatant along with the original recombinant virus, can lead to diminished fluorescence intensity and loss of fluorescence from the ha-tag in certain infected cells. to summarize, this data indicate that the separation of orf and orf and introduction of gp -tag can be stably accommodated within the eav genome and that the ha-tag added to c-terminus of gp is expressed at least up to p . to investigate the molecular weight of gp -ha, the bhk- cells were infected with the eavgp -ha virus. cell lysates were subjected to both non-reducing and reducing sds-page, followed by western blotting with anti-ha antibodies. in the non-reducing conditions, most of the gp -ha protein was present at the band of ~ kda, and only a small fraction migrated to two faint bands of ~ kda ( figure a ). this indicates that the gp -ha in infected cells is present mostly in the monomeric form; however, some of it forms a multimeric structure that might correspond to the gp -ha dimer or the gp -ha associated with other viral or cellular proteins. in reducing conditions, the gp -ha displays a characteristic double-band pattern, which is caused by the heterologous glycosylation of the overlapping nntt sequon, indicating that certain gp -ha molecules have and some have n-glycans [ ] . to confirm that gp -ha in infected cells is indeed n-glycosylated, the cell lysates were digested with peptide n-glycosidase f (pngase f) (cleaves of all types of n-linked carbohydrates) or endo-β-n-acetyl-glucosaminidase (endo h; cleaves only the high-mannose-type carbohydrates) prior to sds-page ( figure a ). the carbohydrate chains from the intracellular gp were completely cleaved by both these enzymes, confirming that gp -ha is retained in er or a cis-golgi compartment, as endo h cannot cleave the n-glycans with additional modifications occurring in the medial or trans-golgi. therefore, the gp -ha expressed in infected cells exhibits the same behavior as in previously published data for the wild-type gp [ , , ] . to demonstrate that the tagged gp -ha is incorporated into the virions, the bhk- cells wer infected with the p eavgp -ha stock. after h post-infection, the supernatants were collected centrifuged at low speed, and purified using filter devices. a major part of the purified virus sampl ( %) was subjected to immunoprecipitation (ip) with anti-ha antibodies, and analyzed usin sds-page under non-reducing and reducing conditions. as expected, in non-reducing conditions the gp -ha was present in virions in small amounts and was barely visible without precipitatio ( figure b ). in the sample subjected to ip, gp -ha was present at a band of kda and a band of kda. these apparent molecular masses correspond to the gp -ha monomer and gp /gp -ha/gp trimer. in reducing conditions, only the monomeric form was present, suggesting that th multimeric form detected in the virions in the non-reducing condition is because of the covalen linkage of gp -ha to other minor proteins of eav. moreover, additional bands were present in th mock samples, which is probably because of ip and western blotting being performed using th to demonstrate that the tagged gp -ha is incorporated into the virions, the bhk- cells were infected with the p eavgp -ha stock. after h post-infection, the supernatants were collected, centrifuged at low speed, and purified using filter devices. a major part of the purified virus sample ( %) was subjected to immunoprecipitation (ip) with anti-ha antibodies, and analyzed using sds-page under non-reducing and reducing conditions. as expected, in non-reducing conditions, the gp -ha was present in virions in small amounts and was barely visible without precipitation ( figure b ). in the sample subjected to ip, gp -ha was present at a band of kda and a band of kda. these apparent molecular masses correspond to the gp -ha monomer and gp /gp -ha/gp trimer. in reducing conditions, only the monomeric form was present, suggesting that the multimeric form detected in the virions in the non-reducing condition is because of the covalent linkage of gp -ha to other minor proteins of eav. moreover, additional bands were present in the mock samples, which is probably because of ip and western blotting being performed using the same antibody. unfortunately, detection of gp -ha with different anti-ha antibody in western blotting failed (mouse monoclonal anti-ha antibody, enzo). in reducing conditions, the kda band is possible an antibody heavy chain; however, in non-reducing conditions, the bands indicated with triangles are probably detected covalently bond light and heavy chains of the antibodies used in ip. it is possible, that those antibody-derived bands might hide some bands coming from the targeting protein or protein complex. therefore, we can assume that at least monomeric and trimeric gp -ha was present in the virion, but other forms cannot be excluded. to investigate if the e protein associated in a virion with the gp /gp -ha/gp , the immunoprecipitates obtained using anti-ha antibodies and purified supernatant lysate (wsl), which is the virion fraction, and the whole cell lysates (wcl) were subjected to reducing sds-page and western blotting using anti-e antibodies ( figure c) . the e protein was expressed in infected cells (wcl) as a double band; however, in the purified virion, only one band was detected, which is a feature that has been previously observed [ ] . however, in the sample immunoprecipitated with anti-ha antibodies, no e protein could be detected, indicating that the e protein does not form any covalent linkages with the gp /gp -ha/gp trimer in virions. we studied the co-localization of the gp -ha, e protein, and n protein in eav-infected bhk- cells using immunofluorescence microscopy ( h p.i.). however, the gp -ha partially co-localized with e and n proteins, and co-localization was higher in the case of e protein (mander's coefficient = . ± . ) compared to that of n protein (mander's coefficient = . ± . ) ( figure a,b) . furthermore, very little co-localization was observed between e and n proteins (mander's coefficient = . ± . ) ( figure c ). same antibody. unfortunately, detection of gp -ha with different anti-ha antibody in western blotting failed (mouse monoclonal anti-ha antibody, enzo). in reducing conditions, the kda band is possible an antibody heavy chain; however, in non-reducing conditions, the bands indicated with triangles are probably detected covalently bond light and heavy chains of the antibodies used in ip. it is possible, that those antibody-derived bands might hide some bands coming from the targeting protein or protein complex. therefore, we can assume that at least monomeric and trimeric gp -ha was present in the virion, but other forms cannot be excluded. to investigate if the e protein associated in a virion with the gp /gp -ha/gp , the immunoprecipitates obtained using anti-ha antibodies and purified supernatant lysate (wsl), which is the virion fraction, and the whole cell lysates (wcl) were subjected to reducing sds-page and western blotting using anti-e antibodies ( figure c) . the e protein was expressed in infected cells (wcl) as a double band; however, in the purified virion, only one band was detected, which is a feature that has been previously observed [ ] . however, in the sample immunoprecipitated with anti-ha antibodies, no e protein could be detected, indicating that the e protein does not form any covalent linkages with the gp /gp -ha/gp trimer in virions. we studied the co-localization of the gp -ha, e protein, and n protein in eav-infected bhk- cells using immunofluorescence microscopy ( h p.i.). however, the gp -ha partially co-localized with e and n proteins, and co-localization was higher in the case of e protein (mander's coefficient = . ± . ) compared to that of n protein (mander's coefficient = . ± . ) ( figure a,b) . furthermore, very little co-localization was observed between e and n proteins (mander's coefficient = . ± . ) ( figure c ). in the next step, we investigated the co-localization of gp -ha with the cellular compartment specific markers. the bhk- cells were infected with recombinant eavgp -ha virus, and the cells ( h p.i.) were fixed and subjected to double immunostaining with rabbit anti-ha antibodies and antibody against a particular cellular compartment. the localization of gp -ha was analyzed using antibodies directed against the following markers of the secretory pathway: protein disulfide-isomerase (pdi) for er; ergic- for er-golgi intermediate compartment, ergic; and membrin for cis-golgi. the gp -ha was co-localized with er (mander's coefficient = . ± . ) and cis-golgi (mander's coefficient = . ± . ) markers ( figure a,c) , and, to a lesser extent, with the ergic compartment marker (mander's coefficient = . ± . ) ( figure b ). respectively. e is shown in green, gp -ha is shown in red. gp -ha shows little co-localization with n protein (b). cells were infected as above; cells ( h p.i.) were then subjected to immunofluorescence with rabbit anti-ha antibodies and mouse anti-n antibodies and secondary alexa fluor -conjugated anti-mouse antibodies and alexa fluor -conjugated anti-rabbit antibodies, respectively. n is shown in red, gp -ha is shown in green. e shows little co-localization with n protein (c). cells were infected as above; cells ( h p.i.) were then subjected to immunofluorescence with rabbit anti-e antibodies and mouse anti-n antibodies and secondary alexa fluor -conjugated anti-mouse antibodies and alexa fluor -conjugated anti-rabbit antibodies, respectively. n is shown in red, e is shown in green. the cell nuclei were stained with dapi. scale bar = µm. in the next step, we investigated the co-localization of gp -ha with the cellular compartment specific markers. the bhk- cells were infected with recombinant eavgp -ha virus, and the cells ( h p.i.) were fixed and subjected to double immunostaining with rabbit anti-ha antibodies and antibody against a particular cellular compartment. the localization of gp -ha was analyzed using antibodies directed against the following markers of the secretory pathway: protein disulfide-isomerase (pdi) for er; ergic- for er-golgi intermediate compartment, ergic; and membrin for cis-golgi. the gp -ha was co-localized with er (mander's coefficient = . ± . ) and cis-golgi (mander's coefficient = . ± . ) markers ( figure a,c) , and, to a lesser extent, with the ergic compartment marker (mander's coefficient = . ± . ) ( figure b) . similarly, we investigated the co-localization of the e protein with the same cellular compartment's markers. bhk- cells, infected with eavgp -ha virus, were subjected to double immunostaining with rabbit anti-e antibodies and antibody against a particular cellular compartment. the e protein co-localized with pdi, marker of er (mander's coefficient = . ± . ) ( figure a ). there was some co-localization with the cis-golgi marker membrin (mander's coefficient = . ± . ) ( figure c ) and minimal co-localization within the ergic (mander's coefficient = . ± . ) ( figure b ). similarly, we investigated the co-localization of the e protein with the same cellular compartment's markers. bhk- cells, infected with eavgp -ha virus, were subjected to double immunostaining with rabbit anti-e antibodies and antibody against a particular cellular compartment. the e protein co-localized with pdi, marker of er (mander's coefficient = . ± . ) ( figure a ). there was some co-localization with the cis-golgi marker membrin (mander's coefficient = . ± . ) ( figure c ) and minimal co-localization within the ergic (mander's coefficient = . ± . ) ( figure b ). immunofluorescence. e partially co-localizes with pdi, marker for er (a). confocal analysis of the immunofluorescence samples stained with rabbit anti-e antibodies and mouse anti-pdi antibodies and secondary antibodies alexa fluor anti-mouse and alexa fluor anti-rabbit antibodies. e is shown in green, pdi is shown in red. e minimally co-localizes with ergic (b). confocal analysis of the immunofluorescence samples stained with rabbit anti-e antibodies and mouse anti-ergic antibody and secondary antibodies alexa fluor anti-mouse and alexa fluor anti-rabbit antibodies, respectively. e is shown in green, ergic is shown in red. e partially co-localizes with membrin, marker for cis-golgi (c). confocal analysis of the immunofluorescence samples stained with rabbit anti-e antibodies and mouse anti-membrin antibodies and secondary antibodies alexa fluor anti-mouse and alexa fluor anti-rabbit antibodies. e is shown in green, membrin is shown in red. the cell nuclei were stained with dapi. scale bar = µm. in this study, we describe the successful design and characterization of recombinant eav that have separated orfs encoding minor structural glycoproteins gp and gp , which were then used to derive recombinant eav with ha-tagged gp . tagging the proteins in viral context might be a good tool to study localization and protein-protein interactions, particularly if the antibodies against those proteins are difficult to obtain. in arteriviruses, tagging the structural proteins is difficult because of the overlapping orfs and nested genome. previously, the separation of the overlapping orfs in eav genome was achieved only for orf and , orf and , and for orfs , , [ ] . moreover, a small aa epitope was successfully added to the n terminus of the m protein (orf ); however, the overlapping sequences between major structural protein coding gens orfs , , and are smaller compared to the overlap between genes coding for minor virion proteins. in the abovementioned studies, the introduction into the eav genome composed of additional - nucleotides and was stable up to p . in our study, we successfully introduced nucleotides between structural virion genes and tested the stability of the expression of the ha-tag, which lasted till at least p . this is a very stable tag expression comparing bigger tags, such as mcherry and gfp, which were introduced in parts of the genome encoding non-structural proteins that had fluorescent protein expression declining after a few passages [ , ] . these infectious clones, peav s / and peav gp -ha, will enable easier mutagenesis on the c-terminus of the gp and n-terminus of the gp as changing the nt sequence of one orf in particular regions will not influence the coding sequence in the other orf. in this study, we demonstrated that the addition of ha-tag to the gp did not affect virus infectivity and replication. gp -ha was incorporated into the virions and was forming a multimeric structure, corresponding to the size of the gp / / trimer. the trimer was not detected in infected cells, only in the virion, thus supporting earlier experiments [ , ] . the recombinant eavgp -ha will enable analysis of the entry of eav as the information on the mechanisms of fusion and uncoating of the arteriviruses is still missing. furthermore, the tagged protein could be used in proteomic experiments that explore host-virus interactions [ ] . information that the eav genome supports a stable tag in the structural protein could be useful for vaccine development of arterivirus, e.g., in the techniques of virion purification or marker vaccine development [ ] . in this study, we used the recombinant eav to study the localization of the gp -ha. previously, the localization of the gp was tested in transfected cells only, and exclusively with the er marker [ ] . as the incorporation of minor proteins is e dependent we also performed detailed co-localization of the e. clearly, these membrane proteins poorly co-localized with n, which was shown to be primarily located in the cytoplasm (inside dmvs) [ ] . the gp -ha and e co-localized to some extent with each other, however we expected higher co-localization of those minor eav proteins. both gp -ha and e localized primarily in the er, and some of the gp -ha was also found in the cis-golgi compartment. the e protein co-localized with cis-golgi to a lesser extent, then gp -ha, which can explain why the co-localization of the gp -ha and e was lower than expected. our study suggests that only a small fraction of the expressed gp -ha and an even smaller fraction of e protein was transported to the golgi apparatus. the gp -ha and e did not co-localize in ergic; however, this does not rule it out as a budding site of arteriviruses. although the gp does not contain any known er retention motif, a majority of the protein was retained in the er in our study. the n-glycans of gp -ha were sensitive to endoglycosidase h, confirming that most of the protein remains in the er. we observed some localization of gp -ha and e in the cis-golgi, possibly because some of the gp -ha and e might have escaped from er because of overexpression and was transported back from the golgi to er [ ] . although expressed in high amounts in infected cells, only a small fraction of the e and minor glycoproteins gp , gp , and gp end up in the virions [ , ] . a few reasons for this phenomenon are as follows. first, minor proteins might not be present at the assembly site. if gp /m, which is indispensable for vlps formation, is located primarily in the golgi, it could be the main budding site. in our study, we have shown that only some fraction of gp -ha is also located in the cis-golgi, while a majority of it seems to be retained in er. only properly folded and assembled transmembrane proteins are exported from the endoplasmic reticulum (er) [ ] . it is possible that the complex gp /gp dimer, as well as gp and e, moved to the golgi, while monomers remained in er. unfortunately, the gp -ha does not form a disulfide-linked trimer in the cell; therefore, we could not perform the fractionation of internal membranes to see if the gp -ha did move from the er to the golgi on oligomerization with gp and gp . disulfide-linked trimer formation is mediated after virion release, preferably at more basic ph [ ] . we also detected the higher forms of gp -ha in non-reducing conditions only in virions. the site of arterivirus assembly is still not fully understood. previous electron microscopy studies suggest budding from intracellular membranes: er or golgi [ , ] . clearly, for the production of vlps, the nucleocapsid n coupled with rna and the gp /m heterodimer formation is essential [ , ] . this is in contrast to the assembly of coronaviridae (which are in the same order as arteriviridae-nidovirales), in which only the m and e proteins drive virion budding [ ] . the failure to achieve budding by co-expression of only n, gp , and m without the eav rna suggests that there are additional factors in the eav particle formation. it is possible that the arterivirus assembly is coupled to viral genome replication, interaction of rna with structural proteins, or to the expression of non-structural proteins. it has been shown primarily for other nidovirales, such as the coronaviruses, that the efficient incorporation of viral proteins into virions depends on two determinants, i.e., protein trafficking and interaction between proteins at the budding site, which in the case of coronaviruses is ergic [ ] . however, the factors that control the site of budding in well-studied coronaviruses are unknown. when expressed independently or during an infection, many coronaviral proteins pass through the ergic and localize in the golgi complex, e.g., the spike s protein is expressed even on the plasma membrane [ , ] . in arteriviruses, gp /m dimers, which are essential for eav budding, were shown to be located primarily in the golgi apparatus (co-localization with mannosidase ii), although at subsequent time points of the infection, the m was also present in the er [ ] and golgi localization was not achieved if the dimer formation was blocked. if the budding occurs where the gp /m is localized, this could be the mechanism of lower incorporation into the virions of minor proteins, i.e., they are not abundant at the assembly site. in this study, we have shown that the gp -ha and e localized mainly in the er. however, the mechanism of lower incorporation of the e and minor glycoproteins in arteriviruses might be different, and not dependent on the localization of the proteins. in coronaviruses the e protein, which is essential for virion formation, remains at the budding site, but is incorporated into virions in small amounts, by unknown mechanism [ ] . in the influenza a virus the m protein is excluded from the budozone, presumably because it localizes at the edges of the lipid rafts at plasma membrane, the site of infuenza a budding [ , ] . further experiments are needed to explain lower incorporation of the minor proteins into eav virions. in this study, we successfully introduced the ha-tag into the minor structural protein gp of the eav. the tagged protein was incorporated into the virion particles, whereas the mutant virus behaved similar to the wt. the tagged gp in the virus context might facilitate research on the biology of eav, e.g., in this study, we analyzed the localization of the gp -ha in the infected cells. the gp -ha primarily localized in the er, some gp -ha was present in cis-golgi, but very little was present in the ergic. this study shows that the arterivirus genome tolerates substantial manipulation within genes coding for structural proteins that are responsible for cellular tropism without eav virus infectivity loss. the separation of orf and orf in reverse genetic plasmids can facilitate mutagenesis on the terminal parts of gp and gp . these results can be also implemented for the development of new arteriviral vaccines. arterivirus molecular biology and pathogenesis equine viral arteritis equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily all subgenomic mrnas of equine arteritis virus contain a common leader sequence membrane proteins of arterivirus particles: structure, topology, processing and function structural protein requirements in equine arteritis virus assembly pathological observations of an experimental infection of geese with goose circovirus the minor envelope glycoproteins gp a and gp of porcine reproductive and respiratory syndrome virus interact with the receptor cd pigs lacking the scavenger receptor cysteine-rich domain of cd are resistant to porcine reproductive and respiratory syndrome virus infection the small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties formation of disulfide-linked complexes between the three minor envelope glycoproteins of equine arteritis virus identification of a novel structural protein of arteriviruses characterization of two new structural glycoproteins, gp and gp , of equine arteritis virus morphological studies on equine arteritis virus electron microscopicstudy of tissue cultures infected with simian haemorrhagic fever virus non-structural proteins and interact to modify host cell membranes during the formation of the arterivirus replication complex discontinuous subgenomic rna synthesis in arteriviruses is guided by an rna hairpin structure located in the genomic leader region an open-source platform for biological-image analysis a guided tour into subcellular colocalization analysis in light microscopy co-translational processing of glycoprotein from equine arteritis virus: n-glycosylation adjacent to the signal peptide prevents cleavage expression of recombinant, tagged gp / / trimer and e protein from equine arteritis virus in eukaryotic system signal peptide cleavage from gp enabled by removal of adjacent glycosylation sites does not impair replication of equine arteritis virus in cell culture, but the hydrophobic c-terminus is essential genetic manipulation of equine arteritis virus using full-length cdna clones: separation of overlapping genes and expression of a foreign epitope an infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene development and characterization of a synthetic infectious cdna clone of the virulent bucyrus strain of equine arteritis virus expressing mcherry (red fluorescent protein) proteomic approaches to uncovering virus-host protein interactions during the progression of viral infection live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction nuclear localization of non-structural protein and nucleocapsid protein of equine arteritis virus quality control in the endoplasmic reticulum the two major envelope proteins of equine arteritis virus associate into disulfide-linked heterodimers nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes incorporation of spike and membrane glycoproteins into coronavirus virions coronavirus m proteins accumulate in the golgi complex beyond the site of virion budding differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins s construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus tropism in cell culture coronavirus envelope (e) protein remains at the site of assembly association of influenza virus proteins with membrane rafts lateral organization of influenza virus proteins in the budozone region of the plasma membrane we would like to thank eric snijder (university of leiden) for providing the anti-e antibody. the authors declare no conflict of interest. key: cord- - iwwsfz authors: lundstrom, kenneth title: alphavirus-based vaccines date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: iwwsfz alphavirus vectors have demonstrated high levels of transient heterologous gene expression both in vitro and in vivo and, therefore, possess attractive features for vaccine development. the most commonly used delivery vectors are based on three single-stranded encapsulated alphaviruses, namely semliki forest virus, sindbis virus and venezuelan equine encephalitis virus. alphavirus vectors have been applied as replication-deficient recombinant viral particles and, more recently, as replication-proficient particles. moreover, in vitro transcribed rna, as well as layered dna vectors have been applied for immunization. a large number of highly immunogenic viral structural proteins expressed from alphavirus vectors have elicited strong neutralizing antibody responses in multispecies animal models. furthermore, immunization studies have demonstrated robust protection against challenges with lethal doses of virus in rodents and primates. similarly, vaccination with alphavirus vectors expressing tumor antigens resulted in prophylactic protection against challenges with tumor-inducing cancerous cells. as certain alphaviruses, such as chikungunya virus, have been associated with epidemics in animals and humans, attention has also been paid to the development of vaccines against alphaviruses themselves. recent progress in alphavirus vector development and vaccine technology has allowed conducting clinical trials in humans. alphaviruses are single-stranded rna viruses with an envelope structure belonging to the family of togaviridae [ ] . certain alphaviruses have been associated with pathogenicity, resulting in global fever epidemics, such as observed recently for chikungunya virus [ ] . furthermore, semliki forest virus (sfv) [ ] and venezuelan equine encephalitis (vee) virus [ ] have been identified as the causes of an outbreak of febrile illness in central africa and an epidemic in horses and humans in south america, respectively. despite this potential concern, several alphaviruses, including sfv [ ] , sindbis virus (sin) [ ] and vee [ ] have been subjected to the engineering of vectors for heterologous gene expression. in these cases, attenuated strains have been employed. several types of vector systems have been engineered. there are three types of replication-deficient vectors consisting of naked rna, recombinant particles and layered dna vectors (figure ). the application of naked rna vectors involves the use of in vitro transcribed rna from an expression vector consisting of the viral nonstructural replicase genes and the foreign gene of interest downstream of the strong subgenomic promoter. the production of recombinant particles requires the co-transfection of in vitro transcribed rna from an expression vector (as described above) and a helper vector supplying the viral structural genes into mammalian cell lines (for example, baby hamster kidney (bhk) cells). the generated particles are capable of one round of infection of a broad range of host cells, but due to the selective packaging of only expression vector rna, no further virus production occurs. the layered dna vector system consists of delivery of a dna vector providing foreign gene expression from a cmv promoter. furthermore, the engineering of vectors with an additional subgenomic promoter to the full-length genome allows for the generation of replication-proficient particles, which can provide improved delivery and extended gene expression. all alphavirus vectors described take advantage of the extremely efficient rna replication, resulting in some , rna copies from each rna molecule. the essential question is: which vector system to use? obviously, replication-proficient particles can provide efficient delivery, but suffer from potential insufficiency related to safety aspects. although replication-deficient particles provide a higher level of safety, there is still a marginal risk of the generation of replication-proficient particles through non-homologous recombination. to minimize any unwanted recombination events, a split helper vector system with capsid and envelope genes expressed from separate helper vectors has been engineered [ ] . so far, alphaviruses have been applied for the expression of a number of topologically different recombinant proteins [ ] . particularly, the use of sfv particles has resulted in high expression levels of integral membrane proteins in various mammalian host cell lines [ ] , in primary neurons [ ] and in vivo [ ] . for vaccine development, vectors based on sfv, sin and vee have been applied as naked rna, recombinant virus particles and layered dna vectors [ ] . in this context, viral and tumor antigens have been administered in various animal models to elicit neutralizing antibodies and protection against challenges with tumor cells or lethal doses of viruses. moreover, non-viral pathogens have been subjected to vaccine development. replicon particles derived from vee have furthermore demonstrated activity as safe and potent systemic, mucosal and cellular adjuvants when co-administered with antigen [ ] . finally, as alphaviruses have been identified as the cause of viral epidemics in animals and humans, a number of approaches have been initiated for immunization against alphavirus-based infections. in this review, the latest development on alphavirus vectors for vaccine production is summarized. due to their immunogenic properties, viral structural proteins have been popular targets for alphavirus-based vaccine development [ ] (table ) . in this context, immunization with sfv particles expressing influenza nucleoprotein (np) elicited a strong immune response in mice [ ] . moreover, vee-based expression of influenza hemagglutinin (ha) provided protection against challenges with h n virus in chicken [ ] . similarly, sfv particles expressing the hiv envelope [ ] and gp [ ] attempts to further improve the immunogenicity of vaccine candidates, the herpes simplex virus type i (hsv- ) vp protein was fused to the h n subtype influenza ha [ ] . the responses of both interleukin- (il- ) of cd + t-cells and interferon-gamma (ifnɣ) of cd + t-cells were observed in vaccinated mice. vee replicon particles expressing the severe acute respiratory syndrome coronavirus (sars-cov) glycoprotein managed to provide protection against challenges with lethal doses of sars-cov in vaccinated mice [ ] . furthermore, vee particles were applied for the expression of glycoproteins from the zoonotic pathogenic hendra virus (hev) and nipah virus (niv), known to cause fatal infections in both animals and humans [ ] . immunization resulted in enhanced induction of cross-reactive neutralizing antibodies. in another study, mice were vaccinated with both dna plasmids and alphavirus replicons expressing rift valley fever virus (rvfv) glycoprotein gn fused to the c d complement protein [ ] . the immunization generated neutralizing antibodies and provided protection against challenges with rvfv, which suggested that plasmid dna and alphavirus replicon approaches, as well as the combined dna prime/replicon boost strategy show great promise for valid rvfv vaccine development. the combined approach included plasmid vaccinations at weeks and followed by a replicon boost at week . in a combined vaccine approach, sfv dna vectors and recombinant adenovirus expressing the classical swine fever virus (csfv) e glycoprotein elicited higher titers of neutralizing antibodies in pigs [ ] . after challenges with the virulent csfv shimen strain, no symptoms of viremia were observed, for the combined vaccine, whereas vaccination with adenovirus alone resulted in viremia in one pig of five. furthermore, sequential immunization with sin and vee replicon particles expressing the type hiv gp envelope (env) and trimeric env protein in mf adjuvant provided partial protection in macaques against intravenous challenges with high doses of simian-human immunodeficiency virus (shiv) [ ] . the administration could be further extended to intramuscular and mucosal delivery [ ] . different degrees of protection were observed against challenges with shiv after mucosal administration. in contrast, intramuscular vaccination rendered macaques to be completely resistant to shiv. in cotton rats, sin dna vectors carrying the hemagglutinin (pmsin-h) and fusion proteins (pmsinh-fdu) elicited neutralizing antibodies, mucosal and systemic antibody-secreting cells, memory b-cells and ifnɤ secreting t-cells [ ] . priming with pmsin-h provided % protection against challenges with pulmonary measles. however, pmsinh-fdu priming was observed only after a boost with live measles virus vaccine. in another study, chimeric vee/sin replicon particles were applied for the expression of measles virus hemagglutinin (h) and fusion (f) proteins, which elicited high-titer neutralizing antibody and ifnɤ-producing t-cells in macaques after intradermal vaccination [ ] . protection from rash and viremia was obtained after challenges with wild-type measles virus - months after vaccination. alphaviruses have also been subjected to the development of smallpox vaccines by the introduction of a r, b r, a l and l r genes into vee particles [ ] . vaccinated mice showed protective immunity. furthermore, vaccination of macaques elicited strong antibody responses and was capable of neutralizing and inhibiting the spread of vaccinia and monkey pox viruses. sin-based dna vaccines have been developed against rabies [ ] . in comparison to a conventional rabies dna vaccine, the sin dna vaccine induced better humoral and cell-mediated immune responses in immunized mice and showed complete protection against challenge with the cvs rabies strain. recently, novel hepatitis c virus (hcv) vaccine candidates were developed by expressing all or a part of the hcv non-structural proteins (nsps) from an sfv vector [ ] . an insert as large as . kb allowed the expression of all nsps leading to a strong and long-lasting ns -specific cd + t-cell response. the level of t-cell response was similar to that observed for the expression of only ns / a. immunization demonstrated significant growth delay of hcv-expressing el tumors in a mouse model. in another study, glycoproteins for either sudan virus (sudv) or ebola virus were expressed from vee replicons and evaluated in vaccinated nonhuman primates [ ] . a single intramuscular injection with vee particles expressing sudv gp provided complete protection against challenges with sudv in cynomolgus macaques. however, vee-sudv gp vaccinated primates were not fully protected against back challenges with ebola virus. on the other hand, co-injection of vee particles expressing sufv gp and ebola virus gp showed protection against both virus types. recently, vee replicon particles generated in vero cells were used to express the e glycoprotein of bovine viral diarrhea virus (bvdv) [ ] . vaccination of bvdv free calves with × iu and × iu, respectively, resulted in neutralizing antibody titers, which were able to cross-neutralize both type and type bvdv genotypes after booster immunizations. vaccination with the higher dose significantly reduced the viral-based leukopenia and showed some protection from clinical disease. similarly, vee replicon particles were engineered to express dengue virus e antigen in two configurations as subviral particles (prme) and soluble dimers (e ) [ ] . immunization of macaques resulted in the rapid production of neutralizing antibodies and demonstrated protection against challenges with dengue virus. moreover, the tetravalent e vee replicon particle vaccine induced a protective response to all four dengue virus serotypes when two immunizations were administered six weeks apart. a novel approach has been to combine alphavirus replicons with pseudotyped baculovirus [ ] . in this context, pseudotyped baculovirus vectors containing the hybrid cm promoter and sfv replicon were used for the expression of gp and m proteins of porcine reproductive and respiratory syndrome virus (prrsv) and compared to a pseudotyped baculovirus vector carrying only a cmv promoter. immunization with the hybrid cmv/sfv showed the induction of strong gp -specific antibodies, and in general, the th -dominant immune response was stronger than the one elicited by the cmv promoter alone. veterinary medicine has also gained from alphavirus vaccine development. in this context, salmonid alphavirus (sav) replicons were applied for the expression of infectious salmon anemia virus (isav) hemagglutinin-esterase (he) [ ] . intramuscular administration of sav replicons provided protection against challenges with isav in atlantic salmon. in contrast, intraperitoneal injection was not successful [ ] . in another study, dna vaccines based on the sav e and e spike proteins were compared to whole virus vaccine in atlantic salmon [ ] . the whole virus vaccine showed superior immunogenicity to the dna vaccine; the latter provided only marginal reduction in viral replication, and the protection against sav challenges was no different from controls. alphavirus-based vaccine development has also been addressed for a number of other infectious pathogens ( table ). for instance, sfv vectors were employed for the expression of the plasmodium falciparum pf antigen, which elicited immunological memory in vaccinated mice [ ] . moreover, vaccination of mice with sin plasmid dna vectors carrying the mycobacterium tuberculosis a antigen (ag a) provided strong immunity and resulted in long-term protection against m. tuberculosis challenges [ ] . in another approach, using sfv dna replicons, the botulinum neurotoxin a hc (bonta-hc) gene provided both antibody and lymphoproliferative responses in vaccinated balb/c mice [ ] . the immunogenicity was enhanced when granulocyte-macrophage colony-stimulating factor (gm-csf) was co-expressed as an adjuvant. additionally, replication-deficient sfv particles were used for the expression of the brucella abortus translation initiation factor (if ) [ ] . immunization of balb/c mice demonstrated significant levels of protection against challenges with the virulent b. abortus strain . similarly, protective antigen (pa) for bacillus anthracis were expressed from sin vectors, resulting in specific and neutralizing antibodies in swiss webster mice and offered some protection against challenges with lethal doses of the pathogenic bacteria [ ] . alphaviruses have found frequent applications in the area of tumor vaccine development (table ) . in this context, naked rna, replication-deficient particles and dna layered vectors have been employed as delivery vehicles. for instance, mice have been subjected to immunization with naked sfv rna replicons carrying the lacz gene [ ] . interestingly, a single injection of only μg of sfv-lacz rna presented complete tumor protection. furthermore, when tumors were administered two days prior to the immunization, the survival was extended by - days. among dna-based tumor vaccine approaches, sin vectors expressing mouse and human tyrosine-related protein- (trp- ) were evaluated in a b mouse melanoma model [ ] . intramuscular injection was capable of breaking immune tolerance and provided protection against melanoma when mice were vaccinated five days prior to cancer challenge. in another study, alphavirus replicon-based expression of melanoma differentiation antigen (mda) tyrosine demonstrated the inhibition of the growth of b transplantable melanoma [ ] . in this context, the vaccine encoding tyrosine related protein (trp- ) relied on a novel immune mechanism, which required the activation of both igg and cd + cell effector responses. furthermore, vaccination with recombinant particles expressing the p a gene [ ] and the human papilloma virus (hpv) e gene [ ] from sfv and vee vectors, respectively, provided protection against further tumor development in mice. attempts have also been made to improve the efficacy of sfv-based hpv vaccines by supplying sfv-based il- expression in mice [ ] . at low doses, il- stimulated antigen-specific ctl responses and enhanced anti-tumor responses after sfv-based hpv -e e immunization. subsequent increases in dosage, however, neither improved the immune responses, nor tumor regression. sin dna vectors have been employed for the expression of the murine melanoma cell adhesion molecule (mcam/muc ) as a vaccine against murine melanoma, which resulted in the induction of humoral and cd + t-cell immune responses against melanoma [ ] . in the context of breast cancer, a dna-based sin vector expressing the neu gene was applied for intramuscular vaccination of mice days prior to the injection of cancer cells overexpressing neu [ ] . the immunization provided strong protection against tumor development. the incidence of lung metastasis from mammary fat pad tumors was reduced. moreover, the number of lung metastases from intravenous injection of neu overexpressing cells decreased. additionally, intradermal vaccination provided tumor protection applying % less plasmid than required for conventional dna vectors. further confirmation of successful cancer vaccination was obtained from the administration of sin vectors expressing neu (psincp/neu) in a murine breast tumor model [ ] . however, in this case, the prophylactic vaccine only showed efficacy when administered prior to the tumor challenge. another approach was comprised of combining alphavirus-based delivery with the chemical anticancer agent, doxorubicin [ ] . when psincp/neu dna and vee/neu particles were administered after injection of mg/kg of doxorubicin, the tumor progression was significantly delayed. this phenomenon did not occur for doxorubicin alone. similarly, a combination therapy with paclitaxel ( mg/kg) and psincp/neu was ineffective. moreover, vee-neu particles were subcutaneously administered in a rat mammary tumor model, which resulted in the elimination of % of pre-existing aggressive mammary tumors [ ] . the combination of dendritic cell (dc)-based cancer immunotherapy with vee-neu particle administration induced both cellular and humoral immunity against neu in transgenic human breast tumor-bearing mice [ ] . moreover, this treatment resulted in the significant inhibition of tumor growth. similarly, both tumor growth and pulmonary metastatic spread were significantly inhibited when mice with pre-existing tumors were subjected to five immunizations with sfv -e vlp expressing the vascular endothelial growth factor receptor (vegfr- ) [ ] . furthermore, co-immunization with sfv particles encoding vegfr- and il- generated significant tumor regression in mice. lung cancer has also been targeted by combined therapy with sfv-il- particles and anti-cd monoclonal antibodies [ ] . syngeneic tc- lung carcinoma was inhibited after intratumoral sfv-il- administration and co-stimulation with anti-cd mabs. in the context of colon cancer vaccines, a sin-based dna vector carrying the lacz gene was compared to conventional plasmid dna vectors in mice with ct .cl tumors [ ] . intramuscular immunization elicited immune responses at doses -to , -fold lower for the sin dna replicon vector compared to the conventional cmv-promoter-based dna-lacz vector. similarly, sfv particles providing vegfr- expressing in vaccinated mice inhibited ct colon carcinoma growth [ ] . closer analysis of microvessel density demonstrated that a significant inhibition of tumor angiogenesis occurred. additionally, when mice were co-immunized with sfv-vegfr- and sfv-il- particles, their survival rate was significantly enhanced. furthermore, oncolytic sfv vectors have been used for immune stimulation in a ct colon tumor model [ ] . intratumoral injections led to an immediate and intense inflammatory reaction and a significant improvement in survival rates. sfv particles expressing hpv e and e have been subjected to prophylactic vaccine development in a murine tc- model for cervical cancer [ ] . pre-immunization with a low dose ( particles) resulted in an hpv-specific ctl response in % of mice, whereas a higher dose ( particles) elicited ctl responses in all animals. furthermore, at a dose of × particles, % of mice were protected from tumor challenges. in another study, the sfv-enhe , particle vaccine showed its potential after intravenous and intramuscular delivery, where exponentially growing tumors completely resolved [ ] . similarly, sin virus rna replicons expressing hpv e were evaluated in a tc- mouse model [ ] . the humoral and cellular immune responses were poor, and no tumor protection was obtained; but, when the hpv e gene was fused to the secretory sig protein and lysosome-associated membrane protein (lamp- ), enhanced e -specific cd + helper t-cell and cd + cytotoxic t-cell activity was observed. moreover, strong in vivo anti-tumor activity was induced. in another study, sin virus particles expressing both hpv e and calreticulin (crt), an endoplasmic reticulum ca + binding transporter, were tested as prophylactic vaccines [ ] . vaccinations generated antigen-specific immune responses, an anti-angiogenic effect and a strong anti-tumor activity. furthermore, intramuscular immunization one week prior to challenge with tc- carcinoma cells provided protection to all treated mice. also vee particles expressing hpv e were subcutaneously injected in mice two weeks prior to cancer cell inoculation, which prevented tumor formation [ ] . furthermore, vaccination induced long-term memory, as protection was observed for challenges three months after the immunization. the therapeutic efficacy was only % of treated tumor-bearing mice. however, co-expression of hpv e and e from the same vector significantly enhanced the therapeutic effect [ , ] . in a prostate tumor model, vee particles expressing human prostate-specific membrane antigen (psma) showed strong cellular and humoral immunity after subcutaneous administration [ ] . furthermore, vee particles have been employed for the expression of the predominantly prostate tissue-specific six transmembrane epithelial antigen of the prostate (steap) [ ] . pre-immunization with vee-steap particles induced a specific immune response and significantly prolonged the overall survival of mice bearing trampc- tumors. when tramp mice were prophylactically immunized with a prostate stem cell antigen (psca) dna plasmid followed by vee-psca particle administration, a specific immune response and anti-tumor protection were observed in % of vaccinated animals [ ] . several vaccine studies have targeted brain tumors. for instance, sfv particles expressing endostatin showed a significant reduction of intratumoral vascularization after intratumoral delivery [ ] . in another approach, bone-marrow isolated dendritic cells (dcs) were transduced with sfv vectors carrying cytokine genes of specific cdnas from melanoma and glioma cells [ ] . pre-vaccination with dcs transduced with sfv-based b and glioma cdnas, respectively, resulted in tumor challenge protection and the prolonged survival of tumor-bearing mice. the combination of dcs transduced with sfv-il- particles and systemically administered il- also provided increased survival rates [ ] . moreover, the expression of human melanoma-associated antigen gp and il- from a sin virus dna vector induced specific anti-tumor ctl responses and provided anti-tumor protection [ ] . vaccination prevented b -hgp tumor formation and demonstrated significant prolongation of survival in mice with established b -hgp tumors. as alphaviruses have been suggested to be responsible for epidemics in various parts of the world, it has also become important to develop vaccines against alphaviruses themselves (table ). in this context, protection against airborne virus was observed in balb/c mice vaccinated with an attenuated vee strain [ ] . in another study, improved protection against vee challenges was achieved by using a live attenuated v vee vaccine [ ] . similarly, when a chimeric eastern equine encephalitis (eee) and western equine encephalitis (wee) virus were applied for the vaccination of c bl/ mice, complete protection was observed against lethal challenges with a virulent eastern equine encephalitis (eee) virus strain [ ] . in attempts to design attenuated alphaviruses for vaccine development, the mechanisms of replication and virus-host interaction have been investigated. in this context, mosquito transmission of chikungunya (chik) virus has been prevented by making their replication dependent on internal ribosome entry sites (ires) [ ] . although replication does not occur in mosquito cells, replication proceeds efficiently in vero cells. in another approach, the non-structural genes of the attenuated vee strain tc- and the naturally attenuated eev strain, respectively, were engineered with the structural genes of chik [ ] . the chimeric vaccine candidates presented a significantly reduced infectivity of the common urban aedes aegypti and a. albopictus vectors for chik, thereby providing a low risk of virus transmission. a new chik virus isolated from an acutely infected human patient was used for the engineering of a synthetic dna vaccine [ ] . when this dna vaccine was electroporated into mice, robust antigen-specific cellular and humoral responses were obtained, which provided protection against further challenges with chik. furthermore, immunization of macaques demonstrated the induction of neutralizing antibodies similar to those elicited in sera from convalescent human patients. in another dna vector approach, the vee s structural genes were expressed and administered as an aerosol in nonhuman primates [ ] . vaccination resulted in no viremia in two macaques and low viremia in one animal, whereas it was high in all control animals. in an approach to target epidermal and dermal antigen presenting cells (apcs), the nanopatch (np) technology has been applied for skin vaccination of west nile virus and chik in mice [ ] . np, comprised of arrays of densely packed projections two orders of magnitude smaller than standard needles, provided efficient delivery of inactivated whole chik vaccine and a dna-based west nile virus vaccine. moreover, as virus-like particles (vlps) consisting of chik capsid and envelope proteins have been demonstrated to protect nonhuman primates against infection of multiple strains of chik, it was of benefit to screen and optimize the solution conditions for stable vaccine formulations [ ] . in this context, sugar, sugar alcohols and polyanions were identified as potential stabilizers. the relatively recent discovery of the commonly occurring phenomenon of gene silencing has also made its impact on vaccine development. in this context, small interfering rnas (sirnas) were engineered against two conserved regions of the nsp and e genes of chik [ ] . a significant reduction of virus titer was observed in vero cells after h ( %) and h ( %), suggesting a potential new therapeutic approach. in another approach, microrna (mirna)-specific target sequences for replicon particle production were introduced into alphavirus helper rnas [ ] . in the presence of mirna-specific inhibitors, chik particles were efficiently generated, whereas in their absence, cellular mirnas downregulated helper rna replication. when mice were inoculated with replicon rna carrying engineered mirna sequences, cellular mirnas prevented the replication of replicon rna, which opens the feasibility of using mirnas as a therapeutic approach for the inhibition of viral replication. moreover, the vee rna-dependent rna polymerase (rdrp) was targeted by artificial mirnas in attempts to inhibit vee replication [ ] . of five mirnas that were evaluated in bhk cells, three showed significant inhibition of vee replication. despite the large number of studies conducted in various animal models, relatively few evaluations have been subjected to humans. a clinical trial conducted with alphaviruses involved intravenous administration of liposome encapsulated sfv particles expressing il- particles to melanoma and kidney carcinoma patients [ ] . high transient expression of recombinant il- with a five-fold increase lasted for - days. as higher doses induced a fever response in patients, the maximum tolerated dose (mtd) was restricted to × encapsulated particles per m . no liposome-or sfv-related toxicity was observed. furthermore, the liposome-encapsulation prevented sfv particles from being recognized by the host immune system and, therefore, allowed repeated systemic administration. the phase i study clearly demonstrated the safe administration of encapsulated sfv particles. a human phase ii, randomized, double-blind, placebo-controlled, safety and immunogenicity study was conducted in healthy adult volunteers on a serially passaged, plaque-purified, live chik vaccine [ ] . a single subcutaneous injection of the chik vaccine was administered to volunteers, and individuals received placebo. there were no adverse events, except for transient arthralgia in five individuals receiving the chik vaccine. of the evaluable vaccinated individuals, ( %) developed chik neutralizing antibodies by day and % still remained seropositive one year later. none of the individuals who received placebo were seropositive. in another vaccine-related alphavirus phase i randomized, double-blind clinical trial for cytomegalovirus (cmv), a two-component vaccine expressing cmv gb or pp / e fusion protein was administered intramuscularly or subcutaneously in cmv seronegative adult volunteers [ ] . the vaccination showed only mild to moderate local reactions and no clinically important changes. it induced neutralizing antibody and multifunctional t-cell responses against cmv antigens. in another clinical trial, alphavirus particles expressing the carcinoembryonic antigen (cea) were repeatedly administered to patients with metastatic cancer [ ] . cea-specific antibodies were able to mediate antibody-dependent cellular cytotoxicity against tumor cells from human colorectal cancer metastases. furthermore, patients with cea-specific antibodies encouragingly showed extended overall survival. similarly, vee replicons expressing the prostate-specific membrane antigen (psma) were applied for a human trial of patients with metastatic cancers at five doses of . × iu or . × iu [ ] . at the lower dose, no psma-specific cellular response was obtained, but a weak psma-specific signal was detected by elisa. disappointingly, the higher dose showed no pmsa-specific response. the trial demonstrated that while there was neither clinical benefit nor robust immune responses, there were no toxicities associated with the immunization, and the vee-pmsa particles were well tolerated. however, as neutralizing antibodies were elicited, the dosing might be suboptimal, requiring some further optimization. a number of vaccine development studies have been conducted using mainly the three most commonly applied alphavirus vectors, sfv, sin and vee. to demonstrate the variety of approaches available, replicon particles, naked rna and layered dna vectors have been employed. each approach has commonly generated responses detected by cellular or humoral responses. in the case of vaccine development against lethal viruses (table ) , non-viral infectious targets ( table ) and tumors ( table ) immunization has provided long-term protection against challenges with the disease-causing agents. moreover, due to the pathogenicity of several alphaviruses, they are themselves credible targets for vaccine development. therefore, a number of studies, particularly for vee and chik (table ) , have provided protection against challenges with virulent alphavirus strains. the potential of alphaviruses causing global epidemics has placed additional concern on the needs of addressing biosafety issues. generally, the strains used for vaccine development are attenuated. furthermore, in the case using alphavirus particles, second generation helper vectors [ ] or split helper systems [ ] have been applied to prevent any production of wild-type-like replication-proficient particles through non-homologous recombination. only recently alphavirus-based vaccines have been subjected to clinical trials. in this context, sfv vectors have been used for delivery of immunostimulatory genes. furthermore, immunizations for the treatment of cmv and vaccinations against prostate and metastatic cancers have been conducted with alphavirus vectors and particles. the strength of applying alphaviruses is the generation of rapid transgene expression and the transient nature of expression. however, the full potential of alphavirus-based vaccines has not been explored yet. in the future, it is anticipated that additional, positive observations, particularly in the form of providing protection against challenges with lethal pathogens and tumors, will attract the enhanced application of alphaviruses for vaccine development. the author declares no conflict of interest. the alphaviruses: gene expression, replication and evolution outbreak of chikungunya in the republic of congo and the global picture an outbreak of semliki forest virus infections in central african republic remergence of epidemic venezuelan equine encephalomyelitis in south america. vee study group a new generation of animal cell expression vectors based on the 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replicon, and packaged sindbis replicon vectors expressing hantavirus structural genes in hamsters outcome of immunization of cynomolgus monkeys with recombinant semliki forest virus encoding human immunodeficiency virus type envelope protein and challenge with a high dose of shiv- virus venezuelan equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with ebolavirus molecular smallpox vaccine delivered by alphavirus replicons elicits protective immunity in mice and non-human primates construction and cellular immune response induction of ha-based alphavirus replicon vaccines against human-avian influenza (h n ) characterization of immune responses elicited in macaques immunized sequentially with chimeric vee/sin alphavirus replicon particles expressing sivgag and/or hivenv and with recombinant hivgp env protein antibody-mediated protection against mucosal simian-human immunodeficiency virus challenge of macaques immunized with alphavirus replicon particles and boosted with trimeric envelope glycoprotein in mf adjuvant enhanced immunogenicity induced by an alphavirus replicon-based pseudotyped baculovirus vaccine against porcine reproductive and respiratory syndrome virus salmonid alphavirus-based replicon vaccine against infectious salmon anemia (isa): impact on infectious route and interactions of the replicon vector superior protection against conferred by inactivated whole virus vaccine over subunit and dna vaccines against salmonid alphavirus infection in atlantic salmon (salmon salar l) comparative immunization study using rna and dna constructs encoding a part of the plasmodium falciparum antigen pf enhanced immunogenicity to mycobacterium tuberculosis by vaccination with an alphavirus plasmid replicon expressing antigen a enhancement of the immunogenicity of dna replicon vaccine of clostridium botulinum neurotoxin serotype a by gm-csf gene adjuvant vaccination with recombinant semliki 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humoral and cellular immunity marked enhancement of antitumor immune responses in mouse brain tumor models by genetically modified dendritic cells producing semliki forest virus-mediated interleukin- induction of therapeutic antitumor antiangiogenesis by intratumoral injection of genetically engineered endostatin-producing semliki forest virus enhancement of antitumor immune response in glioma models in mice by genetically modified dendritic cells pulsed with semliki forest virus-mediated complementary dna induction of antigen-specific immune responses against malignant brain tumors by intramuscular injection of sindbis dna encoding gp and il- dna vaccination against neu reduces breast cancer incidence and metastasis in mice prime-boost vaccination with plasmid and adenovirus gene vaccines control her /neu+ metastatic breast cancer in mice doxorubicin and paclitaxel enhance the antitumor efficacy of vaccines directed against her /neu in a murine mammary carcinoma model vrp immunotherapy targeting neu: treatment efficacy and evidence for immunoediting in a stringent rat mammary tumor model alphaviral vector-transduced dendritic cells are successful therapeutic vaccines against neu-overexpressing tumors in wild-type mice immunization strategy against cervical cancer involving an alphavirus vector expressing high levels of a stable fusion protein of human papillomavirus e and e superior therapeutic efficacy of alphavirus-mediated immunization against human papilloma virus type antigens in a murine tumour model: effects of the route of immunization sindbis virus replicon particles encoding calreticulin linked to a tumor antigen generate long-term tumor-specific immunity eradication of established tumors by vaccination with venezuelan equine encephalitis virus replicon particles delivering human papillomavirus e rna augmentation of alphavirus vector-induced human papilloma virus-specific immune and anti-tumour responses by coexpression of interleukin- cancer immunotherapy using sindbis virus replicon particles encoding a vp -antigen fusion treatment of rapidly growing k-balb and ct mouse tumours using semliki forest virus and its derived vector inhibition of angiogenesis by a semliki forest virus vector expressing vegfr- reduces tumour growth and metastasis in mice induction of a therapeutic antitumor immunological response by intratumoral injection of genetically engineered semliki forest virus to produce interleukin- biology and application of alphaviruses in gene therapy immunotherapeutic synergy between anti-cd mab and intratumoral administration of a cytopathic semliki forest virus encoding il- immunization against muc /mcam, a novel antigen that drives melanoma invasion and metastasis an alphavirus vector overcomes the presence of neutralizing antibodies and elevated numbers of tregs to induce immune responses in humans with advanced cancer a phase i dose escalation trial of vaccine replicon particles (vrp) expressing prostatespecific membrane antigen (psma) in subjects with prostate cancer a novel alphavirus vaccine encoding prostate-specific membrane antigen elicits potent cellular and humoral immune responses in vivo effects of vaccination with six-transmembrane epithelial antigen of the prostate: a candidate antigen for treating prostate cancer prostate stem cell antigen vaccination induces a long-term protective immune response against prostate cancer in the absence of autoimmunity immunotherapy with recombinant sfv-replicons expressing the p a tumor antigen or il- induces tumor regression innovative cancer vaccine strategies based on the identification of tumour-associated antigens induction of p tumor immunity by recombinant semliki forest virus expressing the p a gene enhancement of tumorspecific immune response with plasmid dna replicon vectors genetic immunization against cervical carcinoma: induction of cytotoxic t lymphocyte activity with a recombinant alphavirus vector expressing human papillomavirus type e and e enhancement of sindbis virus self-replicating rna vaccine potency by targeting antigen to endosomal/lysosomal compartments establishment of an hla-a* human papillomavirus type tumor model to determine the efficacy of vaccination strategies in hla-a* transgenic mice antitumor efficacy of venezuelan equine encephalitis virus replicon particles encoding mutated hpv e and e genes induction of an antitumor immunological response by an intratumoral injection of dendritic cells pulsed with genetically engineered semliki forest virus to produce interleukin- combined with the systemic administration of interleukin- an immunological profile of balb/c mice protected from airborne challenge following vaccination with a live attenuated venezuelan equine encephalitis virus vaccine improved mucosal protection against venezuelan equine encephalitis virus is induced by the molecularly defined, live-attenuated v vaccine candidate recombinant chimeric western and eastern equine encephalitis viruses as potential vaccine candidates phase ii safety and immunogenicity study of live chikungunya virus vaccine tsi-gsd- a dna vaccine against chikungunya virus is protective in mice and induces neutralizing antibodies in mice and nonhuman primates design of chimeric alphaviruses with a programmed, attenuated, cell type-restricted phenotype development of a stable virus-like particle vaccine formulation against chikungunya virus and investigation of the effects of polyanions rna interference mediated inhibition of chikungunya virus replication in mammalian cells in vitro and in vivo characterization of micrornatargeted alphavirus replicon and helper rnas role for mucosal immune responses and cell-mediated immune functions in protection from airborne challenge with venezuelan equine encephalitis virus immunogenicity and protective efficacy of a dna vaccine against venezuelan equine encephalitis virus aerosol challenge in nonhuman primates transmission potential of two chimeric chikungunya vaccine candidates in the urban mosquito vectors, aedes aegypti and ae. albopictus artificial micrornas can effectively inhibit replication of venezuelan equine encephalitis virus nanopatch-targeted skin vaccination against west nile virus and chikungunya virus in mice semliki forest virus expression system: production of conditionally infectious recombinant particles key: cord- -h itv authors: mok, darren z. l.; chan, kuan rong title: the effects of pre-existing antibodies on live-attenuated viral vaccines date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: h itv live-attenuated vaccines (lavs) have achieved remarkable successes in controlling virus spread, as well as for other applications such as cancer immunotherapy. however, with rapid increases in international travel, globalization, geographic spread of viral vectors, and widespread use of vaccines, there is an increasing need to consider how pre-exposure to viruses which share similar antigenic regions can impact vaccine efficacy. pre-existing antibodies, derived from either from maternal–fetal transmission, or by previous infection or vaccination, have been demonstrated to interfere with vaccine immunogenicity of measles, adenovirus, and influenza lavs. immune interference of lavs can be caused by the formation of virus–antibody complexes that neutralize virus infection in antigen-presenting cells, or by the cross-linking of the b-cell receptor with the inhibitory receptor, fcγriib. on the other hand, pre-existing antibodies can augment flaviviral lav efficacy such as that of dengue and yellow fever virus, especially when pre-existing antibodies are present at sub-neutralizing levels. the increased vaccine immunogenicity can be facilitated by antibody-dependent enhancement of virus infection, enhancing virus uptake in antigen-presenting cells, and robust induction of innate immune responses that promote vaccine immunogenicity. this review examines the literature on this topic and examines the circumstances where pre-existing antibodies can inhibit or enhance lav efficacy. a better knowledge of the underlying mechanisms involved could allow us to better manage immunization in seropositive individuals and even identify possibilities that could allow us to exploit pre-existing antibodies to boost vaccine-induced responses for improved vaccine efficacy. "it's time to close the book on infectious diseases, declare the war against pestilence won, and shift national resources to such chronic problems as cancer and heart disease" [ ] . contrary to this infamous statement, long misattributed to the former us surgeon general dr. william h. stewart, and despite advances in healthcare and technology, we remain extremely vulnerable to the threat of communicable diseases. in the last ten years alone, we have experienced the pandemic spread of swine-origin h n influenza, the west african ebola epidemic, the resurgence of yellow fever, the zika virus emergency, and the return of a global coronavirus threat [ ] [ ] [ ] [ ] [ ] . with the continued emergence and re-emergence of new and current viral pathogens, it is imperative that we continue to design new strategies to combat their spread and prevent human disease. among the various methods to impede viral transmission, vaccines are widely heralded as one of the most effective medical interventions. indeed, since the pivotal discovery by edward jenner over two hundred years ago, vaccination has seen tremendous success in reducing the burden of viral diseases against wild-type virus infections and not cause worse disease outcomes. for vaccines that are administered to neonates, the presence of passively acquired maternal antibodies within the first six months may also interfere with vaccination [ , ] . the potential effects of pre-existing antibodies on lav and virally vectored antigens will hence be the focus of this review. in this review, we examine the scenarios in which pre-existing antibodies can either enhance or inhibit lav efficacy, as well as the underlying mechanisms involved. a better understanding will allow us to tailor our vaccination schedules or vaccine doses, to ensure that lav efficacy will not be compromised by the presence of pre-existing immunity. prior to the advent of vaccination, the incidence rate of measles was so high that infection by the measles virus was basically considered an inevitability [ , ] . the virus, which is spread by the respiratory route, is highly contagious and infects over % of individuals by age in the pre-vaccination era [ , ] . about seven to eight million children were estimated to have died from measles infection each year during this period, with many others suffering from disease complications [ ] . however, in , society experienced a turning point in the fight against measles with the development of the first live-attenuated measles vaccine (mv) [ ] . the live-attenuated mv is one example of a highly successful lav, and its introduction has transformed measles from a complicated disease into a triviality in most developed countries, although measles still forms a significant disease burden in developing nations [ ] [ ] [ ] . the wild-type virus was first isolated in by dr. john f. enders and his team from the blood of an -year-old boy named david edmonston, who became the namesake of the viral strain that would eventually become the first measles vaccine [ ] . serial passage of the wild-type edmonston strain in human and chicken embryo fibroblast tissue culture resulted in a virus with reduced virulence. however, the ability of the parental strain to induce protective immunity is retained. the mv is highly immunogenic, inducing both humoral and cellular immunity at magnitudes comparable to that of a natural infection, although antibody titers induced are often lower [ , ] . investigators have demonstrated that this protection is highly robust, and could last for as long as years after vaccine administration [ ] . its excellent safety profile, highly immunogenic nature, and low possibility of reversion to virulence has also placed it as a promising candidate for use as a viral vector to deliver heterologous antigens [ ] . some of the first evidence that describes the role of pre-existing immunity on live vaccines comes from the measles vaccine. the efficacy of live mv was found to be often hampered by pre-existing immunity at the time of vaccination, and this effect is best illustrated in infants who are born to measles-immune mothers. during gestation, infants acquire protective antibodies as a result of transplacental transfer of maternal igg antibodies [ ] . these antibodies, while protective against infection, can also suppress infant responses to immunization. indeed, studies have shown that vaccinating infants born to measles-immune mothers before or at the age of six months often results in seroconversion failure [ , ] . by contrast, immunization campaigns with mv between to months of age are relatively successful [ , ] . this is likely explained by waning maternal antibody levels over the period of to months, where antibody titers fall below the inhibitory threshold required for successful vaccination [ ] . moreover, the claims that pre-existing antibody titers can impact mv efficacy are further supported by animal studies. for instance, following the intravenous transfer of varying titers of mv neutralizing antibodies, immunization of mice with a recombinant measles vaccine (rmv) expressing simian immunodeficiency virus (siv) gag protein was significantly inhibited when pre-existing antibody titers were above miu/ml of serum [ ] . likewise, investigations in cynomolgus macaques by van binnendijk et al. revealed that pre-existing antibody titers as low as . iu/ml abrogated the development of antibodies following vaccination with mv or a recombinant vaccinia virus vector expressing measles antigens [ ] . interestingly, while antibody induction by mv is negatively impacted by pre-existing immunity, the effect on cellular responses seem unaffected. for example, infants vaccinated with mv at age or months followed by the measles-mumps-rubella (mmr) vaccine at months generated equivalent t-cell responses to control infants given only mmr at age months [ ] . thus, experimental models and clinical data support that the inhibition of mv vaccination in neonates is more likely due to interference from maternal antibodies rather than the immaturity of the neonatal system. adenoviruses (adv) and adeno-associated viruses have been widely studied as a potential viral vector for cancer gene therapy and infectious diseases. this double-stranded dna virus holds several advantages as a vector, including but not limited to the ability to induce robust cellular and humoral responses, high expression of transgenes, favorable safety profiles, and no risk of integration into the host genome. the most commonly used adenoviral vector is adv serotype (ad ) and has been tested in more than gene therapy trials. however, the large majority of the human population having pre-existing immunity to adv limits its widespread use in the clinics. indeed, a recent international cross-sectional serological survey demonstrated that greater than % of the study participants possessed neutralizing antibodies against ad [ ] . in a similar line of investigation, mast et al. found that . % of their study participants from the us, europe, thailand, africa, and brazil were seropositive for anti-ad neutralizing antibodies [ ] . these pre-existing antibodies have been shown to neutralize the ad vectors after administration, thereby lowering their efficacy and transgene expression. the most convincing evidence is from the large-scale clinical trial (step) that tested an ad -based hiv- vaccine, where reduced efficacy of ad was found to be associated with subjects who had pre-existing immunity to ad [ ] . in support of this theory, the clinical trial for the ad -based ebola vaccine showed that the low-dose vaccine of . × viral particles was weakened by pre-existing immunity, whereas immunogenicity was enhanced at a higher dose of . × - . × viral particles. in a mouse model, pre-existing anti-ad neutralizing antibodies were observed to severely hamper the induction of both cellular and humoral responses by a recombinant ad -ebolavirus glycoprotein vaccine [ ] . likewise, a recombinant ad vector expressing the human immunodeficiency virus- (hiv) gag gene showed diminished, but not complete, abrogation of gag-specific cellular immune responses following the vaccination of rhesus monkeys pre-exposed to an empty ad vector [ ] . nonetheless, there exists conflicting data that pre-existing anti-ad may not affect the induction of cytotoxic t-cell responses, indicating that more studies may be required to resolve these discrepancies [ ] . the high seroprevalence of anti-ad immunity has pivoted the development of genetically-modified adv and the search of rarer adv serotypes for use as vectors, in hopes that these viruses may circumvent the effects of pre-existing anti-ad immunity [ , ] . however, there remains the possibility of cross-reactivity between antibodies against the different adv serotypes, which could in turn potentially influence the efficacy of these rarer adv as vaccine vectors. indeed, investigations by heemskerk et al. have shown that ad -specific cd + t-cells could cross-react with other adv serotypes including but not limited to ad , ad , ad , and ad , suggesting that the different adv serotypes share similar t-cell epitopes [ ] . several studies have attempted to further explore this school of thought, and their results have provided greater insight into this particular topic. ad is one of the rarest human adv serotypes, with a seroprevalence of less than % in the population. in an attempt to determine its potential as an alternative vector to ad , barouch et al. compared the immunogenicity of a rad vaccine expressing simian immunodeficiency virus (siv)-gag versus a rad siv-gag vaccine in ad immune c /bl mice [ ] . indeed, the efficacy of the rad -gag vaccine was unaffected even by high levels of pre-existing ad immunity, indicating the absence of a cross-reactive response between ad and ad . likewise, the presence of anti-ad immunity did not affect the efficacy of a rad -gag vector vaccine in mice. interestingly, pre-existing anti-ad immunity could suppress cellular immune responses elicited by the rad -gag vaccine, suggesting some cross-reactivity between ad and ad immune responses [ ] . the inhibitory effect of pre-existing antibodies to adv remains a challenge for their use as viral vectors, and the development of alternative serotypes remains to be one of the best strategies to circumvent this limitation. the history of mankind is intricately intertwined with that of influenza, and the virus remains one of the top threats to global health. estimates indicate that there are approximately billion cases globally each year, of which to million are severe cases and up to , eventually succumb to the disease [ ] . combined with the economic impact from the loss of work productivity, a proper and robust framework is required to counter this threat. the global influenza strategies recommend vaccination as the most effective intervention to mitigate the impact of influenza [ ] . three types of influenza vaccines are licensed for use: ( ) recombinant, ( ) inactivated, and ( ) live-attenuated. the three vaccines are multivalent and provide protection against selected influenza type a and type b strains that are predicted to spread in the upcoming season [ ] . the former two are capable of inducing only igg responses [ , ] . by contrast, the live-attenuated influenza vaccine (laiv) can generate strain-specific igg antibodies as well as mucosal iga immunity and t-cell responses that are associated with protection from influenza illness [ , ] . indeed, a meta-analysis by ambrose et al. on the efficacy of laiv in children showed that recipients of the live vaccine demonstrated a % reduction in influenza cases compared to those who received the trivalent inactivated vaccine (tiv) [ ] . put together, these studies demonstrate the potential of laiv as a highly efficacious vaccine. yet, efforts to develop such a vaccine have been stymied by the presence of pre-existing immunity gained over a lifetime of exposure to different viral strains either from natural infection or vaccination. indeed, several observational studies point to the possibility that pre-existing immunity can reduce the efficacy of both inactivated and life-attenuated influenza vaccine [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, saito et al. found that children who received tiv for a previous season had reduced vaccine effectiveness for the current seasonal vaccine compared to unvaccinated children [ ] . likewise, sasaki et al. showed lower antibody induction by a laiv in individuals who had prior year tiv vaccination [ ] . furthermore, coelingh et al. demonstrated that younger children aged two to eight as well as baseline seronegative adults had higher fold-induction of serum hemagglutinin inhibition (hai) antibody titers post-laiv vaccination [ ] . however, given how complicated it is to trace the history of an individual's exposure to influenza strains, it is difficult to tease out the exact impact that pre-existing immunity has on vaccine efficacy in human samples. perhaps then, by establishing a good animal model with controlled infection histories, we will be able to better understand these complexities. flaviviruses include a number of clinically important pathogens that are either transmitted by mosquitoes (dengue, zika, yellow fever, west nile, and japanese encephalitis virus) or by ticks (tick-borne encephalitis, powassan virus) [ ] . the recent zika pandemic witnessed more than cases of congenital birth defects linked to zika virus infection in the americas and was declared by the world health organization in february to be a public health emergency [ ] . dengue infections, on the other hand, account for million infections annually, of which million infections are symptomatic [ ] . the antigenic closeness between flaviviruses is such that infection with one flavivirus induces species-specific immunity, as well as cross-reactive antibodies against related serocomplexes [ , ] . however, these cross-reactive antibodies do not necessarily cross-protect. initial insights come from human dengue challenge studies by albert sabin, who provided evidence that a homologous challenge in humans with the same dengue virus (denv) serotype protects against re-infection, but only short-term protection against the heterologous denv serotype challenge for up to six months [ , ] . moreover, when cross-reactive antibodies decline to sub-neutralizing levels, these antibodies can opsonize dengue virus infection, resulting in enhanced virus burden and risk of severe dengue in patients experiencing secondary infection [ ] . indeed, recent cohort studies conducted in nicaragua and thailand provide clinical evidence that a specific range of antibody titers is associated with an increased risk of severe dengue [ , ] . the presence of waning maternal anti-dengue antibodies can also predispose children to dengue hemorrhagic fever, reinforcing the concept that pre-existing antibodies can promote disease pathogenesis [ ] . consistent with the notion that sub-neutralizing cross-reactive antibodies can promote viral infection, the presence of pre-existing cross-reactive antibodies can also increase the immunogenicity of flaviviral lavs. as demonstrated in both dengvaxia ® and takaeda ® dengue vaccine trials, seropositive individuals produced greater neutralizing antibody responses and protection against the wild-type denv infection compared to seronegative vaccinees [ , , ] . these studies were also supported by in vivo studies showing that sequential immunization for flaviviruses with shared cd epitopes that could enhance protection during subsequent heterologous infection [ ] . however, in another study, pre-existing antibodies from yellow fever vaccination can cause impairment of neutralizing antibody responses to tick-borne encephalitis vaccination [ ] . the clinical trial finding that subjects with a limited range of cross-reactive antibodies from a prior japanese encephalitis vaccine were able to enhance yellow fever vaccination, by prolonging vaccine viremia duration that leads to higher antibody titers, thus hints at the possibility that whether pre-existing antibodies inhibit or augment flavivirus infection will depend on both antibody titers and the type/specificity of antibodies produced [ ] . the plausible mechanisms involved are as elaborated below. the primary role of antibodies is antigen binding and interacting with fc-gamma receptors (fcγrs) to modulate subsequent immune responses. the integration of both activating and inhibiting signals is critical for the generation of an effective immune response. in this aspect, fcγrs are an archetype of how such signals influence both innate and adaptive immune functions. functionally, fcγrs can be classified into either activating or inhibiting receptors depending on the pathway they initiate [ ] . activating fcγrs possess an immunoreceptor tyrosine-based activation (itam) motif in their cytosolic domain, or in the case of fcγri and fcγriiia, associate with an itam-containing γ-chain. engagement of activating fcγrs by immune complexes results in the phosphorylation of the γ-chain by src-family kinases in order to create a docking site for spleen tyrosine kinase (syk). subsequent activation of syk results in a signaling cascade, leading to the induction of pro-inflammatory responses and activation of innate immune effector cells [ , ] . by contrast, the inhibitory fcγriib receptor contains an immunoreceptor tyrosine-based inhibition (itim) motif within its intracellular domain. cross-linking of fcγriib enables the recruitment of sh domain-containing inositol polyphosphate phosphatase (ship) and sh domain-containing protein tyrosine phosphatase (shp ) to modulate signals generated by activating fcγrs, thereby regulating the magnitude of inflammatory responses. furthermore, fcγriib is important for controlling b-cell development [ ] [ ] [ ] [ ] [ ] [ ] . indeed, b-cells express fcγriib as the only fcγr on their cell surface, and cross-linking of fcγriib on naïve b-cells could inhibit their proliferation and differentiation into plasma cells [ ] . likewise, cross-linking of fcγriib induces apoptosis in bone marrow plasma cells, suggesting that fcγriib may influence the lifespan of these antibody-producing cells [ ] . the signaling responses triggered by virus-antibody complexes are thus highly dependent on the type of fcγrs with which the immune complexes interact, which can result in either virus inhibition or enhancement of virus infection. virus neutralization occurs when virions are bound by antibodies with stoichiometry exceeding a required threshold. hence, one of the popular explanations to explain the lack of lav efficacy in the presence of pre-existing antibodies is the neutralization of the lav, which could consequently decrease the amount of viral antigens to levels that are below the threshold for immune detection and recognition. antibody concentrations, affinity, and epitope accessibility are critical determinants for virus neutralization [ ] [ ] [ ] . antibody affinity, defined as the fraction of epitopes that are bound by antibodies at non-saturating concentrations, has been shown to correlate with neutralizing activity in vitro. on the other hand, epitope accessibility is defined as the number of epitopes on viruses that are available for binding and can be affected by virus structure, structural dynamics of virus, and virus maturation states [ ] . the epitope availability would thus affect the fraction of epitope occupancy that will be required for virus neutralization. taken together, cross-reactive antibodies that can neutralize virus infection are likely those that can bind to accessible epitopes with considerable affinity. conversely, antibodies that bind weakly and target epitopes with reduced accessibility are unlikely to neutralize viruses, and may instead enhance viral infection via fcγr-mediated uptake. antibodies can neutralize lav strains in a variety of ways, as summarized in figure . they may block virus attachment and entry by either binding to epitopes that are directly involved in virus-receptor interactions or by imposing steric hindrance that prevent virus interaction with host receptors. as most virus structures are dynamic and can change structural conformations at different temperatures, it is thought that antibody binding to these dynamic structures may cause structural changes that can impair virus attachment, thereby causing virus neutralization [ ] . however, the blockade of virus-receptor interactions alone may not be able to completely neutralize the viruses, especially in fcγr-bearing cells, as activating fcγrs can enable entry of virus-antibody complexes by fcγr-mediated uptake. thus, pre-existing antibodies that can block viral fusion and uncoating will likely be more efficient in virus neutralization. in situations where pre-existing cross-reactive antibodies are unable to inhibit viral fusion processes intracellularly, high concentrations of antibodies may enable the formation of viral immune aggregates that influence the types of fcγrs engaged. these large viral aggregates can then inhibit phagocytosis by co-ligating the lowly expressed inhibitory receptor fcγriib that inhibit phagocytosis [ , ] . finally, there have been theories suggesting that fcγr cross-linking by virus immune complexes may increase the production of il- that abolishes innate immune responses [ ] . however, more experimental evidence will be required to support this theory. viruses , , x for peer review of virus maturation states [ ] . the epitope availability would thus affect the fraction of epitope occupancy that will be required for virus neutralization. taken together, cross-reactive antibodies that can neutralize virus infection are likely those that can bind to accessible epitopes with considerable affinity. conversely, antibodies that bind weakly and target epitopes with reduced accessibility are unlikely to neutralize viruses, and may instead enhance viral infection via fcγrmediated uptake. antibodies can neutralize lav strains in a variety of ways, as summarized in figure . they may block virus attachment and entry by either binding to epitopes that are directly involved in virus-receptor interactions or by imposing steric hindrance that prevent virus interaction with host receptors. as most virus structures are dynamic and can change structural conformations at different temperatures, it is thought that antibody binding to these dynamic structures may cause structural changes that can impair virus attachment, thereby causing virus neutralization [ ] . however, the blockade of virus-receptor interactions alone may not be able to completely neutralize the viruses, especially in fcγr-bearing cells, as activating fcγrs can enable entry of virus-antibody complexes by fcγr-mediated uptake. thus, pre-existing antibodies that can block viral fusion and uncoating will likely be more efficient in virus neutralization. in situations where pre-existing cross-reactive antibodies are unable to inhibit viral fusion processes intracellularly, high concentrations of antibodies may enable the formation of viral immune aggregates that influence the types of fcγrs engaged. these large viral aggregates can then inhibit phagocytosis by co-ligating the lowly expressed inhibitory receptor fcγriib that inhibit phagocytosis [ , ] . finally, there have been theories suggesting that fcγr cross-linking by virus immune complexes may increase the production of il- that abolishes innate immune responses [ ] . however, more experimental evidence will be required to support this theory. unlike myeloid cells, b-cells exclusively express fcγriib but not the activating fcγrs [ ] . therefore, cross-linking of the b-cell receptor (bcr) with fcγriib mediated by virus immune complexes can lead to the inhibition of b-cell activation (figure a) . indeed, by adding sheep red blood cell-specific (sbrc) igg to srbcs, b-cell antibody secretion is reduced [ ] . similarly, using the cotton rat model of mv vaccination, maternal antibodies were demonstrated to inhibit b-cells by the cross-linking of bcr and fcγriib [ ] . however, this mechanism of inhibition has not been demonstrated for other viruses. it is conceivable that this mode of inhibition is dependent on the size of the virus-antibody immune complexes, as immunization of small polypeptides can escape maternal antibody inhibition [ ] . more mechanistic studies will hence be required to evaluate the conditions that need to be satisfied to cause b-cell inhibition. viruses , , x for peer review of unlike myeloid cells, b-cells exclusively express fcγriib but not the activating fcγrs [ ] . therefore, cross-linking of the b-cell receptor (bcr) with fcγriib mediated by virus immune complexes can lead to the inhibition of b-cell activation (figure a) . indeed, by adding sheep red blood cell-specific (sbrc) igg to srbcs, b-cell antibody secretion is reduced [ ] . similarly, using the cotton rat model of mv vaccination, maternal antibodies were demonstrated to inhibit b-cells by the cross-linking of bcr and fcγriib [ ] . however, this mechanism of inhibition has not been demonstrated for other viruses. it is conceivable that this mode of inhibition is dependent on the size of the virus-antibody immune complexes, as immunization of small polypeptides can escape maternal antibody inhibition [ ] . more mechanistic studies will hence be required to evaluate the conditions that need to be satisfied to cause b-cell inhibition. another way in which antibodies can inhibit b-cell responses is through epitope masking. this hypothesis postulates that the presence of pre-existing antibodies can mask the exposure of epitopes, thereby prohibiting recognition by the b-cell. this is also termed as epitope-specific suppression, whereby epitopes covered by these antibodies are unable to be recognized by b-cells (figure b) . interestingly, there have been reports of epitope unspecific suppression, where monoclonal antibodies that target only one specific epitope can suppress b-cell recognition of a whole antigen, suggesting the possibility that steric hindrance or obstruction by high concentrations of antibodies can also lead to overall suppression b-cell recognition [ , ] . antibody-dependent enhancement (ade) of viral infection has been documented for many viruses, including flaviviruses, influenza, mv, ross river viruses, hiv, and coronaviruses [ ] . ade can occur when sub-neutralizing levels of cross-reactive antibodies form immune complexes with viruses, opsonizing viral infection in fcγr-bearing myeloid cells including monocytes, macrophages, and dendritic cells via activating fcγr-mediated uptake. the majority of the mechanistic insights about ade of viral infection are gathered from dengue, as waning cross-reactive antibodies that are acquired from a heterotypic denv infection or through maternal-fetal transmission can result in a heightened risk of severe dengue that can be life-threatening [ ] [ ] [ ] . both activating fcγri and fcγriia have been shown to be involved in ade-mediated infection, although increasing studies indicate that fcγriia could be more important than fcγri in enhancing viral infection [ , ] . while the precise mechanisms involved remain unclear, it is possible that the trafficking of immune complexes through fcγriia-mediated uptake is slower, thereby allowing more viral fusion and infection [ , ] . the activation of fcγri, however, may aid to further enhance immunogenic responses to viral antigens by targeting virus immune complexes to the late endosomes or lysosomes for enhanced antigen processing and antigen presentation to the cd + t-cells, thereby increasing b-cell responses [ ] . overall, at sub-neutralizing antibody levels, the presence of pre-existing antibodies can activate both fcγri and fcγriia, which promotes virus uptake, replication, and antigen presentation that consequently augments vaccine immunogenicity. besides promoting viral entry and antigen presentation, the cross-linking of fcγrs may also modulate cellular and host responses that promote viral replication and lav immunogenicity ( figure ). some insights can be obtained from our clinical trial, where subjects were sequentially vaccinated with the inactivated japanese encephalitis virus vaccine followed by the yellow fever vaccine. subjects within a restricted range of cross-reactive antibodies resulted in increased antibody responses, whereas too many or too few antibodies resulted in reduced antibody responses, indicating the possible role of ade in augmented vaccine antibody responses [ ] . in addition to the extended duration of viremia observed in these subjects, enhancing titers of cross-reactive antibodies provoked greater pro-inflammatory responses, including increased innate immune responses and the production of pro-inflammatory metabolites such as arachidonic acid, linoleic acid, and -hete that promote phagocytosis and adaptive immune responses. the co-ligation of both fcγri and fcγriia by virus-antibody immune complexes or cross-linking can also upregulate immune semaphorins such as sema a, sema a, and sema a which are critical for antigen-presenting cell and t-cell interactions [ ] . while the mechanisms of whether upregulation of immune semaphorins by immune complexes leads to increased t-cell proliferation and activation in humans remains to be evaluated, previous studies have shown that sema a can enhance t-cell activation through interaction with tim- , thereby increasing antigen-specific t-cell and antibody responses against t-cell dependent antigens [ , ] . however, it is also noticeable that not all subjects within that specific window of cross-reactive antibody levels exhibited increased vaccine immunogenicity, suggesting that baseline variations, such as genetics, dietary or environmental factors may also influence the outcome of lav immunogenicity [ ] . some recent studies hinted at the possibility that baseline variations in b-cell signatures and gene regulation could influence lav reactogenicity and immunogenicity, which can be potential avenues for future studies [ , ] . viruses , , x for peer review of cell dependent antigens [ , ] . however, it is also noticeable that not all subjects within that specific window of cross-reactive antibody levels exhibited increased vaccine immunogenicity, suggesting that baseline variations, such as genetics, dietary or environmental factors may also influence the outcome of lav immunogenicity [ ] . some recent studies hinted at the possibility that baseline variations in b-cell signatures and gene regulation could influence lav reactogenicity and immunogenicity, which can be potential avenues for future studies [ , ] . immune complexes formed by pre-existing antibodies and viruses can engage fcγrs, resulting in increased viral uptake and fusion through the process of antibody-dependent enhancement that leads to increased vaccine viremia and antigen presentation. activating fcγr-signaling, on the other hand, provokes greater innate immune responses and production of pro-inflammatory metabolites that can enhance innate and adaptive immune responses. in addition, the cross-linking of fcγrs causes increased expression of immune semaphorins, which are critical for antigen-presenting cell and t-cell interaction. overall, this leads to increased t-cell proliferation and activation, which consequently improves lav immunogenicity. in this review, we have highlighted several studies that have shown that the efficacy of some commonly used lavs, such as measles, adenovirus, influenza, and flaviviral vaccines, can be affected by these pre-existing adaptive immune responses. this knowledge will be critical to understanding the limitations of administering lavs in seropositive individuals to reduce the incidence of vaccine failures, as well as in future designs of clinical trials to evaluate the efficacy of lavs. whether preexisting antibodies can inhibit or augment lav immunogenicity depends on the concentration and the type of antibodies that are present (figure ). with high antibody levels or potently neutralizing antibodies, pre-existing antibodies can inhibit lav efficacy by virus neutralization or inhibition of bcell responses. by contrast, at sub-neutralizing titers, pre-existing antibodies can enable viruses to better infect cells and increase innate immune responses that augment lav immunogenicity. moreover, pre-existing immunity can prime dendritic cells and memory t-cells to enhance protection during secondary infection with an antigenically related virus [ ] . we believe that a deeper understanding of the underlying mechanisms involved will help us better understand the circumstances that can allow us to manage immunization in the presence of pre-existing antibodies, and even explore the possibilities of exploiting pre-existing antibodies to promote vaccine immunogenicity and efficacy. it would also be interesting to determine if cross-reactive antibodies immune complexes formed by pre-existing antibodies and viruses can engage fcγrs, resulting in increased viral uptake and fusion through the process of antibody-dependent enhancement that leads to increased vaccine viremia and antigen presentation. activating fcγr-signaling, on the other hand, provokes greater innate immune responses and production of pro-inflammatory metabolites that can enhance innate and adaptive immune responses. in addition, the cross-linking of fcγrs causes increased expression of immune semaphorins, which are critical for antigen-presenting cell and t-cell interaction. overall, this leads to increased t-cell proliferation and activation, which consequently improves lav immunogenicity. in this review, we have highlighted several studies that have shown that the efficacy of some commonly used lavs, such as measles, adenovirus, influenza, and flaviviral vaccines, can be affected by these pre-existing adaptive immune responses. this knowledge will be critical to understanding the limitations of administering lavs in seropositive individuals to reduce the incidence of vaccine failures, as well as in future designs of clinical trials to evaluate the efficacy of lavs. whether pre-existing antibodies can inhibit or augment lav immunogenicity depends on the concentration and the type of antibodies that are present (figure ). with high antibody levels or potently neutralizing antibodies, pre-existing antibodies can inhibit lav efficacy by virus neutralization or inhibition of b-cell responses. by contrast, at sub-neutralizing titers, pre-existing antibodies can enable viruses to better infect cells and increase innate immune responses that augment lav immunogenicity. moreover, pre-existing immunity can prime dendritic cells and memory t-cells to enhance protection during secondary infection with an antigenically related virus [ ] . we believe that a deeper understanding of the underlying mechanisms involved will help us better understand the circumstances that can allow us to manage immunization in the presence of pre-existing antibodies, and even explore the possibilities of exploiting pre-existing antibodies to promote vaccine immunogenicity and efficacy. it would also be interesting to determine if cross-reactive antibodies can impact future development of lavs against newly emerging pandemic viruses, including ebola, severe acute respiratory syndrome coronavirus- (sars cov- ), and zika viruses. can impact future development of lavs against newly emerging pandemic viruses, including ebola, severe acute respiratory syndrome coronavirus- (sars cov- ), and zika viruses. in the presence of high levels of pre-existing antibodies or 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conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord- -ltx w zh authors: zhu, liqian; jiang, xinyi; fu, xiaotian; qi, yanhua; zhu, guoqiang title: the involvement of histone h acetylation in bovine herpesvirus replication in mdbk cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ltx w zh during bovine herpesvirus (bohv- ) productive infection in cell cultures, partial of intranuclear viral dna is present in nucleosomes, and viral protein vp associates with histones and decreases histone h acetylation, indicating the involvement of histone h acetylation in virus replication. in this study, we demonstrated that bohv- infection at the late stage (at h after infection) dramatically decreased histone h acetylation [at residues k (h k ac) and k (h k ac)], which was supported by the pronounced depletion of histone acetyltransferases (hats) including cbp/p (creb binding protein and p ), gcn l (general control of amino acid synthesis yeast homolog like ) and pcaf (p /cbp-associated factor). the depletion of gcn l promoted by virus infection was partially mediated by ubiquitin-proteasome pathway. interestingly, the viral replication was enhanced by hat (histone acetyltransferase) activator ctpb [n-( -chloro- -trifluoromethylphenyl)- -ethoxy- -pentadecylbenzamide], and vice versa, inhibited by hat inhibitor anacardic acid (aa), suggesting that bohv- may take advantage of histone acetylation for efficient replication. taken together, we proposed that the hat-dependent histone h acetylation plays an important role in bohv- replication in mdbk (madin-darby bovine kidney) cells. bovine herpesvirus (bohv- ) is an important pathogen that causes pneumonia, conjunctivitis, genital disorders, and abortions in cattle [ ] . bohv- infection induces severe inflammatory response though diverse mechanisms, such as by overexpression of pro-inflammatory cytokines and reactive oxidative species [ ] [ ] [ ] . the suppression of host immune response by virus infection may render secondary infection by diverse pathogens, such as bovine viral diarrhea viruses (bvdv), bovine respiratory syncytial virus (brsv), parainfluenza- virus (pi v), bovine coronaviruses, mannheimia haemolytica, pasteurella multocida, histophilus somni and mycoplasma spp. [ , ] , and consequently lead to a life-threatening pneumonia known as bovine respiratory disease complex (brdc), one of the costliest ailments in cattle feeding [ , ] . in eukaryotes, dna is packaged into a protein-dna complex called chromatin, with nucleosome as monomeric subunit containing a core of histone proteins (h a, h b, h , and h ) surrounding by~ bp of genomic dna [ ] . the chromatin is dynamically organized into regions of either loosely packaged actively transcribed chromatin (euchromatin) or highly condensed cbp/p rabbit mab (monoclonal antibody) (cat# , : ), pcaf rabbit mab (cat# , : ), gcn l rabbit mab (cat# , : ), histone h rabbit mab (cat# , : ), acetyl-histone h (lys ) rabbit mab (cat# , : ), acetyl-histone h (lys ) rabbit mab (cat# , : ), ubiquitin mouse mab(cat# , : ), hdac (histone deacetylas) mouse mab (cat# , : ), hdac mouse mab (cat# , : ), hdac mouse mab (cat # , : ), hdac rabbit mab (cat # , : ), β-actin rabbit mab(cat# , : ), hrp (horseradish peroxidase) labeled anti-mouse igg (cat# , : ) and hrp labeled anti-rabbit igg (cat# , : ), were purchased from cell signaling technology (beverly, ma, usa). bohv- vp antibody ( : ) is kindly provided by prof. vikram misra at the university of saskatchewan [ ] . anacardic acid (aa) (cat#a ), trichostatin a (tsa) (# ). mg (cat# - ), ammonium chloride (nh cl) (cat# ), were ordered from sigma-aldrich (st. louis, mo, usa). bortezomib (#s ) was obtained from selleckchem.com (houston, tx, usa). n-( -chloro- -trifluoromethyl-phenyl)- -ethoxy- -pentadecyl-benzamide (ctpb) (cat# - - ) was provided by santa cruz biotechnology (dallas, tx, usa). cytotoxicity of indicated chemicals in mdbk cells was assessed by trypan-blue exclusion test, as described by fiorito et al. [ , ] , with modification. in brief, mdbk cells in -well plates were treated with or without chemicals at indicated concentrations for h. then the cells were collected by trypsinization, and an aliquot of the cell suspension was mixed with an equal volume of . % trypan-blue ( . %) (bio-rad, hercules, ca, usa, # ). after incubation for min, cells were counted using a burker chamber under a light microscope. the percentage of cell viability in the chemical treatment groups was calculated by normalization of the number of live cells to that in the control samples. the value of cell viability in the control was arbitrarily set to %. confluent mdbk cells in mm dishes were infected with bohv- (moi = ) for , and h. cell lysates were prepared using lysis buffer ( % triton x- , mm sodium chloride, mm edta, mm egta, mm sodium fluoride, mm sodium pyrophosphate, mm phenylmethylsulfonyl fluoride, . g/ml leupeptin, mm benzamidine, and mm sodium orthovanadate in mm tris-hcl, ph . ). to test the effects of certain inhibitors on the designated signaling, mdbk cells were infected for h along with treatment with indicated chemicals at the designated concentrations. cell lysates were prepared using lysis buffer as described above. cell lysates were separated on or % sds-polyacrylamide gels, and proteins were transferred to a polyvinylidene difluoride (pvdf) membrane (bio-rad, hercules, ca, usa). targeted proteins were detected using respective antibodies. the intensity of immune reactive bands was analyzed with free software image j (https://imagej.nih.gov/ij/download.html). to calculate the relative protein expression levels, the band intensity of target proteins was firstly normalized to β-actin, then normalized to the control lane. for ip studies, mdbk cells in -mm dishes were infected with bohv- at an moi of . at h after infection, cells were lysed with ml of ripa buffer ( × pbs, % np- , . % sodium deoxycholate, . % sds) supplemented with protease inhibitor as described above in western blots analysis. cell lysates were clarified by centrifugation at , rpm for min, and incubated with dynabeads ® (life technologies, carlsbad, ca, usa, cat. no. d), which have been incubated with µl of acetyl-histone h (lys ) rabbit mab (cell signaling technology, cat# ) or gcn l rabbit mab (cell signaling technology, cat# ) for h at room temperature with rotation. after overnight incubation at • c with rotation, the beads were collected with the help of a magnet (dynamag™) (life technologies, cat. no. d). after three washing with pbs, beads were boiled in sds loading buffer and western blots were performed to detect the designated proteins. mdbk cell in -well plates were infected with bohv- (moi of ) along with the treatment of indicated chemicals(paa, anacardic acid, tsa, and ctpb) at the designated concentration for h at • c, after three washing with pbs, fresh medium with designated chemicals was added to each well. at h after infection, viral yields were titrated in mdbk cells. the cell cultures treated with dmso was used as a control. the results are expressed as tcid /ml calculated using the reed-muench formula. confluent mdbk cells in mm dishes were infected with bohv- using an moi of . at , and h post infection(hpi) total rna was purified with trizol ls reagent (ambion, thermo fisher scientific, waltham, ma, usa, cat# ) following the manufacturers' instructions. freshly prepared rna ( µg) was used as a template for the synthesis of the first-strand cdna with commercial random hexamer primers for viral mrna detection using thermoscript™ rt-pcr system kit (invitrogen, carlsbad, ca, usa, cat# - ). the cdna products were used as templates for relative qrt-pcr to measure levels of viral mrna of bicp , and bicp as well as cellular gene glyceraldehyde- -phosphate dehydrogenase (gapdh) with specific primers as previously described in the reference [ ] . analysis of gapdh mrna was used as an internal control. relative qrt-pcr was carried out using the abi fast real-time system (applied biosystems, foster city, ca, usa). separate gapdh amplification was used to normalize gene expression. the data were analyzed using the equation −∆∆ct method. acetylation of histone h is involved in transcription activation, and h k ac is an epigenetic marker for histone acetylation. to test whether bohv- infection alters histone h acetylation, we evaluated the expression levels of h k ac, h k ac as well as h in virus infected mdbk cells at , , and hpi. as a result, virus infection consistently decreased the expression levels of h k ac, h k ac and h , and peaked at hpi ( figure a ). quantitative analysis indicated that the levels of h k ac, h k ac and h were decreased to . %, . % and . % relative to the control, respectively( figure b ), suggesting that bohv- infection reduced histone h acetylation (h k ac and h k ac). histone acetylation and deacetylation are reversible processes regulated enzymatically by hats and histone deacetylases (hdacs). hats such as cbp/p , gcn l , and pcaf, are enzymes that ultraviolet (uv) light-inactivated viruses are replication deficient because it could bind to the receptors and enter the cells, but are unable to express viral genes [ , ] . to further understand whether complete viral replication cycle was required to affect the histone h acetylation, uv-inactivated viral particles were employed for further investigation. as shown in figure c ,d, uv-inactivated virus had no effects on histone h acetylation. thus, these results suggested that de novo viral protein production and/or dna replication seems to be associated with the decreased acetylation of histone h . histone acetylation and deacetylation are reversible processes regulated enzymatically by hats and histone deacetylases (hdacs). hats such as cbp/p , gcn l , and pcaf, are enzymes that acetylate conserved lysine residues on histones. to understand the mechanisms underlying the decreased histone h acetylation by virus infection, we initially detected the protein levels of cbp/p , pcaf, and gcn l following bohv- infection at , , and hpi. virus infection altered the expression of cbp/p , pcaf, and gcn l , only at hpi, all of them were robustly decreased in comparison to the mock infected control (figure a ). the protein levels of cbp/p , pcaf, and gcn l were reduced to approximately . %, . %, and . % relative to the control, respectively ( figure b ). the global decease of hats expression at h after infection may reflect their reduced capability for histone acetylation, which is in agreement with the decreased histone h acetylation. hdacs are a family of enzymes controlling deacetylation of histones. the family of mammalian hdacs is comprised of at least members which are classified into four classes: class i (hdac , , and ), class ii (hdac , , , , and ), class iii (sirt to ) and class iv (hdac ) [ ] . in this study, the expression of hdac - in response to virus infection was detected using western blots. as can be seen in figure c ,d, virus infection altered the expression of both hdac and hdac with distinct manners, while neither hdac nor hdac were apparently affected. at hpi, the expression levels of hdac and hdac were decreased to approximately . % and . % relative to the control, respectively. the unexpected decreased expression levels of both hdac and hdac may undermine the finding that virus infection decreased the acetylation of histone h . taken together, virus infection altered the expression of hats and hdacs with distinct manners. relative to hdacs, the prominently decreased expression of hats strongly supported the reduced acetylation of histone h at hpi. hdacs are a family of enzymes controlling deacetylation of histones. the family of mammalian hdacs is comprised of at least members which are classified into four classes: class i (hdac , , and ), class ii (hdac , , , , and ), class iii (sirt to ) and class iv (hdac ) [ ] . in this study, the expression of hdac - in response to virus infection was detected using western blots. as can be seen in figure c ,d, virus infection altered the expression of both hdac and hdac with distinct manners, while neither hdac nor hdac were apparently affected. at hpi, the expression levels of hdac and hdac were decreased to approximately . % and . % relative to the control, respectively. the unexpected decreased expression levels of both hdac and hdac may undermine the finding that virus infection decreased the acetylation of histone h . taken together, virus infection altered the expression of hats and hdacs with distinct manners. relative to hdacs, the prominently decreased expression of hats strongly supported the reduced acetylation of histone h at hpi. our foregoing results demonstrated that virus infection differentially altered hats and hdacs expression, particularly the depletion of hats correlated with the reduced histone h acetylation. therefore, the role of hats and hdacs in bohv- productive infection was independently investigated using hat inhibitor anacardic acid (aa) and hdac inhibitor trichostatin a (tsa), respectively. aa specifically inhibits the enzymatic activity of hats, such as cbp/p and pcaf, and thereby affects hat-dependent gene transcription [ ] . also, aa has been reported to have multiple other biological effects such as antitumor activity and antioxidant activity [ ] . in this study, we found that the treatment of virus-infected cells with hat inhibitor aa at a concentration of µm and µm resulted in a . -and . -log reduction of the virus titer comparing to that in the mock-treated control, respectively ( figure a) . indeed, µm of aa treatment could inhibit histone h acetylation as demonstrated by the reduced levels of h k ac relative to the control, but aa increased the levels of h k ac in the context of virus infection in comparison to the mock treated but infected cells ( figure e ,f). maybe the significantly decreased progeny virus by aa treatment led to rescuing the depletion of h k ac attributed to virus infection. tsa is an hdac-specific inhibitor that can selectively inhibit the enzymatic activities of class i and ii hdacs, but not class iii hdacs [ ] . interestingly, it was reported that influenza virus infection decreased hdac expression in a cells, and the treatment of infected cells with and µm of tsa resulted in . -fold and . -fold increase of progeny virus relative to the control, respectively [ ] . in this study, we used much fewer concentrations of tsa to investigate its role in bohv- replication. we found that the treatment with nm of tsa could restore histone h acetylation ( figure g ,h), but had no impact on the viral replication because the virus titer was only increased~ . log (equal to -fold), with a difference not statistically significant (p > . ) ( figure b ). of note, all the concentrations used for indicated inhibitors had no cytotoxicity to mdbk cells ( figure e ). but we noticed that in the context of virus infection the cell viability was reduced to approximately . % by tsa ( nm) treatment ( figure i ), which is a possible reason for why tsa treatment could not evidently booster viral replication. these results suggested that the maintenance of hat activities was essential for virus productive infection. ctpb is a potent activator of cbp/p , but not of pcaf activities. to further confirm the role of hat played in the virus infection, ctpb, was employed for further investigation. ctpb at a concentration of µm showing no cytotoxicity to mdbk cells can significantly promote virus productive infection ( figure c ,e). the virus titer was increased~ . log by the treatment with µm of ctpb in comparison with the mock-treated control ( figure c ). this observation further suggested that the histone acetylation by hat played an essential role in bohv- productive infection. considering that histone acetylation regulated by hat is an important factor controlling gene expression, we further investigated the effects of chemical inhibition of hats on viral gene expression. for this purpose, the virus-infected cells were treated with µm of aa or dmso as a control, and the mrna levels of immediate early (ie) genes including bicp and bicp were detected with relative qrt-pcr. in aa treated virus-infected cell cultures, the mrna levels of bicp considering that histone acetylation regulated by hat is an important factor controlling gene expression, we further investigated the effects of chemical inhibition of hats on viral gene expression. for this purpose, the virus-infected cells were treated with µm of aa or dmso as a control, and the mrna levels of immediate early (ie) genes including bicp and bicp were detected with relative qrt-pcr. in aa treated virus-infected cell cultures, the mrna levels of bicp decreased to . % and . % relative to the mock-treated control, at and hpi, respectively, but at hpi the chemical treatment showed minor effects ( figure a ). bicp mrna levels were reduced to . %, . % and . % by aa treatment relative to the control samples, at , and hpi, respectively ( figure b ). though these ie proteins were not detected due to the unavailability of given antibodies, these results of qrt-pcr indicated that the hat inhibitor aa affected the transcription of these ie genes. an additional study was performed to examine the effects of aa on the expression of viral tegument protein vp by western blots using an antibody against vp . as demonstrated in figure c , at hpi vp protein expression levels decreased approximately . -fold by the treatment with aa relative to that in the mock-treated control, indicating that aa affected vp expression. in summary, these findings further suggested that this hat inhibitor affected bohv- productive infection in mdbk cells, and therefore the maintaining of hat activity is essential for virus efficient replication. viruses , , x for peer review of decreased to . % and . % relative to the mock-treated control, at and hpi, respectively, but at hpi the chemical treatment showed minor effects ( figure a ). bicp mrna levels were reduced to . %, . % and . % by aa treatment relative to the control samples, at , and hpi, respectively ( figure b ). though these ie proteins were not detected due to the unavailability of given antibodies, these results of qrt-pcr indicated that the hat inhibitor aa affected the transcription of these ie genes. an additional study was performed to examine the effects of aa on the expression of viral tegument protein vp by western blots using an antibody against vp . as demonstrated in figure c , at hpi vp protein expression levels decreased approximately . -fold by the treatment with aa relative to that in the mock-treated control, indicating that aa affected vp expression. in summary, these findings further suggested that this hat inhibitor affected bohv- productive infection in mdbk cells, and therefore the maintaining of hat activity is essential for virus efficient replication. the virus infected cells were treated with dmso or aa ( µm). total rna was prepared at indicated time points, and qrt-pcr was performed to determine the mrna levels of bicp (a) and bicp (b). (c) mdbk cells in mm dishes were infected by bohv- at an moi of with the treatment of dmso or aa ( µm), at hpi, cell lysate was prepared and subjected to western blots to detect vp protein. the band intensity was analyzed with software image j. and analysis was compared with that of untreated but infected control. data represent three independent experiments. significance was assessed with the student t test (* p < . , ** p < . ). ns: not significant. generally, there are two potential pathways to control protein degradation in eukaryotic cells, one mediated by ubiquitin-proteasome and other mediated by lysosome [ ] , which can be efficiently inhibited by chemical inhibitors, such as mg and nh cl, respectively. to identify whether the ubiquitin-proteasome and/or lysosome pathways are involved in the reduced acetylation of histone h at h after infection, the virus-infected mdbk cells were treated with either proteasome inhibitor mg or lysosome inhibitor nh cl throughout infection as determined elsewhere [ , ] . the treatment of mg ( µm) reversed the depletion of both h k ac and h k ac attributed to virus infection, and rescued their expression to a level higher than that in the uninfected control, whereas nh cl treatment did not show any effect ( figure a,b) . the increased levels of ubiquitinated proteins . total rna was prepared at indicated time points, and qrt-pcr was performed to determine the mrna levels of bicp (a) and bicp (b). (c) mdbk cells in mm dishes were infected by bohv- at an moi of with the treatment of dmso or aa ( µm), at hpi, cell lysate was prepared and subjected to western blots to detect vp protein. the band intensity was analyzed with software image j. and analysis was compared with that of untreated but infected control. data represent three independent experiments. significance was assessed with the student t test (* p < . , ** p < . ). ns: not significant. generally, there are two potential pathways to control protein degradation in eukaryotic cells, one mediated by ubiquitin-proteasome and other mediated by lysosome [ ] , which can be efficiently inhibited by chemical inhibitors, such as mg and nh cl, respectively. to identify whether the ubiquitin-proteasome and/or lysosome pathways are involved in the reduced acetylation of histone h at h after infection, the virus-infected mdbk cells were treated with either proteasome inhibitor mg or lysosome inhibitor nh cl throughout infection as determined elsewhere [ , ] . the treatment of mg ( µm) reversed the depletion of both h k ac and h k ac attributed to virus infection, and rescued their expression to a level higher than that in the uninfected control, whereas nh cl treatment did not show any effect ( figure a,b) . the increased levels of ubiquitinated proteins in mg -treated cells but not in nh cl-treated cells confirmed the efficiency of mg as an inhibitor for the proteasome pathway in mdbk cells ( figure c ). though the concentration of mg used in this study did not show obvious cytotoxicity to mdbk cells ( figure f ), the chemical may have off-target effects. so, another proteasome inhibitor bortezomib was employed to validate the rescued effects of mg . as expected, bortezomib at a concentration of nm showing no cytotoxicity to mdbk cells could reverse bohv- -reduced expression of histone acetylation marker h k ac to a level a little bit higher than that in the uninfected control ( figure d-f) , though bortezomib showed relative lower capability than mg . these results suggested that the ubiquitin-proteasome pathway may contribute to the decreased acetylation of histone h in the virus infected cells. viruses , , x for peer review of in mg -treated cells but not in nh cl-treated cells confirmed the efficiency of mg as an inhibitor for the proteasome pathway in mdbk cells ( figure c ). though the concentration of mg used in this study did not show obvious cytotoxicity to mdbk cells ( figure f ), the chemical may have off-target effects. so, another proteasome inhibitor bortezomib was employed to validate the rescued effects of mg . as expected, bortezomib at a concentration of nm showing no cytotoxicity to mdbk cells could reverse bohv- -reduced expression of histone acetylation marker h k ac to a level a little bit higher than that in the uninfected control ( figure d-f) , though bortezomib showed relative lower capability than mg . these results suggested that the ubiquitinproteasome pathway may contribute to the decreased acetylation of histone h in the virus infected cells. in view that the proteasome inhibitors of both mg and bortezomib could rescue the depletion of h k ac (a marker for histone h acetylation) attributed to virus infection, we investigated whether h k ac was ubiquitinated in the cells with or without infection by ip assay. unexpectedly, when we performed ip with the h k ac specific monoclonal antibody, the ubiquitinated protein bands could not be detected in the cells with or without infection using ubiquitin specific antibody ( figure a ), indicating that h k ac is not ubiquitinated in mdbk cells. so it was highly possible that the depletion of acetylated histone h due to virus infection was not caused by the proteasomemediated h k ac degradation. in view that the proteasome inhibitors of both mg and bortezomib could rescue the depletion of h k ac (a marker for histone h acetylation) attributed to virus infection, we investigated whether h k ac was ubiquitinated in the cells with or without infection by ip assay. unexpectedly, when we performed ip with the h k ac specific monoclonal antibody, the ubiquitinated protein bands could not be detected in the cells with or without infection using ubiquitin specific antibody ( figure a ), indicating that h k ac is not ubiquitinated in mdbk cells. so it was highly possible that the depletion of acetylated histone h due to virus infection was not caused by the proteasome-mediated h k ac degradation. our foregoing results indicated that both pcaf and gcn l were significantly decreased by the virus infection ( figure a) . interestingly, the treatment of virus-infected cells with µm of mg could significantly reverse the depletion of gcn l but not pcaf ( figure b,c) . we speculated that virus infection may promote gcn l degradation via proteasome pathway. therefore, ip was performed with gcn l specific monoclonal antibody, and ubiquitin specific antibody was used to detect the ubiquitination of gcn l in the cell cultures. as a result, the ubiquitinated gcn l could be detected from the ip sample by the immunoblot using ubiquitin specific antibody ( figure d ). this result indicated that virus infection might target gcn l for ubiquitin-mediated degradation, which would partially account for the depletion of acetylated histone h and correlated with the reversed depletion of h k ac by the treatment of mg in the virus-infected cells. our foregoing results indicated that both pcaf and gcn l were significantly decreased by the virus infection ( figure a) . interestingly, the treatment of virus-infected cells with µm of mg could significantly reverse the depletion of gcn l but not pcaf ( figure b ,c). we speculated that virus infection may promote gcn l degradation via proteasome pathway. therefore, ip was performed with gcn l specific monoclonal antibody, and ubiquitin specific antibody was used to detect the ubiquitination of gcn l in the cell cultures. as a result, the ubiquitinated gcn l could be detected from the ip sample by the immunoblot using ubiquitin specific antibody ( figure d ). this result indicated that virus infection might target gcn l for ubiquitin-mediated degradation, which would partially account for the depletion of acetylated histone h and correlated with the reversed depletion of h k ac by the treatment of mg in the virus-infected cells. acetylation is one of the best-characterized covalent modifications of histones. hyperacetylation of histones is associated with an "open chromatin" conformation and transcriptional activation, whilst hypoacetylation of histones is associated with condensed chromatin and gene silencing [ , ] . for some dna viruses, such as hsv- and canine parvovirus, much is known about the effects of histone modification on virus replication. during hsv- productive infection, the viral genomes are associated with histones immediately after injection into the nucleus, and viral proteins icp and vp are required to enhance histone acetylation on the viral genome to enable efficient viral gene expression [ , ] . during canine parvovirus infection, cellular histones are associated with viral dna, and histone acetylation on parvoviral dna is essential for viral gene expression [ ] . bohv- tegument protein vp associates with histones and thereby decreased histone h acetylation in infected cells or vp transfected cells [ ] . in this study, for the first time we demonstrated that histone h acetylation (h k ac and h k ac) was significantly decreased during bohv- infection in mdbk cells, and peaked at h after infection (figure ) , which suggested the involvement of histone h acetylation in bohv- infection. to further elucidate the potential mechanisms for the decreased histone h acetylation in response to the virus infection, the steady state expression of certain hats and hdacs were investigated because they regulate enzymatically histone acetylation with opposite effects. we found that the virus infection led to a global decrease of all the detected hats, including cbp/p , gcn l and pcaf (figure a) , which was clearly in favor of the finding that virus infection decreased histone h acetylation (figure ). however, it seems that downregulation of both hdac and hdac by virus infection was not correlated with the decreased levels of histone h acetylation, which emphasized the complexity of the mechanisms for the regulation of histone acetylation following virus infection. it has been reported that hdac regulates influenza a virus (iav) replication independent of its deacetylation activity [ ] . hdac stimulates host type i interferon antiviral response, therefore iav infection decreases hdac expression for efficient replication [ ] . the treatment with tsa, an hdac inhibitor, increases iav infection via the inhibition of signal transducer and activator of transcription i (stat ) pathway, and the depression of interferon-stimulated genes including ifitm , isg , and viperin in iav-infected cells [ ] . hdac is required for inflammatory gene expression in response to lps stimulation [ ] . so, hdac and hdac can stimulate host antiviral response or inflammatory response, which tend to be depressed by virus infection. moreover, hdac inhibitors promote hsv- productive infection in neural cells [ , ] . likewise, we found that tsa enhanced bohv- virus yield approximately -fold, but the difference was not statistically significant ( figure ). therefore, we assumed that the downregulation of hdac and hadc would be beneficial for bohv- efficient replication independent of their deacetylation activity, which needs further extensive studies, in the future. it has been reported that the ubiquitin-proteasome pathway, generally known to mediate protein degradation, is involved in bohv- productive infection and immune invasion [ , ] . here, we found that the ubiquitin-proteasome inhibitors could reverse the depletion of acetylated h in bohv- infected cells (figure ) . mechanistically, the virus infection targeted gcn l but not acetylated histone h for proteasome-mediated degradation (figure ), which may consequently reduce the acetylation of histone h . previous studies have reported that some bohv- -encoded viral proteins promote the proteasome-mediated degradation of certain cellular proteins. for example, both interferon response factor (irf ) and promyelocytic leukemia (pml) are targeted by viral proteins bicp for proteasome-dependent degradation [ , ] . viral ul . protein is involved in the proteasome-mediated degradation of the transporter associated with antigen presentation [ ] . the bohv- host shutoff protein ul destabilizes the expression of immune responses related genes by ubiquitin-proteasome pathway [ ] . taken together, the above evidence indicates that virus-encoded proteins may target specific cellular proteins for proteasome-dependent degradation. whether a specific viral protein(s) is involved in the depletion of gcn l through proteasome pathway is an interesting subject which is remained to be determined in the future. in this study, we found that bohv- replication was positively correlated with hat activity because the treatment with hat activator (ctbp) increased the virus yield, and vice versa, it was significantly decreased by hat inhibitor (aa) partially through affecting virus gene expression (figures and ) . this finding is supported by a previous report that cbp/p , an hat, enhances bohv- productive infection and transactivation of late viral protein gc promoter [ ] . taken together, these findings suggested that bohv- infection may take advantage of histone acetylation for efficient replication. we speculated that hat-dependent histone h acetylation plays an important role in bohv- replication in mdbk cells. in this study, the expression status of acetylated h including h k ac and h k ac was investigated in the context of virus infection. we demonstrated that histone acetylation played an important 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acetyl-coa binding site inhibition of histone acetyltransferase activity by anacardic acid sensitizes tumor cells to ionizing radiation trichostatin a and trapoxin: novel chemical probes for the role of histone acetylation in chromatin structure and function influenza a virus dysregulates host histone deacetylase that inhibits viral infection in lung epithelial cells the ubiquitin-proteasome pathway and proteasome inhibitors influenza a virus-induced degradation of eukaryotic translation initiation factor b contributes to viral replication by suppressing ifitm protein expression histone acetylation increases chromatin accessibility histone modification patterns during gene activation herpes simplex virus vp , but not icp , is required to reduce histone occupancy and enhance histone acetylation on viral genomes in u os osteosarcoma cells histone deacetylase plays an acetylation-independent role in influenza a virus replication requirement for the histone deacetylase hdac for the inflammatory gene expression program in macrophages histone deacetylase inhibition enhances oncolytic viral replication in glioma histone deacetylase inhibitors induce reactivation of herpes simplex virus type in a latency-associated transcript-independent manner in neuronal cells the infected cell protein encoded by bovine herpesvirus (bicp ) induces degradation of interferon response factor and, consequently, inhibits beta interferon promoter activity regulation of promyelocytic leukemia (pml) protein levels and cell morphology by bovine herpesvirus infected cell protein (bicp ) and mutant bicp proteins that do not localize to the nucleus varicelloviruses avoid t cell recognition by ul . -mediated inactivation of the transporter associated with antigen processing the ul -encoded virion host shutoff (vhs) protein and vhs-independent mechanisms are responsible for down-regulation of mhc class i molecules by bovine herpesvirus bovine herpesvirus immediate-early protein (bicp ) interacts with the histone acetyltransferase p , which stimulates productive infection and gc promoter activity key: cord- -cadyykks authors: felten, sandra; hartmann, katrin title: diagnosis of feline infectious peritonitis: a review of the current literature date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: cadyykks feline infectious peritonitis (fip) is a fatal disease that poses several challenges for veterinarians: clinical signs and laboratory changes are non-specific, and there are two pathotypes of the etiologic agent feline coronavirus (fcov), sometimes referred to as feline enteric coronavirus (fecv) and feline infectious peritonitis virus (fipv) that vary fundamentally in their virulence, but are indistinguishable by a number of diagnostic methods. this review focuses on all important steps every veterinary practitioner has to deal with and new diagnostic tests that can be considered when encountering a cat with suspected fip with the aim to establish a definitive diagnosis. it gives an overview on all available direct and indirect diagnostic tests and their sensitivity and specificity reported in the literature in different sample material. by providing summarized data for sensitivity and specificity of each diagnostic test and each sample material, which can easily be accessed in tables, this review can help to facilitate the interpretation of different diagnostic tests and raise awareness of their advantages and limitations. additionally, diagnostic trees depict recommended diagnostic steps that should be performed in cats suspected of having fip based on their clinical signs or clinicopathologic abnormalities. these steps can easily be followed in clinical practice. feline infectious peritonitis (fip) is a fatal disease that occurs in domestic and wild felids worldwide. the etiologic agent, the feline coronavirus (fcov), occurs in two distinct pathotypes that can be distinguished by their biological behavior, but not by their morphology. some authors use different names for these two pathotypes, although they both belong to the same virus species. the feline enteric coronavirus (fecv) is highly prevalent in multi-cat environments and highly contagious-nearly % of cats that get in contact with fecv from feces of shedding cats become infected. however, infection is mostly asymptomatic or only causes mild and transient diarrhea [ ] [ ] [ ] . the feline infectious peritonitis virus (fipv), in contrast, is not infectious via the fecal-oral route, but arises by mutation from the avirulent fecv within a small percentage of infected cats and then causes the fatal disease feline infectious peritonitis (fip) [ ] [ ] [ ] . it is still unknown which exact genes harbor the mutation(s) leading to fipv development. since promising results using new drugs for treating cats with fip have been published recently, definitive ante mortem diagnosis is crucial in order to correctly identify the population of cats which could benefit from such antiviral treatment. at the same time, definitive diagnosis is challenging, since most existing diagnostic tests cannot differentiate between fecv and fipv, and especially in cats without body cavity effusions, it is often difficult to reach a definitive diagnosis ante mortem. in contrast to what was believed earlier, it is now well-accepted that antibody tests cannot differentiate between antibodies against fecv and fipv, and therefore, even high antibody titers in blood are not a specific indicator for fip [ , ] . additionally, there is evidence of cross-reactivity between fcov and other coronaviruses, such as tgev and canine coronavirus (ccv) [ , ] . a large proportion of the cat population (up to % and more, especially in multi-cat households) has serum antibodies against fcov, but most of these cats never develop fip [ , , [ ] [ ] [ ] [ ] [ ] . moreover, antibodies can be detected in the serum of cats that were vaccinated against fcov [ ] [ ] [ ] . the significance of the presence of antibodies for diagnosing fip in an individual cat therefore is very limited [ , , ] . opinions on the value of antibody measurement for the diagnosis of fip vary, but based on the results of numerous studies, which are shown in the following paragraphs, it is the authors' opinion that antibody measurement is of no use in a cat suspected of having fip. if antibodies are measured in a cat suspected of having fip, then a titer should be determined in any case. especially low and medium titers are of zero diagnostic value for the diagnosis of fip [ , ] . although a rising titer can sometimes be detected during progression of the disease [ , ] , this can also be seen in conjunction with fecv reinfection, and thus is not an indicator for fip [ ] . high and rising titers can also be found in healthy fecv-infected cats [ , ] and should never be used to confirm a suspicion of fip. likewise, a negative antibody test result cannot exclude fip [ , ] . in end-stage fip, especially with fulminant effusions, declining antibody titers are possible and sometimes antibody concentrations can even drop below the limit of detection [ , ] . approximately % of cats with fip do not have serum antibodies [ ] . most likely, the immune-mediated vasculitis in these cats leads to extravasation of blood components including antibodies into the effusion, and as a consequence, they can no longer be detected in the blood by antibody assays. additionally, a large number of antibodies is most likely bound by the high virus load in end-stage fip [ ] [ ] [ ] , and thus, cats with fip without effusions can have negative serum anti-fcov antibody titers [ ] . moreover, it has been demonstrated in longitudinal studies that in healthy cats originating from environments endemic for fip, antibody titers can vary greatly over time [ , ] . therefore, measurement of a single antibody titer at a single random time point does not provide any diagnostic information. the results of studies evaluating the performance of antibody tests for diagnosing fip are summarized in table . sensitivity and specificity partially vary depending on the antibody cut-off titer used. most studies reported the diagnostic value of antibody measurement compared to histopathology [ , , ] . one study included cats with fip in which the diagnosis was established upon a combination of tests performed ante mortem and histopathology [ ] . another study included cats in which fip was suspected clinically, but the diagnosis was not confirmed [ ] . the studies included control groups consisting of either healthy cats [ ] and/or cats with diseases other than fip [ , ] ; in most studies, fip was an important differential diagnosis for all control cats [ , , ] . table . studies evaluating sensitivity and specificity of the detection of serum antibodies in comparison to either histopathology, a combination of diagnostic tests or clinical suspicion of feline infectious peritonitis (fip). control groups either consisted of healthy cats or cats with diseases other than fip (with or without clinical signs consistent with fip). as stated before, the detection of antibodies in plasma or serum (no matter which titer) is not proof for the presence of fip. in an attempt to increase specificity, a few years ago, an antibody assay was developed that only detected antibodies against the fcov b protein. this test was designed based on the erroneous assumption that fipv, but not fecv, contains an intact b gene. however, the test did not result in a better diagnostic performance than other antibody assays, as both cats with fip and fecv-infected cats (healthy or suffering from other diseases) had anti- b antibodies [ ] . nevertheless, measurement of serum antibodies is useful in guiding preventative measures and can be used for fcov control in multi-cat households. as such, antibody detection can be performed to screen cats that are about to be newly integrated into a group, it can help to confirm successful elimination of fcov in groups of cats and can guide separation of infected and non-infected cats [ ] . cats without detectable antibodies most likely do not shed fcov with their feces [ , , ] . however, fecal shedding has been observed in experimentally infected cats despite the absence of serum antibodies [ ] . the diagnostic utility of anti-fcov antibody detection in effusion has been examined in a few studies as well [ , ] . in cats with fip confirmed by histopathology, antibody detection in effusion had a sensitivity of % and a specificity of % [ ] . although these numbers sound more promising than what is reported for the antibody detection in serum, it has to be emphasized that the diagnostic value of antibody measurement in both serum and effusion is similarly low and bears the same limitations. additionally, it has to be mentioned that many studies on antibody detection in effusion suffer from some limitations, such as inclusion of cats without a definitive diagnosis of fip. for example, a comparison of antibody detection in serum and effusion in cats with a clinical suspicion of fip revealed good concordance of test results in both body fluids; the antibody titer measured in effusion was lower than that determined in serum in % of cats. however, fip was not definitively confirmed in any of the cats included [ ] . the same was true for a study in which fip was suspected in cats based on different effusion features (cases had to fulfill all or most of the criteria for fip diagnosis given by the european advisory board of cat diseases recommendations of [ ] ), but the diagnosis was not definitively confirmed in any of the cats. detection of anti-fcov antibodies had a sensitivity of %. the fact that cats without detectable antibodies were positive for fcov rna in their effusion demonstrated that anti-fcov antibody detection alone has only limited diagnostic value [ ] . one study even found an inverse correlation between fcov antibody titers and fcov viral load; therefore, false negative antibody results can occur in cats with high viral rna loads [ ] . in conclusion, antibody detection in effusion is not more useful for the diagnosis of fip than antibody detection in serum (table ) . while the detection of anti-fcov antibodies in the csf of cats with neurologic fip was first regarded as useful for the diagnosis [ ] , subsequent investigations including larger and more appropriate control groups showed that the detection of antibodies in the csf, as in serum and effusion, is not suitable for a definitive ante mortem diagnosis of fip [ ] . sensitivity and specificity were calculated with histopathology as reference standard; the control groups either consisted of only cats with any kind of neurologic disease [ ] or of a combination of cats with neurologic and non-neurologic disorders [ ] (table ) . a recent study included cats with neurological signs suggested to be caused by fip, although the diagnosis was not confirmed by histopathology. in this study, csf from a large number of cats was analyzed and revealed a low sensitivity of the detection of fcov antibodies for the diagnosis of fip. however, the authors hypothesized that the detection of fcov rna in csf would confirm fip and, as they found fcov rna in all cats with a csf antibody titer of : or higher, based on these results concluded that a csf antibody titer of : or higher might be confirmatory for fip. however, the results are of limited value, as control cats were not included in the study and the diagnosis of fip was not confirmed [ ] . due to the correlation of serum and csf antibodies and the fact that antibody titers measured in serum were always higher than those measured in csf, extravasation of antibodies from serum into the csf across an impaired blood-brain barrier is discussed as the most likely explanation for the detection of anti-fcov antibodies in the csf of cats without fip. another possibility seems to be local antibody production by migrating b lymphocytes in cats with fecv infection [ ] , whereas there is no evidence for specific antibody production by intrathecal b lymphocytes [ ] . the production of non-protective antibodies and formation of immune complexes are discussed as key features for the development of typical immune-mediated lesions in cats with fip [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and circulating immune complexes could be detected in serum and effusion of fcov-infected cats [ , , , [ ] [ ] [ ] . compared to histopathology as reference standard, the detection of immune complexes in serum via competitive enzyme-linked immunosorbent assay (elisa) had a sensitivity of % and a specificity of % for diagnosing fip (table ). control cats in that study were suspected of having fip, but another disease was diagnosed [ ] . other investigations confirmed the presence of fcov-specific immune complexes in healthy cats without fip [ , ] . it has been suggested that also in fecv-infected cats, immune complexes seem to circulate, although for a limited period of time only [ ] . in conclusion, detection of immune complexes is not useful for the diagnosis of fip [ ] . monocytes/macrophages are the target cells for fipv replication [ , [ ] [ ] [ ] [ ] [ ] . both fecv and fipv can replicate within monocytes/macrophages and lead to monocyte-associated viremia [ , [ ] [ ] [ ] [ ] . for a long time, it was believed that only fipv can replicate in a sufficient way that allows intracellular antigen detection via immunologic methods [ ] , in which antibodies bind to fcov antigen within its target cells and enzymatic reactions or fluorescent conjugates result in coloring of the antigen. however, this assumption has been questioned recently, as a number of studies, which will be shown in the following paragraphs, have detected positive immunostaining also in cats not suffering from fip. the nature of these false positive results remains unclear; it is possible that not only fipv, but also fecv can be detected intracellularly in macrophages via immunologic staining methods. immunohistochemical staining of fcov antigen within characteristic histopathological tissue lesions is still considered gold standard for the diagnosis of fip [ ] [ ] [ ] , ] . when first reported in , a study described the detection of fcov antigen in epithelial cells of a cat's nictitating membrane by immunofluorescence staining of impression smears [ ] . a different study used an avidin-biotin-complex (abc) method to detect fcov antigen in tissue samples of various organs by enzymatic color change [ ] . since then, successful immunostaining of antigen has been demonstrated several times in different organs from cats with fip-either by indirect immunofluorescence [ , ] or by immunohistochemistry (ihc) within tissue macrophages in histopathologically evident tissue lesions caused by fip [ , , ] . ihc was shown to have excellent sensitivity of - % in cats with histopathologically confirmed fip [ , ] and also specificity of up to % to exclude fip in control cats with other diseases diagnosed by histopathology [ ] . an overview of studies reporting the detection of fcov antigen in tissue samples is shown in table . in many cases, ihc can only be performed post mortem, because ante mortem tissue sample collection via laparotomy or laparoscopy is an invasive procedure that bears several risks for the sick cat. many different organs have to be sampled due to the fact that fcov antigen is variably distributed within the fip-induced tissue lesions [ ] . unfortunately, the use of liver, kidney or lymph node samples obtained by minimally invasive ultrasound-guided fine-needle aspirates (fna) or tru-cut biopsies (tcb) for immunocytochemical or immunohistochemical staining has been shown to result in low sensitivities [ , ] . a possible explanation for this could be inadequate material obtained for analysis due to cellular damage [ ] . additionally, a false positive immunocytochemical result was found in a mesenteric lymph node sample, resulting in a specificity of only % [ ] . therefore, immunocytochemistry (icc) or ihc on samples obtained by fna or tcb cannot reliably confirm or exclude fip as a sole diagnostic test [ , ] . besides its diagnostic use, ihc has also been applied to detect fcov antigen in macrophages in unusual tissues or non-domestic felids. as such, fcov antigen was detected in the skin of two cats with atypical skin lesions caused by fip [ , ] , in the penile tissue of a male cat [ ] , in different tissues obtained from a mountain lion [ ] and in ocular tissues from a lion [ ] . most often, immunofluorescence staining has been performed to detect fcov antigen in effusions. for this, fluorescein-conjugated antibodies are used to detect fcov antigen within the cytoplasm of macrophages [ ] . in earlier studies, this test was reported to have an excellent specificity of %. therefore, it was believed that a positive result would in all cases allow a definitive diagnosis of fip [ , , , ] . however, more recent investigations also showed positive results of immunofluorescence or icc in cats with effusions caused by other diseases than fip. possible explanations for false positive results include binding of the antibody to cellular structures within macrophages (non-specific staining) or systemic spread and subsequent detection of fecv in cats without fip [ , ] . sensitivity of immunostaining in effusion is not absolute; thus, a negative test result cannot exclude fip [ , , [ ] [ ] [ ] . low numbers of macrophages in effusion samples or competitive binding of fcov antigen by circulating antibodies in the effusion are possible reasons for false negative results. one study demonstrated that all cats with false negative test results had anti-fcov antibodies in serum or effusion [ ] . only one study reported a sensitivity of % for the detection of fcov antigen in effusion by immunofluorescence staining [ ] . in contrast to other studies, which examined effusion samples obtained both ante and post mortem [ ] [ ] [ ] , this study only included samples collected ante mortem [ ] . additionally, a monoclonal biotinylated antibody was used, whereas previous studies applied a polyclonal fluorescein-conjugated antiserum in their staining protocols [ , , ] . finally, samples were analyzed within hours after collection [ ] and longer intervals between sampling and analysis could result in decreasing sensitivities. a decreasing sensitivity could be observed with increasing time interval between sample collection and analysis, although fcov antigen could still be detected for at least two days in samples stored at • c or room temperature [ ] . table shows all studies investigating the detection of fcov antigen in effusion. reference standard was either histopathology [ , , , ] and/or ihc [ , ] . control groups consisted of cats with clinical signs consistent with fip but a definitive diagnosis of another disease [ , , [ ] [ ] [ ] [ ] . it is also possible to detect fcov antigen in the csf of cats with fip (with and without neurological signs) [ , ] . the only prospective study to date reported a sensitivity of % and a specificity of % for the immunocytochemical demonstration of fcov antigen in macrophages in the csf of cats with and without neurological signs (table ) . in this study, ihc was the reference standard for the diagnosis of fip; control cats had clinical signs typical for fip, but other diseases were diagnosed histopathologically and ihc was negative in all cats. if only cats with neurological signs were evaluated, sensitivity surprisingly decreased to %, whereas specificity slightly increased to %. if only cats without neurological signs were evaluated, sensitivity was % and specificity only % [ ] . table . sensitivity and specificity from a study evaluating immunostaining for the detection of feline coronavirus (fcov) antigen in cerebrospinal fluid (csf) samples. feline infectious peritonitis (fip) was confirmed by histopathology and immunohistochemistry (ihc) of tissue samples. the control group consisted of cats with diseases other than fip with clinical signs (neurological or non-neurological) consistent with fip. in cats without fip that do not present with effusion, uveitis is common and ocular signs (with or without involvement of the central nervous system) can be noticed in % of the affected cats [ , ] . secondary to the inflammation of ocular structures and breakdown of the blood-ocular barrier, fcov-bearing macrophages are present in the eye and fcov antigen can be detected immunocytochemically within macrophages in the aqueous humor [ , ] . the results of the only prospective study that evaluated icc in aqueous humor in cats with immunohistochemically confirmed fip and control cats with similar clinical signs but histopathologically diagnosed other diseases are depicted in table [ ] . the technique only has a moderate sensitivity and specificity for the diagnosis of fip, and therefore, it cannot be used as a single confirming diagnostic test. since its first application for the detection of fcov [ ] , rt-pcr is frequently used to amplify fcov rna in different materials and to diagnose fip. according to the long-existing hypothesis that only fipv but not fecv is able to spread systemically, it was initially believed that detection of fcov rna via rt-pcr in blood, effusion or any other body fluid or tissue except for the gastrointestinal tract indicates presence of the virulent pathotype fipv [ , , , ] . this hypothesis has, however, been refuted by several studies (tables - ) and it is important to keep in mind that fcov rna can also be amplified outside of the gastrointestinal tract in cats without fip. nevertheless, it is well known that cats with fip exhibit much higher viral loads than healthy fecv-infected cats [ , [ ] [ ] [ ] . therefore, an ideal rt-pcr assay should be able to quantify the viral load in order to facilitate diagnosis. not only are tissue samples from cats with fip more likely to be fcov-positive, but also viral loads are significantly higher in cats with fip compared to healthy fecv-infected cats [ , ] . as an example, in a study analyzing a large number of tissue and fluid samples, % of the tissue samples and % of the fluid samples from cats with fip were positive, whereas only % of the tissue samples and % of the fluid samples from control cats were positive for fcov rna. additionally, relative fcov copy number (a ct value of in rt-pcr was assigned to a relative copy number of , and a relative copy number was then calculated for each rt-pcr-positive sample using the following equation: . ) was significantly higher in tissue samples from cats with fip than from fcov-infected cats with other diseases or healthy fcov-infected cats (median . × vs. median ) [ ] . thus, a positive rt-pcr result with a high viral load is at least very suggestive for fip [ ] . in cats with fip, large quantities of fcov rna can only be found in tissues with inflammatory changes. tissues which are not involved in the disease process contain only small amounts or no viral rna at all. among the organs with highest viral loads are the omentum, mesenteric lymph nodes and spleen, so these tissues are most useful for analysis by rt-pcr. the kidneys, liver, lung, myocardium and popliteal lymph nodes, in contrast, contain little or no viral rna [ ] . studies using rt-pcr to target fcov rna in tissues are summarized in table . however, in veterinary practice, rt-pcr is rarely applied to tissue samples to diagnose fip, as this would usually require invasive tissue sample collection via laparotomy or laparoscopy or has to be done post mortem. therefore, the studies cited here were mainly performed for research purposes and did not have the aim of evaluating rt-pcr in tissue samples as a diagnostic test for fip. when an early study applied rt-pcr to tissue samples (liver, kidney and/or spleen), fcov rna was identified in % cats with clinically suspected fip; % of experimentally infected cats tested positive for fcov by rt-pcr. however, fcov rna was also detected in the tissues from % of cats without fip [ ] , indicating that the detection of fcov rna in tissue is not specific for fip. especially in hemolymphatic tissues (spleen, mesenteric lymph nodes, bone marrow), fcov rna could be detected in all cats with fip but also in % of clinically healthy fecv-infected cats. in cats with fip, real-time rt-pcr was positive in the spleen in % of cases, in mesenteric lymph nodes in % and in the bone marrow in % of cases. in healthy fecv-infected cats, fcov rna was found in the spleen in %, in mesenteric lymph nodes in % and in the bone marrow in % of cases [ ] . as stated before, however, cats with fip usually exhibit higher viral copy numbers in tissues than healthy fecv-infected cats [ , , ] . in order to develop a rt-pcr assay which is more specific for fip, one study evaluated a real-time rt-pcr detecting messenger rna (mrna) (and thus, replicating virus). it was hypothesized that this rt-pcr would only detect fipv from cats with fip but not fecv from healthy fcov-infected cats. indeed, in this study, tissues from healthy fecv-infected cats tested negative for fcov mrna in all cases [ ] , whereas fcov mrna was found in the tissues of % of cats with immunohistochemically confirmed fip. organs with the highest frequency of harboring fcov mrna in these cats were the mesenteric lymph nodes ( %), spleen ( %), lung ( %), liver ( %), bronchial lymph nodes ( %), kidneys ( %) and intestine ( %). recent studies evaluated the use of a quantitative rt-pcr (rt-qpcr) to detect fcov rna in fna samples of the mesenteric lymph nodes and other abnormal tissues of clinical cases [ , ] and hypothesized that this technique would be a useful tool to diagnose fip for veterinary practitioners, especially in cats without effusion. the first study included control cats that were either fcov antibody-negative or antibody-positive but without signs indicative of fip or with a confirmation of another cause of illness/death. additionally, cats with non-effusive fip, that was confirmed either by histopathology or a diagnostic algorithm, were included. in all of the cats, the mesenteric lymph nodes were sampled by minimally invasive ultrasound-guided fna and examined by rt-qpcr. it was found that the detection of fcov rna in mesenteric lymph node fna can be a sensitive and specific method to diagnose fip (even when analyzed after shipping without any preservatives), although fcov rna was also detected in the mesenteric lymph nodes of a small proportion of cats without fip, and therefore, was not completely specific for fip [ ] . the second study retrospectively included eleven cats with fip without effusions and successfully amplified fcov rna from fna samples of various organs in all of the cats. since no control group was included in that study, specificity could not be evaluated [ ] . the same finding was made in a study in which different samples (fna or incisional biopsies (ib)) were obtained from different tissues (mesenteric and popliteal lymph nodes, liver, kidney, spleen, omentum) of cats with immunohistochemically confirmed fip in order to determine the prevalence of fcov rna in different tissues and to potentially evaluate the diagnostic performance of rt-pcr on those tissue samples for diagnosing fip. sensitivity was high in most tissues except for the popliteal lymph nodes and sensitivities did not greatly differ whether fna or ib were used. since no control group was included, specificity could not be determined [ ] . based on the erroneous assumption that only fipv but not fecv is able to infect monocytes/macrophages and disseminate systemically, it was thought that the detection of fcov rna in blood was proof of an infection with fipv, and thus, fip. therefore, rt-pcr was initially developed using primers for the highly conserved '-untranslated region ( '-utr) of the fcov genome in order to detect all known fcov isolates. as a result, the rt-pcr could neither differentiate between fecv and fipv, nor could it distinguish fcov from ccv or tgev. nevertheless, as stated before, it was assumed that only fipv would be detectable in blood. however, over the years, it has been shown that fcov rna could also be detected in the blood of asymptomatic cats and cats with diseases other than fip [ , ] . several studies evaluating the use of rt-pcr in serum, plasma or whole blood confirmed this finding of possible fcov viremia in cats without fip [ , , , [ ] [ ] [ ] . additionally, a recent study demonstrated that fcov viremia does not even predict the development of fip, as none of the healthy cats that tested positive for genomic or replicating fcov developed fip or clinical illness within six months of testing [ ] . all of the studies using rt-pcr on blood samples are summarized in table . if different blood fractions were examined, sensitivity and specificity are presented as a range. most studies used histopathology as reference standard [ , , , , , , ] ; one study also used the detection of fcov antigen in macrophages in effusion [ ] . some studies had the limitation of including cats in which fip was suspected clinically, but the diagnosis was not confirmed [ , , ] . the control groups either consisted of healthy cats [ , , , , , , ] and/or of cats with other diseases than fip, sometimes showing clinical signs consistent with fip [ , , , , , ] . one additional study worth mentioning is not shown in table . this study included serum samples from cats with abdominal clinical signs. since no definitive diagnosis was established in these cats, sensitivity and specificity could not be determined, and therefore, the study was not mentioned in table . nevertheless, it is worth noting that in this study, cats had a positive result in a nested rt-pcr (rt-npcr) assay. one of these cats survived for almost three years after examination; four additional cats lived for at least months. since median survival time for cats with fip is very short (unless new antiviral drugs are applied) [ ] , it is likely that these cats did not have fip and that the rt-npcr was false positive [ ] . since fcov viremia can be detected by rt-pcr in up to - % of fecv-infected cats without fip [ ] , detection of fcov rna in blood is not specific for fip and rt-pcr in blood samples should not be used to confirm a diagnosis of fip. additionally, fcov viral load is low in any blood fraction in experimental fip [ ] and this is most likely also the case in natural infection, leading to a rather low sensitivity of rt-pcr in blood. rt-pcr had better sensitivity when peripheral blood mononuclear cells (pbmc) were used compared to serum [ ] ; however, other studies came to differing results regarding sensitivity in the different blood fractions and most studies are not well comparable [ , , , ] . sensitivity was poor in most of the studies; therefore, due to a rather low virus load and resulting low sensitivities, blood samples are not recommended for analysis by rt-pcr. although fecv can infect monocytes and macrophages, it is not capable of efficiently replicating within them [ , , , ] . in an attempt to increase specificity of rna detection in blood, a rt-pcr specifically amplifying fcov mrna has been developed in order to detect only actively replicating virus in blood. in an initial study, this rt-pcr assay really did not detect fcov mrna in any of the control cats with clinical signs indicative of fip but other confirmed diseases, whereas fcov mrna was found in % of cats with histopathologically confirmed fip [ ] . nevertheless, subsequent studies also identified fcov mrna, and thus, replicating fcov in . - % blood samples from healthy cats [ , , , ] and the detection of replicating virus did not predispose to the development of fip [ ] . therefore, this special rt-pcr assay also is not adequately specific for fip. in general, however, cats with fip exhibit higher viral loads and copy numbers than healthy fecv-infected cats [ , ] and higher viral copy numbers are found in the feces of healthy fecv-infected cats when compared to their blood [ ] . therefore, an rt-pcr that allows simultaneous amplification and quantification of viral mrna could potentially distinguish fecv (with low viral copy numbers) from fipv (with high viral copy numbers). a real-time rt-pcr based on primer-probe energy transfer detecting and quantifying subgenomic fcov mrna showed a very good sensitivity and specificity in one study, but was so far only applied on very small numbers of samples and not used in the field [ ] . loop-mediated isothermal amplification (lamp) is a new molecular method with the advantage of delivering rapid in-house test results. reverse transcriptase lamp (rt-lamp) has been used to amplify fcov rna in blood, effusion, lymph nodes and feces of cats with suspected fip in a recent study, in which rt-npcr was used as reference standard. specificity of rt-lamp was % in all materials; sensitivity, however, was only low to moderate [ ] . in cats with fip that have effusion, virus load is much higher in the effusion than in blood [ ] . sensitivities and specificities of rt-pcr assays for the diagnosis of fip in effusion are listed in table . as shown, sample numbers are relatively low in some and especially in the older studies. most studies used histopathology as reference standard [ , , , , ] ; in some studies, the detection of fcov antigen in effusion [ , ] or tissue [ , ] or laboratory fluid analysis [ ] was the reference standard. one study included cats with a suspicion of fip, of which only had histopathologic confirmation of the disease and / cats had effusion [ ] . another study examined effusion samples from cats with suspected fip based on effusion feature criteria established by the european advisory board on cat diseases in [ ] . the diagnosis was, however, not confirmed by reference standard methods in any of the cats [ ] . the control groups consisted of cats with a clinical suspicion of fip but different diagnoses [ , , [ ] [ ] [ ] . one study examined a large number of effusion samples from cats with clinically suspected fip; the disease was, however, not confirmed. it could also not be determined whether cats with negative rt-pcr results had diseases other than fip. therefore, sensitivity and specificity could not be calculated, but fcov rna was detected in the effusion of / cats ( %) [ ] . in another study, plasma and/or ascites from cats with histopathologically confirmed fip and controls (healthy or with fip-typical clinical signs but other diseases) were examined by rt-npcr. fcov rna was detected in the plasma of % of cats with fip. five additional cats that tested negative in plasma had a positive rt-npcr result in effusion. however, it was not stated how many effusion samples were analyzed in total [ ] ; thus, the study was not included in table . although specificity of rt-pcr in effusion was high in many studies and a positive rt-pcr result can add to a suspicion of fip, it has to be kept in mind that rt-pcr already detects small amounts of viral rna, which can potentially leak from blood into effusion in the face of inflammation (even without fip) in cats with circulating fecv [ ] . especially in recent studies, fcov rna could also be detected in the effusion of cats without fip [ , , , ] . all studies evaluating rt-pcr for the detection of fcov rna in csf have reported specificities of %, meaning that fcov rna could only be detected in the csf of cats with fip (with and without neurological signs) but not control cats (table ) . reference standards used in these studies was either histopathology [ , ] and/or the detection of fcov antigen in effusion [ ] or tissue [ ] . the control groups consisted of cats with neurologic diseases only [ ] or also included cats with non-neurologic diseases but clinical signs consistent with fip [ , ] . although the blood-brain barrier has not specifically been examined in cats with fip yet, it can be assumed that it is impaired secondary to the inflammation caused by fip [ ] . in general, various infectious and inflammatory diseases of the central nervous system lead to the release of cytokines, adhesion molecules, metalloproteases and other mediators, which can damage tight junctions, basal membranes and cerebrovascular endothelium. this enables the leakage of cells and pathogens into the csf across the blood-brain barrier [ ] . therefore, although false positive rt-pcr results have not been reported in csf thus far, it seems theoretically possible that fecv could pass the blood-brain barrier in cats with neurologic diseases other than fip, as it is also suspected for anti-fcov antibodies [ ] . one study detected a correlation between high anti-fcov antibodies and positive rt-pcr in the csf. in that study, fcov rna was found in only % of samples from cats with low, but in % of samples from cats with high csf antibody titers. in contrast, fcov rna was not amplified in any of the antibody-negative cats. however, fip was not confirmed in the cats in this study [ ] . another type of sample material that can be examined for fcov rna by rt-pcr is the aqueous humor. to date, there have been very few studies that evaluated this sample material for analysis by rt-pcr in a small number of cats with fip (table ) . additionally, a case report demonstrated positive rt-pcr in the aqueous humor of / cats with uveitis; however, there were no clear-defined fip and control groups [ ] . fcov replication, like replication of all rna viruses, is prone to error [ ] . multiple individual mutations occur during each cycle of viral replication [ , , , ] . it is hypothesized that specific mutations or a combination of mutations then lead to the development of the virulent pathotype fipv and trigger the tropism switch from enterocytes to macrophages as a key event in fip pathogenesis [ , , ] . several different fcov genes have been analyzed and suggested to harbor the mutation responsible for the fcov pathotype switch. open reading frame (orf) abc, which is coding for the accessory proteins a, b and c, has been sequenced and compared in fcov from cats with fip and healthy cats and differences were detected. for instance, it was found that fipv from tissue or effusion of cats with fip contained a truncated protein c, whereas fecv from feces of healthy cats most often contained an intact protein c [ , , [ ] [ ] [ ] [ ] [ ] . the a and b genes could play a role in fip pathogenesis as well [ ] [ ] [ ] . orf ab, coding for accessory proteins a and b, has also been subject of studies looking into fip pathogenesis, but somewhat contradictory results were found [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] . the fcov spike (s) protein is responsible for viral cell entry. it includes a subunit for receptor binding (s ) and a subunit mediating membrane fusion (s ) [ ] [ ] [ ] . therefore, mutations in the fcov s gene, more specific mutations in the s region and corresponding amino acid substitutions in the s protein, have been suggested to be responsible for the change of viral cell tropism [ , , [ ] [ ] [ ] . as a result, only fipv but not fecv are capable of efficient and sustained replication in macrophages, producing large amounts of viral particles and spreading the infection to adjacent cells [ , ] . therefore, the s gene is of particular interest with regard to determining which mutations are responsible for the transition from fecv to fipv. recently, commercial diagnostic testing was introduced for fcov s gene mutations. when the s genes of a large number of fcov derived from the feces of healthy cats and from tissues or ascites of cats with fip were sequenced, two single nucleotide polymorphisms (snp) were found that only were present in the fcov from cats with fip but not in the fcov from healthy cats. these snp were found at nucleotide position and , which lie in close proximity within the s gene and the detection of one of the snp allowed differentiation between fcov from cats with fip and healthy cats in % of the examined fcov. in all of the sequenced fecal fcov from healthy cats, adenine was found at nucleotide position , whereas thymine or cytosine was detected in % of the fcov from ascites or tissues of cats with fip. both snp led to the substitution of methionine by leucine at amino acid position within the putative fusion peptide of the s protein (m l). in all of the sequenced fecal fcov from healthy cats, thymine was detected at the second nucleotide position , whereas guanine was found in % of the fcov from ascites or tissues of cats with fip. this snp led to the substitution of serine to alanine at position of the s protein (s a) [ ] . after that first study provided evidence of an association of the two s gene mutations with fip [ ] , a number of studies subsequently investigated the prevalence of the mutations in tissue samples from cats with and without fip (table ) . table . sensitivity and specificity from different studies evaluating the detection of feline coronavirus (fcov) spike (s) gene mutations in tissue samples. feline infectious peritonitis (fip) was confirmed by histopathology alone or in combination with immunohistochemistry (ihc), control cats were either healthy cats or cats with diseases other than fip (with or without clinical signs consistent with fip). opinions vary substantially among researchers, especially on the specificity of s gene mutations for the fip phenotype. for example, one recent study corroborated the initial findings by detecting m l in three tissue samples (mesentery, colonic lymph node and omentum) from cats with fip; moreover, the mutation fully discriminated the fcov found in these tissue samples from fcov found in fecal samples from healthy cats [ ] . in contrast, fcov with substitution m l was not specific for the fip phenotype in a different study, which also found the mutation in tissue samples from cats without fip. not only the majority of fcov from tissue samples from cats with fip ( / ; %), but also the majority of those from tissue samples from control cats without fip ( / ; %) had a leucine (and thus, a mutated sequence) at position in that study. on the other hand, the majority of fcov from fecal samples from cats with ( / ; %) and without fip ( / ; %) had a methionine (and thus, a non-mutated sequence) at this position. additionally, a number of tissue samples from cats with fip ( / ; %) also exhibited a methionine at position , which means that they did not contain a mutation despite being from cats with fip. therefore, the authors concluded that substitution m l was indicative of systemic spread of fcov rather than a marker for fip [ ] . in this study, five tissue samples that did not contain the substitution m l were analyzed for substitution s a (the less common s gene mutation) by sanger sequencing, and s a was then detected in / samples from cats with fip but not in the one remaining sample from a control cat [ ] . since the first description, pcr assays detecting mutations m l and s a frequently have been used for diagnostic purposes and a number of studies have evaluated sensitivity and specificity or diagnostic accuracy of the detection of the mutations in the diagnosis of fip (table ) . however, largely contrasting results were also found in these studies, especially in terms of specificity, possibly as a result of different sequencing assays that were used. one study, for example, examined a large number of tissue, fluid and fecal samples from cats with immunohistochemically confirmed fip and controls in which fip was excluded and performed rt-qpcr followed by pyrosequencing of an s gene amplicon in the rt-qpcr-positive samples. in total, tissue samples from cats with fip were analyzed. however, since histopathological data was only available for of the tissue samples, only these numbers were included in the calculation of sensitivity and specificity (table ) . of these samples, were rt-qpcr-positive and s gene mutations were detected in / samples (m l being much more common than s a; mixed mutated and non-mutated fcov or fcov with both m l and s a also present). of the tissue samples from control cats, were rt-qpcr-positive and / were positive for mutation m l. when comparing sensitivity and specificity of rt-qpcr alone with that of rt-qpcr plus s gene sequencing, specificity slightly increased from % to %, whereas sensitivity decreased from % to %. therefore, it was concluded that the detection of fcov s gene mutations did not improve the ability to diagnose fip. mutations were also found in cats without fip, which supported the hypothesis of the mutations being a marker for systemic spread of fcov and not for fip [ ] . a somewhat similar result was obtained a year later when a study compared different laboratory tests in tissue samples (mesenteric lymph nodes, spleen, small intestine, lung) from cats with fip (positive ihc in at least one tissue) and control cats (histopathological diagnosis of another disease plus negative ihc). all samples were examined by rt-npcr targeting the highly conserved '-utr and sequencing was performed to detect s gene mutations. again, the detection of fcov s gene mutations improved specificity of the rt-npcr from % to %, but sensitivity decreased from % to %, and an s gene mutation (type of mutation not shown) was also detected in a cat without fip [ ] . however, contrasting results were obtained when a real-time rt-pcr was developed and evaluated subsequently, which uses specific fluorescent hydrolysis probes to detect either one of the two snp or the wildtype sequence. in that study, none of the cats without fip (with histopathological diagnosis plus negative ihc) tested positive for one of the s gene mutations in their tissues. of the cats with immunohistochemically confirmed fip, tested positive for one of the s gene mutations in at least one of their tissues ( / m l, one mixed genotype). in this study, sensitivity was % and as such comparable to the previous studies; however, specificity was excellent ( %) [ ] . finally, a recent study evaluated the same real-time rt-pcr detecting s gene mutations in cats with immunohistochemically confirmed fip. fna and ib samples were obtained from the mesenteric and popliteal lymph nodes, spleen, liver, kidney and omentum and additionally from different body fluids. samples were first examined by rt-pcr detecting the fcov b gene (detecting all fcov) and rt-pcr-positive samples were subsequently analyzed by real-time rt-pcr detecting s gene mutations. interestingly, fcov with s gene mutations was present in every cat, but the location of mutated fcov varied from cat to cat. sensitivity of the rt-pcr detecting all fcov was good for all tissues except for the popliteal lymph nodes, but sensitivity decreased when s gene mutations were evaluated. since sensitivity of fna and ib did not significantly differ, sampling via minimally invasive fna seems possible in order to obtain adequate samples for rt-pcr testing. specificity was not determined in this study, since a control group was not included in the study protocol. diagnostic accuracy of the detection of fcov s gene mutations has also been evaluated in blood samples (table ). rt-npcr and subsequent s gene sequencing of serum and plasma samples from cats with fip (diagnosed by histopathology ± ihc or by positive immunofluorescence in effusion) and control cats (diagnosed with another disease either ante or post mortem) revealed a sensitivity of only %, which confirms the very low virus load in blood. rt-npcr was negative in all of the blood samples from cats without fip, and thus, specificity of the sequencing step could not be determined [ ] . subsequent studies evaluated the aforementioned real-time rt-pcr detecting s gene mutations by specific fluorescent hydrolysis probes in blood samples (plasma, serum, buffy coat, whole blood) from cats with fip [ ] and/or controls [ ] (reference standard histopathology and ihc) and either did not detect mutated fcov in any of the blood samples or only found very low sensitivity. in some cats with fip, fcov rna was detected, but viral concentrations were too low to allow pathotyping [ ] . this implicates that blood cannot be recommended as sample material. nevertheless, a study comparing different pcr tests including '-utr rt-npcr and a combined approach with rt-pcr followed by s gene sequencing reported a sensitivity of % for the rt-npcr and % for combined rt-pcr and sequencing when whole blood was used. potentially, whole blood can give better results than plasma or serum. although the authors state that specificity of both rt-npcr and combined approach were %, specificity of the sequencing step should not be calculated from that study, since none of the blood samples from cats without fip tested positive for fcov rna at all [ ] . an association of substitutions m l and s a with fip was shown by a study which detected amino acid substitution m l in % fcov extracted from ascites of cats with fip (reference standard for the diagnosis not reported) and a non-mutated sequence (a methionine codon) in % fcov extracted from feces of healthy cats and % fcov extracted from feces of cats with fip [ ] . however, contrasting results of pcr assays detecting fcov s gene mutations were again, as in tissue and blood, found subsequently when analyzing effusion and other fluid samples for diagnostic purposes (table ) . one study including cats with fip confirmed by ihc and control cats classified as non-fip by confirmation of other diseases based on either histopathology and/or the definitive diagnosis of another disease ante mortem showed that rt-qpcr alone had a sensitivity of % and a specificity of %. of the rt-qpcr-positive cats, had a mutated fcov with substitution m l in their effusion detected by sequencing. one cat exhibited mutation s a. this resulted in a sensitivity of % for the detection of s gene mutations. since none of the control cats were rt-qpcr positive, specificity of the sequencing step could not be determined. thus, the majority of effusion samples that generated sequence data contained a mutated fcov, although in that study population, detection of s gene mutations did not improve specificity [ ] . when an rt-npcr and subsequent s gene sequencing was used on effusion samples from cats with fip (diagnosed by histopathology ± ihc or by positive immunofluorescence in effusion) and control cats (diagnosed with another disease either ante or post mortem), a similarly moderate sensitivity of % was detected. of the samples from cats with fip, were rt-npcr-positive, and the majority of those ( / ) contained s gene mutations (m l n = , s a n = ). rt-npcr was negative in all of effusion samples from cats without fip, and thus, specificity of the sequencing step again could not be determined [ ] . evaluations of a real-time rt-pcr detecting the s gene mutations with specific fluorescent hydrolysis probes in effusion from cats with fip and controls (reference standard histopathology and ihc) detected similar sensitivities of - % for the detection of s gene mutations [ , ] . of the samples from cats with fip in one study, were rt-pcr-positive. mutated fcov containing m l was detected in the majority of samples ( / ) . additionally, mixed fcov (with and without s gene mutations) were detected in two cats. in cats, rt-pcr was positive, but rt-pcr detecting s gene mutations was not successful, either because of a low virus load (n = ) or despite a high virus load (n = ). the reason for the latter could be the presence of unknown sequence variations or serotype ii fcov. rt-pcr was also positive in / effusion samples from cats without fip and m l was detected in one ascites sample from a cat with chronic kidney disease [ ] . finally, one recent study collected different body fluids (csf, aqueous humor, ascites, pleural or pericardial effusion) from cats with fip (confirmed by ihc) and cats in which histopathological signs of fip were absent. of the samples from cats with fip, were rt-qpcr-positive. in a subsequent pyrosequencing step, of these (one sample was lost from s gene analysis) contained mutated fcov (m l n = , s a n = , mixed mutated and non-mutated fcov without differentiation of mutation n = ). of the fluid samples from cats without fip, one was rt-qpcr-positive and contained substitution m l (an abdominal fluid sample from a cat with necrotizing pneumonia). since individual results for effusion, csf and aqueous humor are not shown in that study, sensitivity and specificity cannot be calculated individually, but only for all fluid samples together (table ) . as was demonstrated for tissue samples, the detection of s gene mutations in this study led to a decrease in sensitivity ( % for rt-qpcr alone compared with % for combined approach) and an s gene mutation was also detected in a cat without fip [ ] . the detection of fcov s gene mutations in csf was evaluated in two studies presented as conference abstracts thus far (table ) . one study obtained csf samples from cats with fip (confirmed by ihc) and reported a sensitivity of %. control cats were not included in that study [ ] . the other study looked at cats with immunohistochemically confirmed fip (six with neurological signs) and control cats with clinical signs indicative of fip ( with neurological signs) but definitively diagnosed other diseases, and calculated a sensitivity of %. sensitivity increased to % if only cats with neurological signs were included. since fcov rna was not detected in any of the control cats, specificity of the detection of s gene mutations could not be calculated [ ] . ante mortem diagnosis of fip cannot be made based on results of one single diagnostic test. it is important to consider signalment, history, clinical signs and standard clinicopathological abnormalities in every cat that is presented with a suspicion of fip. additional tests, especially tests for direct virus detection, should be utilized depending on the clinical picture. measurement of fcov antibodies is not useful for the diagnosis at all. routine hematology, serum biochemistry and, if present, analysis of effusion should be performed in every cat. in cats with neurological clinical signs, csf analysis should be done as well. diagnostic trees depicting relevant additional diagnostic steps that are recommended depending on a cat's clinical presentation are shown in figure . in cats with effusion, laboratory analysis of the fluid including rivalta's test should be performed. if this test is negative, fip is rather unlikely, especially if the pre-test probability of fip is low. if rivalta's test is positive, however, further diagnostic steps should follow. rt-pcr on effusion can help in establishing a diagnosis, especially if viral copy numbers are high. a negative rt-pcr on effusion makes fip unlikely, unless suspicion is high based on clinical findings and other laboratory test results. high fcov load or detection of s gene mutations in an rt-pcr-positive effusion sample can substantiate a suspicion of fip. additionally, in order to increase sensitivity of the detection of s gene mutations, other sample material, such as fna of mesenteric lymph nodes, spleen and liver and whole blood can be included in the analysis. if, however, s gene mutations cannot be detected in an rt-pcr-positive sample and the virus load is low, but still the suspicion of fip is high in a case, then more invasive diagnostic procedures, such as histopathology and ihc on tissue samples obtained in laparotomy/laparoscopy should be considered in order to confirm or exclude fip. in cats presenting without significant effusion, analysis of different sample types should be combined. ultrasound-guided fna of different organs including the mesenteric lymph nodes, spleen and liver can provide sample material for general rt-pcr, which should be the first diagnostic test in such a case. in cats with neurological clinical signs, diagnostic imaging and csf sampling is an important step in reaching a diagnosis anyway and csf should be submitted for rt-pcr as well. the same is true for cats with uveitis in which the aqueous humor should be integrated in a set of different samples for analysis by rt-pcr. by testing several materials, sensitivity of any given test can be increased. if general rt-pcr is positive in a cat without effusion, then again, the presence of fcov s gene mutations or a high fcov load might aid in cases of uncertainty-if mutations can be detected in an rt-pcr-positive sample, especially if copy numbers and pre-test probability of fip are high, then fip is likely. however, if s gene mutations cannot be detected in the set of samples or if general rt-pcr on the samples submitted is negative or virus load is low, then other causes for fip should be ruled out as far as possible. if the suspicion of fip is still high (based on signalment, clinical and other laboratory findings) in a case with negative rt-pcr or a case lacking s gene mutations, then histopathology and ihc on tissue samples must be considered in order to reach a definitive diagnosis. however, as long as there is uncertainty regarding the putative role of s gene or other mutations in fip pathogenesis, and since specificity of rt-pcr in any given sample is not absolute, an ideal diagnostic test for fip still does not exist. ihc on histopathologically abnormal tissue obtained either post mortem or via laparotomy/laparoscopy still remains the gold standard of diagnosis. fip should be ruled out as far as possible. if the suspicion of fip is still high (based on signalment, clinical and other laboratory findings) in a case with negative rt-pcr or a case lacking s gene mutations, then histopathology and ihc on tissue samples must be considered 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neutralizing/antibody-dependent enhancing epitope on spike protein and b gene of feline infectious peritonitis virus: influences of viral replication in monocytes/macrophages and virulence in cats orf -encoded accessory protein a of feline infectious peritonitis virus as a counteragent against ifn-alpha-induced antiviral response deletions in the a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis field strain feline coronaviruses with small deletions in orf b associated with both enteric infection and feline infectious peritonitis ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex feline coronavirus: insights into viral pathogenesis based on the spike protein structure and function spike protein fusion peptide and feline coronavirus virulence mutation in spike protein cleavage site and pathogenesis of feline coronavirus genotyping coronaviruses associated with feline infectious peritonitis evaluation of a discriminative realtime rt-pcr in cerebrospinal fluid for the diagnosis of feline infectious peritonitis this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license [ ] csf fip (n = ) controls (n = ) only two studies that were presented as conference abstracts have evaluated sensitivity and specificity of the detection of fcov s gene mutations in aqueous humor (table ). these studies included cats with fip confirmed by ihc and reported sensitivities of only - %. specificity, however, could not be calculated in either one of the studies, either because no control cats were included or because none of the control cats were fcov-positive at all [ , ] . key: cord- - qh dsew authors: stegelmeier, ashley a.; van vloten, jacob p.; mould, robert c.; klafuric, elaine m.; minott, jessica a.; wootton, sarah k.; bridle, byram w.; karimi, khalil title: myeloid cells during viral infections and inflammation date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qh dsew myeloid cells represent a diverse range of innate leukocytes that are crucial for mounting successful immune responses against viruses. these cells are responsible for detecting pathogen-associated molecular patterns, thereby initiating a signaling cascade that results in the production of cytokines such as interferons to mitigate infections. the aim of this review is to outline recent advances in our knowledge of the roles that neutrophils and inflammatory monocytes play in initiating and coordinating host responses against viral infections. a focus is placed on myeloid cell development, trafficking and antiviral mechanisms. although known for promoting inflammation, there is a growing body of literature which demonstrates that myeloid cells can also play critical regulatory or immunosuppressive roles, especially following the elimination of viruses. additionally, the ability of myeloid cells to control other innate and adaptive leukocytes during viral infections situates these cells as key, yet under-appreciated mediators of pathogenic inflammation that can sometimes trigger cytokine storms. the information presented here should assist researchers in integrating myeloid cell biology into the design of novel and more effective virus-targeted therapies. the ability of the immune system to recognize invading pathogens and tissue damage, and subsequently respond in a targeted and reproducible manner bestows longevity to our existence. within the diverse cellular network of the immune system, recent research has shown that myeloid cells deserve new-found attention due to their ability to detect and mitigate viral infections and promote inflammation. upon viral infection, there are a number of myeloid cell subsets that play various roles in the subsequent inflammatory, cellular, and humoral responses. myeloid cells are granulocytic and phagocytic leukocytes that traverse blood and solid tissues. when they recognize virus-infected cells or tissues damaged by viruses, these sentinels rapidly initiate an innate immune response [ ] . this multifaceted response involves cellular activation [ ] , signaling cascades [ ] , and the release of cytokines [ ] to guide leukocytes to mount an effective response. evidence is accumulating that two myeloid cell subsets, in particular, are playing a larger role in recognizing and halting viral infections than was previously thought. researchers are discovering that both neutrophils and inflammatory monocytes are intertwined in the immune system's anti-viral response. moreover, they play unique immuno-regulatory roles post-infection, and are critical for restoring homeostasis. neutrophils are the most abundant leukocyte subset in mammals, ranging from - % of white blood cell counts [ ] . they are responsible for both pro-inflammatory and anti-viral responses, and, therefore, constitute a first line of defense against invading pathogens and cell damage [ ] . neutrophils are effector innate cells that live for a relatively brief five days [ ] and exist in one of three states: quiescent, primed, or active. although they are predominately considered cells that target extracellular organisms such as bacteria via phagocytic uptake, their control of other cell subsets enables them to play important indirect roles in clearing viral infections and modulating inflammation. monocytes are large mononuclear leukocytes that are involved with the inflammation and clearance of pathogens. these non-dividing cells are able to further differentiate into other myeloid subsets such as dendritic cells (dcs) and macrophages. monocytes constitute a heterogeneous population that is endowed with a high degree of plasticity, allowing them to respond to environmental cues in tissues. current research is uncovering the role that inflammatory monocytes play during inflammation and viral infections. this subset preferentially traffics to inflamed regions, where they secrete inflammatory cytokines [ ] . however, they can also function as regulatory cells [ ] . for example, alveolar macrophages have been shown to recruit inflammatory monocytes through a type i interferon (ifn)-mediated mechanism [ ] . these monocytes can then provide protection against virus-induced pathology. evidence exists that both neutrophils and monocytes can contribute to viral clearance or exacerbate pathological damage depending on the context of the infection ( figure ). in terms of myeloid cells contributing to virus-induced pathologies, a linkage can be made between the induction of cytokine storms and dysregulated type i ifn responses. in cases where these cells are beneficial, they can be therapeutically boosted, whereas they can also be depleted when viruses have commandeered them towards destructive fates. exploring the pronounced involvement that myeloid subsets have in mitigating viral replication and pathology, therefore, has the potential to create novel therapeutics that are more efficacious against viral infections. the aim of this review is to explore recent advances in our understanding of the roles that neutrophils and inflammatory monocytes play during viral infections. although previous reviews have provided comprehensive coverage on the impact that these myeloid subsets have during bacterial infections [ , , ] , there is no current review with an extensive focus on their contributions to mitigating viral infections. further, this review has a novel focus on the expanding literature discussing the regulatory roles of these cell types during viral infections, as well as a possible link between the virus-mediated blockade of type i interferon signaling and virus-induced cytokine storms. figure . schematic of myeloid cells highlighting their ability to respond to pulmonary viral infections via the initiation and modulation of anti-viral inflammatory activity. lung-resident myeloid cells, such as alveolar macrophages, utilize a complex sensory system to integrate disturbances of pulmonary tissues by viruses such as respiratory syncytial virus (rsv) into the activation of local effector leukocytes. (a) rsv enters and infects the lungs. viral pathogen-associated molecular patterns (pamps), such as double-stranded rna or danger associated molecular patterns (damps), are detected by pattern recognition receptors (prrs) in or on sentinel cells in the lungs, such as tlr in the endosomes of lung-resident macrophages. tlr stimulation activates the nf-κß signaling cascade, resulting in the release of chemokines and inflammatory cytokines. a chemokine gradient forms between the lungs and bone marrow. (b) homeostatic bone marrow tends to retain cxcr + neutrophils and monocytes through endogenous expression of high levels of cxcl . however, the release of pamps, as well as the secretion of cytokines and chemokines as a consequence of pulmonary rsv infections, is sensed by cells in the bone marrow, which in turn allow recruitment of new neutrophils and monocytes from the bone marrow into the lungs. specifically, g-csf downregulates cxcr on neutrophils, triggering their release. similarly, ccl is produced in the bone marrow by endothelial cells following tlr signaling in infected lungs, which is crucial for inflammatory monocyte release into the bloodstream. once in the bloodstream, these cells sense disrupted endothelium from the viral infection, which triggers a complex adhesion cascade. activated ly c hi inflammatory monocytes are recruited to the site of infection by a variety of chemokine receptors including ccr , and , as well as cxcr binding to their respective ligands. (c) once at the site of infection, they differentiate into dendritic cells and macrophages that initiate an inflammatory cascade that includes copious amounts of inflammatory cytokines, in particular il- and ifn-γ, which are potent inducers of th -biased immune responses. once these dendritic cells and macrophages acquire viral antigens, they home to lymph nodes via chemokine receptors, including ccr . monocyte-derived dendritic cells that home to lymph nodes present viral antigens to naïve cd + and cd + t-cells that are required to kill infected cells. (d) the basic neutrophil function of clearing an inflamed area by removing killed pathogens and host cells contributes to reduced inflammation and wound debridement. neutrophils are also capable of promoting tissue repair and increased angiogenesis. further, monocytes can suppress lymphocytes in various clinical scenarios. in lungs, myeloid cells are able to inhibit pro-inflammatory tissue-resident leukocytes through direct cell-to-cell contact through galectin /tim and the effect of tgf-β on nkp in order to regulate tcells and nk cells, respectively. myeloid cells can also exert suppressive functions through secretion of soluble factors such as il- , arginase- and indoleamine , -dioxygenase. (e) we speculate that disruption of the cellular sensing of type i ifn responses can result in excessive production of proinflammatory cytokines, including ifn-γ, il- , il- , and tnf-α, leading to a toxic cytokine storm. figure . schematic of myeloid cells highlighting their ability to respond to pulmonary viral infections via the initiation and modulation of anti-viral inflammatory activity. lung-resident myeloid cells, such as alveolar macrophages, utilize a complex sensory system to integrate disturbances of pulmonary tissues by viruses such as respiratory syncytial virus (rsv) into the activation of local effector leukocytes. (a) rsv enters and infects the lungs. viral pathogen-associated molecular patterns (pamps), such as double-stranded rna or danger associated molecular patterns (damps), are detected by pattern recognition receptors (prrs) in or on sentinel cells in the lungs, such as tlr in the endosomes of lung-resident macrophages. tlr stimulation activates the nf-κß signaling cascade, resulting in the release of chemokines and inflammatory cytokines. a chemokine gradient forms between the lungs and bone marrow. (b) homeostatic bone marrow tends to retain cxcr + neutrophils and monocytes through endogenous expression of high levels of cxcl . however, the release of pamps, as well as the secretion of cytokines and chemokines as a consequence of pulmonary rsv infections, is sensed by cells in the bone marrow, which in turn allow recruitment of new neutrophils and monocytes from the bone marrow into the lungs. specifically, g-csf downregulates cxcr on neutrophils, triggering their release. similarly, ccl is produced in the bone marrow by endothelial cells following tlr signaling in infected lungs, which is crucial for inflammatory monocyte release into the bloodstream. once in the bloodstream, these cells sense disrupted endothelium from the viral infection, which triggers a complex adhesion cascade. activated ly c hi inflammatory monocytes are recruited to the site of infection by a variety of chemokine receptors including ccr , and , as well as cxcr binding to their respective ligands. (c) once at the site of infection, they differentiate into dendritic cells and macrophages that initiate an inflammatory cascade that includes copious amounts of inflammatory cytokines, in particular il- and ifn-γ, which are potent inducers of th -biased immune responses. once these dendritic cells and macrophages acquire viral antigens, they home to lymph nodes via chemokine receptors, including ccr . monocyte-derived dendritic cells that home to lymph nodes present viral antigens to naïve cd + and cd + t-cells that are required to kill infected cells. (d) the basic neutrophil function of clearing an inflamed area by removing killed pathogens and host cells contributes to reduced inflammation and wound debridement. neutrophils are also capable of promoting tissue repair and increased angiogenesis. further, monocytes can suppress lymphocytes in various clinical scenarios. in lungs, myeloid cells are able to inhibit pro-inflammatory tissue-resident leukocytes through direct cell-to-cell contact through galectin /tim and the effect of tgf-β on nkp in order to regulate t-cells and nk cells, respectively. myeloid cells can also exert suppressive functions through secretion of soluble factors such as il- , arginase- and indoleamine , -dioxygenase. (e) we speculate that disruption of the cellular sensing of type i ifn responses can result in excessive production of pro-inflammatory cytokines, including ifn-γ, il- , il- , and tnf-α, leading to a toxic cytokine storm. the fatal outcome of severe lung infections is shown to be correlated with the early persistent production of inflammatory cytokines and chemokines that recruit neutrophils and monocytes. while inflammatory cytokines and chemokines are essential for effective control of viral infections, they can also contribute to the severity of disease and tissue damage. multiple progenitor cell types arise from self-renewing multi-potent hematopoietic stem cells that become committed in the bone marrow to lineage-specific myeloid cells of the immune system [ ] . clonogenic myeloid-primed precursors (cmps) give rise to myeloid cells, which then further differentiate into granulocyte/monocyte progenitors (gmps) [ ] . subsequently, gmps undergo multiple stages of differentiation before they are terminally differentiated into neutrophils or monocytes in the bone marrow [ ] . monocytes require the growth factor colony-stimulating factor- to develop [ ] and high levels of the transcription factor pu. to steer gmps to commit to a monocyte lineage [ ] . monocyte-dc progenitors (mdps) create descendants that are destined to become either dcs or monocytes [ ] . recent discoveries have expanded our understanding of neutrophil development. advances in isolation techniques have elucidated that neutrophils are derived from unique cd b + ly g lo ly b int cd − precursors that possess proliferation capabilities [ ] . indeed, transcriptional profiling coupled with mass cytometry has provided additional information on the process required for gmps to differentiate into neutrophils [ ] . researchers determined the bone marrow possesses three distinct subsets: the aforementioned proliferative precursor cells, as well as non-proliferative immature and non-proliferative mature neutrophils. precursors required the transcription factor c/ebpε to differentiate from gmps. as precursors further shift into non-proliferative populations, they exchange proliferation capacity for increases in effector function and migration [ ] . further experiments on neutrophil precursors demonstrated their ability to expand in the presence of cancers such as melanoma and suppress regulatory t cells [ ] . the role of precursor neutrophils during viral infections has not been determined and represents a novel avenue of research. once viruses such as influenza virus or respiratory syncytial virus (rsv) manage to infect a tissue ( figure a ), type i ifns are released from the infected cells and stimulate the expression of hundreds of genes [ ] , appropriately known as ifn-stimulated genes (isgs), in neighboring cells. this induces an antiviral state within minutes to hours that is characterized by reduced transcription and translation [ ] , the induction of enzymes that degrade viral rnas and proteins, and even the sensitization of cells to apoptosis [ ] . products of isgs, including cytokines and chemokines, also recruit leukocytes, including neutrophils and monocytes to the virally infected tissue [ ] . the induction of the ifn response following viral infections fundamentally changes the bone marrow microenvironment ( figure b) , leading to the enhanced differentiation of myeloid cells [ ] and emigration of neutrophils and monocytes to the site of infection, which is facilitated by chemokine gradients interacting with their cognate receptors ( figure a ) [ ] . murine ly c hi monocytes originate from the bone marrow and travel to sites such as skin, lungs, and lymph nodes [ ] , whereas ly c low monocytes typically scan vasculature and the endothelial cells lining the lumen for damage. the human counterparts to these subsets are cd + cd − and cd low cd + monocytes, respectively [ ] . monocytes require kruppel-like factor- to differentiate into inflammatory monocytes in vivo [ ] . a recent advance by yáñez and colleagues demonstrated that gmps and mdps can independently generate functionally distinct monocytes [ ] . gmps and mdps are both derived from cmp-flt + progenitors but differentiate into the above subsets when either the toll-like receptor (tlr)- agonist lipopolysaccharide (lps) or the tlr agonist cpg dna, respectively, are injected into mice. ly c hi monocytes can be derived from either subset [ ] . therefore, the innate pathogen-associated molecular patterns (pamps) and their cognate receptors, known as pattern recognition receptors (prrs), will dictate which monocyte subset is preferentially generated. infections can affect hematopoiesis and influence the proportions of cell subsets. human immunodeficiency virus (hiv) is capable of infecting bone marrow microvascular endothelial cells and provoking hematopoietic dysfunction [ ] . a plethora of regulatory signals required to differentiate and release myeloid cells, including granulocyte-colony-stimulating factor and interleukin (il)- , can be suppressed by hiv. human t-cell leukemia virus (htlv)- has recently been shown to infect several lineages of hematopoietic stem cells in addition to t-cells [ ] . both neutrophil and monocyte lineages were permissive to infection, as evidenced by the viral tax protein in neutrophils and the ability of monocyte progenitors to become infected. indeed, these infected monocytes were capable of differentiating into dcs and spreading the infection to t-cells. moreover, four subsets of neutrophils have been characterized in infants with viral respiratory infections [ ] . these subsets include suppressive, progenitor, mature, and immature neutrophils, which are present in the blood of infected individuals. however, cd high cd l low suppressive neutrophils were only observed in patients with bacterial co-infections. strikingly, the viral dysregulation of hematopoiesis can lead to numerous diseases [ ] . for example, the epstein-barr virus (ebv) causes infectious mononucleosis, characterized by a dramatic increase in white blood cells in the bloodstream. in rare instances, this virus can cause pancytopenia, which is a severe reduction in the number of platelets, red, and white blood cells [ ] . pancytopenia has also been found in patients who have contracted hepatitis c virus (hcv) [ ] . reducing the number of progenitor cells available to differentiate into lymphoid and myeloid cells may be a reasonably common viral strategy to avoid clearance by the immune system. the functional capacity of myeloid cells to respond to viral particles is influenced by the origin of their precursors. defining the molecular and cellular mechanisms underlying myeloid cell precursor development in viral illnesses will provide a better understanding of the susceptibility of patients to different viruses and the immunological events that may ultimately be exploited for therapeutic benefit. indeed, progenitor cells constitute a promising gene therapy target to treat hiv infections because they can differentiate into multiple cell lineages, all possessing a therapeutic transgene such as an anti-hiv ribozyme [ ] . other viral infections that, in theory, may be successfully treated by targeting hscs with gene therapies include viruses that dysregulate hematopoiesis, such as hcv or ebv. the human body relies on a robust innate sensory system to quickly eliminate many viruses. prrs are present in and on a variety of cells including neutrophils and monocytes to recognize pamps ( figure a ). tlrs are a subset of prrs that recognize pamps. there are multiple tlrs in and on neutrophils and monocytes that specifically recognize viral pamps or danger associated molecular patterns (damps) released from virus-damaged cells. the nucleic acid from rna and dna viruses constitutes a predominant source of viral pamps that can be recognized either via phagocytosis of cellular debris such as epithelial cells, or in cases where viruses infect myeloid cells. within endosomes, tlr recognizes dsrna from viruses (dsrna constitutes the genome of one family of viruses, but is also generated during the life cycle of many viruses) [ ] , ssrna is recognized by tlr and tlr [ ] , whereas tlr recognizes dna viruses while distinguishing from host dna [ ] . monocytes are activated via signaling through surface-bound tlr during varicella-zoster virus [ ] , measles [ ] , and type and herpes simplex virus (hsv) infections [ ] . tlr can recognize a wide range of viral pamps including the glycoproteins gb and gh/gl from hsv [ ] and hemagglutinin from measles [ ] . tlr stimulation after phagocytosis activates the nf-κb signaling cascade, resulting in the release of inflammatory cytokines such as tnf-α, il- , and il- from monocytes [ ] to control virus infections by direct antiviral mechanisms and the recruitment of other leukocytes. direct antiviral mechanisms of monocytes and neutrophils, including phagocytosis and oxidative burst, were reduced in patients who had contracted hcv and were taking ifn-based therapies [ ] . neutrophils also use tlrs to conduct anti-viral surveillance, and express ten out of eleven known human tlrs (they lack tlr ) [ ] . the endosomal tlr is essential for recognition of influenza viruses by neutrophils via sensing viral single-stranded rna when they phagocytose cell debris [ ] . lack of tlrs is associated with increased mortality during viral infections. for example, blocking tlr leads to increased mortalities associated with influenza virus infections by disrupting phagocytosis of infected cells [ ] . although influenza viruses do not contain lps, tlr activation is also involved with delaying fusion between lysosomes and phagosomes, thereby preventing virus entry, and thus has an additional role in innate immunity besides recognition of pamps [ ] . the multifaceted functions of tlrs should, therefore, be studied in greater detail to determine whether additional tlrs have unappreciated mechanisms to mitigate viral infections. another method for host recognition of viruses involves retinoic acid inducible gene-i (rig-i) and melanoma differentiation factor (mda ) [ ] . to clear viral infections, rig-i-like receptors and mda recognize cytosolic viral rnas via the helicase domain [ ] . in contrast to tlrs that are predominately present in leukocyte subsets, these receptors are ubiquitous in human cells. neutrophils and monocytes themselves can become infected by viruses [ ] and therefore possess cytoplasmic and endosomal mechanisms to recognize them, including rig-i and mda signaling cascades in the cytoplasm and endosomal tlrs. in fact, the double-stranded rna mimetic poly(i:c) stimulates neutrophils to increase many antiviral genes, including type i ifn mrna transcripts, ifn-responsive genes, tnf-α, and ifn regulatory factor (irf) [ ] . when infected with encephalomyocarditis virus (emcv), mda -deficient mice mount significantly reduced tnf-α and ifn-β responses [ ] . similar results were also observed after infections with coxsackie b virus (cvb) [ ] and west nile virus (wnv) [ ] . notably, tnf-α and ifn-β have the capacity to upregulate the expression of major histocompatibility complex molecules on antigen-presenting cells, which would make viruses more susceptible to t-cell-mediated clearance. damps are endogenous molecules that are released in response to tissue damage from trauma, including cells killed by viruses, and, like pamps, trigger an immune response ( figure a ). damps can be derived from a variety of cellular components, including the nucleus, cytoplasm, exosomes, plasma, or the extracellular matrix [ ] . damps that promote inflammation and immunogenic cell death [ ] include the chromatin protein high mobility group box (hmgb ) and mitochondrial damps such as mitochondrial dna and formyl peptides [ ] . hmgb interacts with neutrophils and monocytes [ ] by binding to the inflammatory receptor for advanced glycation end-products (rage). this damp causes monocytes to secrete pro-inflammatory cytokines, including il- and tnf-α, reorganize their cytoskeleton, and increases migration across epithelial barriers. monocytes are also capable of secreting hmgb themselves when lysosome exocytosis is induced by the inflammatory lipid lysophosphatidylcholine [ ] . neutrophils, in turn, have upregulated transcription of genes for pro-inflammatory molecules involving the nf-κb, p mapk, and erk / pathways in response to recognition of hmgb [ ] . cell damage from viral infections leads to a release of damps and subsequent detection by myeloid cells. for example, infection of epithelial cells with dengue viruses results in the release of hmgb from necrotic cells [ ] . the interaction between viral pamps and prrs in or on myeloid cells can play an essential survival role in the response to viral infections but may, simultaneously, be responsible for tissue injury associated with severe virus-induced inflammation. in theory, mechanisms involved in the recognition of danger signals by neutrophils and monocytes could be targeted selectively to enhance protection against detrimental viral infections while, simultaneously, preventing exaggerated, pathological innate immune responses. chemokines and their receptors play a critical role in dictating the migration and positioning of myeloid cells. an extensive list of chemokines, their receptors, and their various functions has been described [ ] . neutrophils and monocytes begin their journey to a site of infection by first leaving the bone marrow ( figure b ). neutrophil and monocyte retention in the bone marrow is dictated by steady signaling between the chemokine (c-x-c motif) receptor (cxcr ) and its ligand cxcl expressed on bone marrow stromal cells. during maturation, these cells downregulate cxcr and become less sensitive to cxcl , causing their release into the bloodstream [ ] . cxcr , and mainly cxcr expression, on neutrophils grants an additional form of chemotaxis away from the bone marrow via their respective ligands, cxcl and cxcl [ ] , which are produced by macrophages and mast cells at the site of infection [ ] . however, retention is typically favored in the steady state, as cxcl appears to be constitutively expressed in the bone marrow. inflammation mediated by viral infections that induce g-csf enhances cxcl release and decreases cxcr expression on bone marrow-resident neutrophils, tipping the balance in favor of neutrophil release [ ] . ly c hi inflammatory monocytes appear to require chemokine receptor (ccr ) signaling to efficiently exit the bone marrow and travel to sites of inflammation, whereas ccr signaling appears to be contextually dependent for monocyte emigration from circulation into virus-infected tissues [ ] . more research needs to be conducted to ascertain if ccr signaling is required to respond to viral infections of various tissues. ccr signaling, via ccl binding to the receptors on monocytes, causes the downregulation of cxcr and renders the monocytes less sensitive to cxc , causing their release from the bone marrow [ ] . interestingly, low concentrations of circulating tlrs cause rapid ccl release by mesenchymal stem cells and their progeny in the bone marrow, which triggers the release of monocytes [ ] . the dissemination of neutrophils and monocytes to virally infected tissues involves many complex processes ( figure b) [ , , ] . generally speaking, myeloid cell migration to infected tissues relies on transmigration through vascular endothelium from the blood. this transmigration is dictated by a milieu of cytokines, and chemokines produced by tissue injury and resident sentinel cells in response to damps and viral pamps. the disruption of homeostasis confers a change to the vascular endothelium near sites of infection [ ] . the multitude of changes to the endothelium can happen rapidly, and have been reviewed extensively elsewhere [ ] . in brief, endothelial changes that start and subside within minutes are known as type i activation and can be mediated by factors such as histamine [ ] ). alternatively, type ii activation can last hours to days with substantial changes in gene expression profiles mediated by tumor necrosis factor (tnf)-α [ ] . both forms of activation cause increased blood flow, vascular leakage of plasma proteins, and the recruitment of leukocytes [ ] . these disruptions in endothelium homeostasis can trigger a leukocyte adhesion cascade [ ] that, in harmony with various cytokines released by inflamed endothelium, such as il- and monocyte chemoattractant protein (mcp)- [ ] , initiates the selectin-mediated rolling of leukocytes along the surface of endothelial cells. trafficking of neutrophils and monocytes through the endothelium towards the site of infection is then facilitated by crawling via macrophage-antigen- (mac- /cd b) expressed on monocytes and the intercellular adhesion molecule- (icam- /cd ) expressed on endothelial cells [ ] . crawling appears to facilitate the paracellular (between cells) transmigration of neutrophils and monocytes, which is generally the preferred method of trafficking (occurring - % of the time), as opposed to transcellular (through cells) transmigration [ ] . the dissemination of neutrophils and monocytes from the vasculature into infected tissues is critical for viral clearance. neutrophils are initially recruited to sites of infection by their ability to recognize tissue damage via sensing of h o , dna, n-formyl peptides, adenosine triphosphate, uric acid, and other damps [ ] . further guidance to sites of infection is provided by a family of cxcl chemokines originating from concentrated sites of pamps and damps, including cxcl , cxcl , cxcl , cxcl , cxcl , cxcl , and cxcl (il- ), which are sensed by cxcr and cxcr on neutrophils and monocytes [ ] . within the context of viral infections, experimental data from mice infected with theiler murine encephalomyelitis virus have demonstrated that cxcl released from epithelial cells, macrophages, and neutrophils recruits both neutrophils and monocytes to sites of infection [ ] . macrophages infected with rotaviruses release cxcl to recruit neutrophils [ ] and nipah virus c protein is capable of inducing the release of numerous chemokines, including cxcl , cxcl , and cxcl , from endothelial cells [ ] . pamps from viruses tend to amplify neutrophil recruitment. the inflammatory ly c hi subset and the "patrolling" ly c low cx cr hi subset migrate along luminal and endothelial cell surfaces, with the latter being able to respond rapidly to infections in a cx cr -dependent fashion [ ] . the migration of inflammatory monocytes to tissues is ccr -dependent. however, as mentioned above, this tends to only be required to exit the bone marrow. nonetheless, ccr signaling appears to be critical for inflammatory monocyte recruitment in cases of west nile virus-induced encephalopathies, and influenza virus infections [ , ] . in summary, a range of trafficking signals and endothelial barrier regulatory molecules shape myeloid cell recruitment to virally infected and inflamed tissues. neutrophils are able to lyse and phagocytose virus-infected cells [ ] , and are one of the first leukocyte subsets to enter inflamed tissues ( figure c ). the magnitude of the neutrophil response is a predictor of the host's ability to clear an influenza virus infection with minimal damage [ ] . depleting neutrophils causes greater viral spread and host mortality [ ] , and neutrophils are also crucial to mitigate hsv type- corneal infections in a murine model [ ] . moreover, tate and colleagues demonstrated that neutrophils are critical for limiting the replication of influenza viruses [ ] and that a loss of neutrophils increases disease severity. thus, the antiviral response of neutrophils contributes to clearing viral infections. neutrophils can directly mediate innate immune responses, activate adaptive immunity and recruit lymphoid cells to sites of viral infections [ , ] . a key mechanism of action that enables neutrophils to neutralize invading viruses is the production of neutrophil extracellular traps (nets) [ ] . nets are strands of dna and granule proteins secreted by neutrophils that form around viral particles, preventing their spread [ ] . poxvirus infections in mice were mitigated in liver microvasculature via this mechanism [ ] . in addition to the physical containment of infections, nets are coated with antiviral enzymes that enable neutrophils to concentrate lethal antimicrobial proteins such as histones at sites of infection [ ] . neutrophils are also capable of mediating antibody-dependent cellular cytotoxicity (adcc) or antibody-dependent phagocytosis, which involve the release of cytolytic granules or phagocytosis, respectively, after binding antibodies via fc receptors [ ] . these antibody-dependent processes are critical in the clearance and neutralization of certain viruses such as hiv [ ] . adcc responses peak quickly (i.e., within four hours) and are controlled by the fcγr family of receptors and can also utilize the extracellular release of reactive oxygen intermediates [ ] . reactive oxygen intermediates are also involved in other pathological responses, including exocytosis. exocytosis is a cellular active transport process whereby membrane-bound vesicles transport molecules to the cell surface. neutrophils emit an array of compounds including myeloperoxidase to control sepsis [ ] , antiviral lysozyme with anti-hiv properties [ ] , and n-formyl-methionyl-leucyl phenylalanine (fmlf)-stimulated superoxide release in the presence of periodontitis pathogens [ ] . exocytosis, therefore, expands the neutrophil arsenal to neutralize the array of pathogens they encounter. neutrophils are incredibly diverse in their functions. in addition to trafficking to sites of infection to phagocytize viruses and form nets, they also stimulate virus-specific adaptive immune responses [ ] . neutrophils that have detected viral antigens can home to draining lymph nodes dependent on il- r, where they can act as antigen-presenting cells [ , ] . neutrophils present processed viral antigens to naïve cd + t-cells via the major histocompatibility complex i and t-cell receptor interactions, along with the expression of cd and cd to provide co-stimulation, thereby providing the two signals required to activate t-cells [ ] . furthermore, neutrophils are responsible for the recruitment of effector cd + t-cells to sites of viral infections. the mechanism by which they recruit t-cells during influenza virus infections has been linked to cxcl deposits left behind like a "trail of breadcrumbs". cd + t-cells follow this chemoattractant trail left behind by neutrophil uropods to the sites of influenza virus infections [ ] . rsv causes lung infections that are characterized by neutrophils contributing to host damage [ ] . rsv is capable of delaying the apoptosis of neutrophils and eosinophils, which is hypothesized to delay antigen presentation and increase tissue damage. il- and tlr / binding was determined to contribute to this delay and depended on nf-κb and pi k activation. the authors of this study did not directly examine whether this delay resulted in an increase in host tissue damage in their model, but hypothesized this was the case, constituting an area of future study. during an rsv infection, neutrophils migrate through infected airway epithelial cells [ ] . these neutrophils are characterized by the increased expression of myeloperoxidase and cd b, and their migration promotes epithelial shedding and airway tissue damage. aside from delaying apoptosis, rsv infection has also been shown to increase eosinophil recruitment and degranulation based on the macrophage inflammatory protein (mip) -α and eosinophil cationic protein concentrations measured in lower respiratory airway secretions [ ] . ly c hi monocytes migrate to injured sites, induce inflammation, and eliminate the cause of tissue injury ( figure c ) [ ] . for instance, type i ifns amplify the production of mcp- , the primary chemokine responsible for recruiting inflammatory monocytes to the lungs during influenza virus infections [ ] . these monocytes have been implicated in influenza virus-induced lung injury [ ] . importantly, elevated mcp- levels have been associated with severity of illness in pediatric influenza virus infections [ ] . in mice, the recruitment of monocytes to lungs was shown to be accompanied with an increase in type i ifn production, nlrp inflammasome activation, and alveolar epithelial barrier dysfunction [ ] . it has been identified that increased pro-inflammatory monocytes are a major immunological determinant of severity of disease in previously healthy adults with life-threatening influenza virus infections [ ] . this provides a possible mechanistic cause for disease severity in these patients, a potential early identifier and a modifiable immune pathway for therapeutic targeting. however, there is no role for recruited monocytes in the lungs of mice infected with the natural rodent pathogen, pneumonia virus of mice [ ] , indicating the pathogen-specific functions of these cells. interestingly, monocytes have been proposed to be educated in the bone marrow to promote their tissue-specific functions at sites of persistent challenge [ ] . long-lasting epigenetic alterations in monocyte precursors may account for the "trained immunity" phenomena [ ] . indeed, monocytes have an immunological memory of past insults. thus, this evidence shows that neutrophils and inflammatory monocytes participate in inflammation that is needed for an effective immune response against viruses. a shared feature of neutrophils and monocytes is their ability to synthesize pro-inflammatory cytokines that help the host overcome viral diseases. however, these responses can also be overly robust, thereby contributing to virus-induced tissue damage. future research directions should include a focus on furthering our understanding of the diverse antiviral arsenal of myeloid cells. robust immune responses are critical for protecting hosts against lethal viral infections. it is equally important that immune responses are of adequate magnitude and duration. the capacity for a host to resolve inflammation and return to homeostasis has important consequences for health ( figure d ). the induction of an immune response that is too severe or the failure to return to homeostasis can result in immunopathology [ ] , including tissue and organ damage [ ] , cytokine storms ( figure d ) [ ] , chronic inflammation [ ] , and autoimmune diseases [ ] . as innate immune responders, myeloid cells are key players in orchestrating appropriate inflammatory responses and the return to homeostasis following virus infections. the role of myeloid cells in the regulation of immune responses is complex and involves specialized cellular subsets, suppressive receptors, and cytokines. in addition, much of what we know about the regulatory and immunosuppressive effects of myeloid cells originates from research investigating bacterial, fungal, and sterile inflammation models, but has implications for virus infections. neutrophils possess multiple mechanisms to control inflammation, despite their predominately pro-inflammatory role ( figure d ) [ ] . one mechanism involves the formation of aforementioned nets [ ] . these nets function via serine proteases to degrade excess cytokines and chemokines in areas with high densities of neutrophils [ ] . neutrophils are also capable of reducing lung injury during influenza virus infections [ ] . a neutrophil depletion study in a h n murine model demonstrated that their absence led to weight loss, viremic spread, and increased inflammation. the basic neutrophil function of clearing an inflamed area by removing killed pathogens and host cells contributes to reduced inflammation and wound debridement [ ] . they are also capable of healing mucosal regions of the intestine [ ] , and increasing angiogenesis [ ] . a recent advance in our knowledge of neutrophils concerns their ability to de-prime [ ] . originally considered an irreversible process, neutrophils are capable of returning to quiescence. neutrophils can be spontaneously de-primed in the circulatory system via the degradation of a superoxide anion response [ ] , with a de-priming half-life of approximately forty minutes [ ] , or retained in the bone marrow [ ] to limit the number of primed cells that can traverse the body and cause damaging effects such as lung injury [ ] . recent experimental data have demonstrated that inflammatory monocytes are capable of exhibiting suppressive properties. inflammatory monocytes are recruited to sites of vaccine-mediated inflammation via mcp- [ ] . within the vaccine draining lymph node, monocytes sequester cysteine, resulting in t-cell suppression [ ] . blocking monocyte suppression in this context may prove to be an effective mechanism to improve vaccine effectiveness. monocytes are also capable of suppressing b cells. in vitro studies have demonstrated that monocytes suppress b cell differentiation, proliferation, and ig class distribution [ ] . monocytes, therefore, represent a prime example of a cell type that can be both pro-inflammatory and suppressive, depending on the context. the resolution of immune response is an active regulatory process, which is initiated via the release of soluble mediators such as cytokines and chemokines, as well as through cell-to-cell interactions mediated by surface-expressed ligands and receptors [ ] . evidence has revealed that monocytes that are part of inflammation also can be reprogrammed to cells that are highly anti-inflammatory and contribute to resolution of inflammation [ ] . moreover, during sepsis, human monocytes have been shown to undergo a transition from a pro-inflammatory to an anti-inflammatory status [ ] , although it remains unclear whether the conversion of monocytes from pro-inflammatory to a regulatory phenotype occurs in viral diseases. further studies are needed to understand the mechanisms to explain how monocytes can be switched into suppressor/anti-inflammatory cells during a viral infection, which in turn would allow intervention with targeted therapeutics to control and down-modulate excessive inflammation in viral diseases. an additional myeloid subset of interest is the myeloid-derived suppressor cells (mdscs). mdscs can suppress immune responses in numerous anatomical locations, including tumor microenvironments, virally infected tissues, and sites of inflammation. the subset of mdscs with neutrophil-like properties have been designated polymorphonuclear (pmn)-mdscs or granulocytic (g)-mdscs, while their myeloid counterparts have the nomenclature m-mdscs. viral infections can induce mdscs, as is the case with hcv [ ] . cd + mdscs were upregulated upon co-culture with hcv infected hepatocytes, resulting in t cell suppression mediated by reactive oxygen species. moreover, nk cells are also suppressed by mdscs during hcv infection [ ] . the production of mdsc-derived arginase- resulted in a decrease in ifn-γ production by nk cells. the suppression of key effector cells contributes to viral persistence. hcv is not the only virus to control mdscs to evade the immune system. patients with hiv- have m-mdsc populations that suppress helper t cells [ ] , and elevated levels of these myeloid cells were correlated with increased viral loads. future research should focus on determining whether other viruses engage mdscs to prolong infections. additionally, more research is required to fully determine the position these subclasses have in myeloid cell differentiation. although a recent review concluded that mdscs constitute bona fide alternate lineages [ ] , future studies will be required to cement their status within the field of immunology. myeloid cells are able to translate micro-environmental cues into an effector profile that initiates lymphocyte responses [ ] . innate lymphoid cells (ilcs) react to pathogens indirectly through myeloid or epithelial cell-derived cytokines and other inflammatory mediators including il- , il- , and il- [ ] . ilcs are derived from a lymphoid progenitor but do not contain either a b or t-cell receptor due to the absence of the recombination-activating gene [ ] . there are three major subsets of ilcs: groups , , and . group includes cells that produce ifn-γ and tnf-α and is predominately composed of classical natural killer (nk) cells. ilcs that require gata and rorα to develop and express the cytokines il- and il- are denoted as group , while intestinal ilcs that express nkp and depend on rorγ comprise group [ ] . since evidence shows that ilcs are tissue-resident cell types with limited capacity to directly recognize pamps [ ] , myeloid cells may play a crucial role in controlling ilc homeostasis and function [ ] . in the steady state, monocytes enter tissues and replenish macrophages and dcs [ ] . however, during viral infections they are recruited to infected tissues and mediate direct antiviral activities [ ] . for instance, in mice infected with murine cytomegalovirus, inflammatory monocytes are recruited to the liver and produce mip- a, which recruits nk cells [ ] . nk cells are relevant to viral infections because they target infected cells for destruction. nk cells are cytotoxic ilcs that require il- to develop, differentiate, and survive [ ] . il- is secreted by several cell types, including monocytes after viral recognition [ ] , which therefore places nk cells under the control of myeloid cells. expression of the activating receptor nkg d is upregulated on nk cells in response to il- . il- -activated nk cells show preferential expression of the tnf-related apoptosis-inducing ligand (trail) as well as activation and phosphorylation of erk and , and increases in perforin production [ ] . the increased expression of these activating receptors and effector compounds increases the killing potential of nk cells. many viruses down-regulate the expression of mhc on infected cells to escape detection by cd + t-cells [ ] . therefore, il- secretion by monocytes constitutes a mechanism to upregulate multiple cell receptors. changes in granzyme regulation were not documented in these studies, but represent an area of future investigation due to the role of this compound in the apoptosis of virus-infected cells. human monocytes express membrane-bound il- constitutively, with its expression increased in the presence of ifn-γ [ ] . the monocyte-mediated production of il- was increased in the presence of the anti-inflammatory cytokine il- , but was unaffected by il- or il- [ ] . il- also influences monocytes and can transform them into dcs in airway epithelia [ ] , which has implications for improving the presentation of viral antigens, suggesting a cross-talk between nk cells and myeloid cells under viral inflammatory conditions. recently, ashkar and colleagues [ ] showed that type i ifns produced during a viral infection stimulated vaginal mcp- production, which is a chemoattractant that is responsible for inflammatory monocyte migration to inflamed sites. once recruited, type i ifns stimulate inflammatory monocytes to produce il- , which then signals through the il- receptor expressed by nk cells to induce their production of ifn-γ. interestingly, cytokine il- also promotes the secretion of ifn-γ by nk cells [ ] and neutrophils [ ] . neutrophils can also increase ifn-γ production by nk cells using multiple pathways. the first method is to interact with dcs via icam- to further upregulate il- p [ ] , creating a positive feedback loop. the direct co-stimulation of nk cells also occurs with cd and icam- binding on neutrophils and nk cells, respectively [ ] . our unpublished data (personal observation by karimi k and bridle b) have demonstrated that the induction of viremia in mice, which induces the release of high concentrations of inflammatory cytokines into the circulation, is accompanied by increased numbers of pulmonary ilc subsets and the accumulation of multiple myeloid cell subsets that, interestingly, were type i ifn-dependent (data not shown). additionally, we demonstrated that the induction of inflammation by concanavalin a in mice, which occurs due to macrophage activation downstream of the rapid stimulation of t-cells, led to increased numbers of ilc populations in all organs examined, including the bone marrow, spleen, and liver [ ] (unpublished data). recently, mortha and burrows [ ] discussed how the feedback communication between ilcs and myeloid cells contributes to stabilize immunological homeostasis. further studies are needed to dissect cell-to-cell interactions between myeloid cells and ilcs other than nk cells in viral inflammatory conditions. the concept that neutrophils can initiate, amplify and/or suppress adaptive immune effector responses by establishing direct bidirectional cross-talk with t-cells has garnered attention in the past few years [ ] . a th response can be induced by neutrophils in a murine model [ ] , which increases the number of cd + cytotoxic t-cells available to lyse virally infected cells. indeed, in vivo murine studies have demonstrated that neutrophils can cross-present ovalbumin to cd + t-cells in a tap-and proteasome-dependent manner [ ] . neutrophils can further impact the adaptive immune response by inducing dc maturation, which in turn increases antigen presentation to adaptive cells [ ] . neutrophils have been observed to cluster with immature dcs and bind their mac- to dc-specific intercellular adhesion molecule- -grabbing non-integrin (dc-sign). dc-sign is also referred to as cd and is a prr that recognizes and binds to mannose residues, a conserved pamp associated with a variety of viral infections. however, neutrophil depletion studies have demonstrated an increase in antigen presentation to cd + t-cells. the mechanism by which this phenomenon occurs is thought to be a reduction in competition for viral antigens between neutrophils and dcs [ ] . there are extensive demonstrations that neutrophils in humans and mice can also suppress t-cell responses ( figure d ). suppressive neutrophils that express low levels of cd l are induced after acute inflammation arising from either viral infections or tissue injury [ ] . they have been shown to impair t-cells by releasing hydrogen peroxide into an immunological synapse, which impairs t-cell migration via the cxcl chemokine gradient. ball and colleagues have shown that cxcl -induced migration to sites of infection decreases as the concentration of hydrogen peroxide released into the immunological synapse is increased. results demonstrate the impaired recruitment of th and cd + t-cells to the periphery. ultimately, the mechanistic consequence pertains to defective migration mechanisms rather than tcr:mhc signal transduction. it is also important to note that this interaction required mac- (cd b). additional research has demonstrated that mac- -expressing neutrophils are crucial in limiting pathology caused by t-cells in a murine model of infection with influenza virus, presumably by suppressing t-cell proliferation [ ] . we have demonstrated that a subset of neutrophils function as negative regulators of excessive cytokine production in a mouse model of viremia, in which type i ifn signaling has been disrupted (karimi k and bridle b, unpublished data). altogether, these findings allow us to envision the therapeutic potential of subsets of neutrophils. however, one of the major challenges would be the heterogeneity of immunosuppressive or regulatory neutrophils. future studies taking advantage of flow cytometry technology and next-generation sequencing to phenotypically and functionally define neutrophil subsets will extend our knowledge about the immunoregulatory role neutrophils play in viral infections and inflammation. neutrophils also have an indirect mechanism to modulate t cells during a viral infection. the bacteria mycobacterium tuberculosis is capable of delaying neutrophil apoptosis, which delays an adaptive cd + t-cell response [ ] . although this has not been demonstrated via a viral infection, it nonetheless demonstrates a key effect neutrophils have on controlling a cd + t helper cell response. this response may be delayed because dcs ingest whole infected neutrophils [ ] to acquire antigens and present them to t-cells. additionally, dcs that ingest neutrophils possessing pathogen-derived antigens can migrate to lymph nodes more efficiently [ ] . the differentiation of inflammatory monocytes into cd b + pulmonary dcs is triggered by the presence of respiratory viruses such as influenza virus [ ] . defects in this differentiation delay the clearance of influenza viruses and significantly reduce the activation of cd + t-cells [ ] . while inflammatory monocytes are key regulatory cells in maintaining macrophage and dc populations in healthy tissues, a function of homeostasis, they are quintessential in the clearance of infections due to their ability to induce adaptive immunity and prime a variety of lymphocytes, including t-cells ( figure c ) [ ] . upon viral infection, inflammatory monocytes in the blood are recruited to the primary site of infection or the draining lymph node. cells that traffic to the primary site of infection play a critical role in the recruitment of t-cells and, thereby, the activation of inflammatory responses and cellular immunity [ ] . however, inflammatory monocytes that traffic to draining lymph nodes acquire a dc phenotype that enables them to present viral antigens to naïve t-cells [ ] . in particular, studies have shown that inflammatory monocytes stimulate a th -biased immune response via production of il- that promotes production of ifn-γ by t cells primed in lymph nodes [ ] . this th immunity is critical in the defense against intracellular pathogens, such as viruses [ ] . although memory is traditionally considered a hallmark of the adaptive immune response, recent advances have shed light on the contributions of innate memory. innate memory, also referred to as trained immunity, is a multifaceted response. a recent component of trained immunity involves its modulation of hematopoiesis [ ] . although myeloid cells have a short lifespan in circulation, the administration of the agonist ß-glucan resulted in myeloid progenitor expansion and subsequent improved responses to a secondary challenge with the agonist lps. trained immunity was able to reduce myelosuppression from chemotherapy, and was associated with metabolic shifts in cholesterol biosynthesis and glucose metabolism [ ] . other benefits of innate myeloid memory have been elegantly reviewed by netea and colleagues [ ] . in brief, monocytes are influenced by vaccination and viral infections, and are more responsive upon re-challenge. this innate memory response helps mitigate pathogens via upregulated cytokine production and enhanced pathogen elimination response times. this exciting new field may allow vaccines to be optimized for viruses by targeting the innate memory response. clearly, the cross-talk that is occurring between monocytes, neutrophils, and t-cells constitutes a crucial bridge between innate and adaptive immunity. future investigations are encouraged to examine the full extent of communication between these cells, further elucidate the mechanisms, and the anatomical locations of these interactions. depletion assays will be beneficial to determine which cell subsets can mount effective anti-viral responses, not just by t-cell and apc interactions, but also by direct interactions with neutrophils and monocytes. extensive studies have highlighted the role type i ifns play in initiating an anti-viral state in cells through the inhibition of viral replication [ ] . in some cases, the disruption of this response results in the excessive production of cytokines, leading to a so-called cytokine storm that can be very toxic ( figure e ) [ ] . this is a cause of mortality in cases of severe acute respiratory syndrome (sars) [ ] , infection with some strains of influenza viruses [ ] , ebola virus [ ] , and dengue virus [ ] . during viral infections, the regulation of cytokine networks and the mechanisms by which the cytokines may interact with neutrophils and monocytes are poorly documented. the fatal outcome of severe influenza infections is shown to be correlated with the early persistent production of inflammatory cytokines and chemokines that recruit neutrophils and monocytes [ , ] . lethal outcomes of h n influenza infections in humans correlated with early excessive innate immune response, involving type i ifns followed by prolonged inflammatory responses, and were associated with high viral loads and hypercytokinemia [ , ] . while inflammatory cytokines and chemokines are absolutely essential for the effective control of viral infections, they can also contribute to the severity of disease [ , ] . other fatal viral infections that are hallmarked by dysregulated type i ifn responses and cytokine storms are hantaviruses [ ] and wnv [ , ] . given the dynamic nature of cytokines, the complexity of signaling pathways they interact with, and the fact that their excessive production is often associated with some of the worst clinical outcomes of viral infections, there is a need for much more research into the mechanisms by which virus-induced cytokine storms are triggered or controlled. investigation into the mechanisms involved in host responses to viral infections demonstrates a complex and carefully balanced interaction between type i ifns and inflammatory neutrophils and monocytes. recent analysis of mrnas in the blood of humans responding to infections with influenza viruses revealed that early gene expression patterns of anti-viral molecules, such as the genes encoding for myxovirus resistance protein- (mx ) and isg- , are correlated with the heightened production and activation of type i ifns after viral infections [ ] . late gene expression patterns were also induced by type i ifns, but in contrast to patterns of antiviral molecules being observed, the transcriptional profiles of patients in the late stages of infections were highly reflective of neutrophil and inflammatory molecule activation [ ] , suggesting an important interplay between the secretion of type i ifns and the activation of neutrophils and inflammatory monocytes. it is important to study the receptors mediating the neutrophil antiviral response to reduce aberrant host responses and damage. nlrp is a nucleotide-binding domain leucine-rich repeat protein that is expressed on blood-derived leukocytes, including monocytes, and modulates neutrophil recruitment by increasing the chemokine cxcl through the il- -nlrp axis and increasing vascular permeability [ ] . another activator and recruiter of neutrophils is produced by liver cells and is entitled serum amyloid a (saa) [ ] . injections of saa increased phagocytosis of influenza viruses by neutrophils, resulting in the release of il- . modulating these protein concentrations might represent a promising therapeutic strategy to achieve ideal neutrophil responses to promote elimination of influenza viruses without excessive bystander damage to tissues. neutrophil-mediated antiviral responses have varying effects on the outcome of influenza virus infections, depending on the strain of virus [ ] . neutrophils contributed to terminating infections with h n influenza virus strains of intermediate virulence and h n strains that were highly virulent, while they did not limit the severity of disease during infection with an h n strain of low virulence. the early production of virus-induced type i ifns has been observed to upregulate genes in neutrophils that encode pro-apoptotic molecules, such as ifn-induced dsrna-activated protein kinase, and the oligoadenylate synthase-like proteins and the rnase l system [ ] . experiments with irf- -/x irf- -/double-knockout mice and wnv [ ] concluded that the viral induction of cellular ifn-β secretion depends on interferon-β promoter stimulator- -mediated signaling without requiring the ifn transcription factors irf / , suggesting the essentiality of the immediate and optimal activation of the type i ifn response. sars-coronaviruses are highly pathogenic and cause alveolar damage, fibrin deposition, and tissue necrosis [ ] . the delayed expression of the type i ifn response in mice infected with sars-coronaviruses was implicated in the promotion of inappropriate and chronic inflammatory responses, such as excessive inflammatory monocyte, neutrophil and cytokine accumulation, and impaired virus-specific t-cell responses due to augmented t-cell apoptosis, leading to lung damage [ ] . in contrast, an early type i ifn response reduced the immunopathological damage observed, linking the early activation of the type i ifn response to the control of overly robust inflammation. additionally, type i ifns have been implicated in the regulation of myeloid cell migration during initial exposure to viral infections, heightening inflammatory and virus-specific b and t-cell responses [ , ] . the production of type i ifns by sentinel leukocytes, in particular that of plasmacytoid dcs that serve as a potent source of ifns, upon viral infection initiates a type i ifn-dependent secretion of neutrophil and inflammatory monocyte chemoattractants such as il- α, cxcl and cxcl [ , ] , highlighting the role of virus-induced type i ifns in the regulation of neutrophil and monocyte trafficking. pollara et al. [ ] demonstrated that the secretion of type i ifns by hsv- -infected myeloid dcs results in the activation of uninfected dcs. this process enables the adaptive immune system to become activated even during a viral infection that targets myeloid cells and prevents their maturation, such as in the case of hsv. the protective functions of type i ifns have been associated not only with the recruitment of neutrophils and inflammatory ly c hi monocytes to sites of viral infections, but also with the prevention of excessive monocyte and neutrophil activation, thereby controlling inflammation caused by type ii ifns, such as ifn-γ [ ] . the interplay between type i and ii ifns was crucial for mitigating damage stemming from influenza a virus-induced inflammation in rag -/-, ifnar -/-, ifngr -/and stat -/-c bl/ mice [ ] . both ifns were required to prevent excessive numbers of neutrophils trafficking into lungs. stat was experimentally determined to coordinate inflammation via type i and ii ifn receptors. when type i ifns were absent, ly c lo monocytes transitioned to being more inflammatory than ly c hi monocytes. in the absence of type i ifn signaling, ly c lo monocytes traditionally associated with tissue re-modeling became phenotypically and functionally more pro-inflammatory during infection with influenza a viruses [ ] . notably, infection of trophoblasts with zika virus induced a lower secretion of type i ifns, and higher immunopathological inflammatory immune responses when compared to trophoblasts infected with yellow fever virus and dengue virus [ ] . measurement of immune mediators in nasal fluids from rsv-infected infants indicated that severe disease caused by heightened inflammatory responses was also associated with diminished type i ifn responses [ ] , furthering the idea that a link between type i ifns and the promotion versus suppression of virus-induced inflammation exists. taken together, these findings suggest that type i ifn signaling drives a balance of pro-and anti-inflammatory effects on the functions of monocytes and neutrophils in response to viral infections; providing protective immunity while simultaneously limiting immunopathology. these results suggest that the administration of type i ifns at optimized time points and doses could prove beneficial in the limitation of toxic cytokine storm onset and the control of excessive immunopathological damage. indeed, in vitro evidence suggests that the administration of exogenous type i ifns can mitigate excessive cytokine production induced by sars-coronaviruses [ ] . determining the means by which type i ifns control excessive inflammation while ensuring effective anti-viral responses is required. neutrophils, inflammatory monocytes, and their roles in mitigating bacterial infections have been extensively studied and well characterized. exciting new research in immunology and virology has demonstrated that these first responders of the innate immune system are also crucial in limiting viral infections, replication, and associated off-target pathological damage. a multifaceted range of tactics is utilized to combat an equally diverse range of viruses, including phagocytosis, the formation of extracellular traps, the production of cytokines such as ifns, and modulation of ilcs and lymphocytes. despite rapid advances in the field, many exciting unknown aspects of the involvement of neutrophils and inflammatory monocytes in combating viral infections remain to be clarified. current research has documented the impact of neutrophil/monocyte retention in the bone marrow as it pertains to viral infections, but we still do not completely understand all mechanisms by which myeloid cells are recruited from the blood stream to the primary sites of infection. future studies should aim to elucidate the specific signaling cascades that recruit myeloid cells into infected tissues and the mechanistic consequences of disruptions in these cascades via the chemokine gradient as well as depletions of specific ligands. if the scientific community can determine how different cell subsets can influence the production of chemokine populations and hone in on the essential ligands required for migration into the primary sites of infection, drugs could potentially be developed to exploit this localized production of chemokines. the discovery of pharmaceuticals that could fine-tune myeloid cell trafficking could prove beneficial to inducing rapid antiviral responses. differential ligation versus the blockade of prrs associated with protective versus pathological inflammation constitutes another strategy to balance rapid viral clearance and minimize host damage. current knowledge from myeloid cell studies in bacterial diseases demonstrated that neutrophils are essential for monocyte recruitment and function. additionally, it has been shown that the ratio of neutrophils to lymphocytes is higher in bacterial than viral infections among patients hospitalized for fevers [ ] . it is clear that neutrophils and monocytes work in concert to enhance immune responses against bacterial pathogens. however, future studies are needed to explore the mechanisms by which these myeloid cells collaborate with each other to control viral infections, with the aim of gaining new insights into how they function in virus-infected microenvironments to regulate cell-to-cell communication within the innate and adaptive arms of the immune system. gaining a better understanding of the role of myeloid cells in the pathogenesis of viral diseases will facilitate the design of better therapies. importantly, viruses and virus-mediated tissue damage stimulate both neutrophils and monocytes, triggering a cascade of cytokine/chemokine-mediated innate immune responses. this antiviral activity is not always beneficial for a host and, when improperly regulated, may contribute to immunopathologies such as cytokine storms that have been observed in many severe viral infections and could 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interferons is essential to limit influenza a virus-induced tissue inflammation herpes simplex virus type- -induced activation of myeloid dendritic cells: the roles of virus cell interaction and paracrine type i ifn secretion dengue and yellow fever viruses induce differential anti-viral immune responses in human monocytic and first trimester trophoblast cells differential ability of pandemic and seasonal h n influenzaa viruses to alter the function of human neutrophils characterization of the antiviral effects of interferon-alpha against a sars-like coronoavirus infection in vitro role of neutrophil to lymphocyte and monocyte to lymphocyte ratios in the diagnosis of bacterial infection in patients with fever key: cord- - zgc xrb authors: zhao, shan; smits, constance; schuurman, nancy; barnum, samantha; pusterla, nicola; van kuppeveld, frank; bosch, berend-jan; van maanen, kees; egberink, herman title: development and validation of a s protein-based elisa for the specific detection of antibodies against equine coronavirus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: zgc xrb equine coronavirus (ecov) is considered to be involved in enteric diseases in foals. recently, several outbreaks of ecov infection have also been reported in adult horses from the usa, france and japan. epidemiological studies of ecov infection are still limited, and the seroprevalence of ecov infection in europe is unknown. in this study, an indirect enzyme-linked immunosorbent assay (elisa) method utilizing ecov spike s protein was developed in two formats, and further validated by analyzing paired serum samples (acute and convalescent sera) from horses involved in an ecov outbreak and sera of horses with unknown ecov exposure. both formats showed high diagnostic accuracy compared to virus neutralization (vn) assay. receiver-operating characteristic (roc) analyses were performed to determine the best cut-off values for both elisa formats, assuming a test specificity of %. employing the developed elisa method, we detected seroconversion in . % of horses from an ecov outbreak. among the horse sera, seropositivity varied from . % (young horses) to . % (adult horses) in dutch horse populations. further, sera of icelandic horses were included in this study and a significant number of sera ( %) were found to be positive. overall, the results demonstrated that the ecov s -based elisa has reliable diagnostic performance compared to the vn assay and is a useful assay to support seroconversion in horses involved with ecov outbreaks and to estimate ecov seroprevalence in populations of horses. coronaviruses (covs) are enveloped, positive single-stranded rna viruses that belong to the subfamily orthocoronavirinae in the family coronaviridae of the order nidovirales. they are classified into four genera (alpha-, beta-, gamma-and deltacoronavirus) and infect both mammalian and avian hosts [ , ] . equine coronavirus (ecov) belongs to betacoronavirus species, within the embecovirus subgenus of the betacoronavirus genus, as does human coronavirus oc , hku and bovine coronavirus [ ] . ecov was isolated for the first time from a two-week-old diarrheic foal in north carolina (usa) in , suggesting the role of ecov in causing enteric disease [ ] . since , several cases of ecov infections have also been reported in adult horses from the united states, europe and japan [ ] [ ] [ ] [ ] [ ] . equine coronavirus has been detected in fecal samples from horses with clinical signs that included anorexia, lethargy, fever and, less frequently, diarrhea, colic and neurologic deficits [ , ] . the morbidity rate varies from % to % during outbreaks. mortality is low and has been related to endotoxemia, septicemia or hyperammonemia-associated encephalopathy [ , ] . the outbreaks in adult horses demand further studies on the pathogenesis and epidemiology of ecov infections. for this, diagnostic assays with high sensitivity and specificity are crucial. ecov is known to be associated with enteric infections but can also be detected in a small percentage of horses with respiratory signs. virus shedding can be observed in fecal samples or nasal swabs from sick horses as well as healthy horses, but with a strong association between clinical signs assumed to be related to ecov infection and virus detection in fecal samples suggesting a possible etiological role of ecov [ , ] . recently, real-time quantitative pcr (qpcr) methods have been established and were shown to be able to detect ecov in feces efficiently. however, ecov viral nucleic acid is generally only detectable by qpcr within a limited timeframe of - days post infection, as reported from both field and experimental studies [ , , , ] . on the other hand, serological assays can be used to support the diagnosis of a clinical ecov infection by showing seroconversion or a significant increase in antibody titer in paired serum samples. serological assays are also needed to gain more insight into the transmission rate of infection within animal populations [ ] . antibodies induced by betacoronaviruses persist in blood for a longer period after infection [ , ] . the virus neutralization (vn) assay has long been used as a gold standard to confirm serological responses to coronavirus infections [ ] [ ] [ ] . although the vn assay is highly specific for the detection of antibodies, it is also time-consuming and laborious to perform. alternative high-throughput serologic assays that correlate well with neutralizing antibodies are therefore needed. severe infections of ecov have been shown to be associated with high viral load, but mild or asymptomatic infections may occur with low levels of virus replication being negative in pcr and with variable immune responses [ ] . consequently, specific, sensitive and high-throughput serodiagnostic methods are necessary to avoid the underestimation of prevalence in surveillance studies. the spike protein (s) of coronaviruses is the key mediator in virus cell entry and therefore the major target for neutralizing antibodies. the s ectodomain consists of two functionally interdependent subunits, s and s . the n-terminal s subunit is responsible for receptor binding, while the c-terminal s subunit mediates membrane fusion [ , ] . the s subunit is the most variable immunogenic antigen among coronaviruses, and therefore it is an ideal candidate for the detection of cov species-specific antibodies [ , ] . the objective of the study was to develop and validate an elisa method for the detection of specific antibodies to ecov and provide a tool for the diagnosis and the future estimation of ecov prevalence and incidence in various equine (sub) populations. a total of equine serum samples were included in this study. the details of serum panels a-h (n = ) are shown in table . they were retrieved from the serum bank at gd animal health deventer, the netherlands. all of them were collected for the monitoring of other diseases independent to this study, and their ecov exposure status was unknown. with the exception of panel h (collected from iceland), all serum samples from panel a to g were collected from horses in the netherlands. additionally, panel i included paired (acute-and convalescent-phase) serum samples that were collected during an ecov outbreak in the usa ( ). all samples were stored at − • c until tested. ecov strain nc was propagated and titrated in human rectal adenocarcinoma (hrt- g) cells. hrt- g cells and human embryonic kidney cells stably expressing the sv large t antigen (hek- t) were maintained in dulbecco modified eagle medium (dmem, lonza, basel, switzerland) the sequence of the s subunit of the spike protein of the ecov nc strain (residue - of the amino acid sequence) was derived from genbank (genbank no.: ef . ). human codon-optimized sequences encoding the ecov s subunit were synthesized and fused to the fc domain of mouse igg a, which was subsequently cloned into the pcaggs mammalian expression vector as described before [ ] . for ecov s -fc protein production, expression plasmid was transfected into hek- t cells using polyethyleneimine (polysciences, inc., warrington, pa, usa) in a ratio of : . after h of incubation, the transfection medium was removed and replaced by sfm ii expression medium (gibco ® , life technologies inc., grand island, ny, usa). at six days post transfection, cell culture supernatants were harvested and the soluble s was purified from the culture medium using protein a sepharose beads (ge healthcare bio-sciences ab, uppsala, sweden). subsequently, the proteins were eluted using . m citric acid, ph . , and immediately neutralized with m tris-hcl, ph . . the purity and integrity of proteins were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page) and stained with gelcodeblue stain reagent (thermofisher scientific inc., waltham, ma, usa). purified proteins were quantified by nanodrop spectrophotometry (thermofisher scientific inc., waltham, ma, usa) and by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page) with bovine serum albumin (bsa) as standard, then stored at − • c until further usage. equine sera (n = ) were randomly selected from different serum panels (a-d) and tested for neutralizing antibody titers in an ecov vn assay. heat-inactivated equine sera ( • c for min) were serially diluted -fold in dmem supplemented with % fetal bovine serum and mixed with an equal volume of ecov nc strain ( % tissue culture infective doses (tcid )/well) in -well cell culture plates (corning inc., kennebunk, me, usa). virus-serum mixtures were incubated at • c for min. then µl of the virus-serum mixture was added in duplicate to hrt- g cells monolayers in -well cell culture plates. at six days post infection, a clear cytopathic effect (cpe) was observed and the virus neutralization titers (vnt) were determined. the vnt of sera were expressed as the reciprocals of the highest serum dilution that resulted in % neutralization of cpe. a titer of ≥ was considered to be positive. two different formats were developed employing ecov s protein, a so-called wet format elisa (welisa) and a dry format elisa (delisa). . . . ecov s wet format elisa (welisa) high-binding microtiter plates (greiner bio-one bv, alphen aan den rijn, the netherlands) were coated with ecov s protein ( µl per well) in phosphate buffered saline (pbs, ph . ) overnight at • c. the optimal protein amount and dilution of secondary antibody conjugate were determined by checkerboard titration. the protein concentration in use was . µg/ml. after three washes with pbs containing . % tween- (pbst), the plates were blocked with pbst containing % milk powder (protifar, nutricia, zoetermeer, the netherlands) for h at • c. following blocking, plates were incubated with serum samples diluted : in pbst containing % milk powder for h at • c. after a washing step, µl/well : , diluted horseradish peroxidase (hrp)-conjugated goat anti-horse igg (h&l) (abnova, taiwan, china) was added to detect bound antibodies and plates were incubated for h at • c. subsequently, the plates were washed, and the peroxidase reaction was then visualized via incubating plated with tmb super slow one component hrp microwell substrate (biofx ® , surmodics ivd, inc., eden prairie, mn, usa) for min at room temperature. the reaction was stopped by adding . % sulfuric acid (h so (vwr international bv, amsterdam, the netherlands)) and optical densities (od) were immediately measured at nm using an elisa microplate reader (biotek instruments, inc., winooski, vt, usa). all serum samples were tested in duplicate. . . . ecov s dry format elisa (delisa) high-binding microtiter plates (greiner bio-one bv, alphen aan den rijn, the netherlands) were coated with ecov s protein ( µl per well) in ammoniumcarbonate solution ( . g/l (vwr international bv, amsterdam, the netherlands)) overnight at • c. then µl/well blocking solution ( . g/l ammoniumcarbonate + g/l caseine (vwr international bv, amsterdam, the netherlands) + g/l sucrose (merck and co., inc., kenilworth, nj, usa)) was added and plates were incubated for one hour at room temperature. subsequently, the contents of the plates were discarded, and plates were dried for four hours at • c, vacuum sealed and stored at - • c. the optimal protein amount and dilution of secondary antibody conjugate were determined by checkerboard titration. the protein concentration in use was . µg/ml. plates were incubated with serum samples µl per well and diluted : in pbs + . % tween- + . % dry milk (bio-rad laboratories, inc., hercules, ca, usa) for h at • c. after a washing step (five times with pbst µl/well on a biotek automatic washing station), µl per well : , diluted horseradish peroxidase (hrp)-conjugated goat anti-horse igg (h&l) (abnova, taiwan, china) was added to detect bound antibodies and incubated for h at • c. subsequently, the plates were washed again using the same washing procedure and the peroxidase reaction was then visualized by incubating plates with tmb (idexx laboratories, westbrook, nj, usa) for min at room temperature. the reaction was stopped by adding µl/well sulfuric acid (h so . m (vwr international bv, amsterdam, the netherlands)) and optical densities (od) were immediately measured at nm using an elisa microplate reader (biotek instruments, inc., winooski, vt, usa). all serum samples were tested in duplicate. s/p values were calculated with the formula: s/p = (od sample-od negative control)/(od positive control-od negative control). the correlation between od values scored with two elisa formats was measured by the pearson correlation coefficient using graph pad prism, version . the discriminating power of the two different elisa formats was analyzed by performing receiver operator characteristic (roc) analysis with sera, which were negative (vnt < ) and were positive (vnt ≥ ). the cut-off value, diagnostic specificity and sensitivity were determined by roc analysis using sigmaplot. a minimum specificity of % was chosen for the selection of cut-off values. additionally, the reproducibility of assays was evaluated by testing three samples with different od values. inter-assay coefficients of variation (cv) and intra-assay cv were determined testing each sample in triplicate on three different plates in three different runs and within the same plate, respectively. to identify equine sera containing ecov-neutralizing antibodies, we screened a subset of equine sera, composed of randomly selected serum samples from panels a-c, and all samples from panel d were screened in the vn assay. of the sera, sera were tested as negative (titers < ) and positive samples (titers ranging from to ). additionally, paired samples from horses (n = , panel i) were tested in the vn assay. twenty out of sera collected from the first time point exhibit titers ranging from to . the convalescent serum samples were collected - days following the first round of sample collections, and all of them showed neutralization responses with titers ranging from to . within these horses, seven of them showed seroconversion and showed a significant ( -fold or greater: log ) increase in titer in the vn assay. to confirm the presence of ecov specific igg in icelandic horses, horse sera with positive ecov s elisa results (in panel h, s/p value > . ) were tested in ecov neutralization assays. all of them had neutralizing antibodies with titers varying between and . besides the conventional wet elisa format (welisa) for general laboratory usage, a dry standardized elisa format (delisa) was also developed and validated to facilitate implementation as routine diagnostic method in different laboratories and possibly wider application as an elisa kit. both elisa formats were developed for the detection of ecov-specific antibodies in horse serum samples. the diagnostic performance of both elisas was evaluated using a subset of horse sera with known vn results as described above. the pearson correlation coefficient was calculated to assess the correlation between the od values obtained with the two elisa formats (figure ) . results indicate that od values obtained with both elisas show a high degree of correlation, with correlation and regression coefficients close to (r = . , regression coefficient = . , p < . ). thus, the performance of both elisa formats is very similar. additionally, paired samples from horses (n = , panel i) were tested in the vn assay. twenty out of sera collected from the first time point exhibit titers ranging from to . the convalescent serum samples were collected - days following the first round of sample collections, and all of them showed neutralization responses with titers ranging from to . within these horses, seven of them showed seroconversion and showed a significant ( -fold or greater: log ) increase in titer in the vn assay. to confirm the presence of ecov specific igg in icelandic horses, horse sera with positive ecov s elisa results (in panel h, s/p value > . ) were tested in ecov neutralization assays. all of them had neutralizing antibodies with titers varying between and . besides the conventional wet elisa format (welisa) for general laboratory usage, a dry standardized elisa format (delisa) was also developed and validated to facilitate implementation as routine diagnostic method in different laboratories and possibly wider application as an elisa kit. both elisa formats were developed for the detection of ecov-specific antibodies in horse serum samples. the diagnostic performance of both elisas was evaluated using a subset of horse sera with known vn results as described above. the pearson correlation coefficient was calculated to assess the correlation between the od values obtained with the two elisa formats (figure ) . results indicate that od values obtained with both elisas show a high degree of correlation, with correlation and regression coefficients close to (r = . , regression coefficient = . , p < . ). thus, the performance of both elisa formats is very similar. subsequently, the discriminating power of the welisa and delisa was evaluated via receiver operator characteristic (roc) analysis. the roc curves were plotted based on the previous classification of sera into negative and positive by vn assays (figure a,b) . then the optimal cutoff values, diagnostic specificity and sensitivity of both elisa formats were determined by the established roc curves. the elisa results of the vnt-positive and negative samples are shown in figure c ,d. the diagnostic accuracy of both elisa formats was considered to be high as the same area under the curve (auc) values were observed (auc = . ), with a relative sensitivity and specificity approximately % according to the youden plot of welisa and delisa. therefore, the test characteristics of both elisa formats were assigned the same weight. in this study, a minimum specificity of % was chosen for the threshold of cut-off values for both elisas. accordingly, the optimal cut-off for welisa was an od value of . -for which, the sensitivity was % and the specificity was %. for delisa, the test results were expressed as s/p values. a cut-off at an s/p value of . yielded a sensitivity of % and specificity of %, respectively. subsequently, the discriminating power of the welisa and delisa was evaluated via receiver operator characteristic (roc) analysis. the roc curves were plotted based on the previous classification of sera into negative and positive by vn assays (figure a,b) . then the optimal cut-off values, diagnostic specificity and sensitivity of both elisa formats were determined by the established roc curves. the elisa results of the vnt-positive and negative samples are shown in figure c ,d. the diagnostic accuracy of both elisa formats was considered to be high as the same area under the curve (auc) values were observed (auc = . ), with a relative sensitivity and specificity approximately % according to the youden plot of welisa and delisa. therefore, the test characteristics of both elisa formats were assigned the same weight. in this study, a minimum specificity of % was chosen for the threshold of cut-off values for both elisas. accordingly, the optimal cut-off for welisa was an od value of . -for which, the sensitivity was % and the specificity was %. for delisa, the test results were expressed as s/p values. a cut-off at an s/p value of . yielded a sensitivity of % and specificity of %, respectively. furthermore, the inter-and intra-coefficient of variation (cv) of the three ecov positive sera tested with both elisa formats were lower than %. more specifically, the intra-assay cv of welisa and delisa ranged from . % to . % and from . % to . %, respectively, while the inter-assay cv of welisa and delisa varied from . % to . % and from . % to . %, respectively. overall, these results indicate that the performances of both elisa formats were very much equivalent and that the results of both elisas were strongly correlated to vn results. to determine the diagnostic performance of the ecov s elisa, paired serum samples (panel i) collected from an acute ecov outbreak were investigated by welisa. the horses presented similar clinical signs as described in [ ] , and virus shedding was confirmed by qpcr analysis [ ] . at the acute stage, out of horses were qpcr positive, while at the convalescent stage, this number had decreased to six. serum samples were further validated by vn assay ( figure b ; table s ). seven out of horses showed seroconversion, while another horses showed a significant ( -fold or greater) increase in vnt. performing the welisa (see figure a ; table s ), the same seven out of horses showed seroconversion; acute phase sera were negative (od value < . ) whereas the convalescent phase sera all had od values greater than . ( . - . ). thus, seroconversion rates calculated from welisa and vnt showed a % correlation (table s ). for the horses that showed a -fold or greater increase in vnt (n = ), nine of the acute phase sera had positive od values between . and . ( × background) and also a higher than (n = ) to (n = ) fold increase in the od value in the convalescent serum. five of the vnt positive paired serum samples had od values of > . (twice the background od value) in the acute phase serum. two of these samples with an od value of . and . respectively in welisa also showed a greater than -fold increase in od value. the three vnt positive samples with less than -fold increase in od values already had high furthermore, the inter-and intra-coefficient of variation (cv) of the three ecov positive sera tested with both elisa formats were lower than %. more specifically, the intra-assay cv of welisa and delisa ranged from . % to . % and from . % to . %, respectively, while the inter-assay cv of welisa and delisa varied from . % to . % and from . % to . %, respectively. overall, these results indicate that the performances of both elisa formats were very much equivalent and that the results of both elisas were strongly correlated to vn results. to determine the diagnostic performance of the ecov s elisa, paired serum samples (panel i) collected from an acute ecov outbreak were investigated by welisa. the horses presented similar clinical signs as described in [ ] , and virus shedding was confirmed by qpcr analysis [ ] . at the acute stage, out of horses were qpcr positive, while at the convalescent stage, this number had decreased to six. serum samples were further validated by vn assay ( figure b ; table s ). seven out of horses showed seroconversion, while another horses showed a significant ( -fold or greater) increase in vnt. performing the welisa (see figure a ; table s ), the same seven out of horses showed seroconversion; acute phase sera were negative (od value < . ) whereas the convalescent phase sera all had od values greater than . ( . - . ). thus, seroconversion rates calculated from welisa and vnt showed a % correlation (table s ). for the horses that showed a -fold or greater increase in vnt (n = ), nine of the acute phase sera had positive od values between . and . ( x background) and also a higher than (n = ) to (n = ) fold increase in the od value in the convalescent serum. five of the vnt positive paired serum samples had od values of > . (twice the background od value) in the acute phase serum. two of these samples with an od value of . and . respectively in welisa also showed a greater than -fold increase in od value. the three vnt positive samples with less than -fold increase in od values already had high od values in the acute phase serum as well as high vnt (mean od value = . , mean vnt = . ). for the six horses that did not show a significant rise in vnt, five serum samples collected at the acute stage already had high antibody levels as shown by elisa and neutralization assay (mean od value > . , mean vnt > , table s ). further, the pearson correlation coefficient was calculated to assess the overall correlation between the od values obtained with welisa and vnt (log titers) from acute and convalescent-phase sera of the horses ( figure s ). results indicate that od values and vnt show a good degree of correlation (r = . , p < . ). these data support the use of the welisa as a diagnostic tool in case of suspected ecov outbreaks. od values in the acute phase serum as well as high vnt (mean od value = . , mean vnt = . ). for the six horses that did not show a significant rise in vnt, five serum samples collected at the acute stage already had high antibody levels as shown by elisa and neutralization assay (mean od value > . , mean vnt > , table s ). further, the pearson correlation coefficient was calculated to assess the overall correlation between the od values obtained with welisa and vnt (log titers) from acute and convalescent-phase sera of the horses ( figure s ). results indicate that od values and vnt show a good degree of correlation (r = . , p < . ). these data support the use of the welisa as a diagnostic tool in case of suspected ecov outbreaks. we further set out to determine the seroprevalence in horses with unknown ecov exposure using the delisa format. a total of serum samples (table , panel a-h) were analyzed. with the exception of panel d, all sera were from adult horses (older than months). seroprevalence varied from . % (panel d) to . % (panel c) among these eight serum panels. the lowest number of positive samples was found in panel d which contained young horses ( - months old, average age: . months ( % ci . - . )). in the other four serum panels (panel a, b, e and f) from dutch horses, the historical serum samples (panel g) and samples from iceland (panel h) higher seroprevalences were found ( . - . %). table . prevalence of ecov s -reactive antibodies in equine sera used in this study. we further set out to determine the seroprevalence in horses with unknown ecov exposure using the delisa format. a total of serum samples (table , since the beginning of the st century, ecov infections have been reported in horses, causing fever and enteric diseases [ ] . more recently, infections in adult horses were reported with clinical signs of fever, anorexia, lethargy and, less commonly, specific signs of diarrhea and colic [ , ] . nevertheless, information regarding the circulation of ecov in the equine population, especially in europe, is still limited [ , ] . serological studies are useful tools to investigate ecov prevalence in horse populations. in the present study, our aim was to develop a simple and reliable method for antibody detection against ecov that can be used for diagnostics and sero-epidemiological studies. as compared to virus neutralization assays, the elisa method has the advantage of being reproducible, potentially high-throughput and much less laborious. in our study, we set up an ecov s -based elisa method in two complementary formats. the conventional welisa format is for general laboratory usage with simplified, easy to perform coating procedures. on the other hand, coated plates of the delisa format could be stored for a longer time period, making it ideal for transportation and kit development. we showed that both formats performed equally well, and their results correlated nicely. when comparing with the vn assay by roc analysis, our elisa method with both formats was shown to have high accuracy. in our current study we applied welisa for the analysis of the paired outbreak samples, while the delisa was further validated and used for the high-throughput screening of larger amount of serum samples. we utilize ecov s as the viral antigen for antibody detection in this study. the s chimeric protein was expressed in mammalian cells, and hence both the protein conformation and modification (e.g., glycosylation) are mimicking the s proteins on the surface of virus particles [ ] . as the most divergent and immunodominant component of coronaviruses, s has been widely used in the development of methods for specific coronavirus serological studies [ , , , ] . our findings validate that ecov s is a highly suitable antigen for the detection of antibodies against ecov showing very good agreement between the elisa and vn assays. recently, similar conclusions were also drawn for the role of mers s in mers serology [ ] . with our welisa method, we were able to analyze paired samples that were collected during an ecov outbreak. in the virus neutralization assay seroconversion or a -fold or greater increase in ecov antibody titers could be detected in sera of out of horses within weeks of the initial observation of clinical disease and detection of viral rna in feces. of these positive horses showed seroconversion or a -fold or higher increase in od values in the welisa. the three remaining vnt positive samples had high od values already in the acute phase serum. of the six ecov negative paired samples five had high vn antibody titers and od values already at the acute phase. this might be due to late sampling of these horses or previous exposure to ecov (table s ). this study confirms that the ecov s elisa is a useful diagnostic test for the demonstration of a potential ecov outbreak and should be considered as a useful adjunct to investigation of fecal samples by qpcr. we also determined the seroprevalence of serum samples collected from horses with unknown ecov exposure via our delisa. results showed that the overall seroprevalence in the different cohorts tested is . %- . %. these percentages are in agreement with the study performed by hemida et al. [ ] , in which they detected coronavirus infections in horses in saudi arabia and oman and they found that % of them had detectable neutralizing antibodies to ecov. a lower percentage ( . %) of positive animals was found in another ecov seroprevalence study conducted in the usa [ ] . several factors might contribute to these differences in results. there is only limited information regarding ecov prevalence in europe including the netherlands [ , ] , and it is possible that the overall ecov distribution differs between continents. moreover, our study employs eukaryotically expressed ecov s protein as coating antigens, while in the us study chimeric s protein expressed in escherichia coli was used. the expression in mammalian cells guarantees a more native configuration of the protein, in particular of glycosylated antigens such as the coronavirus spike protein. reports had shown that both coronavirus s and s subunit elicit antibody responses, but the level of immune responses triggered by them may differ [ , ] . furthermore, the criteria for determining the cut-off value are different for the two studies. in our study we defined positive and negative samples on the basis of a vn assay, whereas the us study used negative qrt-pcr and absence of clinical signs as criteria to define horses as ecov negative. in this way, seropositive horses may have contributed to higher cut-off values and potentially a lower sensitivity of the assay. in our study, we noticed differences in seroprevalence between young and adult horses. in the group of young horses (panel d, table ), the lowest seroprevalence was found. young horses may initially be protected against ecov infection by maternal antibodies and may become gradually more susceptible as maternal antibodies wane. the risk of becoming infected increases with age. this hypothesis is further supported by the age distribution of pcr-confirmed ecov infection cases: foals (age - months) have the lowest infection rates, and the infection rate increases with age [ ] . we also observed a significant percentage of seropositive horse serum samples collected back in (panel g, table ). ecov-like viruses were detected in the s and s by electron microscopy in feces of horses with enteric disease, but virus isolation and characterization was not reported [ ] [ ] [ ] [ ] . the history of ecov presence, especially in europe, is possibly much longer than currently understood [ ] . intriguingly, we noticed that icelandic horses also are seropositive against ecov (panel h, table ). twenty-four serum samples showed high ecov elisa reactivity (s/p value > . ) and also had neutralizing antibodies with vnt varying between and . the horse population of iceland has been geographically isolated for more than years and is free from most common equine contagious diseases such as equine influenza, equine herpesvirus , strangles and equine viral arteritis [ ] . to date, no prior studies of ecov prevalence in horses from iceland had been performed. this is the first evidence of the existence of ecov infection in iceland. in conclusion, we developed a high-throughput, reliable and specific elisa method to study humoral immune responses in horses against ecov. with this method, we are able to perform the serodiagnosis of ecov infection and assess the seroprevalence within horse populations in the future. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . table s : detection of antibodies to ecov in equine serum samples during an ecov outbreak by welisa in winter ; figure s . correlation between the od values obtained with welisa and virus neutralization titers (vnt) of horses from acute and convalescent-phase sera. origin and evolution of pathogenic coronaviruses genetic recombination, and pathogenesis of coronaviruses genomic characterization of equine coronavirus characterization of a coronavirus isolated from a diarrheic foal isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the nc strain first detection of equine coronavirus (ecov) in europe emerging outbreaks associated with equine coronavirus in adult horses epidemic of equine coronavirus at obihiro racecourse, hokkaido, japan in the first detection of equine coronavirus in adult horses and foals in ireland enteric coronavirus infection in adult horses frequency of molecular detection of equine coronavirus in faeces and nasal secretions in horses with acute onset of fever disease associated with equine coronavirus infection and high case fatality rate clinical presentation, diagnostic findings, and outcome of adult horses with equine coronavirus infection at a veterinary teaching hospital: cases ( - ) evaluation of equine coronavirus fecal shedding among hospitalized horses experimental inoculation of equine coronavirus into japanese draft horses seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt persistence of antibodies against middle east respiratory syndrome coronavirus experimental reproduction of winter dysentery in lactating cows using bcv-comparison with bcv infection in milk-fed calves porcine epidemic diarrhea virus (pedv) introduction into a naive dutch pig population in middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study sars-cov antibody prevalence in all hong kong patient contacts cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer coronavirus spike protein and tropism changes mechanisms of coronavirus cell entry mediated by the viral spike protein serological assays for emerging coronaviruses: challenges and pitfalls serological screening for coronavirus infections in cats development of an equine coronavirus-specific enzyme-linked immunosorbent assay to determine serologic responses in naturally infected horses detection of equine coronavirus in horses in the united kingdom production of recombinant protein therapeutics in cultivated mammalian cells specific serology for emerging human coronaviruses by protein microarray sensitive and specific detection of low-level antibody responses in mild middle east respiratory syndrome coronavirus infections coronavirus infections in horses in saudi arabia and oman seroprevalence and risk factors for infection with equine coronavirus in healthy horses in the usa identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design coronavirus and gastroenteritis in foals rotavirus and coronavirus associated diarrhoea in domestic animals isolation of coronavirus-like agent from horses suffering from acute equine diarrhoea syndrome concurrent cryptosporidium and coronavirus infections in an arabian foal with combined immunodeficiency syndrome genomic dissection of an icelandic epidemic of respiratory disease in horses and associated zoonotic cases we are grateful to udeni b.r. balasuriya (louisiana animal disease diagnostic laboratory and department of pathobiological sciences, school of veterinary medicine, louisiana state university, baton rouge, louisiana, usa) for providing strain nc and hrt- g cells and to sigríður björnsdóttir (icelandic food and veterinary authority, selfoss, iceland) and vilhjálmur svansson (institute for experimental pathology, university of iceland, reykjavik, iceland) for providing sera from icelandic horses. we also thank heleen zweerus for her practical assistance. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -brgtwxhe authors: fumian, tulio m.; tuipulotu, daniel enosi; netzler, natalie e.; lun, jennifer h.; russo, alice g.; yan, grace j. h.; white, peter a. title: potential therapeutic agents for feline calicivirus infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: brgtwxhe feline calicivirus (fcv) is a major cause of upper respiratory tract disease in cats, with widespread distribution in the feline population. recently, virulent systemic diseases caused by fcv infection has been associated with mortality rates up to %. currently, there are no direct-acting antivirals approved for the treatment of fcv infection. here, we tested compounds from different antiviral classes against fcv using in vitro protein and cell culture assays. after the expression of fcv protease-polymerase protein, we established two in vitro assays to assess the inhibitory activity of compounds directly against the fcv protease or polymerase. using this recombinant enzyme, we identified quercetagetin and ppnds as inhibitors of fcv polymerase activity (ic( ) values of . μm and . μm, respectively). we also demonstrate the inhibition of fcv protease activity by gc (ic( ) of µm). using cell culture assays, ppnds, quercetagetin and gc did not display antivirals effects, however, we identified nitazoxanide and ′-c-methylcytidine ( cmc) as potent inhibitors of fcv replication, with ec( ) values in the low micromolar range ( . μm and . μm, respectively). in conclusion, we established two in vitro assays that will accelerate the research for fcv antivirals and can be used for the high-throughput screening of direct-acting antivirals. feline caliciviruses (fcv) are members of the caliciviridae family (genus vesivirus) and a major pathogen of cats worldwide. the virus has been associated with vesicular and upper respiratory tract disease, especially in multi-cat environments, such as shelters, colonies, and catteries, where fcv is detected in up to % of cats [ ] [ ] [ ] . fcv infections typically cause a variety of clinical manifestations, such as acute respiratory disease and oral ulceration, with less common symptoms including pneumonia and acute arthritis/limping syndrome [ , ] . more recently, highly contagious virulent strains of fcv have emerged and were linked with severe disease (fcv-associated virulent systemic disease (vsd)) and high mortality rates (up to %) [ ] [ ] [ ] . after the first description of fcv-vsd in , outbreaks have occurred in the usa and europe, which were associated with genetically distinct virulent fcv strains that have evolved locally [ ] [ ] [ ] [ ] [ ] [ ] . the severe disease has a marked tropism for endothelial and epithelial cells of the skin and parenchymal organs and adult cats are often more severely affected than kittens [ , ] . % (v/v) fetal bovine serum (sigma-aldrich, st. louis, mo, usa), u/ml penicillin (thermo fisher, waltham, ma, usa) and µg/ml streptomycin (thermo fisher). cells were grown at • c with % co . the fcv strain f- (vr- ™; genbank accession number m ) was purchased from atcc. thirteen antiviral compounds were selected based on the reported in vitro antiviral effects against other caliciviruses [ ] and included nnis, nas, pis, and the broad-spectrum nitazoxanide. the stock solutions for all compounds were prepared in % dimethyl sulfoxide (dmso) and aliquoted before storage at − • c. the pro-pol cds from fcv urbana (genbank accession: l ) was commercially synthesized in the poa-rq vector (life technologies, carlsbad, ca, usa) and then sub-cloned into pet b (merck millipore, burlington, ma, usa) between bamhi and sali restriction sites using forward and reverse primers: -aggtaggatccagtggattataaagacgatg- and -aggtagtcgaccacttcaaacacatcac- to produce pvrl . for the expression of pro-pol containing a c-terminal histidine tag, pvrl -transformed escherichia coli bl (de ) (neb, ipswich, ma, usa) were grown in luria-bertani media ( l) at • c with µg/ml kanamycin until the od was~ . . the culture was induced with . mm isopropyl β-d- -thiogalactopyranoside (iptg) for h at • c with shaking and bacteria pelleted by centrifugation. chemical lysis of the pellet was performed as previously described [ ] , and lysates were loaded onto ni + columns (biorad, hercules, ca, usa) and purified with an imidazole gradient ( - mm) using an akta start dual-buffer system (ge healthcare, little chalfont, uk). the equilibration buffer consisted of mm tris-hcl, mm nacl, mm imidazole, % glycerol (v/v) and . % triton x- (v/v), and the elution buffer was composed of the equilibration buffer with mm imidazole. the purified protein was concentrated using an amicon ® ultra centrifugal filter ( kda cut-off, millipore, tokyo, japan) and dialyzed against three buffers with decreasing nacl concentration ( , , or mm nacl, with mm tris-hcl, % glycerol (v/v); . % triton x- [v/v]). all buffers were prepared at ph . the protein concentration was determined using a bca protein assay kit (life technologies). crfk cells ( . × cells/well, µl/well), were seeded into flat-bottom -well plates and incubated overnight at • c. the cell monolayers were then treated with increasing concentrations of compounds in triplicate ( . µm- µm), followed by h in incubation. dmso (vehicle only, . % (v/v)) was used as a negative control. the cytotoxicity of each compound was measured using the celltitre-blue viability assay kit (promega, madison, wi, usa) according to the manufacturers' instructions. fluorescence was measured on a fluostar optima microplate reader (bmg labtech, ortenberg, germany) and the half maximal cytotoxic concentrations (cc ) were determined with graphpad prism v. (la jolla, ca, usa). rdrp activity was measured by monitoring the formation of double-stranded rna (dsrna) from a single-stranded rna polycytidine template (poly(c)), as described previously [ , ] . rdrp activity was first optimized by increasing concentrations of enzyme ( - ng per reaction), and varying sodium chloride (nacl) concentrations ( - mm). heat-inactivated rdrp was used as the negative control. accumulation of the dsrna product was measured using the fluorescent dye picogreen (life technologies). reactions ( µl) were performed in black-bottomed -well plates containing ng of fcv pro-pol, mm tris-hcl (ph . ), . mm rgtp, ng of poly(c) rna, mm mncl , mm dithiothreitol (dtt), . % tween (v/v) and . % bovine serum albumin (bsa) (v/v). for antiviral screening, fcv pro-pol ( µl) was incubated with µl of each test compound ( µm final concentration in . % dmso) or vehicle ( . % dmso) for min at • c before the addition of µl of the reaction mixture with a further incubation of min at • c. reactions were terminated with mm edta, followed by incubation with picogreen and dsrna quantitation [ , ] . graphpad prism v. was used to plot the half maximal inhibitory concentration (ic ) values. the amino acid (aa) sequence of the cleavage site between the precursor leader capsid (lc) and the mature capsid protein (vp ) of the fcv genome was synthesized as a fluorogenic substrate peptide dabcyl-frle↓addg-edans (genscript, piscataway, nj, usa) and a stock solution ( mm) was prepared in % dmso. protease assays were performed in -well plates using a reaction volume of µl containing mm hepes, ph . , mm dtt, . mm edta, % glycerol (v/v), ng of fcv pro-pol and the fluorogenic substrate. the initial measurements to determine the michaelis-menten constant (k m ) of the substrate were performed using increasing concentrations ( - µm) with incubation for h at • c. the influence of increasing nacl concentration ( - mm) on protease activity as also evaluated. following the determination of the k m , inhibition assays were performed with µm of a substrate with either a pi ( - µm) or the vehicle control ( . % dmso) with incubation for min at • c. upon cleavage of the substrate at the site indicated (↓), the quenching of dabcyl fluorescence by the edans group is abolished and the fluorescence generated was quantified at an excitation wavelength of nm and an emission of nm on a polarstar plate reader. ic and k m values were determined using graphpad prism v. . fcv plaque reduction assays were performed as previously described [ , ] . crfk monolayers ( × cells/well) in -well plates were infected with approximately plaque forming units (pfu) of fcv for h at • c, followed by the addition of semisolid agarose overlays containing different concentrations of compounds. plates were incubated for h, fixed and stained with crystal violet. plaque numbers were determined for each drug treatment and the dmso vehicle control was defined as maximal viral infectivity. to determine whether the combination of nitazoxanide and cmc had synergistic, antagonistic or additive effects, the percentage of inhibition of fcv infection was assessed over a dose-response matrix that included four concentrations of nitazoxanide (ranging from to . µm) and cmc ( to µm). the effects of drug combination were assessed using synergyfinder [ ] and the zero-interaction potency (zip) model [ ] was used to generate synergy scores from a dose-response matrix. synergistic or antagonistic effects are shown as peaks above or below the horizontal plane, respectively. at least two independent experiments with triplicate datasets were performed for each treatment, with results presented as the mean with standard error of the mean (sem). rt-qpcr was used to evaluate the reduction in fcv rna following antiviral treatment. briefly, crfk cells ( × cells/well) in -well plates were infected with fcv at the multiplicity of infection (moi) of . for h. media was then replaced with media containing drug and incubated for a further h. fcv viral rna was extracted from the cells and supernatant using the qiamp viral rna kit (qiagen, hilden, germany). following this, an bp amplicon of the orf region was generated using itaq™ universal sybr ® green one-step kit (biorad) as described in reference [ ] . a standard curve was generated using a serially diluted plasmid (containing the end of the fcv orf ) for genome quantitation. the cycling parameters were • c for min, • c for min and cycles of • c for s and • c for min. all reactions were run in duplicate. statistical calculations were performed using the graphpad prism v. software. data were analyzed using an unpaired t-test with welch's correction. all error bars depict standard errors of the mean (sem), and the level of significance are indicated as: ns, not significant, p > . ; * p ≤ . ; ** p ≤ . ; *** p ≤ . . we successfully expressed the fcv pro-pol polyprotein containing a c-terminal -histidine tag in e. coli bl cells, under the control of the t promoter system. from l of the culture, we purified~ . mg of pro-pol which appeared at the expected molecular mass ( kda) by sds-page. the presence of the his-tag was confirmed by western blotting. to confirm the rdrp activity of the pro-pol dual protein, we tested it using an in vitro fluorescence-based transcription assay, where the dsrna product was detected with picogreen dye [ ] . the fcv transcriptional activity increased with increasing concentrations of rdrp ( - ng per reaction) ( figure a ). furthermore, a decrease in rdrp activity was observed with increasing nacl concentration ( - mm) ( figure b ). the rdrp activity was reduced by % in the presence of mm nacl, and completely inhibited at mm ( figure b) . six nni compounds (quercetagetin, compound , ppnds, beclabuvir, tmc- , and jtk- ) were tested for fcv rdrp inhibition using the picogreen in vitro assay (table ) . at a fixed concentration of µm, only quercetagetin and ppnds demonstrated a significant reduction of rdrp activity compared to mock controls ( . % and . %, respectively) ( figure a ). compound slightly reduced the fcv rdrp activity ( %), while all other compounds (beclabuvir, tmc- , and jtk- ) showed a minimal inhibitory effect (≤ %). dose-dependent inhibitory response curves ( . - µm) were generated to establish the ic values for quercetagetin ( . µm) and ppnds ( . µm) ( figure b,c) . the cc of each nni on crfk cells was determined using the celltitre-blue viability assay (table ) (table ). in addition, ppnds and quercetagetin were examined using an fcv plaque reduction assay, with the inhibitory activity calculated after h relative to a mock control (dmso treatment). at µm, no antiviral activity was observed for both compounds (< % of plaque formation inhibition) ( table ) . previous studies have demonstrated that recombinant fcv pro-pol exhibits a bifunctional activity of polymerase and protease in vitro [ , ] . therefore, we also established an in vitro fret fluorescence-based assay to measure fcv protease activity that cleaves the substrate dabcyl-frle↓addg-edans (corresponding to the lc/vp cleavage site) and demonstrated a k m value of . µm ( figure a) . in contrast to fcv rdrp, the protease activity was not inhibited with increasing nacl concentrations ( figure b ). the development of the in vitro fret assay enabled us to test the inhibitory activity of three previously published pis: gc [ ] , rupintrivir [ ] and chymostatin [ ] . of those, only gc exhibited an inhibitory effect against the fcv protease in vitro with a % inhibition at µm, and further experiments demonstrated an ic of . µm ( figure c and table ). five na compounds were chosen and tested for their antiviral effects against fcv in the cell culture, including; cmc, famciclovir, sofosbuvir, t- , and d m. all nas tested have previously shown antiviral effects against several viral families, such as caliciviruses, herpesvirus, paramyxoviruses, orthomyxoviruses, and flaviviruses (table ) . however, the antiviral efficacy of these compounds against fcv infections has not been evaluated thus far. in addition to these nas, we also tested the broad-spectrum antimicrobial agent, nitazoxanide, whose mechanism of antiviral action has not been fully elucidated [ ] . the dose-response of each compound against fcv was examined using a plaque reduction assay. the compounds cmc and nitazoxanide exhibited dose-response inhibition of fcv plaque formation at low micromolar concentrations with ec s of . µm and . µm ( . µg/ml), respectively ( figure a,b) . the compound cmc demonstrated cc values of > µm, whilst nitazoxanide showed value of . µm (table ) , and the therapeutic index values (ti = cc /ec ) determined were of > and . , respectively. we also performed rt-qpcr to quantify the fcv rna levels after antiviral treatment with different concentrations of nitazoxanide or cmc. as shown in figure c , a decrease of dose-dependency in fcv rna levels was observed after h of treatment for both compounds. nitazoxanide ( . µm) resulted in an % reduction of fcv rna levels, whilst cmc ( µm) reduced the rna levels by % compared to the mock-treated cells ( figure c ). the combined inhibitory effects of nitazoxanide ( to . µm) and cmc ( to µm) were tested over a range of combinations against fcv in the cell culture using the plaque reduction assay. a dose-response matrix was generated and analyzed for synergy using synergyfinder. the zip mode synergy score is presented as the average of all δ-scores across the dose-response landscape, and the peaks above the plane of % synergy in the plot indicate synergism. nitazoxanide and cmc displayed a synergistic antiviral effect against fcv. data were analyzed using an unpaired t-test. ** p < . ; *** p < . ; ns, not significant. duplicate (panels c and d) or triplicate values (panels a and b) from at least two independent experiments are presented, and the mean ± sem are shown for panels a and c. sofosbuvir, t- , and d m showed a modest antiviral activity at µm (< % inhibition of plaque formation). famciclovir exhibited minimal fcv antiviral activity at concentrations as high as µm ( % inhibition of plaque formation). to determine the synergistic effects of nitazoxanide with cmc, we performed plaque reduction assays over several combined concentrations ( figure d ). the synergistic effect is shown as peaks above the horizontal plane, with zip synergy scores varying from to . the interaction of both compounds resulted in a moderate synergistic effect (zip synergy score of . ), with a maximal synergy at concentrations of . : µm for nitazoxanide and cmc, respectively ( figure d ). fcv is a common pathogen of cats and usually associated with acute, mild and self-limiting upper respiratory tract disease, however, more recently highly contagious strains of the virus (fcv-vsd) have been reported in the usa, europe [ , , ] , and three states of australia (personal communication, https://au.virbac.com/home/vet-newsletter/main/vet-newsletter/research-update-fcv-vsd.html). with the lack of an effective vaccine and/or antiviral treatment for fcv infection, there is a clear unmet need to identify an effective antiviral agent to improve the management and control of fcv infections. in the present study, we evaluated different compounds, from four different antiviral classes, using in vitro enzyme-and cell culture-based assays, of which have not previously been evaluated against this virus. we identified the na cmc (ec = . µm) and the broad spectrum antimicrobial compound nitazoxanide (ec = . µm or . µg/ml) as potent inhibitors of fcv replication ( figure a and table ). an na originally designed for use against hcv, cmc is a promising calicivirus antiviral and has previously been tested against human and murine norovirus, with similar results to our current study [ ] [ ] [ ] . using in vitro assays, jin et al. [ ] showed that tri-phosphorylated cmc inhibited human and murine norovirus rdrp activity with ic s of . µm and . µm, respectively. in the same study, cmc was tested against the human gi. norovirus replicon in the cell culture and demonstrated an ec of . µm. in related studies, cmc was also shown to inhibit the murine norovirus (ec ~ µm) and the human norovirus replicon (ec ~ µm) using cell culture-based assays [ , ] . recently, using a b-cell culture system, cmc also effectively inhibited the human norovirus with an ec of . µm [ ] . therefore, our results are consistent with the inhibitory data obtained against other caliciviruses as reported in the above cited studies. valopicitabine, the prodrug form of cmc, was used in pre-clinical studies to treat hcv infections, however, after dose-related gastrointestinal adverse events, the drug has been placed on clinical hold by the us food and drug administration (fda). although promising results were obtained here and in other studies [ , ] , concerns over adverse side-effects may limit its future clinical use to treat calicivirus infections. nitazoxanide is a broad-spectrum antimicrobial compound with activity against anaerobic bacteria, protozoa, and viruses [ ] . it is an fda-approved drug licensed for gastroenteritis caused by the parasites cryptosporidium parvum and giardia intestinalis [ , ] . in cell cultures, nitazoxanide has been evaluated against several viruses, showing inhibition in the replication of rotavirus (ec . µg/ml), adenovirus (ec . µg/ml), canine coronavirus (ec µg/ml), influenza viruses (ec . - . µg/ml), among others [ , ] . in the present study, nitazoxanide demonstrated an ec of . µg/ml ( . µm) against fcv, which is within the range of values found when tested on other viruses. recently, the drug was reported to inhibit gi norovirus replicon replication at µg/ml, and cleared the replicon from the host cells, but was ineffective against murine norovirus [ ] . nitazoxanide has been commercialized in latin american countries and india to treat a broad spectrum of intestinal parasitic infections and is currently in clinical trials to treat norovirus gastroenteritis [ , ] . for example, a large randomized, double-blind, placebo-controlled clinical trial is being conducted using nitazoxanide to treat acute gastroenteritis mainly caused by cryptosporidium parvum, norovirus, and rotavirus in hospitalized aboriginal children in the northern territory, australia [ ] . there is also some published anecdotal evidence that this drug works on norovirus in a small number of case studies [ , ] . in the veterinary field, small animals such as cats and dogs have received nitazoxanide to treat intestinal parasites. gookin et al. [ ] demonstrated the successful use of nitazoxanide in eliminating the shedding of tritrichomonas foetus, a cause of chronic diarrhea in cats. in another study, the successful administration of nitazoxanide to treat giardiasis and cryptosporidiosis in dogs was demonstrated [ ] . given that nitazoxanide displayed a potent inhibition against fcv and is already used in a clinical setting for feline infections, our data illustrate that nitazoxanide could be repurposed for the treatment of fcv infections. however, considering the narrow in vitro therapeutic index of nitazoxanide, and its side-effects (diarrhea and vomiting) observed in cats after nitazoxanide administration [ ] , concerns about the effective dose in vivo should be addressed. the combination of antiviral compounds with additive or synergistic effects is a strategy to improve drug efficacy, reduce antiviral toxicity, and limit the development of viral resistance. here, we demonstrated that the combination of nitazoxanide and cmc in cell cultures had a synergistic inhibitory effect against fcv, with an average delta score of . ( figure d ). as nitazoxanide showed cytotoxicity on crfk cells at a relatively low concentration (cc = . µm), the synergistic effect resulted from the combination with cmc (cc > µm) could be useful in limiting its cytotoxic effects by reducing the effective concentration of nitazoxanide, and overall improving the efficacy of the combination treatment. in the present study, we have expressed the recombinant fcv pro-pol with high yields of active protein. previous studies have demonstrated that the fusion protein is stably expressed in fcv-infected cells and is the primary and active form of the protein, which maintains both protease and polymerase activity [ , ] . as previously shown by wei et al. [ ] , we also demonstrated that high concentrations of nacl ( mm) caused a reduction in the rdrp activity ( figure b) , however, no effect in the protease activity was observed at this concentration ( figure b ). of the six nni compounds tested in the current study, ppnds and quercetagetin showed an inhibition of fcv rdrp activity with ic values in the low micromolar range (figure and table ). in previous studies, ppnds demonstrated potent inhibition of rdrp activity against viruses from three calicivirus genera, norovirus, sapovirus, and lagovirus, with ic values between . and . µm [ , , ] . however, due to cell permeability issues limiting bioavailability and antiviral efficacy in cell cultures, ppnds is not considered a potential antiviral drug candidate [ , ] . while in the current study quercetagetin displayed an ic of . µm in polymerase assays, it did not inhibit fcv plaque formation and therefore is not a suitable fcv antiviral. quercetagetin, a natural flavonoid compound, was first reported as a potent inhibitor of hcv replication in vitro [ ] . the compound demonstrated a potent rdrp inhibition against different hcv genotypes, with ic s between . and . µm, but was less potent in cell cultures against the infectious virus (ec . µm ± . ) [ ] . quercetagetin also showed a moderate inhibitory activity against the chikungunya replicon, with an ic of . µm [ ] . in addition to the polymerase inhibition assay, using the purified fcv pro-pol, we also described a fret protease assay for high throughput screening of fcv protease inhibitors. as with viral polymerases, proteases play a crucial role in the viral replication cycle and are attractive targets for antiviral development. several viral pis are currently approved or under development to treat pathogenic viruses such as hiv, hcv, and the sars coronavirus [ , ] . gc is under development for feline coronavirus infection (feline infectious peritonitis) [ ] . using the fret-based assay, we tested three previously published pis, with only gc demonstrating a moderate inhibition against the protease (ic of . µm) ( figure c ). this compound has previously shown a potent inhibition against the proteases of norovirus, coronaviruses, and picornaviruses, with ic s ranging from . to . µm [ ] . however, against fcv in cell-based assays, an ec value of µm was obtained [ ] , similar to the value obtained in our study. the pis rupintrivir and chymostatin have previously demonstrated an inhibition of the human norovirus protease (genogroup i and ii) in fret-based assays, with ic values of < µm and - µm, respectively [ , ] . however, no inhibitory effect was observed for either pi against fcv protease in this study. among the nas tested, famciclovir is used for the treatment of feline herpesvirus (fhv)-associated clinical disease [ ] . this drug is also commercially used as an fcv and fhv treatment. we tested famciclovir at concentrations up to µm using the cell culture plaque reduction assay with no antiviral effect observed. our data show that the compound is ineffective at inhibiting virus replication and thus is a poor therapeutic option for the treatment of fcv infections. fcv is a highly infectious respiratory pathogen of cats with a global distribution, and more recently fcv-vsd associated high-mortality outbreaks have been reported. despite the availability of a vaccine, the high diversity of the fcv genome plays a key role in vaccine failure and is also the basis for the emergence of virulent strains. in addition, there are currently no approved antivirals to treat the disease. here, we report the establishment of two in vitro assays that allow for the identification of novel inhibitors of the fcv polymerase and protease. the present findings have implications for the development of fcv antivirals, providing a basis to design and select drugs which may be used in the veterinary clinic. using the in vitro assays, we identified quercetagetin and ppnds as potent rdrp inhibitors, and we also demonstrated a moderate inhibition of protease activity by gc . finally, we reported the identification of two compounds (nitazoxanide and cmc) with antiviral activity against fcv in cell culture at low micromolar concentrations with a potential combinational therapeutic utility to treat fcv-infected cats. an etiological investigation of domestic cats with conjunctivitis and upper respiratory tract disease in japan feline calicivirus epidemiologic evaluation of multiple respiratory pathogens in cats in animal shelters molecular virology of feline calicivirus acute arthritis of cats associated with feline calicivirus infection an isolated epizootic of hemorrhagic-like fever in cats caused by a novel and highly virulent strain of feline calicivirus dealing with a potential case of fcv-associated virulent systemic disease two outbreaks of virulent systemic feline calicivirus infection in cats in germany virulent feline calicivirus disease in a shelter in italy: a 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proteinase-polymerase precursor protein haemorrhagic fever, oedema and high mortality associated with fcv infection the viral polymerase inhibitor -c-methylcytidine inhibits norwalk virus replication and protects against norovirus-induced diarrhea and mortality in a mouse model treatment with a nucleoside polymerase inhibitor reduces shedding of murine norovirus in stool to undetectable levels without emergence of drug-resistant variants biochemical evaluation of the inhibition properties of favipiravir and -c-methyl-cytidine triphosphates against human and mouse norovirus rna polymerases inhibition of human norovirus by a viral polymerase inhibitor in the b cell culture system and in the mouse model treatment of diarrhea caused by cryptosporidium parvum: a prospective randomized, double-blind, placebo-controlled study of nitazoxanide nitazoxanide: a new thiazolide antiparasitic agent nitazoxanide in the treatment of viral gastroenteritis: a randomized double-blind placebo-controlled 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rna polymerase: a new mechanism for antiviral intervention antiviral activity of selected flavonoids against chikungunya virus structure-based design, synthesis, and biological evaluation of peptidomimetic sars-cov clpro inhibitors antiviral drug advances in the treatment of human immunodeficiency virus (hiv) and chronic hepatitis c virus (hcv) efficacy of a c-like protease inhibitor in treating various forms of acquired feline infectious peritonitis evaluation of orally administered famciclovir in cats experimentally infected with feline herpesvirus type- we are grateful to dae jong han for his help with fcv construct. we also acknowledge salvatore ferla, andrea brancale and marcella bassetto for the nni, compound . the authors declare no conflict of interest. key: cord- -nidgnvvi authors: medkour, hacène; amona, inestin; akiana, jean; davoust, bernard; bitam, idir; levasseur, anthony; tall, mamadou lamine; diatta, georges; sokhna, cheikh; hernandez-aguilar, raquel adriana; barciela, amanda; gorsane, slim; la scola, bernard; raoult, didier; fenollar, florence; mediannikov, oleg title: adenovirus infections in african humans and wild non-human primates: great diversity and cross-species transmission date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: nidgnvvi non-human primates (nhps) are known hosts for adenoviruses (advs), so there is the possibility of the zoonotic or cross-species transmission of advs. as with humans, adv infections in animals can cause diseases that range from asymptomatic to fatal. the aim of this study was to investigate the occurrence and diversity of advs in: (i) fecal samples of apes and monkeys from different african countries (republic of congo, senegal, djibouti and algeria), (ii) stool of humans living near gorillas in the republic of congo, in order to explore the potential zoonotic risks. samples were screened by real-time and standard pcrs, followed by the sequencing of the partial dna polymerase gene in order to identify the adv species. the prevalence was . folds higher in nhps than in humans. more than / ( . %) of the nhps and / ( . %) of the humans excreted advs in their feces. the positive rate was high in great apes ( %), with a maximum of . % in chimpanzees (pan troglodytes) and . % in gorillas (gorilla gorilla), followed by monkeys ( . %), with . % in barbary macaques (macaca sylvanus) and . % in baboons (seven papio papio and six papio hamadryas). no green monkeys (chlorocebus sabaeus) were found to be positive for advs. the advs detected in nhps were members of human mastadenovirus e (hadv-e), hadv-c or hadv-b, and those in the humans belonged to hadv-c or hadv-d. hadv-c members were detected in both gorillas and humans, with evidence of zoonotic transmission since phylogenetic analysis revealed that gorilla advs belonging to hadv-c were genetically identical to strains detected in humans who had been living around gorillas, and, inversely, a hadv-c member hadv type was detected in gorillas. this confirms the gorilla-to-human transmission of adenovirus. which has been reported previously. in addition, hadv-e members, the most often detected here, are widely distributed among nhp species regardless of their origin, i.e., hadv-e members seem to lack host specificity. virus isolation was successful from a human sample and the strain of the mbo genome, of kb, that was identified as belonging to hadv-d, exhibited close identity to hadv-d members for all genes. this study provides information on the advs that infect african nhps and the human populations living nearby, with an evident zoonotic transmission. it is likely that advs crossed the species barrier between different nhp species (especially hadv-e members), between nhps and humans (especially hadv-c), but also between humans, nhps and other animal species. adenoviruses (advs), members of the adenoviridae family, are dna viruses that naturally infect many vertebrates, including humans and non-human primates (nhps). their name derives from their initial isolation from human adenoids in [ ] . since then, human adenoviruses (hadvs) have been increasingly recognized as major contributors to clinical illness, from mild respiratory infections in young children (known as the common cold) to life-threatening multi-organic diseases in people with weakened immune systems. it is estimated that more than % of the human population is seropositive for at least one serotype of advs [ , ] . illnesses include upper and lower respiratory tract diseases, conjunctivitis, acute hemorrhagic cystitis, meningoencephalitis, diarrhea, intussusceptions, celiac disease, hepatitis, myocarditis and obesity, with certain clinical diseases associated with specific adenoviral species and genotypes [ ] . despite their role as pathogens, some hadvs and simian adenoviruses (sadvs) have been used or proposed as tools for vaccine delivery and gene therapy [ ] . until now, within the genus mastadenovirus (including all human adenoviruses), human adv genotypes have been assigned in high resolution using genomics (http://hadvwg.gmu.edu/). they have been divided into seven distinct species (human mastadenovirus a to human mastadenovirus g, informally hadv-a to hadv-g) that were originally distinguished by their biological, clinical and restriction enzyme digestion properties and were reconfirmed using omics data. advs within the genus mastadenovirus could infect other mammalian hosts, such as nhps, bats, bovines, canines, deer, dolphins, equines, murines, ovines, swine, sea lions, skunks, squirrels and tree shrews [ ] . novel human and animal advs continue to be identified and characterized [ , ] . in addition, there are advs in four other genera within the adenoviridae family that infect animals. these are atadenovirus, type species: ovine atadenovirus d; aviadenovirus, type species: fowl aviadenovirus a; ichtadenovirus, type species: sturgeon ichtadenovirus a; and siadenovirus, type species: frog siadenovirus a (https://talk.ictvonline.org/taxonomy/). as with humans, adv infections in animals can cause diseases that range from asymptomatic to fatal [ ] . for millennia, interactions between humans and their closest living relatives have led to an inherent risk of pathogen transfer. nhps are increasingly implicated as potential sources of emerging zoonotic diseases in humans [ , ] . indigenous groups that depend on wildlife for survival were exposed to the risk of the transmission of nhp pathogens through hunting, consumption of bushmeat [ ] and through other ways; for example, by sharing non-flowing water sources, fruits and plant sources that nhps have used. concerning adenoviruses, virologists have long wondered about the possibility that nhp adenoviruses may one day pose a risk to humans [ ] . although adenoviruses are generally considered to be rather host species-specific viruses, canine adenovirus has a wider host range and can infect members of the canidae, mustelidae and ursidae families [ ] . the host range and zoonotic potential of simian adenoviruses has more recently become an area of interest [ , ] . simian adenoviruses (sadvs) have been described in macaques (sadv- to sadv- ), african green monkeys (sadv- to sadv- and sadv- ), baboons (sadv- ) and chimpanzees (sadv- to sadv- ) [ ] . many more simian advs have been described, like new world monkey and prosimian advs [ ] . in , roy s et al. isolated and characterized novel great ape advs from the feces of chimpanzees, bonobos and gorillas, as well as three macaque advs. all of them were captive nhps, held in facilities and zoos in north america. virus isolation was performed, and complete genomes were sequenced and tentatively named sadv- . to sadv- [ ] . most sadvs have proven to be very similar to hadvs. currently, the naming of a species follows the principle that if an adenovirus species with primate hosts contains at least one type of hadv, the species is called a hadv species, otherwise it is called a sadv species. as a result, most sadvs have been grouped correspondingly into the hadv-b, c, e and g species [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the present study, we sought to investigate the presence and molecular diversity of advs in wild african nhps, including great apes (gorillas and chimpanzees), macaques and other monkeys (baboons, green monkeys), living in close proximity to or outside human settlements. in addition, we assessed the adv infections of a local human population from the republic of congo, who were living near gorillas, in order to evaluate potential zoonotic transmissions. fecal samples were collected and tested by pcr/sequencing, and the adenoviruses were typed based on their dna polymerase partial gene. a human strain was isolated by viral cell culture and described. in senegal, in august , fecal samples of western chimpanzees (pan troglodytes verus) were collected from three sites located within the dindefelo community natural reserve in the kédougou region. site : three degraded stool samples ( • . • e) were collected. for nhps, fresh fecal samples were collected at sleeping sites, feeding sites and places where the primates had been observed. in addition, in , human stool samples were collected after obtaining the verbal consent of all the participants because of their low level of literacy. a total of samples were collected, including from the local population of the village of mbomo, located inside the oknp, and from eco-guards in the llr. all great ape samples were stored in absolute alcohol, and fresh samples and human samples were first stored at − • c before being sent from the republic of congo to france for analysis. in the republic of djibouti, fecal samples of hamadryas baboons (papio hamadryas) were collected in . these baboons lived outside the village of oueah, km from the city of djibouti ( • . " n • . " e). this collection was carried out in partnership with the center for studies and research of djibouti. in algeria, fecal samples were collected from barbary macaques (macaca sylvanus), with the authorization of the management of the chréa national park (cnp), including samples collected from two sites, the stream of monkeys and the gorges of la chiffa in blida province, km north of algiers ( • . " n • . " e), and samples from cap carbon ( • . " n • . " e) in the suburbs of béjaïa, km east of algiers. these primates were synanthropic and lived in close contact with the people who provided them with food. no experimentation was conducted on any of the nhps in our study as all fecal samples were collected from the ground. the sampling was non-invasive and did not disrupt any wild animal. all humans in our study were apparently healthy. in addition, the fact that the nhps' feces were not diarrheic may perhaps be some indication of them also being healthy. all collected samples were taken to the ihu méditérranée infection laboratory, marseille, france, for analysis. they were identified and stored at either − • c or − • c. viral dna extraction was performed using the qiagen virus mini kit ® v . (qiagen, courtaboeuf, france) on biorobot ez (qiagen, courtaboeuf, france), according to the manufacturer's instructions. firstly, about g of each stool was mixed with µl of g buffer and µl of proteinase k (qiagen, courtaboeuf, france). this was subjected to a mechanical lysis with tungsten beads (qiagen, courtaboeuf, france), using a fastprep- tm g grinder, before being incubated overnight at • c. then, viral dna was extracted from µl of supernatant with µl of internal control (enterobacteria phage t ). dna was eluted in µl volume, then aliquoted in individual pcr tubes to an amount of µl of pure extracted dna. then, other aliquots of µl of dna were diluted to : , and finally, one-third of µl of dna diluted to : . dna tubes were stored at − • c until use. the dna extraction and dilutions were controlled by qpcr, targeting the phage t (table ) . based on the results of the qpcr for extraction control, dna which had been diluted to / th was chosen for adenovirus testing. first, all samples were screened using qpcr targeting the t phages [ ] , and after that they were screened by qpcr targeting a conserved region of the hexon gene of all hadv prototypes [ ] . the qpcr amplifications were performed in a cfx real-time system (bio-rad laboratories, foster city, ca, usa). reactions were carried out in a final volume of µl, containing µl of dna template, µl of master mix roche (eurogentec, seraing, belgium), . µl each primer per reaction at a concentration of µm, . µl udg and . µl of each probe at a concentration of µm. the taqman cycling conditions included two hold steps at • c for min, followed by • c for min and cycles of two steps each ( • c for s and • c for s). the pcr systems used for the study are detailed in table . each pcr plate contained wells. dna from the cultured adenovirus was included as a positive control and master mixtures as negative controls in each test. all samples were tested by a sensitive and specific nested pcr (table ) , using outer primers in the first reaction and inner primers in the second. the nested pcr was used for the amplification of the dna polymerase partial gene and the specific diagnostic for adenoviral shedding [ ] . for gene amplification, pcr reactions were performed in a total volume of µl, consisting of µl of amplitaq gold master mix, µl of ultra-purified water dnase-rnase free, µl of each primer ( µm of concentration) and µl of the dna template. in the second amplification, we used only µl of pcr product rather than µl of dna and µl of ultra-purified water rather than µl. the thermal cycling conditions for both pcr amplifications were as follows: incubation step at • c for min, cycles (only cycles for the second pcr) of one minute at • c, s for the annealing at • c, . min ( s for the second pcr) of elongation time at • c. finally, we included an extension step for five minutes at • c. pcr amplification was performed in a peltier ptc- model thermal cycler (mj research inc., watertown, ma, usa). the results of amplification were visualized by electrophoresis on % agarose gel. samples that had ct < in qpcr and a good band of nested pcr were selected for sequencing. amplicons were purified using nucleofast pcr plates (macherey nagel eurl, hoerdt, france), as per the manufacturer's instructions, and sequenced using the big dye terminator cycle sequencing kit (perkin elmer applied biosystems, foster city, ca, usa) with an abi automated sequencer (applied biosystems). the obtained electropherograms were assembled and edited using chromaspro software (chromaspro . , technelysium pty ltd., tewantin, australia) and compared with those available in the genbank database compiled by ncbi blast (https://blast.ncbi.nlm.nih. gov/blast.cgi). the sequences obtained were aligned and compared with each other and with those of the advs available in the genbank database. a maximum likelihood method was used to infer the phylogenetic analyses and tree reconstruction was performed using mega software version (https://www.megasoftware.net/). bootstrap analyses were conducted using replicates. fecal samples from positive nhps and humans for the molecular detection of advs were subjected for virus isolation in cell cultures. the large particles were removed by low speed centrifugation, and the supernatant was filtered through . µm-pore sized syringe filters. a volume of µl of each filtered sample was inoculated into hep and mrc cells which had been grown in a mem medium with % fetal bovine serum (fbs) for days. cultures were observed every days to detect the appearance of a cytopathogenic effect (cpe). qpcr targeting adenovirus was performed at day and day in each culture to confirm that cpe was related to adenovirus growth. virus isolates collected from the cell lines were analyzed using the molecular approaches described above. the isolated viruses were first analyzed using the same molecular approaches described above and then subjected to next generation sequencing (ngs) for the sequencing of the whole adenovirus genome. whole adenovirus genome analysis was performed using dna extracted from the culture isolate using next generation sequencing (ngs). the genomic dna (gdna) of adv mbo was quantified by a qubit assay with the high sensitivity kit (life technologies, carlsbad, ca, usa) to . ng/µl. the genomic dna was then sequenced with the miseq technology (illumina inc., san diego, ca, usa) with the paired end strategy and was barcoded in order to be mixed respectively with other genomic projects prepared with the nextera xt dna sample prep kit (illumina). to prepare the paired end library, a dilution was performed so that ng of each genome was needed as input to prepare the paired end library. the tagmentation step fragmented and tagged the dna. then, limited cycle pcr amplification ( cycles) completed the tag adapters and introduced dual-index barcodes. after purification on ampure xp beads (beckman coulter inc., fullerton, ca, usa), the libraries were normalized on specific beads according to the nextera xt protocol (illumina). normalized libraries were pooled into a single library for sequencing on the miseq. the pooled single strand library was loaded onto the reagent cartridge and then onto the instrument, along with the flow cell. automated cluster generation and paired end sequencing with dual index reads were performed in a single -h run in x -bp. total information of , gb was obtained from a k/mm cluster density with a cluster passing quality control filters of . %. within this run, the index representation for adv mbo was determined to index . . the , , paired end reads were filtered according to the read qualities. the quality of the sequence data obtained was controlled using the clc genomics workbench v (qiagen, https://www.qiagenbioinformatics.com/products/clc-genomics-workbench/) and the assembly was performed by spades v . . [ ] . sequences were trimmed with miseq and trimmomatic [ ] software, whereas untrimmed data were processed only using miseq software. to reduce assembly deviations, we used gapcloser software [ ] . scaffolds that had a nucleotide number < (bp) and scaffolds that had a depth value lower than % of the mean depths were removed. the best assembly was selected using different criteria (number of scaffolds, n , number of n) [ ] . after assembly, we applied a mapping program between our genome and that of the reference genomes, human adenovirus (jn ) and human adenovirus (jn ), to produce consensus sequences in order to ensure the consistency of the methodology using a scaffold builder [ ] with a minimum identity for merging contigs ( %), minimum length for ambiguously mapped contigs ( %) and maximum gap length allowed ( nt) (supp. material s ). finally, the genes (e a, e b, ix, iva , e a, e b, ptp, l -l , e , e , u-exon) were analyzed using a homology search engine, the local blast [ , ] . the sequences were aligned using muscle with default parameters. the dna extraction was validated by the qpcr targeting the t phage: all samples tested positive (ct ≤ where we collected gorilla and human samples simultaneously, advs were more frequent in gorillas ( . %) than humans ( . %) ( / ) (z test, p-value < . ) (figure , table ). (figure , table ). we succeeded in obtaining sequences of ≈ bps of adenoviral dna polymerase genes from the stool of nhps: six sequences from gorillas, ten from chimpanzees, seven from macaques and one from baboons from djibouti. four sequences were obtained from human stool samples from the republic of congo. the sequences were compared with each other and with the dna polymerase sequences available in the genbank database (table s ). seven sequences from chimpanzees, one from gorillas, one from baboons and seven from macaques exhibited at least a % identity with each other and a % identity with sadv- (fj /hb ). they clustered together and with hadv-e we succeeded in obtaining sequences of ≈ bps of adenoviral dna polymerase genes from the stool of nhps: six sequences from gorillas, ten from chimpanzees, seven from macaques and one from baboons from djibouti. four sequences were obtained from human stool samples from the republic of congo. the sequences were compared with each other and with the dna polymerase sequences available in the genbank database (table s ). seven sequences from chimpanzees, one from gorillas, one from baboons and seven from macaques exhibited at least a % identity with each other and a % identity with sadv- (fj /hb ). they clustered together and with hadv-e members from genbank ( figure ) . one sequence from a chimpanzee showed a % identity with sadv- . (fj ) and grouped with hadv-b types. one human sequence was % identical to a hadv-d type (kf ). three human and two gorilla sequences were perfectly similar to each other and two other sequences from gorillas showed > % identity with them. all these sequences clustered together with species hadv-c ( figure ). members from genbank ( figure ) . one sequence from a chimpanzee showed a % identity with sadv- . (fj ) and grouped with hadv-b types. one human sequence was % identical to a hadv-d type (kf ). three human and two gorilla sequences were perfectly similar to each other and two other sequences from gorillas showed > % identity with them. all these sequences clustered together with species hadv-c ( figure ). sequence alignment of the dna polymerase gene of human and simian adenoviruses. the evolutionary history was inferred using the maximum likelihood method, based on the tamura three-parameter model. sequences were aligned by the clustalw method and compared with each other as well as with available dna polymerase sequences from genbank. sequences in this study are indicated by the following colors: brown for humans; green for gorillas; blue for chimpanzees; violet for macaques and red for baboons. sequences from genbank are in black. adv types from hadv-c, -b and -e were detected in african nhps and from hadv-c and -d in humans. hadv-c jumped from humans to gorillas and, inversely, hadv-c strains g and g a jumped from gorillas to humans from the republic of congo. the tree with the highest log likelihood (− . ) is shown. the percentage of trees in which the associated taxa clustered together is shown next to the branches. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. the analysis involved nucleotide sequences. the confidence probability (multiplied by ) for the inside length of the branch is greater than , as estimated by the bootstrap test ( ), which is shown next to the branches. in the republic of congo, we found hadv-c and e members in gorillas and hadv-c and d members in humans, with an abundance of c members in both humans and gorillas (table s ). in senegal, hadv-b and e members were detected in chimpanzees, and the e members were the most prevalent ones. adv-e was also found in baboons from djibouti. in algeria, the macaques were infected by adv-e types. in this study, the green monkeys were negative for advs, probably due to the limited number of samples (only four samples). viral isolation was successful in hep cells inoculated with a human stool sample with ct in the qpcr control at day . no virus was isolated from the nhp stool samples. the mbo strain of the whole genome of a kb length showed a . % identity with hadv- (jn ) and a . % identity with hadv- (jn ) from hadv-d detected in a human from the united states of figure . molecular phylogenetic analysis by maximum likelihood method based on a short ( bps) sequence alignment of the dna polymerase gene of human and simian adenoviruses. the evolutionary history was inferred using the maximum likelihood method, based on the tamura three-parameter model. sequences were aligned by the clustalw method and compared with each other as well as with available dna polymerase sequences from genbank. sequences in this study are indicated by the following colors: brown for humans; green for gorillas; blue for chimpanzees; violet for macaques and red for baboons. sequences from genbank are in black. adv types from hadv-c, -b and -e were detected in african nhps and from hadv-c and -d in humans. hadv-c jumped from humans to gorillas and, inversely, hadv-c strains g and g a jumped from gorillas to humans from the republic of congo. the tree with the highest log likelihood (− . ) is shown. the percentage of trees in which the associated taxa clustered together is shown next to the branches. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. the analysis involved nucleotide sequences. the confidence probability (multiplied by ) for the inside length of the branch is greater than , as estimated by the bootstrap test ( ), which is shown next to the branches. in the republic of congo, we found hadv-c and e members in gorillas and hadv-c and d members in humans, with an abundance of c members in both humans and gorillas (table s ). in senegal, hadv-b and e members were detected in chimpanzees, and the e members were the most prevalent ones. adv-e was also found in baboons from djibouti. in algeria, the macaques were infected by adv-e types. in this study, the green monkeys were negative for advs, probably due to the limited number of samples (only four samples). viral isolation was successful in hep cells inoculated with a human stool sample with ct in the qpcr control at day . no virus was isolated from the nhp stool samples. the mbo strain of the whole genome of a kb length showed a . % identity with hadv- (jn ) and a . % identity with hadv- (jn ) from hadv-d detected in a human from the united states of america. the fifteen recognized genes for advs were identified in the mbo strain and compared to the reference adv species; they were close to the hadv-d types (table s ). the hadv-mbo whole genome was deposited in the genbank database under accession number (lr . ). since the recurring influenza scares (h n since and h n in ) and the recent coronavirus outbreaks (sars-cov in , mers-cov in and sars-cov- in ), the topic of zoonotic viruses, particularly viruses that can cross the interspecies barrier from their natural host and eventually cause disease in humans, has aroused great interest. for any virus, adaptation to a new host must meet several basic requirements and key events. in this study, we detected several species of advs in african great apes (gorillas and chimpanzees) and monkeys (baboons and algerian macaques), as well as in local people residing near gorillas in the republic of congo. interestingly, the positive rate was . folds higher in nhps than in humans. more than one third of the nhps and one tenth of the humans in this study carried and excreted advs in their feces. the high prevalence of advs in nhps could be explained by the fact that these animals are repeatedly infected, although these infections would have to be prolonged and frequent to maintain virus circulation and increase their prevalence. there was much less virus excretion in human feces, which may have been due to a more efficient t-cell response to viruses [ ] . adv infections have been reported at a high prevalence in wild gorilla and chimpanzee populations, as well as in other great apes [ , , [ ] [ ] [ ] [ ] . in the present study, the adv-infection rate in gorillas from the republic of congo was . %, which is close to the previously reported rates in free ranging gorillas in the same area ( . %) [ ] or those from loango national park, gabon ( %) [ ] . captive gorillas in zoos had a much higher prevalence of shedding ( %- %) [ ] . the results in our senegalese chimpanzee study population ( . %) were higher than in the cameroon/democratic republic of congo study ( %) [ ] and in another population of savannah-dwelling chimpanzees from the issa valley in tanzania ( %) [ ] , and they were lower than the reported rate of % in the republic of congo [ ] . these differences seem to be marginal and could be due to differences in sample collection and conservation conditions, study region, seasonality or degree of anthropogenic disturbance of the habitat, but they are less likely to be due to laboratory differences, since all the studies used the same pcr assay [ ] . our results confirm the persistence of adv feces shedding and circulation in african great apes. in addition, the occurrence of advs in macaque feces ranged from % to %: % in oregon-rhesus, % covance-rhesus, % gtp-cynomolgus and % gtp-rhesus [ ] . for the first time, we detected the dna of advs in % of barbary macaques from algeria ( %). it was also reported that african monkeys could be infected with advs [ ] . we found a positive rate of % in the sub-saharan monkeys of our study, including % in baboons (from senegal and djibouti) and % in green monkeys. although this difference in results between the macaques in north africa and the other monkeys in sub-saharan africa was not statistically significant and was related to the difference in sample sizes ( macaques versus only baboons and green monkeys), the difference was not statistically significant. in addition, macaques already live in contact with tourists, which may be the cause of transmission between the two species. although prevalence statistics are presented here, the reality may be underestimated due to the potential deterioration of the sample. great apes seemed to be more infected ( %) than monkeys ( . %) and humans ( . %). on the one hand, apes could be repeatedly infected and, consequently, viruses may be maintained and circulated fluently. on the other hand, the human t-cell response to the viruses is more effective, leading to the limitation of virus shedding. our study revealed a great diversity of advs among african humans and nhps. we identified adv sequences from nhps and four from humans. from nhps, based on the bp of adv dna polymerase gene [ ] , we detected the members of three adv species: these were principally hadv-e in % of cases, followed by hadv-c in % of cases and hadv-b in % of cases. these advs have been previously reported [ , , , , ] . in addition, it could be possible, because of the methodology, that very divergent segments were not picked up by the primers, especially those of monkey advs. therefore, the non-identified advs here could be sadvs or advs from other species which were not amplified by the applied primers. furthermore, the dna polymerase fragment was selected in order to be highly conserved. it is therefore no surprise that it was found to be considerably conserved. as demonstrated in figure , hadv-e members seemed to be well adapted in nhps, since they were present in all nhps in this study, except green monkeys (perhaps because of the small number of samples), and they clustered both with one another and with sadv-e members. although advs are generally considered to be rather host species-specific viruses, there are some exceptions [ ] . indeed, hadv-e could be an exception, since african great apes, as well as macaques or other monkeys in our study, carried hadv-e members. for hadv-e type occurrence in this study, wild senegalese chimpanzees were the most numerous hosts ( % of cases), followed by macaques ( %) and then gorillas and baboons ( . % of cases for each). herein, the hypothesis that nhp adv members of the hadv-e originated from chimpanzees [ , ] , and because chimpanzees, gorillas and baboons all live in sub-saharan africa, we can suppose that the gorilla and baboon hadv-e types of this study were the result of chimpanzee adv transmission. the evolution of hadv-e viruses and their adaptation to new hosts requires further investigation. hadv-c types were detected in the gorilla feces samples from the llr and oknp and eco-guards as well as in humans living around the oknp. these adv-c members consist of three clades: (i) clade : one gorilla isolate from oknp, one gorilla isolate from llr and three human isolates that are fully identical and they are all very similar to sadv- . . in addition, two other isolates from llr gorillas had close similarity with sadv- . ; (ii) clade : a gorilla adv from oknp with perfect similarity to hadv-c k - (lc ); (iii) clade : a gorilla adv from llr, closely related to gorilla beringei graueri adv- [ ] . hadv-c types had already been reported in free ranging gorillas from republic of congo [ ] and gabon [ ] , wild mountain (gorilla beringei beringei) and grauer's (gorilla beringei graueri) gorillas from rwanda [ ] and captive gorillas, as well as captive chimpanzees and bonobos [ ] . according to hoppe et al. [ ] and other studies [ , ] , it seems that all the lineages in hadv-c are host specific. nevertheless, we report here that both a gorilla adv (g ) and human advs (mbo , and ibou ) are related to species hadv-c (figure ) , which is evidence for cross-species and potential zoonotic transmission. as a result, hadv-c types could have a wider range of hosts rather than being strictly host species-specific. wild gorillas and chimpanzees that do not necessarily come into daily contact with humans carry human-associated adenoviruses. the same was true for the local human population, including the eco-guards that carried simian viruses. this suggests that a kind of "jump" has occurred. a hadv-b type detected here in a senegalese chimpanzee was close to sadv- . which was detected in a captive gorilla [ ] . advs belonging to this species have been previously reported in chimpanzees and gorillas from the republic of congo [ ] , wild gorillas from gabon [ ] and both captive and wild gorillas and chimpanzees [ , ] . wevers et al. [ ] reported hadv-b types in gorillas and six chimpanzees and confirmed for wild individuals that members of hadv-b widely infect great apes. however, viruses of the same host species strayed throughout the clade, and several subclades comprising advs of different hosts were visible [ ] . they suggested that human hadv-b originated in great apes. in our study, the hadv-b type that was found in chimpanzees formed a separate lineage from the clade of hadv-b. among human advs, hadv-d is the largest and fastest growing species [ ] . we detected hadv-d members in the stool of humans living around the oknp, thus confirming that the species hadv-d originated in humans [ ] and so far has been exclusively human specific. nkogue et al. reported four different serotypes in a human population around a gorilla park, highlighting the diversity of advs circulating in humans [ ] . furthermore, it has been found that advs from wild chimpanzees clustered in hadv-d and showed a % pairwise hexon nucleic acid identity with hadv- and a % identity with hadv- and - [ ] . as a result of the co-habitation of eco-guards with nhps, the transmission of these viruses from humans to nhps could eventually lead to the extension of hadv-d members in nhp communities. in our study, viral isolation was possible only in a human sample as the nhp samples were not fresh. adv strain mbo was identified as a hadv-d member and was found to be almost identical to hadv- , thus confirming the pcr results. unfortunately, it has not been possible to isolate other advs in humans or nhps in order to better understand their genetic diversity and relationships. in addition to the degraded quality of the nhp samples, failures of the simian virus culture could be related to the lack of a suitable culture system, since the culture was performed using human cell lines. finally, our results show that advs are naturally present among african nhps and the human communities living near them. wild chimpanzees and gorillas of the same population carry a variety of adv genotypes, suggesting that great apes are the origin of many hadvs. these viruses are maintained by the exchange between ape individuals and potentially with human populations that live nearby. one of the strengths of this study was that we analyzed the fecal samples of different nhp species with different origins (north africa and sub-saharan africa) as well as samples from humans living in contact with primates in order to evaluate the potential for zoonotic transmission. moreover, the fact that while the target partial gene seemed to be linked even across species, one organism could be capable of hosting simian and human viruses and recombination events, is not highlighted here, but it is a possibility. we complement and enrich recent knowledge (e.g., [ , , ] ) which has suggested that hadv evolution has been mainly governed by strong, long-term associations with their hominid hosts. adv species (in particular hadv-e) have been found to be represented in several primates, suggesting, most likely, cross-species transmission. moreover, we found evidence of the zoonotic transmission of adenoviruses, particularly in hadv-c members, in the republic of congo. our study reported a rich diversity of adenovirus types in hadv-e, hadv-c and hadv-b among african apes and/or monkeys as well as indigenous human populations (hadv-c and hadv-d). adv shedding was more prevalent in nhps than in humans (infection rate was . folds higher in nhps compared to humans). hadv-e types were the most predominant ones in nhps regardless of their origins, and they seemed to lack strict host specificity. the advs from nhps that were detected here (especially adv-c members) are common in nhps and human populations living nearby, thus suggesting a zoonotic transmission, meaning that these viruses have switched hosts. this has been especially true for crossing the species barrier between different nhp species (especially in the case of the adv-e 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adenovirus infecting western lowland gorillas and humans in and around moukalaba-doudou national park (gabon) chimpanzee adenovirus antibodies in humans, sub-saharan africa adenovirus infection in savanna chimpanzees (pan troglodytes schweinfurthii) in the issa valley adenoviruses isolated from wild gorillas are closely related to human species c viruses we are grateful to clio grimaldier, mboussi vincent, bréchard ludivine, aurelia caputo for their valuable technical assistance in this work. we are grateful to ismail lafri, rahal mohamed and fayçal zeroual, and the parc national de chréa (algeria). we are also grateful to agence congolaise de la the authors declare no conflict of interest. the funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript and in the decision to publish the results. the following are available online at http://www.mdpi.com/ - / / / /s , table s . adenoviruses identified in the present study in african humans and nhps (sequences of ≈ bp dna polymerase gene) and their identity with genbank reference sequences; table s : mbo gene annotation and comparison to other related human mastadenovirus d types. supp. material s : whole genome mbo . key: cord- - o upns authors: pascual-iglesias, alejandro; sanchez, carlos m.; penzes, zoltan; sola, isabel; enjuanes, luis; zuñiga, sonia title: recombinant chimeric transmissible gastroenteritis virus (tgev)—porcine epidemic diarrhea virus (pedv) virus provides protection against virulent pedv date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: o upns porcine epidemic diarrhea virus (pedv) is an enteric coronavirus causing high morbidity and mortality in porcine herds worldwide. although both inactivated and live attenuated vaccines have been extensively used, the emergence of highly virulent strains and the recurrent outbreaks even in vaccinated farms highlight the need of effective vaccines. engineering of genetically defined live attenuated vaccines is a rational approach for novel vaccine development. in this line, we engineered an attenuated virus based on the transmissible gastroenteritis virus (tgev) genome, expressing a chimeric spike protein from a virulent united states (us) pedv strain. this virus (rtgev-rs-spedv) was attenuated in highly-sensitive five-day-old piglets, as infected animals did not lose weight and none of them died. in addition, the virus caused very minor tissue damage compared with a virulent virus. the rtgev-rs-spedv vaccine candidate was also attenuated in three-week-old animals that were used to evaluate the protection conferred by this virus, compared with the protection induced by infection with a virulent pedv us strain (pedv-nvsl). the rtgev-rs-spedv virus protected against challenge with a virulent pedv strain, reducing challenge virus titers in jejunum and leading to undetectable challenge virus rna levels in feces. the rtgev-rs-spedv virus induced a humoral immune response specific for pedv, including neutralizing antibodies. altogether, the data indicated that rtgev-rs-spedv is a promising vaccine candidate against virulent pedv infection. acute infectious diarrhea is a major cause of high morbidity and mortality in piglets worldwide. enteric infections in animals are frequently associated with viruses, including rotaviruses and coronaviruses (covs) [ ] . a metagenomics analysis of diarrheic and healthy samples from china in found porcine covs in % of the diarrheic samples, and only in around % of the healthy samples, highlighting the potential relevance of covs as enteric porcine pathogens [ ] . porcine epidemic diarrhea virus (pedv) was found in more than % of the diarrheic samples [ ] , in agreement with the importance that this virus has for the porcine industry worldwide. pedv was first described in europe in the s and the virus has, since then, remained endemic in the european porcine herds [ ] . in asian countries, pedv was detected in the s, with highly virulent outbreaks observed since viruses , , of in piglets. subsequently, an attenuated chimeric vaccine candidate was developed by introducing duplication of transcription regulating sequences (trss), as previously described by our group [ ] . this attenuated vaccine candidate conferred partial protection to pigs from a challenge with a virulent pedv. baby hamster kidney cells stably transformed with the gene coding for porcine aminopeptidase n (bhk-papn) [ ] were grown in in dulbecco's modified eagle's medium (dmem) supplemented with % fetal calf serum (fcs) and g ( . mg/ml) as selection agent. vero cells were grown in dmem supplemented with heat-inactivated % fcs. a us virulent pedv strain (pedv-nvsl, genbank accession number kf ) was kindly provided by ceva animal health. a recombinant virulent us pedv virus (rpedv), recovered from an infectious cdna engineered by our group (a. pascual-iglesias, l. enjuanes and s. zuñiga, unpublished data), was used as challenge virus in the animal experiments. pedv infectious cdna was maintained as a bac, including pedv usa/iowa/ / sequence (genbank accession number kf ). pedv viruses and recombinant tgev viruses obtained in this work were grown in vero cells supplemented with infection medium (dmem supplemented with . % tryptose phosphate broth, . % yeast extract, and µg/ml trypsin). the culture medium was supplied daily with % of the initial trypsin amount. a dna fragment containing the s gene sequence from virulent pedv usa/iowa/ / (genbank accession number kf ) [ ] was chemically synthesized and purchased from geneart (regensburg, germany). a pgem-t plasmid containing nucleotides , to , of tgev-sc genome (genbank accession aj ) and engineered paci and mlui restriction sites [ ] were used as an intermediate plasmid. overlapping polymerase chain reaction (pcr) was used to fuse pedv and tgev s gene sequences. the pedv s sequence encoding the ectodomain was amplified using purchased s sequence as template and oligonucleotides pac-s-vs ( -ggattaattaagaagggtaagttgctcattagaaataatggtaagttactaaactttggtaa ccacttcgttaacacaccatgaagtctttaacctac- , paci restriction site in italics) and pedv-s- -rs ( -ccacacataccaaggccacttgatgtatgtctc- ). tgev sequence encoding s protein transmembrane domain and endodomain was amplified using pgem-t-tgev-sc intermediate plasmid as a template and oligonucleotides tgev-s- -vs ( -gagacatacatcaag tggccttggtatgtgtgg- ) and tgev-s- -rs ( -tccaacgcgtaagtttag- , mlui restriction site in italics). in all cases, pedv sequences are indicated underlined. the obtained bp and bp pcr products were used as templates and amplified with oligonucleotides pac-s-vs and tgev-s- -rs. the bp pcr product was digested with paci and mlui and cloned into the same sites of pgem-t-tgev-sc plasmid, generating pgem-tgev-spedv plasmid. this plasmid was digested with paci and mlui restriction enzymes and the fragment containing chimeric s gene was cloned into the same sites of pbac-tgev-sc -p-m or pbac-tgev-sc -rs [ ] . all cloning steps were checked by sequencing of the pcr fragments and cloning junctions. for each mutant sequence, two independent cdnas were constructed. bhk-papn cells grown to % confluence in mm plates were transfected using µg of the corresponding pbac and µl of lipofectamine (invitrogen, carlsbad, ca, usa), according to manufacturer's specifications. at h post-transfection (hpt), bhk-papn transfected cells were trypsinized, washed twice with dmem, and plated over confluent vero monolayers grown in -mm-diameter plates. one hour later, trypsin was added to a final concentration of µg/ml. after a -day incubation period, the cell supernatants were harvested (passage ) [ ] . the rtgevs were cloned by limiting dilution method, grown, and titrated as previously described [ ] . twenty-one five-day-old suckling piglets, born from tgev and pedv seronegative sows, were randomly divided in two groups of nine piglets and one group of three animals. the nine-piglet groups were orally inoculated with tcid /animal of the different rtgevs in phosphate buffered saline (pbs). the three piglets in the other group were mock inoculated. infected animals were monitored daily to detect signs of enteric disease, and body weights were determined each day. three animals per group were euthanized and necropsied at days , , and after inoculation. intestinal macroscopic lesions were evaluated. front, mid, and end sections of jejunum were collected in all cases. samples were kept frozen for subsequent virus titration, stabilized with rnalater stabilization reagent (life technologies, carlsbad, ca, usa) for rna extraction, or fixed in zinc formaline fixative (sigma-aldrich, st. louis, mo, usa) for histopathology. fixed jejunum sections were washed twice with pbs and stored in % ethanol at • c. paraffin embedding, sectioning, and hematoxylin-eosin staining were performed by the histological laboratory (autopsy path kft., budapest, hungary). samples were examined with a zeiss axiophot fluorescence microscope. determination of the jejunum damage was obtained from unbiased measurement of three full-length perceived villi and crypt per location. at least two different locations per section per animal were measured. the villous height to crypt depth (vh/cd) ratio was calculated from the measurements. twenty-one -day-old piglets, born from tgev and pedv seronegative sows, were randomly divided in four groups ( table ) . piglets of groups and were orally inoculated with tcid /animal of rtgev-rs-spedv or pedv-nvsl, respectively. piglets of groups and were mock inoculated. three weeks after the immunization, piglets of groups , , and were challenged with tcid of rpedv per animal by oral route. infected animals were monitored daily to detect signs of enteric disease, and body weights were determined every days. fecal swabs were collected at days , , , , , and post-first inoculation. serum samples were taken at days , , , and post-immunization. saliva samples were collected, using salivette tubes (sarstedt, nümbrecht, germany), at days , , and post-first inoculation. three animals per group were euthanized and necropsied at days and post-challenge ( and post-immunization, respectively). intestinal macroscopic lesions were evaluated. front, mid, and end sections of jejunum were collected in all cases. samples were kept frozen for subsequent virus titration, stabilized with rnalater stabilization reagent (life technologies) for rna extraction, or fixed in zinc formaline fixative (sigma-aldrich) for histopathology. antibodies induced against tgev and pedv viruses were detected by elisa as described before [ ] . briefly, elisas were performed using . µg per well of partially purified tgev, pedv-nvsl, or rpedv viruses. antigens were bound to -well microplates, saturated with % bovine serum albumin (bsa) in pbs for h at • c, and incubated with serial dilutions of the serum sample in wash buffer ( . % bsa, . % tween in pbs) for min at • c. microplates were washed three times with wash buffer. bound antibodies were detected by incubation with peroxidase-conjugated protein a, goat anti pig igg (fc domain), or goat anti pig iga (biorad, hercules, ca, usa), diluted : , in pbs with . % bsa. elisa was developed with k-blue tmb substrate (neogen, lexington, ky, usa) for min at room temperature. reactions were stopped with . m h so , and the absorbance was read at nm. the elisa values of the sera were expressed as sample to positive ratio [sp-ratio = (od of sample − od of negative control)/(od of positive control − od of negative control)]. heat-inactivated sera were incubated for h at • c in the presence of pfus of pedv virus in dmem containing % fcs. serial dilutions of the mixtures were added to confluent vero cells in -well plates. after one hour of incubation at • c, medium was removed and infection medium was added. after h, cells were fixed with % formaldehyde in pbs, stained with a crystal violet solution, and virus titer was determined. the neutralization index was defined as the logarithm of the ratio of tcid in the presence of medium or in the presence of serum sample. total intracellular rna was extracted from vero cells or tissue samples. total rna was purified with rneasy mini kit (qiagen, hilden, germany), according to the manufacturer's specifications. to isolate viral rna from fecal swabs, the swab was resuspended in µl of pbs with antibiotics ( u/ml penicillin-streptomycin, µg/ml gentamicin, µg/ml amphotericin b). rna was isolated from µl of that solution using qiaamp viral mini kit (qiagen) following the manufacturers' instructions. in all cases, total cdna was synthesized using ng of total rna as a template, random hexamers, and the high-capacity cdna transcription kit (life technologies), following the manufacturers' recommendations. the viral genome region from b gene to utr (nt. - ) was sequenced in all cases. to that end, overlapping pcrs were performed using oligo pairs described in table . the pcr fragments were then sequenced using specific oligonucleotides. pedv genomic rna (grna) levels were measured by rt-qpcr analysis using a custom taqman assay detecting a conserved orf a region (taqman probe -fam-tgtact ggcttactggtgtt-mgb; forward primer -tgttgctatgtttgtgcattgg- ; reverse primer -tctgaatcactaggctgacctttg- ). tgev grna was evaluated using a custom taqman assay set up in our laboratory [ ] . the porcine β-glucuronidase (gusb) gene (taqman code ss _u ) was used as a reference housekeeping gene, since its expression remains constant in infected cells compared to that in non-infected cells [ [ ] and data not shown]. data were acquired with a viruses , , of real-time pcr system (applied biosystems, foster city, ca, usa) and analyzed with software v . . . the relative quantifications were performed using the −∆∆ct method [ ] . all experiments and data analysis were miqe (minimum information for publication of quantitative real-time pcr experiments guidelines) compliant [ ] . reverse size (bp) two-tailed, unpaired student t tests were used to analyze the difference in mean values between groups. all results were expressed as means ± the standard deviations of the means. p values < . were considered significant. a chimeric s protein (spedv-tgev) was designed. the n terminus of the protein contained the exposed domain of pedv s protein from a virulent us strain ( figure a ), including the epitopes recognized by neutralizing antibodies, and the heptad-repeats domains (aa to ). the c-terminus of the protein was that from the enteropathogenic tgev sc virus [ ] , including the transmembrane domain and c-terminus (aa to ). the chimeric protein was cloned in the tgev infectious cdna [ ] , replacing tgev s protein (rtgev-spedv, figure b ). in addition, chimeric spedv-tgev protein was cloned in an infectious cdna from an attenuated tgev, which contained duplications of transcription regulating sequences (trss) and engineered unique restrictions sites (rs) to avoid the overlapping of consecutive genes [ ] (rtgev-rs-spedv, figure c ). recombinant viruses rtgev-spedv and rtgev-rs-spedv were recovered after transfection of vero cells with the wild-type and attenuated cdnas, respectively. these viruses required trypsin to efficiently infect vero cells, and form syncytia, similarly to pedv virus. growth kinetics in cell culture was analyzed by infecting vero cells at two multiplicities of infection (moi)moi of . and . . both viruses reached peak titers at or hpi, depending on the moi ( figure a ). nevertheless, rtgev-rs-spedv virus peak titers were five-to -fold lower than those for parental rtgev-spedv virus ( figure a ). vero cells with the wild-type and attenuated cdnas, respectively. these viruses required trypsin to efficiently infect vero cells, and form syncytia, similarly to pedv virus. growth kinetics in cell culture was analyzed by infecting vero cells at two multiplicities of infection (moi)moi of . and . . both viruses reached peak titers at or hpi, depending on the moi (figure a ). nevertheless, rtgev-rs-spedv virus peak titers were five-to -fold lower than those for parental rtgev-spedv virus ( figure a ). to analyze the virulence of control rtgev-spedv and attenuated rtgev-rs-spedv viruses, fiveday-old piglets were orally inoculated with tcid /animal. interestingly, despite the difference in viral titers observed in cell cultures, viral titers in the jejunum of infected piglets were similar for both viruses at and dpi ( figure b ). at dpi, rtgev-spedv titers were slightly lower than those for rtgev-rs-spedv virus, suggesting a decreased number of gut epithelial cells available for reinfection. piglets inoculated with parental rtgev-spedv virus lost weight after infection (figure to analyze the virulence of control rtgev-spedv and attenuated rtgev-rs-spedv viruses, five-day-old piglets were orally inoculated with tcid /animal. interestingly, despite the difference in viral titers observed in cell cultures, viral titers in the jejunum of infected piglets were similar for both viruses at and dpi ( figure b ). at dpi, rtgev-spedv titers were slightly lower than those for rtgev-rs-spedv virus, suggesting a decreased number of gut epithelial cells available for reinfection. piglets inoculated with parental rtgev-spedv virus lost weight after infection ( figure a ) and % died ( figure b ). this data indicates that the chimeric rtgev-spedv control virus was virulent. in contrast, animals infected with rtgev-rs-spedv virus maintained weight ( figure a ) and all of them survived ( figure b ), indicating that the virus was attenuated. interestingly, virus shedding in the feces of piglets infected with attenuated rtgev-rs-spedv virus was up to -fold lower than that observed in the animals infected with the virulent parental rtgev-spedv virus ( figure c ). a) and % died ( figure b ). this data indicates that the chimeric rtgev-spedv control virus was virulent. in contrast, animals infected with rtgev-rs-spedv virus maintained weight ( figure a ) and all of them survived ( figure b ), indicating that the virus was attenuated. interestingly, virus shedding in the feces of piglets infected with attenuated rtgev-rs-spedv virus was up to -fold lower than that observed in the animals infected with the virulent parental rtgev-spedv virus ( figure c ). in agreement with the observed clinical signs, epithelial degeneration and exfoliation in the different parts of the jejunum was observed in animals infected with the virulent rtgev-spedv virus, accompanied by inflammatory infiltration and shortening of the villi ( figure a ). interestingly, the jejunum damage was significantly lower in animals infected with the attenuated rtgev-rs-spedv virus ( figure b ). altogether, the data indicate that rtgev-rs-spedv virus was partially attenuated. in agreement with the observed clinical signs, epithelial degeneration and exfoliation in the different parts of the jejunum was observed in animals infected with the virulent rtgev-spedv virus, accompanied by inflammatory infiltration and shortening of the villi ( figure a ). interestingly, the jejunum damage was significantly lower in animals infected with the attenuated rtgev-rs-spedv virus ( figure b ). altogether, the data indicate that rtgev-rs-spedv virus was partially attenuated. the main concern for the use of modified live vaccines based on attenuated viruses is their biosafety. reversion to virulence or recombination with circulating strains is in the basis for the emergence of novel virulent pedv strains, especially in asia [ , ] . the rtgev-rs-spedv virus genetic stability, which may influence its safety as vaccine candidate, was evaluated in cell culture. after passages in cell culture, viral genome region comprising from b gene to utr (nucleotides to ) was sequenced. no changes appeared in the passed virus compared with the parental one, strongly suggesting that the engineered virus was genetically stable in cell culture. to address virus stability in vivo, the -ends (nt to ) of rtgev-spedv and rtgev-rs-spedv viruses were sequenced, using rna isolated from the jejunum of five-day-old infected piglets at six days post-infection. no modifications were found in the parental rtgev-spedv virus. in the rtgev-rs-spedv virus, modifications were only detected in the engineered trs duplicated sequences ( figure ). small deletions and point mutations were found between e and m genes, eliminating the engineered fsei restriction site, but maintaining duplicated sequence ( figure ). extensive deletion was observed between m and n genes, reverting the sequence to that of the wild-type virus ( figure ). no changes were observed in the engineered mutations between n and genes ( figure ). interestingly, viruses , , of when viral rna was isolated from feces of -day-old piglets at seven days post-vaccination (see below) and rtgev-rs-spedv virus was sequenced, the same modifications were observed. this data strongly suggests that the rtgev-rs-spedv genome recovered from piglets may represent the in vivo evolution of the engineered virus. it is worth noting that tgev attenuation was similar with the three trss duplications and with just the duplication between n and genes [ ] , suggesting that in vivo evolved rtgev-rs-spedv virus may still be attenuated. the main concern for the use of modified live vaccines based on attenuated viruses is their biosafety. reversion to virulence or recombination with circulating strains is in the basis for the emergence of novel virulent pedv strains, especially in asia [ , ] . the rtgev-rs-spedv virus genetic stability, which may influence its safety as vaccine candidate, was evaluated in cell culture. after passages in cell culture, viral genome region comprising from b gene to ′utr (nucleotides to ) was sequenced. no changes appeared in the passed virus compared with the parental one, strongly suggesting that the engineered virus was genetically stable in cell culture. to address virus stability in vivo, the ′-ends (nt to ) of rtgev-spedv and rtgev-rs-spedv viruses were sequenced, using rna isolated from the jejunum of five-day-old infected piglets at six days no changes were observed in the engineered mutations between n and genes ( figure ) . interestingly, when viral rna was isolated from feces of -day-old piglets at seven days postvaccination (see below) and rtgev-rs-spedv virus was sequenced, the same modifications were observed. this data strongly suggests that the rtgev-rs-spedv genome recovered from piglets may represent the in vivo evolution of the engineered virus. it is worth noting that tgev attenuation was similar with the three trss duplications and with just the duplication between n and genes [ ] , suggesting that in vivo evolved rtgev-rs-spedv virus may still be attenuated. pedv infects animals of all ages, although the severity of the clinical signs is age-dependent [ ] . pregnant sows are the most suitable models to evaluate the effect of pedv vaccine candidates, as the target animals are neonatal piglets, and lactogenic immunity has an important role in protection [ ] . nevertheless, availability, cost, and housing resources limited its use in preliminary vaccine candidate trials [ , ] , and a young pig model has been proposed for preliminary vaccine efficacy trials [ , ] . therefore, to evaluate the protection conferred by rtgev-rs-spedv virus, -day-old pigs were used. group (g ) was vaccinated with rtgev-rs-spedv virus, group (g ) was vaccinated with a virulent us pedv strain (pedv-nvsl), and two groups of animals (g and g ) were not vaccinated ( table ) . twenty-one days after vaccination, animals from g , g , and g were challenged with a virulent recombinant pedv strain (rpedv) ( table ) . as expected, due to the animal age, no clinical sings of enteric disease such as diarrhea, vomiting, or dehydration were observed after vaccination or challenge (data not shown). nevertheless, animals vaccinated with the virulent pedv-nvsl strain showed a delay in weight gain the first week after vaccination, compared with non-infected animals or those vaccinated with rtgev-rs-spedv virus ( figure ). similar observations were reported for other virulent us isolates [ ] . these data indicate that rtgev-rs-spedv virus was also attenuated in -day-old piglets. virus titers were evaluated in the jejunum of challenged animals. the challenge virus replicated efficiently in the gut of non-vaccinated animals ( figure a ). in contrast, the titers were reduced more than -fold or -fold in animals vaccinated with rtgev-rs-spedv and pedv-nvsl viruses, respectively ( figure a ). similar results were obtained when viral rna presence was evaluated, with up to -fold reduction in the accumulation of pedv rna ( figure b ). interestingly, after challenge, pedv virus was only shed by non-vaccinated animals ( figure c ). altogether, these data indicate that the challenge virus did not efficiently infect the vaccinated animals. furthermore, these results indicate that rtgev-rs-spedv protected the animals against virulent pedv challenge. pigs were used. group (g ) was vaccinated with rtgev-rs-spedv virus, group (g ) was vaccinated with a virulent us pedv strain (pedv-nvsl), and two groups of animals (g and g ) were not vaccinated (table ) . twenty-one days after vaccination, animals from g , g , and g were challenged with a virulent recombinant pedv strain (rpedv) ( table ) . as expected, due to the animal age, no clinical sings of enteric disease such as diarrhea, vomiting, or dehydration were observed after vaccination or challenge (data not shown). nevertheless, animals vaccinated with the virulent pedv-nvsl strain showed a delay in weight gain the first week after vaccination, compared with non-infected animals or those vaccinated with rtgev-rs-spedv virus ( figure ). similar observations were reported for other virulent us isolates [ ] . these data indicate that rtgev-rs-spedv virus was also attenuated in -day-old piglets. figure . weight gain in vaccinated animals. three-week-old pigs were divided into four groups. animals from two groups were vaccinated with tcid /animal of rtgev-rs-spedv virus (red) or a virulent us pedv strain (pedv-nvsl, green). three weeks after vaccination ( dpv indicated in red), non-vaccinated animals were either mock infected (mock, grey) or challenged with tcid /animal of a virulent pedv strain (non vaccinated, black). vaccinated animals were also challenged with the same dose as non-vaccinated animals. clinical signs and weight were observed daily after vaccination and challenge, and once per week during the course of the experiment. average daily gain represents the mean from six different animals. error bars represent the standard deviation for each value; p value, ** < . . virus titers were evaluated in the jejunum of challenged animals. the challenge virus replicated efficiently in the gut of non-vaccinated animals ( figure a ). in contrast, the titers were reduced more than -fold or -fold in animals vaccinated with rtgev-rs-spedv and pedv-nvsl viruses, respectively ( figure a ). similar results were obtained when viral rna presence was evaluated, with up to -fold reduction in the accumulation of pedv rna ( figure b ). interestingly, after challenge, pedv virus was only shed by non-vaccinated animals ( figure c ). altogether, these data indicate that the challenge virus did not efficiently infect the vaccinated animals. furthermore, these results indicate that rtgev-rs-spedv protected the animals against virulent pedv challenge. figure . weight gain in vaccinated animals. three-week-old pigs were divided into four groups. animals from two groups were vaccinated with tcid /animal of rtgev-rs-spedv virus (red) or a virulent us pedv strain (pedv-nvsl, green). three weeks after vaccination ( dpv indicated in red), non-vaccinated animals were either mock infected (mock, grey) or challenged with tcid /animal of a virulent pedv strain (non vaccinated, black). vaccinated animals were also challenged with the same dose as non-vaccinated animals. clinical signs and weight were observed daily after vaccination and challenge, and once per week during the course of the experiment. average daily gain represents the mean from six different animals. error bars represent the standard deviation for each value; p value, ** < . . the antibody response specific for pedv was evaluated in the sera from vaccinated and nonvaccinated animals. before challenge ( dpv), vaccination elicited pedv-specific antibodies to a higher level in pedv-nvsl vaccinated animals than in rtgev-rs-spedv vaccinated ones ( figure a ). this result was expected, as the elisa test was developed against the whole pedv virus, and the chimeric rtgev-rs-spedv virus only expressed pedv s protein. after challenge at and dpv, pedv total antibodies increased in all challenged groups, as expected. interestingly, the increase was slower in the vaccinated pigs, in agreement with a decreased challenge virus replication in these animals. total anti-tgev antibodies were also evaluated by elisa. all animals were seronegative at the time of vaccination. when challenge was performed, the level of tgev antibodies was not significant in any group, and the all the animals remained seronegative after pedv challenge. this result was expected, as s protein is the main inducer of antibodies against tgev and it was not present in any of the viruses used for vaccination or challenge. in addition, it has been shown that the antibody response specific for pedv was evaluated in the sera from vaccinated and non-vaccinated animals. before challenge ( dpv), vaccination elicited pedv-specific antibodies to a higher level in pedv-nvsl vaccinated animals than in rtgev-rs-spedv vaccinated ones ( figure a ). this result was expected, as the elisa test was developed against the whole pedv virus, and the chimeric rtgev-rs-spedv virus only expressed pedv s protein. after challenge at and dpv, pedv total antibodies increased in all challenged groups, as expected. interestingly, the increase was slower in the vaccinated pigs, in agreement with a decreased challenge virus replication in these animals. total anti-tgev antibodies were also evaluated by elisa. all animals were seronegative at the time of vaccination. when challenge was performed, the level of tgev antibodies was not significant in any group, and the all the animals remained seronegative after pedv challenge. this result was expected, as s protein is the main inducer of antibodies against tgev and it was not present in any of the viruses used for vaccination or challenge. in addition, it has been shown that there is no cross-reactivity between tgev and pedv [ ] . to further analyze the humoral response specific for pedv, the levels of pedv-specific iggs ( figure b ) and igas ( figure c ) were evaluated. the data were similar to those obtained for total anti-pedv antibodies. it has been demonstrated that protection against pedv infection correlates with iga levels [ , , , ] . it is worth noting that iga levels in the sera of vaccinated animals, after challenge, were significantly reduced compared with non-vaccinated animals ( figure c ), strongly suggesting that challenge virus did not elicit a potent iga response in the gut of vaccinated animals, most likely due to reduced replication in the enteric tract, correlating with protection. the neutralization capability of the antibodies elicited was evaluated. as expected, non-vaccinated animals only induced the production of neutralizing antibodies after challenge (figure ). in contrast, pigs vaccinated with both pedv-nvsl and rtgev-rs-spedv virus induced a significant level of neutralizing antibodies before challenge, which were slightly increased after challenge (figure ). the neutralization capability of the antibodies elicited was evaluated. as expected, nonvaccinated animals only induced the production of neutralizing antibodies after challenge (figure ). in contrast, pigs vaccinated with both pedv-nvsl and rtgev-rs-spedv virus induced a significant level of neutralizing antibodies before challenge, which were slightly increased after challenge (figure ). . neutralizing antibodies produced in vaccinated animals. virus neutralization assay was performed using serum samples, collected before ( dpv) or after ( dpv) challenge, from rtgev-rs-spedv virus (red) or pedv-nvsl (green) vaccinated animals, and from non-vaccinated and challenged animals (black). the values represent neutralization index from three different animals. error bars represent the standard deviation for each value. altogether, the data indicate that rtgev-rs-spedv virus conferred protection against pedv infection in the young pig model and could be the basis for an effective vaccine candidate. an attenuated chimeric rtgev virus expressing the ectodomain of a virulent us pedv s protein (rtgev-rs-spedv) was engineered as vaccine candidate for pedv and evaluated in a young piglet model system. the rtgev-rs-spedv virus elicited a neutralizing humoral response specific for pedv. in fact, vaccinated animals were protected against challenge with a virulent pedv virus. the attenuated live vaccine candidates are the best option for pedv vaccination because nonreplicative antigens, as in inactivated vaccines, do not induce mucosal immunity and protection [ , ] . traditionally, attenuated live vaccines were developed for classical pedv strains, consisting of serial passages of the virulent virus in cell culture, as tissue culture adaptation led to attenuation in vivo. these vaccines were not effective to control the infection, as they were extensively used in countries were novel pedv strains emerged [ , ] . as a consequence, many groups have developed traditionally attenuated live vaccines based on novel epidemic pedv strains. in general, it has been shown that more than passages of the virulent virus in cell culture are required to attenuate pedv in vivo [ ] [ ] [ ] [ ] [ ] [ ] . it is worth noting that in our rtgev-rs-spedv vaccine candidate, the attenuating mutations were specifically designed and introduced in several locations of the viral genome. in contrast, traditionally attenuated vaccines contain non-controlled mutations throughout the genome figure . neutralizing antibodies produced in vaccinated animals. virus neutralization assay was performed using serum samples, collected before ( dpv) or after ( dpv) challenge, from rtgev-rs-spedv virus (red) or pedv-nvsl (green) vaccinated animals, and from non-vaccinated and challenged animals (black). the values represent neutralization index from three different animals. error bars represent the standard deviation for each value. altogether, the data indicate that rtgev-rs-spedv virus conferred protection against pedv infection in the young pig model and could be the basis for an effective vaccine candidate. an attenuated chimeric rtgev virus expressing the ectodomain of a virulent us pedv s protein (rtgev-rs-spedv) was engineered as vaccine candidate for pedv and evaluated in a young piglet model system. the rtgev-rs-spedv virus elicited a neutralizing humoral response specific for pedv. in fact, vaccinated animals were protected against challenge with a virulent pedv virus. the attenuated live vaccine candidates are the best option for pedv vaccination because non-replicative antigens, as in inactivated vaccines, do not induce mucosal immunity and protection [ , ] . traditionally, attenuated live vaccines were developed for classical pedv strains, consisting of serial passages of the virulent virus in cell culture, as tissue culture adaptation led to attenuation in vivo. these vaccines were not effective to control the infection, as they were extensively used in countries were novel pedv strains emerged [ , ] . as a consequence, many groups have developed traditionally attenuated live vaccines based on novel epidemic pedv strains. in general, it has been shown that more than passages of the virulent virus in cell culture are required to attenuate pedv in vivo [ ] [ ] [ ] [ ] [ ] [ ] . it is worth noting that in our rtgev-rs-spedv vaccine candidate, the attenuating mutations were specifically designed and introduced in several locations of the viral genome. in contrast, traditionally attenuated vaccines contain non-controlled mutations throughout the genome introduced during passages in cell cultures that, after passages in vivo, may lead to vaccine virus reversion to virulence. the engineered rtgev-rs-spedv vaccine candidate has the advantage of replicating in the jejunum of infected piglets as efficiently as a virulent control virus, without causing significant pathology, and most likely leading to a protective iga response. indeed, vaccinated animals were protected against virulent pedv challenge. in contrast, traditionally attenuated vaccines, as a consequence of adaptation to cell culture, have a dramatically reduced replication in the enteric tract [ ] and, as a consequence, these vaccines do not actively replicate to induce good iga and lactogenic immune responses [ , , ] . recently, a swine enteric coronavirus (secov) that is a recombinant between tgev and pedv has been described in europe [ ] [ ] [ ] . the secov contains a genome background identical to tgev, but expresses the s protein from pedv. in addition, recombination led to extensive deletions in the coding sequence and in trs of tgev gene a, and, as a consequence, protein a may not be expressed by secov. it is worth noting that secov was isolated from diarrheic samples in all cases, suggesting that it is virulent. the engineered parental rtgev-spedv virus resembles the recombinant secov and, as shown by our data, is virulent in suckling piglets. in contrast, the engineered virus (rtgev-rs-spedv) used as a vaccine candidate was attenuated. there are some genetic differences that may eventually allow the discrimination between secov and the rtgev-rs-spedv vaccine candidate as (i) rtgev-rs-spedv virus did not contain the full pedv s gene sequence, (ii) it expressed tgev protein a, and (iii) because of the tgev strain used to engineer the infectious cdna, it did not express tgev protein b [ ] . the rtgev-rs-spedv vaccine candidate was genetically stable in tissue culture; nevertheless, it evolved in vivo. therefore, its biosafety could be improved by adding an additional safety guard at the -end of pedv virus genome (in the replicase gene), at a distal position from the attenuating mutations introduced by duplicating sequences closed to m, n and a genes ( figure ) [ ] [ ] [ ] [ ] . the optimized vaccine candidate should keep a balance between virus replication attenuation and eliciting an efficient immune response. strategies for design and application of enteric viral vaccines viral metagenomics analysis demonstrates the diversity of viral flora 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coronavirus from diseased pigs in central eastern europe in sequence motifs involved in the regulation of discontinuous coronavirus subgenomic rna synthesis identification of the mechanisms causing reversion to virulence in an attenuated sars-cov for the design of a genetically stable vaccine a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking '-o-methyltransferase activity mutagenesis of coronavirus nsp reveals its potential role in modulation of the innate immune response we thank ceva animal health for providing vero cells and pedv-nvsl virus. we are grateful to dora katona and máté halas (prophyl ltd., hungary) for in vivo experiments. we thank marga gonzalez (cnb-csic) for her technical assistance.conflicts of interest: z.p. is the global director of bio r&d at ceva animal health. the authors declare no other conflict of interest. key: cord- -rrx r n authors: fan, wensheng; tang, ning; dong, zhihua; chen, jiming; zhang, wen; zhao, changrun; he, yining; li, meng; wu, cuilan; wei, tianchao; huang, teng; mo, meilan; wei, ping title: genetic analysis of avian coronavirus infectious bronchitis virus in yellow chickens in southern china over the past decade: revealing the changes of genetic diversity, dominant genotypes, and selection pressure date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: rrx r n the high mutation rates of infectious bronchitis virus (ibv) pose economic threats to the poultry industry. in order to track the genetic evolutionary of ibv isolates circulating in yellow chickens, we continued to conduct the genetic analyses of the structural genes s , e, m, and n from ibv isolates in southern china during – . the results showed that the dominant genotypes based on the four genes had changed when compared with those during – . based on the s gene phylogenetic tree, lx -type (gi- ) was the most dominant genotype, which was different from that during – . the second most dominant genotype was ldt -a-type, but this genotype disappeared after . new-type (gvi- ) isolates showed increasing tendency and there were four aa (qkep) located in the hypervariable region (hvr) iii and one aa (s) insertion in all the new-type isolates. both the analyses of amino acid entropy and molecular evolutionary rate revealed that the variations from large to small were s , e, m, and n. purifying selection was detected in the s , e, m, and n gene proteins, which was different from the positive selection during – . six isolates were confirmed to be recombinants, possibly generated from a vaccine virus of the / -type or ldt -a-type and a circulating virus. the estimated times for the most recent common ancestors based on the s , e, m, and n genes were the years of , , , and , respectively. bayesian skyline analysis revealed a sharp decrease in genetic diversity of all the four structural genes after and since late , the viral population rapidly rose. in conclusion, the ibvs circulating in southern china over the past decade have experienced a remarkable change in genetic diversity, dominant genotypes, and selection pressure, indicating the importance of permanent monitoring of circulating strains and the urgency for developing new vaccines to counteract the emerging lx -type and new-type ibvs. infectious bronchitis (ib) is one of the major viral diseases affecting the poultry industry globally. the causative agent avian infectious bronchitis virus (ibv) is a member of the genus gammacoronaviruses, subfamily coronavirinae, family coronaviridae and is prone to mutate. there are multiple genotypes and serotypes of ibv isolates identified worldwide and limited cross-protection confers between serotypes of ibvs [ ] [ ] [ ] [ ] [ ] [ ] , which poses great challenge to the control of ib by vaccination. the ibv has a single-stranded rna genome of approximately . kb in length [ ] and encodes four structural proteins: the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins [ , ] . the s protein is cleaved into subunits s and s by proteases [ , ] . the function of the four structural proteins has been extensively reviewed by others [ ] [ ] [ ] . the genetic analysis based on the s gene has become the primary method of classifying ibv strains because of variability and functional importance [ ] . however, those previous ibv molecular characterizations that were merely focused on the analysis of the s gene or partial s gene sequence could not explain the changes in the serotypes and pathotypes of ibv variants [ ] . sometimes, single gene analysis even is misleading. therefore, it is necessary to analyze all the structural protein-coding genes s , e, m, and n simultaneously in order to obtain comprehensive genetic information and molecular mechanism of variation of circulating ibv isolates. a variety of ibv genotypes and variants are distributed globally. so far, a total of seven genotypes comprising distinct viral lineages have been defined worldwide based on the complete s gene sequences [ ] . ibv strains within a certain country or region are unique even though many countries share some common antigenic types. for example, two distinct lineages that fall in two different genotypes-gi- and gii- -were identified as unique to europe. gi- , gi- , and giv- -genotypes have been implicated in widespread disease disseminations and persistent virus infections in north american [ , , ] . gi- , gi- , and gi- are currently circulating in south american flocks [ ] . lx -type (qx-type or which was firstly isolated in china in spread westward invading russia, middle east, and europe, and then becoming the most prevalent genotype in many countries, such as korea, russia, iran, italy, uk, malaysia, sudan, and so on [ , , [ ] [ ] [ ] [ ] [ ] . nowadays, lx -type and ck/ch/lsc/ i-type (gi- ) appear to be the dominant viruses based on the s gene in china [ ] . because of differences in breeding variabilities and feeding patterns of chickens in china, the characteristics of circulating ibvs at different times and in different regions are variable. hence, it is necessary to conduct long-term tracking of the ibv circulating isolates in specific geographic regions or countries for the effective control of ib [ , , ] . the appearance of ibv variants was related to high mutation and recombination rates, which result in the generation of genetic diversity and phenotypic heterogeneity. however, ibv evolution is not driven by genetic drift alone. evolution involves two fundamental steps-i.e., generation of genetic diversity and selection [ ] . the selection process was affected by multiple factors, such as immune responses, the microenvironment of infected hosts, physical and biosafety conditions [ , ] . vaccines not only give rise to new variants through recombination, but also impose selection pressure on the evolution of field strains [ ] [ ] [ ] . it is essential for appropriately controlling and prevention of the disease to understand the evolution of ibv [ ] . southern china is the major production region of yellow chickens ( . billion in and made a proportion of % of the total chickens) in the country. guangxi province, located in southwest of china, has the biggest production of local breeds of chickens ( . billion birds in ) [ ] , and most of the birds are free-range and raised for a longer time (about -day-old) at a rather high density, and the chicks are raised in relatively closed environment and lack of ventilation during brooding [ , ] . also, flocks of various companies, with different chicken breeds and differing ages and vaccination programs located in the same areas are common [ ] . these situations surely increase the odds of multiple-infection of several ibv strains including the vaccine strains or/and field strains in flocks of birds [ ] . despite the widespread use of mass-type (massachusetts genotype) (h , h , ma , m , and w ), / , ltd -a live vaccines and inactivated vaccines, ib has been a continuing problem in vaccinated flocks in these regions [ ] . our previous studies found the serotype and genotype diversity of guangxi ibvs from to and the important role of vaccine strains in the emerging of new ibv strains via recombination [ , , ] . however, the comprehensive genetic information of circulating ibv strains in this region was unavailable over the past decade. hence, we continued to carry out the genetic analysis of ibv isolates during - . the aim was to track the genetic evolutionary trends of ibv field strains circulating over the past decade and their possible causes through relatively new and comprehensive analyses of virus genes, and then provide valuable reference and countermeasure against ibv field breakouts in southern china. sixty-four ibv strains isolated from flocks of yellow chickens during - were analyzed in the present study (table ). all ibv field isolates were obtained from the birds of previously vaccinated flocks with h , ldt -a and/or / vaccines and experienced clinical signs of the ibv infection. all ibvs were isolated and propagated as previously described [ ] . nanning jx jx jx kj gx-gl guilin jx jx jx kj gx-gl guilin jx jx jx kj gx-gl n/a guilin jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn nanning jx jx jx kj gx-yl yulin jx jx jx kj gx-yl yulin jx jx jx kj gx-nn nanning jx jx jx kj gx-nn table . cont. days of age locations a genbank accession numbers s n m e gx-nn nanning mk mk mk mk gx-qz qinzhou mk mk mk mk gx-yl yulin mk mk mk mk gx-lz liuzhou mk mk mk mk gx-yl yulin mk mk mk mk gx-lz liuzhou mk mk mk mk gx-yl yulin mk mk mk mk gx-yl yulin mk mk mk the entire s , e, m, and n genes were amplified for each ibv strain and the primers used were designed as previously described [ , ] . the anticipated amplification segments for the s , e, m, and n genes are bp, bp, bp, and bp in lengths, respectively. . . rna extraction and amplification of s , e, m, and n genes viral rna was extracted and the first cdna strand was synthesized as previously described [ ] . the pcr conditions for the s , m, and n gene amplification were the same as previously described [ ] . the pcr conditions for the e gene amplification were • c for min, cycles of • c for s, • c for s, and • c for s, followed by • c for min. the pcr products were analyzed on . % agarose-gel electrophoresis. the pcr products of s , e, m, and n genes were sequenced by beijing genomics institute (bgi) (shenzhen, china) after cloning. the open reading frames of ibvs were determined and their nucleotide sequences were submitted to genbank database and assigned accession numbers ( table ) . sequences of reference ibv strains (with the exception of strains for e gene) retrieved from the genbank database were used (supplementary table s ). the ibvs isolated during - in guangxi [ ] were also analyzed together in order to get a general profile of ibv evolution. the nucleotide and deduced amino acid (aa) sequences of the s , e, m, and n genes obtained from the ibv isolates were aligned using the editseq program in the lasergene package (dnastar inc., madison, wi, usa) and compared to those of ibv reference strains representing the main well-established lineages and genotypes using the megalign program in the same package. to ensure the scientificity and reliability of the results, we extracted the s , e, m, and n genes from the complete genome sequences of reference strains. phylogenetic trees based on the aa sequences of s , e, m, and n genes were constructed using mega version . according to previous description with nodal support values obtained by posterior probabilities and bootstrap replicates [ ] . aligned nucleotide sequences of s , e, m, and n genes were subjected to the recombination detection program (rdp version . ) to detect potential recombination events by seven algorithms (rdp, geneconv, bootscan, maxchi, chimera, siscan and seq) in rdp . [ ] . the detection of recombination breakpoints by at least four of these methods were considered as confirmation of any putative recombination event. the potential recombination events and breakpoints were further verified by similarity plots (simplots) analysis in simplot version . . [ , ] . entropy of amino acid sequences within the s , e, m, and n proteins of ibv isolates was calculated by bioedit version . . . in order to understand the variation degree of these four structural protein genes [ ] . positive selection and positively selected sites within the s , e, m, and n proteins were analyzed by the single-likelihood ancestor counting (slac), fixed effects likelihood (fel), and internal fixed effects likelihood (ifel) methods of datamonkey version (http://www.datamonkey.org/ / / ) [ ] to detect whether these proteins have undergone positive selection. the recombinants were excluded in order to reduce a false detection of positive selection. the potential n-glycosylation sites were predicted within the s , e, m, and n proteins. the analysis was performed using netnglyc server . software available at http://www.cbs.dtu.dk/services/ netnglyc [ ] . the results of aligned sequences were computed by multiple alignment with fast fourier transformation (mafft) [ ] . the nucleotide substitution process was modelled independently for each partition with gtr (general time reversible) + g (gamma distribution with a discrete) + i (proportion of invariant sites) based on aic by the jmodel test . . . bayesian tree reconstructions were performed in beast version . . . a bayesian skyline coalescent model and strict molecular clock were selected. the bayesian markov chain monte carlo (mcmc) chains of the s , e, m, and n genes were run for million, million, million, and million generations, respectively. results were analyzed using tracer version . and confirmed convergence of mcmc chains with % of each chain discarded as burn-in and sampled every , steps [ ] . statistical uncertainty (reflected calculating % high probability density (hpd) values) and convergence (reflected calculating effective sample size) in parameter estimates were evaluated in tracer version . program. the posterior sets of trees were summarized as a maximum clade credibility (mcc) tree using tree annotator version . . with % burn-in and then displayed the created mcc tree using figtree version l. . [ ] . the mutation rates and the most recent common ancestor (tmrca) of the aligned sequences were estimated. the change of effective population size over time was inferred by bayesian skyline plots (bsp). the nucleotide and deduced aa sequence similarities of the s , e, m, and n genes among the isolates during - were . - % and . - %, . - % and . - %, . - % and . - %, and . - % and . - %, respectively. compared with h , the isolates gx-qz , gx-lz , and gx-nn have higher amino acid sequence similarities of . - . %, . - %, . - %, and . - % in the s , e, m, and n genes, respectively. within the s gene, there were different nucleotide lengths (from to bp), and the most common lengths were bp ( / isolates, . %) and bp ( / isolates, . %). there were eight types of s protein cleavage site motifs found: rrfrr ( / ), hrrrr ( / ), rrlrr ( / ), rrsrr ( / ), hrrkr ( / ), hrikr ( / ), hrskr ( / ), and rkrkr ( / ) among the isolates. there were four aa (qkep) (located in the hypervariable region (hvr) iii) and one aa (s) insertion found in the s genes of six isolates (gx-nn , gx-nn , gx-nn , gx-nn , gx-qz , and gx-qz ), respectively (supplementary figure s ). the s gene phylogenetic tree showed that the ibv isolates during - were divided into eight distinct groups (figure a ). counts of , , , , and out of ibv isolates during - belonged to lx -type (qx or gi- ), ldt -a-type (gi- ), / -type (gi- ), mass-type (gi- ), and ck/ch/lsc/ i-type (gi- ), respectively. five isolates (gx-nn , gx-nn , gx-nn , gx-nn , and gx-nn ) in recent years were grouped with taiwan reference strains tw / as taiwan-i-type (gi- ). six isolates (gx-nn , gx-nn , gx-nn , gx-nn , gx-qz , and gx-qz ) and two isolates (gx-nn and gx-yl ) showed considerable low similarities ( . - . %, . - . %) with the above genotypes and belonged to two separate groups new-type (gvi- ) and new-type (gvii- ). viruses , , x for peer review of ( / ), hrrkr ( / ), hrikr ( / ), hrskr ( / ), and rkrkr ( / ) among the isolates. there were four aa (qkep) (located in the hypervariable region (hvr) iii) and one aa (s) insertion found in the s genes of six isolates (gx-nn , gx-nn , gx-nn , gx-nn , gx-qz , and gx-qz ), respectively (supplementary figure s ). the s gene phylogenetic tree showed that the ibv isolates during - were divided into eight distinct groups (figure a ). counts of , , , , and out of ibv isolates during - belonged to lx -type (qx or gi- ), ldt -a-type (gi- ), / -type (gi- ), mass-type (gi- ), and ck/ch/lsc/ i-type (gi- ), respectively. five isolates (gx-nn , gx-nn , gx-nn , gx-nn , and gx-nn ) in recent years were grouped with taiwan reference strains tw / as taiwan-i-type (gi- ). six isolates (gx-nn , gx-nn , gx-nn , gx-nn , gx-qz , and gx-qz ) and two isolates (gx-nn and gx-yl ) showed considerable low similarities ( . - . %, . - . %) with the above genotypes and belonged to two separate groups new-type (gvi- ) and new-type (gvii- ). the phylogenetic trees of e, m, and n genes of the isolates were segregated into six, four, and six unique groups, respectively (figure b-d). and their phylogenetic trees exhibited considerably different topology compared with that of the s gene. no obvious geographic differences were found among the isolates, while there was a high degree of sequence identity among the isolates in the same period of time (supplementary tables s -s ) . the percentages of different genotypes based on s , e, m, and n genes of ibv isolates in different years were summarized in figure . based on the s gene, the ck/ch/lsc/ i-type was the predominant genotype during - , but the lx -type was the predominant genotype circulating in the field during - . the ldt -a-type was the second most dominant genotype, but this genotype disappeared after . based on the e and m genes, the ck/ch/lsc/ i-type was the predominant genotype during - , while the ck/ch/lsc/ i-type and lx -type were the predominant genotypes during - . based on the n gene, the ck/ch/lsc/ i-type and the lx -type were the predominant genotypes during - , but only the lx -type was the predominant genotype during - . therefore, our results demonstrated that the ck/ch/lsc/ i-type isolates were the predominant ibvs according to the phylogenetic study of the s , e, m, and n genes from to . thereafter, the proportion of lx -type, ldt -a-type, and the phylogenetic trees of e, m, and n genes of the isolates were segregated into six, four, and six unique groups, respectively (figure b-d) . and their phylogenetic trees exhibited considerably different topology compared with that of the s gene. no obvious geographic differences were found among the isolates, while there was a high degree of sequence identity among the isolates in the same period of time (supplementary tables s -s ) . the percentages of different genotypes based on s , e, m, and n genes of ibv isolates in different years were summarized in figure . based on the s gene, the ck/ch/lsc/ i-type was the predominant genotype during - , but the lx -type was the predominant genotype circulating in the field during - . the ldt -a-type was the second most dominant genotype, but this genotype disappeared after . based on the e and m genes, the ck/ch/lsc/ i-type was the predominant genotype during - , while the ck/ch/lsc/ i-type and lx -type were the predominant genotypes during - . based on the n gene, the ck/ch/lsc/ i-type and the lx -type were the predominant genotypes during - , but only the lx -type was the predominant genotype during - . therefore, our results demonstrated that the ck/ch/lsc/ i-type isolates were the predominant ibvs according to the phylogenetic study of the s , e, m, and n genes from to . thereafter, the proportion of lx -type, ldt -a-type, and new-type strains increased over time. recombination events of the s , e, m, and n genes of isolates were examined using the rdp in the study. the results showed that recombinant events were found in the s gene of six isolates ( figure and table ). gx-nn was derived from recombination between the two lx -type strains gx-nn (major parent) and gx-nn- (minor parent). gx-nn was derived from recombination between lx -type strain qx (major parent) and ck/ch/lsc/ i-type strain saibk (minor parent). gx-nn was derived from recombination between lx -type strain gx-hc (major parent) and / strain (minor parent). gx-nn was found to be a recombinant between the new-type isolate gx-nn (major parent) and the vaccine strain ldt -a (minor parent). gx-nn was found to be a recombinant isolate formed by a major parent isolate gx-yl (ldt -a-type) and a minor parent isolate gx-yl (ck/ch/lsc/ i-type). the potential parents of gx-yl were proved to be a major parent isolate gx-nn (tw / -type) and a minor parent isolate gx-nn ( / -type). in addition, it was found that a-t rich hotspot sequence atttt (t/a) was near the breakpoint site of the s subunit gene in all of recombinant isolates except gx-yl (supplementary figure s ) . in order to confirm the results of the rdp analysis, genomic sequences analyses of the six ibv isolates were carried out by the simplot software, and the results were consistent with those of the rdp analysis ( figure ) . therefore, the dominant recombinants were the lx -type isolates from to . recombination events of the s , e, m, and n genes of isolates were examined using the rdp in the study. the results showed that recombinant events were found in the s gene of six isolates ( figure and table ). gx-nn was derived from recombination between the two lx -type strains gx-nn (major parent) and gx-nn- (minor parent). gx-nn was derived from recombination between lx -type strain qx (major parent) and ck/ch/lsc/ i-type strain saibk (minor parent). gx-nn was derived from recombination between lx -type strain gx-hc (major parent) and / strain (minor parent). gx-nn was found to be a recombinant between the new-type isolate gx-nn (major parent) and the vaccine strain ldt -a (minor parent). gx-nn was found to be a recombinant isolate formed by a major parent isolate gx-yl (ldt -a-type) and a minor parent isolate gx-yl (ck/ch/lsc/ i-type). the potential parents of gx-yl were proved to be a major parent isolate gx-nn (tw / -type) and a minor parent isolate gx-nn ( / -type). in addition, it was found that a-t rich hotspot sequence atttt (t/a) was near the breakpoint site of the s subunit gene in all of recombinant isolates except gx-yl (supplementary figure s ) . in order to confirm the results of the rdp analysis, genomic sequences analyses of the six ibv isolates were carried out by the simplot software, and the results were consistent with those of the rdp analysis ( figure ) . therefore, the dominant recombinants were the lx -type isolates from to . the entropy of the amino acid sequences of s , e, m, and n genes showed that there were many high entropy amino acid sites within the s gene, but a few high entropy amino acid sites were scattered within the e, m, and n genes (supplementary figure s ) . the average entropy values sorted from large to small as follows: s ( . ), e ( . ), m ( . ), and n ( . ). the percentages of entropy value which was bigger than . in s , e, m, and n genes were . % ( / ), . % ( / ), . % ( / ), and . % ( / ), respectively. therefore, the largest variation was observed in the s gene, and the e gene was also more variable than the m and n genes. the selection profile of s , e, m, and n proteins of totally (with deleted recombinant strains) ibvs were showed in table . the dn/ds ratio of s , e, m, and n proteins of eighty isolates were . , . , . , and . , respectively, indicating that the s , e, m, and n proteins of these ibv isolates had evolved under purifying selection (table ) . however, a few positively selected sites were detected although most sites were under neutral selection and purifying selection (table and supplementary table s ). aa residues , , , , , and of the s protein were consistently highlighted by positive selection models (slac, fel, and ifel) as positive selection sites. similarly, residue of the e protein, residues , , , , and of the m protein, residues , , , , and of the n protein were identified as positively selected sites. in addition, all of the positively selected sites within s , e, m, and n genes had high entropy values of larger than . (supplementary table s ). the results showed that there were - , - , - , - potential glycosylation sites within the s , e, m, and n proteins (except that gx-nn strain did not have a glycosylation site in the n protein). the comparison of the estimated n-glycosylation sites of s protein between the isolates and the reference strains showed that all the strains presented one n-glycosylation site at the residue / (nfsd) (except ck/ch/lsc/ i-type (gi- ) strains). and at the residue / (nitl), all the strains presented one n-glycosylation site (except taiwan-type (gi- ) and new-type (gvi- )). similarly, all the strains presented one n-glycosylation site at e protein residue / (ngsf) and residue / (nktl) (except ck/ch/lsc/ i-type and conn-type), all the strains presented one n-glycosylation site at m protein residue / / (nctl) and at n protein residue (nasw) (except gx-nn strain) (table and supplementary figure s -s ). the mean substitution rates for the s , e, m, and n genes of the epidemic isolates during - were calculated to be . × − , . × − , . × − , and . × − substitutions/site/year (s/s/y), respectively ( table ), indicating that s gene is the most easily mutated and n gene is the most stable among the four structural genes. the estimated times for most recent common ancestor (tmrca) of s , e, m, and n genes were before . , . , . , and . , respectively ( figure ). based on s , genotypes of ck/ch/lsc/ i (gi- ), new (gvi- ), mass (gi- ), lx (gi- ), / (gi- ), taiwan (gi- ), ldt -a (gi- ), and new (gvii- ) were dated back to . , . , . , . , . , hpd, highest probability density. viruses , , x for peer review of the reconstruction of population history was assessed using a bayesian skyline plot coalescent model. results showed that the effective population size of s , e, m, and n genes of ibvs was featured by a continuous and slow reduction in viral population size between s and s. a more obvious decrease was observed between s and s base on the s gene ( figure a ). remarkably, a sudden and sharp decline in the effective population size was observed after s base on the s , e, m, and n genes. however, since late , the viral population rapidly rose ( figure ). the reconstruction of population history was assessed using a bayesian skyline plot coalescent model. results showed that the effective population size of s , e, m, and n genes of ibvs was featured by a continuous and slow reduction in viral population size between s and s. a more obvious decrease was observed between s and s base on the s gene ( figure a ). remarkably, a sudden and sharp decline in the effective population size was observed after s base on the s , e, m, and n genes. however, since late , the viral population rapidly rose ( figure ). the reconstruction of population history was assessed using a bayesian skyline plot coalescent model. results showed that the effective population size of s , e, m, and n genes of ibvs was featured by a continuous and slow reduction in viral population size between s and s. a more obvious decrease was observed between s and s base on the s gene ( figure a ). remarkably, a sudden and sharp decline in the effective population size was observed after s base on the s , e, m, and n genes. however, since late , the viral population rapidly rose ( figure ). southern china is the largest region of yellow chickens produced in china. despite extensive vaccination, ib continues to be a serious problem. in the present study, strains of ibv were isolated from diseased chicken flocks in southern china during - and the genetic properties of the entire s , e, m, and n genes analyzed. to increase our insight into the comprehensive epidemiological situation and evolutionary trend of ibv in southern china, ibv isolates from to in southern china were also used to analyze together. the representative reference strains included the live vaccine strains commonly used and the prevalent strains in china and other countries worldwide, which represented the main well-established lineages and genotypes in recent years in china [ ] . our results indicated that there was a remarkable change in genetic diversity, dominant genotypes, and selection pressure of ibv strains in southern china over the past decade compared with the previous period of - . the co-circulation of multiple ibv genotypes and the increasing of ibv variants have resulted in great challenges for the controlling ib through vaccination. in our study, multiple ibv genotypes were also identified in guangxi over the past decade. a total of eight, six, four, and six genotypes were identified based on the s , e, m, and n genes, respectively. therefore, there is still ongoing genetic diversity of ibvs in southern china, which is the same as previous epidemics in this region and other parts of china [ , , ] . interestingly, the predominant genotype changed from ck/ch/lsc/ i-type lx -type was the most dominant genotype in our study, which agreed with many previous descriptions [ , , , ] . surprisingly, the second most dominant genotype of ibv circulating in southern china was ldt -a-type, which was a pandemic type and frequently isolated from chicken flocks in china [ , , ] . ldt -a commercial live vaccine has been issued by the official authority in china since [ ] . the re-isolation of vaccine strain is possible when ldt -a live vaccine strain is extensively used. we noticed that among the ldt -a type isolates during - , four, eight and two strains were isolated in , , and respectively. however, only one ldt -a type strain was isolated during - . we are not sure whether or not the strains of ldt -a type isolated between and are re-isolation of vaccine strains, but it seems that the ldt -a type vaccine could provide sufficient protection against the circulating homologous field strains in southern china because none of ldt -a genotype strains were isolated after . at the same time, we found the e genes of most ldt -a type isolates belonged to the ck/ch/lsc/ i-type, and the m and n genes of most ldt -a type isolates belonged to lx (qx)-type. these results implied that ldt -a-type might isolate the recombinants from ldt -a vaccine strain and other circulating ibv strains. considering that the ldt -a types were occasionally isolated in other regions of china recently [ ] , the ldt -a type isolates in southern china still need to be further monitored. the taiwan-type ibvs were firstly isolated in taiwan in s and have been divided into the taiwan-i and taiwan-ii subgroups [ , ] . more studies reported that an increasing number of taiwan-type strains have been isolated in southern china in recent years [ , , , ] . taiwan-ii-type strains (gx-xd and gx-g) were isolated in and no taiwan-ii-type strain was isolated in guangxi after that [ ] , but five taiwan-i-type strains were isolated during - . in our study, . % ( / ), . % ( / ), and % ( / ) of taiwan-i-type strains were isolated in , , and , respectively. it seemed that the taiwan-i-type strains were increasing in recent years. evidences proved that the taiwan-i-type strains were widely spread from taiwan to guangdong, guangxi, fujian, sichuan hunan, zhejiang, yunnan province and so on [ , , [ ] [ ] [ ] , and the increasing isolates demonstrated that the currently used vaccines were lacking protective efficacy against the taiwan-i-type strains. the recombinants between taiwan-i-type and lx (qx) causing severe economic losses have been reported [ ] . how the taiwan-type strains spread from taiwan to mainland china remains unknown. recently, wild birds have been of concern as natural ibv carriers, since infected birds have been showed to carry the viruses over long distances [ , , , ] . therefore, monitoring of wild birds should be needed in further. of course, ibv live vaccines and poultry products will also need to be followed. the earliest strain of new-type (gvi- ) was isolated in [ ] and has in recent years spread to many chicken flocks [ , , , , ] . surprisingly, majorities of the new-type strains from other studies were isolated from south china [ , , , ] . recently, another novel genotype vii (gvii- ) was reported [ ] . both these two new-type viruses were identified in our study and the total number of their strains showed new-type was the third most dominant genotype. we found that there was a four aa (qkep) insertion located in the hvr iii and one aa (s) insertion in all the new-type isolates. interestingly, the four aa (qkep) were predicted b cell epitopes by the bepipred /iedb tool and half of the new-type viruses' serotype were different from that of / or other vaccine strains [ ] . the aa mutation in the hvr of the s gene maybe lead to the occurrence of new serotype and immunity escape. the origin of new-type viruses in china remains unknown. interestingly, the "novel" isolates were shown to undergo recombination in our study. the new-type isolate gx-nn was a major parent to the recombinant ldt -a-type isolate gx-nn . to the best of our knowledge, this first report of a recombination event needs to be further investigated. analyzing the complete-genome and identifying the antigenicity and pathogenicity of new-type isolates will be further studied. it is uncertain whether the new-type would become the predominant type in china in further, but it continues circulating in the field suggests that the new-type ibvs are still endemic in china and more attention should be paid to them. high rates of recombination result in ibv variability. many recombinants were confirmed. some recombination occurred between field and vaccine viruses [ , , , ] , some occurred between field isolates [ , , ] . six recombinants were identified in this study and three had a major parent of the lx -type, and two with minor parents of / -type genotype. our previous studies confirmed five recombinants, which were between the vaccine strain / and the ck/ch/lsc/ i-type field strain gx-yl during - [ ] . therefore, we have identified recombinants so far and recombinants were derived from the / -type strain during - . high frequencies of recombination between vaccine and field strains have been reported frequently worldwide [ , , , , , ] . the / vaccine strain has been commonly used in china for a long time, so it is not surprising that / -type recombinants were found in the field. however, there were more recombinants involved in the / -type vaccine strain when compared to the mass-type vaccine strains' recombinants according to our and other previous reports [ , , , , ] , although both mass-type and / -type vaccine strains were widely used in china. the phenomenon may have the following three explanations. firstly, incomplete protection of / -type vaccine strain against heterologous strains might result in co-infection of vaccine and heterologous strains in the same birds and eventually led to recombination. secondly, the / -type vaccine strain recombinants may be more likely to escape vaccine immunization than mass-type vaccine strain recombinants. thirdly, the / -type vaccine strain persisted longer in the immunized birds than the mass-type vaccine strain, increasing the likelihood of co-infection with the latter infected strain/strains, as a previous report showed that / vaccine could persist in birds for days [ ] . the complete genome, pathogenicity, and immunogenicity of / -derived recombinants should be assessed in further studies. the isolation ratio of / (gi- ) genotype strains was relatively stable recently. therefore, in order to reduce the recombination occurred between field and vaccine viruses, the multi-valent live vaccine combined with / strain or other new vaccines (such as ldt -a strain, lx strain, and so on) should be used with caution and thoughtful consideration. in order to understand the variation degree of the s , e, m, and n genes and the correlation between gene variation and selection pressure, the analyses of entropy of amino acid sequences, molecular evolutionary rate, and positive selection were carried out in our study. the results showed that mean substitution rates were between . × − and . × − substitutions per site per year in different genes, which is comparable with previous description [ ] . both the average entropy and mean substitution rates sorted from large to small as follows: s , e, m, and n genes, and the dn/ds ratios of s , e, m, and n genes were . , . , . , and . , respectively. the dn/ds ratios of s , e, m, and n genes were less than , meaning that the analyzed region were under purifying selection. therefore, our results indicated that the degree of variation sorted from large to small as follows: s , e, m, and n genes and there was a positive correlation between selection pressure and gene variation. it is known that s genes had the largest variation, but the mutation of e gene has not been paid enough attention. a recent study reported that the e gene evolved at the fastest rate among four structural protein-coding genes [ ] . the e gene mutation needs to be focused on in the future. to our knowledge, it is the first report to analyze systematically the variation degree of the four structural genes of ibvs and correlation between gene variation and selection pressure. as mentioned above, the s , e, m, and n genes of guangxi ibvs during - were under purifying selection. despite that the four structural genes underwent purifying selection, there were six, one, five, and five positively selected sites within the s , e, m, and n genes respectively. the dn/ds value and positively selected sites of guangxi isolates were different from those of isolates from other countries and regions [ , , ] . in addition, all of the positively selected sites in s , e, m, and n genes had high entropy values of bigger than . . the accumulation of amino acid variation will have an important effect on the gene characteristics and evolutionary direction of viruses [ ] . therefore, more attention should be paid to those positively selected sites with high entropy values. in the present study, positive selection was not detected in the s , e, m, and n proteins of ibv isolates in southern china during - although there were high number of mutations. zhao et al. also found that purifying selection was the main evolutionary pressure in the protein-coding regions in china over the past two decades [ ] . however, positive selection was detected in the s , m, and n proteins of ibv isolates in southern china during - according to our previous investigation [ ] . therefore, positive selection in the s , m, and n proteins in different periods in southern china was changeable. this phenomenon was also seen in some other previous reports [ ] [ ] [ ] . the change in selection pressures may be due to immune responses induced by multiple types of vaccines, the microenvironment of infected hosts, or physical and biosafety conditions [ ] . molecular evolutionary rate analysis can accurately estimate the molecular dating and it was used to determine successfully the timing of ancestors of classical swine fever virus (csfv) [ ] , sars virus [ ] , and ibv [ , ] . according our previous investigation [ ] , the first ibv strain was isolated in , demonstrating that the ibv was already present at that time. this study further backdated the ibvs emergence of more than years (during - ) and indicated that the most recent ancestor of guangxi ibv isolates based on the genes s , e, m, and n existed in the early , , , and , respectively. different structural protein genes of the ibvs had different time of the most recent ancestor. this phenomenon was also reported in other viruses [ , , ] . the data of tmrca estimated from our study suggested ibv has been already circulating in field for a period before its first outbreak. interestingly, the evolution rates sorted from large to small as follows: s , e, m, and n genes; the similarity plot data showed high genetic divergence and recombination was found in the s gene of ibv isolates, which agreed with the dates of most recent ancestor. although ibv was discovered in s, it is thought that it may have a long history as a species. the similar results were reported in other viruses [ , ] . in our early study, maybe unavailability of advanced diagnostic and sequencing technologies, the backyard poultry farming, inefficient surveillance systems, and limited financial resources resulted in the non-detection of ibv isolates for such a long time in guangxi. bayesian skyline analysis of s , e, m, and n genes of ibvs revealed that a persistent and slow decrease in relative genetic diversity between s and s, mirroring the implementation of mass-or / -based vaccine was effective in controlling the virus in field conditions to some extent, although not being able to eradicate the pathogen. analysis of s showed a more obvious decrease between s and s. the live vaccine h has been used in china for many years and the / -like live vaccine was also commonly used in china since s even without official authorization. therefore, the / -like vaccine was effective between s and s. surprisingly, a sudden and sharp decrease in genetic diversity base on the s , e, m, and n genes was observed after , mirroring the implementation with a newly registered ldt -a-based vaccine. in our study, the ldt -a-type disappeared after . ldt -a-like strains were also rarely isolated in other recent studies [ , ] . it was reported that the change of viral population size had a strong association with the vaccine administration/withdrawal [ , ] . therefore, it can be speculated that the ldt -a vaccine could provide a better protection against the circulating strains in china. however, since late , a sharp increase was observed in viral population size, which may suggest the implementation of current vaccines could not provide higher protection against the circulating strains. the lx -type isolates had become the predominant ibvs since in our study. the proportion of lx -like genotype strains increased over time in china [ ] . increasing of lx genotype viruses over time agreed with the rapid rise in the viral population since late . our previous study showed that the h , / , or ldt -a vaccines could not provide complete protection against the prevalent local strains of ibv (such as lx genotype isolates) in southern china [ ] . therefore, it is predicted that the epidemic trend of lx type strain will not decline in the next few years and will become more and more intense. therefore, new lx type vaccine was urgently needed. lx live vaccine was officially authorized in china last year, but has not yet come into the market. lx commercial vaccine urgently needs to go to the market in china. taken together, our results of genetic analyses of the four structural protein genes s , e, m, and n clearly demonstrated that there has been a remarkable genetic change of ibv in southern china over the past decade. the coexistence of multiple genotypes of ibv, the changes of dominant genotypes in different periods, the emergence of new genotypes, the recombination of vaccine strains with the field strains, the rapid evolving rate of ibv, the ever-changing of viral population size, and the presence of multiple positively selected sites in the structural proteins suggest a complexity of ibv epidemic strains in southern china. this epidemiological complexity may be caused by multiple factors: simultaneous vaccination of multiple live vaccines, illegal use of live vaccines, large number of avian varieties, raising of chickens at a rather high densities and the patterns of raising (free-range style and lack of biosecurity), etc. the complexity also suggests the importance of molecular epidemiological investigation and permanent monitoring of circulating strains as well as more serious challenges for ib prevention and control. based on the present results, it is predicted that lx -type will remain to be dominant genotype in near future and new-type ibv isolates will be more and more prevalent in the future. therefore, it is necessary to choose and/or develop new vaccines to counteract the ibvs of these two genotypes. in addition, multivalent new vaccines should be developed and rational modified vaccination strategies and the strict biosafety should be observed. to our knowledge, this is the first report showing the changes of genetic diversity, dominant genotypes, and selection pressure of ibv strains. the present study extends our knowledge about past and present ibv variability in southern china, providing valuable reference and countermeasures against ibv field breakouts in china, also emphasizing the importance of constant dynamic surveillance of the circulating isolates. in conclusion, our results indicate that the ibv strains in southern china have experienced a remarkable change in genetic diversity, dominant genotypes, and selection pressure, indicating the importance of permanent monitoring of circulating strains and the urgency for developing new vaccines which counteract the emerging lx -type and new-type ibvs and should be resistant to recombination and mutation. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : ibv reference strains used in this study; table s : estimates of evolutionary distances over sequence pairs between groups of s gene; table s : estimates of evolutionary distances over sequence pairs between groups of e gene; table s : estimates of evolutionary distances over sequence pairs between groups of m gene; table s : estimates of evolutionary distances over sequence pairs between groups of n gene; table s : the selection profile and entropy values of s , e, m, and n proteins of guangxi ibv isolates; figure s : alignment of the deduced amino acid sequences of the s subunit of the spike protein of ibv isolates (new-type or gvi- ) and other strains. the insertion of critical amino acids , , , , and are highlighted in red; figure s : the sequences were aligned using the clustal w method in the bioedit version . . . package. the hotspot sequence atttt(t/a) (green underline) was found near the breakpoint site (red box) of the s subunit gene in all of recombinant isolates except gx-yl . the gx-yl isolate hotspot sequence and breakpoint site were marked in blue box; figure s : entropy plot of amino acid of s (a), e (b), m (c), and n (d) protein genes of ibvs. x-axis shows the amino acid sites of s , e, m, and n genes; y-axis shows the entropy of each amino acid site; figure s : the location of positive selection sites and putative glycosylation sites of the s subunit of ibv isolates. the positively selected sites and n-glycosylation site are highlighted in red and blue separately; figure s : the location of positive selection sites and putative glycosylation sites of the e subunit of ibv isolates. the positively selected sites and n-glycosylation site are highlighted in red and blue separately; figure s : the location of positive selection sites and putative glycosylation sites of the m subunit of ibv isolates. the positively selected sites and n-glycosylation site are highlighted in red and blue separately; figure s : the location of positive selection sites and putative glycosylation sites of the n subunit of ibv isolates. the positively selected sites and n-glycosylation site are highlighted in red and blue separately. serotype and genotype diversity of infectious bronchitis viruses isolated during - in guangxi continuous evolution of avian infectious bronchitis virus resulting in different variants co-circulating in southern china complete genome sequences of two chinese virulent avian coronavirus infectious bronchitis virus variants molecular characterization of major structural protein genes of avian coronavirus infectious bronchitis virus isolates in southern china analysis of s gene of avian infectious bronchitis virus isolated in southern china during - immune protection conferred 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of the creative commons attribution (cc by) license the authors declare no conflicts of interest. key: cord- -fw w nm authors: friedman, stephanie d.; snellgrove, wyatt c.; genthner, fred j. title: genomic sequences of two novel levivirus single-stranded rna coliphages (family leviviridae): evidence for recombinationin environmental strains date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: fw w nm bacteriophages are likely the most abundant entities in the aquatic environment, yet knowledge of their ecology is limited. during a fecal source-tracking study, two genetically novel leviviridae strains were discovered. although the novel strains were isolated from coastal waters km apart (north carolina and rhode island, usa), these strains shared % nucleotide similarity and – % amino acid similarity. when the novel strains were compared to nine levivirus genogroup i strains, they shared – % similarity among the maturation, capsid and lysis proteins, but only – % in the rna-dependent rna polymerase gene. further bioinformatic analyses suggested a recombination event occurred. to the best of our knowledge, this is the first description of viral recombinants in environmental leviviridae ssrna bacteriophages. bacteriophages have played a major role contributing to our knowledge of molecular biology, not only in the role of model viruses but also as tools to investigate mrna, genes, genetic codes and genomes. the first sequenced genomes were the rna bacteriophage ms [ ] and the dna bacteriophage Ф-x [ ] . as important as drosophila was in shaping the field of genetics and tobacco mosaic virus was in advancing the study of virology and biochemistry, the rna phage ms (family leviviridae) was fundamental in laying the foundation of molecular biology. thus, it is important to continue adding to the basic understanding of phages. the observations presented in this study were rather serendipitous, in that the focus was not on searching for natural recombinant bacteriophages. nonetheless, evidence of a recombination event was revealed during a ssrna bacteriophage sequencing project [ ] . male-specific ssrna (frna) coliphages belong to the family leviviridae. they are classified into two genera (levivirus and allolevivirus) which are subdivided into four genogroups (genogroups i and ii in levivirus and genogroups iii and iv in allolevivirus). investigating the genetic diversity of frna phages vinjé et al [ ] conducted a phylogenetic analysis of levivirus field strains using a bp replicase gene fragment. this study revealed three main clusters: genogroup i, genogroup ii and a potential novel group, designated js, which clustered between genogroup i and genogroup ii. the putative js group, represented by phages, wwtp _ and gi , had a > % sequence diversity in the bp replicase gene sequence when compared to strains from genogroups i and ii. as these strains were isolated from widely separated geographical regions (massachusetts and south carolina) vinjé et al., [ ] proposed that js may form a stable lineage. this report suggested further genomic sequencing and serological data were needed to confirm whether these strains formed a novel genogroup or whether they were the result of recombination or rearrangement events [ ] . in its simplest form, recombination occurs when two disparate dna or rna strands exchange or merge stretches of their sequences whereas mutation involves the substitution, deletion or insertion of a nucleotide resulting in the change of the nucleotide sequence of a gene or an amino acid sequence of a protein. in some rna viruses, rna recombination events can occur when two or more strains infect the same host. proposed models for the formation of novel rna sequences include (i) cleavage and ligation in rna molecules or rna secondary structures [ ] , (ii) replicative template switching whereby the rna-dependent rna polymerase (replicase) switches from one template to another rna template, also known as copy choice [ , ] , and (iii) rna transesterification which occurs when the polymerase adds a separate rna fragment to the ' terminus of the original rna template [ ] . historically, experiments with ssrna coliphage mutants failed to provide evidence for recombination and the investigators concluded that rna phages would not undergo recombination [ ] . a potential flaw in the conclusion may have been that the study occurred at the time when frna phages were thought to possess only three genes, not four. in all likelihood, laboratory-applied selective pressure failed to detect or generate a specific recombinant. this failure may not necessarily reflect the lack of recombination or responsible mechanisms that could occur under actual environmental conditions encountered by ssrna coliphages. the first indication of rna recombination in a male-specific frna phage was the report of small, non homologous, recombinant rna molecules produced from a purified template-free qβ replicase molecule [ ] . the investigators noted similar rna molecules were present in e. coli cells infected with phage qβ. chetverin et al., [ ] studied this phenomenon by observing the formation of novel sequences in rna molecules which suggested that this recombination event occurred as a transesterification reaction catalyzed by a conformation acquired by qβ replicase during rna synthesis [ , ] . nucleotide sequences of recombined rna molecules non-homologous to the parent rna were formed in the absence of dna intermediates, demonstrating an rna recombination mechanism in the presence of qβ replicase [ ] . therefore, it was plausible to have recombination in environmental ssrna male-specific coliphage (leviviridae) isolates. in the present study, two js strains, dl and dl , were isolated during an environmental genotyping study of leviviridae frna phages [ , ] . as in the vinjé study [ ] , strains dl and dl were isolated from separate coastal waters. these phages were placed into the putative js subgroup using the genotyping methods of vinjé et al. [ ] . the objective of this study was to determine whether the existence of a novel js-like subgroup representing a third levivirus cluster as proposed by vinjé et al., [ ] could be verified. the approach taken here was to compare sequences from the js strains to nucleotide and amino acid sequence data from entire genomes of levivirus genogroup i strains and levivirus genogroup ii strains [ ] . analysis of the novel js strains provided evidence to determine whether these levivirus strains clustered to genogroup i, ii, a combination of groups i and ii or a unique genogroup. to further understand the phylogeny of these js strains, complete genomic sequencing, amino acid composition, phylogenetic, bioinformatic and statistical analyses were performed. preliminary analysis of nucleotide sequences from a replicase bp amplicon placed the two novel strains, dl and dl , into a "js-like" subgroup [ ] . reverse-line blot hybridization failed to genotype the two strains into genogroups i or ii [ ] . a total of strains (table ) were used to examine the relationships among nucleotides and amino acids in the levivirus genus. the first strains in genogroup i, table , i.e., ms , st , dl , dl , dl , dl , r , m and j , were referred to as "ms -like." genogroup i ms -like strains open reading frame (orf) start and stop codons were located at identical or very similar nucleotide positions as previously reported for strain ms . the js strains also had identical orf start and stop codon positions as the ms- like strains ( table ) . nucleotide pairwise comparisons of full-length genomes were made between all strains within the levivirus genome, including strains within genogroups i, js and genogroups ii. within the nine strains of ms -like genogroup i, full-length nucleotide sequence similarity was - % [ ] whereas the two js strains, dl and dl , shared . % sequence similarity to each other. in comparison, the js nucleotide sequences were more similar to ms -like genogroup i ( - %) than to the genogroup i strain fr ( %) or to genogroup ii strains ( - %) ( table a) . despite their sequence similarities, genome lengths for js strains ( nt) were shorter than all genogroup i strains ( - nt) ( table ) but longer than genogroup ii ( - nt) [ ] . numerous deletions in the ' untranslated region and a portion of orf (replicase) in js strains accounted for the decreased genome length (data not shown) but did not alter the orf positions when the genogroup i strains were aligned ( table ) . analysis of the replicase gene revealed a nt insertion at the nucleotide region when counting orf start site as nucleotide (figure ). this insertion occurred upstream from the orf stop codon. beginning approximately nt downstream from the replicase orf stop codon and continuing to the ' termini, nt deletions were present in the js strains when aligned to ms -like genomes. nucleotide alignment of the replicase and nontranslated regions (ntr) revealed numerous nt deletions in the js strains when compared to genogroup i strains accounting for the change in amino acid composition. however, js strains shared the ' terminal "signature", accaccca, present in levivirus genogroups i and ii [ ] . initially, nucleotide pairwise analyses of full-length genomes were made comparing all strains within the levivirus genome, including genogroups i, js and ii; an - % nucleotide similarity between js strains and the ms -like strains was observed (table a ). in comparison, the amino acid sequences of the maturation, capsid and lysis proteins of the js strains were very similar to those of the ms -like genogroup i strains, sharing - %, - % and - % sequence similarities, respectively (table b ). genogroup i strain fr, when compared to ms -like and js genogroup i strains, only shared an amino acid similarity to the maturation, capsid and lysis proteins ranging from . - . % (table b ). in contrast, the replicase protein sequences of the js strains were quite dissimilar to the replicase protein sequences of the ms -like genogroup i strains, displaying a similarity range of - % (table c) . however, a similarity of - % was observed among the highly conserved replicase genes for the ms -like strains. strain fr shared a % replicase similarity to js strains and approximately - % similarity to ms -like strains. genogroup ii replicase was approximately - % similar to js strains, - % to ms -like and fr strains and - % similar to other genogroup ii strains (table c) . table . (b) percent similarity in amino acid sequences between levivirus js strains and genogroup i maturation, capsid and lysis proteins. amino acid pairwise computations were performed in bionumerics. all genogroup i strains, including fr, and the two js strains had a replicase protein length of amino acids (table ) [ ] . however, js replicase differed from genogroup i replicase as it had one amino acid insertion at replicase position and one amino acid deletion at the ' termini of the stop codon, but maintained a total of amino acids (data not shown). identical to genogroup i strains, the replicase catalytic domain in the js strains occurred between amino acid positions - , thereby adding confidence to placing the grouping of js into genogroup i [ ] . beginning at amino acid number within the replicase gene, js strains were unique in amino acid composition and diverged from the ms -like strains. individual proteins from js strains dl and dl were grouped to protein families by pfam analysis. the maturation protein generated "phage_mat-a" domain including all leviviridae strains plus three additional bacteriophage, prr , pp and ap . the capsid protein resulted in a "levi-coat" domain including all leviviridae strains plus bacteriophage prr . the lysis protein only generated results in a pfamb search matching the genus levivirus strains from both genogroups i and ii including ku , jp , m , fp , ms , jp , fr, th , sd, ga, bo , tl and zr. replicase protein matched "rna_replicase_b" domain within the leviviridae family plus the additional bacteriophages prr , pp and ap . common protein motifs such as casein kinase ii phosphorylation, camp and cgmp-dependent protein kinase phosphorylation and protein kinase c phosphorylation occurred in dl and dl when compared to the levivirus strains [ ] . interestingly, every amino acid motif position in all four genes was identical among these two js strains. cophenetic correlations showed the genogroup i strains, the js subgroup strains, and the genogroup ii strains all formed faithful clusters with correlations of , and , respectively. the cluster cutoff method, however, showed only two relevant clusters being the genogroup i strains, which included fr and js, and genogroup ii strains (figure ). when referring to nucleotide or amino acid positions within the replicase gene, the numbering is in reference to the start codon as being position . in all analysis programs, the nucleotide or amino acid sequences were aligned to other strains and were therefore approximate positions on the replicase gene. all recombination programs used, simplot, rat, rdp and recco, statistically predicted recombination in both js strains, dl and dl , when compared to genogroup i ms -like strains. no recombination, however, was detected when dl and dl were compared to genogroup i strain fr and all genogroup ii strains. the simplot and bootscan analyses of the replicase nucleotides from js strains dl compared to levivirus genogroup i strains dl , dl , dl , dl , dl , st , r , j and ms is shown in figure a . since the replicase nucleotide sequences in strain dl were % similar to strain dl , dl was chosen as the query. the simplot analysis revealed the first recombination breakpoint occurred in the replicase from strain dl at nt positions - (approximate amino acid - ) where the χ changes from . to . (sum χ of . ). the second breakpoint occurred at nt positions - (approximate amino acid - ) where the χ changes from . to . (sum χ of . ). however, simplot amino acid analysis (figure b) with strain dl showed a divergence at approximate amino acid position region which is in agreement with the manual alignment ( figure ). rdp predicted dl and dl as the recombinant strains using several detection methods and analysis algorithms (table ) and suggested dl as a minor parent strain. breakpoint nucleotides for strains dl and dl (when aligned to genogroup i strains) occurred between nt - and - , respectively ( figure a, b) , corresponding to the approximate amino acid breakpoint positions of - within the replicase gene. manual alignment in bioedit of the replicase nucleotides, counting the atg start codon of the replicase gene as nt , showed an insertion of the nucleotides ya beginning at position (figure ) whereas the amino acid composition of the js strains diverged from the other genogroup i strains slightly upstream from this insertion at amino acid position (nucleotide ). alignment also revealed numerous nt deletions as discussed in the "sequence analyses and orf" section. the recco p-value inspector predicted strain dl had recombined with strain dl (figure a ). in dl , the recombinant region spanned from amino acids - whereas the dl region spanned from - with resulting sequence p-values of . and . , respectively. recco parametric cost curves predicted the highest preference for recombination in strains dl and dl (cost of . - ) whereas the remaining genogroup i strains did not show a preference for recombination (cost of - ) (figure b) . rat, rdp and recco all predicted recombination breakpoints ranging from amino acid positions - whereas simplot agreed most closely with the manual alignment of and , respectively. also in agreement with the manual alignment was the crossover region between dl and dl occurring in the approximate amino acid region of - (figure a) . the predicted breakpoint regions occurred either upstream or downstream from the highly conserved catalytic domain amino acid positions - in levivirus genogroup i [ ] . reported here are whole genome sequence data and bioinformatic analyses supporting the hypothesis that two novel frna isolates, dl and dl , were the result of natural recombination. initially classified as a js subgroup of genogroup i within the levivirus genus (family leviviridae), these strains were isolated from seawater approximately km apart in the rachel carson reserve, beaufort, nc, and narragansett bay, ri. findings that js strains were highly similar to three out of four genes (maturation, capsid and lysis) in genogroup i ms -like strains, shared the catalytic site location in the rna-dependent rna polymerase (replicase) gene, had an identical ' signature [ ] and then greatly diverged along a stretch of the replicase gene all supported the occurrence of a recombination event. in this study, two js strains shared > % amino acid identity in three (maturation, capsid and lysis) levivirus genogroup i ms -like genes but only an - % amino acid identity to the otherwise, highly conserved replicase protein. in comparison, genogroup i strain fr was uniformly different from all other genogroup i strains in all four proteins [ ] . cophenetic correlations and bootstrap analysis strengthen the possibility that js strains were recombinants as the js strains were only a subgroup of genogroup i and not a novel genogroup. throughout the leviviridae family, subgroups emerge within genogroups, however, subgroup strains differ in all four genes from the parent genogroup [ ] . it is therefore plausible to propose natural recombination in these two novel js-like frna coliphages as data presented here suggested a specific genetic rearrangement or recombination event in the replicase gene. interestingly, different leviviridae subgrouped strains originating from across the globe display high amino acid similarity among subgrouped strains [ ] . recombination may explain why leviviridae strains circulate as discrete subgroups independent of geographical location. although the two unique js-like strains were isolated from nc and ri, they shared . % nucleotide similarity across the entire genome. thus, either a single, natural recombination event occurred as a de novo mutation in each strain or identical, natural recombinations along a hot spot in these genomes formed these strains. in either case discovering that geographically-separated js-like strains acquired the same recombination event is intriguing. largely responsible for the diversity of rna viruses [ ] rna-rna recombination was observed in several positive-sense, ssrna human and animal viral taxa including caliciviruses, coronaviruses, hepatitis, dengue, enteroviruses and astroviruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, genetic exchange in ssrna viruses was first demonstrated in polioviruses [ , ] . recombination events frequently alter the rna-dependent rna polymerase region. human noroviruses, a positive sense ssrna virus with a genome length of - nt, are considered to belong to a prototype strain if they share approximately % overall nucleotide sequence identity and a high amino acid sequence identity (> %) in the polymerase gene [ ] . a naturally occurring human norovirus strain shared % amino acid sequence identity with the capsid sequences from a mexico cluster and % amino acid identity to the polymerase in a lordsdale virus cluster. sequences from the natural strain were obtained from one viral isolate. the combination of sequences in the one strain being complementary to two distinct human norovirus clusters led to the proposition that this strain was a naturally occurring recombinant [ ] . genetic recombination is known to occur in certain enteroviruses, a positive ssrna virus having an approximate nt genome. poliovirus recombination occurs in vaccine-derived strains [ ] in the human population as a single infected individual excretes a high proportion of recombinants [ ] . to determine if other enteroviruses undergo natural recombination, isolates of echoviruses were collected from a meningitis outbreak. nucleotide sequences were clustered based on a capsid protein (vp ) and rna-dependent rna polymerase ( d). dendrogram relatedness of the echovirus strains grouped the vpi sequences to the prototype strains. however, the rna polymerase sequences did not cluster to the prototype strains, suggesting genetic recombination among the outbreak strains [ ] . human astroviruses are positive sense, ssrna with a genome length of approximately , nucleotides [ ] and a polyadenylated ' tail [ ] . two sets of strains were investigated for recombination; one set was identified from a child care center in houston, tx, and the two other strains were found in stool samples from two children in mexico city. the pool of strains shared > % nucleotide sequence similarity in two out of three genomic regions. the novel strain clustered to one group based on the capsid region. when the rna-dependent rna polymerase was analyzed, the novel strain clustered to a separate human astrovirus group. the strains were identified as naturally occurring recombinants on the evidence of high sequence similarity to a few genes of one prototype and similarity to different genes in a second prototype. a total of additional human astroviruses lacked these novel traits [ ] . an enteric turkey astrovirus is a non-enveloped, positive sense ssrna virus with a polyadenylated ' tailed genome of approximately kb. the most conserved gene in the avian and mammalian astrovirus is the rna-dependent rna polymerase or replicase. genetic analysis of capsid and polymerase sequences from twenty-three turkey astrovirus strains resulted in clusters for the capsid gene and two phylogenetic clusters for the rna polymerase gene. computer-generated analyses identified polymerase gene recombination in strains of turkey astrovirus [ ] . in this study, four different recombination detection programs along with manual alignment predicted strains dl and dl as recombinants although the exact amino acid and/or nucleotide breakpoint varied somewhat along the rna-dependent rna polymerase gene. as expected the breakpoints did not occur within the catalytic-site domain. the sliding window approach as used with many recombination programs is based on an arbitrarily chosen window length, thus affecting the sensitivity and accuracy when pinpointing the precise breakpoint [ ] . overall, the use of a variety of recombination algorithms provided a stronger, more rigorous scientific case. when comparing the levivirus strains, manual alignment provided a more accurate picture of where the recombination event occurred along the genome but it did not provide a statistical analysis. therefore, statistical analysis in combination with manual alignment resulted in a more confident assessment of recombination. evidence for recombination among positive ssrna viruses exists within the rna-dependent rna polymerase (replicase) gene for numerous viruses as described here and supports the data that natural recombination can occur within the leviviridae family. frna phage strains ciceet and ciceet were isolated and placed into the putative js subgroup [ , ] using the genotyping methods of vinjé et al., [ ] . ciceet , renamed dl , was isolated from estuarine waters in rachel carson w reserve (beaufort), nc, and ciceet , renamed dl , and was isolated from narragansett bay, ri (table ) . each strain was plaque purified and further enriched using escherichia coli hs(pfamp)r as host [ ] . approximately - ml aliquots of the purified viral supernatant were frozen at & °c. coliphage rna was extracted from purified virus as described [ ] using a qiaamp viral rna mini kit (qiagen, valencia, ca, usa). purified rna was stored frozen at & °c. full-length genome sequencing was performed by the "primer walking" approach as described [ ] . nucleotide and amino acid sequences from js strains dl and dl were compared to nucleotide and amino acid sequences from genogroup i strains (ms , dl , dl , dl , dl , st , r , j , m , fr) and genogroup ii strains (t , dl , dl , ga, kui) [ ] . nucleotide sequences from three individual clones were imported and aligned using bioedit v . . [ ] followed by basic local alignment search tool (blast, national center for biotechnology information) analyses for sequence and phylogenetic confirmation. completed sequences from all strains were aligned with full-length prototype strains (genbank) using bioedit clustalw application. for each strain, the open reading frames (orfs) were mapped using bioedit. deduced amino acid sequences for each of the four genes were determined using a computer-generated dna-to-protein translation tool, expasy (http://ca.expasy.org/). prediction of protein sequence motifs were identified by prosite (http://ca.expasy.org/) and protein families and domains were modeled in pfam (http://pfam.janelia.org). sequence data were analyzed using bionumerics software v. . (applied maths, saint-martens-latem, belgium). phylogenetic trees were built by global cluster analysis performed on multiple aligned sequences and clustered by unweighted pair group method using arithmetic averages (upgma). a bootstrap analysis, based on , substitutions, was used to measure cluster significance. the reliability of each cluster was expressed on a percentage basis [ ] . nucleotide percent similarity and dendrograms were constructed using bionumerics software v. . (applied maths, saint-martens-latem, belgium). phylogenetic trees were built by global cluster analysis performed on multiple aligned sequences and clustered by upgma using the jukes and cantor correction [ ] . cophenetic correlations and cluster cutoff method were employed to measure faithfulness and relevancy of the clusters (applied maths, saint-martens-latem, belgium). average similarities with standard deviations were calculated for the relevant clusters. various approaches were used to examine recombination in levivirus frna strains, using aligned nucleotides and aligned amino acids, as follows: (i) manual alignment using bioedit, (ii) bootscan analysis in simplot v . . [ ] (iii) recombination analysis tool v . [ ] , (iv) recombination detection program v . [ ] , and (v) recombination analysis using cost optimization (recco) v . [ ] frna strains used in analyses were genogroup i strains dl , dl , dl , dl , st , r , j , ms , fr; js strains dl and dl ; and genogroup ii strains ga, ku , dl , dl and t . simplot analyses was determined with a sliding window of bp wide and a step size between plots of bp when comparing reference strains to the queried sequences. recombination events by simplot bootscan analysis occurred when the χ value changes signifying a breakpoint position. aligned replicase amino acids and/or nucleotide sequences were analyzed in recombination analysis tool (rat), recombination detection program (rdp ) and recco. rat uses a distance-based method of recombination in both dna and protein multiple alignments [ ] . unless stated otherwise, default settings were used with each program. the rat default settings were window size of % of the sequence length and an increment size being half of the window size. both settings of "auto search" and "test sequence search" were used with rat. recombination detection program v (rdp ) uses a number of recombination detection algorithms such as rdp, bootscan, geneconv, maximum chi square, chimaera, sister scanning (siscan) and seq [ ] . the rdp program sorts the analyses from these various algorithms and statistical data to determine the unique recombination events. rdp used an alignment of all genogroup i and js replicase nucleotide sequences and queried to dl , ms , dl and dl . the recco p-value inspector was set at and the permutation was set at . recco uses an algorithm that locates putative recombination points based on cost minimization. recco compares the cost of mutation relative to recombination as represented by α. the accession numbers for dl and dl are jq and jq , respectively. the results of this study provide genetic evidence, bioinformatic and statistical analyses suggesting a natural recombination event in the formation of a genogroup i subgroup js-like levivirus, represented by two strains, dl and dl . there was high nucleotide and amino acid identity in three genes, the maturation, capsid and lysis genes (≥ %) but a lack of similarity in the replicase gene ( - %) when js strains were compared to genogroup i ms -like strains. four different recombination programs demonstrated one or two breakpoint regions in the replicase gene, signifying a recombination event. the recombination event occurred downstream of the replicase catalytic site thereby maintaining viral integrity and replication function. thus, primers for oligonucleotide hybridization probes targeting the replicase beyond the catalytic site would not hybridize to js strains. in contrast, molecular assays targeting the maturation, capsid or lysis sequences would presumptuously place js strains as an ms- like genogroup. phylogenetic tree analysis produced a cophenetic correlation which showed (i) ten genogroup i strains, including strain fr, (ii) the js subgroup strains, and (iii) the genogroup ii strains all formed faithful clusters with correlations of , and , respectively. the cluster cutoff method, however, revealed only two relevant clusters, (i) genogroup i strains, which included fr and js, and (ii) genogroup ii strains. therefore, the novel js strains are not a unique levivirus genogroup. the proposed classification of js strains is genogroup i subgroup "js-like". although both js strains were prepared for sequencing in the same laboratory, these strains were field-collected by different investigators and shipped to another location where they were plaque-purified and preliminarily classified. therefore, the possibility that contamination resulted in false recombinants seems unlikely. likewise, the possibility of cloning and/or pcr errors contributing to the nucleotide and amino acids changes would not have led to both js strains being almost identical in the non recombinant regions as seven genogroup i strains (dl , dl , dl , dl , r , j and st ), three genogroup ii strains (dl , dl , t ) and two js strains (dl and dl ) were sequenced in this lab in no certain strain or fragment order using the same methods and sequencing company [ ] . finally, to the best of our knowledge, this is the first description of recombinant viruses from natural isolates in ssrna leviviridae bacteriophages. complete nucleotide sequence of bacteriophage ms rna: primary and secondary structure of the replicase gene nucleotide sequence of bacteriophage Ф-x dna gene mapping and phylogenetic analysis of the complete genome from single-stranded rna male-specific coliphages (family leviviridae) molecular detection and genotyping of male-specific coliphages by reverse transcription-pcr and reverse line blot hybridization the puzzle of rna recombination nonenzymatic recombination of rna: possible mechanisms for the formation of novel sequences viral rna-directed rna polymerases use diverse mechanisms to promote recombination between rna molecules genetic studies of rna phages an in vivo recombinant rna capable of autocatalytic synthesis by qβ replicase f+rna coliphages as source tracking viral indicators of fecal pollution. a final report submitted to the noaa/unh cooperative institute for coastal and estuarine environmental technology (ciceet) evaluation of rt-pcr and reverse line blot hybridization for detection and genotyping f+ rna coliphages from estuarine waters and molluscan shellfish rna recombination in animal and plant viruses evidence of structural genomic region recombination in hepatitis c virus phylogenetic analysis of turkey astroviruses reveals evidence of recombination phylogenetic evidence for recombination in dengue virus evidence for frequent recombination within species human enterovirus b based on complete genomic sequences of all thirty-seven serotypes random nature of coronavirus rna recombination in the absence of selection pressure natural genetic recombination between co-circulating heterotypic enteroviruses molecular characterization of a novel recombinant strain of human astrovirus associated with gastroenteritis in children characterization of a novel human calicivirus that may be a naturally occurring recombinant detection and genetic differentiation of human astroviruses: phylogenetic grouping varies by coding region genetic recombination with newcastle disease virus, polioviruses, and influenza genetic recombination with poliovirus type studies of crosses between a normal horse serum-resistant mutant and several guanidine-resistant mutants of the same strain outbreak of poliomyelitis in hispaniola associated with circulating type vaccine-derived poliovirus rb-finder: an improved distance-based sliding window method to detect recombination breakpoints sequence variation among group iii f-specific rna coliphages from water samples and swine lagoons bioedit: a user-friendly biological sequence alignment editor and analysis program for windows / /nt evolution of protein molecules full-length human immunodeficiency virus type genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination recombination analysis tool (rat): a program for the high-throughput detection of recombination rdp : recombination detection and analysis from sequence alignments recombination analysis using cost optimization this research was funded, in part, through epa's new england regional applied research effort (rare). we gratefully acknowledge the assistance of jack paar, iii, u.s. epa new england regional laboratory for initiating and sponsoring this program.we wish to thank jan vinjé, centers for disease control and prevention, atlanta, ga, for his intellectual contribution. an acknowledgement is extended to syed muaz khalil for providing a portion of the sequence data. we thank greg lovelace and david love (johns hopkins university) for isolating and providing some of the strains used in this study and emilie cooper (cdc) for reviewing the manuscript.the information in this document has been funded wholly (or in part) by the u.s. environmental protection agency. it has been subjected to review by the national health and environmental effects research laboratory and approved for publication. approval does not signify that the contents reflect the views of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. the authors declare no conflict of interest. key: cord- -xtsi k authors: ortiz-riaño, emilio; cheng, benson y. h.; de la torre, juan c.; martínez-sobrido, luis title: d g mutation in lcmv-np affects its ability to self-associate and results in a dominant negative effect in viral rna synthesis date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: xtsi k arenaviruses merit significant interest because several family members are etiological agents of severe hemorrhagic fevers, representing a major burden to public health. currently, there are no fda-licensed vaccines against arenaviruses and the only available antiviral therapy is limited to the use of ribavirin that is partially effective. arenavirus nucleoprotein (np) is found associated with the genomic rna forming the viral ribonucleoproteins (vrnps) that together with the polymerase (l) direct viral replication and transcription. virion formation requires the recruitment of vrnps into budding sites, a process in which the arenavirus matrix-like protein (z) plays a major role. therefore, proper np-np and np-z interactions are required for the generation of infectious progeny. in this work we demonstrate the role of the amino acid residue d in the self-association of lymphocytic choriomeningitis virus nucleoprotein (lcmv-np). amino acid substitutions at this position abrogate np oligomerization, affecting its ability to mediate replication and transcription of a minigenome reporter plasmid. however, its ability to interact with the z protein, counteract the cellular interferon response and bind to dsrna analogs was retained. additionally, we also document the dominant negative effect of d g mutation on viral infection, suggesting that np self-association is an excellent target for the development of new antivirals against arenaviruses. arenaviruses cause rodent infections with a worldwide distribution [ ] . infections of humans are common and can occur by direct contact between infectious materials and abraded skin or inhalation of aerosol forms of the virus [ ] . arenaviruses are divided, according to serologic, genomic and geographic distribution, into two groups: old world (ow) and new world (nw) arenaviruses [ ] . members of both groups can cause hemorrhagic fever (hf) disease in humans and pose a serious public health problem in their endemic regions [ , ] . the ow lassa virus (lasv), the causative agent of lassa fever (lf), is the hf arenavirus of highest impact in public health, with some , infections estimated yearly, which are associated with high morbidity and significant case fatality rates [ , ] . moreover, increased travel to and from endemic regions has resulted in the importation of lf cases into non-endemic regions [ ] . in addition, evidence indicates that the globally distributed prototypic arenavirus lymphocytic choriomeningitis virus (lcmv) is likely a neglected human pathogen associated with congenital infection leading to hydrocephalus, mental retardation and chorioretinitis in infants [ , ] . lcmv also poses a threat to immuno-compromised individuals, as shown by reported lethal cases of lcmv infection after organ transplantation [ , ] . public health concerns posed by human pathogenic arenaviruses are aggravated by the lack of food and drug administration (fda)-licensed vaccines and current antiarenaviral therapy limited to off-label use of the nucleoside analog ribavirin, which is only partially effective and associated with significant side effects [ − ] . additionally, several arenaviruses have been classified as national institute of allergy and infectious disease category a priority pathogens because of their potential misuse as agents of bioterrorism [ ] . therefore, the development of novel effective antiviral strategies to combat human pathogenic arenaviruses are urgently needed, a task that would be facilitated by a better understanding of the arenavirus molecular and cell biology. on the other hand, lcmv has proven very useful as an experimental model system in the fields of viral immunology and pathogenesis [ , ] . arenaviruses are enveloped, negative-sense rna viruses with bisegmented genomes [ ] . each segment uses an ambisense coding strategy to direct the synthesis of two proteins in opposite orientation [ ] . the large (l) segment encodes for the rna-dependent rna polymerase (l) and a small ring finger protein (z) that has matrix-like functions [ , ] , including directing the budding process [ ] . the small (s) segment encodes the glycoprotein precursor (gpc) and the nucleoprotein (np). gpc is posttranslationally processed by the cellular site protease to produce gp- and gp- [ , ] , which associate to form the glycoprotein complex (gp) that constitute the spikes on the surface of the virion structure responsible for receptor recognition and cell entry [ ] . np encapsidates the viral genome, and together with the l protein, are the minimal components required for viral transcription and replication [ , ] . recent publications have underscored the variety of roles played by np during infection. besides its involvement in virus replication and transcription, np counteracts the host type i interferon (ifn-i) response by preventing activation of the interferon regulatory factor (irf ) and subsequent induction of ifn-i production and interferon-stimulated genes (isgs) [ , ] . np-mediated inhibition of irf activation has been shown to correlate with np's ability to interact with ikkε, thus preventing phosphorylation of irf [ ] . the anti-ifn-i function of np has been mapped to its c-terminal region that contains also a functional ′- ′ exoribonuclease domain, believed to be responsible for the anti-ifn-i properties of np [ − ] . furthermore, the same domain and amino acid residues shown to play a critical role in np anti-ifn-i activity have been recently shown to also mediate inhibition of nuclear translocation and transcriptional activity of nuclear factor kappa b (nf-κb) [ ] . these results suggest that arenaviruses may use a common mechanism to evade the host ifn-i and inflammatory responses, which likely plays a key role in arenavirus pathogenesis and virulence [ ] . mutation-function studies have also mapped the np domains involved in np self-association and np-z interaction at the n-and c-terminal regions, respectively [ − ] . interestingly, the crystal structure of np revealed the presence of head-to-tail np-np interactions, suggesting the potential role of both, the n-and the c-terminal regions of np in self-association [ , ] . however, the contribution of specific residues to np self-association has not been determined. in this work, we provide evidence that an aspartic residue (d) at position in lcmv-np affects np self-association and its ability, together with l, to replicate an lcmv minigenome (mg). however, mutations at d did not affect np-z interaction, np binding to double-stranded (ds)rna analogs or np's ability to counteract the cellular ifn-i antiviral response. moreover, we also show that lcmv-np with mutation d g acts as a dominant-negative mutant that results in decreased viral replication in cell culture. together, our findings suggest that np-np interaction might be an excellent target candidate for the development of new antivirals for the treatment of arenavirus infections. the ability of np to self-associate has been recently demonstrated by biochemical studies [ , ] . this np oligomerization likely plays a major role in the encapsidation of viral rna and in the formation, together with l, of vrnps that direct both replication and transcription of the viral genome [ , ] . to identify single amino acid residues involved in self-association of lcmv-np, we screened a previously documented battery of lcmv-np mutants [ ] for their self-association properties. our initial screen identified a double mutant containing q r and d g substitutions that showed impairment in np-np interaction, as determined by co-immunoprecipitation (co-ip) ( figure a ) and the mammalian two-hybrid (m h) ( figure b ) assays. to assess the individual contribution of q r and d to np-np interaction, we generated np mutant forms containing only mutation d g or q r and tested them in our co-ip and m h assays. for co-ip assays, we co-transfected t cells with n-terminal flag-tagged wild-type (wt) lcmv-np (flag-np), together with c-terminal ha-tagged versions of the lcmv-np single (q r and d g) or double (q r/d g) mutants. wt ha-tagged lcmv-np was used as a validated control [ ] . at hours post-transfection (hpt), cell lysates were prepared and analyzed by western blot (wb). np mutant q r was expressed at similar levels as wt np, while d g and d g/q r np mutants were expressed at slightly lower levels ( figure a , i). cell lysates were incubated with anti-flag affinity agarose gel and immunoprecipitates analyzed by wb using anti-ha or anti-flag antibodies. band intensities were quantified and normalized with respect to total np expression. d g, but not q r, amino acid substitution disrupted np-np interaction ( figure a , ii). to confirm and further characterize the effect of d g substitution in np-np interaction, we used the m h system ( figure b ). for this, we co-transfected t cells with pcaggs (pc) plasmids expressing wt or mutant nps fused to the n-terminus of vp (pc-np-vp ) [ ] , together with plasmids expressing wt np fused to the n-terminal of gal (pc-np-gal ) [ ] and the reporter plasmid encoding a green fluorescent protein (gfp) and firefly luciferase (ffl) fusion protein, pg gfp/ffl. to normalize transfection efficiencies we included the plasmid prl sv . we used pc-vp as a negative control to show specificity of the assay [ , ] . we detected np self-association of wt and q r nps as determined by both gfp (figure b , i) and ffl ( figure b , ii) reporter gene expressions. in contrast, d g and the double mutant q r/d g were severely impaired in their ability to interact with wt lcmv-np. all vp fusion constructs were expressed at similar levels as determined by wb using an anti-vp polyclonal antibody ( figure b , iii). these results uncovered a key role of residue d in np-np interaction. co-ip: human t cells were co-transfected with μg of the pc-lcmv-np wt, single (q r and d g), or double (q r/d g) amino acid mutants ha-tagged, together with μg of pc-lcmv-np wt flag-tagged expression plasmid. as controls, lcmv-np tagged versions were expressed individually together with μg of empty pc to keep constant the total amount of transfected dna. at hpt, cell lysates were prepared and analyzed for protein expression levels by wb using anti-ha or anti-flag polyclonal antibodies (i). gapdh was used as a loading control. cell lysates were immunoprecipitated with anti-flag affinity agarose beads (ii) and analyzed by wb with the indicated antibodies (left). numbers at the bottom of each wb lane represent the quantification of band intensities normalized to the signal of cells co-transfected with haand flag-tagged wt nps, as described in material and methods. (b) m h: human t cells were co-transfected in triplicate with μg of the indicated wt, single, or double amino acid mutant pc-vp -tagged expression plasmids, together with μg of lcmv-np-gal expression plasmid, along with μg of the pg gfp/ffl dual reporter plasmid, and . μg of the prl sv expression plasmid to normalize transfection efficiencies. at hpt, gfp expression was assessed using fluorescence microscopy and cell extracts were prepared to determine the strength of np-np interaction using the promega dual-luciferase reporter assay and a lumicount luminometer. as negative control, we transfected cells with pc-np-gal together with pc-vp . representative fields of transfected cells are illustrated (i). reporter gene activation (ffl) is shown as percentage (%) of lcmv np-np wt interaction (pc-np-vp and pc-np-gal ) after normalization of transfection efficiencies with the renilla luciferase expression plasmid prl sv (ii). cell lysates were used to detect expression of lcmv-np wt and mutants by wb using an anti-vp polyclonal antibody (iii). gapdh was used as a loading control. numbers at the bottom of each wb lane represent the quantification of band intensities normalized to wt np, as described in material and methods. arenavirus z protein has been shown to be the driving force of virus budding and in the absence of other viral proteins, mediates the formation of viral-like particles (vlp) [ , − ] . cell egress of arenavirus infectious progeny requires the interaction between z and np present in the vrnp. accordingly, np has been shown to be incorporated into z-derived vlp [ , , ] . the lcmv-np domain involved in the interaction with z was mapped to the c-terminal of viral np [ ] . to determine if residue d played a key role in the np-z interaction, we examined its interaction with z using both vlp ( figure a ) and m h ( figure b ) assays. for the vlp assay, a flag-tagged lcmv-z pc expression plasmid was co-transfected in t cells alone or in combination with pc expression plasmids of ha-tagged wt or mutant nps. at hpt, tissue culture supernatants (tcs) and cell lysates were collected. wb analysis of cell lysates showed that all samples expressed similar levels lcmv-np and lcmv-z proteins (figure a , i). vlp were purified through a % sucrose cushion, and vlp-containing pellets were analyzed by wb [ ] . all np mutants tested were incorporated into the lcmv-z-mediated vlp, although we observed a slight reduction for the q r/d g np mutant (figure a , ii). as expected, np was not detected in the tcs of t cells transfected only with pc-np, demonstrating the specific np incorporation into z-derived vlp [ ] . to further characterize the np-z interaction, we performed a m h assay [ ] ( figure b ). in this case, t cells were co-transfected using pc plasmids expressing np-vp fusion proteins containing wt or mutant forms of np together with pc plasmids expressing gal -z, the reporter plasmid pg gfp/ffl, and prl sv to normalize transfection efficiencies. all mutants tested interacted at similar levels with z as determined by gfp ( figure b , i) and ffl ( figure b , ii) reporter gene expression. all np-vp fusion proteins were expressed at similar levels as determined by wb ( figure b , iii). taken together, these results indicate that lcmv-np residue d does not play a critical role in np-z interaction. figure . d g amino acid substitution in lcmv-np does not affect its interaction with lcmv-z. (a) vlp assay: human t cells were co-transfected with μg of the indicated pc wt or mutant nps ha-tagged, together with μg of pc-lcmv-z flagtagged expression plasmid. empty pc plasmid was included to normalize the total amount of transfected dna. at hpt, cell lysates were prepared and analyzed for protein expression levels for np (α-ha) and z (α-flag) (i). gapdh was used as a loading control. tcs from same transfected cells were used for isolation of vlp to evaluate np incorporation (α-ha) into z-mediated vlp (α-flag) (ii). numbers at the bottom of each wb lane represent the quantification of band intensities normalized to np and z wt expression levels. (b) m h assay: human t cells ( . x ) were co-transfected in triplicate ( -well plate format) with μg of the indicated pc-vp -tagged wt or mutant nps, together with μg of pc-gal -tagged z expression plasmid as described in figure b . at hpt, lcmv np-z interaction was determined by gfp expression (i) and by luciferase activity (ii). pc-vp expression plasmid was used as negative control. reporter gene activation (ffl) is shown as percentage of wt interaction (pc-np-vp and pc-gal -z) after normalization of transfection efficiencies with the renilla luciferase expression plasmid prl sv . expression levels of wt and mutant np were determined by wb using an anti-vp polyclonal antibody (iii). gapdh was used as a loading control. numbers at the bottom of each wb lane represent the quantification of band intensities normalized to wt np lane as described in material and methods. np plays a critical role in arenavirus rna replication and gene transcription [ , ] , but whether monomers or multimers of np are required to direct these rna biosynthetic processes has not yet been determined. we therefore examined the activity of d g np mutant in an lcmv minigenome (mg) rescue assay [ ] . in order to perform this task, we generated a dual reporter lcmv mg, where pur-gfp and gluc substituted for the lcmv-np and -gpc open reading frames (orfs), respectively, within the s segment that was flanked by the mouse pol-i promoter and terminator sequences (ppoli gfp-pur/gluc). bhk- cells were co-transfected with ppoli gfp-pur/gluc together with pc plasmids expressing l and ha-tagged versions of wt or mutant nps and psv -cluc to normalize transfection efficiencies. at hpt, reporter gene expressions were assessed by fluorescence microscopy ( figure a ) and luciferase activities from tcs ( figure b ). cells lysates were prepared and analyzed by wb to determine protein expression ( figure c ). np mutants containing the d g substitution failed in their ability to replicate and transcribe the lcmv mg, while np containing substitution q r showed a minimal defect on replication and transcription. although we observed some variation in the expression levels among the different ha-tagged versions, the total absence of reporter gene expression associated with substitution d g could not be attributed to the modest differences in protein expression levels observed by wb, indicating that np-np interaction is required for the function of np in virus rna replication and gene transcription. were determined. cell lysates were used to determine expression levels of wt and mutant nps by wb using an anti-ha antibody (c). gapdh was used as a loading control. empty pc was used as a negative control. percentages of relative luciferase units (% rlus) were normalized with respect to the activity of the wt np, after normalization of transfection efficiencies based on cluc luminescence values. numbers at the bottom of each wb lane represent the quantification of band intensities normalized to wt np lane as described in material and methods. the anti-ifn-i activity of arenavirus np [ ] was mapped to the c-terminal region of the viral protein [ ] , whereas np self-association was mapped to an n-terminal region of np that did not overlap with the anti-ifn-i domain, suggesting that monomeric forms of np can inhibit induction of ifn-i [ ] . since d plays a critical role in np-np interaction and is located within the c-terminal region of lcmv-np responsible for the anti-ifn-i activity of np, we examined whether substitution d g that disrupted np-np association affected also the anti-ifn-i activity of np ( figure ). to that end, we co-transfected t cells with pifnβ-gfp and pifnβ-ffl [ ] reporter plasmids and the indicated pc-np ha-tagged expression plasmids, together with the prl sv , to normalize transfection efficiencies. at hpt cells were infected (moi = ) with sendai virus (sev), [ ] and at hours post-infection (hpi), reporter gene expression was determined by fluorescence microscopy ( figure a ) and luminescence ( figure b ) assays. both d g and q r/d g np mutants were expressed to slightly lower levels than wt and q r mutant nps as determined by wb ( figure c ), but wt and all np mutants tested similarly counteracted sev-mediated induction of ifnβ, confirming that self-association of lcmv-np is not required for counteracting the ifn-i response [ ] . to induce activation of the ifnβ promoter, and hours later, gfp expression was assessed by fluorescence microscopy (a). cell lysates were prepared for luciferase activities (b), and to detect expression of wt and mutant nps by wb using an anti-ha antibody (c). gapdh was used as a loading control. reporter gene activation is shown as % of rlus of a sev-infected, empty-vector transfected control, after normalization by rl luminescence values. numbers at the bottom of each wb lane represent the quantification of band intensities normalized to wt np lane, as described in material and methods. we have previously shown that self-association of lcmv-np is mediated by rna [ ] . furthermore, we have recently suggested the possibility that two different types of interactions mediate np-np self-association [ ] . one interaction, requiring the participation of rna, involves mainly a region located at the n-terminal region of np [ ] . the other interaction consists of an rna-independent head-to-tail protein-protein interaction involving both the n-and c-terminal regions of np [ , ] . this model would predict that np-np interaction mediated by the c-terminal domain of np could be separated from the ability of np to bind rna. to test this, we evaluated the binding of wt and mutant nps to the dsrna analog poly i:c ( figure ). as a control, we included lcmv-z, which was predicted to not bind rna. for this, we co-transfected t cells with the indicated pc-np or pc-z ha-tagged expression plasmids and hpt, cells lysates were prepared and analyzed by wb ( figure a ). cell lysates were then incubated overnight with poly i:c sepharose beads. as an additional internal control and to demonstrate the specificity of np-dsrna binding, cell extracts were incubated with sepharose beads lacking poly i:c (data not shown). the beads were then washed and pull-down proteins were analyzed by wb. all lcmv-nps, but not lcmv-z, were pulled down in the presence of poly i:c sepharose beads ( figure b ). as expected, none of the proteins tested were detected in the pull-down lacking polyi:c (data not shown). these results demonstrate that the lcmv-np d g mutation does not affect its ds rna binding capability, and suggest that the np self-association is not required for dsrna binding. human t cells were co-transfected with . μg of the indicated pc-lcmv-np wt or amino acid mutants ha-tagged. pc-lcmv-z ha-tagged was used as negative control for binding. at hpt, cell lysates were prepared and analyzed for np expression levels by wb using an anti-ha antibody (a). gapdh was used as a loading control. cell lysates were used in pull-down assays with poly i:c bound to sepharose beads and immunoprecipitates analyzed by wb (b). numbers at the bottom of each wb lane represent the quantification of band intensities normalized to wt np lane, as described in material and methods. to further characterize the key role of residue d in the np-np interaction and rna synthesis, but not in np-z interaction or anti ifn-i activity, we introduced additional amino acid substitutions at position (d e and d a) and evaluated their contribution to np-np and np-z interactions, as well as their role in replication and expression of the lcmv mg and anti-ifn-i activity of np ( figure ). the conservative change d e resulted in reduced levels of np self-association ( figure a ) without affecting np-z interaction ( figure b) . interestingly, the d e substitution allowed replication and transcription of the lcmv mg, although to lower levels compared to wt np ( figure c ). likewise, d e was not affected in its ability to inhibit sev-mediated induction of ifn-i ( figure d) . unexpectedly, the d a substitution impeded both np-np and np-z interactions, as well as np functions in replication and transcription of the lcmv mg and the ability of np to counteract sev-mediated induction of the host ifn-i response, suggesting that change d to a at position might have affected the overall structure of np without affecting its stability as reflected by its unchanged expression levels determined by wb using anti-vp ( figures a and b ) or anti-ha ( figures c and d) antibodies. human t cells were co-transfected as described in figure b with μg of the indicated pc wt or mutant np-vp fusion proteins, together with μg of np-gal expression plasmids. at hpt, cell extracts were prepared to determine the strength of the interaction. vp expression plasmid was used as negative control. reporter gene activation (ffl) is shown as percentage of wt interaction (pc-np-vp and pc-np-gal ) after normalization of transfection efficiencies based on levels of renilla luciferase activity driven by plasmid prl sv . cell lysates were used to detect expression of wt and mutant nps by wb using an anti-vp polyclonal antibody. gapdh was used as a loading control. (b) np-z interaction: human t cells were co-transfected as described in figure b with μg of the indicated pc wt or mutant np-vp , together with μg of gal -z expression plasmids. at hpt, cell extracts were prepared to determine the strength of the interaction and protein expression. reporter gene activation (ffl) is shown as percentage of wt interaction (pc-np-vp and pc-gal -z) after normalization of transfection efficiencies based on renilla luciferase values. cell lysates were used to detect expression of wt and mutant nps by wb using an anti-vp polyclonal antibody. gapdh was used as a loading control. (c) replication and transcription activity: bhk- cells were co-transfected with the lcmv mg as described in figure together with expression plasmids for the viral polymerase (l) and wt or indicated mutant nps, and psv -cluc expression vector to normalize transfection efficiencies. at hpt, tcs were collected for luciferase assay and cell lysates were prepared for protein detection. empty pc was used as a negative control. rlus (%) were calculated based on the replication and transcription activity mediated by wt np, after normalization by cluc luminescence values. expression levels of wt and mutant nps were determined by wb using an anti-ha antibody. gapdh was used as a loading control. (d) inhibition of induction of ifn-i: human t cells were co-transfected as described in figure with . μg of the indicated pc-np ha-tagged expression vectors together with the ifnβ reporter plasmids. at hpt, cells were infected with sev (moi= ) to induce activation of the ifnβ promoter, and hours later cell lysates were prepared for luciferase assay and detection of protein expression. luciferase values were normalized with respect to those obtained in cells transfected with empty pc and infected with sev, after adjusting for renilla luminescence values. expression of wt and mutant nps were determined by wb using an anti-ha antibody. gapdh was used as a loading control. consistent with its lack of self-association, lcmv-np d g was not able to promote replication and expression of the lcmv mg. however, based on the dsrna-binding assay, np d g may potentially keep its interaction with the viral rna, thus possibly interfering with viral replication and transcription by blocking the viral polymerase extension on the rna or its interaction with l. this would predict a dominant negative phenotype of np d g in the lcmv mg rescue assay. to examine this possibility, bhk- cells were co-transfected with either empty pc or pc np d g together with our mg reporter plasmid ppoli gfp-pur/gluc, along with pc l and increasing amounts of pc wt np-ha to establish a linear response range. transfection efficiencies were normalized using psv -cluc. at hpt, reporter gene expressions were assessed by fluorescence microscopy ( figure a ) and luciferase activities from tcs ( figure b ). transfected cells containing lcmv-np d g mutant showed strong reduction in the replication and transcription of the lcmv mg at the different concentration tested compared to cells transfected with empty plasmid. these results suggest a dominant negative effect of d g mutant on the mg assay, possibly by interfering with the replication and transcription function. the observed dominant negative effect on the replication and transcription in the mg assay led us to hypothesize that mutation d g in lcmv-np would also interfere with viral infection. supporting this hypothesis, lcmv-np d g mutant might still interact with the z protein, therefore interfering with the z-vrnp interaction required for formation of matured infectious progeny [ , ] . to test this, we generated bhk- stable cell lines expressing ha-tagged versions of wt or d g nps ( figure a , b) and examined their susceptibility to lcmv in comparison to parental bhk- cells. for this, we infected the different cell lines at low moi ( . ) with lcmv and determined levels of infectious progeny in tcs at , and hpi ( figure c ). cells expressing d g mutant displayed reduced viral titers at all time points tested. intriguingly, at hpi virus titers in tcs were higher in cells expressing wt np than in bhk- parental, but peak titers in cells expressing wt np did not reach the titers obtained in parental bhk- cells, suggesting that over-expression of lcmv-np might interfere with optimal production of infectious progeny. this dominant negative effect, however, was not observed in cells infected with a vesicular stomatitis virus expressing gfp (vsv-gfp, figure d ), confirming that the stable cell line expressing d g np has a specific effect on lcmv viral proliferation. representative merged images are illustrated. cells lysates were prepared and μg of total cellular protein content were analyzed by wb using an anti-ha antibody (b). gapdh was used as a loading control. numbers at the bottom of each wb lane represent the quantification of band intensities normalized to wt np lane, as described in material and methods. kinetics of lcmv (c) and vsv-gfp (d) propagation: parental and np-ha expressing bhk- cell lines were infected with lcmv (moi = . ) or vsv-gfp (moi = . ) in triplicates. tcs at the indicated hpi were titrated using a focus forming unit assay on vero cells as described in materials and methods. dashed lines indicate the limit of detection for the assay. baby hamster kidney (bhk- ) cells (atcc ccl- ) and human embryonic kidney ( t) cells (atcc crl- ) were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs), l-glutamine ( mm), penicillin ( units/ml) and streptomycin ( μg/ml), in a % co atmosphere at °c [ , ] . lcmv (armstrong strain, arm b) was produced by infecting bhk- cells (moi = . ) and harvesting the tissue culture supernatants (tcs) at hours post-infection (hpi) as previously described [ ] . vesicular stomatitis virus expressing gfp (vsv-gfp) was produced by infecting bhk- cells (moi = . ) and harvesting the tcs at hpi [ ] . hemagglutinin (ha)-and flag-tagged versions of lcmv-np and -z in pcaggs (pc) [ , ] , as well as lcmv-np and -z open reading frames (orfs) fused to vp and gal in pc [ , ] , have been described. lcmv-np single amino acid mutants (q r, d g, d e, and d a) and the double mutant (q r and d g) were generated by site-directed mutagenesis (stratagene) and then sub-cloned into pc-ha and -vp expression plasmids to generate the corresponding c-terminal fusion proteins [ ] . the pg ffl reporter plasmid (promega) was modified fusing the green fluorescent protein (gfp) orf to the n-terminal region of firefly luciferase (ffl) coding sequence to generate pg gfp/ffl [ ] . for the mg reporter assays, a fusion construct of puromycin (pur) and green fluorescent protein (gfp) was amplified by pcr from the p kprom-puro-egfp plasmid, kindly provided by drs. g. chen and j. roberts [ ] , with primers containing bbsi restriction sites and cloned into the lcmv s segment backbone ppoli gpc/bbsi plasmid [ ] to generate the ppoli gpc/pur-gfp. similarly, gaussia luciferase (gluc) was amplified by pcr from the pc-gluc plasmid [ ] with primers containing bsmbi restriction sites and cloned into the lcmv s segment backbone ppoli bsmbi/np plasmid [ ] to generate the ppoli gluc/np. both plasmids (ppoli gpc/pur-gfp and ppoli gluc/np) were then combined using bglii restriction sites to generate the dual lcmv mg reporter plasmid ppoli gluc/pur-gfp, where the pur-gfp fusion replaces the np orf and gluc replaces the gpc orf and the '-and ' non-coding untranslated regions (utrs) of the lcmv s segment viral rna [ ] were flanked by the mouse polymerase i promoter and terminator sequences, respectively [ ] . pifnβ-gfp/cat and pifnβ-ffl, for the ifn-i induction assay, have been previously described [ ] . primers used to generate the described plasmids are available upon request. constructs orfs were verified by dna sequencing and protein expression confirmed by western blot (wb). human t ( x cells) were co-transfected in suspension ( -well tissue culture plates format) with μg total of ha-and flag-tagged np pc expression plasmids using lipofectamine (invitrogen), according to manufacturer's instructions [ ] . empty pc plasmid was included to maintain a constant total ( µg) amount of transfected plasmid dna. at hours post-transfection (hpt), cells were collected by centrifugation at , rpm for minutes (min) at °c and lysed with μl of lysis buffer ( mm tris-hcl ph . , mm edta, mm nacl, % np- , complete cocktail of protease inhibitors, roche) for min on ice. cell lysates were clarified by centrifugation at , rpm for min at °c. aliquots ( µl; % of total cell lysate) were analyzed for protein expression by gel electrophoresis and wb. for immunoprecipitation, μl of ezview red anti-flag m affinity gels (sigma) were pre-incubated for min with lysis buffer at room temperature and used to immunoprecipitate μl ( . % of total cell lysates) of cleared lysates overnight at °c. affinity gels were washed four times with μl of lysis buffer and immunoprecipitated samples were resuspended in μl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) loading buffer. a μl aliquot of each immunoprecipitated sample was analyzed by wb [ ] . to generate vlps, human t cells were co-transfected in suspension ( -well plate format, x cells/well) with μg of the pc-z-flag expression plasmid and μg of wild type (wt) or mutant lcmv-np expression plasmids [ ] using μg of lipofectamine per μg of dna. empty pc plasmid was used to keep constant the total amount of transfected plasmid dna. at hpt, cells and tcs were collected. tcs were clarified at , rpm for min and then layered on top of a % sucrose cushion and centrifuged at , rpm on an sw- rotor for . hours. vlp-containing pellets were resuspended in μl of x phosphate-buffered saline ( x pbs), and μl were analyzed by wb. cell pellets were lysed with μl of lysis buffer ( mm tris-hcl, ph . , mm edta, mm nacl, % np- , complete cocktail of protease inhibitors; roche) for min on ice. cell lysates were clarified by centrifugation at , rpm for min at °c. aliquots ( μl; % of total cell lysates) from each sample were analyzed by wb [ ] . bhk- cells ( x ) were co-transfected in monolayer ( -well plate format, triplicates) with . μg of pc-l, . μg of the dual lcmv mg reporter plasmid ppoli gluc/pur-gfp, indicated amounts of pc-lcmv-np ha-tagged, and . μg of a mammalian expression vector encoding the secreted cypridina luciferase (cluc) under the control of the simian virus (sv ) constitutive promoter (psv -cluc, new england biolabs) to normalize transfection efficiencies, using . μg of lipofectamine per well. at hpt, pur-gfp expression was assessed by fluorescence microscopy using a zeiss fluorescent microscope and luciferase activities in tcs determined using a lumicount luminometer (packard). representative images of gfp expression are shown. gluc activity was determined using the dual luciferase reporter assay (promega) and cluc activity by the biolux gaussia kit (new england biolabs). reporter gene activation (gluc) is indicated as a percentage of relative light units (% rlus) of lcmv-np wt or x-fold induction over negative transfected-control, where pc-lcmv-np ha-tagged expression plasmid was replaced by empty pc, after normalization with the cluc luminescence values mean value and standard deviation were calculated using microsoft excel software [ ] . human t cells ( . x ) were co-transfected in suspension in -well tissue culture plates, using lipofectamine . each well contained μg of the indicated pc-vp and gal expression plasmids, μg of the reporter pg gfp/ffl plasmid, and . μg of a renilla luciferase (rl) expression plasmid under the control of the sv promoter (prl sv , promega) to normalize transfection efficiencies. at hpt, representative fluorescence images were obtained to evaluate protein-protein interaction by gfp expression using a zeiss fluorescent microscope. upon imaging, cell lysates were prepared to determine luciferase activities using the promega dual-luciferase reporter assay and a lumicount luminometer (packard). percentage of interaction of lcmv-np mutants with wt lcmv-np or lcmv-z, after normalization of transfection efficiencies with the rl expression plasmid prl sv , was calculated based on wt np-np interaction (pc-np-vp and pc-np-gal ) and wt np-z interaction (pc-np-vp and pc-gal -z), respectively. m h experiments were performed in triplicate. the mean and standard deviation were calculated using microsoft excel software. protein expression was determined by wb as described [ ] . mean value and standard deviation were calculated using microsoft excel software [ ] . for dsrna pull down assays, human t cells ( x , -well plate format) were co-transfected in suspension, harvested, and cell lysates prepared as described for co-ip assays. aliquots ( μl; % of total cell lysates) were analyzed for protein expression by gel electrophoresis and wb. for dsrna binding, polyinosine-polycytidylic acid (poly i:c, sigma) was bound to cnbr-activated sepharose b (ge healthcare) following manufacturer's recommendation. poly i:c sepharose beads ( μl) were pre-incubated for min with lysis buffer at room temperature and used to pull-down μl ( % of total cell lysates) of cleared lysates overnight at °c. sepharose beads were washed four times with μl of lysis buffer and pull-down samples were resuspended in μl of sds-page loading buffer. a μl aliquot of each pull-down sample was analyzed by wb. to control for specific binding, sepharose beads without poly i:c bound were included during the pull-down assay (data not shown). human t cells were co-transfected ( x cells/well, -well plate format, triplicate) using a calcium phosphate method with . μg of each of the ifnβ reporter plasmids (pifnβ-gfp/cat and pifnβ-ffl [ ] ) and . μg of the pc-lcmv-np ha-tagged expression plasmids, together with . μg of prl sv to normalize transfection efficiencies. after overnight transfection, cells were infected with sendai virus (sev, moi= ), and hpi, fluorescence images were obtained to determine ifnβ promoter activation by gfp expression [ ] . after imaging, cell lysates were prepared for luciferase activities and protein expression [ ] . luciferase activities were measured by using the dual-luciferase kit (promega) as recommended by the manufacturer. reporter gene activation is shown as % rlus of the infected empty-plasmid-transfected control. protein expression was determined by wb as previously described [ ] . mean value and standard deviation were calculated using microsoft excel software [ ] . proteins were separated by . % or % sds-page and then transferred onto nitrocellulose membranes (bio-rad) overnight at °c. after blocking for one hour at room temperature with % dry milk in x pbs, membranes were incubated with polyclonal primary antibodies against ha and flag (sigma h and f , respectively), a polyclonal antibody against vp (sigma v ), or a monoclonal antibody against glyceraldehyde- -phosphate dehydrogenase (gapdh, abcam ab ) for one hour at room temperature. membranes were then washed three times with x pbs containing . % tween- , and probed with secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin (ig) antibodies (ge healthcare uk) for one hour at room temperature. after three washes with x pbs containing . % tween- , proteins were detected using a chemiluminescence kit and autoradiography films from denville scientific inc. protein band intensities were quantified using imagej software (national institutes of health, nih). band quantifications for inputs of co-ip and vlp assays, m h assay, mg assay, and ifn-i induction assay were normalized according to gapdh expression and assigning % intensity to wt lcmv-np. lcmv-np mutants expressions were then normalized by their relative intensity to wt lcmv-np. bhk- cells constitutively expressing carboxy-terminal ha-tagged lcmv-np wt or the d g mutant were generated as previously described [ , , ] . briefly, bhk- cells were co-transfected with pc-lcmv-np wt or d g ha-tagged, together with the puromycin resistance vector ppur (clontech) ( : ratio) using lipofectamine . at hpt, cells were plated into -cm dishes at low density and cell clones were selected in the presence of . μg/ml of puromycin (cellgro, - -ra). puromycin resistant clones were screened for np expression by immunofluorescence and wb using a polyclonal antibody against the ha epitope (sigma h ). the best lcmv-np expressing bhk- clones were maintained in dmem supplemented with % fbs, penicillin/streptomycin, and . μg/ml of puromycin. growth kinetic analyses of lcmv and vsv-gfp were performed in parental and np-expressing bhk- cell lines ( -well plate format). sub-confluent cell monolayers ( x cells/well) were infected in triplicates with lcmv arms b and vsv-gfp at a low moi ( . and . , respectively). after min of adsorption at °c for lcmv and min at room temperature for vsv-gfp, virus inoculum was removed, cell monolayers were washed twice with pbs, and maintained in post-infection media ( : mixture of opti-mem reduced serum medium and dmem containing % fbs for lcmv and dmem containing . % of bovine albumin for vsv-gfp). at the indicated times post-infection, tcs were collected, clarified by centrifugation, and titrated. virus titers (focus-forming units [ffu]/ml) in tcs were determined by immunofocus assay [ ] for lcmv and by fluorescent focus assay for vsv-gfp. briefly, for lcmv, vero cells were infected with -fold serial dilutions of tcs for min at °c. at hpi, cells were fixed with % formaldehyde in x pbs for min, permeabilized with . % triton x- in x pbs for min at room temperature, and blocked overnight with . % bovine serum albumin (bsa) in x pbs. lcmv for a variety of negative strand rna viruses, self-association of the virus nucleoprotein has been shown to be required for the formation of functional vrnp that directs replication and transcription of the viral genome [ − ] . in this work, we have strongly suggested the role of the residue d in the self-association of np from the prototypic arenavirus lcmv. the substitution d to g at position abrogated the self-association property of lcmv-np, affecting its ability to promote replication and transcription of an lcmv mg. however, the d g mutant was not affected in its ability to interact with z, counteract the sev induced ifn-i response or bind to dsrna analogs. amino acid substitution to a similarly charged e residue partially restored the properties lost by the d to g substitution, while d to a substitution disrupted all np's interactions and functions tested, probably by affecting the overall np structure. in addition, we have documented the dominant negative effect of the d g mutant on virus multiplication, suggesting that np self-association is a potential target for the development of new therapeutics against arenaviruses. we have shown that the mutation d g in lcmv-np disrupts its ability to self-associate by using co-ip ( figure a ) and m h ( figure b) approaches. however, this amino acid mutation is localized in the c-terminal region of lcmv-np, which is outside of the previously described domain required for np oligomerization [ , ] . analysis of the only available arenavirus np crystal structure, that of lasv-np [ ] ( figure a ) shows that the residue in lasv-np corresponding to d in lcmv is not located in the proposed protein-protein interaction interface, but its side chain points towards the intramolecular n-c interface. np sequence alignment from representative members of the arenaviridae family shows the highly conserved nature of the d residue ( figure b ), suggesting its structural or functional, or both, importance across the different arenaviruses. interestingly, it has been suggested that lasv-np has a possible gating mechanism that allows protein structural rearrangement upon rna binding [ ] . however, the c-terminal region organization has not been determined for this newly adopted conformation. based on biochemical and structural analysis we proposed that the np self-association might involve both the n-terminal and the c-terminal region. it is plausible that in the newly formed np open conformation following rna binding to the n-terminal domain of np, residue d becomes engaged in np oligomerization via direct protein-protein interaction. previous studies documented that n-terminal deletions affecting np-np interaction abrogated np's ability to promote rna replication and gene expression of an lcmv mg [ , ] . however, we did not have available a single amino acid mutant affecting np self-association to corroborate its effect on the replication and transcription function. confirming our hypothesis, the d g mutant failed to replicate and transcribe a mg reporter plasmid (figure ) , when co-transfected with the l protein. supporting our data, mutations in amino acids predicted to be in the np-np interface have been described to also abrogate the replication and transfection function [ ] . taken together, these results suggest that proper oligomerization of np is necessary for viral replication and transcription, where the overall structural protein integrity is required. np-mediated encapsidation of arenavirus genome and antigenome rna species, which involves np-rna interaction, is strictly required for replication and gene expression of viral genome. biochemical and structural data indicate that np self-association involves a rna-dependent protein-protein interaction mediated by the n-terminal region of np [ ] . in the absence of viral rna, np self-association can be mediated by cellular rna [ ] . the mechanisms whereby in virus-infected cells np discriminates between viral from cellular rna remain to be elucidated. likewise, the structural features of the rna substrate recognized by np to mediate encapsidation have not been defined. however, as with other ns rna viruses, arenaviruses exhibit sequence complementarity between the '-and '-termini of their genomes and antigenomes, which is predicted to result in the formation of a panhandle structure that could provide the structural scaffold for both the virus promoter and nucleation site for rna encapsidation [ , ] . our results have shown that d g mutant np, deficient in np-np interaction, was capable of binding to the dsrna analog poly i:c ( figure ), suggesting that the initial np-rna interaction is not required for the self-association of np. the contribution of residue d to the multiple functions associated with np was further supported by the finding that substitution d a disrupted all tested np interactions and functions ( figure ). this likely reflected the limited rotational flexibility of a compared to g [ ] , which could have resulted in the disruption of the overall structure in d a mutant np. the d g mutant np, defective in np-np interaction, exhibited a dominant negative phenotype in the mg assay ( figure ) and interfered with virus multiplication (figure ). this might be explained because np (d g) can still bind rna, and it could compete with wt np for the rna substrate during the initial phase of viral rna encapsidation. likewise, np (d g) is likely to interact with l [ ] and compete with np (wt)-l interaction required for the formation of the functional virus polymerase complex. moreover, np (d g) has the ability to interact with the z protein and thereby compete with the z-vrnp interaction required for production of matured infectious virions. it should be noted that for a variety of ns rna viruses, mutations affecting np-np interaction have been documented to exhibit dominant negative phenotypes [ ] . these findings support the feasibility of targeting np-np interaction as a novel antiviral strategy to combat arenavirus infections flev, flexal virus (nc_ . ) sabv, sabia virus (nc_ . ) chapare virus (nc_ . ) amapari virus (nc_ . ) cpxv, cupixi virus (nc_ . ), latv, latino virus (af . ) oliveros virus (nc_ . ). clustalw program was used for alignment amino acid are colored according to chemical properties: red (hydrophobic and aromatic amino acids), blue (acidic), magenta (basic), green (hydroxyl and amine containing), as specified by the embl-ebi clustalw . . multiple sequence alignment program however, we have previously shown that n-terminal deletion mutants of np, which were defective in np-np interaction we have shown that the d g mutation affected np-np interaction without disrupting the np's anti-ifn-i activity (figure ). likewise, although the c-terminal region of lcmv-np was implicated in np-z interaction [ ], mutation d g did not affect the ability of np to interact with the z protein in both vlp (figure a) and m h (figure b) assays. together, these findings strongly suggest the presence of domains moreover, results obtained with the d g np mutant further support the conclusion that monomers of np are sufficient to counteract the host ifn-i response lms laboratory is partially funded by the nih grants ro ai , r ns - , r ai - a , and hhsn c. research at jct laboratory is supported by grants ro ai , ro ai , and ro ai from nih/niaid. references and notes arenaviridae: the viruses and their replication phylogenetic analysis of the arenaviridae: patterns of virus evolution and evidence for cospeciation between arenaviruses and their rodent hosts human infection with arenaviruses in the americas arenaviruses other than lassa virus lassa virus viral hemorrhagic fever hazards for travelers in africa main syndrome of prenatal infection caused by lymphocytic choriomeningitis virus -hydrocephalus and chorioretinal degeneration transmission of lymphocytic choriomeningitis virus by organ transplantation a new arenavirus in a cluster of fatal transplant-associated diseases treatment of bolivian hemorrhagic fever with intravenous ribavirin effective therapy with ribavirin ribavirin prophylaxis and therapy for experimental argentine hemorrhagic fever biology and pathogenesis of lymphocytic choriomeningitis virus infection lymphocytic choriomeningitis virus and immunology arenaviruses: genomic rnas, transcription, and replication characterization of the lassa virus matrix protein z: electron microscopic study of virus-like particles and interaction with the nucleoprotein (np) complementarity in the supramolecular design of arenaviruses and retroviruses revealed by electron cryomicroscopy and image analysis the small ring finger protein z drives arenavirus budding: implications for antiviral strategies molecular and cell biology of the prototypic arenavirus lcmv: implications for understanding and combating hemorrhagic fever arenaviruses the lassa virus glycoprotein precursor gp-c is proteolytically processed by subtilase ski- /s p role of the virus nucleoprotein in the regulation of lymphocytic choriomeningitis virus transcription and rna replication np and l proteins of lymphocytic choriomeningitis virus (lcmv) are sufficient for efficient transcription and replication of lcmv genomic rna analogs inhibition of the type i interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus differential inhibition of type i interferon induction by arenavirus nucleoproteins arenavirus nucleoprotein targets interferon regulatory factor-activating kinase ikk{varepsilon} identification of amino acid residues critical for the anti-interferon activity of the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus cap binding and immune evasion revealed by lassa nucleoprotein structure structure of the lassa virus nucleoprotein reveals a dsrna-specific ' to ' exonuclease activity essential for immune suppression arenavirus nucleoproteins prevent activation of nuclear factor kappa b inhibition of the type i interferon antiviral response during arenavirus infection the c-terminal region of lymphocytic choriomeningitis virus nucleoprotein contains distinct and segregable functional domains involved in np-z interaction and counteraction of the type i interferon response self-association of lymphocytic choriomeningitis virus nucleoprotein is mediated by its n-terminal region and is not required for its anti-interferon function identification of two functional domains within the arenavirus nucleoprotein a role for the c terminus of mopeia virus nucleoprotein in its incorporation into z proteininduced virus-like particles crystal structure of the lassa virus nucleoprotein-rna complex reveals a gating mechanism for rna binding myristoylation of the ring finger z protein is essential for arenavirus budding lassa virus z protein is a matrix protein and sufficient for the release of virus-like particles the role of myristoylation in the membrane association of the lassa virus matrix protein z identification of the lymphocytic choriomeningitis virus (lcmv) proteins required to rescue lcmv rna analogs into lcmvlike particles dual role of the lymphocytic choriomeningitis virus intergenic region in transcription termination and virus propagation use of single-cycle infectious lymphocytic choriomeningitis virus to study hemorrhagic fever arenaviruses murine coronavirus delays expression of a subset of interferon-stimulated genes improved knockout methodology reveals that frog virus mutants lacking either the k immediate-early gene or the truncated vif- alpha gene are defective for replication and growth in vivo generation of recombinant lymphocytic choriomeningitis viruses with trisegmented genomes stably expressing two additional genes of interest a cell-based luciferase assay amenable to high-throughput screening of inhibitors of arenavirus budding reverse genetics system for uukuniemi virus (bunyaviridae): rna polymerase i-catalyzed expression of chimeric viral rnas hemagglutinin-pseudotyped green fluorescent protein-expressing influenza viruses for the detection of influenza virus neutralizing antibodies a novel high-throughput cell-based method for integrated quantification of type i interferons and in vitro screening of immunostimulatory rna drug delivery quantification of lymphocytic choriomeningitis virus with an immunological focus assay in -or -well plates crystal structure of the rabies virus nucleoprotein-rna complex crystal structure of a nucleocapsid-like nucleoprotein-rna complex of respiratory syncytial virus structure of the influenza virus a h n nucleoprotein: implications for rna binding, oligomerization, and vaccine design crystal structure of the borna disease virus nucleoprotein structure of the vesicular stomatitis virus nucleoprotein-rna complex web services at the european bioinformatics institute characterization of the genomic promoter of the prototypic arenavirus lymphocytic choriomeningitis virus molecular organization of junin virus s rna: complete nucleotide sequence, relationship with other members of the arenaviridae and unusual secondary structures conformations of amino acids in proteins cross-species analysis of the replication complex of old world arenaviruses reveals two nucleoprotein sites involved in l protein function functional mapping of the nucleoprotein of ebola virus we thank members in lms laboratory for their discussions and snezhana dimitrova for technical support. we thank drs. g. chen and j. roberts (department of microbiology and immunology at university of rochester medical center) for providing us with the plasmid p kprom-puro-egfp. eor is a fulbright-conicyt fellowship recipient (bio ). byhc was supported by grant number the authors declare no conflict of interest. key: cord- - qydcc e authors: kumar, asit; kodidela, sunitha; tadrous, erene; cory, theodore james; walker, crystal martin; smith, amber marie; mukherjee, ahona; kumar, santosh title: extracellular vesicles in viral replication and pathogenesis and their potential role in therapeutic intervention date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qydcc e extracellular vesicles (evs) have shown their potential as a carrier of molecular information, and they have been involved in physiological functions and diseases caused by viral infections. virus-infected cells secrete various lipid-bound vesicles, including endosome pathway-derived exosomes and microvesicles/microparticles that are released from the plasma membrane. they are released via a direct outward budding and fission of plasma membrane blebs into the extracellular space to either facilitate virus propagation or regulate the immune responses. moreover, evs generated by virus-infected cells can incorporate virulence factors including viral protein and viral genetic material, and thus can resemble noninfectious viruses. interactions of evs with recipient cells have been shown to activate signaling pathways that may contribute to a sustained cellular response towards viral infections. evs, by utilizing a complex set of cargos, can play a regulatory role in viral infection, both by facilitating and suppressing the infection. ev-based antiviral and antiretroviral drug delivery approaches provide an opportunity for targeted drug delivery. in this review, we summarize the literature on evs, their associated involvement in transmission in viral infections, and potential therapeutic implications. cells mediate intercellular communication and modulation of immune responses through shedding and release of extracellular vesicles (evs) [ ] . these evs are diverse and originate from plasma membrane and endosomes and include exosomes, micro-vesicles (mvs, also known as microparticles), and apoptotic bodies. they are categorized based on their biogenesis, release pathways, size, content, and function [ ] . evs shed from plasma membranes are generally referred to as mvs [ ] [ ] [ ] , while vesicles that are generated by inward budding of endosomes to form multivesicular bodies (mvbs) that fuse with the plasma membrane, and release into the extracellular environment, are known as exosomes [ , ] ; whereas, cells undergoing apoptosis can release vesicles or cell filaments exclusively from the plasma membrane, called apoptotic bodies [ , ] . depending on their biogenesis pathway and cellular origin, exosomes are vesicles of endocytic origin and their size usually ranges from - nm [ ] . exosomal markers include tetraspanins (tspan and tspan , escrt components, and tsg ). the invasion of the plasma membrane inwards forms the early endosome and the limiting membrane of the later endosome sprouts further to form the mvbs. mvbs are characterized by the invagination of the inner body membrane, which results in the formation of intraluminal vesicles (ilvs) [ ] . during this process, cytoplasmic components and certain peripheral proteins are integrated into them. the ilvs accumulated in the mvb lumen have two routes. one is to diffuse with the lysosomes, which causes the contents of the vesicles to degrade, and the other is fusion with the cytoplasmic membrane and release of the vesicles to the extracellular space by exocytosis, referred as "exosomes" [ ] . loading of biological cargos into ilvs involves the endosomal sorting complexes required for transport (escrt) complexes (escrt- , -i, -ii, -iii and the vps ) and other accessory proteins such as alix/pdcd ip, tsg , hrs, etc. [ , ] . in addition to escrt, other mechanisms can also produce exosomes of certain biochemical components. for instance, in some cells production of exosomes requires lipid ceramide and neutral sphingomyelinase [ ] , an enzyme that converts sphingomyelin to ceramide, and related proteins including phospholipase d that hydrolyzes phosphatidylcholine into phosphatidic acid and dgk alpha [ , ] . another mechanism of exosome release relies on small gtpases such as rab a/b [ ] , rab , , , and in some cells, or soluble n-ethylmaleimide-sensitive factor attachment protein receptor (snare) family proteins like ykt [ , ] , vesicle-associated membrane protein (vamp ) [ , ] , cd , and cd . these proteins are involved in exosome biogenesis and are commonly used as markers unlike exosomes and microvesicles, which are released during normal cellular processes, apoptotic bodies are formed only during programmed cell death [ , ] . apoptotic bodies' size ranges from - nm. during apoptosis, the cell undergoes morphological changes and shrinks to a smaller size with densely packed cytoplasm and other organelles, and eventually their nucleus disintegrates [ ] . further, the cells form blebs on its surface and disintegrate into small fragments called apoptotic bodies. these are characterized by the presence of organelles within the vesicles [ ] and are cleared from the body by phagocytosis by specific mechanisms [ , ] . the most commonly used identifiers of apoptotic bodies are annexin v, thrombospondin, and c b [ ] . limited knowledge exists in the literature regarding the role of apoptotic bodies in cell-cell communication during viral infection and their contribution to viral pathogenesis. to understand their possible role and function in intercellular communication, numerous in-depth studies are warranted in the future. uptake of ev seems to depend on the type of recipient cell, its physiological state, and recognition of ligands or receptors on the recipient cell and evs. cells broadly internalize evs either by fusion with the plasma membrane or via endocytosis. internalization of evs by recipient cells occurs by various mechanisms of endocytosis including clathrin-dependent and clathrin-independent mechanisms such as caveolin-mediated uptake, macro-pinocytosis, phagocytosis, and lipid raft-mediated internalization [ , ] . ev uptake is an energy-dependent process [ ] . neurons internalize oligodendrocyte-derived exosomes by clathrin-mediated endocytosis [ ] , whereas microglia internalize exosomes by micropinocytosis [ ] . epithelial cells internalize exosomes by caveola-dependent endocytosis [ ] , while dendritic cells internalize evs through lipid raft domains [ ] . different methods are employed to detect ev uptake, among which the most used viruses , , of method is the use of fluorescent lipid membrane dyes to stain ev membranes. examples of such dyes include pkh , pkh , rhodamine b, dii, and did [ , , , ] . the internalization of evs by recipient cells can be measured using methods such as flow cytometry and confocal microscopy [ , ] . evs and viruses are highly heterogeneous in size, structure, and biogenesis, and therefore they cause apparent difficulties in distinguishing and separating evs from viruses. even though evs and viruses overlap in size and biophysical properties, evs far outnumber high-titer viruses during infection [ ] . in the past decade, a multitude of isolation and purification methods for evs and virus particles have been developed. differential centrifugation/ultracentrifugation (uc) technique is widely used for the isolation of evs from cell cultures' media and biological fluids that contain viruses [ ] . although this technique is considered as the gold standard of ev isolation, it often coprecipitates with proteins and lipoproteins that can affect sample purity and may interfere with downstream analysis [ , ] , limiting its use in hospital settings. this limitation can be overcome by including multiple isolations and characterization techniques such as antibody-based immunoaffinity purification, tangential flow filtration (tff), and nano-flow cytometry (nfcm) [ ] [ ] [ ] [ ] . however, each of these methods has its limitations, which need to be considered before planning ev isolation and purification. for instance, ev isolation using the antibody-based immunoaffinity purification method provides a refined ev population but is limited by the sample volume and amount of final product [ ] . moreover, the expression level of ev markers such as cd , cd , and cd can vary depending on the ev origin and physiological condition, requiring a combination of markers to be used. compared to uc, the tff method can be effective in obtaining ev-enriched formulations from a large volume of samples. however, tff is likely to cost higher than conventional ev isolation methods. further studies are required to explore the utilization of tff for clinical studies [ ] . due to limitations associated with isolation procedures, and lack of a standardized isolation process, a validated good manufacturing practice (gmp)-compliant procedure is desperately needed. bari et al. employed conditioned media from mesenchymal stem/stromal cells for the secretome/ev isolation. a key aspect of their study is a large-scale secretome or ev isolation process using uc and tff that complies with gmp, which allows standardized and pharmaceutical grade products suitable for clinical applications [ , ] . the use of nfcm is reported as a new benchmark for quality assessment of evs. phenotyping of single particles is possible through nfcm using immunofluorescent labeling of evs [ ] . however, the limitations in resolution and detection varied depending on the criteria used to define the ev populations based on markers [ ] that have excluded many researchers widely utilizing this technique. besides, an nfcm based method can be challenging to develop and to validate ev characterization, given the specific ev population measurement and due to the lack of standard guidelines for handling and analyzing a variety of samples with appropriate normative controls in nfcm. li k et al. have developed an approach termed cushioned-density gradient ultracentrifugation (c-dguc), a variant of ultracentrifugation, for ev refinement [ ] . in this approach, samples were processed through a density gradient cushion such as iodixanol (optiprep™) and centrifugal force to maximizes ev recovery followed by density gradient ultracentrifugation steps that eventually provide high-purity purification of evs by effectively removing protein aggregates. however, evs can lose integrity while isolated from a fixed density range [ ] . polyethylene glycol (peg) precipitation followed by iodixanol density separation has recently become a useful method to pull down evs, viruses, and proteins or protein-rna aggregates within a sample, followed by an additional centrifugation step. this method results in a significantly higher yield of evs in comparison to the conventional uc method [ ] . the contents of evs vary greatly depending upon the condition of the parent cell. thus, apart from characterizing the vesicles, identifying these contents reveals a breadth of information regarding the parent cells. the international society for extracellular vesicles (isev) guidelines should be followed when isolating evs from cells or plasma/biological fluids for drug encapsulation. the most pragmatic approach appears to be viruses , , of the isolation of evs using a commercial kit and size exclusion chromatography (sec; also known as gel filtration) methods followed by microfiltration of samples using filters with pore diameters of . , . , or . µm depending on the size of vesicles required. in sec, evs are separated from other material according to differences in sizes (hydrodynamic radii) [ ] that gives this technique the upper edge over conventional methods and can be effectively used for a variety of complex biological samples such as body fluid, blood/plasma, urine, and breast milk [ ] [ ] [ ] [ ] . isolation of high-purity evs from samples containing virions is challenging since both evs and some viruses, in this case, retroviruses, are similar in size. as of now, no validated protocol is available to specifically separate evs from virions that are similar in size and carry the same markers [ ] . however, a study has demonstrated that defective viruses could be separated from naturally occurring viruses based on differences in buoyant densities [ ] . evs loaded with drugs to treat viral diseases require them to target majorly infected cells or tissues. when considering evs as personalized therapeutic carriers, surface engineering of evs is required that can be performed using covalent and noncovalent modification [ ] [ ] [ ] . it is important to optimize the method of isolation for evs for drug loading on a case-to-case basis. upon loading drugs to these evs, the evs can be further separated using a sucrose gradient that utilizes iodixanol and characters each fraction for loading efficiency and total loading. the ev fractions with optimally loaded drugs can be further characterized by their size, shape, and marker proteins for further use. evs released by virus-infected cells can incorporate protein molecules, derived from viral genes involved in viral assembly. delivery of the ev-associated virulence molecules affects recipient cells by rendering them particularly vulnerable to viral infection (table ) . moreover, incorporating viral proteins can trigger cell death of non-participating immune cells [ ] that would contribute to the heavy loss of immune cells during the early stages of viral infection or low viral load. intercellular transfer of viral proteins and viral cell surface receptors by evs not only facilitates evasion of the host's immune response by suppressing antibody production in lymphocytes but also makes immune cells susceptible to viral infection [ , ] . however, while evidence indicates that evs can, directly and indirectly, mediate the antiviral response, their role in regulating immune response is not yet fully elucidated in vivo. hiv-infected cell-derived exosomes carrying negative regulatory factor (nef) induces apoptosis in t-lymphocytes; nef-transfected microglia-released nef+exosomes reduce the blood-brain barrier (bbb) integrity [ , ] chemokines and receptors ccr , cxcr , mcp- facilitate the entry of hiv [ ] proinflammatory markers il- , tnf-β, il- hiv-infected cells derived exosome containing tar rna plays a role in the increase of il- and tnf-β in macrophages. hiv-infected u macrophages upon cigarette smoke condensate (csc) treatment enhanced the packaging of il- in evs; il- served as a biomarker for hiv patients with altered immune function due to alcohol and tobacco abuse [ , , ] host protein apobec g inhibit replication of viral infectivity factor (vif) -deficient and wild-type hiv- in recipient cells [ ] mirna vmir- and vmir- triggers endosomal toll-like receptor (tlr) and nuclear factor-κb (nf-κb) signaling, stimulating the release of tnfα by delivering ev to bystander macrophages, and may contribute to chronic immune activation [ ] oxidative stress factors cellular markers cyp ( a , b , and a ), sod , cat gfap induce hiv replication. hiv-infected u macrophages upon csc treatment promotes differential packaging of cyps and aoes in evs increased levels of glial fibrillary acidic protein (gfap) in plasma evs from hiv subjects can serve as a potential biomarker [ , , ] contribute to viral immune-evasion and act in concert to promote tumor development through the interaction with multiple cellular proteins [ , ] mirna mir- , - b, and let- b cancer-associated, cellular pathways targeted by these mirnas. induce tumorigenesis through the effect of these micrornas on their targets [ ] mir- plays a role in cervical carcinogenesis, notably through the downregulation of p and phosphatase and tensin homolog deleted on chromosome (pten) [ ] mir- - p favors cell proliferation [ ] mir- a- p possesses anti-apoptotic properties [ hbv viral proteins large s, core and p proteins hepatocytes secreted exosomes participate in virus replication [ ] viral mirnas hbv-mir- represses viral protein production and hbv replication [ ] htlv- viral proteins gp , tax, and hbz increase cell-to-cell contact and promote a potential increase in viral spread [ ] zika viral genetic material and protein rna and zikv-e evs derived from infected c / cells promote infection and activation of monocytes with enhanced tnf-α mrna expression. [ ] in hiv, evs are thought to play an important role in disease progression through multiple mechanisms. viral components may be packaged in evs, which can then be delivered to uninfected cells, modulating the systemic inflammatory status. for instance, hiv-infected cell-derived exosomes carry viral protein nef that induces apoptosis in immune cells and reduces the blood-brain barrier (bbb) integrity to spread viral infection in the brain [ , ] . it has been shown that evs released during hiv infection are heterogeneous including size variability. a study has shown that treatment-naïve people living with hiv/aids (plwha) contain evs larger in size and numbers compared to plwha who were either virally suppressed, elite controllers, or healthy controls [ ] . additionally, cd counts and the abundance of evs in the blood were inversely correlated, with low cd counts associated with more abundant evs. interestingly, there was no relationship between cd counts and ev size. both size and abundance were also inversely correlated with neutrophils and platelet counts, as well as the cd /cd ratio, all of which are markers of disease progression [ ] . this suggests that evs may function as a biomarker for hiv disease progression. other studies have observed similar findings. in cells treated with antiretroviral drugs (arvs), increases in relative ev production has been observed [ ] , along with decreased loading of genomic, but not non-coding, rna into evs from cells, which were treated with arvs, as opposed to untreated cells. additionally, treatment with interferon-alpha increased the packaging of viral rna into evs. the authors suggest that this occurs because arv or interferon prevents the release of viral particles from cells, which then allows for viral rna to be packaged into evs due to the increased presence of viral rna in the cell. in addition to viral rna, a variety of molecules, e.g., viral & host proteins, cellular markers, mirna, inflammatory molecules such as oxidative stress markers, chemokines and cytokines can also be packaged into evs [ , [ ] [ ] [ ] , ] . a study showed that the viral envelop (env) protein can be packaged into evs from infected cells [ ] . the env-containing evs can increase susceptibility to viral infection in cell culture experiments, and depletion of env-containing evs showed decreased susceptibility to viral infection. altered levels of proteins in plasma evs are often described upon viral infection. for example, various examples of significantly altered expression of proteins, and markers associated with cellular stress, have been reported in plasma evs derived from hiv and htlv- infected patients. however, the mechanism of specific packaging of these proteins and markers in evs and their role in intercellular communication was not elucidated [ , ] . blood plasma can be considered as disease biomarkers since it contains glycoproteins and cellular markers carried in evs [ ] . dysregulation of cytokines and chemokines is often associated with hiv infection and subsequently contribute to the viral pathogenesis [ , , ] . moreover, the use of substances such as alcohol, tobacco, and drugs is prevalent among hiv-infected individuals [ ] [ ] [ ] [ ] . circulating inflammatory cytokines have been found to be elevated in hiv-positive substance users [ , , , ] . in prior studies, we demonstrated that exosomes derived from hiv-infected monocytes/macrophage cells exert a protective effect against cytotoxicity and viral replication in hiv-infected macrophages. however, exosomes derived from hiv-infected cells lost their protective capacity that could be due to the selective packaging of cytochrome p (cyps) and antioxidant enzyme (aoe) mrnas in exosomes [ ] . similar to the previous study, exposure to cigarette smoke condensate (csc) increased the packaging of cytokines, especially il- and cyps ( a and b ) in evs isolated from hiv-infected u macrophages [ ] . conversely, ev packaging of aoes (sod- and catalase) decreased in hiv-infected u macrophages more than in uninfected u macrophages [ ] . recently, our group showed that the astrocytic and neuronal-specific markers (gfap and l cam) can be packaged in evs and circulate in plasma, which is further elevated in the presence of hiv infection, alcohol, and/or tobacco [ ] . human cytidine deaminase apobec g (a g) can be packaged in evs and inhibit hiv replication with its potential dna-editing activity [ ] . hpv-infected cells release evs that make other cells more susceptible to infection as they deliver proteins that affect viral expression, and subsequently tumor development [ , , ] . to enhance protein delivery and hpv replication, hpv-infected cells hijack ev signaling pathways to control the quantitative and qualitative release of evs from hpv-infected cells [ , [ ] [ ] [ ] . as tumor genes and proteins are persistently expressed from evs, this contributes to hpv cancer cell growth [ ] , thereby making the signaling pathways of evs harmful to the host. the oxidative stress released from hpv-infected cells into evs should also be considered detrimental to the host as this stress has the potential to induce viral replication of other viruses such as hiv- [ ] . to make matters more complex, the signaling pathways of evs are not limited to increased hpv replication as the release of evs can also promote an adaptive immune response that becomes beneficial to the host [ ] . for example, in the setting of hpv replication and tumor progression, evs have prompted immune activation in head and neck cancers and are being considered as biomarkers for improved clinical outcomes [ ] [ ] [ ] [ ] . besides, endogenously engineered evs are being considered as a novel method to deliver anti-hpv immunotherapy [ ] , thus making them yet another way to improve clinical outcomes. unique mirna signatures were found in evs released from cervical cancer affected cells that were associated with hpv status [ ] [ ] [ ] [ ] ] . during influenza virus infection, evs carrying host mirna or viral epitopes are thought to be integral to antigen transfer, reducing virus spread, and immune regulation [ ] . for example, influenza virus hemagglutinin (ha) epitopes enclosed within exosomes on mhcii molecules have been shown to improve the efficiency of antigen delivery to immune cells [ ] . further, exosomal-like vesicles carrying mucin molecules such as muc , muc , and muc can bind sialic acids and neutralize influenza viruses [ ] , which may help reduce virus dissemination. virus replication can also be blocked by some highly upregulated exosomal mirnas, such as the type i interferon-inducing hsa-mir- and mir- - p [ , ] . also, these evs excite other proinflammatory cytokines, such as il- , tnf-α, and ifn-β [ , ] , although their efficacy may be dependent on cell source, maturity, and mhc molecules. macrophages have been shown to produce thousands of proteins within exosomal vesicles in response to influenza infection. these evs included a variety of host factors, including cytokines and proteins involved in copper metabolism and autophagy [ ] . interestingly, proinflammatory cytokines from macrophages and dendritic cells were suppressed by vaccine-induced evs (e.g., mir- a, mir- , or mir- ) [ ] . although much of the current work has focused on single influenza virus strains, important strain specific ev dynamics have begun to be identified. in one study, nearly half of exosomal mirnas were conserved between h n and h n infection in a cells [ ] . of the differentially expressed evs, they were > -fold during infection with the highly pathogenic h n than with uninfected samples. a better understanding of these dynamics and temporal-and strain-specific differences could provide important insight into pathogenicity and pinpoint new therapeutic and universal influenza vaccine targets. hcv belongs to a family of human virus called flaviviridae characterized by positive-sense single-stranded rna that encodes precursor polyprotein that is cleaved into three structural proteins comprising of core protein p with envelope glycoprotein e & e , and seven non-structural proteins that play a role in viral pathogenesis [ , ] . the chronic viral infection leads to hepatic inflammation that is associated with increased production of pro-inflammatory cytokines and chemokines from liver residential immune cells and immune cells recruited to the liver [ ] . evs are observed as major modifiers of cellular crosstalk between hcv-infected hepatocytes & immune cells [ ] . in hcv pathogenesis evs act as a double edge power by: ( ) delivering vireo-independent hcv rna and ( ) obtaining antiviral immune responses [ ] . the cellular vesicular pathway is exploited by hcv to congregate and release viral particles. this happens by releasing vesicles containing envelope glycoprotein e & e , entire hcv genome & viral particles. when the vesicles containing these components enter the target cells, this helps to establish infection [ ] . in systemic alteration of an immune response, major regulators commonly known as specifically enriched micro rnas (mirnas) are delivered by evs. these are loaded into evs and are involved in post-transcriptional regulation of gene expression, which is known to be influential for hcv replication [ , ] . this confirms that evs have peculiar mirna expression isolated from the sera of chronic hcv patients. exosomes derived from hcv infected cells are responsible for developing infection to other uninfected cells. these exosomes carried viral rna in complex with mir- , ago , and hsp that support virus replication [ ] . evs, isolated from sera of patients with acute or chronic hcv or interferon-stimulated macrophage cultures, mediate inhibitory effects on hcv replication [ ] . in co-culture models, the immunoregulatory effects of evs were assessed on the replication of hcv. stimulation with type i & ii interferon n, which is a fast but short-lasting ev-derived antiviral, leads to the production of macrophages by secreting various cytokines resulting in innate immunity. thus, hcv replication in macrophages derives ev-mediated long-lasting inhibitory effects [ ] . evs released by hcv infected cells contain viral rna that might trigger plasmacytoid dendritic cells to produce ifnα [ ] . the emergence of the life-threatening "atypical pneumonia" caused by severe acute respiratory syndrome coronavirus (sars-cov) in the early st century has led to renewed interest in coronaviruses [ ] . coronaviruses belong to the family of rna viruses and possess the largest genome among them. similar to other viruses, their genome contains essential genes encoded for open reading frames a and b (orf ab), and viral structural proteins, which are required for virus replication, transcription, and virus assembly [ ] . a newly emerged coronavirus disease in (covid- ) is caused by a novel severe acute respiratory syndrome coronavirus- (sars-cov- ). sars-cov- infection spread within a few months after the first outbreak reported in december in china, which later became a worldwide crisis. with high morbidity, the disease is often characterized by an atypical severe pulmonary pneumonia [ , ] . the novel sars-cov- is closely related to sars-cov- coronavirus responsible for the sars outbreak that emerged in late in china. its subsequent worldwide spread had caused cases and deaths by july [ ] . sars-cov- infections, which has already infected > million people and caused the death of~ , people world-wide, are presently occurring and represent an ongoing threat to public health. out of cases in china reported having at least one comorbidity [ ] . the risk of serious adverse outcomes of covid- is especially pronounced in patients with comorbidities such as hypertension, diabetes, kidney, and cardiovascular diseases [ , ] . sars-cov encodes four structural proteins; spike glycoprotein (s), nucleocapsid protein (n), membrane protein (m) & small envelope glycoprotein (e) & several nonstructural proteins of unknown functions [ ] . sars-cov- spike (s) glycoprotein interacts with angiotensin-converting enzyme (ace- ), the same receptor used by sars-cov to enter the target cells, in particular lung alveolar epithelial cells [ ] . it has been demonstrated that evs released by sars-cov- infected lung epithelial cells contain viral rna fragments that were subsequently detected in the cardiomyocytes, suggesting viral rna transmission via evs [ ] . sars-cov- is a positive-stranded rna virus in an envelope with a genome of , nucleotides [ ] . the spike protein s of sars-cov- (sars-s) facilitates the viral fusion that can be triggered following the fusion-mediated conformational changes in the target cell receptor that mediates the entry of the virus into the target cells. once inside the cell, a virus may utilize the exosome secretion pathway to enhance its pathogenesis and viral spread [ ] . to find a vaccine against sars-cov- , researchers performed exosome-based research, where they constructed chimeric s protein of the sars by replacing cytoplasmic and transmembrane domains of sars-s with g protein of the vesicular stomatitis virus. this chimeric s-protein was readily expressed on the cell surface, allowed entry of pseudotyped retroviral vectors, and was incorporated into exosomes. subsequently, chimeric s protein-containing exosomes have been tested as a novel protein for vaccine immunogenicity against sars-cov in mouse models [ ] . recently, preclinical studies have uncovered a therapeutic role of msc-derived secretome or evs in lung regeneration [ ] , which could offer a new therapeutic approach in treating severe covid- infection [ , ] . intravenous transplantation of ace -negative mesenchymal stem cells (mscs) promoted recovery of patients from severe covid- [ ] , thus supporting the hypothesis that binding of sars-s protein through ace expressed on msc-derived small evs could limit the viral infection through competitively inhibit the binding of sars-s to ace expressed on alveolar type ii cells [ ] . epstein-barr virus (ebv) is one of the herpes viruses that hijack its host evs. ebv infected cells release evs that contain ebv-coding/non-coding mirnas and transfer it to uninfected cells including b lymphocytes and epithelial cells [ , ] . the transfer of ebv-coding mirnas to b lymphocytes, especially the akata-lymphoblastoid cell lines-derived evs, causes inflammatory responses of monocytes/macrophages and induces severe lymphoproliferative disease (lpd) [ ] . ebv viral reactivation was recently detected in co-cultured latently ebv-infected bl cells in response to the transfer of evs that contain epithelium-specific mirnas from oropharyngeal epithelial cells [ ] . ebv-infected cells can transfer non-coding rnas such as bart and bhrf mirnas via evs to the target cells. upon entry, mirnas can be directed to cellular sites of mirna-mediated gene repression, causing repression of their target genes cxcl and lmp [ ] . ebv-infected nasopharyngeal carcinoma cells release evs containing galectin- protein that interacts with the tim membrane receptor and induces apoptosis in t cells [ ] . similarly, exosomes released by these cells convey the viral protein latent membrane protein (lmp ) that provoke intrinsic t-cell inhibitory activity and thus modulate immune response mechanisms [ ] . herpes simplex virus (hsv- ) is another herpes virus that hijacked its host evs. hsv- -infected cells release evs with different components based on their stage in the infection cycle [ ] . early in the lytic cycle, hsv- proteins cause remodeling to evs' cargos, which in turn cause virion egress from infected cells to uninfected cells [ ] . hsv- evs contain coding and non-coding rnas and more importantly immune components, such as the stimulator of interferon genes (sting) [ ] . a recent study demonstrated that sting-containing evs play an important role in inhibiting viral replication during the lytic cycle, as well as inhibiting viral gene expression during the latent stage [ ] . another recent report illustrated that mir-h and mir-h are being expressed late in the virus infection cycle and transferred to uninfected cells via evs [ ] ; mirna- induces the formation of gamma interferon (ifn-γ) which blocks viral replication in uninfected cells but not in infected cells [ ] . ifn-γ loaded evs maximize viral transmission between individuals by diminishing the spread from infected cells to uninfected cells [ ] . a study reported that hsv- encoded glycoprotein b (gb) modulates the immune response by manipulating the mhc class ii processing pathway by diverting human leukocyte antigen-dr (hla-dr) molecules into the exosome pathway [ ] . an ev vaccine for the hepatitis b virus (hbv) is currently under investigation. as in most of the viruses, evs carry hbv viral proteins such as large s, core and p proteins which participate in viral replication [ ] . they also play many roles in hbv infection; they are responsible for hbv replication, innate immune response during infection, a biomarker for its diagnosis, and development of a possible vaccine [ , ] . a recent study elucidated that unmodified evs can be attractive coadjutants to hepatitis b recombinant antigen (hbsag), because it triggers the healthy mice immune response due to an increased ifn-γ concentration and accelerates the production of igg antibodies [ ] . hepg . . cells with integrated hbv genome release evs containing hbv-mir- which represses viral protein production and hbv replication [ ] . moreover, the study elucidated that engineered evs that are loaded with exosome-anchoring protein nef mutant (nefmut) and hbv core protein can induce cytotoxic t lymphocyte (ctl) immunization in animals for hbv infection [ ] . on the one hand, evs are responsible for infection transfer from one cell to another. on the other hand, evs are also responsible for antiviral response initiation by inducing the uninfected cells' immune response [ , ] . due to their abilities to activate the innate and adaptive immune response, evs can be the future pathway for the treatment of many viral infections. so far, viruses that impair their host immune response such as human t-lymphotropic virus (htlv- ) only use their host's evs to use viral proteins such as gp , tax, and hbz to increase cell-to-cell contact and promote a potential increase in viral infection [ ] . htlv- evs were found to contain a protein called tax that is implicated with the dysregulation of the recipient cells' immune response [ , ] . interestingly, there are viruses that not only hijack host evs, but also boost the production of evs such as in zika virus (zikv). evs released from zikv-infected (c / ) cells carry viral rna and zikv-e protein that can trigger monocyte activation to induce mrna expression of tnf-α [ ] . zikv-infected cells have incrementation in their neutral sphingomyelinase (nsmase)- /smpd gene expression and activity, which provokes the production and excretion of evs in neurons. treatment of zikv requires the hindrance of ev production through the inhibition of smpd s in neurons to prevent further neuronal death and virus spreading [ ] . with the introduction of antiretroviral therapy (art), the morbidity and mortality associated with hiv infection have drastically reduced [ ] . however, due to the presence of latent reservoirs and inadequate drug concentration in the central nervous system (cns), the virus continues to replicate and causes a wide range of cns pathologies, including hiv-associated neurocognitive disorders (hand) [ ] . therefore, new drug delivery systems that facilitate drug passage across the bbb to effectively suppress the virus in cns, with minimal/tolerable neurotoxicity need to be developed. evs can be used as a potential drug delivery system as they can cross the bbb [ , ] with less immunogenicity. further, in preclinical studies, ev-based drug delivery platforms have been shown to carry therapeutic small molecules across the bbb to help alleviate multiple cns diseases, including parkinson's disease and brain cancer [ ] [ ] [ ] . evs that can be used as a drug delivery platform are mainly derived from exosomes that linked to an endolysosomal pathway. exosomes released from dendritic cells are considered vaccine candidates for immunotherapy in diseases such as cancer. these exosomes can be further taken up by dendritic cells leading to a presentation of mhc-i or peptide complexes [ ] [ ] [ ] . arvs can be loaded into evs to deliver them across the bbb to achieve viral suppression in the cns [ ] . since the autoclaved exosomes show intrinsic stability at a physiological temperature [ ] , sterile drug-loaded evs can be formulated. large scale production of ev drug formulation can be achieved using an endogenous drug-loading method that uses cells to release evs with target drugs encapsulated in vitro. evs with encapsulated drugs are capable of targeting the diseased cell or tissue, with targeting characteristics [ ] . this inherent feature could be used to deliver drugs selectively to their intended targets while abrogating off-target side effects. virus-targeting antiviral drugs can include protease inhibitors (pis), integrase inhibitors, nucleoside and nucleotide reverse transcriptase inhibitors, and nonnucleoside reverse-transcriptase inhibitors. an ev-based drug delivery platform with either hiv pis alone or in combination with ritonavir is used as a pharmaco-enhancer or second line of therapy for the treatment of hiv [ ] . evs can also be used as a vehicle for delivery of crispr-associated endonuclease (cas ) and potentially as the guide rna (grna) to target nucleotide sequences within viral genomes [ , ] . another therapeutic use of evs is vaccination against infectious diseases and viral infection. ev-mediated delivery of mrna encoding pathogenic proteins required for viral infection might be a vaccine candidate that can induce t helper (th )-type immune responses and cell-mediated immunity, without the need to attenuate and inactivate pathogenic viruses or bacteria [ , ] . for the ongoing pandemic of covid- , anti-hiv pis, other pis, or other antiviral and antibacterial drugs can either be encapsulated in evs derived from various cell lines using endogenous loading technique or from the plasma of patients using exogenous loading method for personalized therapy [ ] . repurposing fda-approved antiviral drugs using evs could be a fast way to get tested through clinical trials. although the clinical research done on evs seem promising for therapeutic application, several factors must be considered before translating evs into clinics. at present, available ev isolation methods, such as ultracentrifugation, density gradient centrifugation, precipitation, size exclusion chromatography, affinity, and novel microfluidic techniques are not sensitive enough to distinguish evs subpopulation due to lack of specificity, physical and chemical biomarkers [ ] ; therefore, a high level of standardization is required to compare ev protocols and results used across different laboratories before the adoption of ev therapy to various clinical applications. also, evs' pharmacokinetics, half-life, and plasma stability, as well as the interaction of encapsulated drugs with ev components, ev-targeting, and immune clearance of evs, are other limitations that need to be overcome before realizing the clinical applications of evs in drug delivery. a growing body of evidence suggests that virus-infected cells produce evs, encapsulated with viral proteins and parts of viral genetic material, and in some cases they carry the full infectious viral genome that facilitates viral infection and mediates immune responses ( figure ) . notably, evs can enhance viral infection by: ( ) mediating transfer of chemokine co-receptors or cell surface proteins to null-target cells that do not express endogenous viral co-receptors; ( ) helping viruses to evade the host immune system; ( ) transferring of viral components (viral proteins and rnas) to recipient cells, which induce cytotoxic effects on infected cells, leading to progressive loss of immune cells resulting from the apoptosis of uninfected bystander cells. here, we aimed to shed light on how evs potentially impact infection and the pathogenesis of various viruses. we also evaluated the potential utilization of evs in antiviral and antiretroviral therapy, and in drug delivery. characterizing evs from virus-infected cells and their functional analyses could aid not only in the understanding of the mechanisms of viral infection but also in the utilization of evs as a delivery system for therapeutic agents. overcome before realizing the clinical applications of evs in drug delivery. a growing body of evidence suggests that virus-infected cells produce evs, encapsulated with viral proteins and parts of viral genetic material, and in some cases they carry the full infectious viral genome that facilitates viral infection and mediates immune responses ( figure ) . notably, evs can enhance viral infection by: ( ) mediating transfer of chemokine co-receptors or cell surface proteins to null-target cells that do not express endogenous viral co-receptors; ( ) helping viruses to evade the host immune system; ( ) transferring of viral components (viral proteins and rnas) to recipient cells, which induce cytotoxic effects on infected cells, leading to progressive loss of immune cells resulting from the apoptosis of uninfected bystander cells. here, we aimed to shed light on how evs potentially impact infection and the pathogenesis of various viruses. we also evaluated the potential utilization of evs in antiviral and antiretroviral therapy, and in drug delivery. characterizing evs from virusinfected cells and their functional analyses could aid not only in the understanding of the mechanisms of viral infection but also in the utilization of evs as a 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delivery by extracellular vesicles in mammalian cells and its applications the crispr/cas genome editing methodology as a weapon against human viruses therapeutic applications of extracellular vesicles: clinical promise and open questions extracellular vesicles move toward use in clinical laboratories this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors are grateful to kelli anne gerth (ph.d. student; university of tennessee health science center) for critical reading and editing of the manuscript. the authors declare no conflict of interest.acknowledgments: the authors are grateful to kelli anne gerth (ph.d. student; university of tennessee health science center) for critical reading and editing of the manuscript. the authors declare no conflict of interest. key: cord- -lbr q qh authors: chinchar, v. gregory; yu, kwang h.; jancovich, james k. title: the molecular biology of frog virus and other iridoviruses infecting cold-blooded vertebrates date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: lbr q qh frog virus (fv ) is the best characterized member of the family iridoviridae. fv study has provided insights into the replication of other family members, and has served as a model of viral transcription, genome replication, and virus-mediated host-shutoff. although the broad outlines of fv replication have been elucidated, the precise roles of most viral proteins remain unknown. current studies using knock down (kd) mediated by antisense morpholino oligonucleotides (asmo) and small, interfering rnas (sirna), knock out (ko) following replacement of the targeted gene with a selectable marker by homologous recombination, ectopic viral gene expression, and recombinant viral proteins have enabled researchers to systematically ascertain replicative- and virulence-related gene functions. in addition, the application of molecular tools to ecological studies is providing novel ways for field biologists to identify potential pathogens, quantify infections, and trace the evolution of ecologically important viral species. in this review, we summarize current studies using not only fv , but also other iridoviruses infecting ectotherms. as described below, general principles ascertained using fv served as a model for the family, and studies utilizing other ranaviruses and megalocytiviruses have confirmed and extended our understanding of iridovirus replication. collectively, these and future efforts will elucidate molecular events in viral replication, intrinsic and extrinsic factors that contribute to disease outbreaks, and the role of the host immune system in protection from disease. : sizes and coding potentials of iridovirid genomes. iridovirus iiv- , gq iiv- aside from differences in virion and genome sizes, the organization of virus particles within the family is generally similar. while transmission electron microscopy (tem) has been conducted using fv and other iridovirids, detailed cryo-electron microscopic (cryoem) analyses (discussed below) were performed with iiv- and provide the most detailed view of virion structure. virions exist in two forms: non-enveloped particles ~ - nm in diameter, and enveloped particles that acquire an envelope by budding from the plasma membrane. in addition to the external envelope, some iridovirids contain a fringe of fine fibers (fibrils) that extend outward from the capsid subunits [ , ] . non-enveloped particles are composed of three distinct layers: an outer capsid composed of multiple copies of an ~ kda major capsid protein (mcp), an internal lipid membrane possibly derived from the endoplasmic reticulum (er) or other cellular membranes, and an inner core containing the viral genome and associated virus-encoded proteins [ , ] . the mcp shows marked sequence conservation within all members of the family [ ] . sequence identity within the mcp gene allows primer-based pcr amplification of viral dna and provides an easy way to determine whether a given virus is a member of the family and to which genera it belongs [ ] . although the mcp is the primary structural protein and comprises % of the total virion protein content, an additional proteins have been identified following gel analysis of purified virions [ ] . while many of these proteins likely represent virus-encoded catalytic proteins that play various roles in replication, three minor capsid proteins have been identified by cryoem analysis of iiv- and designated as finger, zip, and anchor proteins [ ] . finger and zip proteins were suggested to stabilize the virus by acting as intercapsomer cross-links, whereas the anchor proteins appear to be transmembrane proteins that extend into the internal lipid membrane and provide further stabilization. in addition to these proteins, a fifth protein, a putative myristoylated protein (fv orf r), has been suggested to interact with cellular membrane fragments and play a role in virion assembly [ ] . as shown in table , iridovirid genomes range in size from - kbp. ranavirus genomes occupy the low end of this range and fall into three groups: the genomes of fv , atv, tfv, and stiv are - kbp, ehnv is kbp, whereas sgiv and giv, likely isolates of the same viral species, are kbp in size. consistent with their sizes, ranaviruses contain between and close-packed, predominantly non-overlapping orfs of amino acids or greater [ , [ ] [ ] [ ] . repetitive regions are common and may explain the high rate of recombination seen within these viruses. in addition, a small number of putative micrornas of unknown function have been detected [ ] . amino acid conservation is marked among iridovirid genes, but gene order, even within members of the same genus, is not conserved suggesting that expression is likely determined by individual promoter elements closely associated with each gene. moreover, although viral genes are expressed in an ordered temporal cascade (consisting of immediate early (ie), delayed early (de), and late (l) genes) nucleotide sequences corresponding to ie, de, and l promoters have not yet been identified. the observation that gene order is not conserved supports the view that genes can be reshuffled through recombination without adversely affecting expression. microarray analysis suggests that fv contains ie, de, and l genes [ ] . moreover, the classes assigned in that study were generally similar to those assigned to known sgiv genes in two earlier studies [ , ] . although all ranaviruses contain a core set of genes [ ] , unique genes are found within specific viral species. genes held in common (e.g., viral dna polymerase, mcp, the large and small subunits of the viral rna transcriptase, etc.) are thought to be those required for replication in all cell types, whereas those unique to a given viral species may represent specific host adaptations that contribute to virulence, host range, and immune evasion. early studies of iridovirid replication were conducted almost exclusively using fv , whereas recent studies have included additional ranaviruses such as atv and sgiv and several megalocytivirus isolates that have been linked to massive die-offs in mariculture facilities in japan and south-east asia. below we summarize events in fv -infected cells and highlight recent work that has appeared since the last comprehensive reviews [ , , , ] . fv virions, and presumably virions of all other iridoviruses, exist in two forms: non-enveloped (i.e., naked) particles and enveloped virions. although both forms are infectious, enveloped virions were shown to have higher specific infectivity [ , ] . with regard to entry, enveloped particles likely enter cells by receptor-mediated endocytosis in a ph-dependent manner and require clathrin-coated pits, whereas naked particles enter by fusion at the plasma membrane [ , ] . recently this model has been questioned by guo and co-workers who argued that tiger frog virus (tfv), a ranavirus nearly identical to fv , enters cells by an atypical, ph-dependent, caveola-mediated endocytic pathway [ ] . their conclusion was based on experiments using chlorpromazine and over-expression of a dominant-negative form of esp that inhibited assembly of clathrin-coated pits, but did not affect entry. consistent with a role for caveolae, endocytosis of tfv was dependent on membrane cholesterol and was blocked by caveolin- scaffolding domain protein. given that fv and tfv are nearly identical in nucleotide sequence [ , ] , it is surprising that their modes of entry are so different. because the reported differences in entry mechanisms between fv and tfv may be due to infection of cells from different non-physiological hosts, e.g., rats and hamsters, resolution of this issue will require direct comparison of tfv and fv entry using the above approaches coupled with tem. regardless of whether uncoating takes place at the plasma or nuclear membranes, the viral genome enters the nucleus. unlike herpesviruses, iridovirid genomes are not infectious, indicating that virion-associated proteins are required to initiate viral gene transcription [ ] . accordingly, immediate-early (ie) and delayed early (de) viral transcripts are synthesized in reactions mediated by putative virion-associated (for ie transcripts) and virus-encoded (for de mrna) transcriptional trans-activators and, at least for ie transcription, host rna polymerase ii [ ] [ ] [ ] . among the products of early transcription is a viral dna polymerase which catalyzes the synthesis of unit-sized copies of the viral genome within the nucleus [ ] . additional ie and de proteins include proteins that may play roles in blocking host immune responses such as a virus-encoded, card (caspase activation and recruitment domain) motif-containing protein (vcard), β-hydroxysteroid dehydrogenase (βhsd), and a rnase iii-like protein, catalytic proteins involved in nucleic acid synthesis (proliferating cell nuclear antigen [pcna], dna methyltransferase [dmtase], the large and small subunits of the viral homolog of cellular rna polymerase ii [vpol-iiα and -iiβ], transcription factor iis), catalytic proteins that may act to increase dttp pool sizes and influence host range (deoxyuridine triphosphatase [dutpase], deoxynucleotide kinase, the large and small subunits of ribonucleotide reductase), and proteins non-essential for replication in vitro, but needed for growth in vivo (the k protein) [ ] . following its synthesis, unit-sized viral dna is transported into the cytoplasm where it is methylated by a virus-encoded dmtase and, following a second round of dna synthesis, converted into large concatameric molecules that are thought to be the substrate from which viral genomes are derived [ ] [ ] [ ] . virion formation takes place in electron-lucent, morphologically-distinct vas. vas contain viral dna and the structural and non-structural proteins that give rise to virions, but are devoid of ribosomes, mitochondria, and other cellular organelles [ , ] . although little is known about the precise process of virion morphogenesis, by analogy to african swine fever virus and by study of various iridovirids, it appears that host membranes, perhaps derived from the er, serve as a scaffold to which capsid and shell proteins bind [ , ] . as progressively larger amounts of viral proteins bind the membrane scaffold, crescent-shaped structures that resemble icosahedral vertices are formed. ultimately both full and empty icosahedral virus-like particles are detected. however, it is unclear precisely how the genome is encapsidated. while packaging via a headful mechanism explains the circularly-permuted terminal redundancy detected within all iridovirids [ ] , it is not known whether nearly complete virions bind viral dna and internalize it through a virion portal as seen in some viral systems [ ] [ ] [ ] , or whether developing crescents/icosahedrons engulf the viral genome as they assemble. following their formation, virions remain within vas, accumulate within cytoplasmic paracrystalline arrays, or bud from the plasma membrane. at late times after infection, virions are sometimes seen within the nucleus and elongated tubular structures can be detected in vas, but these are likely artifacts that reflect breakdown of the nuclear membrane and the disruption of the assembly process due to the shortage of key structural proteins and the dysregulation of cellular and viral macromolecular synthesis. a transmission electron micrograph of an fv -infected fathead minnow cell is shown in figure a , and an enlargement of the vas, displaying full and empty virions, is shown in figure b . the identities of virus-encoded proteins involved in virion assembly remain to be determined. twelve complementation groups defective in the ability to synthesize infectious virions have been identified through study of temperature-sensitive (ts) mutants (see below), but it is unclear whether the gene products encoded by these complementation groups represent scaffold proteins that are required for virion assembly, but which are not incorporated into mature particles, authentic viral structural proteins, or virion-associated accessory proteins required for viral replication [ ] . aside from the mcp, only fv orf r, which encodes a putative myristoylated membrane protein, has been linked to virion assembly. knock down studies using either asmos or artificial micrornas [ , ] demonstrated that r was required for virion assembly, whereas in vitro studies showed r was associated with virus factories and the virion membrane [ ] . by analogy to asfv, r may play a role in recruiting er-derived membranes into virus factories where they serve as precursors of the inner viral lipid membrane and/or act as a scaffold protein. as with other nuclear and cytoplasmic, large dna viruses (ncldv) late viral gene expression is dependent upon full viral dna synthesis, and is catalyzed by a virus-encoded or virus-modified transcriptase [ ] . temperature-sensitive mutants defective in viral dna synthesis show markedly reduced levels of late gene expression as do cells treated with drugs (phosphonoacetic acid [paa], cytosine arabinoside [arac]) that block dna synthesis [ , ] . fv and other iridovirids encode homologs of the two largest subunits of rna polymerase ii and likely use these proteins, and perhaps others, to catalyze late viral gene expression [ ] . knock down of the expression of the largest subunit of the viral homolog of rna polymerase ii (i.e., vpol-iiα) results in a marked reduction in the synthesis of late viral proteins and a corresponding reduction in virion formation and supports the notion that vpol-iiα, and other viral proteins, are responsible for late viral gene expression [ ] . aside from the mcp, other late proteins tentatively involved in dna packaging and virion assembly include orf r, a putative packaging protein, and evr /alr, a protein involved in the formation of disulfide bonds [ ] . to illustrate the above, a schematic diagram of the viral replication cycle is shown in figure . [ ] . used with permission. as with other viruses, fv infection markedly inhibits host macromolecular synthesis and triggers apoptosis [ ] [ ] [ ] [ ] . both phenomena are mediated by heat-or uv-inactivated virus indicating that the inhibitory agent is a virion-associated protein [ , ] . viral infection appears to trigger activation of pkr, a double-stranded rna-activated protein kinase, which leads to the phosphorylation of the largest subunit of eukaryotic initiation factor (eif- α) and the subsequent inhibition of protein synthesis [ ] . viral translation may be spared due to the synthesis of large amounts of highly efficient viral transcripts and the presence of a virus-encoded protein, e.g., a viral homolog of eif- α (vif- α), that acts as a pseudosubstrate and prevents phosphorylation/inactivation of eif- α [ , [ ] [ ] [ ] . most ranaviruses, with the exception of fv , contain a full-sized vif- α gene [ , ] . however, because the absence of a full-sized vif- α gene in fv does not adversely affect viral protein synthesis, other viral proteins may also play roles in maintaining viral protein synthesis in infected cells. aside from the aforementioned vif- α protein, fv and other ranaviruses encode a number of putative immune evasion gene products including βhsd, vcard, and an rnase iii-like protein. by analogy to a similar protein in vaccinia virus, βhsd is thought to trigger glucocorticoid synthesis in infected animals and enhance virus replication by suppression of the overall immune response [ , ] . likewise, vcard may inhibit the induction of interferon and/or apoptosis. induction of ifn and/or apoptosis follows interaction of rig-i or mda , cellular proteins that bind dsrna, with downstream effectors such as mavs and isp- [ ] [ ] [ ] [ ] [ ] [ ] [ ] . since rig-i/mda and mavs/isp- interactions are mediated by card motifs, it is possible that a viral card-containing protein might bind either one or both of these proteins and short-circuit the ifn induction pathway [ ] . the rnase iii-like protein is found within all iridoviruses and has been postulated to block sirna-mediated interference, or to play a role in the processing of viral mrnas [ ] . alternatively, viral rnase iii could act like vaccinia virus e l and bind and/or degrade dsrna and block the activation of pkr, the induction of interferon, and the inhibition of protein synthesis [ ] . as with vaccinia virus [ ] [ ] [ ] , iridoviruses that infect ectothermic animals encode a series of proteins whose function is to ensure an adequate supply of nucleotides for viral dna synthesis. these enzymes include the two subunits of ribonucleotide reductase [ , ] , thymidine kinase [ ] , thymidylate synthase, dutpase [ ] , purine nucleoside phosphorylase (pnp), and a dihydrofolate reductase homolog [ , ] . not all of these putative enzymes are found within each viral species, and it is postulated that specific enzymes might be needed in certain hosts. for example, pnp is found only within giv and it has been suggested that because giv's natural host, the grouper, might not be able to supply the purine nucleotides required for viral replication the virus encodes its own pnp [ ] . currently there are seven completely-sequenced ranavirus genomes ranging in size from - kbp (table ) . whole genome dot plot analyses show that there are three distinct genomic organizational profiles among ranaviruses that are based on the conservation of gene order [ ] : fv -like viruses (i.e. fv , tfv and stiv), atv-like viruses (i.e. atv and ehnv) and giv-like viruses (i.e. sgiv and giv). dot plot comparisons of viruses within each group show complete co-linearity. however, comparison of atv-like ranaviruses with fv -like ranaviruses detected two large inversions, whereas comparing either of these groups to giv-like viruses revealed very little conservation of gene order [ , ] . in the latter case, gene order is so jumbled between fv /atv-like viruses and sgiv/giv-like viruses that any trace of the degree diagonal line, indicative of complete sequence alignment between genomes in dot plots, is lost. interestingly, ranavirus genomic organization is markedly different from that of poxviruses. for example, all members of the subfamily chordopoxvirinae share a conserved genomic core structure wherein gene order, with the single exception of avipoxviruses, is conserved [ ] . however, despite the conservation of gene order, global sequence alignments show that sequence identity among members of the chordopoxvirinae is only - % [ , ] . ranaviruses, on the other hand, share a much greater sequence identity (~ - % within the mcp) yet have three widely divergent genomic organizations. moreover, as additional ranavirus genomes are sequenced, we may discover novel genomic morphotypes that will elucidate the evolutionary relatedness of ranaviruses. examination of complete ranavirus genomic sequences, including whole genome dot plot and phylogenetic analyses of ranavirus core genes, suggested that ranaviruses have undergone a number of evolutionarily-recent host shifts [ , ] . phylogenetic analysis using the core iridovirid genes identified by eaton and her co-workers [ ] support whole genome dot plot analysis and indicate that fv -like viruses (fv , tfv, stiv), atv-like viruses (atv and ehnv), and giv-like viruses (sgiv and giv) cluster on separate branches of the ranavirus tree ( figure ). collectively, these data suggest that for the greater part of their evolutionary history, ranaviruses were restricted to infecting fish. in addition, the shallow branch lengths of fv -like and atv-like viruses, which infect amphibians and fish, suggest that this group of viruses has recently, at least in evolutionary terms, radiated to infect a wide-range of poikilothermic vertebrates [ ] . this hypothesis is supported by studies showing that ranaviruses are multi-host pathogens [ ] . as additional ranaviruses are sequenced, particularly those isolates that infect multiple host species, a better understanding of ranavirus evolution, host shifts, and the molecular determinants of ranavirus host range and pathogenesis will be achieved. pathological and immunological aspects of infection by fv and various iridovirids will be dealt with by chen and robert [ ] and miller et al. [ ] in this special issue of viruses. thus, only a brief overview of this topic will be provided here. although iridovirids, in contrast to the chytrid fungus batrachochytrium dendrobatidis (bd), have not been reported to drive species extinction, fv -like viruses have caused deaths in several amphibian culture facilities and in nature, and megalocytiviruses have triggered wide-spread mortality in mariculture operations in south-east asia [ , , [ ] [ ] [ ] [ ] . ranavirus infections range from inapparent to fulminant and involve a variety of tissues including the skin, kidney, liver, and intestine [ ] . using xenopus laevis as a model host, robert and his colleagues have shown that fv infection occurs in both larval and adult animals [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, whereas infections of immunocompetent adult frogs are confined to the kidney and cleared within a few weeks, infection of tadpoles or immunocompromised adults results in widespread infection that spreads to multiple internal organs and often leads to death. protection from fv infection appears to involve both humoral and cell-mediated immunity and both antiviral antibodies and cytotoxic t cells have been identified as protective. vaccination may prove useful in protecting susceptible species and vaccines have been developed to protect commercially important fish from megalocytivirus infection [ , ] . likewise, infection of bullfrog tadpoles (rana catesbeiana) with less pathogenic fv protected them against death following infection with the more pathogenic rana catesbeiana virus z [ ] . as with the above section, the ecology of ranavirus infections will be more fully discussed by miller and co-workers in their contribution to this issue [ ] . suffice it to say iridovirus infections have a marked effect on lower vertebrates. as green and co-workers observed most mortality events seen among amphibians from - were attributable to iridovirus infections [ ] . in addition, whereas lymphocystivirus and megalocytivirus infections have, to date, only been detected in fish species, ranavirus infections affect three classes of ectothermic vertebrates: bony fish, amphibians, and reptiles [ ] . results from experimental challenges as well as natural infections suggest that ranaviruses isolated from one species can not only infect different host species within the same genus, but also host species from different genera, orders, and classes. for example, amphibian ranaviruses such as bohle iridovirus, fv , and atv infect a variety of fish and amphibian species, and at least one insect virus appears to also infect reptiles [ , , , - ]. early studies with fv attempted to elucidate viral replicative events and the function of viral gene products using a variety of inhibitors, e.g., cycloheximide to confine viral gene expression to ie transcription, α-amanitin to block host rna polymerase ii activity, phosphonacetic acid to block viral dna synthesis, azacytidine to block the methylation of viral dna, flurophenylalanine to block the expression of late viral proteins, elevated temperatures ( °c) to confine viral gene expression to early transcripts and proteins, etc. (reviewed in [ , ] ). in addition, panels of ts mutants were generated and subsequently characterized [ , , ] . ts mutants proved useful in confirming and identifying various aspects of the virus life cycle, e.g., the two-stages of viral dna synthesis [ ] . ultimately, ts mutants were organized into complementation groups comprising three classes [ ] . class i mutants ( complementation groups) appeared to contain mutants with defects in virus assembly. these mutants synthesized early and late viral proteins and viral dna, but did not generate infectious virions [ ] . recently, tem analysis suggested that this class could be further sub-divided into at least two subclasses, those that failed to assemble icosahedral particles and those that formed ostensibly normal, but non-infectious, icosahedral particles at the non-permissive temperature [ ] . the remaining complementation groups appeared to contain defects in proteins associated with viral transcription (class ii) and viral dna synthesis (class iii). consistent with an early study suggesting that only early viral proteins were required for assembly site formation [ ] , tem analysis suggested that full viral dna synthesis was not required for the formation of viral assembly sites as two ts mutants that synthesized markedly reduced levels of viral dna were able to form large vas, that were devoid of viral dna, most viral proteins, and viral particles [ , ] . however, although ts mutants have contributed to our understanding of fv replication, it has not been possible to associate a given mutant phenotype with a specific viral orf, and thus link a specific viral protein with its function. to address this issue, we have recently developed approaches that target individual viral genes and have used them to determine viral gene function by observing changes in phenotype. to link specific viral proteins with their functions, viral gene expression was knocked down (kd) using antisense morpholino oligonucleotides (asmos) and gene function was inferred by changes in phenotype. asmos are dna-like macromolecules, optimally nucleotides in length, whose backbone contains morpholine rings instead of deoxyribose rings and non-ionic, phosphorodiamidate bonds in place of phosphodiester links [ ] . moreover, because phosphorodiamidate bonds are resistant to degradation by cellular nucleases, asmos are remarkably stable in cell culture [ ] . furthermore, in contrast to sirnas that sometimes trigger off-targeting effects by activating pattern recognition receptors such as toll-like receptor (tlr- ), asmos are not recognized by cellular dna receptors such as tlr- or aim- and thus do not induce innate immune responses. asmos bind complimentary sequences within the ' non-translated region, or the region immediately surrounding the aug initiation codon, of the targeted mrna and are thought to block scanning of the s ribosome by steric hindrance. in addition, asmos also block gene expression by inhibiting splicing or preventing interaction of regulatory proteins with their specific target sequence [ ] . collectively asmos have been used to block cellular and viral gene expression both in vitro and in vivo [ ] [ ] [ ] . asmos have been used to ascertain the function of several fv genes including those encoding the mcp, vpol-iiα, an kda immediate-early viral gene product ( k), a putative myristoylated membrane protein ( r), and two ie proteins of unknown function: a kda protein designated r, and kda protein termed k [ , , , ] . in an initial proof-of-concept study, sample et al. [ ] showed that asmo treatment knocked down mcp expression by greater than % and resulted in a corresponding drop in the production of infectious virions without any collateral adverse effects on the expression of other viral gene products. moreover, mcp knock down was accompanied by the appearance of atypical elements within vas suggesting that in the absence of full mcp expression aberrant viral structures, perhaps representing altered products of virion assembly, were generated. in the same study, kd of vpol-iiα was shown to result in a marked reduction in the synthesis of late viral proteins. this result provided the first formal evidence that viral homologs of host rna polymerase ii played a role in the synthesis of late viral transcripts. lastly, sample and co-workers observed that kd of the k ie protein had no observable effect on viral gene expression or replication in vitro suggesting that, at least in fathead minnow cells, k was not required for the production of infectious virions. subsequent kd studies of r, a putative virus-encoded myristoylated membrane protein, r, and k confirmed an essential role for each of these proteins in fv biogenesis [ , ] . kd of r resulted in the appearance of putative non-encapsidated viral dna cores within vas and supported the view that r plays a major role in capsid formation. kd of r and k, two ie proteins, did not affect the synthesis of other viral proteins, but resulted in a > % reduction in virion formation. tem study showed that cells treated with asmos targeting either r or k formed viral assembly sites, but failed to form virions or recognizable assembly intermediates. studies targeting the virus-encoded rnase iii-like protein (orf l), a putative ntpase (orf l), the largest subunit of ribonucleotide reductase (orf r), a putative viral membrane protein (orf l), a dna packaging protein (orf r), a putative serine/threonine kinase (orf r), a putative rad -like dna repair protein (orf r), a kda protein of unknown function (orf r), and the viral dmtase (orf r) are ongoing and have demonstrated reductions in viral yields ranging from - % [ ] . however, unlike the first study in which kd of mcp, vpol-iiα, and k was confirmed by d sds-polyacrylamide gel electrophoresis, we have been unable to identify unambiguously the targeted protein in these latest studies, and thus do not know if the partial reductions in virus yields reflect the non-essential nature of the gene product or incomplete kd of the targeted protein. to overcome this problem, recombinant viral proteins will be generated and used to produce polyclonal antibodies for western blot and immunofluorescent analyses. lastly, although asmos can be powerful tools for uncovering viral gene function, their use is potentially limited by the nature of some ranavirus mrnas. because many fv transcripts possess very short, au-rich ' non-translated regions, and because sequences surrounding the aug initiation codon may be unfavorable for targeting due to, for example, high au content or secondary structure, asmo-mediated kd approaches may not succeed with all targeted genes. in these cases, sirna-mediated kd or ko approaches, discussed below, provide alternative approaches. nevertheless, despite these limitations, asmos have provided insight into the roles of several fv genes and will continue to be a useful tool for elucidating viral gene function in vitro. together with anti-viral antibodies to confirm knock down and identify intracellular locations, these studies will broaden our understanding of the complex story of virus-host interactions. as an alternative to asmos, sirnas have also been used in a limited number of cases to knock down iridovirid gene expression [ , , , ] . while knock down has been achieved, we observed that inhibition of fv gene expression only took place if cells were infected at low multiplicities of infection, i.e., < . pfu/cell. similar to the situation in some other viral systems, the inverse relationship between multiplicity of infection and sirna knockdown suggests that a virion-associated or virus-encoded protein may block sirna-mediated knockdown by either binding or degrading sirna [ ] . to date, expression of fv transcripts encoding mcp, dmtase, and vpol-iiα have been knocked down by sirna and resulted in a marked inhibition of replication. a powerful and potentially more useful approach to elucidate viral gene function involves the generation of knock out (ko) mutants since, in contrast to transient kd triggered by asmos or sirnas, the phenotypes of ko mutants can be evaluated both in vitro and in vivo. this feature makes ko mutants especially useful for identifying viral genes that play roles in immune evasion and virulence. ko mutants have been used extensively to elucidate the function of various poxviruses genes [ ] [ ] [ ] , and methodology developed here has been adapted to ranaviruses. however, despite using poxviruses as a guide, ranavirus ko mutants, using homologous recombination to replace the targeted gene with a selectable marker, have only recently been isolated. the reasons for the difficulty in applying this approach to ranaviruses are not clear, but may reflect the presence of a ranavirus-encoded endonuclease that cleaves unmethylated dna [ , ] . if correct, introduction of unmethylated plasmid dna, bearing the selectable marker, into a virus-infected cell may lead to degradation of the majority of plasmid dna and make recombination unlikely. as was done previously in a recombinant biv vector [ , ] , positioning the selectable marker, e.g., the neomycin-resistance gene, downstream of the promoter for the k immediate early (ie) gene drove high levels of expression of the resistance gene and permitted isolation of rare recombinants. the first ranavirus ko mutant targeted the atv-encoded homolog of eukaryotic translational initiation factor α (vif- α) [ ] . as discussed above, following virus infection, host cells activate pkr, an interferon (ifn) inducible double-stranded rna activated protein kinase [ ] , that, in turn, phosphorylates the α subunit of eif- and, as a consequence, inhibits both host and viral protein synthesis. to prevent translational shut-off, several viruses, including most ranaviruses, encode an eif- α homolog that has been proposed to function as a pseudosubstrate for pkr [ ] . in vaccinia virus this homolog is designated k l, whereas in ranaviruses it is termed vif- α. although k l and vif- α differ markedly in size, they share a common sequence motif (vdrvdrekgyvdl) that is likely required for activity. in this scenario, pkr binds k l/vif- α instead of eif- α, and as a consequence eif- α is not phosphorylated and protein synthesis is maintained. to determine if atv vif- α (orf r) is functionally similar to k l, the ranavirus gene was replaced with a selectable marker, the neomycin resistance (neor) gene. to generate an atv ko mutant, neor was inserted downstream of the atv k ie promoter and this construct was bracketed with sequences identical to those in the flanking region of the targeted gene. a pcr product bearing this construct was then transfected into bf- cells and infected with wild-type (wt) atv. recombinant virus was isolated by selective growth in the presence of neomycin, and replication of the ko mutant was compared to wt virus in vitro and in vivo. the ko mutant, designated atv∆ r, replicated to titers comparable to that of wt atv in vitro, but had a small plaque phenotype. in addition, atv∆ r was -fold more sensitive to ifn than wt atv. the increased ifn sensitivity of atv∆ r was correlated with the increased phosphorylation of eif- α and the lack of pkr degradation. in contrast, wt atv degraded pkr and inhibited cellular eif- α phosphorylation. in addition, atv∆ r was less pathogenic than wt atv following infection of tiger salamanders (ambystoma tigrinum) indicating that vif- α was a likely viral virulence/immune evasion protein. thus the atv vif- α gene is a putative ifn-resistance gene that inhibits cellular innate immune responses by degrading pkr and maintaining high levels of viral protein synthesis. in an effort to improve the ko methodology, chen et al. [ ] recently developed a potentially more powerful dual selection method to generate ko mutants within fv . in this system, the gene of interest is replaced, via homologous recombination, with a gene encoding the puromycin-resistance gene fused to the gene for enhanced green fluorescent protein (egfp). in initial experiments ko mutants targeting the truncated vif- α protein (fv -∆vif- α) and the k ie protein (fv -∆ k) were generated as well as a control, "knock in" mutant in which the puromycin-resistance/egfp gene was inserted into a non-coding portion of the genome. while all three recombinant viruses grew well in vitro, the vif- α and k ko mutants, but not the control "knock in" mutant, showed a % drop in virus levels in x. laevis tadpoles. these results confirmed the previous observation that the k protein was not required for replication in vitro [ ] , and indicate that k plays a role, albeit unknown, in replication in vivo. the finding that the truncated vif- α protein of fv was also required for replication in vivo was surprising because the fv homolog of vif- α is missing the n-terminal two-thirds of this protein, including the region homologous to vaccinia virus k l and eukaryotic translational initiation factor α. inspection of the fv nucleotide sequence indicated that fv vif- α is a chimeric protein resulting from deletion and in-frame fusion of a small upstream orf with the larger, downstream vif- α orf. the resulting product contains amino acids from the n-terminus of the upstream orf and the last amino acids of the full-length vif- α protein. the reduced replication of fv -∆vif- α in vivo suggests that the c-terminal end of the fv vif- α homolog is required for full replication in vivo. in addition to the above ko mutants, a recombinant soft-shelled turtle iridovirus has recently been constructed that expresses egfp. egfp-expressing mutants may provide an alternative, simplified strategy for screening antiviral substrates through the visualization and quantification of fluorescent virus [ ] . in contrast to the above studies, a fourth approach for determining viral gene function involves the synthesis of recombinant viral proteins and assessment of their functions in vivo and in vitro. several investigators have used this approach and representative examples are discussed below. a putative homolog of fv icp was identified in sgiv [ ] . icp (mol wt . kda) is an ie gene product that is distributed predominantly within the cytoplasm but is also found within virions. a plasmid expressing icp was introduced into grouper embryonic (gp) and fathead minnow cells and stably transfected cells were characterized. over expression of sgiv icp resulted in higher cell densities and increased growth of monolayer cultures. sgiv replication was compared in gp cells transfected with an empty vector and in gp cells expressing icp . sgiv replicated more rapidly and reached titers that were -fold higher in icp expressing cells than in control cells. although conserved domains suggestive of function were not detected within icp , the authors speculate, based on the above results, that icp might encode a protein involved in cell growth control. the authors suggested that icp , like ie proteins in other viral systems, might control host cell growth by regulating the cell cycle, preventing growth arrest, or delaying apoptosis and play a critical role in promoting a proliferative environment that enhances virus replication. isknv (genus megalocytivirus) orf r encodes a viral protein with marked similarity to vascular endothelial growth factor (vvegf) [ ] . microinjection of a plasmid expressing vvegf into zebrafish one-cell embryos resulted in pericardial edema and dilation of the tail region suggesting that vvegf triggers vascular permeability. moreover, vvegf binds and up-regulates the expression of flk- and together both proteins likely contribute to the proliferative and highly vascularized nature of isknv lesions in its natural host, the chinese mandarin fish. although these results suggest that isknv orf r plays a vital role in host infection and viral replication, its precise function remains unclear. following orf virus (family poxviridae) infection, the vvegf homolog aids in scab formation and wound healing and may enhance transmission since scabs contains substantial amounts of infectious virus. a novel histone-binding protein was identified in sgiv by structural analysis of the orf l gene product [ ] . x-ray diffraction analysis of recombinant l determined the crystal structure at a resolution of . Å, and revealed that l exhibited partial structural resemblance to the histone-binding region of anti-silencing factor (asf ), a histone h /h chaperon. using recombinant l protein, interaction was demonstrated between l and histone h /h complexes and h by isothermal titration calorimetry. the authors suggested that l may be involved in both the regulation and expression of histone h and h methylation, events which allow the virus to control host cell gene expression and facilitate viral replication. sgiv orf r encodes a homolog of the fv k immediate early protein. although kd studies using an asmo targeted against k [ ] and an k knock out mutant [ ] indicated that expression of k was not required for replication in either fhm or bhk cells, replication of the k ko in xenopus laevis tadpoles was reduced about -fold compared to wt virus or a "knock in" mutant. to determine the intracellular location of the k gene product, xia et al. [ ] generated recombinant sgiv k protein and used it to produce rabbit anti- k serum. immunofluorescent assay showed sgiv k to be distributed within the cytoplasm adjacent to vas and nuclei, and western blotting using purified and disrupted virions indicated k was a non-envelope virion-associated protein. transfection of uninfected grouper cells with a vector expressing k enhanced cellular growth rates and led to an increase in the density of monolayer cultures. moreover, sgiv replication in cultures expressing k were about -fold higher than in cultures transfected with an empty vector. the authors suggest that k may be a protein that plays a role in the control of cellular growth and thus indirectly enhances sgiv replication. however, because virus yields were expressed as tcid /ml, it was not clear if the increase in sgiv yield reflected higher virus production per cell or simply equivalent cellular yields in cultures containing varying numbers of cells. rothenburg et al. [ ] cloned the vif- α gene from rana catesbeiana virus z (rcv-z) into an expression vector and examined its function in a yeast-based assay system. yeast expressing human pkr failed to grow due to the toxicity of the pkr protein. however, yeast expressing vif- α, or the vaccinia virus k l protein, suppressed the toxic effects of human pkr indicating that vif- α was functionally equivalent to the vaccinia virus protein. subsequent work showed that whereas k l was effective only against human pkr, vif- α suppressed the toxic effects of both human and zebrafish pkr suggesting that pkr antagonists evolved to protect physiologically-relevant/phylogeneticallyrelated targets. in addition, a study using vectors expressing the various domains of vif- α, i.e., n-terminal, central/helical, and c-terminal, demonstrated that the n-terminal and central/helical domains were sufficient for suppressing pkr toxicity. collectively, these results provide the first formal proof that vif- α functions as a virus-encoded pkr antagonist. recombinant versions of thymidylate synthase (lcdv-china), erv (rana grylio virus, rgv), dutpase (rgv), rnase iii (rbiv), and litaf (sgiv) were generated and evaluated for their ability to influence cellular growth, enhance virus replication, induce apoptosis, and cleave dsrna [ , , , ] . recombinant thymidylate synthase from lcdv-china was found to promote cell cycle progression and produced a transformed phenotype [ ] . antibodies directed against rana gyrlio virus (rgv) erv detected erv expression within the cytoplasm and nucleus, but not within vas; transcript analysis indicated that erv was a late viral gene product [ ] . rgv dutpase was determined to be a de gene product that was localized to the cytoplasm. ectopic expression did not enhance virus replication, however, its effect on cellular proliferation was not examined [ ] . recombinant rnase iii from rbiv cleaved dsrna, but the salt optimum for cleavage was inconsistent with a physiological effect [ ] . the authors speculated that rnase iii might play a role in the suppression of rna interference, as has been suggested for other viral dsrna-binding proteins, or could be involved in the processing of viral rna. lastly, huang et al. [ ] demonstrated that the sgiv homolog of litaf (lipopolysaccharide-induced tnf-α factor) was a de viral gene product. litaf was located within the cytoplasm and was associated with mitochondria. when over-expressed ectopically, it led to the activation of caspase and the induction of apoptosis. collectively, ts mutants, asmo-and sirna-mediated knock down, gene knock out, and recombinant protein studies are slowly elucidating the function of iridovirid genes and providing a clearer and more complete picture of how those genes enhance viral replication and modulate cellular functions. future studies of ranaviruses and other iridoviruses infecting ectothermic animals will focus on the following areas: ( ) identifying and elucidating the function of replicative genes and those contributing to virulence, host range, and immune evasion; ( ) understanding the correlates of antiviral immunity in an effort to understand the cellular and molecular basis of anti-viral immunity and to protect endangered and commercially important species from infection; ( ) determining the impact of iridovirus infections in nature, defining viral host range, identifying susceptible hosts and reservoir species; ( ) understanding how intrinsic (e.g., host immune suppression, host mhc repertoire, etc.) and extrinsic (e.g., habitat disruption, environmental stress, introduction of invasive species, etc.) factors influence the clinical outcomes of iridovirus infection. for example, determining whether iridovirids encode viral immune evasion proteins and how these proteins circumvent host immunity will highlight events at the interface of virology and immunology, and may identify possible targets for viral attenuation. successful completion of these studies will involve the interactive efforts of virologists, immunologists, population biologists, ecologists, and veterinary pathologists. the global ranavirus consortium [ ] is but one interactive group of scientists interested in the role of 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subcellular distribution of a rana grylio virus late gene encoding erv homologue identification and characterization of a putative lipopolysaccharide-induced tnf-alpha factor (litaf) homolog from singapore grouper iridovirus constitutive expression of thymidylate synthase from lcdv-c induces a transformed phenoptype in fish cells we would like to thank robert sample for the electron micrographs shown in figure and the artwork depicted in figure . the work was partially supported by nsf award no. ios - . the authors declare no conflict of interest. key: cord- -eumuid r authors: widagdo, w.; okba, nisreen m. a.; richard, mathilde; de meulder, dennis; bestebroer, theo m.; lexmond, pascal; farag, elmoubasher a. b. a.; al-hajri, mohammed; stittelaar, koert j.; de waal, leon; van amerongen, geert; van den brand, judith m. a.; haagmans, bart l.; herfst, sander title: lack of middle east respiratory syndrome coronavirus transmission in rabbits date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: eumuid r middle east respiratory syndrome coronavirus (mers-cov) transmission from dromedaries to humans has resulted in major outbreaks in the middle east. although some other livestock animal species have been shown to be susceptible to mers-cov, it is not fully understood why the spread of the virus in these animal species has not been observed in the field. in this study, we used rabbits to further characterize the transmission potential of mers-cov. in line with the presence of mers-cov receptor in the rabbit nasal epithelium, high levels of viral rna were shed from the nose following virus inoculation. however, unlike mers-cov-infected dromedaries, these rabbits did not develop clinical manifestations including nasal discharge and did shed only limited amounts of infectious virus from the nose. consistently, no transmission by contact or airborne routes was observed in rabbits. our data indicate that despite relatively high viral rna levels produced, low levels of infectious virus are excreted in the upper respiratory tract of rabbits as compared to dromedary camels, thus resulting in a lack of viral transmission. middle east respiratory syndrome coronavirus (mers-cov) is a novel pathogen that is known to infect dromedary camels and humans [ , ] . seroepidemiological studies indicate that this virus has been circulating in dromedary camels in the arabian peninsula and africa for decades [ ] [ ] [ ] . mers-cov sequences obtained from these camels are largely similar to those obtained from human mers cases in corresponding regions, thus providing evidence that dromedary camels act as the zoonotic source of this virus [ , ] . however, many primary human mers cases do not have a history of direct contact with these animals [ ] . this suggests the presence of unidentified routes of human-to-human transmission or the involvement of other animal species in spreading the virus to humans. besides dromedary camels, other animal species, i.e. llamas, alpacas, and pigs have been shown to be susceptible and develop upper respiratory tract infection upon experimental intranasal mers-cov inoculation [ ] [ ] [ ] . this is in line with the expression of the mers-cov receptor, dipeptidyl peptidase- (dpp ), in their nasal epithelium [ ] . mers-cov-seropositive llamas and alpacas have been reported in the field, and mers-cov-experimentally-inoculated alpacas have also been shown to transmit the virus via contact [ ] [ ] [ ] . to further understand the zoonotic potential of mers-cov, it is crucial to delineate the factors involved in the spread of the virus among dromedaries as well as other animal species. in order to gain insight into these factors, we performed mers-cov transmission experiments in rabbits. we have previously shown that rabbits are susceptible to mers-cov and develop both upper and lower respiratory tract infection upon virus inoculation [ ] . naïve recipient rabbits were housed with mers-cov-inoculated donor rabbits either in the same or in adjacent cages to determine whether mers-cov can be transmitted via contact or airborne routes, respectively [ ] . donor rabbits were found to shed high levels of viral rna but a limited amount of infectious virus thus potentially restricting mers-cov transmission in these animals. in vivo experiments in this study were performed using passage human isolate mers-cov emc strain (hcov-emc/ ) and passage isolate mers-cov (qatar / ; genbank acc. no. mk ) that were propagated in vero cells as described earlier [ ] . qatar was isolated from a years old qatari man that developed severe pneumonia and was pcr confirmed to have a mers-cov infection [ ] . animal experiments were approved and performed according to the guidelines from the institutional animal welfare committee (no. approved on july , - - approved on september , and avd -wp approved on november ). the studies were performed under biosafety level (bsl ) conditions. to compare whether different routes of mers-cov inoculation generate similar clinical outcomes, twelve -month-old new zealand rabbits (oryctolagus cuniculus), specific pathogen free, and seronegative for mers-cov were divided into four groups. animals were inoculated under ketamine-medetomidine anesthesia either (a) intranasally with µl of × tcid /ml mers-cov; (b) intranasally with ml × tcid /ml mers-cov; (c) intranasally with µl of × tcid /ml mers-cov and intratracheally with ml × tcid /ml mers-cov; or (d) intranasally with ml pbs. the intranasal inoculums were divided equally over both nostrils. nasal and throat swabs were obtained daily from day up to day post inoculation. these animals were then sacrificed on day and respiratory tract tissues were collected. to compare the clinical outcomes of the mers-cov emc strain and the qatar strain, ten new zealand rabbits were divided into two groups and inoculated with ml of × tcid /ml of each mers-cov strain intranasally. nasal and throat swabs were obtained from day up to day post inoculation and these animals were sacrificed on day . to study mers-cov transmission, a modified version of the previously described influenza a virus ferret transmission set-up was used. this set-up consists of two clear polymethyl methacrylate cages of different sizes. donor rabbits and direct contact recipients were housed in a cage of cm × cm × cm (w × h × l), whereas airborne recipients were housed in a cage of cm × cm × cm (w × h × l). these cages are separated by two stainless steel grids cm apart to prevent direct contact but still allow airflow from the donor rabbit to the airborne recipient rabbit. these transmission cages allow the experiment to be conducted in negatively pressured isolators in the bsl facility, with hepa-filtered airflow < . m/s [ ] . since these cages were too small for new zealand rabbits, we chose a smaller-sized breed, the netherland dwarf rabbits (oryctolagus cuniculus), for the mers-cov transmission experiment. we used both male and female rabbits with an age viruses , , of range of months- years in these experiments. first, three mers-cov seronegative netherland dwarf rabbits were inoculated intranasally with ml of × tcid /ml mers-cov qatar strain ( µl per nostril) to show that they are equally susceptible to mers-cov as the new zealand rabbits. nasal and throat swabs were obtained daily up to days post inoculation. these animals were then sacrificed on day and their respiratory tract tissues were collected. for the virus transmission experiment, twelve netherland dwarf rabbits were randomly distributed into four individually housed groups. one naïve rabbit from each group was inoculated intranasally with ml of × tcid /ml mers-cov ( µl per nostril), thus acting as donor rabbits. the other two rabbits were used as direct contact and airborne recipients, respectively. donor and direct contact animals were of the same sex. all animals were sacrificed at days post exposure and blood was collected to assess seroconversion. nasal swabs, throat swabs, and respiratory tract tissue samples were evaluated for the presence of infectious virus by virus titration, and for viral rna by rt-qpcr against the upe gene as previously described [ ] . samples with a cycle threshold less than forty were considered as positive for mers-cov rna. viral rna was quantified as genome equivalents (ge) using mers-cov strain emc (containing tcid /ml) as a calibrator. virus titration was performed in serial -fold dilutions on vero cells. cells were monitored under a light microscope at day for cytopathic effect. the amount of infectious virus in swab samples was calculated by determining the tcid . statistical analysis was performed using the graphpad prism program (la jolla, ca, usa). kruskal-wallis test was applied due to the non-normal distribution of our data as priorly determined by shapiro-wilk test. the significant difference between groups was determined at a p-value < . . respiratory tract tissue samples were collected in formalin and embedded in paraffin for pathological analysis. hematoxylin-eosin staining was performed for histopathological analysis. the presence of mers-cov nucleoprotein and mers-cov rna was detected by immunohistochemistry and in-situ hybridization, respectively, using previously published protocols [ ] . the localization of dpp in the respiratory tract of non-infected new zealand rabbits was analyzed using an optimized immunohistochemical assay [ , ] . collected serum samples were tested for mers-cov neutralizing antibodies using a virus neutralization assay and for mers-cov s -specific antibodies using mers-cov s elisa according to the previously published protocols [ ] . goat anti-rabbit igg conjugated with hrp ( : , dako, glostrup, denmark) was used as a secondary antibody in the elisa. rabbits are the smallest animal species that can be naturally infected by mers-cov. we previously reported that they develop both upper and lower respiratory tract infection upon mers-cov inoculation [ ] , suggesting the expression of the viral receptor at these locations. using immunohistochemistry, we analyzed the dpp expression in rabbit respiratory tract tissues. in the upper respiratory tract, dpp is strongly expressed at the apical surface of both nasal respiratory and olfactory epithelium ( figure ). in the lower respiratory tract, dpp is present in both bronchiolar and alveolar epithelial cells, although some variation in dpp expression was observed throughout the lungs. dpp is either absent, limitedly expressed on alveolar type ii cells, or expressed on both alveolar type i and ii cells ( figure ). thus, these data highlight a broad dpp expression in the respiratory tract different human mers-cov strains have been isolated since the emc/ strain was first characterized [ , ] . however, studies that evaluate phenotypic differences between these strains in animals are currently lacking. we investigated whether a more recent mers-cov strain (qatar / ) replicates differently in rabbits in comparison to the emc strain. we found that rabbits inoculated with the mers-cov emc strain and those with the qatar strain developed an equally mild infection and shed similar levels of viral rna in their nasal and throat swabs ( figure ). following this result, the mers-cov transmission experiment was performed using the qatar strain, the more recent strain of these two. type ii cells are indicated with arrows, and type i cells with an arrowhead. nasal epithelium and bronchiolar epithelium pictures were taken at a × magnification, and alveolar epithelium at ×. the isotype control showed no background signal in our assay. in our previous study, we inoculated rabbits both intranasally and intratracheally [ ] . intratracheal inoculation is quite invasive, and thus requires a skillful operator to minimize procedure-related damage in the respiratory tract. in contrast, intranasal inoculation is less invasive and had been used in other studies to infect rabbits with mers-cov [ , ] . here we investigated whether intranasal mers-cov inoculation is sufficient to induce both upper and lower respiratory tract infection in rabbits, in comparison to combined intranasal and intratracheal inoculation. three new zealand rabbits (oryctolagus cuniculus) were inoculated with mers-cov emc strain either intranasally with µl of × tcid /ml (group a); intranasally with ml of × tcid /ml (group b); intranasally with µl of × tcid /ml and intratracheally with ml of × tcid /ml (group c); or intranasally with ml of pbs as a negative control (group d). all three groups of mers-cov inoculated rabbits developed minimal clinical manifestations and histopathological lesions. the amount of viral rna shed in the nasal and throat swabs did not vary among the inoculated groups (figure a,b) . however, in the lungs of the rabbits, the amount of viral rna was significantly lower in group a than in groups b and c ( figure c ). in line with these observations, fewer mers-cov-infected cells were observed in the lungs of group a animals compared to groups b and c ( figure d ). based on these results, we decided to use intranasal inoculation with ml of × tcid /ml mers-cov for our subsequent experiments. pictures were taken at a × magnification, and alveolar epithelium at ×. the isotype control showed no background signal in our assay. different human mers-cov strains have been isolated since the emc/ strain was first characterized [ , ] . however, studies that evaluate phenotypic differences between these strains in animals are currently lacking. we investigated whether a more recent mers-cov strain (qatar / ) replicates differently in rabbits in comparison to the emc strain. we found that rabbits inoculated with the mers-cov emc strain and those with the qatar strain developed an equally mild infection and shed similar levels of viral rna in their nasal and throat swabs (figure ). following this result, to study mers-cov transmission, an experimental set up previously used to investigate influenza a virus transmission between ferrets was used. this set up consists of two polymethyl methacrylate cages separated with two steel grids, cm apart [ ] . because this set-up was too small to house new zealand rabbits, we used a smaller-sized breed, the netherland dwarf rabbits. prior to the virus transmission experiment, netherland dwarf rabbits were inoculated with mers-cov qatar strain to determine their susceptibility to the virus. similar to the new zealand rabbits [ ] , netherland dwarf rabbits shed viral rna in the nasal and throat swabs as well as in the respiratory tract tissues upon intranasal inoculation ( figure a,b) . identical to the new zealand rabbits [ ] , the mers-cov-inoculated netherland dwarf rabbits did not develop any clinical signs, including nasal discharge, and showed minimal histopathological lesions and immune cell infiltration in the respiratory tract. to study mers-cov transmission, four netherland dwarf rabbits were intranasally inoculated with mers-cov. six-hours later, each of them was co-housed with one naïve rabbit in the same cage, and h later another one was co-housed in an adjacent cage to determine whether mers-cov could be transmitted through contact and/or airborne routes. nasal and throat swabs were collected every other day up to day or post inoculation/exposure for the donor and direct contact rabbits, respectively, and day post exposure for the airborne recipient ones. both viral rna and infectious virus were quantified in these samples. we found that all donor rabbits shed high loads of viral rna in both the nasal swabs (~ - tcid ge/ml) and the throat swabs (~ - tcid ge/ml). the nasal and throat swabs were obtained from day (before inoculation) until day post inoculation. the amount of viral rna is displayed in genome equivalents per ml (ge/ml). dashed lines depict the detection limit of the assay. all error bars represent standard deviations. to study mers-cov transmission, an experimental set up previously used to investigate influenza a virus transmission between ferrets was used. this set up consists of two polymethyl methacrylate cages separated with two steel grids, cm apart [ ] . because this set-up was too small to house new zealand rabbits, we used a smaller-sized breed, the netherland dwarf rabbits. prior to the virus transmission experiment, netherland dwarf rabbits were inoculated with mers-cov qatar strain to determine their susceptibility to the virus. similar to the new zealand rabbits [ ] , netherland dwarf rabbits shed viral rna in the nasal and throat swabs as well as in the respiratory tract tissues upon intranasal inoculation ( figure a,b) . identical to the new zealand rabbits [ ] , the mers-cov-inoculated netherland dwarf rabbits did not develop any clinical signs, including nasal discharge, and showed minimal histopathological lesions and immune cell infiltration in the respiratory tract. donor rabbits at day post inoculation, and in one of the donors up to day . in the throat swabs, infectious virus was only detected in two donors on day , up to day in one of them. in contrast, none of the swabs from recipient rabbits was positive for virus titration (figure c,d) . serological analysis of samples collected days after exposure showed that only the donor rabbits seroconverted and developed neutralizing antibodies ( figure a,b) . the antibody response of these directly inoculated rabbits was relatively low, confirming the results of previous studies [ , ] . this indicates that these rabbits developed mers-cov infection while the contact and airborne-exposed rabbits did not, supporting the results of the virus titration. to study mers-cov transmission, four netherland dwarf rabbits were intranasally inoculated with mers-cov. six-hours later, each of them was co-housed with one naïve rabbit in the same cage, and h later another one was co-housed in an adjacent cage to determine whether mers-cov could be transmitted through contact and/or airborne routes. nasal and throat swabs were collected every other day up to day or post inoculation/exposure for the donor and direct contact rabbits, respectively, and day post exposure for the airborne recipient ones. both viral rna and infectious virus were quantified in these samples. we found that all donor rabbits shed high loads of viral rna in both the nasal swabs (~ - tcid ge/ml) and the throat swabs (~ - tcid ge/ml). the amount of viral rna shed by the inoculated rabbits remained high until day post inoculation. on the other hand, recipient rabbits housed in the same cage (direct contact recipients), or adjacent cage (airborne recipients), shed limited amounts of viral rna (~ tcid ge/ml) in both nasal and throat swabs. among four animals in each group, only two direct contact recipient and two airborne recipient rabbits had detectable viral rna up to day post inoculation in the nasal swabs, while in the throat swabs, viral rna was only detected in one direct contact recipient and one airborne recipient rabbit at day post inoculation ( figure a,b) . infectious virus was detected at low level (~ tcid /ml) both in the nasal and throat swabs of the donor rabbits; in the nasal swabs of all donor rabbits at day post inoculation, and in one of the donors up to day . in the throat swabs, infectious virus was only detected in two donors on day , up to day in one of them. in contrast, none of the swabs from recipient rabbits was positive for virus titration ( figure c,d) . serological analysis of samples collected days after exposure showed that only the donor rabbits seroconverted and developed neutralizing antibodies ( figure a,b) . the antibody response of these directly inoculated rabbits was relatively low, confirming the results of previous studies [ , ] . this indicates that these rabbits developed mers-cov infection while the contact and airborne-exposed rabbits did not, supporting the results of the virus titration. current data indicate that mers-cov is highly endemic in dromedary camels in the arabian peninsula and africa and has been circulating in these animals for decades [ ] [ ] [ ] ] . this suggests that this virus is easily transmitted between dromedary camels. from an epidemiological point of current data indicate that mers-cov is highly endemic in dromedary camels in the arabian peninsula and africa and has been circulating in these animals for decades [ ] [ ] [ ] ] . this suggests that this virus is easily transmitted between dromedary camels. from an epidemiological point of view, it is important to know whether other animal species in the region may also spread the virus to humans or other animal species. in vitro, mers-cov has been found to infect cells from a broad range of animal species including old and new world camelids as well as primates, bats, cows, sheep, goats, pigs, horses, and rabbits [ , , ] . the dpp viral receptor of these species, especially rabbits, has high similarity to that of humans and dromedary camels, especially in the region that interacts with the spike protein, and thus can facilitate mers-cov infection [ ] [ ] [ ] . the new world camelids, i.e. llamas and alpacas, have been shown to seroconvert to mers-cov when present in regions where mers-cov is circulating and may transmit the virus [ ] [ ] [ ] . it is currently unclear why, besides camelids, other livestock animals do not seem to transmit the virus to humans [ , [ ] [ ] [ ] . to further understand the transmission potential of mers-cov, we performed virus transmission experiments using rabbits as animal model. to perform the virus transmission experiments, we housed mers-cov-inoculated rabbits together with naïve contact rabbits either in the same or adjacent cages. rabbits developed both upper and lower respiratory tract infection upon mers-cov inoculation [ ] , either via intranasal or combined intranasal and intratracheal routes, in line with the localization of dpp in their respiratory tract epithelium. the amount of viral rna being shed by the inoculated rabbits during the first three days post inoculation is almost as high as that of the mers-cov-inoculated dromedary camels [ , ] . however, none of the direct contact and airborne-exposed rabbits developed any clinical signs, shed significant levels of viral rna, shed infectious virus, nor did they seroconvert. one possible reason for this lack of transmission is the limited amount of infectious virus being shed by the inoculated rabbits [ ] . previous studies have shown that in the nasal and lung tissues of experimentally infected rabbits, infectious virus was generally detected in a limited amount despite the abundant presence of viral rna and virus nucleoprotein [ , ] . alternatively, low levels of infectious virus transmitted to recipient animals may be unable to initiate a productive infection due to the presence of host proteins that restrict replication. comparable to rabbits, mers-cov-infected pigs and goats develop minimal clinical signs, hardly shed infectious virus, and barely spread the virus to naïve animals [ , ] . in contrast, mers-cov-infected dromedary camels develop nasal discharge and shed a high amount infectious virus ( - tcid /ml), almost equal to the amount of viral rna being shed ( - ge/ml), during the first days post inoculation [ , ] . these interspecies differences may indicate presence of nasal discharge and infectious virus shedding as critical factors in mers-cov transmission. in humans, levels of infectious virus shed by mers-cov patients have rarely been reported. however, mers-cov patients that transmit the virus have been shown to shed a higher amount of viral rna in their swabs compared to those that do not, supporting the quantity of virus shed as an important factor in the transmission of mers-cov between humans [ ] . for influenza a viruses, infectious virus shedding has been documented as one of the main determinants of airborne virus transmission. using ferrets as an animal model, it has been reported that a reduction in infectious virus shedding in the nasal swabs can subsequently limit virus transmission [ ] [ ] [ ] . our results show that despite the presence of dpp in the upper respiratory tract, accompanied by mers-cov replication at this site, a limited amount of infectious virus was shed. similarly, titration of rabbit lung homogenates that show high levels of viral rna and presence of nucleoprotein ( figure c ,d) resulted in only low levels of infectious virus, in line with earlier observations [ , ] . at this stage, it is not clear which host mediated mechanisms limit the production of infectious virus while allowing viral rna to still be shed at high levels. since restriction of infectious virus shedding in the rabbits already occurred one day post inoculation, activation of host innate immune responses, including type i interferon induction, may be relevant. in vitro studies have shown that mers-cov is relatively sensitive to type i interferon-mediated antiviral activities [ , ] . in human plasmacytoid dendritic cells, mers-cov inoculation leads to secretion of large amount of type i and iii interferons and production of viral rna, but hardly any infectious virus is being produced [ ] . it is also possible that most infectious virus shed by these rabbits is defective, lacking the capacity to efficiently infect target cells. further studies are needed to elucidate the mechanisms that restrict mers-cov replication in rabbits compared to dromedary camels. potentially, some of the mers-cov accessory proteins shown to antagonize immune responses including production of interferon, may not work efficiently in some mers-cov susceptible species, including rabbits. it is intriguing to investigate whether a similar phenomenon occurs in some human mers-cov infections and whether this is linked to the development of asymptomatic to mild clinical manifestations [ ] . this might partly explain why mers-cov transmission in humans is rather inefficient in comparison to dromedary camels [ , ] , and why camelids that secrete high levels of infectious virus are the only known zoonotic source of mers-cov [ , , , ] . deciphering these mechanisms could potentially offer insight into understanding mers-cov transmission as well as developing novel treatments to tackle the ongoing outbreaks. isolation of a novel coronavirus from a man with pneumonia in saudi arabia isolation of mers coronavirus from a dromedary camel middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia geographic distribution of mers coronavirus among dromedary camels zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases replication and 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orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels middle east respiratory syndrome coronavirus experimental transmission using a pig model risk factors for transmission of middle east respiratory syndrome coronavirus infection during the outbreak in south korea impact of prior seasonal h n influenza vaccination or infection on protection and transmission of emerging variants of influenza a(h n )v virus in ferrets efficacy of seasonal live attenuated influenza vaccine against virus replication and transmission of a pandemic h n virus in ferrets predicting "airborne" influenza viruses: (trans-) mission impossible? mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment inhibition of novel beta coronavirus replication by a combination of interferon-alpha b and ribavirin high secretion of interferons by human plasmacytoid dendritic cells upon recognition of middle east respiratory syndrome coronavirus world health organization mers situation update transmission of mers-coronavirus in household contacts mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -pgxxkfc authors: wang, cong; hua, chen; xia, shuai; li, weihua; lu, lu; jiang, shibo title: combining a fusion inhibitory peptide targeting the mers-cov s protein hr domain and a neutralizing antibody specific for the s protein receptor-binding domain (rbd) showed potent synergism against pseudotyped mers-cov with or without mutations in rbd date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: pgxxkfc middle east respiratory syndrome coronavirus (mers-cov) has continuously posed a threat to public health worldwide, yet no therapeutics or vaccines are currently available to prevent or treat mers-cov infection. we previously identified a fusion inhibitory peptide (hr p-m ) targeting the mers-cov s protein hr domain and a highly potent neutralizing monoclonal antibody (m ) specific to the s spike protein receptor-binding domain (rbd). however, m was found to have reduced efficacy against mers-cov strains with mutations in rbd, and hr p-m showed low potency, thus limiting the clinical application of each when administered separately. however, we herein report that the combination of m and hr p-m exhibited potent synergism in inhibiting mers-cov s protein-mediated cell–cell fusion and infection by mers-cov pseudoviruses with or without mutations in the rbd, resulting in the enhancement of antiviral activity in contrast to either one administered alone. thus, this combinatorial strategy could be used in clinics for the urgent treatment of mers-cov-infected patients. middle east respiratory syndrome (mers) coronavirus (mers-cov), a lineage c beta-coronavirus, was reported to cause severe respiratory tract infection [ , ] . to date, laboratory-confirmed cases of infection with mers-cov, including mers-cov associated deaths, have been reported to the world health organization (who) from countries. currently, no effective therapeutics or vaccines are available to treat or prevent mers-cov infection. the spike (s) protein of mers-cov plays important roles in virus attachment, fusion, and entry into the target cell [ ] [ ] [ ] . similar to other coronaviruses, the s protein of mers-cov consists of s and s subunits. the s subunit is responsible for the binding of the virion by its receptor binding domain (rbd) to the cellular receptor, dipeptidyl peptidase- (dpp ), while the s subunit mediates the fusion the t cell line was obtained from atcc (manassas, va, usa), and the huh- cell line was from the cell bank of the chinese academy of sciences (shanghai, china). these two cell lines were propagated in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs). peptide hr p-m was synthesized by solid phase peptide synthesis at syn inc. (shanghai, china), and human mab m was provided by prof. tianlei ying at fudan university, shanghai, china. recombinant plasmids encoding the mers-cov s protein with d g, d g, q h, or i t mutations were kindly provided by dr. lanying du at the new york blood center, ny, usa. mers-cov pseudoviruses were constructed as described previously [ , ] . briefly, t cells were plated in a t tissue culture flask and incubated at • c for h. cells were cotransfected with plasmids pnl - .luc.re encoding env-defective, luciferase-expressing hiv- and pcdna . -mers-cov-s encoding s protein with or without mutation in rbd at mass ratio of : using vigofect (vigorous biotechnology, beijing, china), according to the manufacturer's recommendation. the supernatant was replaced with fresh dmem at - h post-transfection and harvested after incubation for an additional h. in order to remove cell debris, the supernatant was centrifuged at viruses , , of rpm for min, followed by filtration through a . µm filter. mers-cov pseudovirus in the supernatant was quantified by testing p content in the product of mers-cov pseudovirus. a mers-cov pseudovirus inhibition assay was performed as previously described [ , , ] . briefly, huh- cells were seeded ( cells/well) into a -well plate and incubated overnight at • c. mers-cov pseudovirus was incubated with a serially diluted inhibitor for min at • c, followed by the addition of huh- cells. the cells were incubated with or without pseudovirus as virus control and cell control, respectively. the culture was replaced with fresh medium h post-infection and incubated for an additional h. cells were lysed using lysis reagent (promega, madison, wi, usa), and cell lysates were transferred to a -well costar flat-bottom luminometer plate (corning costar, new york, ny, usa), followed by the addition of luciferase substrate (promega) to measure luminescence using an infinite m pro (tecan, grödig, austria). mers-cov s protein-mediated cell-cell fusion was performed as previously described [ ] . briefly, plasmid paav-ires-mers-egfp encoding the mers-cov s protein was transfected into t cells ( t/mers/egfp) using the transfection reagent, vigofect (vigorous biotechnology, beijing, china). the target huh- cells expressing dpp were incubated at × cells/well in wells of a -well plate for h. the effector t/mers/egfp cells that express mers-cov s protein and egfp or the control t/egfp cells that express egfp only were preincubated at cells/well with an inhibitor at the indicated concentration or phosphate buffered saline (pbs) as control at • c for min. the mixture of t/mers/egfp cells and an inhibitor or pbs were added to huh- cells in the wells, followed by a co-culture at • c for h. the t/mers/egfp cells fused or unfused with huh- cells were fixed with % pfa and counted under an inverted fluorescence microscope (nikon, tokyo, japan). the fused cell showed much larger size and weaker fluorescence intensity than the unfused cell because of the diffusion of egfp from one cell to more cells (figure ). almost no fused cells could be observed in the groups of negative control (pbs+ t/egfp+huh- ) or peptide treatment (hr p-m + t/mers/egfp+huh- ) (figure ). the concentration for % inhibition (ic ) was calculated using calcusyn software kindly provided by dr. t.c. chou [ ] . viruses , , x for peer review of was centrifuged at rpm for min, followed by filtration through a . µm filter. mers-cov pseudovirus in the supernatant was quantified by testing p content in the product of mers-cov pseudovirus. a mers-cov pseudovirus inhibition assay was performed as previously described [ , , ] . briefly, huh- cells were seeded ( cells/well) into a -well plate and incubated overnight at °c. mers-cov pseudovirus was incubated with a serially diluted inhibitor for min at °c, followed by the addition of huh- cells. the cells were incubated with or without pseudovirus as virus control and cell control, respectively. the culture was replaced with fresh medium h post-infection and incubated for an additional h. cells were lysed using lysis reagent (promega, madison, wi, usa), and cell lysates were transferred to a -well costar flat-bottom luminometer plate (corning costar, new york, ny, usa), followed by the addition of luciferase substrate (promega) to measure luminescence using an infinite m pro (tecan, grödig, austria). mers-cov s protein-mediated cell-cell fusion was performed as previously described [ ] . briefly, plasmid paav-ires-mers-egfp encoding the mers-cov s protein was transfected into t cells ( t/mers/egfp) using the transfection reagent, vigofect (vigorous biotechnology, beijing, china). the target huh- cells expressing dpp were incubated at × cells/well in wells of a -well plate for h. the effector t/mers/egfp cells that express mers-cov s protein and egfp or the control t/egfp cells that express egfp only were preincubated at cells/well with an inhibitor at the indicated concentration or phosphate buffered saline (pbs) as control at °c for min. the mixture of t/mers/egfp cells and an inhibitor or pbs were added to huh- cells in the wells, followed by a co-culture at °c for h. the t/mers/egfp cells fused or unfused with huh- cells were fixed with % pfa and counted under an inverted fluorescence microscope (nikon, tokyo, japan). the fused cell showed much larger size and weaker fluorescence intensity than the unfused cell because of the diffusion of egfp from one cell to more cells ( figure ). almost no fused cells could be observed in the groups of negative control (pbs+ t/egfp+huh- ) or peptide treatment (hr p-m + t/mers/egfp+huh- ) (figure ). the concentration for % inhibition (ic ) was calculated using calcusyn software kindly provided by dr. t.c. chou [ ] . six-week-old female specific-pathogen-free (spf) balb/c mice (bodyweight about g) were divided into groups of mice each. mice in group , , and were intraperitoneally (i.p.) injected with m ( . mg in µl pbs) alone, hr p-m ( mg in µl pbs) alone, and the combination of m ( . mg in µl pbs) and hr p-m ( mg in µl pbs), respectively. mice were sedated with nembutal ( mg/kg body weight) before and h after injection of the inhibitors, respectively, and bled retro-orbitally. the blood was centrifuged at rpm for min after standing at room temperature for h. the sera were collected and heat-inactivated at • c for min. inhibitory activity of the inhibitors on mers-cov pseudovirus was evaluated in serum as described above. to assess the potential synergistic effect, hr p-m and m were mixed at the indicated molar concentration ratio, while hr p-m alone and m alone were included as controls. the mixtures were serially diluted and tested for their inhibitory activity on mers-cov pseudovirus infection as described above. each sample was tested in triplicate, and data were analyzed for synergistic effect by calculating the combination index (ci), using the calcusyn program. ci values of < and > indicate synergy and antagonism, respectively, and synergy was divided into different strengths, according to ci values, as follows: < . indicates very strong synergism; . - . indicates strong synergism; . - . indicates synergism; . - . indicates moderate synergism; and . - . indicates slight synergism [ , ] . fold of potency enhancement was calculated with the ratio of concentrations of inhibitor testing alone and in combination. to determine the significance of difference in sensitivity between wild-type and mutant viruses to inhibitors and the inhibitory activity detected in sera from balb/c mice treated with inhibitors alone or combination, statistical analyses were performed using a two-tailed unpaired student's t-test, using graphpad prism, version . . values with p < . and p < . were considered statistically significant and very significant, respectively. we first investigated the potential cooperative effects of combining hr p-m with m on mers-cov pseudovirus infection. in our preliminary study, we found that ic values of hr p-m and m for inhibiting mers-cov pseudovirus infection were about nm and . nm, respectively. therefore, we tested the inhibitory activity of hr p-m alone, m alone, and hr p-m /m in combination at a molar concentration ratio of , : , respectively. as shown in figure and table , combining hr p-m and m resulted in strong synergistic inhibitory activity against mers-cov pseudovirus infection with ci values of . - . for - % inhibition, including potency enhancement of . -to . -fold for m and . -to . -fold for hr p-m . this result suggested that the mers-cov fusion inhibitory peptide hr p-m and the mers-cov neutralizing mab m could be used in combination to enhance anti-mers-cov activity. note: each sample was tested in triplicate, and the mean values are presented. ratio of molar concentration of hr p-m and m in combination is , : . next, we tested the potential synergistic activity of the hr p-m /m combination on mers-cov s protein-mediated cell-cell fusion. we adjusted the molar concentration ratio of hr p-m and m in the combination to : , since the ic values of hr p-m and m for inhibiting mers-cov s protein-mediated cell-cell fusion in our preliminary studies were about nm and . nm, respectively. as shown in figure and table , the combination also exhibited strong synergism against mers-cov s protein-mediated cell-cell fusion (ci = . ) with enhancement of -fold for m and -fold for hr p-m . this result confirms that combining hr p-m , a mers-cov fusion inhibitor, with m , a human neutralizing mab, results in strong synergism on s protein-mediated membrane fusion because they target the different stages of mers-cov fusion and entry processes. the blue curves represent inhibitors used alone, and the red curves represent each inhibitor used in combination. the width between two curves represents the fold of enhancement between an inhibitor used alone and in combination. note: each sample was tested in triplicate, and the mean values are presented. ratio of molar concentration of hr p-m and m in combination is , : . next, we tested the potential synergistic activity of the hr p-m /m combination on mers-cov s protein-mediated cell-cell fusion. we adjusted the molar concentration ratio of hr p-m and m in the combination to : , since the ic values of hr p-m and m for inhibiting mers-cov s protein-mediated cell-cell fusion in our preliminary studies were about nm and . nm, respectively. as shown in figure and table , the combination also exhibited strong synergism against mers-cov s protein-mediated cell-cell fusion (ci = . ) with enhancement of -fold for m and -fold for hr p-m . this result confirms that combining hr p-m , a mers-cov fusion inhibitor, with m , a human neutralizing mab, results in strong synergism on s protein-mediated membrane fusion because they target the different stages of mers-cov fusion and entry processes. note: each sample was tested in triplicate, and the mean values are presented. the molar concentration ratio of hr p-m and m in combination is : . du et al. have previously shown that mers-cov pseudoviruses with mutations in rbd, such as d g and d g detected in some mers-cov strains isolated from different regions and at different times [ , ] , are resistant to the neutralizing activity of an rbd-specific mouse mab mersmab [ ] . in the present study, the sensitivity of pseudotyped mers-cov strains with key mutations in rbd, as identified in some mers-cov mutants isolated during the - outbreaks [ ] , including d g, d g, q h, and i t, along with wild-type mers-cov, was compared between the inhibitory activity of hr p-m peptide alone and m neutralizing mab alone. as shown in table , the resistance of mers-cov mutants to the neutralizing activity of m is about -to -fold, whereas the pseudoviruses with or without mutations were equally sensitive to fusion inhibitory activity of hr p-m . this result suggested that use of mab m alone is unable to control the infection by mers-cov strains with mutations in rbd. table . sensitivity of mers-cov pseudoviruses with or without mutations in the receptor-binding domain (rbd) to the inhibitory activity of m (nm) and hr p-m (µm) separately. [ , ] , are resistant to the neutralizing activity of an rbd-specific mouse mab mersmab [ ] . in the present study, the sensitivity of pseudotyped mers-cov strains with key mutations in rbd, as identified in some mers-cov mutants isolated during the - outbreaks [ ] , including d g, d g, q h, and i t, along with wild-type mers-cov, was compared between the inhibitory activity of hr p-m peptide alone and m neutralizing mab alone. as shown in table , the resistance of mers-cov mutants to the neutralizing activity of m is about -to -fold, whereas the pseudoviruses with or without mutations were equally sensitive to fusion inhibitory activity of hr p-m . this result suggested that use of mab m alone is unable to control the infection by mers-cov strains with mutations in rbd. table . sensitivity of mers-cov pseudoviruses with or without mutations in the receptor-binding domain (rbd) to the inhibitory activity of m (nm) and hr p-m (µm) separately. to determine whether the combination of hr p-m and m also exhibited synergistic antiviral activity against infection of mers-cov strains with mutations in rbd or in the hr domain, we constructed pseudoviruses bearing mers-cov s protein with mutations in rbd, including d g, d g, q h, or i t, and those in the hr domain, including q h and q r [ , ] . we then tested their sensitivity to the inhibition of hr p-m alone, m alone, and the hr p-m /m combination. as shown in table , combining m with hr p-m exhibited strong synergism against infection by pseudotyped mers-cov strains with or without mutations in the rbd or hr domain with ci value less than . and potency enhancement in the range of -to -fold, suggesting that this combinational therapy has potential to be further developed for treatment of patients infected by different mers-cov strains, including those with resistance to rbd-specific neutralizing antibodies. table . combination index and fold of enhancement for inhibiting mers-cov pseudoviruses with or without mutations in rbd in s subunit and hr in s subunit of mers-cov s protein by hr p-m and m . to determine whether the hr p-m /m combination could sustain its efficacy in vivo compared to hr p-m or m alone, we tested the anti-mers-cov pseudovirus activity of the inhibitors in sera of mice treated with i.p. injection of hr p-m , m , and the hr p-m /m combination, respectively. as shown in figure , the inhibitory activity detected in sera from mice treated with hr p-m or m alone was significantly higher than that detected in sera from mice before inhibition of any inhibitor. on the other hand, the anti-mers-cov activity detected in sera from mice treated with the hr p-m /m combination was significantly more potent than that detected in sera of mice administered with hr p-m or m alone. this result confirms that combining hr p-m with m affords synergism against mers-cov s infection, both in vitro and in vivo. note: the molar concentration ratio of hr p-m and m in combination against wildtype virus, viruses with mutations in rbd, and those in hr is , : , : , and , : , respectively. to determine whether the hr p-m /m combination could sustain its efficacy in vivo compared to hr p-m or m alone, we tested the anti-mers-cov pseudovirus activity of the inhibitors in sera of mice treated with i.p. injection of hr p-m , m , and the hr p-m /m combination, respectively. as shown in figure , the inhibitory activity detected in sera from mice treated with hr p-m or m alone was significantly higher than that detected in sera from mice before inhibition of any inhibitor. on the other hand, the anti-mers-cov activity detected in sera from mice treated with the hr p-m /m combination was significantly more potent than that detected in sera of mice administered with hr p-m or m alone. this result confirms that combining hr p-m with m affords synergism against mers-cov s infection, both in vitro and in vivo. the high mortality of mers-cov-infected patients [ ] [ ] [ ] calls for the development of highly effective anti-mers-cov therapeutics. although we and others have previously identified a mers-cov fusion inhibitory peptide (hr p-m ) targeting the mers-cov s protein hr domain and a highly potent human neutralizing mab (m ) targeting the mers-cov s protein rbd data are presented as means ± sd. **, and *** represent p < . , and p < . , respectively. the high mortality of mers-cov-infected patients [ ] [ ] [ ] calls for the development of highly effective anti-mers-cov therapeutics. although we and others have previously identified a mers-cov fusion inhibitory peptide (hr p-m ) targeting the mers-cov s protein hr domain and a highly potent human neutralizing mab (m ) targeting the mers-cov s protein rbd [ , , ] , their further development is limited by low potency in the case of hr p-m and low efficacy to neutralize mers-cov strains with rbd mutations in the case of m [ , [ ] [ ] [ ] . the combinatorial use of drugs with different mechanisms of action, i.e., cocktail regimen, has been widely applied in clinics [ ] . for example, the combinatorial use of hiv reverse transcriptase (rt) inhibitors and protease inhibitors, known as highly active anti-retrovirus therapy (haart), has shown significant synergism in inhibiting hiv- infection, reducing adverse effects and delaying the emergence of drug resistance, thus extending the lifespan of millions of hiv/aids patients [ ] [ ] [ ] . moreover, we previously showed that combining hiv- attachment inhibitors with rt inhibitors, or combining the st, nd, and/or rd generation hiv fusion inhibitors that target different sites in the hiv- gp hr domain, exhibited synergistic and complementary effect against infection by a broad spectrum of hiv- strains, including those resistant to hiv attachment inhibitors, fusion inhibitors, and rt inhibitors [ ] [ ] [ ] . in this study, we compared the anti-mers-cov activity of hr p-m alone and m alone with that of hr p-m /m in combination and found that the inhibitory activity of the hr p-m /m combination was significantly more potent than either one administered alone against mers-cov s protein-mediated cell-cell fusion and mers-cov pseudovirus infection, suggesting synergistic activity based on the dual mechanisms of action whereby hr p-m targets the s subunit hr domain for inhibiting s -mediated virus-cell or cell-cell fusion [ ] and m targets the s subunit rbd for inhibiting virus-cell binding or virus attachment [ ] . it has been well known that drug synergism can be expected when drugs that act by different mechanisms of action are mixed together [ ] . while mers-cov pseudoviruses with mutations in rbd were resistant to the rbd-specific mab m , they were equally sensitive to hr -targeting peptide hr p-m . notably, however, the hr p-m /m combination exhibited strong synergistic antiviral activity against all pseudotyped mers-cov strains, including those with mutations in rbd of s protein, which are even resistant to an rbd-specific mouse mab mersmab [ ] . we also demonstrated that sera from mice treated with the hr p-m /m combination revealed significant efficacy in inhibiting mers-cov pseudovirus infection compared to hr p-m or m alone. collectively, these results suggest that the combinatorial strategy overcomes the weaknesses of hr p-m peptide and m mab, while, at the same time, takes advantage of the unique mechanism of action of each to provide, by the sum of both, much more effective inhibitory activity against mers-cov infection than either peptide or mab used alone. the strong synergy of the combination is expected to reduce the dosage of the individual inhibitor in such combinational therapy, resulting in decreased cost and toxicity, thus making the final product more affordable and safer. therefore, this combinational therapy shows promise for further clinical development. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus 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a newly discovered coronavirus associated with acute respiratory distress syndrome in humans hospital outbreak of middle east respiratory syndrome coronavirus urgent development of effective therapeutic and prophylactic agents to control the emerging threat of middle east respiratory syndrome (mers) middle east respiratory syndrome coronavirus (mers-cov) entry inhibitors targeting spike protein a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov mutations in the spike protein of mers-cov transmitted in korea increase resistance towards antibody-mediated neutralization quantifying residual hiv- replication in patients receiving combination antiretroviral therapy antiviral effect of double and triple drug combinations amongst hiv-infected adults: lessons from the implementation of viral load-driven antiretroviral therapy the challenge of finding a cure for hiv infection combination of candidate microbicides cellulose acetate , -benzenedicarboxylate and uc has synergistic and complementary effects against human immunodeficiency virus type infection combinations of the first and next generations of human immunodeficiency virus (hiv) fusion inhibitors exhibit a highly potent synergistic effect against enfuvirtidesensitive and -resistant hiv type strains synergistic efficacy of combination of enfuvirtide and sifuvirtide, the firstand next-generation hiv-fusion inhibitors we thank lanying du at the new york blood center for providing the plasmid encoding mers-cov s protein with mutation in rbd and tianlei ying at fudan university for providing mab m . the authors declare no competing financial interests.viruses , , key: cord- - pkrg mx authors: mcbride, ruth; van zyl, marjorie; fielding, burtram c. title: the coronavirus nucleocapsid is a multifunctional protein date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: pkrg mx the coronavirus nucleocapsid (n) is a structural protein that forms complexes with genomic rna, interacts with the viral membrane protein during virion assembly and plays a critical role in enhancing the efficiency of virus transcription and assembly. recent studies have confirmed that n is a multifunctional protein. the aim of this review is to highlight the properties and functions of the n protein, with specific reference to (i) the topology; (ii) the intracellular localization and (iii) the functions of the protein. coronaviruses (covs) have a global distribution and infect a variety of human and animal hosts, causing illnesses that range from mostly upper respiratory tract infections in humans to gastrointestinal tract infections, encephalitis and demyelination in animals; and can be fatal [ , ] . the international committee for taxonomy of viruses (ictv) reports four coronavirus genera, namely alphacoronaviruses, betacoronaviruses, gammacoronaviruses and deltacoronaviruses [ ] . covs are enveloped single-stranded, positive-sense rna viruses with genomes ranging between . - . kb, the largest among known rna viruses [ ] . this large, capped and polyadenylated genome contains seven open access common coronavirus genes in the following conserved order: '-orf a-orf b-s-orf -e-m-n- ' [ ] . orf a/b encompasses two-thirds of the genome and produces a genome-length mrna (mrna ) that encodes two overlapping viral replicase proteins in the form of polyproteins a (pp a) and pp ab [ ] . these polyproteins are formed as a result of a - ribosomal frame shift that involves a complex pseudoknott rna structure [ ] and are then proteolytically processed by virally encoded proteases into mature nonstructural proteins (nsp to nsp ), which assemble to form a membrane-associated viral replicase-transcriptase complex (rtc) [ , , ] . the last third of the genome produces subgenomic (sg) mrnas that encode the four structural proteins, spike (s), envelope (e), membrane (m), and nucleocapsid (n), as well as a number of accessory proteins [ , ] . amino acid sequence comparisons have shown that cov n proteins have three distinct and highly conserved domains: two structural and independently folded structural regions, namely the n terminal domain (ntd/domain ) and c-terminal domain (ctd/domain ), which are separated by a intrinsically disordered central region (rna-binding domain/domain ) ( figure ); all three domains have been shown in different covs to bind with viral rna [ ] [ ] [ ] [ ] [ ] [ ] . [ , , ] . the ntd is divergent in both sequence and length. it has been mapped for infectious bronchitis virus (ibv)-n to aa - [ ] , for severe acute respiratory syndrome human coronavirus (sars)-n to aa - [ ] , and for mouse hepatitis virus (mhv)-n to aa [ ] . the n-termini of these three covs have been found to associate with the ' end of the viral rna genome, possibly through electrostatic interactions [ , ] . there are several common characteristics of cov n protein ntds, including predicted secondary structures such as a central β-sheet platform flanked by α-helices [ ] , with a basic rna binding groove along the β-platform and an extended β-hairpin. the ntd is enriched in aromatic and basic residues and the folded shape resembles a hand with basic fingers that extend far beyond the protein core, a hydrophobic palm, and an acidic -wrist‖ [ ] . it has been proposed that the flexible, positively charged finger-like β-hairpin extension in the ntd of both ibv and sars-cov n protein is able to grasp rna by neutralizing its phosphate groups, while the base moieties can make contact with exposed aromatic residues from the hydrophobic palm [ , ] . more precise mapping of the rna-binding site locations has been determined for sars-and ibv-n protein. within the ntd of sars-cov-n, positively charged lysine and arginine residues have been proposed to bind a nucleotide stem-loop structure located at the ' end of the sars-cov rna genome [ ] . site-directed mutagenesis studies on ibv-n have identified two residues that are critical for rna binding; namely tyr- and arg- [ ] . tyr- is located in strand β of the four-stranded anti-parallel β sheet; arg- is located in the immediate vicinity of tyr- , at the base of the extended flexible hairpin loop [ ] . it is however likely that, since no single mutation totally disrupts rna binding, other aromatic/basic residues at the surface of the ntd contribute to nucleic acid binding by creating a broad surface that comes into contact with the viral genomic rna [ ] . the ntd possesses some features similar to those of other rna-binding proteins that form a rnp. for example, the u a spliceosomal protein [ ] and the coat protein of ms bacteriophage [ ] bind viral rna with residues arising from the surface of a four-stranded anti-parallel β sheet. seemingly, strands β , β , and the flexible β-hairpin from the ibv n protein could fulfill a comparable role by interacting with phosphate groups on the viral rna [ ] . the arg- and tyr- residues in the ibv n protein are well conserved across the whole cov family, and may structurally correspond to the arg- and tyr- residues in the sars-cov n protein [ ] , meaning that arg- and tyr- may therefore be critical for sars n-rna binding. the crystal structure of mhv n (residues - ) adopts a u-shaped β-platform containing five short β-strands (arranged β -β -β -β -β ) across the platform with an extended β '-β ' hairpin similar to ntds from other cov n proteins [ ] . interestingly, the crystal structure of the mhv ntd shares a similar overall and topology structure with that of sars-cov and ibv but varies in its potential surface, indicating a possible difference in rna-binding module [ ] . it has been shown that n , an mhv-a n domain protein fragment that contains the folded ntd and the immediately adjacent intact linker region (lkr; residues - ), binds to the trs in the viral genome body (trs-b) and complementary trs (ctrs) with high affinity to form a n -trs duplex [ ] . mhv trs binds across the β-platform of ntd in a defined orientation, with the '-end of trs near β and the '-end of trs near β ; this n binding to single-stranded rnas-containing the trs or ctrs-uses base stacking interactions between aromatic side chains on the β-platform with a triple adenosine motif within the trs, '-gaaucuaaacu- ' [ ] . furthermore, due to its potent helix-destabilizing activity, n is able to efficiently melt an rna duplex between the template trs and nascent ctrs strand into component single strands that may be transiently formed during discontinuous transcription of viral sgrna by the coronaviral replicase complex [ ] . three residues on the β-platform have been shown to play key roles in trs binding and helix destabilization: arg- and tyr- on the β strand and tyr- on the β strand, suggesting that the aaa motif in the '-end of the trs is anchored here [ ] . these three residues are completely invariant in betacoronavirus n proteins and occupy precisely analogous positions on the fold of each ntd, and are therefore likely to define similar rna binding grooves in the closely related sars ntd [ ] . the duplex formation and duplex trs unwinding activity exhibited by n therefore implicates mhv ntd in template switching during discontinuous sgrna transcription [ , ] . moreover, the ability of the ntd to melt dsrna may also play a role in rna packaging or other steps in the viral life cycle where rna remodeling is needed [ ] . for example, mutations that cripple duplex unwinding are defective in stimulating cov replication in bhk-r cells, and are lethal, providing evidence of a critical role for ntd in viral replication [ , ] . cov n proteins have also been recognized as rna chaperones [ , ] , which, as part of their chaperone activities, anneal nucleic acids, and so rna duplex destabilization activity may be important in cov n ntds role in assisting viral rna in reaching its functional three-dimentional structure. viral nucleocapsid and replication accessory proteins from other viruses have also been shown to function as rna chaperones and facilitate helix destabilization, for example hiv- ncp protein [ ] , and adenovirus dna binding protein [ ] . the ntd is separated from the ctd by an intrinsically disordered middle region referred to as the linker region (lkr). the charged lkr is also known as the sr-domain because it is rich in serine and arginine residues [ ] , and it is involved in cell signaling [ , , ] . the flexible lkr is capable of direct interaction with rna under in vitro conditions [ ] . potential phosphorylation sites have been mapped to the ser/arg-rich portion of the lkr of sars-cov n [ ] [ ] [ ] . these lkr phosphorylation sites are thought to function in binding m protein, heteronuclear ribonucleoprotein (hnrnp-a ) and rna to the n protein with high binding affinity [ , [ ] [ ] [ ] . there are conflicting reports regarding the involvement of the lkr in n protein oligomerization. some studies has suggested that the lkr is directly involved [ ] and that through electrostatic effects, hyperphosphorylation of the lkr reduces the total positive charge on the sars-cov n protein and leads to enhanced oligomerization of di-domain constructs [ ] . other studies have, however, reported that the lkr interferes with oligomerization when the ctd is present [ ] or if the lkr is phosphorylated [ ] . despite almost no structural information being available for the lkr, possibly due to its high positive charge and flexible nature [ ] , there is evidence in support of the functional importance of intrinsically disordered regions in proteins for modulating transcription, translation, post-translational modifications such as phosphorylation, and cell signaling [ ] . rna chaperones often have structural flexibility because the rna-protein recognition process often requires conformational changes in the rna, the protein or both [ ] . an interaction between n protein and a subunit of the viral replicase-transcriptase complex, namely non-structural protein (nsp ), has been described and key binding determinants localize to the lkr [ , ], highlighting the importance of this unstructured region for a number of potential interactions, such as viral infectivity [ ] . it has also been proposed that nsp binding induces a conformational change in the lkr, potentially regulating the intracellular localization of n to the site of replication [ ] and/or other rna binding functions of n. the ctd, which is a hydrophobic, helix-rich terminal, has been mapped for sars-n to aa - [ ] , and for ibv-n to aa - [ , ] . this domain is also referred to as the dimerization domain because it contains residues responsible for self-association to form homodimers, as well as homo-oligomers through a domain-swapping mechanism [ , , , [ ] [ ] [ ] [ ] [ ] . oligomerization of n protein is necessary to produce a stable conformation because in its monomeric form, the ctd folds into an extended conformation with a large cavity in its center, making it unstable [ ] . sequence comparison shows that the dimerization domain of the n protein is conserved at least among the alpha, beta and gamma groups of covs, suggesting a common structural and functional role for this domain [ ] . the monomer of csars-n, a crystalized c-terminal construct of sars-n that contains residues - , comprises five short α-helices, one helix, and two β-strands [ ] . the general shape of the monomer resembles the letter c, with one edge formed by a β-hairpin extending away from the rest of the molecule [ ] . this structure is similar to the crystalline structure of another sars n ctd monomer (np - ), which consists of eight α-helices and two β-strands [ ] . the csars-n dimer interface is formed largely by insertion of the β-hairpin of one subunit into the cavity of the opposite subunit, resulting in the four β-strands of the two subunits forming an anti-parallel β-sheet that is superposed by two long alpha helices [ ] . due to the extensive hydrogen bond formation between the two hairpins, together with hydrophobic interactions between the beta-sheet and the alpha helices, this interface is highly stable [ ] , and these interactions suggests that the dimeric structure may in fact represent the functional unit of the n protein [ ] . the crystal structure of np - , a sars-cov ctd spanning residues - , revealed that the n protein dimer has the shape of a rectangular slab in which the four-stranded β-sheet forms one face of the slab and the α-helices form the opposite face [ ] . similarly to csars n, the dimerization interface of np - is composed of four β-strands and six α-helices, with each protomer contributing one β-hairpin and helices α , α and α . the two β-hairpins form a four-stranded intermolecular β-sheet that is stabilized through extensive hydrogen bonding. the other part of the dimerization interface is composed of helices α and α , where strong hydrophobic interactions involving trp , ile , pro , phe and phe were observed. the dimer is further stabilized by hydrophobic interactions between the longest helix, α , and the intermolecular β-sheet [ ] . similarly to csars-n and np - , a nuclear magnetic resonance (nmr) study has reported secondary structural assignments of a sars n protein construct whose dimeric interface also consists of a four-stranded anti-parallel β-sheet and two α-helices [ ] . self-association of the n protein has been observed in many viruses, and is required for the formation of the viral capsid which protects the viral genome from extracellular agents [ ] . in addition to the ability of n protein to oligomerize, viral capsid formation also requires rna-binding ability [ ] . studies revealed that sars-cov n protein fragments containing the dimerization domain (residues - ) can bind to a putative packing signal within the viral rna, with the most likely rna binding site being within the basic region between residues - [ ] . nmr studies then showed that the rna-binding site between residues - formed part of the complete dimerization domain structure [ ] . it was not until the crystal structure of sars n ctd was resolved that the molecular basis of rna-binding activity and organization of the ctd octamer was determined. the ctd spanning residues - (np - ) revealed that, due to the presence of the eight positively charged lysine and arginine residues, amino acids - form a positively charged groove, one of the most positively charged regions of the n protein [ ] . this groove is similar to that in ibv-n ctd, except that the positively charged surface area is larger in the sars-cov construct than in the ibv [ ] , due in part to the presence of additional negatively charged residues in the ibv n protein and in part due to the absence of residues - from the ibv construct, which contain two lysine residue in the sars-cov construct [ ] . the np - construct, which contains both the charge-rich region (residues - ) and dimerization core (residues - ) of the dimerization domain, is capable of binding to single-stranded rna (ssrna), single-stranded dna (ssdna) and double-stranded dna (dsdna), and np - has stronger nucleic acid-binding activity than the ntd [ , ] . the strong electrostatic character of residues - and the fact that both ssrna, ssdna and dddna bind to np - , strongly indicates that oligonucleotide binding is based on non-specific charge interactions between the positively charged protein and the negatively charged nucleic acid backbone [ , ] . by keeping the rna-binding domains in close proximity to the ctd, the formation of a large helical nucleocapsid core is therefore possible [ ]. association of the n protein dimers is necessary for further assembly of the core. the full-length dimeric n protein has a tendancy to form tetramers and higher molecular weight oligomers in vitro [ ] , and a serine/arginine-rich motif (residues - ) has been shown to be important for n protein oligomerization [ ] . two dimers arrange themselves into a butterfly-shaped tetramer, while two butterfly-shaped tetramers unite to form an octamer in the asymmetric unit of the ctd crystal [ ] . the octamer is held together through hydrophobic interactions and hydrophilic contacts among the four dimers, and networks of inter-dimer hydrogen bonds further help stabilize the octamer [ ] . crystallography studies have demonstrated that cov-sars ctd (np - ) packs as an octamer which stacks to form a helical supercomplex structure with a continuous positively charged surface that could potentially allow viral rna strands to bind and wrap around the helical oligomer structure through electrostatic interactions [ ] . the existence of transient self-association between dimers in solution was confirmed using a disulphide trapping technique, and it was shown that by neutralization of excessive charges on the protein, either through environmental charge screening or charge modifications, this transient self-association can be regulated [ ] . this proposed biophysical mechanism whereby electrostatic repulsion between n protein molecules acts as an oligomerization switch has implications for understanding how nucleocapsid assembly is then subsequently modulated [ ] . in addition, the ctd residues of the mhv n protein have been shown to be the major determinant for interaction with the m protein [ ] , and so association of the n protein with the m protein may also play a role in the assembly of the nucleocapsid core into a progeny virion [ ] . oligomerization via the ctd has also been reported in human coronavirus e (hcov- e) and a recent study has shown that the c-terminal tail peptide, an intrinsically disordered domain that flanks the ctd, plays an important role in dimer-dimer association [ ] . the c-terminal tail interferes with oligomerization of the ctd and has an inhibitory effect on viral titer of hcov- e; and further understanding this mechanism of oligomerization may provide insight into the viral assembly process and could identify additional targets for drugs to combat covs through the disruption of the n protein self-association [ ] . the ctd of sars-n (aa - ) is also responsible for stress granule localization that occurs as part of an integrated stress response in arsenite-treated hela cells [ ] . once sequestered in these granules, the n protein can induce host translational shutoff. the ntd and the ctd are interspersed by intrinsically disordered regions (idrs) [ , ] . intrinsically disordered proteins (idps) or idrs lack a tertiary structure and have no fixed -dimentional shape in the native form. however, idps and idrs play a role in various biological functions including dna, rna and protein binding with the disordered regions facilitating access to these binding sites [ ] [ ] [ ] . in fact, the three idrs in sars-n (aa - , - and - ) have all been shown to modulate the rnd-binding activity of the ntd and ctd [ , ] . moreover, both the middle and c-terminal idrs (figure ) have been implicated in the oligomerization of the n protein [ , ] , with the middle idr also associated with n protein functionality and n-m interaction [ , , , ] . it would be interesting to determine whether the presence of three disordered regions in sars n, compared to the one disordered region in hcov-nl n for example, would result in sars n having a higher binding affinity to viral, as well as host cellular proteins. if indeed so, could this then indicate a probable basis for the increased pathogenicity of sars-cov compared to hcov-nl ? in order for cov n proteins to package the viral genome with structural proteins to form ribonucleoprotein (rnp) complexes for viral assembly, two key activities are required: the interaction between protein and nucleic acid and the ability of the complex to oligomerize [ ] . the n proteins of sars-cov, ibv and mhv have all been shown to perform both these functions. sars-cov-n protein interacts with rna at multiple sites, with all three domains having charged regions [ ] . the crystal structures of the ntd and ctd domains of the n protein from sars-cov, ibv and mhv all share a similar overall and topology structure, which corroborates a conserved mechanism of nucleocapsid formation for covs [ ] . furthermore, despite a lack of significant sequence similarity, the csars-n had a similar fold to that of the n protein of porcine reproductive and respiratory syndrome virus, a member of arteriviridae family, suggesting an evolutionary link between coronaviridae and arteriviridae in which the n proteins of both viruses have a common origin [ ] . in fact, due to their similar genome organization and viral replication mechanisms, the coronaviridae and arteriviridae were united to form the relatively new order nidovirales. in virus-infected cells, cov n proteins can localize to the cytoplasm alone or to the cytoplasm and nucleolus [ ] . proteins that are able to localize to the cytoplasm, nucleus and/or nucleolus require multiple signals to determine their subcellular localization [ ] . cov n proteins commonly localize in the nucleolus, and although nucleolar localization/retention signals (norss) and pathways are not well characterized, nucleolar localization usually requires regions in the protein that are rich in arg residues and is likely cell-cycle dependent [ , , ] . the n protein of ibv was found to localize in the cytoplasm alone or to co-localize in both the cytoplasm and nucleolus [ , , ] . ibv n protein contains a functional nuclear export signal (nes) to traffic n protein to the cytoplasm [ , ] , and an amino acid nors motif at its ntd and is necessary and sufficient for nucleolar retention [ ] . it is hypothesized that the localization of ibv-n to the nucleolus forms part of a virus strategy to control sgrna synthesis in both the host cell and virus by associating with ribosomal subunits [ ] and interacting with nucleolar proteins, nucleolin and fibrillarin [ ] . importantly, this interaction is not direct, but mediated through rna and could therefore simply be an artifact of the proteins having rna-binding domains [ ] . thus, the nucleolar localization could simply be due to a high density of the host rna attracting a viral rna-binding protein. even so, it has been postulated that the nuclear localization of the n protein may interfere with cellular machinery and thus lead to triggering of apoptosis [ ] . the localization of n to nucleoli alone might be cell cycle dependent, because the number and size of nucleoli differ at different stages of the cell cycle: at the beginning of g phase, multiple nucleoli can be found, but only single nucleoli can be seen at later g , s and g phases [ , ] . it was also found that domain of ibv-n predominantly localizes in the nucleus, but when fused with domain (ctd) it localizes to the cytoplasm and thus supports the findings of other studies done on ibv-n localization [ , ] . the ability for nucleolar localization varies between n proteins of different covs. unlike other cov n proteins, sars-cov n protein is mostly distributed to the cytoplasm [ , , ] . this cytoplasmic localization is somewhat unexpected because there is at least one nors in domain and eight putative nuclear localization signal (nls) motifs within domains i and ii of the sars-cov n protein [ ] , of which the short lysine-rich sequence ( - ) near the carboxy-terminus is a putative bipartite nls that is unique to sars-cov n [ , ] . as a reason for this, it has been suggested that signals for nuclear and nucleolar targeting of sars-cov n protein are poorly accessible to nuclear import machinery due to phosphorylation or conformation restraints [ ] . a cytoplasmic nes may be involved in also over-riding the nls, resulting in significantly less n protein (only %) being localized to the nucleolus [ ] . shuttling of n protein from the nucleus to the cytoplasm occurs through phosphorylated-dependent binding of sars-cov to - - , with the absence/inhibition of this - - molecule resulting in increased nuclear localization of sars-n [ ] . also, the deletion of the sr-rich domain contained within the middle region of sars-n can result in dramatic changes in sub-cellular localization of n compared to wild-type n [ ]. these results indicate that the localization of n protein to the nucleus or nucleolus is not a conserved property of nidovirales [ ] . the primary role of cov n protein is to package the genomic viral genome into long, flexible, helical ribonucleoprotein (rnp) complexes called nucleocapsids or capsids (table ) . the nucleocapsid protects the genome and ensures its timely replication and reliable transmission. the filamentous nucleocapsids are to nm in diameter and several nm in length, and these macromolecular structures are visible using electron microscopy [ ] . within the nucleocapsid there are both n-rna interactions as well as intermolecular association between disulfide-linked n protein multimers [ ] . the n-rna interaction is mediated by binding signals contained within the leader rna sequences [ ] . during the virus life cycle, multiple copies of the n protein interact with grna and sgrna molecules, indicating a role for n protein in viral transcription and translation [ , ] . the basic building block for cov nucleocapsid formation is a dimeric assembly of n protein [ ] , and it is the ctd of n protein that possesses dimerization function [ ] . a structural model of cov proposes that n protein is not only present in the helical nucleocapsid but also in the internal spherical/icosahedral core [ ] . the internal core consists of n protein, rna and the ctd of m protein. the m protein is the main core shell component and a amino acid domain (aa - ) on the ctd of m protein binds directly to n protein via an ionic interaction, leading to specific genome encapsidation in the budding viral particle [ ] [ ] [ ] .the n protein therefore plays an essential structural role in the cov virion through a network of interactions with (i) the genomic rna; (ii) m protein and (iii) other n proteins. assembly of virus particles is an essential step for a productive viral replication cycle. cov virions contain three envelope proteins, m, e and s, and a viral nucleocapsid, which consists of genomic rna and n protein, within the viral envelope. [ , ] . it is believed that the interaction of the nucleocapsid with envelope proteins drives the incorporation of the nucleocapsid in enveloped viruses [ ] , and such protein-protein interactions are critical for viral assembly, as has been shown for alphaviruses [ , ] . n and m proteins are the two major structural proteins in cov virions [ ] . the m protein is anchored by its three transmembrane domains to the viral envelope and its large carboxy-terminal tail in the virion interior interacts with the nucleocapsid [ ] . the nucleocapsid consists of the positive strand genomic rna, mrna , helically encapsidated by n protein monomers, and the n protein region that interacts with the c-terminus of the m protein domain seems to be cov specific. the intracellular sites of virus assembly also vary among different viruses [ , ] . in mhv, the large carboxy-terminal domain of the m protein interacts with the ctd of the n protein [ ] . newly synthesized, unglycosylated m protein interacts with n protein at the er membrane, which is a pre-golgi compartment that is also the site of mhv budding [ ] , suggesting that the site of interaction overlays with mhv budding sites [ ] . the m protein-nucleocapsid interaction is thought to be initiated by a direct binding of m protein to genomic rna that is mediated by a nucleotide (nt) packaging signal (ps) present only on the mrna association [ , ] . these ps is located about kb from the ' end of mhv mrna [ , ] , is necessary and sufficient for packaging rna into mhv particles [ ] , and it has been suggested that the m protein-ps interaction could lead to the association of m protein with n protein, thereby stabilizing the complex between m protein and the nucleocapsid [ ] . although the m protein-nucleocapsid interaction could theoretically also be initiated by direct binding of n protein to genomic rna, this is unlikely because n protein interacts with all mrnas [ , ] , which makes it difficult to explain how the formation of n protein-mrna rnp complex might lead to specific packaging of genomic rna, and not sgrna, into virus particles [ ] . it has since been conclusively demonstrated that m protein selectively interacts with ps-containing rna in the absence of n protein, indicating that the mechanism of m-ps recognition does not require the formation of rnp complex by n protein, and in fact, n protein is not required for rna packaging in that model [ ] . mhv m protein was the first example of a viral transmembrane protein that could bind to a specific viral rna element in the absence of any other viral structural proteins [ ] , and a proposed model for rna packaging in mhv suggests that once m protein accumulates and oligomerizes in the intermediate compartment between the er and golgi complex, m protein binds to ps-mrna , and only thereafter does n protein associated with mrna interact with oligomerized m protein [ ] . although n protein appears to be dispensable for mhv rna packaging, n-m interaction might be important in compensating for viral envelope defects that occur due to m protein mutation. the m protein carboxy terminus is extremely sensitive to mutations, and removal of even only the last two amino acid residues from the tail of the m protein appears to be lethal [ ] . interestingly, n protein becomes mutated in its ctd, and these changes then compensate for the loss of the two m protein residues, either by increasing the affinity of an adjacent interaction or by providing a new contact point between n and m to stabilize the virion [ ] . for the porcine transmissible gastroenteritis coronavirus (tgev), an interaction between the carboxy terminus of m and nucleocapsid has been mapped to residues - of the tgev m protein [ ] . this segment corresponds to residues - of the mhv m protein [ ] , which overlaps with only one of the critical residues identified in the mhv m protein [ ] . this region of the two m proteins is, however, poorly conserved and the apparent disagreement between the tgev and mhv results may relate to differences in the respective folds of the m proteins, or differences in how these residues influence those folds [ ] . sars-cov is markedly different from other members of the coronaviridae family in the sense that there is only %- % amino acid identity with other known covs, with both the n and m proteins having low sequence homology [ , ] . one might therefore expect that there could be differences in the mechanism of viral assembly. a mammalian two-hybrid system, which is performed in vivo so that viral proteins will adopt their native state and therefore be more likely to interact in a biologically accurate manner [ ] , confirmed that n-m protein interactions occur in vivo [ ] . moreover, this study identified a stretch of amino acids in the middle of the n gene that may be critical for n-m protein interaction [ ] . this stretch of amino acids spans the lkr and dimerization domain in the ctd, suggesting that this region may be essential in maintaining correct n protein conformation for both self-association and n-m protein interaction [ ] . despite sars-cov having the closest genetic resemblance to mhv, the m proteins of these viruses bind to different domains on the n protein. covs assemble and bud intracellularly at the er-golgi complex [ , ] , and association of the nucleocapsid with this organelle may reflect a role in virus budding. the formation of the virion envelope requires expression of only m-and e-protein, and not n protein, as has been observed for mhv [ ] , ibv [ ] , tgev [ ] , and bcov [ ] . it was recently noted however, that these studies all used vaccinia-based expression systems, where overexpression of viral membrane proteins may lead to release in microvesicles, complicating the interpretation of virus-like particle (vlp) experiments [ ] . subsequent experiments using transient transfection to express the proteins from plasmids have shown that, at least for mhv [ ] , sars-cov [ ] and ibv [ ] , the presence of n protein can greatly increase vlp yield. therefore, while n protein is not necessarily required for envelope formation, n protein plays an important role in forming a complete virion [ ] . n protein binds to both full-length genomic rna (grna) as well as all six sgrnas but displays an increased affinity for grna [ ] . the grna functions as a template for the viral rna-dependent rna polymerase as well as a message for translation [ ] . during infection, grna is initially transcribed by an early polymerase activity into a genome-sized negative-stranded rna [ ] and then a late polymerase activity transcribes the negative-stranded rna into a full length grna that is bound to polysomes [ ] and detected in nucleocapsid structures [ ] . numerous studies have demonstrated that n protein is required for optimal cov replication [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the participation of n protein in an early event in rna synthesis is implied by at least two things: firstly mhv-and sars-cov n protein colocalize intracellularly with replicase components at early stages of infection [ ] [ ] [ ] [ ] ; and secondly, stimulation of grna infection is dependent upon n protein translation [ , ] . a more clearly defined role for n protein in grna synthesis was delineated when an interaction between the sr region of the mhv n protein and a region in the amino terminal segment of the nsp subunit of the viral replicase was discovered [ , ] . the n-nsp interaction has been specifically mapped to the ubiquitin-like domain (ubl ) of nsp , an essential domain to the virus [ ] . moreover, it was also shown that this n-nsp interaction is required for n protein to promote optimal infectivity of grna [ ] . it was proposed that the formation of an initiation complex at the ' end of the genome requires n protein to tether grna to the newly translated replicase via its interaction with nsp [ , ] . considering that the start of negative-strand synthesis is the first step in both genomic replication and transcription [ , ] , it is likely that this n-nsp interaction is important in both of these rna-synthetic processes [ ] . the role of n protein in mhv grna synthesis may not necessarily be limited to early time points of infection as it has also been proposed that n protein sustains ongoing transcription throughout the course of infection [ , ] . the role of cov n protein in rna synthesis remains controversial as n protein is not essential for tgev rna replication but rather for efficient transcription [ ] . rna chaperone proteins assist the proper folding of nucleic acids [ , ] . chaperone activity has been postulated as a general activity of all cov n proteins, and has been demonstrated for tgevand sars-cov n proteins [ ] . it has been proposed that amino acids - in the central disordered domain (the lkr domain) of the tgev n protein has chaperone activity [ ] , and that this region facilitates template switching in vitro by decreasing the energy barrier needed to dissociate the nascent minus rna chain from the grna template during discontinuous rna transcription [ , ] . this facilitation of template switching is required for efficient transcription and may explain how the presence of n protein therefore increases rna synthesis. deregulation of the cell cycle is a common strategy adopted by many rna (and dna) viruses aimed at exploiting host cell machinery in order to create a more favorable environment for their survival. one of the mhv nonstructural proteins, p , induces g /g cell cycle arrest by inhibiting hyperphosphorylation of retinoblastoma protein (rb), a step which is necessary for cell cycle progression through late g into the s phase [ ] [ ] [ ] . a model was proposed whereby expressed cytoplasmic p induces stabilization of p , and accumulation of p causes transcriptional upregulation of p , a cyclin-dependent kinase (cdk) inhibitor, leading to suppression of cyclin e/cdk activities, and the resulting reduction in g cyclin-cdk complexes and cdk activities ultimately inhibits rb hyperphosphorylation [ ] . certain herpes simplex virus gene products [ , ] , cytomegalovirus gene products [ , ] , and zta of epstein-barr virus [ ] have similarly also been shown to arrest cell cycle progression at the g phase. ibv infection also perturbs cell cycle progression and arrests cells in the s and g /m phases, and this occurs partly through the interaction of ibv nsp and dna polymerase delta [ ] . the n protein of sars-cov also modulates the host cell cycle by regulating cyclin-cdk activity such that s phase progression is halted. the n protein bears a signature cyclin box-binding region (rxl motif), can be phosphorylated by cdk and is therefore a substrate for the cyclin-cdk complex [ ] . n protein has been shown to have an inhibitory effect on s phase cyclins cdk and to a lesser extent cdk [ ] . the inhibition of kinases means that rb remains hypophosphorylated and cannot release e f , and the transcription of s phase genes is halted [ ] . similarly to mhv p , sars-cov n protein also inhibits cdk activity, meaning that by blocking the activity of both g and s phase cyclins, n protein doubly ensures the blockage of s phase progression [ ] . n protein mediated inhibition of cdk activity; leading to inhibition of rb phosphorylation, is independent of cdk inhibitors (cdkis) such as p , and so it has been suggested that the n protein mimics the role of cdkis by acting as a competitive inhibitor to cdk and cdk [ ] . the ability of n protein to inhibit cdk activity was dependent on the rxl motif, whereas its ability to inhibit cdk requires at least two mechanisms: the cdk phosphorylation motif and dependence on host cell signaling pathways [ ] . it has been postulated that, p -mediated (mhv) and n protein-mediated (sars) blocking of the s phase, allows these virus enough time to utilize the cellular raw materials that have been synthesized ahead of the s phase, for replication of its genome, as well as for assembly and budding of progeny particles [ ] . these studies provide evidence that even within the cov family, different proteins can deregulate the cell cycle and that those proteins, in the case of sars-n protein, can employ multiple mechanisms to achieve this. in response to environmental stress, eukaryotic cells reprogram their mrna metabolism to adapt to stress-induced damage. translationally stalled mrnas together with a number of translational initiation factors and rna-binding proteins are selectively deposited in cytoplasmic stress granules (sgs) [ ] . covs, like many other viruses, induce host translational shutoff, while maintaining synthesis of their own gene products, as a means to interfere with cellular defense mechanisms [ ] . mhv-induces a host translational shutoff that is reminiscent of a cellular stress response; sgs appear, numerous mrnas including protein translation-related factors are downregulated and levels of phosphorylated translation initiation factors increase [ ] . it is important to remember, though that this translational shutoff could also simply be the host cell's response to the infection. in sars-infected hela cells that are exposed to arsenite treatment, n protein translocates to cytoplasmic sgs where it colocalizes with poly(a)-binding protein (parb) and transiently expressed tia- [ ] , both of which are components of sgs [ ] . this localization and pattern was enhanced when the protein was hypophosphorylated (nΔrs), perhaps because nΔrs induced larger ribonucleoprotein (rnp) complex formation [ ] . the sequestration of n protein in sgs possibly indicates a role in translational suppression. the nucleocapsid protein of both porcine and respiratory syndrome and rubella virus also interact with parb, which promotes protein synthesis by circularizing mrnas, and inhibit host protein synthesis by sequestration of parb through a stoichiometric mechanism [ , ] . it has been proposed that, at least in the rubella virus, that n-dependent inhibition of translation via parb binding may facilitate the switch from viral translation to packaging rna into nucleocapsids [ ] . more specifically, binding of the capsid to parb late in the virus cycle, when the levels of newly synthesized genomes and viral proteins are high, and there is little need for additional replicase components, could prevent recruitment of ribosomes to the nascent s rna. this scenario would favour packaging of the s genomic rna into nucleocapsids [ ] . considering that sars-n and mhv-n protein has been shown to interact with the parb hnrnp-a [ , ] , it is possible that the n-parb interaction also acts as a switch that redirects viral activity from rna synthesis to nucleocapsid formation. the translational suppressive effect of the n protein could also be through its ctd interaction with elongation factor α (ef α), a major translational factor in mammalian cells [ ] . n protein of sars-cov binds directly to and induces aggregation of ef α, resulting in inhibition of protein translation [ ] . moreover, ef α is a multifunctional protein and has unconventional functions related to its association with the cytoskeleton, including interaction with filamentous (f) actin, promotion of f actin bundling and formation of a contractile ring during cytokinesis [ ] [ ] [ ] [ ] . n-ef α therefore blocks ef α-mediated f actin bundling and the formation of the contractile ring, leading to the formation of multinucleated cells by inhibiting cytokinesis [ ] . multinucleated syncytia of macrophages have been observed in late-phase sars-cov but not in other cov-infected patients [ ] . the n protein of hcov- e also binds to ef α, but with significantly lower affinity than sars-cov n, and induces multinucleation much more slowly in a substantially smaller fraction of transfected cells [ ] . in addition, n-protein mediated inhibition of cytokinesis leads to inhibition of cell proliferation in human peripheral blood lymphocytes (pbls) [ ] . since actively dividing lymphocytes are a major cell target of sars-cov [ ] , sars-cov n may decelerate lymphocyte proliferation and therefore interfere with immune system function [ ] . sars-n protein has also been shown to cause cytoskeletal changes in response to a different type of cell stress, namely serum deprivation [ ] . n protein induces actin reorganization in cos- monkey kidney cells by down regulating focal adhesion kinase and fibronectin via p mapk pathway activation [ ] . n protein plays an important role in viral pathogenesis since anti-n monoclonal antibodies protect mice from lethal infection of jmhv [ ] . coronaviruses seem to have developed mechanisms to overcome the host innate immune response, including the interferon response, a major component of innate immunity. sars-cov infected cells do not allow production of interferon and so it seems likely that sars-cov, like many other viruses, has developed mechanisms to subvert the interferon response [ ] . sars-cov n protein is one of three β interferon (ifβ) antagonists, with orf b and orf being the other two, and inhibition of ifβ synthesis by n protein is due to inhibition of irf- and nf-kb, two of the transcription factors required for if gene expression [ ] . sars-cov n protein inhibits the synthesis of type- interferon ( fn) and the ctd of n has been shown to be critical for antagonism of fn induction [ ] . this inhibition of the interferon response likely contributes to the pathogenesis of sars-cov. cov infection is likely to activate a diversity of host cell signal transduction pathways and kinases, which would lead to phosphorylation of n protein [ ] . n protein phosphorylation would therefore only occur at a relatively late stage of the viral cell cycle and may explain why more phosphorylated n protein is incorporated into ibv virions [ ] . it has been demonstrated that the sars-cov n protein can bind to dna in vitro [ ] . recently, the n protein of hcov-oc was shown to interact with the transcription factor nuclear factor-kappa b (nf-kappab) [ ] . it was reported that hcov-oc n protein potentiates nf-kb activation as a direct result of the ability of the nucleocapsid to bind microrna mir- , a negative regulator of nf-kb. it was suggested that this previously undescribed interaction between virus and host is a potential mechanism of immune evasion in rna viruses because nf-kb required for if gene expression [ , ] . ] , which could act as a switch that redirects viral activity from rna synthesis to nucleocapsid formation.  interaction of n protein ctd with elongation factor α (ef α), a major translational factor in mammalian cells, can suppress translation [ ] .  n protein plays an important role in viral pathogenesis. mice infected with jmhv protected by anti-n monoclonal antibodies [ ] .  synthesis of type- interferon ( fn) inhibited by sars-cov n [ ] .  the ctd of n has been shown to be a critical antagonist of fn induction [ ] . . signal transduction  activation of host cell signal transduction pathways and kinases leads to phosphorylation of n [ ] . nf-kb is also involved in sars-cov mediated activation of cyclooxygenase- (cox- ), more specifically, n protein binds to nf-kb and ccaat/enhancer binding protein binding sites on the cox- gene promoter [ ] . activation of cox- , a proinflammatory factor, by sars-cov n protein is a likely cause of lung inflammation in sars-cov infected patients [ ] . sars-cov n protein is also known to activate the activator protein (ap ) signal transduction pathway by increasing the amount of transcription factors binding to promoter sequences of c-fos, atf , creb- , and fosb [ ] . sars-cov n protein induces apoptosis of cos- monkey kidney cells in the absence of growth factor by down-regulating extracellular-signal-regulated kinase (erk) and up-regulating c-jun n-terminal kinase (jnk) and p mitogen-activated protein kinase (mapk) pathways, with activation of p mapk also causing actin reorganization in serum-deprived cells [ ] . the coronavirus n protein is abundantly produced within infected cells. n has multiple functions, including binding to viral rna to form the ribonucleocapsid and has also been proposed to have roles in virus replication, transcription and translation. in host cells, n proteins have been shown to cause deregulation of the cell-cycle [ , ] , inhibit the production of interferon [ ] , up-regulate the production of cox [ ] , up-regulate the activity of ap [ ] , and induce apoptosis in serum deprived cells [ ] -of all which may have possible pathological consequences [ ] . several excellent reviews on the coronavirus n protein have previously been published [ , ] , including one on the structure and function of the sars-cov n and its interaction with nucleic acids [ ] . notwithstanding these reviews, the manner in which coronavirus n proteins carry out its roles during the viral life cycle is still not clearly understood. an important piece of missing information lies in the difficulty in resolving the atomic structure of the rnp complex, which has been hampered by low solubility of the rnp complex and the labile nature of the full-length n protein. in addition, in order to determine whether the rnps from various coronaviruses share a common structural code, the structure of different coronavirus rnps need to be resolved [ ] . direct intraviral protein-n interactions identified to date include the interaction between n and m [ , , ] and n and nsp a, a component of the viral replicase [ , ] . additionally, in mhv-infected cells, monoclonal anti-n antibody co-immunoprecipitates both m and s proteins; this n-s interaction is not a direct one though. rather, it is due to the interaction between s and m protein [ ] , where the s protein forms complexes with m protein in the endoplasmic reticulum (er) [ , ] . the identification of host proteins targeted by viral proteins during the infection process provides important insights into mechanisms of viral protein function [ ] . to date, the interaction of n with numerous host cell proteins have been identified, including hcypa [ ] , proteasome subunit p [ ] , the b phosphoprotein [ , ] , smad [ ] , nrnp-a [ ] , the chemokine cxcl [ ] , translation elongation factor- alpha [ ] , cellular pyruvate kinase protein [ ] , - - [ ] and nucleolin [ , ] . more recently, a study using high-throughput mass spectrometry identified a list of cellular proteins that could potentially interact with the ibv n protein [ ] . comparative studies between various coronavirus n protein interactions could provide valuable information on host specificity and evolution of the interactions between n and host cell proteins. in turn, this may offer insight into the development of novel antiviral therapeutics that target interactions between host cell proteins and the n protein [ , ] . sars-cov n protein is extremely antigenic. sars-cov infection causes a highly restricted, immunoglobulin g-dominated antibody 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multifarious activities background paper. functions of the coronavirus nucleocapsid protein protein interactions during coronavirus assembly envelope glycoprotein interactions in coronavirus assembly discovery of host-viral protein complexes during infection nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a interactions of sars coronavirus nucleocapsid protein with the host cell proteasome subunit p co-localization analysis between porcine epidemic diarrhea virus nucleocapsid protein and nucleolar phosphoprotein b . . wei sheng wu xue bao the nucleocapsid protein of sars-associated coronavirus inhibits b phosphorylation severe acute respiratory syndrome-associated coronavirus nucleocapsid protein interacts with smad and modulates transforming growth factor-beta signaling cxcl interact with sars-cov n protein in and out cell sars-cov nucleocapsid protein interacts with cellular pyruvate kinase protein and inhibits its activity the cellular interactome of the coronavirus infectious bronchitis virus nucleocapsid protein and functional implications for virus biology viruses and interactomes in translation antibody response of patients with severe acute respiratory syndrome (sars) targets the viral nucleocapsid generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity immunoreactivity characterisation of the three structural regions of the human coronavirus oc nucleocapsid protein by western blot: implications for the diagnosis of coronavirus infection burtram c. fielding receives funding from the senate research fund, university of the western cape and the national research foundation, south africa. any opinion, findings and conclusions or recommendations expressed in this material are those of the author and therefore the nrf does not accept any liability in regard thereto. the authors apologize to any author whose work was inadvertently omitted from this review. the authors declare no conflict of interest. key: cord- -t v pu w authors: yang, yiming; gaspard, gerard; mcmullen, nichole; duncan, roy title: polycistronic genome segment evolution and gain and loss of fast protein function during fusogenic orthoreovirus speciation date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: t v pu w the reoviridae family is the only non-enveloped virus family with members that use syncytium formation to promote cell–cell virus transmission. syncytiogenesis is mediated by a fusion-associated small transmembrane (fast) protein, a novel family of viral membrane fusion proteins. previous evidence suggested the fusogenic reoviruses arose from an ancestral non-fusogenic virus, with the preponderance of fusogenic species suggesting positive evolutionary pressure to acquire and maintain the fusion phenotype. new phylogenetic analyses that included the atypical waterfowl subgroup of avian reoviruses and recently identified new orthoreovirus species indicate a more complex relationship between reovirus speciation and fusogenic capacity, with numerous predicted internal indels and ’-terminal extensions driving the evolution of the orthoreovirus’ polycistronic genome segments and their encoded fast and fiber proteins. these inferred recombination events generated bi- and tricistronic genome segments with diverse gene constellations, they occurred pre- and post-orthoreovirus speciation, and they directly contributed to the evolution of the four extant orthoreovirus fast proteins by driving both the gain and loss of fusion capability. we further show that two distinct post-speciation genetic events led to the loss of fusion in the waterfowl isolates of avian reovirus, a recombination event that replaced the p fast protein with a heterologous, non-fusogenic protein and point substitutions in a conserved motif that destroyed the p assembly into multimeric fusion platforms. the fusogenic reoviruses are rare examples of non-enveloped viruses that induce cell-cell fusion and syncytium formation [ ] . this diverse group of non-enveloped viruses, with segmented, dsrna genomes, are members of two distinct but related genera in the reoviridae family: the orthoreovirus genus, whose members infect a wide range of vertebrate hosts, and the aquareovirus genus, whose hosts are restricted to freshwater and saltwater fish [ ] . three of the five recognized and sequenced species in the aquareovirus genus are fusogenic. of the seven currently recognized species of orthoreoviruses, only mammalian orthoreovirus (mrv) and piscine orthoreovirus (prv) lack isolates that are fusogenic. isolates of reptilian orthoreovirus (rrv), mahlapitsi orthoreovirus, (marv), nelson bay orthoreovirus (nbv), baboon orthoreovirus (brv), and avian orthoreovirus (arv) all induce syncytium formation [ , ] . furthermore, three additional orthoreovirus species have been proposed, all of which are fusogenic: broome orthoreovirus (brrv) from a bat, neoavian orthoreovirus (arvn) from wild birds, and testudine orthoreovirus (rrvt) from a tortoise ( figure a ). the preponderance of fusogenic over orthoreoviruses are members of the spinareovirinae subfamily, whose virions have large turrets at the icosahedral vertices. the double-layered capsid contains eight structural proteins, five of which comprise the transcriptionally active core particle containing the viral rna-dependent rna polymerase (rdrp) [ , ] . two of the three remaining proteins, the outer clamp and outer shell proteins, assemble into heterohexameric complexes and form the outer capsid shell [ ] . during reovirus endocytic cell entry, cathepsins proteolytically degrade the outer clamp protein to expose the underlying outer shell protein that mediates the exit of the core particle from the endocytic pathway into the cytoplasm [ ] . the rdrp functions from within the core particle to transcribe mostly table s for a list of viruses, proteins, and accession numbers. lowercase labels denote isolates within a species: gal and ans are landfowl and waterfowl isolates of avian orthoreovirus (arv); bu and co are bulbul and corvid isolates of neoavian orthoreovirus (arvn). bootstrap values ( replicates) are shown for branch points where the associated taxa clustered together in less than % of the replicate trees. the tree with the highest log likelihood is shown. branch lengths are to scale and measured by the number of substitutions per site. (b) diagrams of the polycistronic s and s genome segments from the indicated orthoreovirus species, drawn to approximate scale. open reading frames are depicted by rectangles, labeled to indicate the protein product and color-coded to depict functional or evolutionary protein relationships: yellow-fast protein; blue-fiber protein; green-potential cell cycle inhibitory protein; purple-homologous proteins with no defined function; pink-nucleocytoplasmic shuttling protein. numbers refer to mrna length. (c) diagrams of the orthoreovirus fast proteins, depicting their topology in the plasma membrane. structural motifs contained within their n-terminal ectodomains and c-terminal cytoplasmic endodomains are color-coded as described in the legend at the bottom. numbers indicate the number of residues in each protein. orthoreoviruses are members of the spinareovirinae subfamily, whose virions have large turrets at the icosahedral vertices. the double-layered capsid contains eight structural proteins, five of which comprise the transcriptionally active core particle containing the viral rna-dependent rna polymerase (rdrp) [ , ] . two of the three remaining proteins, the outer clamp and outer shell proteins, assemble into heterohexameric complexes and form the outer capsid shell [ ] . during reovirus endocytic cell entry, cathepsins proteolytically degrade the outer clamp protein to expose the underlying outer shell protein that mediates the exit of the core particle from the endocytic pathway into the cytoplasm [ ] . the rdrp functions from within the core particle to transcribe mostly monocistronic mrnas from the genome segments that are capped but not polyadenylated by the turret protein during the exit from the core particle [ , ] . at some stage, interactions between the viral rdrp, viruses , , of packaging signals in the viral mrna, and the core shell protein result in the coordinated replication of the mrna and the encapsidation of the dsrna genome segments inside the progeny virus core particles [ , ] . these transcriptionally active progeny core particles drive mrna synthesis and amplification of the virus replication cycle. the three outer capsid proteins eventually assemble on the core particles to generate infectious progeny virions. during the progeny core assembly stage of the replication cycle in cells which have been co-infected with two different reoviruses, heterologous viral mrnas can be encapsidated and replicated inside a single progeny core particle to generate reassortant viruses. the genomes of these reassortants are mosaics of the parental virus genome segments and, as with antigenic shift in influenza viruses [ ] , genome segment reassortment is a major driver of reovirus evolution [ ] . the final structural protein is the fiber protein, referred to as σ in mrv and σc in the fusogenic reoviruses, that forms trimeric spikes at the capsid vertices and serves as the cell adhesin [ ] [ ] [ ] . the reovirus fiber protein comprises a long n-terminal filamentous tail and an~ -residue c-terminal globular head that folds into a compact, eight-stranded b-barrel structure with receptor binding activity [ , ] . the filamentous tail contains three structurally distinct subregions: the n-terminal - residues interact with the turret protein to anchor the spike to the virion capsid; a long α-helical coiled coil domain formed by heptad repeats zips together the trimeric fiber; and a region of triple β-spiral repeats connects the tail to the head [ ] [ ] [ ] . in those fusogenic reoviruses that encode a fiber ( figure b) , the σc fiber is shorter than the mrv σ fibers (~ versus - residues, respectively), due primarily to a reduced number of β-spiral repeats [ ] . in some reovirus species (marv, brv, brrv), the fiber protein orf has been completely replaced by a heterologous sequence encoding a non-structural p protein of no defined function ( figure b ). it is unclear how these fiberless virus particles attach to cell receptors. the genomes of all fusogenic aqua-and orthoreoviruses contain an additional open reading frame (orf) which is not found in their non-fusogenic counterparts. this orf is present at the 'end of a bi-or tricistronic mrna and encodes the fusion-associated small transmembrane (fast) protein responsible for cell-cell fusion and syncytium formation ( figure b ) [ ] . fast proteins are a unique family of viral membrane fusion proteins that are structurally and evolutionarily unrelated to the fusion proteins that are responsible for enveloped virus entry into cells. these "accessory" proteins are non-essential for productive virus infection and they are non-structural proteins, hence they are not involved in cell entry. fast proteins are small, integral membrane proteins expressed inside reovirus-infected cells where they localize at adherens junctions and mediate cell-cell, not virus-cell, membrane fusion [ ] . in so doing, fast proteins promote the rapid cell-cell transmission of the infection, a possible explanation for the positive selection pressure on acquiring or maintaining a fast protein. natural fusogenic reovirus infections are frequently associated with overt disease. arv is responsible for outbreaks of enteritis, tenosynovitis, and myocarditis in commercial poultry flocks [ ] , rrv is associated with pneumonia and neurological dysfunctions in snakes [ , ] , brv causes meningoencephalomyelitis in nonhuman primates [ , ] , and nbv has been associated with acute respiratory infections in humans. studies in different experimental systems link fast proteins to the pathogenic potential of these viruses [ ] [ ] [ ] [ ] . there are currently six members of the aqua-and orthoreovirus fast protein family that share limited sequence conservation but share several hallmark biochemical features [ , ] . the remainder of this discussion will focus on the orthoreovirus fast proteins ( figure c ). all fast proteins are small (~ - residues), acylated (shown or predicted to be palmitoylated or myristoylated), single-pass membrane proteins that position a very small (< residues) n-terminal ectodomain external to the plasma membrane and an extended (~ - residues) c-terminal endodomain in the cytoplasm [ ] [ ] [ ] . fast proteins appear to have evolved via the assembly of diverse membrane interaction motifs into three small domains (ecto-, endo-, and transmembrane) that function as fusion modules. the ectodomains are amphiphilic, structurally dynamic peptides, with remarkably diverse structures, that function as fusion peptides to destabilize lipid bilayers [ , , [ ] [ ] [ ] . fast protein tm viruses , , of domains contain different motifs, adjacent to the ectodomain-transmembrane domain boundary, that are required for cell-cell fusion (e.g., polyglycine in p , polyserine and hydrophobic β-branched residues in p ) [ , , ] . the fast protein endodomains all contain a juxtamembrane cluster of basic residues and an adjacent amphipathic helix that are essential for syncytium formation. the polybasic motif functions as a golgi export signal for the rrv p fast protein [ , ] , while the amphipathic helices in the p and brv p fast proteins partition into highly curved membranes to promote pore formation [ ] . the c-terminal half of the fast protein endodomains are predicted to be disordered [ ] , a common feature of protein regions that interact with multiple partners; annexin a is one such p partner [ ] . lastly, fast proteins are modular fusogens, meaning that the exchange of modules between different fast proteins can generate functional recombinants, although not all combinations are tolerated. the noteworthy mechanistic implication of the above discussion is that specific combinations of different structural motifs can be assembled in order to generate a functional fast protein [ , ] . the modular nature of fast proteins and the diversity of the functional motifs that they employ to drive membrane fusion raises interesting questions regarding the evolution of these non-enveloped virus fusogens. previous phylogenetic studies that included the six available sequenced species of orthoreoviruses suggested two separate gain-of-fusion events caused by a common ancestral, non-fusogenic reovirus led to the extant fusogenic aqua-and orthoreoviruses [ ] . the evolutionary relationship between the orthoreovirus fast proteins was unclear. these studies also overlooked the waterfowl subgroup of arv typified by the muscovy duck reoviruses (mdrv). the mdrv infection of young ducklings results in high morbidity and mortality ( %- % mortality), with necrotic focus formation in the liver, spleen, and kidneys [ ] . early isolates of mdrv were reported to be syncytiogenic like their landfowl counterparts [ ] , but no sequence information is available for these isolates. all the more recent mdrv isolates lack syncytium-inducing capabilities [ , ] . mdrvs are divided into two subgroups, the "classical" and "novel" mdrvs. classical muscovy duck reoviruses (mdrvc) possess a bicistronic s genome segment encoding a truncated fiber protein of residues and a p protein that lacks sequence similarity to the p fast proteins of arv, arvn, and nbv ( figure b ) [ ] . the more recently identified novel subgroup (mdrvn) has a tricistronic s genome segment similar to other arvs [ ] , encoding a p fast protein homologue, a p protein of unknown function, and a -residue fiber protein ( figure b ). there has been little discussion in the literature regarding the loss of fusion activity in mdrvs or the genetic events underlying the evolution of the orthoreovirus polycistronic genome segments. as we now show, using phylogenetic analysis that includes the current seven orthoreovirus species, three new proposed species, and previously excluded non-fusogenic mdrvs, the evolution of the orthoreovirus polycistronic genome segment from an inferred ancestral, monocistronic fiber-encoding genome segment involved polymorphic, species-specific indels at a "genetic hotspot" in the fiber orf. these genome segments further evolved by the addition of '-terminal extensions that added additional orfs upstream of the fiber orf, one of which encoded a fast protein homologue. evidence suggests these '-terminal recombination events occurred multiple times, both pre-and post-speciation, to generate the constellation of extant orthoreovirus polycistronic genome segments and fast proteins. we further directly demonstrate that point substitutions in a nine-residue multimerization motif are solely responsible for the loss of fusion capability in mdrvn isolates. these results highlight the underappreciated role of recombination, not just genome segment reassortment, in reovirus evolution, and support the concept that fast proteins evolved from small ancestral membrane proteins by independently acquiring the correct assembly of diverse membrane interaction motifs needed to generate a fusogenic fast protein. qm cells were maintained in medium with earle's salts containing u/ml of penicillin and streptomycin and % heat-inactivated fetal bovine serum. monoclonal mouse anti-flag (sigma-aldrich) and rabbit anti-myc (sigma-aldrich) antibodies, horseradish peroxidase (hrp)-conjugated goat anti-rabbit (santa cruz) and goat-anti mouse (santa cruz) antibodies, alexa fluor -conjugated goat anti-mouse (invitrogen) and alexa fluor -conjugated goat anti-rabbit (invitrogen), sulfo-nhs-lc-biotin (thermo scientific), maliemide-peg -biotin (thermo scientific), neutravidin agarose resin (thermo scientific), and polyethylimine (pei, polysciences inc.) were purchased from the indicated commercial sources. arv p , mdrv p , and md-cm (mdrv p containing the -residue conserved motif of arv p created using sequential pcr reactions with custom oligonucleotide primers) were subcloned into pcdna mammalian expression vectors between the hindiii and ecori sites. a triple flag or single myc tag was added to the n-terminus, or egfp or mcherry were added to the c-terminus of the indicated p constructs by pcr amplification and cloning. custom oligonucleotide primers were purchased from integrated dna technologies (idt), and all constructs were confirmed by sequencing. qm cell monolayers at % confluency in -well plates were transfected with . µg of plasmid dna using pei as per the manufacturer's instructions. at h post-transfection, the transfection mix was replaced with medium (gibco), supplemented with % fetal bovine serum (sigma-aldrich). at the indicated times post-transfection, cells were fixed with methanol and wright-giemsa stained (siemens healthcare diagnostics), and the stained monolayers were imaged using a nikon diaphot microscope at × magnification. the numbers of syncytial nuclei were counted in five random fields of triplicate wells (n = independent experiments) and the syncytial index reported as the mean ± sem. sds-page and western blotting were carried out as previously described [ , ] . a : dilution of primary anti-flag or anti-myc antibodies, followed by a : , dilution of hrp-conjugated secondary antibody was used to measure overall protein expression levels. western blots were developed using clarity western ecl substrate (bio-rad) and imaged on a chemidoc imaging system (bio-rad). qm cells grown to approximately % confluence on coverslips were transfected with flag-or myc-tagged versions of arv or mdrv p constructs, as described above. at h post-transfection, cells were fixed with . % paraformaldehyde for min at room temperature, blocked with % bovine serum albumin (bsa) in phosphate buffered saline (pbs) for h, and then incubated with : dilutions of mouse anti-flag and rabbit anti-myc monoclonal antibodies overnight at • c. cells were thoroughly washed with pbs and then incubated with : dilutions of alexa -conjugated goat anti-mouse antibody and alexa -conjugated goat anti-rabbit antibody for h at room temperature. coverslips were then mounted using prolong gold anti-fade reagent (invitrogen) and imaged using a zeiss lsm axiovert m confocal microscope. qm cells grown in cm dishes (corning) to % confluency were transfected with the indicated flag-tagged constructs. at h post-transfection, cells were washed three times with ice-cold pbs (ph . ), then incubated with shaking in mg/ml sulfo-nhs-lc-biotin (a membrane impermeable biotinylation reagent) for h at • c to biotinylate primary amino groups on the cell surface. cells were washed with pbs containing mm glycine to quench and remove excess biotin reagent. cells were washed twice with pbs, then lysed in a ripa buffer ( mm tris ph . , mm nacl, mm edta, % np , . % na desoxycholate) with protease inhibitors (pierce). cell lysates were incubated overnight with neutravidin agarose resin to pull down the biotinylated proteins, and pellets were boiled in a protein sample buffer with mm dithiothreitol (dtt) to release the biotinylated proteins. samples were then analyzed by sds-page and western blotting. to detect the presence of an intramolecular disulfide bond in the p ectodomain, qm cells transfected with myc-tagged p constructs were washed twice with hanks buffered saline solution (hbss) at h post-transfection and then incubated in hbss with or without . mm dtt for five min. cells were washed three times with hbss, then incubated with shaking in mg/ml maliemide-peg -biotin (a membrane impermeable biotinylation reagent) for min at • c to biotinylate free thiol groups on the cell surface. cells were washed four times with hbss to remove excess biotin reagent and once with hbss containing % bsa to quench residual biotinylation reagent. cells were washed twice with pbs, resuspended with mm edta in pbs, and lysed in the ripa buffer with protease inhibitors, and the lysate was incubated overnight with neutravidin agarose resin to pull down biotinylated proteins. pellets were boiled in a protein sample buffer with mm dtt to release biotinylated proteins and analyzed by sds-page and western blotting. a zeiss lsm confocal microscope was used in wide field mode to detect sensitized emission fluorescence resonance energy transfer (fret) from qm cells transfected with egfp-and/or mcherry-tagged versions of the indicated p constructs, as previously described [ , ] . briefly, donor and acceptor spectral bleed-through (sbt) values were minimized using cells expressing egfp or mcherry alone, and donor and acceptor sbt ratios were modeled using exponential relationships with fluorophore intensity, after the exclusion of aberrant background values at low intensities and the application of a gaussian blur. using this microscope setup and the sbt values, three images were acquired from each cell that was imaged: ( ) a donor image (donor excitation, donor emission), ( ) an acceptor image (acceptor excitation, acceptor emission), and ( ) a sensitized emission fret image (donor excitation, acceptor emission). the background subtraction and gaussian blur of the donor, acceptor, and fret channels were performed on each image stack prior to analysis. ten images were acquired for each sample from each of the two independent experiments (total of twenty images per sample). the pixfret image j plugin [ , , ] was used to normalize the fret intensity of each pixel to the donor and acceptor expression levels by dividing the fret-channel pixel intensity by the square-root of the product of the corresponding donor-and acceptor-channel pixels, using the following equation: the gaussian distribution with the highest calculated r value for the pixel amplitude distributions generated by pixfret from the -bit normalized fret (nfret) images was used to calculate the mean nfret (mnfret) for each image. phylogenetic analysis used clustal muscle to generate multiple sequence alignments of homologous proteins from the different orthoreovirus species that were analyzed for evolutionary relationships, using mega . and the maximum likelihood method based on the jones-taylor-thornton (jtt) matrix-based model [ , ] . these methods were applied to concatenated protein sequences for the seven homologous orthoreovirus structural proteins from either the orthoreovirus species or the proposed species (excluding the highly variable fiber protein), to the sigma-class outer capsid clamp proteins of either the eight fusogenic orthoreovirus species or the proposed species, and to the fast proteins encoded by these eight virus species. accession numbers are provided in the figures or figure legends and in the supplementary tables. phylogenetic analysis revealed species-specific features of the different orthoreovirus fiber proteins and the polycistronic genome segments encoding these proteins. the orthoreovirus fiber proteins share the same overall trimeric structure but with considerable variation in their lengths, ranging from to residues ( figure a ). all orthoreovirus fiber-encoding genome segments are polycistronic except the prv s genome segment. the prv genome segment is~ nucleotides shorter than the corresponding s genome segment of mrv due to an internal indel of~ - nucleotides within the region of the fiber orf where the coiled-coil tail joins the β-spiral repeats ( figure a) . consequently, prv contains - predicted heptad repeats in its coiled coil tail and only two β-spiral repeats while mrv fibers contain - . heptads and seven β-spiral repeats [ , , , ] . viral polymerase errors frequently generate substitutions, but indels also occur at about a four-fold lower frequency [ ] . excluding mrv, all other orthoreovirus species, including those whose hosts are more ancient (i.e., fish, reptiles, and birds), contain homologous short versions of the trimeric fiber. the logical inference is the ancestral fiber genome segment resembled that of prv, encoding a short fiber on a monocistronic mrna. the fiber orf indels are polymorphic but species-specific in their length, implying indels altered the fiber protein several times during orthoreovirus speciation. the extant fiber proteins cluster into four groups based on their total lengths and the approximate number of heptad and β-spiral repeats: the prv/arv/mdrvn/arvn/nbv fiber group ( - predicted heptad and two predicted β-spiral repeats), the mdrvc fiber protein ( predicted heptad and two predicted β-spiral repeats), the rrv/rrvt clade ( predicted heptad and three potential β-spiral repeats), and the mrv fibers ( - . heptads and seven β-spiral repeats) ( figure a ) [ , ] . three error-prone polymerase events, giving rise to indels with variable lengths, primarily during the synthesis of the β-spiral repeat region, would explain the evolution of the prv/arv/mdrvn/arvn/nbv fiber protein into the extant mrv, rrv/rrvt, and mdrvc fiber proteins ( figure b ). it would appear that this structural motif is highly tolerant to changes in the number of repeats without losing protein function, namely the cell attachment capacity. as occurs with recombination events in many rna viruses [ ] , undefined rna secondary structures in this region might also play a role in making this region a hotspot for such genetic events. sequence analysis suggests these fiber orf indels continue to occur, as evidenced by an additional -residue indel detected in the fiber orf of a recent mdrvn isolate that creates a -residue mdrvn fiber protein lacking an additional β-spiral repeat [ ] . figure a) . consequently, prv contains - predicted heptad repeats in its coiled coil tail and only two β-spiral repeats while mrv fibers contain - . heptads and seven β-spiral repeats [ , , , ] . viral polymerase errors frequently generate substitutions, but indels also occur at about a four-fold lower frequency [ ] . excluding mrv, all other orthoreovirus species, including those whose hosts are more ancient (i.e., fish, reptiles, and birds), contain homologous short versions of the trimeric fiber. the logical inference is the ancestral fiber genome segment resembled that of prv, encoding a short fiber on a monocistronic mrna. the fiber orf indels are one major contributor to the variable lengths of the different polycistronic genome segments. the second is what can only be inferred are '-terminal extensions, assuming that the last common ancestor of the extant fiber-encoding genome segments resembled that of prv. these~ - nucleotide extensions in the fusogenic orthoreoviruses added additional reading frames upstream of the fiber orf, one of which encoded a fast protein homologue (figure a ). template-switching as the rdrp replicates the plus-strand into a minus-strand would generate such genetic hybrids with '-extensions, as previously reported in plant viruses [ ] . this copy choice mechanism of recombination is common in many plant viruses and in plus-strand rna animal viruses including retroviruses, enteroviruses, and coronaviruses, and may simply reflect the stochastic association and dissociation of the rdrp from the rna template [ , ] . rna recombination usually viruses , , of occurs between regions of high sequence similarity. such homologous recombination between cognate genome segments has been noted in arvs, where as many as twelve recombination events between multiple cognate genome segments of different circulating virus strains have been predicted [ ] . however, non-homologous recombination can also occur, with genetic exchange taking place between different virus genomes [ ] or, more rarely, between viral and cellular rna [ ] [ ] [ ] . the high degree of sequence divergence in the polycistronic orthoreovirus mrnas and the likelihood that ancient, non-homologous recombination events generated these '-terminal extensions confounds similarity searches to define specific breakpoints or even the potential source of the alternate template rna. however, additional analyses discussed below support the concept that such genetic events occurred more than once and were major evolutionary drivers of the fusogenic orthoreovirus polycistronic genome segments and the fast proteins that they encode. the distinct features of the p fast proteins and their tricistronic genome segments compared to the other orthoreovirus fast proteins and their bicistronic genome segments imply that at least two distinct recombination events occurred during the fusogenic reovirus evolution. despite their distinct host ranges (i.e., birds and bats), arv, arvn, and nbv form a monophyletic clade ( figure a ) and they share a tricistronic gene arrangement comprising three sequential, partially overlapping orfs encoding a p fast protein, a non-structural p protein, and the σc fiber protein ( figure b ). this includes the mdrvn, but not the mdrvc, subgroup of waterfowl arv isolates; the latter have a bicistronic gene arrangement and are further discussed below. the arv, arvn, and nbv p fast proteins also form a monophyletic clade that is distinct from the p , p , and p fast proteins, with a % bootstrap value at that branchpoint ( figure c ). in multiple sequence alignments, the p fast proteins of the three species (arv, arvn, and nbv) are essentially colinear (two small, terminal indels) and share~ %- % amino acid identity in pairwise comparisons ( figure a ). this is not the case for alignments of p with the other fast proteins, where~ indels are needed to attain amino acid identities of only~ %- % (data not shown). the p proteins also contain several conserved features which are not found in the other orthoreovirus fast proteins-an unusual ectodomain cystine noose fusion peptide, a -residue ectodomain conserved motif, a palmitoylated endodomain di-cysteine motif instead of n-terminal myristoylation, and a short cytoplasmic tail ( figure c ) [ , , , ] . based on the above considerations, we infer that the predicted '-extension that led to the evolution of the p fast proteins occurred after the rrv/brrv/brv/marv and arv/nbv orthoreovirus clades diverged from each other ( figure a ). in addition to p , the arv/arvn/nbv clade acquired a second orf upstream of the fiber orf encoding a p /p protein. the arv p protein is a nucleocytoplasmic shuttling protein that suppresses the cell cycle ( [ , ] , but similar attributes have not been ascribed to other reovirus p proteins or to the mdrvn p protein. alignments of the p proteins from the nbv, arvn, and landfowl isolates of arv are mostly colinear ( figure b ), and in pairwise comparisons of the more distantly related species these proteins still share~ %- % amino acid identity which is suggestive of a homologous relationship. however, this degree of amino acid conservation is considerably lower than that between the nbv and arv/arvn p proteins (~ %- % identity). the p protein of mdrvn shares even less similarity with p ; protein alignments require - internal indels, totaling > nucleotides, in order to generate only~ %- % amino acid identity (data not shown). it is therefore unclear whether one recombination event simultaneously added precursors of the p /p /p orfs that then evolved at very different rates or independent recombination events added these orfs separately ( figure a ). p proteins also contain several conserved features which are not found in the other orthoreovirus fast proteins-an unusual ectodomain cystine noose fusion peptide, a -residue ectodomain conserved motif, a palmitoylated endodomain di-cysteine motif instead of n-terminal myristoylation, and a short cytoplasmic tail ( figure c) [ , , , ] . based on the above considerations, we infer that the predicted '-extension that led to the evolution of the p fast proteins occurred after the rrv/brrv/brv/marv and arv/nbv orthoreovirus clades diverged from each other ( figure a) . the brrv, marv, rrv, rrvt, and brv virus species form a monophyletic clade, as do their encoded p , p , and p fast proteins ( figure a-c) . these three diverse fast proteins are encoded by the first orfs of bicistronic genome segments that have two distinct gene arrangements. all reptilian orthoreovirus isolates, including the tortoise isolate of the new proposed rrvt species, encode homologous p fast proteins using the first orf and retain a truncated σc fiber protein orf ( figure b) . brv, brrv, and marv encode p , p , or p fast proteins, but their fiber orfs have been replaced by an unrelated orf encoding a p protein of no defined function. aside from terminal indels to accommodate length differences, these p proteins are mostly colinear (four single residue indels) and share %- % amino acid identity clustered in subregions (figure c ), indicating a shared common ancestor. brv, marv, and brrv form a monophyletic clade when comparing their sigma-class outer clamp capsid proteins ( figure a ) or lambda-class core shell proteins [ ] , consistent with a single recombination event replacing the fiber orf with the p orf prior to speciation of these viruses. since the three viruses are paraphyletic when comparing other homologous proteins ( figure a) , lateral gene transfer via genome segment reassortment between these evolving virus species presumably occurred after the p recombination event. recombination events and genome segment reassortment are well-linked to intra-species arv evolution, with some suggestion that similar inter-species genetic events may have occurred between orthoreovirus species [ ] , possibly early post-speciation. a single recombination event that extended the '-end of the fiber orf with an orf encoding a shared ancestral protein could explain the evolution of the brrv, marv, rrv, rrvt, and brv p , p , and p fast proteins ( figure a ). evidence does support the divergent evolution of the brrv p and reptilian reovirus p fast proteins from a shared, fusogenic ancestor. phylogenetic trees based on fast protein sequences have weak bootstrap values at branchpoints separating the single isolates of brv p , marv p , and brrv p , making evolutionary relationships unclear ( figure c ). however, the p fast proteins of rrv, rrvt, and marv are clear homologues. these proteins share conserved sequence motifs, including a conserved n-terminal decapeptide that is sensitive to substitution and contains the n-terminal myristoylation consensus sequence ( figure d ) [ ] , and they share~ %- % amino acid identity. brrv p fast protein possesses the same motifs as p , including the n-terminal decapeptide but excluding a cytoplasmic ppxy motif of no defined function ( figure d ), and in mostly colinear, pairwise sequence alignments it shares~ %- % sequence identity with marv and rrv p . the hypothesis that the p and p fast proteins are homologues is consistent with all phylograms that show a monophyletic relationship between brrv, marv, rrv, and rrvt and their encoded p and p fast proteins ( figures a and a) . as indicated by the fast protein and concatenated sequence phylograms, brv and the p fast protein present as outliers. pairwise alignments of p with p or p require - large indels, totaling - residues, to generate~ %- % sequence identity. a similar level of sequence conservation (~ % amino acid identity in alignments containing four large indels) is observed between brv p and a recently identified p fast protein (referred to as nsp - , the first orf on a bicistronic mrna) of rotavirus species b (rvb), a virus from a distantly related genus and different subfamily of reoviridae [ ] . the brv p fast protein also contains several features not associated with p or p ; p lacks the conserved n-terminal decapeptide sequence present in p and p , but it does contain a functional gxxxs myristoylation site [ , ] . the~ -residue ectodomain is markedly truncated and contains a unique polyproline motif that forms a left-handed polyproline type ii helix required for fusion activity [ ] , and the p endodomain is two to three times longer than the c-terminal cytoplasmic tails of the other orthoreovirus fast proteins ( figure d ). the absence of significant sequence conservation and dramatically different structural features of p relative to p /p , coupled with the more distant evolutionary relationships between the parental viruses encoding these proteins, suggests p may have arisen by an independent gain-of-fusion event ( figure a ). additional sequenced isolates of these interrelated orthoreoviruses are needed in order to more robustly test this hypothesis. the concept of independent gain-of fusion regarding p /p /p is not intended to exclude the possibility of recombination leading to acquisition of a shared non-fusogenic ancestor followed by an independent, post-speciation gain-of-fusion capability. for example, we previously speculated that fast proteins may have evolved from viroporins or other small, non-structural, non-fusogenic viral proteins that contain diverse membrane remodeling motifs (e.g., transmembrane domains, polybasic motifs, amphipathic helices) [ , , ] . in the above discussion, a single recombination event could have added an orf encoding a non-fusogenic, viroporin-like fast protein precursor to the ancestral fiber-encoding genome segment and this precursor independently acquired fusion capability and evolved to become the p /p and p fast proteins. while there have been reports of fusogenic mdrv isolates [ ] , there are no recent reports of sequenced isolates that induce syncytium formation. as noted previously [ , ] , the mdrvc p protein is not a fast protein homologue, implying that the loss of mdrvc fusion capacity reflects a post-speciation recombination event that replaced the arv p fast protein orf with a heterologous orf ( figure a ). this is not the case for mdrvn, which clearly encodes a p fast protein homologue, sharing the primary sequence conservation and almost all the hallmark features of a p fast protein. the exception is a nine-residue ectodomain conserved motif (cm; aggdlqats) present in all other arv and nbv p sequences ( figure a ). the mdrvn isolates have two sequence variants of the cm with two to three of the first four residues matching the consensus sequence (ahgnidsyt and asgdidsyt: boldface indicates sequence conservation with consensus motif; lowercase indicates mdrv sequence variants). the residues flanking the cm are also conserved between arv and mdrv p proteins ( figure a ). it seems most likely that a point substitution occurred in the cm after the classical and novel mdrvs diverged, destroying p function and allowing the remaining sequence to rapidly devolve from the consensus. whatever selective pressure there might be to acquire and maintain a fast protein, it did not prevent two distinct genetic events that resulted in the loss of fusion capability in mdrvs. the fact that mdrvn has retained almost all the hallmark fast protein features but is non-functional for syncytium formation suggests the loss of fusion capacity may have been a recent genetic event, consistent with earlier reports of fusogenic mdrvs [ ] . we further explored the hypothesis that the loss of the cm is solely responsible for the absence of mdrvn's syncytiogenesis capacity. to generate syncytia, p proteins need to traffic to the plasma membrane in the correct n out /c in membrane topology. transfected cells expressing n-terminal flag-tagged versions of arv and mdrvn p were treated with membrane impermeable sulfo-nhs-lc-biotin to label the externally exposed lysine sidechains of plasma membrane proteins, and the western blots of the cell lysates were probed with anti-flag or neutravidin. as shown ( figure c ), both proteins were expressed and detected on the cell surface at similar levels. functional p proteins also need to form an intramolecular disulfide bond between the two conserved ectodomain cysteine residues in order to generate an -residue cystine noose fusion peptide ( figure a) , and the formation of this structure is highly sensitive to substitutions within and adjacent to the noose [ , , ] . a previously described assay applying a membrane-impermeable biotinylation reagent (maliemide-peg -biotin) to biotinylate free thiol groups on the cell surface [ , ] determined that mdrv p forms a cystine noose; plasma-membrane localized mdrv p was only labeled if cells were treated with dtt prior to the thiol-specific biotinylation reagent ( figure d ). these results confirm that the formation of the p intramolecular disulfide bond occurs independent of the cm [ ] and that the null syncytiogenic phenotype of novel mdrvs is not due to aberrant protein expression, trafficking, or formation of the essential cystine noose fusion peptide structure. substitution occurred in the cm after the classical and novel mdrvs diverged, destroying p function and allowing the remaining sequence to rapidly devolve from the consensus. whatever selective pressure there might be to acquire and maintain a fast protein, it did not prevent two distinct genetic events that resulted in the loss of fusion capability in mdrvs. the fact that mdrvn has retained almost all the hallmark fast protein features but is non-functional for syncytium formation suggests the loss of fusion capacity may have been a recent genetic event, consistent with earlier reports of fusogenic mdrvs [ ] . in arv and nbv p proteins, the cm governs the cholesterol-dependent, reversible assembly of p multimeric fusion platforms in the plasma membrane [ ] . single, conservative point substitutions in this motif, which presumably provides a p multimerization binding interface, ablated multimer formation. to determine whether this motif serves the same function in mdrv p , and whether the absence of this motif is solely responsible for the cell-cell fusion defect in novel mdrvs, we constructed a recombinant mdrv p (md-cm) containing the nine-residue conserved motif (i.e., ahgnidsyt was converted to aggdlqats) ( figure a) . a time-course analysis of the syncytium formation revealed that the replacement of the cm restored mdrv syncytiogenesis to robust levels with only slightly reduced kinetics relative to arv p ( figure a,b) . furthermore, cell-surface immunofluorescence microscopy, using cells co-transfected with n-terminally flag-tagged and n-terminally myc-tagged p constructs, indicated that mdrv p failed to form the dual-color cell surface puncta that are typical of arv p fusion platforms, but replacement of the cm in md-cm p resulted in the extensive punctate colocalization of recombinant p proteins ( figure c ). to confirm the punctate staining pattern of the md-cm p reflected assembly of multimeric fusion platforms, we employed in cellula fret, as previously described [ , ] . briefly, arv, mdrv, and md-cm p proteins were c-terminally tagged with either egfp or mcherry. donor and acceptor spectral bleed-through (sbt) values and normalized fret (nfret) intensities were calculated using the pixfret image-j plug-in and pixel-by-pixel analysis of sensitized emission fret [ ] , and mean nfret (mnfret) values were determined for cells in each of the two separate experiments. as shown in the fret images ( figure a ) and by mnfret quantification (figure b ), arvp formed the expected homomultimers in cells but mdrvp failed to multimerize. replacement of the cm in mdrv p generated positive fret signals equivalent to those observed with arv p , indicating that point substitutions leading to the loss of a multimerization motif required for the fusion platform assembly destroyed the ability of novel mdrvs to induce syncytium formation. to confirm the punctate staining pattern of the md-cm p reflected assembly of multimeric fusion platforms, we employed in cellula fret, as previously described [ , ] . briefly, arv, mdrv, and md-cm p proteins were c-terminally tagged with either egfp or mcherry. donor and acceptor spectral bleed-through (sbt) values and normalized fret (nfret) intensities were calculated using the pixfret image-j plug-in and pixel-by-pixel analysis of sensitized emission fret [ ] , and mean nfret (mnfret) values were determined for cells in each of the two separate experiments. as shown in the fret images ( figure a ) and by mnfret quantification (figure b ), arvp formed the expected homomultimers in cells but mdrvp failed to multimerize. replacement of the cm in mdrv p generated positive fret signals equivalent to those observed with arv p , indicating that point substitutions leading to the loss of a multimerization motif required for the fusion platform assembly destroyed the ability of novel mdrvs to induce syncytium formation. viruses , , x for peer review of current analyses support the concept that multiple recombination events were primary evolutionary drivers of the constellation of polycistronic orthoreovirus genome segments and directly contributed to the diversity of their encoded fiber and fast proteins. the four "classes" of extant orthoreovirus fiber proteins most likely arose via three polymorphic internal recombination events in a genetically tractable region of the fiber orf that encodes a structurally malleable repeat motif ( figure b ). genome segment reassortment may have influenced early inter-species evolution and the precise order of events, but nonhomologous copy choice recombination events are the most plausible explanation for the addition of '-extensions that generated fast protein-encoding polycistronic genome segments. one such event led to the tricistronic, p -encoding arv/arvn/nbv clade, with a subsequent post-speciation recombination event leading to the heterologous replacement of the p fast protein orf and the loss of fusion capacity by mdrvc ( figure a) . a second independent genetic exchange led to the p -encoding rrv/rrvt/marv clade and likely the related p -encoding brrv. there is some evidence to suggest that brv may have acquired the p precursor from a third independent recombination event. at some point, a 'proximal genetic exchange generated the brrv/marv/brv bicistronic s genome segments that have the entire fiber orf replaced with a heterologous p orf of no known function. lastly, we show that the extant mdrvs lost fusion capacity via two independent post-speciation genetic events: the recombinant loss of the entire p fast protein orf by mdrvc and point substitutions in the mdrvn cm that ablated multimeric fusion platform assembly. together, these results highlight the genetic tractability of the orthoreovirus polycistronic genome segments and the underappreciated role of recombination in dsrna virus evolution and the evolution of the fast protein family. current analyses support the concept that multiple recombination events were primary evolutionary drivers of the constellation of polycistronic orthoreovirus genome segments and directly contributed to the diversity of their encoded fiber and fast proteins. the four "classes" of extant orthoreovirus fiber proteins most likely arose via three polymorphic internal recombination events in a genetically tractable region of the fiber orf that encodes a structurally malleable repeat motif ( figure b ). genome segment reassortment may have influenced early inter-species evolution and the precise order of events, but nonhomologous copy choice recombination events are the most plausible explanation for the addition of '-extensions that generated fast protein-encoding polycistronic genome segments. one such event led to the tricistronic, p -encoding arv/arvn/nbv clade, with a subsequent post-speciation recombination event leading to the heterologous replacement of the p fast protein orf and the loss of fusion capacity by mdrvc ( figure a) . a second independent genetic exchange led to the p -encoding rrv/rrvt/marv clade and likely the related p -encoding brrv. there is some evidence to suggest that brv may have acquired the p precursor from a third independent recombination event. at some point, a '-proximal genetic exchange generated the brrv/marv/brv bicistronic s genome segments that have the entire fiber orf replaced with a heterologous p orf of no known function. lastly, we show that the extant mdrvs lost fusion capacity via two independent post-speciation genetic events: the recombinant loss of the entire p fast protein orf by mdrvc and point substitutions in the mdrvn cm that ablated multimeric fusion platform assembly. together, these results highlight the genetic tractability of the orthoreovirus polycistronic genome segments and the underappreciated role of recombination in dsrna virus evolution and the evolution of the fast protein family. reovirus fast proteins: virus-encoded cellular fusogens bioinformatics of 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encouragement and helpful comments from other members of the duncan laboratory. the authors declare that they have no conflicts of interest with the contents of this article. key: cord- -roxa ix authors: i. sardi, silvia; h. carvalho, rejane; c. pacheco, luis g.; p. d. almeida, joão p.; m. d. a. belitardo, emilia m.; s. pinheiro, carina; s. campos, gúbio; r. g. r. aguiar, eric title: high-quality resolution of the outbreak-related zika virus genome and discovery of new viruses using ion torrent-based metatranscriptomics date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: roxa ix arboviruses, including the zika virus, have recently emerged as one of the most important threats to human health. the use of metagenomics-based approaches has already proven valuable to aid surveillance of arboviral infections, and the ability to reconstruct complete viral genomes from metatranscriptomics data is key to the development of new control strategies for these diseases. herein, we used rna-based metatranscriptomics associated with ion torrent deep sequencing to allow for the high-quality reconstitution of an outbreak-related zika virus (zikv) genome ( , nt), with extended ′-utr and ′-utr regions, using a newly-implemented bioinformatics approach. besides allowing for the assembly of one of the largest complete zikv genomes to date, our strategy also yielded high-quality complete genomes of two arthropod-infecting viruses co-infecting c / cell lines, namely: alphamesonivirus strain salvador ( , nt) and aedes albopictus totivirus-like ( nt); the latter likely represents a new viral species. altogether, our results demonstrate that our bioinformatics approach associated with ion torrent sequencing allows for the high-quality reconstruction of known and unknown viral genomes, overcoming the main limitation of rna deep sequencing for virus identification. arthropode-borne viral infections (arboviroses), transmitted to mammals by hematophagous arthropod vectors, have recently emerged as one of the most important threats to human health. due to their worldwide occurrence and fast adaptation to environmental changes, mosquitoes from the aedes genus play an important role in the transmission of viral etiological agents of emerging human infections, including dengue virus (denv), zika virus (zikv), chikungunya virus (chykv) and of yellow fever virus (yfv) [ , ] . many epidemic events have been reported, which are associated with the lack of efficient vaccines, the rapid evolution of viral genomes, and inefficient control strategies. one of the last episodes was the zika outbreak in brazil in the period - , which impacted more than , individuals by the end of [ ] . the impact of this outbreak was amplified due to the zika virus infection's relationship to many neurological disorders, such as guillain-barre and microcephaly syndromes, which led the world health organization to declare the brazilian zika outbreak as one of international concern by . different from african lineage, the asian lineage of zika virus, supposedly the origin of the strains circulating in brazil, exhibits a different profile with lower pathogenicity and prolonged infections [ ] . these differences have been attributed to mutations in the viral genome, mainly in untranslated regions [ , ] . the long-term infection, which could facilitate mosquito transmission, associated with the high susceptibility of the population likely explains the rapid spread of the virus that reached almost all brazilian states in only a few months, different, for example, to the dengue virus whose spread took decades [ ] . in addition to arboviruses, aedes mosquitoes are also known to carry a vast range of viruses, most of them insect-specific [ ] [ ] [ ] . although they do not cause direct harm to human health, they have been described as impacting mosquito susceptibility to other viral infections and vector competence [ ] [ ] [ ] . therefore, the knowledge of the viruses circulating in mosquitoes (virome) is important in understanding mosquito-arbovirus interactions as well as in developing new strategies of control, and in the prevention of epidemic events. in this context, metagenomics associated with deep sequencing dramatically increased the resolution of viral genomes and the identification of new viruses [ , ] . however, while dna sequencing restricts the identification of viruses that use dna intermediates in the replication cycle, rna sequencing allows for the identification of all classes of viruses, since almost all of them generate rna intermediates during their replication. indeed, metatranscriptomics has been successfully applied to the identification of rna and dna viruses in a vast range of organisms, such as plants, insects, fungi, and mammals [ , [ ] [ ] [ ] . despite being a powerful strategy, there are technical limitations that hamper confident identification of whole viral genomes, such as length of sequenced reads, repetitive genomes, differential expression of virus segments, and natural variation in the virus population that make the confident assembly of contiguous sequences a challenging task [ , ] . here, we applied rna-based metatranscriptomics associated with ion torrent deep sequencing and a newly developed bioinformatics approach to the high-quality reconstitution of viral genomes. using this strategy, we successfully reconstituted an outbreak-related zika virus genome with extended -utr and -utr regions and the genomes of two arthropod-infecting viruses co-infecting c / cell lines. altogether, our data demonstrate that our bioinformatics approach associated with ion torrent sequencing allows for high-quality reconstruction of known and unknown viral genomes. c / cells (aedes albopictus) were maintained in leibovitz's l medium (gibco ® , ca, usa), supplied with % fetal bovine serum (fbs) (gibco ® ), % tryptophan broth (sigma-aldrich ® , boston, ma, usa) at • c. the cells were inoculated with a zika virus strain (partial fragment of e protein deposited at genbank kr from zika virus isolate br/ufba/labviro/ex -see figure b ) derived from a serum sample collected from a patient in the brazilian outbreak of with moi , and after observation of the cytophatic effect ( days), the supernatant of the infected cells was centrifugated ( . g at • c for min) and then submitted to ultracentrifugation ( . g at • c for . h). the pellet was resuspended in phosphate saline solution (pbs) and used to perform rna extraction through the qiaamp viral rna mini kit (qiagen, hilden, germany). quantification of the total extracted rna was performed by fluorimetry using qubit equipment (life technologies, carlsbad, ca, usa). the rna library was constructed from the extracted rna using ion total rna-seq kit v viruses , , of according to the manufacturer's protocols. sequencing was performed using an ion ™ chip kit (life technologies) in the ion onetouch™ system and the ion s instrument. sequencing data were deposited at the sra database under accession number: srr . a. albopictus genome was downloaded from vectorbase (www.vectorbase.org). all complete bacterial genomes used to remove possible contamination in sequenced reads were downloaded from the refseq database [ ] . sequence and metadata of the zika virus were downloaded from virus pathogen resource (vipr) [ ] , and only complete genomes were analyzed. for phylogenetic analysis, top blast hits for each new viral contig were retrieved from the ncbi database ordered by score. pre-processing: reads derived from rna deep sequencing were submitted to quality checking and filtering using fastqc [ ] and fastx toolkit [ ] ; reads with an average phred quality below were discarded. to enrich the viral sequences, filtered reads were aligned against the a. albopictus genome, and bacterial genomes present on the refseq database using bowtie [ ] . only unaligned reads were used for the subsequent analysis. assembly: assemblies were performed using spades assembler [ ] with the following variations: ( ) standard: filtered reads were used as input and standard parameters kept; ( ) meta: filtered reads were analyzed as derived from the metagenomics sample, using the parameter "-meta"; and ( ) meta (trusted) + regular: viral contigs assembled in "meta" strategy were used as trustable contigs, the parameter "-trusted-contigs," together with filtered reads. all strategies were run with the parameter "-iontorrent." in strategies and , the parameter "-cov-cutoff" was set to "auto." in strategy , "-cov-cutoff" was set to "off." all contigs with significant hit with viral sequences were submitted to manual curation. manual curation of assembled genomes: assembled viral genomes were curated based on structure (presence of orfs, conserved domains, secondary structure) and rna coverage (evaluation of continuous coverage along segment). the viral genome sequences produced in this work were deposited on genbank under accession numbers mn -mn . characterization of viral contigs: assembled contigs were submitted to sequence similarity searches using blast software [ ] against non-redundant ncbi databases of nucleotide (nt) and aminoacids (nr), requiring minimum e-values of e − and e − , respectively. contigs that showed significant similarity to viral sequences were further analyzed. conserved domains were analyzed using the hmmer tool [ ] with the pfam database. orf prediction: the prediction of the open read frame of the assembled sequences was performed using the ncbi orf finder (https://www.ncbi.nlm.nih.gov/orffinder/) with "standard" genetic code defining minimum orf length to nt. rna coverage analysis: reads were re-aligned to assembled viral genomes with bowtie ; sam files were processed and quantified using samtools [ ] and bedtools [ ] , and for visual inspection of reads coverage we used igv [ ] . secondary structure analysis: analyses of the secondary structure of and utr of the zika virus genomes were performed using the rnafold webserver from the vienna platform [ ] with standard parameters. phylogenetic and genotypic analysis: phylogenetic analysis was performed using mega software [ ] . maximum likelihood phylogenetic trees were constructed based on the whole viral genome with the kimura -parameter method, or rdrp protein using the poisson substitution model for alphamesonivirus and totivirus-like virus, respectively. the trees were drawn with branch sizes measured in the number of substitutions per site. the percentage of trees in which the associated taxa clustered together in the bootstrap test ( replicates) is shown next to the branches. investigation of the zika virus genotype was performed using well defined phylogenetic clusters with information of geographic region integrated in the zika virus typing tool [ ] . rna coverage analysis: preprocessed reads were compared to each reference sequence using bowtie , allowing a maximum of two mismatches. bowtie 's sam output was used to compute per base coverage with the bedtools package. per base coverage was plotted using r language [ ] with the ggplot package [ ] . it is important to highlight that this viral genome extended in bp ( bp in ′-utr and bp in ′-utr including coding regions) is the closest viral sequence derived from the outbreak, the zika virus strain bahia , that shows ~ % of coverage with ~ % of identity (figure b) [ ] . since confident assembly of utr regions is challenging due to the lower number of reads in comparison to non-utr regions, we further manually cured these regions by investigating aligned reads using a genome browser. as expected, we noted that utr regions presented a considerably lower number of reads in comparison to non-utr regions, , compared to , , , respectively. however, although the number of reads was restricted, we observed concordantly alignments in both utr regions, and , for ′ and ′ extremities, respectively ( figure s ) . furthermore, analysis of the utr region sequences suggests that both of them form secondary structures, which has been shown previously to be essential to the virus driving viral fitness and pathogenesis ( figure ) [ ] . this demonstrates that our strategy based on ion torrent metatranscriptomics associated with de novo assembly is able to reconstitute whole viral genomes. indeed, performing a global overview of complete zika virus genomes, it is noticeable that our newly total rna from c / culture cells was extracted using qiamp ® rna minikit and submitted to one-step rt-pcr using the following primers (f: acgtgccagctgtgtctatg and r: acaccagccatcaaggacg) and the access rt-pcr system (promega, ma usa). cycling conditions were defined as follows: • c for min, • c for min, and cycles of • c for s, • c for s, and • c for min, the final extension step was at • c for min. the primers amplified a fragment of bp when visualized in % agarose gel, stained with ethidium bromide and detected by an ultraviolet-transilluminator system. rt-pcr product was cleaned up using the qiaquick pcr purification kit, according to the manufacturer's instructions (qiagen, germany). sequencing was performed by the genetic analyzer using the big dye terminator method, and it was carried out in ng of purified rt-pcr product and . pmol of the same primers used for the rt-pcr. rna deep sequencing produced , , reads. after preprocessing, , , were kept for posterior analysis. since the goal was to reconstitute the zika virus genome that only has a small fragment of the protein e available, we discarded all sequences derived from mosquito genome ( , , ) . the remaining reads ( , , ) were used to perform assembly using different strategies described in figure a , in the top panel. we first tried to use spades with the default parameters (highlighted in gold in figure a ) referred here as 'regular.' although we identified many contigs derived from the zika virus, we were not able to reconstitute the viral genome as a single segment. since our data are derived from deep sequencing of all rnas present in the cell, we performed the assembly using the "-meta," a variant of the assembler optimized for metagenomic data. using this strategy, we considerably improved the assembly of the zika virus genome, reducing the number of contigs from to , and increasing size average from . to nt (highlighted in dark blue in figure a ). although we assembled longer contigs, the genome was still fragmented. interestingly, we observed that fragmentation in the zika genome correlated with the positions that had high variability in the density of coverage (figure a-bottom panel) . this is consistent with the literature showing that differences in coverage of overlapping sequences can lead to the split of supposedly contiguous sequences into different contigs [ ] . this is a problem mainly when assembling viral genomes from rnas since viral genes can show different transcription profiles [ , ] . to overcome this limitation, we decided to use contigs derived from the "meta" strategy as trusted contigs trying to extend these contiguous sequences. this new strategy, which is hereafter referred to as "meta (trusted) + regular," resulted in the complete reconstitution of the zika virus genome containing , nt (figure a ,b-top panel). an overview of the final approach is shown in figure s and described in detail in the material and methods section. it is important to highlight that this viral genome extended in bp ( bp in -utr and bp in -utr including coding regions) is the closest viral sequence derived from the outbreak, the zika virus strain bahia , that shows~ % of coverage with~ % of identity (figure b) [ ] . since confident assembly of utr regions is challenging due to the lower number of reads in comparison to non-utr regions, we further manually cured these regions by investigating aligned reads using a genome browser. as expected, we noted that utr regions presented a considerably lower number of reads in comparison to non-utr regions, , compared to , , , respectively. however, although the number of reads was restricted, we observed concordantly alignments in both utr regions, and , for and extremities, respectively ( figure s ). furthermore, analysis of the utr region sequences suggests that both of them form secondary structures, which has been shown previously to be essential to the virus driving viral fitness and pathogenesis ( figure ) [ ] . this demonstrates that our strategy based on ion torrent metatranscriptomics associated with de novo assembly is able to reconstitute whole viral genomes. indeed, performing a global overview of complete zika virus genomes, it is noticeable that our newly assembled genome figures are among the top in length out of more than viral sequences available, which seems to be independent of collection year or country (figure c) . indeed, recent work has shown that strategies based on amplicon are still preferentially used to reconstruct zikv genomes, which create a bias to the coding region since amplicons are mostly designed to anchor at genic regions with lower mutation rates [ ] . viruses , , x for peer review of assembled genome figures are among the top in length out of more than viral sequences available, which seems to be independent of collection year or country (figure c) . indeed, recent work has shown that strategies based on amplicon are still preferentially used to reconstruct zikv genomes, which create a bias to the coding region since amplicons are mostly designed to anchor at genic regions with lower mutation rates [ ] . similarly, for the previous zika virus strains derived from the outbreak in , the viral strain assembled in this work clustered together with other strains with asian genotype (figure d ). comparative analysis among all complete sequences deposited at the vipbrc database revealed that differences among viral genomes are mainly in untranslated regions, which is consistent with the literature [ , , ] . curiously, untranslated regions have been linked to differences in virulence of different arboviruses [ , , ] . in addition, these regions are also suggested to be involved in other viral characteristics, such as host preference and tissue tropism [ , , ] . this result highlights the need for achieving complete viral genomes to better our understanding of virus-host interactions. similarly, for the previous zika virus strains derived from the outbreak in , the viral strain assembled in this work clustered together with other strains with asian genotype (figure d ). comparative analysis among all complete sequences deposited at the vipbrc database revealed that differences among viral genomes are mainly in untranslated regions, which is consistent with the literature [ , , ] . curiously, untranslated regions have been linked to differences in virulence of different arboviruses [ , , ] . in addition, these regions are also suggested to be involved in other viral characteristics, such as host preference and tissue tropism [ , , ] . this result highlights the need for achieving complete viral genomes to better our understanding of virus-host interactions. notably, besides the identification of contigs showing sequence similarity to the zika virus, with the new assembly strategy, we were also able to reconstruct two large contigs showing similarities to viral sequences. one contig of , nt showed a sequence similarity at the nucleotide level ( % identity with e-value = ) with viruses from alphamesonivirus genus while one contig with nt presented a similarity at the protein level (~ % identity with e-value = e- ) with viruses from the totiviridae family, which likely represents a new viral species. phylogenetic analysis based on the nucleotide sequence revealed that the alphamesonivirus identified in c / cell lines is a new strain of a previously identified alphamesonivirus from the mesoniviridae family, a group of single-stranded rna viruses that have been described in mosquitoes (figure a ) [ ] . this is the first report of this viral family in a. albopictus mosquitoes. it was named alphamesonivirus strain salvador. phylogenetic analysis based on rdrp protein of contig related to the totiviridae family suggested its relationship to a newly proposed family, totivirus-like, which is different from totiviruses that have fungi as natural hosts, and have been described in a vast range of organisms (figure b ) [ ] [ ] [ ] [ ] [ ] . this new virus was named aedes albopictus totivirus-like. for both of the two new viruses identified, we observed an orf structure consistent with their closely related viruses (figure c,d) . in addition, we observed a considerable number of reads aligned with the totivirus and alphamensonivirus assembled genomes, , and , respectively, representing . % and . % of the library. furthermore, rna density along viral genomes showed continuous coverage, suggesting that they were correctly assembled (figure c,d) . to confirm the detection of the newly identified totivirus-like sequence, experimental validation was performed by rt-pcr, and we were able to detect the viral genome in the same c / population we used to perform deep sequencing. we also retrieved a fragment of its genome through sanger sequencing in order to compare with the contiguous sequence obtained through metatranscriptomics ( figure s a,b) . we observed a high similarity between the fragment retrieved through sanger sequencing and the assembled using deep sequencing ( % identity), suggesting the high accuracy of our metatranscriptomics strategy to reconstitute viral genomes. assembled in this work clustered together with other strains with asian genotype (figure d ). comparative analysis among all complete sequences deposited at the vipbrc database revealed that differences among viral genomes are mainly in untranslated regions, which is consistent with the literature [ , , ] . curiously, untranslated regions have been linked to differences in virulence of different arboviruses [ , , ] . in addition, these regions are also suggested to be involved in other viral characteristics, such as host preference and tissue tropism [ , , ] . this result highlights the need for achieving complete viral genomes to better our understanding of virus-host interactions. altogether, our results indicate that our strategy based on rna deep sequencing using ion torrent technology allows the confident reconstitution of known and unknown viruses. using this strategy, we were able to reconstruct the complete genome of the outbreak-related zika virus genome, including and utr regions, that would not be possible using standard approaches. furthermore, we also assembled the full genomes of two unknown viruses co-infecting the stock, alphamesonivirus and the newly described totivirus-like, highlighting the potential of our strategy for monitoring viral contaminations. therefore, our work highlights the importance of the development of new unbiased metagenomic approaches for the identification of viral sequences. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : bioinformatics approach to reconstruct viral genomes based on ion torrent data; figure s : overview of reads aligned to utr regions of the zika virus genome assembled using igv genome browser; figure the global distribution of the arbovirus vectors aedes aegypti and ae. albopictus. elife global risk mapping for major diseases transmitted by aedes aegypti and aedes albopictus zika virus outbreak differential virulence between asian and african lineages of zika virus zika virus genome biology and molecular pathogenesis comparative genomic analysis of pre-epidemic and epidemic zika virus strains for virological factors potentially associated with the rapidly expanding epidemic the zika virus epidemic in brazil: from discovery to future implications sequence-independent characterization of viruses based on the pattern 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rna deep sequencing and pcr techniques reveal the presence of a potential new virus genome assembly reborn: recent computational challenges accurate viral population assembly from ultra-deep sequencing data reference sequence (refseq) database at ncbi: current status, taxonomic expansion, and functional annotation virus pathogen database and analysis resource (vipr): a comprehensive bioinformatics database and analysis resource for the coronavirus research community. viruses a quality control tool for high throughput sequence data fast gapped-read alignment with bowtie spades: a new genome assembly algorithm and its applications to single-cell sequencing basic local alignment search tool hmmer web server: interactive sequence similarity searching the sequence alignment/map format and samtools bedtools: a flexible suite of utilities for comparing genomic features integrative genomics viewer (igv): high-performance genomics data visualization and exploration vienna rna secondary structure server molecular evolutionary genetics analysis version . genome detective: an automated system for virus identification from high-throughput sequencing data a language and environment for statistical computing; r foundation for statistical computing elegant graphics for data analysis biology of negative strand rna viruses: the power of reverse genetics rna virus replication, transcription and recombination zika virus evolution and spread in the americas functions of the and genome rna regions of members of the genus flavivirus secondary structure of the -untranslated region of yellow fever virus: implications for virulence, attenuation and vaccine development virus pathogenesis and tissue tropism mesoniviruses are mosquito-specific viruses with extensive geographic distribution and host range a novel totivirus-like virus isolated from bat guano co-infection by two distinct totivirus-like double-stranded rna elements in chalara elegans (thielaviopsis basicola) virome of > thousand culex mosquitoes from throughout california southern tomato virus: the link between the families totiviridae and partitiviridae expanding our understanding of the seaweed holobiont: rna viruses of the red alga delisea pulchra this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank all the members of virology and allergy and acarology laboratories. the authors declare no conflict of interest. key: cord- -v o ees authors: nieto-torres, jose l.; verdiá-báguena, carmina; castaño-rodriguez, carlos; aguilella, vicente m.; enjuanes, luis title: relevance of viroporin ion channel activity on viral replication and pathogenesis date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: v o ees modification of host-cell ionic content is a significant issue for viruses, as several viral proteins displaying ion channel activity, named viroporins, have been identified. viroporins interact with different cellular membranes and self-assemble forming ion conductive pores. in general, these channels display mild ion selectivity, and, eventually, membrane lipids play key structural and functional roles in the pore. viroporins stimulate virus production through different mechanisms, and ion channel conductivity has been proved particularly relevant in several cases. key stages of the viral cycle such as virus uncoating, transport and maturation are ion-influenced processes in many viral species. besides boosting virus propagation, viroporins have also been associated with pathogenesis. linking pathogenesis either to the ion conductivity or to other functions of viroporins has been elusive for a long time. this article summarizes novel pathways leading to disease stimulated by viroporin ion conduction, such as inflammasome driven immunopathology. cells maintain optimum subcellular compartment ionic conditions, different to those of the extracellular media by controlling ion transport through lipid membranes. different cell organelles present particular ion compositions. these asymmetric distributions of ions among biological membranes generate electrochemical gradients, essential for the proper cell functioning [ ] . crucial aspects of the cell are governed by the membrane potential, ca `s tores in the endoplasmic reticulum (er) and the golgi apparatus, and different ph conditions found in the organelles of the secretory pathway, which benefit from those ion gradients. the coordinate action of a multitude of ion channels and transporters generates and tightly controls these ionic milieus found within cells. it is well known that viruses exploit and modify host-cell ion homeostasis in favor of viral infection. to that purpose, a wide range of viruses encode viroporins [ ] . viroporins constitute a large family of multifunctional proteins broadly distributed in different viral families, and are mainly concentrated in rna viruses [ ] . highly pathogenic human viruses, such as influenza a virus (iav), human immunodeficiency virus (hiv- ), hepatitis c virus (hcv), several picornaviruses, respiratory syncytial virus (rsv), and coronaviruses (covs), such as the one responsible for the severe acute respiratory syndrome (sars-cov), and the etiologic agent of middle east respiratory syndrome (mers-cov), encode at least one viroporin [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these are transmembrane proteins that stimulate crucial aspects of the viral life cycle through a variety of mechanisms. noticeably, these proteins oligomerize in cell membranes to form ion conductive pores, which generally display mild ion selectivity, indicating that viroporins do not show preference for particular ionic species. the measurements of channel conductance are in accordance with the formation of relatively wide pores, supporting the non-specificity of viroporins. the influence of the lipid charge in channel function is a distinctive feature of some viroporins, as reported in the case of sars-cov e protein [ , ] . ion channel (ic) activity is relevant for virus propagation and may have a great impact on host-cell ionic milieus and physiology [ , , ] . once inserted on cell membranes, viroporins tune ion permeability at different organelles to stimulate a variety of viral cycle stages that will be described below. ic activity ranges from almost essential, to highly or moderately necessary for viruses to yield properly. besides modifying cellular processes to favor virus propagation, the loss of ion homeostasis triggered by viral ic activity may have deleterious consequences for the cell, from stress responses to apoptosis [ , , ] . that is why cells have evolved mechanisms to sense the ion imbalances caused by infections and elaborate immune responses to counteract viruses. interestingly, the ic activity of several viroporins triggers the activation of a macromolecular complex called the inflammasome, key in the stimulation of innate immunity [ ] [ ] [ ] [ ] [ ] [ ] . inflammasomes control pathways essential in the resolution of viral infections. however, its disproportionate stimulation can lead to disease. in fact, disease worsening in several respiratory viruses infections is associated with inflammasome-driven immunopathology [ , ] . taking into consideration the relevance of ic activity in viral production, and its direct effect in pathology and disease, ion conductivity and its pathological stimulated pathways can represent targets for combined therapeutic interventions. in general, viroporins are small proteins (less than amino acids) with at least one amphipathic helix that constitutes its transmembrane domain, spanning lipid membranes [ ] . larger viroporins have also been described in covs. this is the case of sars-cov a protein, porcine epidemic diarrhea virus (pedv) protein, or human coronavirus e (hcov- e) a protein [ ] [ ] [ ] . to form the ion conductive pore, viroporins self-assemble and oligomerize, which is a key feature of this family of proteins. structural studies for either the transmembrane domain or for full-length viroporins have revealed the molecular architecture of these viral ion channels, which can present different oligomerization statuses. iav m , picornavirus b and chlorella virus kcv form tetrameric structures [ ] [ ] [ ] [ ] , whereas pentamers have been described for hiv- vpu, sars-cov and mers-cov e proteins, and rsv sh protein [ , [ ] [ ] [ ] [ ] . hcv p and human papillomavirus e proteins form hexameric channels [ , ] . in addition, most measurements of channel conductance are in accordance with the formation of relatively wide pores, again in agreement with the non-specificity of viroporins [ , , , ] . ion selectivity of ion channels indicates the preference of the pore for a specific ion and defines the functional roles that the channel may display. it is known that iav m protein channels are highly selective for protons and ion conductance is activated at low ph [ ] [ ] [ ] . likewise the iav m channel, the kcv protein of chlorella virus is another highly selective channel, which contains a conserved k`selectivity filter [ ] . nevertheless, most viroporins usually show mild ion selectivity, which means that, in general terms, these channels do not display preference for a particular ion. hiv- vpu protein displays a mild cationic selectivity in nacl and kcl electrolyte solutions [ ] . similarly, hcv p channels are selective for monovalent cations (na`and k`) over monovalent anions (cl´) [ ] . moreover, functionally relevant h`transport has also been identified for this protein in cell culture [ ] . orf a protein of hcov- e forms a channel that prefers cations over anions but does not show a clear specificity for a particular type of cation [ ] . still, a lot of electrophysiology experiments remain to be done for a proper characterization of viroporin selectivity. setting aside a few highly proton selective viroporins, the vast majority display a weak selectivity, either cationic or anionic, which is strongly dependent on the lipid charge of their host membrane, as discussed below in detail. interestingly, under some circumstances the selectivity of these channels can be modulated. sh protein exhibits a poor cationic selectivity at neutral ph, which turns into anionic at acidic ph [ ] . this is consistent with the titration of histidines, the only titratable residues of the sh protein. the presence of ca `i n selectivity experiments performed in kcl solutions reduced the cationic preference of p channels, which may indicate that ca `a ffect the selectivity filter [ ] . probably, the most striking mechanism influencing ic selectivity and conductance has been recently reported for sars-cov e protein [ , ] . it was observed that the lipids are an integral component of the pore structure because electrophysiological measurements proved that the lipid charge modulates the ion transport properties of the sars-cov e protein. these findings suggested that viroporins can assemble into alternative complex structures, forming protein-lipid pores ( figure ). viruses , figure . ion channels formed by viroporins. left depiction represents a channel exclusively formed by protein monomers (blue cylinders) inserted on a lipid membrane. schematic on the right shows a protein-lipid pore. in this latter case, the lipid head groups (cyan circles) are oriented towards the channel pore, modulating ion conductance and selectivity. the ic activity of a number of transmembrane proteins, as well as of small peptides and antimicrobial peptides, is strongly dependent on the lipid environment. the case of sars-cov e protein is a clear example of how lipid membrane charge influences the main ion transport properties of an ic. e protein behaves quite differently when reconstituted in neutral or negatively-charged membranes ( figure ). depictions represent e protein channels inserted in non-charged membranes (left) or negatively-charged membranes (right), under low solute concentrations and neutral ph. in these circumstances, each e protein monomer presents two negative charges provided by glutamic acid residues, and a positive charge conferred by an arginine. when reconstituted in fully or partially negatively-charged membranes, lipid head groups provide additional negative charges to the pore, which makes e protein channel more selective for cations and more conductive. conductance experiments showed that in non-charged phosphatidylcholine membranes, the e protein channel acts as a non-selective neutral pore, since the channel conductance changes linearly with bulk solution conductivity [ ] . in contrast, in negatively charged phosphatidylserine membranes the variation of channel conductance with solution concentration follows different regimes depending on the salt concentration; a characteristic trait of charged pores [ , , ] . these experiments, together with ion selectivity measurements [ ] , suggest that e protein behaves as a charged pore in negativelycharged membranes. parallel observations are made when reconstituting sars-cov e protein in membranes containing a small percentage of charged lipids and, in particular, in membranes containing figure . ion channels formed by viroporins. left depiction represents a channel exclusively formed by protein monomers (blue cylinders) inserted on a lipid membrane. schematic on the right shows a protein-lipid pore. in this latter case, the lipid head groups (cyan circles) are oriented towards the channel pore, modulating ion conductance and selectivity. the ic activity of a number of transmembrane proteins, as well as of small peptides and antimicrobial peptides, is strongly dependent on the lipid environment. the case of sars-cov e protein is a clear example of how lipid membrane charge influences the main ion transport properties of an ic. e protein behaves quite differently when reconstituted in neutral or negatively-charged membranes ( figure ). left depiction represents a channel exclusively formed by protein monomers (blue cylinders) inserted on a lipid membrane. schematic on the right shows a protein-lipid pore. in this latter case, the lipid head groups (cyan circles) are oriented towards the channel pore, modulating ion conductance and selectivity. the ic activity of a number of transmembrane proteins, as well as of small peptides and antimicrobial peptides, is strongly dependent on the lipid environment. the case of sars-cov e protein is a clear example of how lipid membrane charge influences the main ion transport properties of an ic. e protein behaves quite differently when reconstituted in neutral or negatively-charged membranes ( figure ). depictions represent e protein channels inserted in non-charged membranes (left) or negatively-charged membranes (right), under low solute concentrations and neutral ph. in these circumstances, each e protein monomer presents two negative charges provided by glutamic acid residues, and a positive charge conferred by an arginine. when reconstituted in fully or partially negatively-charged membranes, lipid head groups provide additional negative charges to the pore, which makes e protein channel more selective for cations and more conductive. conductance experiments showed that in non-charged phosphatidylcholine membranes, the e protein channel acts as a non-selective neutral pore, since the channel conductance changes linearly with bulk solution conductivity [ ] . in contrast, in negatively charged phosphatidylserine membranes the variation of channel conductance with solution concentration follows different regimes depending on the salt concentration; a characteristic trait of charged pores [ , , ] . these experiments, together with ion selectivity measurements [ ] , suggest that e protein behaves as a charged pore in negativelycharged membranes. parallel observations are made when reconstituting sars-cov e protein in membranes containing a small percentage of charged lipids and, in particular, in membranes containing depictions represent e protein channels inserted in non-charged membranes (left) or negatively-charged membranes (right), under low solute concentrations and neutral ph. in these circumstances, each e protein monomer presents two negative charges provided by glutamic acid residues, and a positive charge conferred by an arginine. when reconstituted in fully or partially negatively-charged membranes, lipid head groups provide additional negative charges to the pore, which makes e protein channel more selective for cations and more conductive. conductance experiments showed that in non-charged phosphatidylcholine membranes, the e protein channel acts as a non-selective neutral pore, since the channel conductance changes linearly with bulk solution conductivity [ ] . in contrast, in negatively charged phosphatidylserine membranes the variation of channel conductance with solution concentration follows different regimes depending on the salt concentration; a characteristic trait of charged pores [ , , ] . these experiments, together with ion selectivity measurements [ ] , suggest that e protein behaves as a charged pore in negatively-charged membranes. parallel observations are made when reconstituting sars-cov e protein in membranes containing a small percentage of charged lipids and, in particular, in membranes containing the same amount of charged lipid (" %) as the ergic-golgi membranes, where e protein channel is presumably localized [ ] . these observations support the view that some polar lipid heads line the pore lumen together with the protein transmembrane helices and that both, protein and lipids, contribute to channel selectivity. an additional proof reinforcing this hypothesis is provided by electrophysiology experiments using lipids with negative intrinsic curvature. it is known that the protein-lipid pore formation is favored in membranes with positive curvature [ ] . in fact, when e protein is reconstituted in dioleoyl phosphatidylethanolamine membranes, a lipid with intrinsic negative curvature, it is much more difficult to achieve channel insertion because the membrane negative curvature becomes energetically unfavorable for the assembly of a proteolipidic structure [ ] . the e protein-lipid channel may not constitute a unique case, as poliovirus b permeabilization displayed a strict requirement for anionic phospholipids in the membrane composition [ ] , which may indicate that lipid molecules are involved in the pore formation. regulatory mechanisms, such as gating, operate in some viroporins. histidine residues present in the iav m transmembrane domain become protonated when encountering low ph conditions, and experience a conformational change that opens the channel pore allowing h`flux [ ] . the influence of the histidine residues in the ic is also observed in the sh protein. in this protein the conductance is ph-dependent and consistent with the titration of these residues [ ] . viroporins could be regulating the transport capacity of cellular ion transporters, besides showing their intrinsic ic activity. a well-defined case is that of human papillomavirus oncoprotein e , which interacts with vacuolar h`atpase (v-atpase) modulating its function and leading to cell transformation [ , ] . interestingly, other viroporins interact with cellular ion pumps. sars-cov e protein binds the alpha subunit of na`/k`atpase in infected cells [ ] . iav m protein binds na`/k`atpase beta subunit [ ] . it is well known that the ion transport capacity of cellular pumps such as na`/k`atpase and sarcoendoplasmic reticulum ca `a tpase (serca) may be influenced by the interaction with other proteins. fxyd proteins and phospholamban are well-characterized examples [ , ] . these regulatory proteins are small transmembrane proteins that oligomerize and eventually form ics when reconstituted in artificial lipid membranes [ ] , as happens for viroporins. some examples of viroporins modifying the activity of cellular ion channels have also been reported. hiv- vpu interacts with the k`channel task- promoting viral release [ ] . in addition, indirect inhibition of epithelial sodium channels has been reported for iav m and sars-cov e proteins [ , ] . the possibility of a dual role of ion modulation by viroporins, depending both on their intrinsic transport properties and on their possible regulatory activity on key cellular ion transporters, increases the potential relevance of this family of proteins. viroporins are involved in processes relevant for virus production. in general, these proteins do not affect viral genome replication, but stimulate other key aspects of the viral cycle such as entry, assembly, trafficking and release of viral particles [ , ] . as a consequence, partial or total deletion of viroporins usually leads to significant decreases in viral yields. hcv p viroporin is indispensable for virus propagation, as no infectious viruses are recovered when p protein is eliminated [ ] . other viruses are more tolerant to viroporin deletion. thus, iav or hiv- lacking m and vpu, respectively, can be efficiently rescued. nevertheless, in general, viroporin defective viruses show significantly lower virus yields, ranging from -to -fold [ , ] . the relevance of e viroporin in coronavirus production seems to be species-specific. deletion of e gene in transmissible gastroenteritis virus (tgev) and mers-cov generates replication-competent propagation-deficient viruses [ , ] . e gene is not essential for virus production in sars-cov and mouse hepatitis virus (mhv), although in its absence viral titters are reduced from -to -fold, respectively [ , ] . silencing of sars-cov a protein, pedv protein, and hcov- e a protein expression in virus infection cause a reduction in virus titers [ , , ] , supporting that these viroporins are involved in virus production. in the case of sars-cov a protein, this has also been shown in mice infected with a sars-cov variant missing a protein (c. castaño-rodriguez, j.l. nieto-torres and l. enjuanes, unpublished results). in some cases, viroporin deletion induces a growth restriction that can be cell or tissue specific. thus, rsv lacking the sh gene, shows an efficient growth in cell culture, but a limited production in the nasal turbinates of infected mice or chimpanzees, as compared with the wild type virus [ , ] . collectively, previous data support that viroporins stimulate virus propagation. a key issue is to understand the relevance of their ic conductance on this activity. both viroporin ic activity and other functions not related to ion conductivity apparently affect virus propagation. some non-conductive viroporin domains are important players in viral morphogenesis. the cytoplasmic tail of several viroporins actively participates in virus production. a few examples are now briefly described. cov e protein c-terminal domain interacts with the viral membrane (m) protein during the morphogenesis process [ ] . in fact, the last nine amino acids of sars-cov e protein c-terminus stimulate virus growth [ ] . this protein sequence includes a pdz-binding motif involved in interactions with cellular proteins and virulence. iav m cytoplasmic domain interacts with the m protein to favor virus assembly at the site of budding [ ] . an amphipathic helix located in the cytoplasmic tail of the iav m protein induces bending of cellular membranes, necessary for budding, and excision of the nascent particles [ ] . this functional domain works independently to the m ion conductive properties. hiv- vpu also facilitates budding termination of newly formed viruses in an ic independent manner. vpu deleted viruses are less efficiently released and remain attached to the plasma membrane of infected cells [ ] . in this case, the transmembrane domain of vpu accounts for this phenotype by establishing critical interactions with cellular proteins such as one with the interferon-stimulated protein tetherin. this binding prevents tetherin from inhibiting the release of viral particles at the plasma membrane of infected cells [ , ] . both ic activity and tetherin inhibitory property are concentrated in the vpu transmembrane domain, nevertheless these functions work independently [ ] . accumulating reports support that viroporin ion conductivity favors virus yields. inhibition of viroporin ic properties, through subtle mutations that may not interfere with other functions of the protein, affects viral growth to different extents. point mutations that blocked hcv p ic activity either completely abrogated virus production or resulted in a -fold restriction in virus yields, depending on the virus strain [ , ] . influenza viruses lacking m ion conductivity, presented either a -fold reduction of viral titer in tissue culture [ ] , or showed a standard production in cell culture but a restricted growth in the nasal turbinates of infected mice [ ] . sars-cov e protein ion conductivity also supports viral production and fitness. e protein ic activity was knocked down by introducing point mutations in key residues of its transmembrane domain [ ] . although not essential for virus production, e protein ic activity stimulated viral propagation. viruses lacking e protein ion conductivity were outcompeted by others displaying this function [ ] . in fact, ic defective viruses tended to restore ion conductance by introducing compensatory mutations in the transmembrane domain of the protein both in cell cultures and in mice [ ] . in agreement with these data, iav lacking m ic activity showed fitness defects, as it was outgrowth by the parental virus, in an even faster manner than that observed in sars-cov [ ] . the relevance of ion conductivity in boosting virus production remains unknown for the moment in other viral systems. initial findings argued that scrambling of the hiv- vpu transmembrane domain inactivated ion conductivity and partially inhibited viral production [ ] . however, this alteration of the transmembrane domain affected the vpu-tetherin interaction and ic activity. recent reports showed that mutations that specifically inhibited ion conductivity, but not vpu-tetherin interaction, did not affect vlp production; therefore, this interaction with tetherin seems to be responsible for the observed phenotype [ ] . pharmacological inhibition of viroporin ion conductance further supports the role of ic in virus propagation. several compounds inhibit viroporin ion conductivity in artificial lipid membranes, and some of them efficiently reduce viral growth when administered to infected cells. inhibition of m protein ic activity mediated by amantadine prevents or decreases viral growth in a strain specific manner [ , ] . amantadine also inhibits hcv growth [ ] . hcov- -e and mhv growth is restricted by hexamethylene amiloride, which blocks e protein ion conductivity [ ] . the viral growth inhibitory properties of these compounds encourage its use as potential antivirals. this aspect will be further addressed below. widely conserved pathways influenced by viroporin ion conductivity, and others that seem to be species-specific are now described and graphically summarized to facilitate their overview ( figure ). early key aspects of virus cycle, such as viral entry, can be boosted through ion conductivity. non-enveloped viral particles lack viroporins as structural components, and these proteins are poorly represented in the membrane of enveloped viruses [ , [ ] [ ] [ ] . nevertheless, the viroporin molecules embedded in the viral envelope can actively participate in viral entry; iav m protein is a well characterized example. iav is internalized within the cell through an endocytic process. endosomes containing viral particles fuse with acidic organelles to form endolysosomes. the h`atpase of these organelles pumps h`inside their lumen lowering the ph. the few m copies present in the iav envelope are activated by the low ph conditions and allow the flux of h`inside the viral particle. the acidification of the endocytosed virion drives a series of conformational changes in the viral hemagglutinin (ha) protein, leading to the exposure of a fusion peptide [ , ] . in parallel, the m protein layer, which underlies the viral envelope, and protects the viral ribonucleoproteins, is disassembled under these ph conditions [ ] . the fusion peptide finally triggers the fusion of the viral and endosomal membranes, resulting in the release of viral ribonucleoproteins in the cell cytoplasm. inhibition of m protein ic activity by amantadine results in incomplete uncoating of the virion in some iav strains [ , ] . this mechanism does not seem relevant for hcv virus, as its entry is independent of p ic activity [ ] . whether or not this pathway, or related ones, participate in the entrance of other viral species remains to be established. viruses , figure . pathways stimulated by viroporin ion channel activity leading to virus production. viral-membrane embedded viroporins (red ellipses) transport h + inside the endocytosed virion. this causes structural changes in fusion and matrix proteins facilitating the uncoating of viral ribonucleoproteins ( ); viroporin-mediated ions leak from intracellular organelles such as the endoplasmic reticulum (er) or the golgi apparatus towards the cytoplasm causes a blockade of vesicle transport and/or hijacking of autophagic membranes. these processes finally result in the accumulation of membranous structures that will serve as platforms for viral replication and morphogenesis. blue, red and green structures show the viral replicase ( ); in addition, equilibration of golgi and secretory pathway organelles' ph protect both viral proteins involved in entry (blue structures and green ellipses) and newly formed virions that can be sensitive to acidic environments ( ); viroporins (red and blue ellipses), locate in the budding neck of some enveloped viruses. these proteins may interact and oligomerize, rearranging the formation of channels, which additionally could facilitate virion scission ( ). early key aspects of virus cycle, such as viral entry, can be boosted through ion conductivity. non-enveloped viral particles lack viroporins as structural components, and these proteins are poorly represented in the membrane of enveloped viruses [ , [ ] [ ] [ ] . nevertheless, the viroporin molecules embedded in the viral envelope can actively participate in viral entry; iav m protein is a well characterized example. iav is internalized within the cell through an endocytic process. endosomes containing viral particles fuse with acidic organelles to form endolysosomes. the h + atpase of these organelles pumps h + inside their lumen lowering the ph. the few m copies present in the iav envelope + figure . pathways stimulated by viroporin ion channel activity leading to virus production. viral-membrane embedded viroporins (red ellipses) transport h`inside the endocytosed virion. this causes structural changes in fusion and matrix proteins facilitating the uncoating of viral ribonucleoproteins ( ); viroporin-mediated ions leak from intracellular organelles such as the endoplasmic reticulum (er) or the golgi apparatus towards the cytoplasm causes a blockade of vesicle transport and/or hijacking of autophagic membranes. these processes finally result in the accumulation of membranous structures that will serve as platforms for viral replication and morphogenesis. blue, red and green structures show the viral replicase ( ); in addition, equilibration of golgi and secretory pathway organelles' ph protect both viral proteins involved in entry (blue structures and green ellipses) and newly formed virions that can be sensitive to acidic environments ( ); viroporins (red and blue ellipses), locate in the budding neck of some enveloped viruses. these proteins may interact and oligomerize, rearranging the formation of channels, which additionally could facilitate virion scission ( ). modification of the ionic content of intracellular compartments can ultimately result in functional and morphological transformations. the ion conductivity of several viroporins affects protein transport. alteration of the ionic content of the golgi apparatus and the endosomes interfere with cellular protein processing and sorting [ ] . iav m causes a lag in protein transport through the golgi apparatus [ ] . this delay on transport can be inhibited by the addition of amantadine, an ic activity inhibitor of m protein. furthermore, monensin, an h`/na`antiporter, causes similar defects. both m and monensin induce dilation of golgi cisternae, although to different extents. infectious bronchitis virus (ibv) e protein also affects protein transport through the secretory pathway, whereas the mutant predicted to inhibit ic activity does not [ ] . coxsackievirus b protein disturbs ph and calcium homeostasis in the golgi and the er, thereby inhibiting protein transport. mutations inhibiting b ion conductivity restored proper protein trafficking [ ] . ca `i s involved in membrane fusion events, and its leakage to the cell cytoplasm causes anterograde vesicle trafficking blockage. sars-cov a protein is both necessary and sufficient for sars-cov golgi fragmentation and promotes the accumulation of intracellular vesicles that may be used for virus formation or for non-lytic release of virus particles [ ] . the exact relevance of protein transport delay on virus production remains to be established. it has been speculated that alteration of ionic homeostasis in transport vesicles affects anterograde trafficking leading to conglomeration of cell membranes that will serve as the platforms for viral replication and budding [ ] . in addition, ionic efflux from intracellular organelles can lead to pathways triggering the accumulation of autophagic vacuoles as platforms for virus replication. in fact, rotavirus nsp viroporin allows ca `e fflux form er thereby triggering autophagy in favor of viral infection [ ] . in many cases, viral proteins, and even virions, have to progress through the secretory pathway, encountering the low ph found within the golgi apparatus lumen [ ] . these acidic conditions may inactivate forming virions, which eventually are acid-sensitive [ ] . viroporin ion conductivity is critical in viral progeny protection and, occasionally, it can be trans-complemented by heterologous viroporins. iav m ic activity is thought to keep the ph of the golgi apparatus and its associated vesicles above a threshold, in order to avoid conformational changes in ha which may lead to its premature activation rendering non-infectious viruses. in fact, blocking of m ion conductivity by amantadine induces irreversible activation of ha [ ] . this effect can be overcome by the treatment of infected cells with monensin that alkalizes the golgi in an analogous manner to m protein. similarly to what has been shown for m protein, hcv p protein mediates a h`leak from intracellular organelles, and this alkalinization is required for the production of infectious viruses [ ] . mutant viruses lacking p ion conductivity were not rescued, and treatment with amantadine greatly diminished the production of wild type infectious virions. furthermore, in the absence of p ic activity, alternative approaches to balance the ph of intracellular organelles result in partial recovery of infectious viruses. either bafilomycin a treatment, an inhibitor of a h`vacuolar atpase (v-atpase), or expression of iav m , but not the m ic mutant, rescued virus production [ ] . the increase of cellular permeability by viroporins, ic activity may ultimately result in cell lysis, a requisite for the egress of non-enveloped viruses [ ] . it has also been speculated that disruption of ion gradients may trigger the membrane fusion events necessary for budding termination and release of enveloped viruses [ ] . nevertheless, in the latter case other non-ion conductive viroporin functions, as those previously described for m and vpu, may also be relevant in the release process. in addition, we propose that viroporins, located in the budding neck of forming viruses [ ] , could oligomerize to form an ic facilitating membrane fusion and budding termination. besides their key role in virus propagation, viroporins are also virulence factors in different viral systems. total or partial deletion of non-essential viroporins usually leads to attenuated phenotypes. interestingly, these viroporin-deleted viruses have been successfully used as potential vaccine candidates. mice infected with an iav lacking m protein showed no weight-loss, nor pathology, and were protected against the wild type virus [ ] . an rsv defective for sh protein showed growth attenuation in the upper respiratory tracts of mice and chimpanzees and caused mild disease [ , ] . a classical swine fever virus (csfv) missing full-length or partial-length p protein, similar to hcv p viroporin, lacked virulence [ ] . a sars-cov lacking the full-length e protein (sars-cov-∆e) was attenuated in hamsters, transgenic mice expressing the human sars-cov receptor (hace ), and in both young and elderly mice. sars-cov-∆e induced increased stress and limited nf-κb driven inflammation [ , , ] . in addition, elimination of defined e protein regions of - amino acids in its carboxy-terminus leads to viral attenuation [ , ] . several of these mutants are promising potential vaccine candidates for sars-cov. sars-cov a protein regulates host-cellular responses involved in the activation of pro-inflammatory genes such as c-jun and nf-κb [ ] [ ] [ ] , and in the production of pro-inflammatory cytokines and chemokines such as il- or ccl [ ] . furthermore, a protein ic activity has been linked to its pro-apoptotic function [ ] . pedv protein has also been involved in pathogenesis as a pedv with a -nucleotide deletion in gene lacks ic activity and is attenuated [ ] . viroporins are frequently associated with virus propagation, as their deletion from the viral genome usually causes viral titer drops that, by themselves, could contribute to the attenuated viral phenotype. however, viroporin removal can modulate cellular signaling pathways leading to virulence. it has been proposed that the protein transport blockage exerted by the ic activity of viroporins, such as that from coxsackievirus b and coronavirus e proteins causes a delay in the transport of major histocompatibility complex class i (mhc-i) molecules towards the plasma membrane [ , , ] . this mhc-i trafficking defect allows the evasion of adaptive immune responses, leading to more productive infections (figure ) . over stimulation of immune responses can also lead to pathogenesis. indeed, ic activity is an important trigger of immunopathology, as demonstrated for sars-cov e protein. mutant viruses lacking ion conductivity, which showed proficient replication in mice lungs, presented an attenuated phenotype. animals infected with the sars-covs lacking e protein ic activity showed a reduced mortality in comparison with those inoculated with the parental virus [ ] . interestingly, mice infected with the ic defective viruses presented much less edema accumulation in the lung airways, the ultimate cause of acute respiratory distress syndrome (ards) [ ] . flooded bronchioles and alveoli fail to interchange gases, leading to hypoxemia and eventually to death. noticeably, there was a good correlation between edema accumulation and the disassembly of the airway epithelia which, when intact, drive edema resolution through an ion mediated water reabsorption. edema accumulation and airway epithelia damage is accompanied by an exacerbated proinflammatory response in the lung parenchyma [ ] . animals infected with the ic deficient viruses presented decreased levels of il- β, tnf and il- in the lung airways, key mediators of the ards progression. il- β is a key orchestrator of the inflammatory response and plays a critical role in counteracting invading viruses, however overstimulation of this pathway can lead to unwanted deleterious effects for the organism. in fact, over production of il- β is related with important pathologies such as gout, atherosclerosis, diabetes, ards and asthma [ ] [ ] [ ] [ ] . as a consequence, il- β production is tightly regulated in the organism, through the inflammasome multiprotein complex. figure . pathways stimulated by viroporin ion channel activity leading to pathology. molecular patterns associated with viral infections are recognized by cellular sensors (signal ), which activate the transcription and translation of the nlrp inflammasome components (nlrp , asc and procaspase- ) and the inactive pro-il- β. viroporins inserted in the intracellular organelles, such as the endoplasmic reticulum (er) or the golgi apparatus, favor the leak of ca `a nd h`ions that move following their electrochemical gradient into cell cytoplasm. this ionic imbalance (signal ) induces the assembly of the inflammasome complex, which triggers the maturation of pro-il- β into il- β through the action of caspase- . secreted il- β mediates a potent pro-inflammatory response that can be deleterious for the cell and the organism, when overstimulated. in addition, alteration of ionic milieus in intracellular compartments comes along with a protein transport delay or blockage. this results in a decrease of the levels of mhc-i molecules (blue rectangles) at the plasma membrane, preventing the infected cell to be recognized by the immune system. protein transport blockage also diminishes the levels and activity in the cell surface of ion channels and transporters, crucial in the resolution of edema accumulation. epithelial sodium channels (green structure) and na`/k`atpase (purple rectangles) impairment have been related to the worsening of viral respiratory diseases such as those caused by sars-cov, iav or rsv. how can viral ic activity stimulate this exacerbated inflammatory response leading to pathology and disease? inflammasomes participate in pathogen recognition by sensing disturbances in cellular milieus, including intracellular ionic concentrations [ , ] . the nlrp inflammasome is one of the most extensively studied [ ] . two signals are generally required to activate this complex: the first one is usually triggered by molecular patterns associated to the viral infection, such as double-stranded rna. this leads to the transcription and translation of the different components of the inflammasome and the immature pro-il- β [ ] . only when a second signal is simultaneously present, the components of the inflammasome are assembled, leading to the cleavage and activation of caspase- . this protein processes pro-il- β into its active form il- β that is released to the extracellular media to promote proinflammation [ ] . interestingly, viroporin ic activity represents the second signal required for inflammasome activation, inducing the release of active il- β (figure ). in general, viroporins induce an efflux of ions, such as h`and ca `t hat move from their intracellular stores into the cytoplasm following strong electrochemical gradients, triggering the nlrp inflammasome. iav m was the first noticed ion conducting viral protein activating this pathway [ ] . nowadays, several other viroporins stimulating the inflammasome have been identified. rsv sh, human rhinovirus b, encephalomyocarditis virus b, hcv p , and iav pb -f , among others, are additional examples [ ] [ ] [ ] [ ] , ] . this mechanism of overstimulation of il- β production seems to have key consequences in pathogenesis. the severity of human rhinovirus infection is linked to overinflammation and release of cytokines, including il- β. rhinovirus b protein causes a ca `l eakage from the er and golgi apparatus essential for inflammasome activation and il- β production [ ] . similarly, rsv sh activates this pathway resulting in immunopathology [ ] . given the wide variety of effects that viroporin ic activity has on viral propagation and its influence on pathogenesis, inhibition of ic conductivity has been a promising target for therapeutic interventions. a growing list of compounds interfering with viroporin ic activity of several viruses such as iav, hcv, hiv- , coronaviruses, and rsv, has been described [ , , [ ] [ ] [ ] . despite being active in artificial lipid bilayers, and sometimes in cell culture, the pharmacological use of antagonists of ic activity in humans is still limited to a reduced number of cases. amantadine was the first inhibitor of viroporin ic activity approved for use in humans, and it has been utilized in clinics for around years in the treatment of iav [ ] . amantadine binds m protein at the n-terminal lumen of the channel and at the c-terminal surface of the protein with high and low affinities, respectively, blocking ion conductance and interfering with viral replication [ ] . however, iav variants containing mutations in the m transmembrane domain that conferred resistance to amantadine emerged [ , ] . amantadine and hexamethylene amiloride are effective inhibitors of hcv p ic activity as shown using in vitro systems [ , ] . hcv shows a genotype-dependent sensitivity to amantadine, when the drug is applied at high doses, which may explain its inefficacy in infected patients, limiting the application of amantadine in hcv disease treatment [ ] . similarly, high concentrations of hexamethylene amiloride are required to inhibit p conductivity in cell culture, which increased its toxicity, making this drug unsuitable for clinical administration [ ] . however, drug screenings have identified other promising compounds interfering p ic activity, such as long alkyl iminosugars derivatives and bit , with the latter showing modest but successful restriction of hcv load in infected patients [ , ] . these compounds may represent the basis for a therapeutic treatment of hepatitis c. taking into consideration the fast selection of drug-resistant viruses under drug pressure, the simultaneous inhibition of ic activity, and other signaling pathways stimulated by ion conductivity leading to pathology, may represent a better treatment option. interfering with these pathological pathways constitutes a valuable approach, with the advantage that it is independent of the appearance of drug resistant viruses. targeting exacerbated inflammatory responses, such as those triggered by ic activity leading to inflammasome activation in various respiratory infections, may reduce disease worsening and progression [ ] . novel specific inhibitors of nlrp inflammasome showing promising results for inflammatory diseases could be applied for these purposes [ ] . viroporins have been known for a long time as key contributors to virus propagation and stimulators of pathogenesis. several reports have dissected the crucial impact that ic activity has in these processes. it is 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inflammasomes in health and disease critical role for calcium mobilization in activation of the nlrp inflammasome k(+) efflux is the common trigger of nlrp inflammasome activation by bacterial toxins and particulate matter initiation and perpetuation of nlrp inflammasome activation and assembly hepatitis c virus induces interleukin- beta (il- beta)/il- in circulatory and resident liver macrophages the p protein of hepatitis c virus forms an ion channel that is blocked by the antiviral drug, amantadine antivirals for the treatment and prevention of epidemic and pandemic influenza. influenza other respir amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by hiv- protein vpu structure of the amantadine binding site of influenza m proton channels in lipid bilayers combination chemotherapy for influenza genotype-dependent sensitivity of hepatitis c virus to inhibitors of the p ion channel a novel hepatitis c virus p ion channel inhibitor, bit , inhibits bovine viral diarrhea virus in vitro and shows synergism with recombinant interferon-alpha- b and nucleoside analogues a small-molecule inhibitor of the nlrp inflammasome for the treatment of inflammatory diseases the work done by the authors was supported by grants from the government of spain the authors declare no conflict of interest. key: cord- -ksodnkic authors: xu, shengnan; jiao, cuicui; jin, hongli; li, wujian; li, entao; cao, zengguo; shi, zhikang; yan, feihu; zhang, shengnan; he, hongbin; chi, hang; feng, na; zhao, yongkun; gao, yuwei; yang, songtao; wang, jianzhong; wang, hualei; xia, xianzhu title: a novel bacterium-like particle-based vaccine displaying the sudv glycoprotein induces potent humoral and cellular immune responses in mice date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ksodnkic sudan virus (sudv) causes severe lethal hemorrhagic fever in humans and nonhuman primates. the most effective and economical way to protect against sudan ebolavirus disease is prophylactic vaccination. however, there are no licensed vaccines to prevent sudv infections. in this study, a bacterium-like particle (blp)-based vaccine displaying the extracellular domain of the sudv glycoprotein (egp) was developed based on a gram-positive enhancer matrix-protein anchor (gem-pa) surface display system. expression of the recombinant gem-displayed egp (egp-pa-gem) was verified by western blotting and immunofluorescence assays. the sudv blps (sblps), which were mixed with montanide isa vg plus poly (i:c) combined adjuvant, could induce high sudv gp-specific igg titers of up to : , and robust virus-neutralizing antibody titers reached : . the sblp also elicited t-helper (th ) and t-helper (th ) cell-mediated immunity. these data indicate that the sblp subunit vaccine has the potential to be developed into a promising candidate vaccine against sudv infections. ebolaviruses, members of the family filoviridae, cause severe hemorrhagic fever in primates and respiratory disease in pigs, and induce high morbidity and mortality [ ] [ ] [ ] . the outbreak in western africa was the largest ebola virus disease (evd) outbreak in history, resulting in , deaths in insect cells and biochemically synthesized (sangon biotech, shanghai, china). the egp-pa fusion gene was amplified by pcr using synthetic oligonucleotide primers as listed in table and cloned into xbaiand kpni-linearized pfastbac vector by clonexpress ultra one step cloning kit (vazyme biotech). pfastbac -egp-pa was then transformed into escherichia coli dh bac competent cells to generate recombinant bacmids. spodoptera frugiperda (sf ; gibco, grand island, ny, usa) insect cells were transfected with the recombinant bacmids using cellfectin ii reagent following the bac-to-bac expression systems manual (invitroge, waltham, ma, usa). recombinant baculoviruses (rbv-egp-pa) were harvested at days post transfection and defined as the first passage (p ) premaster virus. these viruses were expanded in sf cells to generate virus stocks. the sequences of restriction enzyme sites are underlined and italicized. the middle linker (gly-gly-ser-gly) x base sequences are underlined. for indirect immunofluorescence assay (ifa) analysis, sf cells were grown in -well plates and infected with rbv-egp-pa for h and then fixed with % cold acetone for min at room temperature (rt). subsequently, the cells were incubated with a : dilution of a mouse anti-sudv-gp monoclonal antibody (prepared and stored in our laboratory; only reacts specifically with sudv gp) in a buffer containing % bovine serum albumin (bsa) for h at rt. after three washes with phosphate buffered saline (pbs) containing . % tween (pbst), a : dilution of a fluorescein isothiocyanate (fitc)-labeled goat anti-mouse igg antibody (sigma, st. louis, mo, usa) was added with . % evans blue for h at rt. the cells were observed with a fluorescence microscope after washing. for western blotting (wb), expressed fusion protein (egp-pa) was separated by % sds-page under denaturing conditions, and the proteins were transferred to a nitrocellulose (nc) membrane (ge healthcare life sciences, freiburg, germany) for immunoblot analysis with the mouse anti-sudv-gp monoclonal antibody. detection was then performed with a horseradish peroxide (hrp)-conjugated goat anti-mouse antibody and enhanced chemiluminescence. the preparation of gem particles has been described in detail elsewhere [ ] . briefly, l. lactis mg cells were cultured in m broth (oxoid) supplemented with . % glucose at • c. gem particles were obtained by boiling harvested l. lactis in % trichloroacetic acid (tca) for min, followed by extensive washing with pbs. one unit (u) was defined as . × gem particles. finally, the gem particles were resuspended in pbs and stored at − • c until use. preparation of the gem-based vaccine was conducted as follow: supernatants, following supersonic schizolysis containing the egp-pa fusion protein, were mixed with gem particles for min at rt. after binding, the egp-pa-gem complexes were collected, washed five times with sterile pbs, and resuspended in pbs to produce sblp, which were the gem particles displaying the egp antigen on their surface. the target was determined by using a gp-specific antibody for wb. the amount of bound egp-pa was compared to bsa standards by analysis of the sds-page results using software quantity one. for the sds-page and wb analyses of the gem particles, the egp-pa-gem complexes were treated with × sds loading buffer for min at • c, separated using % sds-page gel, and then transferred onto a nitrocellulose (nc) membrane for wb analysis with the mouse anti-sudv-gp monoclonal antibody. for ifa analysis, gem particles with bound egp-pa were blocked with % bsa for min at • c. then, incubations with the primary antibody (mouse anti-sudv-gp monoclonal antibody) and secondary antibody (fitc-labeled goat anti-mouse igg) were performed as previously described (section . ), and the particles were viewed and imaged using a zeiss microscope with incident uv illumination and a zeiss axiovision digital imaging system (zeiss, oberkochen, germany). in total, two batches of balb/c mice (six-to eight-weeks-old females) were purchased from the changchun institute of biological products co., ltd. (changchun, china) and immunized. poly in batch i, mice were randomly divided into groups and immunized as shown in table . in batch ii, mice were randomly divided into groups and then vaccinated with µg egp-pa-gem alone or with isa vg plus poly (i:c) compound adjuvant. in the two batches of animal experiments, all of the mice in the control group received both the same volume of pbs at the same time points. immunizations were performed on study days and . blood samples were collected at two, four, and five weeks post immunization. immune sera were collected and analyzed for sudv-specific antibodies by indirect enzyme-linked immunosorbent assay (elisa). briefly, the assays were performed in -well polystyrene microtiter plates (corning costar, usa) that were precoated with purified gp proteins at a concentration of µg/ml overnight at • c and blocked for . h at • c. serial dilutions of the serum samples were incubated at • c for . h, and the secondary antibodies included hrp-conjugated goat anti-mouse igg, hrp-conjugated igg , and hrp-conjugated igg a, which were incubated at • c for h. to develop the colorimetric reaction, the substrate tetramethylbenzidine (sigma, usa) was added to each well and then the reaction was stopped with m h so . the absorbance was read at nm. titers were determined as the highest dilution at which the mean absorbance of the sample was -fold greater than the mean absorbance of the same dilution of control serum. the neutralizing activity of sera from vaccinated mice against pseudoviruses containing an sudv gp, based on a human immunodeficiency virus backbone, was analyzed as described in previous studies [ ] . briefly, diluted serum samples were added to huh cells, followed by the addition of × % tissue-culture infective dose (tcid ) of pseudotype virus (prepared in a volume equal to that of the serum samples), which were incubated at • c for h before addition to the huh cells. after a h incubation, the medium was replaced with dmem containing % fbs. the plates were incubated for h at • c and luciferase activity was measured using infinite m . neutralizing activity is expressed as the percentage reduction in luciferase activity between the sample wells and control wells: ((luciferase activity in the control well -luciferase activity in the sample well)/(luciferase activity in the control well)) × %. the % neutralization dose (nd ) was calculated using graphpad prism. splenocytes were isolated from vaccinated mice at days after the second vaccination. the cells were cultured in roswell park memorial institute (rpmi) medium (gibco, san diego, ca, usa) containing % fbs and then stimulated with or without purified sudv gp antigen ( µg/ml). following an incubation at • c in % co for h, the splenocytes producing interferon-gamma (ifn-γ), interleukin (il- ) or tumor necrosis factor alpha (tnf-α) were measured using mouse enzyme-linked immunospot (elispot) kits (mouse ifn-γ/il- elispot kit, mabtech ab, stockholm, sweden) according to the manufacturer's instructions. the spot-forming cells (sfcs) were counted using an automated elispot reader (aid elispot reader-ispot, aid gmbh, ger). to detect the levels of the cytokines interleukin (il- ), il- , interleukin (il- ), ifn-γ, and tnf-α, splenocytes were isolated from vaccinated mice at days after the second vaccination and then cultured ( × cells/ml) with stimulation as described above. after h, the supernatants of the stimulated cells were evaluated using murine il- , il- , il- , ifn-γ, and tnf-α elisa kits (mabtech ab, sweden) according to the manufacturer's instructions. statistical analysis was performed using graphpad prism software (graphpad software, la jolla, ca, usa). significant differences between two means were determined by an unpaired student's t-test. data are presented as the mean ± standard error unless otherwise indicated. statistical significance is indicated as * p < . , ** p < . , *** p < . , and **** p < . . as shown in figure a , according to sequence preference characteristics, we designed a new synthetic microconsensus gp based on all strains of sudv from - that were available on the national center for biotechnology information (ncbi) website. the strategy for designing the egp-pa fusion protein, in which egp is fused to the pa with a linker, is shown in figure b . ifa results showed that, compared to control cells, sf cells expressing the egp-pa fusion protein emitted strong green fluorescence ( figure c ). wb showed that egp-pa was successfully expressed and present as a kda protein in supernatants and precipitates after supersonic schizolysis ( figure d ,e). furthermore, as shown in figure d , the mouse anti-sudv-gp monoclonal antibody only reacted specifically with sblp. all these results indicated that the recombinant egp-pa protein reacted with anti-sudv-gp monoclonal antibodies with good antigenicity ( figure c -e). the production process for sblp is shown in figure a . a cell-free extract containing egp-pa was mixed with gem particles, and, after washing, the particles were subjected to sds-page analysis ( figure b ). analysis by quantity one indicated that u of gem particles could bind approximately . μg of the egp-pa fusion protein (data not shown). the surface location of egp-pa on the gem particles was also analyzed by wb ( figure c ) and ifa ( figure d ) with anti-sudv-gp monoclonal antibody. the wb results showed that the egp-pa fusion protein was bound to gem particles. moreover, microscopic observation indicated that, compared with the gem particles alone, the combination of the gem particles and egp-pa fusion protein emitted strong green fluorescence. therefore, the above results indicated that the egp-pa fusion protein was anchored to gem particles. the production process for sblp is shown in figure a . a cell-free extract containing egp-pa was mixed with gem particles, and, after washing, the particles were subjected to sds-page analysis ( figure b ). analysis by quantity one indicated that u of gem particles could bind approximately . µg of the egp-pa fusion protein (data not shown). the surface location of egp-pa on the gem particles was also analyzed by wb ( figure c ) and ifa ( figure d ) with anti-sudv-gp monoclonal antibody. the wb results showed that the egp-pa fusion protein was bound to gem particles. moreover, microscopic observation indicated that, compared with the gem particles alone, the combination of the gem particles and egp-pa fusion protein emitted strong green fluorescence. therefore, the above results indicated that the egp-pa fusion protein was anchored to gem particles. to select the most effective adjuvant from among isa vg plus poly (i:c), poly (i:c), alum, and isa vg for sblp immunization, weeks after immunization, sera samples were obtained and analyzed ( figure a) . the results showed that all mice immunized with sblp had significantly stronger sudv gp-specific igg and neutralizing antibody titers than mice immunized with pbs or gem. in particular, the sblp + + p group had significantly higher anti-sudv gp igg titers than the sblp alone group (p < . ) ( figure b ). furthermore, when the sera samples from vaccinated mice were tested for neutralizing activity, compared with other adjuvant groups, the group immunized with isa vg plus poly (i:c) as an adjuvant had greater neutralization across the most dilutions. (figure b, c) . therefore, the isa vg plus poly (i:c) compound adjuvant was selected for further vaccination experiments in mice. then, we further compared the antibody responses against sudv gp induced by sblp with isa vg plus poly (i:c) and those induced by sblp. pseudotyped virus neutralization assay data showed that the sblp + + p group exhibited stronger neutralizing antibodies than the sblp group ( figure d) . elisa results showed that sera from mice immunized with sblp with isa vg plus poly (i:c) strongly reacted with the sudv gp protein after receiving a second immunization, reaching endpoint titers of up to : , ( figure e ). by contrast, the antibody levels in sera from sblp immunized mice showed no significant differences between the samples harvested at week or weeks after the second immunization, indicating that the antibody response plateaued ( figure e) . a similar phenomenon was also found for sera sudv gp-specific igg ( figure f ) and igg a ( figure f ) antibodies at weeks post immunization in mice immunized with sblp with isa vg plus poly (i:c) and sblp. interestingly, the ratios of igg a/igg of sblp with isa vg plus poly (i:c) were no significant differences than those of sblp ( figure g ), indicating that the sblp induced a mixed t-helper (th ) and t-helper (th ) immune responses. to select the most effective adjuvant from among isa vg plus poly (i:c), poly (i:c), alum, and isa vg for sblp immunization, weeks after immunization, sera samples were obtained and analyzed ( figure a) . the results showed that all mice immunized with sblp had significantly stronger sudv gp-specific igg and neutralizing antibody titers than mice immunized with pbs or gem. in particular, the sblp + + p group had significantly higher anti-sudv gp igg titers than the sblp alone group (p < . ) ( figure b ). furthermore, when the sera samples from vaccinated mice were tested for neutralizing activity, compared with other adjuvant groups, the group immunized with isa vg plus poly (i:c) as an adjuvant had greater neutralization across the most dilutions. (figure b,c) . therefore, the isa vg plus poly (i:c) compound adjuvant was selected for further vaccination experiments in mice. then, we further compared the antibody responses against sudv gp induced by sblp with isa vg plus poly (i:c) and those induced by sblp. pseudotyped virus neutralization assay data showed that the sblp + + p group exhibited stronger neutralizing antibodies than the sblp group ( figure d) . elisa results showed that sera from mice immunized with sblp with isa vg plus poly (i:c) strongly reacted with the sudv gp protein after receiving a second immunization, reaching endpoint titers of up to : , ( figure e ). by contrast, the antibody levels in sera from sblp immunized mice showed no significant differences between the samples harvested at week or weeks after the second immunization, indicating that the antibody response plateaued ( figure e) . a similar phenomenon was also found for sera sudv gp-specific igg ( figure f ) and igg a ( figure f ) antibodies at weeks post immunization in mice immunized with sblp with isa vg plus poly (i:c) and sblp. interestingly, the ratios of igg a/igg of sblp with isa vg plus poly (i:c) were no significant differences than those of sblp ( figure g ), indicating that the sblp induced a mixed t-helper (th ) and t-helper (th ) immune responses. after confirming that sblp with isa vg plus poly (i:c) successfully induced an enhanced antibody responses in mice, we next evaluated the t-cell responses in mice following vaccination. the ifn-γ, il- , and tnf-α secretion by mouse splenocytes was measured in elispot assays. as shown in figure , the sfcs indicative of ifn-γ, il- , or tnf-α production by splenocytes from mice immunized with sblp with isa vg plus poly (i:c) were significantly more than those from mice immunized with sblp, indicating that both the th and th arms of adaptive immunity were activated. after confirming that sblp with isa vg plus poly (i:c) successfully induced an enhanced antibody responses in mice, we next evaluated the t-cell responses in mice following vaccination. the ifn-γ, il- , and tnf-α secretion by mouse splenocytes was measured in elispot assays. as shown in figure , the sfcs indicative of ifn-γ, il- , or tnf-α production by splenocytes from mice immunized with sblp with isa vg plus poly (i:c) were significantly more than those from mice immunized with sblp, indicating that both the th and th arms of adaptive immunity were activated. . elispot analysis of ifn-γ, il- , and tnf-α secretion by mouse splenocytes. the splenocytes were collected from each group days after the second immunization treated and analyzed. the secretion of (a) ifn-γ, (b) il- , and (c) tnf-α was measured by using elispot kit. data are shown as the mean ± sd and were analyzed using one-way anova (* p < . , ** p < . , *** p < . ). to further investigate antigen-specific cellular immune responses, the cytokines secreted by splenocytes were assayed using commercial elisa kits. the levels of the cytokine il- , il- , il- , ifn-γ, and tnf-α secreted by splenocytes from the mice in the sblp with isa vg plus poly (i:c) or sblp alone group were significantly higher than those secreted by splenocytes from the mice in the pbs group ( figure ) . the secretion of ifn-γ, tnf-α, and il- was associated with a th profile, whereas the secretion of il- and il- were associated with a th immune response. these data demonstrated that the sblp vaccine enhanced the secretion of both type cytokines and type cytokines in splenocytes, especially in the presence of adjuvants. . elispot analysis of ifn-γ, il- , and tnf-α secretion by mouse splenocytes. the splenocytes were collected from each group days after the second immunization treated and analyzed. the secretion of (a) ifn-γ, (b) il- , and (c) tnf-α was measured by using elispot kit. data are shown as the mean ± sd and were analyzed using one-way anova (* p < . , ** p < . , *** p < . ). to further investigate antigen-specific cellular immune responses, the cytokines secreted by splenocytes were assayed using commercial elisa kits. the levels of the cytokine il- , il- , il- , ifn-γ, and tnf-α secreted by splenocytes from the mice in the sblp with isa vg plus poly (i:c) or sblp alone group were significantly higher than those secreted by splenocytes from the mice in the pbs group ( figure ). the secretion of ifn-γ, tnf-α, and il- was associated with a th profile, whereas the secretion of il- and il- were associated with a th immune response. these data demonstrated that the sblp vaccine enhanced the secretion of both type cytokines and type cytokines in splenocytes, especially in the presence of adjuvants. the resurgence of the evd epidemic in eastern drc reminds us that there is still the threat of filovirus infection around the world. subunit vaccines are a promising platform to prevent ebov infection due to their relative safety, induction of effective immune responses, and available methods for high-level production [ ] . gp is a promising candidate antigen for an ebov protein vaccine. currently, most ebov vaccines target the viral gp antigen. notably, previous studies have shown that subunit vaccines based on egp were able to protect vaccinated mice against lethal ebov challenge [ , ] . bazzill, j. d. et al. [ ] proved that a recombinant ebov antigen (egp) incorporated into lipid-based nanoparticles could efficiently generate germinal center b cells and polyfunctional t cells while eliciting robust neutralizing antibody responses. furthermore, the absence of the signal sequence and transmembrane domain facilitated protein expression [ ] . considering the immune effect and protein expression, we chose the soluble extracellular domain of the gp protein as the target immunogen in this study. the novel exogenous antigen delivery system in this study was based on nonliving, nongenetically modified l. lactis cells designated gem particles. l. lactis has a long history of use in foods and is recognized as safe [ ] . these particles can bind to externally-added heterologous antigens by means of pa with a high loading capacity and high affinity [ ] . in addition, chimeric anchor fusion proteins can efficiently, strongly, and selectively bind to gem particles in culture medium at rt within a short time period without the need for additional purification steps [ ] . it is easier to obtain purified antigens in a blp-based vaccine than in a virus-like particle (vlp) vaccine [ ] . therefore, the cost of production for vaccines using the gem-pa surface display system will be low. based on the above advantages, the gem-pa surface display system has been applied for the development of a variety of vaccines [ ] . in the present study, we developed a novel sblp vaccine by using the gem-pa surface display system. we found that the sblp were immunogenic, especially in promoting th -and th -type immune responses. however, some studies have shown that the resurgence of the evd epidemic in eastern drc reminds us that there is still the threat of filovirus infection around the world. subunit vaccines are a promising platform to prevent ebov infection due to their relative safety, induction of effective immune responses, and available methods for high-level production [ ] . gp is a promising candidate antigen for an ebov protein vaccine. currently, most ebov vaccines target the viral gp antigen. notably, previous studies have shown that subunit vaccines based on egp were able to protect vaccinated mice against lethal ebov challenge [ , ] . bazzill, j. d. et al. [ ] proved that a recombinant ebov antigen (egp) incorporated into lipid-based nanoparticles could efficiently generate germinal center b cells and polyfunctional t cells while eliciting robust neutralizing antibody responses. furthermore, the absence of the signal sequence and transmembrane domain facilitated protein expression [ ] . considering the immune effect and protein expression, we chose the soluble extracellular domain of the gp protein as the target immunogen in this study. the novel exogenous antigen delivery system in this study was based on nonliving, nongenetically modified l. lactis cells designated gem particles. l. lactis has a long history of use in foods and is recognized as safe [ ] . these particles can bind to externally-added heterologous antigens by means of pa with a high loading capacity and high affinity [ ] . in addition, chimeric anchor fusion proteins can efficiently, strongly, and selectively bind to gem particles in culture medium at rt within a short time period without the need for additional purification steps [ ] . it is easier to obtain purified antigens in a blp-based vaccine than in a virus-like particle (vlp) vaccine [ ] . therefore, the cost of production for vaccines using the gem-pa surface display system will be low. based on the above advantages, the gem-pa surface display system has been applied for the development of a variety of vaccines [ ] . in the present study, we developed a novel sblp vaccine by using the gem-pa surface display system. we found that the sblp were immunogenic, especially in promoting th -and th -type immune responses. however, some studies have shown that immunization with unadjuvanted gp nanoparticle vaccines by intramuscular (im) injection induces igg antibodies but not igg a antibodies against gp [ , , ] . therefore, we assumed that this outcome might be related to the presence of gem particles, which can induce th -type immune responses when used as a vaccine adjuvant [ ] [ ] [ ] . this skewing might be the result of an interaction with toll-like receptor (tlr)- by the peptidoglycan present in the gem particles as tlr- activation can shift the immune response toward a th -type response [ , ] . as far as we know, this is the first report that an egp-based sudv subunit vaccine can induce a mixed th /th immune response without an adjuvant. the importance of a mixed th and th response in mediating protection against lethal ebov infection has been demonstrated in several reports [ ] [ ] [ ] [ ] [ ] . to further improve the immune effect of the sblp vaccine, we used isa vg mixed with poly (i:c) as an adjuvant. isa vg, which was developed for commercial products made with water-in-oil-in-water emulsions, can effectively improve immune response and protection [ ] . poly (i:c) has been demonstrated to be a potent adjuvant with the ability to enhance host innate and adaptive immune responses [ , , ] . immunization of mice with isa vg mixed with poly (i:c)-adjuvanted sblp resulted in significant increases in sudv gp-specific igg, igg , and igg a and sudv pseudotyped virus-neutralizing antibody titers, which are considered to be important correlates of protection [ , ] . we used the ratio of igg to igg a as an indirect method to evaluate induced th -and th -type biases, respectively, in immune responses. interestingly, the ratio of igg a/igg showed no significant difference between sblp alone and with an adjuvant. however, in most studies, isa vg and poly (i:c) adjuvants induced a th -polarized immune response [ , ] . we assumed that the presence of the gem particles may change this bias. further investigations are warranted to confirm the immunological adjuvant function of the gem particles in the sblp subunit vaccine. ebov vaccines should simultaneously stimulate the specific humoral and cellular immunity essential for effective vaccination [ ] [ ] [ ] . the protective cellular immune response is associated with the production of several cytokines, including ifn-γ, tnf-α, il- , il- , and il- [ , ] . our results showed that sblp resulted in significant increases in the secretion of these cytokines and that these levels also significantly increased in the presence of the isa vg plus poly (i:c) adjuvant. our results further proved that sblp can boost immune responses through th and th pathways, which are considered to be important correlates of protection [ ] [ ] [ ] [ ] [ ] . in conclusion, we successfully constructed an sblp vaccine displaying the egp protein antigen in this study. our results clearly demonstrated that sblp with isa vg plus poly (i:c) had high immunogenicity and could elicit robust specific humoral and cellular immunity in vaccinated mice. while protective efficacy evaluation of sblp vaccines in animal models must be performed in a future study, our results strongly support the potential of gem-pa particles as a display and delivery system for subunit vaccine development. additionally, efforts are underway to 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of host immune responses in ebola virus infections marburg virus-like particles by co-expression of glycoprotein and matrix protein in insect cells induces immune responses in mice this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results key: cord- -iv dcpqj authors: li, su; feng, shuo; wang, jing-han; he, wen-rui; qin, hua-yang; dong, hong; li, lian-feng; yu, shao-xiong; li, yongfeng; qiu, hua-ji title: eef a interacts with the ns a protein and inhibits the growth of classical swine fever virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: iv dcpqj the ns a protein of classical swine fever virus (csfv) is involved in the rna synthesis and viral replication. however, the ns a-interacting cellular proteins engaged in the csfv replication are poorly defined. using yeast two-hybrid screen, the eukaryotic elongation factor a (eef a) was identified to be an ns a-binding partner. the ns a–eef a interaction was confirmed by coimmunoprecipitation, glutathione s-transferase (gst) pulldown and laser confocal microscopy assays. the domain i of eef a was shown to be critical for the ns a–eef a interaction. overexpression of eef a suppressed the csfv growth markedly, and conversely, knockdown of eef a enhanced the csfv replication significantly. furthermore, eef a, as well as ns a, was found to reduce the translation efficiency of the internal ribosome entry site (ires) of csfv in a dose-dependent manner, as demonstrated by luciferase reporter assay. streptavidin pulldown assay revealed that eef a could bind to the csfv ires. collectively, our results suggest that eef a interacts with ns a and negatively regulates the growth of csfv. classical swine fever virus (csfv), together with bovine viral diarrhea virus (bvdv) and border disease virus (bdv), is a member of the genus pestivirus within the family flaviviridae and an important pathogen of pigs that often causes huge economic losses to the pig industry worldwide. csfv is a small enveloped virus with a single-stranded, positive-sense rna genome of . kb. the rna genome contains a single large open reading frame (orf) coding for a polyprotein, flanked by a -untranslated region (utr) and a -utr. under cellular and viral protease processing, the polyprotein is processed into mature proteins, including four structural proteins (c, e rns , e and e ) and seven nonstructural proteins (n pro , p , ns - , ns a, ns b, ns a and ns b) [ ] [ ] [ ] . the -utr contains an internal ribosome entry site (ires) that is responsible for translation initiation of the viral genome [ ] . the -utr is in charge of regulating the pestiviral genome replication [ , ] . the csfv ns a comprises amino acids and is a component of the replication complex. the conserved sequence c -c -c -c in the ns a protein has been proved to be important for viral growth and rna synthesis [ ] . it has been shown that ns a regulates viral rna replication through binding to ns b (viral rna-dependent rna polymerase [rdrp] ) and -utr [ ] . the ns a protein of hepatitis c virus (hcv), another member of the family flaviviridae, is an essential component of the replication complex [ ] and plays a role in regulating cellular and viral mrna translation [ , ] . the hcv ns a-core interaction is critical for the subcellular localization of ns a and the production of infectious virus [ ] . the hcv ns a also plays an essential role in the switch from genome replication to particle assembly [ , ] . in addition, the csfv ns a decreases ires-mediated translation in a dose-dependent manner and the key sites (k , t , e and l ) are also found in bvdv, bdv and hcv [ ] . to date, studies have been focused on the roles of the csfv ns a protein in viral genomic replication and translation [ , ] , but knowledge of ns a-interacting host proteins and their impacts on csfv replication is limited. the eukaryotic elongation factor a (eef a), one of the most abundant protein synthesis factors, constitutes % to % of the total soluble proteins in actively dividing cells [ , ] . the canonical function of eef a is to bind aminoacyl-trna (aa-trna) in a gtp-dependent manner and deliver it to the a site on the ribosome during protein synthesis [ ] . eef a is also involved in non-canonical functions including turnover of misfolded proteins, nuclear export events, binding and bundling of the actin cytoskeleton, apoptosis and the viral life cycle [ , ] . eef a contains three well-defined domains: the domain i binds to gtp; the domain ii binds to the aminoacyl end of aa-trna, the domains i and ii bind to the eef b complex, and the domain iii is linked to actin binding [ ] . different groups of viruses, such as tombusvirus (tbsv), human immunodeficiency type (hiv- ) and west nile virus (wnv), utilize eef a as cofactor for viral transcription, translation and assembly. for tbsv, eef a facilitates the assembly of the tbsv replicase and stimulates minus-strand synthesis [ ] . the hiv- nef protein enhances the resistance to stress-induced apoptosis in primary human macrophages through the nuclear-cytoplasmic transport of eef a and trnas. moreover, eef complex subunits are critical hiv- reverse transcription cofactors [ , ] . in addition, the bvdv ns a was also shown to interact with eef a [ ] . considering csfv, bvdv, hcv and wnv are all flaviviridae members, we suppose that the csfv ns a protein possibly interacts with eef a or affects the csfv growth in host cells. in this study, we identified the host protein eef a as a novel ns a-interacting partner that negatively regulates the growth of csfv. in the yeast two-hybrid screen, only one protein, eef a, was identified to be a potential binding protein for ns a ( figure a ). to confirm the interaction between ns a and eef a, glutathione s-transferase (gst) pulldown assay was performed with the gst-tagged ns a protein expressed in escherichia coli and the myc-tagged eef a protein expressed in hek t cells. the results showed that gst-ns a but not gst interacts with eef a ( figure b ). to further verify the interaction between ns a and eef a, coimmunoprecipitation (co-ip) assay was performed with hek t cells overexpressing ˆflag-tagged ns a and myc-tagged eef a. after incubation with anti-flag monoclonal antibody (mab) and protein g-agarose, the myc-tagged eef a was found to coprecipitate with the ˆflag-tagged ns a ( figure c) . furthermore, the ˆflag-tagged ns a was shown to coprecipitate with myc-eef a when the cell lysate was incubated with anti-myc mab and protein g-agarose ( figure d ). considering that both eef a and ns a show a strong affinity for nucleic acids, we investigated whether the ns a-eef a interaction might be mediated through a nonspecific rna bridge. the results showed that the ns a-eef a interaction was not influenced by rnase treatment, indicating that the interaction is not due to nonspecific rna-mediated binding ( figure e ). to examine the colocalization of ns a protein with eef a, the subcellular localization of ˆflag-ns a and myc-eef a was examined by confocal microscopy. both ˆflag-ns a and myc-eef a were colocalized in the cytoplasm ( figure f ). taken together, the results demonstrated that eef a is an interacting partner of the csfv ns a protein. to determine which domain(s) of eef a is responsible for the association with ns a, a series of myc-tagged deletion mutants of eef a were constructed ( figure a ) and tested for the interaction with ns a by co-ip assay. the results showed that eef a( - ) and eef a( - ) were capable of binding ns a ( figure b ), indicating that the domain i is critical for the ns a-eef a interaction. in a yeast two-hybrid (y h) system. yeast y hgold strain was cotransformed with pgbkt -ns a (bd-ns a) as a bait and pgadt -eef a (ad-eef a) as a prey. as a positive control, pgbkt -p (bd-p ) and pgadt -t (ad-t) were cotransformed into y hgold strain. cotransformation of pgbkt -lamin c (bd-lamin c) and ad-t was used as a negative control. all the yeast colonies cotransformed with the above plasmids were grown on synthetically defined (sd) medium lacking his and leu (sd/- ), sd medium lacking his, leu, trp and ade (sd/- ) and sd/- medium containing -bromo- -chloro- -indolyl-α-d-galactopyranoside (x-α-gal) and aureobasidin a (aba) (sd/- /x-α-gal/aba); (b) glutathione s-transferase (gst) pulldown assay. the gst or gst-ns a fusion proteins expressed in escherichia coli bl (de ) were purified with glutathione sepharose b resin and incubated with the lysate of hek t cells overexpressing the myc-tagged eef a. after washing with cold pbs, the bound proteins were subjected to sulfate-polyacrylamide gel electrophoresis (sds-page) ( %) and western blotting using the anti-gst polyclonal antibody (pab) ( : ) and the anti-myc monoclonal antibody (mab) ( : ); (c) coimmunoprecipitation (co-ip) analysis of myc-tagged eef a and flag-tagged ns a. hek t cells were cotransfected with the indicated plasmids (+) or empty vectors (-) for h. the transfected cells were lyzed and incubated with a mouse anti-flag mab, followed by incubation with the protein g-agarose for h at ˝c. the immunoprecipitate was analyzed by western blotting using the anti-flag mab ( : ) and a rabbit anti-myc pab ( : ); (d) co-ip analysis of eef a and ns a by the anti-myc mab. hek t cells were cotransfected with the indicated plasmids (+) or empty vectors (´), the cell lysate was collected at h post-transfection (hpt), incubated with the anti-myc mab and protein g-agarose. the immunoprecipitate was examined by western blotting using the anti-flag mab ( : ) and the anti-myc pab ( : ); (e) effects of rnase a/t treatment on the ns a-eef a interaction. extracts of hek t cells overexpressing different proteins were treated or untreated with rnase a/t for h prior to co-ip and immunoblotted with the indicated antibodies. the total rna in the immunoprecipitate was visualized by ethidium bromide-staining; (f) colocalization of ns a protein with eef a. hek t cells were cotransfected with p ˆflag-ns a and pmyc-eef a. cells were fixed at hpt and subjected to indirect immunofluorescence assay to detect ˆflag-ns a (green) and myc-eef a (red) with mouse anti-flag and rabbit anti-myc antibodies. the position of the nucleus is indicated by dapi (blue) staining in the merged image. samples were imaged and magnified times on the leica sp confocal system fitted with a ˆobjective lens. scale bar = . µm. to investigate the effects of eef a on the csfv replication, stable pk- cell line overexpressing egfp-eef a (pk-egfp-eef a) or egfp (pk-egfp) was generated and infected with the csfv shimen strain at a multiplicity of infection (moi) of . . the n pro expression in the csfv-infected pk-egfp-eef a cells was decreased at and h post-infection (hpi), compared with that in the pk-egfp cells ( figure a ,b). real-time rt-pcr analysis revealed that the viral genome copies in the pk-egfp-eef a cells were also reduced at and hpi, compared with the pk-egfp cells ( figure c ). additionally, yields of infectious virus in culture supernatant of pk-egfp-eef a cells were decreased at and hpi ( figure d ). the results indicated the eef a negatively modulates the csfv replication. to determine the effects of eef a on csfv replication, recombinant lentiviruses expressing short hairpin rna (shrna) against eef a (sheef a) or non-targeting negative control shrna (shnc) to investigate the effects of eef a on the csfv replication, stable pk- cell line overexpressing egfp-eef a (pk-egfp-eef a) or egfp (pk-egfp) was generated and infected with the csfv shimen strain at a multiplicity of infection (moi) of . . the n pro expression in the csfv-infected pk-egfp-eef a cells was decreased at and h post-infection (hpi), compared with that in the pk-egfp cells ( figure a ,b). real-time rt-pcr analysis revealed that the viral genome copies in the pk-egfp-eef a cells were also reduced at and hpi, compared with the pk-egfp cells ( figure c ). additionally, yields of infectious virus in culture supernatant of pk-egfp-eef a cells were decreased at and hpi ( figure d ). the results indicated the eef a negatively modulates the csfv replication. to further determine the effects of eef a on csfv replication, recombinant lentiviruses expressing short hairpin rna (shrna) against eef a (sheef a) or non-targeting negative control shrna (shnc) were generated and transduced into pk- cells, resulting in efficient knockdown of the eef a expression ( figure a ). twenty-four hours after lentivirus-mediated shrnas transduction, pk- cells were infected with the csfv shimen strain at an moi of . . the viral replication was analyzed by real-time rt-pcr, virus titration and western blotting. in comparison with the shnc control, the viral genome copies in the eef a-knocked down cells were increased at and hpi ( figure a ). similarly, the csfv titers were significantly increased after eef a knockdown in pk- cells ( figure b) , and the n pro protein expression in the sheef a-transduced cells was increased at and hpi ( figure c ). the results indicate that the eef a negatively modulates the csfv replication. viruses , were generated and transduced into pk- cells, resulting in efficient knockdown of the eef a expression ( figure a ). twenty-four hours after lentivirus-mediated shrnas transduction, pk- cells were infected with the csfv shimen strain at an moi of . . the viral replication was analyzed by real-time rt-pcr, virus titration and western blotting. in comparison with the shnc control, the viral genome copies in the eef a-knocked down cells were increased at and hpi ( figure a ). similarly, the csfv titers were significantly increased after eef a knockdown in pk- cells ( figure b ), and the n pro protein expression in the sheef a-transduced cells was increased at and hpi ( figure c ). the results indicate that the eef a negatively modulates the csfv replication. a previous study showed that ns a decreases the csfv ires-mediated translation in a dose-dependent manner [ ] . to examine the effects of eef a on the ns a-mediated inhibition of the csfv ires activity, the luciferase reporter assay was used in this study. the results showed that eef a ( figure a ), as well as ns a ( figure b ), inhibited the csfv ires activity in a dose-dependent manner. to further investigate the inhibitory effects of eef a and ns a coexpression, eef a-and ns a-expressing plasmids were cotransfected with the luciferase reporter plasmids into hek t cells. the results indicated that the eef a did not antagonize the inhibition of the ires activity by ns a when ns a and eef a were coexpressed ( figure c ). a previous study showed that ns a decreases the csfv ires-mediated translation in a dose-dependent manner [ ] . to examine the effects of eef a on the ns a-mediated inhibition of the csfv ires activity, the luciferase reporter assay was used in this study. the results showed that eef a ( figure a ), as well as ns a ( figure b ), inhibited the csfv ires activity in a dose-dependent manner. to further investigate the inhibitory effects of eef a and ns a coexpression, eef a-and ns a-expressing plasmids were cotransfected with the luciferase reporter plasmids into hek t cells. the results indicated that the eef a did not antagonize the inhibition of the ires activity by ns a when ns a and eef a were coexpressed ( figure c ). hek t cells were transfected with pmyc-eef a, pmyc-ns a or pcmv-myc. the cell lysate was incubated with the biotinylated csfv ires or prrsv ʹ-utr, followed by incubation with streptavidin beads. bound proteins were analyzed by immunoblotting using the anti-myc monoclonal antibody (mab) ( : ); (b) eef a interacts with the csfv ires in a dose-dependent manner. hek t cells grown in the -well plate were transfected with an increased amount of pmyc-eef a ( , , and μg), followed by streptavidin pulldown assay as described above; (c) competitive rna pulldown assay. hek t cells grown in the -well plate were transfected with the indicated pmyc-eef a ( μg). the cell lysate was incubated with an increased amount of the ires rna ( , . , . and . μg) for h at °c and incubated with the biotinylated ires rna and streptavidin beads. bound proteins were subjected to immunoblotting using the anti-myc mab. or pmyc-ns a. the cell lysate was incubated with the biotinylated csfv ires or porcine reproductive and respiratory syndrome virus (prrsv) -utr, followed by incubation with streptavidin beads. the bound proteins were analyzed by immunoblotting using the anti-myc monoclonal antibody (mab) ( : ); (b) eef a interacts with the csfv ires in a dose-dependent manner. hek t cells grown in the -well plate were transfected with an increased amount of pmyc-eef a ( , , and µg), followed by streptavidin pulldown assay as described above; (c) competitive rna pulldown assay. hek t cells grown in the -well plate were transfected with the indicated pmyc-eef a ( µg). the cell lysate was incubated with an increased amount of the ires rna ( , . , . and . µg) for h at ˝c and incubated with the biotinylated ires rna and streptavidin beads. the bound proteins were subjected to immunoblotting using the anti-myc mab. the observation that overexpression of eef a resulted in the inhibition of the csfv ires activity prompted an examination of the interaction between eef a and ires of csfv. streptavidin pulldown assay showed that the biotinylated csfv ires, but not the biotinylated porcine reproductive and respiratory syndrome virus (prrsv) -utr, could bind to eef a in a dose-dependent manner ( figure a,b) . competitive rna pulldown assay indicated that the association of eef a with the csfv ires could be inhibited by the unlabeled csfv ires ( figure c ). the results demonstrated that eef a binds to the csfv ires and inhibits its activity. the csfv ns a functions as the modulation of viral rna replication via interacting with ns b and -utr [ ] . in addition, the ns a protein can inhibit the ires-mediated translation through binding to the ires located in the -utr. a previous study showed that the ns a-interacting cellular proteins were screened using y h analysis, and the identified proteins are mostly related to gene transcription, protein degradation, protein folding and metabolism [ ] . recently, heat shock protein has been identified to interact with the csfv ns a and enhance viral rna replication [ ] . in this study, eef a was identified as a novel ns a partner and a negative regulator of csfv replication. eef a had not been identified in the previous study, probably due to the different cell types (endothelial cells vs. primary macrophages) used to construct the cdna library [ ] . we showed that ns a and eef a were colocalized in the cytoplasm. as ns a is the component of the replication complex, whether eef a is also localized in the replication complex needs further investigation. the domain i of eef a was shown to be critical for the ns a-eef a interaction, implying that it may be a potential target of anti-csfv strategies. we also showed that eef a was able to bind to the ires located in the -utr and inhibit its activity. in addition, we investigated the effects of eef a on the inhibition of the ires activity by ns a; however, we demonstrated that eef a did not antagonize the inhibition of the ires activity by ns a, indicating that ns a and eef a perhaps bind to distinct parts of the ires. several viral proteins have been observed to associate with eef a. the nucleocapsid protein of severe acute respiratory syndrome coronavirus (sars-cov) could modulate cell cytokinesis and proliferation via interacting with eef a [ ] . eef a also interacts with the nucleocapsid protein of transmissible gastroenteritis virus (tgev) and regulates the virus replication [ ] . furthermore, eef a can be utilized as the cofactors of the viral rna synthesis for several viruses, such as hiv- [ , ] , tbsv [ ] and wnv [ ] . previous studies on turnip mosaic virus (tumv), tobacco mosaic virus (tmv) and tbsv have shown that eef a interacts primarily with the viral rdrp complex, and thus acts as a chaperone and supports viral replication [ ] [ ] [ ] . as members of flaviviridae, both bvdv ns a and hcv ns a interact with eef a [ , ] . thereinto, the exact role of eef a on the bvdv replication remains unclear [ ] . the hcv ns a inhibits the viral ires-mediated translation, and the translation inhibitory effect of ns a was relieved by the addition of purified recombinant eef a in the in vitro translation system [ ] . however, we showed here that eef a negatively regulates the csfv growth. we conclude that the different roles of eef a in viral replication may vary depending on the distinct interactions between eef a and different viruses. in this study, the eef a was shown to bind to the ires and inhibit its activity, thus it is possible to suppress the csfv replication. since eef a binds to aa-trna among mammalian rna species [ ] , it is possible for eef a to bind to the similar rna structures located in -or -utr of the viral genome. it has been shown that interaction between eef a and the -terminal stem-loop of the wnv genomic rna facilitates the viral minus-strand rna synthesis [ , , ] . in the present study, we demonstrated that eef a binds to ires located in the -utr of csfv. whether eef a binds to -utr of csfv and influences rna replication requires further study. we showed that eef a inhibits csfv growth and reduces ires activity of csfv. thus, we speculate that eef a affects the process of the viral protein translation via interacting with the csfv ires. in summary, we demonstrated that eef a interacts with ns a and negatively modulates the csfv growth. the possible mechanism of the negative effect of eef a on the csfv growth is that eef a interacts with the csfv ires and inhibits its activity. further studies are needed to define the detailed interplay among eef a, ns a and ires in the life cycle of csfv. pk- or sk cells (porcine kidney cell lines) and hek t cells were grown in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) confirmed to be free of bvdv and anti-bvdv antibodies. the csfv shimen strain was propagated in pk- or sk cells. viral titers in the culture supernatant of csfv-infected cells were determined as described previously [ ] . table . primers used in this study. sequences ( the swine eef a gene (genbank accession no. nm_ . ) and its various domains were amplified by pcr and cloned into the pcmv-myc vector (clontech, palo alto, ca, usa) to generate pmyc-eef a, pmyc-eef a( - ), pmyc-eef a( - ), pmyc-eef a( - ) and pmyc-eef a( - ), respectively. the csfv ns a gene (genbank accession no. eu . ) was amplified by pcr and cloned into the p ˆflag-cmv- vector (sigma-aldrich, st. louis, mo, usa) or pcmv-myc to generate p ˆflag-ns a or pmyc-ns a. the primers for amplifying eef a and ns a genes are listed in table . a matchmaker two-hybrid system (catalog no. ; clontech) was used to screen host proteins that interact with ns a. briefly, the bait construct pgbkt -ns a (bd-ns a) was transformed into the yeast strain y hgold and hybridized with a cdna library derived from porcine macrophages [ ] . transformants were selected for growth on synthetically defined (sd) medium lacking his, leu, trp and ade (sd/- ) for to days at ˝c. the clones were then transferred to sd/- medium containing -bromo- -chloro- -indolyl-α-d-galactopyranoside (x-α-gal) and aureobasidin a (aba) (sd/- /x-α-gal/aba). blue colonies were selected and inoculated with ml of sd/- medium for shaking culture at ˝c for to days. yeast plasmids were extracted using a yeast plasmid kit (catalog no. d ; omega, guangzhou, china) according to the manufacturer's instructions, and the target inserts were verified by sequencing using the gal ad and ad primers. to validate the interaction between ns a and the cellular proteins, the bait and prey plasmids were cotransformed into the y hgold yeast strain using a yeast transformation system (catalog no. ; clontech). the interaction between murine p and sv large t-antigen was used as a positive control, and the human lamin c protein, which does not interact with the sv large t-antigen, was included as a negative control. hek t cells grown in -well plates (nunc, waltham, ma, usa) were cotransfected with µg of pmyc-eef a and µg of p ˆflag-ns a using an x-tremegene hp dna transfection reagent (catalog no. ; roche, penzberg, germany) according to the manufacturer's instructions. briefly, the cells were transfected with µl of serum-free dmem containing µg of plasmids and µl of x-tremegene hp dna transfection reagent. at h post-transfection (hpt), the transfection mixture was replaced with dmem supplemented with % fbs and incubated for an additional h before being assayed. for expression of the gst-tagged ns a protein, the ns a gene was subcloned into the pgex- p- vector (catalog no. - - ; ge healthcare, piscataway, nj, usa), creating pgex-ns a. the gst or gst-ns a fusion protein expressed in e. coli bl (de ) were purified with the glutathione-sepharose b resin (catalog no. ; ge biosciences, piscataway, nj, usa) according to the manufacturer's instructions. briefly, the expression of gst or gst-ns a protein was induced by addition of mm isopropylthiogalactoside (iptg) (catalog no. st ; beyotime, shanghai, china). the bacterial cells were harvested and resuspended in cold phosphate-buffered saline (pbs) containing mg/ml protease inhibitor cocktail (catalog no. ; roche, penzberg, germany), followed by mild sonication. insoluble components were removed by centrifugation at , ˆg for min. subsequently, the soluble gst or gst-ns a protein was incubated with the glutathione-sepharose b resin for h at ˝c. the resins were washed four times with cold pbs and incubated with µl of the lysate of the hek t cells transfected with pmyc-eef a for h at ˝c. after an extensive wash with pbs, the bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) followed by immunoblotting using the anti-myc mab ( : ) (catalog no. m ; sigma-aldrich) and an anti-gst polyclonal antibody (pab) ( : ) (catalog no. ab ; tiangen, beijing, china). for co-ip assay, the p ˆflag-ns a and pmyc-eef a were cotransfected into hek t cells as described above. the cells were collected at hpt, washed two times with cold pbs, and lyzed with the chaps lysis buffer ( % chaps, mm nacl, mm mgcl and mm tris-hcl [ph . ]) containing mm pmsf (catalog no. st ; beyotime) and mg/ml protease inhibitor cocktail at ˝c for h. the cell lysate was centrifuged at , ˆg for min at ˝c. the clarified lysate was treated with a mixture of rnase a ( u/ml) (catalog no. ; takara, dalian, china) and rnase t ( u/µl) (catalog no. pf ; fermentas, burlington, canada) for h at room temperature prior to co-ip analysis. the lysate was precleared with the protein g-agarose (catalog no. ; roche) at ˝c for h followed by incubation with the indicated antibodies at ˝c for h, and the protein g-agarose was then added and incubated at ˝c for h. the protein g-agarose was washed three times with pbs, and the bound proteins were separated by sds-page followed by western blotting using the indicated antibodies. hek t cells were cotransfected with pmyc-eef a ( µg) and p ˆflag-ns a ( µg). after -h incubation, the cells were fixed with % paraformaldehyde in pbs for min and permeabilized with . % triton x- for min. the cells were then incubated with an anti-myc pab ( : ) (catalog no. c ; sigma-aldrich) or anti-flag mab ( : ) (catalog no. f ; sigma-aldrich) for h. the cells were incubated with goat anti-mouse igg (whole molecule)-fluorescein isothiocyanate (fitc) antibody ( : ) (catalog no. f ; sigma-aldrich) and goat anti-rabbit igg (whole molecule)-tetramethyl rhodamine isocyanate (tritc) antibody ( : ) (catalog no. t ; sigma-aldrich). subsequently, the cells were stained with , -diamidino- -phenylindole (dapi) ( : ) (catalog no. d ; sigma-aldrich) for min and examined using a leica sp confocal system (leica microsystems, gmbh, wetzlar, germany). to construct a stable cell line overexpressing eef a, the swine eef a gene was subcloned into the lentivirus vector pfugw (addgene, cambridge, ma, usa). hek t cells were cotransfected with the recombinant plasmid pfugw-eef a or the empty vector pfugw, and the packaging plasmids pspax (addgene) and pmd .g (addgene). at hpt, the medium was replaced with dmem containing % fbs. after -h incubation, the supernatant of the cell culture was harvested and ultra-centrifuged to concentrate the recombinant lentiviruses. subsequently, pk- cells grown on -well plates were transduced with the lentiviruses of transduction units (tu) per cell. the expression of egfp-eef a in the transduced pk- cells was examined by western blotting using a rabbit anti-eef a mab ( : ) (catalog no. ab ; abcam, cambridge, uk). to knock down the expression of eef a in pk- cells, a lentivirus vector-mediated shrna targeting eef a was constructed. briefly, the upper and lower sequences of shrnas for eef a and non-targeting negative control (nc) are listed in table . the oligonucleotides were annealed and cloned into the lentivirus vector plvx-shrna (clontech). the resulting recombinant plasmids plvx-eef a-shrna , plvx-eef a-shrna or plvx-nc-shrna were cotransfected with the packaging plasmids pspax and pmd .g into hek t cells in -cm cell dishes. at hpt, the lentiviruses in the culture supernatant were concentrated at ˆg for min at ˝c using an amicon ultra- centrifugal filter unit with ultracel- membrane (catalog no. ufc ; millipore, billerica, ma, usa). the lentiviuses were titrated and transduced into pk- cells as described above. knockdown of eef a was validated by western blotting using the anti-eef a mab ( : ). total rna was extracted from the culture supernatant of csfv-infected pk- cells using a trizol reagent (catalog no. ; invitrogen, waltham, ma, usa). the isolated rna was then reverse transcribed to cdna with reverse transcriptase xl (amv) (catalog no. ; takara, dalian, china) according to the manufacturer's instructions. genomic copies of csfv were quantified by a real-time rt-pcr assay as described previously [ ] . the experiment was carried out in triplicates. in luciferase reporter assay, the reporter plasmid pfluc/ires/rluc [ ] , a gift from prof. belsham, was used, which contains the firefly luciferase (fluc) gene under the control of the t promoter and the renilla luciferase (rluc) gene under the control of the csfv ires. the plasmid plxsn-t expressing the t rna polymerase [ ] was also used in the assay. for the luciferase reporter assay, hek t cells grown on -well plates were cotransfected with ng of pfluc/ires/rluc, ng of plxsn-t and different amounts of p ˆflag-ns a or pmyc-eef a. after -h incubation, the reporter gene activity was analyzed with a dual-luciferase reporter assay system (catalog no. e ; promega, madison, wi, usa) and measured with the td- / luminometer (turner designs, morgan hill, ca, usa) according to the manufacturer's instructions. the data represent the rluc activity normalized to the fluc activity. three independent experiments were carried out in duplicates. the csfv ires or prrsv -utr fragment was amplified and cloned into the pcdna . (+) vector to generate pcdna-ires or pcdna-prrsv- -utr. subsequently, the plasmids were linearized by ecorv and transcribed in vitro using a ribomaxtm large scale rna production system (catalog no. p ; promega) according to the manufacturer's instructions. the resulting rna was labeled with photobiotin (catalog no. a ; baoman, shanghai, china) using a mercury vapor lamp for min. hek t cells grown on -well plates were transfected with the indicated plasmids (pmyc-ns a and pmyc-eef a) and lyzed with the lysis buffer. the lysate collected from two wells of the -well plate (lyzed with µl of lysis buffer per well) was incubated with the biotinylated ires rna ( µg) for h at ˝c, and then incubated with streptavidin beads (catalog no. d; invitrogen) for another min at room temperature. the beads were washed three times with lysis buffer and analyzed by immunoblotting with the anti-myc mab ( : ). for competitive rna pulldown, the cell lysate was incubated with an increased amount of unlabeled ires rna ( , . , . and µg) for h at ˝c and then incubated with the biotinylated ires and streptavidin beads. the experiments were carried out in triplicates. statistical analysis was performed using the spss . software. student's t-test or one-way analysis of variance was used to compare the csfv genomic copies. a p-value of < . was considered significant. introduction to classical swine fever: virus, disease and control policy genus pestivirus (flaviviridae). in the springer index of viruses pestivirus internal ribosome entry site (ires) structure and function: elements in the -untranslated region important for ires function specific interaction between the classical swine fever virus ns b protein and the viral genome essential and nonessential elements in the nontranslated region of bovine viral diarrhea virus characterization of ns , ns a and ns b of classical swine fever virus through mutation and complementation analysis classical swine fever virus ns a regulates viral rna replication through binding to ns b and utr the ns a protein of hepatitis c virus is a zinc metalloprotein hepatitis c virus (hcv) ns a protein downregulates hcv ires-dependent translation hepatitis c virus ns a protein down-regulates the expression of spindle gene aspm through pkr-p signaling pathway interaction of hepatitis c virus nonstructural protein a with core protein is critical for the production of infectious virus particles domain iii of ns a contributes to both rna replication and assembly of hepatitis c virus particles regulation of the production of infectious genotype a hepatitis c virus by ns a domain iii influence of ns a protein of classical swine fever virus (csfv) on csfv internal ribosome entry site-dependent translation determination of the amounts of the protein synthesis initiation and elongation factors in wheat germ elongation factor alpha, translation and the cytoskeleton thinking outside the ribosome actin bundling and polymerisation properties of eukaryotic elongation factor alpha (eef a), histone h a-h b and lysozyme in vitro the many roles of the eukaryotic elongation factor complex translation elongation factor a facilitates the assembly of the tombusvirus replicase and stimulates minus-strand synthesis inhibition of er stress-mediated apoptosis in macrophages by nuclear-cytoplasmic relocalization of eef a by the hiv- nef protein eukaryotic elongation factor complex subunits are critical hiv- reverse transcription cofactors the ns a protein of bovine viral diarrhoea virus interacts with the alpha subunit of translation elongation factor- evaluation of a multiplex real-time rt-pcr for quantitative and differential detection of wild-type viruses and c-strain vaccine of classical swine fever screening of cellular proteins that interact with the classical swine fever virus non-structural protein a by yeast two-hybrid analysis heat shock protein is associated with csfv ns a protein and enhances viral rna replication the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor alpha ef a interacting with nucleocapsid protein of transmissible gastroenteritis coronavirus and plays a role in virus replication identification of cis-acting nucleotides and a structural feature in west nile virus -terminal rna that facilitate viral minus strand rna synthesis eukaryotic elongation factor a interacts with turnip mosaic virus rna-dependent rna polymerase and vpg-pro in virus-induced vesicles in vivo interaction between tobacco mosaic virus rna-dependent rna polymerase and host translation elongation factor a translation elongation factor a is a component of the tombusvirus replicase complex and affects the stability of the p replication cofactor hepatitis c virus ns a inhibits cap-dependent and the viral ires-mediated translation through interacting with eukaryotic elongation factor a rna-mediated response to heat shock in mammalian cells translation elongation factor- alpha interacts with the stem-loop region of west nile virus genomic rna interaction between the cellular protein eef a and the -terminal stem-loop of west nile virus genomic rna facilitates viral minus-strand rna synthesis a simple method of estimating fifty percent endpoints thioredoxin is a novel e -interacting protein that inhibits the replication of classical swine fever virus modulation of translation initiation efficiency in classical swine fever virus rapid recovery of classical swine fever virus directly from cloned cdna we thank graham j. belsham from the national veterinary institute, technical university of denmark, for kindly providing the plasmid pfluc/ires/rluc.we are grateful to lihong liu at national veterinary institute, sweden, for helpful suggestions. this study was supported by the natural science foundation of china (grant ) and the natural science foundation of heilongjiang province of china (grants zd , c and qc ). hua-ji qiu conceived and designed the study. su li, shuo feng, jing-han wang, wen-rui he, hua-yang qin, hong dong, lian-feng li, shao-xiong yu and yongfeng li performed the experiments. su li, shuo feng and hua-ji qiu wrote the manuscript. the authors declare no conflicts of interest. key: cord- -yok mh authors: andreata-santos, robert; alves, rúbens prince dos santos; pereira, sara araujo; pereira, lennon ramos; de freitas, carla longo; pereira, samuel santos; venceslau-carvalho, alexia adrianne; castro-amarante, maria fernanda; favaro, marianna teixeira pinho; mathias-santos, camila; amorim, jaime henrique; ferreira, luís carlos de souza title: transcutaneous administration of dengue vaccines date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: yok mh in the present study, we evaluated the immunological responses induced by dengue vaccines under experimental conditions after delivery via a transcutaneous (tc) route. vaccines against type dengue virus particles (denv new guinea c (ngc) strain) combined with enterotoxigenic escherichia coli (etec) heat-labile toxin (lt) were administered to balb/c mice in a three-dose immunization regimen via the tc route. as a control for the parenteral administration route, other mouse groups were immunized with the same vaccine formulation via the intradermic (id) route. our results showed that mice vaccinated either via the tc or id routes developed similar protective immunity, as measured after lethal challenges with the denv ngc strain. notably, the vaccine delivered through the tc route induced lower serum antibody (igg) responses with regard to id-immunized mice, particularly after the third dose. the protective immunity elicited in tc-immunized mice was attributed to different antigen-specific antibody properties, such as epitope specificity and igg subclass responses, and cellular immune responses, as determined by cytokine secretion profiles. altogether, the results of the present study demonstrate the immunogenicity and protective properties of a dengue vaccine delivered through the tc route and offer perspectives for future clinical applications. infection with one of the four dengue virus serotypes (denv - ) may cause a spectrum of diseases ranging from an acute, self-limiting febrile illness (df) characterized mainly by fever, retro-orbital headache, rash, arthralgia, and to more severe, life-threatening, conditions that may include hemorrhagic manifestations, increased vascular permeability, thrombocytopenia, and shock [ ] [ ] [ ] . in fact, it is estimated that . billion people in countries are at risk of infection [ , ] . denv causes approximately million infections, of which , cases develop into severe forms, making denv infection one of the most economically and epidemiologically relevant arthropod-borne diseases the murine model elected for this study was based on the balb/c mouse line. male mice from to weeks old were supplied by the parasitology department animal house at the university of são paulo. the animals were considered free of pathogens and routinely subjected to standard monitoring. aedes albopictus cells of the c / strain were used for viral propagation, while cercopithecus aethiops kidney epithelial cells (vero) were used for plaque assays. briefly, vero cells ( × /well) were plated in -well culture plates and incubated at • c in a co incubator overnight. denv supernatant aliquots ( µl) were -fold serially diluted in medium, added to the vero cells, and incubated at • c for h. the viral supernatant was aspirated, and a prewarmed solution of % carboxymethyl cellulose medium (synth, diadema, brazil) was added to each well. after days incubation, the cells were fixed with % paraformaldehyde solution for min at room temperature (rt), washed with water and stained with a % crystal violet solution for min. the staining solution was removed and the plates washed to remove residual staining. finally, after drying on the bench, plaque forming units (pfu) were counted to obtain the virus titers, which were expressed in pfu/ml. the denv new guinea c (ngc) strain was previously isolated and adapted to infect and kill immunocompetent mice after intracranial (i.c.) administration [ ] . the lt was purified from a recombinant e. coli k strain transformed with a bspks (-) vector carrying the sequence encoding the toxin originally expressed by enterotoxigenic e. coli [ ] . the recombinant lt was purified by affinity chromatography with immobilized d-galactose, as previously described [ ] . c / cells were grown in leibovitz- (l- ) medium (vitrocell, campinas, brazil) supplemented with % fetal bovine serum (fbs) (vitrocell, campinas, brazil) until they reached approximately % confluence in plastic culture bottles (corning, new york, ny, usa). then, the cells were washed and infected with the denv ngc at a standard moi equal to , and when the cells displayed syncytial characteristics, the supernatant was harvested. the cells were centrifuged (eppendorf, hamburg, ger) at × g for min and then lysed by freezing at − • c for min and melting through the liquid state three times. after being lysed, the cells were centrifuged one more time, and the supernatant containing the released virus was added to the cell culture supernatant. the whole supernatant was mixed with polyethylene glycol (peg- ) (synth, diadema, brazil) in a proportion of . ml of peg to ml of supernatant and incubated overnight at − • c. after incubation, the supernatants were centrifuged at × g for min, the supernatant was discarded, and the precipitate viruses were suspended in mem supplemented with hepes buffer. the concentrated viruses (up to -fold) were titrated, and the content of total protein was also determined using an epoch take spectrophotometer (biotek, winooski, vt, usa) and maintained at − • c until being used for immunization assays. the purification of denv virus particles was performed by ion exchange chromatography using the akta fplc chromatograph (amersham pharmacia biotech, little chalfont, uk) associated with a ml hitraptm anx (high sub) (gelifesciences, boston, ma, usa) anion exchange column previously loaded with mm sodium phosphate buffer (ph . ). a total of ml of infected cell culture supernatant (vero cell previously infected for days with denv , moi of . ) was used for purification. the elution procedure was carried out by applying a linear gradient from to mm of sodium phosphate (ph . ) in column volumes (cv). the virus-containing fractions were combined, added with % sterile glycerol and stored at − • c for later use. for the id immunizations, balb/c mice (n = ) were immunized with different vaccine formulations containing µg/ml of polymyxin b, or µg of denv ngc virus particles administered alone or with µg of purified lt . the vaccination regimen consisted of three doses given at two-week intervals ( days). serum samples were collected through the submandibular plexus before each immunization (days − , , , ). the administration was performed in the dorsal region on the back of the mice with a mm gauge needle loaded with µl of the vaccine formulation. the same vaccine formulations were used in the tc immunizations. the dorsum of balb/c mice (n = /group) was shaved with an electric clipper and depilatory cream. before the administration of the vaccine patches, a slight abrasion with fine-grained sandpaper was applied to the shaved skin to disrupt the stratum corneum (sc). the patches were prepared with a double-sided waterproof adhesive film (smith&nephew, london, uk) cut in squares of approximately . cm . an absorbent sterile gauze piece (approximately cm ) was placed in the center of the adhesive film to form a pad that received the vaccine. the vaccines were placed on the back of the animals for h, and bandages were applied to prevent their removal by the animals ( figure s ). mouse sera were individually tested for the presence of the virus-specific abs by elisas. maxisorp plates (nunc, roskilde, dk) were coated with pbs containing ng of denv ngc virus particles and incubated overnight at • c. the plates were blocked with % gelatin in pbs at • c for h. serum samples were then serially diluted in % gelatin diluted in . % pbs-tween (pbst) and incubated at • c for . h. after three washes with pbst, the plates were treated with goat anti-mouse igg ( : ), igg ( : ) or igg a ( : ) conjugated with horseradish peroxidase (sigma-aldrich, san luis, mo, usa) at • c for . h. after a new wash cycle with pbst, a solution containing orthophenylenediamine dihydrochloride (opd) (sigma-aldrich, san luis, mo, usa) and h o was added for a final volume of µl per well. the plates containing the developer solution were maintained in the dark for min, at which time, the reaction was stopped by the addition of µl of n h so . the od nm was measured by an epoch take spectrophotometer (biotek, winooski, vt, usa), and the ab concentration values were calculated by comparison with a reference igg preparation at defined concentrations that had been added to each plate. antigen avidity with serum abs was determined with serum pools collected after the last vaccine dose was administered and carried out as previously reported [ ] . after incubation with normalized concentrations of antivirus sera, plates were washed and exposed to phosphate-buffered saline (pbs)-diluted ammonium thiocyanate sigma-aldrich, san luis, mo, usa ranging from to m. the plates were allowed to stand for min at room temperature before being washed. the concentrations of the ammonium thiocyanate required to dissociate % of the bound abs were determined. the percentage of binding was calculated as follows: od nm in the presence of ammonium thiocyanate x /od nm in the absence of ammonium thiocyanate. the values are expressed as percentages compared with those of the sample not subjected to the ammonium thiocyanate treatment. mice were sacrificed, and the spleens were aseptically harvested. splenocytes were counted after red blood cell lysis and suspended in % fbs/rpmi medium. splenocytes were plated ( . × cells/well) and stimulated with µg of thermally inactivated denv for h. the levels of secreted il- , tnf-α, ifn-γ, il- , and il- were determined with a cytometric bead array (cba) mouse th /th /th cytokine kit (bd, franklin lakes, nj, usa) according to the manufacturer's protocol. the measurements were performed using bd lsrfortessa (bd, franklin lakes, nj, usa), and the results were processed with system software. three technical repeats were carried out for each animal, and the results were statistically evaluated using one-way anova with bonferroni's post-test. two weeks after receiving the last vaccine dose, the immunized mice were first anesthetized with a mixture of ketamine and xylazine and subsequently challenged with × plaque forming units (pfus) of the ngc strain to a final volume of µl administered through the intracranial (i.c.) route, as previously described [ ] . these inoculated mice were monitored for days for survival. all statistical analyses were calculated using prism and software (graphpad software inc, la jolla, ca, usa), and differences with p ≤ . were considered significant. to compare results generated in several groups, a two-way anova test was applied in association with bonferroni's post-test. a comparison of the results generated in three groups was performed using one-way anova in association with bonferroni's post-test. statistical significance based on the mortality curves was determined by mantel-cox tests. male balb/c mice received three vaccine doses via the tc or id route with either or µg of concentrated denv particles (ngc strain) with or without purified lt ( figure s e ). as shown in figure , the mice inoculated with denv mounted a low antigen-specific igg response following the tc and id immunizations ( figure a ,b). the addition of lt to the vaccines significantly enhanced the denv -specific serum igg responses elicited in the mice immunized via the tc or id route, with the maximal values reached two weeks after the third vaccine dose ( figure a ,b). similar serum antibody responses were also measured in mice immunized virus particles purified from cell culture supernatants by anionic chromatography ( figure s ). male balb/c mice received three vaccine doses via the tc or id route with either or μg of concentrated denv particles (ngc strain) with or without purified lt ( figure s e ). as shown in figure , the mice inoculated with denv mounted a low antigen-specific igg response following the tc and id immunizations ( figure a ,b). the addition of lt to the vaccines significantly enhanced the denv -specific serum igg responses elicited in the mice immunized via the tc or id route, with the maximal values reached two weeks after the third vaccine dose ( figure a ,b). similar serum antibody responses were also measured in mice immunized virus particles purified from cell culture supernatants by anionic chromatography ( figure s ). the results also demonstrated that the serum denv -specific igg responses increased according to the amount of inoculated antigen in the id-immunized mice but not in the mice immunized via the tc route ( figure a ,b). the tc-immunized mice that received the higher antigen dose ( μg) combined with lt showed an increased serum igg response two weeks after the third dose ( figure c ). the id-immunized mice showed significant differences in denv -specific serum igg responses in the presence of lt , particularly in the group immunized with μg of the tested antigen ( figure d ). in conclusion, tc administration of the anti-denv vaccine induced serum antigen-specific antibody responses but at lower levels than those elicited by the id administered vaccine. the results also demonstrated that the serum denv -specific igg responses increased according to the amount of inoculated antigen in the id-immunized mice but not in the mice immunized via the tc route ( figure a,b) . the tc-immunized mice that received the higher antigen dose ( µg) combined with lt showed an increased serum igg response two weeks after the third dose ( figure c ). the id-immunized mice showed significant differences in denv -specific serum igg responses in the presence of lt , particularly in the group immunized with µg of the tested antigen ( figure d ). in conclusion, tc administration of the anti-denv vaccine induced serum antigen-specific antibody responses but at lower levels than those elicited by the id administered vaccine. the vaccinated mice were also monitored for different physiological aspects in a search of possible vaccine-induced adverse effects ( figure s ). in general, there was no indication of significant hematological alterations or tissue damage, such as in a tissue damage marker like ldh, in mice immunized via the tc or id route, with the exception of decreased hematocrit values and increased platelet numbers in the id-immunized mice receiving lt and µg of the vaccine antigen ( figure s b ). no other adverse reactions, such as weight loss or altered animal behavior, were observed in the mice in which the vaccine regimens were tested [ ] . collectively, these results indicate that the anti-denv vaccines were safe under the experimental test conditions. we first measured the igg-specific subclass responses in the mice subjected to different immunization regimens. the serum igg subclass response prevailed in the mice immunized without adjuvant two weeks after they received the third dose. a higher igg /igg a ratio was observed among the mice immunized via the tc route compared with that of the mice immunized via the id route (figure a,b) . the mice immunized with lt also developed a predominant igg response, but those immunized with the higher antigen dose developed a more balanced igg /igg a ratio, particularly the animals immunized via the id route (figure a,b) . aiming for an initial characterization of the antibodies capable of binding conformational epitopes in the mice immunized with the tested vaccine formulations, we measured the specificity of denv -specific antibodies against intact viral epitopes. for this experiment, we used heat-denatured and intact virus particles in the solid phase. as shown in figure c ,d, igg was increased in the mice immunized via the tc or id route, but significantly fewer igg molecules were bound to the denv particles after heat-denaturation treatment. the reduction in the anti-denv titers for groups inoculated with lt adjuvant and was greater in the tc-immunized mice ( % to %) than it was in the id-immunized mice ( % to %) ( figure s a ,b). these results suggest that a significant fraction of antibodies generated in mice immunized with intact virus particles target conformational epitopes on the virus surface. in addition, the results indicate that the proportion of antibodies targeting conformational epitopes was higher (p = . ) in the mice immunized via the tc route than it was in the mice immunized via the parenteral route. finally, we measured the avidity of the denv-specific antibodies in the mice subjected to different vaccine regimens tested. for this experiment, we followed a previously described elisa protocol with an additional dissociation step performed with ammonium thiocyanate. taken together, the results presented in figure e ,f demonstrate that the addition of lt steadily promoted an increase in the avidity of the antibodies to virus particles administered through both routes. however, in accordance with the measurements of the antibody titers, the amount of antigen had an impact only in the id-immunized mice, which presented enhanced avidity after the mice received higher antigen doses ( figure e ,f and figure s c ,d). finally, we measured the avidity of the denv-specific antibodies in the mice subjected to different vaccine regimens tested. for this experiment, we followed a previously described elisa protocol with an additional dissociation step performed with ammonium thiocyanate. taken together, the results presented in figure e ,f demonstrate that the addition of lt steadily promoted an increase in the avidity of the antibodies to virus particles administered through both routes. however, in accordance with the measurements of the antibody titers, the amount of antigen had an impact only in the id-immunized mice, which presented enhanced avidity after the mice received higher antigen doses ( figure e ,f and figure s c ,d). to analyze the immune responses elicited in the vaccinated mice, we assessed the cytokine secretion profile of spleen cells collected two weeks following the administration of the last vaccine dose. as indicated in figure , different cytokine secretion patterns were observed according to the tested administration route. mice immunized via the tc route showed higher tumor necrosis factor alpha (tnf-α) and interleukin (il- ) levels than shown by those immunized via the id route ( figure b,d) . in contrast, id-immunized mice mounted higher il- and il- responses than shown by those immunized via the tc route ( figure c,e) . nonetheless, an increase in interferon gamma (ifn-γ) was found following immunization via both routes. flow-cytometry assays were also performed with spleen cells after the immunization protocols prior to challenge, but no significant difference among immunized versus nonimmunized mice was observed [ ] . alpha (tnf-α) and interleukin (il- ) levels than shown by those immunized via the id route ( figure b,d) . in contrast, id-immunized mice mounted higher il- and il- responses than shown by those immunized via the tc route ( figure c,e) . nonetheless, an increase in interferon gamma (ifn-γ) was found following immunization via both routes. flow-cytometry assays were also performed with spleen cells after the immunization protocols prior to challenge, but no significant difference among immunized versus nonimmunized mice was observed [ ] . mice immunized via the tc or id route were subjected to a lethal i.c. challenge with the denv ngc strain. as shown in figure , the immunized mice developed partial or complete protective immunity to a lethal challenge with the denv strain. in the tc-immunized mice, the conferred protection was dependent on the addition of lt and the antigen amount, with maximal survival values observed in the mice immunized with μg of denv combined with lt ( % survival in comparison with % survival in the sham-treated group) ( figure a ). among the id-immunized mice, the group immunized with the higher antigen dose and lt were fully protected against the lethal challenge ( figure b ). we also looked for signs of morbidity in the mice challenged with the denv ngc strain ( figure c,d) . among the immunized mice, only those immunized with the higher antigen dose combined with lt displayed the maximal protection compared to that shown by the sham-treated animals ( figure d ). at the end of the observation period, all the mice immunized via the id route and % of the mice immunized via the tc route showed no signs of mice immunized via the tc or id route were subjected to a lethal i.c. challenge with the denv ngc strain. as shown in figure , the immunized mice developed partial or complete protective immunity to a lethal challenge with the denv strain. in the tc-immunized mice, the conferred protection was dependent on the addition of lt and the antigen amount, with maximal survival values observed in the mice immunized with µg of denv combined with lt ( % survival in comparison with % survival in the sham-treated group) ( figure a ). among the id-immunized mice, the group immunized with the higher antigen dose and lt were fully protected against the lethal challenge ( figure b ). we also looked for signs of morbidity in the mice challenged with the denv ngc strain ( figure c,d) . among the immunized mice, only those immunized with the higher antigen dose combined with lt displayed the maximal protection compared to that shown by the sham-treated animals ( figure d ). at the end of the observation period, all the mice immunized via the id route and % of the mice immunized via the tc route showed no signs of morbidity. altogether, these results indicated that the administration of the tested vaccine formulation administered via either the tc or id route induced protective immunity against denv . viruses , , of viruses , , x for peer review of morbidity. altogether, these results indicated that the administration of the tested vaccine formulation administered via either the tc or id route induced protective immunity against denv . despite a number of reports describing the use of tc for the delivery of different antibacterial and antivirus vaccines [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , thus far, only two studies addressed the performance of denv vaccines, delivered with microneedle pads, that were tested under experimental conditions [ , ] . here, we showed mice immunized with whole denv particles through the use of adhesive patches applied on the surface of the skin and compared the results with regard to mice immunized via a parenteral route (id). the results demonstrated that induction of dose-dependent serum virus-specific antibody responses required the incorporation of an adjuvant (lt ). in addition, although mice immunized via the tc route developed lower serum igg responses, the protective immunity elicited in both mouse groups was similar. such results indicate that differences in the properties of antigen-specific antibodies or activation of differential cellular immune responses contribute to the protective immunity induced in tc-immunized animals. there are several ways to perform tc immunizations that include the use of different technologies to break the skin barrier. microneedle devices represent an alternative to deliver the antigens right below the sc [ ] . on the other hand, tc immunization may be performed with adhesive patches soaked with the antigen and applied directly on the surface skin after a gentle superficial abrasion [ ] . the use of adhesive patches has several inherent advantages over parenteral routes, such as the lack of needles and pain and reduced risks of contamination, but precise determination of the amount of antigen actually delivered into the host is not possible. a precise definition of the efficiency of the tc administration route would, therefore, require comparison of immune responses and protective immunity induced after immunization with the same vaccine delivered via a parenteral route. in the present study, we performed, for the first time, such a comparison with denv antigens and demonstrated that antigen delivered via the tc route induced similar immune responses and, more relevantly, protective immunity to denv, thus representing an alternative for the administration of denv vaccines. the recent report of microneedle-based tc immunization with a live-attenuated denv vaccine further supported the use of this delivery route for the administration of live virus vaccines [ ] . previous reports demonstrated that the tc route is a safe and efficient immunization method [ ] [ ] [ ] [ ] [ ] [ ] [ ] , ] . in particular, the tc administration route prevents the toxicity of certain adjuvants, such as lt , which may induce local and systemic side effects when delivered via parenteral routes [ , ] . indeed, our results demonstrate that administration of denv particles admixed with lt and delivered through the tc route led to the generation of specific antibody and cellular responses without any significant deleterious effects or local inflammatory reactions. studies describing the use of viral antigens administered via the tc route usually exhibit a wide range of antigen doses [ , , , , ] . the antigen loads tested in the present study were designed to permit comparisons with mice immunized under the same conditions via a parenteral (id) route. interestingly, similar amounts of denv envelope (e) protein were shown to be immunogenic and protect against challenge after skin application of the microneedle device [ ] . immunization of mice with the same vaccine via two different administration routes allowed for comparisons of the impact on the antigen-specific immune responses and the induction of protective immunity. under the test conditions, the induction of anti-denv serum igg responses was lower in the mice subjected to tc immunization than it was in the mice immunized via the id route. this difference was clear after the third immunization dose and may be ascribed to the natural limitations of the naked ablated skin to efficiently deliver antigens compared with that delivered via parenteral inoculations. despite such differences, mice immunized via the tc or id route developed similar protective immunity after challenge with the denv ngc strain. the immune responses elicited in the mice immunized via the tc route, therefore, confer a more efficient protective immunity against denv. antibodies increased in the mice immunized via the tc route displayed a different reactivity toward conformation-dependent epitopes presented on the virus particles. heat denaturation of the virus particles is expected to disrupt conformational epitopes present on structural proteins, while linear epitopes would not be affected to the same extent after similar exposure to the protein denaturation step. indeed, results from previous studies based on human monoclonal abs (hmabs) generated from patients infected with denv demonstrated that protective antibodies react preferentially to tertiary and quaternary conformational epitopes of the e protein [ ] . thus, the lower reactivity of the antibodies to the heat-denatured viruses in the tc-immunized mice may reflect differences in epitope specificity. antigen avidity has been reported to affect the capacity of antibodies to neutralize viruses [ ] . although mice immunized via the id route developed antibodies with higher antigen avidity than those immunized via the tc route, particularly the mice immunized with the higher antigen concentration ( µg/dose), the protective immunity to denv was similar in both groups. thus, antigen avidity did not seem to play a relevant role in the immune protection against denv observed under the test conditions. the classic protection correlate for denv infection consists of the induction of neutralizing antibodies, but several results generated under experimental and clinical conditions indicated that the cellular arm of the immune system also plays a relevant role in the development of antiviral immunity [ , ] . modulation of immune responses, particularly the production of th -related cytokines, such as ifn-γ and tnf-α, enables the activation of immune cells and control of the intracellular replication of viruses through both direct destruction of infected host cells and noncytotoxic pathways [ ] . the abundant presence of intraepithelial antigen-present cells (apcs), such as langerhans cells (lcs), capable of processing exogenous antigens and priming naïve t cells, is expected to improve the performance of vaccines delivered via this route [ ] . furthermore, a previous report showed that, in contrast to other antigen-presenting cells, few lcs are infected by denv [ ] , indicating the more efficient activation of b and t lymphocytes. in this study, the mice immunized via the tc route secreted high amounts of ifn-γ and, particularly, tnf-α. on the other hand, spleen cells collected from the id-immunized mice secreted ifn-γ and il- . the production of ifn-γ and tnf as associated with the expression of cd a was previously correlated with protection mediated by cd + t cell responses [ , ] . moreover, ifn-γ, tnf-α and il- production was correlated with subclinical denv secondary infections [ ] and may contribute to protection through multifunctional cd + t cells after vaccination [ ] . the same pattern was observed in hiv patients with lower viral loads, indicating that proinflammatory responses are relevant for viral control [ ] . spleen cells collected from the mice immunized via the tc route also secreted higher amounts of il- and had lower production of il- in comparison with the mice immunized via the id route. both il- and il- were previously correlated with severity increases in denv diseases [ ] [ ] [ ] . il- is associated with the upregulation of denv-targeted receptors, leading to an increase in denv infectivity in dermal apcs [ ] . the production of il- is modulated by denv during infection, which explains the crescent levels after the increase in viral particle administration or adjuvant-mediated inflammation. il- also induces the transformation of b cells in plasma cells [ ] , increasing the amount of specific antibodies produced, as observed at a level of significance in the group that received µg of denv . il- is usually associated with the suppression of pathogen-mediated inflammation and is found in high amounts in the sera of denv-infected mice and humans [ , ] . the presence of il- + monocytes after yellow fever (yfv) vaccination was associated with a reduction in immune responses and immune system adverse reactions [ , ] . accordingly, higher levels of il- were expected to be observed after id inoculation with lt . furthermore, il- + cd + t cells showed higher cytolytic activity than did the il- -cd + t cells during the control of coronavirus-induced acute encephalitis [ ] , suggesting possible enhancement of the survival and activation of cd + t cells during acute inflammation. collectively, these results demonstrated that the same vaccine formulation delivered by either the id or tc routes triggered different cellular immune responses, as indicated by the cytokine secretion pattern of total spleen cells collected from the vaccinated animals. the experimental virus challenge model used in the present study depended on the i.c. inoculation of a mouse-adapted denv strain (ngc). although this infection route did not correspond to the infection process observed under natural conditions, the mouse-adapted virus strain represents a classic tool for the determination of protective immunity elicited in immunocompetent mice [ ] [ ] [ ] . it has been demonstrated that serum antibody responses are only partial contributions to the encephalitis developed in i.c. challenge with denv viruses [ ] . furthermore, the depletion of cd + or cd + t lymphocytes drastically reduced the vaccine-based protection observed in the encephalitis model [ , ] . thus, the high protective status induced by the tested vaccine administered via both inoculation routes suggests the relevant role of cellular immune responses. this study adds further experimental evidence that, similar to other virus vaccines, the tc route represents a promising route for vaccine administration capable of inducing protective immunity without the concerns associated with needle-delivered vaccines. despite difficulties in standardizing the amount of antigen effectively delivered into the host tissues, this vaccine administration route is safer and a similarly effective alternative to parenterally administered denv vaccines. further studies based on preclinical models should contribute to the translation of the present results into an alternative for the clinical testing of denv vaccines in humans. the following are available online at http://www.mdpi.com/ - / / / /s , figure s : antigens and transcutaneous immunization procedures adopted in the study. figure s : serum igg responses in mice immunized with purified denv particles. figure s : hematological analyses of mice submitted to tc or id immunizations with concentrated denv particles. figure s : binding of anti-denv antibodies to viral particles following antigen heat denaturation or treatment with ammonium thiocyanate. cost of dengue outbreaks: literature review and country case studies the global distribution and burden of dengue assessing the economics of dengue: results from a systematic review of the literature and expert survey world health organization (who) dengue and severe dengue transcutaneous immunization with cholera toxin protects mice against lethal mucosal toxin challenge transcutaneous immunization: a human vaccine delivery strategy using a patch a low-invasive and effective transcutaneous immunization system using a novel dissolving microneedle array for soluble and particulate antigens topical application of escherichia coli-vectored vaccine as a simple method for eliciting protective immunity skin-resident t cells: the ups and downs of on site immunity transcutaneous and intradermal vaccination transcutaneous antigen delivery system needle-free vaccine delivery advances in transcutaneous vaccine delivery: do all ways lead to rome? effective transcutaneous immunization against hepatitis b virus by a combined approach of hydrogel patch formulation and microneedle arrays simultaneous approach using systemic, mucosal and transcutaneous routes of immunization for development of protective hiv- vaccines particle-based transcutaneous administration of hiv- p protein to human skin explants and targeting of epidermal antigen presenting cells transcutaneous immunization by lipoplex-patch based dna vaccines is effective vaccination against japanese encephalitis virus infection transcutaneous immunisation with herpes simplex virus stimulates immunity in mice a combined approach of vesicle formulations and microneedle arrays for transcutaneous immunization against hepatitis b virus new mouse model for dengue virus vaccine testing functional diversity of heat-labile toxins (lt) produced by enterotoxigenic escherichia coli: differential enzymatic and immunological activities of lt (hlt) and lt (plt) production and release of heat-labile toxin by wild-type human-derived enterotoxigenic escherichia coli functional and immunological characterization of a natural polymorphic variant of a heat-labile toxin (lt-i) produced by enterotoxigenic escherichia coli (etec): research article a genetic and pathologic study of a denv clinical isolate capable of inducing encephalitis and hematological disturbances in immunocompetent mice targeting the non-structural protein from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge other adverse reactions, such as weight loss or altered animal behavior, were observed in the mice in which the vaccine regimens were tested preferential amplification of cd effector-t cells after transcutaneous application of an inactivated influenza vaccine: a randomized phase i trial transcutaneous yellow fever vaccination of subjects with or without atopic dermatitis coated microneedle arrays for transcutaneous delivery of live virus vaccines transcutaneous anti-influenza vaccination promotes both cd and cd t cell immune responses in humans efficient delivery of dengue virus subunit vaccines to the skin by microprojection arrays microneedle-based intradermal delivery of stabilized dengue virus transcutaneous immunization with bacterial adp-ribosylating exotoxins, subunits, and unrelated adjuvants transcutaneous delivery and thermostability of a dry trivalent inactivated influenza vaccine patch successful induction of protective antibody responses against haemophilus influenzae type b and diphtheria after transcutaneous immunization with the glycoconjugate polyribosyl ribitol phosphate-cross-reacting material vaccine genetic diversity of heat-labile toxin expressed by enterotoxigenic escherichia coli strains isolated from humans microneedle arrays for the transcutaneous immunization of diphtheria and influenza in balb/c mice mechanistic study of broadly neutralizing human monoclonal antibodies against dengue virus that target the fusion loop high-avidity and potently neutralizing cross-reactive human monoclonal antibodies derived from secondary dengue virus infection protective role of cross-reactive cd t cells against dengue virus infection cells can mediate short-term protection against heterotypic dengue virus reinfection in mice gamma interferon can prevent herpes simplex virus type reactivation from latency in sensory neurons antibodies are not required to a protective immune response against dengue virus elicited in a mouse encephalitis model a protective role for dengue virus-specific cd + t cells cells are not required for the induction of dengue virus-specific cd + t cell or antibody responses but contribute to protection after vaccination intracellular cytokine production by dengue virus-specific t cells correlates with subclinical secondary infection primary vaccination with low dose live dengue virus generates a proinflammatory, multifunctional t cell response in humans presence of hiv- gag-specific ifn-γ + il- + and cd + il- + cd t cell responses is associated with nonprogression in hiv- infection vascular endothelium: the battlefield of dengue viruses differing influences of virus burden and immune activation on disease severity in secondary dengue- virus infections thrombocytopenia in dengue fever dermal cd + dendritic cell and macrophage infection by dengue virus is stimulated by interleukin- il- levels in dengue patients: some findings from the exceptional epidemiological conditions in cuba vascular endothelial growth factor kdr receptor signaling potentiates tumor necrosis factor-induced tissue factor expression in endothelial cells innate immunity phenotypic features point toward simultaneous raise of activation and modulation events following dd live attenuated yellow fever first-time vaccination characterization of main cytokine sources from the innate and adaptive immune responses following primary dd yellow fever vaccination in adults highly activated cytotoxic cd t cells express protective il- at the peak of coronavirus-induced encephalitis protection against dengue type virus induced in mice immunized with a dna plasmid encoding the non-structural (ns ) gene fused to the tissue plasminogen activator signal sequence production of a recombinant dengue virus ns protein and potential use as a vaccine antigen immunogenic and protective response in mice immunized with a purified, inactivated, dengue- virus vaccine prototype made in fetal rhesus lung cells flow-cytometry assays were also performed with spleen cells after the immunization protocols prior to challenge we thank mariana cintra and pietro pisani for technical support in tc immunizations and eduardo gimenes martins for the administrative and technical support. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- - zf qtlh authors: farag, elmoubasher; sikkema, reina s.; vinks, tinka; islam, md mazharul; nour, mohamed; al-romaihi, hamad; al thani, mohammed; atta, muzzamil; alhajri, farhoud h.; al-marri, salih; alhajri, mohd; reusken, chantal; koopmans, marion title: drivers of mers-cov emergence in qatar date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: zf qtlh mers-cov (middle east respiratory syndrome corona virus) antibodies were detected in camels since , but the first human case was only detected in . this study sought to identify and quantify possible drivers for the mers-cov emergence and spillover to humans. a list of potential human, animal and environmental drivers for disease emergence were identified from literature. trends in possible drivers were analyzed from national and international databases, and through structured interviews with experts in qatar. the discovery and exploitation of oil and gas led to a -fold increase in qatar gdp coupled with a -fold population growth in the past years. the lifestyle gradually transformed from bedouin life to urban sedentary life, along with a sharp increase in obesity and other comorbidities. owing to substantial governmental support, camel husbandry and competitions flourished, exacerbating the already rapidly occurring desertification that forced banning of free grazing in . consequently, camels were housed in compact barns alongside their workers. the transition in husbandry leading to high density camel farming along with increased exposure to humans, combined with the increase of camel movement for the racing and breeding industry, have led to a convergence of factors driving spillover of mers-cov from camels to humans. emerging infectious diseases are a cause for increasing global concern, because of their impact on global health and economics [ ] . the ebola outbreak in west africa during - showed that pathogens which previously caused small and easy to control outbreaks had the potential to infect thousands of people under the right circumstances [ ] . this is also a concern for the middle east respiratory syndrome coronavirus (mers-cov), which until now has been the cause of sporadic cases and hospital outbreaks [ ] . to date, there have been confirmed laboratory cases worldwide, with deaths [ ] . all mers index cases are linked to the arabian peninsula. dromedary camels have been identified as a reservoir of mers-cov with occasional zoonotic transmission to humans [ , ] . human-to-human transmission is also common, with around % of the mers cases reported to who being health care associated [ , ] . however, the source of infection of many index cases remains unclear [ , ] . studies have shown that mers-cov, or related viruses have been circulating among camels at least since [ ] . since that period, massive changes have occurred in people's lives and in animal husbandry across the arabian peninsula. understanding these changes may help to reconstruct the events that led to the emergence of mers-cov as a human disease. past research identified several drivers of emerging zoonoses, such as urbanisation, population growth and demography, and environmental and agricultural changes [ ] [ ] [ ] . the drivers which could have potentially influenced the mers-cov emergence in humans have only sporadically been investigated [ , ] . by reviewing changes involving humans and camels over the past years in qatar, this study sought to identify the key drivers of the emergence and spread of mers-cov. potential drivers for disease emergence were identified from literature and from discussions with national and international experts in mers-cov. the final list had the following categories: economic development; human demography and behavior; international travel, commerce, sports and leisure; political environment; agriculture and food industry change, including camel demography, husbandry and movement; changes in climate and land use. data from onwards were collected from national and international databases. if multiple data sources were available, data from both sources were collected. all data were entered in an excel datasheet and reviewed and discussed with the project team (supplementary ). qualitative information and remaining data gaps were addressed by interviews with a group of experts and stakeholders from qatar. criteria to select experts included years or more experience in a camel-related business (farming, trading and racing) or professional services related to camels and being familiar with cultural aspects of the qatari community. using a structured interview guide (supplementary ) and a moderator, a series of interviews were conducted in arabic, each lasting approximately for hours. the main themes that were covered during the interviews included: (changes in) people's living conditions; customs and purposes of camel ownership; cultural habits related to camels; educational level and personal behaviors of camel owners and workers; camel movement; demographic distribution of camels in qatar; camel farming practices: feeding, grazing, and slaughter. a detailed transcript was shared with the experts for authentication. a literature search was done to complement findings from the quantitative and qualitative study, using pubmed, google scholar and the local sources of information including the ministry of public health (moph), ministry of municipality and environment (mme), ministry of development and planning statistics (mdps), and qatar statistical authority (qsa). the funder had no role in study design, data analysis, data interpretation, or writing of the review. historically, qatari inhabitants were mostly bedouins along with a few settled people [ , ] . the bedouins owned limited numbers of camels, sheep, and goats [ ] . camels were used as a source of food (milk and meat) and means for transportation. in , oil and natural gas resources were discovered. however, large-scale exploitation started in the s [ ] . from the s onwards, qatar's economy has been steadily growing. however, the year marked a significant turning point as qatar's gdp almost increased by more than -fold during the period - ( figure a ) [ , ] . qatar is currently considered to be one of the wealthiest countries in the world [ ] . the thriving economy was paralleled by major demographic and life style changes. in the late s, around , people lived in qatar [ ] . in response to demands for a larger workforce after the exploitation of oil and gas began, foreign laborers started to migrate to qatar from countries in the region, like palestine, oman, iran, and the kingdom of saudi arabia (ksa). later, immigrants from pakistan, india, nepal, sri lanka, bangladesh, the philippines, and indonesia joined the older migrant populations, increasing the number of inhabitants to , by and recently to , , ( figure a ) [ ] . in , non-qatari males made up % of the residents of working age ( - ) and non-qatari made up more than % of the total number of qatar inhabitants older than years of age ( figure b ,c) [ ] . most recent estimations of the origins of the non-qatari population are that % is indian, % bangladeshi, % nepali, % filipinos, % egyptian, % pakistani, and % iranian [ ] . the total number of males in qatar increased from . % of the total population in to . % in ( figure b ). in , almost % of residents were between and years old, and this has risen to more than % in ( figure a ). detailed accounts on age distribution were not available before [ ] . most people in qatar live in urban areas. the percentage of residents living in cities increased from . % in , to . % in , and . % in [ ] . doha, the capital and the biggest city of qatar, hosts the greatest number of people. however, there has also been a large increase in number of people living in the al-rayan area, where most of the camel farms are located. the number of tourists visiting qatar also increased, especially since . most tourists came from other gcc countries, but the number of visitors from europe and america were also increasing ( figure d ) [ , ] . according to experts, the economic development and population increase coincided with major changes in life style. the bedouin nomadic lifestyle gradually decreased as most of the qatari tribes shifted to an urban, settled lifestyle; cars and planes rapidly replaced camels as transportation means. this transformation to a more sedentary lifestyle is reflected in the profile of comorbidities. more than % of adults are overweight and almost half of them obese [ ] . male obesity increased from % in to % in , which is extremely high compared to the current % prevalence in men worldwide [ ] . in , % of residents above years were hypertensive, rising to % in , and % in [ , ] . prevalence of high blood sugar among adults in was %, compared to a worldwide prevalence of % [ ] . the qatar stepwise report reported in that % of adults were daily smokers. yet, qatar has a low death rate: . / , compared to the worldwide death rate of . / , and its healthcare system has developed rapidly over the past twenty years [ , , ] . the increase in the number of dromedary camels reflects the increasing popularity of camels as sports animals ( figure b ). with the changing life style and increasing wealth, the purchase and breeding of (expensive) racing camels came within reach of an increasingly large segment of the qatari national population. according to experts, although camel racing has traditionally been part of the bedouin culture, the organized racing business went through major changes over the past decades. this was partly due to financial and regulatory support from the qatari government. this support increased the social and economic value of camels in qatar, further stimulating their popularity. the al-shehaniya camel-racing track, one of the biggest tracks in the gulf, was opened in [ ] . the camel farms that are located near the al-shehaniya camel racing area are mostly used for racing camels. there are about racing camel holdings at the al-shehaniya camel racing area. some of the camels in qatar are used to compete in camel beauty contests that are organized around the arabian peninsula. according to the fao, in there were about , camels in qatar. this rose to over , in , , in , and more than , in ( figure b ). more than % of the animals are currently kept for racing [ ] . across the gulf region, qatar has the highest camel density, with . units/km , compared to . units/km in united arab emirates (uae) and . units/km in the ksa [ ] . in , the total number of camel farms was and by it had increased to [ ] . as a result of the loss of traditional methods of rangeland management, the vegetation coverage decreased from % to only % of total land cover. overgrazing of the green areas due to the increased population of camels and other livestock accelerated the desertification of qatar [ ] . therefore, the government decided to assign natural protected areas in [ , ] , and started to sanction the free grazing of livestock since [ ] . by , open grazing was completely banned [ ] . according to the experts' opinions, this led to changes in farming practices, as herds were then moved outside of qatar to areas where free grazing remained possible. moreover, in qatar, camels are now raised in closed systems and within of designated farming areas (camel complexes) in the residential districts. camel workers also live on the premises of the camel complexes. typically, a camel complex has a reception room (majlis) for social activities of the camel owners. the al-rayyan municipality, where the al-shehaniya camel racing area is also located, currently holds about % of the total camel population and % of camel holdingss (figure ) [ , ] . according to the experts, this newly adopted closed farming system led to the increase of disease incidence, especially of parasitic diseases. however, we did not find any disease statistics to substantiate these findings. the increasing focus on camel race competitions caused big changes in camel farming practices. previously, the calves were weaned when the next calf was born. currently, weaning occurs at around months of age. after being weaned, young camels are directly taken for acclimatization (during the period mid-july through mid-august) from the general livestock farms (located across the region) to the racing farms, mostly located within the al-shehaniya area. this involves drastic changes in feeding systems, intense training for races, and mock races alongside camels from other farms and older training camels. the off-season for camel racing is during summer (mid-april to august) ( figure ). during this time, most of the owners travel abroad, the frequency of visits to the farms substantially decreases, and workers are permitted to take annual vacations. from september onward, training intensifies, in preparation of the racing season, which lasts from mid-september through mid-april. during that time, , registered camels from different origins, ages, gender, nationalities, and breeds compete together at the al-shehaniya camel-racing track. during the racing season, up to rounds take place, approximately five days per week. an unprecedented, increasingly intensified mobility of camels inside and outside qatar has been seen over the recent decades. the domestic and cross border mobility does not only involve camels, but also people who look after the camels to provide care along the journey. import and export of camels have especially increased since the year ( figure c ). the imported camels mainly come from the uae and ksa ( figure d) . the dynamics and travel patterns of qatari camels are complex (figure ). camels are transported to and from different locations, for a variety of purposes, and with a noticeable seasonal pattern. mobility gets more intensive during the racing and trading season (september to april). experts believe that the ban of open grazing in qatar played a key role in the intensity and frequency of camel movements. they mention that there has been a remarkable increase after in numbers of camel workers and owners who cross the borders to and from ksa along with their animals, although this recently stopped with the ksa-qatar political situation. the ban of open grazing stimulated camel owners to establish farms in ksa and uae where open grazing is still permitted. therefore, camels are moved through gulf countries, particularly during the winter season. camel races and beauty contests that are routinely organized in nearly all gulf countries are another factor that boost the national and international movement of camels. compared to other types of camels, racing camels dominate in terms of numbers and frequency of mobility both across borders and domestically, particularly between september and april. as per the records of the camel racing committee, in the racing competitions, , camels from qatar and camels from the other gcc countries contested [ ] . however, owing to the lack of standardized identification system, it was difficult to determine the exact figures and the extent of these movements. camels are also being mobilized for reproduction purposes (figure ). mating season (also known as camels' honeymoon) starts in the middle of august and continues through february of the next year, with the high season in the september-october period. female camels are usually taken from their own location to other farms where selected males are kept particularly for reproduction purposes. about , female camels are annually being moved for mating. they spend around week at a breeding farm with male camels before they are taken back to their original farms. programmed mating is exclusively being practiced for race and show camels. the mating season is another seasonal activity that entails intensive movements of camels, camel owners, workers, car drivers and veterinarians. the doha wholesale market constitutes the primary hub for camel trading. in parallel with the increased number of camel races, al-shehaniya city also grew as a market and has become a hub for trade of racing and beauty show camels in qatar. the wholesale market in doha hosts camels and other types of livestock from countries all over the gulf region. the camels typically stay at the market until they are sold. camel workers live at the market premises. camels that are being sold (calves in particular) serve a variety of purposes. they are sold to be slaughtered at the doha wholesale market abattoir, for breeding purposes, to be trained as racing camel, or to be prepared for camel show competitions. in recent years, the doha wholesale market has been surrounded by rapidly growing residential areas. animals in the market are now in close proximity to the residents. as of , slaughter practices were banned inside residential premises, and can only be performed in official slaughterhouses and exclusively by licensed persons. camel meat and milk are no longer part of the daily diet of most qatar inhabitants. nonetheless, camel meat is a fundamental ingredient of qatari social events and family celebrations. production of camel meat and milk has remained stable in the past years. camel milk is generally kept for personal use, particularly for the perceived therapeutic merits of raw camel milk, as well as camel urine. experts state that there is an unshakable belief that the regular consumption of camel milk helps to prevent and control diabetes. it is also widely believed in the qatari community that camel urine and milk can heal skin lesions and other diseases. camel urine is also regularly used to whiten the skin and face and lighten the hair. the majority of camel owners offer camel milk and urine for free, as a practice of generosity. the role of camels in the transmission of mers-cov is well documented [ ] . despite the fact that mers antibodies have already been detected in camels since [ ] and human contact with animals is not new, human mers cases were only detected in [ ] . based on institutional and literature data and in-depth interviews with key professionals, this study sought to examine the changes involving the human, animal, and environmental drivers that may have contributed to the spread and virus spillover to humans. our reconstruction of events over the past decades, based on available literature, statistics, and expert opinions, lead to the conclusion that the discovery of oil and natural gas resources has been the starting point of a chain of events that ultimately led to conditions favoring the emergence of mers-cov ( figure ). this discovery led to massive economic growth. owning a camel represents the wealth and status of its owner in arabic culture. governmental sponsorship of camel ownership and camel racing further stimulated the camel industry, especially the camel-racing sector. this in turn lead to an accelerating increase of the camel population, a change in camel farming, and a concomitant increase in the number of camel workers [ ] . the human population of qatar has increased by -fold over the last decades [ ] . this is unlike other high-income countries, that have a yearly overall population growth of only . % [ ] . population growth and high population density have been shown previously to be important risk factors for disease emergence [ ] . moreover, consistent with the disease profile of wealthy countries where sedentary lifestyle prevails, the prevalence of chronic diseases increased in qatar in accordance with the increasing gpd, ultimately rendering the qatar population not only vulnerable to virus transmission, but also to its deadly complications [ ] . the intimate nature and number of interactions between camels and humans has also increased significantly in the past years, increasing the risk of any zoonotic spillover. at camel complexes, workers intimately reside, sleep, and eat with their camels. camel owners, on the other hand, pay regular visits to their barns and stay there for considerable hours every day (even longer during weekends, holidays, and winter season) in the majlis built at the corner of their barns. owners, who are often of advanced age with multiple comorbidities, enjoy drinking fresh camel milk and entertaining guests. those who suffer certain diseases tend to visit the camel barns to use camel urine or drink fresh camel milk for its perceived curative properties. among the variety of changes that involved camel husbandry in qatar, the shift from open grazing to close housing systems seems to be most significant. opportunities for camel-to-camel and camel-to-human spread have greatly increased since then. it is possible that housing camels in barns, with poor biosecurity and hygienic standards, turned these barns into 'melting pots' for the virus that ultimately acquired the ability to cross the human-animal barrier. the increase of cross-border movement of camels increased chances and frequency of (international) virus spread. camels are transported freely across borders for a variety of purposes through multiple routes and means of transportation. when camels and the humans that accompany them, arrive at the site of a race or beauty event in qatar, they are housed with the local camels. owners are welcomed in the majlis at the camel complexes. the mixing of camel and human of different origins further increase chances of virus transmission. viruses , , x; doi: for peer review www.mdpi.com/journal/viruses species variation in dpp [ ] . as such, the increasing human-animal interface that is described in this paper may have facilitated the adaptation of the spike protein to human ddp . however, much remains unknown, also in view of the findings that mers-cov from east africa were not phenotypically different from the viruses from the middle east, while human mers patients have not been reported from the african continent [ ] . finally, the changes in animal husbandry practices, earlier weaning, frequent grouping and transportation of animals, and the introduction of an entirely new feeding system, may induce stress in the camels. these changes and movements often involve young weaned animals, at the same time as maternal antibodies are waning, which are linked to the shedding of the virus [ , ] . most of the limitations of this study were related to the availability of data. firstly, statistics on animals, import and export, animal workers, and land use were only found since onwards, limiting the chance although much effort was made to study mers-cov viral sequences and mers-cov transmission between dromedary camels and humans, it is still unknown which genetic mechanisms have caused the viral spillover of dromedary camels to humans. however, the most important determinant of host specificity seems to be the spike s protein, that recognizes and binds to host-cell receptor dpp [ ] . recently it has been shown that the mers-cov spike can rapidly adapt to species variation in dpp [ ] . as such, the increasing human-animal interface that is described in this paper may have facilitated the adaptation of the spike protein to human ddp . however, much remains unknown, also in view of the findings that mers-cov from east africa were not phenotypically different from the viruses from the middle east, while human mers patients have not been reported from the african continent [ ] . finally, the changes in animal husbandry practices, earlier weaning, frequent grouping and transportation of animals, and the introduction of an entirely new feeding system, may induce stress in the camels. these changes and movements often involve young weaned animals, at the same time as maternal antibodies are waning, which are linked to the shedding of the virus [ , ] . most of the limitations of this study were related to the availability of data. firstly, statistics on animals, import and export, animal workers, and land use were only found since onwards, limiting the chance to 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region-dependent genetic diversity key: cord- -cpn j authors: chen, jun; xia, sisi; yang, xiangmin; chen, huizi; li, fanni; liu, fenyong; chen, zhinan title: human cytomegalovirus encoded mir-us - - p attenuates cd /emmprin-mediated early antiviral response date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: cpn j cellular receptor-mediated signaling pathways play critical roles during the initial immune response to human cytomegalovirus (hcmv) infection. however, the involvement of type-i transmembrane glycoprotein cd /emmprin (extracellular matrix metalloproteinase inducer) in the antiviral response to hcmv infection is still unknown. here, we demonstrated the specific knockdown of cd significantly decreased hcmv-induced activation of nf-κb and interferon-beta (ifn-β), which contribute to the cellular antiviral responses. next, we confirmed that hcmv-encoded mir-us - - p could target the ′ utr (untranslated region) of cd mrna, and thus facilitate hcmv lytic propagation at a low multiplicity of infection (moi). the expression and secretion of cyclophilin a (scypa), as a ligand for cd and a proinflammatory cytokine, were up-regulated in response to hcmv stimuli. finally, we confirmed that cd mediated hcmv-triggered antiviral signaling via the scypa-cd -erk (extracellular regulated protein kinases)/nf-κb axis signaling pathway. these findings reveal an important hcmv mechanism for evading antiviral innate immunity through its encoded microrna by targeting transmembrane glycoprotein cd , and a potential cause of hcmv inflammatory disorders due to the secretion of proinflammatory cytokine cypa. human cytomegalovirus (hcmv) is a widespread human β-herpesvirus with seroprevalence from to %, which is closely related to socioeconomic and geographical conditions [ ] . life-long latent or persistent hcmv infection can be established in immunocompetent populations after the primary infection, and productive replication can occur from spontaneous reactivation due to immune abnormalities. thus, hcmv is a major viral cause of mental retardation and sensorineural deafness in newborns, and particularly causes morbidity and mortality in transplant recipients and aids patients with an immunocompromised state [ ] . current studies also tie hcmv to multiple common chronic herpes simplex virus (hsv- ) was provided by fenyong liu (university of california, berkeley, ca, usa). to generate the u -cd overexpressing (u -cd ) and u -control (u -c) cell lines, u mg cells were transfected with the pcdna . -cd plasmid or the pcdna . (+) (invitrogen, carlsbad, ca, usa) empty vector control plasmid using lipofectamine tm , as prescribed by the manufacturer (life technologies, waltham, ma, usa). after a -h incubation, transfected cells were selected for resistance in cell culture medium containing µg/ml g (life technologies). after two to three weeks of selection, single clones were randomly isolated, and each was plated separately. the cd levels in individual selected clones were determined by western blot analysis. the maintenance medium for cell lines was supplemented with µg/ml g . the bacterial artificial chromosome (bac)-derived clinical strain nr- with enhanced green fluorescent protein (egfp) expression cassette, provided by dr. ke zen (nanjing university, nanjing, china), was used in this study [ ] . hcmv mutant (mus - - p) and the revertant wild type (rvwt) was generated using a bacterial recombineering method, as previously described [ ] . in brief, approximately bp of the mir-us - flanking region was cloned into the pcdna . (+) vector. the three nucleotides in the seed region of mir-us - - p were mutated using site-directed polymerase chain reaction (pcr). then, the pcr-amplified products containing point mutations of mir-us - - p were used in the second round of recombination to replace the tet/str cassette that inserted in the region of mir-us - - p at first. the amplified dna fragments were transformed into e. coli el cells by electroporation (bio-rad, hercules, ca, usa). the bacteria harboring the rescued bacs were selected in the presence of streptomycin. mutants were confirmed using bac dna sequencing. similarly, the bac of rvwt was generated based on the newly constructed mus - - p, with two rounds of recombination. hcmv wt, mus - - p, and rvwt were propagated in hff cells, and virus stocks were stored in dmem supplemented with % fetal bovine serum (fbs) and . % bovine serum albumin (bsa) at − • c. cyclosporin a (csa) reagent was obtained from sigma-aldrich (st. louis, mo, usa). cd antibodies (hab , igg ) were prepared in our laboratory [ ] . dylight -conjugated secondary antibody, used for immunofluorescence, was from life technology (san jose, ca, usa). we also used anti-hcmv ie / mouse mab (ab , abcam, cambridge, uk), rabbit anti-human cypa mab (ab , abcam), phospho-mek / (ser / ) rabbit mab (# , cell signaling technology (cst), danvers, ma, usa), erk / rabbit mab (# , cst), irf- rabbit mab (# , cst), phospho-irf- (ser ) rabbit mab (# , cst), nf-κb p rabbit mab (# , cst), phospho-nf-κb p (ser ) rabbit mab (# , cst), and anti-gapdh mouse mab ( - -ig, proteintech, rosemont, il, usa). the following plasmids were used: cd plko. lentiviral shrna (a ) and non-target shrna control plasmid (plko. -ntc) were purchased from open biosystems (ge healthcare, little chalfont, uk). hcmv-encoded mir-us - - p plko. lentiviral shrna (plko. -us - - p) was constructed in this study. pcdna . (+) empty vector was obtained from invitrogen (carlsbad, ca, usa). full-length cd -expressing plasmid pcdna . -cd was constructed in our laboratory [ ] . then, the extracellular domain (residues - of cd ) or intracellular domain (residues - of cd ) were deleted to generate pcdna . -cd -decd and pcdna . -cd -dicd, respectively. the nf-κb-response promoter reporter plasmid (pnf-κb-luc) and ifn-β promoter reporter plasmid (pifn-β-luc) were obtained from beyotime (shanghai, china). dual luciferase mirna target expression vector (pmirglo) and the renilla luciferase control reporter plasmid (prl-tk) were purchased from promega (madison, wi, usa). the pmirglo-cd utr plasmid was made by inserting the utr of the human cd gene into the pmirglo empty vector using the primers as follows: (forward) -aagctagcggcaggtggcccgaggacgctccctg- and (reverse) viruses , , of -agtctagagagggtggaggtgggggcgatc- . site-directed mutagenesis was performed using a quikchange lightning multi site-directed mutagenesis kit (stratagene, san diego, ca, usa) on the pmirglo-cd utr to generate a pmirglo-cd utrm plasmid with the following primers: (forward) -agtcatggccgggtagacagcacagccttct- and (reverse) -agaaggct gtgctgtc tacccggccatgact- . cells that were grown on chambered cover slips were infected with hcmv strain nr- at a multiplicity of infection (moi) of . at h posttransfection, cells were fixed with % formaldehyde and blocked with % bovine serum albumin (bsa) in pbs and stained with primary mouse ie / antibody (ab , abcam, cambridge, uk), and then incubated with the secondary antibody dylight anti-mouse igg (life technology). cell nucleus was stained with , -diamidino- -phenylindole (dapi) (invitrogen). images were captured with a nikon eclipse te microscope (diagnostic instruments, inc., sterling heights, mi, usa) [ ] . the digital images were subsequently merged using fv asw v . software (olympus, tokyo, japan). the cells were co-transfected with the two packaging plasmids (pspax and pmd g), together with a control or rnai plko. lentiviral plasmid using lipofectamine tm (invitrogen). the sequence for cd shrna was: -cccatcatacacttccttctt- (sicd ); the sequence for hcmv-mir-us - - p shrna was: -ccgctcagtggctcggacc- (mir-us - - p). after h, cells were incubated with fresh medium without antibiotics for another h. the medium containing the recombinant virus was collected and filtered, and then added to hff or u cells in the presence of mg/ml polybrene. the infected cells were selected by adding puromycin ( - mg/ml) to the culture medium for days before additional experiments. the silencing of expression was verified by qpcr and western blot. cells ( × ) were seeded on -well plates and the following day were transfected using lipofectaminetm (invitrogen) and the indicated plasmids. for transfection efficiency normalization, . µg renilla luciferase reporter plasmids (prl-tk) were added to each transfection. the total amount of transfeced dna was maintained at a consistent level by adding empty vector dna. then, -h post-transfection, cells were lysed with µl passive lysis buffer (promega). supernatants clarified by centrifugation were used to perform luciferase assays using the dual luciferase assay kit (promega). the values of firefly luciferase activities were normalized to renilla luciferase activities. all of the reporter assays were repeated at least three times in triplicate. data shown are average values ± standard deviation (sd) from one representative experiment. viral dna was extracted from mock or hcmv-infected cells using a dneasy tissue kit (qiagen, hilden, germany). intracellular viral dnas were quantified using primers and a probe for amplifying the hcmv ul dna region, as previously described [ ] . the reaction was performed in a -µl amplification mixture ( µl of dna extract, µl of × premix ex taq tm (takara, dalian, china), . µl each of primer at µm, . µl of the fluorogenic probe at µm, . µl × rox reference dye ii) using an abi device (applied biosystems inc., foster city, ca, usa). pcr cycling conditions were as follows: • c for s, cycles at • c for s and • c for s. viral genome copies were normalized to cellular rnase p with thepreviously described primers and probe [ ] . unknown sample values were determined with a standard curve of known copy numbers of ul (nr- bac) and rnase p. each set of assays was repeated three times in triplicate, and data that are shown are average values ± sd from one representative experiment. total rna was isolated with trizol reagent (invitrogen) and cdna was reverse transcribed using a primescripttm rt reagent kit (takara). sybr-green real-time pcr (rt-pcr) was performed with the abi device (applied biosystems inc.) using sybr premix ex taq ii ( ×) (takara). cellular gapdh (glyceraldehyde- -phosphate dehydrogenase) was used to normalize the rna inputs. relative changes in gene expression were analyzed using the −∆∆ct method. all primers were synthesized by shanghai genepharma co., ltd., and are listed as following: cd the chemically synthetic sirna, targeting the cd mrna cds region (mir-cd , -guucuucgugaguuccuctt- ) [ ] , hcmv mir-us - - p (mirbase, mimat ), mir-us - - p (mirbase, mimat ), and other screened hcmv-encoded mature micrornas (http://www.mirbase.org/) were synthesized by ribobio co., ltd. (guangzhou, china), along with the silencer negative control sirna (c-sirna) molecules. the sirnas were transfected into cells using lipofectamine tm (invitrogen), according to the manufacturer's instructions. cells ( × ) were washed with cold pbs and lysed with ml np lysis buffer with mm β-mercaptoethanol and protease inhibitor cocktail (roche, basel, switzerland) for min on ice. cell lysates were centrifuged at , rpm for min, and supernatants were collected. the protein concentration was determined by bca assay. proteins were separated by % sds-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (pvdf) membrane (ge healthcare). block in % bsa was performed for h in tbs with . % triton x- (tbst), followed by incubation with primary antibody solution overnight at • c. then, the membrane was rinsed - times for min with tbst. after rinsing, the membrane was incubated with the horse radish peroxidase (hrp)-conjugated secondary antibody solution for h at room temperature. after another rinsing, the membrane was subsequently reacted with the chemiluminescent substrate, according to the recommendation of the ecl western blot detection reagent kits (thermo scientific, waltham, ma, usa), and were quantitated chemiluminescent signals using a ccd camera-based imager (bio-rad, hercules, ca, usa). quantitation was performed in the linear range of protein detection and analyzed by image lab . software (bio-rad). the experiments were repeated at least three times in triplicate, and a representative result is shown. cells ( × ) were either mock or hcmv infected at a moi of . to in . ml dmem with % fetal bovine serum. the medium was replaced with % fetal bovine serum containing dmem after h of incubation. protein extracts prepared from the infected cells at to days post-infection, were analyzed by western blot analysis. to determine viral growth level, cells ( × ) were infected with hcmv at an moi of . or . viral stocks were prepared with the total cells and the medium was harvested to days post-infection by adding an equal volume of % (w/v) skim milk, followed by ultrasonication. the titers of the viral stocks were determined in × hff cells, with standard virus viruses , , of plaque assay and the number of plaques were counted to days after viral infection. the values obtained were averages ± sd from triplicate experiments [ ] . all of the statistical analyses were performed with graphpad prism version . (graphpad software, san diego, ca, usa). experiments were repeated at least three times in triplicate. data are expressed as mean ± standard deviation. means between two groups were compared by using unpaired t-test or one-way analysis of variance (anova) with post hoc bonferroni t-test. differences were considered statistically significant at p < . . many transmembrane receptor proteins have been proposed to mediate signal transduction pathways that activate the early antiviral immune to hcmv entry [ ] . here, we have investigated the role of cd /emmprin in this response. we established two stable knockdowns of cd , with a cd -specific short hairpin rnas (sicd ) targeting the utr of cellular cd mrna, and two corresponding non-specific rnai stable cells as controls (sicontrol) via lentiviral-based transduction of human astrocytoma u cells (u cells) and human foreskin fibroblasts cells (hff cells). cd expression was markedly reduced in sicd -transduced cells when compared to the level in sicontrol cells, as observed with immunoblotting ( figure s ). then, we measured the effect of cd knockdown on hcmv-induced activation of the ifn-β and nf-κb promoters using a dual-luciferase reporter assay in u cells. cd knockdown significantly reduced the activation of nf-κb and ifn-β promoters that were stimulated by infection with hcmv, but not with herpes simplex virus (hsv- ) (figure a ). in addition, the ectopic expression of cd -wt, but not the extracellular domain (residues - ) or intracellular domain (residues - ) deletion mutants of cd (cd -decd and cd -dicd), restored promoter activity that was impaired by cd -specific rnai. these results confirm the specific contribution of cd to the induction of nf-κb and ifn-β by hcmv infection (figure b) . we next examined whether the endogenous cd was required for hcmv-induced antiviral signaling under physiological conditions. we found that the knockdown of cd expression in hff cells decreased the activation of ifnb and interferon-stimulated isg gene expression induced by hcmv infection, as well as expression of the nf-κb downstream genes il- and tnf-α ( figure c ). together, these data suggest that cd might block the hcmv-triggered early antiviral immune responses in a virus-specific manner. the graphical data are presented as the means ± the sds (n = ); ns denotes not significant (p > . ); * denotes p < . , ** denotes p < . and *** denotes p < . . from the results above, one might assume that cd acts as a receptor for hcmv infection. however, cd had no significant effect on hcmv entry, either when tested with a function-blocking antibody against cd to inhibit hcmv infection of hff cells, or when the expression of cd was knocked down in u cells ( figure s ). in our previous report that agreed well with the findings of other studies, we found that cypa can bind directly to the ectodomain of cd , and could therefore be responsible for the pro-inflammatory effect of cd [ ] . thus, we had reason to speculate that hcmv infection might promote the expression and release of cellular cypa, which could then lead to the activation of innate immune signaling through the interaction with cd . to test this hypothesis, we analyzed the kinetics of cypa rna expression by quantitative real-time pcr, intracellular cypa (incypa), and secreted cypa (scypa) protein levels by western blot analysis following hcmv infection of hff cells. as shown in figure a , the relative mrna levels of cypa rna expression were gradually up-regulated after hcmv infection. additionally, both incypa and scypa were highly expressed at post-infection (hpi), followed by a gradual increase in expression level with prolonged hcmv infection (figure b ). (a) luciferase assay analyzing nf-κb and ifn-β promoter activity in non-targeting control sirna (sicontrol) or cd -specific short hairpin rna (sicd ) stable u cells ( × ) transfected for h with a plasmid encoding an nf-κb or ifn-β firefly luciferase reporter ( . µg), along with a renilla luciferase reporter control plasmid (prl-tk, . µg), then left uninfected (ui) or infected for h with hcmv or hsv- (multiplicity of infection (moi) = . ); (b) luciferase assay analyzing nf-κb and ifn-β promoter activity in sicontrol or sicd stable u cells ( × ) transfected for h with a plasmid encoding an nf-κb or ifn-β firefly luciferase reporter ( . µg) and an expression plasmid for the wildtype cd (cd -wt), or the mutants (cd -decd and cd -dicd) ( . µg), along with a renilla luciferase reporter control plasmid (prl-tk, . µg), then left uninfected (ui) or infected for h with hcmv before the luciferase assays were performed (moi = . ); and, (c) quantitative real-time polymerase chain reaction (rt-pcr) analysis of ifnb , isg , il- and tnf mrna in sicontrol or sicd stable human foreskin fibroblast (hff) cells with hcmv uninfected (ui) or infected (moi = . ) for h. the graphical data are presented as the means ± the sds (n = ); ns denotes not significant (p > . ); * denotes p < . , ** denotes p < . and *** denotes p < . . from the results above, one might assume that cd acts as a receptor for hcmv infection. however, cd had no significant effect on hcmv entry, either when tested with a function-blocking antibody against cd to inhibit hcmv infection of hff cells, or when the expression of cd was knocked down in u cells ( figure s ). in our previous report that agreed well with the findings of other studies, we found that cypa can bind directly to the ectodomain of cd , and could therefore be responsible for the pro-inflammatory effect of cd [ ] . thus, we had reason to speculate that hcmv infection might promote the expression and release of cellular cypa, which could then lead to the activation of innate immune signaling through the interaction with cd . to test this hypothesis, we analyzed the kinetics of cypa rna expression by quantitative real-time pcr, intracellular cypa (incypa), and secreted cypa (scypa) protein levels by western blot analysis following hcmv infection of hff cells. as shown in figure a , the relative mrna levels of cypa rna expression were gradually up-regulated after hcmv infection. additionally, both incypa and scypa were highly in previous studies, cypa has been demonstrated to activate nf-κb by the erk / pathway through the cypa-cd interaction, which promotes an inflammatory effect in monocytes and macrophages [ ] . the transcription factors p and irf play key roles in the induction of cellular antiviral responses. phosphorylation of p and irf is required for their activity and constitutes a key checkpoint. thus, we next determined whether cd -mediates hcmv-triggered antiviral signaling by the activation of erk in hff cells. as shown in figure c , the phosphorylation levels of erk / (p-erk / ), nf-κb p subunit (p-p ), and irf (p-irf ) were increased in response to hcmv infection when compared to mock infection with the medium only. however, when endogenous cd expression was knocked down by lentiviral-based transduction of cd -specific short hairpin rna (sicd ), hcmv-induced phosphorylation levels of erk / and p were dramatically reduced relative to the sicontrol group. the decreased activation of erk / and p phosphorylation were more obvious than the level of irf , potentially emphasizing the importance and leading role of erk and nf-κb activation in signal transduction. furthermore, cd -blocking mab and cyclosporine (csa), a novel inhibitor of cypa that can specifically block in previous studies, cypa has been demonstrated to activate nf-κb by the erk / pathway through the cypa-cd interaction, which promotes an inflammatory effect in monocytes and macrophages [ ] . the transcription factors p and irf play key roles in the induction of cellular antiviral responses. phosphorylation of p and irf is required for their activity and constitutes a key checkpoint. thus, we next determined whether cd -mediates hcmv-triggered antiviral signaling by the activation of erk in hff cells. as shown in figure c , the phosphorylation levels of erk / (p-erk / ), nf-κb p subunit (p-p ), and irf (p-irf ) were increased in response to hcmv infection when compared to mock infection with the medium only. however, when endogenous cd expression was knocked down by lentiviral-based transduction of cd -specific short hairpin rna (sicd ), hcmv-induced phosphorylation levels of erk / and p were dramatically reduced viruses , , of relative to the sicontrol group. the decreased activation of erk / and p phosphorylation were more obvious than the level of irf , potentially emphasizing the importance and leading role of erk and nf-κb activation in signal transduction. furthermore, cd -blocking mab and cyclosporine (csa), a novel inhibitor of cypa that can specifically block the direct binding of cypa to cd , also inhibited the hcmv-induced phosphorylation of erk / (figure d ). together, these results suggest that hcmv infection triggers the secretion of cypa, which then activates the phosphorylation of erk and downstream nf-κb through interaction with cd , which ultimately promotes cellular antiviral signaling pathways. due to the potential role that cd may play in the hcmv early antiviral signaling mentioned above, we next investigated the influence of hcmv infection on the expression kinetics of cellular cd during different phases of the hcmv replication cycle. the protein expression levels of cd were dramatically down-regulated after hpi of hcmv infection (figure a ), corresponding to a decrease in the relative rna expression (figure b ). it is well known that micrornas encoded by hcmv play essential roles in all aspects of the hcmv life cycle through the manipulation of many cellular signaling pathways. thus, we next investigated whether the expression of cd was targeted and down-regulated by one of the hcmv-encoded mature mirnas previously identified in the reactome database (www.reactome.org). using rnahybrid (http://bibiserv.techfak.uni-bielefeld.de/ rnahybrid/submission), we screened hcmv-encoded mature mirna candidates that might target the utr region of cd . to experimentally determine which hcmv-encoded mirnas targets cd , a pmirglo dual luciferase reporter plasmid containing the utr sequence of human cd downstream of the firefly luciferase gene was generated. transfection of human embryonic kidney cells ( cells) with each of the individual putative hcmv mirna constructs, and subsequent luciferase assay, suggested that cd was most likely targeted by mir-us - - p (figure c ). the most likely target site for mir-ul - - p was identified by rnahybrid in the utr region of cd mrna (figure d) . we next performed site-directed mutagenesis on the seed sequence of the predicted target site in the pmirglo-cd utr reporter construct. as indicated by luciferase assays, the mutation of this site almost completely rescued the knockdown of cd by mir-ul - - p in the wild type (wt) construct ( figure e ). we then tested whether transfection of the mir-ul - - p mimic could target endogenous cd in hcmv permissive u cells. when compared to cells that were transfected with a non-targeting control sirna (nc) or mir-ul - - p mimic, the cd protein level was appreciably reduced after the transfection of the mir-ul - - p mimic. notably, the efficiency was almost comparable to the commercial sirna against cd (mir-cd ) (figure f ). these results indicate that mir-ul - - p efficiently targets and down-regulates endogenous cd via a specific location site in the utr sequence. (d) schematic of mir-us - - p-binding sites on cd ′ utr (cd ′utr ) and its mutant cd ′utrm , with the seed-recognizing site marked in red. the predicted hybrid free-energy of mir-us - - p is indicated; (e) confirmation of the predicted target site for mir-us - - p. the t cells were co-transfected with mir-us - - p mimic and the luciferase reporter pmirglo-cd ′utr (wt) or pmirglo-cd ′utrm (mut) for h before dual luciferase reporter assay; and, (f) confirmation that mir-us - - p ( p), but not mir-us - - p ( p) specifically down-regulates endogenous cd in u cells transfected as indicated and harvested two days after transfection for western blot analysis. commercial non-targeting control sirnas (nc) and cd -specific sirnas (sicd ) were used for comparisons. the graphical data are presented as the means ± the sds (n = ); ns denotes not significant (p > . ); ** denotes p < . . a luciferase reporter plasmid pmirglo-cd utr was tested; the putative hcmv mimics are outlined in the abbreviations. stars indicate potential down-regulated relative luciferase activities and the corresponding p value; (d) schematic of mir-us - - p-binding sites on cd utr (cd utr ) and its mutant cd utrm , with the seed-recognizing site marked in red. the predicted hybrid free-energy of mir-us - - p is indicated; (e) confirmation of the predicted target site for mir-us - - p. the t cells were co-transfected with mir-us - - p mimic and the luciferase reporter pmirglo-cd utr (wt) or pmirglo-cd utrm (mut) for h before dual luciferase reporter assay; and, (f) confirmation that mir-us - - p ( p), but not mir-us - - p ( p) specifically down-regulates endogenous cd in u cells transfected as indicated and harvested two days after transfection for western blot analysis. commercial non-targeting control sirnas (nc) and cd -specific sirnas (sicd ) were used for comparisons. the graphical data are presented as the means ± the sds (n = ); ns denotes not significant (p > . ); ** denotes p < . . we next determined if mir-us - - p had the same effect as the cd -specific short hairpin rna on the cd -mediated early innate immune response to hcmv. mir-us - - p stably transfected hff cells were constructed using the lentiviral-based transduction method. as shown in figure s b , the expression of cd obviously decreased in mir-us - - p transduced cells when compared with the level in sicontrol cells that was observed by western blot analysis. we then confirmed that knockdown of the cd expression by mir-us - - p could specifically reduce the activation of ifnb and interferon-related gene isg and the nf-κb downstream genes il- and tnf-α induced by hcmv early infection. these effects were similar to the effect of the cd -specific sirna studied above (figure ). more importantly, activation of these innate immune related genes by hcmv infection was also dramatically impaired in the sicontrol group when the culture medium was treated with cyclosporin a (csa), a cypa specific inhibitor that has been proven to block the cypa-cd interaction [ ] . mir-us - - p and csa appeared to have redundant roles in the inhibition of the cd -mediated early innate immune response to hcmv infection, since the addition of csa to the mir-us - - p transduced cell group had no effect on the transcriptional up-regulation of these genes. taken together, these findings suggest that mir-us - - p could function as a cd -specific short hairpin rna to inhibit the cd -mediated early innate immune response to hcmv infection, and further supports the importance of the scypa-cd interaction in this process. we next determined if mir-us - - p had the same effect as the cd -specific short hairpin rna on the cd -mediated early innate immune response to hcmv. mir-us - - p stably transfected hff cells were constructed using the lentiviral-based transduction method. as shown in figure s b , the expression of cd obviously decreased in mir-us - - p transduced cells when compared with the level in sicontrol cells that was observed by western blot analysis. we then confirmed that knockdown of the cd expression by mir-us - - p could specifically reduce the activation of ifnb and interferon-related gene isg and the nf-κb downstream genes il- and tnf-α induced by hcmv early infection. these effects were similar to the effect of the cd -specific sirna studied above (figure ). more importantly, activation of these innate immune related genes by hcmv infection was also dramatically impaired in the sicontrol group when the culture medium was treated with cyclosporin a (csa), a cypa specific inhibitor that has been proven to block the cypa-cd interaction [ ] . mir-us - - p and csa appeared to have redundant roles in the inhibition of the cd -mediated early innate immune response to hcmv infection, since the addition of csa to the mir-us - - p transduced cell group had no effect on the transcriptional up-regulation of these genes. taken together, these findings suggest that mir-us - - p could function as a cd -specific short hairpin rna to inhibit the cd -mediated early innate immune response to hcmv infection, and further supports the importance of the scypa-cd interaction in this process. quantitative rt-pcr analysis of ifnb , isg , il- , and tnf mrna in sicontrol or mir-us - - p stable hff cells with hcmv uninfected (ui) or infected (moi = . ) for h and csa present (hcmv + csa) or absent in the culture medium. the graphical data are presented as the means ± the sds (n = ); ns denotes not significant (p > . ); * denotes p < . , ** denotes p < . . to further evaluate the biological significance of hcmv mir-us - - p targeting of cd , we generated a mutant hcmv (mus - - p) by hcmv bac mutagenesis. to prevent any potential artifacts resulting from full sequence deletion, the mutants were constructed, by substituting only three nucleotides of the seed sequence of mir-us - - p, corresponding to the putative cd mrna utr target site (figure a) . a revertant virus (rvwt) was constructed as a control. in addition, we constructed a u cell line that stably overexpresses cd , as well as control u cell line. effective loss of function in the interference of cd expression after infection with hcmv-mus - - p ( figure s a) , rescued down-regulation of cd with the rvwt (figure s b) , and sustained overexpression of cd in pcdna . -cd stably-transfected u cells for at least days in comparison to control cells ( figure s a) , were confirmed by western blot analysis. artifacts resulting from full sequence deletion, the mutants were constructed, by substituting only three nucleotides of the seed sequence of mir-us - - p, corresponding to the putative cd mrna ′ utr target site (figure a) . a revertant virus (rvwt) was constructed as a control. in addition, we constructed a u cell line that stably overexpresses cd , as well as control u cell line. effective loss of function in the interference of cd expression after infection with hcmv-mus - - p ( figure s a) , rescued down-regulation of cd with the rvwt (figure s b) , and sustained overexpression of cd in pcdna . -cd stably-transfected u cells for at least days in comparison to control cells ( figure s a) , were confirmed by western blot analysis. next, we examined the effect of hcmv-mus - - p and ectopic expression of cd , relative to hcmv-wt (wild type), on different phases of the hcmv replication cycle. multistep growth and single-step growth curves were analyzed in the constructed u cells that were infected with hcmv-mus - - p, rvwt, or hcmv-wt, at a low moi of . or at a high moi of , respectively. the supernatant and cell-associated virus were harvested together at the dpi indicated. hcmv titers were determined by virus plaque assay in hff cells. as seen in figure b , at a low moi of . , when multiple replication cycles of infection were measured, both hcmv-mus - - p in control u cells, and hcmv-wt in cd overexpressing u cells, displayed a similar slight delay in viral growth with statistical significance, but ultimately reached the same titer plateau as hcmv-wt in empty vector stably-transfected u cells. hcmv wild-type and the revertant rvwt showed similar growth kinetics, and reached the replication plateau at almost seven days post-infection (dpi) in u -c cells. however, at the higher moi, no differences in virus yield were observed, either in cells infected with the hcmv mutant virus mir-us - - p, or in cells overexpressing cd ( figure c ). similar results were observed when the mutant hcmv-mus - - p and wild type viruses were used to infect hff cells. these results indicate that hcmv mir-us - - p targeting of cd might promote the course of hcmv lytic propagation at low moi. were infected with the hcmv wild-type virus, rvwt or hcmv-mus - - p mutant virus (mus - - p) and cd -overexpressing cells (u -cd ) were infected with hcmv wild-type virus at an moi of (b) . or (c) . cell-associated and supernatant virus were harvested at the dpi indicated. titers were determined by plaque assay. the values obtained were averages ± sd from triplicate experiments. * denotes p < . , ** denotes p < . . next, we examined the effect of hcmv-mus - - p and ectopic expression of cd , relative to hcmv-wt (wild type), on different phases of the hcmv replication cycle. multistep growth and single-step growth curves were analyzed in the constructed u cells that were infected with hcmv-mus - - p, rvwt, or hcmv-wt, at a low moi of . or at a high moi of , respectively. the supernatant and cell-associated virus were harvested together at the dpi indicated. hcmv titers were determined by virus plaque assay in hff cells. as seen in figure b , at a low moi of . , when multiple replication cycles of infection were measured, both hcmv-mus - - p in control u cells, and hcmv-wt in cd overexpressing u cells, displayed a similar slight delay in viral growth with statistical significance, but ultimately reached the same titer plateau as hcmv-wt in empty vector stably-transfected u cells. hcmv wild-type and the revertant rvwt showed similar growth kinetics, and reached the replication plateau at almost seven days post-infection (dpi) in u -c cells. however, at the higher moi, no differences in virus yield were observed, either in cells infected with the hcmv mutant virus mir-us - - p, or in cells overexpressing cd (figure c ). similar results were observed when the mutant hcmv-mus - - p and wild type viruses were used to infect hff cells. these results indicate that hcmv mir-us - - p targeting of cd might promote the course of hcmv lytic propagation at low moi. numerous cell surface glycoproteins, such as β microglobulin, annexin ii, cd , platelet-derived growth factor receptor-α (pdgfr-α), epithelial growth factor receptor (egfr), and β integrins, have been suggested to act as receptors facilitating hcmv entry [ ] . however, none of the suggested glycoproteins has been validated as a bona fide receptor that is necessary for hcmv infection of all the susceptible cell types. the in vivo broad tissue tropism of hcmv infection and differences in permissiveness for different cell types suggest that hcmv likely uses distinct cell surface receptors depending on the target cells. thus, identifying the specific hcmv cellular receptors is important for understanding the pathogenesis of hcmv. cd /emmprin is a transmembrane receptor glycoprotein that is belonging to the immunoglobulin superfamily and expressed at varying levels in many tissue cells. notably, several bacterial pathogens, such as neisseria meningitidis and plasmodium falciparum, as well as many viruses, including hiv- , hbv, sars-cov, and measles virus (mev), have been previously found to infect target cells via cd , indicating that it might constitute an evolutionarily conserved pathogen receptor for infection and spread within tissues and organisms [ ] . unfortunately, neither the infection-blocking experiment using cd -specfic antibody, nor the viral infection test with cd -knockdown cells, showed any difference in facilitating hcmv entry ( figure s ). since a low passage clinical hcmv strain nr- was used in these experiments, we assumed that cd does not function as a broad-spectrum receptor for hcmv infection, at least in typical in vitro permissive cell lines. in addition to the pathogen recognition receptor function, most transmembrane receptor proteins have been proposed to mediate signal transduction pathways immediately upon pathogenic particle attachment and penetration. cd belongs to the type i membrane proteins and as a candidate tumor biomarker, its expression is up-regulated in breast cancer tissue, lung carcinoma tissue, and bladder cancer tissue, along with a tumor's malignance, invasiveness, and metastasis [ ] . cd has been extensively studied since the discovery of its function in tumor progression and metastasis. here, we have explored a previously unknown role for cd in the host innate immune defense against hcmv invasion. cells detect hcmv infection as early as four to eight hours post-infection (hpi), responding by producing nf-κb-dependent cytokines and an early peak of antiviral ifn [ , ] . our experiments indicate that endogenous cd is required for the maximum activation of nf-κb and ifn-β-associated antiviral signaling that is triggered by hcmv infection, possibly having a redundant effect with other cell surface receptor glycoproteins (figure ). many tumor-related proteins, such as programmed death (pd- ) and pten, are known to play key roles in regulating the innate immunity against viral and bacterial infection [ , ] . however, the involvement of cd in early antiviral immunity appears to be specific to hcmv infection, since it does not extend to other viruses, like hsv- . since the signaling pathways of cd remain to be well defined, we next demonstrated that the phosphorylation of erk / and then nuclear translocation of nf-κb might play a leading role in cd -mediated hcmv-triggered antiviral signal transduction, with a subsequent activation of the irf phosphorylation and the orchestrated nf-κb-dependent cytokines, irf plus nf-κb-dependent interferon responses ( figure ). this finding can be supported by the fact that nf-κb is a key transcriptional enhancesome-component of the ifn-β, according to previous research [ , ] . research has increasingly suggested that the modulation of the host immune microenvironment is not only important for tumor virus-induced inflammatory carcinogenesis, but it is also critical for persistent and latent viral infection. for example, cmvil- , a viral mimic of human il- acquired from the cellular genome, binds with a high affinity to the human il- receptor, and limits the innate and adaptive immune responses even more efficiently than il- itself [ ] . in this study, we have demonstrated that hcmv infection leads to the expression and secretion of a cellular chemokine, cypa. studies show that cypa, as an extracellular ligand for its receptor cd , can be secreted by various cell types, including vascular smooth muscle cells, macrophages, and fibroblasts, and triggers a cascade of inflammatory responses [ ] . we demonstrated that cd -mediated hcmv-triggered phosphorylation of erk / , and the activation of nf-κb depends on the release of cypa from hcmv infected cells and its interaction with cd ( figure ). although hcmv is highly species-specific with no animal model yet known to support its replication, we speculate that secreted extracellular cypa, triggered by hcmv infection, acts through a paracrine mechanism, to stimulate uninfected cells to defend against a second wave of infection by the progeny virus through the interaction with cd . viruses , , of macrophages, and fibroblasts, and triggers a cascade of inflammatory responses [ ] . we demonstrated that cd -mediated hcmv-triggered phosphorylation of erk / , and the activation of nf-κb depends on the release of cypa from hcmv infected cells and its interaction with cd ( figure ). although hcmv is highly species-specific with no animal model yet known to support its replication, we speculate that secreted extracellular cypa, triggered by hcmv infection, acts through a paracrine mechanism, to stimulate uninfected cells to defend against a second wave of infection by the progeny virus through the interaction with cd . figure . general model for hcmv mir-us - - p in attenuating cd /emmprin-mediated hcmv-triggered early antiviral response. upon hcmv infection, intracellular cypa is released. as a paracrine proinflammatory cytokine, cypa interacts with cd of those uninfected cells and tissues, and stimulates the phosphorylation of erk / . the activation of nf-κb then occurs, followed by a subsequent activation of the irf phosphorylation and the orchestrated nf-κb-dependent, irf plus nf-κb-dependent immune responses. hcmv-encoded mir-us - - p targeting cd , cd -blocking mab, and an inhibitor of cypa (csa) can specifically inhibit this process. moreover, we found that endogenous cd was targeted by hcmv-encoded mir-us - - p at the ′ utr, which could replace cd -specific sirna mimics in inhibiting the cd -mediated early innate immune response to hcmv infection ( figure ) . mutation of the hcmv mir-us - - p seed sequence or ectopic expression of cd resulted in delayed multistep growth curves at low moi, suggesting that mir-us - - p targeting of cd is biologically significant (figure b ). micrornas (mirnas) are small noncoding rna molecules that generally target ′ untranslated regions ( ′ utr) of the mrnas. since the discovery of hcmv-encoded mirnas, at least mature mirna species encoded by pre-mirnas have been identified and reported to regulate multiple aspects of viral and cellular processes, including viral replication, immune evasion, formation of the virion, and eukaryotic translation [ ] . the pre-hcmv-mir-us - , from which the mature mir-us - - p is derived, is encoded by the hcmv us and us intergenic regions [ ] . mir-us - - p has been reported to be transcribed upon infection and highly expressed at hpi, followed by a gradually increased expression, during the course of hcmv infection [ ] . one might expect that the cells would respond differently to infection with the mutant virus . general model for hcmv mir-us - - p in attenuating cd /emmprin-mediated hcmv-triggered early antiviral response. upon hcmv infection, intracellular cypa is released. as a paracrine proinflammatory cytokine, cypa interacts with cd of those uninfected cells and tissues, and stimulates the phosphorylation of erk / . the activation of nf-κb then occurs, followed by a subsequent activation of the irf phosphorylation and the orchestrated nf-κb-dependent, irf plus nf-κb-dependent immune responses. hcmv-encoded mir-us - - p targeting cd , cd -blocking mab, and an inhibitor of cypa (csa) can specifically inhibit this process. moreover, we found that endogenous cd was targeted by hcmv-encoded mir-us - - p at the utr, which could replace cd -specific sirna mimics in inhibiting the cd -mediated early innate immune response to hcmv infection ( figure ) . mutation of the hcmv mir-us - - p seed sequence or ectopic expression of cd resulted in delayed multistep growth curves at low moi, suggesting that mir-us - - p targeting of cd is biologically significant (figure b ). micrornas (mirnas) are small noncoding rna molecules that generally target untranslated regions ( utr) of the mrnas. since the discovery of hcmv-encoded mirnas, at least mature mirna species encoded by pre-mirnas have been identified and reported to regulate multiple aspects of viral and cellular processes, including viral replication, immune evasion, formation of the virion, and eukaryotic translation [ ] . the pre-hcmv-mir-us - , from which the mature mir-us - - p is derived, is encoded by the hcmv us and us intergenic regions [ ] . mir-us - - p has been reported to be transcribed upon infection and highly expressed at hpi, followed by a gradually increased expression, during the course of hcmv infection [ ] . one might expect that the cells would respond differently to infection with the mutant virus (hcmv-mus - - p), at late times of hcmv infection, when the expression levels of cd were obviously down-regulated by mir-us - - p. however, we found no difference in the expression of isgs after infection with the mutant virus. based on previous studies and our overall findings, the reasons might be complex. at the late stage of infection, hcmv often uses multi-faceted and redundant strategies to regulate immune responses. thus, the effect of natural mir-us - - p may not be able to be detectable at late times of hcmv infection. we speculate that secreted extracellular cypa, which is triggered by hcmv infection, acts primarily to defend uninfected cells against progeny hcmv virus released from neighboring infected cells. as a result, the observed lack of differences in virus yield at the higher moi, irrespective of the mutation of mir-us - - p in the targeting of cd mrna utr or ectopic expression of cd , are understandable (figure c ). in summary, we have provided the first evidence that cd , a non-hcmv receptor glycoprotein, is targeted by hcmv-encoded mir-us - - p. this may be a tactic that is acquired by hcmv to antagonize the early innate immune response to benefit hcmv chronic infection. additionally, hcmv-induced paracrine effects of the hcmv-induced expression of cypa could cause an immune microenvironment called "smoldering inflammation", which could potentially contribute to the development of many hcmv inflammatory disorders. supplementary materials: the following are available online at www.mdpi.com/ - / / / /s . the cytomegaloviruses: ubiquitous agents with protean clinical manifestations does cytomegalovirus play a causative role in the development of various inflammatory diseases and cancer? systematic microrna analysis identifies atp v c as an essential host factor for human cytomegalovirus replication modulation of host innate and adaptive immune defenses by cytomegalovirus: timing is everything human cytomegalovirus activates inflammatory cytokine responses via cd and toll-like receptor altered cellular mrna levels in human cytomegalovirus-infected fibroblasts: viral block to the accumulation of antiviral mrnas human cytomegalovirus elicits a coordinated cellular antiviral response via envelope glycoprotein b interferon successfully inhibited refractory cytomegalovirus infection and resulted in cd + t-cells increase in a patient with aids effect of human exogenous leukocyte interferon in cytomegalovirus infections human cytomegalovirus immediate-early gene expression blocks virus-induced β interferon production human cytomegalovirus tegument protein ul inhibits sting-mediated signaling to evade antiviral immunity human cytomegalovirus envelope glycoproteins b and h are necessary for tlr activation in permissive cells the human tumor cell-derived collagenase stimulatory factor (renamed emmprin) is a member of the immunoglobulin superfamily extracellular cyclophilins contribute to the regulation of inflammatory responses active site residues of cyclophilin a are crucial for its signaling activity via cd basigin (cd ), a multifunctional transmembrane glycoprotein with various binding partners cyclophilin a (cypa) induces chemotaxis independent of its peptidylprolyl cis-trans isomerase activity: direct binding between cypa and the ectodomain of cd modulation of the cellular distribution of human cytomegalovirus helicase by cellular factor snapin a hsp chaperone protein interacts with and modulates the cellular distribution of the primase protein of human cytomegalovirus human cytomegalovirus mir-ul d facilitates latent viral infection by targeting host cell immediate early response gene functional profiling of a human cytomegalovirus genome epitope mapping of series of monoclonal antibodies against the hepatocellular carcinoma-associated antigen hab g/cd the involvement of hab g/cd in regulation of store-operated calcium entry and metastasis of human hepatoma cells real-time reverse transcription-pcr assay for detection of mumps virus rna in clinical specimens extracellular membrane-proximal domain of hab g/cd binds to metal ion-dependent adhesion site (midas) motif of integrin β to modulate malignant properties of hepatoma cells receptors and immune sensors: the complex entry path of human cytomegalovirus pro-inflammatory activities induced by cypa-emmprin interaction in monocytes peptidylproline cis-trans-isomerases: immunophilins early events in human cytomegalovirus infection cd immunoglobulin superfamily receptor function and role in pathology is cd a new biomarker reflecting histological malignancy of gliomas? multifaceted evasion of the interferon response by cytomegalovirus the tumor suppressor pten has a critical role in antiviral innate immunity role played by the programmed death- -programmed death ligand pathway during innate immunity against mycobacterium tuberculosis defining emerging roles for nf-κb in antivirus responses: revisiting the interferon-β enhanceosome paradigm virus induction of human ifn β gene expression requires the assembly of an enhanceosome attenuation of innate immunity by cytomegalovirus il- establishes a long-term deficit of adaptive antiviral immunity cyclophilin a and emmprin (cd ) in cardiovascular diseases human cytomegalovirus encoded micrornas: hitting targets identification and function of human cytomegalovirus micrornas human cytomegalovirus microrna mir-us - - p inhibits viral replication by targeting multiple cellular genes during infection the authors have declared no competing financial interests exist. key: cord- -bt xo iq authors: magassouba, n’faly; koivogui, enogo; conde, sory; kone, moussa; koropogui, michel; soropogui, barrè; kekoura, ifono; hinzmann, julia; günther, stephan; keita, sakoba; duraffour, sophie; fichet-calvet, elisabeth title: a sporadic and lethal lassa fever case in forest guinea, date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: bt xo iq lassa fever is a rodent-borne disease caused by lassa virus (lasv). it causes fever, dizziness, vertigo, fatigue, coughing, diarrhea, internal bleeding and facial edema. the disease has been known in guinea since but only anectodical acute cases have been reported to date. in january , a -year-old man, a wood merchant from kissidougou, forest guinea, presented himself at several health centers with persistent fever, frequent vomiting and joint pain. he was repeatedly treated for severe malaria, and died three weeks later in mamou regional hospital. differential diagnosis identified lasv as the cause of death. no secondary cases were reported. the complete lasv genome was obtained using next-generation sequencing. phylogenetic analysis showed that this strain, namely the kissidougou strain, belongs to the clade iv circulating in guinea and sierra leone, and is thought to have emerged some years ago. due to the similarity of symptoms with malaria, lassa fever is still a disease that is difficult to recognize and that may remain undiagnosed in health centers in guinea. lassa fever is a haemorrhagic fever due to lassa virus (lasv) that was discovered in in nigeria [ , ] . the disease is endemic in west africa, particularly in guinea, sierra leone, liberia, mali, côte d'ivoire, togo, benin and nigeria. the majority of cases are registered in sierra leone and nigeria, while the other countries have registered sporadic cases [ ] [ ] [ ] . in guinea, the disease is widespread in the south, as evidenced by seroprevalence rates in humans ranging from % to % in contrast to the % to % observed in the north [ ] . ecological studies on rodent reservoirs show a similar distribution of the lassa virus [ ] [ ] [ ] [ ] [ ] . however, guinea has very few reported acute human cases, and only a few studies have a posteriori described acute lassa fever cases in the prefectures of kindia, faranah, kissidougou, guekedou, macenta and n'zérékoré in and - [ ] [ ] [ ] . therefore, the emergence of one acute case in in macenta [ ] and another one in in kissidougou [ ] has prompted great interest, necessitating immediate investigation. the epidemic potential of lassa fever was recently revised by the word health organization, which has listed lassa fever among the top five zoonoses to be monitored, along with ebola, mers-cov, nipah and zika (https://www.who.int/teams/blueprint). in this article, we describe both the epidemiological and molecular investigation of the kissidougou case and his contacts. field investigations were managed by the agence nationale de sécurité sanitaire (anss) in consultation with members of the ministry of health, the gamal-nasser university of conakry, the word health organization (who), the ngo médecins sans frontières (msf) and the centre of disease control (cdc). a first investigation took place - february in the prefecture of kissidougou where the patient lived. a second investigation took place - february in the mamou prefecture where the patient died ( figure ). contacts in health centres and hospitals were identified and their blood was collected for pcr testing. the tests were carried out in the laboratory on january for the patient and from to february for the contacts (table s ). all subjects and the family of the deceased patient gave their informed consent. the protocol has been approved by the ethics committee for health research in guinea (permit n • /cners/ , approval october ). field investigations were managed by the agence nationale de sécurité sanitaire (anss) in consultation with members of the ministry of health, the gamal-nasser university of conakry, the word health organization (who), the ngo médecins sans frontières (msf) and the centre of disease control (cdc). a first investigation took place - february in the prefecture of kissidougou where the patient lived. a second investigation took place - february in the mamou prefecture where the patient died ( figure ). contacts in health centres and hospitals were identified and their blood was collected for pcr testing. the tests were carried out in the laboratory on january for the patient and from to february for the contacts (table s ). all subjects and the family of the deceased patient gave their informed consent. the protocol has been approved by the ethics committee for health research in guinea (permit n° /cners/ , approval october ). [ ] and this study. the presence of antibodies against hiv- /hiv- and yellow fever virus (yfv) was tested using the insti kit (biolytical, richmond, canada) and elisa (pasteur institute, dakar, senegal) respectively. the presence of the hepatitis b virus (hbv) was tested using a rapid screening kit (capitalbio technology, beijing, china). the presence of the ebola virus (ebov) and lasv was investigated by reverse transcription-polymerase chain reaction (rt-pcr). viral rna was extracted from serum samples using the qiamp viral rna kit (qiagen, hilden, germany). in conakry, the ebov test was carried out by real-time rt-pcr, using the realstar ® filovirus screen rt-pcr kit . (altona diagnostics, hamburg, germany) on a smart cycler ii system (cepheid, sunnyvale, ca, usa). the lasv test was carried out using a conventional rt-pcr targeting the glycoprotein (gp) with the primers lvs + ( -accggggatcctaggcattt- ) and lvs -( -gttctttgtgcaggamaggggcatkgtcat- ) [ ] . the ebov and lasv tests were performed on both the patient and contacts in conakry (table s ). a confirmatory real-time rt-pcr lasv test using the realstar ® lassavirus rt-pcr kit . (altona diagnostics) on the rotor-gene q platform (qiagen, hilden, germany) was performed on the serum sample of the patient in our satellite laboratory in gueckedou. the lasv-specific rt-pcr kit targets both the s-and l-segments of lasv. the serum sample from the patient was dried on a filter paper and sent to the bernhard nocht institute for tropical medicine in hamburg, germany. extraction and metagenomic library preparation for next generation sequencing (ngs) on the illumina platform were performed as described previously [ ] . briefly, viral rna was extracted directly from dried serum on filter paper using the qiamp viral rna kit (qiagen), further digested with dnase (turbo dnase, thermo fisher scientific, carlsbad, usa), randomly reverse-transcribed and amplified using a sequence-independent single-primer amplification (sispa) approach. the illumina sequencing library was prepared using the nextera xt v kit (illumina, san diego, usa) with ng of sispa-amplified cdna, according to the manufacturer's instructions, with a total of cycles in the library amplification pcr, and further sequenced on a × bp illumina miseq run. majority consensus was obtained with bases called at a minimum depth of x and a support fraction of %. any base location that did not fulfil the depth and support fraction was assigned an "n" iupac ambiguity notation. the complete sequences were submitted to genbank under the names and accession numbers lasv/gui/kis- -l-segment #mt and lasv/gui/kis- -s-segment #mt . the nucleotide sequences of full-length glycoprotein precursor (gpc), nucleoprotein (np) and polymerase (l) were aligned separately in three data sets including sequences for each. the sequences were chosen to be representative of their clusters after a preliminary analysis done with np sequences only and elimination of similar or clone sequences. the alignment of nucleotides was realized according to the position of amino acids in the protein alignment. the phylogeny was inferred by the bayesian markov chain monte carlo (mcmc) method implemented in the beast software, version . . [ ] . with the view to performing a time-calibrated phylogeny, the parameters were set in beauti as follow: the tip dates at the year level, the substitution model as gtr + gamma and codon partition with positions , , , the clock model as strict or uncorrelated relaxed. a coalescent tree with a constant size population was set as prior. the length of the chain was million, with echo states and log parameters every , steps. the xml files issued from beauti were run in beast and checked in tracer. after checking the effective sample size to be above for all the parameters, the consensus trees were obtained in treeannotator, and then visualized through figtree (beast packages, https://beast.community/programs). on january , a -year-old man consulted a clinic in kissidougou because of fever accompanied by chills, dizziness, very frequent bilious vomiting and joint pains. malaria was diagnosed and the patient was treated with paracetamol and quinine. despite complying with the malaria treatment, symptoms remained unchanged for one week. he returned to the same clinic, which further referred him to the regional hospital located in kissidougou on january . at the end of the consultation, a diagnosis of malaria and typhoid fever was made. he received a perfusion and a recommendation of hospitalization was made. however, the patient refused to be hospitalized and came back home. after three days of treatment, and feeling that there was no improvement, he went to another health centre. three days later, he decided to travel to mamou, where he stayed two days with his family. noting that there was no improvement in his status, his parents decided to bring him to the regional hospital of mamou on january , where he was admitted at p.m. to the emergency room at the epidemic treatment centre (ct-epi). at the time of admission, the symptoms were as follows: fever and chills, dizziness, vomiting, joint pain, diarrhoea and prostration. a diagnosis of severe malaria was given and he received an intensive supportive antimalarial treatment (rehydration, artesunate, ampicillin, paracetamol, dicynone). at p.m., the patient started bleeding with a cough tinged with blood and the epistaxis started. the physician in charge of the intensive care unit raised the suspicion of a viral haemorrhagic fever (vhf) such as the ebola virus disease. a blood sample was taken and packed for dispatch to the reference laboratory in conakry. the patient died at p.m. with a picture of toxic-infectious shock and diffuse bleeding. the body was transported to the morgue and transferred to the red cross for a dignified and secure burial. on january , the laboratory received the blood sample of the case and proceeded to various testing. differential diagnosis included malaria, serology of hiv and yfv, and acute infection by hbv, ebov and lasv. only the presence of the lasv was confirmed on january by conventional lasv rt-pcr. it was further re-tested by real-time lasv rt-pcr, and lasv ct values of . for the s-segment and . for the l-segment were found. according to the equation of the standard curve for the gpc assay (y = . x + . where y = log (rna copies/ml plasma) and x = ct), we can estimate the number of copies/ml to be . e+ . the confirmation of a lassa fever case launched field investigations and contact tracing activities. the investigation in kissidougou revealed that the patient was an entrepreneur who was a timber trader. before falling ill, he had spent a few days in dandou, a village km from kissidougou, where he frequently went to do business. a total of individuals in contact with the case in kissidougou (n = ), mamou (n = ) and conakry (n = ) were identified by the field teams, and further sampled for lasv rt-pcr as per ministry of health national strategy guidelines in the case of vhf suspicion even in the absence of symptoms. none of them were found positive for ebov or lasv by rt-pcr (table s ). phylogenetic analysis shows that the lasv strain from the case, further named the kissidougou strain, belongs to clade iv of the lassa mammarenavirus genus. this clade includes lasv known to circulate in faranah (upper guinea) and in macenta (forest guinea). trees based on the combined complete glycoprotein (gp) and nucleoprotein (np) (figure a ) and polymerase ( figure b) show that the kissidougou strain is very closely related to a lasv strain identified in liberia in (lib- ). nucleotide similarities between these two strains are . % for gp, . % for np and . % for the polymerase. the amino acid translation gives similarities of . % for gp, . % for np and . % for the polymerase. the time of the most recent common ancestor (tmrca) of the kissidougou and lib- cluster is estimated at ( % highest posterior density (hdp) interval: - ) years using the gpc and np, and at ( % hpd interval: - ) years using the polymerase. in addition, the analysis on the partial np, including four more sequences published in bowen et al. [ ] , further supports that the kissidougou cluster is different from that of faranah, macenta and n'zérékoré ( figure s ). viruses , , x for peer review of [ ] , further supports that the kissidougou cluster is different from that of faranah, macenta and n'zérékoré ( figure s ). in guinea, numerous lasv sequences have been largely described in rodents, notably in the region of faranah ( sequences), kindia ( sequences) and guékedou ( sequences) [ ] [ ] [ ] , ] . however, only sequences are derived from humans, complete from faranah and macenta [ , ] , and partials from kissidougou and nzérékoré [ ] . our report represents thus the sixth description of a lasv strain isolated from humans in guinea. it demonstrates the circulation of lasv in the surroundings of kissidougou, which was already observed in among missionaries living in telekoro near kissidougou. indeed, that historical study revealed that three individuals who had fever with long convalescence, with or without deafness, harboured lasv neutralizing antibodies [ ] . the child of one of the missionaries also had lasv-neutralizing antibodies without signs of a febrile episode. from to , an investigation among patients hospitalized in kissidougou revealed that six of them were lassa seropositive [ ] . they came from villages around the town (figure ). this therefore indicates that lasv has been circulating for years in the kissidougou area. phylogenetic analysis of the polymerase indicates an older origin, dating back ( % hpd interval: - ) years. the strain described here is clearly related to the sequence lib- observed in in liberia. it may be thus speculated that trades, including those related to the timber trade between forest guinea and liberia, and commercial business favour lasv circulation between kissidougou and liberia. the case was, indeed, a logger merchant who used to sell wood in forest guinea without prior travel history to liberia. yet, a clear geographical origin (i.e., liberia or guinea-kissidougou) for this sub-cluster remains speculative due to a lack of information about lib- , as well as a lack of additional sequences. thus, the name "kissidougou cluster" is to highlight that it is different to that of the macenta cluster but that it is also still part of the lasv known to circulate in guinea. the macenta cluster also includes three sequences, which, although identified in liberia, originate from macenta, guinea, a town bordering liberia. for example, the case described in , the sequence of which is referenced under number lib-lf (mh in wiley et al. ) , was a woman working at soguipah, near macenta. she was diagnosed in ganta, the neighbouring town in liberia. the other large "liberia" cluster, composed of sequences identified in, and originating from lofa, bong and nimba counties in liberia, forms a cluster independent from that of guinea. altogether, the description of this new strain allows us to speculate about a "macenta" and a "kissidougou" cluster circulating in forest guinea. these clusters appear to be more recent than the ones observed in faranah, upper guinea, and in liberia. this last country is probably the entry point of the virus in the mano river region [ ] . in three weeks, the patient went through two health centres, one in kissidougou and one in mamou. moreover, despite returning to the same health care facility in kissidougou at a one week interval with worsened symptoms, no suspicion of vhf was considered. even following his admission in a critical state at the ct-epi of mamou, the diagnosis of severe malaria continued to be considered. this indicates that the accurate and rapid clinical diagnosis of vhf in endemic areas is challenging, despite the - ebola virus disease epidemic in the country. this could be improved by providing regular awareness training on vhf case definitions and access to adequate communication tools (e.g., posters, flyers, drawings etc.). on the other hand, the turnaround time for differential diagnosis was short, around two days (table s ). this included sample collection, transportation to the laboratories in conakry and guéckédou, analysis, results interpretation and reporting to the anss and who. since the ebola outbreak, the diagnostic chain has greatly improved and two days are now required to provide a diagnostic result of samples collected in remote areas. similarly, field investigations and contact tracing activities, which have been in place since the creation of the anss in july , four months after the start of the ebola crisis in guinea, have been rapidly launched following the alert and no secondary cases have been identified. finally, the absence of secondary infections at the health centres visited by the case also indicates adequate infection prevention and control practices. this case report shows that early identification of vhf cases remains challenging in remote areas. regular awareness training may facilitate the implementation of improved field surveillance and early detection. the molecular description supports a new sub-lineage of lasv in forest guinea. isolation and antigenic characterization of lassa virus lassa fever, a new virus disease of man from west africa risk maps of lassa fever in west africa lassa virus metagenomic sequencing at the epicenter of the nigeria lassa fever outbreak lassa virus activity in guinea: distribution of human antiviral antibody defined using enzyme-linked immunosorbent assay with recombinant antigen lassa virus-infected rodents in refugee camps in guinea: a looming threat to public health in a politically unstable region. vector borne zoonotic dis fluctuation of abundance and lassa virus prevalence in mastomys natalensis in guinea, west africa. vector borne zoonotic dis spatial and temporal evolution of lassa virus in the natural host population in upper guinea mastomys natalensis and lassa fever new hosts of the lassa virus lassa fever in guinea: i. epidemiology of human disease and clinical observations. vector borne zoonotic dis eléments de recherches clinico-épidémiologiques et de laboratoire sur les fièvres hémorragiques en guinée genetic diversity among lassa virus strains lassa fever-west africa ( ): liberia ex guinea lassa fever-west africa ( ): guinea (mm) lassa fever virological and serological studies improved detection of lassa virus by reverse transcription-pcr targeting the ' region of s rna bayesian phylogenetics with beauti and the beast . households as hotspots of lassa fever? assessing the spatial distribution of lassa virus-infected rodents in rural villages of guinea determining ancestry between rodent-and human-derived virus sequences in endemic foci: towards a more integral molecular epidemiology of lassa fever within west africa direct submission lassa mammarenavirus direct submission lassa mammarenavirus lassa virus circulating in liberia: a retrospective genomic characterisation we are grateful to the staff of the laboratoire des fièvres hémorragiques en guinée, namely mory cherif, amadou doré, mamadou alpha baldé and aïssatou sow for their technical support during the field investigation and the differential diagnosis. we also thank the pasteur institute of dakar, regional reference laboratory, for having provided the reagents allowing the yellow fever serology by elisa. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- - jmqd authors: yuan, yuan; zhang, zhi-peng; he, yi-ning; fan, wen-sheng; dong, zhi-hua; zhang, li-hua; sun, xin-kuan; song, li-li; wei, tian-chao; mo, mei-lan; wei, ping title: protection against virulent infectious bronchitis virus challenge conferred by a recombinant baculovirus co-expressing s and n proteins date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jmqd avian infectious bronchitis virus (ibv) is the causative agent of infectious bronchitis, which results in considerable economic losses. it is imperative to develop safe and efficient candidate vaccines to control ibv infection. in the current study, recombinant baculoviruses co-expressing the s and n proteins and mono-expressing s or n proteins of the gx-yl strain of ibv were constructed and prepared into subunit vaccines rhbm-s -n, rhbm-s and rhbm-n. the levels of immune protection of these subunit vaccines were evaluated by inoculating specific pathogen-free (spf) chickens at days of age, giving them a booster with the same dose days later and challenging them with a virulent gx-yl strain of ibv days post-booster (dpb). the commercial vaccine strain h was used as a control. the ibv-specific antibody levels, as well as the percentages of cd + and cd + t lymphocytes, were detected within days post-vaccination (dpv). the morbidity, mortality and re-isolation of the virus from the tracheas and kidneys of challenged birds were evaluated at five days post-challenge (dpc). the results showed that the ibv-specific antibody levels and the percentages of cd + and cd + t lymphocytes were higher in the rhbm-s -n vaccinated birds compared to birds vaccinated with the rhbm-s and rhbm-n vaccines. at dpc, the mortality, morbidity and virus re-isolation rate of the birds vaccinated with the rhbm-s -n vaccine were slightly higher than those vaccinated with the h control vaccine but were lower than those vaccinated with the rhbm-s and rhbm-n vaccines. the present study demonstrated that the protection of the recombinant baculovirus co-expressing s and n proteins was better than that of recombinant baculoviruses mono-expressing the s or n protein. thus, the recombinant baculovirus co-expressing s and n proteins could serve as a potential ibv vaccine and this demonstrates that the bivalent subunit vaccine including the s and n proteins might be a strategy for the development of an ibv subunit vaccine. avian infectious bronchitis (ib) is a highly contagious disease of chickens caused by the infectious bronchitis virus (ibv), belonging to the gammacoronavirus genus within the coronaviridae family [ ] . ibv affects chickens of all ages and types and primarily infects the respiratory and urogenital systems of chickens, causing massive economic losses [ ] [ ] [ ] . there are dozens of ibv serotypes related to the extensive variability of the spike (s) protein. there is little or no cross-protection between different serotypes of ibvs or circulating variant viruses, frequently leading to immune failures and making it extremely difficult to control the disease [ ] [ ] [ ] . control of the disease currently relies on conventional live-attenuated and inactivated vaccines [ ] [ ] [ ] . however, live-attenuated vaccines can result in the occurrence of vaccine-like viruses with increased virulence and persistence due to point mutation and recombination with other vaccines or field strains [ ] [ ] [ ] . inactivated vaccines are not only high in cost but also do not establish long-term immunity. the application of inactivated vaccines alone has frequently failed to induce strong cellular immunity, resulting in little or no protection [ , ] . despite the widespread application of these conventional vaccines, ib continues to cause severe economic losses in many countries, underscoring the need to develop new safer and more effective candidate vaccines for the practical control of ibv. among new vaccines, subunit vaccines have been showed to elicit strong humoral and cellular immune responses [ ] [ ] [ ] . subunit vaccines have significant advantages, such as efficient antigenic presentation, high stability and flexibility in protein or epitope selection, compared with live-attenuated and inactivated vaccines [ ] . additionally, no infectious viral particles are involved in vaccine production, so they are very safe. subunit vaccines are genetically engineered and are thus easy to mass produce [ ] . subunit vaccines also allow the creation of multivalent vaccines. thus, subunit vaccines may have the potential to act as safe and effective vaccine candidates. ibv consists of four structural proteins. they are the spike (s), envelope (e), membrane (m) and nucleocapsid (n) proteins [ ] . the s protein is post-translationally cleaved into s and s subunits [ ] . the s protein determines the antigenicity and immunogenicity of the virus and plays an important role in the induction of neutralizing and hemagglutination inhibition antibodies, tissue affinity and cell adsorption, all involved in the humoral immune response [ ] [ ] [ ] . the n protein plays an extremely important role in the replication and assembly of ibv, affecting its immunogenicity and is thus involved in the cellular immune response [ ] [ ] [ ] . hence, the s and n proteins are the most promising subunit vaccine candidates against ibv. therefore, the development of subunit vaccines including both the s and n proteins has promising prospects. the baculovirus expression system (bes) is an efficient system for gene expression because of its good safety, high expression and excellent protein processing ability compared to other expression systems. a major advantage of the bes is the ease of scaling up from the laboratory to a large-scale production system [ , ] . in addition, baculoviruses are insect pathogens and thus non-pathogenic for vertebrates. therefore, the bes is currently widely used for protein expression and vaccine production. there have been a few studies describing the expression of ibv proteins by bes [ , [ ] [ ] [ ] [ ] but none of these studies have focused on developing and evaluating the immune response of a subunit vaccine from the baculovirus which co-expresses s and n proteins of ibv. this study therefore set out to measure the protective effect of the subunit vaccine co-expressing both the s and n proteins of ibv. moreover, we compared the immune protection offered by this vaccine with those induced by recombinant subunit vaccines expressing s protein or n protein alone and h inactivated vaccine. the aim is to obtain safer and more effective subunit vaccines, thus providing a novel subunit vaccine candidate for ibv. sf insect cells were purchased from qiyin biological technology co., ltd. (jiangyin, china) and cultured in serum-free sf ii medium (gibco, grand island, ny, usa) at • c. the virulent gx-yl strain of ibv was isolated from broilers with nephritis disease and is the representative dominant serotype (different from the h strain) isolate in southern china and shared heterologous serotype with the h strain [ ] . the % tracheal organ culture infection dose (toc-id ) of the ibv gx-yl strain was determined as previously described [ ] . the euokaryotic expression vector pfastbac tm dual was the product of invitrogen (waltham, ma, usa). in order to increase the expression and yield of heterologous secreted proteins in insect cells, the honeybee melittin (hbm) signal peptide recognized by insect cells was introduced into the bes. the coding mature s ( - bp) (accession number fj . ) and n ( - bp) (accession number j . ) protein genes of the ibv gx-yl strain were amplified by reverse transcription polymerase chain reaction (rt-pcr). the insect signal peptide hbm gene was amplified by pcr using a pair of primers with a partial overlapping sequence. then the fusion genes hbm-s and hbm-n were obtained by fusion pcr, cloned into the peasy-t vector and then sub cloned into the transfer vector pfastbac tm dual ( figure a ) at the bamh i/pst i sites as well as the xho i/kpn i sites under the control of ph and p promotors, respectively ( figure b ,c) to obtain recombinant transposon vectors pfast-hbm-s and pfast-hbm-n. a ×his tag and a tobacco etch virus (tev) protease cleavage site were added before the stop codon to facilitate the identification and purification of the expressed proteins. the hbm-n gene was then directly sub cloned into pfast-hbm-s to yield the recombinant transposon vector pfast-hbm-s -n. this involved the s gene being inserted under the control of promotor ph and the n gene being inserted under the control of promoter p ( figure d ). all the recombinant transposon vectors were verified by pcr, restriction endonuclease digestion and sequencing. the verified recombinant transposon vectors were then transformed into dh bac tm escherichia coli cells to generate recombinant bacmids rhbm-s , rhbm-n and rhbm-s -n, which were identified by pcr with m primers. the purified recombinant bacmids rhbm-s , rhbm-n and rhbm-s -n were obtained after several instances of screening. they were then transfected into sf insect cells to obtain recombinant baculoviruses through lipofectin-mediated transfection, following the manufacturer's instructions (invitrogen). the recombinant baculoviruses rhbm-s , rhbm-n and rhbm-s -n were identified by pcr with ibv specific primers and m primers the recombinant baculovirus titers were determined using end-point dilution analysis. all the primers used in this study are shown in table s . in order to increase the expression and yield of heterologous secreted proteins in insect cells, the honeybee melittin (hbm) signal peptide recognized by insect cells was introduced into the bes. the coding mature s ( - bp) (accession number fj . ) and n ( - bp) (accession number j . ) protein genes of the ibv gx-yl strain were amplified by reverse transcription polymerase chain reaction (rt-pcr). the insect signal peptide hbm gene was amplified by pcr using a pair of primers with a partial overlapping sequence. then the fusion genes hbm-s and hbm-n were obtained by fusion pcr, cloned into the peasy-t vector and then sub cloned into the transfer vector pfastbac tm dual ( figure a ) at the bamh i/pst i sites as well as the xho i/kpn i sites under the control of ph and p promotors, respectively ( figure b ,c) to obtain recombinant transposon vectors pfast-hbm-s and pfast-hbm-n. a ×his tag and a tobacco etch virus (tev) protease cleavage site were added before the stop codon to facilitate the identification and purification of the expressed proteins. the hbm-n gene was then directly sub cloned into pfast-hbm-s to yield the recombinant transposon vector pfast-hbm-s -n. this involved the s gene being inserted under the control of promotor ph and the n gene being inserted under the control of promoter p ( figure d ). all the recombinant transposon vectors were verified by pcr, restriction endonuclease digestion and sequencing. the verified recombinant transposon vectors were then transformed into dh bac tm escherichia coli cells to generate recombinant bacmids rhbm-s , rhbm-n and rhbm-s -n, which were identified by pcr with m primers. the purified recombinant bacmids rhbm-s , rhbm-n and rhbm-s -n were obtained after several instances of screening. they were then transfected into sf insect cells to obtain recombinant baculoviruses through lipofectin-mediated transfection, following the manufacturer's instructions (invitrogen). the recombinant baculoviruses rhbm-s , rhbm-n and rhbm-s -n were identified by pcr with ibv specific primers and m primers the recombinant baculovirus titers were determined using end-point dilution analysis. all the primers used in this study are shown in table s . the hbm-s gene was sub cloned into the vector pfastbac tm dual under the control of ph promotor; (c) the hbm-n gene was sub cloned into the vector pfastbac tm dual under the control of p promotor; (d) hbm-s and hbm-n genes were sub cloned into the vector pfastbactm dual under the control of ph and p promotors, respectively. the resulting recombinant transposon vectors were named pfast-hbm-s , pfast-hbm-n and pfast-hbm-s -n. the expression of recombinant proteins s , n and s -n in sf cells were detected using indirect immunofluorescence assay (ifa) and western blot. for ifa, sf cells were infected with the recombinant baculoviruses rhbm-s , rhbm-n and rhbm-s -n at a multiplicity of infection (moi) of , respectively and then fixed with % paraformaldehyde at h post-infection. mouse anti-his igg ( : dilution, cwbio, beijing, china) was used as the primary antibody, while fluorescein schematic diagram of s , n and s -n genes baculovirus expression systems. (a) the schematic diagram of the transfer vector pfastbac tm dual; (b) the hbm-s gene was sub cloned into the vector pfastbac tm dual under the control of ph promotor; (c) the hbm-n gene was sub cloned into the vector pfastbac tm dual under the control of p promotor; (d) hbm-s and hbm-n genes were sub cloned into the vector pfastbactm dual under the control of ph and p promotors, respectively. the resulting recombinant transposon vectors were named pfast-hbm-s , pfast-hbm-n and pfast-hbm-s -n. the expression of recombinant proteins s , n and s -n in sf cells were detected using indirect immunofluorescence assay (ifa) and western blot. for ifa, sf cells were infected with the recombinant baculoviruses rhbm-s , rhbm-n and rhbm-s -n at a multiplicity of infection (moi) of , respectively and then fixed with % paraformaldehyde at h post-infection. mouse anti-his igg ( : dilution, cwbio, beijing, china) was used as the primary antibody, while fluorescein isothiocyanate (fitc)-labeled goat anti-mouse antibody igg ( : dilution, cwbio, beijing, china) was used as the secondary antibody. specific fluorescent signals in sf cells were observed using fluorescent microscopy. at h post-infection, cell culture supernatants and cell lysates infected with the recombinant baculoviruses rhbm-s , rhbm-n and rhbm-s -n were collected for the analysis of recombinant proteins using western blot. protein samples were separated by % sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), transferred onto polyvinylidene fluoride (pvdf) membranes and blocked in % skim milk with phosphate buffer solution tween- (pbst) buffer. mouse anti-his igg ( : dilution, cwbio, beijing, china) was used as the primary antibody and horseradish peroxidase (hrp)-labeled goat anti-mouse antibody igg ( : dilution, cwbio, beijing, china) was used as the secondary antibody. normal sf cells and sf cells infected with the same amount of wild-type baculovirus were set as negative controls. the proteins were visualized using a diaminobenzidine (dab) substrate kit as recommended by the supplier. the specific-pathogen-free (spf) chickens used in this study were hatched from fertilized white leghorn spf eggs (beijing merial vital laboratory animal technology co., ltd., beijing, china) in our facilities and the chickens were housed in separate isolation units until reaching days of age. fourteen-day-old spf chickens were assigned randomly into six groups (n = chickens/group). each bird in the rhbm-s -n, rhbm-s and rhbm-n groups was injected subcutaneously with . ml ( µg) s -n, s and n recombinant proteins, respectively. these proteins were emulsified in freund's complete adjuvant on day and boosted with the same dose of proteins emulsified in freund's incomplete adjuvant on day . birds in the h group, set as a positive control, were injected with the h inactivated vaccine. the wt-b group and cell group, serving as negative controls, were set up by replacing the recombinant proteins with emulsified wild-type baculovirus and normal sf cell lysates, respectively. at days post-booster (dpb), all the birds from each group were challenged with toc-id of the ibv gx-yl strain in . ml by the nasal-ocular route. they were monitored daily for clinical signs, morbidity and mortality and they were then euthanized at days post-challenge (dpc). any birds that died during the observation period were immediately necropsied. any remaining birds were euthanized at the end of the observation period. the trachea and kidney samples were collected from each bird aseptically for the re-isolation of ibv. these animal experiments were approved by the animal care & welfare committee of guangxi university (approval number gxu - , th november ) and were performed in accordance with animal ethics guidelines and approved protocols. blood samples were collected from the wing vein of birds in each group prior to prime immunization ( day) and at , , and days post-vaccination (dpv) for the detection of the ibv-specific igg antibody using commercial enzyme-linked immunosorbent assay (elisa) kits (idexx laboratory, inc., westbrook, me, usa) according to the manufacturer's recommendations. peripheral blood samples were collected from the wing vein of birds in each group at , , , and dpv. peripheral blood lymphocytes were isolated and adjusted to × cells/ml, stained with µl mouse anti-chicken cd -pe and cd a-fitc antibodies (wuhan amyjet scientific inc., wuhan, china) and analyzed for the percentages of cd + and cd + t lymphocytes by a bd accuritm c flow cytometer (becton, dickinson and company, minneapolis, mn, usa). all birds were euthanized at dpc. trachea and kidney mixtures were collected for virus re-isolation through inoculation into -day-old spf chicken embryos via the allantoic cavity route. allantoic fluid was harvested at h post-inoculation. three passages in embryonated eggs were conducted. viral rna in allantoic fluid was extracted using the easypure rna purification kit (transgen biotech, beijing, china) and subjected to a reverse transcriptase polymerase chain reaction targeting the m gene of ibv. the absence of detectable virus in the trachea and kidney was considered to be correlated with the protection of the subunit vaccines against the challenge. all data were shown as mean values ± standard deviation (mean ± sd) and were compared by a one-way analysis of variance (anova) and student's t-test using spss . (spss inc., chicago, il, usa) biostatistics software. p-values less than . were regarded as significant and those less than . were regarded as highly significant. the results of pcr, restriction enzyme digestion analysis ( figure s ) and sequencing showed that recombinant transposon vectors pfast-hbm-s , pfast-hbm-n and pfast-hbm-s -n were successfully constructed. the recombinant bacmids rhbm-s , rhbm-n and rhbm-s -n were obtained by transforming the verified recombinant transposon vectors to dh bac tm escherichia coli cells and identified using pcr. the results of pcr identification showed that the recombinant bacmids rhbm-s , rhbm-n and rhbm-s -n were generated ( figure s ). the purified recombinant bacmids rhbm-s , rhbm-n and rhbm-s -n were transfected into sf insect cells to obtain the recombinant baculovirus. the recombinant baculovirus rhbm-s , rhbm-n and rhbm-s -n were identified using pcr with ibv specific primers and m primers. the sizes of the amplified bands matched those of the predicted products ( figure s ). strong specific immunofluorescence signals were observed in the sf cells infected with the recombinant baculovirus rhbm-s (figure a) , rhbm-n ( figure b ) andrhbm-s -n ( figure c ) at h post-infection but no fluorescence was detected in the sf cells infected with wild-type baculovirus ( figure d ) and normal sf cells ( figure e ). the expected sizes of ( figure a ) and kda ( figure b ) proteins were detected in the culture supernatant and cell lysate infected with rhbm-s and rhbm-n, respectively. two specific bands of and kda ( figure c ) proteins were detected in both the culture supernatant and cell lysate infected with rhbm-s -n. no bands were observed in the culture supernatant or cell lysate infected with wild-type baculovirus and normal sf cells. the results showed that the sizes of expressed recombinant proteins are consistent with the sizes of native s or n proteins and these recombinant proteins retained their antigenicity. at dpv, ibv-specific antibody levels in birds immunized with subunit vaccines rhbm-s -n, rhbm-s , rhbm-n and inactivated vaccine strain h started to rise (p > . ). the antibody levels of the vaccinated groups increased notably at and dpv (i.e., and dpb) and were significantly higher than those of the negative control groups (p < . ) (figure ) . the highest ibv antibodies in each vaccinated group were observed at dpb. the antibody levels in the rhbm-s -n group were always higher than those in the rhbm-s and rhbm-n groups but the difference was not significant (p > . ). similarly, the antibody titers in rhbm-s groups were always slightly higher than those in the rhbm-n group (p > . ). chickens in the h group developed the at dpv, ibv-specific antibody levels in birds immunized with subunit vaccines rhbm-s -n, rhbm-s , rhbm-n and inactivated vaccine strain h started to rise (p > . ). the antibody levels of the vaccinated groups increased notably at and dpv (i.e., and dpb) and were significantly higher than those of the negative control groups (p < . ) (figure ) . the highest ibv antibodies in each vaccinated group were observed at dpb. the antibody levels in the rhbm-s -n group were always higher than those in the rhbm-s and rhbm-n groups but the difference was not significant (p > . ). similarly, the antibody titers in rhbm-s groups were always slightly higher than those in the rhbm-n group (p > . ). chickens in the h group developed the at dpv, ibv-specific antibody levels in birds immunized with subunit vaccines rhbm-s -n, rhbm-s , rhbm-n and inactivated vaccine strain h started to rise (p > . ). the antibody levels of the vaccinated groups increased notably at and dpv (i.e., and dpb) and were significantly higher than those of the negative control groups (p < . ) (figure ) . the highest ibv antibodies in each vaccinated group were observed at dpb. the antibody levels in the rhbm-s -n group were always higher than those in the rhbm-s and rhbm-n groups but the difference was not significant (p > . ). similarly, the antibody titers in rhbm-s groups were always slightly higher than those in the rhbm-n group (p > . ). chickens in the h group developed the highest antibody titers throughout the experimental period but the difference was not significant (p > . ) compared with those in the rhbm-s -n, rhbm-s and rhbm-n groups. viruses , , x for peer review of highest antibody titers throughout the experimental period but the difference was not significant (p > . ) compared with those in the rhbm-s -n, rhbm-s and rhbm-n groups. the results showed that the percentages of cd + and cd + t lymphocytes in vaccinated birds were rising at dpv ( figure ). at and dpv (i.e., and dpb), the percentages of cd + and cd + t lymphocytes in the rhbm-s -n and rhbm-n groups were significantly higher than those of the negative groups (p < . ). the percentages of cd + t lymphocytes in the rhbm-s group were significantly higher than those of the negative controls (p < . ) and the percentages of cd + t lymphocytes were significantly higher (p < . ) compared with the negative controls. based on percentages of cd + and cd + t lymphocytes in the vaccinated groups at and dpv (i.e., and dpb), the groups sorted from highest to lowest as follows: the rhbm-s -n group, the rhbm-n group, the h group and the rhbm-s group. there was a significant difference between the rhbm-s -n and rhbm-s groups (p < . ), while no significant difference was noted between other groups (p > . ). the results showed that the percentages of cd + and cd + t lymphocytes in vaccinated birds were rising at dpv ( figure ). at and dpv (i.e., and dpb), the percentages of cd + and cd + t lymphocytes in the rhbm-s -n and rhbm-n groups were significantly higher than those of the negative groups (p < . ). the percentages of cd + t lymphocytes in the rhbm-s group were significantly higher than those of the negative controls (p < . ) and the percentages of cd + t lymphocytes were significantly higher (p < . ) compared with the negative controls. based on percentages of cd + and cd + t lymphocytes in the vaccinated groups at and dpv (i.e., and dpb), the groups sorted from highest to lowest as follows: the rhbm-s -n group, the rhbm-n group, the h group and the rhbm-s group. there was a significant difference between the rhbm-s -n and rhbm-s groups (p < . ), while no significant difference was noted between other groups (p > . ). the data are shown as mean values ± standard deviation (mean ± sd) in each group. statistically significant differences are indicated by * (p < . ) or ** (p < . ) (n = chickens/group). at dpc, chickens began to show clinical signs or died. morbidity, mortality, virus re-isolation rate and protection rate at dpc were summarized in table . most of the chickens in the two negative control groups (wt-b and cell groups) showed typical signs of ibv infection, such as coughing, sneezing, gasping, tracheal rale and wet droppings. in these groups, there was a - % morbidity rate and a - % mortality rate. in contrast, %, %, % and % morbidity rates as well as %, %, % and % mortality rates were observed in the rhbm-s -n, rhbm-s , rhbm-n and h groups, respectively. birds in the four vaccinated groups exhibited milder clinical signs and gross lesions compared with those in the two negative control groups. in the necropsy at dpc, the kidneys of infected birds were swollen and pale and they exhibited a white sludge indicating urate deposition. some birds showed trachitis with exudates. in order to further evaluate the level of protection, the collected trachea and kidney from the challenged birds at dpc were inoculated into -day-old spf chicken embryos for three passages for virus re-isolation. viral rna was detected from the infected allantoic fluid of the embryonated eggs using qrt-pcr. the virus re-isolation rates for the birds in the rhbm-s -n, rhbm-s , rhbm-n and h groups were %, %, % and % respectively. those in the wt-b and cell groups had virus re-isolation rates of % and % respectively (table ) . birds in the rhbm-s -n, rhbm-s and rhbm-n groups developed higher levels of protection (i.e., %, % and %, respectively) than those in the wt-b and cell groups (i.e., % and %, respectively) but lower levels of protection than that in the h group ( %). no protection was observed in the cell group and a low level of protection ( %) was observed in the wt-b group. the data are shown as mean values ± standard deviation (mean ± sd) in each group. statistically significant differences are indicated by * (p < . ) or ** (p < . ) (n = chickens/group). at dpc, chickens began to show clinical signs or died. morbidity, mortality, virus re-isolation rate and protection rate at dpc were summarized in table . most of the chickens in the two negative control groups (wt-b and cell groups) showed typical signs of ibv infection, such as coughing, sneezing, gasping, tracheal rale and wet droppings. in these groups, there was a - % morbidity rate and a - % mortality rate. in contrast, %, %, % and % morbidity rates as well as %, %, % and % mortality rates were observed in the rhbm-s -n, rhbm-s , rhbm-n and h groups, respectively. birds in the four vaccinated groups exhibited milder clinical signs and gross lesions compared with those in the two negative control groups. in the necropsy at dpc, the kidneys of infected birds were swollen and pale and they exhibited a white sludge indicating urate deposition. some birds showed trachitis with exudates. in order to further evaluate the level of protection, the collected trachea and kidney from the challenged birds at dpc were inoculated into -day-old spf chicken embryos for three passages for virus re-isolation. viral rna was detected from the infected allantoic fluid of the embryonated eggs using rt-pcr. the virus re-isolation rates for the birds in the rhbm-s -n, rhbm-s , rhbm-n and h groups were %, %, % and % respectively. those in the wt-b and cell groups had virus re-isolation rates of % and % respectively (table ) . birds in the rhbm-s -n, rhbm-s and rhbm-n groups developed higher levels of protection (i.e., %, % and %, respectively) than those in the wt-b and cell groups (i.e., % and %, respectively) but lower levels of protection than that in the h group ( %). no protection was observed in the cell group and a low level of protection ( %) was observed in the wt-b group. rhbm-s -n ( / ) ( / ) ( / ) ( / ) rhbm-s ( ib is a highly contagious and acute viral disease of chickens which causes massive economic losses. live-attenuated and inactivated vaccines are commonly used to control this disease. however, live-attenuated vaccines may lead to the occurrence of new serotypes or variants of ibv due to mutation and recombination [ ] [ ] [ ] . also, inactivated vaccines often fail to induce strong cellular immunity and have the disadvantage of high manufacturing costs [ , ] . hence, there is an urgent need to develop new, safe and effective vaccines to control the disease. recombinant subunit vaccines have been shown to elicit strong humoral and cellular immune responses and are very safe [ ] [ ] [ ] . therefore, subunit vaccines may serve as potential vaccine candidates in the future. in the current study, significantly higher antibody levels, higher percentages of cd + and cd + t lymphocytes and higher protection rates were demonstrated when using the subunit vaccines rhbm-s -n, rhbm-s and rhbm-n compared to the wt-b and cells at and dpb. therefore, the recombinant subunit vaccines rhbm-s -n, rhbm-s and rhbm-n could induce the immunized chickens to produce humoral and cellular immunity to resist and eliminate the ibv infection, indicating that the recombinant bacculovirus vaccines expressing the s and n proteins of ibv may be effective subunit vaccines for future use. the immune efficacy of subunit vaccine rhbm-s containing the s protein was stronger than that of the subunit vaccine rhbm-n containing the n protein in the generation of humoral immune responses. however, the immune efficacy of subunit vaccine rhbm-n was stronger than that of rhbm-s in the generation of cellular immune responses. the results confirmed that the s protein and n protein play a major role in humoral and cellular immune responses, respectively, which is in agreement with the previous investigations [ , , ] . the immune efficacy of subunit vaccine rhbm-s -n was better than that of subunit vaccines rhbm-s or rhbm-n in generating humoral and cellular immune responses. the reason for this was that rhbm-s -n expressed both the s and n proteins of ibv and stimulated both the humoral and cellular immune responses simultaneously. therefore, the subunit vaccine rhbm-s -n performed better in the activation of virus-specific immune responses. although the s subunit is the major inducer of neutralizing antibodies, vaccination with the s protein did not confer adequate protection against challenge [ ] . a previous study even found that the antigenicity of the n protein is better than that of the s protein [ ] . in another study, the cell-mediated immune response induced by the n protein was higher than that induced by either the s protein or h [ ] . these results and ours indicate the importance of generating a subunit vaccine that contains both the s and the n genes. a previous study showed that the bacmam virus ac-cmv-s , which expresses the s glycoprotein of ibv-m , was deficient in the induction of ibv-specific antibody compared with that induced by the inactivated vaccine [ ] . in our study, the antibody levels in the groups rhbm-s -n, rhbm-s and rhbm-n were slightly lower than that of the h group at and dpb, which agreed with the previous study [ ] . a possible reason for this was that the h vaccine was derived from the whole virus and was comprised of almost all the epitopes of ibv. another reason may be related to the fact that the coated antigen of the elisa kit used in the study was the mass-type virus strain. based on the percentages of cd + and cd + t lymphocytes, the groups ordered from highest to lowest are the rhbm-s -n group, rhbm-n group, h group and rhbm-s group. it was surprising that the cellular immune response in the rhbm-n group was higher than that in the h group, although the difference was not found to be statistically significant. a previous study showed that ac-cmv-s induced a significantly better cellular immune response in spf chickens compared with that induced by the inactivated vaccine [ ] , which was consistent with our results. the cell-mediated immune response induced by rhbm-n was higher than that induced by either h or rhbm-s . one possible explanation was a more efficient presentation of n peptides by antigen-presenting cells following effective uptake, processing, or presentation on mhc receptors of these cells [ ] . therefore, the necessity of co-expressing the s and n proteins is again stressed. in the present study, chickens in the h group did not obtain % protection against the ibv gx-yl strain challenge. this was possibly because the strain used as the h vaccine was heterologous to the gx-yl strain which was used as the challenge strain [ ] . this was consistent with previous studies [ ] [ ] [ ] . another reason could be the absence of live priming for the inactivated h vaccine [ , ] . in addition, the protection rates of subunit vaccines rhbm-s -n ( %), rhbm-s ( %) and rhbm-n ( %) were lower than that of the traditional vaccine h ( %). a previous description reported % protection against the virulent m challenge from the constructed baculovirus expressing the s protein of the ibv m strain, which was lower than that for the inactivated vaccine ( %) [ ] . only % protection was obtained against the homologous km strain challenge after three immunizations with the ibv s glycoprotein expressed by a recombinant baculovirus [ ] . a similar result was shown in another study [ ] . it was surprising that the protection rate of the rhbm-s -n vaccine ( %) against the homologous gx-yl strain challenge was lower than that of the h vaccine ( %) against the heterologous gx-yl strain challenge. there are some possible reasons for this. firstly, the rhbm-s -n subunit vaccines contained only part of the whole immunogenicity of the virus. this could not lead to an immune effect that was as good as the whole-virus vaccine h . secondly, the cell lysates were used to prepare the oil emulsion vaccines for the chickens' immunizations and it is unknown whether the complicated composition of the cell lysate had an effect on the immunized birds. thirdly, the immune dose of µg/bird may be not enough. doses of µg/bird were used to immunize chickens in other studies [ , ] . finally, two immunizations may be not enough to elicit an effective immune response. a previous study pointed out that at least four immunizations with purified s glycoprotein were needed to induce protection against the homologous n / strain challenge [ ] . similarly, three immunizations were performed and only % protection was obtained against the homologous ibv in the previous study [ ] . in this study, we only immunized the chickens twice with the recombinant proteins; this may be a reason for the low protection conferred by the subunit vaccines. live attenuated vaccines are applied from day-old to achieve early protection and are boosted with the inactivated vaccines in the case of future layers and breeders. although the subunit vaccine rhbm-s -n could not provide complete protection against ibv infection, it conferred higher protection than rhbm-s and rhbm-n vaccines. thus, it still can be used as an alternative vaccine for boosting the primary vaccination with traditional vaccine/vaccines against this disease. as we know, ibv continuously evolves and there are scores of serotypes. little or no cross-protection confers between different serotypes of ibvs [ , ] . therefore, universal ibv vaccines that protect against varying serotypes of ibv are imperative. our results should enable the generation of multivalent vaccines to prevent more serotypes of ibv, especially the newly emerging virus strains. the vaccine rhbm-s -n can also be applied by combining it with traditional vaccines to reduce the occurrence of variants caused by virus mutations and recombination. therefore, the subunit vaccine rhbm-s -n is an alternative to the traditional ibv vaccine. our study also provides reference and ideas for the development of subsequent new vaccines. further study is needed to determine the effectiveness of combining the subunit vaccines with traditional vaccines. in this study, a % protection rate was conferred following ibv challenge in the wild-type baculoviruses control group and the protection rate of the wild-type baculoviruses control group was slightly higher than that of the cell control group, which was consistent with previous reports [ , ] . a possible reason could be that the baculovirus induced innate immunity through the toll-like receptor and myd -dependent signaling pathway and stimulates the production of various inflammatory cytokines [ ] . therefore, vaccination with baculovirus alone may result in non-specific immunity, which could provide only a small amount of protection against an ibv challenge. it is critical to obtain high-level secreted expressions of recombinant proteins with native activities. hbm is an insect-derived signal peptide. it has been reported that the introduction of the hbm signal peptide could increase the expression of foreign proteins, enhance the activity of expressed proteins and achieve the secretion of foreign proteins in the baculovirus system [ ] [ ] [ ] . there have been a few studies about expressing ibv proteins using bes [ , [ ] [ ] [ ] [ ] but none of these studies have focused on introduction of the hbm signal peptide into bes to express ibv proteins. therefore, the hbm signal peptide was introduced in the present study. the results showed that the recombinant proteins s -n, s and n could be expressed both in the culture supernatant and cell lysate. thus, the introduction of the hbm signal peptide into bes can be applied to express other proteins from other pathogens. in summary, the current study showed that the constructed recombinant baculoviruses could elicit both cellular and humoral immune responses to a certain degree and the protection offered by the recombinant baculovirus co-expressing s and n proteins was better than that of recombinant baculoviruses containing the s or n protein alone. to our knowledge, this is the first report that proved that bivalent subunit vaccines, including s and n proteins of ibv, could induce higher immune responses and provide greater protection against infection. the recombinant baculovirus co-expressing s and n proteins could serve as a potential ibv vaccine and a bivalent subunit vaccine including the s and n proteins might be a strategy for the development of an ibv subunit vaccine. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : identification of recombinant transposon vectors pfast-hbm-s , pfast-hbm-n and pfast-hbm-s -n by pcr and restriction endonuclease digestion. figure s : identification of recombinant bacmids rhbm-s , rhbm-n and rhbm-s -n by pcr with m primers. figure s : identification of recombinant baculovirus by pcr with ibv specific primers and m primers. table s : all the primers used in this study. the authors have no conflict of interest to declare. infectious bronchitis virus variants: a review of the history, current situation and control measures coronavirus avian infectious bronchitis virus efficacy of infectious bronchitis virus vaccinations in the field: association between the alpha-ibv igm response, protection and vaccine application parameters review of infectious bronchitis virus around the world emergence of avian infectious bronchitis virus and its variants need better diagnosis, prevention 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china evaluation of the protection conferred by commercial vaccines and attenuated heterologous isolates in china against the ck/ch/ldl/ i strain of infectious bronchitis coronavirus evaluation of the protection conferred by several avian infectious bronchitis attenuated vaccines against the field strain ck/ch/ldl/ i in china baculovirus induces an innate immune response and confers protection from lethal influenzavirus infection in mice immunogenicity of a recombinant pseudotype baculovirus expressing the s protein of infectious bronchitis virus in specific pathogen free (spf) chickens immunogenicity evaluation of s glycoproteins from infectious bronchitis virus isolates js/ / and sd/ / expressed by recombinant baculoviruses infectious bronchitis virus poly-epitope-based vaccine protects chickens from acute infection the s glycoprotein but not the n or m proteins of avian infectious bronchitis virus induces protection in vaccinated chickens involvement of the toll-like receptor signaling pathway in the induction of innate immunity by baculovirus rat acid phosphatase: overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties andcrystallization expression and characterization of soluble human parainfluenza virus type hemagglutinin-neuraminidase glycoprotein expression of recombinant proteinase , the autoantigen in wegener's granulomatosis, in insect cells key: cord- -e is g authors: klein, steffen; müller, thorsten g.; khalid, dina; sonntag-buck, vera; heuser, anke-mareil; glass, bärbel; meurer, matthias; morales, ivonne; schillak, angelika; freistaedter, andrew; ambiel, ina; winter, sophie l.; zimmermann, liv; naumoska, tamara; bubeck, felix; kirrmaier, daniel; ullrich, stephanie; barreto miranda, isabel; anders, simon; grimm, dirk; schnitzler, paul; knop, michael; kräusslich, hans-georg; dao thi, viet loan; börner, kathleen; chlanda, petr title: sars-cov- rna extraction using magnetic beads for rapid large-scale testing by rt-qpcr and rt-lamp date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: e is g rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus (sars-cov- ). the standard diagnostic pipeline for testing sars-cov- presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral rna is extracted using commercial kits, followed by reverse transcription and quantitative pcr detection. as a result of the large demand for testing, commercial rna extraction kits may be limited and, alternatively, non-commercial protocols are needed. here, we provide a magnetic bead rna extraction protocol that is predominantly based on in-house made reagents and is performed in -well plates supporting large-scale testing. magnetic bead rna extraction was benchmarked against the commercial qiacube extraction platform. comparable viral rna detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (rt-lamp) using a primer set targeting the n gene, as well as rt-qpcr using a primer set targeting the e gene, showing that the rna extraction protocol presented here can be combined with a variety of detection methods at high throughput. importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot. severe acute respiratory syndrome coronavirus (sars-cov- ), the causative agent of coronavirus disease (covid- ) , was first described in the city of wuhan in china in december and spread globally thereafter causing pandemic. to slow its spread, large-scale diagnostics and the enforcement of strict public health measures were implemented in many countries. the current standard test for sars-cov- detection and diagnosis is based on viral rna extraction from a pharyngeal swab followed by highly sensitive reverse transcription and quantitative pcr (rt-qpcr). several primer sets targeting one or more of the sars-cov- genes-nucleocapsid (n), envelope protein (e), s glycoprotein (s), or rna-dependent rna polymerase (rdrp)-have been used [ ] . a two-step testing procedure using primer sets targeting the e gene for initial screening followed by the rdrp gene to confirm positive samples is recommended by the german consiliary laboratory for coronaviruses [ ] . the unprecedented global demand for commercial rna extraction kits and ensuing shortage of these reagents [ ] led to the establishment of several diagnostic workflows performed on patient samples with or without an intermediate rna extraction step [ ] [ ] [ ] [ ] . viral rna isolation from clinical samples depends on the rapid inactivation of viral particles, typically by detergent solubilization, and on the denaturation of omnipresent rnases [ ] . the latter may be accomplished by the use of chaotropic chemicals, such as guanidinium salts [ ] or non-specific proteases that are active on both native and denatured proteins, such as proteinase k. in either case, after virus particle lysis, rna must be purified, since guanidinium salts, proteinase k and organic solvents inhibit the subsequent rt-qpcr step. rna can be separated from proteins either by liquid phase separation using chloroform-aqueous emulsions after lysis with commercially available trizol (a mixture of guanidinium thiocyanate and acid phenol) or by means of solid-phase separation using silica [ ] . nucleic acid-binding to negatively charged silica (sio ) is facilitated by guanidinium salts and the basic ph of the lysis buffer [ ] . to achieve a high nucleic acid binding capacity, silica-based nucleic acid extraction methods use either porous silica matrices that are embedded in a column (spin column) [ ] , a tip (trutips) [ ] , or a suspension of microparticles. microparticles can be separated from the lysate either by centrifugation or by a magnetic field provided that the microparticles' dense iron-containing cores are coated with porous silica [ ] . the protocol established in this study aimed at extracting sars-cov- rna from respiratory patients' swabs (oropharyngeal and nasopharyngeal) and is based on the magnetic bead-based nucleic acid extraction protocol that was published by he et al. [ ] and on the protocol from chemicell gmbh, who provided the simag-n-dna magnetic beads. we opted for silica magnetic beads because of their relatively easy manufacturing and sustainable availability, and because specialized plasticware (spin columns, modified tips) is not required to perform their separation. magnetic bead rna extraction was performed in -well plates in combination with a magnet plate optimized for deep-well plates. the portable manual pipetting system liquidator , which is less expensive than automated pipetting robots, was used to minimize the pipetting and handling errors ( figure a ). here, we show that the magnetic bead-based protocol yields rna extracts comparable to the commercially available qiacube viral rna extraction kit, as determined by the commonly applied detection methods rt-qpcr and reverse transcription loop-mediated isothermal amplification (rt-lamp) [ ] . a selection of upper respiratory tract specimens (flocked swabs in amies medium, eswab copan, brescia, italy) sent to the diagnostics laboratory of the heidelberg university hospital between april and may for sars-cov- pcr were used for the study. surplus material from a total of swab samples were collected from sars-cov- positive and negative patients, which were used for sars-cov- testing by rt-qpcr. the swab samples were either used at the day of collection (two positive, negative) or were frozen, stored at − °c, and thawed just before the rna extraction ( positive, negative). positive and negative samples were further diluted and used in replica to generate positive and negative samples in order to facilitate replicate testing. the following steps were performed in a biosafety level (bsl- ) laboratory according to standard microbiological and diagnostic practices. to extract sars-cov- rna from pharyngeal swabs, a lysis buffer containing m guanidinium thiocyanate, mm dithiothreitol, µ g/ml glycogen, % triton x- , and buffered with mm sodium citrate to ph was used. internal control (ic) for rt-qpcr ( µl/sample) (tib-molbiol, berlin, germany) was added into the lysis buffer just before use. µ l lysis buffer and µ l sample were vigorously vortexed in a . ml eppendorf tube for sec and incubated for min at room temperature inside a bsl- laminar flow cabinet to ensure both rapid virus deactivation and rnase denaturation. lysates ( µl) were transferred into a deep-well plate (greiner ag, kremsmünster, austria) with a maximum working volume of µ l per well, which is compatible with the liquidator pipetting system. just before the rna extraction, simag-n-dna magnetic beads (chemicell, berlin, germany) were washed three times in rnase-free water. the aqueous magnetic bead stock solution of µ g/µ l a selection of upper respiratory tract specimens (flocked swabs in amies medium, eswab copan, brescia, italy) sent to the diagnostics laboratory of the heidelberg university hospital between april and may for sars-cov- pcr were used for the study. surplus material from a total of swab samples were collected from sars-cov- positive and negative patients, which were used for sars-cov- testing by rt-qpcr. the swab samples were either used at the day of collection (two positive, negative) or were frozen, stored at − • c, and thawed just before the rna extraction ( positive, negative). positive and negative samples were further diluted and used in replica to generate positive and negative samples in order to facilitate replicate testing. the following steps were performed in a biosafety level (bsl- ) laboratory according to standard microbiological and diagnostic practices. to extract sars-cov- rna from pharyngeal swabs, a lysis buffer containing m guanidinium thiocyanate, mm dithiothreitol, µg/ml glycogen, % triton x- , and buffered with mm sodium citrate to ph was used. internal control (ic) for rt-qpcr ( µl/sample) (tib-molbiol, berlin, germany) was added into the lysis buffer just before use. µl lysis buffer and µl sample were vigorously vortexed in a . ml eppendorf tube for sec and incubated for min at room temperature inside a bsl- laminar flow cabinet to ensure both rapid virus deactivation and rnase denaturation. lysates ( µl) were transferred into a deep-well plate (greiner ag, kremsmünster, austria) with a maximum working volume of µl per well, which is compatible with the liquidator pipetting system. just before the rna extraction, simag-n-dna magnetic beads (chemicell, berlin, germany) were washed three times in rnase-free water. the aqueous magnetic bead stock solution of µg/µl was added into absolute ethanol in order to obtain a working solution of µg/µl. using a multichannel pipette, µl of magnetic bead solution was transferred into the deep-well plate containing the pharyngeal sample lysates. to facilitate the adsorption of the nucleic acid onto the magnetic beads, the -deep-well plate was placed on an orbital shaker ms (ika, staufen, germany) for min at - rpm, before the mixture was resuspended ( ×) using a liquidator , model µl (mettler toledo, columbus, oh, usa) ( figure a ,b) and placed again on the orbital shaker for additional min. subsequently, the plate was placed on a magnet plate for -deep-well plates (magtivio, nuth, the netherlands) ( figure c ) for min to allow the magnetic beads to form rings on the bottom of the wells ( figure d ,e). the -well plate was visually inspected to ensure that all of the pellets were formed. the clear supernatant was discarded using the liquidator and magnetic beads were washed three times with µl % ethanol. for each washing step, the -well plate was removed from the magnet and pellets were resuspended ( ×) using a liquidator . the plate was placed back on the magnet for min until the beads formed visible rings. after three washing steps, the magnetic beads were briefly rinsed with µl rnase-free water in order to remove any residual ethanol while the plate was kept on the magnet. the -well plate was visually inspected to ensure that none of the pellets was removed. finally, nucleic acids adsorbed onto the surface of the magnetic beads were eluted: µl rnase-free water was added to each well, the -well plate was removed from the magnet, resuspended ( ×), and vortexed for - min. the -well plate was placed back on the magnet until rings of magnetic beads were formed and µl eluate was transferred to a new -well pcr plate. a detailed step-by-step procedure and a complete list of all materials and instruments used for this magnetic bead rna extraction protocol can be found in the supplementary materials. the qiaamp viral rna body fluid kit was carried out with manual lysis according to the manufacturer's protocol to compare the performance of the magnetic bead rna extraction (qiagen, hilden, germany). the sample input volume was µl, the volume of ic per sample was µl, and the elution volume was set to µl. for rt-qpcr detection of the sars-cov- envelope protein gene (e gene), we adopted a widely used protocol based on corman et al. [ ] . for the mastermix, . µl of primer/probe lightmix ® modular sars and wuhan cov e-gene (tib-molbiol, berlin, germany) and . µl of lightmix ® modular eav rna extraction control (tib-molbiol, berlin, germany) was mixed with . µl rnase-free water, µl lightcycler ® multiplex rna virus master (roche, basel, switzerland), and . µl reverse transcriptase enzyme (supplied with lightcycler ® multiplex rna virus master kit, berlin, germany) per sample. the following primers and probe were used (provided by tib-molbiol, germany): fwd -acaggtacgttaatagttaatagcgt- , rev -atattgcagcagtacgcacaca- , probe fam-acactagccatccttactgcgcttcg-bbq; these primers are specific for a bp amplicon from position - of the sars-cov- genome (genbank nc_ ). µl of mastermix was distributed into a -well pcr plate and µl of purified rna from patient samples was added using the liquidator , model µl. rt-qpcr was performed using a lightcycler ® instrument ii (roche, basel, switzerland) with min of reverse transcription at • c, initial denaturation at • c for min, and subsequent amplification cycles with • c for sec, • c for sec, • c for sec, and finally cooling to • c for sec. cycle threshold (ct) was determined, where the fluorescence signal of the amplification reaction was above the background fluorescence using the lightcycler software (roche). data analysis on raw cts was performed in excel and graphpad prism (graphpad software, san diego, ca, usa), and % "exact" clopper-pearson confidence intervals were calculated using medcalc (medcalc software, ostend, belgium). five µl diluted ms rna (roche, basel, switzerland) containing . × to . × molecules was spiked into µl lysis buffer before a µl patient sample was added. magnetic bead rna extraction was performed as described in section . . rt-qpcr for ms was performed with a one-step rt-qpcr reaction (tib-molbiol, berlin, germany) using the following primers and probe: fwd -gagtgtttacagttccgaa- , rev -cccctttctggaggtacatattcata- , and probe cy -aatagatcgggctgcctgtaaggagc-bbq. µl of rna was used per rt-qpcr, covering a range from to ms rna molecules per reaction. rt-qpcr cycling program for ms rna amplification was performed as described in section . . rt-lamp detection of the sars-cov- n gene was based on the protocol by dao thi et al. [ ] . the rt-lamp primer set used in this study was targeted against the sars-cov- n gene [ ] . the sequences and concentrations of all oligonucleotides in the x primer mix used for the rt-lamp assays can be found in our recent publication [ ] . for the colorimetric lamp assay, the master mix consisted of . µl warmstart ® colorimetric lamp x master mix m (new england biolabs, ipswich, ma, usa) and . µl × lamp primer mix targeting the n gene [ ] per reaction. immediately afterwards, . µl of the freshly prepared reaction mix was distributed into -well plates and µl of purified rna was added using a liquidator , model µl. the plate was sealed with a transparent adhesive foil and subsequently incubated at • c for min in a -well-pcr block with heated lid ( • c). absorbance was measured at nm and nm wavelengths in a spark ® cyto or infinite m plate reader (tecan, männedorf, switzerland). phenol red absorbance spectra change in response to the acidification of the reaction (the absorbance at nm wavelength is increased and the absorbance at nm wavelength is decreased). to measure ph changes during the reaction, the difference between optical densities was calculated (∆od = od nm − od nm ) and samples with ∆od < . were classified as negative. for the fluorescent lamp assay, the same primer set as described in . was used. the master mix consisted of . µl warmstart ® lamp kit x master mix e (new england biolabs, ipswich, ma, usa), . µl × lamp primer mix targeting the n gene [ ] and . µl of × fluorescent dye (syto- , supplied with the rt-lamp kit) per reaction. . µl of the mix was distributed in a -well plate and . µl purified rna was added before the plate was sealed and incubated at a constant temperature of • c using a lightcycler ® instrument ii (roche, basel, switzerland). real-time fluorescence was detected with intervals of min for a duration of min. time of amplification (toa) was determined based on a fluorescence threshold, where the fluorescence signal of the amplification reaction was above the background fluorescence, using the lightcycler software. samples with toa > min were classified as negative. the presented work was done with the intention to support sars-cov- diagnostics and improve emergency preparedness and response to covid- . pseudo-anonymized surplus material from samples that had been collected for sars-cov- testing were used to establish and validate the protocol presented here. this work complies with the german act concerning the ethical review of research involving humans, permitting use of patient samples collected to perform the testing in question, for development and improvement of diagnostic assays. the magnetic bead rna extraction protocol was established in a -well plate format as part of the detection workflow ( figure ). the complete workflow from rna extraction to rna detection can be conducted in less than - h. we first applied both protocols on one sars-cov- positive pharyngeal swab sample, which was diluted in rnase-free water prior to rna isolation in a -fold dilution series up to fold, in order to compare the magnetic bead rna extraction protocol with the commercial qiacube extraction method. the extracted rna was subjected to rt-qpcr using e gene primers. viral rna was detected in samples diluted up to fold after either qiacube or magnetic bead rna extraction ( figure we first applied both protocols on one sars-cov- positive pharyngeal swab sample, which was diluted in rnase-free water prior to rna isolation in a -fold dilution series up to fold, in order to compare the magnetic bead rna extraction protocol with the commercial qiacube extraction method. the extracted rna was subjected to rt-qpcr using e gene primers. viral rna was detected in samples diluted up to fold after either qiacube or magnetic bead rna extraction ( figure a we first applied both protocols on one sars-cov- positive pharyngeal swab sample, which was diluted in rnase-free water prior to rna isolation in a -fold dilution series up to fold, in order to compare the magnetic bead rna extraction protocol with the commercial qiacube extraction method. the extracted rna was subjected to rt-qpcr using e gene primers. viral rna was detected in samples diluted up to fold after either qiacube or magnetic bead rna extraction ( figure a ,c). rt-qpcr of the extracted rna by either of the two methods showed approximately equidistant amplification curves with an interval of ct values for each -fold dilution step ( figure b ,d). a dilution series of rna from ms bacteriophage was added to the swab sample prior to magnetic bead rna extraction and subjected to rt-qpcr using ms primers to further evaluate linearity of the magnetic bead rna extraction method across a broad range of defined rna inputs ( figure e,f) . ct values were linear over five orders of magnitude with a goodness of fit of r = . . the lower limit of detection (~ct ) was less than rna copies/reaction ( figure e ). in order to evaluate the magnetic bead rna extraction protocol on larger sample sets, sars-cov- positive and negative samples were generated from sars-cov- positive patients and from persons negative for sars-cov- , respectively. the samples were subjected to three independent magnetic bead rna extractions as well as to qiacube extractions. ct values of positive samples obtained by rt-qpcr using primers for the e gene and ic values from either magnetic bead or qiacube extraction were compared. in the remaining six samples, ct values for the e gene were not detected after magnetic bead rna purification but detected ct values for ic confirmed successful rna extraction. noteworthy, all six samples were previously frozen and had a ct value higher than as determined by rt-qpcr after the qiacube rna extraction. the e gene ct values that were obtained from the magnetic bead and qiacube rna extraction were in good agreement as shown by the slope of the linear regression of . ( figure a ). the average ic ct of . (sd = . , n = ) after magnetic bead extraction was the same as compared to the average ic ct of . from the qiacube rna extractions (sd = . , n = ) ( figure b,c) , showing that the magnetic bead rna extraction protocol is robust. these results also illustrate that quickly washing the beads with water to remove residual ethanol (which could inhibit pcr) does not lead to a substantial loss of rna. the sd of ct values for ic should be calculated to determine false negatives due to rna loss and judge the quality of the rna extraction. the sd of the ct values for ic should be smaller than ct values and samples with ic ct values with × sd lower ic ct values than the average ic ct should be repeated. because assays performed in a -well plate format can be prone to cross-contaminations, we evaluated the sensitivity and specificity of the three independent magnetic bead rna extractions. the samples were distributed in small groups of positive and negative samples on the -well plate. after magnetic beads rna extraction and rt-qpcr or rt-lamp analysis the results were compared to the results of the rt-qpcr after qiacube extraction to determine true and false positives (tp and fp) and true and false negatives (tn and fn) . for the magnetic beads extracted samples, a ct cutoff of was used to determine if a sample is defined as positive. the sensitivity was calculated (tp/(tp+fn)) for subsets of samples in specific ct ranges ( - , - , - , - ) ( figure d ). for all subsets up to ct , no false negatives were detected, which leads to a sensitivity of % with a % confidence interval (ci) of - %. in the range between ct - , six false negatives were counted leading to a sensitivity of % (ci = - %). these results indicate that false negatives were samples with high ct values and the negative results might be due to fluctuation of the viral load around the limit of detection. in total, three false positives were detected, resulting in a specificity of % (ci = - %) ( figure e ). to determine sensitivity and specificity, qiacube rt-qpcr results were used as a reference. rt-qpcr after magnetic bead rna extraction provides close to % sensitivity and specificity at a cutoff ct of . data are shown as mean with indicated % clopper-pearson confidence intervals (supplementary table s ). we also recently explored the rt-lamp assay as a valid alternative rna detection method due to shortages in rt-qpcr reagents [ ] . we compared the standard pipeline qiacube rna extraction followed by rt-qpcr detection of the e gene with magnetic rna extraction followed by rt-lamp detection using primer sets targeting the n gene, with either colorimetric (figure a -c, supplementary table s ) or fluorescent ( figure d-f, supplementary table s ) read-out. in total, we tested rna extracted from positive and negative swab samples. to determine sensitivity and specificity, qiacube rt-qpcr results were used as a reference. rt-qpcr after magnetic bead rna extraction provides close to % sensitivity and specificity at a cutoff ct of . data are shown as mean with indicated % clopper-pearson confidence intervals (supplementary table s ). we also recently explored the rt-lamp assay as a valid alternative rna detection method due to shortages in rt-qpcr reagents [ ] . we compared the standard pipeline qiacube rna extraction followed by rt-qpcr detection of the e gene with magnetic rna extraction followed by rt-lamp detection using primer sets targeting the n gene, with either colorimetric (figure a -c, supplementary table s ) or fluorescent ( figure d-f, supplementary table s ) read-out. in total, we tested rna extracted from positive and negative swab samples. during dna synthesis, the formation of a phosphodiester bond results in the release of a molecule of pyrophosphate and a proton causing a gradual acidification of the reaction mix. the detection principle of colorimetric rt-lamp is based on monitoring ph changes during the dna amplification, which only occurs in positive samples. typically, phenol red, which changes color from red to yellow when the ph is lowered, is used as a ph indicator [ ] . the color change can be determined by measuring the difference between the wavelengths of the two absorbance maxima of phenol red. the majority of isolated rna samples with a ct ≈ value obtained from rt-qpcr using the e gene yielded a color change with ∆od values between . and . as shown in figure a . therefore, we used a ∆od value of . as a threshold for positive samples. the majority of samples with higher ct values, especially between ct and ct , scored negative (∆od < . ), while all negative rt-qpcr samples also scored negative in the rt-lamp assay. using a ct cutoff of , the overall sensitivity of the colorimetric assay with magnetic bead-isolated rna was % ( % ci = - %) ( figure b) , with a specificity of % ( % ci = - %) ( figure c ). table s ). during dna synthesis, the formation of a phosphodiester bond results in the release of a molecule of pyrophosphate and a proton causing a gradual acidification of the reaction mix. the detection principle of colorimetric rt-lamp is based on monitoring ph changes during the dna amplification, which only occurs in positive samples. typically, phenol red, which changes color from red to yellow when the ph is lowered, is used as a ph indicator [ ] . the color change can be determined by measuring the difference between the wavelengths of the two absorbance maxima of phenol red. the majority of isolated rna samples with a ct ≈ value obtained from rt-qpcr using the e gene yielded a color change with Δod values between . and . as shown in figure a . therefore, we used a Δod value of . as a threshold for positive samples. the majority of samples with higher ct values, especially between ct and ct , scored negative (Δod < . ), while all negative rt-qpcr samples also scored negative in the rt-lamp assay. using a ct cutoff of , the overall sensitivity of the colorimetric assay with magnetic bead-isolated rna was % ( % ci = - %) ( figure b) , with a specificity of % ( % ci = - %) ( figure c ). the fluorescent rt-lamp assay is based on a fluorescent dye that intercalates into amplifying dna strands, allowing for their detection in real time. magnetic bead isolated samples with ct ≈ table s ). the fluorescent rt-lamp assay is based on a fluorescent dye that intercalates into amplifying dna strands, allowing for their detection in real time. magnetic bead isolated samples with ct ≈ led to a fluorescent signal at early time points (approx. - min) as compared to negative samples (> min). therefore, we used the time point min as a threshold for positive samples. similar to the colorimetric read-out, the majority of samples with higher ct values, especially between ct and ct , scored negative (> min) ( figure d ). all negative rt-qpcr samples also scored negative (> min). using a ct cutoff of , the overall sensitivity of the fluorescent assay was % ( % ci = - %) ( figure e) , with a specificity of % ( % ci = - %) ( figure f ). these results are well in agreement with the recently reported sensitivity cutoff at ct for the rt-lamp assay [ ] and, thus, confirm that rna purified by the presented magnetic bead extraction is compatible with rt-lamp detection without lowering its sensitivity. within the last decades, the frequency of emerging virus outbreaks has increased globally [ , ] , possibly as a result of cumulative anthropogenic environmental changes that increase the risk of zoonotic transmission [ ] . due to globalization, many of the outbreaks have increased pandemic potential and pose a burden on society and health systems. the currently ongoing sars-cov- pandemic emphasizes the urgency of appropriate preparedness and response. before a therapeutic or vaccine exists, the early identification and isolation of positive patients remains the most effective way to inhibit further human-to-human spread and mitigate the disease outbreak. in the case of a respiratory viral disease, such as influenza or covid- , pharyngeal swabs are collected and tested for the presence of viral rna. rna isolation prior to detection is a pivotal step to ensure high specificity and sensitivity of detection methods. to this end, robust and high-throughput nucleic acid isolation methods with high manufacturing and distribution capacity must be available. however, the majority of commercially available rna purification kits are cost-ineffective and often rely on multiple components that are not easily replaceable, and their supply can often not be guaranteed. in addition, buffer compositions are not provided. thus, overall, commercial kits do not offer enough flexibility and availability when it comes to a large epidemic or pandemic. here, we provide a magnetic bead-based rna extraction protocol that is, to a large extent, producer independent, does not rely on unique components that are difficult to replace, and is scalable for mass testing. because of the large surface binding capacity and rapid separation in solution by magnetic field, silica coated magnetic beads are compatible with a variety of plasticware and they can be used in a high throughput multiwell format [ ] as well as in small scale testing [ ] . in addition, magnetic beads can be prepared from widely available chemicals in a laboratory with basic equipment [ ] . recently, a magnetic bead rna purification protocol has been established for automated, high throughput sars-cov- diagnostics by the covid- crick consortia [ ] . however, a sophisticated infrastructure is required to carry out the protocol, and the protocol is partially dependent on commercial buffers. our aim was to provide a robust protocol that can be rapidly implemented and carried out in most of the laboratories around the world equipped with a magnetic plate, a multichannel pipette, or with a manual pipetting device. we established a magnetic bead rna isolation protocol that takes approximately min using a single -well plate as part of a workflow for sars-cov- diagnostic. although it is feasible to perform four extractions per day for one person, the rate-limiting and the most labor-intensive step of the presented workflow is the sample transfer from swab tubes to -well plates. special care should be taken regarding sample handling prior to rna isolation, and the samples should be kept at • c and processed at the day of collection. a single freeze-thawing cycle of a sars-cov- positive swab sample that was × diluted results in decreased sensitivity of rt-qpcr by approximately ct values (ct frozen -ct fresh = . ; sd = . , n = ). in contrast to other magnetic bead based nucleic acid purification protocols [ , ] that rely on an air-drying step to remove residual ethanol before elution, we rinse magnetic beads with a small amount of rnase-free water. the introduction of this step made our protocol more robust, since air-drying in a -well plate is slow and uneven. furthermore, air-drying did not lead to complete removal of ethanol, which interfered with the subsequent rt-qpcr even at low concentrations. we report a similar rna extraction yield when compared to the commercial qiacube extraction kit and validate that the quality of the rna extract is suitable for rt-qpcr and rt-lamp detection. rt-lamp relies on a different dna polymerase than rt-qpcr and, thus, provides a supplier-independent alternative to rt-qpcr. additionally, rt-lamp is faster and cheaper when compared to rt-qpcr and it does not require a thermocycler [ ] . as we have recently reported [ ] , rt-lamp to detect sars-cov- rna has a decreased sensitivity compared to rt-qpcr: rt-lamp with the primer set against the n gene as used in this study failed to detect most of the samples with a ct value over measured by rt-qpcr using the e-sarbeco primers. efforts are underway to increase the sensitivity of the rt-lamp assay, but this is out of the scope of this work. here, we showed that the presented magnetic bead extraction is compatible with rt-lamp and does not lower its sensitivity when compared to a qiacube purification method performed in our previous study [ ] . either rt-qpcr or rt-lamp can be used as an independent detection method and both methods do not have to be run in parallel. due to higher sensitivity, we recommend using rt-qpcr as a detection method after magnetic bead rna extraction. if resources are limited or large-scale testing is needed, rt-lamp offers an alternative detection approach; however, its lower sensitivity must be considered for now. although it is possible to perform rt-qpcr [ , ] and rt-lamp on unpurified patient samples [ , ] , the combination with our magnetic bead rna extraction protocols increases the sensitivity. due to the low cost, high-throughput compatibility, and independence from sophisticated laboratory equipment such as automated rna extraction kits and rt-qpcr machines, the combination of magnetic bead rna extraction and rt-lamp offers a framework for diagnostic preparedness in the case of mass-scale testing. in conclusion, we report a detailed step-by-step rna extraction protocol from pharyngeal swabs, which is based on magnetic beads and does not depend on commercial extraction kits and reagents. our protocol was validated in a -well plate format by the detection of sars-cov- rna by rt-qpcr and rt-lamp and provides reliable rna extraction for a diagnostic pipeline that can be rapidly deployed during the sars-cov- pandemic and is also accessible to regions with insufficient laboratory capacities. this might be of importance in case of possible future shortages of commercial rna extraction kits and enables diagnostic laboratories to be well prepared in case of a new rise of sars-cov- cases. our protocol can be easily adapted to fully automated liquid handling robotic systems and is very likely suitable for isolation and downstream detection assays for any kind of rna virus isolated from pharyngeal swabs. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , list of materials and instruments for magnetic beads extraction, detailed step-by-step procedure for rna extraction using magnetic beads, supplementary table s : specificity and sensitivity of qpcr as a pool of independent magnetic bead rna extractions, supplementary analytical sensitivity and efficiency comparisons of sars-cov- rt-qpcr primer-probe sets detection of novel coronavirus ( -ncov) by real-time rt-pcr. eurosurveillance rna extraction kits for covid- tests are in short supply in us. the scientist. available online: /www.the-scientist.com/news-opinion/rna-extraction-kits-for-covid- -tests-are-in-short-supplyin-us- the crick covid- consortium scalable and robust sars-cov- testing in an academic center fast sars-cov- detection protocol based on rna precipitation and rt-qpcr in nasopharyngeal swab samples a simple magnetic nanoparticles-based viral rna extraction method for efficient detection of sars-cov- a colorimetric rt-lamp assay and lamp-sequencing for detecting sars-cov- rna in clinical samples a modular method for the extraction of dna and rna, and the separation of dna pools from diverse environmental sample types the single-step method of rna isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on rapid and simple method for purification of nucleic acids multiphasic dna adsorption to silica surfaces under varying buffer, ph, and ionic strength conditions dna purification on homemade silica spin-columns high-throughput, automated extraction of dna and rna from clinical samples using trutip technology on common liquid handling robots preparation of iron oxide silica particles for zika viral rna extraction integrated dna and rna extraction using magnetic beads from viral pathogens causing acute respiratory infections loop-mediated isothermal amplification of dna rapid molecular detection of sars-cov- (covid- ) virus rna using colorimetric lamp visual detection of isothermal nucleic acid amplification using ph-sensitive dyes global trends in emerging infectious diseases global rise in human infectious disease outbreaks the consequences of human actions on risks for infectious diseases: a review simple synthesis of functionalized paramagnetic beads for nucleic acid purification and manipulation rapid direct nucleic acid amplification test without rna extraction for sars-cov- using a portable pcr thermocycler massive and rapid covid- testing is feasible by extraction-free sars-cov- rt-qpcr sars-cov- detection using an isothermal amplification reaction and a rapid, inexpensive protocol for sample inactivation and purification this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank all diagnostics employees for their technical support and advice. we thank jan-philipp mallm, karsten rippe and vladimir benes for advice on magnetic bead nucleic acid purification and for lending us their instruments. we would also like to acknowledge susanne horner und fabian finger for it support. we would like to thank different institutes on heidelberg campus for providing devices and their employees for support, namely in alphabetical order: johannes backs, nina beil, marco binder, christine engeland, holger erfle, stefan fröhling, richard harbottle, joshua hartmann, christel herold-mende, angret joester, dominik niopek, simon john ogrodnik, katrin pfütze, steffi sandke, claudia scholl, rolf warta, ellen wiedtke. the authors declare no conflict of interest.viruses , , key: cord- -cik ino authors: corder, brigette n.; bullard, brianna l.; poland, gregory a.; weaver, eric a. title: a decade in review: a systematic review of universal influenza vaccines in clinical trials during the decade date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: cik ino on average, there are – million severe cases of influenza virus infections globally each year. seasonal influenza vaccines provide limited protection against divergent influenza strains. therefore, the development of a universal influenza vaccine is a top priority for the nih. here, we report a comprehensive summary of all universal influenza vaccines that were tested in clinical trials during the – decade. of the studies found, eligible clinical trials, which investigated vaccines, were included in this review. information from each trial was compiled for vaccine target, vaccine platform, adjuvant inclusion, clinical trial phase, and results. as we look forward, there are currently three vaccines in phase iii clinical trials which could provide significant improvement over seasonal influenza vaccines. this systematic review of universal influenza vaccine clinical trials during the – decade provides an update on the progress towards an improved influenza vaccine. globally, seasonal influenza virus epidemics are estimated to cause - million cases of severe infection and result in , - , deaths annually [ , ] . mortality is increased in the elderly over years, children under years, and people in developing countries [ , ] . in the united states alone, influenza virus infects between . - . million people each year, leading to , - , hospitalizations [ ] . these annual influenza epidemics result in an estimated total economic loss of $ . billion each year due to direct medical costs and indirect costs such as projected lost earnings and loss of life [ ] . while the disease burden for seasonal influenza epidemics is substantial, this is significantly increased during influenza pandemics. for example, it is estimated that % of the worldwide population was infected during the h n swine influenza pandemic [ ] . a substantial challenge in the development of an effective influenza vaccine is the significant viral population diversity. the current influenza vaccine can be either trivalent or quadrivalent. the trivalent vaccine contains a h n , h n , and an influenza b strain, with the quadrivalent vaccine including both yamagata and victoria influenza b lineage strains [ , ] . the strains contained in the seasonal influenza vaccine are updated yearly to include those predicted to circulate in the upcoming influenza season. although the current influenza vaccine is effective at reducing morbidity and mortality due to seasonal influenza infections [ ] , vaccine effectiveness estimates only range from to % [ , ] . the vaccine effectiveness is lowest when there is poor antigenic match to the circulating influenza strains [ , ] . relevant information was extracted from the clinicaltrials.gov database including phase, vaccine target, vaccine platform, and results. if information was not available, relevant publications were analyzed. a summary of this data is reported in table . all data were analyzed using graphpad prism . software. figures were designed in adobe illustrator ( . . ). seasonal influenza vaccines provide limited protection and are updated annually to incorporate circulating strains. a vaccine which induces broad cross-protection against influenza remains a top priority for the national institute of health (nih). here we report a comprehensive review of universal influenza vaccine (uiv) clinical trials that were active between january and december . in the last decade, clinical trials investigating vaccines were performed ( figure ) . these trials include a variety of viral targets, vaccine platforms, and adjuvants to boost the immune response to vaccination. table reports a chronological summary for each uiv clinical trial. the unique id for each trial is used for identification in subsequent figures. importantly, several uivs were tested in up to clinical trials. since the trends may be skewed by these vaccines, we differentiate between vaccines and total clinical trials throughout this paper. influenza vaccines typically target specific viral antigens to maximize the immune response to vaccination. vaccination aims to induce a strong adaptive immune response which results in both t and b cell activation. these immune cells produce cytotoxic t cells and antibodies which can protect against future infection. vaccines targeting internal viral proteins, such as nucleoprotein (np) and matrix (m ), can induce strong t cell responses [ ] . viral surface (external) antigens, hemagglutinin (ha) and neuraminidase (na), are targeted by neutralizing antibodies [ ] . traditionally, a robust antibody response has been the goal of influenza vaccination and has been the basis upon which vaccines have been tested and licensed [ , ] . however, these antibodies provide limited protection against divergent influenza strains. since there are strengths for both internal and external strategies, many vaccines include multiple antigens to induce a strong humoral and cellular immune response. over the past decade, both internal and external influenza proteins were utilized in uiv clinical trials ( figure ). other strategies which target whole virus or attenuated virus through gene deletion have also been investigated. however, recent vaccines have focused on external proteins, specifically ha. internal influenza proteins are attractive vaccine candidates since they are more conserved than the external glycoproteins [ ] . this may result in broader cross-protection induced by the vaccine. one example is fp- . , a peptide-based vaccine which includes several cd + and cd + t-cell epitopes conjugated to a fluorocarbon chain. these epitopes are derived from internal influenza proteins including np, m , polymerase basic (pb ), and polymerase basic (pb ). four trials were performed in the past decade utilizing participants - years old. one phase i trial demonstrated that vaccination with fp- . induced strong cellular responses in % of participants with a median response of spot forming cells (sfc)/million peripheral blood mononuclear cells (pbmc) as measured using ifnγ elispot assay [ ] . this cellular response was activated against several heterologous h n and h n strains indicating broad cross-reactivity [ ] . another uiv targeting internal proteins is ovx , which is a recombinant np vaccine. in preclinical trials, vaccinated mice were protected against three lethal influenza a virus (iav) challenges and induced stronger immunogenicity than wild-type np alone [ ] . protection was further improved if mice were immunized with a combination of the seasonal inactivated vaccine and ovx . in the past decade, of the vaccines in uiv clinical trials targeted external glycoproteins. although the ha protein has a high amount of diversity in the globular head, the ha stalk region is more conserved [ ] . vaccines targeting the stalk region of ha have shown promise during development and are being investigated in several uiv clinical trials [ , , , , ] . one such vaccine is ch / n , h / n , which utilizes a prime-boost immunization strategy to promote an viruses , , of immune response towards the ha stalk domain. the phase trial included individuals between and years of age. two viruses were modified with chimeric ha containing a homologous ha stalk and heterologous ha heads. these were administered as live attenuated or inactivated vaccines and boosted with a heterologous ha head vaccine days later [ ] . an oil-in-water adjuvant, as , was included with the inactivated vaccine. after the prime vaccination, only the adjuvanted groups induced strong igg antibody responses. however, all groups demonstrated . to . -fold increases in ha stalk specific igg antibodies after a heterologous boost. these h ha antibodies were cross reactive to h , h , and h ha, indicating broad cross-protection against group ha [ ]. influenza vaccines typically target specific viral antigens to maximize the immune response to vaccination. vaccination aims to induce a strong adaptive immune response which results in both t and b cell activation. these immune cells produce cytotoxic t cells and antibodies which can protect against future infection. vaccines targeting internal viral proteins, such as nucleoprotein (np) and matrix (m ), can induce strong t cell responses [ ] . viral surface (external) antigens, hemagglutinin (ha) and neuraminidase (na), are targeted by neutralizing antibodies [ ] . traditionally, a robust antibody response has been the goal of influenza vaccination and has been the basis upon which vaccines have been tested and licensed [ , ] . however, these antibodies provide limited protection against divergent influenza strains. since there are strengths for both internal and external strategies, many vaccines include multiple antigens to induce a strong humoral and cellular immune response. over the past decade, both internal and external influenza proteins were utilized in uiv clinical trials ( figure ). other strategies which target whole virus or attenuated virus through gene deletion have also been investigated. however, recent vaccines have focused on external proteins, specifically ha. another vaccine utilized the full-length h ha protein in an oral recombinant adenovirus type (ad ) vectored vaccine, ad -h -vtn. three clinical trials have enrolled participants between and years of age to investigate this avian h influenza vaccine. three immunizations with ad -h -vtn resulted in low seroconversion, % for vaccinees and % for placebo [ ] . participants were boosted with an inactivated h n vaccine, which resulted in % seroconversion for vaccinees compared to % in the placebo group. vaccination with ad -h -vtn induced a significant t cell response after a single vaccination with a median sfc/million pbmc. no serious adverse events were reported although vaccinees experienced higher rates of self-limited abdominal pain ( . % vs. . %), diarrhea ( . % vs. . %), and nasal congestion ( . % vs. . %) compared to the placebo. na was included in a dna vaccine, vgx- x [ ] . the dna vaccine included plasmids containing na, ha, and m e-np from h n avian influenza. the vaccine was administered intramuscularly to over participants - years of age during clinical trials [ , [ ] [ ] [ ] . no results have been posted to date. interestingly, na was only investigated in combination with other influenza proteins. furthermore, besides the vgx- x vaccine, na was only included in whole inactivated and vlp vaccine strategies. the na protein should be further investigated for its cross-protective potential against influenza [ ] . antigens are presented to the immune system in different ways depending on the vaccine platform. most seasonal influenza vaccines utilize attenuated or inactivated wild-type viruses. these viruses display the external influenza proteins and stimulate strong antibody responses [ ] . although this strategy has been utilized since , it has consistently shown low efficacy for protection against mismatched influenza strains [ , ] . therefore, a variety of vaccine platforms were investigated during the last decade to further improve influenza vaccination. although many vaccine platforms have been investigated, no single platform has thus far been demonstrated to show superior protection against influenza. a common platform for uiv clinical trials is viral vectors ( . %), which utilize viral machinery to package, deliver, or display the vaccine antigen ( figure ). viral vectors have been commonly used as molecular biology tools and are approved for several gene therapies [ ] . one of these vaccines is mva-np+m , which is a modified vaccinia virus ankara (mva) viral vector expressing the nucleoprotein (np) and matrix protein (m ) genes from an h n influenza strain [ ] . in the past decade, nine clinical trials investigated mva-np+m enrolling over participants years or older. results from these trials report an increase in t cell response to vaccination, which remained significant above baseline for weeks in - -year-old participants. however, the response was only significant for weeks in subjects - years old and weeks for participants over years [ ] . a subsequent trial using healthy subjects reported no significant difference in t cell response days post-vaccination [ ] . the antibody response to vaccination was not reported. to further boost the immune response to mva-np+m , a heterologous boost with a simian adenovirus viral vector chadox -np+m was investigated [ ] . in this study involving participants, both the mva-np+m and chadox -np+m vaccines were shown to boost t cell responses when administered individually or together. a heterologous boost, regardless of the order, increased t cell responses -fold. another study investigating this heterologous strategy reported a significant increase in t cell responses at day after chadox -np+m vaccination, but a decreased response by day [ ] . vaccinees were boosted with mva-np+m , which again increased the t cell response; however, this response was not significant days following the boost vaccination. a recent viral vectored vaccine is nasovax, an intranasal adenoviral vectored vaccine. though no results have been posted for this clinical trial, data presented at the world vaccine congress reported strong immunogenicity and protection [ ] . indeed, vaccination with nasovax induced % seroconversion, which was maintained for over year. the newest vaccine platform utilizes nanoparticles to deliver viral antigens [ ] . one vaccine utilizing this method is val- and val- , which are mrna ha from h n and h n influenza strains delivered in a lipid nanoparticle (lnp) [ ] . two trials were performed utilizing participants aged - years. for the h n mrna vaccine, vaccination resulted in mild to moderate systemic adverse events including injection site pain ( . - . % vs. . - . %) and myalgia ( . - . % vs. . - . %) compared to the placebo. antibody responses were increased in a dose-dependent manner for the h n lnp vaccine reaching % seroconversion at µg compared to . % for the placebo group. these levels remained seropositive (hai ≥ ) for months after immunization. the h n lnp vaccine induced strong antibody titers for all vaccine doses with . % seroconversion for the µg dose group. participants vaccinated with the h vaccine displayed mild injection site pain compared to the placebo ( . - % vs. . - . %). mva-np+m and chadox -np+m vaccines were shown to boost t cell responses when administered individually or together. a heterologous boost, regardless of the order, increased t cell responses ~ -fold. another study investigating this heterologous strategy reported a significant increase in t cell responses at day after chadox -np+m vaccination, but a decreased response by day [ ] . vaccinees were boosted with mva-np+m , which again increased the t cell response; however, this response was not significant days following the boost vaccination. other nanoparticle vaccines include vrc-flunpf - -vp, which is a recombinant ha vaccine delivered in a ferritin nanoparticle, and vrc-flunpf - -vp, which is a ha stalk protein delivered in a ferritin nanoparticle. although neither trial has posted results, influenza ferritin nanoparticle vaccines have shown strong immunogenicity in mice and ferrets during preclinical trials [ ] . another common vaccine platform utilizes recombinant protein of a viral antigen ( . %). due to low immunogenicity, recombinant protein vaccines typically require the use of an adjuvant to enhance the immune response to vaccination [ , ] . panblok is a recombinant ha protein administered with a novel stable emulsion adjuvant. four clinical trials were performed which enrolled participants - years old. in one adjuvant dose-dependent trial targeting h influenza, results demonstrated that all adjuvanted vaccines ( . µg, . µg, or µg) increased seroconversion from % in the unadjuvanted group to % for participants who received an adjuvanted vaccine [ ] . however, another trial targeting h influenza reported low seroconversion regardless of the adjuvant dose [ ] . despite low antibody detection using hai assay, antibodies against h influenza were detected using elisa. passive transfer of these antibodies resulted in protection against lethal h challenge in mice. additionally, these antibodies were cross-reactive to h , h , h , h , h , and h , indicating broad immunity against both group and group influenza [ ] . some vaccines deliver conserved immunogenic peptides of viral antigens. one such vaccine is flu-v, a peptide-based vaccine containing conserved epitopes from influenza a and b viruses and adjuvanted with montanide isa- . over the past decade, clinical trials were performed involving participants between and years of age. vaccination with flu-v increased ifn-γ cellular responses -fold but did not induce antibody responses as expected [ ] . in another study, seronegative males were vaccinated with flu-v and then challenged with h n influenza virus [ ] . participants vaccinated with flu-v showed reductions in viral load and symptoms as well as an -fold increase in ifn-γ cellular responses. nonstructural protein (ns ) is an influenza protein that antagonizes the immune system by downregulating antiviral host proteins [ ] . several attenuated virus vaccines in clinical trials have deleted this viral gene to improve the immune response to vaccination. both ghb l and ghb l are intranasal live attenuated viruses with the ns gene deleted, but neither clinical trial has published results from these studies. matrix protein (m ) is an essential structural viral protein for influenza replication. the m sr vaccine includes a virus that lacks the m protein resulting in non-infectious viral progeny, essentially a single-cycle virus [ ] . all three m sr vaccine trials are currently active. an ideal uiv will provide highly effective and long-lasting protection. this can be difficult to achieve when targeting internal proteins or using poorly immunogenic vaccine platforms. adjuvants are compounds that stimulate the immune system and improve vaccine efficacy [ ] . this is commonly achieved by oil-in-water emulsions, which recruit immune cells to the site of vaccination [ ] . another common group of adjuvants are toll-like receptor (tlr) agonists. these adjuvants bind and activate cellular host pathways, which leads to increased immune activation [ ] . new adjuvants continue to be discovered and explored, but few are licensed for use in the united states [ ] . over the past decade, most adjuvants in uivs have been oil-in-water emulsions ( %) (figure ). m- is a recombinant protein vaccine that contains common b and t cell epitopes from the ha, np, and m influenza proteins. seven trials were performed over the past decade, which enrolled , individuals over years old. this vaccine was combined with an adjuvant, montanide isa vg, which increased igg titers -fold against the m- protein [ ] . strong t cell responses to m- were shown for all groups regardless of adjuvant inclusion. a subsequent trial reported m- could be used as a stand-alone or priming vaccine for the seasonal influenza vaccine [ ] . when compared to seasonal vaccination alone, participants primed with m- before seasonal vaccination showed elevated antibody responses for matched h n ( -fold vs. . -fold) and h n ( . -fold vs. . -fold), but not influenza b ( . -fold vs. . -fold). additionally, m- vaccination increased both cd + and cd + t cell responses to h n , h n , and influenza b strains compared to baseline. another unpublished clinical trial reported that % of m- -vaccinated participants had a -fold increase in hai titers compared to % for the control group [ ] . this vaccine has moved into phase iii clinical trials and was scheduled for primary completion in may . immunose flu is an inactivated split vaccine with a novel lipid adjuvant, endocine. the immunogenicity of immunose flu was not reported, but vaccination resulted in serious adverse events in of participants including erysipelas and gastroenteritis [ ] . mild to moderate adverse events were recorded in . % and . % of vaccinated participants compared to . % in the saline placebo control group. another common group of adjuvants are toll-like receptor (tlr) agonists. these adjuvants bind and activate cellular host pathways, which leads to increased immune activation [ ] . an example is vax , which is a recombinant ha protein fused to the tlr ligand, flagellin. four clinical trials were performed using participants over the age of . vaccine doses over µg resulted iñ -fold elevated hai titers, % seroconversion, and % seroprotection rates for h n influenza [ ] . however, dose escalation over µg and µg was stopped due to serious adverse events [ ] . vaccine doses ≥ . µg resulted in an average -fold increase in hai titer, % seroprotection, and % seroconversion against a matched h n influenza strain [ ] . viruses , , x for peer review of immunose flu is an inactivated split vaccine with a novel lipid adjuvant, endocine. the immunogenicity of immunose flu was not reported, but vaccination resulted in serious adverse events in of participants including erysipelas and gastroenteritis [ ] . mild to moderate adverse events were recorded in . % and . % of vaccinated participants compared to . % in the saline placebo control group. another common group of adjuvants are toll-like receptor (tlr) agonists. these adjuvants bind and activate cellular host pathways, which leads to increased immune activation [ ] . an example is vax , which is a recombinant ha protein fused to the tlr ligand, flagellin. four clinical trials were performed using participants over the age of . vaccine doses over µ g resulted in ~ -fold elevated hai titers, % seroconversion, and % seroprotection rates for h n influenza [ ] . however, dose escalation over µ g and µ g was stopped due to serious adverse events [ ] . vaccine doses ≥ . µ g resulted in an average -fold increase in hai titer, % seroprotection, and % seroconversion against a matched h n influenza strain [ ] . another tlr adjuvant is double-stranded rna (dsrna), which binds tlr and activates inflammatory pathways [ ] . vxa-a . utilizes this adjuvant by encoding dsrna and an h n ha transgene in a recombinant adenovirus type (ad ) vector. this oral vaccine has been studied in clinical trials with participants between and years of age. one trial reported increased antibody responses to matched h n strains with an average of . -fold increases in hai titers and -fold increases in microneutralization titers after vaccination [ ] . vaccination resulted in mild side effects at similar rates to the placebo group. phase clinical trial participants were immunized with vxa-a . or the seasonal qiv vaccine and then challenged with an h n influenza strain [ ] . vaccination with vxa-a . resulted in % protection compared to % with the seasonal vaccine. another tlr adjuvant is double-stranded rna (dsrna), which binds tlr and activates inflammatory pathways [ ] . vxa-a . utilizes this adjuvant by encoding dsrna and an h n ha transgene in a recombinant adenovirus type (ad ) vector. this oral vaccine has been studied in clinical trials with participants between and years of age. one trial reported increased antibody responses to matched h n strains with an average of . -fold increases in hai titers and -fold increases in microneutralization titers after vaccination [ ] . vaccination resulted in mild side effects at similar rates to the placebo group. phase clinical trial participants were immunized with vxa-a . or the seasonal qiv vaccine and then challenged with an h n influenza strain [ ] . vaccination with vxa-a . resulted in % protection compared to % with the seasonal vaccine. interestingly, although alum is one of the most commonly used fda-approved adjuvants, only one clinical trial in utilized this adjuvant [ ] . hai- is a recombinant h ha protein vaccine that is produced in a plant-expression system, nicotiana benthamiana [ ] . this trial enrolled individuals between and years of age and investigated the dose response of hai- with alum. interestingly, any combination of hai- ( , , and µg) with alum resulted in minimal antibody titers while hai- alone ( µg) induced the greatest antibody response ( . -fold increase). this suggests the hai- induced low immunogenicity that was not improved by the addition of an adjuvant. in the us, new drugs and vaccines must complete four phases of clinical trials to be licensed and marketed for public use. phase i trials investigate the safety and dosage of the vaccine. typically, phase i trials have limited numbers of participants and do not assess efficacy due to low statistical power [ ] . phase ii trials assess the dose response, efficacy, and side effects of the new vaccine. these trials include more study participants and can last longer than phase i trials. occasionally, phases i and ii can be combined into one clinical trial, phase i/ii. phase iii trials include a large sample size and assess participants for vaccine efficacy and adverse reactions. at this point, the new vaccine or drug may be approved for the market [ ] . lastly, phase iv clinical trials involve post-marketing surveillance of the efficacy and safety of the new vaccine. importantly, not all clinical trial results are reported or published. it is common for results to be posted several years after the completion of a trial ( figure ). over the past decade, only half of completed trials reported their findings ( figure e ). this delay is consistent regardless of clinical trial phase ( figure d ). one clinical trial in utilized this adjuvant [ ] . hai- is a recombinant h ha protein vaccine that is produced in a plant-expression system, nicotiana benthamiana [ ] . this trial enrolled individuals between and years of age and investigated the dose response of hai- with alum. interestingly, any combination of hai- ( , , and µ g) with alum resulted in minimal antibody titers while hai- alone ( µ g) induced the greatest antibody response ( . -fold increase). this suggests the hai- induced low immunogenicity that was not improved by the addition of an adjuvant. in the us, new drugs and vaccines must complete four phases of clinical trials to be licensed and marketed for public use. phase i trials investigate the safety and dosage of the vaccine. typically, phase i trials have limited numbers of participants and do not assess efficacy due to low statistical power [ ] . phase ii trials assess the dose response, efficacy, and side effects of the new vaccine. these trials include more study participants and can last longer than phase i trials. occasionally, phases i and ii can be combined into one clinical trial, phase i/ii. phase iii trials include a large sample size and assess participants for vaccine efficacy and adverse reactions. at this point, the new vaccine or drug may be approved for the market [ ] . lastly, phase iv clinical trials involve post-marketing surveillance of the efficacy and safety of the new vaccine. importantly, not all clinical trial results are reported or published. it is common for results to be posted several years after the completion of a trial ( figure ). over the past decade, only half of completed trials reported their findings ( figure e ). this delay is consistent regardless of clinical trial phase ( figure d ). as expected, most uiv clinical trials performed over the past decade were phase i trials ( . %) ( figure ). of the vaccines, have progressed past phase i ( . %); however, only vaccines ( %) have been tested in phase iii clinical trials. the first phase iii trial investigated inflexal v, a trivalent adjuvanted virus-like particle (vlp) vaccine [ ] . this study included children between and months and was completed in november [ ] . all participants were immunized with a single full dose ( . ml) or with two doses ( . ml) of the inflexal v vaccine. results suggest that both vaccine groups demonstrated improved seroprotection and seroconversion rates. participants who received two . ml doses weeks apart showed higher seroprotection rates for h n ( . ), h n ( . ), and influenza b ( . ). for h n and h n , the two-dose regimen resulted in higher seroconversion and geometric mean titer (gmt) fold increases than the single-shot regimen. half of participants from each group experienced non-serious adverse events including pyrexia, malaise, rhinitis, cough, otitis media acute, as well as adverse events at the injection site including erythema, induration, pain, or hemorrhage. the second uiv tested in a phase iii clinical trial was m- . this vaccine is a synthetic recombinant protein containing common linear influenza epitopes [ ] . as discussed above, the adjuvanted m- vaccine has shown promising immunogenicity and the phase iii trial was scheduled for primary completion in may [ , ] . the third vaccine tested in a phase iii clinical trial is nanoflu. this vaccine is a recombinant ha protein delivered in a nanoparticle with a saponin-based matrix-m adjuvant [ ] . although results for the phase ii trial have not been posted, a press release from novavax stated that nanoflu induced superior hai antibody responses against homologous and drifted strains compared to the seasonal influenza vaccine. a phase iii clinical trial involving participants over years of age was scheduled for primary completion in december . this systematic review documents uivs that were tested in clinical trials from january to december . although many papers have discussed strategies for uivs, few review papers address the translation of uiv strategies to clinical trials [ , ] . this is the first systematic review of uivs in clinical trials. the definition of a "universal" influenza vaccine is highly debated [ , ] . in , the niaid announced that a uiv should ( ) be at least % effective, ( ) protect against group i and ii iav, ( ) have durable protection that lasts at least year, and ( ) be suitable for all age groups [ ] . since this standard was put forward towards the end of the decade, our definition of a uiv remains broader than the niaid requirements. here, we have defined a uiv as a vaccine that aims to induce better cross-protection than seasonal influenza vaccines. therefore, "supra-seasonal vaccines" which cover a large subset of influenza strains and vaccines against specific subtypes of influenza have been included in this analysis. the influenza diversity targeted by each vaccine varied. only % of universal vaccines were designed to protect against both influenza a and b viruses. other strategies focused on iav ( %) or a single subtype of iav ( %). importantly, no vaccines focused on influenza b virus (ibv) alone. furthermore, the current niaid requirements for a universal influenza vaccine do not require cross-protection against ibv. notably, the cdc reports that ibv is responsible for % of influenza cases reported for children and young adults each year [ ] . overall, approximately % of annual influenza cases can be attributed to ibv [ ] . the significant burden of ibv should be addressed in the design of universal influenza vaccines. some limitations to this review should be noted. first, information about clinical trials can be limited until the results are published. specifically, not all clinical trial summaries include information on vaccine design and mechanism. in these cases, previous publications and press releases for the vaccines were consulted. additionally, most results reported safety information and homologous vaccine efficacy, providing limited information on the cross-reactivity of each vaccine. second, we searched clinical trials registered through clinicaltrials.gov, which could potentially exclude some studies. there are other clinical trial databases such as eu clinical trials register, however, the clinicaltrials.gov database reports more accurate and updated information for clinical trials [ ] . despite limited information, this review provides a comprehensive summary of the uivs tested in clinical trials. indeed, this is the first comprehensive review to also discuss efficacy and trends in vaccine development for influenza. the field of influenza vaccine development is ever progressing. this is reflected in new vaccine targets and platforms such as ha stalk and nanoparticles. researchers over the past decade have produced many promising influenza vaccines, each with strengths and limitations. the efficacy of a vaccine may induce strong protection against matched strains, but an effective uiv must induce strong cross-protection as well. this review identifies vaccines that report efficacy against matched strains alone. importantly, these vaccines may provide cross-protection if delivered in combination with vaccines targeting other influenza subtypes. however, this would require further research and investigation. influenza virus remains a major global pathogen despite the general widespread use of seasonal vaccines due to varying efficacy to drifted strains. a uiv remains a top priority for the nih and world health organization. this review provides an update on the progress towards a better influenza vaccine. with this information, researchers and clinicians can remain informed about the status and limitations of universal influenza vaccines. universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases influenza (seasonal) estimates of global seasonal influenza-associated respiratory mortality: a modelling study global role and burden of influenza in pediatric respiratory hospitalizations, - : a systematic analysis annual estimates of the burden of seasonal influenza in the united states: a tool for strengthening influenza surveillance and preparedness the annual impact of seasonal influenza in the us: measuring disease burden and costs estimating age-specific cumulative incidence for the influenza pandemic: a meta-analysis of a(h n )pdm serological studies from countries. 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university of oxford. a study to determine the safety and immunogenicity of co-administration of the candidate influenza vaccine mva-np+m and seasonal influenza vaccine a phase i study of candidate influenza vaccines mva-np+m and chadox np+m heterologous two-dose vaccination with simian adenovirus and poxvirus vectors elicits long-lasting cellular immunity to influenza virus a in healthy adults safety and immunogenicity of co-administration of candidate influenza vaccine mva-np+m and viroflu®seasonal influenza vaccine a study to determine the safety and immunogenicity of the candidate influenza vaccine mva-np+m safety and immunogenicity of the heterosubtypic influenza a vaccine mva-np+m manufactured on the age .cr.pix avian cell line. vaccines improved novel vaccine combination influenza study a phase iib study to determine the safety and efficacy of candidate influenza vaccine mva-np+m in combination with licensed inactivated influenza vaccine in adults aged years and above 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vaccine and as a h n vaccine primer in adults two doses of multimeric- (m- ) followed by influenza vaccine biondvax pharmaceuticals ltd. a pivotal trial to assess the safety and clinical efficacy of the m- as a standalone universal flu vaccine safety and immunogenicity of vax influenza vaccine in community-living adults >= years of age induction of a potent immune response in the elderly using the tlr- agonist, flagellin, with a recombinant hemagglutinin influenza-flagellin fusion vaccine (vax , stf .ha si) comparative safety and immunogenicity of vax a, vac b and vax c novel h n influenza vaccine in healthy adults development of vax , a recombinant hemagglutinin (ha) influenza-flagellin fusion vaccine with improved safety and immune response safety and immunogenicity of a novel h n influenza vaccine in healthy adults age - years superior efficacy of a recombinant flagellin:h n ha globular head vaccine is determined by the placement of the globular head within flagellin study of the safety and immunogenicity of a novel h n influenza vaccine in healthy adults age - safety and immunogenicity of an oral, replicating adenovirus serotype vector vaccine for h n influenza: a randomised, double-blind, placebo-controlled, phase study safety and immunogenicity of replication-competent adenovirus -vectored vaccine for avian influenza h n niaid); national institutes of health clinical center (cc) niaid); national institutes of health clinical center (cc) dose finding study of single dose ghb l in healthy adults phase b influenza vaccine study in healthy subjects synthetic influenza vaccine (flu-v) stimulates cell mediated immunity in a double-blind, randomised, placebo-controlled phase i trial influenza vaccine challenge study in healthy subjects caparros-wanderley, w. meta-analysis and potential role of preexisting heterosubtypic cellular immunity based on variations in disease severity outcomes for influenza live viral challenges in humans a synthetic influenza virus vaccine induces a cellular immune response that correlates with reduction in symptomatology and virus shedding in a randomized phase ib live-virus challenge in humans peptcell limited; national institute of allergy and infectious diseases (niaid) peptcell limited; seventh framework programme double-blind, placebo-controlled phase iib trial to test flu-v vaccine evaluation of the immunogenicity and safety of different doses and formulations of a broad spectrum influenza vaccine (flu-v) developed by seek: study protocol for a single-center, randomized, double-blind and placebo-controlled clinical phase iib trial study of vgx- x, h n avian influenza virus dna plasmid + electroporation in healthy adults inovio biomedical h n avian influenza dna vaccine receives korean approval to begin clinical trials. in first component of inovio's syncon(tm) universal flu vaccine to be tested in healthy volunteers inovio pharmaceuticals. study of vgx- , h n avian flu virus plasmid dna with electroporation device in healthy adult males a follow-on study with an h influenza vaccine for subjects who participated in study flu- a study of dna vaccine with electroporation for the prevention of disease caused by h and h influenza virus immune targeting systems ltd.; hammersmith medicines research. a study to evaluate the safety, tolerability and immunogenicity of a universal influenza a vaccine a novel peptide-based pan-influenza a vaccine: a double blind, randomised clinical trial of immunogenicity and safety tolerability and immunogenicity of two different formulations of an influenza a vaccine (fp- . ) immune targeting systems ltd. influenza a vaccine (fp- . ) formulated with and without adjuvant, in the presence or absence of a single administration of a trivalent inactivated influenza virus vaccine in older adults efficacy and immunogenicity of an influenza a vaccine (fp- . ) in healthy volunteers following virus challenge immunogenicity and safety of a single . ml dose of inflexal v with a . ml -dose regimen of inflexal v inflexal v a trivalent virosome subunit influenza vaccine: production. vaccine safety and immunogenicity of a recombinant h n vaccine in adults safety and immunogenicity of a plant-produced recombinant hemagglutinin-based influenza vaccine (hai- ) derived from a/indonesia/ / (h n ) influenza virus: a phase randomized, double-blind, placebo-controlled, dose-escalation study in healthy adults study of single dose ghb l trivalent influenza vaccine in healthy adults muster, t. phase i/ii trial of a replication-deficient trivalent influenza virus vaccine lacking ns protein sciences corporation. safety and immunogenicity of panblok influenza vaccine in healthy adults stable emulsion (se) alone is an effective adjuvant for a recombinant, baculovirus-expressed h influenza vaccine in healthy adults: a phase trial trial to evaluate the immunogenicity and safety of panblok®(h rha) in healthy adults aged and older vaccination with a recombinant h 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phase influenza a challenge study following oral administration of an h n ha ad-vector seasonal flu vaccine pharmacodynamic open-label trial with vxa-a . oral h vaccine in healthy adults a(h n ) vlp antigen dose-ranging study with matrix-m ™ adjuvant nova laboratories limited; the emmes company, llc. the safety, tolerance, and immunogenicity of mas- -adjuvanted seasonal inactivated influenza vaccine (mer ) mrna vaccines against h n and h n influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase randomized clinical trials tolerability, and immunogenicity of val- in healthy adult subjects tolerability, and immunogenicity of val- in healthy adult subjects safety and immunogenicity study of h n m sr monovalent influenza vaccine in healthy volunteers safety and immunogenicity study of an influenza vaccination strategy including a h n m sr prime followed by a seasonal quadrivalent inactivated vaccine boost in a pediatric population - years old flugen inc. safety and immunogenicity of the bris m sr and sing m sr h n monovalent influenza vaccines novel influenza vaccine m sr protects against drifted h n and h n influenza virus challenge in ferrets with pre-existing immunity study to assess the safety, tolerability and immune response following vaccination with immunose™ flu study to assess the safety, tolerability and immune response following vaccination with immunose™ flu in older adults nitto denko corporation. evaluation of the safety and immunogenicity of a sublingual influenza vaccine nsv in healthy male volunteers a study to evaluate the reactogenicity, safety and immunogenicity of glaxosmithkline (gsk) biologicals' investigational supra-seasonal universal influenza vaccines-inactivated (suivs) (gsk a) in healthy adults aged to years single-ascending-dose study of the safety and immunogenicity of nasovax safety and immunogenicity of nasovax, a novel intranasal influenza vaccine sybil tasker to present on april at : p.m. eastern time novavax. evaluation of the safety and immunogenicity of a recombinant trivalent nanoparticle influenza vaccine with matrix m- adjuvant (nanoflu). available online novavax announces positive phase nanoflu results in older adults phase pivotal trial of nanoflu™ in older adults immunogenicity of chimeric haemagglutinin-based, universal influenza virus vaccine candidates: interim results of a randomised, placebo-controlled, phase clinical trial icahn school of medicine at mount sinai; children's hospital medical center, cincinnati; duke university; the emmes company, llc.; glaxosmithkline. safety and immunogenicity of a live-attenuated universal flu vaccine followed by an inactivated universal flu vaccine influenza ha ferritin vaccine, alone or in prime-boost regimens with an influenza dna vaccine in healthy adults self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h n antibodies ferritin in the field of nanodevices vaccinate for the next h n pandemic now niaid); national institutes of health clinical center (cc). dose, safety, tolerability and immunogenicity of an influenza h stabilized stem ferritin vaccine, vrcflunpf - -vp, in healthy adults hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection safety and immune response of increasing doses of ovx after intramuscular or intranasal administrations in healthy subjects ovx a recombinant nucleoprotein vaccine inducing cellular responses and protective efficacy against multiple influenza a subtypes safety and immune response of one dose of ovx at two dose levels, in comparison to influvac tetratm, after intramuscular administration in healthy subjects aged - years health ministry of the russian federation. the study of the safety, reactogenicity and immunogenicity of the gamfluvac a double-blind randomized placebo-controlled study study of the safety, reactogenicity and immunogenicity of the gamfluvac influenza and memory t cells: how to awake the force host immune response to influenza a virus infection nucleoprotein of influenza a virus is a major target of immunodominant cd + t-cell responses emerging influenza viruses and the prospect of a universal influenza virus vaccine influenza infection in humans induces broadly cross-reactive and protective neuraminidase-reactive antibodies comparisons of the humoral and cellular immune responses induced by live attenuated influenza vaccine and inactivated influenza vaccine in adults history and evolution of influenza control through vaccination: from the first monovalent vaccine to universal vaccines viral vectors in gene therapy nanoparticle vaccines against infectious diseases recombinant vaccines and the development of new vaccine strategies. braz correlates of adjuvanticity: a review on adjuvants in licensed vaccines functions of the influenza a virus ns protein in antiviral defense mechanisms of action of adjuvants toll-like receptor agonists: are they good adjuvants? adjuvants help vaccines work better safety and immunogenicity of multimeric- -a novel universal influenza vaccine dsrna-activation of tlr and rlr signaling: gene induction-dependent and independent effects what are clinical trials and studies? available online the hurdles from bench to bedside in the realization and implementation of a universal influenza vaccine universal influenza virus vaccines and therapeutic antibodies impact of influenza b lineage-level mismatch between trivalent seasonal influenza vaccines and circulating viruses prevalence of clinical trial status discrepancies: a cross-sectional study of , trials registered on both clinicaltrials.gov and the european union clinical trials register key: cord- -jja wcfz authors: voigt, kathleen; hoffmann, markus; drexler, jan felix; müller, marcel alexander; drosten, christian; herrler, georg; krüger, nadine title: fusogenicity of the ghana virus (henipavirus: ghanaian bat henipavirus) fusion protein is controlled by the cytoplasmic domain of the attachment glycoprotein date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jja wcfz the ghana virus (ghv) is phylogenetically related to the zoonotic henipaviruses nipah (niv) and hendra virus. although ghv uses the highly conserved receptor ephrin-b , the fusogenicity is restricted to cell lines of bat origin. furthermore, the surface expression of the ghv attachment glycoprotein (g) is reduced compared to niv and most of this protein is retained in the endoplasmic reticulum (er). here, we generated truncated as well as chimeric ghv g proteins and investigated the influence of the structural domains (cytoplasmic tail, transmembrane domain, ectodomain) of this protein on the intracellular transport and the fusogenicity following coexpression with the ghv fusion protein (f). we demonstrate that neither the cytoplasmic tail nor the transmembrane domain is responsible for the intracellular retention of ghv g. furthermore, the cytoplasmic tail of ghv g modulates the fusogenicity of ghv f and therefore controls the species-restricted fusogenicity of the ghv surface glycoproteins. viral rna of the ghana virus (ghv, formerly termed kumasi virus, genbank accession no.: hq . ) was detected in a straw-colored fruit bat (eidolon helvum) in [ ] . ghv is phylogenetically related to hendra (hev) and nipah (niv) virus, two highly pathogenic members of the genus henipavirus within the paramyxoviridae family. hev and niv emerged from their reservoir host pteropid bats [ ] [ ] [ ] causing severe and often fatal diseases in humans and domestic animals such as pigs and horses [ ] [ ] [ ] [ ] . due to their zoonotic potential, their high pathogenicity, and the lack of approved therapeutics and a vaccine licensed for use in humans, hev and niv are classified as biosafety level pathogens. the zoonotic potential of ghv is unknown and so far, no cases of henipavirus infections of livestock or humans have been reported in africa; but nevertheless, serological studies provided evidence that fruit bats, domestic animals, and humans in this region harbor neutralizing antibodies reactive against henipaviruses [ , [ ] [ ] [ ] [ ] [ ] . henipaviruses express two surface glycoproteins: the fusion (f) and the attachment glycoprotein (g) that mediate the viral entry process. the g protein is a type ii membrane protein and is responsible for binding to the cellular receptor ephrin-b [ ] [ ] [ ] [ ] [ ] [ ] . the f protein, a type i membrane protein, mediates the fusion between viral and cellular membranes as well as the fusion between infected and neighboring cells to enable the release of the viral genome into the cytoplasm and to facilitate the spread of infection, respectively [ ] . after synthesis, the f protein is transported to the cell surface as an inactive precursor that has to undergo a recycling step via clathrin-mediated endocytosis to be proteolytically cleaved by the cellular proteases cathepsin l or b within the endosomal compartment [ ] [ ] [ ] [ ] [ ] . furthermore, the f protein has to undergo conformational changes that are triggered by the interaction between f and g [ ] [ ] [ ] . upon binding to the cellular receptor, the g protein undergoes three conformational changes in the head and stalk regions that induce the refolding of f into its fusion-active conformation [ , ] . to gain information about the functionality of the two surface glycoproteins of ghv, previous studies focused on the directed expression of these two viral proteins in cell culture systems, as neither viral isolate nor recombinant ghv are available. in this way, it has been shown that the ghv g protein interacts with ephrin-b , the cellular receptor of niv and hev, to mediate binding to target cells [ ] [ ] [ ] [ ] . however, striking differences between niv and ghv were observed when the fusogenicity of f was investigated: in the case of niv, coexpression of f and g resulted in the formation of multinucleated giant cells, so-called syncytia, in different mammalian cell lines. in contrast, the ghv f and g proteins induced significantly smaller syncytia the presence of which was further restricted to bat cell lines [ ] [ ] [ ] [ ] . in previous studies, it has been shown that the transport of ghv g to the cell surface is significantly reduced compared to niv g and that the majority of the ghv g protein accumulates in the endoplasmic reticulum (er), suggesting that the amount of surface-expressed ghv g is not efficient enough to trigger conformational changes in the f protein that are required to acquire the fusion-active form of f [ , ] . in this study we addressed the question: which domain of ghv g contributes to the accumulation in the er and to the reduced fusogenicity of ghv f? therefore, we generated truncated as well as chimeric ghv g proteins and analyzed their intracellular localization and the fusogenicity of ghv f following coexpression. we provide evidence that the truncation of the cytoplasmic domain (cd) results in a ghv g that enhances fusion activity of ghv f and abolished the species restriction, though the cellular localization did not differ from that of wildtype ghv g. vero (african green monkey, kindly provided by andrea maisner), hek t (human, dsmz no. acc- ), bhk- (human, dsmz no. acc- ), eidni/ (straw-colored fruit bat, eidolon helvum) [ ] , and hypni/ . (hammer-headed fruit bat, hypsignathus monstrosus) [ ] cells were maintained in dulbecco's modified eagle's medium (dmem; thermofisher scientific, waltham, ma, usa) supplemented with % or % fetal calf serum (fcs; pan-biotech, aidenbach, germany) and % penicillin/streptomycin (paa laboratories gmbh, pasching, austria). cells were cultivated in cm tissue culture flasks (greiner bio-one, frickenhausen, germany) at • c and % co . expression plasmids coding for epitope-labelled full-length niv and ghv f and g proteins have been described previously [ ] . here, we generated truncated versions of ghv g in which to amino acid (aa) residues of the n-terminus were deleted (ghv g∆ , ∆ , ∆ , ∆ , ∆ , ∆ , ∆ , ∆ ) ( figure ) . furthermore, chimeric g proteins consisting of the transmembrane domain (tm) and ectodomain (ed) of ghv g and the cytoplasmic domain (cd) of niv g (ghv g cd-niv), hev g (ghv g cd-hev), moijang henipavirus (mojv) g (ghv g cd-mojv), cedar henipavirus (cedv) g (ghv g cd-cedv), or measles virus (mev) h protein (ghv g cd-mev) were generated. in addition, niv g tm and ed were fused to the cd of ghv g (niv g cd-ghv). further, we generated chimeric ghv and niv g proteins in which the tm was replaced by the corresponding section of the other protein (ghv g tm-niv, niv g tm-ghv). proteins consisting of ghv g cd and tm and the ed of niv g (ghv g ed-niv) and vice versa (niv g ed-ghv) have been described previously [ ] . to allow detection by immunostaining, all g proteins were fused to a flag-epitope at the c-terminal end, while niv f and ghv f contained an ha-epitope at their corresponding c-terminus. all plasmids were verified by automated sequencing (eurofins genomics gmbh, ebersberg, germany). to determine the intracellular localization, cotransfection experiments with fluorescence-labeled cellular compartment markers for the endoplasmic reticulum (er) were performed (egfp-er, provided by frank van kuppeveld). differences with respect to syncytium formation were obtained when the truncated g proteins were coexpressed with ghv f in bat (eidni/ ) and non-bat (vero and bhk- ) cells. consistent with previous publications, coexpression of wildtype ghv f and g mediates syncytium formation only in bat cell lines [ ] [ ] [ ] [ ] and the truncated g proteins ghv g∆ , ∆ , ∆ , ∆ , ∆ , and ∆ displayed the same phenotype as the full-length ghv g protein. in contrast to that, the deletion of the terminal and aa residues of the cd of ghv g resulted in an enhanced fusogenicity in bat cells as indicated by the increased number as well as the increased size of syncytia (table , figure a ). this effect was most pronounced in the case of ghv g∆ (figure b ). most remarkably and in contrast to the parental g protein, ghv g∆ and ghv g∆ were able to induce cell-to-cell fusion also in the non-bat cell lines vero and bhk- ( figure c ). cells were grown on coverslips and transfected using the icafectin tm transfection reagent (in-cell-art, nantes, france) according to the manufacturer's protocol. cells were fixed with % paraformaldehyde (pfa) and either permeabilized with . % triton x- or untreated to detect intracellular or surface-expressed g proteins, respectively. f and g proteins were detected by incubation with antibodies directed against the ha-(dilution : , rabbit, sigma-aldrich, st. louis, mo, usa) or the flag-epitope (dilution : , mouse, sigma-aldrich, st. louis, mo, usa), respectively. next, the incubation with anti-mouse igg alexa fluor -(dilution : , life technologies, carlsbad, ca, usa) and anti-rabbit igg fluorescein isothiocyanate-conjugated (dilution : , sigma-aldrich, st. louis, mo, usa) secondary antibodies was performed. finally, the cells were incubated with , -diamidino- -phenylindole (dapi; carl roth gmbh + co. kg, karlsruhe, germany) and mounted in prolong gold antifade reagent (thermo fisher scientific, waltham, ma, usa). immunofluorescence analysis was performed using a nikon eclipse ti microscope and nis elements ar software (nikon instruments, tokyo, japan). to analyze the fusogenicity following co-expression of f and g proteins, the total number of syncytia per coverslip as well as the number of nuclei in each syncytium were counted. based on these two parameters, the cumulative number of nuclei in syncytia per coverslip was calculated and used as an indicator for fusogenicity. syncytia were defined as multinucleated cells that are positive for the expression of both viral proteins and harbor at least three nuclei. hek t cells were grown in -well plates and transfected for the expression of g proteins by calcium-phosphate precipitation. at h p.t., cells were washed and resuspended in pbs, transferred to reaction tubes, and pelleted by centrifugation at × g for min at • c. the cells were subsequently resuspended in np lysis buffer with protease inhibitors and incubated on ice for min. afterwards, the cell lysates were centrifuged for min at , × g and • c. the supernatant was transferred to a new reaction tube and mixed with × lds sample buffer (life technologies) in a ratio of to . subsequently, dithiothreitol (dtt, . m final concentration) was added and the samples were heated for min at • c, before the lysates were loaded on an sds gel ( % sds) and further subjected to sds-page and western blotting. g proteins were detected by incubation with an anti-flag antibody (dilution : , mouse, sigma-aldrich, st. louis, mo, usa) and anti-mouse horseradish peroxidase (hrp, dilution : , dako gmbh, jena, germany). for the visualization of protein bands, membranes containing the immobilized proteins were incubated with super signal west dura extended duration substrate (thermo fisher scientific, waltham, ma, usa), placed in a chemidoc imager (bio-rad, hercules, ca, usa), and analyzed with the image lab software (bio-rad, hercules, ca, usa). hek t cells were grown in -well plates and transfected for the expression of g proteins by calcium-phosphate precipitation. at h p.t., cells were lysed as described above. cell lysates were loaded onto µl of protein-a sepharose beads (sigma-aldrich, st. louis, mo, usa) mixed with recombinant mouse ephrin-b -fc (r&d systems, minneapolis, mn, usa) and incubated overnight at • c on an overhead shaker. the samples were centrifuged for min at , g and • c. the supernatant was discarded, and the pellet was washed by the addition of µl np lysis buffer. this washing procedure was repeated three times. afterwards, µl of × lds sample buffer (life technologies, carlsbad, ca, usa) were added to the pellet and incubated for min at • c. finally, the samples were centrifuged for min at , rpm at room temperature, before the supernatants were loaded onto an sds gel. sds-page, western blotting, and protein detection by incubation with an anti-flag antibody and anti-mouse hrp were performed as mentioned before. hek t cells were grown in -well plates and transfected for the expression of g proteins by calcium-phosphate precipitation. at h p.t., cells were washed and resuspended in pbs, transferred to reaction tubes, and pelleted by centrifugation at × g for min at • c. the cells were subsequently resuspended in facs-buffer (pbs without calcium and magnesium ions (pbsm)/ % bovine serum albumin (bsa)/ mm edta) and centrifuged again before they were resuspended in facs-buffer containing anti-flag antibodies (dilution : , mouse, sigma-aldrich, st. louis, mo, usa), followed by incubation for h on an overhead shaker at • c. afterward, the cells were washed. for this, the cells were pelleted by centrifugation and resuspended in facs-buffer. then the cells were pelleted again, resuspended in facs-buffer containing biotin-conjugated anti-mouse antibodies (dilution : , sigma-aldrich, st. louis, mo, usa), and rotated for min at • c. after an additional washing step, cells were resuspended in facs-buffer containing phycoerythrin-conjugated streptavidin (dilution : , bio-rad, hercules, ca, usa) and rotated for min at • c. next, the cells were washed and subsequently fixed by incubation with % pfa for min. finally, the cells were washed again to remove residual pfa and resuspended in µl facs-buffer, before the mean fluorescence intensity was quantified by flow cytometry using an attune nxt flow cytometer (thermo fisher scientific, waltham, ma, usa). the data analysis was performed by flowjo®(flowjo llc, ashland, or, usa). the ghv g protein consists of three major domains: the n-terminal cytoplasmic domain (cd), the transmembrane domain (tm), and the c-terminal ectodomain (ed). the latter can be subdivided into a stalk and a head domain (figure a) . despite a similar structural organization, the cd of ghv g is longer compared to the cd of niv and hev ( vs aa residues, respectively, figure b ). in general, the sequence of this domain is not well conserved among the henipavirus species ranging from approximately to % aa identity (table ) . to determine whether the cd of ghv g-in particular the three potential retention motifs-may contribute to the intracellular retention of this protein and thereby lead to the limited fusogenicity of ghv f and g in comparison to coexpression of niv f and g, truncated versions of ghv g were generated in which up to aa residues of the cd were deleted in a stepwise fashion (figure c ). differences with respect to syncytium formation were obtained when the truncated g proteins were coexpressed with ghv f in bat (eidni/ ) and non-bat (vero and bhk- ) cells. consistent with previous publications, coexpression of wildtype ghv f and g mediates syncytium formation only in bat cell lines [ ] [ ] [ ] [ ] and the truncated g proteins ghv g∆ , ∆ , ∆ , ∆ , ∆ , and ∆ displayed the same phenotype as the full-length ghv g protein. in contrast to that, the deletion of the terminal and aa residues of the cd of ghv g resulted in an enhanced fusogenicity in bat cells as indicated by the increased number as well as the increased size of syncytia (table , figure a ). this effect was most pronounced in the case of ghv g∆ (figure b ). most remarkably and in contrast to the parental g protein, ghv g∆ and ghv g∆ were able to induce cell-to-cell fusion also in the non-bat cell lines vero and bhk- ( figure c ). table . syncytium formation following coexpression of ghv f and truncated g proteins. bat cells non-bat cells similar to niv and hev g, the cd of ghv g contains two lysine-rich motifs at the aa positions - (kktl) and - (kkqknq) and a tyrosine-based motif ( - yfgl), all of which have been implicated to be involved in the retention of diverse proteins within the er [ , ] (figure b) . by truncation of the cd of ghv g we obtained modified versions that lacked either the first lysine-rich motif (ghv g∆ ), the first lysine-rich motif and the tyrosine-based motif (ghv g ∆ , ∆ ), or all three motifs combined (ghv g∆ , ∆ , ∆ ) (figure c ). to investigate whether these motifs contribute to er-retention, the surface expression levels and the intracellular expression pattern were analyzed. all truncated g proteins showed a total expression level comparable to that of full-length ghv g as indicated by similar amounts of fluorescence-positive cells (analyzed by immunofluorescence analysis of permeabilized cells) and no increased surface expression was observed for any of the g proteins as exemplarily shown for ghv g∆ and ∆ (figure a) . furthermore, the truncation of the cd did not affect the intracellular localization and all truncated versions of ghv g showed a clear colocalization with the er marker, indicating that they are still intracellularly retained (figure b) . when comparing the surface expression level of full-length ghv g to that of ghv g∆ and ∆ by flow cytometry, no significant differences were obtained (figure c ) although the latter proteins increased the fusogenicity of ghv f (figure ). in addition, to investigate whether the truncation affects the conformation of ghv g, the ability of truncated g proteins to interact with the receptor ephrin-b as well as their ability to dimerize has been analyzed. both truncated g proteins were able to form high-order multimers and to bind to ephrin-b (figure d ). contribute to er-retention, the surface expression levels and the intracellular expression pattern were analyzed. all truncated g proteins showed a total expression level comparable to that of full-length ghv g as indicated by similar amounts of fluorescence-positive cells (analyzed by immunofluorescence analysis of permeabilized cells) and no increased surface expression was observed for any of the g proteins as exemplarily shown for ghv gΔ and Δ (figure a) . furthermore, the truncation of the cd did not affect the intracellular localization and all truncated versions of ghv g showed a clear colocalization with the er marker, indicating that they are still intracellularly retained (figure b) . when comparing the surface expression level of full-length ghv g to that of ghv gΔ and Δ by flow cytometry, no significant differences were obtained ( figure c ) although the latter proteins increased the fusogenicity of ghv f (figure ). in addition, to investigate whether the truncation affects the conformation of ghv g, the ability of truncated g proteins to interact with the receptor ephrin-b as well as their ability to dimerize has been analyzed. both truncated g proteins were able to form high-order multimers and to bind to ephrin-b ( figure d) . (c) surface expression of truncated ghv g proteins was quantified by flow cytometry and the mean fluorescence intensity obtained for ghv g∆ and ∆ was normalized to that of wildtype ghv g, which was set as %. the graph shows mean and sem of three independent trials. statistical significance was tested by paired, two-tailed student's t-tests with % confidence intervals (no significant differences were obtained: p > . ). (d) cell lysates of g expressing cells were loaded onto protein-a sepharose beads mixed with fc-conjugated mouse ephrin-b . after immunoprecipitation, sds-page and western blotting were performed. proteins were visualized by incubation with anti-flag antibodies (mouse) and anti-mouse igg horseradish peroxidase (hrp). numbers on the left indicate molecular weight (kda). exposure time: sec. after having shown that the truncated proteins ghv g∆ and ∆ led to an increased fusogenicity of ghv f and abolished species restricted syncytium formation, we analyzed whether the replacement of the cd of ghv g by the corresponding domain of niv g (ghv g cd-niv) and vice versa (niv g cd-ghv) has any effect on the fusogenicity of the respective f proteins. in addition, chimeric ghv g proteins harboring the cd of the g proteins of the related henipaviruses hev, cedv, and mojv g, or the h protein of the unrelated paramyxovirus measles virus (mev, genus rubulavirus) were generated. all chimeric ghv g proteins showed an intracellular localization similar to that of the parental g protein (figure a) . furthermore, the replacement of the cd did not increase the surface expression level of ghv g (figure b) . however, none of these g proteins was capable of inducing the fusion activity of coexpressed ghv f in bat or non-bat cell lines. in contrast, chimeric niv g harboring the cd of ghv g showed an expression pattern comparable to that of parental niv g (figure a) . furthermore, coexpression of niv g cd-ghv and niv f resulted in the formation of large syncytia as observed after coexpression of parental niv g and f ( table ). the heterologous expression of chimeric ghv g and niv f or chimeric niv g and ghv f did not mediate syncytium formation. the mean fluorescence intensity obtained for the chimeric ghv g proteins was further normalized to wildtype ghv g, which was set as %. the graph shows mean and sem of three independent trials. statistical significance was tested by paired, two-tailed student's t-tests with % confidence intervals (no significant differences were obtained: p > . ). table . syncytium formation following coexpression of ghv or niv f and chimeric g proteins. the mean fluorescence intensity obtained for the chimeric ghv g proteins was further normalized to wildtype ghv g, which was set as %. the graph shows mean and sem of three independent trials. statistical significance was tested by paired, two-tailed student's t-tests with % confidence intervals (no significant differences were obtained: p > . ). of the domains (figure b ). in contrast, the replacement of the tm of niv g by the corresponding domain of ghv g led to a partial retention of the chimeric g protein within the er (figure a, lower lane) and significantly decreased the cell surface expression level (figure b ), which might have resulted from suboptimal compatibility of the three protein domains (cd, tm, ed) in this particular organization. consistent with previous findings, no differences were detected when comparing the parental niv g and niv g cd + tm-ghv [ ] . the mean fluorescence intensity obtained for chimeric g proteins was further normalized to that of the corresponding wildtype g proteins, which were set as %. the graph shows mean and sem of three independent trials. statistical significance of differences in surface expression of mutant versus wildtype g proteins was tested by paired, twotailed student's t-tests with % confidence intervals (*: p ≤ . ). the mean fluorescence intensity obtained for chimeric g proteins was further normalized to that of the corresponding wildtype g proteins, which were set as %. the graph shows mean and sem of three independent trials. statistical significance of differences in surface expression of mutant versus wildtype g proteins was tested by paired, two-tailed student's t-tests with % confidence intervals (*: p ≤ . ). table . syncytium formation following coexpression of ghv or niv f and chimeric g proteins. ghv g wt after replacing the cd of ghv and niv g, we focused on the remaining domains and generated chimeric g proteins in which only the tm (ghv g tm-niv, niv g tm-ghv) or the tm in conjunction with the cd (ghv g cd + tm-niv, niv g cd + tm-ghv) were replaced. all chimeric g proteins were analyzed for their localization and their fusogenicity following coexpression with homo-or heterologous f proteins, given that the exchanged domains may be necessary for the specific interaction between the corresponding f and g proteins. none of the chimeric g proteins was able to mediate cell-to-cell fusion regardless of the origin of the f protein or the cell line used ( table ). all chimeric ghv g proteins showed the same expression pattern as parental ghv g and were predominantly localized in the er (figure a, upper lane) . further, the surface expression level of ghv g was slightly reduced by the replacement of any of the domains (figure b ). in contrast, the replacement of the tm of niv g by the corresponding domain of ghv g led to a partial retention of the chimeric g protein within the er (figure a , lower lane) and significantly decreased the cell surface expression level (figure b ), which might have resulted from suboptimal compatibility of the three protein domains (cd, tm, ed) in this particular organization. consistent with previous findings, no differences were detected when comparing the parental niv g and niv g cd + tm-ghv [ ] . a hallmark of the henipaviruses niv and hev is their strong fusion activity, which requires the interaction between the viral surface proteins f and g. however, whereas the glycoproteins of niv and hev induce cell-to-cell fusion in a broad variety of cells from different host species, syncytium formation upon expression of ghv f and g proteins is restricted to certain bat cells [ ] [ ] [ ] [ ] . this difference was believed-at least in part-to be due to an inefficient surface transport of the g protein of ghv, as the restricted surface expression has been shown to be less pronounced in bat cells compared to non-bat cells [ ] . here we analyzed the importance of the functional domains of the g protein of ghv for ghv g surface transport and for triggering the fusogenic activity of ghv f. intracellular retention is also a characteristic feature of other viral glycoproteins and is often associated with intracellular budding of the respective enveloped viruses, such as coronaviruses, e.g., transmissible gastroenteritis virus, or pestiviruses, e.g., bovine viral diarrhea virus (bvdv) [ , ] . further, viruses from different families express glycoproteins that are retained at intracellular structures, e.g., the envelope proteins e and e of rubella virus [ , ] , the e and e rns proteins of bvdv [ , ] , the e protein of hepatitis c virus [ ] , the gn and gc proteins of bunyamwera virus [ ] , or the spike glycoprotein (s) of certain coronaviruses [ ] [ ] [ ] [ ] . for the latter, lysine-and/or tyrosine-based motifs have been identified to cause accumulation of the s protein within intracellular compartments of the secretory pathway [ , ] . the cytoplasmic tail of the ghv g protein contains two lysine-based motifs ( -kktl- and -kkqknq- ) as well as a tyrosine-based motif ( -yfgl- ), but none of them appears to serve as a retention signal. this is evident from the comparison of the deletion mutants that lacked one, two, or all three motifs; none of them showed an increased surface expression. retention in the er may also be mediated by the tm domain of viral glycoproteins as shown for the e protein of bvdv [ , ] . here an arginine residue within the hydrophobic amino acid sequence of the transmembrane anchor is crucial for the intracellular localization of this surface glycoprotein. however, the tm domain of the ghv g protein lacks a comparable amino acid sequence. when chimeric g proteins were analyzed, ghv g containing the tm of niv-g did not show enhanced surface expression, while the tm of ghv in the context of niv g led to an intracellular accumulation of the resulting g protein. therefore, the information for the intracellular retention of ghv g resides mainly or exclusively within the ed. the presence of putative retention signals within the cd portion of g may raise the question whether these motifs may have a synergistic effect and support the retention signal in the ed. however, the results obtained with the chimeric proteins argue against this possibility, because they did not provide any evidence that cd of ghv-g contains an er retention signal. although the ultimate reason for intracellular retention of ghv g could not be uncovered, we could exclude a pivotal role of cd and tm for this retention. moreover, our data suggest that the ed-more precisely, aa motifs/residues within the ed-might be the cause for ghv g accumulation within the er. once attempts to isolate ghv or to generate it by dna technology have been successful, it will be interesting to find out whether the virus matures at the cell surface or whether the budding process takes place at an intracellular compartment. other remarkable features of ghv surface glycoproteins that are distinct from niv f and g are the dramatically reduced fusogenicity and the host restriction of cell-to-cell fusion upon coexpression of ghv f and g [ ] [ ] [ ] . the less efficient fusion activity of the surface proteins of ghv-compared to that of the corresponding proteins of niv-may be explained by differences in both the f [ ] and the g protein [ ] . as far as the g protein is concerned, a major difference between ghv and niv is the aforementioned inefficient surface expression of ghv g. however, surface expression is not the only determinant of the g protein for interacting with the f-protein and inducing membrane fusion. both the cd and the tm domain may affect the fusion activity, even in the absence of increased transport of the g protein to the cell surface. the importance of cd for the fusion activity is evident from our analysis of the deletion mutants g∆ and ∆ . although the lack of the major part of the cytoplasmic tail did not result in a detectable increase of the surface expression or binding efficiency to ephrin b , it enhanced the fusion activity in bat cells and more importantly, abolished the species restriction to bat cells. thus, the cd portion has some restricting effect. it might affect the conformation of g that is required for the interaction with the f protein or it impedes conformational changes within the g protein that are necessary to render the f protein fusion-active [ , ] . this restriction appears to be a peculiar feature of ghv g because a chimeric protein containing ed and tm of niv g and cd of ghv g was highly efficient in inducing the fusion activity of niv f. the results obtained with chimeric proteins further suggest that not only cd but also the tm domain affects the role of the g protein as a modulator of the fusion activity of henipavirus f proteins. while cd of ghv g did not affect the fusion activity of niv glycoproteins, replacement of tm by the corresponding domain of ghv completely abolished the fusion activity of niv f + g. as discussed for cd, the tm domain may affect the conformation of the g protein and thus the interaction with the f protein. an alternative explanation is that there is a direct interaction between the tm domains of the niv f and g proteins and that this interaction does not tolerate the replacement of the niv g-tm domain by ghv-g-tm. in sum, we demonstrate that neither the cd nor td cause intracellular retention of ghv g, which suggests that the structural cause for intracellular retention resides within the ed. moreover, we identified the cd of ghv g as a modulator of ghv f fusogenicity and, most importantly, as the factor controlling species restriction of f-mediated membrane fusion. future efforts will be undertaken to unravel which aa motif/residues are responsible for intracellular ghv g retention 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functional bioactivity of an african bat henipavirus surface glycoprotein type i interferon reaction to viral infection in interferon-competent, immortalized cell lines from the african fruit bat eidolon helvum differential sensitivity of bat cells to infection by enveloped rna viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum intracellular targeting signals contribute to localization of coronavirus spike proteins near the virus assembly site greiser-wilke, i. localization of viral proteins in cells infected with bovine viral diarrhoea virus structure and intracellular assembly of the transmissible gastroenteritis coronavirus targeting of a heterodimeric membrane protein complex to the golgi: rubella virus e glycoprotein contains a transmembrane golgi retention signal characterization of an endoplasmic reticulum retention signal in the rubella virus e glycoprotein retention and topology of the bovine viral diarrhea virus glycoprotein e a new type of intracellular retention signal identified in a pestivirus structural glycoprotein the transmembrane domain of hepatitis c virus glycoprotein e is a signal for static retention in the endoplasmic reticulum mapping the golgi targeting and retention signal of bunyamwera virus glycoproteins incorporation of spike and membrane glycoproteins into coronavirus virions the spike protein of infectious bronchitis virus is retained intracellularly by a tyrosine motif contribution of trafficking signals in the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection the cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds copi and promotes interaction with membrane protein a novel sorting signal for intracellular localization is present in the s protein of a porcine coronavirus but absent from severe acute respiratory syndrome-associated coronavirus expression of the surface glycoprotein e of bovine viral diarrhea virus by recombinant vesicular stomatitis virus we thank andrea maisner (institute of virology, philipps university marburg) and frank van kuppeveld (department of infectious diseases and immunology, faculty of veterinary medicine, utrecht university) for providing the vero cell line and the egfp-er expression plasmid. the authors declare no conflict of interest.viruses , , key: cord- - g klpyg authors: guajardo-leiva, sergio; chnaiderman, jonás; gaggero, aldo; díez, beatriz title: metagenomic insights into the sewage rna virosphere of a large city date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: g klpyg sewage-associated viruses can cause several human and animal diseases, such as gastroenteritis, hepatitis, and respiratory infections. therefore, their detection in wastewater can reflect current infections within the source population. to date, no viral study has been performed using the sewage of any large south american city. in this study, we used viral metagenomics to obtain a single sample snapshot of the rna virosphere in the wastewater from santiago de chile, the seventh largest city in the americas. despite the overrepresentation of dsrna viruses, our results show that santiago’s sewage rna virosphere was composed mostly of unknown sequences ( %), while known viral sequences were dominated by viruses that infect bacteria ( %), invertebrates ( %) and humans ( . %). interestingly, we discovered three novel genogroups within the picobirnaviridae family that can fill major gaps in this taxa’s evolutionary history. we also demonstrated the dominance of emerging rotavirus genotypes, such as g and g , that have displaced other classical genotypes, which is consistent with recent clinical reports. this study supports the usefulness of sewage viral metagenomics for public health surveillance. moreover, it demonstrates the need to monitor the viral component during the wastewater treatment and recycling process, where this virome can constitute a reservoir of human pathogens. viruses are the most abundant biological entities on earth, with an estimated particles worldwide [ ] . urban environments impacted by human activity, such as wastewater treatment plants (wwtps) and sewage are not an exception. sewage and wwtps form an ecosystem that supports thriving microbial communities (prokaryotic and eukaryotic), plants and animals, such as rodents, birds and bats [ ] . in these environments, viruses associated with the biological waste of a city are mixed with viruses from all the organisms living in the wwtp, thus forming an untapped source of viral diversity [ , ] . in general, the sewage virosphere can be considered a mixture of human viruses excreted in the feces, urine and skin peeling, and viruses from animals, invertebrates, plants, fungi and bacteria [ , ] . sewage has been historically used to monitor known human viral pathogens, such as noroviruses, hepatitis viruses, enteroviruses, rotaviruses and adenoviruses [ , ] . the presence of viral pathogens in wastewater reflects ongoing infections being transmitted in the human population served by the given wwtp [ ] [ ] [ ] . likewise, sewage can reveal new and unknown viral genomes that could, in the future, be associated with idiopathic human diseases [ ] . nowadays, decreased water availability due to global warming and increased human water consumption have turned water scarcity into a cyclical problem. to solve this, recycled water derived from wwtp effluent has been intensively used for industrial operations, agricultural irrigation and even recreational activities [ ] . in this way, recycling of treated sewage generates a potential public health risk, as well as a critical risk for agricultural and animal production industries, due to insufficient removal of pathogenic viruses [ , , ] . current regulations have promoted improved treatment guidelines, which now combine mechanical, biological and chemical processes, such as flocculation, sedimentation, filtration, chlorination and uv-radiation [ , , ] . these treatments have significantly reduced microbiological contamination by inactivating and removing bacteria and protozoa, but they have little effect on human viruses, such as adenoviruses and enteroviruses, which are later dispersed in effluent waters [ ] [ ] [ ] . in latin america, the sanitary systems, which are still under development, have a low sewage treatment coverage ( %), generating a potential health risk for human and animal populations [ ] . chile is a middle-income country with a population of million inhabitants, of which approximately million inhabitants are concentrated in the capital, santiago de chile, making it the seventh largest city in the americas. most of santiago's sewage is decontaminated by three wwtps: el trebal, la florida and la farfana. el trebal serves . million equivalent inhabitants, and water decontaminated by this wwtp provides irrigation for , agricultural hectares of land that is mainly used for the production of fruits and vegetables consumed in santiago and exported according to international environmental standards. the sewage virosphere has been monitored world-wide using molecular techniques, such as pcr and quantitative pcr (qpcr) [ , ] . these methods can only provide information about the presence and abundance of known and characterized viruses because there is no universal marker for viruses, such as the s rrna gene for bacteria [ , ] . despite the increasing application of high-throughput sequencing techniques (hts) for viral metagenomics, their use in identifying viruses in sewage has not been well explored [ , , ] . however, the few existing studies have demonstrated that the study of the viral metagenomics of sewage is an excellent tool to monitor, identify and explore the diversity of the viral communities circulating among human and livestock populations [ , , , , , ] . this is especially important for rna viruses that, in the last decade and along with viral metagenomics, have undergone a revolution in terms of their discovery, thereby contributing to our understanding of viral diversity [ ] . to our knowledge, few studies [ , ] have used viral metagenomics to investigate rna viral communities in sewage around the world, none of which have been conducted in latin america. with this in mind, we conducted a pilot study to investigate the rna virosphere using a single sample equivalent to the sewage treated during one day in santiago (el trebal wwtp) using viral metagenomics. our main results, despite bias related to the overrepresentation of dsrna viruses (in comparison to other studies), show that the rna virosphere of el trebal is mainly composed of unknown sequences (microbial and viral dark-matter). the known viral sequence fraction was dominated by viruses that infect bacteria and invertebrate hosts. a high diversity and novelty were discovered within the picobirnaviridae family, which can fill major gaps in the evolutionary history of this group. likewise, we unveiled abundant and emergent rotavirus a genotypes never before recorded in chile, thus representing changes in the prevalence of the classical genotypes. the latter discovery provides evidence for the benefits of using viral metagenomics to aid public health surveillance based on excreted viruses in sewage. additionally, this study reveals the importance of analyzing viral dark matter through self-clustering of the sequences independent of their direct comparison to databases, which can result in the discovery and classification of new viral sequences. el trebal (hereafter referred to as trebal) is a wwtp ( • . " s • . " w) located in santiago, chile (figure ), that serves a population equivalent to . million inhabitants. a composite sample ( l), representing h of raw sewage, was obtained on jun . the sample was sequentially filtered through -and -µm pore size polycarbonate filters (isopore, mm diameter, millipore, milford, ma, usa) using a swinex filter holder (millipore) and then a . -µm pore size filter (sterivex pes, millipore). particles in the . -µm filtrate were concentrated by ultracentrifugation to a final volume of approximately ml, as described in [ ] . briefly, the . -µm filtered sample was centrifuged at , × g for h. the pellet containing viral particles was resuspended in glycine buffer and then incubated on ice for min. finally, after an additional ultracentrifugation at , × g for h, viruses were recovered by resuspending the viral pellet in ml of pbs. viruses , , x for peer review of el trebal (hereafter referred to as trebal) is a wwtp ( ° ′ . ″ s ° ′ . ″ w) located in santiago, chile (figure ), that serves a population equivalent to . million inhabitants. a composite sample ( l), representing h of raw sewage, was obtained on jun . the sample was sequentially filtered through -and -μm pore size polycarbonate filters (isopore, mm diameter, millipore, milford, ma, usa) using a swinex filter holder (millipore) and then a . -μm pore size filter (sterivex pes, millipore). particles in the . -μm filtrate were concentrated by ultracentrifugation to a final volume of approximately ml, as described in [ ] . briefly, the . -μm filtered sample was centrifuged at , × g for h. the pellet containing viral particles was resuspended in glycine buffer and then incubated on ice for min. finally, after an additional ultracentrifugation at , × g for h, viruses were recovered by resuspending the viral pellet in ml of pbs. the resuspended viral particles ( ml) were treated with dnase i ( u) to remove the remaining free dna from the cellular fraction. the mixture was incubated for h at °c, followed by inactivation at °c for min. viral rna was extracted using the high pure viral rna kit (roche, basel, switzerland) according to the manufacturer's instructions, but without the use of the poly(a) carrier. bacterial dna contamination was checked by s rrna gene pcr amplification using a universal bacterial primer set ( f: '-gtgycagcmgccgcggtaa- ' and r: '-ggactacnvgggtwtctaat- ') https://earthmicrobiome.org/protocols-and-standards/ s/. bacterial (e. coli jm ) dna was used as a pcr spike control to check for pcr inhibition of viral rna. the purified rna sample was then sequenced using illumina hiseq technology (roy j. carver biotechnology center, urbana, il, usa). briefly, the rnaseq library was prepared with the illumina truseq stranded mrna sample prep kit (illumina, san diego, ca, usa). the library was quantitated by qpcr and sequenced from one end of the fragment in a single lane for cycles on a hiseq . fastq files were generated and demultiplexed with the bcl fastq v . . . conversion software (illumina). raw metagenomic reads were quality filtered using cutadapt v . [ ] , leaving only sequences longer than bp (-m ), and conducting ′ end trimming for bases with a quality below (-q ) and hard clipping of the first five leftmost bases (-u ). finally, sequences representing simple the resuspended viral particles ( ml) were treated with dnase i ( u) to remove the remaining free dna from the cellular fraction. the mixture was incubated for h at • c, followed by inactivation at • c for min. viral rna was extracted using the high pure viral rna kit (roche, basel, switzerland) according to the manufacturer's instructions, but without the use of the poly(a) carrier. bacterial dna contamination was checked by s rrna gene pcr amplification using a universal bacterial primer set ( f: -gtgycagcmgccgcggtaa- and r: -ggactacnvgggtwtctaat- ) https://earthmicrobiome.org/protocols-and-standards/ s/. bacterial (e. coli jm ) dna was used as a pcr spike control to check for pcr inhibition of viral rna. the purified rna sample was then sequenced using illumina hiseq technology (roy j. carver biotechnology center, urbana, il, usa). briefly, the rnaseq library was prepared with the illumina truseq stranded mrna sample prep kit (illumina, san diego, ca, usa). the library was quantitated by qpcr and sequenced from one end of the fragment in a single lane for cycles on a hiseq . fastq files were generated and demultiplexed with the bcl fastq v . . . conversion software (illumina). raw metagenomic reads were quality filtered using cutadapt v . [ ] , leaving only sequences longer than bp (-m ), and conducting end trimming for bases with a quality below (-q ) and hard clipping of the first five leftmost bases (-u ). finally, sequences representing simple repetitions, which are usually due to sequencing errors, were removed using prinseq v . . [ ] at a dust threshold of (-lc_method dust, -lc_threshold ). details of the obtained sequences are shown in supplementary table s . viral rna metagenomes were assembled using de bruijn graphs, as implemented in the megahit v . . [ ] and idba-ud v . assemblers [ ] in metagenomic mode. only contig sequences > pb were further analyzed. both assemblies were merged by clustering contigs at % identity and % coverage of the shortest sequence, leaving the largest contig using the nucmer algorithm implemented in mummer v [ ] . after assembly, prodigal software [ ] was used to predict protein-coding regions, with options (-p meta -n). the predicted proteins were aligned against the ncbi nr database using diamond v . . [ ] (-e-value . ) and parsed using the lowest common ancestor algorithm in megan [ ] (lca score = ) using ncbi taxonomy tree to obtain the taxonomic annotation of each viral protein. species classification of viral proteins was used to infer putative hosts based on the virus-host db [ ] , as described in [ ] . the abundance of mapped proteins was quantified through read recruitment via bowtie [ ] , with parameters (-end-to-end-very sensitive-n ). the resulting sam file was parsed by the bbmap pileup script (bushnell b.-sourceforge.net/projects/bbmap/) and the relative protein abundances were normalized by gene length. the predicted proteins were functionally annotated using the pfam v database [ ] through hmmscan options (-cut_ga) implemented on hmmer [ ] . proteins annotated as rna-dependent rna polymerase (rdrp; predicted proteins) were taxonomically identified as described before, and the species classification was used to infer putative hosts. pairwise genetic distances were calculated for trebal rdrp nucleotide sequences and refseq (release ) using word-based alignment-free distances implemented on alfree software [ ] , with options (-s -d braycurtis-v counts). the pairwise distances between samples were analyzed by hierarchical clustering (hclust function in r) using a minimal increase in the sum of squares method (ward's method). the resulting dendrogram was cut (cutree function in r) into groups that represent the main clusters based on the "unrooted" dendrogram representation. the trebal proteins annotated as rdrp (> aa) using the pfam database [ ] and classified as picobirnaviridae were aligned by mafft v [ ] using default parameters, with the option -globalpair. a phylogenetic rdrp gene tree was constructed using the maximum likelihood method implemented with iqtree (-bb , -nm , -alrt -abayes) and , ultrafast bootstraps to evaluate branch robustness [ ] . the amino acid substitution model used in the phylogenetic analysis (vt + f + i + g ) was determined by modelfinder [ ] . reference sequences were obtained from ncbi refseq based on the phylogenetic analysis of the picobirnaviridae family from the international committee on taxonomy of viruses (ictv) [ ] , and a reference genome of the genus alphapartitivirus was used as an outgroup. a ribosomal binding site (rbs) prediction of the rdrp-predicted proteins used in the phylogenetic analysis was performed using prodigal software [ ] , as described before. nucleotide sequences of trebal proteins annotated as rotavirus vp , vp and vp using the pfam database [ ] were aligned against the ncbi nt database using blastn [ ] . the best hit (e-value < . ) classification of each nucleotide sequence was used to assign rotavirus species and type (g and p). raw sequences are available under ncbi sra bioproject prjna . assembled contigs and their annotation are available in the mg-rast server under the accession number mgm . and can be accessed at the following link https://www.mg-rast.org/linkin.cgi? project=mgp . the trebal rna viral fraction, sequenced on the illumina hiseq platform, yielded~ m high-quality reads (supplementary table s ). assembly of the rna metagenomic reads resulted in , contigs, which included . m reads and , predicted proteins. as with most viral metagenomes, only a small fraction (~ %) of the viral proteins matched to large databases [ , ] such as ncbi nr, representing~ % of the assembled reads. the high number of unmapped contigs ( % of the assembled reads) are most likely derived from novel and uncharacterized microbial and viral sequences, as has been observed in other wastewater studies [ , ] . a taxonomic survey of the predicted proteins shows that the known trebal reads ( figure a ) were assigned mostly to the virus domain ( %), followed by bacteria ( %) and eukarya ( %). cellular contamination of viral enriched fractions is a common feature of viral metagenomes [ ] [ ] [ ] . in this study, this could correspond to cellular transcripts, probably due to the lack of rnase treatment during nucleic acid preparation, or the presence of some residual dna after the dnase treatment, although the s rrna gene was not amplified by pcr. viral sequences can also be misannotated to homologous cellular genes [ , ] , which relies on the low number and diversity of viral sequences in the databases. additionally, horizontal gene transfer between viral and host genomes can lead to incorrect annotation based on the closest homologous sequences [ , ] . misleading annotation is a frequent phenomenon in underexplored communities, such as environmental rna viruses where new schemes of classification are needed [ ] . viruses , , x for peer review of "unclassified rna viruses shim- " category in the ncbi taxonomy (~ % abundance; figure b ) and totiviriade family were also highly abundant in treated and untreated sewage samples from the eu [ , ] . the cystoviridae family (~ % abundance; figure b ) is the only ictv-recognized bacteriophage family detected at more than % abundance. these bacteriophages are known to be abundant in the gastrointestinal tract of vertebrates and as part of raw sewage samples [ ] . only recently was this family reported in metagenomic assessments of sewage samples using previously published viral metagenomes from pittsburgh, barcelona and addis ababa [ , ] . finally, the reoviridae family, represented by the genus rotavirus, accounted for ~ . % abundance. these human pathogenic viruses are routinely found in raw sewage samples using amplification techniques such as rt-pcr and rt-qpcr [ , ] ; however, they have been detected by hts in a recent investigation in wales, uk [ ] . since some human pathogenic viruses are seasonal (e.g., rotavirus and norovirus), their presence in wastewater can also vary, which could explain the absence of rotavirus in previous metagenomic surveys of sewage [ ] . the assignment of viral species through the last common ancestor allowed us to classify the known viral sequences by their putative host using the virus-host database [ ] , as described in [ ] . most of the sequences belong to viruses putatively infecting bacteria ( %) and invertebrates (~ %) in further sections, we only analyze proteins classified as having viral origin. most of the viral proteins in the trebal rna viral metagenome ( figure b ) were classified as belonging to the picobirnaviridae family ( %), followed by partitiviridae (~ %) and other families, such as totiviridae (~ %), cystoviridae (~ %) and reoviridae (~ %). in summary, most of classified viral sequences ( %) belong to families comprising group iii (dsrna viruses) of the baltimore classification. nevertheless, ssrna (e.g., virgaviridae and leviviridae), dsdna (e.g., myoviridae and siphoviridae), and ssdna (microviridae) viral families were detected, but their relative abundances were below % (supplementary table s ). it is important to emphasize that since we did not perform any amplification step (e.g., mda) before library construction, it is not expected that relative viral abundances were affected by amplification bias in any steps before library preparation. thus, a possible explanation for the low abundance of ssrna viruses is that the inclusion of an inactivation step using dnase at • c could potentially enhance the effect of natural rnases present in the sample, as has been described before [ ] . however, this must be confirmed experimentally using an ssrna virus as spike control subjected to the indicated conditions in the same type of matrix. despite possibly missing some viral types during the extraction procedure, the characterized viral rna metagenome still harbors a vast diversity of viruses within each family. in general, rna viral families identified here have been identified in other previous studies that describe the rna virus diversity of wastewater [ , , , [ ] [ ] [ ] . however, the viral community's specific taxonomic composition in the trebal has not been reported in other sewage studies. picobirnaviruses (pbvs) were dominant in sewage influent samples from wales, uk [ ] . likewise, pbvs were prevalent in sewage samples across the usa [ ] and have been proposed as a potential marker of fecal pollution [ ] . viral sequences identified as partitiviridae-like viruses included in the "unclassified rna viruses shim- " category in the ncbi taxonomy (~ % abundance; figure b ) and totiviriade family were also highly abundant in treated and untreated sewage samples from the eu [ , ] . the cystoviridae family (~ % abundance; figure b ) is the only ictv-recognized bacteriophage family detected at more than % abundance. these bacteriophages are known to be abundant in the gastrointestinal tract of vertebrates and as part of raw sewage samples [ ] . only recently was this family reported in metagenomic assessments of sewage samples using previously published viral metagenomes from pittsburgh, barcelona and addis ababa [ , ] . finally, the reoviridae family, represented by the genus rotavirus, accounted for~ . % abundance. these human pathogenic viruses are routinely found in raw sewage samples using amplification techniques such as rt-pcr and rt-qpcr [ , ] ; however, they have been detected by hts in a recent investigation in wales, uk [ ] . since some human pathogenic viruses are seasonal (e.g., rotavirus and norovirus), their presence in wastewater can also vary, which could explain the absence of rotavirus in previous metagenomic surveys of sewage [ ] . the assignment of viral species through the last common ancestor allowed us to classify the known viral sequences by their putative host using the virus-host database [ ] , as described in [ ] . most of the sequences belong to viruses putatively infecting bacteria ( %) and invertebrates (~ %) ( figure c ). other relevant groups belong to known viruses that infect humans (~ . %). finally, there was a small contribution of viruses infecting plants, fungi, unicellular eukaryotes, non-mammal vertebrates and non-human mammals ( figure c ). most bacterial viruses belong to the picobirnaviridae ( %), cystoviridae (~ . %) and levoviridae (~ . %) families, while invertebrate viruses are associated with the partitiviridae-like sequences in the "unclassified rna viruses shim- " group and totiviridae family. human viruses were composed exclusively of sequences from the reoviridae family. viruses , , of as discussed above, most of these viral families and their hosts have been reported in previous metagenomic wastewater studies [ , , , , ] . interestingly, the most abundant viruses in these studies belong to the virgaviridae family and the caudovirales order that infect plants and prokaryotes, respectively; however, these viruses appeared in low abundance in our sample [ , ] . the only rna-based metagenomic study that was methodologically similar to our study also reported a high abundance of pbvs related sequences and rotaviruses [ ] . the presence of pbvs sequences in the trebal wastewater, which were genetically close to those found in animal feces, could be related to farm runoff, since the location of this wwtp (figure ) is outside the urban zone of santiago and surrounded by many irrigation channels. even though farm waste should not end up in the trebal, whose exclusive purpose is the treatment of sewage from santiago city, negligent handling of this waste could cause this result. the presence and high abundance of bacterial viruses (bacteriophages) is frequent in wwtps. their numbers are in part due to the release of phages that infect intestinal bacteria by human or animal defecation, but also from new infections of bacteria whose natural niche is the sewage ponds and sludge [ ] . bacterial rna viruses are poorly understood in comparison with their dna counterparts that are commonly found in sewage viral metagenomic studies [ , ] . the international committee on taxonomy of viruses (ictv) has only recognized two rna bacteriophage families, the ssrna family leviviridae and the dsrna family cystoviridae [ ] . both families are represented by only genomes in the ncbi refseq database (january ), which is low when compared to the viral dna genomes in the same database. however, it has been proposed that the picobirnaviridae is a new family of rna bacteriophages based on the presence of bacterial ribosome binding sites (rbss) upstream of the coding sequences, and also due to the lack of any consistent epidemiologic association with animal and human diseases [ , , ] . in contrast to previous studies [ , ] , most of the sequences associated with rna bacteriophages that we found correspond to the ictv-unrecognized picobirnaviridae family and the cystoviridae family, with only a small fraction associated with the leviviridae family. all the known members of the cystoviridae family infect pseudomonas species, which are commonly present in eutrophic environments, such as sewage and the human body [ ] . therefore, the abundance of these viruses in the trebal metagenome can expand the known sequence space associated with this family (only genomes are currently available in the ncbi database) and contribute to a better understanding of the bacteriophage biology related to rna genomes. invertebrate virus categories mostly include sequences of those that putatively infect annelids and arthropods, which was expected since these phyla have high densities in sewage stabilization ponds and aquatic environments of wwtps [ , ] . in previous studies, a high prevalence of these invertebrate rna viruses was not reported since only recently was their vast diversity discovered and their sequences made available in the databases [ ] . human viruses (which in trebal correspond to rotavirus a and c) are common in sewage and come from feces, urine, and respiratory secretions of infected hosts [ ] . the most commonly identified viral pathogens in wastewater are adenovirus, enterovirus, hepatitis a and e viruses, norovirus, sapovirus, and rotavirus a [ ] . these viruses are considered a potential public health risk because they are usually found at high concentrations in raw sewage, and their removal efficiency in wwtps is not commonly assessed [ , ] . moreover, since wastewater is composed of the excreta of thousands to millions of inhabitants, it is a representative sample that can be used for epidemiological surveillance purposes [ ] . therefore, metagenomic analysis of wastewater can highlight the presence of viral strains that circulate within a population, while enabling the discovery of new viruses that spread between humans and that are outside surveillance programs. to uncover the untapped viral diversity present in the trebal metagenome, which was not possible to recover through direct mapping of viral sequences against databases, we searched for rdrp homologous sequences using hidden markov models (hmms) for the protein. subsequently, we estimated the genetic diversity of the rna viruses using an alignment-free comparison of the retrieved rdrp sequences with those available in the ncbi refseq database (figure ). the rdrp is the most essential and conserved protein in rna viruses [ ] . it catalyzes rna synthesis from rna templates and is responsible for viral genome replication and transcription processes [ ] . viruses , , x for peer review of figure . hierarchical clustering analysis of rna-dependent rna polymerase (rdrp) predicted protein sequences from trebal and ncbi refseq database based on bray-curtis amino acid distance (k = ). dendrogram was divided in main cluster based on the "unrooted" dendrogram. pie charts represent the frequency of sequences in each cluster classified by the putative host trough lca algorithm. bar charts represent the source (ncbi or trebal) from which sequences were retrieved inside each cluster. to assess the novelty of the most abundant viral sequences observed in trebal, namely those of the picobirnaviridae family, we performed a phylogenetic analysis. first, we identified the rdrp protein sequences inside the picobirnaviridae family and then filtered them by size to include only proteins ≥ aa, which is the size of the smaller full-length pbv rdrp in the ncbi nr database. next, we reconstructed a phylogenetic gene tree (figure ) using rdrps that met the filtering criteria and reference pbv sequences. our results show that seven trebal sequences were associated with the known pbv genogroups [ ] i and ii associated with vertebrate stools. interestingly, the rest of the environmental sequences ( ) formed three separate monophyletic clades. two of these exclusive trebal monophyletic groups, tg and tg ( sequences), could be considered sister clades of the known pbv genogroup three (giii) associated with invertebrate samples [ ] . in contrast, the third monophyletic group, tg , which contains ten trebal sequences, is between the pbv genogroups one (gi) and two (gii) [ ] , but closer to gii. therefore, tg can represent a highly divergent version of viruses that infect bacteria of the gastrointestinal tract from other vertebrates, such as domestic, farm or wild animals. this is highly probable since pbvs are ubiquitous in the feces of a vast range of animal species worldwide [ , , ] , including cattle, monkeys, dogs, cats, bats, horses, poultry and chickens [ ] . for instance, pbv rdrps sequences recovered from a metagenomic survey of bat stools in cameroon showed a large group of highly divergent sequences that were closely related to the pbv figure . hierarchical clustering analysis of rna-dependent rna polymerase (rdrp) predicted protein sequences from trebal and ncbi refseq database based on bray-curtis amino acid distance (k = ). dendrogram was divided in main cluster based on the "unrooted" dendrogram. pie charts represent the frequency of sequences in each cluster classified by the putative host trough lca algorithm. bar charts represent the source (ncbi or trebal) from which sequences were retrieved inside each cluster. we identified predicted proteins as rdrps using protein hmms in the pfam database [ ] . most of the rdrp sequences ( %) were classified as unknown since they do not align with any known protein in the ncbi nr database under standard cutoff parameters (e-value ≤ × − and score ≥ ). the latter is expected since the software that was used is designed to detect remote homologs in the most sensitive way, based on the strength of the underlying probability models (hmms) [ ] . the remaining % of the rdrps corresponded to known viruses that putatively infect invertebrates ( %), bacteria ( %), unicellular eukaryotes ( %), fungi ( %), humans ( %), and plants ( %). hierarchical clustering analysis based on the genetic distances of rdrps showed well-defined genetic clusters, five of which were formed exclusively by ncbi sequences and five of which were formed mostly by the trebal sequences ( figure ) . moreover, the sequences were clustered by a single host only in three of the clusters (e.g., human viruses of cluster c , and plant viruses of clusters c -c ). additionally, most of the rdrps formed a continuous sequence space represented by highly heterogeneous clusters (e.g., c -c and c -c ). the latter feature was expected due to the orthologous nature of viral rdrps and their degree of structural conservation inside the riboviria viruses , , of realm [ , ] . despite this, a closer inspection of the clusters with stricter cutoffs could reveal more specific associations. the animal virus cluster c was formed exclusively by ncbi reference sequences inside the astroviridae and coronaviridae families, but no further precision regarding the host was feasible. astroviruses are a commonly known cause of viral gastroenteritis in animals and humans [ ] . specifically, sequences of cluster c corresponded to avian astrovirus associated with poultry and wild aquatic birds from a study in asia [ ] . coronaviruses have a global distribution and infect a wide range of mammals and birds. they can cause respiratory and enteric infections that are usually mild, but severe infections of the respiratory system can develop, such as severe acute respiratory syndrome (sars) and the infection currently responsible for a global pandemic, sars-cov- [ ] . coronaviridae sequences from cluster c were described in two studies from that investigated the viral population in bats in china and vietnam [ , ] . plant virus clusters c -c exclusively represent ncbi reference sequences of the luteoviridae (c ) and bromoviridae (c ) families. both viral families have a global distribution and are transmitted by specific aphid vectors [ , ] . these two viral families have a broad host range of genera within many plant families, causing necrosis in most of their hosts [ , ] . finally, the human virus cluster c was formed exclusively by ncbi sequences of the norovirus genogroup ii (nov gii). noroviruses are a genetically diverse genus within the caliciviridae family that can cause acute gastroenteritis in mammalian hosts [ ] . most of the human noroviruses belong to genogroups gi and gii, where nov gii is usually the causal agent of acute gastroenteritis outbreaks [ , ] . these pandemic characteristics are probably related to the nov gii epidemiology, which resembles that of influenza a viruses, with the emergence of new variants every - years that replace the previously established variant [ ] . interestingly, bacterial viruses form two clusters, c and c , which group picobirnaviridae reference sequences from the ncbi, recovered from animal stools and trebal new pbvs, together with unknown sequences that escaped our analyses using direct mapping against ncbi databases. this reflects the great diversity within the picobirnaviridae family, which is not represented in current databases. taken together, our results show that metagenomic surveys of rna viruses in sewage samples and the use of hmms could uncover extraordinary viral diversity through the detection of remote homologs in these human-impacted environments. additionally, the use of alignment-free genetic distances, such as the bray-curtis distance used here [ ] , can provide a reliable method for clusterization and classification based on related sequences for a large number of sequences, such as those generated by metagenomics methods. to assess the novelty of the most abundant viral sequences observed in trebal, namely those of the picobirnaviridae family, we performed a phylogenetic analysis. first, we identified the rdrp protein sequences inside the picobirnaviridae family and then filtered them by size to include only proteins ≥ aa, which is the size of the smaller full-length pbv rdrp in the ncbi nr database. next, we reconstructed a phylogenetic gene tree ( figure ) using rdrps that met the filtering criteria and reference pbv sequences. our results show that seven trebal sequences were associated with the known pbv genogroups [ ] i and ii associated with vertebrate stools. interestingly, the rest of the environmental sequences ( ) formed three separate monophyletic clades. two of these exclusive trebal monophyletic groups, tg and tg ( sequences), could be considered sister clades of the known pbv genogroup three (giii) associated with invertebrate samples [ ] . in contrast, the third monophyletic group, tg , which contains ten trebal sequences, is between the pbv genogroups one (gi) and two (gii) [ ] , but closer to gii. s ). we found that all except one of the full sequences (those not predicted in contig edges) contain an rbs, being aggagg and aggag, which are the most frequent motifs. this matches with other sewage pbvs that have the aggagg motif in % of the full rdrp [ ] . this result is relevant because only prokaryotic viral families contain species whose genomes are highly enriched in rbs sequences [ , ] . finally, pbvs have been assumed to be animal pathogens based on inferences from a few studies reporting the virus in diarrhea stool samples. however, they have not been cultured in animal cell lines, nor do they have any consistent epidemiologic association with diarrhea [ ] . rotavirus species are classically defined by their major capsid protein vp , whereas rotavirus genotypes are defined based on their outer capsid proteins vp and vp [ ] . in chile, over the last therefore, tg can represent a highly divergent version of viruses that infect bacteria of the gastrointestinal tract from other vertebrates, such as domestic, farm or wild animals. this is highly probable since pbvs are ubiquitous in the feces of a vast range of animal species worldwide [ , , ] , including cattle, monkeys, dogs, cats, bats, horses, poultry and chickens [ ] . for instance, pbv rdrps sequences recovered from a metagenomic survey of bat stools in cameroon showed a large group of highly divergent sequences that were closely related to the pbv giii [ ] , as is the case of tg and tg . this last evidence provides a probable origin for tg and tg since sewage ponds are known to be a feeding area for insectivorous bats [ ] . new evidence of pbv sequences from sewage samples shows that sewage-recovered rdrps were spuriously distributed between many pbv genogroups [ ] . the latter reinforce the idea that pbvs do not infect mammals but are a new family of rna bacteriophages, due to the consistent presence of bacterial rbss upstream of the coding sequences [ , , ] . to test this hypothesis, we searched for prokaryotic rbs motifs in the rdrp sequences from the pbvs (supplementary table s ). we found that all except one of the full sequences (those not predicted in contig edges) contain an rbs, being aggagg and aggag, which are the most frequent motifs. this matches with other sewage pbvs that have the aggagg motif in % of the full rdrp [ ] . this result is relevant because only prokaryotic viral families contain species whose genomes are highly enriched in rbs sequences [ , ] . finally, pbvs have been assumed to be animal pathogens based on inferences from a few studies reporting the virus in diarrhea stool samples. however, they have not been cultured in animal cell lines, nor do they have any consistent epidemiologic association with diarrhea [ ] . rotavirus species are classically defined by their major capsid protein vp , whereas rotavirus genotypes are defined based on their outer capsid proteins vp and vp [ ] . in chile, over the last ten years, globally common rotavirus genotypes, such as g p ( ), g p( ), g p( ) and g p( ) have alternated in their dominance, while emerging genotypes, such as g p ( ), have only recently been reported [ ] . here, using pfam annotation of the trebal predicted proteins, we recovered vp , vp and vp human rotavirus proteins. local alignment-based classification ( figure ) shows that the most abundant rotavirus species belongs to human rotavirus a, which is the most common cause of hospitalization due to viral gastroenteritis worldwide [ ] . however, it is interesting to note that the presence (in low abundance; . %) of human rotavirus c sequences that are closely associated to asian strains, have, to date, only been reported in chile through personal communication. likewise, the most abundant rotavirus genotypes were g ( %), g ( %) and g ( %) and p ( %) and p ( %). the segmented nature of rotavirus genomes does not allow us to infer the g-p genotype classification because we do not know which combinations of vp and vp were inside the same viral particle. however, it is highly probable that the most abundant g genotypes were combined with the most abundant p genotypes-for example, g p( ), g p( ) or g p ( ) . of these genotypes, g p( ) was recently reported as an emergent rotavirus strain in a medium-sized city near santiago between and [ ] . this strain has not been previously reported in south or north america, but has a highly similar identity to sequences detected between and in asia [ ] . in the same line of evidence, the g sequences found in trebal also shared a % nucleotide identity with sequences detected in in thailand [ ] and between and in japan [ ] . the rotavirus g genotype is also an emergent strain, with most of the reports concentrated in europe between - , but spurious circulation has been reported since the s [ , ] . in our analysis, the g recovered from the trebal sequences are closely related ( % nucleotide identity) to a g p( ) strain detected in germany in and to a g p( ) strain detected in belgium in ; yet, to our knowledge, this is the first report of rotavirus g in chile. these results emphasize the relevance of sewage viromes as epidemiological surveillance tools. likewise, our study demonstrates the advantage of using viral metagenomics for this task, which, despite using short sequences, can deliver reliable results that can later be confirmed by other methodologies. the latter point is especially relevant for rotavirus since surveillance based on pcr has shown serious bias related to primer specificity [ ] . these results are especially significant for chile, as it is one of the few south american countries that has not implemented a national rotavirus vaccination program [ ] . therefore, identification of genotypes that have not been previously reported in chile represents a first step in rotavirus prevention. furthermore, this can be used to generate valuable information to improve or implement new vaccination programs against this disease worldwide. this study explored the use of viral metagenomics to discover rna viruses in sewage and it is the first insight into the wastewater virosphere from a large city in south america. we have demonstrated the utility of metaviromics for the discovery of new groups and viral genotypes associated with known families. this is especially important due to the underrepresentation of pbvs in databases and the presence of uncommon rotavirus genotypes that are usually beyond the range of pcr-based surveillance. 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diarrhea in bulgaria this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we want to thank to christina ridley for her help in the language editing of this manuscript and for her valuable opinion on it. we are grateful to aguas andinas staff, marcela etcheberrigaray, jacqueline pizarro, and christian sepulveda, for their invaluable support in obtaining the sewage samples. the authors declare no conflict of interest. key: cord- -mameyzih authors: shi, da; lv, maojie; chen, jianfei; shi, hongyan; zhang, sha; zhang, xin; feng, li title: molecular characterizations of subcellular localization signals in the nucleocapsid protein of porcine epidemic diarrhea virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: mameyzih the nucleolus is a dynamic subnuclear structure, which is crucial to the normal operation of the eukaryotic cell. the porcine epidemic diarrhea virus (pedv), coronavirus nucleocapsid (n) protein, plays important roles in the process of virus replication and cellular infection. virus infection and transfection showed that n protein was predominately localized in the cytoplasm, but also found in the nucleolus in vero e cells. furthermore, by utilizing fusion proteins with green fluorescent protein (gfp), deletion mutations or site-directed mutagenesis of pedv n protein, coupled with live cell imaging and confocal microscopy, it was revealed that, a region spanning amino acids (aa), – in region of the n protein was sufficient for nucleolar localization and r and r were critical for its function. we also identified two nuclear export signals (nes, aa – , and – ), however, only the nuclear export signal (aa – ) was found to be functional in the context of the full-length n protein. finally, the activity of this nuclear export signal (nes) was inhibited by the antibiotic lepomycin b, suggesting that n is exported by a chromosome region maintenance -related export pathway. porcine epidemic diarrhea (ped) was first recognized as a devastating enteric disease in feeder and fattening pig, resembling transmissible gastroenteritis (tge) in pigs in the united kingdom, in . it is a member of coronavirinae, which are single-stranded, positive-sense rna viruses with the largest genome that are known to infect humans, other mammals, and birds, usually causing subclinical or respiratory and gastrointestinal diseases. the porcine epidemic diarrhea virus (pedv) subgenomic mrnas, which are transcribed from the genome, produce viral proteins, such as the spike (s, - kda), envelope (e, ~ . kda), membrane (m, - kda), nucleoprotein (n, [ ] [ ] [ ] [ ] , and several other proteins of unknown function [ ] [ ] [ ] . among the proteins, n, as the rna-binding protein, play an important role in both virus rna synthesis and modulating host cell processes, and phosphorylation may regulate these processes by exposing various functional motifs [ , ] . several other functions have been postulated for the coronavirus n protein throughout the virus life cycle, including encapsidation, packaging, correct folding of the rna molecule, the deregulation of the host cell cycle [ ] [ ] [ ] , inhibition of interferon production [ , ] , up-regulation of cox production [ , ] , up-regulation of ap activity [ ] , induction of apoptosis [ ] [ ] [ ] , association with host cell proteins [ ] , and rna chaperone activity [ ] . therefore, it is clear that n is a multifunctional protein involved in biological processes related to the survival of pedv. the nucleolus was one of the first subcellular structures to be identified by early users of the light microscope, appearing as a highly refractive black dot(s) in the nucleus of the cell [ ] . the nucleolus is a highly specialized structure that participates in regulation of several host cell processes, including ribosome subunit biogenesis, rna processing, control of cell growth and response [ ] . interestingly, a cytoplasmic-nucleolar distribution pattern has been reported for the n proteins of several coronaviruses, including representative members of alphacoronavirus (transmissible gastroenteritis virus, tgev), betacoronavirus (mouse hepatitis virus, mhv; and severe acute respiratory syndrome coronavirus, sars-cov), and gammacoronavirus (infectious bronchitis virus, ibv) [ , , ] . further study indicated that n protein co-localize with major nucleolar proteins, including nucleolin, fibrillarin, and nucleophosmin [ ] [ ] [ ] . how viral and cellular proteins traffic to the nucleolus and what determines their sub-nucleolar localization is not clearly understood, but proteins that localize to the cytoplasm and nucleus or nucleolus contain multiple signals that determine their subcellular localization [ ] , such as nucleolar localization signal (nols). active nuclear import of proteins is mediated by nuclear localization signals (nlss), which are then recognized by proteins of the importin super-family (importin α and β) that mediate the transport across the nuclear envelope using ran-gtp [ ] . similar to nuclear import, export of a protein from nucleus depends on the presence of a specific nuclear export signal (nes) [ ] . the chromosome region maintenance (crm ; also known as exportin or xpo ) has been identified as an export receptor that interacts with the predominant nes, the so-called leucine-rich nes, which is found in a large variety of nucleocytoplasmic shuttling proteins [ , ] . in fact, some of these ness are not necessarily leucine rich but rather characterized by several hydrophobic residues. the pharmacological compound leptomycin b (lmb) directly interacts with crm and blocks nes-mediated protein export [ ] . therefore, the proteins can shuttle between the nucleus and the cytoplasm with their subcellular localization signals. it was reported previously that n protein nucleolar localization is a common feature in coronaviruses, however, there are different results regarding n subcellular localization in a strain of sars-cov [ ] . within the alphacoronavirus coronaviruses, the precise nols and nes of pedv n and its traffic mechanism are still elusive. therefore, we have attempted to characterize these signals, and the molecular mechanism responsible for its subcellular localization. in this study, we examined the intracellular localization of the pedv n protein in pedv-infected and transfected cell lines using mouse polyclonal antisera and confocal microscopy. by generating a series of deletion and mutagenesis constructions, we found that amino acids - in region were sufficient for nucleolar retention and we also identified two ness (aa - and - ), but only the nes (aa - ) was found to be functional in the context of the full-length n protein. the nucleocytoplasmic shuttling of n and the nuclear export of gfp-nes could be blocked by lmb, an inhibitor of the crm , which is the receptor for exportin- -dependent nuclear export. a polyclonal antibody specifically against the n protein was produced to determine its intracellular localization. we generated anti-n mouse antisera using an e coli-produced fusion protein as the antigen. the antigenicity of the recombinant n protein was confirmed by immunoreactivity with pedv pigs sera using elisa assay, which showed high sensitivity and specificity (data not show). to examine the reactivity and specificity of the mouse antiserum, blot results demonstrated that mouse antisera notably reacted with n protein from cv strain pedv, and the cell lysate from vero e cells transfected with pcdna . -n showed a band with the same molecular mass to n protein, whereas no band was detected from samples of cells uninfected pedv and transfected with an empty vector alone ( figure a ,b). our results showed that n protein was localized predominantly in the cytoplasm in pedv cv -infected cells, while in a few cells fluorescence was also observed in the nucleus (or nucleolus) ( figure a ). no significant fluorescence was observed in uninfected cells (data not shown). a similar observation was also found in vero e cells transfected with plasmid expressing full length n protein ( figure b ). the n protein was observed to localize mainly in the cytoplasm with some protein in the nucleus (or nucleolus). the results suggested that the n protein localized to a subnuclear structure and may contain functional signals. to identify predicted nuclear (or nucleolar) localization signals, and whether they participate in this process or not, we conducted further experiments. after h, infected or transfected, cells were fixed and analyzed by indirect immunofluorescence using mouse anti-n polyclonal antibody (green) and stained with pi (red) to visualize the nuclear dna. differentially fluorescing images were gathered separately using confocal microscopy. images were obtained with a × oil objective. to identify whether there were subcellular localization signals in pedv n protein, we first conducted a bioinformatics analysis on the protein using existing motif prediction algorithms. predict nls [ ] and psort ii [ ] were used to identify potential nlss, and the nes predictor (net nes) [ ] was used to identify potential ness. predict nls found no nlss, whereas psort ii indicated that pedv n protein contained a pat motif ( pkknksr ). net nes found no nes. in other studies, coronaviruses, such as tgev, mhv, ibv, and sars-cov showed a common characteristic, that of nls-rich in c-terminal. so through amino acid sequence comparison among groups we also found a basic amino acid-rich short peptide ( rkkekknkre ) in c-terminal. we presumed it might play a role in n protein localization as a new localization signal, and named it patx. although n protein contains putative nuclear (or nucleolar) localization signals (n (or no) ls), it is not known whether they are functional or not. to investigate whether these and other unknown signals operated to determine the subcellular trafficking of n protein, we utilized acgfp as a fusion marker to observe the localization characteristics of n and truncated n protein in live cells. the b . -dsred fusion protein was used to tag the nucleolus, so we could analyze nucleolar localization properties and colocalization in cotransfected cells by live cell imaging (direct fluorescence) or confocal microscopy. at h post-cotransfection, live cell imaging indicated that as previously shown, acgfp evenly distributed throughout the cytoplasm and the nucleoplasm but not the nucleolar, on the contrary acgfp-n protein localized to both the cytoplasm and nucleolus but not the nucleus in vero e cells ( figure ). from the above results we hypothesize that there is a nols in n, which can guide exogenous protein to the nucleolus. to determine the exact nols, the acgfp-tagged truncated different regions of the n protein were transfected into vero e cells, which did not destroy the integrity of the signals. in our observation, acgfp-nr mainly localized to nucleolar structures, acgfp-nr localized predominantly to the cytoplasm and appeared also to accumulate in the nucleolus to the low level as the cytoplasm, whereas acgfp-nr predominantly localized to the cytoplasm; acgfp-nr + protein same to acgfp-n and localized to the cytoplasm and nucleolus, acgfp-nr + protein localized predominantly in cytoplasmic ( figure ). this evidence demonstrated that region targeted to the nucleolus with weaker enrichment, while region localized to the nucleolus with stronger enrichment. thus, we speculated that region have an effect on the nucleolar localization of n. this data also suggested that region and may contain ness because region and was mainly directed to the cytoplasm. interestingly, although region and contained predicted pat and patx motifs, respectively, they could either have been submissive to the nes or not functional. none of these fusion proteins had a distribution similar to acgfp only. to further identify the sequence for nucleolar localization in detail, a series of expression constructs containing fragments of region were constructed. vero e cells were cotransfected with, either pacgfp-nr - , pacgfp-nr - , or pacgfp-nr - , and pdsred-b . , analyzed using live cell imaging and confocal microscopy at h post-transfection. the data indicated that acgfp-nr - colocalized with b . , whereas the other two fusion proteins did not ( figure ). to further refine the amino acids involved in nucleolar localization, amino acid overlapping motifs encompassing amino acids - were cloned into downstream of acgfp, creating plasmids pacgfp-nr - , pacgfp-nr - , pacgfp-nr - , and pacgfp-nr - for the expression of recombinant fusion proteins. vero e cells were cotransfected with these constructs and pdsred-b . , at h post-cotransfection analyzed using live cell imaging and confocal microscopy ( figure ). the data indicated that acgfp-nr - localized to the nucleolus and colocalized with b . . therefore, the amino acids at positions - in pedv n protein were capable to localize in the nucleolus. comparison of the pedv n protein nols with known cellular and viral nolss showed that the r and r of the pedv n protein nols might be conserved, although some cellular and viral nolss in this site did not contain basic amino acids ( figure ). current work is directed at further resolving pedv nols sequence, including the contribution of individual amino acid residues. red squares indicate the amino acids of conservation. the cellular and viral nolss are described in nols ibv n protein [ ] , nols prrsv n [ ] , nols htlv- rex [ ] , nols hsv gamma . [ ] , nols mdm [ ] , nols mdv meq [ ] , nols nf-kappa [ ] , nols nuclear vcp-like protein (nvl ) [ ] , nols p [ ] , nols surviving-deltaex [ ] , nols (ggnnv) protein alpha [ ] , bhv- bicp [ ] , earning-associated protein - [ ] , hsv- icp [ ] , nols angiogen [ ] , nols fibroblast growth factor- [ ] , nols herpes-mareks meq [ ] , nols hic p [ ] , nols hiv- rev [ ] , nols hiv- tat [ ] . to map the amino acid sequence of region responsible for its nuclear export, similar to the approach used to identify the nols in region , region was subdivided into two distinct components. amino acids - and - were placed downstream of acgfp, creating expression vectors pacgfp-nr - and pacgfp-nr - . these expression plasmids were transfected into vero e cells, the nuclear was stained with dapi at h post-transfection indicated that amino acids - directed acgfp to the cytoplasm and nucleus and had a subcellular localization similar to acgfp. in contrast, amino acids - directed acgfp to the cytoplasm; further investigation revealed that amino acids - and - , when fused to acgfp, directed this protein to the cytoplasm and nucleus, whereas amino acids - directed acgfp to the cytoplasm (figure ) . to further define the amino acids involved in cytoplasm trafficking, we conducted a tetra-alanine substitution mutagenesis of amino acids - . these were placed down stream of acgfp, creating expression plasmids, pacgfp-nr - dlva-aaaa , pacgfp-nr - avkd-aaaa , pacgfp-nr - alks-aaaa , pacgfp-nr - lgig-aaaa and pacgfp-nr - enpd-aaaa . therefore, in some cases, the wild-type alanine was not substituted. the data indicated that substituting - dlva-aaaa, - alks-aaaa, and - lgig-aaaa abolished cytoplasm trafficking. the remaining tetra-alanine substitutions had no effect on cytoplasm trafficking (figure ), indicating that amino acids dlvaavkdalkslgig were involved in cytoplasm trafficking. to test whether this amino acid sequence was involved in directing the cytoplasm trafficking of n protein, this motif was deleted in the context of full-length n protein tagged to acgfp (plasmid pacgfp-n ∆ - ). this plasmid was transfected into vero e cells and the subcellular localization of the resulting fusion protein acgfp-n ∆ - investigated using confocal microscopy. there was no difference at h post-transfection ( figure ) compared with cells expressing acgfp-n protein. this data also indicated that the nes identified in region was not necessary for cytoplasm trafficking in pedv n protein. as region of pedv n protein localized to the cytoplasm, thus, the similar approach was used to identify the nes in region . the data indicated that amino acids - directed acgfp to the cytoplasm; further investigation revealed that amino acids - directed acgfp to the cytoplasm ( figure ). taken together, we proposed that amino acids - are necessary and sufficient to direct n protein to the cytoplasm, and no other signals are involved. to determine if the pedv n protein was exported via the crm -mediated pathway, the subcellular localization of the pacgfp-n, pacgfp-nr or pacgfp-nr - , was compared between vero e cells left untreated or treated with . ng/ml lmb. as shown in figure , the nucleocytoplasmic shuttling of pedv n, nr and nr - was completely inhibited by lmb. the finding indicates that pedv n shuttling activity was affected by lmb and, thus, suggests that pedv n protein was transported via the classical crm -dependent pathway. pedv is an important pathogen causing viral diarrhea in the swine industry. although much research has been carried out on the general characteristics of pedv, few reports have been reported on the functions of the pedv structural proteins, especially n protein. n protein plays an important role in virus replication and modulation of host cellular machinery, which should be a result of its self-interaction and interaction with other viral and cellular proteins and with virus and host cell nucleic acids. therefore, it is important to understand the subcellular localization properties of the pedv n protein. figure . the nuclear export mechanism of pedv n. vero e cells were transiently transfected with plasmids encoding pacgfp-n, pacgfp-nr , pacgfpnr - , with or without treatment with lmb, and examined live h after transfection by confocal microscopy. each image is representative of the majority of the cells observed in the same cells. the nucleolus (no) is arrowed where appropriate. to enter and export from the nucleus, all molecules or cargoes must traverse a large macromolecular structure called the nuclear pore complex (npc), which is located in the nuclear envelope. small molecules up to - kda or less than nm in diameter can passively diffuse through the npc, but if proteins are larger than this size-exclusion limit and/or are required to move against a concentration gradient, then transport requires energy-driven mechanisms [ ] . in this case, most proteins should contain the appropriate trafficking motifs, such as nls. the rules and signals that govern the nuclear localization of proteins are well defined. nlss can be classified into several categories, including the pat and pat motifs and bipartite nlss, are composed of basic amino acids of a given sequence length. the pat motif consists of a continuous stretch of (usually) four basic amino acids, and the pat motif starts with a proline residue and is followed by six amino acids [ , ] . by contrast, the signals that govern nucleolar localization and retention are not well defined [ ] . the motifs involved are usually rich in arginine and lysine residues; however there is no immediately obvious consensus sequence or structure. proteins that localize to the nucleolus can also have nuclear-import motifs. the nolss that have been identified so far can be grouped into those that contain single motif and those that contain multiple motifs [ ] . in this study, the living cells fluorescence microscopy and confocal microscopy were employed for investigating the subcellular localization and nuclear import and export mechanisms of pedv n protein. it is well known that living cells fluorescence microscopy and confocal microscopy has an advantage over conventional in vitro nuclear transport assays in that cells are not physically damaged by microinjection, detergent, or mechanical perforation, this means that cellular components important for trafficking, such as nuclear import and export receptors, the mt network, intact [ , ] . our results indicated that pedv n protein mainly distributes throughout the cytoplasm with localization to a substructure within the nucleolus in infected cells. similar results were also obtained in transfected cells. to eliminate the influence of charged protein migration to the nucleolus post-fixation, and to investigate the nuclear (or nucleolar) localization of pedv n protein and the functions of these motifs in more detail, we generated constructs that express the protein (or parts of the protein) as a fusion with enhanced green fluorescent protein. the protein could then be detected by direct fluorescence using both live-cell and confocal microscopy. no difference in the localization of either protein was observed between different cell lines, and the presence of a fluorescent tag at either the n terminus or c terminus of n protein did not affect the localization of the fusion protein compared with native protein [ , ] . furthermore, the b . gene was amplified from the vero e cells and then fused with dsred in order to visualize the nucleolus. transfection assay results indicated that acgfp localized predominately to the cytoplasm and the nucleus, but not the nucleolus. the characteristics of acgfp-n protein localized to either the cytoplasm alone or the cytoplasm and nucleolus, with a maximum of % of transfected cells exhibiting this phenotype at h. our studies indicated that fusing acgfp with pedv n protein increased the molecular weight of this protein ( kda) above the size exclusion limit of the nuclear pore complex, and it could not diffuse passively through the npc. this result suggested that there must be some signals in n protein that determined the nucleolar localization. a number of viruses and viral proteins can disrupt nucleolar architecture [ ] , and the n protein of coronavirus can also localize to the nucleolus in a cell cycle dependent manner and this may be related to dynamic trafficking [ ] . a previous study of sars-cov indicated the n protein inhibited b phosphorylation and might influence ribosome biogenesis to suppress host gene expression and create a more favorable milieu for virus survival [ ] , so the n protein of pedv might take part in some cellular process. to investigate whether pedv n protein contained a nols, the protein was expressed as a series of single and overlapping regions. this preliminary analysis indicated that pedv n protein contained a nols in region . deletion mutagenesis delineated amino acid pedv n - , a aa motif that modulated nucleolar localization. furthermore, comparison with cellular and viral nolss, the data indicated that r and r may be critical for the nucleolar localization. for the site of replication and virus assembly is the cytoplasm, if the subcellular localization of the nucleocapsid protein is in the nucleolar, then the protein is not available for rna synthesis, encapsidation and assembly, and therefore progeny virus production might be less efficient. however, if such proteins do target the nucleolar as part of a virus replication strategy then they will contain appropriate targeting signals. thus, these will include not only nlss and nolss, but perhaps more importantly nes. by using a series of n deletion mutants fused to the acgfp and tetra-alanine substitution mutagenesis, we identified two nes site in region (aa - ) and region (aa - ), but nes in region was deleted in the context of full length n protein did not affect cytoplasm trafficking, suggesting that the nes presented in region submissive to the nes in region or not function. furthermore, pedv n protein nucleocytoplasmic shuttling was specifically blocked by lmb treatment suggested that pedv n protein is transported via the classical crm -dependent pathway. vero e was grown and maintained in dulbecco's modified eagle's medium supplemented with % heat-inactivated fetal calf serum and penicillin-streptomycin, and incubated at °c in % co . vero e cells were infected with pedv strain cv kindly provided by pensaert m b at moi = , and were seeded and after h for both indirect immunofluorescence assay and western blot analysis. all enzymes used for cloning procedures were purchased from takara (dalian, china) except t dna ligase from new england biolabs (ipswich, ma, usa). pdsred-b . encodes a fusion of dsred protein to the amino terminus of b . and pacgfp-n encodes a c-terminally gfp-epitope-tagged version of pedv n and has been previously described [ ] . the regions and subregions were amplified by pcr using pacgfp-n as a template. primers incorporated ' bamh i site and ' xho used for cloning into pet a; primers incorporated ' kpn i site and ' xho i site used for cloning into pcdna . and the other primers incorporated ' xhol i site and ' kpn i site used for cloning into pacgfp-c . the cloning procedures for constructing most of recombinant plasmids were similar and all primers are listed in table . at all times, numbers used in primer or construct names denoting amino acid numbers refer to their position on the full length n protein. for the tetra-alanine substitution, each pair of oligonucleotide strands was annealed, and the resultant double-stranded dna fragments were cloned into the vector pacgfp-c . additionally, the nes of nr was deleted in the context of full-length n protein by overlapping pcr using forward primer -u and reverse primer -l (table ) to generate one pcr product and forward primer -u and reverse primer -l (table ) to generate the second pcr product. a second round of pcr was performed using both pcr products as templates and -u and -l as the forward and reverse primer, respectively. the resulting product was subcloned into pacgfp-c . all plasmid were confirmed by sequencing analysis. the recombinant × his-tagged n protein was expressed in e. coli bl (de ) cells after induction with mm iptg for h in lb-medium at °c . the recombinant protein was purified by a gravitraptm affinity column (ge healthcare, bio-sciences, piscataway, nj, usa) according to the manufacturer's instructions. balb/c mice were immunized with . ~ . mg of purified recombinant n protein injected subcutaneously at multiple sites on the back. booster injections were given two and four weeks later. blood was drown from the mouse at the fifth week following the immunization, and the blood was allowed to clot at °c and the antiserum was recovered by centrifugation at , × g for min at °c . a control serum was made by injecting normal saline under the same conditions. the animal experiment was approved by harbin veterinary research institute and performed in accordance with animal ethics guidelines and approved protocols. the animal ethics committee approval number is heilongjiang-syxk- - . on the day before transfection, . ~ × cells grown on mm culture dishes so that %- % confluent cell monolayers were transfected with plasmid dna with lipofectamine™ (invitrogen, groningen, netherlands) according to the instructions of the manufacturer. protein samples were collected h after transfection by direct lysis of the cells in × sds protein sample buffer with % -mercaptoethanol. the samples were boiled for min, resolved with % sds-page, and blotted with the following antibodies. for the infection and transfection protein samples: the n protein polyclonal antiserum was the first antibody and irdyetm dx conjugated affinity purified anti-mouse igg (h&l)(mouse) (li-cor biosciences, lincoln, ne, usa) was the second antibody; the images were acquired by the odysseytm infrared imaging system (li-cor). vero e cells were grown on coverslips and fixed h post-infection (or post-transfection) with % methanol- % acetone for analysis by indirect immunofluorescence using mouse anti-pedv n polyclonal sera ( : dilution) followed by fluorescein isothiocyanate (fitc)-labeled goat anti-mouse antibody (sigma, st louis, mo, usa). the cells were washed twice with phosphate-buffered saline (pbs) and subjected to perforation using . % triton x- . then the cells were stained with propidium iodide (pi) ( μg/ml) (sigma) to visualize nuclear dna. samples were analyzed using fluorescence microscopy. for acgfp and dsred fusion expression constructs, co-transfection analyses were carried out. at h post-transfection, subcellular localization properties in living cells were analyzed by laser confocal scanning microscope (leica laser technik, heidelberg, germany) with appropriate filters. in order to better display the results at the time of identify the nes in the regions of nr and nr , at h after transfection, cells on glass cover slips were rinsed with pbs and subjected to fixation using % methanol- % acetone for min and permeabilized with . % triton x- . then the nuclear was stained with ', -diamidino- -phenylindole (dapi) ( . μg /ml) (sigma) and analyzed by confocal microscope. cells were transfected with pacgfp-n, pacgfp-nr , or pacgfp-nr - . at h post-transfection, cells were washed with pbs followed by either treated with . ng/ml lmb diluted with medium or left in unsupplemented cell culture medium for a further h, then analyzed by used confocal microscope. a nols (aa - ) and a functional nes (aa - ) of pedv n protein were identified for the first time. additionally, the n protein was demonstrated to transport between the nucleolus and the cytoplasm through the nes by crm -dependent pathway. characterization of the structural proteins of porcine epizootic diarrhea virus, strain cv the coronavirus infectious bronchitis virus nucleoprotein localizes 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function effects of a highly basic region of human immunodeficiency virus tat protein on nucleolar localization classical nuclear localization signals: definition, function, and interaction with importin alpha transport into and out of the nucleus. microbiol ppar γ mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance function of dynein and dynactin in herpessimplex virus capsid transport p is associated with cellular microtubules and is transported to the nucleus by dynein rna viruses: hijacking the dynamic nucleolus cell cycle dependent localization of the coronavirus nucleocapsid protein co-localization analysis between porcine epidemic diarrhea virus nucleocapsid protein and nucleolar phosphoprotein b . this work was supported by grants from the national natural science foundation of china ( ), the natural science foundation for distinguished young scholars of heilongjiang province (jc ), the agricultural scientific and technological transformative project ( gb ), and higher school science and technology innovation team project of heilongjiang province ( td ). the sponsors had no role in study design, collection, analysis, and interpretation of the data, writing the report, and in the decision to publish the results of the study. the authors declare no conflicts of interest. key: cord- - b authors: ianevski, aleksandr; yao, rouan; fenstad, mona høysæter; biza, svetlana; zusinaite, eva; reisberg, tuuli; lysvand, hilde; løseth, kirsti; landsem, veslemøy malm; malmring, janne fossum; oksenych, valentyn; erlandsen, sten even; aas, per arne; hagen, lars; pettersen, caroline h.; tenson, tanel; afset, jan egil; nordbø, svein arne; bjørås, magnar; kainov, denis e. title: potential antiviral options against sars-cov- infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: b as of june , the number of people infected with severe acute respiratory coronavirus (sars-cov- ) continues to skyrocket, with more than . million cases worldwide. both the world health organization (who) and united nations (un) has highlighted the need for better control of sars-cov- infections. however, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against sars-cov- can be time-consuming and costly. convalescent sera and safe-in-man broad-spectrum antivirals (bsaas) are readily available treatment options. here, we developed a neutralization assay using sars-cov- strain and vero-e cells. we identified the most potent sera from recovered patients for the treatment of sars-cov- -infected patients. we also screened safe-in-man broad-spectrum antivirals against the sars-cov- infection in vero-e cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. we found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. finally, we developed a website to disseminate the knowledge on available and emerging treatments of covid- . every year, emerging and re-emerging viruses such as sars-cov- , sars-cov, middle east respiratory syndrome coronavirus (mers-cov), zika virus (zikv), ebola virus (ebov), influenza a virus (fluav) and rift valley fever virus (rvfv) surface from natural reservoirs to infect people [ , ] . as of june , the number of people infected with sars-cov- continues to rise, with more than . million cases worldwide. we selected subjects among healthy individuals, in-and outpatients, as well as patients recovered from sars-cov- or endemic coronavirus infections. hospitalization was determined by whether a patient was able to manage symptoms effectively at home, according to local guidelines. icu admittance was evaluated consistently with the who interim guidance on "clinical management of severe acute respiratory infection when covid- is suspected" (who/ -ncov/clinical/ . ). the patients gave their informed consent through the koronastudien.no website. for icu patients, consent for sample collection was received after treatment or from relatives. for children, consent for sample collection was received from their parents. donors were recruited through information at the blood collection centers websites and through national media. nasopharyngeal swabs (nps) and blood samples were collected. all patients were treated in accordance with good clinical practice, following study protocols. the study was approved by the national ethical committee (clinical trial: nct ; rek: ). human telomerase reverse transcriptase-immortalized retinal pigment epithelial (rpe), lung adenocarcinoma a , non-small-cell lung cancer calu- and epithelial colorectal adenocarcinoma caco- cells were grown in dmem-f supplemented with µg/ml streptomycin and u/ml penicillin (pen-strep), mm l-glutamine, % fbs and . % sodium bicarbonate (sigma-aldrich, st. louis, mo, usa). human neural progenitor cells derived from ips cells were generated and maintained as described previously [ ] . human large-cell lung carcinoma nci-h , colon cancer sw , colorectal carcinoma sw and ht cells were grown in rpmi medium supplied with % fbs and pen-strep. human adenocarcinoma alveolar basal epithelial a , human embryonic kidney hek- cells and kidney epithelial cells extracted from an african green monkey (vero-e ) were grown in dmem supplemented with % fbs and pen-strep. the cell lines were maintained at • c with % co . the sars-cov- strains were isolated and propagated in a biological safety level (bsl- ) facility. two hundred microliters of nasopharyngeal swabs (nps) in universal transport medium were diluted times in culture medium (dmem) supplemented with . % bovine serum albumin (bsa), . µg/ml penicillin, µg/ml streptomycin and mm l-glutamine and inoculated into vero-e cells. after days of incubation, the media were collected, and the viruses were passaged once again in vero-e cells. after days, a clear cytopathic effect (cpe) was detected, and the virus culture was harvested. virus concentration was determined by rt-qpcr and plaque assays. viral rna was extracted using the ntnu_mag_v protocol, a modified version of the bomb-protocol [ ] . the eluate ( . or µl) was analyzed by rt-qpcr using a cfx real-time thermal cycler (bio-rad, hercules, ca, usa) as described elsewhere [ ] , with some modifications. one -µl reaction contained µl of qscript xlt one-step rt-qpcr toughmix ( ×) (quanta biosciences, beverly, ma, usa), µl each of the primer and probe with final respective concentrations of . and . µm and µl of molecular-grade water. thermal cycling was performed at • c for min for reverse transcription, followed by • c for min and then cycles of • c for s and • c for s. for testing the production of infectious virions, we titered the viruses as described in our previous studies [ ] [ ] [ ] . in summary, media from the viral culture were serially diluted from − to − in serum-free dmem containing . % bovine serum albumin. the dilutions were applied to a monolayer of vero-e cells in or -well plates. after one hour, cells were overlaid with virus growth medium containing % carboxymethyl cellulose and incubated for h. the cells were fixed and stained with crystal violet dye, and the plaques were calculated in each well and expressed as plaque-forming units per ml (pfu/ml). viral rna was extracted using the ntnu_mag_v protocol, a modified version of the bomb-protocol. libraries were prepared using a nugen trio rna-seq kit. sequencing was performed on the nextseq instrument (ns ; setup: pe × bp + single index ( bp)) using a nextseq mid sequencing kit and nextseq mid flow cell (ncs version: . . . ). reads were aligned using the bowtie software package version . . . to the reference viral genome wuhan-hu- / . sequence alignments were converted to binary alignments and sorted using samtools version . . the consensus fastq sequences were obtained with bcftools and vcfutils.pl (from samtools) and converted to fasta using seqtk (https://github.com/lh /seqtk). viral genomes in fasta formats were submitted to www.gisaid.org. the accession numbers of these genomes are: epi_isl_ (hcov- /norway/trondheim-e / ), epi_isl_ (hcov- /norway/trondheim-e / ), epi_isl_ (hcov- /norway/trondheim-s / ), epi_isl_ (hcov- /norway/trondheim-s / ), epi_isl_ (hcov- /norway/ trondheim-s / ), epi_isl_ (hcov- /norway/trondheim-s / ) and epi_isl_ (hcov- /norway/trondheim-s / ). transmission of the strains was visualized using https: //nextstrain.org/ncov/global?f_author=aleksandr% ianevski% et% al. the virus (multiplicity of infection, moi . ) was aliquoted in eppendorf tubes and incubated at − , − , , , and • c for h or at • c for min. alternatively, the virus was aliquoted in wells of a -well plate without a lid. the virus was exposed to uvc light (λ = nm, ≥ µw/cm ) for , , , , , and s using a germicidal lamp in a biosafety cabinet. the thermo and uvc stability of the viral rna was analyzed by rt-qpcr. subsequently, vero-e cells were infected with the virus for h, and cell viability was measured using a celltiter-glo assay (promega, madison, wi, usa). luminescence was read using a plate reader. approximately × vero-e cells were seeded per well in -well plates. the cells were grown for h in dmem supplemented with % fbs and pen-strep. serum samples were diluted -fold, and protein concentrations were quantified using nanodrop. serum samples were prepared in -fold dilutions at different concentrations, starting from mg/ml in the virus growth medium (vgm) containing . % bsa and pen-strep in dmem. virus hcov- /norway/trondheim-e / was added to the samples to achieve a moi of . and incubated for h at • c. . % dmso was added to the control wells. the vero-e cells were incubated for h with vgm. after the incubation period, the medium was removed, and a celltiter-glo assay was performed to measure viability. we have previously published a library of safe-in-man bsaas [ ] . supplementary materials table s lists these and other potential bsaas, their suppliers and catalogue numbers. the control wells. the cells were mock-or hcov- /norway/trondheim-e / -infected at a moi of . . after h of infection, the medium was removed from the cells, and a celltiter-glo assay was performed to measure viability. the half-maximal cytotoxic concentration (cc ) for each compound was calculated based on viability/death curves obtained on mock-infected cells after nonlinear regression analysis with a variable slope using graphpad prism software version . a (graphpad software, san diego, ca, usa). the half-maximal effective concentrations (ec ) were calculated based on the analysis of the viability of infected cells by fitting drug dose-response curves using the four-parameter ( pl) logistic function f (x): where f (x) is a response value at dose x, a min and a max are the upper and lower asymptotes (minimal and maximal drug effects), m is the dose that produces the half-maximal effect (ec or cc ) and λ is the steepness (slope) of the curve. a relative effectiveness of the drug was defined as the selectivity index (si = cc /ec ). the threshold of the si used to differentiate between active and inactive compounds was set to . area under the dose-response curve auc was quantified as: using the numerical integration implemented in the mess r package (bell laboratories, murray hill, nj, usa), where x max and x min are the maximal and minimal measured doses. serum sensitivity score (sss) was quantified as a normalized version of the standard auc (with the baseline noise subtracted and normalization of the maximal response at the highest concentration often corresponding to off-target toxicity) as where activity threshold t equals %. vero-e cells were treated with different concentrations of a combination of two bsaas. after h, cell viability was measured using a celltiter-glo assay. to test whether the drug combinations acted synergistically, the observed responses were compared with expected combination responses. the expected responses were calculated based on the zip reference model using synergyfinder web application, version [ ] . we measured the igg and igm in human serum using epitope diagnostics enzyme-linked immunosorbent assays (elisa) according to manufacturer specifications (epitope diagnostics, san diego, ca, usa). background-corrected optical density values were divided by the cutoff to generate signal-to-cutoff (s/co) ratios. samples with s/co values greater than . were considered positive. the pearson correlation coefficients were calculated by means of the stats r package, with the significance determined using a student's t-test. vero-e cells were treated with nelfinavir, amodiaquine or both drugs at indicated concentrations. cells were infected with the hcov- /norway/trondheim-e / strain at moi . or mock. after h, total rna was isolated using rneasy plus mini kit (qiagen, hilden, germany). libraries were prepared and sequenced on a nextseq (ns ) instrument (set up: pe × bp + single index bp) using a nextseq mid sequencing kit, nextseq mid flow cell, ncs version: . . . . reads were aligned using the bowtie software package version . . . to the ncbi reference sequence for sars-cov- (nc_ . ) and to the human grch genome. number of mapped and unmapped reads that aligned to each gene were obtained with the featurecounts function from rsubread r-package version . . the gff table for the reference sequence was downloaded from https://ftp.ncbi.nlm.nih.gov/genomes/all/gcf/ / / /gcf_ . _asm v /gcf_ . _asm v _genomic.gff.gz and flattened to gtf format and given as an additional argument to the rsubread function. the heatmaps were generated using the pheatmap package (https://cran.r-project.org/web/packages/pheatmap/index.html) based on log -transformed profiling data. we reviewed the current landscape of the available diagnostic tools, as well as the emerging treatment and prophylactic options for the sars-cov- pandemic and have summarized the information in a database that can be freely accessed at https://sars-coronavirus- .info. the information in the database was obtained from pubmed, clinicaltrials.gov, drugbank, drugcentral, the chinese clinical trials register (chictr) and eu clinical trials register databases [ ] [ ] [ ] , as well as other public sources. the website was developed with php v technology using d .js v (https://d js.org/) for visualization. the covid- statistics are automatically exported from the covid- data repository by the center for systems science and engineering (csse) at johns hopkins university (https://github.com/cssegisanddata/covid- ). we isolated seven sars-cov- strains from nps samples of covid- patients using vero-e cells. the rt-qpcr cycle threshold (ct) values were - before and - after propagation of the viruses in vero-e cells (figure a ,b). we sequenced seven strains and found that the sequences differed from the reference hcov- /wuhan/wiv / strain by a few missense mutations ( figure c) . phylogenetic analysis showed a close relationship between the strains (figure d ). in cross-referencing our sequence data with the pathogen-tracking resource nextstrain.org, we determined that the sars-cov- strains isolated in trondheim originated from china, denmark, the usa and canada (figure e) . in addition, we tested several cell lines and found that vero-e was the most susceptible for virus-mediated death and virus amplification (figure s a-c). to establish the rationale for safe work, we incubated the virus at different temperatures for h or exposed to uvc radiation for different time periods. the resulting virus preparations were analyzed by rt-qpcr and used to infect vero-e cells. virus incubation at • c for h or uvc exposure for sec destabilized the virus and rescued infected cells from virus-mediated death ( figure s ). we evaluated the neutralization capacity of seven serum samples obtained from patients recovered from covid- . we also used sera from patients recovered from endemic coronavirus infections and from healthy blood donors as controls. five samples from patients recovered from covid- had serum sensitivity scores (sss) > and rescued > % cells from virus-mediated death at mg/ml (figure a,b) . notably, that serum from a recovered patient with sss = . neutralized all seven sars-cov- strains ( figure s ). our neutralization test of samples (table s ) showed a moderate positive correlation with igg (r = . , p < . ) and igm (r = . , p = . ) s/co values obtained using commercial elisa kits that recognize the sars-cov- n protein (figure c,d) . however, the correlation between the igg and igm elisa results was only r = . , p = . . furthermore, we found a moderate negative correlation between ssss and time intervals between the sars-cov- diagnosis and serum collection for samples (- . , p < . ; figure d ). altogether, these results suggest that patients diagnosed with covid- produce different immune responses to the sars-cov- infection and that the neutralization capacity of convalescent sera declines with time. we evaluated the neutralization capacity of seven serum samples obtained from patients recovered from covid- . we also used sera from patients recovered from endemic coronavirus infections and from healthy blood donors as controls. five samples from patients recovered from covid- had serum sensitivity scores (sss) > and rescued > % cells from virus-mediated death at mg/ml (figure a,b) . notably, that serum from a recovered patient with sss = . neutralized all seven sars-cov- strains ( figure s ). our neutralization test of samples (table s ) showed a moderate positive correlation with igg (r = . , p < . ) and igm (r = . , p = . ) s/co values obtained using commercial elisa kits that recognize the sars-cov- n protein (figure c,d) . however, the correlation between the igg and igm elisa results was only r = . , p = . . furthermore, we found a moderate negative correlation between ssss and time intervals between the sars-cov- diagnosis and serum collection for samples (− . , p < . ; figure d ). altogether, these results suggest that patients diagnosed with covid- produce different immune responses to the sars-cov- infection and that the neutralization capacity of convalescent sera declines with time. through the literature review, we made a database to summarize safe-in-man bsaas (https://drugvirus.info/). recently, we have expanded on the spectrum of activities for some of these agents [ ] [ ] [ ] , ] . some of these agents could be repositioned for the treatment of a sars-cov- infection. we tested agents against sars-cov- in vero-e cells. remdesivir was included as a positive control [ ] and nicotine as a negative control. seven different concentrations of the compounds were added to virus-infected cells. cell viability was measured after h to determine compound efficiency. after the initial screening, we identified apilimod, emetine, amodiaquine, obatoclax, homoharringtonine, salinomycin, arbidol, posaconazole and nelfinavir as compounds that rescued virus-infected cells from death (auc from to ; table s ). the compounds we identified possessed a structure-activity relationship (figure a) . auc for remdesivir was . interestingly, µm of nicotine rescued cells from virus-mediated death but altered the cell morphology (auc = ; figure s ). we repeated the experiment with hit compounds, monitoring their toxicity and efficacy. we confirmed the antiviral activity of emetine, amodiaquine, obatoclax, homoharringtonine, salinomycin and nelfinavir (figure b ,c). importantly, amodiaquine had a superior si over its analogs chloroquine, hydroxychloroquine, quinacrine and mefloquine ( figure s ). thus, we identified and validated anti-sars-cov- activities for six bsaas in vero-e cells. to test for potential synergism among the validated hits, we treated cells with varying concentrations of a two-drug combination and monitored the cell viability (figure a) . the observed drug combination responses were compared with the expected combination responses calculated by means of the zero-interaction potency (zip) model [ , ] . we quantified synergy scores, which represent the average excess response due to drug interactions (i.e., % of cell survival beyond the expected additivity between single drugs has a synergy score of ). we found that combinations of nelfinavir with salinomycin, amodiaquine, homoharringtonine and obatoclax, as well as the combination of amodiaquine and salinomycin, were synergistic (most synergistic area scores > ; figure b ). moreover, the nelfinavir-amodiaquine treatment was effective against all seven sars-cov- strains (figure c ). thus, we identified synergistic drug combinations against sars-cov- infections. we next profiled transcriptional responses to nelfinavir, amodiaquine or both drugs in virus-or mock-infected vero-e cells at h. we showed that the addition of nontoxic but effective concentrations of drugs slightly affected the transcription of immune-related genes in virus-infected cells ( figure s a ). these genes (cxcl , cxcl , cxcl , cxcl , cxcl , cxcl , oasl, ifnl , mx and herc ) are needed for alarming neighboring cells about the ongoing infection and for the protection of the organism from repeated infections. amodiaquine and its combination with nelfinavir lowered the transcription of viral genomic and sub-genomic rnas ( figure s b) . (a) structure-antiviral activity relation of broad-spectrum antivirals (bsaas). the compounds were clustered based on their structural similarity calculated by ecpf fingerprints and visualized using the d javascript library. the anti-sars-cov- activity of the compounds was quantified using the auc and shown as bubbles. bubble size corresponds to compounds aucs. (b) vero-e cells were treated with increasing concentrations of a compound and infected with the hcov- /norway/trondheim-e / strain (moi, . ) or mock. after h, the viability of the cells was determined using the celltiter-glo assay. mean ± sd; n = . (c) table showing half-maximal cytotoxic concentration (cc ), the half-maximal effective concentration (ec ) and selectivity indexes (si = cc /ec ) for selected anti-sars-cov- compounds calculated from ctg and plaque assays. mean ± sd; n = . figure . anti-sars-cov- activity of safe-in man broad-spectrum antivirals in vero-e cells. (a) structure-antiviral activity relation of broad-spectrum antivirals (bsaas). the compounds were clustered based on their structural similarity calculated by ecpf fingerprints and visualized using the d javascript library. the anti-sars-cov- activity of the compounds was quantified using the auc and shown as bubbles. bubble size corresponds to compounds aucs. (b) vero-e cells were treated with increasing concentrations of a compound and infected with the hcov- /norway/trondheim-e / strain (moi, . ) or mock. after h, the viability of the cells was determined using the celltiter-glo assay. mean ± sd; n = . (c) table showing half-maximal cytotoxic concentration (cc ), the half-maximal effective concentration (ec ) and selectivity indexes (si = cc /ec ) for selected anti-sars-cov- compounds calculated from ctg and plaque assays. mean ± sd; n = . to rapidly respond to the covid- outbreak, we developed a freely accessible website summarizing novel anti-sars-cov- developments and currently approved diagnostic options around the globe. it also tracks the development of therapeutic/antiviral drugs and vaccines. the "treatment" section of the website summarizes in-progress and completed clinical trials that test the efficacy of therapeutic agents to treat covid- or complications that arise from covid- . these trials include over unique therapeutic agents in varying combinations and applications. importantly, we list clinical trials that are already completed or are projected to be completed by the end of june . of note, among these are trials of remdesivir, favipiravir, lopinavir/ritonavir, hydroxychloroquine, to rapidly respond to the covid- outbreak, we developed a freely accessible website summarizing novel anti-sars-cov- developments and currently approved diagnostic options around the globe. it also tracks the development of therapeutic/antiviral drugs and vaccines. the "treatment" section of the website summarizes in-progress and completed clinical trials that test the efficacy of therapeutic agents to treat covid- or complications that arise from covid- . these trials include over unique therapeutic agents in varying combinations and applications. importantly, we list clinical trials that are already completed or are projected to be completed by the end of june . of note, among these are trials of remdesivir, favipiravir, lopinavir/ritonavir, hydroxychloroquine, dipyridamole and interferons alpha and beta, which are all phase or clinical trials scheduled to be currently completed. the "prevention" section summarizes current vaccine trials taking place around the globe. although vaccine development lags considerably behind drug development, several repurposed vaccine options have also emerged. this includes trials of the cross-reactivity of the mmr (measles, mumps and rubella) vaccine, as well as several trials of the bacillus calmette-guérin (bcg) vaccine among high-risk populations, such as healthcare workers. finally, the "testing" section of the website provides a summary of currently available laboratory-based and point-of-care diagnostic tests that are approved for clinical diagnosis in at least one country. the website also includes predictions of experimental and approved drugs effective against sars-cov- , as well as provides a summary of information about the coronavirus pandemic. the website allows interactive exploration of the data with built-in feedback and is available in several languages. the website is updated as soon as novel anti-sars-cov- options emerge, or the statuses of existing ones are updated. here, we reported the isolation of seven sars-cov- strains from samples of patients suffering from covid- . full-genome sequencing revealed that the strains were highly similar ( . %) to one another and to the strains circulating in china, denmark, the usa and canada. all seven strains contain d g in the s protein. strains with this mutation began spreading in europe in early february and became dominant in other regions (https://doi.org/ . / . . . ). we screened cell lines for their susceptibility for the sars-cov- infection and virus replication. vero-e cells appeared to be the most susceptible cell line for virus-mediated cell death and virus propagation. this cell line has been widely used in toxicology, virology and pharmacology research, as well as in the production of vaccines and diagnostic reagents. the cell line is interferon-deficient; i.e., unlike normal mammalian cells, it does not secrete interferon alpha or beta when infected by viruses [ ] . moreover, this cell line was used routinely in anti-sars-cov- research [ ] [ ] [ ] [ ] . we also showed that > sec of uvc radiation or h incubation at • c neutralized sars-cov- , establishing a rationale and methodology for safe work in the laboratory. these results are consistent with previous studies showing that physical factors destabilize sars-cov- and other viruses [ ] [ ] [ ] [ ] . neutralization tests are crucial tools for the assessment of previous sars-cov- exposure and potential immunity [ , ] . we have developed a test to assess the neutralization capacity of serum samples from patients recovered from sars-cov- infections, patients with endemic coronavirus infections and healthy blood donors. our results suggest that covid- patients respond differently to the sars-cov- infection. moreover, the neutralization capacity of convalescence sera declined with time. thus, the neutralization test allowed us to identify the most potent sera from patients recovered from covid- for the treatment of sars-cov- -infected patients. moreover, results from our neutralization test positively correlated with those from commercial elisa assays. however, the correlation between the igg and igm elisa results was only moderate. the difference could be associated with the time of the sample collection, production of the immunoglobulins or sensitivity that can be attributed to the technique and the antigen in use (i.e., igm is the first immunoglobulin to be produced in response to an antigen and can be detected during early onset of disease, whereas igg is maintained in the body after initial exposure for the long-term response and can be detected after the infection has passed). there were certain concerns regarding the antibody-dependent cell-mediated toxicity of convalescent sera [ ] . however, the recent safety study on hospitalized patients transfused under the u.s. food and drug administration's national expand access program for covid- revealed no toxicity (https://doi.org/ . / . . . ) . drug repurposing, also called drug repositioning, is a strategy for generating an additional value from an existing drug by targeting diseases other than that for which it was originally intended [ , ] . this has significant advantages over new drug discoveries, since chemical synthesis steps, manufacturing processes, reliable safety and pharmacokinetic properties have already been studied in preclinical (animal model) and early clinical developmental phases (phase , i and iia). therefore, drug repositioning for covid- provides unique translational opportunities, including a substantially higher probability of success to the market as compared with developing new virus-specific drugs and vaccines, as well as significantly reduced costs and timelines to clinical availability [ , , ] . we tested safe-in-man bsaas against sars-cov- in cell cultures. we identified salinomycin, obatoclax, amodiaquine, nelfinavir, emetine and homoharringtonine as having anti-sars-cov- activity, which we put forward as potential anti-sars-cov- drug candidates. nelfinavir (viracept) is an orally bioavailable inhibitor of human immunodeficiency virus hiv- ( mg per os (po) q hr). it targets hiv protease for the treatment of hiv infections [ ] . molecular docking studies predict that nelfinavir binds to the sars-cov- protease [ ] . nelfinavir could also inhibit cell fusion caused by the sars-cov- s glycoprotein (https://doi.org/ . / . . . ) [ ] . it also inhibits chikungunya virus (chikv), dengue virus (denv), hepatitis c virus (hcv), herpes simplex virus (hsv- ) and sars-cov infections (https://doi.org/ . / c ra h) [ ] [ ] [ ] . amodiaquine is a medication used to treat malaria. the recommended dose for a course of amodiaquine is mg amodiaquine base/kg body weight over three days, i.e., mg/kg/day [ ] . [ ] [ ] [ ] [ ] [ ] [ ] . importantly, amodiaquine showed more potent antiviral activity than its analogs chloroquine and hydroxychloroquine. obatoclax was originally developed as an anticancer agent. several phase ii clinical trials have investigated the use of obatoclax in the treatment of leukemia, lymphoma, myelofibrosis and mastocytosis. a continuous h infusion of obatoclax - mg/day for three days in two-week cycles or h infusions in a -day cycle have previously been evaluated in cancer patients [ ] . in addition, obatoclax showed antiviral activity against fluav, zikv, wnv, yfv, sinv, junín virus (junv), lasv, herpes simplex virus (hsv- ), echovirus (ev ), human metapneumovirus (hmpv), rvfv and lymphocytic choriomeningitis virus (lcmv) in vitro [ , , , , ] . it was shown that obatoclax inhibited the viral endocytic uptake by targeting the cellular mcl- protein [ ] . emetine is an antiprotozoal drug. it is administered by intramuscular or deep subcutaneous injection in a dose of mg/kg/day (maximum mg/day) for days [ ] . emetine is also used to induce vomiting. in addition, it possesses antiviral effects against zikv, ebov, rabv, cytomegalovirus (cmv), hcov-oc , hsv- , ev , hmpv, rvfv, fluav, hiv- and sars-cov- [ , , , [ ] [ ] [ ] [ ] [ ] (https://doi.org/ . / . . . ). emetine was proposed to inhibit viral polymerases, though it could have some other targets [ ] . homoharringtonine is an anticancer drug that is indicated for the treatment of chronic myeloid leukemia ( mg/m iv daily × ). it also possesses antiviral activities against hepatitis b virus (hbv), mers-cov, hsv- , ev , vzv and sars-cov- in vitro [ , , [ ] [ ] [ ] [ ] . homoharringtonine binds to the s ribosome and inhibits viral protein synthesis by interfering with the chain elongation [ ] . salinomycin is an orally bioavailable antibiotic that is used against gram-positive bacteria in animal husbandry ( . mg/kg body weight (bw) po). it also inhibits fluav, respiratory syncytial virus (rsv) and cmv infections [ , ] . salinomycin was proposed to disrupt the endosomal acidification and to block entry of the viruses into cells [ , ] . our results have uncovered several existing bsaas that could be repositioned to sars-cov- infections. since pharmacokinetic/pharmacodynamic and toxicology studies have already been performed on these compounds, in vivo efficacy studies could be initiated immediately, saving time and resources. combination therapies became a standard for the treatment of hiv and hcv infections. the reasons for using combinations rather than single antiviral are better efficacy, decreased toxicity and the prevention of resistance emergence. here, we found that combinations of nelfinavir with salinomycin, amodiaquine, obatoclax, emetine or homoharringtonine were synergistic against sars-cov- in vero-e cells. interestingly, synergistic interactions occurred between compounds belonging to different sar clusters (i.e., nelfinavir belongs to a separate cluster than amodiaquine or obatoclax-emetine-homoharringtonine). in particular, the synergy was achieved when a virus-directed drug was combined with host-directed ones. this observation agrees with other studies on such combinations and virus-host interactions [ , , , ] (https://doi.org/ . / . . . ). according to the available pharmacological data for these drugs, the most potent combination could be a combination of orally available nelfinavir and amodiaquine. this was also the combination that exhibited the highest synergy of all the drug combinations we tested, with the synergy score at the most synergistic area being . (i.e., . % of cell survival beyond the expected additivity between the single drugs). there are no guidelines of what is considered a good synergy, but it is very common to consider synergy > as true (significant) synergy. thus, the amodiaquine and nelfinavir combination could result in better efficacy and decreased toxicity for the treatment of sars-cov- and perhaps other viral infections. our future goal is to complete preclinical studies and translate our findings into trials in patients. the most effective and tolerable bsaas or their combinations will have a global impact, improving the protection of the general population from emerging and re-emerging viral infections or coinfections and allowing the rapid management of drug-resistant strains. our bigger ambition is to assemble a toolbox of bsaas for the treatment of emerging and re-emerging viral infections. this toolbox can be offered to the who as a means for the fast identification of safe and effective antiviral options. we have summarized the information about the status of currently available and emerging anti-sars-cov- options on the freely accessible website (https://sars-coronavirus- .info). the website is updated regularly and incorporates novel anti-sars-cov- options as they emerge or changes the statuses of existing ones as updates occur. in conclusion, sera from recovered patients, bsaas and combinations of bsaas, as well as other available and emerging treatments, could have pivotal roles in the battle against covid- and other emerging and re-emerging viral diseases. further development of these options could save time and resources that are required for the development of alternative virus-specific drugs and vaccines. this could have a global impact by decreasing morbidity and mortality, maximizing the number of healthy life years, improving the quality of life of infected patients and decreasing the costs of patient care curtailing to the impact of the current sars-cov- pandemic, as well as future viral outbreaks. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s : table s : compounds, their suppliers, catalogue numbers and aucs. table s . results of neutralization and elisa assays. figure s . propagation of hcov- /norway/trondheim-e / in cell cultures. figure s . effects of temperature and uv radiation on the infectivity of the hcov- /norway/trondheim-e / strain. figure s . the effects of the serum from patients recovered from the sars-cov- infection on the viability of vero-e cells infected with sars-cov- strains. figure s . the effects of nicotine on the viability and morphology of mock-and sars-cov- -infected vero-e cells. figure s : the comparison of anti-sars-cov- activities of amodiaquine and its analogs. figure s . transcriptomic analysis of mock-and sars-cov- -infected vero-e cells treated with nelfinavir, amodiaquine or both drugs. author contributions: all authors contributed to the methodology, software, validation, formal analysis, investigation, resources, data curation, writing and review and editing of the manuscript. d.k. conceptualized, supervised and administrated the study and acquired funding. all authors have read and agreed to the published version of the manuscript. funding: this research was funded the european regional development fund, the mobilitas pluss project mobtt (to d.k.). available online: www.who.int/medicines/ebola-treatment/who-list-of-top-emerging-diseases/en emerging virus diseases: can we ever expect the unexpected? emerg. microbes infect. , , e infectious disease. combating emerging viral threats srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target experimental drugs poised for use in ebola outbreak an off-target effect of bx blocks herpes simplex virus type infection of the eye repurposing of kinase inhibitors as broad-spectrum antiviral drugs the future of antivirals: broad-spectrum inhibitors approaches for identification of hiv- entry 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influenza viral infection: reviving old drugs to fight against a long-lived enemy drug repurposing screens and synergistic drug-combinations for infectious diseases circulating metabolites of the human immunodeficiency virus protease inhibitor nelfinavir in humans: structural identification, levels in plasma, and antiviral activities binding site analysis of potential protease inhibitors of covid- using autodock the anti-hiv drug nelfinavir mesylate (viracept) is a potent inhibitor of cell fusion caused by the sarscov- spike (s) glycoprotein warranting further evaluation as an antiviral against covid- infections nelfinavir inhibits maturation and export of herpes simplex virus inhibition of intracellular hepatitis c virus replication by nelfinavir and synergistic effect with interferon-alpha hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus amodiaquine dosage and tolerability for intermittent preventive treatment to prevent malaria in children identification of broad-spectrum antiviral compounds by targeting viral entry arbidol and other low-molecular-weight drugs that inhibit lassa and ebola viruses novel amodiaquine derivatives potently inhibit ebola virus infection high-content screening in hpsc-neural progenitors identifies drug candidates that inhibit zika virus infection in fetal-like organoids and adult brain repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection amodiaquine, an antimalarial drug, inhibits dengue virus type replication and infectivity a multicenter phase i/ii study of obatoclax mesylate administered as a -or -hour infusion in older patients with previously untreated acute myeloid leukemia the reframe library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors obatoclax inhibits alphavirus membrane fusion by neutralizing the acidic environment of endocytic compartments a phase i study of emetine hydrochloride (nsc ) in solid tumors high-throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry efficacy and mechanism of action of low dose emetine against human cytomegalovirus natural plant alkaloid (emetine) inhibits hiv- replication by interfering with reverse transcriptase activity emetine inhibits replication of rna and dna viruses without generating drug-resistant virus variants anti-varicella-zoster virus activity of cephalotaxine esters in vitro the natural compound homoharringtonine presents broad antiviral activity in vitro and in vivo a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs effect of cantharidin, cephalotaxine and homoharringtonine on "in vitro" models of hepatitis b virus (hbv) and bovine viral diarrhoea virus (bvdv) replication targeting intracellular ion homeostasis for the control of respiratory syncytial virus wnt modulating agents inhibit human cytomegalovirus replication salinomycin inhibits influenza virus infection by disrupting endosomal acidification and viral matrix protein function identification of antiviral drug candidates against sars-cov- from fda-approved drugs jnj inhibits influenza a virus replication without altering cellular antiviral responses in vitro testing of combined hydroxychloroquine and azithromycin on sars-cov- shows synergistic effect acknowledgments: this study is devoted to wenliang li, a chinese doctor who tried to warn about coronavirus, as well as to many other doctors and covid- patients. we thank koit aasumets, sergio miguel castañeda zegarra, qindong zhang, simona komarova, nikki upfold, miriam khider, hege hval and kasia kolasa for translation of the sars-coronavirus- .info website to different languages. we also thank maxim bespalov, sergei shiryaev, pavel uvarov, evgeny kulesskiy and many other people for sharing their ideas on drug candidates. in addition, we thank wei wang, marit bugge and nadra j. nilsen for the cell lines. the authors declare no conflicts of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -pd s y authors: lu, chien-yi; hour, mann-jen; wang, ching-ying; huang, su-hua; mu, wen-xiang; chang, yu-chun; lin, cheng-wen title: single-round infectious particle antiviral screening assays for the japanese encephalitis virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: pd s y japanese encephalitis virus (jev) is a mosquito-borne flavivirus with a positive-sense single-stranded rna genome that contains a big open reading frame (orf) flanked by ′- and ′- untranslated regions (utrs). nearly , je cases with , deaths are still annually reported in east asia. although the jev genotype iii vaccine has been licensed, it elicits a lower protection against other genotypes. moreover, no effective treatment for a je case is developed. this study constructed a pbr -based and cytomegaloviruses (cmv) promoter-driven jev replicon for the production of jev single-round infectious particles (srips) in a packaging cell line expressing viral structural proteins. genetic instability of jev genome cdna in the pbr plasmid was associated with the prokaryotic promoter at ′ end of the jev genome that triggers the expression of the structural proteins in e. coli. jev structural proteins were toxic e. coli, thus the encoding region for structural proteins was replaced by a reporter gene (enhanced green fluorescent protein, egfp) that was in-frame fused with the first eight amino acids of the c protein at n-terminus and the foot-and-mouth disease virus (fmdv) a peptide at c-terminus in a pbr -based jev-egfp replicon. jev-egfp srips generated from jev-egfp replicon-transfected packaging cells displayed the infectivity with cytopathic effect induction, self-replication of viral genomes, and the expression of egfp and viral proteins. moreover, the combination of jev-egfp srip plus flow cytometry was used to determine the half maximal inhibitory concentration (ic ) values of antiviral agents according to fluorescent intensity and positivity of srip-infected packaging cells post treatment. mj- , a quinazolinone derivative, significantly inhibited jev-induced cytopathic effect, reducing the replication and expression of jev-egfp replicon in vitro. the ic value of . µm for mj- against jev was determined by the assay of jev-egfp srip infection in packaging cells plus flow cytometry that was more sensitive, effective, and efficient compared to the traditional plaque assay. therefore, the system of jev-egfp srips plus flow cytometry was a rapid and reliable platform for screening antiviral agents and evaluating antiviral potency. srips were generated in the cells co-transfected with pbr -jevrep-egfp and pflag-cmv -cprme. the infectivity of jev-egfp srips was characterized by the replication of viral subgenomes and the expression of egfp as well as viral proteins using real-time pcr and fluorescent microscopy. finally, the antiviral screening platform was performed according to comparing the fluorescence intensity of egfp in srip-infected cells with and without treatment by flow cytometry. accuracy and reproducibility of the half maximal inhibitory concentration (ic ) of anti-jev agent mj- by jev-egfp srip plus flow cytometry were further evaluated using plaque-reduction assay. viruses , , of virus (fmdv) a peptide, and then cloned and in-frame fused with ns ~ coding regions in pbr plasmid-based jev-egfp replicon (named as pbr -jevrep-egfp). subsequently, jev-egfp srips were generated in the cells co-transfected with pbr -jevrep-egfp and pflag-cmv -cprme. the infectivity of jev-egfp srips was characterized by the replication of viral subgenomes and the expression of egfp as well as viral proteins using real-time pcr and fluorescent microscopy. finally, the antiviral screening platform was performed according to comparing the fluorescence intensity of egfp in srip-infected cells with and without treatment by flow cytometry. accuracy and reproducibility of the half maximal inhibitory concentration (ic ) of anti-jev agent mj- by jev-egfp srip plus flow cytometry were further evaluated using plaque-reduction assay. human medulloblastoma te cells were cultured in minimum essential medium (mem, ge healthcare life sciences, pittsburgh, pa, usa) with % fetal bovine serum (thermofisher, waltham, ma, usa), glutamine, pyruvate, and penicillin/streptomycin at °c in an atmosphere containing % carbon dioxide. jev t p strain (genbank: af . ) used in this study was propagated in bhk- cells, as described in a prior report [ ] . plasmid pbr -linker was derived from pbr (thermofisher, waltham, ma, usa) by the insertion of a -bp nucleotide linker containing ecori, kpni, noti, xhoi, and ecori into ecori/bamhi sites of pbr ( figure s ). resultant pbr -linker was used as the vector for assembling fragments of jev-egfp replicon consisting of five nucleotide (nt) fragments, including cmv immediate-early promoter, nt - of jev t p strain (genbank: af . ), egfp/fmdv human medulloblastoma te cells were cultured in minimum essential medium (mem, ge healthcare life sciences, pittsburgh, pa, usa) with % fetal bovine serum (thermofisher, waltham, ma, usa), glutamine, pyruvate, and penicillin/streptomycin at • c in an atmosphere containing % carbon dioxide. jev t p strain (genbank: af . ) used in this study was propagated in bhk- cells, as described in a prior report [ ] . plasmid pbr -linker was derived from pbr (thermofisher, waltham, ma, usa) by the insertion of a -bp nucleotide linker containing ecori, kpni, noti, xhoi, and ecori into ecori/bamhi sites of pbr ( figure s ). resultant pbr -linker was used as the vector for assembling fragments of jev-egfp replicon consisting of five nucleotide (nt) fragments, including cmv immediate-early promoter, nt - of jev t p strain (genbank: af . ), egfp/fmdv a peptide, nt - , of t p strain/hepatitis delta virus ribozyme (hdvr), and sv polya (figure a ). each fragment viruses , , of was amplified using platinum ® pcr supermix high fidelity reaction (life technology, carlsbad, ca, usa) with indicated primer pairs and templates listed in supplemental table s . forward primer of fragment has an extra nucleotide sequence for flag tag and clai restriction site. reverse primers of fragments and contain extra nucleotide sequences for fmvd a peptide and hdvr, respectively. the in-frame fusion of fragments and were produced using a jumping pcr method ( figure s ), and cloned into kpni and noti sites of pbr -linker as pbr -f f . since a unique apai restriction site at nt of jev, fragment was ligated with pbr -f f after apai and noti double digestion, as pbr -f f f . subsequently, fragments and were cloned into xhoi/bamhi and noti/xhoi sites of pbr -f f f . the assembled jev-egfp replicon with these five fragments was propagated in e. coli dh α cells, and then verified by restriction enzyme analysis plus sequencing. figure a ). each fragment was amplified using platinum ® pcr supermix high fidelity reaction (life technology, carlsbad, ca, usa) with indicated primer pairs and templates listed in supplemental table s . forward primer of fragment has an extra nucleotide sequence for flag tag and clai restriction site. reverse primers of fragments and contain extra nucleotide sequences for fmvd a peptide and hdvr, respectively. the in-frame fusion of fragments and were produced using a jumping pcr method ( figure s ), and cloned into kpni and noti sites of pbr -linker as pbr -f f . since a unique apai restriction site at nt of jev, fragment was ligated with pbr -f f after apai and noti double digestion, as pbr -f f f . subsequently, fragments and were cloned into xhoi/bamhi and noti/xhoi sites of pbr -f f f . the assembled jev-egfp replicon with these five fragments was propagated in e. col dh α cells, and then verified by restriction enzyme analysis plus sequencing. a. b. construction of pbr -based jev replicon and pflag-cmv -cprme. (a) the dna fragments of cmv promoter, jev genome cdna, egfp and sv -polya were amplified using pcr, and then assembled into the indicated restriction sites of the pbr plasmid; (b) the cdna sequence encoding structural proteins was amplified using pcr and cloned into the pflag-cmv with ecori and xbai double digestion. the detailed procedure was described in the section of materials and methods. for examining the synthesis of positive-and negative-sense rna subgenomes, total rnas of the transfected te cells with jev-egfp replicon were extracted using purelink mini total rna purification kit (thermofisher, waltham, ma, usa), reverse transcripted into cdna with specificcapture primers, and followed by measuring positive-and negative-sense rna subgenomes using sybr green-based real time pcr with jev-specific primer pairs ( figure s ). relative levels of rna subgenomes were normalized by glyceraldehyde -phosphate dehydrogenase (gapdh), described in a prior report [ ] . to explore the expression of egfp and jev proteins, transfected cells were rinsed once with pbs, fixed with % formaldehyde in pbs at room temperature for min, and permeabilized . % triton x- , % bsa in pbs at room temperature for hr. after blocking with figure . construction of pbr -based jev replicon and pflag-cmv -cprme. (a) the dna fragments of cmv promoter, jev genome cdna, egfp and sv -polya were amplified using pcr, and then assembled into the indicated restriction sites of the pbr plasmid; (b) the cdna sequence encoding structural proteins was amplified using pcr and cloned into the pflag-cmv with ecori and xbai double digestion. the detailed procedure was described in the section of materials and methods. for examining the synthesis of positive-and negative-sense rna subgenomes, total rnas of the transfected te cells with jev-egfp replicon were extracted using purelink mini total rna purification kit (thermofisher, waltham, ma, usa), reverse transcripted into cdna with specific-capture primers, and followed by measuring positive-and negative-sense rna subgenomes using sybr green-based real time pcr with jev-specific primer pairs ( figure s ). relative levels of rna subgenomes were normalized by glyceraldehyde -phosphate dehydrogenase (gapdh), described in a prior report [ ] . to explore the expression of egfp and jev proteins, transfected cells were rinsed once with pbs, fixed with % formaldehyde in pbs at room temperature for min, and permeabilized . % triton x- , % bsa in pbs at room temperature for hr. after blocking with % bovine serum albumin in pbs, permeabilized cells were stained using primary rabbit polyclonal antibodies, including anti-jev e protein, anti-jev ns , and anti-jev ns b (genetex, inc., irvine, ca, usa), followed by the incubation with secondary af goat anti-rabbit igg (thermofisher, waltham, viruses , , of ma, usa) in dark box for h. after washing with pbs, stained cells were photographed using the immunofluorescence microscopy (olympus, bx , tokyo, japan). to construct the recombinant plasmid for expressing jev structural proteins in mammalian cells, the coding region for jev structural proteins was amplified using pcr, and then cloned into ecori and xbai sites of the expression plasmid pflag-cmv ( figures b and b ). resultant plasmid pflag-cmv -cprme was analyzed using restriction enzyme analysis and sequencing. all primers for sequencing were listed in supplemental table s . no mutation in pflag-cmv -cprme was detected. to create a packaging cell line expressing jev structural proteins, te cells were grown to % confluence in a six-well plate, and then transfected with µg of pflag-cmv -cprme using lipofectamine ltx (invitrogen, carlsbad, ca, usa) according to the manufacturer's guidelines. the transfected cells were treated with µg/ml of g h post transfection; a stable transfected cell line was established after a -day-selection, in which the expression of jev structural proteins was validated by immunofluorescence staining. next, the packaging cell line was grown to % confluence, and then transfected with µg of pbr -jev-egfp replicon using lipofectamine ltx. jev-egfp srips were collected from the cultured media and lysate supernatant of transfected packaging cells. finally, viral subgenome synthesis and viral protein expression in infected cells were examined using real time rt-pcr and immunofluorescent staining, as described above. to construct the recombinant plasmid for expressing jev structural proteins in mammalian cells, the coding region for jev structural proteins was amplified using pcr, and then cloned into ecori and xbai sites of the expression plasmid pflag-cmv ( figures b and b ). resultant plasmid pflag- figure . quantitative analysis of positive-and negative-sense jev subgenomes using strand-specific real time rt-pcr. total rnas were extracted from mock cells, packaging cells, replicon-transfected mock cells and replicon-transfected packaging cells; relative copy numbers of positive-(a) and negative-sense (b) subgenomes were quantitated using real-time pcr, and then normalized by gapdh mrna. *, p-value < . ; ***, p-value < . compared with mock cells. to determine the infectivity of jev-egfp srips, cprme-packaging cells were infected with srips at tcid , harvested h post infection, and washed with pbs. after centrifuging at rpm for min, cell pellets were dissolved with µl pbs, and then fixed in ml % alcohol at − • c overnight. after washing twice with pbs the next day, over , resuspended cells were analyzed by bd facsaria (becton dickinson, franklin lakes, nj, usa) with excitation at nm and emission at nm. mock-infected cprme packaging cells served as negative controls and their fluorescent intensity was set as the threshold. the results were recorded by a ratio of egfp-positive cells to total cells and the green fluorescent intensity of positive cells, respectively. compound mj- (methyl -(( -( -methoxyphenyl)- -(pyrrolidin- -yl)quinazolin- -yl)oxy)acetate) was synthesized based on a lead compound mj- ( figure s ), an antineoplastic quinazolinone derivative [ ] , and then introduced a functional group, such as alkoxyl or aniline moiety, to the -position of quinazolinoe for decreasing cytotoxicity. hmj- ( . g, . mmol), synthesized according to the published procedure [ ] , was dissolved in ml n,n-dimethylformamide (dmf) and treated with sodium hydride (nah) ( % dispersion in paraffin oil, merck) at • c for - min. methyl chloroacetate ( . g, . mmol, lancaster) was added to the above suspension with stirring at • c for about h. the mixture was poured into ice water ( ml) and then filtered to collect the brown precipitate. the precipitate was washed with water, dried, and purified by column chromatography (silica gel; dichloromathane/n-hexane) and recrystallization with % ethanol to afford mj- (yield . g ( . %); brown crystals). the identity and purity of mj- were confirmed by nuclear magnetic resonance spectroscopy and mass spectrum. melting points were determined with a yanaco mp- d melting point apparatus and are uncorrected. nuclear magnetic resonance (nmr) spectra were obtained on a bruker avance drx- nmr spectrometer (waltham, ma, usa) in dimethyl sulfoxide (dmso)-d cytotoxicity of mj- to te cells was measured using mtt assay. cells were treated with mj- ( , , , , and µm) in quadruplicate at • c in % co for h, incubated with µl of mg/ml mtt solution for h, and then dissolved by dmso ( µl/well). absorbance (od - ) was detected using a micro-elisa reader. the survival rate was calculated as a ratio of od - of treated cells to od - of mock cells. cytotoxic concentration giving % (cc ) was calculated by a computer program, as described in a prior report [ ] . the expression of egfp and viral proteins as well as the synthesis of positive-and negative-sense rna subgenomes in mj- -reated infected cells were determined using fluorescent microscope, immunofluorescent staining and real-time rt-pcr assays, as described above. jev cprme-expressing packaging cells were infected with jev-egfp srips at tcid in the presence and absence of mj- . after -h incubation, treated and infected cells were washed trypsinized, collected by the centrifugation at rpm for min, fixed in ml % alcohol at − • c overnight, and then were analyzed by bd facsaria (becton dickinson) with excitation at nm and emission at nm. ic values were calculated according to a change in ratio of green fluorescence intensity and green fluorescence positivity of treated/infected cells to those of un-treated infected cells, respectively. monolayer of bhk- cells in -well plates was infected with jev ( pfu) and simultaneously treated with mj- ( , , , , , , and µm). after -h incubation ( • c, % co ), plaque was quantified after staining by naphthol blue-black dye. inhibitory concentration showing % jev plaque reduction (ic ) was calculated using plaque reduction data from three independent experiments. in the virucidal assay, the mixture of jev ( pfu per reaction) with indicated concentrations of mj- was incubated for h at • c. the -fold dilution of the mixture was added onto a bhk- cell monolayer, and then followed by the protocol of plaque assay. in the virus attachment assay, the mixtures of jev ( pfu per well) with mj- ( , , , or µm) were immediately added onto a bhk- cell monolayer at • c for h. after washing with cold pbs, the cell monolayer was followed by the protocol of plaque assay. virucidal and attachment inhibition activities were calculated as residual plaques. each piece of data from three independent experiments was represented as mean ± standard deviation (s.d.). each group was evaluated by a student t-test. if its p-value was lower than . , the comparison was recognized as a statistical significance. a jev-egfp replicon consisting of five fragments self-replicated in e. coli and was verified using restriction endonuclease fragmentation and nucleotide sequence analyses. the sizes of resultant restriction fragments using single-and double-digestion matched the given restriction map of jev-egfp replicon. moreover, nucleotide sequence analysis with sequence-specific primers (table s ) indicated that synonymous mutations and nonsynonymous mutations were identified in a jev-egfp replicon, which was nearly identical to the jev t p parent strain by the nucleotide identity of . % and amino acid identity of . % (tables s and s ) . to examine the functional properties of jev-egfp replicon in vitro, viral genome transcription and translation of the replicon in human medulloblastoma te cells were further characterized (figures and ) . relative copy numbers of positive-and negative-sense jev subgenomes were measured using real-time reverse transcription-pcr with specific capture and amplification primers, and normalized by housekeeping gene gapdh (figures and s ) . the copy numbers of positive-sense jev subgenomes viruses , , of were . × copies in transfected cells with jev-egfp replicon at h post transfection. meanwhile, the copy numbers of negative-sense jev subgenomes were detectable at late stage of jev replication in replicon-transfected cells. the ratio of positive-to negative-sense rna synthesis was greater than , implying highly efficient self-replication of viral genomes in replicon-transfected cells. in addition, fluorescent microscopy revealed the green fluorescence in the replicon-transfected cells, indicating the expression of replicon-driven egfp. immunofluorescent staining also demonstrated the synthesis of viral proteins like ns b in transfected cells ( figure ) . interestingly, the cytopathic effect (cpe) was observed in replicon-transfected mock cells and replicon-transfected packaging cells, but not in mock cells and packaging cells (figure ). in addition, the cpe level was higher in replicon-transfected packaging cells than replicon-transfected mock cells, which was associated with a lower amount of e and ns b in replicon-transfected packaging cells compared to replicon-transfected mock cells. jev non-structural proteins like ns b were more abundant than egfp at late stage of jev replicon replication in mock cells. the results showed that pbr plasmid-based jev replicon enabled production of the self-replicated jev rna genomes and viral non-structural proteins in cells. to examine the functional properties of jev-egfp replicon in vitro, viral genome transcription and translation of the replicon in human medulloblastoma te cells were further characterized (figures and ) . relative copy numbers of positive-and negative-sense jev subgenomes were measured using real-time reverse transcription-pcr with specific capture and amplification primers, and normalized by housekeeping gene gapdh (figure , figure s ). the copy numbers of positivesense jev subgenomes were . × copies in transfected cells with jev-egfp replicon at h post transfection. meanwhile, the copy numbers of negative-sense jev subgenomes were detectable at late stage of jev replication in replicon-transfected cells. the ratio of positive-to negative-sense rna synthesis was greater than , implying highly efficient self-replication of viral genomes in replicon-transfected cells. in addition, fluorescent microscopy revealed the green fluorescence in the replicon-transfected cells, indicating the expression of replicon-driven egfp. immunofluorescent staining also demonstrated the synthesis of viral proteins like ns b in transfected cells (figure ) . interestingly, the cytopathic effect (cpe) was observed in replicon-transfected mock cells and replicon-transfected packaging cells, but not in mock cells and packaging cells (figure ). in addition, the cpe level was higher in replicon-transfected packaging cells than replicon-transfected mock cells, which was associated with a lower amount of e and ns b in replicon-transfected packaging cells compared to replicon-transfected mock cells. jev non-structural proteins like ns b were more abundant than egfp at late stage of jev replicon replication in mock cells. the results showed that pbr plasmid-based jev replicon enabled production of the self-replicated jev rna genomes and viral non-structural proteins in cells. to generate the packaging cell line for the production of jev-egfp srips, a stably transfected te cell line with pflag-cmv -cprme was selected after a long-period screening with g . the expression of jev structural proteins, such as e protein, in a stably transfected packaging cell line viruses , , of was detected using immunofluorescent staining ( figure ). subsequently, pbr -based jev-egfp replicon was transfected into the packaging cell line. real-time rt-pcr and immunofluorescent staining demonstrated jev-egfp replicon-driven viral positive-and negative-sense rna genomes were synthesized, as well as replicon-driven egfp and viral non-structural proteins being expressed in the packaging cell line (figures and ) . the copy numbers of positive-sense jev subgenomes were . × copies in packaging cells transfected with jev replicon at h post transfection. the copy numbers of negative-sense jev subgenomes were also detectable at late stages of jev replication in replicon-transfected packaging cells. the ratio of positive-to negative-sense rna synthesis was greater than , implying higher efficient self-replication of replicon-driven viral subgenomes in packaging cells compared to mock cells. immunofluorescent staining indicated that the amount of viral proteins, such as e and ns b, was lower in packaging cells transfected with jev-egfp replicon than un-transfected packaging cells or mock cells transfected with replicon, which was associated with the cytopathic effect in replicon-transfected packaging cells due to the generation of jev-egfp srips. therefore, jev-egfp srips were harvested from the supernatant and lysate of replicon-transfected packaging cells h post transfection. to examine the infectivity of jev-egfp srips in vitro, the packaging cells were infected with jev-egfp srips at tcid , and then srip-induced cytopathic effect and srip-driven egfp in packaging cells were observed h post infection ( figure a ). the green fluorescent intensity of mock-infected and srip-infected cells was quantitated using flow cytometry ( figure b-d) . srip-infected cells had a significant increase in the ratio of green fluorescence-positive cells and the accumulated fluorescence intensity compared to mock-infected cells ( figure c,d) . the results demonstrated the production of infectious srips in co-transfected cells and quantitative analysis of srip infectivity using flow cytometry. to generate the packaging cell line for the production of jev-egfp srips, a stably transfected te cell line with pflag-cmv -cprme was selected after a long-period screening with g . the expression of jev structural proteins, such as e protein, in a stably transfected packaging cell line was detected using immunofluorescent staining ( figure ). subsequently, pbr -based jev-egfp replicon was transfected into the packaging cell line. real-time rt-pcr and immunofluorescent staining demonstrated jev-egfp replicon-driven viral positive-and negative-sense rna genomes were synthesized, as well as replicon-driven egfp and viral non-structural proteins being expressed in the packaging cell line (figures and ) . the copy numbers of positive-sense jev subgenomes were . × copies in packaging cells transfected with jev replicon at h post transfection. the copy numbers of negative-sense jev subgenomes were also detectable at late stages of jev replication in replicon-transfected packaging cells. the ratio of positive-to negative-sense rna synthesis was greater than , implying higher efficient self-replication of replicon-driven viral subgenomes in packaging cells compared to mock cells. immunofluorescent staining indicated that the amount of viral proteins, such as e and ns b, was lower in packaging cells transfected with jev-egfp replicon than un-transfected packaging cells or mock cells transfected with replicon, which was associated with the cytopathic effect in replicon-transfected packaging cells due to the generation of jev-egfp srips. therefore, jev-egfp srips were harvested from the supernatant and lysate of replicontransfected packaging cells h post transfection. to examine the infectivity of jev-egfp srips in vitro, the packaging cells were infected with jev-egfp srips at tcid , and then srip-induced cytopathic effect and srip-driven egfp in packaging cells were observed h post infection ( figure a ). the green fluorescent intensity of mock-infected and srip-infected cells was quantitated using flow cytometry ( figure b-d) . sripinfected cells had a significant increase in the ratio of green fluorescence-positive cells and the accumulated fluorescence intensity compared to mock-infected cells ( figure c,d) . the results demonstrated the production of infectious srips in co-transfected cells and quantitative analysis of srip infectivity using flow cytometry. b. to access whether jev-egfp srips could be applicable in the antiviral assays and substitute for jev virions, a combination of jev-egfp srips and flow cytometry was used to evaluate the ic values of mj- compound against jev, compared to a plaque reduction assay with wild type virions (figures and ) . mj- was a quinazolinone derivative with a low cytotoxicity ( figure a , supplemental figure s ). mj- served as a significant inhibition on the jev-induced cytopathic effect in te cells, respectively ( figure b ). mj- had no effect on virucidal activity and virus attachment inhibition ( figure s ) to access whether jev-egfp srips could be applicable in the antiviral assays and substitute for jev virions, a combination of jev-egfp srips and flow cytometry was used to evaluate the ic values of mj- compound against jev, compared to a plaque reduction assay with wild type virions (figures and ) . mj- was a quinazolinone derivative with a low cytotoxicity ( figure a , supplemental figure s ). mj- served as a significant inhibition on the jev-induced cytopathic effect in te cells, respectively ( figure b ). mj- had no effect on virucidal activity and virus attachment inhibition ( figure s ) in the plaque assay (d), cell monolayer was infected with jev ( pfu), immediately treated with mj- , overlaid with ml of a methylcellulose medium for days, and then stained with naphthol blue-black dye. plaque reduction was calculated from ratio of experimental data to un-treated control. **, p-value < . ; ***, p-value < . compared with mock control. the cmv promoter-launched replicon did not need either an in vitro transcription step required for the t promoter-launched replicon, or co-transfection of plasmid containing t rna polymerase and t promoter-launched replicon [ , ] . moreover, dna-dependent rna polymerase had a higher fidelity than rna-dependent rna polymerase. thus, this study constructed the cmv promoter-launched jev replicon carrying a green fluorescence reporter (egfp) (figures and ) . however, the cloning process of jev full-length infectious clones and replicons revealed that nonsense mutations (nt c t and nt c t) and the deletion with nt - appeared in the onethird (nt - ) of the jev genome at ′-end (data not shown). importantly, the nonsense mutations and deletion identified resulted in the production of truncated m protein and the loss of e protein in the cells. our findings indicated that the prokaryotic promoter activity of the ′ one-third of jev genome directed the synthesis of the structural proteins c, prm/m and e that might be toxic genes or proteins in bacteria. the toxic sequences, especially the region encoding the prm protein, correlated with the genetic instability of jev infectious clones and replicons in e. coli. our findings were similar to previous reports that several e. coli promoter sequences were identified within the ′ end of jev genome, particularly nt - [ , ] . since a restriction enzyme apai site was located with jev genome nt - , the nucleotides to of jev genome encoding eight amino acids of the c in the plaque assay (d), cell monolayer was infected with jev ( pfu), immediately treated with mj- , overlaid with ml of a methylcellulose medium for days, and then stained with naphthol blue-black dye. plaque reduction was calculated from ratio of experimental data to un-treated control. *, p-value < . ; **, p-value < . ; ***, p-value < . compared with mock control. the cmv promoter-launched replicon did not need either an in vitro transcription step required for the t promoter-launched replicon, or co-transfection of plasmid containing t rna polymerase and t promoter-launched replicon [ , ] . moreover, dna-dependent rna polymerase had a higher fidelity than rna-dependent rna polymerase. thus, this study constructed the cmv promoter-launched jev replicon carrying a green fluorescence reporter (egfp) (figures and ) . however, the cloning process of jev full-length infectious clones and replicons revealed that nonsense mutations (nt c t and nt c t) and the deletion with nt - appeared in the one-third (nt - ) of the jev genome at -end (data not shown). importantly, the nonsense mutations and deletion identified resulted in the production of truncated m protein and the loss of e protein in the cells. our findings indicated that the prokaryotic promoter activity of the one-third of jev genome directed the synthesis of the structural proteins c, prm/m and e that might be toxic genes or proteins in bacteria. the toxic sequences, especially the region encoding the prm protein, correlated with the genetic instability of jev infectious clones and replicons in e. coli. our findings were similar to previous reports that several e. coli promoter sequences were identified within the end of jev genome, particularly nt - [ , ] . since a restriction enzyme apai site was located with jev genome nt - , the nucleotides to of jev genome encoding eight amino acids of the c protein were fused with the egfp reporter gene in the cmv promoter-launched jev replicon. in addition, the addition of restriction enzyme sequences noti and clai before encoding partial c-terminal transmembrane ( amino acids) of e protein allowed two unique restriction enzyme sites in the dna-based jev replicon that could be used as the expression vector in mammalian cells. therefore, the cmv promoter-launched jev-egfp replicon did not contain the toxic sequence encoding the prm protein, allowing the stability of jev cdna clones in bacteria. in addition, recombinant plasmid with the nucleotide sequence - of jev genome encoding structural proteins c, prm/m and e was stable in bacteria due to lacking of the prokaryotic promoter at nt - . the pflag-cmv-cprme exhibited the plasmid stability in e. coli. the pbr plasmid, a low copy number plasmid, was chosen as the jev replicon vector. the plasmid pbr -based jev-egfp replicon was genetically stable in e. coli, exhibiting high nucleotide ( . %) and amino acid ( . %) identities (tables s and s ). the pbr -based replicon also had a higher yield in e.coli than cosmid vectors and bacterial artificial chromosomes [ ] , offering an effective and efficient approach for manipulating and cloning the viral genes. among non-silent mutations in the replicon compared to the original genome sequence of jev t p strain, eight mutated residues appeared within ns and three mutations occurred within ns (table s ). these eight mutations in the replicon did not relate with active sites and conserved motifs of ns protease and helicase as well as ns methyltransferase and rna-dependent rna polymerase based on the structure and functionality analysis of flaviviral non-structural proteins [ , ] . in addition, the other amino acid substitutions in the replicon sporadically existed within ns , ns a, ns b, ns a and ns b did not match with the reported mutation sites that altered jev replication [ , ] . the finding suggested that mutations in the replicon might have no significant effect on virus replication. functional characterization of jev-egfp replicon indicated a significant increase of green fluorescence intensity in replicon-transfected cells (figure ) . moreover, the percentage and yellow fluorescent intensity of ns b-positive cells were is much higher than the percentage and green fluorescent intensity of egfp-expressing cells post transfection with jev-egfp replicon. the results might be associated with the loss or quenching effect of egfp fluorescence by the fixation process of stained cells with paraformaldehyde, ethanol or methanol [ ] . moreover, the synthesis of positive-and negative-sense rna subgenomes as well as the expression of jev proteins were detected in replicon-transfected cells (figures and ) . jev-egfp replicon contained cmv immediate-early promoter at the -end, as well as hdvr and sv termination and polyadenylation sequences at the -end, which allowed the production of functional jev subgenomes (figures - ) . the pflag-cmv-cprme was utilized to establish the packaging cell line that continuously expressed c, prm/m, and e proteins to allow the production of jev srips h post a transient transfection with jev-egfp replicon (figures - ) . therefore, producing jev srips was an alternative approach to overcoming the obstacles in the genetic instability of jev infectious clones using the full-length cdna sequence. although there is still a question about whether these mutations affect the genome replication efficiency, jev-egfp replicon and srip generated in this study could be used to screen antiviral agents. then, the antiviral activity of identified agents might be further confirmed using wild type jev virions. egfp reporter was the real-time functional marker as the genome replication and viral protein expression of jev-egfp replicon in mock cells and srip in packaging cells, detected using fluorescent microscope (figures - ) . the fluorescent intensity and positivity in srip-infected cells were further quantitated using flow cytometry ( figure ). the reasons for low egfp-positive percentage and fluorescent intensity in un-treated srip-infected cells could be ( ) a low infectious dose with tcid srips; ( ) the loss of egfp fluorescence with the fixation with ethanol, and ( ) a highly stringent threshold for the positivity of green fluorescence in the infected cells. for increasing the egfp-positive cells without treatment, the elevation of moi was used in the assay and the fluorescent intensity in the infected cells were directly measured using a fluorescence microplate reader under the condition without the fixation agents. in the screening assay to identify antiviral agents, figure c based on the total fluorescent intensity showed the near line with a negative slope in antiviral activity of mj- against jev, but not figure b based on the green fluorescence positivity. since plaque reduction assay was labor-intensive, time-consuming and safe keeping, the combination of jev-egfp srip and flow cytometry might be easy, rapid, and safer approaches to determining the ic values of antiviral agents. comparison of ic values of mj- against jev using plaque reduction and jev-egfp srip plus flow cytometry indicated the ic value ( . ~ . µm) of mj- by jev-egfp srip plus flow cytometry was lower than plaque reduction assay ( . µm) (figure ) . the result demonstrated that the assay of jev-egfp srip plus flow cytometry was more sensitive than plaque reduction assay, showing the potential application in antiviral drug screening and neutralizing assays. in addition, the system of jev-egfp srip in packaging cells plus flow cytometry was easily observed using a fluorescent microscope and quantitated by flow cytometry, suggested as a better tool compared to the luciferase reporter system [ , ] . the approach for the design of jev-egfp srip plus flow cytometry might be applicable to other flaviviruses, such as dengue and zika viruses. moreover, the alternative approaches to quantitate replicon-driven reporter activity might be applicable for high-throughput screening antiviral agents, such as the combination of jev-egfp srip and fluorescence microplate reader. mj- (methyl -(( -( -methoxyphenyl)- -(pyrrolidin- -yl)quinazolin- -yl)oxy)acetate), a quinazolinone derivative, processed the potently anti-jev activity, in which an ic value of . µm was determined by the assay of jev-egfp srip in packaging cells plus flow cytometry ( figure ) . moreover, mj- reduced the synthesis of viral genomes and the expression of viral proteins ( figure c ,d), implying that mj- significantly decreased the efficient self-replication of replicon-driven viral subgenomes. however, mj- affected neither irreversible loss of jev infectivity (virucidal activity), nor virus attachment to host cells (supplemental figure s ) . therefore, we suggested that anti-jev mechanism(s) of mj- could be related with the influence on late stages of jev replication cycle, e.g., viral transcription and translation. interestingly, several quinazolinone derivatives had identified their antiviral activity against a broad range of dna and rna viruses, such varicella-zoster virus, cytomegalovirus, influenza a virus, and hepatitis c virus [ ] [ ] [ ] . a -pyridinyl- ( h)-quinazolinone derivative potently inhibited the replication of influenza a virus in vitro via suppressing virus neuraminidase and cellular nf-κb signal [ ] . quinazolinone-containing macrocycles were considered as next-generation hcv ns / a protease inhibitors [ ] . therefore, mj- 's antiviral ability might be further examined against other flaviviruses. in conclusion, the jev-egfp replicon, avoiding genetic instability of the -end one-third of the full-length jev genome cdna, was constructed into the low-copy plasmid pbr , regulated under the control of cmv promoter, and transfected into a packaging cell line expressing viral structural proteins for the generation of jev-egfp srips. the reporter gene egfp, correlating with the infectivity of jev-egfp srips in packaging cells, were easily and rapidly utilized to screen antiviral agents using fluorescent microscopes. the combination of jev-egfp srip plus flow cytometry was a reliable, effective, and efficient assay for measuring the ic values of antiviral agents like mj- based on the fluorescent intensity and positivity of srip-infected packaging cells post treatment. supplementary materials: the following are available online at www.mdpi.com/ - / / / /s , figure s : modification of multiple cloning sites by the insertion of the linker kpni-noti-xhoi into the pbr with ecori and bamhi double digestion, figure s : preparation of chimeric sequences of cmv promoter and jev '-seq ( - nt) using jumping pcr, figure s : primer design for quantitative analysis of positive-and negative-sense rna genome using sybr-green rt-pcr, figure s : general experimental procedures for mj- synthesis. nah, sodium hydride; dmf, n,n-dimethylformamide, figure s : survival rate of treated te cells with mj- . te cells cultured on -well plates were treated with mj- , incubated for h, and then followed by mtt assay, figure s : virucidal and attachment inhibitory activities of mj- against jev, table s : primers for constructing pbr -based jev-egfp replicon and pflag-cmv-cprme plasmid, table s : primers for sequencing pbr -based jev-egfp replicon and pflag-cmv-cprme plasmid, table s : list of synonymous substitutions in the jev-egfp replicon, table s : list of nonsynonymous substitutions in the jev-egfp replicon. present and future arboviral threats zika virus: new clinical syndromes and its emergence in the western hemisphere origin and evolution of japanese encephalitis virus in southeast asia a conserved internal hydrophobic domain mediates the stable membrane integration of the dengue viruscapsid protein a single amino acid substitution in the m protein attenuates japanese encephalitis virus in mammalian hosts envelope protein mutations l f and e k are important for neurovirulence attenuation for japanese encephalitis virus sa - - strain genotype-specific neutralization determinants in envelope protein: implications for the improvement of japanese encephalitis vaccine immunolocalization of the dengue virus 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dna vaccine candidate japanese encephalitis virus replicon-based vaccine expressing enterovirus- epitope confers dual protection from lethal challenges -alkylamino-and , -dihydro- -methoxy- -phenyl- -quinazolinones and related compounds: their synthesis, cytotoxicity, and inhibition of tubulin polymerization construction of an infectious molecular clone of japanese encephalitis virus genotype v and its derivative subgenomic replicon capable of expressing a foreign gene genetic instability of japanese encephalitis virus cdna clones propagated in escherichia coli successful propagation of flavivirus infectious cdnas by a novel method to reduce the cryptic bacterial promoter activity of virus genomes structure and functionality in flavivirus ns-proteins: perspectives for drug design crystal structure of the full-length japanese encephalitis virus ns reveals a conserved methyltransferase-polymerase interface an amino acid substitution (v i) in the japanese encephalitis virus ns a protein increases its virulence in mice, but not its growth rate in vitro recovery of a chemically synthesized japanese encephalitis virus reveals two critical adaptive mutations in ns b and ns a fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains new isoxazolidine-conjugates of quinazolinones-synthesis, antiviral and cytostatic activity -pyridinyl- ( h)-quinazolinone: a scaffold for anti-influenza a virus compounds p -quinazolinones and bis-macrocycles as new templates for next-generation hepatitis c virus ns / a protease inhibitors: discovery of mk- and mk- the authors declare no conflict of interest. key: cord- -i oxm mr authors: kautz, tiffany f.; jaworski, elizabeth; routh, andrew; forrester, naomi l. title: a low fidelity virus shows increased recombination during the removal of an alphavirus reporter gene date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: i oxm mr reporter genes for rna viruses are well-known to be unstable due to putative rna recombination events that excise inserted nucleic acids. rna recombination has been demonstrated to be co-regulated with replication fidelity in alphaviruses, but it is unknown how recombination events at the minority variant level act, which is important for vaccine and trans-gene delivery design. therefore, we sought to characterize the removal of a reporter gene by a low-fidelity alphavirus mutant over multiple replication cycles. to examine this, gfp was inserted into tc- , a live-attenuated vaccine for the alphavirus venezuelan equine encephalitis virus, as well as a low-fidelity variant of tc- , and passaged until fluorescence was no longer observed. short-read rna sequencing using clickseq was performed to determine which regions of the viral genome underwent recombination and how this changed over multiple replication cycles. a rapid removal of the gfp gene was observed, where minority variants in the virus population accumulated small deletions that increased in size over the course of passaging. eventually, these small deletions merged to fully remove the gfp gene. the removal was significantly enhanced during the passaging of low-fidelity tc- , suggesting that increased levels of recombination are a defining characteristic of this mutant. rna viruses are notoriously error-prone due to the lack of an rna-dependent rna polymerase (rdrp) proofreading enzyme (with the unique exception of proofreading exonucleases present in coronavirus genomes). this error results in approximately one mutation per round of genome replication [ ] , which due to the rapid replication of rna viruses, culminates in a broad cloud of related virus genomes, commonly referred to as viral intra-host diversity, and in specific cases as a quasispecies. if the intra-host viral population is either too diverse or too clonal, this can impact the fitness of the virus population, which commonly results in a reduction in host tropism or virulence [ ] [ ] [ ] [ ] . while the quasispecies theory has been studied extensively in silico, in vitro, and in vivo by analyzing the ratios of nucleotides at specific genomic sites [ ] [ ] [ ] , the contribution of recombination between rna virus minority variants and the effects on genome maintenance and evolution have been comparatively understudied. rna virus recombination primarily occurs when the rdrp switches from the template rna strand to an acceptor rna strand (i.e., copy choice recombination) or, less frequently, by ligation of cleaved rna strands [ ] . both homologous recombination, where the rdrp switches to a template with high sequence similarity, and nonhomologous recombination, where the rdrp switches to a template with low sequence similarity, can occur [ ] . the molecular determinants of rna recombination are not well-defined, but rna recombination has been described for viruses such as poliovirus and brome-mosaic virus to arise more frequently in regions with high amounts of rna secondary structure and au-rich sequences [ , ] . additionally, other factors, such as sequence homology between rna strands and altered replication kinetics have been implicated in changing the rate of rna recombination [ ] . rates of rna recombination vary between different families of rna viruses. for example, picornaviruses have relatively high rates of recombination [ ] compared to families such as flaviviridae where viable recombination is less common [ ] . venezuelan equine encephalitis virus (veev) is an alphavirus from the family togaviridae. alphavirus genomes are composed of a positive-sense, monopartite single-stranded rna that is approximately . kb in length. the first two-thirds of the genome encodes the nonstructural proteins, while the last third of the genome encodes the structural proteins under a subgenomic promoter [ ] . inter-strain of inter-species rna recombination is not thought to be common for members of the alphavirus genus due to superinfection exclusion [ ] [ ] [ ] , which occurs rapidly upon cellular infection and precludes the high multiplicity infections believed to be required for frequent recombination events between co-infecting viral genomes [ ] . nonetheless, alphavirus coinfection has been described [ ] , and it is thought that the recombination of sindbis and eastern equine encephalitis virus-like ancestors, two different alphaviruses, produced western equine encephalitis virus [ , ] . conversely, the formation of defective viral genomes (dvgs) of alphaviruses has been well described for numerous family members. dvgs are generated due to nonhomologous rna recombination events either intra-molecularly or between two copies of a viral genome resulting in shorter recombined versions of the parental genome missing critical regions required for autonomous replication. we have extensively characterized rna recombination in the alphavirus, chikungunya virus, and demonstrated that rna recombination events are plentiful even under low moi and low passage conditions resulting in the synthesis of dvgs de novo after viral infection [ ] . beyond the role that rna recombination plays during the natural replication-cycle of rna viruses both in vitro and in vivo, it is important to characterize the role of rna recombination in the stability of recombinantly engineered strains of rna virus containing inserted reporter genes (e.g., green fluorescent protein (gfp), luciferase). increasing the understanding of where these recombination events occur may allow for a more rational design of how to insert foreign genetic material into viral genomes to increase their genetic stability, as well as how to reduce the impact that these insertions commonly have on virus fitness. here, we inserted gfp into a live-attenuated vaccine for venezuelan equine encephalitis virus, tc- , and passaged the virus using a physiologically relevant low multiplicity of infection (moi). illumina sequencing using clickseq library preparation [ ] was used to determine minority variant recombination events in the genome. we found a slow but persistent removal of the gfp gene that was highly specific to the inserted gene. additionally, we found that a low-fidelity variant of tc- [ ] removed the gfp reporter gene much more efficiently. vero (african green monkey kidney) cells were obtained from the american type cell collection (atcc, bethesda, md, usa) and maintained in dmem (gibco, gaithersburg, md, usa) supplemented with % fetal bovine serum (atlanta biologicals, flowery branch, ga, usa), and µg/ml gentamycin (corning, corning, ny, usa) in a • c % co incubator. a tc- gfp infectious clone was kindly provided by dr scott weaver (utmb), and standard cloning protocols were used to insert the gfp gene into the low-fidelity tc- genome. as described previously [ ] , low-fidelity tc- contains three coding mutations to the rdrp, which were found to lower the mutation frequency produced by this mutant during replication. following this, the plasmids were transcribed, and the virus was rescued by electroporation (ep) using bhk cells. titers of the ep stock virus were determined by the plaque assay using vero cells [ ] . one day prior to infection, a six-well plate was seeded with , vero cells. the day of infection, the tc- gfp or tc- x gfp (low-fidelity tc- ) virus was diluted , -fold for an moi of approximately . . once diluted, media was decanted from the six-well vero cell plate, and µl was used to infect each well. these infections were done using three replicates per virus. following infection, the plate was transferred to a • c % co incubator for h with occasional rocking. when the incubation was complete, the inoculum was removed, and ml of dmem (gibco, gaithersburg, md, usa) supplemented with % fbs (atlanta biologicals, flowery branch, ga, usa) and µg/ml gentamycin (corning, corning, ny, usa) was added to each well. the plates were then incubated for h in a • c % co incubator. following this, the cell supernatant was removed, transferred to a tube with fbs (atlanta biologicals, flowery branch, ga, usa) to a final concentration of % fbs, and centrifuged at for min at . rcf to remove debris. virus aliquots were stored at − • c. plaque assays were used to determine virus titer [ ] . ten total passages were performed for each virus and replicate. to determine pfu:gfp, plaque assays were performed as per the usual protocol [ ] . after h, the cells were visualized under a fluorescent microscope, and fluorescent plaques were counted. following this, the cells were fixed and stained following the standard plaque assay protocol, and the number of plaques was counted. the number of gfp to plaque forming units (pfu) was then calculated as a ratio (i.e., # plaques/# gfp fluorescent foci) to determine the amount of gfp fluorescence lost over the course of passaging. viral rna was extracted using the zymo direct-zol rna mini kit as per the manufacturer's protocol (zymo, irvine, ca, usa). rna sequencing libraries were made following the standard clickseq protocol using ng of viral rna, as previously described [ , ] . clickseq was used instead of the traditional illumina library procedures to avoid the errant recombination that frequently occurs during the typical fragmentation and ligation steps. cdna libraries between - bp were excised and purified using the zymo research gel recovery kit (zymo, irvine, ca, usa). sequencing was performed by the university of texas medical branch next-generation sequencing core using an illumina nextseq obtaining × bp reads. cutadapt [ ] was used to remove adaptor sequences. following this, the fastx toolkit [ ] was used to remove the random hexamer sequences generated during cdna synthesis, as well as sequences with a phred score below . bowtie [ ] was used to align the virus sequences to the reference genome (accession number, l ), allowing for one mismatch. those sequences that did not align using bowtie were processed using virema [ ] , a viral recombination mapper, to determine the incidence and location of recombination events. these reads were normalized to the average number of reads aligning to the virus genome. m-fold [ ] was used from nucleotide position to the end of the virus genome to determine the secondary structure of the virus genomes and how this related to the observed recombination events. raw fastq data are available on the ncbi small read archive under bioproject id prjna . to examine reporter gene stability, three independent replicates of tc- gfp and low-fidelity tc- gfp were passaged times on vero cells using an approximate moi of . ( figure a ). over the course of this passaging, reduced gfp expression for tc- was observed beginning at passage ( figure b ). in contrast, gfp loss was observed much earlier for the low-fidelity tc- replicates, typically by passage ( figure c ). viruses , , x for peer review of to examine reporter gene stability, three independent replicates of tc- gfp and low-fidelity tc- gfp were passaged times on vero cells using an approximate moi of . ( figure a ). over the course of this passaging, reduced gfp expression for tc- was observed beginning at passage ( figure b ). in contrast, gfp loss was observed much earlier for the low-fidelity tc- replicates, typically by passage ( figure c ). to quantify this loss, the number of fluorescent plaques was compared to the number of overall plaques for passages - . until passage , the majority of tc- plaques were fluorescent, and the largest pfu:gfp difference was approximately : , which was only observed for one replicate during the final passage ( figure d ). in contrast to this, all low-fidelity tc- replicates experienced an approximately - -fold decrease in fluorescence by passage , which was orders of magnitude higher than the ratios observed for the tc- gfp passage replicates ( figure e ). as a low-fidelity sindbis virus mutant was previously found to produce defective interfering (di) particles at high rates [ , ] , we hypothesized that the low-fidelity tc- was also recombining at higher rates than tc- , resulting in rapid gfp gene removal. to quantify this loss, the number of fluorescent plaques was compared to the number of overall plaques for passages - . until passage , the majority of tc- plaques were fluorescent, and the largest pfu:gfp difference was approximately : , which was only observed for one replicate during the final passage ( figure d ). in contrast to this, all low-fidelity tc- replicates experienced an approximately - -fold decrease in fluorescence by passage , which was orders of magnitude higher than the ratios observed for the tc- gfp passage replicates ( figure e ). as a low-fidelity sindbis virus mutant was previously found to produce defective interfering (di) particles at high rates [ , ] , we hypothesized that the low-fidelity tc- was also recombining at higher rates than tc- , resulting in rapid gfp gene removal. clickseq was used to generate illumina rna sequencing libraries, allowing for the determination of recombination hotspots as well as the frequency of virus deletion mutants. all three tc- gfp replicates contained deletion variants present at a low frequency that were able to selectively remove the gfp reporter gene by passage (figure a) . the deletion inside the gfp gene was higher than for any other gene and occurred as soon as passage . in support of our hypothesis, low-fidelity tc- gfp was much more efficient at removing the gfp gene, as the frequency of deletions within the gfp gene was far higher than those of tc- gfp, which was consistent among all replicates ( figure b ). the first replicate of each virus was chosen for further analysis, as these both had median levels of decreased gfp fluorescence in figure d ,e. for both viruses, peak recombination first appeared in the middle of the gfp gene, and progressively removed additional nucleotides over the course of passaging until the entire gfp gene was removed (figure ). low-fidelity tc- was able to efficiently remove the entire gfp fragment by passage , while tc- required five additional passages to reach similar levels of gfp removal. this clearly demonstrates that low-fidelity tc- gfp produces recombination variants more frequently than tc- gfp. figure . virema recombination results for tc- gfp and low-fidelity tc- gfp. each passage of the first replicate of tc- gfp and low-fidelity tc- gfp was sequenced using clickseq libraries and analyzed using virema. the x-axis shows genome position, while the y-axis shows the recombination frequency (i.e., normalized to the total reads that map to low-fidelity tc- gfp). below the recombination charts, a schematic shows the approximate location of the nonstructural protein, gfp, and structural genes. "p" represents passage. to determine if the regions where recombination occurred most frequently were conserved between replicates, the top recombination events for each virus and replicate were identified (supplemental file ). with few exceptions, all of the most common recombination events were deletions found within and around the gfp gene, regardless of the virus backbone. of the top five recombination events for each passage and replicate, all the replicates of low-fidelity tc- predominately exhibited a deletion from nucleotide positions - for the first five passages. this happened concurrently with another deletion event, - , which occurred slightly less frequently than the - deletion. following the appearance of these two deletions, a much larger removal occurred from nucleotides - , which encompassed the entire gfp gene ( figure b ). in one replicate, this deletion was found to be one of the most predominant deletions after only one passage, and in another replicate, this became the predominate deletion by passage . by passage , this - deletion quickly became the most common deletion event by - orders of magnitude for all low-fidelity tc- gfp replicates. unlike the low-fidelity mutant, tc- did not show the same conservation of deletions between replicates and passages, but, like low-fidelity tc- , the - deletion was one of the most frequently observed. the other two deletions observed during low-fidelity tc- passaging were also sporadically found as one of the five most common recombination events, but not consistently between replicates. however, while not always the most common of mutations, these three mutations still occurred during tc- gfp passaging and at levels comparable to the low-fidelity tc- virus ( figure a) . conversely, the level of the variance observed between the replicates of tc- gfp and low-fidelity tc- gfp was large, with higher variances observed between the replicates for tc- gfp than for low-fidelity tc- gfp. other than these three deletions, all tc- gfp replicates produced the majority of the deletions surrounding nucleotides - . the length and frequency of these deletions were variable but suggested that this region of the gfp gene was the most amenable to deletions and insertions of various forms. to better understand the mechanisms underlying the conserved removal of the low-fidelity tc- gfp gene, m-fold was used to determine the rna secondary structure of the virus genome ( figure c-e) . the - and - deletions were found close together in the primary rna sequence, and the sequence found between - was identical to the sequence found between - , which was indicative of homologous recombination. this five base pair sequence (ggcaa) was similarly structured between - and - , with three g-c nucleotides forming a small stem that led into a loop. as previously stated, once these two deletions occurred, a - deletion surrounding the gfp gene appeared and rose to prominence. like the other two deletions, a nearly identical sequence was found at the beginning and end of the deletion (cuaga vs. cuaaga), showing micro-homology at the site of the recombination juncture. unlike the previous deletion, these sequences were found in a loop structure and were far away from each other in the predicted secondary structure. viruses , , x for peer review of figure . frequency of the three most common deletion events during tc- gfp and low-fidelity tc- gfp passaging and rna structure locations of these events in low-fidelity tc- gfp. as tc- gfp (a) and low-fidelity tc- gfp (b) were passaged, three deletions ( - ,  - , and  - ) rose to prominence, especially in the low-fidelity passages. frequency of the recombination events was determined by dividing the number of recombination events by the total number of sequencing reads. if one of the three deletion events depicted above was not found for a passage replicate (i.e., below the limit of detection), no data point was depicted. the rna structure of low-fidelity tc- was determined using m-fold (g = − . ) (c-e). the gfp gene (nucleotides - ) is highlighted in yellow (c). during passages - , the nucleotide deletions - and - (yellow highlight) concurrently arose in all replicates (d). an identical nucleotide sequence, ggcaa, was found at the beginning and end of these initial deletions. after these deletions, a - nucleotide deletion (yellow highlight) arose and became the dominant deletion mutant in the population, resulting in the total excision of the gfp gene (e). a nearly identical sequence was found at the beginning and end of this deletion juncture (cuaga vs. cuaaga). figure . frequency of the three most common deletion events during tc- gfp and low-fidelity tc- gfp passaging and rna structure locations of these events in low-fidelity tc- gfp. as tc- gfp (a) and low-fidelity tc- gfp (b) were passaged, three deletions (∆ - , ∆ - , and ∆ - ) rose to prominence, especially in the low-fidelity passages. frequency of the recombination events was determined by dividing the number of recombination events by the total number of sequencing reads. if one of the three deletion events depicted above was not found for a passage replicate (i.e., below the limit of detection), no data point was depicted. the rna structure of low-fidelity tc- was determined using m-fold (∆g = − . ) (c-e). the gfp gene (nucleotides - ) is highlighted in yellow (c). during passages - , the nucleotide deletions - and - (yellow highlight) concurrently arose in all replicates (d). an identical nucleotide sequence, ggcaa, was found at the beginning and end of these initial deletions. after these deletions, a - nucleotide deletion (yellow highlight) arose and became the dominant deletion mutant in the population, resulting in the total excision of the gfp gene (e). a nearly identical sequence was found at the beginning and end of this deletion juncture (cuaga vs. cuaaga). it is well known that the virus reporter genes (e.g., gfp, luciferase) commonly used to answer questions related to virus lifecycles are unstable, with papers commonly reporting a - % loss of fluorescence upon one virus passage [ ] [ ] [ ] [ ] [ ] . however, it is not well understood how reporter gene removal occurs in virus populations because previous research has typically been limited to the use of low-throughput sequencing methods, generally by sanger sequencing of a handful of virus clones surrounding a small area of the virus genome. rnaseq library synthesis methods are often prone to the generation of chimeric short-reads, giving rise to artifactual recombination in the output data obfuscating the detailed analysis of rare and low-frequency rna recombination [ ] . however, the clickseq method for rnaseq library preparation is specifically designed to reduce this artifact, thus allowing for the robust analysis of minority recombination events across the entire virus genomes. in this paper, we used clickseq to better understand how gfp is removed when inserted into an alphavirus genome under a double subgenomic promoter. this technique allowed us to visualize the thousands of recombination events that occurred during virus passaging, which were predominantly found within the inserted gfp reporter gene. we originally hypothesized that gfp removal would primarily occur across the homologous promoter sequences surrounding the gfp gene. however, while true in later passages, the first gfp deletion events actually occurred in the middle of the gfp gene via the removal of smaller segments, and thus inactivating gpf expression before spreading outwards towards the promoter sequences. this removal was enhanced in a low-fidelity tc- variant. perhaps counterintuitively, the specific nucleotide deletions within the gfp gene were more conserved among the low-fidelity tc- gfp replicates than the wild type virus. this is similar to previous research with a low-fidelity sindbis virus where a conserved deletion mutant was found at elevated levels during its passaging at a high moi of [ ] . the wild type sindbis virus demonstrated no such conservation under similar passaging conditions. additionally, a high-fidelity variant of poliovirus appeared to only use one type of deletion to ablate the utility of a detrimental inserted mirna, while the wild type virus displayed little conservation in the mirna sequence deletion [ ] . together, these results suggest that high and low-fidelity rna virus mutants are more predictable in their methods of genome pruning than their wild type counterparts, which is ideal for the use of these mutants during live-attenuated vaccine and transgene delivery design. these results also suggest that altered recombination potential may be common among fidelity variants. this is unsurprising as altering fidelity also changes the speed and processivity of the rdrp [ ] [ ] [ ] [ ] [ ] [ ] [ ] , which has been linked to changes in polymerase stuttering and recombination frequency [ ] . however, there is evidence that at least some fidelity mutants do not exhibit large changes in recombination frequency or vice-versa [ , ] , although this is important to reexamine using updated techniques. in the future, using clickseq to analyze virus sequence diversity and recombination would allow both of these aspects to be examined using the same dataset, which will clarify the link between replication fidelity and recombination. the mechanism of gfp removal during low-fidelity tc- gfp passaging primarily occurred through the formation of three deletions. the first two deletions, ∆ - and ∆ - , are found within the gfp gene and were in the highest quantities for all three replicates early during passaging. the other deletion, ∆ - , fully encompasses gfp and the surrounding promoters and occurred with reduced frequency until later passages. while these deletions were also found during tc- gfp passaging, their fixation in the population was obscured by other deletions. by later tc- gfp passages, almost all of the most common deletions were found between nucleotides - , which are within the gfp subgenomic promoter. while the most frequent deletions for tc- gfp and low-fidelity tc- gfp were different, our results show that the most common deletions were not usually due to sequence homology between the two identical promotors surrounding the gfp gene but were instead due to multiple small deletions. to characterize the role of rna structure of the virus genome in rna recombination site selection, we modeled the three most common deletions that occurred at high levels during low-fidelity tc- gfp passaging. the first two deletions, ∆ - and ∆ - , were close together in the primary sequence and shared an identical sequence and structure at the and ends of their shared deletion space in a clear act of homologous recombination. in the future, it would be interesting to see if disruption of the homologous recombination sites shared by ∆ - and ∆ - could help stabilize the gfp gene, as other deletions appeared to require more replication cycles to accumulate. the larger recombination event, ∆ - , demonstrated microhomology, with nearly identical sequences around the and end of the deletion. in our study, we only examined the stability of the gfp gene when placed between two identical subgenomic promoters. in the future it would be interesting to determine how placement of the gfp gene within the virus genome could change the most common deletions within and around gfp. additionally, it would be worthwhile to examine other fidelity variants using clickseq to determine whether our observations were conserved among other fidelity mutants. however, here we demonstrate that deletions within the gfp gene are initially more common than deletions spanning the entire gfp gene, even when the gene is surrounded by identical promoter sequences. additionally, some of these deletions are common between replicates, which could be used in the future to change the secondary structure of the gfp gene while maintaining the protein sequence to aid in increasing the stability of the gene. in summary, our research has provided clear evidence for the utility of clickseq both translationally for use in understanding reporter gene stability as well as enhancing our understanding of the changes in recombination that occur during low-fidelity virus replication. funding: research reported in this publication was supported by the niaid of the national institutes of health under award numbers r -ai - a and r -ai - . alr and ej were supported by start-up funds from utmb. tfk was supported by a predoctoral fellowship from the utmb mclaughlin endowment as well as a predoctoral fellowship from the nih/niaid emerging and tropical infectious diseases training program (t ai ). the authors wish to report no conflicts of interest. viral mutation rates selforganization of matter and the evolution of biological macromolecules quasispecies theory and the behavior of rna 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stability of expression an infectious west nile virus that expresses a gfp reporter gene analysis of in vivo dynamics of influenza virus infection in mice using a gfp reporter virus engineering attenuated virus vaccines by controlling replication fidelity a speed-fidelity trade-off determines the mutation rate and virulence of an rna virus ribavirin and lethal mutagenesis of poliovirus: molecular mechanisms, resistance and biological implications design of a genetically stable high fidelity coxsackievirus b polymerase that attenuates virus growth in vivo attenuation of foot-and-mouth disease virus by engineered viral polymerase fidelity sequence-specific fidelity alterations associated with west nile virus attenuation in mosquitoes vaccine-derived mutation in motif d of poliovirus rna-dependent rna polymerase lowers nucleotide incorporation fidelity coxsackievirus b mutator strains are attenuated in vivo signatures of nucleotide analog incorporation by an rna-dependent rna polymerase revealed using high-throughput magnetic tweezers rna recombination enhances adaptability and is required for virus spread and virulence key: cord- - o dsf authors: hong, seung-min; an, se-hee; lee, chung-young; song, chang-seon; choi, kang-seuk; kim, jae-hong; kwon, hyuk-joon title: pathobiological and genomic characterization of a cold-adapted infectious bronchitis virus (bp-cakii) date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: o dsf we established a cold-adapted infectious bronchitis virus (bp-cakii) by passaging a field virus through specific pathogen-free embryonated eggs times at °c. we characterized its growth kinetics and pathogenicity in embryonated eggs, and its tropism and persistence in different tissues from chickens; then, we evaluated pathogenicity by using a new premature reproductive tract pathogenicity model. furthermore, we determined the complete genomic sequence of bp-cakii to understand the genetic changes related to cold adaptation. according to our results, bp-cakii clustered with the kii genotype viruses k and km , and showed less pathogenicity than k , a live attenuated vaccine strain. bp-cakii showed delayed viremia, resulting in its delayed dissemination to the kidneys and cecal tonsils compared to k and km , the latter of which is a pathogenic field strain. a comparative genomics study revealed similar nucleotide sequences between bp-cakii, k and km but clearly showed different mutations among them. bp-cakii shared several mutations with k (nsp , , and ) following embryo adaptation but acquired multiple additional mutations in nonstructural proteins (nsp , and ), spike proteins and nucleocapsid proteins following cold adaptation. thus, the establishment of bp-cakii and the identified mutations in this study may provide insight into the genetic background of embryo and cold adaptations, and the attenuation of coronaviruses. infectious bronchitis virus (ibv) is a pathogenic gamma coronavirus that causes respiratory symptoms, egg drops, nephritis and proventriculitis in domestic fowls [ ] . due to the persistent infection ibvs are difficult to eradicate and easy to mutate into new recombinant viruses in infected flocks [ , ] . the single-stranded, positive-sense rna genome is approximately kb and encodes rna polymerase/transcriptase ( ab), spike (s), envelope (e), membrane (m), nonstructural ( a, b, a, b), and nucleocapsid (n) proteins [ ] . approximately nonstructural proteins (nsp) are generated from the large a and ab proteins by viral proteases, and these nsp play roles in virus replication and pathogenicity [ , ] . the spike protein is a protective antigen that is essential for virus infection in host cells [ ] . in korea, several genotypes of ibv, including k-i, km -like, new cluster (nc ), qx-like and recurrent qx-like, have been reported [ ] [ ] [ ] . among them, nephropathogenic km -like viruses were first reported in and became prevalent in the field. an inactivated oil emulsion vaccine and an embryo-adapted, attenuated live vaccine (k strain) have been used to prevent km -like virus infection, but occasionally, km -like viruses have been isolated in the field [ ] . cold adaptation has been used for the attenuation of various viruses, and a cold-adapted influenza a virus (iav) has been successfully applied to the generation of attenuated human vaccine strains [ , ] . cold-adapted ibv strains have also been established and characterized; however, the biological traits and genetic backgrounds of these cold-adapted ibvs have not been completely elucidated [ ] [ ] [ ] [ ] . ibvs replicate firstly in the trachea then disseminate into internal organs. the temperature of the trachea is lower than internal organs, and it may be valuable to characterize the traits of cold-adapted ibvs. furthermore, in comparison with respiratory and kidney pathogenicity tests, reproductive organ pathogenicity tests have not been easy to perform due to the long periods of observation necessary for the sexual maturation of hens [ ] . therefore, a time-saving and reproducible animal model to test reproductive organ pathogenicity is required [ ] . in this study, we established a cold-adapted ibv (bp-cakii), and we characterized its pathogenicity in embryos and chickens, tissue tropism, and persistence of infection. we applied a premature reproductive tract pathogenicity model to the differentiation of the pathogenicity of ibvs and performed comparative genomics to shed light on the genetic background of the embryo adaptation of k and the cold adaptation of bp-cakii. snu was isolated from pooled tissues of the cecal tonsil and trachea of commercial layers ( -day-old) and was sent to the laboratory of avian diseases of seoul national university for diagnosis in . a live attenuated commercial vaccine strain, k was previously established by times passages of embryonated eggs [ ] . a nephropathogenic field strain, km that had been passaged times through embryonated eggs was kindly provided by avian disease division, animal and plant quarantine agency in korea. the viruses were propagated by inoculating into -day-old spf embryonated eggs (valo biomedia, adel, ia, usa) via the allantoic cavity route and incubated for to h, after which they were chilled at • c overnight. the allantoic fluid was harvested, and the supernatant was stored at − • c after centrifugation at rpm ( × g) for min. a primer set for real-time rt-pcr was designed based on the conserved region of the nsp genes of reference strains in the genbank (table ). the reference strains used for the primer design are as follows: lx, peafowl/gd/kq , km , qia- , qia-kr/d / , qia-q , snu- , snu- , m , turkey coronavirus, snu- , ita/ / , and arkdpi . their accession numbers are available in figure . primers for genome amplification and sequencing were described previously and additional new primers were summarized in table [ ] . viral genomic rna was extracted from µl of infectious allantoic fluid using the viral gene-spin kit (intron biotechnology, seongnam, korea). sybr real-time one-step rt-pcr (real-time rt-pcr) was performed using an applied biosystems stepone real-time pcr machine and a one-step rt-pcr kit (kapa-biosystems, boston, ma, usa) according to the manufacturer's instructions. total reaction volumes were adjusted to µl and µl of the extracted rna used. conventional rt-pcr for genome amplification was performed using a one-step rt-pcr kit (qiagen gmbh, hilden, germany) according to the manufacturer's instructions. the rt-pcr conditions were as follows: cdna synthesis at • c for min, inactivation of the reverse transcriptase at • c for min, cycles of denaturation at • c for s, annealing at • c for s, and extension at • c for min, with a final extension at • c for min. the amplicons were purified using a pcr purification kit (mega-quick-spin total fragment dna purification kit, intron biotechnology) and sequenced with pcr and sequencing primers using an abi automatic sequencer (macrogen co., seoul, korea). the overlapping gene fragments were assembled to obtain a single complete genome sequence using chromaspro version . (technelysium pty ltd., brisbane, australia). nucleotide (nt) and amino acid (a.a) identity estimates and amino acid translations were obtained using bioedit (ver. . . . .). the full genome sequences of bp-cakii and k were submitted to the genbank database. phylogenetic analyses of the complete s gene of the ibv strains were conducted using mega software (ver. . . , neighbor-joining method with tamura-nei distance and repeats of bootstrapping) [ ] . snu , propagated via three blind passages, was diluted by − and inoculated into -day-old spf embryonated eggs (valo biomedia, adel, ia, usa) via the allantoic cavity route; the embryonated eggs were incubated at • c for h, after which they were chilled at • c overnight. the allantoic fluid was harvested, and the supernatant was stored at − • c after centrifugation at rpm ( × g) for min. the allantoic fluid harvested from the inoculated embryonated eggs was used for the real-time rt-pcr. the allantoic fluid with the lowest ct value was inoculated into the embryonated eggs, and the same procedure was repeated times to become bp-cakii. to test virus titer and embryo pathogenicity, bp-cakii was inoculated into -day-old spf embryonated eggs (valo biomedia) via the allantoic cavity route and incubated at • c for h. the infected allantoic fluid was harvested after chilling at • c overnight. then, bp-cakii and k titers were determined by inoculating -fold serial dilutions ( − - − ) of the virus into five -day-old spf embryonated eggs via the allantoic cavity. the inoculated embryonated eggs were observed for death and dwarfism for days at • c, and the % chicken embryo infectious dose (eid /ml) was calculated using the reed-muench formula [ ] . the mortality rate of bp-cakii and k was measured by mean death time (mdt), and absolute lethal dose (ald). mdt was measured as previously reported [ ] [ ] [ ] . briefly, bp-cakii and k were diluted within the range of to eid / µl and µl was injected into -day-old spf embryonated eggs via the allantoic cavity. five embryonated eggs per dilution were inoculated at a.m., and inoculation was repeated at p.m. with the same dose. the candling of embryonated eggs for mean death time was performed at eight-hour intervals within one day. ald was determined by the highest dilution at which all embryos died. bp-cakii and k diluted to × eid / µl were injected into -day-old spf embryonated eggs (valo biomedia, osterholz-scharmbeck, germany) via the allantoic cavity route and incubated at • c and • c for h. after , , , , , , and h, three eggs inoculated with each virus were chilled at • c overnight. the allantoic fluid of all the eggs was harvested and viral rna was extracted for the real-time pcr. the ct values of samples were measured, and the ∆ct was calculated by subtract ct value of each sample from that of the h sample. in addition, we performed regression analysis with ct values of -fold diluted bp-cakii and k viruses and fitted linear equations of bp-cakii and k . theoretically unit of ∆ct is the -fold difference of rna copies, but we calculated the real fold difference of rna of each virus by using the slope value of the linear equation (bp-cakii, y = . x + . , r = . , . -fold difference/ct; k , y = . x + . , r = . , . -fold difference/ct). the independent experiment was repeated and each sample was tested in triplicate in an experiment. to determine tissue tropism and the persistence of ibv strains, we performed experimental viral infection using -day-old spf chicks that were assigned to groups (n = per group). all chicks were treated with pbs (negative control), bp-cakii, k , or km ( . × eid / µl/chick) via ocular and intranasal routes, and observed for clinical signs and mortality for weeks after the challenge. each week, five chicks from each group were euthanized by cervical dislocation, and the pathological lesions of internal organs were observed throughout necropsy. the tracheal, cecal tonsil and kidney tissue samples were collected and examined using the real-time rt-pcr without virus titer calculation. to investigate the influence of ibv on the premature reproductive organs of the chicks, we induced the sexual precocity by des (sigma) treatment [ ] . a total of -day-old spf female chicks (biopoa co., yongin, korea) were distributed into four different groups. three groups were inoculated with bp-cakii, k , and km ( . eid / µl/chick) via ocular and intranasal routes, and a des-control group was not inoculated with virus. a total of birds were assigned to each group. after inoculation with each virus, the dead chicks were excluded when comparing reproductive tract lesions. at , and days after virus infection, all chicks were inoculated with des intramuscularly ( mg/ µl) and were observed for clinical signs and mortality for weeks after the challenge and were euthanized by cervical dislocation at weeks post-infection. during the experiment, all chicks were reared in air-filtered isolators (threeshine, daejeon, korea) and feed and water were provided ad libitum. gross lesions of the reproductive tract were examined based on cyst formation (four-grade-scoring: negative, moderate, + (cyst covering less than % of oviduct), marked, ++ (covering % to %), and severe, +++ (covering more than % of oviduct)), and the presence or absence of caseous material in the oviduct. the significance of growth kinetics and embryo pathogenicity were evaluated by two way analysis-of-variance (anova) and a man-whitney test, respectively. the difference in virus frequency in tissues and lesions in the premature reproductive tract pathogenicity model was assessed using chi-squared distribution ( % confidence intervals). all animal experiments were performed at biopoa co. (suwon, south korea) following a protocol that adhered to the national institutes of health's public health service policy on the humane care and use of laboratory animals. the protocol was reviewed and approved by the institutional animal care and use committee (iacuc) of biopoa co. (tissue tropism and persistence test, identification code: bp- - - ; date of approval: january ; premature reproductive tract pathogenicity test, identification code: bp- - - ; date of approval: january ). snu was passaged times through three embryonated eggs by inoculating allantoic fluid with high virus titer, and bp-cakii was established. out of the three allantoic fluids of each passage, the allantoic fluid with the highest virus titer after the real-time rt-pcr was selected for the next passage. the growth kinetics of bp-cakii in embryonated eggs at • c and • c were compared with those of k ( figure ). according to our results, bp-cakii grew to higher titers at • c than at • c for the first h, but then this result was reversed for the last h. however, k showed significantly higher titers at • c than at • c and after h. therefore, bp-cakii showed a tendency to grow more efficiently at • c than at • c, and bp-cakii grew to a higher titer than k at • c. snu was passaged times through three embryonated eggs by inoculating allantoic fluid with high virus titer, and bp-cakii was established. out of the three allantoic fluids of each passage, the allantoic fluid with the highest virus titer after the real-time rt-pcr was selected for the next passage. the growth kinetics of bp-cakii in embryonated eggs at °c and °c were compared with those of k ( figure ). according to our results, bp-cakii grew to higher titers at °c than at °c for the first h, but then this result was reversed for the last h. however, k showed significantly higher titers at °c than at °c and after h. therefore, bp-cakii showed a tendency to grow more efficiently at °c than at °c, and bp-cakii grew to a higher titer than k at °c. x-and the y-axis, respectively. fold changes were calculated by multiplying the ddct value and real fold-change of each virus calculated from regression analysis. * significant difference of k - °c from bp-cakii- °c and k - °c; ** significant difference of bp-cakii- °c from others (p < . ). ibvs tend to acquire increased embryonic pathogenicity during embryonated eggs passages, and we compared the embryonic pathogenicity of bp-cakii with that of k . bp-cakii and k showed . ald and . ald, respectively, and bp-cakii showed higher ald than k . in addition, the standard deviation (±sd) of mdt of bp-cakii was . ± . h, which is slightly longer than the ibvs tend to acquire increased embryonic pathogenicity during embryonated eggs passages, and we compared the embryonic pathogenicity of bp-cakii with that of k . bp-cakii and k showed . ald and . ald, respectively, and bp-cakii showed higher ald than k . in addition, the standard deviation (±sd) of mdt of bp-cakii was . ± . h, which is slightly longer than the . ± . h of k (p < . ). bp-cakii showed significant difference from k in mdt, but not in mortality (table ) . a ald: absolute lethal dose (the lowest dose causing % mortality). b mdt: mean death time. * significant difference of standard deviation (±sd) from k (p < . ). the tissue tropism and persistence of bp-cakii have been compared with those of k and km strains via the detection of viral rna in the trachea, kidneys and cecal tonsils of infected and uninfected spf chickens at weeks of age. except for the negative control group, the bp-cakii, k -and km -inoculated groups showed different positive rates over the time period of observation (table ) . bp-cakii, k and km showed / - / , / - / and / - / positive rates in chickens, respectively, during the first weeks, which disappeared at week-post-inoculation (wpi) in the trachea samples. in the kidneys, bp-cakii was detected in out of chickens ( - wpi) only at wpi, but k and km were detected in - chickens ( wpi) at - wpi and - chickens at - wpi, respectively. in cecal tonsils, bp-cakii was detected in out of chickens only at wpi, but k and km were detected in - chickens at - wpi and - chickens at - wpi, respectively. only km showed significantly different positive rates from k and bp-cakii in the half of cases (p < . ). the premature reproductive tract pathogenicity model was used to compare the pathogenicity of bp-cakii, k and km , and all the chicks treated with des showed a premature phenotype in the reproductive tract. (table , figure ). some chicks in all groups died during the experiment due to weakness or viral infection, and only the surviving chicks were included for lesion scoring. bp-cakii-infected chicks did not show any cyst or caseous material in the oviduct, but % ( / ) of k -infected and % ( / ) of km -infected chicks did show the lesions. only the frequency of lesions in the km challenge group was significantly higher than that in other groups (p < . ). to understand the genetic background of the cold adaptation and the decreased pathogenicity of bp-cakii, we determined the complete genomic sequence of bp-cakii (mf ) and performed phylogenetic analysis with the s gene. unexpectedly, we found that bp-cakii was classified into the kii genotype (figure ) , which was clearly different from the nc i genotype of the parent strain, snu [ ] . therefore, we determined the complete genome sequences of e -, e -and e passaged viruses and compared their amino acid sequences together with bp-cakii and snu , using km as a reference [ ] . out of the different amino acids in km , the amino acids that were different between the passaged viruses are summarized in supplementary table . only one or two amino acids changed during e -e passages, and most of the changes had already been acquired at the e passage. to understand the genetic background of the cold adaptation and the decreased pathogenicity of bp-cakii, we determined the complete genomic sequence of bp-cakii (mf ) and performed phylogenetic analysis with the s gene. unexpectedly, we found that bp-cakii was classified into the kii genotype (figure ) , which was clearly different from the nc i genotype of the parent strain, snu [ ] . therefore, we determined the complete genome sequences of e -, e -and e -passaged viruses and compared their amino acid sequences together with bp-cakii and snu , using km as a reference [ ] . out of the different amino acids in km , the amino acids that were different between the passaged viruses are summarized in supplementary table s . only one or two amino acids changed during e -e passages, and most of the changes had already been acquired at the e passage. figure . phylogenetic analysis of the s genes of infectious bronchitis viruses. phylogenetic trees are based on the s amino acid sequence of ibvs, where the bp-cakii strain is marked with a red filled circle. phylogenetic trees were constructed with the neighbor-joining method using mega . . the bootstrap values were determined from replicates of the original data. the branch number represents the percentage of times that the branch appeared in the tree. bootstrap values greater than % are shown. the p-distance is indicated by the bar at the bottom of the figure. for comparative genomics study, we determined the genomic sequence of k (mf ). km was used as a reference, and the length and gc content of the genomes, different nucleotide sequences in the ′-and ′-utrs, and different amino acid sequences within each coding gene are summarized in tables and . the length of the genomic sequence of bp-cakii was and nucleotides shorter than that of k and km due to the deletions of codons, f del in s and i del in nsp . the gc content was very similar across viruses, ranging from . % to . %. the amino acids that differ between km and both k and bp-cakii are summarized, and the different mutations between k and bp-cakii strains are represented in bold in tables and . in comparison with km , k showed no amino acid changes in a, b, e, m, nsp , nsp and nsp , but it did show multiple amino acid changes in other genes, especially s, nsp , nsp ( . %), nsp ( . %) and nsp ( . %) ( table ). in comparison with k , bp-cakii shared a similar or single amino acid different sequence for nsp , nsp , nsp , nsp , nsp , nsp , nsp , b, c, a, e, and m. therefore, bp-cakii and k shared embryo-adaptation-related multiple amino acid changes in nsp , , , , and s. however, bp-cakii also showed multiple amino acid changes in s, n, nsp , nsp , and nsp ( table ). all of the mutations were unreported previously, and their effects on protein function were unknown. we summarize the location of each mutation in the functional domain, subunit, or specific region in tables and . figure . phylogenetic analysis of the s genes of infectious bronchitis viruses. phylogenetic trees are based on the s amino acid sequence of ibvs, where the bp-cakii strain is marked with a red filled circle. phylogenetic trees were constructed with the neighbor-joining method using mega . . the bootstrap values were determined from replicates of the original data. the branch number represents the percentage of times that the branch appeared in the tree. bootstrap values greater than % are shown. the p-distance is indicated by the bar at the bottom of the figure. for comparative genomics study, we determined the genomic sequence of k (mf ). km was used as a reference, and the length and gc content of the genomes, different nucleotide sequences in the -and -utrs, and different amino acid sequences within each coding gene are summarized in tables and . the length of the genomic sequence of bp-cakii was and nucleotides shorter than that of k and km due to the deletions of codons, f del in s and i del in nsp . the gc content was very similar across viruses, ranging from . % to . %. the amino acids that differ between km and both k and bp-cakii are summarized, and the different mutations between k and bp-cakii strains are represented in bold in tables and . in comparison with km , k showed no amino acid changes in a, b, e, m, nsp , nsp and nsp , but it did show multiple amino acid changes in other genes, especially s, nsp , nsp ( . %), nsp ( . %) and nsp ( . %) ( table ). in comparison with k , bp-cakii shared a similar or single amino acid different sequence for nsp , nsp , nsp , nsp , nsp , nsp , nsp , b, c, a, e, and m. therefore, bp-cakii and k shared embryo-adaptation-related multiple amino acid changes in nsp , , , , and s. however, bp-cakii also showed multiple amino acid changes in s, n, nsp , nsp , and nsp ( table ). all of the mutations were unreported previously, and their effects on protein function were unknown. we summarize the location of each mutation in the functional domain, subunit, or specific region in tables and . table . %/ . % table ab / . %/ . % table . %/ . % passages of pathogenic ibvs through embryonated eggs resulted in virus attenuation and efficient virus replication, and all of the attenuated ibv vaccine strains, such as h , k and yx p , have been established by embryonic passages [ , , ] . cold adaptation was another virus attenuation method, and a cold-adapted iav vaccine strain has been used to generate vaccine strains against seasonal flu [ , ] . in a previous study, cold adaptation of ibv was reported, but the attenuation of pathogenicity was incomplete, and the genomic backgrounds of the attenuated strains were unavailable [ ] . in this study, we established a cold-adapted, attenuated ibv strain, bp-cakii, and characterized its pathobiological and genomic traits. although we started embryo passages with a snu isolate that had been classified into the new cluster i genotype, the e , e , e and bp-cakii strains were classified into the km -like genotype. snu is a recombinant virus possessing the ab gene of the km -like genotype and a recombinant s gene containing a partial segment from the qx-like virus [ ] . therefore, bp-cakii was unlikely to have originated from snu . although the k strain has been used commercially since , it might have been under clinical evaluation in the field for governmental approval for commercial use during this period, including [ ] . the multiple shared mutations between k and bp-cakii, and the fact that both strains shared almost the same mutational patterns following cold-passaging ( times), indicate that the k and bp-cakii strains may be closely related. although bp-cakii showed less pathogenicity in embryonated eggs than k they showed more delayed viral dissemination to the kidneys and cecal tonsils than did km . km was detected and persisted in the trachea and kidneys for the first weeks and in the cecal tonsils throughout the whole period of observation ( weeks). pathogenic field isolates of qx-like and variant ibvs also persisted for a long time in the trachea, proventriculus, kidneys and cecal tonsils of infected chickens [ ] . therefore, embryo and cold adaptation apparently decreased pathogenicity and changed the tissue tropism of k and bp-cakii in embryonated eggs and chickens. however, in certain conditions, even vaccine strains may persist for a long time before being excreted into the trachea and cloaca [ ] . thus, vaccine strains cleared within a short period of time, but with enough time to induce protective immunity, may be preferable to minimize the conversion of pathogenicity and the occurrence of recombinant viruses. to date, various pathogenicity models of ibvs have been developed, but testing pathogenicity on reproductive organs requires a long period before the sexual maturation of hens occurs [ ] . des has been known to induce abnormalities in female reproductive tracts and to induce a premature phenotype in the reproductive tracts of female chicks [ , ] . previously, the premature reproductive tract pathogenicity model was established and used to evaluate ibv pathogenicity [ ] . in this study, we verified the pathogenicity of bp-cakii, k and km by using this model. km showed a significantly higher pathogenicity than bp-cakii and k , but the pathogenicity of bp-cakii was insignificantly different from k . considering the differences in pathogenicity in embryos and premature reproductive tracts, as well as the differences in tissue tropism, bp-cakii may be less pathogenic than k . the comparative genomics study of bp-cakii and k revealed mutations acquired during embryo and cold adaptation. k acquired relatively more missense mutations in nsp , nsp , nsp and nsp genes than other genes during embryo adaptation. nsp is multifunctional, and its rna ' triphosphatase and helicase domains are located on its n-and c-terminals, respectively [ , ] . the helicase domain of nsp is subdivided into the reca and reca domains, and four out of the five mutations identified were located in reca [ ] . nsp is composed of the n-terminal exonuclease (exon) and c-terminal adomet-dependent guanosine n -methyltransferase (n -mtase) domains, and all the mutations were located in both domains [ ] [ ] [ ] . exon and n -mtase play roles in the correction of replication errors and in mrna capping, respectively [ , ] . nsp is an adomet-dependent '-o-methyltransferase ( '-o-mtase) that plays a role in the capping of viral rnas, which helps in the evasion of innate immunity [ ] . nsp is an endoribonuclease that plays an important role in the evasion of dsrna sensors, such as rig and mda , in infected cells [ ] . the mutations shared by k and bp-cakii in the nsp , , and genes may facilitate or impair innate immunity evasion in embryos and chickens, respectively. however, the exact innate immunity differences between embryos and chickens have never been elucidated. to date, embryo-adapted strains of ibvs have shown high mutation rates in the nsp genes, including a large nucleotide deletion in the ' end of the n gene and partial coding region of gene [ , ] . therefore, mutations for embryo adaptation may be strain-specific. bp-cakii acquired multiple additional missense mutations in the nsp , , , s and n genes during cold adaptation, but the exact functions of these mutations were unknown. enzyme activity may be affected at different temperatures. nsp has multiple domains, and three out of the four mutations were located in the pl (papain-like protease, l f) and ecto (nsp -ectodomain, i del and v i) domains [ ] . nsp is an rna-dependent rna polymerase (rdrp), and the p l mutation and the r c and p i mutations are located in the nidovirus rdrp-associated nucleotidyltransferase (niran) and the rdrp domains, respectively. nsp is involved in the formation of the replication and transcription complex (rtc) with nsp , and nsp is an essential component of the rtc. therefore, further study on the roles of the multiple mutations of nsp , and seen during viral replication at the lower temperature may be interesting. the r m mutation in s ntd of k and bp-cakii is located in the partial ceiling region of the receptor binding site, and v a of bp-cakii is located in the vicinity of the r m mutation site [ ] . evolutionarily, coronaviruses developed a ceiling to protect the receptor binding site in s ntd from host immune surveillance, and the r m and v a mutations may be related to the function of the partial ceiling [ ] . the accumulation of multiple mutations in the n gene was interesting, but their functions in cold adaptation are unclear. the cold-adapted iav showed mutations in its nucleoprotein, as well as the polymerase genes [ ] . the rna 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nonstructural protein are associated with attenuation in avian coronavirus infectious bronchitis virus nsp of coronaviruses: structures and functions of a large multi-domain protein cryo-em structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins identification of sequence changes in the cold-adapted, live attenuated influenza vaccine strain, a/ann arbor/ / (h n ) characterization of a critical interaction between the coronavirus nucleocapsid protein and nonstructural protein of the viral replicase-transcriptase complex x-ray structures of the n-and c-terminal domains of a coronavirus nucleocapsid protein: implications for nucleocapsid formation structure of the sars coronavirus nucleocapsid protein rna-binding dimerization domain suggests a mechanism for helical packaging of viral rna key: cord- - tbc x authors: rustmeier, nils h.; strebl, michael; stehle, thilo title: the symmetry of viral sialic acid binding sites—implications for antiviral strategies date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: tbc x virus infections are initiated by the attachment of the viral particle to protein or carbohydrate receptors on the host cell. sialic acid-bearing glycan structures are prominently displayed at the cell surface, and, consequently, these structures can function as receptors for a large number of diverse viruses. structural biology research has helped to establish the molecular bases for many virus–sialic acid interactions. due to the icosahedral point group symmetry that underlies many viral capsids, the receptor binding sites are frequently arranged in a highly symmetric fashion and linked by five-fold, three-fold, or two-fold rotation axes. for the inhibition of viral attachment, one emerging strategy is based on developing multivalent sialic acid-based inhibitors that can simultaneously engage several of these binding sites, thus binding viral capsids with high avidity. in this review, we will evaluate the structures of non-enveloped virus capsid proteins bound to sialylated glycan receptors and discuss the potential of these structures for the development of potent antiviral attachment inhibitors. the cell membranes of eukaryotes are decorated with a large number of chemically and structurally diverse carbohydrates. these so-called glycans form a protective layer at the interface between a cell and its environment. components of this layer are synthesized and assembled by a large set of enzymes that differ among species, thus helping to provide host-specific glycan structures. despite some differences, the carbohydrate building blocks of the glycan layer (monosaccharides) are largely identical in all cases. commonly found monosaccharides in glycan structures are glucose (glc), galactose (gal), their n-acetylated forms n-acetylglucosamine (glcnac) and n-acetylgalactosamine (galnac), and mannose (man). these building blocks constitute the bulk of most glycans. however, two particularly important sugar classes are missing here: fucoses and sialic acids. while glc(-nac) and gal(-nac) are usually components of the scaffold, fucose (fuc) and sialic acids are often added as head groups to a glycan. while fucose is, for example, an important determinant in histo-blood group antigens (hbga), sialic acids are major components of the glycan portions of gangliosides and the glycan structures of many membranous proteins in eukaryotes. sialic acids are based on a nine-carbon acidic α-keto sugar framework ( figure ). due to their anionic nature, sialic acids contribute to the negative net charge of the cell surface. sialic acids are abundant in the animal kingdom and are found in different forms. their most common form is n-acetylneuraminic acid (neu ac), which also constitutes the chemical basis for other sialic acids. the hydroxylation of the n-acetyl group of neu ac gives rise to n-glycolylneuraminic acid (neu gc), another commonly found sialic acid in animals. while neu gc cannot be synthesized in humans due to a gene defect, humans glycans terminating in sialic acid are prominently expressed at the cell surface. they are, therefore, often easily accessible and serve as the initial contact points for many viruses in different families [ , ] . while some of these glycans function as attachment receptors to simply tether a virus to the target cell membrane, others act as entry receptors and mediate binding of the virus, as well as the delivery of the viral genome into the cytoplasm. in the latter case, the attachment step is often followed by the recruitment of secondary (co-)receptors and endocytosis factors, eventually leading to cell entry of the virus particle or its components and infection of the cell. among the enveloped viruses that recognize sialic acid-containing receptors are members of the families coronaviridae, paramyxoviridae and orthomyxoviridae [ ] [ ] [ ] [ ] [ ] . in non-enveloped viruses, sialic acid-containing glycans serve as attachment receptors for members of the parvoviridae, picornaviridae, caliciviridae, polyomaviridae, reoviridiae, and adenoviridae [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . structural biology has provided precise views of how these pathogens interact with sialylated glycans, and although the binding modes differ among the viruses listed above, several common principles have emerged. (i) the viral binding sites for sialylated glycans are typically surface-exposed and feature a small number of contacts. the affinities of the interactions are, therefore, quite low (in the millimolar range) [ ] [ ] [ ] [ ] . firm adhesion of the virus to the cell surface is achieved through the engagement of multiple receptors via identical binding sites, which is known as avidity. (ii) in all cases investigated to date, the sialic acid itself mediates the majority of contacts with the viral capsid, with a smaller number of additional contacts formed to neighboring monosaccharides. (iii) most viruses are highly specific in the context in which sialic acid is presented; that is, they only recognize sialylated glycans featuring, for example, α- , -linked sialic acid but do not engage sialylated glycans carrying α- , -linked or α- , -linked sialic acid. (iv) although the database remains small, some viruses can discriminate between the many different modifications of sialic acids, and, as some of these modifications, are species-specific, this phenomenon can contribute to the ability of a virus to only infect species that express a particular sialic acid modification. the available structural information on virus-receptor interactions is crucial to enable the rational design of therapeutic compounds. due to the surface-exposed binding mode and the weak individual interactions between sialic acids and their cognate virus proteins, modifying sialic acid to achieve high-affinity binding is challenging. however, viruses possess many identical binding sites that are often linked by symmetry operators, and thus multivalent and symmetric ligands that target several binding sites could result in high-affinity interactions. the strategy of employing a carbohydrate-based, multivalent, and symmetric inhibitor that matches the symmetry of the binding sites in a multimeric target protein was first applied in the context of the bacterial shiga-like toxin (slt). slt consists of an enzymatic domain a and a pentameric, cell-binding domain b [ ] . the crystal structures revealed that the b domain pentamer recognizes the p k trisaccharide portion (αgal - βgal - βglc) of its physiologic ganglioside receptor, globotriaosylceramide (gb ) [ , ] . in order to achieve high affinity binding, kitov et al. [ ] designed the starfish compound, a quasi-symmetric, pentavalent molecule with a central glucose motive carrying five linkers that terminate in dimeric p k trisaccharides ( figure ). x-ray crystallography of the toxin-inhibitor complex revealed a sandwich-like arrangement of two slt b-pentamers intercepted by one starfish molecule. all five b-pentamer binding sites were simultaneously occupied by the inhibitor. in line with this, affinity measurements showed an increase in the inhibition potency from a millimolar affinity for the monovalent receptor (p k trisaccharide) to a subnanomolar affinity for the starfish compound. this concept of targeting multiple, symmetric receptor binding sites by multivalent inhibitors is also applicable for many viruses, since viral capsids are often icosahedral and, therefore, highly symmetric structures. figure . an example of a tailored multivalent inhibitor. the globotriaosylceramide-binding b-subunit of shiga-like toxin (slt) forms pentamers and serves as target for the pentavalent inhibitory compound starfish, which has been functionalized with the p k trisaccharide. the starfish compound exploits the symmetric structure of its target and binds to slt with a subnanomolar affinity [ ] . the slt pentamer is shown as a protein surface with single protomers colored in grey, yellow, pink, green, and light blue, respectively. the starfish compound is shown in stick representation with carbon, nitrogen and oxygen atoms colored in orange, dark blue and red, respectively. missing parts of the scaffold structure are schematically indicated as orange lines (pdb id qnu). all protein representations in the figures of this review were generated using pymol (schrödinger inc.). in this chapter, we will introduce some universal concepts of virus capsid geometry and architecture, focusing in particular on non-enveloped viruses that bind sialic acid-based receptors. we will highlight the local symmetries that relate the sialic acid binding sites in different viral attachment proteins to each other. these local symmetries can serve as a useful framework for the rational design of multivalent virus-targeting inhibitors, similar to the approach used to develop the starfish compound. this strategy has been successfully applied to several viruses, as we will show in chapter . however, in order to evaluate this approach, we first need to introduce the symmetry elements that guide viral capsid assembly. typically, small viral genomes can only encode a low number of structural capsid proteins. these capsid proteins often form multimers (capsomers), which, again, assemble into a stable virus capsid that can house the viral genome and associated components (e.g., nucleoproteins or enzymes). thus, the assembled virus particles comprise many copies of capsid proteins and display a high (quasi-) symmetry that is often based on an icosahedron [ ] . in icosahedral capsids, the particle architecture can be expressed in terms of the triangulation number. an icosahedron is a polyhedron consisting of identical triangular faces that intersect at twelve vertices with five-fold rotational symmetry. three-fold rotational axes are located in the center of each triangular face. the edges between the faces are intersected by two-fold rotational axes. the combination of these symmetry operations gives rise to the point group of an icosahedron ( figure , lower right). in the context of an icosahedral virus particle, the triangulation number (t = h + hk + k ) can be interpreted as a measure of capsid size. it is calculated by the numbers of inter-capsomeric steps (iterated in h and k) that one has to traverse via (quasi-) six-fold symmetric capsomers, from one five-fold vertex to another. five-fold vertices and the associated capsid proteins are highlighted in blue. a schematic view of an icosahedron in the same orientation is shown on the lower right, with two-fold, three-fold and five-fold axes indicated as ellipse, triangle and pentagon, respectively. pdb ids b t (adenovirus), cse (orthoreovirus), kz (rotavirus), sid (polyomavirus), and q w (coxsackievirus). the smallest and simplest virus capsids have an architecture corresponding to a triangulation number of t= . in a t= capsid, each of the twelve five-fold symmetric vertices are occupied by a single capsid protein pentamer. neighboring capsomers are pentamers that are also associated with the five-fold icosahedral vertices. no additional capsomers are present, which gives rise to a total number of × = capsid proteins in the particle. to our knowledge, the only example of a t= sialic acid binding capsid is found in the adeno-associated virus (aav) of the parvoviridae family. sialic acid binding in aav was reported to occur at the interface between two single capsid protein monomer chains, resulting in a total number of binding sites in the capsid. the symmetry of the local binding site is defined by the shortest distance of a single binding site towards the rotational axis. the aav capsid proteins that are responsible for sialic acid binding form pentamers. however, the binding sites themselves locate closer to the icosahedral three-fold symmetry axis than the five-fold capsomer/vertex axis. this implies that the sialic acid binding sites of the aav capsid are arranged in twenty local three-fold rather than twelve local five-fold symmetries [ ] . virus particles comprising more than twelve capsomers must, by definition, have a higher triangulation number than t= . picornaviridae family members (such as coxsackieviruses, rhinoviruses, polioviruses, or enteroviruses) are viruses possessing three surface-exposed capsid proteins. the icosahedral five-fold penton positions are occupied by vp pentamers, which are bridged by one pseudo-hexon capsomer (vp -vp heterohexamer), giving rise to pseudo-t= geometry ( figure ). in the sialic acid-binding human coxsackievirus, a variant (cva v), the binding site is located at the interface of two vp protomers, close to the five-fold icosahedral axis. vp and vp do not bind sialic acid, which results in a total of binding sites, arranged in twelve local five-fold symmetries (figure a ) [ ] . the distance between an individual sialic acid binding site and the local five-fold rotation axis of the vp pentamer amounts to ca. . nm. in the members of both papillomaviridae and polyomaviridae, single capsid proteins named l and vp , respectively, constitute the outer capsid. both proteins exclusively form pentamers, which can occupy pentavalent and hexavalent positions in the mature particle, thus deviating from the quasi-equivalence principle proposed by caspar and klug in [ ] . here, a total number of pentameric capsomers are arranged in a t= d fashion, with a diameter of ca. nm in the mature particles ( figure ) [ ] [ ] [ ] . the formation of smaller, non-viable lower-symmetry t= virus-like particles (vlps) has also been described for both papillomaviruses and polyomaviruses [ ] [ ] [ ] . many members of the polyomavirus family bind sialic acid-based glycans using their vp proteins, so the binding sites on individual pentamers are always linked by local five-fold symmetry (figure a , tspyv). in all structures of polyomavirus-sialyl-oligosaccharide complexes, the majority of contacts between the protein and receptor can be attributed to sialic acid, with a small number of augmenting contacts to other saccharides providing specificity for a given glycan structure [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in contrast, papillomaviruses do not bind sialylated receptors, but instead interact with glycosaminoglycans to adhere to cells [ ] [ ] [ ] . reoviridae are a large family including, among others, the genera of orthoreoviruses and rotaviruses. members of the reoviridae family are double-shelled particles, in which the inner core layer consists of two core proteins arranged in a t= * order and the outer capsid possesses t= geometry [ ] [ ] [ ] . the diameter of mature virions is about - nm (figure ). in the case of mammalian reoviruses, the icosahedral vertex positions are occupied by the trimeric attachment protein sigma , which markedly protrudes as a thin fiber from the virion surface. the sigma protein is about nm long and has a head-and-tail morphology, with a globular head domain and an elongated tail that has flexible regions and partially inserts into the virion [ ] . the location of sialic acid binding is type dependent. type reoviruses bind sialylated receptors in the protruding head domain while type reoviruses use a binding site in a region near the mid-point of the tail (figure b , reov t d and t l) [ , ] . both sites primarily engage sialic acid with a small number of contacts, and in both cases, three-fold rotational symmetry is found between the binding sites within a single sigma trimer. thus, sigma trimers give rise to sialic acid binding sites in orthoreoviruses. rotaviruses also have protruding domains that recognize carbohydrate receptors, which are also dimers of the virus protein (vp ) [ ] . while some of the more pathogenic human viruses bind hbgas, animal rotaviruses primarily engage sialic acid-based receptors [ ] [ ] [ ] . each rotavirus vp subunit carries a single sialic acid binding site, which is related to the two-fold rotational symmetry in the dimer (figure a , rrv) [ ] . in further contrast to orthoreoviruses, these fibers do not coincide with the five-fold vertices but rather associate at the margins of the five capsomers around the five-fold vertices, resulting in a total number of vp dimers and sialic acid binding sites [ ] . adenoviridae members are large viruses with a diameter of about nm and a t= icosahedral capsid ( figure ) [ , ] . they display trimeric fibrous attachment proteins, known as the fiber, at their icosahedral vertices. similar to the reovirus sigma , the adenovirus fiber has a head-and-tail morphology and features a globular head domain (the knob) that projects from the virus surface, and a tail (the shaft) that inserts into the virus particle [ ] . the sialic acid binding sites of the different structurally characterized adenovirus types are located in the fiber knob domain [ , ] . while these sites differ in location in different adenoviruses, they are all linked by three-fold symmetry and lie in close proximity to each other (figure a, hadv and hadv ) . although this review focuses on non-enveloped viruses, it should still be mentioned that some of the concepts described above also apply to enveloped viruses, such as coronaviruses, paramyxoviruses, and orthomyxoviruses. while global symmetry measures for these viruses are elusive (since their attachment proteins are membrane-bound, somewhat mobile, and do not follow easily-appreciated assembly rules), the existing local binding site symmetries within multimeric attachment proteins can still be exploited for rational multivalent inhibitor design. a prominent example is the influenza a virus hemagglutinin, which is a homotrimeric protein bearing three individual sialic acid binding sites [ ] . these binding sites are related by local three-fold rotational symmetry, with a distance of . nm from the three-fold axis (figure a, iav) . the sialic acid moieties of glycosylated proteins and/or glycolipids are required for the attachment, entry, and productive infection of many viruses. in the majority of cases, the viral binding sites are surface-exposed and engage terminal sialic acid residues with a range of hydrophilic and hydrophobic contacts. the available structural data for receptor-ligand interactions at an atomic resolution can inform the synthesis of high affinity inhibitors via the chemical modifications of sialic acids. a prominent example of this approach is the inhibition of influenza virus neuraminidases. these enzymes recognize and hydrolyze terminal sialic acids of cell surface glycans and are vital for the release of viral progeny [ , ] . an early non-selective prototype inhibitor of neuraminidases was the sialic acid derivate -deoxy- , -didehydro-n-acetylneuraminic acid (neu ac en, dana) [ , ] . dana can engage and block the binding sites of neuraminidase of both ortho-and paramyxoviruses, thereby suppressing the activity of these enzymes. in the early s, mark von itzstein and colleagues made substantial advances towards selective and highly affine influenza a virus inhibitors. they discovered that the derivatization of the -o-hydroxyl group of dana with a guanidine moiety drastically increases affinity to the influenza a virus neuraminidase, which resulted in the anti-influenza drug zanamivir [ , ] . subsequently, the structures of the planar sialic acid transition state during the neuraminidase reaction became available. the analogous compounds of sialic acid's transition state based on benzoic and shikimic acids, and subsequently the drug oseltamivir [ ] [ ] [ ] [ ] , benefit from the increased affinity to neuraminidase compared to the native neu ac structure [ , ] . unfortunately, the family of compounds related to zanamivir or oseltamivir do not act against viruses that do not possess neuraminidase activity but instead engage undistorted sialic acids via a hemagglutinin (e.g., many non-enveloped viruses discussed in chapter of this review). in cases where affinities between sialylated glycans and hemagglutinins have been measured, the interactions were shown to have dissociation constants in the millimolar range [ ] [ ] [ ] [ ] . the firm cell attachment of these viruses is usually driven by high avidity, relying on a large number of identical binding sites that can engage receptors. in terms of the inhibition of attachment, monovalent sialic acid analogues are generally poor choices, as they are not able to engage a single binding site in a viral attachment protein with high affinity. therefore, polyvalent sialic acid-based compounds can sometimes be more effective. in parallel to the development of monovalent sialic acid transition state analogues against the influenza virus neuraminidases, polymeric or nanoparticle based sialic acid conjugates were developed to also prohibit influenza virus infection by extensive binding to the viral hemagglutinin, thereby shielding the virus particle. the influenza virus hemagglutinin can be blocked by multivalent sialosides that vary in chemical composition, size, branching complexity, and ligand density [ ] [ ] [ ] [ ] [ ] . particularly the effects of the nature of the scaffold (or platform) and spatial sialic acid distribution in polyvalent viral attachment inhibitors are still being investigated [ ] . recently, excellent reviews covering the composition and biophysical properties of multivalent sialosides were published by bhatia et al. [ ] and lu et al. [ ] , so we will not discuss this topic further here. next to the design of monovalent inhibitors or highly complex polyvalent macromolecular structures, a third and rather minimalistic design strategy makes use of the internal symmetry of the addressed targets. as elucidated earlier in this review, many viral attachment proteins employ a local symmetry of their receptor binding sites that represent a convenient framework for the rational design of small, tailored inhibitors. in contrast to monovalent inhibitors, oligovalent compounds can benefit from cooperative binding or avidity. nevertheless, they are small enough for nephric clearance and display a higher bioavailability than most macromolecular polyvalent structures [ ] . the design of oligovalent inhibitors is usually based on available crystal structures, taking the symmetry and topology of the target into account. the accessibility of the individual ligand binding sites and their distances from an eventual symmetry axis should be considered. for binding sites in close proximity to the local symmetry axis, it can be feasible to start from a central molecule, which can be derivatized by ligand terminating linkers, resulting in an inhibitor with a radial structure. for binding sites that are far from the local symmetry axis or in a recessed part of the target protein, it may be viable to directly link ligand molecules to each other instead of using a central scaffold. direct linking of ligand molecules may eventually result in the design of a circular oligovalent inhibitor. so far, structural information on attachment proteins bound by oligovalent inhibitors is scarce. x-ray crystallography analysis is usually hampered by the high flexibility of spacer groups and scaffolds. these non-binding parts of the inhibitors typically do not assume defined conformations, resulting in weak and uninterpretable electron density. this was the case in the study of the shiga-like toxin inhibitor starfish, described in the introduction of this review, where interpretable electron density was only present for the bound ligand moieties of the compound. still, the structural characterization of virus-inhibitor interactions in order to design or optimize anti-viral compounds has been the subject of extensive research. recently, a research paper by lu et al. [ ] reported on the trivalent design of sialic acid bearing inhibitors against influenza a virus hemagglutinin, showing a greater than -fold increase in affinity compared to the monovalent ligand. however, structural data of the interactions between the inhibitor and hemagglutinin were not reported in that study. one study of a central group-based anti-viral inhibitor, which contains structural data, can be found for adenoviruses. adenoviridae members carry trimeric fibers terminating in the knob domain, which engages sialic acid-based receptors in some adenoviruses. human adenovirus (hadv ), which causes epidemic keratoconjunctivitis (ekc), carries three individual binding sites for sialic acids in its fiber knob, which are located at a distance of Å from the local three-fold symmetric axis (figure a ) [ ] . the physiologic receptor of hadv is the glycan portion of ganglioside gd a. this glycan is a branched hexasaccharide with two terminal sialic acid moieties, both of which were shown to simultaneously occupy two of the three available sialic acid binding sites in the same fiber knob [ ] . based on this observation, spjut et al. ( ) [ ] designed and synthesized a symmetric, tridentate sialylated inhibitor, which is capable of occupying all three binding sites of the fiber knob at once. the first generation of these inhibitors was designed around a central tris( -aminoethyl) amine group. it utilized flexible spacers between the central group and the sialic acid ligand, in order to minimize the chance of steric hindrance of the inhibitor docking. the resulting compounds demonstrated a potency increase of four orders of magnitude compared to monomeric sialic acid [ ] . recently, in the second-generation inhibitors, the binding affinity could be improved even further by using a shorter, triazole-based linker structure, which was based on the results of the first-generation complex structures. the more compact and rigid design resulted in an additional -fold increase in potency [ ] . the crystal structures of the second-generation inhibitor molecules in a complex with adenovirus fiber knob proteins verify the trivalent binding mode by also displaying the electron density of the linkers (figure a,b, left) . an example of directly-linked ligand moieties was shown in a study of potential polyomavirus inhibitors, in which baier et al. [ ] synthesized so-called divalent sialylated glycooligopeptides. they solved the structures of two glycooligopeptide compounds in a complex with the major capsid protein (vp ) pentamer of the trichodysplasia spinulosa-associated polyomavirus (tspyv), which is associated with abnormal skin growth in immunocompromised patients. the vp pentamer carries five individual sialic acid binding sites at a distance of Å between neighboring sites [ , ] . the glycooligopeptide-vp complex structures displayed a similar ligand binding mode that was reported for sialic acid in an earlier study [ ] and showed, for the compounds, that the linker between the ligand and the scaffold occupies the space that is usually targeted by the natural glycan receptor moieties (figure a,b, right) . however, the interconnectivity of functional receptors by the scaffold remained undetermined (probably due to their flexibility) and are, therefore, the average of several potential bridging modes on top of the pentamers [ ] . many viral lectins or attachment proteins rely on the recognition of sialic acids. due to the high symmetry of viral particles and the occurrence of local symmetry within commonly multimeric viral proteins, sialic acid binding often occurs in a symmetrical context, too. this symmetry is a convenient framework for the design of tailor-made inhibitory ligands competing with the high avidity of virus-cell interactions. structural biology techniques, such as x-ray crystallography and single-particle electron cryo-microscopy (cryo-em), can now tackle the visualization of viral attachment and carbohydrate interactions with unprecedented scope and detail. the resulting structural data can be used for the optimization of anti-viral compounds, which could be developed further into high-affinity drug candidates. however, challenges in compound design remain. for example, the higher rigidity of a multivalent ligand does not necessarily translate into improved binding. in the case of had , a rigid compound bound -fold less well to the fiber knob than a related compound that had higher flexibility [ ] . this suggests that the perfect positioning of all sialic acids in the binding site, especially for larger inhibitor molecules, is difficult to achieve, and a certain degree of flexibility might help with the high-affinity binding of the inhibitor. another limiting factor for oligovalent inhibitors is the positioning of the binding pockets. in the case of the trivalent adenovirus inhibitor, the binding sites are on the very top of the knob domain, so there is enough space for linkers and a central core (figure a ). in contrast, the sialic acid binding sites of the reovirus sigma fibers are located on the side of the protein (figure b) , which makes it challenging to design an appropriate oligovalent inhibitor. additional challenges are the long-term stability, convenient synthesis, and, for later application, reasonable bioavailability of multivalent compounds. n-glycolylneuraminic acid deficiency in humans chemical diversity in the sialic acids and related α-keto acids: an evolutionary perspective diversity in cell surface sialic acid presentations: implications for biology and disease essentials of glycobiology sialic acid tissue distribution and influenza virus tropism structural features of glycan recognition among viral pathogens glycan engagement by viruses: receptor switches and specificity human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses sialic acids as receptor determinants for coronaviruses role of sialic acid-containing molecules in paramyxovirus entry into the host cell: a minireview structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid sialic acid species as a determinant of the host range of influenza a viruses binding of adeno-associated virus type to , -linked sialic acid is required for gene transfer adeno-associated virus serotype (aav ) and aav both require sialic acid binding for hemagglutination and efficient transduction but differ in sialic acid linkage specificity sialic acid functions in enterovirus binding and infection coxsackievirus a variant uses sialic acid-containing o-linked glycoconjugates as cellular receptors on human ocular cells sialic acid-dependent cell entry of human enterovirus d tulane virus recognizes sialic acids as cellular receptors infection of glial cells by the human polyomavirus jc is mediated by an n-linked glycoprotein containing terminal α( - 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Å resolution x-ray crystal structure of the rotavirus inner capsid particle at . a resolution sigma protein of mammalian reoviruses extends from the surfaces of viral particles the gm glycan serves as a functional coreceptor for serotype reovirus crystal structure of reovirus attachment protein sigma in complex with sialylated oligosaccharides three-dimensional visualization of the rotavirus hemagglutinin structure comparison of human, simian, and bovine rotaviruses for requirement of sialic acid in hemagglutination and cell adsorption role of sialic acids in rotavirus infection sialic acid dependence in rotavirus host cell invasion the rhesus rotavirus vp sialic acid binding domain has a galectin fold with a novel carbohydrate binding site atomic model of an infectious rotavirus particle the structure of the adenovirus capsid image reconstruction reveals the complex molecular organization of adenovirus crystal structure of species d adenovirus fiber knobs and their sialic acid binding 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inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity synthesis of a carbocyclic sialic acid analogue for the inhibition of influenza virus neuraminidase evidence for a sialosyl cation transition-state complex in the reaction of sialidase from influenza virus a study of the active site of influenza virus sialidase: an approach to the rational design of novel anti-influenza drugs polyacrylamides bearing pendant α-sialoside groups strongly inhibit agglutination of erythrocytes by influenza-virus effective inhibitors of hemagglutination by influenza virus synthesized from polymers having active ester groups. insight into mechanism of inhibition generation and in situ evaluation of libraries of poly(acrylic acid) presenting sialosides as side chains as polyvalent inhibitors of influenza-mediated hemagglutination inhibition of viral adhesion and infection by sialic-acid-conjugated dendritic polymers polymeric inhibitor of influenza virus attachment protects mice from experimental influenza infection linear polysialoside outperforms dendritic analogs for inhibition of influenza virus infection in vitro and in vivo pathogen inhibition by multivalent ligand architectures carbohydrate-protein interactions and multivalency: implications for the inhibition of influenza a virus infections properties of the glomerular barrier and mechanisms of proteinuria enhanced inhibition of influenza a virus adhesion by di-and trivalent hemagglutinin inhibitors the gd a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis a potent trivalent sialic acid inhibitor of adenovirus type infection of human corneal cells divalent sialylated precision glycooligomers binding to polyomaviruses and the effect of different linkers this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank the swiss institute of bioinformatics for the provision of the expasy viralzone and the scripps institute for the provision of the viper data bank-two very helpful resources of virus knowledge. we apologize to our many colleagues whose work could not be discussed and cited here due to space considerations. the authors declare no conflict of interest. key: cord- -xhfmio k authors: fagre, anna c.; kading, rebekah c. title: can bats serve as reservoirs for arboviruses? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: xhfmio k bats are known to harbor and transmit many emerging and re-emerging viruses, many of which are extremely pathogenic in humans but do not cause overt pathology in their bat reservoir hosts: henipaviruses (nipah and hendra), filoviruses (ebola and marburg), and coronaviruses (sars-cov and mers-cov). direct transmission cycles are often implicated in these outbreaks, with virus shed in bat feces, urine, and saliva. an additional mode of virus transmission between bats and humans requiring further exploration is the spread of disease via arthropod vectors. despite the shared ecological niches that bats fill with many hematophagous arthropods (e.g., mosquitoes, ticks, biting midges, etc.) known to play a role in the transmission of medically important arboviruses, knowledge surrounding the potential for bats to act as reservoirs for arboviruses is limited. to this end, a comprehensive literature review was undertaken examining the current understanding and potential for bats to act as reservoirs for viruses transmitted by blood-feeding arthropods. serosurveillance and viral isolation from either free-ranging or captive bats are described in relation to four arboviral groups (bunyavirales, flaviviridae, reoviridae, togaviridae). further, ecological associations between bats and hematophagous viral vectors are characterized (e.g., bat bloodmeals in mosquitoes, ingestion of mosquitoes by bats, etc). lastly, knowledge gaps related to hematophagous ectoparasites (bat bugs and bed bugs (cimicidae) and bat flies (nycteribiidae and streblidae)), in addition to future directions for characterization of bat-vector-virus relationships are described. bats and the viruses they harbor have been of interest to the scientific community due to the unique association with some high consequence human pathogens in the absence of overt pathology. virologic and serologic reports in the literature demonstrate the exposure of bats worldwide to arboviruses (arthropod-borne viruses) of medical and veterinary importance [ ] . however, the epidemiological significance of these observations is unclear as to whether or not bats are contributing to the circulation of arboviruses. historically, a zoonotic virus reservoir has been considered a vertebrate species which develops a persistent infection in the absence of pathology or loss of function, while maintaining the ability to shed the virus (e.g., urine, feces, saliva) [ ] [ ] [ ] . haydon et al. extended this definition of a reservoir to include epidemiologically-connected populations or environments in which the pathogen can be permanently maintained and from which infection is transmitted to the defined target population. the significance of the relative pathogenicity of the infectious agent to the purported reservoir host has been debated [ ] . in the case of bats as a reservoir species, rigorous field and experimental evidence now exist to solidify the role of the egyptian rousette bat (rousettus aegyptiacus) as the reservoir for marburg virus [ ] [ ] [ ] . considering arboviruses, additional criteria must be met in order to consider a particular vertebrate species a reservoir. reviewed by kuno et al., these criteria include the periodic isolation of the infectious agent from the vertebrate species in the absence of seasonal vector activity, and the coincidence of transmission with vector activity [ ] . further, the vertebrate reservoir must also develop viremia sufficient to allow the hematophagous arthropod to acquire an infectious bloodmeal [ ] in order for vector-borne transmission to occur. bats have long been suspected as reservoirs for arboviruses [ ] , but experimental data that would support a role of bats as reservoir hosts for certain arboviruses remain difficult to collect. here we synthesize what information is currently known regarding the exposure history and permissiveness of bats to arbovirus infections, and identify knowledge gaps regarding their designation as arbovirus reservoirs. the order bunyavirales is divided into eight families, four of which pose threats to public health and veterinary medicine-families nairoviridae, peribunyaviridae, phenuiviridae, and hantaviridae [ ] . while bats have been demonstrated to host hantaviruses, these viruses do not rely on an arthropod in their transmission cycle and thus will not be discussed [ ] . viruses in order bunyavirales that have been experimentally examined in bats or described in field studies are descried in table . members of the genus orthonairovirus of medical and veterinary significance include crimean congo hemorrhagic fever virus (cchfv) and nairobi sheep disease virus (nsdv) [ ] . cchfv is transmitted by ticks in genera rhipicephalus and hyalomma [ ] . while neither live virus nor nucleic acid of cchfv has been detected from bats, serologic evidence suggests past infection of populations of bats across a diverse geographic range [ ] [ ] [ ] . further, bats are often parasitized by both soft and hard ticks, which occupy a diverse range of ecological niches in endemic countries [ ] [ ] [ ] . a seroprevalance study by müller and colleagues examining african bat species (n = , ) found that the prevalence of antibodies against cchfv was much higher in cave-dwelling bats ( . %- . %, depending on species) than foliage-living bats ( . %- . %) [ ] . they also screened , serum samples by rt-pcr, but all were negative for cchfv nucleic acid [ ] . experimental studies to assess the ability of bats to support replication of cchfv have not been published. members of the genus orthobunyavirus include many viruses of importance to human and veterinary medicine, including bunyamwera virus, california encephalitis virus, jamestown canyon virus, kaeng khoi virus, and la crosse encephalitis virus [ ] , but limited evidence exists regarding the exposure or potential involvement of bats in the circulation of viruses in this family. kaeng khoi virus (kkv) has been isolated from cimicid bugs (order: hemiptera, family: cimicidae) (stricticimex parvus and cimex insuetus) and from suckling wrinkle-lipped bats (tadarida plicata ) in caves in thailand, but was not isolated from soft ticks tested in the same area (ornithodorus hermsi) [ ] . additionally, kkv has been implicated in the case of several mine workers who reported illness and were discovered to have seroconverted [ ] , demonstrating spillover of this virus to humans in association with the cave environment, and suggesting that cimicids may play a role in vectoring virus between bat and human hosts. to date, no experimental data have been generated to address this hypothesis. spence and colleagues attempted to experimentally infect jamaican fruit bats (artibeus jamaicensis) via intramuscular injection with nepuyo virus (group c serogroup), yet no infectious virus was subsequently recovered from the bats [ ] . this is interesting considering two strains of nepuyo virus were isolated from jamaican fruit bats (artibeus jamaicensis) and great fruit-eating bats (artibeus literatus) in honduras, and protective sera were found in jamaican fruit bats in trinidad. [ , ] . bats of undetermined species were involved in a large serosurvey in brazil that examined antibodies in wildlife against the gamboa serogroup orthobunyaviruses, though none were found to be positive [ ] . seven and twelve species of trinidadian bats were examined for antibodies by hi against caraparu (group c serogroup) and maguari (bunyamwera serogroup) viruses, respectively, and were all found to be negative [ ] . viruses in the genus phlebovirus (family phenuiviridae) of importance to human and animal health include rift valley fever virus (rvfv) and severe fever with thrombocytopenia syndrome virus (sftsv) [ ] . bats of the species miniopterus schreibersii (n = ) and eptesicus capensis (n = ) were experimentally infected with rvfv and the m. schreibersii bat's urine and liver tested positive for antigen [ ] . a recent study by balkema-buschmann and colleagues experimentally infected egyptian rousette bats (rousettus aegyptiacus) with vaccine strain mp- and recovered infectious virus from spleen and liver of some animals [ ] . oelofsen & van der ryst ( ) examined samples from field-caught bats in africa, yet none were positive for antigen by use of elisa [ ] . kading et al ( ) detected neutralizing antibodies against rvfv in egyptian rousette bats and little epauletted fruit bats (epomophorus labiatus) in uganda, a country that has recently experienced human cases of rvfv [ , ] . whether or not bats serve as a reservoir of rvfv during interepidemic periods remains to be determined. bangui virus (bgiv) is an unclassified bunyavirus and was isolated from an unidentified bat in the central african republic (car) [ ] . mojuí dos campos virus (mdcv) is another ungrouped bunyavirus isolated from an unidentified bat species [ , ] . the family flaviviridae includes many high-consequence emerging arboviruses, including zika virus (zikav), yellow fever virus (yfv), and dengue virus (denv). flaviviruses associated with bats that do not appear to utilize an arthropod vector ("no-known vector flaviviruses") have been reviewed elsewhere [ ] . viruses in family flaviviridae that have been experimentally examined in bats or described in field studies are descried in table . interestingly, despite denv isolations from artibeus spp. bats in the wild, experimental infections of great fruit-eating bats (a. intermedius) with denv- and jamaican fruit bats with denv serotypes and resulted in low levels of viremia, low rates of seroconversion, and lack of detection of viral rna in the organs via rt-pcr, indicating that bats may not act as a suitable reservoir host [ ] [ ] [ ] . experimental infection of the indian flying fox (pteropus giganteus) resulted in no viremia or clinical signs, but intracerebral inoculation of little brown bats (myotis lucifugus) resulted in irritability, paralysis, and death [ , ] . denv nucleic acid and anti-denv antibodies have been detected in mexican bats on the gulf and pacific coast, and nucleic acid has been detected in the liver and/or sera of wild-caught bats in french guiana [ , ] . anti-denv antibodies have been detected in multiple bat species in uganda [ ] . however, a survey in central and southern mexico analyzing individuals representing bat species by rt-pcr resulted in no detection of viral nucleic acid [ ] . a study by vicente-santos and colleagues examined bat species from costa rica and found a cumulative seroprevalence of . % ( / ) by prnt and a prevalence of . % ( / ) in organs tested by rt-pcr [ ] . no infectious virus was isolated and viral loads were considered too low for the bats to function as amplifying hosts. rather, vicente-santos and colleagues surmised a spillover event from humans to bats, with bats functioning as a dead-end host [ ] . the serum of jamaican fruit bats (artibeus jamaicensis) and great fruit-eating bats (a. literatus) from grenada (n = ) were also tested for antibodies against denv , , , and , and none were seropositive [ ] . while field evidence supports the exposure of bats to denv in multiple geographic areas, experimental infections conducted to date are consistent in that bats are not likely to support denv replication and circulation to levels high enough to infect blood-feeding mosquitoes. multiple studies conducted experimental infections of insectivorous bats with japanese encephalitis virus (jbev) and found that bats were susceptible to infection with this virus. three species of bats (big brown bats (eptesicus fuscus), little brown bats (myotis lucifigus) and eastern pipistrelles (pipistrellus subflavus)) were inoculated with jbev in the laboratory and maintained infection while held under simulated hibernation conditions. bats infected prior to hibernation were viremic upon arousing from hibernation, with circulating virus detectable as long as days after the initial infection [ ] . big brown bats also demonstrated recurrent viremia in the absence of clinical signs in a subsequent study [ ] . importantly, researchers demonstrated a mosquito-bat-mosquito transmission cycle and postulated this may be an overwintering mechanism for jbev since mosquitoes did successfully transmit jbev to bats at low temperatures [ ] . eastern pipistrelles also became infected with jbev after consumption of infected mosquitoes, demonstrating that bats could be infected orally as well as through a mosquito bite [ ] . no demonstrable pathologic effects noted during infection of three bat species [big brown bats (eptesicus fuscus), little brown bats (myotis lucifigus) and mexican free-tailed bats (tadarida brasiliensie mexicana) with various strains of jbev or st. louis encephalitis virus (slev) [ ] . no pathology nor viremia was appreciated when pipistrelles (pipistrellus abramus) were infected with jbev [ ] . while experimental data demonstrated that some bat species can sustain jbev infections and support mosquito-borne transmission of this virus, the epidemiological significance of these observations in the field remains unclear. jbev has been isolated from wild-caught bats in taiwan (miniopterus fuliginosus and hipposideros armiger terasensis [ , ] , china (rousettus leschenaultia and murina aurata [ , ] , japan (miniopterus schreibersi fuliginosus and rhinolophus cornutus cornutus [ ] . antibodies against jbev have been detected in wild-caught bats in indonesia (unspecified species) [ ] , china (rousettus leschenaultia, cynopterus sphinx, taphozous melanopogon, miniopterus schreibersii, pipistrellus abramus, rhinolophus macrotis and miniopterus fuliginosus [ , ] , australia (pteropus scapulatus and pteropus gouldi) [ ] , taiwan (unspecified species) [ ] , india (pteropus giganteus, hipposideros pomona, hipposideros speoris, hipposideros bicolor, hipposideros cineraceus, megaderma lyra, cynopterus sphynx, and rhinolophus rouxi) [ ] [ ] [ ] , and japan (miniopterus schreibersi fuliginosus, rhinolophus ferrum equinum nippon, vespertilio superans, myotis macrodactylus, rhinolophus cornutus cornutus, pipistrellus abramus, myotis mystacinus, plecotus auritus sacrimontis, and murina leucogaster hilgendorfi) [ ] . multiple isolations of jbev from locations where the virus is endemic, in addition to the fact that genetic characterization of isolates has supported their similarity to strains identified from human and mosquito isolates, support the role of bats in ongoing circulation of jbev [ ] . another medically-important flavivirus with both field-obtained information and in vivo experimental inoculation is slev. a study by herbold and colleagues demonstrated that % of wild-caught eptesicus fuscus and myotis lucifugus (n = ) in ohio possessed neutralizing antibodies to slev [ ] . other serosurveillance efforts in north america and grenada focused on detection of slev in free-ranging bat populations have resulted in largely negative findings [ , ] . following experimental infection, viremia and transplacental transmission (albeit infrequent) was appreciated in mexican free-tailed bats (tadarida brasiliensis) [ , ] . the viremia in these bats reached log units, likely too low a titer to facilitate transmission to a blood-feeding mosquito [ ] . upon inoculation, little brown bats (myotis lucifugus) appear to be resistant or only slightly susceptible to slev [ ] . herbold and colleagues ( ) demonstrated that inoculation of eptesicus fuscus with slev results in infection and virus was maintained throughout hibernation ( days), with viremia developing four days following arousal ( days post-infection) [ ] . low levels of viremia upon experimental inoculation in conjunction with low seroprevalence data indicate this virus likely does not utilize bats as a reservoir host in nature. to date, biosurveillance testing of bats in central america for wnv have turned up negative results. grenadian artibeus jamaicensis and artibeus literatus (n = ) bats were negative for wnv neutralizing antibodies by prnt [ ] , trinidadian bat species (n = ) were negative by elisa for wnv antibodies [ ] , and mexican bat species (n = ) tested for wnv rna by rt-pcr were negative [ ] . in north america, results have been negative or indicative of low levels of circulation in bat populations tested. tissues from field-collected bats representing seven species in illinois tested by rt-pcr were all negative for wnv, and the same study reported one big brown bat (eptesicus fuscus) with neutralizing antibodies (n = ) [ ] . a field survey taking place in new jersey and new york reported one big brown bat and one northern long-eared bat (myotis septentrionalis) with neutralizing antibodies to wnv (n = ) [ ] . in another field study, only two of free-tailed bats (tadarida brasiliensis) possessed neutralizing antibodies against wnv [ ] . in uganda, kading et al. ( ) detected neutralizing antibodies to wnv in / african straw-colored flying foxes (eidolon helvum), and / little epauletted fruit bats (epomophorus labiatus) [ ] . simpson and o'sullivan ( ) demonstrated experimental inoculation of african straw-colored flying foxes did not result in viremia though two of three bats developed neutralizing antibody. in the same study, two of three egyptian rousette bats were infected but only trace viremia was detected and seroconversion was not appreciated [ ] . experimental inoculation of free-tailed bats (tadarida brasiliensis) did not result in viremia, and infection of big brown bats resulted in low titers ( - pfu/ml) [ ] , not capable of supporting transmission to feeding mosquitoes [ ] . attempts to experimentally infect vampire bats (desmodus rotundus) and black mastiff bats (molossus rufus) by mosquito bite (aedes aegypti) were unsuccessful [ ] . experimental inoculation of multiple bat species (eumops perotis, carollia perspicillata, phyllostomus hastatus and bats in the genus mollosus) were similarly unsuccessful [ ] . still, kading et al. detected a significant neutralizing antibody titer against yfv in one egyptian rousette bat in uganda in , indicating bats are exposed to this virus in nature [ ] . uganda has experienced outbreaks of yfv in recent years [ ] . while multiple african bat species (eidolon helvum, rousettus aegyptiacus, and rousettus angolensis) demonstrated viremia following inoculation with zikav, mops condylurus did not become viremic, although did contain low virus titers in the kidney [ , ] . experimentally-infected little brown bats were susceptible to the zikav by the intraperitoneal, intradermal, intracerebral and intrarectal routes of exposure, but not susceptible intranasally [ ] . however, it is unclear how zikav could circulate in bat populations. kading et al. ( ) did not detect neutralizing antibodies to zikav among ugandan bats screened. flavivirus infections of bats with an emphasis on the potential role in zika virus ecology has been reviewed elsewhere [ ] . flavivirus serology has been historically challenging due to the cross-reactivity of viral epitopes to circulating antibodies [ ] . therefore, the results of serologic surveillance studies must be interpreted cautiously [ , ] . further, multiple methods exist for antibody detection (e.g., hi, prnt, elisa), and the biological significance of neutralizing vs. non-neutralizing antibodies must be taken into account. in , the serum of mexican bats from three species (glossophaga soricina, artibeus jamaicensis, and artibeus literatus) was assayed by prnt using wnv, slev, and denv - , and were positive for flavivirus-specific antibodies ( %). none of the titers exceeded , and all samples were also negative when tested for flavivirus nucleic acid by rt-pcr [ ] . in a serosurvey, eight bats ( . %) displayed non-specific hemagglutination-inhibition (hi) results indicating cross-reactivity or antibodies against an undetermined flavivirus [ ] . kading and colleagues performed a serosurveillance study in ugandan bats and identified . % ( / ) had non-specific flavivirus antibodies by plaque reduction neutralization assay (chaerephon pumilus, hipposideros ruber, mops condylurus, nycteris macrotus, eidolon helvum, epomophorus minor, and rousettus aegyptiacus) [ ] . still, results generally supported the widespread exposure of bats in uganda to flaviviruses [ ] . in , sotomayor-bonilla and colleagues reported that liver and spleen samples from mexican bat species tested negative using pan-flavivirus ns primers [ ] . a recent study in brazil suggested a lack of arboviral circulation in bat populations, as individuals from species were tested for molecular and serologic evidence of alphavirus and flavivirus infection and all were negative [ ] . results of experimental infection of egyptian rousette bats with wnv and of angolan free-tailed bats (mops condylurus) with ntaya virus resulted in very low levels of viremia, while infection of african straw-colored fruit bats with ntaya virus resulted in neither pathology nor detectable viremia [ ] . few studies have examined the presence of viruses in genus coltivirus in bat populations, and to date, a single isolation has been made (table ) [ ] . a study by chastel and colleagues failed to detect antibodies to eyach virus (reoviridae, colorado tick fever group) in the serum of two field-caught bats [ ] . to date, five orbiviruses have been isolated from wild-caught bats and serologic evidence exists for exposure of australian and south american bats to orbiviruses (table ) . while no evidence of human exposure exists for these bat-associated orbiviruses, bukakata (bukv) and fomede (fomv) appear to be strains of the chobar gorge species [ ] . cgv was isolated from ornithodoros species ticks in nepal, and serum from nearby humans and ruminants possessed anti-cgv antibodies, indicating past exposure [ ] . further investigation is warranted to determine the true vector-host association of these viruses and their zoonotic potential. viruses in family reoviridae that have been experimentally examined in bats or described in field studies are descried in table . viruses in genus alphavirus (family togaviridae) that have been experimentally examined in bats or described in field studies are descried in table . enzootic circulation of chikv is understood to occur among non-human primates and forest-dwelling mosquitoes [ ] , but other vertebrates including rodents, bats, reptiles and amphibians have been shown to support chikv replication [ , ] . the range of peak viremia developed by big brown bats was relatively low, but within the range of infectivity to blood feeding mosquitoes [ , ] . when indian flying foxes (pteropus giganteus) and big brown bats were experimentally infected with chikv, bats developed viremia but no clinical signs of disease, indicating they could play a role in the natural transmission of this virus [ , ] . experimental infection of african straw-colored flying foxes did not result in viremia or seroconversion to chikv, supporting a separate study which reported lack of viremia in experimentally infected egyptian rousette bats and african straw-colored flying foxes [ , ] . in , the serum of wild-caught grenadian bats (genus artibeus) were subjected to prnt and ( %) were found to possess neutralizing antibody to chikv [ ] . chikv has been circulating in central and south america since [ ] . whether or not bats are contributing to the natural circulation of chikv in endemic areas or areas of introduction remains to be determined. serological evidence exists supporting exposure of bats to encephalitic alphaviruses in the field, and experimental data demonstrate the susceptibility of bats to infection with alphaviruses including veev. four mexican bat species were examined for molecular evidence of infection with venezuelan equine encephalitis virus (veev), western equine encephalitis virus (weev), and eastern equine encephalitis virus (eeev). no individual bats were positive for weev or eeev, but % ( / ) representing all four species were positive for veev [ ] . field-caught jamaican fruit bats (artibeus jamaicensis) and great fruit-eating bats (artibeus literatus) were negative by prnt for eeev and weev antibodies, but . % ( / ) had neutralizing antibodies to veev [ ] . similarly, the serum of bats representing species was subjected to elisa, and . % ( / ) contained veev-specific antibodies. elisa and hi assays for eeev and weev antibodies, respectively, were all negative [ ] . four species of wild-caught bats from the northeastern united states were tested for neutralizing antibody against eeev and weev. samples were negative for antibodies against weev, but . % of the bats tested did possess eeev-neutralizing antibody [ ] . bats of the genera myotis and eptesicus were experimentally infected with eeev, and developed viremia but failed to develop neutralizing antibodies. infection of big brown bats by bite of culiseta melanura and aedes aegypti mosquitoes was successful. more non-hibernating than hibernating bats were seropositive for eeev [ ] . in a recent serosurveillance study, / bats were seropositive by plaque reduction neutralization assay to babanki virus (bbkv) and / egyptian rousette bats had non-specific alphavirus antibodies (table ) [ ] . multiple isolates of bbkv were obtained from cx. perfuscus mosquitoes collected from multiple locations in uganda during this same sampling period as when bats were sampled [ ] . mosquito blood meals from bats comprised . % of the total blood meals identified from the species cx. perfuscus [ ] . it is unclear whether bats contribute to the transmission cycle of bbkv or are merely incidentally exposed through mosquito bites ten pteropus poliocephalus bats were experimentally infected with ross river virus, and five developed low (log . tcid / µl) detectable and short-lived ( days) viremia. still, % of the colonized mosquitoes (aedes vigilax) that fed on the bats between days - post-infection became infected [ ] . kading et al. ( ) modeled that for viremias <log . /ml, the probability of a mosquito becoming infected was around . or less given the low circulating titer and the volumetric constraints of a small mosquito blood meal; therefore, at least mosquitoes would need to feed on an animal with a low viremia in order for one mosquito to ingest virus [ ] . in the case of rrv, published data demonstrate that infection of mosquitoes fed on rrv-viremic bats is still possible despite a viremia and low titer [ ] . therefore, if bat and mosquito populations are in high numbers, % of bats develop a detectable viremia, and % of mosquitoes become infected, mosquito-borne transmission could take place even though experimentally-determined efficiencies are low [ ] . antibodies against mayaro virus were detected by hi in trinidadian bat species tested [ ] . a number of hematophagous arthropods feed on bats, including bat flies (genera nycteribiidae and streblidae), bat bugs and bed bugs (family cimicidae), and ticks (families argasidae and ixodidae) [ , , , [ ] [ ] [ ] [ ] [ ] [ ] . viruses of medical and veterinary significance have also been isolated from these arthropods [ , [ ] [ ] [ ] . however, the contribution that these ectoparasites play in the circulation of medically important viruses among bats and other hosts is unclear and necessitates further investigation. kading and schountz ( ) reviewed instances in the literature where mosquito blood meals have been identified as originating from bats [ ] . information on primary mosquito vectors feeding on bats is very limited. tiawsirisup et al. collected mosquitoes from five genera inside a bat cave in thailand to investigate sylvatic circulation of jbev. while these collections included arbovirus vectors cx. quinquefasciatus and cx. tritaenhiorhynchus, the only blood-fed mosquitoes collected from the cave were cx. quinquefasciatus, at least of which had fed on leschnault's rousette (rousettus leschenaulti) bats [ ] . culiseta morsitans theobald mosquitoes (vector of eastern equine encephalitis virus) were found to have fed on eastern pipistrelle bats (pipistrellus subflavus), but these blood meals comprised only % of the total blood meals identified from this mosquito species [ ] . no information was found on any blood meals from bats being detected in aedes (stegomyia) species, but it is unclear how much investigation has been done in this area. sixteen of field-collected ae. funereus mosquitoes (vector of rrv) had fed on pteropus alecto bats [ ] . in africa, mosquito species in which bat blood meals have been identified and are known to be associated with a number of medically-important arboviruses include: coquillettidia (cq.) fuscopennata (theobald) (yfv, sindbis, chikungunya viruses), culex (cx.) perfuscus edwards (wnv, oropouche, sindbis, wesselsbron, usutu, babanki viruses), cx. (cx.) neavei theobald (wnv, babanki, spondweni, sindbis, koutango viruses), and cx. (cx.) decens group (wnv, chikungunya, babanki viruses [ , ] . while mosquitoes in the subgenus cx. (cx.) are recognized as primary vectors of wnv, sindbis, babanki, and usutu viruses, only for babanki virus has additional field data been collected so far that support a potential role for bats in virus circulation (discussed above) [ ] . bat flies (order: diptera; families: nycteribiidae, streblidae) are highly host-specific obligate hematophagus ectoparasites of bats [ , ] . a novel fusogenic orthoreovirus, tentatively named mahlapitsi virus (mahlv), was discovered in bat flies (eucampsipoda africana) associated with egyptian rousette bats [ ] . a novel orthobunyavirus, tentatively named wolkberg virus (wbv), was isolated from the same species of bat flies in a similar geographic region [ ] . dengue virus rna was detected by rt-pcr in bat flies (strebla wiedemanni, trichobius parasiticus) associated with common vampire bats (desmodus rotundus) [ ] . bartonella spp. bacteria have also been found infecting both fruit bats and the bat flies parasitizing them in madagascar, providing a documented example of a pathogen-vector-bat association involving bat flies [ ] . more research in this area is needed to elucidate the role of bat ectoparasites in spreading pathogens among individual bats in a roost. in addition to parasitizing humans by infesting their dwellings, bed bugs and other arthropods in family cimicidae are found in close association with bat populations [ ] . some cimicids are known to play a role in alphavirus transmission. cliff swallow bugs (oeciacus vicarious) transmit fort morgan virus and buggy creek virus [ , , , ] among passerine birds. tonate virus, which is closely related to veev, and also causes fatal encephalitis in man, has also been isolated from swallow bugs in the 's [ ] . as previously described, bat bugs (cimex insuetus and strcticimex parvus) host the orthobunyavirus kkv which likely causes disease in humans [ , ] , but this also remains a largely understudied research area. ticks and mites are known to parasitize bats and they both fill similar ecological niches. many studies have identified viruses in bats that cluster phylogenetically with other arboviruses transmitted by ticks. other isolations are indicative of bats playing a role in viruses isolated from either hard-bodied or soft-bodied ticks. for example, estero real virus was first isolated from ticks (ornithodoros tadaridae) obtained from a palm tree colonized by cuban bats [ ] . due to the close ecological association between egyptian rousette bats (r. aegyptiacus) and soft-bodied ticks in caves, the argasid tick ornithodoros faini was screened for marburgvirus rna but all pools were found to be negative [ ] . to date, evidence does not support the role of arthropods in the transmission of filoviruses in nature. bats are also known to host mite species in families macronyssidae, dermanyssidae, and spinturnicidae, though the significance of these arthropod's role in virus transmission between bats and other vertebrates is largely unknown [ ] [ ] [ ] . species in macronyssidae and dermanyssidae have also been demonstrated to efficiently transmit arboviruses (further reviewed in [ ] ). a recent study explored viruses of potential arthropod-origin in the fruit bat hypsignathus monstrosus and identified five viruses: one dicistrovirus (family dicistroviridae, order picornavirales), one nodavirus (family nodaviridae), and two tombus-like viruses. they also detected a related tombus-like virus isolated from fig wasps and primitive crane flies co-habitating with bats, suggesting the ability of arthropods to host "bat-associated" viruses [ ] . tombus viruses typically infect plants, but tombus-like viruses infect a wide range of hosts, including arthropods and marine invertebrates [ ] [ ] [ ] . ingestion of arthropods by fruit bats has been documented [ , ] , and could be a potential mechanism explaining the association between viromes of arthropods and frugivorous bat species. with regard to experimental infections, one must take into account the method of inoculation (e.g., intracerebral, subcutaneous, intraperitoneal, intramuscular), the inoculating titer, as well as whether or not vector bites were associated with the infection. in analyzing serologic results, one must take into account whether the assay is detecting neutralizing or non-neutralizing antibodies, and consider the dynamics of antibody decay which may impact detection sensitivity [ ] . arthropod saliva has been shown to potentiate arbovirus infection, affecting not only the magnitude of peak viremia but also the viral loads in certain organs [ ] . the inoculating viral strain used is also important, as it should align geographically with where the species under study resides to truly characterize their potential as a reservoir species. komar et al. ( ) presented detailed methods for estimating the reservoir competence of vertebrate species for mosquito-borne viruses [ ] , including calculating a reservoir competence index based on the susceptibility to infection, mean daily infectiousness, and the duration of infectiousness. this reservoir competence index is interpreted as the relative number of infectious vectors that derive from a particular vertebrate species [ ] , and would be easily transferrable to evaluating the reservoir competence of bats. further, bats are very long-lived (up to years) compared to other small mammals, potentially increasing the chances of exposure and subsequent transmission to other vertebrates or invertebrate vectors [ ] . cell culture also offers a viable starting point for initial assessments of viral replication in a particular host species, particularly when in vivo studies are not feasible. to date, many cell lines derived from the organs of bats have been established [ ] . importantly, the intent of this review is not to vilify bats, but to analyze the existing literature and its support for bats as arboviral reservoirs, in addition to identifying areas for future study. bats provide vital ecosystem services, such as arthropod suppression, pollination, and seed dispersal [ ] . past depopulation efforts in response to viral outbreaks resulted in increased viral spillover, and are not a viable means of disease control and have even resulted in higher virus infection rates in the bat species when colonies repopulated from neighboring roosts [ ] . further work should encompass directed field surveillance complemented by both in vitro and in vivo approaches, with field surveillance efforts focused on optimization of non-lethal and non-invasive sampling [ ] [ ] [ ] [ ] . when possible, longitudinal sampling of identifiable animals through use of marking or tagging allows for monitoring of seroconversion and viremia persistence, providing a chance to better characterize transmission periodicity and seasonal changes in viremia profiles. to truly elucidate the role of bats as reservoirs for arboviruses, field surveillance studies documenting natural infection and transmission dynamics among vector and vertebrate species must be supplemented with experimental infections to characterize viremia profiles and infectiousness to vectors, virus-induced pathology, and immune kinetics following infection. with bats, these tasks are not trivial, and carry significant challenges in both the field and the lab. these challenges are evidenced by the few arboviruses for which there are substantial field and laboratory data involving the infection of bats. while many studies have presented serologic data indicative of past exposure of free-ranging bats to arboviruses of medical and veterinary importance, these studies should be followed up with laboratory assessments of reservoir host competence to shed light on the true epidemiological significance of the field data. further, the detection of viral nucleic acid in free-ranging bats does not necessarily implicate the species as an arboviral reservoir. rather, recovery and isolation of live virus at biologically relevant titers, and demonstrating the persistence of the pathogen in nature among connected populations of a potential reservoir species is more definitive. unfortunately, few established bat colonies exist for use in vivo viral pathogenesis studies, limiting the achievability of these studies. to fulfill the vertebrate reservoir host paradigm, in vivo infections must support findings in the field. the isolation of marburg virus from egyptian rousette bats in uganda in addition to experimental infections demonstrating viremia and shedding in the absence of overt pathology support the role of this bat species as the reservoir for marburg virus [ , , ] . for arboviruses, the combination of field work and in vivo pathogenesis studies are lacking. still, several examples have emerged from this review that point to bats as potentially competent amplifying hosts for arboviruses. kaeng khoi virus was isolated from both bats and cimicid bugs in thailand, and was also shown to be the causative agent behind sick mine workers [ ] . some bat species did develop a viremia at a level that would be infectious to mosquitoes when inoculated experimentally with chikv, and bats have been found exposed to chikv during field investigations [ , ] . experimental evidence supporting transmission of rrv from pteropus poliocephalus to recipient mosquitoes in addition to identification of rrv-seropositive bats trapped in australia and indonesia highlights the need for further investigation into the role of bats in the ecology of this disease [ , , ] . a mosquito-bat-mosquito transmission cycle was established in the lab for jbev [ ] . serological evidence from the field documenting bat exposure to jbev, the sustained viremia in bats during hibernation, and demonstration of mosquito transmission 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optimizing non-invasive sampling of an infectious bat virus experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera key: cord- -fhie m u authors: mazaleuskaya, liudmila; veltrop, rogier; ikpeze, nneka; martin-garcia, julio; navas-martin, sonia title: protective role of toll-like receptor -induced type i interferon in murine coronavirus infection of macrophages date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: fhie m u toll-like receptors (tlrs) sense viral infections and induce production of type i interferons (ifns), other cytokines, and chemokines. viral recognition by tlrs and other pattern recognition receptors (prrs) has been proven to be cell-type specific. triggering of tlrs with selected ligands can be beneficial against some viral infections. macrophages are antigen-presenting cells that express tlrs and have a key role in the innate and adaptive immunity against viruses. coronaviruses (covs) are single-stranded, positive-sense rna viruses that cause acute and chronic infections and can productively infect macrophages. investigation of the interplay between covs and prrs is in its infancy. we assessed the effect of triggering tlr , tlr , tlr , and tlr with selected ligands on the susceptibility of the j a. macrophage cell line to infection with murine coronavirus (mouse hepatitis virus, [mhv]). stimulation of tlr , tlr , or tlr did not affect mhv production. in contrast, pre-stimulation of tlr with polyinosinic-polycytidylic acid (poly i:c) hindered mhv infection through induction of ifn-β in macrophages. we demonstrate that activation of tlr with the synthetic ligand poly i:c mediates antiviral immunity that diminishes (mhv-a ) or suppresses (mhv-jhm, mhv- ) virus production in macrophages. coronaviruses (covs), a genus in the coronaviridae family, order nidovirales, are emerging rna pathogens of many animal species, including humans [ ] . currently there are no approved treatments or completely successful vaccines against cov infections. mice infected with different strains of mouse hepatitis virus (mhv), the prototype of betacoronaviruses, provide animal models for human diseases. the neurotropic strains, mhv-jhm and mhv-a , are commonly used to study viral encephalitis and virus-induced chronic demyelination, respectively [ ] . mhv-a also triggers mild to moderate hepatitis. the highly hepatovirulent strain mhv- provides a model of fulminant viral hepatitis [ ] . the first few days after infection with mhv are characterized by a strong innate immune response with infiltration of macrophages, neutrophils, and natural killer cells to the site of infection. it is widely documented that the host immune response plays a dual role in cov infection. on the one hand, it limits virus spread and replication and initiates adaptive immunity; on the other hand, it triggers overproduction of cytokines and chemokines, thus contributing to the severity of the disease [ ] [ ] [ ] [ ] [ ] . macrophages are productively infected by murine covs [ ] [ ] [ ] and represent the largest group of innate immune cells that infiltrate the central nervous system (cns) after infection with neurotropic mhv strains [ ] and the lungs of patients infected with severe acute respiratory syndrome (sars)-cov [ ] . the adaptive immune response that occurs during cov infection is well characterized [ , ] , but our understanding of the interaction of covs with the innate immune system of the host is still emerging [ , ] . type i interferon (ifn) (ifn-α and ifn-β) is crucial for the control of mhv infection in vivo [ ] [ ] [ ] . in most cell lines, murine covs are poor inducers of type i ifn and are barely sensitive to pretreatment with ifn [ ] . in primary cells, however, mhvs trigger ifn-α in plasmacytoid dendritic cells (pdcs) [ ] and ifn-β in macrophages [ , ] and are sensitive to pre-treatment with ifn-β in macrophages [ ] . therefore, interaction between murine covs and the type i ifn response depends on the cell type. the importance of type i ifn in cov infection is highlighted by a number of countermeasures and evasion mechanisms that covs in general and mhvs in particular developed to suppress signaling or prevent induction of the ifn response [ ] [ ] [ ] . induction of type i ifn can occur in all nucleated cells on tlrs activation [ ] . tlrs comprise a family of pattern recognition receptors (prr) that sense conserved molecular motifs of pathogens and trigger innate immunity and prime the adaptive immune response [ ] . triggering of tlrs induces complex signaling cascades initiated by the toll/interleukin- receptor (tir) domain in the cytoplasmic tail of the tlr. tir domain-containing adaptor molecules, myd , which is utilized by all tlrs except for tlr , as well as tirap, trif, and tram (for tlr ), are recruited to the receptor and activate a complex containing iraks and trafs which signal through nf-kb leading to the expression of a variety of genes encoding pro-inflammatory cytokines, chemokines and/or type i interferons (ifns) that orchestrate anti-bacterial and anti-viral responses [ ] . in the context of rna virus infection, tlr , tlr , tlr , tlr , and tlr can potentially be activated. cell surface tlr and tlr may recognize viral structural components, whereas endosomal tlr and tlr / may sense viral double-stranded and single-stranded rna, respectively [ ] . all of the above-mentioned tlrs were shown to induce type i ifn through activation of transcription factors and interferon regulatory factors (irfs); the magnitude of response, however, depends on the stimulus and the cell system. tlr , tlr and tlr are known to be potent inducers of the ifn response depending on the cell type [ ] . in contrast, tlr has been considered until recently a poor inducer of ifn response, despite triggering of tlr with bacteria-derived ligands induces strong pro-inflammatory cytokine response. in this regard, emerging evidence suggests that tlr and tlr activation induces pro-inflammatory cytokine and type i ifn responses from distinct sub-cellular sites: the plasma membrane and the endolysosomal compartments, respectively [ , ] . interestingly, only a particular monocyte subset has been reported to induce type i ifn through tlr in response to viral ligands [ ] . once secreted, ifn-α/β act through the jak-stat signaling pathway that triggers an "antiviral state" and help to eliminate viral infection [ , ] . the ability of tlrs to trigger antiviral immunity makes them a promising target for antiviral therapeutics. stimulation with tlr agonists has been shown to provide protection from some viral infections, such as hepatitis b virus (through tlr , tlr , tlr , tlr , or tlr ) [ ] , herpes simplex virus encephalitis (through tlr ) [ ] , lethal influenza virus (through tlr or tlr ) [ ] , hiv strains bal and jago (through tlr ) [ ] , and hepatitis c virus (through tlr ) [ ] . this study was undertaken to assess the effect of ligand-mediated, tlr activation of macrophages on their susceptibility to infection with murine cov. we profiled tlr , tlr , tlr , and tlr agonists (heat-killed listeria monocytogenes (hklm), poly i:c, lipopolysaccharide (lps), and imiquimod, respectively) and observed differential effects of these ligands on mhv production in macrophages. of all the ligands tested, only the triggering of tlr with poly i:c induced a strong antiviral response. mechanistically, the antiviral effect of poly i:c was promoted in a type i ifn-dependent manner. ligand-mediated activation of tlrs has been reported to affect the infectivity of various viruses [ , [ ] [ ] [ ] [ ] [ ] [ ] . the potential immunomodulatory and antiviral effects of triggering tlrs against cov infections in macrophages have not yet been investigated. macrophages are antigen-presenting cells that express tlrs and play a pivotal role in cov pathogenesis. the goal of this study was to investigate the effect of activation of tlr , tlr , tlr , or tlr with selected ligands on macrophage susceptibility to infection with murine coronavirus. we chose these tlrs on the basis of their potential role in the recognition of mhv by macrophages. tlr has been shown to recognize mhv- in peritoneal macrophages [ ] ; tlr has been implicated in protection and pathogenesis in mhv- -induced respiratory infection [ ] . despite the fact that tlr is a sensor of dsrna and could sense cov intermediate replicative forms in infected cells, its role in the recognition of covs or in their pathogenesis has not yet been established. tlr senses mhv-a in pdcs [ ] . first, we developed an in vitro model suitable for this study. the mouse macrophage cell line j a. was profiled for tlr - gene expression by quantitative real-time polymerase chain reaction (pcr) using predeveloped taqman gene expression assays (appliedbiosystems life technologies corp, carlsbad, ca) ( figure a ). expression levels of target genes were normalized to the housekeeping gene β-actin (Δct). gene expression values were calculated based on the ΔΔct method, with data for all samples analyzed against the mean value of four replicates. tlr showed a -fold greater expression than that of tlr and tlr (student's t test, p < . ), and the latter two had a more than -fold greater expression than tlr , , , , and (student's t test, p < . ). tlr and tlr transcripts were expressed to the highest and lowest levels, respectively (student's t test, p < . ). the expression of tlr , tlr , tlr , and tlr proteins was analyzed using flow cytometry (facs). as shown in figure b , facs data demonstrated robust expression of cell-surface tlr and tlr and intracellular tlr and tlr in naïve j a. cells. next we determined whether tlr , tlr , tlr , and tlr are functional in j a. cells. activation of the cells with a tlr ligand (hklm, cells/ml), a tlr agonist (poly i:c, μg/ml), a tlr ligand (lps, μg/ml), or a tlr agonist (imiquimod (r ), μg/ml) for , , and h resulted in the robust production of il- and tnf-α ( figure a ). this result indicates that, in j a. macrophages, tlr - and tlr are fully functional and signal with cytokine secretion after stimulation. we assessed type i ifn mrna induction in tlr-activated j a. macrophages. expression of ifn-α and ifn-β genes was up regulated by poly i:c, lps, and r ligands in j a. cells with different kinetics ( figure b ). lps-induced ifn-β and ifn-α mrnas peaked at h and h post-stimulation, respectively. r -induced ifn-β and ifn-α mrnas peaked at h post-stimulation. the induction of type i ifn gene expression after lps and r was not sustained (in contrast to ifn-β gene expression after poly i:c stimulation). ifn-α and ifn-β levels were determined by elisa in cell-free supernatants collected after h of prestimulation with hklm ( cells/ml), lps ( μg/ml), r ( μg/ml), and poly i:c ( . μg/ml). interestingly, activation of tlr but not of tlr , tlr or tlr triggered robust production of ifn-β following pre-stimulation for h in macrophages ( figure c ). similar to ifn-β, ifn-α was secreted only in tlr -activated cells, albeit to a much lesser degree ( figure c ). these contrasting results suggest that type i ifn response may be regulated differentially on tlr stimulation at the post transcriptional level in j a. cells. their lack of ifn secretion (as measured by elisa) in response to stimulation with the bacterial ligands hklm and lps is somewhat unpredicted and deserves further investigations. in addition, although it is well established that type i ifn response against rna viruses is mainly mediated by pdcs via a tlr -dependent pathway, the role of tlr in macrophage activation remains poorly understood. in this regard, the absence of ifn-β secretion after r stimulation of tlr in j a. cells may suggest differences in the regulation of the tlr pathway and/or its effectors between macrophages and pdcs. we are currently investigating the role of macrophage tlr in the antiviral response against rna viruses. furthermore, mhvs have been shown to productively infect j a. cells [ ] . by combining these results, we established a valid in vitro model in which to investigate the effect of triggering tlrs with selected ligands on mhv infectivity in macrophages. effect of triggering tlr , tlr , tlr , and tlr on virus production in mhv-infected j a. macrophages. j a. cells were prestimulated with tlr ligands for h, infected with mhv-a , mhv-jhm or mhv- ( moi) by adsorption for h in the absence of the ligands, and activated for up to h p.i. with the appropriate tlr agonist. tlr ligands were used as follows: hklm (tlr ) at cells/ml; lps (tlr ) at μg/ml; imiquimod (r ) (tlr ) at μg/ml. poly i:c (tlr ) was tested at a range of concentrations ( . , . , and . μg/ml). because poly i:c triggered a comparable effect on mhv production at all concentrations (data not shown), viral titers at . μg/ml were included in the plot. cells incubated with the basal medium before and during infection served as a negative control for the effect of tlr triggering on virus production. mhv titers were assessed in cell-free supernatants using a plaque assay on l fibroblasts. the data shown are the mean viral titers of three independent experiments, each done in duplicate ± standard deviation (*p value relative to virus alone, p < . student's t test). to test whether treatment with the tlr ligands hklm, poly i:c, lps, and r affected the replication of mhv, j a. cells were prestimulated with the appropriate ligand at the above-mentioned concentrations for h; and cells were infected with mhv-a , mhv-jhm, or mhv- at a multiplicity of infection (moi) of by adsorption for h in the absence of the ligands and stimulated again for up to h postinfection (p.i.) with the appropriate tlr agonist. therefore, there were two challenges with tlr ligands: one before and one after virus adsorption. activation of macrophages with tlr , tlr and tlr did not noticeably affect mhv production in j a. macrophages ( figure ). conversely, the triggering of tlr with poly i:c significantly inhibited mhv-a , mhv-jhm, and mhv- production relative to virus alone (student's t test, p = . ; figure ). complete suppression was observed only in poly i:c-treated mhv-jhm-and mhv- -, infected macrophages, although all mhv strains showed a dramatic -log reduction in virus production. the lack of complete suppression in mhv-a infected cells could be explained by the ability of mhv-a to grow to higher titers ( - log) than mhv-jhm and mhv- in macrophages. additionally, these results may also suggest that mhv-a counteracts the tlr pathway in j a. macrophages. indeed, our data shows that tlr -mediated, ifn-β secretion is significantly reduced in mhv-a -infected macrophages ( figure ). interestingly, poly i:c triggered comparable antiviral effect regardless of its concentration ( . , . , and . μg/ml; data not shown). it will be of interest to determine the minimal antiviral concentration of poly i:c in future experiments. the optimal concentration range for poly i:c was selected based on the highest rate of cytokine production (il- elisa) and minimal cytotoxicity (ldh cytotoxicity assay) in j a. macrophages activated with poly i:c at various doses (data not shown). collectively, these data demonstrate that, depending on the receptor, ligand-mediated tlr stimulation exerts differential effects on mhv production. triggering tlr with poly i:c, but not activation of tlr , tlr , or tlr with their respective ligands, impairs mhv replication in macrophages with a comparable magnitude of suppression of viral titers for mhv-a , mhv-hjm, and mhv- strains. given that all four tlr ligands induced strong il- and tnf-α proinflammatory responses (figure a ), we concluded that the inability of tlr , tlr and tlr agonists to protect macrophages from mhv infection is not due to the lack of signaling through these receptors, rather it stems from the absence of ifn-α and ifn-β production after h of stimulation with their ligands ( figure c ). in contrast, the antiviral effect mediated by activation of tlr with poly i:c is associated with a sustained transcriptional upregulation and secretion of ifn-α and ifn-β. we investigated the optimal conditions for poly i:c antiviral effects in j a. macrophages infected with a recombinant mhv-a expressing the gfp protein (ra -gfp) ( moi) and treated as follows: ( ) prestimulated with poly i:c, with no drug present during infection (poly i:c +/−); ( ) treated with poly i:c only after virus adsorption (poly i:c −/+); ( ) treated with poly i:c before and after virus adsorption (poly i:c +/+). the tlr ligand was used at concentrations of . to . μg/ml for h of prestimulation and/or h p.i. a profound suppression of gfp expression in cells stimulated with . μg/ml poly i:c was observed with all of the above-mentioned treatments relative to infected macrophages in the absence of the drug ( figure a ). thus, a single challenge with the tlr ligand before or after virus adsorption was sufficient to trigger a robust antiviral effect comparable to cells challenged with poly i:c twice. to determine the level of mhv production, released virus was quantified by plaque assay in cell-free supernatants from macrophages stimulated with . and . μg/ml poly i:c as above and in the absence of the drug ( figure b ). regardless of the concentration of poly i:c, the triggering of tlr with poly i:c resulted in a -log reduction in ra -gfp titers in prestimulated and coactivated macrophages (poly i:c +/+) relative to infected cells in the absence of the drug ( figure b , p < . ). cells challenged with poly i:c once before (poly i:c +/−) or h after mhv adsorption (poly i:c −/+) also exhibited a significant suppression (p < . ) of virus production comparable to that of prestimulated and coactivated macrophages (poly i:c +/+). interestingly, the triggering of tlr before adsorption with mhv (poly i:c +/−) resulted in significantly lower virus production relative to coactivated macrophages (poly i:c −/+) (p = . and p = . for . and . μg/ml poly i:c, respectively), suggesting that a single challenge with poly i:c prior to infection dramatically reduces macrophage susceptibility to mhv infection. taken together, these results indicate that . μg/ml of poly i:c is sufficient to trigger a profound tlr -mediated antiviral effect and that prestimulation alone is enough to protect macrophages from infection with mhv. to investigate the mechanism of the poly i:c-triggered antiviral effect in mhv-infected macrophages, we wanted to determine if the tlr ligand induced soluble factors that mediated protective immunity against cov infection. we pretreated j a. cells for h with conditioned medium (cm) from macrophages prestimulated with tlr - and tlr ligands for h (figure a ). next, cells were infected with ra -gfp ( moi) for h adsorption in the absence of tlr ligands and incubated for additional h in basal medium. ra -gfp virus titers determined by plaque assay are shown in figure . as expected, cm from mock macrophages (unstimulated, uninfected) did not affect virus production. treatment with cm from macrophages stimulated with hklm, r , and lps did not affect virus production ( figure ). remarkably, there was a -log reduction in ra -gfp titers in cells pretreated with poly i:c cm ( figure , p < . ) that correlated with the inhibition of gfp expression in these cells (data not shown). i. cells pretreated with the cm from mock cells or with fresh basal medium served as negative controls for the tlr-triggered effect. ra -gfp titers were assessed in cell-free supernatants using a plaque assay on l fibroblasts. error bars represent the standard error of the mean of two replicates (p value relative to cells pretreated with cm from mock, p < . , student's t test). overall, these data suggest that prestimulation with poly i:c but not hklm, lps, or r triggers the production of soluble factors that further protect macrophages from infection with mhv on subsequent exposure. in addition to soluble factors, residual poly i:c in the cm may have also contributed to the antiviral effect in cells pretreated with tlr -stimulated supernatants. our data in figure c demonstrated that after h of stimulation with hklm, lps, r , and poly i:c, ifn-β and ifn-α were secreted only in tlr -activated j a. cells. these results together with the antiviral effect of poly i:c (figure ) suggested that the protective role of tlr against coronavirus infection in j a. macrophages may be mediated by type i ifn. to investigate the role of type i ifn in the poly i:c-mediated inhibition of murine cov production in macrophages, we focused on mhv-a and mhv-jhm strains. we hypothesized that the differential effect of tlr ligands on mhv production is due to their variable ability to induce type i ifn crucial for triggering an "antiviral state" and protecting cells from virus infection [ , ] . to test this hypothesis, we assessed type i ifn production in tlr-stimulated and/or mhv-infected j a. a second challenge with poly i:c for h resulted in a robust secretion of ifn-α and ifn-β ( figure ). in contrast, a single challenge with poly i:c for h induced lower levels of ifn-β and levels of ifn-α that were close to the limit of detection of the elisa assay ( figure c ). such a pattern of induction of type i ifn in cells treated with dsrna (like poly i:c) is consistent with the activation of two types of type i ifn genes, immediate-early and delayed-type genes (reviewed in ref. [ ] ). immediate-early genes, mostly ifn-β and some ifn-α (only in murine cells), are induced by a protein-synthesis-independent pathway. secreted ifn signals in both an autocrine and paracrine fashion through the type i ifn receptor and triggers delayed-type ifns (including other ifn-α subtypes). expression of the delayed-type ifn depends on de novo protein synthesis and results in amplification of the ifn response. similarly, poly i:c-activated j a. macrophages exhibit high levels of ifn-β and a modest secretion of ifn-α on induction of the immediate-early gene. later, these cells secrete comparably high levels of both ifn-α and -β as a result of delayed-type ifn gene expression. figure ). the effect of infection with mhv on poly i:c-triggered ifn-β induction was previously assessed in ci- murine fibroblasts [ ] . in that study, neither mhv-a nor mhv-jhm inhibited ifn-β induction after poly i:c was transfected into fibroblasts (a way to activate rig-i and mda cytoplasmic helicases but not endosomal tlr ). thus, the ability of mhv to counteract poly i:c-induced ifn-β is cell type-specific and depends on the mode of delivery of poly i:c into the cell. targeting rig-i and mda helicases by poly i:c transfection with a lipid carrier as reported by [ ] , did not result in mhv-mediated inhibition of poly i:c-induced ifn-β secretion. in contrast, our data suggest that mhv-a might counteract the ifn-β response when macrophages are stimulated with soluble poly i:c to trigger the tlr pathway. further experiments are needed to define how mhv-a might counteract the tlr pathway in macrophages. interestingly, mhv-a has been reported to develop various measures to counteract the type i ifn response [ ] [ ] [ ] . besides poly i:c, the tlr ligand r induced a modest level of secretion of ifn-β with h stimulation; tlr and tlr agonists did not promote type i ifn secretion in j a. macrophages as measured by elisa ( figure ). overall, these findings are in agreement with our hypothesis that poly i:c triggers an antiviral effect via ifn-α/β, whereas the lack of a strong type i ifn production during tlr , tlr , or tlr signaling is responsible for uncontrolled mhv production in j a. macrophages. figure . il- and tnf-α production in cell-free supernatants from j a. macrophages stimulated with tlr ligands, mhv-infected, and/or coactivated during mhv infection. j a. cells were prestimulated for h with hklm ( cells/ml), lps ( μg/ml), r ( μg/ml), and poly i:c ( . μg/ml); media was removed and: ) a second challenge of the corresponding tlr ligand (same concentrations) was added to the cells for h; ) cells were prestimulated for h with the tlr ligands as above; media was removed and cells infected with mhv-a or mhv-jhm at . moi for h of adsorption in the absence of the ligands; after virus adsorption cells were stimulated with a second challenge of the tlr ligands using the same concentrations as during prestimulation and samples were taken at h; ) cells were not tlr activated and only infected with mhv-a or mhv-jhm at . moi for h of adsorption in the absence of the ligands; fresh media was added to the cells for h. non-stimulated, non-infected j a. cells were used as mock control. cell-free supernatants were taken at h from tlr-activated alone, infected alone, and tlr-activated and infected and assessed for il- and tnf-α using elisa. error bars represent the standard error of the mean of two independent experiments, each done in duplicate. to further rule out the potential antiviral effect of il- and tnf-α in infected and co-stimulated cells, we measured proinflammatory cytokines in the same samples as above. stimulation for h resulted in a very high induction of both cytokines after lps (a tlr ligand that based on our data does not induce antiviral effect against infection with mhv in j a. macrophages) and poly i:c (albeit to a lesser extent than with lps) (figure ) . overall, these data argue against the potential antiviral effects of il- and tnf-α in tlr -activated macrophages. tnf-α levels were reduced in mhv-a and mhv-jhm infected macrophages relative to mock cells (figure ) . although it was not the focus of the present study, the inhibitory effect of mhv on the basal tnf-α levels may be mediated through the action of the anti-inflammatory cytokine il- . il- is a known negative regulator of tnf-α production and function in macrophages (reviewed in ref. [ ] ). mhv-a was reported to induce il- in infected primary bone marrow-derived macrophages [ ] , therefore, one could speculate that covs suppress basal macrophage tnf-α secretion through triggering of il- . future studies will be designed to test this hypothesis. although tnf-α was reported to induce a strong antiviral response against various influenza strains in lung epithelial cells [ ] , our data demonstrated that tlr-induced tnf-α does not affect mhv production in macrophages. collectively, these results indicate that il- and tnf-α are not responsible for and do not contribute to a poly i:c-triggered antiviral effect in mhv-infected macrophages. considering that soluble factors mediate the poly i:c-triggered antiviral effect ( figure ) and that poly i:c potently induces type i ifn before and during virus infection ( figures c and ) , we further confirmed the role of ifn-β in tlr -triggered mhv suppression in macrophages ( figure a-c) . we focused on ifn-β because unlike ifn-α, ifn-β was profoundly induced in prestimulated macrophages at h poststimulation ( figure c ), and cm from macrophages treated with poly i:c for h exhibited inhibition of mhv-a production sufficient to reduce viral infection ( figure ). titration of anti-ifn-β antibody (ab) was done to establish the optimal ab concentration for neutralization of poly i:c-stimulated ifn-β. j a. macrophages were stimulated with . μg/ml poly i:c in the presence or absence of anti-ifn-β ab at various concentrations. we chose . μg/ml poly i:c because prestimulation with such a low concentration of the tlr ligand was sufficient to promote a strong antiviral effect (figure ). poly i:c-triggered ifn-β was significantly reduced by u/ml to u/ml of the ifn-β neutralizing ab, and it was suppressed in the presence of a higher concentration ( u/ml) ( figure a ). after stimulation, cells were infected with ra -gfp ( moi) by adsorption for h and incubated in fresh medium for up to h p.i. cm from mock or ifn-β ab-treated macrophages did not affect mhv-a virus production in these cells ( figure b,c) . as expected on the basis of our previous results, pretreatment with poly i:c-conditioned medium resulted in a drastic reduction in ra -gfp expression and in a -log reduction in mhv production in infected macrophages ( figure b,c) . importantly, mhv-a infection was significantly restored (p = . ) in j a. cells incubated with the supernatant from macrophages activated with poly i:c in the presence of the ifn-β neutralizing polyclonal ab (poly i:c + ifn-β ab cm) ( figure b,c) . this result indicates that ifn-β is a crucial mediator in the antiviral response against mhvs elicited by triggering tlr with poly i:c in macrophages. murine covs are sensitive to pretreatment of macrophages with recombinant ifn-β [ ] . in the present study prestimulation of j a. macrophages with poly i:c, a potent type i ifn inducer, resulted in a strong ifn-β response that triggered antiviral immunity and protected macrophages from mhv infection before and after virus adsorption. in support of our data, a poly i:c analog ampligen tm (poly i:poly c u) was successfully tested in sars-cov animal models [ , ] . balb/c mice were treated with ampligen tm intraperitoneally (i.p.) h before sars-cov infection and then the virus titers in the lungs were assessed days after virus exposure. sars-cov titers in the lungs were below the limit of detection suggesting that poly i:poly c u completely protected mice from viral infection [ ] . in a different study, ampligen tm was given intraperitoneally to balb/c mice h before they were infected with the mouse-adapted sars-cov strain v . treated mice exhibited complete survival, suppressed virus titers in the lungs, significantly reduced lung scores and weight loss [ ] . these studies did not investigate the mechanism of the ampligen tm -triggered antiviral effect in sars-cov-infected mice; type i ifn, however, was proposed as a mediator of antiviral immunity. ampligen tm is indeed a potent type i ifn inducer that acts through tlr [ ] and triggers protection from hiv [ , ] , coxsackie virus [ ] , punta toro virus [ ] , venezuelan equine encephalitis virus [ ] , and influenza virus [ ] infections. overall, our data demonstrates that tlr triggered type i ifn inhibits murine cov infection of macrophages. j a. murine macrophages (attc, tib- ) were cultured in dulbecco's modification of eagle's medium (dmem) (cellgro, mediatech, manassas, va, usa) supplemented with % v/v heat-inactivated fetal bovine serum (hifbs) (hyclone, thermo scientific, rockford, il, usa), u/ml penicillin (cellgro), μg/ml streptomycin (cellgro), . g/l sodium bicarbonate (biowhittaker, lonza, walkersville, md, usa) and mm glutamine (cellgro). cells were incubated in a humidified atmosphere at c with % co . the dualtropic mhv-a , and neurotropic mhv-jhm strains were previously characterized [ , ] . the hepatotropic mhv- strain was provided by dr. julian leibowitz (texas a&m university). recombinant mhv-a virus expresses the gfp in place of the orf [ ] . cells were infected at moi by adsorption for h in the basal medium. then, excess virus inoculum was removed and cells were incubated for up to h p.i. plaque assays were performed on l murine fibroblasts [ ] . % confluent monolayers of l cells were infected with -fold dilutions of samples in dmem supplemented with % v/v hifbs. after virus adsorption, cells were overlaid with a : mixture of . % agarose and % fbs in dmem and incubated for h at c with % co . to visualize viral plaques, infected cells were overlayed with equal parts of . % agarose, % fbs in dmem and / neutral red (harleco, emd chemicals inc., gibbstown, nj, usa) and incubated for h at c with % co . all tlr agonists were purchased from invivogen (san diego, ca, usa). the following tlr ligands were used: hklm for tlr at cells/ml; poly i:c for tlr at . to μg/ml; ultrapure lps from e. coli for tlr at μg/ml; and imiquimod (r ), an imidazoquinoline amine analogue of guanosine, for tlr at μg/ml. cells were prestimulated with the appropriate tlr ligand for h, infected as necessary and coactivated during infection with the corresponding tlr agonist for up to h p.i. mouse il- and tnf-α were quantified by elisa (ebioscience, san diego, ca, usa) according to the manufacturer's instructions. limits of detection: . - pg/ml for il- and . - pg/ml for tnf-α. verikine mouse ifn-α and verikine mouse ifn-β elisa kits were purchased from pbl interferon source (thermoscientifc, rockford, il, usa). limits of detection: . - pg/ml for ifn-β, and . - pg/ml for ifn-α. to titrate anti-ifn-β ab, j a. macrophages were activated with . μg/ml poly i:c in the presence or absence of anti-ifn-β ab at , , or u/ml for h. rabbit polyclonal anti-ifnβ ab were purchased from calbiochem (emd chemicals inc.). poly i:c-triggered ifn-β with or without neutralizing ab was assessed with a verikine ifn-β elisa kit. cell-free cm from poly i:cactivated and/or u/ml anti-ifn-β ab-incubated macrophages was used to pretreat j a. cells for h. then macrophages were infected with moi mhv-a by adsorption for h and incubated in fresh medium for up to h p.i. total rna was isolated using trizol ® reagent (invitrogen, carlsbad, ca, usa). total rna ( ng) of was transcribed into cdna with the superscript ii reverse transcriptase kit (invitrogen), using a total reaction mix volume of μl; . μl cdna was combined with . μl of taqman universal master mix (applied biosystems, life technologies, carlsbad, ca, usa), μl diethyl polycarbonate (depc)-treated water, and . μl murine tlr to tlr taqman gene expression assays (applied biosystems). dna was amplified using the applied biosystems real-time pcr machine, and cycle threshold values (c t ) were recorded. basal tlr - mrna levels were expressed as ΔΔc t values relative to s rrna [ΔΔc t = Δc t(tlr) − Δc t( s rrna) ]. tlr mrna expression levels were expressed as fold changes relative to mock values, using the variable −ΔΔct . ifn- α and ifn-β gene expression was quantified as above using specific pre-developed taqman gene expression assays (applied biosystems). expression of cell surface tlr and tlr , and endosomal tlr and tlr was determined using a facscalibur flow cytometer (bd biosciences, san jose, ca) and data were processed using flowjo software (treestar, ashland, or). cells were stained and analyzed by using a fixation & permeabilization kit (ebioscience) following the recommended protocols. specific antibodies against murine tlr , , , and , fluorescence-labeled secondary antibodies, isotype controls, and anti-fcrr (for blocking) were purchased from invivogen, imgenex (san diego, ca), and ebioscience (san diego, ca). gfp expression was analyzed in live or % paraformaldehyde fixed cells. cells were incubated with nm dapi ( ', '-diamidino- -phenylindole dilactate; invitrogen, molecular probes, eugene, or, usa) for nuclei stain for min at rt. images were obtained with an olympus x motorized inverted fluorescence microscope (center valley, pa, usa) using digital microscopy software (slidebook™ . software, intelligent imaging innovations, denver, co). an unpaired two-tail student's t test was used to determine statistical significance. all data were analyzed with graphpad prism (graphpad software, inc., ca, usa) our goal was to investigate the effects of triggering tlr , tlr , tlr and tlr with selected ligands on j a. macrophages susceptibility to mhv infection. our data demonstrates that triggering of tlrs with hklm (tlr agonist), lps (tlr agonist), and r (tlr agonist) does not affect mhv-a , mhv-jhm, and mhv- production. the absence of tlr -, tlr -, and tlr -mediated antiviral effects is not explained by their lack of expression or activation in j a. macrophages; rather, it is based on the weak induction of type i ifn. in contrast, stimulation of macrophages with poly i:c added to the cells to trigger endosomal tlr , induces a strong type i ifn-dependent antiviral response. neutralization of ifn-β successfully restored the poly i:c-inhibited production of mhv-a in macrophages. taken together, activation of tlr by poly i:c may be a successful antiviral approach against covs in vivo; its therapeutic potential as a curative drug remains to be established in future investigations. coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol pathogenesis of murine coronavirus in the central nervous system coronaviruses post-sars: update on replication and pathogenesis coronavirus infection of the central nervous system: host-virus stand-off pathogenesis of acute and chronic central nervous system infection with variants of mouse hepatitis virus, strain jhm the pathogenesis of murine coronavirus infection of the central nervous system murine coronavirus mouse hepatitis virus is recognized by mda and induces type i interferon in brain macrophages/microglia macrophage interleukin- and tumour necrosis factor-alpha are induced by coronavirus fixation to toll-like receptor /heparan sulphate receptors but not carcinoembryonic cell adhesion antigen a autocrine interferon priming in macrophages but not dendritic cells results in enhanced cytokine and chemokine production after coronavirus infection lung pathology of fatal severe acute respiratory syndrome sars coronavirus and innate immunity control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd t cells type i ifn-mediated protection of macrophages and dendritic cells secures control of murine coronavirus infection murine coronavirus cell type dependent interaction with the type i interferon response mouse hepatitis coronavirus a nucleocapsid protein is a type i interferon antagonist deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene induction of type i interferon by rna viruses: cellular receptors and their substrates the role of pattern-recognition receptors in innate immunity: update on toll-like receptors toll-like receptors and their crosstalk with other innate receptors in infection and immunity toll-like receptors: key players in antiviral immunity murine toll-like receptor activation induces type i interferon responses from endolysosomal compartments tram couples endocytosis of toll-like receptor to the induction of interferon-beta toll-like receptor on inflammatory monocytes induces type i interferon in response to viral but not bacterial ligands interferon regulatory factors: the next generation toll-like receptor signaling inhibits hepatitis b virus replication in vivo effect of pretreatment with toll-like receptor agonists in a mouse model of herpes simplex virus type encephalitis activation of toll-like receptor signaling pathway for protection against influenza virus infection a critical function of toll-like receptor- in the induction of anti-human immunodeficiency virus activities in macrophages activation of anti-hepatitis c virus responses via toll-like receptor tlr / triggering exerts opposing effects in acute versus latent hiv infection tlr signaling is either protective or pathogenic for the development of theiler's virus-induced demyelinating disease depending on the time of viral infection activation of toll-like receptor impairs the dengue virus serotype replication through induction of ifn-beta in cultured hepatoma cells decreased dengue replication and an increased anti-viral humoral response with the use of combined toll-like receptor and / agonists in macaques toll-like receptor deficiency increases disease and mortality after mouse hepatitis virus type infection of susceptible c h mice murine coronavirus replication-induced p mitogen-activated protein kinase activation promotes interleukin- production and virus replication in cultured cells master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production accessory protein a is a major antagonist of the antiviral action of interferon against murine coronavirus plp of mouse hepatitis virus a (mhv-a ) targets tbk to negatively regulate cellular type i interferon signaling pathway regulation of interferon and toll-like receptor signaling during macrophage activation by opposing feedforward and feedback inhibition mechanisms tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells evaluation of immunomodulators, interferons and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice a new mouse-adapted strain of sars-cov as a lethal model for evaluating antiviral agents in vitro and in vivo tlr is essential for the induction of protective immunity against punta toro virus infection by the double-stranded rna (dsrna), poly(i:c u), but not poly(i:c): differential recognition of synthetic dsrna molecules mismatched dsrna (ampligen) induces protection against genomic variants of the human immunodeficiency virus type (hiv- ) in a multiplicity of target cells mismatched double-stranded rna (polyi-polyc( )u) is synergistic with multiple anti-hiv drugs and is active against drug-sensitive and drug-resistant hiv- in vitro the interferon inducer ampligen [poly(i)-poly(c u)] markedly protects mice against coxsackie b virus-induced myocarditis hen mouse model for the evaluation of antiviral agents for the treatment of venezuelan equine encephalitis virus infection polyi:polyc u adjuvant-combined intranasal vaccine protects mice against highly pathogenic h n influenza virus variants replicase genes of murine coronavirus strains a and jhm are interchangeable: differences in pathogenesis map to the ' one-third of the genome enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system murine coronavirus evolution in vivo: functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein this article is an open access article distributed under the terms and conditions of the creative commons attribution license the authors would like to thank drs. paul s masters, julian leibowitz, and susan r weiss for reagents. this work was supported in part by public health service grants ns , ai and dk to s.n.m, and ns to j.m.g., from the national institute of neurological disorders and stroke (ns), the national institute of allergy and infectious disease (ai), and the national institute of diabetes and digestive and kidney diseases; and by drexel university college of medicine's internal funds to snm. the founders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. pamela fried (drexel university college of medicine academic publishing services) is acknowledged for manuscript editing. the authors declare no conflict of interest. key: cord- - j zmuub authors: hunt, catherine l.; lennemann, nicholas j.; maury, wendy title: filovirus entry: a novelty in the viral fusion world date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: j zmuub ebolavirus (ebov) and marburgvirus (marv) that compose the filovirus family of negative strand rna viruses infect a broad range of mammalian cells. recent studies indicate that cellular entry of this family of viruses requires a series of cellular protein interactions and molecular mechanisms, some of which are unique to filoviruses and others are commonly used by all viral glycoproteins. details of this entry pathway are highlighted here. virus entry into cells is initiated by the interaction of the viral glycoprotein( ) subunit (gp( )) with both adherence factors and one or more receptors on the surface of host cells. on epithelial cells, we recently demonstrated that tim- serves as a receptor for this family of viruses, but the cell surface receptors in other cell types remain unidentified. upon receptor binding, the virus is internalized into endosomes primarily via macropinocytosis, but perhaps by other mechanisms as well. within the acidified endosome, the heavily glycosylated gp( ) is cleaved to a smaller form by the low ph-dependent cellular proteases cathepsin l and b, exposing residues in the receptor binding site (rbs). details of the molecular events following cathepsin-dependent trimming of gp( ) are currently incomplete; however, the processed gp( ) specifically interacts with endosomal/lysosomal membranes that contain the niemann pick c (npc ) protein and expression of npc is required for productive infection, suggesting that gp/npc interactions may be an important late step in the entry process. additional events such as further gp( ) processing and/or reducing events may also be required to generate a fusion-ready form of the glycoprotein. once this has been achieved, sequences in the filovirus gp( ) subunit mediate viral/cellular membrane fusion via mechanisms similar to those previously described for other enveloped viruses. this multi-step entry pathway highlights the complex and highly orchestrated path of internalization and fusion that appears unique for filoviruses. filoviruses (family mononegavirales, genera ebolavirus (ebov) and marburgvirus (marv)) are single-stranded, negative-sense rna viruses that exhibit a unique heterogeneous filamentous structure. both ebov and marv infect a wide variety of mammals and this wide tropism has complicated the identification of cellular proteins required for viral entry. a hemorrhagic fever is caused by these viruses in humans, non-human primates and perhaps other mammals and is associated with high morbidity and mortality during outbreaks. no therapeutic drugs or vaccines are currently available to treat or prevent filoviral infection. because of this and the high lethality associated with infection, filoviruses are considered category a priority pathogens by niaid and, in recent years, much research has focused on understanding how these viruses bind to and enter permissive cells. the ebov genome encodes seven genes, including the glycoprotein (gp) gene. the gp gene encodes for two known soluble gp forms (sgp and ssgp) [ , ] . the precise functions of the soluble forms of ebov gp are unknown, but it is speculated that they are involved in immune evasion, as most of the antibodies during a natural zaire ebov (zebov) infection are against sgp [ ] . full-length ebov gp is not directly encoded by the gp gene, but is produced by a transcriptional editing event that shifts the reading frame of about % of the gp transcripts. in contrast, while marv also encodes seven genes, its gp is directly encoded by the gp gene and soluble forms of marv gp are not thought to be synthesized from viral transcripts. precursor gp is cleaved by the host enzyme furin in the golgi apparatus, resulting in the formation of two gp subunits, gp and gp . gp contains the receptor binding domain (rbd) and is responsible for interacting with one or more cellular receptors. this interaction is believed to mediate virus entry into the endosomal compartment. gp contains a fusion loop, heptad repeat regions, the transmembrane domain and a short cytoplasmic tail. while furin processing of the filovirus gp routinely occurs within the golgi apparatus before the glycoprotein is expressed on the plasma membrane, proteolytic clipping is not required for virion infectivity [ ] . the cleaved subunits are linked by a disulfide bond to generate a gp , heterodimer that is located on the surface of virions and is approximately kda in size [ ] . as a class i viral fusion protein, gp , exists as a trimer of gp and gp heterodimeric subunits and is found in its full-length, heavily glycosylated, pre-fusion form on the surface of newly budded virions. this pre-fusion class i glycoprotein conformation is thought to be a metastable state, that upon the appropriate set of triggers, will convert to its post-fusion, low-energy form [ ] . the conformational change from a pre-fusion to post-fusion structure provides the energy to permit the viral and cellular membranes to fuse and thereby release the viral core into the cytoplasm. the events that are required for filovirus/cellular membrane fusion to occur have yet to be completely elucidated, but current studies are unraveling these steps and are highlighted here. several ebov gp crystal structures have been instrumental in understanding the conformational alterations that are required as the glycoprotein changes from the metastable, pre-fusion state to the low-energy, post-fusion form. lee et al. elucidated the trimeric structure of pre-fusion, mucin domain-deleted ebov gp , ectodomain [ ] , whereas, two groups independently solved the trimeric gp six helix bundle structure that is formed during fusion events [ , ] . mature gp is composed of three distinct domains: the rbd, the glycan cap and a heavily o-linked glycosylated mucin-like domain. the rbd in mature zebov gp is located from amino acid ~ to and composed of a base region and a region that interacts with one or more receptors on the surface of cells ( figure ) [ ] . while rbds of the different ebov strains are relatively conserved, only % identity between ebov and marv rbds exists. nonetheless, ebov and marv gp pseudovirions compete with each other for filoviral gp , -dependent entry into permissive cells, indicating that a common receptor or receptors are used by both viruses [ , ] . amino acids through and three additional short downstream regions interact with gp , serving as the base of the rbd. several linearly discontinuous regions from amino acids to sit above the base forming a series of beta sheets. residues both in beta sheets and adjacent loops have been implicated in cell binding, leading to the conclusion that the receptor binding site (rbs) is located in this region of the rbd [ , ] . gp residues to encode for a "glycan cap" that is extensively n-linked glycosylated and sits distal to the rbd from the surface of the virion. the glycan cap may protect the rbs from antibodies [ ] . this glycan cap also interacts with two regions of gp , including the internal fusion loop of gp that is critical for gp -mediated membrane fusion [ ] . the glycan cap/gp interaction restricts the availability of the fusion peptide, preventing pre-mature fusion events. finally, filovirus gp contains a mucin-like domain at the carboxy terminus from amino acids to . this region is heavily glycosylated with both n-and o-linked glycans [ ] . while the ebov mucin domain is not required for virus entry [ , ] , several roles for this domain have been suggested. like the glycan cap, it may shield gp rbs residues from immune recognition on free virus [ ] . in addition, the mucin domain causes cell rounding, masking of a number of cell surface markers and cytotoxicity that is not observed upon expression of mucin domain-deleted ebov gp [ , ] . the shielding effect of the bulky mucin domain of the rbd of gp is thought to also obstruct rbs interactions with adherence factors and receptors since removal of this domain enhances ebov titers. similar attempts to delete the marv mucin domain have proved unsuccessful [ ] . two of the three trimers are shown as space filling structures with gp in lighter grey and gp as dark grey/black. the third gp , heterodimer of the trimer is depicted as a ribbon structure with gp shown in teal and the gp subunit shown in tan. (c,d) ribbon diagrams of a single heterodimer of gp , . domains in gp are highlighted in c, whereas domains in gp are highlighted in d. in panel c, the base domain of gp that interacts with gp is shown in royal blue, the head domain is shown in teal with the beta-strands and adjacent loop region containing the rbs highlighted in red and the glycan cap is shown in gold. gp is shown in grey. in panel d, the internal fusion loop (ifl) that is flanked by beta-strands is shown in dark brown and heptad repeat region is shown in tan. the interaction of the ifl with gp residues from an adjacent subunit is evident in panel a. all ebov gp graphics (pdb accession number csy) were produced with pymol. filovirus gp is similar to other class i viral glycoproteins that mediate fusion events. gp is composed of a fusion loop found near the amino terminus followed by a n-terminal heptad repeat region, a c-terminal heptad repeat region, a transmembrane region and a short cytoplasmic tail [ , , ] . in the pre-fusion form on the surface of newly budded virions, the fusion loop and n-terminal heptad region are integral regions of the gp /gp trimeric structure, forming a platform or base upon which gp sits ( figure ). in contrast, the c-terminal heptad repeat region in the pre-fusion form may contain little structure and was deleted in the crystallization studies and it is not thought to contribute to the metastable, pre-fusion form of gp [ ] . not surprisingly, both the fusion loop and n-terminal heptad repeat are conserved (> % identity) between zebov and the musoke strain of marv, but the remaining portions of gp have limited identity. site directed mutagenesis studies of ebov or marv gp [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] suggest a similar chain of events to those previously reported for other class i viral glycoproteins leads to viral/cell membrane fusion [ , , ] . these events will be discussed in detail below. while progress is being made to identify cell surface proteins that enhance filovirus transduction/infection, the advancement of this area of research has been slow. in part this is due to the broad tropism of filoviruses for a variety of different cell types as well as the ability of these viruses to infect cells from a wide range of species [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . one classical approach to identifying virus receptors that has been used is the introduction of a cdna library from a permissive cell into a cell that is not permissive for the virus [ ] [ ] [ ] . however, for reasons that are not entirely clear, this type of study has not been successful in identifying cell surface proteins that directly interact with ebov gp to mediate virus entry [ , ] . instead, another screening approach that correlated gene expression in a large panel of human cells with ebov gp-dependent transduction proved more productive and allowed us to identify a surface receptor for these viruses [ ] . the cell surface protein we identified, tim- , as well as other cell surface proteins that enhance filovirus infectivity are discussed in detail below. c-type lectin family members l-sign, dc-sign and hmgl have been shown to enhance filovirus entry [ , [ ] [ ] [ ] [ ] [ ] . studies have demonstrated that both the mucin domain and the glycan cap of gp interact with c-type lectins [ , ] . however, as both of these regions can be deleted from ebov gp without loss of viral transduction efficiency [ , [ ] [ ] [ ] , it is likely that c-type lectins increase filovirus attachment to cells rather than serving as cellular receptors that mediate internalization of the virus into endosomes [ ] . a similar adherence function for c-type lectins has been identified for other enveloped viruses such as hiv [ ] . early studies found that surface expression of some integrins was down regulated upon transfection of full-length ebov gp [ , , ] . in addition, ebov gp-mediated pseudovirion entry is reduced by either antibodies targeting β integrins or a soluble form of β integrins [ ] . these characteristics suggested that one or more integrin subunits might serve as receptors for filoviruses. however, no direct interaction between any portion of ebov gp and a member of the β integrin family has been identified. more recent studies implicate β integrin in stimulation of endosomal protease events that are required for productive ebov transduction, thus reducing the likelihood of β integrins serving as bona fide filoviral receptors [ ] . the tam family member axl was first implicated in filovirus entry through a cdna screen that introduced vero e cell cdna into poorly permissive jurkat t cells [ ] . axl is a tyrosine kinase receptor that is found on the plasma membrane in a variety of different cell types and enhances cell migration, division and viability upon activation [ ] . shimojima et al. demonstrated that anti-axl antibodies blocked ebov transduction of some cells, whereas these antibodies had no effect on transduction of other cells [ ] . mapping studies indicated that amino acid residues in both the ectodomain and the cytoplasmic tail of axl were required for filovirus entry enhancement [ ] . subsequently, a screen performed in our laboratory also identified axl as being important in ebov gp-dependent entry [ ] . through the use of multiple biochemical inhibitors, sirna and anti-axl antibodies, we defined a role for axl in ebov uptake [ ] , demonstrating that axl expression enhances macropinocytosis in some cells. as macropinocytosis is a principal uptake mechanism of filoviruses [ , ] , increased axl surface expression leads to greater virus internalization. however, axl does not appear to interact directly with ebov gp to promote viral internalization and therefore is unlikely to serve as a filoviral receptor [ ] . our lab performed a comparative genomic analysis (cga) screen [ ] to identify cellular genes whose expression highly correlated with ebov pseudovirion transduction [ ] . this screen showed a positive correlation between ebov transduction and expression of a series of cellular proteins that were previously appreciated to enhance ebov transduction (c-type lectins, integrins and axl). interestingly, expression of the t-cell immunoglobulin mucin domain- (tim- ) gene proved to be one of the strongest positive correlations. subsequent studies demonstrated that expression of tim- in poorly permissive cells enhanced ebov entry and loss of surface-expressed tim- in highly permissive cells abrogated filovirus infection/transduction. furthermore, tim- and the mucin domain-deleted ebov gp interacted, and removal of the glycan cap enhanced the specificity of gp interaction with tim- -expressing cells [ ] . in total, these findings have led us to propose tim- as a cell surface receptor for filoviruses. as epithelial cells are the only relevant cell type that expresses tim- , it is likely that other as of yet unidentified surface receptors will also prove to be important in mediating filovirus entry. the cellular uptake pathways mediating filovirus entry remain controversial despite numerous studies. the three most common and well studied endocytic pathways-caveolin-dependent endocytosis, clathrin-dependent endocytosis and macropinocytosis-have all been implicated in filovirus entry. early studies reported that the caveolae vesicular system and/or lipid raft domains were important for ebov gp-mediated entry [ ] [ ] [ ] . however, it was demonstrated that overexpression of caveolin in the poorly permissive lymphocytic cell line cem did not enhance levels of ebov gp-dependent transduction, suggesting that caveolae may not play a role in filovirus entry [ ] . since that time several groups have also implicated clathrin-dependent entry mechanisms in filovirus entry [ ] [ ] [ ] [ ] . in parallel, other groups using virus-like particles (vlps) and/or infectious filovirions identified macropinocytosis as a main and perhaps sole mechanism of filovirus uptake [ , ] . several groups have found that ebov gp-dependent entry can occur through a variety of different uptake mechanisms including caveolae, clathrin-coated pits and through actin-and microtubule-dependent pathways such as macropinocytosis [ , , ] within the same cell population. a number of these uptake studies that identified caveolin and/or clathrin as important in entry used ebov gp pseudotyped lentiviruses or vesicular stomatitis virus (vsv) as the cargo and an argument has been made that these smaller viral particles do not accurately represent the size constraints that may limit the uptake options of a filovirion that can be greater than µm in length [ ] . while a role for caveolin-and clathrin-dependent pathways in infectious filoviral entry remains to be demonstrated, it should be noted that several large, intracellular bacteria or parasites have been found to use either caveolin-or clathrin-dependent pathways, suggesting that the size of these vesicles can be expanded to meet the size of the cargo [ ] [ ] [ ] . in addition, it is likely that the mechanism of ebov uptake may be cell-dependent as both green african monkey cells (vero) [ , ] and human neuroblastoma cells (snb- ) [ ] both primarily use macropinocytosis for ebov uptake. however, the signaling pathways that are required for virus entry is cell-specific since vero cells require activation of the pi k/akt pathway, whereas snb- cells require plc activation [ ] . unlike some other class i viral glycoproteins, no evidence exists that ebov gp undergoes conformational changes upon binding to a cell surface receptor. presumably receptor/virus interactions lead directly to virus internalization into endosomes; however, this has not been demonstrated directly to date. characterization of the endosomal vesicles mediating ebov uptake is limited. saaed et al. have demonstrated that the early endosome antigen (eea ) colocalizes with ebov virus particles in endosomes early during infection of vero cells [ ] , whereas nanbo et al. have shown association of ebov particles lacking vp associated with the sorting nexin (snx ) within minutes of transduction [ ] . early and late endosomal proteins, rab and rab , respectively, have also been shown to be required for productive infection [ , ] . several gtpases previously implicated in endocytosis, rhob, rac and cdc , are also important for ebov gp-dependent transduction, providing additional insights into trafficking pathways used by these viruses [ , ] . as the vesicles traffic into the cell, they become acidified. for some viral fusion proteins, the combination of receptor engagement and endosomal acidity is sufficient for conformational changes that lead to viral/cellular membrane fusion [ ] [ ] [ ] . however, that is not the case for filoviruses. instead, multiple low ph-dependent endosomal and lysosomal proteolytic proteins are involved in ebov gp processing, priming a multi-step process that ultimately results in a small ebov gp , trimer that serves as the fusion-ready form of the glycoprotein. it is believed that this processed, fusion-ready form is then triggered by additional events to a conformationally stable state, resulting in fusion. as endosomal vesicles mature into late endosomes and the vesicular ph drops, activation of endosomal cysteine proteases cathepsin l and b occurs. these cathepsins sequentially process ebov gp into smaller forms [ , ] . cathepsin l proteolysis first removes a substantial portion of ebov gp , generating an approximate kda gp , species that lacks both the glycan cap and mucin domain of gp [ , ] . subsequently, gp is further trimmed by both cathepsin l and b to generate a much smaller form of gp . the exact size of this smaller gp remains controversial, but is between and kda [ , ] . irrespective of the exact size of the processed form, prevention of endosomal acidification or inhibition of cathepsin b activity abolishes ebov infectivity [ ] [ ] [ ] ] . interestingly, this processing may be insufficient for productive ebov infection as studies have demonstrated that the smaller - kda cathepsin l and b-cleaved form cannot infect cells entirely lacking cathepsins [ ] . thus, it has been proposed that additional cathepsin-dependent gp processing is required to generate the fusion-ready form of the glycoprotein [ ] . the size and composition of this smaller form is not known. in contrast to these studies, a recent study demonstrated that a thermolysintrimmed gp that is believed to generate a gp that is similar to the cathepsin l and b-cleaved form can be triggered to bind to liposomes at elevated temperatures under low ph and mildly reducing conditions [ ] . this new study suggests that at least under these conditions this gp conformation is a fusion-ready form. given the apparent importance of cathepsin cleavage for the generation of a fusion-ready form of the filovirus glycoprotein, it is surprising that studies have demonstrated that cathepsin l and b cleavage events can be sidestepped by the virus [ , ] . martinez et al. have shown that in monocyte-derived dendritic cells, cathepsin b is required for ebov infection, but not cathepsin l [ ] . these observations were confirmed in vero cells in a recent study by wong et al. [ ] . this group also extended the finding by selecting an infectious, recombinant vsv encoding ebov gp over several passages to become resistant to the cathepsin b inhibitor ca [ ] . six different mutants were identified that conferred cathepsin b independence. many of these mutations sit at the interface of gp and gp and the selected mutations were thought to alter the gp structure such that enhanced proteolysis by one or more currently undefined cysteine proteases was possible. thus, while cathepsin b and l independence can be achieved by filoviruses, processing by one or more additional cysteine proteases is still required for production of the fusion-ready form. most recently, two groups independently identified a novel host protein essential for ebov infection. cote et al. screened a library of small chemical molecules to find those that inhibited ebov gp pseudovirion entry into vero cells [ ] , whereas carette et al. utilized a genome-wide screen in mutagenized haploid human cells to look for those cells resistant to ebov gp-dependent entry [ ] . each group was able to deduce that disruption of one endosomal/lysosomal membrane protein, niemann-pick c (npc ), could significantly reduce ebov entry into a variety of cell populations. npc is primarily a membrane bound, late endosomal/lysosomal protein that is critical for cholesterol absorption and homeostasis. those individuals lacking a functional npc exhibit an abnormally high accumulation of cholesterol in the lysosomes of their cells, leading to altered protein and lipid trafficking with most cases resulting in fatality by months of age [ ] . cells where npc function has been biochemically disrupted or cells lacking npc showed a resistance to ebov infection [ , ] . clearance of cholesterol from npc null cells by cultivation in lipoprotein-depleted media did not rescue ebov infection, indicating that the npc protein itself, and not aberrant cholesterol transport, was important for ebov entry [ ] . cote et al. also showed that the expression of npc and not its cholesterol transport activity were critical for ebov entry [ ] . as the npc protein is primarily located on the endosomal and lysosomal membranes, npc has been proposed to serve as an entry factor downstream of ebov gp engagement of attachment factors/receptor(s) at the cell surface. consistent with a vesicular role for npc , cote et al. showed that a soluble form of thermolysin-cleaved ebov gp, but not uncleaved gp containing the glycan cap, bound to lysosomal membranes of npc -transfected cho cells [ ] . thus, at least in this over expression system, proteolytically-processed ebov gp appeared to interact with npc -containing membranes, suggesting that these interactions may be important for filovirus entry events that occur in late endosomes and/or in lysosomes. ebov gp-mediated attachment and entry into early endosomes was unaffected in npc -defective cells; however, electron micrographs of npc null cells infected with ebov gp pseudotyped virus show the accumulation of perinuclear vesicles laden with ebov gp pseudovirions that were positive for the lysosomal marker lamp [ ] . therefore, carette et al. have proposed that ncp is crucial for viral membrane fusion and escape from the lysosomal vesicle [ ] . at present, the precise role of npc during the ebov entry process remains to be fully elucidated. in addition, investigations into the precise location of filovirus fusion events within endosomal compartments will provide important insights into these events. interestingly, an inhibitor of npc , u a, has recently been shown to block entry of several pathogens, including the flavivirus dengue [ ] . this inhibitor also inhibited entry of hepatitis c virus [ ] as well as prions [ ] , suggesting that this cholesterol transporter may be critical for passage of a number of viruses, and perhaps other pathogens, through endosomes. most recently, a more definitive role for npc in hepatitis c virus entry has been determined through the use of additional biochemical agents [ ] . future studies exploring lipid accumulation and changes in lipid composition within the endosomal pathway could significantly enhance the understanding of the novel role of npc specifically in filoviral entry and more generally in endosomal trafficking of a number of enveloped viruses. as described above, the gp portion of ebov gp , allows delivery of the filovirion to an endosome where conditions become progressively more favorable for generating the fusion-ready form of gp , . trimming of gp , by host cathepsins (or artificially by thermolysin) enhances interaction of gp with tim- [ ] and permits npc binding [ ] . whether ebov gp interaction, with either of these molecules, directly mediates gp fusion events remains to be determined. filovirus gp contains an n-terminal internal fusion loop of residues defined by a disulfide linkage between cysteines and [ , ] . a core hydrophobic sequence of amino acids within this loop is thought to insert into host endosomal membranes, initiating membrane fusion events. within the intact gp heterodimer, the hydrophobic, internal fusion loop is flanked by antiparallel β-strands composed of the most n-terminal portion of the internal fusion loop and the n-terminal region of first heptad repeat region. the fusion loop is restrained by gp residues from a neighboring subunit, preventing premature fusion events [ , , ] . cathepsin-dependent processing alone is insufficient to trigger insertion of the fusion loop into liposomes [ , ] . when the ebov internal fusion loop interacts with liposomal membrane mimetics, lipid mixing is promoted with a parallel structural change in the loop [ ] . in a neutral ph, lipid environment, the antiparallel β-strands that flank the fusion loop lose their structure, generating a more alpha helical content with a flattened extended loop structure. under low ph conditions (<ph . ) in the presence of lipids, this flattened loop structure broadens out, forming a more hook-like structure [ ] . it has been previously shown that specific proline residues contained within the central portion of the fusion loop facilitate this lipid membrane interaction [ , , , ] . the insertion of gp into host membranes causes an extension of the gp trimers into an energetically unfavorable state. this also causes the two heptad repeat regions (hrs) within gp to be fully exposed to the physiological conditions within the acidified host endosome, which may aid in further triggering of gp to promote final collapse of the two hrs [ ] . the n-terminal gp heptad repeat region (hr ) is a highly ordered, alpha-helical structure that serves as a platform or base for gp and contains residues that are necessary for interactions with other gp hr s to form the trimeric structure of the pre-fusion gp [ , ] . in the pre-fusion form, hr does not interact with the carboxy terminal heptad repeat region (hr ). within the intact, pre-fusion trimer, hr appears to be disordered and was not included in the crystallized structure [ ] . following insertion of ebov gp fusion loop into the host membrane, the ebov gp trimeric heptad repeats collapse and form a six-helix bundle containing three hr and hr domains. the collapse into a coiled coil structure draws the two membranes into proximity allowing partial fusion (hemifusion) of the viral membrane and the host endosomal membrane [ , ] . hemifusion eventually leads to complete fusion of the viral and host endosomal membranes, and an opening through which the viral rna and its associated proteins can be released into the host cell cytoplasm, where the viral life cycle continues. this complex set of entry events summarized in figure provides numerous potential avenues for the development of antiviral therapies against filovirus infection. these possibilities include: ( ) interfering with adherence to permissive cells by blocking c-type lectin (or other adherence factor) interactions with the filoviral gp; ( ) blocking binding of gp to tim- and/or other cellular receptors that are identified in the future; ( ) preventing virus uptake by blocking macropinocytosis; ( ) interfering with cathepsin activity; ( ) inhibiting availability of npc within the lysosomal compartment and ( ) blocking hr /hr (coiled coil) interactions. it is likely that some of these steps will be more amendable than others to the development of antivirals that have minimal or no cytotoxicity. regardless, elucidation of these entry events provides clear targets for the development of drugs that may prevent both filovirus infection and disease. model for filoviral entry. trimers of filoviral gps on virions interact with both attachment factors (c-type lectins) and receptors (tim- ) on the surface of permissive cells. attachment factors are likely to concentrate virions on cells before receptor engagement and virion internalization by macropinocytosis. macropinocytosis is enhanced by tyrosine kinase receptors such as tam family members. following endosomal acidification, cathepsins l and b trim the ebov gp to a smaller form that needs at least one as yet undetermined factor to elicit gp fusion with host endosomal membranes. this smaller form of gp is able to interact with both tim- and the endosomal portion of the npc protein; however, whether gp and tim- interact within endosomes is not known. the energetically unfavorable insertion of the ebov gp fusion loop into host endosomal membranes (i) is followed by the energetically favorable collapse of ebov gp into a six-helix bundle (ii) allowing for lipid mixing and hemifusion of host and viral membrane lipids (ii). finally, the hemifused host and viral membranes resolve and a complete pore is formed (iii) through which the viral genomic complex passes into the cytoplasm, allowing the viral replication cycle to continue. structure-function analysis of the soluble glycoprotein, sgp, of ebola virus a new ebola virus nonstructural glycoprotein expressed through rna editing ebolavirus glycoprotein structure and mechanism of entry reverse genetics demonstrates that proteolytic processing of the ebola virus glycoprotein is not essential for replication in cell culture biochemical analysis of the secreted and virion glycoproteins of ebola virus structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme structure of the ebola virus glycoprotein bound to an antibody from a human survivor core structure of the envelope glycoprotein gp from ebola virus at . -a resolution crystal structure of the ebola virus membrane fusion subunit, gp , from the envelope glycoprotein ectodomain conserved receptor-binding domains of lake victoria marburgvirus and zaire ebolavirus bind a common receptor characterization of marburg virus glycoprotein in viral entry ebola virus glycoprotein : identification of residues important for binding and postbinding events the primed ebolavirus glycoprotein ( -kilodalton gp , ): sequence and residues critical for host cell binding covalent modifications of the ebola virus glycoprotein identification of the ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury ebola virus glycoprotein toxicity is mediated by a dynamin-dependent protein-trafficking pathway effect of flanking residues on the conformational sampling of the internal fusion peptide from ebola virus functional importance of the coiled-coil of the ebola virus glycoprotein involvement of viral envelope gp in ebola virus entry into cells expressing the macrophage galactose-type c-type lectin permeabilization of the plasma membrane by ebola virus gp distribution of hydrophobic residues is crucial for the fusogenic properties of the ebola virus gp fusion peptide mutational analysis of the putative fusion domain of ebola virus glycoprotein phosphatidylinositol-dependent membrane fusion induced by a putative fusogenic sequence of ebola virus cell entry of enveloped viruses the role of the transmembrane and of the intraviral domain of glycoproteins in membrane fusion of enveloped viruses ebola virus glycoprotein: proteolytic processing, acylation, cell tropism, and detection of neutralizing antibodies the glycoproteins of marburg and ebola virus and their potential roles in pathogenesis ebola virus: new insights into disease aetiopathology and possible therapeutic interventions pathogenesis of ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines distinct cellular interactions of secreted and transmembrane ebola virus glycoproteins a search for ebola virus in animals in the democratic republic of the congo and cameroon: ecologic, virologic, and serologic surveys, - . ebola virus study teams distinct mechanisms of entry by envelope glycoproteins of marburg and ebola (zaire) viruses apoptosis induced in vitro and in vivo during infection by ebola and marburg viruses association of ebola-related reston virus particles and antigen with tissue lesions of monkeys imported to the united states folate receptor alpha and caveolae are not required for ebola virus glycoprotein-mediated viral infection dc-sign and dc-signr bind ebola glycoproteins and enhance infection of macrophages and endothelial cells a putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection characterization of a human gene conferring sensitivity to infection by gibbon ape leukemia virus a receptor for subgroup a rous sarcoma virus is related to the low density lipoprotein receptor tyro family-mediated cell entry of ebola and marburg viruses folate receptor-alpha is a cofactor for cellular entry by marburg and ebola viruses t-cell immunoglobulin and mucin domain (tim- ) is a receptor for zaire ebolavirus and lake victoria marburgvirus c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans the asialoglycoprotein receptor is a potential liver-specific receptor for marburg virus lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus human macrophage c-type lectin specific for galactose and n-acetylgalactosamine promotes filovirus entry dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus modulation of virion incorporation of ebolavirus glycoprotein: effects on attachment, cellular entry and neutralization endosomal proteolysis of the ebola virus glycoprotein is necessary for infection role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity c-type lectins do not act as functional receptors for filovirus entry into cells the multiple facets of hiv attachment to dendritic cell lectins ebola virus glycoproteins induce global surface protein down-modulation and loss of cell adherence downregulation of beta integrins by ebola virus glycoprotein: implication for virus entry alpha beta -integrin controls ebolavirus entry by regulating endosomal cathepsins tam receptor tyrosine kinases: biologic functions, signaling, and potential therapeutic targeting in human cancer the mechanism of axl-mediated ebola virus infection tyrosine kinase receptor axl enhances entry of zaire ebolavirus without direct interactions with the viral glycoprotein the tyro receptor kinase axl enhances macropinocytosis of zaire ebolavirus cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner epidermal growth factor receptor is a co-receptor for adeno-associated virus serotype studies of ebola virus glycoprotein-mediated entry and fusion by using pseudotyped human immunodeficiency virus type virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha lipid raft microdomains: a gateway for compartmentalized trafficking of ebola and marburg viruses association of the caveola vesicular system with cellular entry by filoviruses differential requirements for clathrin endocytic pathway components in cellular entry by ebola and marburg glycoprotein pseudovirions ebola virus uses clathrin-mediated endocytosis as an entry pathway analysis of filovirus entry into vero e cells, using inhibitors of endocytosis, endosomal acidification, structural integrity, and cathepsin (b and l) activity ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis interactions with the host cell macropinocytosis: an endocytic pathway for internalising large gulps vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization role of caveolae in leishmania chagasi phagocytosis and intracellular survival in macrophages rho gtpases modulate entry of ebola virus and vesicular stomatitis virus pseudotyped vectors receptor binding and low ph coactivate oncogenic retrovirus envelope-mediated fusion endocytosis and a low-ph step are required for productive entry of equine infectious anemia virus alpharetrovirus envelope-receptor interactions a forward genetic strategy reveals destabilizing mutations in the ebolavirus glycoprotein that alter its protease dependence during cell entry cathepsin cleavage potentiates the ebola virus glycoprotein to undergo a subsequent fusion relevant conformational change zaire ebola virus entry into human dendritic cells is insensitive to cathepsin l inhibition small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection ebola virus entry requires the cholesterol transporter niemann-pick c niemann-pick type c disease: molecular mechanisms and potential therapeutic approaches u a, an intra-cellular cholesterol transport inhibitor, inhibits dengue virus entry and replication hepatitis c virus egress and release depend on endosomal trafficking of core protein prevention of prion propagation by dehydrocholesterol reductase inhibitors in cultured cells and a therapeutic trial in mice identification of the niemann-pick c -like cholesterol absorption receptor as a new hepatitis c virus entry factor structure and function of the complete internal fusion loop from ebolavirus glycoprotein ollmann saphire, e. ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion structure of the ebola fusion peptide in a membrane-mimetic environment and the interaction with lipid rafts roles of a conserved proline in the internal fusion peptide of ebola glycoprotein we would like to thank sven moller-tank, bethany rhein and andrew kondratowicz for critically reading the manuscript and their helpful suggestions and discussions. the authors declare no conflict of interest. key: cord- -s ci r w authors: andersen, petter i.; krpina, klara; ianevski, aleksandr; shtaida, nastassia; jo, eunji; yang, jaewon; koit, sandra; tenson, tanel; hukkanen, veijo; anthonsen, marit w.; bjoras, magnar; evander, magnus; windisch, marc p.; zusinaite, eva; kainov, denis e. title: novel antiviral activities of obatoclax, emetine, niclosamide, brequinar, and homoharringtonine date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: s ci r w viruses are the major causes of acute and chronic infectious diseases in the world. according to the world health organization, there is an urgent need for better control of viral diseases. repurposing existing antiviral agents from one viral disease to another could play a pivotal role in this process. here, we identified novel activities of obatoclax and emetine against herpes simplex virus type (hsv- ), echovirus (ev ), human metapneumovirus (hmpv) and rift valley fever virus (rvfv) in cell cultures. moreover, we demonstrated novel activities of emetine against influenza a virus (fluav), niclosamide against hsv- , brequinar against human immunodeficiency virus (hiv- ), and homoharringtonine against ev . our findings may expand the spectrum of indications of these safe-in-man agents and reinforce the arsenal of available antiviral therapeutics pending the results of further in vitro and in vivo tests. every year, emerging and re-emerging viruses, such as ebola virus (ebov), marburg virus (marv), and rift valley fever virus (rvfv), surface from natural reservoirs and kill people [ , ] . in addition, influenza a virus (fluav), human immunodeficiency (hiv- ), herpes simplex (hsv), and other viruses regularly infect human population and represent substantial public health and economic burden [ , ] . the world health organization (who) and the united nations (un) have called for better control of viral diseases (https://www.who.int/blueprint/priority-diseases/en/; https://sustainabledevelopment.un.org/). developing novel virus-specific vaccines and antiviral drugs can be time-consuming and costly [ , ] . in order to overcome these time and cost issues, academic institutions and pharmaceutical companies have focused on the repositioning of existing antivirals from one viral disease to another, considering that many viruses utilize the same host factors and pathways to replicate inside a cell [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . broad-spectrum antiviral agents (bsaas) are small-molecules that inhibit a wide range of human viruses. we have recently reviewed approved, investigational and experimental antiviral compounds and identified bsaas, whose pharmacokinetics (pk) and toxicity had been studied in clinical trials [ ] . we tested of these bsaas against human metapneumovirus (hmpv), hepatitis c virus (hcv), cytomegalovirus (cmv), and hepatitis b virus (hbv). we demonstrated novel antiviral effects of azacytidine, itraconazole, lopinavir, nitazoxanide, and oritavancin against hmpv, as well as cidofovir, dibucaine, azithromycin, gefitinib, minocycline, oritavancin, and pirlindole against hcv [ ] . we also tested bsaas, including these , against fluav, rvfv, echovirus (ev ), zikv, chikv, rrv, hiv- and hsv- . we identified novel activities for dalbavancin against ev , ezetimibe against hiv- and zikv, and azacytidine, cyclosporine, minocycline, oritavancin and ritonavir against rvfv [ ] . here, we evaluated the efficacy of bsaas, which do not overlap with agents we tested before. we identified novel in vitro activities of obatoclax and emetine against hsv- , ev , hmpv and rvfv. moreover, we demonstrated novel antiviral effects of emetine against fluav, niclosamide against hsv- , brequinar against hiv- , and homoharringtonine against ev in vitro. to identify potential bsaas, we have reviewed approved and investigational safe-in-man antiviral agents using drug bank and clinical trials websites, respectively. in addition, we reviewed investigational and approved safe-in-man antibacterial, antifungal, antiprotozoal, antiemetic, etc. agents, for which antiviral activities have been reported in pubmed. by excluding vaccines and interferons, we identified molecules that inhibit the replication of several viruses in man [ ] . most recently, novel antiviral activities have been reported for some of these agents [ ] . forty-three compounds were used in this study, and their suppliers and catalogue numbers are summarized in table s . to obtain mm stock solutions compounds were dissolved in dimethyl sulfoxide (dmso, sigma-aldrich, steinheim, germany) or milli-q water. the solutions were stored at − • c until use. madin-darby canine kidney (mdck, american type culture collection (atcc)) and african green monkey kidney epithelial (vero-e , atcc) cells were grown in dulbecco's modified eagle's medium (dmem; gibco, paisley, scotland) supplemented with u/ml penicillin and µg/ml streptomycin mixture (pen/strep; lonza, cologne, germany), mm l-glutamine, and % heat-inactivated fetal bovine serum (fbs; lonza, cologne, germany). human telomerase reverse transcriptase-immortalized retinal pigment epithelial (rpe, atcc) cells were grown in dmem-f medium supplemented with pen/strep, mm l-glutamine, % fbs, and . % sodium bicarbonate (sigma-aldrich, st. louis, usa). ach- cells, which possess a single integrated copy of the provirus hiv- strain lai (nih aids reagent program), were grown in rpmi- medium supplemented with % fbs and pen/strep. tzm-bl cells were grown in dmem supplemented with % fbs and pen/strep. human lung adenocarcinoma epithelial a cells were cultured in dmem medium containing % fbs and pen/strep. a -npro cells (kindly provided by prof. steve goodbourn, university of london), which stably express bvdv npro protein, which inhibits ifn production, were cultured in dmem containing % fbs, pen/strep, and µg/ml puromycin. all cell lines were grown in a humidified incubator at • c in the presence of % co . hsv- strain g was from the atcc. ev (farouk strain; atcc) was from prof. marjomäki (university of of jyväskylä) [ ] . rvfv encoding the far-red fluorescent protein instead of non-structural (ns) protein (rvfv-rfp) was from profs. hartmut hengel and friedemann weber (university medical center freiburg) [ ] . hmpv nl/ / strain, encoding green fluorescent protein (hmpv-gfp), was from vironovative and erasmus mc [ ] . the gfp-expressing influenza a/pr/ -ns -gfp strain (fluav-gfp) was generated by drs. andrej egorov (vienna) [ ] . all the experiments with viruses were performed in compliance with the guidelines of the national authorities using appropriate biosafety laboratories under appropriate ethical and safety approvals. fluav-gfp was amplified in a monolayer of mdck cells in dmem containing pen/strep, . % bovine serum albumin, mm l-glutamine, and µg/ml l- -tosylamido- -phenylethyl chloromethyl ketone-trypsin (tpck)-trypsin (sigma-aldrich, st. louis, usa). hmpv-gfp, rvfv-rfp and the wild-type hsv- strain were amplified in a monolayer of vero-e cells in the dmem medium containing pen/strep, . % bovine serum albumin, mm l-glutamine, and µg/ml tpck-trypsin. ev was amplified in a monolayer of a cells in the dmem media containing pen/strep, . % bovine serum albumin, and mm l-glutamine. for the production of hiv- , × ach- cells were seeded in ml medium. virus production was induced by the addition of nm phorbol- -myristate- -acetate. the cells were incubated for h. the hiv- -containing medium was collected. the hiv- concentration was estimated by measuring the concentration of hiv- p in the medium using anti-p -elisa, which was developed in-house. recombinant purified p protein was used as reference. the virus stocks were stored at − • c. approximately × rpe cells were seeded per well in -well plates. the cells were grown for h in dmem-f medium supplemented with % fbs, and pen/strep. the medium was replaced with dmem-f medium containing . % bovine serum albumin, mm l-glutamine, and µg/ml tpsk-trypsin. the compounds were added to the cells in -fold dilutions at seven different concentrations starting from or µm. saliphenylhalamide, abt- and dmso were added to the control wells. saliphenylhalamide is an inhibitor of cellular vacuolar atpase, which protects cells from virus-mediated death, whereas abt- is an inhibitor of anti-apoptotic bcl- proteins, which facilitates death of cells with viral nucleic acids [ ] [ ] [ ] [ ] [ ] [ ] . rpe cells were infected with hsv- , fluav-gfp, hmpv-gfp or rvfv-rfp viruses at multiplicity of infections (moi) of . , . , . and , respectively. hsv- -infected rpe cells were imaged after h in the phase-contrast mode. rvfv-mediated rfp expression and fluav-mediated gfp expression were visualized after h, whereas hmpv-mediated gfp expression was recorded after h using fluorescent microscopy (zeiss observer z , zaventem, belgium). image j software (v.ij . r, nih) was used to determine fluorescent intensities. rpe cells were treated with bsaas or control compounds as described above and infected with hsv- , ev , fluav-gfp, hmpv-gfp or rvfv-rfp viruses at multiplicity of infections (moi) of . , . , . , . and , respectively. after h of infection, the medium was removed from the cells. the viability of mock-and virus-infected cells were measured using cell titer glow assay (ctg; promega, madison, usa). the luminescence/fluorescence were read with a pherastar fs plate reader (bmg labtech, ortenberg, germany). for testing compound toxicity and efficacy against hiv- , approximately × tzm-bl cells were seeded in each well of a -well plate. tzm-bl cells express firefly luciferase under control of hiv- long terminal repeat (ltr) promoter allowing quantitation of the viral infection (tat-protein expression by integrated hiv- provirus) using firefly luciferase assay. the cells were grown for h in cell growth medium. compounds were added to the cells in three-fold dilutions at seven different concentrations starting from µm. no compounds were added to the control wells. the cells were infected with hiv- (corresponding to ng/ml of hiv- p ) or mock. at hpi, the media was removed from the cells, the cells were lysed, and firefly luciferase activity was measured using the luciferase assay system (promega, madison, wi, usa) and pherastar fs plate reader. in a parallel experiment, cell tox green reagent (ctxg; promega, madison, wi, usa) was added to the cells and fluorescence was measured with a plate reader. the half-maximal cytotoxic concentration (cc ) for each compound was calculated based on viability/death curves obtained on mock-infected cells after non-linear regression analysis with a variable slope using graphpad prism software version . a. the half-maximal effective concentrations (ec ) were calculated based on the analysis of reporter protein expression or the viability/death of infected cells by fitting drug dose-response curves using four-parameter ( pl) logistic function f (x): where f (x) is a response value at dose x, a min and a max are the upper and lower asymptotes (minimal and maximal drug effects), m is the dose that produces the half-maximal effect (ec or cc ), and λ is the steepness (slope) of the curve. a relative effectiveness of the drug was defined as selectivity index (si = cc /ec ). the threshold of si used to differentiate between active and inactive compounds was . rpe cells were treated with combinations of increasing concentrations of obatoclax and emetine. the cells were infected with fluav-gfp at moi . . after h, gfp fluorescence was recorded using fluorescent microscopy. in a parallel experiment, the viability of infected cells was measured using the ctg assay. to test whether the drug combinations act synergistically, the observed responses were compared with expected combination responses. the expected response of the emetine-obatoclax drug combination on the viability of fluav-and mock-infected rpe cells was calculated using bliss reference model [ ] . for testing the production of hsv- and ev viruses in compound-treated and non-treated rpe cells, the media from the cells were serially ( -fold) diluted, starting from − to − in serum-free growth media containing . % bovine serum albumin, and applied to a monolayer of a -npro cells in -well plates. after one hour, cells were overlaid with growth medium containing % carboxymethyl cellulose and % fbs and incubated for h. the cells were fixed and stained with crystal violet dye. the plaques were calculated in each well. the titers were expressed as plaque-forming units per ml (pfu/ml). forty-three safe-in-man bsaas used in this study reached different stages of drug development process (figure , tables s and s ). altogether, these bsaas inhibit the replication of viruses belonging to (−) single-stranded (ss)rna, (+)ssrna, ssrna-reverse transcriptase (rt), ssdna, double-stranded (ds)dna, or dsdna-rt virus groups. we tested bsaas against wild-type hsv- in rpe cells. seven different concentrations of the compounds were added to virus-or mock-infected cells. cell viability was monitored by microscopy and the ctg assay. after the initial screening, we identified four compounds (obatoclax, emetine, niclosamide and ganciclovir) that at none-cytotoxic concentrations rescued cells from virus-mediated death (figure a) . to determine the efficiency and toxicity of these hsv- inhibitors, we measured the viability of mock-and virus-infected cells after h using the ctg assay ( figure b ). the sis for obatoclax, emetine, niclosamide and ganciclovir were , , and > , respectively (table s ) . we titrated hsv- produced from drug-treated and non-treated cells in a -npro cells ( figure c ). the experiment revealed that . µm obatoclax, . µm emetine, . µm niclosamide and . µm ganciclovir lowered the production of hsv- in rpe cells. thus, we identified novel anti-hsv- activities of obatoclax, emetine and niclosamide and confirmed the known anti-hsv- activity of ganciclovir. similarly, we examined bsaas against wild-type ev in rpe cells. we monitored the viability of mock-and virus-infected cells by microscopy and the ctg assay. after the initial screening, we identified three compounds (obatoclax, emetine, and homoharringtonine), which rescued cells from virus-mediated death at none-cytotoxic concentrations. to determine the efficiency and toxicity of novel ev inhibitors, we measured the viability of mock-and virus-infected cells after h using the ctg assay ( figure a ). the sis for obatoclax, emetine, and homoharringtonine were , > , and > , respectively (table s ) . we titrated ev produced from drug-treated and non-treated cells in a -npro cells ( figure b ). the experiment revealed that all three compounds lowered the production of ev , confirming novel antiviral activities of obatoclax, emetine, and homoharringtonine. we also examined the toxicity and antiviral activity of bsaas against hiv- -mediated firefly luciferase expression. the firefly luciferase open reading frame is integrated into the genome of tzm-bl cells under the hiv- ltr promoter. our primary screen identified two compounds (brequinar and suramin) that suppressed hiv- -mediated firefly luciferase expression without detectable cytotoxicity. we performed a validation experiment with anti-hiv- compounds and calculated selectivity. the si for brequinar was > , whereas the si for suramin was > (figure ; table s ). thus, we identified novel activity of brequinar and confirmed known activity of suramin. in addition, we examined the toxicity and antiviral activity of bsaas against rfp-expressing rvfv in rpe cells. both fluorescent microscopy and the ctg assay showed that obatoclax and emetine inhibited rvfv-mediated rfp expression at non-cytotoxic concentrations ( figure a-c) . the si for obatoclax was > , and the si for emetine was > (table s ). next, we tested bsaas against gfp-expressing hmpv. hmpv-mediated gfp expression and cell viability were measured after h. after the initial screening, we identified two compounds, obatoclax and emetine, which lowered gfp expression without detectable cytotoxicity. replicate experiments confirmed these hits ( figure a-c) . the si for obatoclax was six and for emetine was (table s ). next, we tested bsaas against gfp-expressing fluav in rpe cells. after the initial screening, we identified two compounds, obatoclax and emetine, which lowered gfp expression without detectable cytotoxicity. we repeated the experiment with these compounds and confirmed initial hits ( figure a -c). the si for obatoclax was and for emetine was > (table s ) . thus, we identified novel anti-fluav activity of emetine and confirmed known activity of obatoclax. to test whether the obatoclax-emetine combinations act synergistically against fluav-mediated gfp expression and rescue infected cells from death, the observed responses were compared with expected combination responses. the deviations in observed and expected responses showed no synergistic effect ( figure d ,e; figure s ). this result indicates that obatoclax and emetine target distinct cellular pathways essential for virus infection. here, we tested safe-in-man bsaas against (−)ssrna, (+)ssrna, rt-ssrna and dsdna viruses and identified novel activities for five agents (table , figure s ). we identified novel activities of niclosamide against hsv- , brequinar against hiv- , homoharringtonine against ev , obatoclax against hsv- , ev , hmpv and rvfv, and emetine against hsv- , ev , hmpv, rvf and fluav. we also confirmed antiviral activities of ganciclovir against hsv- , suramin against hiv- , and obatoclax against fluav [ , , ] . our results pointed out that an evasion mechanism observed in one virus could be relevant for other viruses and that existing bsaas could be re-positioned to other viral infections. obatoclax was originally developed as an anticancer agent. several phase ii clinical trials were completed that investigated the use of obatoclax in the treatment of leukemia, lymphoma, myelofibrosis, and mastocytosis. in addition, its antiviral activity was reported against fluav, zikv, wnv, yfv, sinv, junv, lasv, and lcmv in vitro [ , , , ] . it was shown that obatoclax inhibited viral endocytic uptake by targeting cellular induced myeloid leukemia cell differentiation protein mcl- [ ] . given that obatoclax also inhibits rvfv, ev , hmpv and hsv- , it could be pursued as a potential bsaa candidate. emetine is an anti-protozoal drug. it is also used to induce vomiting. in addition, it possesses antiviral effects against zikv, ebov, rabv, cmv, hcov-oc and hiv- [ ] [ ] [ ] [ ] [ ] . it was proposed that emetine can directly inhibit viral polymerases, though it may have some other targets as well [ ] . given that emetine also inhibits fluav, rvfv, ev , hmpv and hsv- , it may represent a promising bsaa candidate. niclosamide is an orally bioavailable anthelmintic drug and potential antineoplastic agent. in addition, it inhibits the broadest range of viruses, including hsv- , in vitro and, in some cases, in vivo [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it was shown that niclosamide induces endosomal neutralization and prevents virus entry into host cells. this supports the further development of niclosamide as a bsaa. homoharringtonine is an anticancer drug which is indicated for treatment of chronic myeloid leukemia. it also possesses antiviral activities against hbv, mers-cov, hsv- and vzv [ ] [ ] [ ] [ ] . homoharringtonine binds to the s ribosome and inhibits viral protein synthesis by interfering with chain elongation [ ] . given that homoharringtonine also inhibits ev , it may represent a promising bsaa candidate. brequinar is an investigational anticancer agent (phase i/ii). brequinar attenuates the replication of denv, wnv, yfv, lasv, junv, lcmv, vsv, hiv- , and powv (nct ) [ , ] . it inhibits dihydroorotate dehydrogenase, thereby blocking de novo pyrimidine biosynthesis, which is essential for the transcription and replication of viral rna. given that brequinar also inhibits hiv- , it may represent a promising bsaa candidate. the human non-malignant rpe cell line represents an excellent model system for studying the infection of different viruses [ , , , ] . in addition, different viral strains expressing reporter proteins, such as rvfv-rfp, hmpv-gfp and fluav-gfp, are excellent tools for drug screening [ , , , ] . however, the number of novel and confirmed antiviral activities of bsas could be higher if we had used other cell lines and viral strains, different virus loads, different measurement endpoints, a different time of compound addition, as well as a higher purity and concentration range of bsaas. moreover, antiviral properties of bsaas detected in cell-line-based assays might not be reproduced in vivo because systemic mechanisms may compensate the blocked target effect. thus, follow-up studies are needed to validate our initial hits. altogether, we expanded the spectrum of antiviral actions of niclosamide, brequinar, homoharringtonine, obatoclax and emetine in vitro. importantly, pk and safety studies have been performed on these compounds in laboratory animals and humans. this information could be used to initiate efficacy studies in vivo, saving time and resources. the most effective and tolerable bsaas or their combinations will have a global impact, improving the preparedness and protection of the general population from emerging and re-emerging viral threats and the rapid management of drug-resistant strains, as well as being used for first-line treatment or for prophylaxis of viral co-infections. bsaas could have a pivotal role in the battle against emerging and re-emerging viral diseases. the development of novel bsaas could save time and resources which are required for the development of their alternatives-virus-specific drugs and vaccines. in future, bsaas could have a global impact by decreasing morbidity and mortality from viral and other diseases, maximizing the number of healthy life years, improving the quality of life of infected patients, and decreasing the costs of patient care. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . table s : compounds, their suppliers and catalogue numbers; table s : developmental status of broad-spectrum antivirals used in the study; table s : human viruses and associated diseases; figure s : the expected response of the emetine-obatoclax drug combination on the viability of fluav-and mock-infected rpe cells, as measured with the ctg assay using the bliss reference model; figure s available online: wwwwhoint/medicines/ebola-treatment/who-list-of-top-emerging-diseases/en emerging virus diseases: can we ever expect the unexpected? emerg. microbes infect global, regional, and national incidence, prevalence, and years lived with disability for diseases and injuries for countries and territories, - : a systematic analysis for the global burden of disease study global, regional, and national disability-adjusted life-years (dalys) for diseases and injuries and healthy life expectancy (hale) for countries and territories, - : a systematic analysis for the global burden of disease study approved antiviral drugs over the past years infectious disease. combating emerging viral threats srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target experimental drugs poised for use in ebola outbreak an off-target effect of bx blocks herpes simplex virus type infection of the eye repurposing of kinase inhibitors as broad-spectrum antiviral drugs the future of antivirals: broad-spectrum inhibitors approaches for identification of hiv- entry inhibitors targeting gp pocket drug repurposing: progress, challenges and recommendations editorial: drug repositioning: current advances and future perspectives review of drug repositioning approaches and resources expanding the activity spectrum of antiviral agents common nodes of virus-host interaction revealed through an integrated network analysis. front novel activities of safe-in-human broad-spectrum antiviral agents internalization of echovirus in caveolae t rna polymerase-dependent and -independent systems for cdna-based rescue of rift valley fever virus an improved plaque reduction virus neutralization assay for human metapneumovirus rescue of influenza virus expressing gfp from the ns reading frame antiviral properties of chemical inhibitors of cellular anti-apoptotic bcl- proteins saliphenylhalamide, and gemcitabine inhibit influenza a virus infection anticancer compound abt- accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism the proton translocation domain of cellular vacuolar atpase provides a target for the treatment of influenza a virus infections inhibition by cellular vacuolar atpase impairs human papillomavirus uncoating and infection synergyfinder: a web application for analyzing drug combination dose-response matrix data the anti-parasitic drug suramin potently inhibits formation of seminal amyloid fibrils and their interaction with hiv- ]methyl)guanine: a selective inhibitor of herpes group virus replication the reframe library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors obatoclax inhibits alphavirus membrane fusion by neutralizing the acidic environment of endocytic compartments high-throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry efficacy and mechanism of action of low dose emetine against human cytomegalovirus natural plant alkaloid (emetine) inhibits hiv- replication by interfering with reverse transcriptase activity emetine inhibits replication of rna and dna viruses without generating drug-resistant virus variants identification of broad-spectrum antiviral compounds by targeting viral entry arbidol and other low-molecular-weight drugs that inhibit lassa and ebola viruses the antiparasitic drug niclosamide inhibits dengue virus infection by interfering with endosomal acidification independent of mtor niclosamide rescues microcephaly in a humanized in vivo model of zika infection using human induced neural stem cells niclosamide inhibits lytic replication of epstein-barr virus by disrupting mtor activation antiviral activities of niclosamide and nitazoxanide against chikungunya virus entry and transmission identification of three antiviral inhibitors against japanese encephalitis virus from library of pharmacologically active compounds niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects thiazolides as novel antiviral agents. . inhibition of hepatitis c virus replication inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide anti-varicella-zoster virus activity of cephalotaxine esters in vitro the natural compound homoharringtonine presents broad antiviral activity in vitro and in vivo a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs effect of cantharidin, cephalotaxine and homoharringtonine on "in vitro" models of hepatitis b virus (hbv) and bovine viral diarrhoea virus (bvdv) replication characterization of dengue virus resistance to brequinar in cell culture we thank jelena kiprovskaja and laivi karu for ordering consumables and handling mtas. we thank andres merits for creating excellent infrastructure to work with viruses at tuit. the authors declare no conflict of interest. key: cord- -cfv qn authors: paillot, romain title: special issue “equine viruses”: old “friends” and new foes? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: cfv qn the food and agriculture organization of the united nations has recently estimated that the world equid population exceeds million (faostat ).[...]. the food and agriculture organization of the united nations has recently estimated that the world equid population exceeds million (faostat ) . working equids (horses, ponies, donkeys, and mules) remain essential to ensure the livelihood of poor communities around the world. in many developed countries, the equine industry has a significant economical weight, with around million horses in europe alone. the close relationship between humans and equids, and the fact that the athlete horse is the terrestrial mammal that travels the most worldwide after humans, are important elements to consider in the transmission of pathogens and diseases, amongst equids and to other species. the potential effect of climate change on vector ecology and vector-borne diseases is also of concern for both human and animal health. with this special issue, which assembles a collection of communications, research articles, and reviews, we intend to explore our understanding of a panel of equine viruses, looking at their pathogenicity, their importance in terms of welfare and potential association with diseases, their economic importance and impact on performance, and how their identification can be helped by new technologies and methods. beyond their potential risk to other species, including humans, equine viruses may also represent an interesting model for reproducing virus infection in the host species. dennis et al. [ ] contributed a review on african horse sickness (ahs). this disease, caused by the orbivirus ahs virus (ahsv), induces a very high mortality rate that can exceed % in its most severe form. this disease mostly occurs in southern african countries, but its transmission by culicoides biting midges is of great concern in the current context of global warming and its consequence on displacement of vector populations. in the absence of treatment, prevention is essential, and dennis et al. also provide a comprehensive review of the different vaccine strategies and technologies available and in current development against ahsv. while live attenuated and inactivated ahsv vaccines have played a role to reduce the impact and occurrence of ahs in affected areas, the number of ahsv serotypes in circulation and their lack of diva markers (differentiating infected from vaccinated animals) is a drawback that leads to the development of a new generation of vaccines, such as poxvirus-vectored or reverse genetics vaccines. lecollinet et al. [ ] reviewed major viruses inducing encephalitis in equids and their growing importance as a threat to the european horse population. amongst them, equid herpesviruses (ehvs) are some of the most frequently isolated equine viruses worldwide. the equine herpesvirus type (ehv- ) is of particular interest to the equine industry because of the different forms of disease it can induce, from a mild respiratory infection to abortion, neonatal death, and myeloencephalopathy (ehm) [ ] . a communication from preziuso et al. [ ] and an article from sutton et al. [ ] specifically focused on ehv- strain characterization in order to better understand ehv circulation in italy and france. different approaches were compared, from the single-nucleotide point (snp) mutation in orf (historically associated with abortive or neuro-pathogenic strains), to other orf gene sequences and the newly described multilocus strain typing methods (mlst; [ ] ). the mlst method is an interesting new approach for ehv- and a potential epidemiological tool that could provide an alternative until the development of more accessible ehv whole-genome sequencing methods. ehv- strain characterization by sutton et al. allowed to conclude that the surge of ehv- outbreaks reported in france in was not linked to the introduction and/or circulation of a new ehv- strain in the french horse population. the origin of this crisis could be linked to a shortage of ehv vaccine and a subsequent reduced rate of ehv vaccination in the preceding years [ ] . lecollinet et al. also reviewed less frequently isolated encephalitis viruses, which may be zoonotic, such as rabies virus, borna disease virus, and west nile virus (wnv). in the case of wnv, both horses and humans are highly susceptible to viral infection through infected mosquitoes. unprecedented circulations of wnv have been observed in several european countries in the last decade, with a potential role of climatic and environmental conditions. both species are considered as dead-end hosts. however, the horse could be used as a sentinel species to monitor and control vector-borne virus activity. other enzootic flaviviruses were also reviewed, such as tick-borne encephalitis virus (tbev) and louping ill virus (liv). in europe, vaccination is only available against some of these pathogens (i.e., ehv- and , rabies virus, and wnv), which highlights the importance of surveillance. taking into account that most of these viruses will induce similar clinical signs of disease, the development of discriminative diagnostic tools is also of increasing importance. finally, the review presents some other vector-borne (mosquitoes or midges) equine encephalitis viruses, not currently circulating in europe, from the flaviridae family (i.e., the japanese encephalitis virus (jev), saint louis encephalitis virus (slev), and murray valley encephalitis virus (mvev)) or the alphaviruses from the togaviridae family (i.e., eastern, western, and venezuelan equine encephalitis viruses; eeev, weev, and veev, respectively). in relation to veev, rusnak et al. [ ] presented systematic approaches for strain selection and propagation of virus and challenge material for the development and approval of a veev vaccine under the fda animal rule and the different animal models available (rodents and non-human primates). altan et al. [ ] have used metagenomics to identify viruses in horses with neurological and respiratory diseases. the equine hepacivirus (eqhv) was detected in the plasma from several neurological cases. this virus, which was first reported in horses in [ ] , was further investigated by badenhorst et al. [ ] , with a specific focus on its circulation in austria and the potential role of mosquitoes in its transmission. the prevalence of eqhv in the austrian horse population studies reached % (based on serological evidence), with around % of samples positive for eqhv rna. no eqhv rna was found in mosquitoes collected across austria, raising questions about its methods of transmission. some aspects of this particular question of eqhv transmission were treated by pronost et al. [ ] , who presented evidence to support a potential in utero transmission of eqhv from the mare to the foal, based on three positive clinical cases amongst dead foals screened for the presence of eqhv rna (prevalence of . %). altan et al. [ ] also detected two copiparvoviruses, the equine parvovirus-hepatitis (eqpv-h) and a new one named eqcopivirus by the authors, with no specific and/or statistical association with disease. equine parvovirus-hepatitis was also the subject of the article from meister et al. [ ] , which reported an eqpv-h infection occurrence in a quarter of the actively breeding thoroughbred horse population from northern and western germany. eqpv-h prevalence reached % and % (eqpv-h dna positive detection and seroconversion, respectively). this study concerned mostly thoroughbred brood mares, which represented % of the analyzed cohort. concerning thoroughbred stallions, li et al. [ ] identified a new equine papillomavirus (ecpv ) in the semen from an australian thoroughbred stallion suffering from a genital wart. the clinical significance of this new equine papillomavirus remained to be determined and will require further investigation. a similar question was raised by nemoto et al. [ ] , who reported the first detection of equine coronavirus (ecov) in irish equids suffering from diarrhea. at five occasions, ecov rna was detected in feces from more than equids with enteric diseases. however, the association with disease remains to be substantiated. while ecov prevalence in irish equids was . % when measured by rrt-pcr in feces samples, evidence of ecov infection was significantly higher when measured by serology in serum samples from dutch horses, serums from icelandic horses, and paired serum samples from an ecov outbreak in the usa. zhao et al. [ ] developed and validated an s -protein-based elisa for this purpose. seroprevalence ranged from % in young horses to nearly % in adults. the authors highlighted the potential use of this elisa as a diagnostic test to confirm ecov outbreaks, as a complement to feces samples analysis by qrt-pcr. the study from back et al. [ ] shed some light on the potential role of equine rhinitis a virus (erav) infection in poor performance. this longitudinal study, which involved thoroughbred racehorses, significantly associated seroconversion to erav and subsequent failure to attend races. however, similarly to eqpv-h and ecov infections previously reported in this special issue, a direct association of erav infection with clinical signs of disease could not be confirmed in this study. finally, fatima et al. [ ] investigated the antiviral activity of the equine interferon-mediated host factors myxovirus (mx) protein (eqmx ) against a range of influenza a viruses (iavs). the authors highlight the potential protective role of eqmx , which primarily targets the virus nucleoprotein (np), against the transmission of new iavs in horses (i.e., eqmx could only inhibit the polymerase activity of iavs of avian and human origin but remained inactive against the equine iavs tested). introduction of a new iav in the equine population is considered a rare event. in , an equine influenza epizootic was reported in the jilin and heilongjiang provinces of northeastern china, with up to % mortality, which is quite high when compared with conventional equine influenza outbreaks. the iav strain representative of this outbreak (i.e., a/equine/jilin/ / ) was closely related to an avian h n iav [ ] . the authors show that the iav strain a/equine/jilin/ / bears two adaptive np mutations that confer resistance to eqmx . to date, equine influenza virus remains one of the most important respiratory pathogens of horses worldwide, with a potential damaging impact on the equine industry, as clearly illustrated in in australia and in in europe [ , ] . we hope this special issue helps to highlight the diversity of equine viruses and their importance, in terms of welfare and/or economic impact, to equids and humans. african horse sickness: a review of current understanding and vaccine development viral equine encephalitis, a growing threat to the horse population in europe? viruses ehv- : a constant threat to the horse industry equid alphaherpesvirus from italian horses: evaluation of the variability of the orf , orf , orf and orf genes molecular surveillance of ehv- strains circulating in france during and after the major molecular characterisation of equine herpesvirus isolates from cases of abortion, respiratory and neurological disease in ireland between equine vaccines: how, when and why? report of the vaccinology session, french equine veterinarians association approach to strain selection and the propagation of viral stocks for venezuelan equine encephalitis virus vaccine efficacy testing under the animal rule viruses in horses with neurologic and respiratory diseases serology-enabled discovery of genetically diverse hepaciviruses in a new host no evidence of mosquito involvement in the transmission of equine hepacivirus (flaviviridae) in an epidemiological survey of austrian horses further evidence for in utero transmission of equine hepacivirus to foals characterization of equine parvovirus in thoroughbred breeding horses from germany identification of a novel equine papillomavirus in semen from a thoroughbred stallion with a penile lesion the first detection of equine coronavirus in adult horses and foals in ireland development and validation of a s protein-based elisa for the specific detection of antibodies against equine coronavirus equine rhinitis a virus infection in thoroughbred racehorses-a putative role in poor performance? viruses equine mx restricts influenza a virus replication by targeting at distinct site of its nucleoprotein characterization of a new avian-like influenza a virus from horses in china the use of a recombinant canarypox-based equine influenza vaccine during the australian outbreak: a systematic review and summary success and limitation of equine influenza vaccination: the first incursion in a decade of a florida clade equine influenza virus that shakes protection despite high vaccine coverage acknowledgments: i wish to express my sincere thanks to all authors for their contribution to the special issue "equine viruses". i am also pleased to acknowledge all the editorial office team from viruses for their great help and support and all reviewers involved in the peer-review process. the author declares no conflict of interest. viruses , , key: cord- -ssnvr nj authors: berry, michael; gamieldien, junaid; fielding, burtram c. title: identification of new respiratory viruses in the new millennium date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ssnvr nj the rapid advancement of molecular tools in the past years has allowed for the retrospective discovery of several new respiratory viruses as well as the characterization of novel emergent strains. the inability to characterize the etiological origins of respiratory conditions, particularly in children, led several researchers to pursue the discovery of the underlying etiology of disease. in , this led to the discovery of human metapneumovirus (hmpv) and soon following that the outbreak of severe acute respiratory syndrome coronavirus (sars-cov) promoted an increased interest in coronavirology and the latter discovery of human coronavirus (hcov) nl and hcov-hku . human bocavirus, with its four separate lineages, discovered in , has been linked to acute respiratory tract infections and gastrointestinal complications. middle east respiratory syndrome coronavirus (mers-cov) represents the most recent outbreak of a completely novel respiratory virus, which occurred in saudi arabia in and presents a significant threat to human health. this review will detail the most current clinical and epidemiological findings to all respiratory viruses discovered since . viral infections of the upper and lower respiratory tract are among the most common illness in humans. children and infants bear the major burden of infection, typically presenting with to episodes annually [ ] . these infections are often associated with significant patient morbidity and related mortality. for this reason, urtis and lrtis represents the leading cause of death in children younger than five years of age worldwide [ , ] ; this accounts for approximately million deaths annually [ ] . acute respiratory tract disease is the leading cause of hospitalization in children and febrile episodes in infants younger than three months of age [ , ] . bacteria only represent approximately % of all upper respiratory tract infections with the subsequent % of infections caused by respiratory viruses [ ] . despite the viral aetiological origin of most respiratory infections, antibiotics are often prescribed in the treatment of such diseases [ ] , exacerbating antibiotic abuse. the morbidity and fiscal implications associated with respiratory infections are significant, with approximately million cases reported in the united states alone each year with subsequent direct and indirect costs to the us economy estimated at $ billion annually [ ] . the burden of respiratory tract infections is increased in patients with chronic comorbidities or clinical risk factors including asthma [ ] , chronic obstructive pulmonary disease (copd) [ ] , young, elderly [ ] and immunocompromised [ , ] . the viruses primarily associated with upper respiratory tract infections commonly include rhinoviruses, enteroviruses, adenoviruses, parainfluenza viruses (piv), influenza viruses, respiratory syncytial viruses (rsv) and coronaviruses [ , , , ] . in recent years six new human respiratory viruses have been reported including human metapneumovirus (hmpv) [ ] , bocavirus and four new human coronaviruses including severe acute respiratory syndrome coronavirus (sars-cov), human coronavirus nl (hcov-nl ), hcov-hku and middle east respiratory syndrome coronavirus (mers-cov). this review will detail these newly discovered and emerging respiratory viruses. coronaviruses affect a diverse group of animal hosts, and cause a plethora of diseases in animals including progressive peritonitis, acute and chronic hepatitis, gastroenteritis, nephritis, and encephalitis [ ] . in humans coronavirus infection results in respiratory tract complications with varying degree of severity and have been associated with gastroenteritis. four human coronaviruses (hcov- e, hcov-oc , hcov-nl and hcov-hku ) are endemic in the human population and are mainly associated with mild, self-limiting respiratory illnesses. another two human coronaviruses, namely sars-cov and mers-cov cause severe respiratory syndromes and present a significant threat with their high fatality rates. the first human coronaviruses were identified in the s by tyrell and bynoe who passaged a virus, named b , in human embryonic tracheal organ cultures. when this virus was inoculated intranasally into human volunteers, common cold-like symptoms were produced [ , ] . hamre and procknow ( ) later isolated a virus, which they were able to grow in tissue culture, from subjects presenting with symptoms of the common cold. the virus was later named human coronavirus e (hcov- e) [ ] . seroepidemiological studies have shown that % of wild animal traders and % of people responsible for the slaughtering of animals in the region where human sars was thought to originate, were seropositive for sars-cov, although all cases were asymptomatic. this indicates that these people were previously exposed to a sars-like-cov, which resulted in asymptomatic infection [ ] . sars presents as an atypical pneumonia [ , ] , with pneumocytes being the primary target of infection. infection results in haemorrhagic inflammation in most pulmonary alveoli with alveolar thickening, diffuse alveolar damage, desquamation of pneumocytes, formation of hyaline membranes and multinucleated pneumocytes with capillary engorgement and microthrombosis [ ] . approximately % of patients deteriorated in the second week of infection, presenting with persistent fever, dyspnoea and oxygen desaturation [ ] . approximately %- % of patients were subsequently admitted to intensive care, where mechanical ventilation was necessary [ ] . a surprising finding with the sars outbreak was that it was not as great a threat to infants and children [ , ] . clinical presentation was less severe in infants and no children aged between and required intensive care or mechanical ventilation [ , ] . this is in sharp contrast to the age related burden of other respiratory infections and the underlying biological mechanism remains unclear [ ] . a family of viruses that were previously understood to cause mild, self-limiting upper respiratory tract infections was showcased by the sars-cov outbreak to be a significant threat to global public health. the economic losses brought on by the sars pandemic was estimated to be in the region billion dollars [ ] with hong kong bearing a significant proportion of the losses. tourism, entertainment and restaurant industries in the area recorded up to % loss in business. the pressure on healthcare facilities in affected areas was substantially exacerbated by the spread to healthcare workers. several hospitals were forced to close to new admissions as large numbers of staff became infected with sars-cov. to view this as an isolated incidence would be naï ve and the potential for the emergence and re-emergence of new and existing infectious agents poses a probable risk. understanding the sars-cov outbreak has provided immense knowledge and an excellent model to replicate in the event of further outbreaks of communicable diseases. in a month old child presenting with bronchiolitis and conjunctivitis was screened for several respiratory viruses to identify the causative agent, with all diagnostics yielding negative results. the group led by lia van der hoek then used a modified cdna amplified restriction fragment-length polymorphism (cdna-aflp) technique (virus-discovery-cdna-aflp or vidisca), to identify the causative agent. briefly, the technique utilizes reverse transcription-pcr of viral rna with subsequent restriction digest of the cdna using frequently cutting restriction enzymes. since the restriction sites are selected and therefore known, the resultant "sticky ends" can be ligated into anchors for amplification and sequencing with specific primers. the results showed highest sequence similarities with known coronaviruses, but with significant sequence divergence indicating the discovery of a new coronavirus species, later named human coronavirus nl [ ] . at about the same time, two other independent groups identified essentially the same virus [ , ] . shortly after the van der hoek paper [ ] , a novel coronavirus that replicated efficiently in tertiary monkey kidney and vero cells, was retrospectively isolated from a nose swab sample collected in from an month-old boy presenting with pneumonia. this virus was reported to be similar to hcov-nl and named hcov-nl [ ] . in , also reported the identification of a novel coronavirus isolated in new haven, connecticut, which was named hcov-nh. this novel virus was identified by pcr which was adapted to amplify a conserved region within the replicase a or pol gene [ ] . subsequent sequence analysis showed that these three viruses were essentially the same virus or variants thereof [ , ] . it is obvious that hcov-nl has been circulating in the human population since before [ ] . in fact, molecular clock analyses have shown that hcov-nl and hcov- e diverged from a most recent common ancestor, in a zoonotic event, approximately to years ago [ ] . hcov-nl later diverged into two lineages with subsequent recombination of the two lineages during co-infection. this frequent recombination has given hcov-nl a mosaic structured genome with multiple recombination sites [ ] . a year old male patient from china was admitted to hospital with pneumonia in january . viral cultures, rt-pcr and direct antigen detection from nasopharyngeal aspirate were all negative for respiratory viruses. a pan-coronavirus rt-pcr targeting a conserved region of the pol gene confirmed the presence of a coronavirus however attempts to culture the causative agent were all unsuccessful. sequencing the gene segment amplified by the pan-coronavirus assay indicated a high homology to other viruses of the βcov genus including hcov-oc but of novel origin. the human coronavirus, termed hcov-hku , was later isolated from another female patient. efforts to culture the virus had posed complicated and the complete genome was isolated, amplified and sequenced directly from rna extracted from a nasopharyngeal aspirate [ ] . successful propagation of the virus was achieved recently in human ciliated airway epithelial cells [ ] but culturing of hcov-hku still remains a daunting task. the presence of the he gene further characterizes hcov-hku as belonging to the βcov genus. since the discovery of hcov-hku , its prevalence has shown a global distribution and retrospective analysis on stored nasopharyngeal aspirates have confirmed its existence since [ ] ; however, phylogenetic analysis suggests a much earlier divergence. june saw the most recent emergence of a completely novel strain of human coronavirus. a sputum sample was collected from a year old male patient presenting with severe respiratory disease in the dr. soliman fakeeh hospital in jeddah, saudi arabia. viral assays frequently used could not identify any aetiological agent responsible for the disease. the sputum sample was sent to dr. ron fouchier at the erasmus medical college in rotterdam, netherlands, where the virus was identified as a novel coronavirus, provisionally termed hcov-emc (human coronavirus erasmus medical college). the patient later succumbed to the disease with acute pneumonia and subsequent renal failure [ ] . a retrospective study further traced the virus back to april where an outbreak of pneumonia, resulting in two fatalities, occurred in health care workers in an intensive care unit in zarqa, jordan [ ] . since the initial discovery, several new isolates were identified and described in scientific literature, databases and media under various names. this provoked the convening of the coronavirus study group to introduce a naming convention and avoid confusion within the research field, health care authorities, governments and general public. the name middle east respiratory syndrome coronavirus (mers-cov) was coined and has been widely accepted by the discoverers of the virus and pioneers of the field, the who and saudi ministry of health [ ] . primary infections, to which all cases were linked directly or indirectly, occurred in middle eastern countries including saudi arabia, qatar, jordan, oman and united arab emirates and subsequently spread to united kingdom, tunisia, france, italy and germany with egypt and the united states recently reporting their first laboratory confirmed cases [ , ] . the potential of widespread pandemic outbreak is thought to be low as human-to-human transmission is largely inefficient [ ] and only reported in close, sustained contact [ ] such as within families [ ] , healthcare workers [ ] and as nosocomial transmission [ ] , especially if the patient represents with comorbidities. there has been no evidence to suggest sustained community transmission [ ] . the ever increasing case numbers and countries affected by the disease however suggests mers-cov does represent potential for widespread, global outbreak [ ] . from the first reported case in june , until february , a total of cases had been reported with fatalities. according to the latest figures available from the who, as of june , the virus had resulted in a total of laboratory confirmed cases with a total of fatalities [ ] . these statistics indicate that within a month period case numbers had more than trebled suggesting the virus is far from controlled. the fatality rate of % is also substantially increased in patients with comorbidities, with a large number of reported cases being identified in immunocompromised patients or those with underlying disease [ , ] and a severe threat of nosocomial transmission has been demonstrated [ ] . clinical presentation is similar to sars and includes a spectrum of respiratory diseases with the most common symptoms including cough, fever and gastrointestinal symptoms [ ] before progressing to pneumonia [ ] . acute respiratory distress syndrome (ards), renal failure, pericarditis and disseminated intravascular coagulation have also been reported [ ] . the potential of widespread pandemic outbreak is thought to be low as human-to-human transmission is largely inefficient [ ] and only reported in close, sustained contact [ ] such as within families [ ] , healthcare workers [ ] and as nosocomial transmission [ ] , especially if the patient presents with comorbidities. there has been no evidence to suggest sustained community transmission [ ] . the ever increasing case numbers and countries affected by the disease however suggests that mers-cov does represent potential for widespread, global outbreak [ ] . the inefficient spread between infected patients strongly suggests zoonotic transmission, however over two years on from the initial discovery of mers-cov it is still unclear where it originated from and how it infects humans. during the outbreak of sars-cov the identification of the wet markets of china as the probable source greatly contributed to the control of the disease [ ] . this highlights the importance in identifying the origins of disease outbreak. phylogenetic analysis places it in the same genus as sars-cov but within a new lineage, c. the closest detectable relatives suggests origins from bat coronavirus species with relative high sequence homology between bat-cov hku and hku in china [ ] , vm in the netherlands [ ] and a recently discovered isolate from south africa [ ] . assuming that bats also form the immediate source is improbable and as with sars-cov an intermediate animal host species for transmission to humans is of greater likelihood [ ] . high levels of neutralizing antibodies, viral rna and infectious viruses have been discovered in dromedary camels suggesting a potential for camels to form the source for human transmission [ ] [ ] [ ] [ ] . a recent study identified identical single nucleotide polymorphisms in a sequence of mers-cov isolated from an infected patient and camel, which was cared for by the patient. phylogenetic analysis of these two sequences supported the conclusion that transmission occurred between patient and camel, however the direction of transmission could not be confirmed [ ] . it has since been proven that a patient who succumbed to mers-cov infection obtained the virus directly from his camel herd. the direction of transmission was confirmed as camel-to-human by serological analysis [ ] . these facts strongly contribute to an increasingly popular hypothesis that mers-cov infections in humans are directly acquired from camels, however very few patients have had reported contact with camels. many residents of the arabian peninsula frequently consume unpasteurized camel milk and this has been suggested as a potential source as the virus has been detected and can remain viable for prolonged periods in milk [ , ] . the who, saudi arabia and qatar have recently issued warnings and recommended against consuming unpasteurized camel milk and to be cautious when interacting with dromedary camels [ ] . the outbreak of sars-cov in and mers-cov less than years later highlights the significant threat of coronaviruses to humans and confirms that the sars outbreak was not an isolated incident. with the ever increasing diversity of animal coronavirus species, especially within bats, the likelihood of recombination leading to future outbreaks is high and the threat of potential pandemics is real as highly pathogenic coronaviruses continue to spill over from zoonotic sources into the human population. misdiagnosis of these outbreaks pose a further substantial threat to healthcare workers with nosocomial spread to other patients putting further pressure on an already strained healthcare system. understanding the dynamics and molecular characteristics of human coronaviruses currently in circulation and how they emerge, infect and cause disease, we can be better prepared for future pandemics allowing for improved response, management and treatment of related conditions. the clinical presentation of human coronaviruses clearly follows two distinct lines of progression. sars-cov and mers-cov present with severe respiratory complications and often multisystem involvement with renal failure and enteric symptoms. the high fatality rates associated with these infections is not reflected in the remaining four human coronaviruses, hcov- e, hcov-oc , hcov-nl and hcov-hku . the clinical importance of sars-cov and mers-cov are evident and discussed in detail in previous sections allowing for the presentation of the involvement of the remaining non-severe human coronaviruses and there implications in human health. the clinical presentation of these four non-severe human coronaviruses is largely identical and indistinguishable symptomatically, commonly presenting with rhinorrhoea, sore throat, cough and fever [ , ] . majority of infections are associated with self-limiting upper respiratory tract disease or "the common cold" but can also present with high morbidity outcomes of the lower respiratory tract including bronchiolitis, pneumonia, [ ] [ ] [ ] , asthmatic exacerbations [ ] acute exacerbations of chronic obstructive pulmonary disease (copd) [ ] and croup in hcov-nl infected patients [ ] . a report by esper et al. found a correlation between hcov-nl infections and kawasaki disease [ ] , although other studies could not replicate the association [ ] [ ] [ ] . febrile seizures have been reported for most human coronaviruses but the significance of hcov-hku is alarming with one study indicating that % of patients infected with hcov-hku experience febrile seizures [ ] . human coronaviruses affect all age groups [ , ] but elicit more serious disease in young, elderly and immunecompromized [ , , ] , frequently resulting in hospitalization. reports on the prevalence of human coronaviruses and their association with upper and lower respiratory tract disease vary but range between . % and % [ , , , ] . over % of the general public has seroconverted towards all four non-severe human coronaviruses with primary infection shown to occur in childhood [ ] and reinfection occurring throughout life [ ] . given the high prevalence of respiratory infections, human coronaviruses represent a substantial disease burden which is exacerbated by the high implications of healthcare workers in coronavirus outbreaks [ ] . high rates of co-infection with other respiratory viruses are commonly reported [ , , , ] . viruses frequently associated with co-infection include enterovirus, rhinovirus and piv [ ] however reports of co-infection with two human coronaviruses are limited. dijkman et al. recently demonstrated that hcov-oc and hcov-nl may elicit immunity that protects against hcov-hku and hcov- e, respectively [ ] . clinical progression and outcomes of disease in patients presenting with co-infection are however similar to patients presenting with mono-infection [ , ] . there is also no substantial difference in coronaviral load between co-infected and mono-infected patients. no substantial difference in disease progression in co-infected versus mono-infected patients has been reported and therefore understood to have little impact; however, the role in facilitation of infection of one respiratory virus by another is still speculative [ ] . all four human coronaviruses are endemic worldwide but the prevalence, regional distribution and pathogenicity of individual human coronaviruses is unclear and highly subjective on study conducted with parameters including population sampled, respiratory sample collected, sensitivity of diagnostic assay used, region where study was conducted and duration of the study playing a substantial role in epidemiological findings. a common finding in majority of studies conducted is the increased prevalence of human coronaviruses during winter months in temperate climates [ , ] . however even this has shown to be geographically dependent with a spring/summer predominance in subtropical and tropical climates [ , ] . biennial outbreaks are frequently reported [ , , , ] for all strains of non-severe human coronaviruses. in a previously undiscovered virus was identified in epidemiologically distinct patients in the netherlands. patient symptoms were similar to those infected with rsv and, several patients required hospitalization and mechanical ventilation. viral isolates were cultured in tertiary monkey kidney (tmk) cells and cytopathic effects caused by the virus were largely identical to those caused by rsv. electron microscopy of infected cell supernatants revealed paramyxovirus-like particles; however, rt-pcr assays to detect known paramyxoviruses were all negative. the low stringency of the assays used indicated a currently unknown, genetically distinct virus. a rap-pcr assay was then utilized to obtain sequence information of the unknown virus and fragments amplified by the rap-pcr allowed for further sequencing of the '-end of the genome. based on the sequence homology and gene organization, the unidentified virus displayed closest homology with avian pneumovirus, but to be a tentative new member of the metapneumovirus genus and the first virus in the genus to infect humans, provisionally termed human metapneumovirus (hmpv) [ ] . symptomatic differentiation between hmpv and other respiratory viruses cannot be made as there is a significant overlap in clinical presentation [ , ] . the most common presentation of hmpv in children includes complications of the upper respiratory tract with rhinorrhoea, cough and fever [ ] . acute otitis media is also frequently reported [ , ] and conjunctivitis, rash, diarrhea and vomiting are reported but infrequently [ ] . bronchiolitis, pneumonia, croup and asthmatic exacerbations are the most frequently associated lower respiratory tract complications [ ] and viral load is directly associated with disease severity [ ] . hmpv infection in the young and elderly frequently requires hospitalization and fatalities have been reported in the elderly [ , ] . an increased morbidity in elderly patients with a delayed clearance of symptoms has been reported and is likely related to the age related impairment of the innate and adaptive immunity [ ] or an over stimulated immune response leading to inflammation [ ] . elderly patients requiring hospitalization most frequently present with acute bronchitis, copd exacerbations, pneumonia and congestive heart failure [ ] . in healthy adults asymptomatic infections or cold-and flu-like symptoms are the most prevalent presentation [ ] . the pathogenesis of hmpv infection is strongly affected by bacterial coinfections with pneumococcus. one study has shown that administration of a conjugate pneumococcal vaccine is sufficient to reduce the incidence of hmpv infection of the lower respiratory tract and the incidence of clinical pneumonia in both hiv positive and negative patients [ ] . these finding suggest that the incidence of hospitalizations in hmpv infections may be decreased by vaccination with a conjugate pneumococcal vaccine. another case report of severe respiratory failure was found to be caused by coinfection with hmpv and streptococcus pneumonia in a year old patient [ ] . both in vitro and in vivo studies have shown that infection with hmpv facilitates adhesion of pneumococcal bacteria, which may provide an explanation for the coinfection with pneumococcal strains and hmpv [ ] . viral coinfections between hmpv and rsv have been reported, but remain a contentious issue. the typical seasonal overlap of the two viruses has been suggested to promote viral coinfection. one study reported a -fold increase in risk of admission to an intensive care unit in pediatric patients coinfected with rsv and hmpv and associated the dual infection as capable of augmenting severe bronchiolitis [ ] . other studies do not support this finding and further report a decreased correlation between hmpv-rsv coinfections and hospitalization and additionally lists dual infection, along with breastfeeding, as having protective effects [ ] . although hmpv was only discovered in , it has been shown by phylogenetic analysis to have been in existence for approximately years [ , ] . soon after the discovery of hmpv, it was evident that two lineages, a and b, existed. these two lineages were further subdivided into two sublineages per lineage, a -a and b -b [ ] . a recent report analyzing sequence divergence of the attachment and fusion surface glycoproteins indicates the presence of five sublineages, namely a , a a, a b, b and b [ ] . from long term retrospective studies it was evident that these lineages are not restricted to certain locations or times and that multiple lineages can exist in the same location and period [ , ] . it has also become evident that old sublineages may be replaced by new variants [ ] . disease progression or varying clinical outcomes related to different lineages of hmpv has become a contentious issue. several studies have reported that lineage a presents with more severe clinical outcomes [ ] [ ] [ ] where the same is reported for lineage b by other groups [ , ] . it has been further reported that there is no difference in disease outcomes related to the two lineages [ , , ] . between % and % of all cases of respiratory infections in children are caused by hmpv, in both hospitalized and outpatients [ , , ] and has been reported to be the second most frequently identified virus in respiratory tract infections [ ] . extrapolation of consensus data suggests a total of hospital and one million clinic visits annually in the us among children younger than [ ] . children hospitalized with hmpv infections are also more likely to present with pneumonia or asthma and required longer stay in intensive care units with supplemental oxygen, when compared to other respiratory viruses [ ] . seroprevalence studies indicates that % of young adults are seropositive for hmpv with stable neutralizing titres, which further suggests that reinfection occurs throughout life [ ] , with a potential for genetic variation between clades promoting reinfection [ ] . hmpv has a worldwide distribution and affects all age groups but predominantly affects young, elderly and immunocompromised patients [ ] , with children younger than five years of age being most susceptible to infection [ ] . children and adults with underlying or chronic conditions such as asthma, chronic lung disease, congenital heart disease, cancer or copd are more likely to be hospitalized with hmpv infection [ ] . infection with hmpv occurs throughout the year but seasonal prevalence in late winter and spring has been observed and coincides with the peak of rsv infection [ , [ ] [ ] [ ] . the first human bocavirus (hbov) was discovered in from nasopharyngeal aspirates of patients with unresolved lower respiratory tract infections in sweden. researchers utilized a novel technique which included steps of dnase treatment to exclude contaminating, or non-viral, nucleic acids followed by pcr amplification by nonspecific primers. the pcr-products were subsequently cloned with large-scale sequencing of the clones. bioinformatic analysis of generated sequence data yielded the discovery of a new parvovirus with a high homology to bovine and canine minute parvoviruses. the genus name bocavirus was in fact derived from the species infected by the known virus strains, namely bovine and canine. the new virus was named hbov and was the first virus to be discovered by molecular virus screening [ ] . three additional species of hbov were later discovered in and added to the genus; these were named hbov , hbov and hbov [ ] [ ] [ ] . hbov is a respiratory pathogen affecting all regions of the globe and is associated with approximately %- % of all upper and lower respiratory tract conditions [ ] [ ] [ ] . hbov productively infects human airway epithelium cell cultures and leads to damage of airway epithelial cells [ ] [ ] [ ] , which supports clinical observations that infection does result in respiratory disease. in contrast, hbov - , are found in the gastrointestinal tract and hbov , and possibly hbov , are associated with gastroenteritis [ , [ ] [ ] [ ] . interestingly, hbov is the only enteric bocavirus to be isolated from nasopharygeal aspirates and may, therefore, also be associated with respiratory disease [ , ] . hbov is detected in all age groups, but predominantly in young children between the ages of - months [ , , ] and is rarely detected in adults [ ] [ ] [ ] [ ] [ ] . transmission and infection occurs throughout the year, but predominantly during winter and spring months [ , [ ] [ ] [ ] . seroprevalence studies suggest that maternal antibodies, which provide protection, are present in infants younger than months of age [ , ] , after which seropositivity decreases with low levels of detection until - months. virtually % of children aged are seroconverted for hbov and as reinfection occurs throughout life this remains into adulthood [ , [ ] [ ] [ ] [ ] . the presence of the three enteric bocaviruses does however complicate the findings of seroconversion as cross-reactivity does exist [ ] . as with many respiratory viruses, clinical differentiation with hbov infection is not possible by symptomatic presentation [ ] . common features of infection of the upper respiratory tract include common cold-like symptoms with cough, rhinorrhoea and acute otitis media [ ] . infection of the lower respiratory tract in children is associated with pneumonia, acute wheezing, asthmatic exacerbations and bronchiolitis [ , , [ ] [ ] [ ] , but life-threatening complications are rare with hbov infection [ ] . although hbov has been isolated from stool samples, there is no statistical evidence to associate hbov with gastrointestinal disease [ ] . hbov has not only been found in the upper and lower respiratory tract and gastrointestinal specimens, but also in urine samples, serum, saliva, and tonsils [ ] . rather than having a role in disease pathogenesis, this viraemia and systemic spread may be a feature common to all parvoviruses as they require proliferating host cells for replication [ ] . interestingly, hbov appears to be more than just a respiratory or gastrointestinal virus. in a recent study, hbov was identified in . % of lung (n = / ) and . % of colorectal (n = / ) tumors screened. unfortunately, the study did not investigate whether the hbov genomes were in fact incorporated into the host genome as reported for other known parvoviruses. therefore, based on their observations as well as previous studies on other parvoviruses, the authors speculate that hbov could contribute to the development of some lung and colorectal tumors. however, they do also acknowledge that these tumors could simply be providing an optimal environment for hbov replication and more conclusive studies are required to resolve this issue [ ] . hbov has been associated with a prolonged period of persistence in the mucosa of the respiratory tract. this prolonged presence has possibly led to a high frequency of coinfection found with hbov infections of the both the upper and lower respiratory tract [ , , ] . the high rate of detection of multiple respiratory viruses within up to % of respiratory specimens, and the presence of asymptomatic hbov infections, does complicate the determination of the actual pathogenic role of hbov [ ] . high viral load is statistically associated with symptoms [ ] and this may therefore be a better indication of coinfections which are related to disease severity or symptomatic presentation. it has been further suggested that patients presenting with viraemia are better candidates to assess the symptoms of disease when compared to investigations of respiratory secretions [ ] . the effects and mechanisms of latency, persistence, reactivation and reinfection are however poorly understood and therefore its effects on coinfection and its contribution to active disease cannot be accurately stated [ ] . the etiological agents of %- % of lower respiratory tract infections still remain to be identified [ , ] . these results may vary significantly depending on the sensitivity of the diagnostic assay used, respiratory site sampled and even geographical location of the study but it does suggest that many uncharacterized respiratory pathogens could still remain elusive awaiting discovery. the vast improvements in molecular techniques within the past decade have however led to the discovery of four previously circulating respiratory viruses and also the rapid characterization of two completely novel viruses, namely sars-cov and mers-cov. all these viruses have varying but significant impact on human health and the potential for outbreak of completely novel, emergent respiratory viruses, seen with sars and mers, poses their own unique threats. lessons learned from these viruses, and others currently in circulation, provide health care authorities and scientists with suitable expertise and knowledge to rapidly identify and combat novel respiratory viruses and as our techniques improve we will be in a position to characterize those viruses that are currently difficult to isolate and identify. burtram c. fielding receives funding from the national research foundation, south africa. any opinion, findings and conclusions or recommendations expressed in this material are those of the author and therefore the nrf does not accept any liability in regard thereto. mb wrote the earlier drafts of the manuscript. jg edited and reviewed the early drafts of the manuscript. bcf conceptualized the paper and wrote the submission draft of the manuscript. the authors declare no conflict of interest. viral upper respiratory tract infection and otitis media complication in young children the economic burden of non-influenza-related viral respiratory tract infection in the united states the clinical impact 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a multi-omics study of chicken infected by nephropathogenic infectious bronchitis virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qh y chicken gout resulting from nephropathogenic infectious bronchitis virus (nibv) has become a serious kidney disease problem in chicken worldwide with alterations of the metabolic phenotypes in multiple metabolic pathways. to investigate the mechanisms in chicken responding to nibv infection, we examined the global transcriptomic and metabolomic profiles of the chicken’s kidney using rna-seq and gc–tof/ms, respectively. furthermore, we analyzed the alterations in cecal microorganism composition in chickens using s rrna-seq. integrated analysis of these three phenotypic datasets further managed to create correlations between the altered kidney transcriptomes and metabolome, and between kidney metabolome and gut microbiome. we found that genes and metabolites were deferentially expressed or accumulated in the kidney during nibv infection processes. these genes and metabolites were linked to nibv-infection related processes, including immune response, signal transduction, peroxisome, purine, and amino acid metabolism. in addition, the comprehensive correlations between the kidney metabolome and cecal microbial community showed contributions of gut microbiota in the progression of nibv-infection. taken together, our research comprehensively describes the host responses during nibv infection and provides new clues for further dissection of specific gene functions, metabolite affections, and the role of gut microbiota during chicken gout. gout is a urate crystal deposition disease that occurs when supersaturation of individual tissues with urate arises, leading to the construction of monosodium urate (msu) crystals in and around the joints, abdomen, and organs [ , ] . it is worth noting that in addition to its occurrence in humans, gout is one of the common diseases that plague poultry and causes huge economic losses to the poultry industry worldwide [ ] . avian gout is commonly divided into visceral gout and joint gout, and the typical clinical pathology of visceral gout is hyperuricemia. according to reports, various aviaries from all over the world have visceral gout, which has become one of the most commonly diagnosed causes of fatality in poultry [ , ] . in the poultry industry, visceral gout can be caused by many factors, including vitamin a deficiency, high dietary calcium, renal insufficiency, chicken diseased room (dis, n = ). birds in each experimental room were randomly divided into three subgroups ( birds for each subgroup), with ad libitum access to food and water. each chicken of dis groups was intranasally injected with . ml median embryo lethal doses of strain sx at days of age [ ] , while the con group intranasally received . ml of sterile physiological saline. at days of age, four chicken randomly chosen per subgroups were euthanized by carbon dioxide inhalation, then dislocated their cervical vertebra. the samples in a group were pooled and dead birds were not used for analysis. ten serum samples were randomly collected from surviving chickens in the con and dis groups before euthanasia that were used for uric acid test. six biological replicates of kidney samples were extracted from each group making a total of samples that were used for gc-tof/ms analysis. four biological replicates of kidney samples were collected from each group giving a total of eight samples that were used for rna-seq analysis. six biological replicates of cecal contents from each group were collected giving a total of samples that were used for s rrna gene sequencing analysis ( figure a shows the experimental design). the isolated kidney tissues were fixed by immersion in % neutral formalin at room temperature for over h. tissues were then routinely processed; h&e staining was performed and a section per chicken was observed under the optical microscope. metabolite extraction, metabolite derivatization, metabolite detection, and data analysis followed those of yang et al. [ ] . first, methanol (v methanol :v chlorofrom = : ) was used as an extraction liquid, and l- -chlorophenylalanine ( mg/ml stock in dh o) was added as an internal standard. the metabolites are then derivatized with the methoxy amination hydrochloride ( mg/ml in pyridine) and the stfa regent ( % tmcs, v/v). finally, gc-tof/ms analysis was performed using an agilent gas chromatograph system coupled with a pegasus ht time-of-flight mass spectrometer. the energy was - ev in electron impact mode. after . min of solvent delay, mass spectrometry data were acquired in full-scan mode with an m/z range of - at a rate of spectra per second. chroma tof . x software (leco corporation, st. joseph, mi, usa) and the leco-fiehn rtx database were used for raw peak exacting, data baseline filtering and calibration, peak alignment, deconvolution analysis, peak identification, and integration of the peak area. simca software package (umetrics, umea, sweden) was used for further data analysis, including principal component analysis (pca) and orthogonal projections to latent structures-discriminate analysis (opls-da). in addition, commercial databases including kyoto encyclopedia of genes and genomes (kegg, http://www.genome.jp/kegg/) and national institute of standards and technology (nist, http:// www.nist.gov/index.html) were utilized to search for metabolic pathways. metaboanalyst (http: //www.metaboanalyst.ca) was used for pathway enrichment analysis. rna preparation, library preparation, rna-sequencing, and data analysis followed those of yang et al. [ ] . first, total rna was extracted from each kidney sample individually using trizol reagent (invitrogen, burlington, on, canada). we further used the nanodrop (thermo, waltham, ma, usa) to measure the rna concentration, and the agilent bioanalyzer system (agilent technologies, ca, usa) to assess the rna integrity. library preparation was performed as described by li et al. [ ] . briefly, a total amount of µg rna per sample was used for the rna sample preparations. sequencing libraries were generated using nebnext ultratm rna library prep kit for illumina (neb, ipswich, ma, usa) and index codes were added to attribute sequences to each sample. the library fragments were purified with ampure xp system (beckman coulter, beverly, ca, usa). at last, pcr products were purified (ampure xp system) and library quality was assessed on the agilent bioanalyzer system. finally, the constructed cdna libraries were sequenced by an illumina hiseq xten platform. clean data of high quality were obtained from the raw data through in-house scripts by removing those containing adapters or poly-n sequences. all clean reads were aligned to the reference genome (https://www.ncbi.nlm.nih.gov/assembly/gcf_ . /) using tophat [ ] . cufflinks was used to calculate and analyze the gene expression levels, and fpkm (fragments per kilobase of exon per million fragments mapped) values of each gene were calculated based on the length of the gene and the fragments count mapped to this gene. differential expression genes (degs) analysis was performed by using the deseq r package ( . . ). only those genes with a fc (fold change) ≥ and fdr (false discovery rate) < . were defined as degs. furthermore, gene ontology (go) enrichment analysis of degs was implemented by the goseq r packages [ ] and the enrichment analysis of degs in kegg pathways was performed using kobas software [ ] . in addition, the target seven degs in response to nibv infection were chosen for validation using real-time quantitative pcr (rt-qpcr). the primer pairs for the selected genes were designed using primer and are shown in table s . total genome dna from samples was extracted from the cecal contents using ctab/sds method. dna concentration and quality were determined and diluted to ng/µl using sterile water. the s rrna genes of each sample v -v region were amplified. the primer set corresponding to primers f- r with the unique barcode was applied for amplification (forward f: cctaygggrbgcascag and reverse r: ggactacnngggtatctaat). all pcr reactions were carried out with phusion ® high-fidelity pcr master mix (new england biolabs, usa). the pcr products were detected on % agarose gel electrophoresis and bands of the desired size (approximately - bp) were chosen for further experiments. sequencing libraries were generated using trueseq dna pcr-free sample preparation kit (illumina, san diego, ca, usa) and index codes were added. next, sequencing was performed on an illumina hiseq platform (novogene company, beijing, china). based on their unique barcode, the paired-end reads were granted to samples. those files were demultiplexed and quality filtered with quantitative insights into microbial ecology (qiime) software (version . . ) [ ] . the chimera sequences were removed by using uchime algorithm and then the effective tags finally obtained [ ] . sequences were clustered to the same otus at % similarity by uparse software (uparse v . . ). moreover, the greengene database was used to annotate taxonomic information and the multiple sequence alignment was conducted using the muscle software (version . . ). further, otus abundance information was normalized and subsequent analysis were all performed based on this output normalized data including alpha diversity, beta diversity, and linear discriminant analysis (lda) effect size (lefse) analysis [ ] . heatmap for metabolomics, transcriptomics, and microbiomics data was generated using the pheatmap package. corrplot package in r was used to visualize the correlations coefficient (spearman method). availability of data and materials: rna-seq raw data are accessible through ncbi' database: bioproject: prjna ; s rrna gene sequencing raw data are accessible through ncbi' database: bioproject: prjna . the institutional animal care and use committee of jiangxi agricultural university approved these animal experiments and all animal experiments adhered rigorously to the animal care guidelines of jiangxi agricultural university (approval id: jxaull- ; approval date: march ). all the birds were sacrificed using carbon dioxide euthanasia, and all attempts were carried out to minimize the suffering of the animals. obvious enlargement and urate deposition in the kidney of a chick infected with nibv at dpi (right). (d) histopathological changes in the kidney of chickens infected with nibv (h&e staining). the black arrow shows the shedding of kidney tubular epithelial cells and the white arrow shows the interstitial expansion and prominent inflammatory cell infiltration. the black asterisk shows the brush border that was lost in some segments of proximal tubules. the black delimited area shows the loss of the kidney tubular structure. to explore the metabolic pathway alterations associated with nibv infection, we used a gc-tof/ms-based metabolomics method to examine metabolite alterations in the kidney. a total of valid peaks were identified in the total ion current profiles. to compare the metabolite composition of the con and dis groups, pca models were tested (figure a ). opls-da was conducted to determine whether nibv infection influenced the metabolic pattern ( figure b ) and a -fold cross validation was further applied to estimate the robustness and predictive ability to validate the model (figure c ). the results of pca and opla-da analysis showed that there was an obvious separation between the content of the con and dis groups, obvious enlargement and urate deposition in the kidney of a chick infected with nibv at dpi (right). (d) histopathological changes in the kidney of chickens infected with nibv (h&e staining). the black arrow shows the shedding of kidney tubular epithelial cells and the white arrow shows the interstitial expansion and prominent inflammatory cell infiltration. the black asterisk shows the brush border that was lost in some segments of proximal tubules. the black delimited area shows the loss of the kidney tubular structure. the chicks inoculated with strain sx showed obvious clinical signs from to "day post-infection" (dpi). the nibv-infected chickens were listless, huddled together, and displayed ruffled feathers, while no clinical symptoms similar to the above were observed in the con group. the dpi survival rate of all nibv-infected chickens under analysis was %, and the uric acid level in the serum of the dis group was much higher than that of the con group (~ vs.~ µmol/ml, n = , p < . , student's t-test, figure b ). we observed that kidney lesions were present in all dis group chickens infected with sx . at dpi (mortality peak), the kidney parenchyma of the dead birds were pale, swollen, and mottled ( figure c ). histological examination revealed remarkable injuries in the kidney, including tubular epithelial cell detachment, loss of the kidney tubular structure, as well as interstitial expansion and prominent inflammatory cell infiltration ( figure d ). these results indicate that the sx strain has strong renal tissue tropism and successfully replicates the chicken visceral gout model. to explore the metabolic pathway alterations associated with nibv infection, we used a gc-tof/ms-based metabolomics method to examine metabolite alterations in the kidney. a total of valid peaks were identified in the total ion current profiles. to compare the metabolite composition of the con and dis groups, pca models were tested (figure a ). opls-da was conducted to determine whether nibv infection influenced the metabolic pattern ( figure b ) and a -fold cross validation was further applied to estimate the robustness and predictive ability to validate the model (figure c ). the results of pca and opla-da analysis showed that there was an obvious separation between the content of the con and dis groups, revealing significant changes in the concentrations of metabolites in the kidney induced by nibv infection. all samples fell within the % (hotelling's t-squared ellipse) confidence interval. the differential metabolites between dis and con groups were the key to explaining the occurrence of gout in chickens under nibv infection. a total of metabolites displayed significantly different levels based on vip > (variable importance for the projection, opls-da model) and p-value < . (student's t-test). the lists of differential metabolites are shown in table s and its volcano plot are shown in figure d . there were annotated metabolites in all differential metabolites, and its categories are shown in figure e . as figure e shows, the differential metabolites were mainly classified as amino acids, carbohydrates, fatty acids, and conjugates. among those annotated differential metabolite profiles which were displayed in heat maps ( figure s ), metabolites were significantly upregulated and were significantly downregulated in the dis group compared to the con group. in addition, the pathway enrichment results are shown in figure f . the analysis revealed that the differential metabolites participated in six target pathways (p < . ), including valine, leucine and isoleucine biosynthesis, arginine and proline metabolism, alanine, aspartate and glutamate metabolism, d-glutamine and d-glutamate metabolism, aminoacyl-trna biosynthesis, and beta-alanine metabolism. among them, there are two pathways with an impact factor > , including valine, leucine and isoleucine biosynthesis (impact value = . ), and arginine and proline metabolism (impact value = . ). these pathways are related to amino acid metabolism and glycometabolism, which are involved in protein synthesis and energy production, respectively. moreover, metabolomics highlighted n-formyl-l-methionine, a well-known agent for kidney injury for its effects on the increase of the vascular tone/resistance and reduction of renal perfusion [ ] , as the most relevant metabolite alteration in nibv-infected chickens (vip = . ). undoubtedly, the uric acid level in the dis group was significantly upregulated compared with the con group (vip = . , . -fold). to study the gene expression alterations of chicken's kidney under nibv infection, cdna libraries from two groups were subjected to illumina sequencing. a total of . gb clean data was obtained, and the q base percentage of each sample was not less than . %. from the mapping results, the mapping efficiency between the reads and the reference genome of each to study the gene expression alterations of chicken's kidney under nibv infection, cdna libraries from two groups were subjected to illumina sequencing. a total of . gb clean data was obtained, and the q base percentage of each sample was not less than . %. from the mapping results, the mapping efficiency between the reads and the reference genome of each sample was between . % and . %. the detail of the sequencing and mapping results is provided in table s . pca analysis of rna-seq replicates from the kidneys of dis and con group revealed a great deal of variability (figure a) . a total of genes were differentially expressed in the chicken's kidney with nibv infection compared to con group, in which genes were upregulated and genes were downregulated. then, we screened seven degs for rt-qpcr analysis to validate the accuracy of the rna-seq data, and the result (figure b) showed that the general trends of the selected genes were consistent which proved the reliability of our rna sequencing profiling. go (http://www.geneontology.org) project was applied to explore the potential biological functions of degs. in this study, the most enriched go terms classified by molecular function (mf), cellular components (cc), and biological processes (bp) terms were listed in figure c . among them, heparin binding, oxidoreductase activity, external side of plasma membrane, extracellular space, membrane, integral component of membrane, oxidation-reduction process, and immune response were the significantly enriched go terms (p adj < . ) in chickens with nibv infection. to further study the biochemical metabolic pathway and signal transduction pathways related to nibv infection, kegg analysis and pathway enrichment analysis were conducted in the kegg pathway database. there were of the degs that were annotated to pathways in kegg analysis. the level- kegg classes are shown in figure d , and the significant enriched pathways (corrected p-value < . as determined by a fisher's exact test) are marked with red number. in environmental information processing pathways which include cytokine-cytokine receptor interaction and cell adhesion molecules (cams) were dramatically regulated. in cellular processes pathway, peroxisome was dramatically affected. in metabolism pathways, metabolism of xenobiotics by cytochrome p , drug metabolism-cytochrome p , pyruvate metabolism, arginine and proline metabolism, glycolysis/gluconeogenesis, and fatty acid degradation were significantly regulated. in organismal systems pathways, intestinal immune network for iga production and toll-like receptor signaling pathway were significantly regulated. furthermore, most of these pathways of the upregulated genes enriched were immune system-related pathways, and "cytokine-cytokine receptor interaction" was the most represented pathway. among these pathways we found upregulated, "toll-like receptor signalling pathway", "nod-like receptor signalling pathway" and "rig-i-like receptor signalling pathway" are pattern recognition signal receptor pathways involved in innate immunity. simultaneously, the transcriptome showed that the "peroxisome" and amino acid metabolite pathways were suppressed in chickens infected with nibv. based on the above results, we predicted a schematic diagram of important pathways for chickens in the nibv infection processes (figure e) . signalling is activated by monosodium urate (msu) and reactive oxygen species (ros). the activation of tlrs and the ros accumulation activates the nlrp inflammasome, resulting in proteolytic cleavage of caspase- and the maturation of il- and il- β, and msu has been identified to activate inflammasome complex. considering that the intestinal tract is an important organ for lowering serum uric acid concentrations, s rrna sequencing was performed and demonstrated marked alterations of the gut microbial communities in the dis group. the rarefaction analysis curves for each group were near saturation, revealing that the sequencing data had a great quality and that certainly few new species were present ( figure s ). nmds (nonmetric multidimensional scaling, figure a (figure g,h) . lefse was performed to identify significant microbiota composition differences between the groups. seven differentially represented core major groups were identified (figure i,j) . differentially abundant phylum detected showed that proteobacteria (lda = . ) phylum was most dominantly present in dis group. at the genus level, the microbiota of the con group was enriched with bacteroides (lda = . ) from the bacteroidetes phylum and lactobacillus (lda = . ) from the firmicutes phylum. at the species level, the microbiota of the con group was enriched with bacteroides vulgatus (lda = . ) from the bacteroidetes phylum. the above data describe that nibv infection alters kidney gene expression and metabolic pathways (figure a ). we filtered out five overlapping pathways in the transcriptome and metabolome pathway enrichment analysis, including valine, leucine and isoleucine biosynthesis, arginine and proline metabolism, taurine and hypotaurine metabolism, alanine, aspartate and glutamate metabolism, and glutathione metabolism. in addition, the degs in significantly enriched pathways associated with innate immunity and differential metabolites with vip > . were also chosen for correlation analysis (tables s and s , respectively). differences in those pathways-associated with degs and differential metabolites were shown with heatmap in figure b . then, we analyzed the spearman correlation between genes and metabolites in those pathways, and the result is shown in figure c . as shown in figure c , there was a positive correlation between tlr (tlr, toll-like receptor) and proline (r = , p = ), isoleucine (r = . , p = . ), threonine (r = . , p = . ), and valine (r = . , p = . ). conversely, strong negative correlations were found between glutathione synthetase (gss) and glutamine (r = − , p = ). interestingly, we observed that most genes related to innate immune responses had strong positive correlations with differentially abundant metabolites, such as amino acids and fatty acids. the gut microbiota is deliberated a massive "organ" able to perform complex functions and thereby produce a myriad of differentially abundant metabolites. to investigate the functional correlation between the gut microbiome changes and host metabolome alterations, a correlation plot was visualized by calculating spearman's correlation coefficients (figure d ). the result indicated that clear correlations could be identified between altered metabolic profiles and modulated gut microbiomes (table s ) . of particular note, some metabolites, including trans- -hydroxy-l-proline, guanine, and , -anhydro-d-galactose, which decreased in the kidney of nibv-infected chickens, were negatively correlated with the presence of the phylum proteobacteria. furthermore, other metabolites, including canavanine, , -diaminobutyric acid, , -dihydrouracil, malonamide, thymine, phenylalanine, , -diaminopropane, -acetamidobutyric acid, proline, threonine, isoleucine, valine, and oxalacetic acid, which increased in the kidney of nibv-infected chickens, were positively correlated with the presence of the phylum proteobacteria. these observations indicated that the significantly modulated gut microbiota was correlated with host metabolic disorders. in our study, the three pathways included in the activation of the innate immune system, the toll-like receptor signalling, rig-i-like receptor signalling, and nod-like receptor signalling pathways, that are the most important three parts of the pattern-recognition receptor (prr) signalling pathway are usually activated in response to infections to stimulate inflammatory responses [ ] [ ] [ ] . in the toll-like receptor signalling pathway (figure f, signal ) , a series of upregulated genes were noticed, including tlr , tlr , myd , irf , traf , and irf . it was already reported that tlr primarily recognizes single-stranded rna (ssrna) sequences of rna viruses that enter endosomes by endocytosis [ ] [ ] [ ] . nibv used in this experiment is a single-stranded positive sense rna virus. thus, the expansion of tlr is pivotal for the recognition of nibv and variable functions of the toll-like receptor signalling pathway. moreover, a number of viral glycoproteins have been shown to act as viral pamps (pathogen-associated molecular pattern) that bind to and activate tlr , leading to ifn-β and/or proinflammatory cytokine expression (such as sars coronavirus) [ ] . ru liu-bryan's study has established that host expression of tlr , tlr , and their intracellular adapter protein myd is a major mediator of msu crystal-induced inflammation [ ] . this explains the reason for the increase in tlr transcription levels in this experiment. in addition, the transcriptomic analysis showed that nibv infection also activated the rig-i-like receptor signalling pathway (figure f , signal ), which included the transcriptional upregulation of genes such as mda , ips- , traf , and iκb. this induction may be due to mda acting as a double-stranded rna (dsrna) sensor to trigger an innate immune response against viral infection [ ] [ ] [ ] , while coronaviruses can produce dsrna intermediates during replication [ ] . rlrs can not only be expressed in cells infected with various viruses but also directly recognize and perceive virus products and virus particles present in the cytosol outside of the endosomes [ ] . therefore, we suspect that the rig-i-like receptor signalling pathway has a greater role than toll-like receptor signalling in recognizing nibv infection. the activation of the toll-like receptor signalling and rig-i-like receptor signalling pathways results in the production of chemokines and other cytokines that induce a proinflammatory response and tissue destruction. in our study, several features that are familiar to chickens infected with nibv are consistent with the immunopathological disease. these features include the pathological damage of kidney and the presence of increased transcription of chemokines and other cytokines, such as il- (interleukin ), il- (interleukin ), and tgf-β (transforming growth factor β). peroxisomes act to eliminate a microbial infection by modulating the canonical innate immunity pathways through ros signalling [ ] . several enzymes with antioxidant activity in the peroxisome are involved in neutralizing ros to protect the cells from ros damage. among these enzymes, catalase (cat), superoxide dismutase (sod ), peroxiredoxin (prdx ), glutathione s-transferase kappa (gstk ), dehydrogenase/reductase member (dhrs ), and epoxide hydrolase (ephx ) are included [ , ] . multiple viruses have been shown to use different mechanisms to reduce peroxisome numbers or interfere with their functions. in our study, the "peroxisome" was the most important pathway according to the downregulated gene enrichment analysis, which includes cat, sod , dhrs , and ephx that belong to the peroxisome antioxidant defence system, while the catalase activity was decreased both in kidney and serum ( figure s ). thus, decreased abundance and/or impaired function of the peroxisome could potentially cause endogenous elevation of ros. ros may either straightly trigger nlrp inflammasome assemblage or be indirectly sensed through cytoplasmic proteins that modulate inflammasome activity (figure f, signal ) . the nlrp inflammasome senses pathogens or danger signals to promote the maturation of cytokines such as il- [ ] . the release of active il- engages il- receptor-harbouring cells and promotes inflammatory responses [ ] . in addition, uric acid has been reported as another well-established activator of nlrp that is usually generated via xanthine oxidase (xod), accompanying the generation of o •− [ , ] . consistently, in this study, we detected a significant increase in xod activity in serum ( figure s ) and a significant increase in serum uric acid levels (figure b) . these results indicate that nibv infection can activate the inflammatory response by inducing severe ros accumulation. the kidney is responsible for the elimination of % of the daily ua production [ ] . atp-binding cassette transporter, sub-family g, member (abcg ) is a high-capacity urate exporter that is located in the brush border membrane of kidney proximal tubule cells (s segment). abcg dysfunction results in extra-renal urate under-excretion and is a common mechanism of hyperuricemia [ ] [ ] [ ] . in the present study, the abcg mrna was downregulated in the model group chicken kidneys, partially explaining the significantly increased uric acid levels caused by nibv infection. in addition to being caused by insufficient urate excretion, hyperuricemia can also be caused by excessive production of uric acid [ ] . we found a high level of glutamine, which enter the purine metabolic pathway as a raw material for uric acid synthesis. the high level of glutamine can be explained by two reasons. first, the lack of mrna abundance of the gene gss in the kidney tissue of the model group inhibited the conversion of glutamine to glutathione. a significant negative correlation between gss and glutamine has confirmed this finding. second, the transcription level of the gene glutamic pyruvate transaminase (gpt ) in the model group chickens' kidneys increased significantly. gpt is a pyridoxal enzyme that promotes the conversion of α-ketoglutarate to glutamate [ ] . of note, α-ketoglutarate is an intermediate in the tca cycle, and glutamate can be reversibly converted to glutamine by glutamine synthetase. together, these data point to key genes and metabolites related to elevated uric acid synthesis, which provides us with new insights into host response to nibv infection. the present study highlights the correlation between differential expression of genes and differential abundance of metabolites in significantly enriched pathways, and the results showed that those differentially abundant metabolites that map in amino acid metabolism pathways have strongly positive correlations with degs related to innate immune responses. it is well known that organisms fuel or instruct the immune response to pathogen threats by modulating metabolic pathways [ ] . innate and acquired immune systems are regulated by a highly interactive network of chemical communications, which include the synthesis of the antigen-presenting machinery, immunoglobulins, and cytokines. this network is highly dependent on the sufficient availability of amino acids. thus, amino acids affect immune responses either directly or indirectly through their metabolites [ ] . in our study, the correlation analysis highlighted a significant positive correlation between tlr and proline, isoleucine, threonine, and valine. according to reports, isoleucine could maintain the development of immune organs and cells and stimulate the secretion of immune molecule substances in humans and animals [ , ] , valine could improve dendritic cell function [ ] , and it has been proved in vitro that threonine plays a key role in lymphocyte proliferation and immunoglobulin production [ ] . we observed an increase in the level of amino acids enriched in proline, leucine, and isoleucine biosynthesis pathway, including isoleucine, valine, and threonine. therefore, changes in these amino acid metabolism pathways may occur through the activation of innate immunity to enhance the body's antiviral response. although there are many studies on amino acids and immunity, the modulation of amino acid metabolism in innate immune responses is still poorly known and deserves further study. previous research has confirmed that the dysfunction of the gut microbiome is tightly associated with kidney and liver diseases, including liver cirrhosis [ ] , liver cancer [ ] , and chronic kidney disease [ ] . the gut microbiome incorporates a wide variety of commensal microbiota and has a large effect on the health of individuals. a community-wide effect involving the gain and loss of microbial populations and changes in general metabolic functions always occurs under pathological conditions. our results showed that the abundance of bacteroides, lactobacillus, and bacteroides vulgatus were significantly reduced in the cecal microbiota of nibv-infected chickens based on lefse analysis. similar to many other bacteroides species, especially bacteroides vulgatus which was shown to promote intestinal homeostasis, the bacteroides vulgatus-mediated induction of semimature dendritic cells is associated with inflammation silencing [ , ] . thus, decreased abundance of bacteroides vulgatus promotes intestinal flora imbalance and activation of the immune system. furthermore, it is well accepted that lactobacillus may lower serum uric acid levels by reducing intestinal absorption of purines in humans [ ] . therefore, a decrease in the abundance of lactobacillus in the caecum of the model group infected with nibv further promoted an increase in uric acid levels in serum. the results of our correlation analysis further confirm this point, that is, the negative relationship between the abundance of bacteroides vulgatus, lactobacillus and the uric acid concentration. however, the mechanism of nibv infection leading to a decrease in their abundance still needs further study. taken together, multitudinous studies to date endorse the concept that a bloom of proteobacteria in the gut reflects gut dysbiosis or an unstable gut microbial community [ ] . in view of balanced gut microbiota with high stabilization that has symbiotic relationships with the immune system of the host, which is capable of suppressing the uncontrolled expansion of proteobacteria, the increase in the abundance of proteobacteria in this experiment may be closely related to the immune and metabolic changes caused by nibv infection. this report is the first time that multi-omics approach was employed to profile the metabolic changes and immune responses in kidney, as well as the effects on the intestinal microbiome during nibv infection. our results showed that nibv significantly increased uric acid synthesis, inhibited the function of peroxisomes, and significantly elevated the pattern recognition receptor signalling pathways. in summary, our study comprehensively describes the host responses during nibv infection and provides new clues for further dissection of specific gene functions, metabolite affections, and the role of gut microbiota during chicken gout. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . figure s : heat map of annotated significant differential metabolites. note: the increasing gradient of red color indicate higher upregulation, while an increasing gradient of blue color indicate higher downregulation. figure s : rarefaction curves of the species number in the con and dis groups. the curve in each group is nearly smooth when the sequencing data set is sufficiently large (n = per group). figure s : the catalase activity (a, b), xanthine oxidase activity (c, d) in kidney and serum. table s : primers of selected genes for rt-qpcr. table s : differentially abundant metabolites identified by gc-tof/ms. significant differences were declared at the level of p-value < . and vip > . table s : basic summary of sequence and sequencing reads mapping to reference genome. table s : degs in enriched pathways for correlation analysis. table s : differentially abundant metabolites in enriched pathways for correlation analysis. table s : the relative abundance of seven discriminative features (lda score ≥ ) identified by lefse analyse. author contributions: conceptualization, x.g.; methodology, x.g. and p.l.; software, c.z. and y.s.; validation, q.w., p.x., and y.y.; formal analysis, p.x., c.z., and p.l.; resources, g.l., p.l., and x.g.; data curation, p.x. and x.g.; writing-original draft preparation, p.x. and p.l.; writing-review and editing, x.g., p.l., g.l., and g.h.; visualization, p.x., c.z., and y.s.; supervision, g.h.; project administration, x.g.; funding acquisition, x.g. funding: this research was funded by the national natural science foundation of china, grant number . acknowledgments: all authors thank all members of the team for their help in the experimental process in clinical veterinary medicine laboratory in the college of animal science and technology, jiangxi agricultural university. we also thank vincent latigo for the language help rendered in reviewing the revised manuscript. the authors declare no conflict of interest. gout induced by intoxication of sodium bicarbonate in korean native broilers clinicopathology of gout in growing layers induced by high calcium and high protein diets nephropathogenic infectious bronchitis in 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gut-associatedbacteroides vulgatusmpk outer membrane vesicles blebbing contributes to b. vulgatus mpk-mediated immune response silencing evaluation of purine utilization by lactobacillus gasseri strains with potential to decrease the absorption of food-derived purines in the human intestine proteobacteria: microbial signature of dysbiosis in gut microbiota this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - vkigjv authors: osterrieder, nikolaus; bertzbach, luca d.; dietert, kristina; abdelgawad, azza; vladimirova, daria; kunec, dusan; hoffmann, donata; beer, martin; gruber, achim d.; trimpert, jakob title: age-dependent progression of sars-cov- infection in syrian hamsters date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: vkigjv in late , an outbreak of a severe respiratory disease caused by an emerging coronavirus, sars-cov- , resulted in high morbidity and mortality in infected humans. complete understanding of covid- , the multi-faceted disease caused by sars-cov- , requires suitable small animal models, as does the development and evaluation of vaccines and antivirals. since age-dependent differences of covid- were identified in humans, we compared the course of sars-cov- infection in young and aged syrian hamsters. we show that virus replication in the upper and lower respiratory tract was independent of the age of the animals. however, older hamsters exhibited more pronounced and consistent weight loss. in situ hybridization in the lungs identified viral rna in bronchial epithelium, alveolar epithelial cells type i and ii, and macrophages. histopathology revealed clear age-dependent differences, with young hamsters launching earlier and stronger immune cell influx than aged hamsters. the latter developed conspicuous alveolar and perivascular edema, indicating vascular leakage. in contrast, we observed rapid lung recovery at day after infection only in young hamsters. we propose that comparative assessment in young versus aged hamsters of sars-cov- vaccines and treatments may yield valuable information, as this small-animal model appears to mirror age-dependent differences in human patients. emerging coronaviruses have caused serious global public health concerns in the past two decades and cause infections that lead to severe respiratory and occasionally systemic disease [ ] . these include severe acute respiratory syndrome (sars)-cov as well as middle east respiratory syndrome (mers)-cov, both of which resulted in high morbidity and mortality in infected humans [ , ] . similar to other emerging cov, the novel sars-cov- likely arose from an ancestor in bats and amplified in a yet unknown animal reservoir before making its jump into the human population [ ] . sars-cov- has pushed global health systems to the brink of breakdown. the remarkably fast and unexpected spread of sars-cov- can be attributed to efficient replication in the upper respiratory tract and robust human-to-human transmission. while covid- is primarily a respiratory syndrome, it can induce quite variable clinical signs including fatigue, headache, and gastrointestinal symptoms, which can in severe cases result in fatality [ ] . it has become evident that differences in the type and severity of sars-cov- -induced disease and its sequelae seem to be strongly correlated with the age of patients and exacerbated by pre-existing medical conditions (e.g., chronic obstructive pulmonary disease, heart disease, diabetes, obesity) [ , [ ] [ ] [ ] . the availability of reliable animal models is of critical importance for pathogenesis studies as well as the development and preclinical evaluation of vaccines and therapeutics [ , , ] . for sars-cov- , the susceptibility of several animal species was predicted by in silico analysis based on comparisons of the entry receptor for sars-cov and sars-cov- , angiotensin converting enzyme (ace ). these predictions are of relevance because the ace sequence is deemed an important factor governing susceptibility [ ] . more specifically, the interaction of the viral spike (s) glycoprotein receptor binding domain with its ace counterpart was examined [ , ] , and in some cases confirmed in vivo [ ] . productive sars-cov- infection was shown in non-human primates, which developed respiratory disease recapitulating moderate disease as observed in humans [ ] [ ] [ ] [ ] . mice are not naturally susceptible to sars-cov- , but mouse-adapted virus strains have been developed and used in balb/c mice [ , ] . moreover, transgenic mice expressing human ace represent a lethal sars-cov- infection model resulting in significant weight loss and permitting robust virus replication in the respiratory tract including the lungs [ ] . ferrets have provided valuable data in the case of sars-cov [ , ] , and two studies describe the infection of ferrets with sars-cov- and successful transmission to in-contact animals without clinical signs [ , ] . first and preliminary studies also focused on the assessment of a syrian hamster model that had previously been used successfully in sars and mers research [ , , , ] . it was suggested that hamsters are highly susceptible, although they were reported to show no or only moderate respiratory signs and body weight losses. however, it is important to note that only young male hamsters of to weeks of age were used in these studies [ , ] . we sought to explore age-related differences in the course of sars-cov- infection in syrian hamsters and to establish a small-animal model that resembles the more severe sars-cov- -infection observed particularly in elderly patients. we conducted all animal work in compliance with relevant national and international guidelines for care and humane use of animals. the animal use protocol for the experiments reported here was approved by the landesamt für gesundheit und soziales in berlin, germany (approval number / ; approved on . . ). virus stocks were prepared from a previously published sars-cov- isolate (betacov/germany/bavpat / ) [ ] , which was kindly provided by drs. daniela niemeyer und christian drosten, charité berlin, germany. the isolate, referred to as sars-cov- münchen (sars-cov- m) [ ] , was handled under the appropriate safety precautions in a bsl- facility (freie universität berlin, institut für virologie) and propagated on vero e cells (atcc crl- ) in minimal essential medium (mem; pan biotech, aidenbach, germany) supplemented with % fetal bovine serum (pan biotech), iu/ml penicillin g and µg/ml streptomycin (carl roth, karlsruhe, germany). thirty-six -or -to- -week-old female and male syrian hamsters (mesocricetus auratus; breed rjhan:aura, janvier labs, saint-berthevin, france) were kept in individually ventilated cages (ivcs; tecniplast, buguggiate, italy) in an approved bsl- facility. ivcs were equipped with enrichment (carfil, oud-turnhout, belgium). all animals had unrestricted access to food and water and were allowed to acclimate to the conditions for seven days prior to infection. cage temperatures and relative humidities were recorded daily and ranged from - • c and - %, respectively. the -week-old hamsters were randomly distributed into two groups: mock (n = , -week-old) and young infected (n = , -week-old). the third group represents the aged infected hamsters (n = , - -week-old). iptt- transponders (biomedic data systems, seaford, de, usa) were subcutaneously implanted into all hamsters days prior to infection to allow the identification and monitoring of body temperatures. animals were mock-infected with µl medium from uninfected vero e cells or infected with × pfu sars-cov- m in µl by intranasal instillation. for transponder implantation, hamsters were sedated with butorphanol ( . mg/kg; cp-pharma, burgdorf, germany) and midazolam ( mg/kg; braun, melsungen, germany). for infections, hamsters were sedated with ketamine ( mg/kg; serumwerk bernburg, bernburg, germany) and midazolam ( mg/kg; braun). on , , and days post-infection (dpi), three randomly assigned hamsters of each group were euthanized by exsanguination under medetomidine ( . mg/kg; pharma-partner, hamburg, germany), midazolam ( mg/kg), and butorphanol ( . mg/kg) anesthesia [ ] . blood, nasal washes, bucco-laryngeal swabs, lungs (left and right), kidneys, spleens, duodenums, and blood sera were collected for (histo)pathological examinations and/or virus titrations, rt-qpcr, and serological examination. during the -day experiment, body temperatures, body weights, and clinical signs of all animals were monitored twice daily. animals that had a body weight loss of more than % weight over a h period were euthanized in compliance with the animal use protocol. such humane termination applies to the two hamsters euthanized dpi. for histopathology and in situ hybridization (ish), the left lung lobe was carefully removed, immersion-fixed in formalin, ph . , for h, embedded in paraffin, and cut in µm sections. for histopathology, slides were stained with hematoxylin and eosin (he) after dewaxing in xylene and rehydration in decreasing ethanol concentrations. lung sections were microscopically evaluated in a blinded fashion by a board-certified veterinary pathologist to assess the character and severity of pathologic lesions using lung-specific inflammation scoring parameters as described for other lung infection models before [ ] . three different scores were used that included the following parameters: ( ) lung inflammation score including severity of (i) interstitial pneumonia (ii) bronchitis, (iii) epithelial necrosis of bronchi and alveoli, and (iv) hyperplasia of type ii-alveolar epithelial cells; ( ) immune cell infiltration score taking into account the presence of (i) neutrophils, (ii) macrophages, and (iii) lymphocytes in the lungs as well as (iv) perivascular lymphocytic cuffing; and ( ) edema score including (i) alveolar edema and (ii) perivascular edema. ish was performed as reported previously [ ] using the viewrna™ ish tissue assay kit (invitrogen by thermo fisher scientific, darmstadt, germany) following the manufacturer's instructions with minor adjustments. probes for the detection of n gene rna of sars-cov- (ncbi database nc_ . , nucleotides , to , assay id: vpnkrhm) and the mouse housekeeping gene eukaryotic translation elongation factor- α (ef a; assay id: vb - -vt, affymetrix, inc., santa clara, ca, usa), which shares % sequence identity with the syrian hamster orthologue, were designed. lung sections ( µm thickness) on adhesive glass slides were dewaxed in xylol and dehydrated in ethanol. tissues were incubated at • c for min with subsequent protease digestion for min. sections were fixed with % paraformaldehyde in phosphate-buffered saline (alfa aesar, thermo fisher, kandel, germany) and hybridized with the probes. amplifier and label probe hybridizations were performed according to the manufacturer's instructions using fast red as the chromogen, followed by counterstaining with hematoxylin for s, washing in tap water for min, and mounting with roti ® -mount fluor-care dapi ( , -diaminidino- -phenylindole; carl roth). for negative and morphologically intact controls, lungs from uninfected hamsters of each group (n = ) were included. in addition, an irrelevant probe for the detection of pneumolysin was used as a negative control for unspecific reactions. he-stained and ish slides were analyzed and images were taken using an olympus bx microscope with a dp microscope digital camera and the cellsens™ imaging software, version . (olympus corporation, münster, germany). for the display of overviews of whole lung lobe sections, slides were automatically digitized using the aperio cs slide scanner (leica biosystems imaging inc., vista, ca, usa), and image files were generated using the image scope software (leica biosystems imaging inc.). the percentages of lung tissues affected by inflammation were determined histologically by an experienced board certified experimental veterinary pathologist (k.d.) as described previously [ ] . lung inflammation scores were determined as ( ) absent, ( ) minimal, ( ) mild, ( ) moderate, or ( ) severe, and quantified as described previously [ ] . immune cell influx scores and edema scores were rated from ( ) absent to ( ) sporadic, ( ) mild, ( ) moderate, or ( ) severe. ish signals were digitally quantified on scanned whole slides of each animal using the aperio positive pixel count algorithm (leica biosystems imaging inc.) with minor adjustments. specifically, a positivity score encompassing the number of positive pixels in relation to the total number of pixels per tissue scan as well as the total intensity of positive pixels were determined ( figure s ). for an assessment of virus titers from mg of lung tissue, tissue homogenates were serially diluted and plated on vero e cells in -well cell culture plates (sarstedt, nümbrecht, germany). at dpi, cells were fixed in % formalin, stained with . % crystal violet (in % methanol), and plaques were counted. rna was extracted from nasal washes and tracheal swabs with the rtp dna/rna virus mini kit (stratec, birkenfeld, germany) according to the manufacturer's instructions. the innuprep virus dna/rna kit (analytic jena, jena, germany) was used for rna extractions from tissue samples. viral rna was quantified using a one-step rt qpcr reaction with the neb luna universal probe one-step rt-qpcr (new england biolabs, ipswitch, ma, usa) and the -ncov rt-qpcr primers and probe (e_sarbeco) [ ] on a steponeplus realtime pcr system (thermo fisher scientific, waltham, ma, usa) according to the manufacturer's instructions. viral rna copies were then normalized to cellular rpl as previously described [ ] . all primers and probes are listed in table s . standard curves for absolute quantification were generated from serial dilutions of sars-cov- rna obtained from a full-length virus genome cloned as a bacterial artificial chromosome and propagated in e. coli or from serial dilutions of the purified hamster rpl- pcr product. the latter was generated by pcr using rpl- qpcr-primers (table s ) and a cdna template obtained from hamster lung tissue. in lung tissue, viral rna copies were calculated per × hamster rpl- transcripts. for serum neutralization assays, µl of medium containing . tissue culture infectious doses (tcid ) of sars-cov- m were mixed with µl of diluted serum. each sample was tested in triplicate. after h incubation at • c, the mixture was transferred to confluent vero e cells in a -well plate (sarstedt, nümbrecht, germany). viral replication was assessed after days by the detection of cytopathic effects. statistical analyses were performed using graph-pad prism v (graphpad software inc., san diego, ca, usa). the statistical details of all analyzed experiments can be found in the respective figure legend. for our experiments, we used a total of female and male syrian hamsters (mesocricetus auratus), which were either (n = ) or to weeks old (n = ). hamsters were kept in individually ventilated cages (ivcs) and randomly assigned to three groups: mock (n = , -week-old), young infected (n = , -week-old) and aged infected (n = , -to -week-old). animals were mock-infected with supernatants of cell culture medium taken from uninfected vero e cells or infected with × plaque-forming units of sars-cov- münchen (sars-cov- m; betacov/germany/ bavpat / ) [ ] . during the -day experiment, body temperatures, body weights, and clinical signs were recorded daily. animals were euthanized and sampled at different time points after infection to assess virus titers in various organs and to examine pathological changes in the lungs (figures and ) . each group (n = for , , and dpi, n = for dpi and n = for dpi; bar = . cm) (b) affected areas of inflammation were identified histologically for each lung and compared between the groups. (c) lung inflammation score taking into account (i) severity of pulmonary inflammation; (ii) bronchitis (iii) bronchial and alveolar necrosis; iv) hyperplasia of alveolar epithelial cells type ii. (d) immune cell influx score taking into account the infiltration of lung tissue with (i) neutrophils; (ii) macrophages; (iii) lymphocytes; (iv) perivascular lymphocytic cuffing; and (e) edema score including (i) alveolar and (ii) perivascular edema. scores and parameters in c to e were graded as absent ( ), minimal ( ), mild ( ), moderate ( ), or severe ( ) as described [ ] . (f) time-dependent distribution of sars-cov- rna signals in young and adult hamsters representative of each group as detected by in situ hybridization (group sizes as in a; for digital image analysis of the differences, see figure s ; bar = . cm). first, we observed age-dependent sars-cov- -induced body weight losses, with more pronounced weight reductions in aged compared to young hamsters ( figure a-c) . mean body weight losses peaked at to days post-infection (dpi), with partial recovery until dpi in both infected groups. there were no differences in body temperatures between the infected groups or between infected and mock-infected animals ( figure d ). next, we determined viral titers and sars-cov- rna copy numbers in various tissues by rt-qpcr [ , ] and performed virus titrations from lung homogenates using vero e cells ( figure e -h). results were similar between age groups and confirmed high viral loads in respiratory samples at early time points after infection, but a relatively rapid clearance of infection. while we did not identify any other sex-specific differences, it seems important to note that the rt-qpcr of blood samples revealed viremia in two male individuals from both age groups with viral rna copy numbers of > at and dpi, respectively ( figure i ). in these individuals, we also detected relatively high levels of viral rna in the spleen, kidneys, and duodenum, indicating a systemic spread of sars-cov- in some cases (table s ). to further investigate the potential dissemination of infection, we tested the aforementioned organs from all animals sacrificed at dpi and found them to be either negative for sars-cov- rna or to contain only low levels of viral rna (table s ). viral loads in bucco-laryngeal swabs and nasal washes appeared to be a reliable surrogate of the presence or absence of virus in the lungs in both age groups. the average loads ranged between and copies, respectively, at early times after infection, indicating that these sampling techniques can be used to monitor sars-cov- replication in syrian hamsters ( figure g,h) . at dpi, hamsters had mounted a humoral immune response as evidenced by relatively high titers of neutralizing antibodies. it is worth noting that antibody titers appear to be higher in young when compared to aged hamsters in the animals tested (table s ) . histopathology revealed clear age-dependent differences, with young hamsters launching an earlier and stronger immune cell influx into the lungs associated with a faster recovery than their aged counterparts (figure a-e) . at dpi, young hamsters developed a marked necro-suppurative bronchointerstitial pneumonia with strong alveolar and interstitial influx of neutrophils and macrophages as well as perivascular lymphocytic cuffing, which was much milder or absent in the aged group ( figure s a, right panel) . in contrast, only aged animals developed pronounced alveolar and perivascular edema indicating vascular leakage at dpi ( figure e and figure s c) . a more diffuse, severe bronchointerstitial pneumonia was similarly present in both groups at dpi with an onset of tissue regeneration, including hyperplasia of the bronchial epithelium ( figure s c , arrowhead) and type ii alveolar epithelial cells. only at the dpi time point was the arterial and venous endothelium of animals in both groups swollen and vacuolated, with necrotic endothelial cells separated from the underlying basement membrane by the presence of subendothelial lymphocytes and neutrophils ( figure s c, left) , which was consistent with what has been described as endothelialitis in human sars-cov- infection [ ] . at dpi, recovery as indicated by the marked hyperplasia of bronchial epithelial cells and type ii alveolar epithelial cells were seen in both groups ( figure s a ). interestingly, lung tissues had almost recovered in young hamsters at day , while the aged animals still had persistent tissue damage and active inflammation ( figure s b ). from dpi onwards, sars-cov- rna was detected by in situ hybridization in bronchial epithelial cells, debris in the bronchial lumen, alveolar epithelial cells type i and type ii, as well as macrophages in both groups, again with clear age-dependent differences over time ( figure f , figures s d and s ) . young animals had high amounts of viral rna in numerous bronchial epithelial cells and within the bronchial lumen that was accompanied by marked spreading through the lung parenchyma on and dpi. in contrast, aged animals had less virus rna present in the bronchi. we detected only a scattered pattern of infected bronchial epithelial cells and sporadic areas of parenchymal infection at and dpi. at dpi, viral rna was undetectable in the bronchi of young hamsters, and only small infected areas containing low levels of rna with a patchy distribution were detected. it is noteworthy that aged animals, at the same time after infection, had increased numbers of infected areas with a similarly patchy distribution throughout the lungs as well as copious amounts of viral rna associated with cellular debris in the bronchial lumen. using this technique, no viral rna was detected at dpi in either group. in summary, our study examined the suitability of a small animal model to study sars-cov- infections and expands on determining the role that age might play in the differences with respect to disease progression. intranasal infection of syrian hamsters resulted in weight loss and robust virus replication in the upper and lower respiratory tract. a limitation of this study is the lack of an age-matched mock group for the older hamsters, which we are unable to include for legal reasons as we are bound under german law to follow the r principle. we further demonstrate that aged -to -week-old hamsters experienced higher and more consistent weight loss after intranasal infection, while body temperatures and virus replication in upper airways and lungs were similar between both age groups. furthermore, we show that, using in situ hybridization, viral rna was detectable in bronchial epithelial cells, type i and type ii alveolar epithelial cells, and macrophages. all these cell types are potential targets of sars-cov- in human lung tissue; hence, the infection of hamsters of different ages seems to closely reflect what has been reported for human patients [ , ] . in contrast to sars-cov- titers, histopathological changes differed markedly between young and aged syrian hamsters over time: younger animals launched more severe reactions at early time points after infection, while lesions and inflammation in the lungs became more pronounced and widespread at later time points in the elderly. notably, age-related differences of sars-cov- infections have also been observed in non-human primates [ ] , and while this manuscript was under revision, in hamsters [ ] . the study by imai et al. also describes infections of syrian hamsters of different age groups, and they also observed 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immune responses of the coronavirus sars-cov- in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures syrian hamsters as a small animal model for sars-cov- infection and countermeasure development this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors acknowledge the excellent technical assistance by ann reum, annett neubert, and simon dökel. we would like to thank carfil inc. for their generous support of our animal husbandry. the authors declare no conflict of interest. key: cord- -ie ok lz authors: yeh, ming te; capponi, sara; catching, adam; bianco, simone; andino, raul title: mapping attenuation determinants in enterovirus-d date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ie ok lz enterovirus (ev)-d has been associated with epidemics in the united sates in , and . this study aims to identify potential viral virulence determinants. we found that neonatal type i interferon receptor knockout mice are susceptible to ev-d infection via intraperitoneal inoculation and were able to recapitulate the paralysis process observed in human disease. among the ev-d strains tested, strain us/mo- - caused no observable disease in this mouse model, whereas the other strains caused paralysis and death. sequence analysis revealed several conserved genetic changes among these virus strains: nucleotide positions and in the ′-untranslated region (utr); amino acid position in vp ; , , and in vp ; in a; in a. a series of chimeric and point-mutated infectious clones were constructed to identify viral elements responsible for the distinct virulence. a single amino acid change from isoleucine to valine at position in vp attenuated neurovirulence by reducing virus replication in the brain and spinal cord of infected mice. enterovirus d (ev-d ), previously known as rhinovirus [ ] , is a single-stranded, plus-sense rna virus belonging to the genus enterovirus of the picornaviridae family. the viral genome encodes a polyprotein with a single open reading frame with untranslated regions at both ends. secondary structures form an internal ribosome entry site (ires) in the -untranslated region (utr) and mediate virus translation [ ] . the polypeptide contains a p region that encodes the structural proteins (vp , vp , vp and vp ) and p and p regions that encode non-structural proteins ( a, b, c, a, b, c and d) that are important to virus replication. although genetically closer to enteroviruses, ev-d possesses key characteristics of rhinovirus: a lower optimal growth temperature at • c and acid sensitivity which limits its survival through the alimentary tract. ev-d is regarded as a respiratory pathogen that causes symptoms including sneezing, cough, runny nose and, in some cases, wheezing and difficulty breathing. ev-d was first isolated from children with pneumonia and bronchiolitis in california in [ ] but has remained silent for the past few decades. starting with sporadic clusters of acute respiratory infections around the world in the s [ ] [ ] [ ] [ ] , ev-d caused an outbreak with at least confirmed cases in the us in [ ] and was associated with acute flaccid paralysis [ ] . infections were also infectious cdna clones of ev-d strains , and have been banked in bei resources (catalog numbers: nr- , nr- and nr- ). all animal experiments were conducted in accordance with the guidelines of the laboratory animal center of national institutes of health. the institutional animal care and use committee of university of california, san francisco, approved all animal protocols on january , (approved protocol number an - a). c bl/ -derived tg mice with type i interferon receptor knockout (tg /ifnr-ko) [ ] were obtained from dr. satoshi koike (tokyo metropolitan institute of medical science, tokyo, japan). mice were bred in-house in sterilized cages and maintained in a / light cycle with standard chow diet in the aaalac-certified animal facility at ucsf. specific pathogen-free (spf), - -day-old mice were used in this study. ev-d viruses were obtained from bei resources (table ) and used to extract viral rna for cdna synthesis. viral rna was extracted with zr viral rna extraction kit (zymo research, irvine, ca, usa), following the manufacturer's protocol. to construct full-length infectious cdna clones of wild-type strain , rt-pcr was performed using the superscript iii one-step rt-pcr system with platinum taq dna polymerase (invitrogen, carlsbad, ca, usa) with primers fecori and rt sali to amplify viral genome. pcr product was gel-purified, digested with ecori and sali (new england biolabs, ipswich, ma, usa) and ligated to pre-digested pbr vector (new england biolabs, usa). for wild-type strains and , viral genomic fragments were amplified as described above, with primer sets f-infus/r and f/rt infus for strain and f-infus/r and f/rt infus for strain , and cloned into puc (new england biolabs, usa) by using in-fusion hd cloning kit (takara bio usa, mountain view, ca, usa). full-length sequences of the resulting infectious cdna clones were confirmed with sanger sequencing and used for our sequence analysis. sequences of primers are provided in table s in the supplementary materials. infectious cdna clones of chimeric ev-d were constructed by replacing -utr or p gene of strains nr- and with corresponding sequences of strain . briefly, viral gene fragments were pcr amplified from each wild-type infectious cdna clone, gel-purified and cloned into puc by using in-fusion hd cloning kit (takara bio, usa). nucleotide and amino acid variations between virulent and non-virulent strains were introduced into the infectious cdna clone of strain by pcr-mutagenesis. plasmid dna of strain nr- was pcr amplified with primers carrying desired nucleotide or amino acid change. pcr product was digested with dpni (new england biolabs, usa) and then transformed into sure competent cells (agilent technologies, santa clara, ca, usa). these sequence variations were introduced individually into infectious cdna clones of strain , except vp -k r and vp -g e, which were introduced together. infectious viral rna was transcribed from sali-linearized plasmid dna with megascript t transcription kit (invitrogen), following the manufacturer's instruction, and examined on a standard agarose gel for rna integrity. viral rna was electroporated into rd cells with a btx electroporator (btx, holliston, ma, usa) using the following settings: v, µf, Ω in a . cm electro-cuvette (btx). electroporated cells were incubated h at • c and then frozen at − • c and thawed at room temperature times to release the virus. cell lysate was cleared at × g for min under • c and supernatant was stored as passage (p ) virus stock. rd cells were infected with p virus at m.o.i. (multiplicity of infection) of . at • c for h and harvested as described above for the p virus used in this study. virus titers were determined by a standard tcid assay [ ] and calculated with the spearman and karber method, as previously described [ ] . full-length viral genomic sequence was confirmed with sanger sequencing. tg /ifnr-ko mice were intraperitoneally (i.p.) or intracranially (i.c.) inoculated with either wild-type, chimeric, mutant viruses or viral media as a mock control group. i.p. inoculation was performed by injecting µl of inoculum delivering × - × tcid of virus per mouse ( - mice per virus dosage). i.c. inoculation was performed to deliver × - × tcid of virus in a -µl inoculum per mouse ( - mice per virus dosage). mice of , and days old were used to test susceptibility to ev-d and days old for identification of virulence determinant. neurovirulence test for vp -i v mutant virus was performed with -day-old mice. infected mice were monitored daily for signs of disease onset including ruffled hair, hunched back, reduced mobility and paralysis. mice were euthanized upon appearance of paralysis on both posterior limbs, the humane endpoint. seven-day-old tg /ifnr-ko mice were i.p. inoculated with µl of inoculum carrying × tcid of wild-type or mutant ev-d virus. for i.c. inoculation, µl of inoculum was injected to deliver × tcid of wild-type or mutant ev-d virus. at days , and post-inoculation, mice from each virus group were euthanized, perfused with x pbs (ucsf ccf), and selected tissues were collected aseptically, weighted and stored at − • c. tissue samples were homogenized in viral medium, disrupted by freeze-thaw cycles and cleared at × g for min at • c. muscle, brain and spinal cord were harvested from i.p. and i.c. infected mice, while blood, spleen, heart, kidney, small intestine and liver were harvested only from i.p. inoculated mice. virus titer in cleared supernatant was determined by a standard tcid assay [ ] and calculated with the spearman and karber method, as previously described [ ] . rd, sk-n-sh and neuro- a cells were infected with wild-type or mutant ev-d at m.o.i. (multiplicity of infection) of . for h at • c and then covered with growth medium. cells were harvested at , , and h post-infection by freezing at − • c. cells were frozen and thawed times and cleared at × g for min under • c. virus titers in supernatant were determined by a standard tcid assay [ ] and calculated with the spearman and karber method, as previously described [ ] . experiment was performed in triplicate and results shown as mean with s.d. (standard deviation). data preparation and statistical analysis were performed with prism (graphpad). log-rank test was used to compare ev-d virulence in mouse model. student's t-test was performed to compare virus titers at collected time points. all multiple test correction was performed using the holm-sidak method [ ] . statistical significance was defined as a p value less than . . to carry out the simulations of the wild-type ev-d protomer (vp , vp , vp and vp ) and its i v mutant, we employed the atomic coordinates extracted from the human enterovirus d crystal structure (pdb id cv ) [ ] . a number of residues from the crystal structure [ ] in the beta sheets' connecting link were unresolved and we modeled these into the structure using charmm-gui software [ ] . we provided as input to charmm-gui software the pdb-format structure file downloaded from the protein data bank website (https://www.rcsb.org/); we patched the n-terminus of each capsid protein protomer with an acetylated (ace) group and the c-terminus with a standard c-terminus patching group and modeled the missing residues by checking a box in charmm-gui (charmm-gui uses galxyfill). to generate the mutated structure, we repeated this protocol and selected the "mutation" box within charmm-gui. we used charmm-gui [ ] for modeling the missing residues of the ev-d protomer crystal structure and for generating the i v mutant. we used the same procedure to simulate the two systems and described it in the following. we solvated the system and added ions to maintain charge neutrality. to minimize the system, we used the conjugate gradient algorithm for steps and gradually heated the simulated cell from to k. to equilibrate the system, we ran short nvt (constant number of particles n, volume v and temperature t) and short npt (constant number of particles n, pressure p and temperature t) simulations of nanosecond (ns) in which we applied harmonic restraints to the protein backbone, water and ions. such restraints were released gently during the consecutive runs. after the last run of the equilibration procedure, we carried out a simulation run of ns in the npt ensemble. all simulations were performed using namd . [ ] , with the charmm m force field for the protein and ions [ ] and the tip p model for water [ ] . we used a langevin dynamics scheme to keep the temperature constant at k and anisotropic coupling in conjunction with the nosé-hoover-langevin piston algorithm to keep the pressure constant at atm [ , ] . periodic boundary conditions were applied in three dimensions. we employed the smooth particle mesh ewald (pme) summation method to calculate the electrostatic interactions [ , ] and the short-range real-space interactions were cut off at Å using a switching function between and Å. the equations of motion were integrated with a time step of femtosecond (fs) for the long-range electrostatic forces, fs for the short-range non-bonded forces and fs for the bonded forces by means of a reversible, multiple time-step algorithm [ ] . the shake algorithm [ ] was used. coordinates were saved every picoseconds (ps). the simulations were visualized using vmd software [ ] . to test the susceptibility of tg /ifnr-ko mice to ev-d , -, -and -day-old mice were inoculated with various dosages ( × - × tcid per mouse) of ev-d strain (us/mo/ - ) via the i.p. route. survival of -day-old infected mice was % for all three tested virus doses, and a delayed onset of death was observed at the lowest dose ( × tcid ) ( figure a ). while × and × tcid of virus led to % and % survival for -day-old mice ( figure b ), only the highest dose, × tcid , of virus caused disease and a % survival in -day-old mice ( figure c ). the survival of infected mice correlated with mouse age and virus dosage. in addition, infected mice showed paralysis on hindlimbs ( figure d ), recapitulating the neurovirulence of ev-d . we also tested the susceptibility of -day-old, immunocompetent tg mice to ev-d and found no observable disease with an i.p. injection of up to × tcid of virus (data not shown). thus, neonatal tg /ifnr-ko mice are susceptible to ev-d and we set out to use this mouse model to study ev-d pathogenicity. to compare the virulence of our collected ev-d strains (listed in table ), -day-old tg /ifnr-ko mice were infected i.p. with × tcid of strains , , or ca/ - . strains , and ca/ - caused death between days and post-infection and resulted in a survival percentage between % and . % (figure a ). on the other hand, strain caused no observable disease in this mouse model. we also used replication analysis to clarify whether the non-pathogenic phenotype of strain was due to impaired replication ability and found that it replicated as efficiently as the pathogenic strains ( figure b ). although some significant titer differences were found between strains and at , and h; between and at h; between and at h, and between and at h, all these ev-d strains replicated relatively efficiently in rd cells, with~ log increase in virus titers after h of infection. thus, the non-pathogenic phenotype of strain in mice was not due to loss of replication ability. we then focused on mapping the viral genetic elements responsible for the distinct virulence. to identify the potential virulence determinant, sequences of nr- , nr- , nr- and ca/ - were aligned to reveal conserved genetic changes that we defined as nucleotides in -utr and amino acids in the coding region that are present only in the non-pathogenic strain but not in pathogenic ones (supplementary data s and s ). in the viral -utr, two positions were revealed as the conserved nucleotide difference: cytosine:uridine (pathogenic:non-pathogenic strain) at position and adenine/guanine:cytosine at position . in addition, seven conserved amino acid differences in four viral proteins were revealed. these conserved genetic changes are listed in table . table . conserved nucleotide and amino acid differences in virulent and non-virulent ev-d strains. glutamine, glycine or lysine (q, g, k) glutamic acid (e) to locate viral genetic elements causing the distinct virulence, we generated several chimeric ev-d viruses by swapping viral genes between the virulent strains and and the non-virulent strain and also viruses carrying various single mutations to better characterize their effect on virulence attenuation. we started with the -untranslated region (utr) and generated a chimeric virus, - utr- , in which the -utr of strain was replaced with the sequence from strain and also two viruses carrying single mutation, c u or g c, in the -utr with as backbone ( figure a ). replication analysis confirmed that all these engineered viruses showed log increase in virus titer, suggesting efficient replication in rd cells ( figure b ). the amount of injected virus was increased to × tcid to better identify attenuation determinant. by i.p. infecting -day-old tg /ifnr-ko mice with × tcid of virus, no mouse infected with , - utr- , -c u or -g c survived after day post-injection. we constructed infectious clones carrying the same swapped -utr and single mutation with as a template and generated viruses to repeat this experiment. we obtained similar results (data not shown), suggesting that the -utr of ev-d was not involved in the attenuation phenotype of ( figure c ). figure b . results of triplicates are shown as mean ± s.d. no significant difference in virus replication ability compared with strain was detected by student's t test (p < . ). (c) seven-day-old tg /ifnr-ko mice were i.p. inoculated with × tcid of virus and monitored for days to compare the virulence of the ′-utr-modified ev-d viruses. viruses , - utr ( - utr- ), - utr-u c and - utr-g c were tested with litters of pups. asterisk represents significantly different survival curve comparing with that of -infected mice detected by using log-rank test (p < . ). next, we examined the effect of amino acid variations in the coding regions on ev-d virulence by generating engineered ev-d viruses carrying swapped p or vp gene or the mutations described above ( figure a ). mutations in the non-structural region (nsr), a-t a and a-h r, were introduced to the pathogenic strain . cell-culture replication of the two these viruses was similar to the parental strains and ( figure b ). however, while - a-t a was as virulent as , around % of mice survived infection with - a-h r ( figure c ). figure b . results of triplicates are shown as mean ± s.d. no significant difference in virus replication ability compared with strain was detected by student's t test (p < . ). (c) seven-day-old tg /ifnr-ko mice were i.p. inoculated with × tcid of virus and monitored for days to compare the virulence of the -utr-modified ev-d viruses. viruses , - utr ( - utr- ), - utr-u c and - utr-g c were tested with litters of pups. asterisk represents significantly different survival curve comparing with that of -infected mice detected by using log-rank test (p < . ). next, we examined the effect of amino acid variations in the coding regions on ev-d virulence by generating engineered ev-d viruses carrying swapped p or vp gene or the mutations described above ( figure a ). mutations in the non-structural region (nsr), a-t a and a-h r, were introduced to the pathogenic strain . cell-culture replication of the two these viruses was similar to the parental strains and ( figure b ). however, while - a-t a was as virulent as , around % of mice survived infection with - a-h r ( figure c ). since multiple amino acid variations within the structural region of the ev-d genome were identified, we generated p or vp swapped viruses to test their effects on virulence ( figure a ). replication analysis with rd cells and a virulence test with tg /ifnr-ko mice were also performed, as described above. both the vp and p swapped viruses replicated efficiently in rd cells, with a - log increase in virus titers after h of infection, although virus titers were - log lower than for strains and at several time points ( figure b ). in addition, virus vp - had virulence comparable to in mice and caused % survival for the infected mice, and p - caused no disease, like ( figure c ). the attenuation by the p gene of was confirmed by swapping the p gene of : the resulting p - virus caused % survival and no observable disease in infected mice (data not shown). this result suggested that the mutation i v in vp is responsible for the attenuation phenotype of since vp swap had little effect on virulence. since multiple amino acid variations within the structural region of the ev-d genome were identified, we generated p or vp swapped viruses to test their effects on virulence ( figure a ). replication analysis with rd cells and a virulence test with tg /ifnr-ko mice were also performed, as described above. both the vp and p swapped viruses replicated efficiently in rd cells, with a - log increase in virus titers after h of infection, although virus titers were - log lower than for strains and at several time points ( figure b ). in addition, virus vp - had virulence comparable to in mice and caused % survival for the infected mice, and p - caused no disease, like ( figure c ). the attenuation by the p gene of was confirmed by swapping the p gene of : the resulting p - virus caused % survival and no observable disease in infected mice (data not shown). this result suggested that the mutation i v in vp is responsible for the attenuation phenotype of since vp swap had little effect on virulence. at later time points, -vp -l p replicated to titers comparable to that of at later time points, but no further increase in virus titer was observed for -k e/g e, suggesting its significant impact on virus replication ( figure d ). results from the mouse virulence test showed that mutations vp -v a and vp -k r/g e did not affect virulence as they caused death in % of infected mice within days. while mutation vp -l p allowed a % survival of infected mice, vp -i v did not cause disease, suggesting its role as major virulence determinant for ev-d in this mouse model ( figure e ). to further confirm the potent attenuation effect on virulence by the mutation vp -i v, a neurovirulence test was performed by injecting -day-old mice with or -vp -i v via the i.c. route. survival rate was % for mice infected with × tcid of and . % for the lower dose of × tcid of virus with delayed death. consistent with i.p. inoculation, -vp -i v caused no disease at both tested doses ( figure ). viruses , , x for peer review of its significant impact on virus replication ( figure d ). results from the mouse virulence test showed that mutations vp -v a and vp -k r/g e did not affect virulence as they caused death in % of infected mice within days. while mutation vp -l p allowed a % survival of infected mice, vp -i v did not cause disease, suggesting its role as major virulence determinant for ev-d in this mouse model ( figure e ). to further confirm the potent attenuation effect on virulence by the mutation vp -i v, a neurovirulence test was performed by injecting -day-old mice with or -vp -i v via the i.c. route. survival rate was % for mice infected with × tcid of and . % for the lower dose of × tcid of virus with delayed death. consistent with i.p. inoculation, -vp -i v caused no disease at both tested doses ( figure ). these results suggest that the amino acid change of vp -i v, vp -l p and a-h r reduced ev-d virulence in mice, and, among them, vp -i v showed the strongest effect. thus, we further characterized the vp -i v mutant. to determine why vp -i v attenuated ev-d virulence, the tissue distribution of the injected virus was examined by i.p. inoculating -day-old mice with × tcid of or -vp -i v virus. various tissues were collected at days , and post-infection for a tcid assay to determine virus titers. virus replicated efficiently, with an increase of . logs in muscle, . in spinal cord and ~ logs in brain during the days, but no significant virus replication was observed in other collected tissues ( figure a ). on the other hand, virus titers of -vp -i v increased . log in muscle, . log in spinal cord and was detected at a very low titer without notable increase in the brain ( figure a ). these results suggest that the amino acid change of vp -i v, vp -l p and a-h r reduced ev-d virulence in mice, and, among them, vp -i v showed the strongest effect. thus, we further characterized the vp -i v mutant. to determine why vp -i v attenuated ev-d virulence, the tissue distribution of the injected virus was examined by i.p. inoculating -day-old mice with × tcid of or -vp -i v virus. various tissues were collected at days , and post-infection for a tcid assay to determine virus titers. virus replicated efficiently, with an increase of . logs in muscle, . in spinal cord and~ logs in brain during the days, but no significant virus replication was observed in other collected tissues ( figure a ). on the other hand, virus titers of -vp -i v increased . log in muscle, . log in spinal cord and was detected at a very low titer without notable increase in the brain ( figure a ). to further clarify whether vp -i v mutation affected viral entry from peripherals to the central nervous system (cns) or viral ability to replicate in the cns, viruses or -vp -i v were i.c. injected directly into the brains of -day-old mice. brain, spinal cord and muscle were collected at days , and post-infection. virus replicated extensively, with an increase in virus titer of . logs in brain and . logs in spinal cord during the days, but it was detected at very low titer without a significant increase in muscle. virus -vp -i v was detected at very low titers in these tissues ( figure b ). results from tissue distribution analysis suggest that the vp -i v mutation reduced the replication ability of ev-d in mouse tissues and, more importantly, in the cns. to further clarify whether vp -i v mutation affected viral entry from peripherals to the central nervous system (cns) or viral ability to replicate in the cns, viruses or -vp -i v were i.c. injected directly into the brains of -day-old mice. brain, spinal cord and muscle were collected at days , and post-infection. virus replicated extensively, with an increase in virus titer of . logs in brain and . logs in spinal cord during the days, but it was detected at very low titer without a significant increase in muscle. virus -vp -i v was detected at very low titers in these tissues ( figure b ). results from tissue distribution analysis suggest that the vp -i v mutation reduced the replication ability of ev-d in mouse tissues and, more importantly, in the cns. since and the -vp -i v mutant showed distinct replication ability in mice, we examined their replication in cultured cells. rd (human rhabdosarcoma cell), sk-n-sh (human neuroblastoma cell) and neuro- a (mouse neuroblastoma cell) were infected at m.o.i. of . and harvested at , , and h post-infection for titration. the viruses replicated with comparable kinetics in rd, sk-n-sh and neuro- a cells, suggesting that the vp -i v mutation had no effect on the replication ability of ev-d in these tested cells (figure ). kinetics in rd, sk-n-sh and neuro- a cells, suggesting that the vp -i v mutation had no effect on the replication ability of ev-d in these tested cells (figure ). to gain insights into the reduced virulence of ev-d caused by the i v mutation in vp , we turned to full-atom molecular dynamics (md) simulations, which allow examination at atomistic resolution of the effects of this amino acid change on the structure. vp , vp and vp capsid proteins (protomers) associate to form a pentamer ( figure a) . the protomers share a wedge-shaped, eightstranded, antiparallel, beta-barrel topology where the connecting loops are structurally different, depending on the specific protomer to which they belong [ ] [ ] [ ] . vp -i is located in the cd loop of vp , between the c beta strand and the αa helix ( figure a ), in proximity to the interface between vp and vp of two different promoters and of the vp c-terminus. the i points inward, toward the αa helix and the cavity enclosed by the eight antiparallel beta strands ( figure b ). such geometry favors interactions with r in the αa helix and with f in the gh loop, which likely makes contact with vp of the adjacent protomer ( figure a, inset) . interestingly, r interacts with d and d , which are involved in the sialic acid binding site [ , ] . to identify the effects of the vp -i v mutation, we examined the simulated vp -i and vp -i v structures (see methods). to gather information on the dynamics, longer simulations are required; nonetheless, our simulations provide useful insights into structural changes upon mutating isoleucine to valine in vp . in figure c , we display the final frame of the simulations of vp -i (red) and vp -i v (pink) structures. compared to the vp -i structure, the mutated structure exhibits important changes in the cd and gh loops. in the cd loop, such changes cause d to point away from r , disrupting this interaction. the gh loop appears to move away from the c and h beta strands, breaking the interaction between k and y in the vp -i structure and forming a new one between d and n . similar conformational changes in the vp gh loop occur during particle expansion of the to gain insights into the reduced virulence of ev-d caused by the i v mutation in vp , we turned to full-atom molecular dynamics (md) simulations, which allow examination at atomistic resolution of the effects of this amino acid change on the structure. vp , vp and vp capsid proteins (protomers) associate to form a pentamer ( figure a) . the protomers share a wedge-shaped, eight-stranded, antiparallel, beta-barrel topology where the connecting loops are structurally different, depending on the specific protomer to which they belong [ ] [ ] [ ] . vp -i is located in the cd loop of vp , between the c beta strand and the αa helix ( figure a ), in proximity to the interface between vp and vp of two different promoters and of the vp c-terminus. the i points inward, toward the αa helix and the cavity enclosed by the eight antiparallel beta strands ( figure b ). such geometry favors interactions with r in the αa helix and with f in the gh loop, which likely makes contact with vp of the adjacent protomer ( figure a, inset) . interestingly, r interacts with d and d , which are involved in the sialic acid binding site [ , ] . to identify the effects of the vp -i v mutation, we examined the simulated vp -i and vp -i v structures (see methods). to gather information on the dynamics, longer simulations are required; nonetheless, our simulations provide useful insights into structural changes upon mutating isoleucine to valine in vp . in figure c , we display the final frame of the simulations of vp -i (red) and vp -i v (pink) structures. compared to the vp -i structure, the mutated structure exhibits important changes in the cd and gh loops. in the cd loop, such changes cause d to point away from r , disrupting this interaction. the gh loop appears to move away from the c and h beta strands, breaking the interaction between k and y in the vp -i structure and forming a new one between d and n . similar conformational changes in the vp gh loop occur during particle expansion of the fully native virion and may precede ev uncoating [ ] . finally, the distance between f and v is . Å greater than that between f and i , suggesting the major mobility of this residue in the mutated structure. overall, our structural analysis shows that vp i v mutation affects the network of interactions among protomer residues in an extremely sensitive region of vp . these structural alterations resemble the conformational changes during ev uncoating, thus suggesting that the i v mutation could prime the expansion and uncoating of the native virus prematurely and resulting in reduced virulence. fully native virion and may precede ev uncoating [ ] . finally, the distance between f and v is . Å greater than that between f and i , suggesting the major mobility of this residue in the mutated structure. overall, our structural analysis shows that vp i v mutation affects the network of interactions among protomer residues in an extremely sensitive region of vp . these structural alterations resemble the conformational changes during ev uncoating, thus suggesting that the i v mutation could prime the expansion and uncoating of the native virus prematurely and resulting in reduced virulence. in this study, we found that type i ifnr-ko mice were susceptible to ev-d infection and recapitulate the paralysis observed in severe human infection. we then determined that strain is non-pathogenic (as it causes no observable disease or paralysis) and that the other tested strains are pathogenic in this mouse model. sequence comparisons revealed several nucleotide and amino acid changes between the pathogenic and non-pathogenic ev-d strains. by constructing a series of infectious clones to generate chimeric and point-mutated ev-d viruses, an amino acid change at position in vp was shown to be the major virulence determinant for the attenuation phenotype, in addition to the other two weaker factors, vp -l p and a-h r. mutation vp -i v reduced virus replication in the cns of mice but showed little effect on that in cell lines, including rd, sk-n-sh and neuro- a cells. in this study, we found that type i ifnr-ko mice were susceptible to ev-d infection and recapitulate the paralysis observed in severe human infection. we then determined that strain is non-pathogenic (as it causes no observable disease or paralysis) and that the other tested strains are pathogenic in this mouse model. sequence comparisons revealed several nucleotide and amino acid changes between the pathogenic and non-pathogenic ev-d strains. by constructing a series of infectious clones to generate chimeric and point-mutated ev-d viruses, an amino acid change at position in vp was shown to be the major virulence determinant for the attenuation phenotype, in addition to the other two weaker factors, vp -l p and a-h r. mutation vp -i v reduced virus replication in the cns of mice but showed little effect on that in cell lines, including rd, sk-n-sh and neuro- a cells. we noticed some sequence discrepancies for us/mo/ - between our infectious cdna clone ( ) and the two records from genbank, km and mh . among them, two differences are relevant to our study: amino acid position in vp (vp - ) and position in vp (vp - ). the nucleotide sequence encoding the vp -v is "gta" in our infectious cdna clone and km but "ata" in mh ; the codon is "cca" for vp -p in our infectious cdna clone and mh but "cta" in km . since we found that the amino acid change from isoleucine to valine at vp - (vp -i v) and leucine to proline at vp - (vp -l p) attenuates ev-d virulence in our mouse model, this sequence discrepancy may cause confusion. we constructed infectious cdna clones using viral rna extracted from viruses from bei resources without further passage in cell culture and our approach did not involve modifications at these two positions, so we believe it is highly unlikely that the two variations were introduced during the cloning. thus, we continued our study with the infectious clones constructed. our finding of vp -i v as a major virulence determinant for ev-d in mice is consistent with a recent report in which a non-pathogenic ev-d strain us/mo/ - ( in our study) became pathogenic after passages in ag mice [ ] . a change from valine to isoleucine at amino acid in vp in the mouse-adapted virus was reported as one of the potential mutations contributing to the increased virulence. here, we demonstrate that, by changing the same amino acid of a pathogenic strain to yield an attenuated mutant virus, another potential virulence determinant vp -l p was revealed in their mouse adaptation and our virulence test. however, we found the nucleotides coding vp -p as "cca" in our infectious clone , which is different from "cta" that encodes leucine in the genbank sequence (accession number: km ), as discussed above. this vp -l p mutation was found in the mouse-adapted with increased virulence in mice, but the same vp -l p in our study partially attenuated the virulence of strain in mice. we also examined the prevalence of this vp -i v in clinical isolates by analyzing ev-d sequences from the virus pathogen resource (vipr) [ ] and found that of the collected sequences have isoleucine but only two have valine at position of vp . as viruses face higher selection pressures when disseminating from tissues to tissues in infected animals, the dominant mutations in the adapted virus population may help to increase viral fitness, adapt quickly to different micro-environments or antagonize immune responses in vivo. analysis of the structure through the simulations showed that the mutation of isoleucine to valine in vp caused important rearrangements in the cd and gh loops and broke the native network of interactions among vp residues in a region that is crucial for viral activities because it has been identified as the sialic acid binding site [ , ] . in the work by y. liu et al. [ ] , the crystal structure of ev-d , in association with sialylated glycan receptor analogues, shows that the sialic acid moiety of these ligands binds to the virus canyon by making interactions with several residues located in proximity to i and causes conformational changes in the loops connecting the sialic acid binding site and the vp hydrophobic pocket and the canyon, apparently determining the expulsion of the pocket factor. in a more recent study [ ] , the authors identified sulfated glycosaminoglycans as receptors in absence of sialylated glycans and, by performing structural analysis, confirmed the binding site of the sialic acid in the canyon and the displacement of the pocket factor. our simulations show that i is located right under the sialic acid binding site, and the structural changes caused by the i v mutation likely facilitate the accommodation of the sialic acid and reduce the enthalpic cost of its binding. interestingly, the motion of the vp cd loop along with that of the vp gh loop and vp gh loop upon binding of sialylated receptor analogues provokes displacement of the pocket factor site, the mechanism suggested to be initiated by the electrostatic interactions between q and the n-acetylneuraminic acid [ ] . even if our systems lack the pocket factor, the conformational changes in the simulated vp -i v structure with respect to the vp -i structure suggest that the i v mutation destabilizes the virus in a similar way when sialic acid binds and the pocket factor is ejected to prime the virus for uncoating [ ] . in addition, cryo-em information suggests that the vp gh loop motion can be considered one of the molecular determinants involved in the process of ev uncoating [ ] . our structural analysis shows that the i v mutation affects the native structure of the vp gh loop in a manner consistent with our experimental results. however, because the vp gh loop is located at one of the protomer corners in contact with vp of another protomer and at the interface between two different pentamers, longer simulations with at least two pentamers would be required in order to fully examine the loop dynamics and its effects on the interactions between pentamers and to formulate broad conclusions on the whole ev uncoating process. capsid proteins of ev-d may have undiscovered functions or interactions with host proteins that affect virus replication, infectivity or virulence [ , ] . another proposed mechanism of interaction involves the neuro-expressed intracellular adhesion molecule (icam- ). icam- may be required for successful ev-d infection [ ] . the pre- fermon strain requires sialic acid, but the strains and (us/ky/ - , not included in this study) can infect cells in the absence of sialic acid [ ] . icam- neutralizes ev-d in vitro by catalyzing the conversion of mature virions to a-particles [ ] . while attempts to determine the structure of the ev-d -icam- complex have been unsuccessful, structures of the complex of ev-d and neutralizing antibodies that also precipitate the a-particle state bind with the bc, de, ef and hi loops of vp [ ] . both ev-d strains and grow in neuro- a cells with or without neuraminidase treatment, which removes sialic acid from the cell surface, suggesting that differential virulence in mouse models may be due to another cellular factor [ ] . if cell factors, such as icam- , are involved in neurovirulence, there may be functional differences in the interaction between murine and human icam- and their interactions with varying strains of ev-d that determine successful infection. this interaction may be blocked by the vp -i v and thus prevent virus replication in mice. to validate this hypothesis, expression levels of viral proteins and viral rna copy number in mouse tissues may be useful indicators. if this hypothesis holds, identification of this host factor may be important as it could serve as a target for antiviral development. the combination of reverse genetics and a small animal model provides a straightforward approach for the ferreting of virulence determinants of pathogens. as the severe acute respiratory syndrome coronavirus (sars-cov- ) is devastating the globe, knowledge obtained from such studies can be valuable. rapid reconstruction of sars-cov- [ ] and susceptible receptor (human angiotensin-converting enzyme , hace )-transgenic mouse models have been reported [ , ] , making such studies possible. however, difficulties are expected. the sars-cov- strain hb- causes only slight bristled fur and body weight loss in hace transgenic mice but not other clinical symptoms [ ] . if a sars-cov- strain that causes virulence greater than the hb- strain is not available, an alternative can be to generate mouse-adapted viruses that display increased virulence [ ] [ ] [ ] . in addition, bioinformatic analysis has been applied to elucidate potential viral determinants for pathogenicity. similar to virulence, gussow et al. analyzed coronaviruses of high and low case fatality rate (cfr), and reported features of enhanced nuclear localization signal (nls) in the nucleocapsids and insertions in the spike protein as shared by the high cfr coronaviruses [ ] . results from such bioinformatic analysis could provide valuable candidates to be further validated in animal models. even with these limitations, knowledge obtained from such studies could still help to further our understanding of viral pathogenicity and thus promote the development of antivirals and vaccines. in conclusion, we report an amino acid change from isoleucine to valine at position in vp of ev-d as a major virulence determinant in a type i ifnr-deficient mouse model. this vp -i v mutation reduces ev-d virulence and virus replication in the brain and spinal cord of mice but has little effect on virus replication in cultured cells. the vp -i v completely attenuated the pathogenic strain by improving the survival rate for infected mice from % to % and also removed its neurovirulence, based on results from tissue distribution analysis. however, virus replication in cell lines, including human muscular and neuronal cells and mouse neuronal cells, was not affected by the vp -i v. our structural analysis shows that the i v mutation generates conformational changes in the native structure that are consistent with structural intermediates observed experimentally during the ev uncoating process. however, the mechanism for vp -i v attenuation in mice remains unknown. one clear difference between cell culture and infection in animals is the immune system. while we used animals that were defective in type i inf responses, vp -i could also suppress other arms of the antiviral defense mechanism. further studies are required in order to determine the mechanism of action that determines neurovirulence. in addition, infectious cdna clones for ev-d constructed in this study can be great tools for evaluating the effect of individual mutation on virus phenotypes. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . table s : primer sequence for the construction of infectious cdna clones, data s : alignment of -utr sequences, data s : alignment of polypeptide sequences. human rhinovirus and enterovirus represent a unique serotype with rhinovirus and enterovirus features evolutionary and functional diversity of the untranslated region of enterovirus d : increased activity of the internal ribosome entry site of viral strains during the s. viruses a probable new human picornavirus associated with respiratory diseases emergence and epidemic occurrence of enterovirus respiratory infections in the netherlands in enterovirus among children with severe acute respiratory infection, the philippines molecular and epidemiological study of enterovirus d in taiwan detection and whole genome sequence analysis of an enterovirus cluster acute flaccid myelitis associated with enterovirus d : a review current status of enterovirus d worldwide and in taiwan systematic community-and hospital-based surveillance for enterovirus-d in three canadian provinces epidemiological and clinical characteristics of patients infected with enterovirus d high frequency of enterovirus d in children hospitalised with respiratory illness in norway enterovirus d infection in a cluster of children with acute flaccid myelitis upsurge of enterovirus d , the netherlands the emergence of enterovirus d in england in autumn and the necessity for reinforcing enterovirus respiratory screening low-level circulation of enterovirus d -associated acute respiratory infections continued biennial circulation of enterovirus d in colorado enterovirus d subclade b strain circulating and causing an outbreak in the united states in enterovirus d -associated respiratory and neurological illness in spain enterovirus d seroepidemiology in taiwan an increase in reports of acute flaccid paralysis (afp) in the united kingdom nasal infection of enterovirus d 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charmm m: an improved force field for folded and intrinsically disordered proteins comparison of simple potential functions for simulating liquid water constant pressure molecular dynamics simulation: the langevin piston method constant pressure molecular dynamics algorithms particle mesh ewald: an n·log(n) method for ewald sums in large systems a smooth particle mesh ewald method generalized verlet algorithm for efficient molecular dynamics simulations with long-range interactions numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes vmd: visual molecular dynamics structure of a human common cold virus and functional relationship to other picornaviruses three-dimensional structure of poliovirus at . a resolution structure and inhibition of ev-d , a virus that causes respiratory illness in children sialic acid-dependent cell entry of human enterovirus d molecular basis for the acid-initiated uncoating of human enterovirus d vipr: an open bioinformatics database and analysis resource for virology research mutations in vp and vp capsid proteins increase infectivity and mouse lethality of enterovirus by virus binding and rna accumulation enhancement foot-and-mouth disease virus capsid protein vp interacts with host ribosomal protein sa to maintain activation of the mapk signal pathway and promote virus replication icam- /telencephalin is a functional entry receptor for enterovirus d atomic structures of enterovirus d in complex with two monoclonal antibodies define distinct mechanisms of viral neutralization neurotropism of enterovirus d isolates is independent of sialic acid and is not a recently acquired phenotype rapid reconstruction of sars-cov- using a synthetic genomics platform the pathogenicity of sars-cov- in hace transgenic mice infectivity, virulence, pathogenicity, host-pathogen interactions of sars and sars-cov- in experimental animals: a systematic review mouse-adapted mers coronavirus causes lethal lung disease in human dpp knockin mice a single nucleotide in stem loop ii of -untranslated region contributes to virulence of enterovirus in mice a mouse-adapted enterovirus strain causes neurological disease in mice after oral infection genomic determinants of pathogenicity in sars-cov- and other human coronaviruses this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank charles chiu for providing ev-d strain ca/ . we also thank members of the andino lab for helpful discussion regarding this project. the authors declare no conflict of interest. key: cord- -xbwilp w authors: kin, nathalie; miszczak, fabien; lin, wei; ar gouilh, meriadeg; vabret, astrid title: genomic analysis of human coronaviruses oc (hcov-oc s) circulating in france from to reveals a high intra-specific diversity with new recombinant genotypes date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: xbwilp w human coronavirus oc (hcov-oc ) is one of five currently circulating human coronaviruses responsible for respiratory infections. like all coronaviruses, it is characterized by its genome’s high plasticity. the objectives of the current study were to detect genetically distinct genotypes and eventually recombinant genotypes in samples collected in lower normandy between and . to this end, we sequenced complete nsp , s, and n genes of molecular isolates of hcov-oc from clinical samples and compared them to available data from the usa, belgium, and hong-kong. a new cluster e was invariably detected from nsp , s, and n data while the analysis of nsp and n genes revealed the existence of new f and g clusters respectively. the association of these different clusters of genes in our specimens led to the description of thirteen genetically distinct genotypes, among which eight recombinant viruses were discovered. identification of these recombinant viruses, together with temporal analysis and tmrca estimation, provides important information for understanding the dynamics of the evolution of these epidemic coronaviruses. ay ), coding for the glomerular part of the s protein (sub-unit s ) of hcov-oc s from clinical samples collected in at the university hospital of caen. they observed four genetically distinct subgroups. one of the subgroups constitutes an outlier group located between hcov-oc and bovine coronavirus (bcov), containing a -nucleotide deletion in common with bcov but not with other hcov-oc s [ ] . more recently, four genetically distinct hcov-oc genotypes have been identified from respiratory specimens sampled at the public hospital of china over a -year period ( to ) [ ] . in this study, lau et al. based their genotype definition upon the complete sequence of nsp , s and n genes [ ] . genotype a contains the prototype vr strain isolated in . genotypes b and c are two naturally circulating hcov-oc s. genotype d is a recombinant virus of genotypes b and c, obtained from a specimen from [ ] . based on a bootscan analysis of the complete genome of the hcov-oc s belonging to the circulating genotypes b, c, and d, it was assumed that a hot spot was likely located between the nsp and s genes, more precisely at the nsp /nsp junction. our objective was to investigate the presence, the temporal distribution and the recombination patterns of hcov-oc in lower normandy over years ( to ), using the methodology and the hcov-oc genotype definition elaborated by lau et al. in [ ] . this study focuses on the sequences of the nsp , s, and n genes of hcov-oc s detected in respiratory specimens sampled from to . of the hcov-oc s and the prototype strain vr , six, eight, and three overlapping sequences were obtained for the nsp , s, and n genes, respectively. after assembly, these overlapping fragments encompassed the full nsp , s, and n genes. in this study, all hcov-oc s including the vr prototype strain are associated with three accession numbers in genbank, for nsp , s, and n genes ( table ). the sequences of hcov-oc s were named according to the following nomenclature: virus/fra-epi/location of sampling/year of sampling/specimen number. epi is an abbreviation for epicorem consortium. for the prototype strain, the specimen number is replaced by vr . the phylogenetic analysis was conducted by comparing the topology of the three phylogenetic trees obtained from the nsp , s, and n genes. figure shows the three trees obtained by the neighbor joining method [ ] . on each tree, five to six clusters were observed, including an outlier group compounded with the three bcovs sequences mebus, kakegawa, and quebec that root hcov-oc sequences [ ] [ ] [ ] . the clustering is supported with high bootstrap values. we used part of the nomenclature of genotypes proposed by lau et al. in [ ] . these authors described the genotypes a, b, c, and d from nsp , s and n genes. we used this nomenclature to name each genotype using three letters corresponding to clusters in which the different sequences are located in nsp , s, and n trees, respectively. following this nomenclature, genotype d is a recombinant genotype b/c/c. the three previously described clusters-a, b, and c-are observed in our three gene trees in addition to a newly described cluster e. the nsp tree depicts a new cluster we named "f" that roots all other hcov-oc s. according to the n gene, a sixth cluster we named "g" separates from cluster e and roots clusters a, b, and c. among the set of sequences obtained from the hcov-oc s of our study, three are distributed in two non-recombinant genotypes as follows: the vr sequence belongs to the genotype aaa; hcov-oc /fra-epi/caen/ / and hcov-oc /fra-epi/caen/ / belong to genotype bbb. the other sets of sequences are allocated among six recombinant genotypes as follows: five genotypes bcc, defined as genotype d by lau et al.; two genotypes cee, three genotypes cbe, one genotype bcg, one genotype cce, and one genotype ceb. among the american sets of sequences, seven are distributed into two non-recombinant genotypes as follows: one ccc and six eee. the seven other sets of sequences are distributed among four recombinant genotypes as follows: three genotypes cca, two genotypes fcb, one genotype ccb, and one genotype cee. among the two sets of sequences from hong-kong, one is a non-recombinant genotype ccc and the other is a recombinant genotype bcc. among the two hcov-oc s from belgium, one is a non-recombinant genotype bbb and the other is a recombinant genotype bcc. finally, the two last sequences of hcov-oc vr (accession number: ay and nc ) belong to the non-recombinant genotype aaa [ , ] . the results of the phylogenetic analysis are summarized in table . the two alignments constructed from the dated sequences of s and n genes have been used to set up a molecular clock calibration in order to estimate the date of emergence of mean clusters. figure shows bayesian trees inferred from the alignments of s and n genes. the dates of emergence of the clusters and the corresponding % highest posterior density ( % hpd) are indicated. based on the sequence data of the s gene, the tmrca of bcov and hcov-oc is estimated in , with a % hpd from to . for the hcov-oc , cluster e is predicted to have emerged in ( % hpd to ), and genotype a is estimated to have emerged in ( % hpd, to ). genotypes b and c are predicted to have emerged from their tmrca in ( % hpd, to ). the molecular clock conducted from sequence data of n gene allows us to estimate the trmca of bcov and hcov-oc in ( % hpd, to ). genotypes e and a are predicted to have emerged in ( % hpd, to ) and ( % hpd, to ), respectively. genotypes b and c are predicted to have emerged from their tmrca in ( % hpd, to ). phylogenetic analysis of the complete nsp , s, and n genes of the hcov-oc strains and bcov strains. the phylogenetic trees were constructed by the neighbor joining method [ ] . bootstrap values were calculated from replicates. bootstrap values over % are shown [ ] . the evolutionary distances were computed using the kimura -parameter method (kimura) and units are the number of base substitutions per site. evolutionary analyses were conducted in mega, version . . [ ] . blue triangle, isolates from caen; red circle, isolates from usa; green circle, isolates from hong-kong; black diamond, isolates from belgium; purple square, atcc-vr strains, empty black square, bcov strains. results of bayesian analysis based on s and n gene sequence data. inferences were calculated with the one parametric coalescent model with a constant size, under the tn +g substitution parameter, using the beast package (version . . ) [ ] . the length of mcmc was fixed at states for s and n genes. the dates of emergence of mean clusters are indicated, associated with the % hpd. the hcovs-oc , e, nl , and hku -belong to the viral molecular panel tested during the virological routine diagnostics of acute respiratory infections in humans. among these four circulating hcovs, oc and nl seem to show the greatest prevalence and incidence. these viruses also proved to be the viruses encountered at the earliest age of childhood [ ] . these hcovs circulate widely in epidemic form in the general population, causing infections that are most often benign. infants, immunosuppressed patients, and very elderly patients may however develop very severe pathologies. these four circulating hcovs must be distinguished from the emerging hcovs, sars-cov, and mers-cov, causes of much graver and potentially fatal respiratory pathologies. control of the latter hcovs requires the implementation of international health measures [ ] [ ] [ ] . the evolutionary potential of coronaviruses brings into play point mutations and recombination events. such genetic diversity generated in this way may have an impact on the performance of molecular detection techniques used in the virological diagnostic process. the study of intra-specific diversity is thus useful in the monitoring of means of detection. specifically, it allows for the detection of new circulating variants, and provides information about the evolutionary dynamics of the family of respiratory viruses being monitored, with special attention given to coronaviruses. our study focuses solely on the hcov-oc . the first analyses of the hcov-oc s gene were conducted in and revealed a potential spatial and temporal distribution of genetic clusters [ ] . in , s. lau and his colleagues were the first to propose a genotypic classification of hcov-oc from the complete sequencing of three genes: nsp (rdrp), s (spike), and n (nucleocapsid). four genotypes-a, b, c, and d-were identified: genotype a corresponds to the sequences of the hcov-oc prototype strain vr , adapted to culture cell line hrt- (human adenocarsinoma rectal) or rd (rhabdomyosarcoma); genotypes b and c correspond to contemporary circulating strains, and genotype d is described as a recombinant b/c virus. this study was performed on hcov-oc s detected in respiratory samples collected in hong kong over a period of years ( to ), in a non-homogenous temporal distribution. more precisely, the majority of these samples ( of ) were collected in , and the others ( of ) fall between and [ ] . our study defines the genotype of hcov-oc s detected from upper respiratory samples frozen at − °c, collected over a period of years from to at the rate of one to two samples per year. the hcov-oc s were detected in patients hospitalized in our university hospital. of these patients, were male and five were female, at ages from months to years. the symptomatology proved variable ( table ). the infections were essentially located in upper and lower respiratory airways. the presence of associated gastrointestinal symptoms in some patients should be noted. the prototype strain hcov-oc atcc vr , grown on cell line hrt- , was used as a control, allowing for the validation of the methodology used by lau et al. [ ] . this methodology had to be adapted since some of the primers published by the authors did not allow for amplification of all the hcov-oc s. of these sequences, we selected that reflect the genetic diversity and temporal distribution of the whole, and we introduced them into the alignment. in the end, three respective alignments of complete genes-nsp , s, and n-of hcov-oc s identified in three different regions of the world (hong kong, europe, and the usa) over a period of years (from to ) were generated and analyzed. they served as the basis for the construction of phylogenetic trees. within the three phylogenetic trees-nsp , s, and n-we identified six different clusters named a, b, c, e, f, and g. a comparative study of the topology of phylogenetic trees corresponding to several genes of the same virus is often used in phylogeny to define recombinant viruses [ , , ] . we thus compared the topology of nsp , s, and n trees by observing the contextual position of the corresponding sequences of the same hcov-oc molecular isolate. the complexity of the results of our analysis led us to establish a nomenclature allowing for the simplest expression of the genotypes: each genotype is expressed in the form of a three-letter code, linking each one to the member cluster of the different nsp , s, and n sequences. from here, we defined different genotypes, of which four were non-recombinant (aaa, bbb, ccc, and eee). the nine remaining genotypes correspond to different recombinant genotypes (table ) . among the hcov-oc sequences examined in this study, hcov-oc s of so-called non-recombinant genotypes were found: three hcov-oc s of genotype aaa corresponding to prototype strains vr ; three hcov-oc s of genotype bbb (origin, france and , and belgium ); two hcov-oc s of genotype ccc (origin, usa and hong kong ); and lastly, six hcov-oc s of genotype eee, all originating in the usa, and having circulated from to . the foremost important point to underline is that these results suggest a high level of recombination among circulating hcov-oc epidemic strains. the limited number of sequences studied in time and space does not allow for conclusive evidence of a possible temporal and spatial distribution of different genotypes hcov-oc . however, it should be noted that the e cluster has not yet been described. in the present study, it is identified only in hcov-oc sequences from the usa and france and data suggest that it has been circulating since the s after being the first known group to differentiate from bcov. the analysis of alignments shows that cluster e is characterized by a deletion of nucleotides in the s gene, more precisely in the s gene that codes the glomerular part of the s protein (position , - , , genbank accession number nc_ ). this deletion of nucleotides in the coding phase results in a deletion of amino acids, tqdg, in the corresponding protein sequence. this deletion was also present in the bovine coronavirus strains used in the alignment, as shown in figure . the analysis of s bcov sequences available in genbank shows that this deletion is expressed in all bcovs, and described in bovine coronaviruses associated with both enteric and respiratory tropisms. this deletion is therefore not a specific marker of viral tropism. it is located in the lectin domain of the s protein of bcov, and is involved in the attachment of the virus to the cellular receptor, identified as a derivative of neuraminic acid (n-acetyl- -o-acetylneuraminic acid or neu , ac ). no protein receptor has yet been described for the bcov or, more broadly, for betacoronavirus bovine-like clade a. we analyzed all the sequences of this s hcov-oc region available in genbank, for a total of sequences, and the deletion of nucleotides is found in only of them. of these sequences, correspond to hcov-oc s detected in respiratory samples from the usa and sequenced by town et al. four correspond to hcov-oc s detected in france (three in the current study, and one published by our team in ); two correspond to hcov-oc sequences deposited by browlin et al. under patent in the united kingdom (ep and wo ), and finally, the remaining two correspond to hcov-oc s adapted to vero cells and mdck i, published in by f. künkel and herrler g [ ] . the hcov-oc e cluster is phylogenetically close to the bcov cluster, and the presence of the genetic marker common to these two clusters provides an argument supporting the hypothesis put forth by vijgen in that would identify a crossing of the species barrier occurring from cattle to human [ ] . a molecular clock analysis was inferred using the beast software version . . (http://beast.bio.ed.ac.uk) based on sequence alignments of s and n genes. sequences of the nsp gene were not used because they were uninformative due to the low variability of this gene. results were compared with those previously obtained by vijgen from betacoronavirus clade a sequences (nine bcovs, three phevs, and eight hcov-oc s). it should be noted that eight sets of hcov-oc sequences used in the analysis of in , wertheim et al. demonstrated that a strong purifying selection could lead to the underestimation of the evolutionary rate of coronaviruses. in this study, the authors proposed an alternative theory of origin for all coronaviruses. they estimate the tmrca of coronaviruses infecting mammalian species (alpha-and betacoronavirus genus) and avian species (gamma-and deltacoronavirus genus) at about million years, as opposed to , years as previously estimated [ ] . these authors found a general agreement in the inferred branch lengths between evolutionary models for the shorter branches (inferior to . substitution per site). as expected in the phylogeny of two species of the same coronavirus genus involved in a relatively recent zoonotic transmission event, all the branches of neighbor joining and bayesian trees were shorter than . substitutions per site, as shown in figures and . it can be speculated that the phylogenetic analyses presented in this study did not underestimate the date of emergence of the different clusters nor the estimation of tmrca for hcov-oc and bcov. the molecular clock analysis indicated that hcov-oc and bcov strains have emerged from their tmrca around ( - ), which was consistent with the data published by vijgen et al. in [ ] . the emergence of cluster e, characterized by a tqgd amino acid deletion, is estimated at around , and would have occurred at the same time as that of cluster a, in the - time range. the cluster a includes the first hcov-oc strain identified in from a respiratory sample [ ] . the b and c clusters would have circulated later, since the s. the molecular clock results therefore suggest that, since their emergence in the late nineteenth century, the evolution of circulating hcov-oc epidemic strains would have been marked by the occurrence of an insertion of four amino acids, tqdg, in the n-terminal region of the s attachment protein, as opposed to the occurrence of the deletion of these four amino acids. the pursuit of this study should lead to a means of determining the functional impact of this insertion, especially with regard to tropism and the clinical expression of infection by hcov-oc . note that the frequent presence of gastrointestinal symptoms in the clinical landscape of hcov-oc respiratory infections has yet to be accounted for. in , hu et al. studied the genetic diversity of the hcov-oc circulating in beijing, china [ ] . the authors performed partial sequencing of the s and n genes ( , - , and , - , for s and n respectively, using the hcov-oc sequence associated with the accession number nc_ as reference) of hcov-oc s detected in the respiratory samples, all taken in . they included the sequences published by lau et al. in their analysis in , and used the nomenclature established by these same authors to identify various clusters obtained [ , ] . in addition to the a and b clusters, they identified other groups, designated unt , unt(b), unt(c/d), and (c/d), respectively. several clusters were found to correspond to those previously described by lau et al. in [ ] . the unt(b) cluster or "untyped b" is a group close to the b cluster. the c/d cluster corresponds to the c cluster, named "c/d", due to the presence of the hk _ strain defined by lau et al. as a genotype d, referring to a recombination virus belonging to the b cluster on the nsp sequence and to the c cluster on the s and n sequences [ ] . the unt(c/d) cluster or "untyped (c/d)" corresponds to a group close to the preceding cluster. finally, the unt cluster or "untyped ", which is close to the bcov sequences included in this analysis, might correspond to the e cluster describe in the current study. in the report by hu et al. ( ) , the unt cluster includes only five hcov-oc s (accession numbers z , hc , cq , dd , and z ) [ ] . we have verified that they all have the characteristic nucleotide deletion of nucleotides. this very recent report supports the results determined in the current study in that they both demonstrate a high genetic diversity of circulating hcov-oc s. the present study highlights the difficulty of investigating intraspecific genetic variability of hcov-oc s, though the analysis of partial viral genomes (complete genes) allows the description of clear recombination patterns. nevertheless, due to the high evolutionary potential of hcov-oc , analyzing complete genomes would reveal a more detailed and perhaps more complex recombination pattern. in addition, the data reported and analyzed here illustrate the dynamic diversification of hcov-oc following its emergence in human. this remarkable diversification contrasts with the relative stability of bcov in cattle (unpublished data). the present study therefore draws attention to the importance of increasing our knowledge of the evolution dynamism of coronavirus in combating the permanent emergence risk that they pose in the human population, evidenced by the sars pandemic of - and the recently identified mers-coronavirus in the middle-east. fifteen human respiratory specimens (upper and lower) positive for hcov-oc using molecular detection, sampled from to (one to two for each year) were chosen among the specimens available at the virology department of the university hospital of caen. these specimens were sampled from patients suffering from acute upper or lower respiratory diseases, and were sent to our laboratory for a viral diagnosis. the detection of hcov-oc was performed using an in-house real-time quantitative rt-pcr, for which the original primers may be found in table . clinical signs, age, and sex of patients were investigated and compiled in table . we also used a culture supernatant of the total nucleic acids of μl of clinical specimens and vr cultures supernatant were extracted using the qiasymphony automate and the qiasynphony virus/bacteria kit (qiagen, holden, germany), following instructions of manufacturer. to determine the genomic sequence of nsp , s, and n genes, a set of overlapping rt-pcr products, from to nucleotides, were generated. these rt-pcr products encompassed the entire three genes. twelve primers were used to amplify and sequence the nsp gene, composed of nucleotides and located from nucleotides , to , (reference to vr , nc ). eighteen primers were used to amplify and sequence the s gene composed of nucleotides. this gene is located in the last third of the hcov-oc genome, between bases , and , (reference to hcov-oc vr , nc ). six primers were used to amplify and sequence the n gene composed of nucleotides and located at the ' end of the hcov-oc genome, from nucleotide , to , (reference to hcov-oc vr , nc ). for both rt-pcr and sequencing reactions, primers were selected in conserved domains from an alignment of the previously published hcov-oc . some of the selected primers were published by lau in , but others were designed using primer-blast software (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) [ ] . all of them were tested in silico and in vitro with the vr prototype strain to test their efficacy. in vitro experimentations have also been conducted using the vr strain to test the primer abilities to provide us with quality sequencing products. table , associated with the corresponding targeted gene and with the fragment size. rt-pcr reactions were performed using onestep rt-pcr system (qiagen, holden, germany), from . μl of nucleic acid in a final volume of μl. the same primers were used to perform the complete bidirectional sequencing of the rt-pcr products, with a sanger derived method. . μl of each rt-pcr product were first purified by adding μl of exosap-it (usb corporation, cleveland, oh, usa), and by heating it at °c for min, followed by min at °c to disable enzymatic activities. two sequencing reactions were performed for each rt-pcr product with either a forward or a reverse primer. to μl of purified rt-pcr product were used in a final volume of μl of reaction mixture, made up with the bigdye terminator cycle sequencing kit, version . (applied biosystems, foster city, ca, usa), in addition to μl of one of the corresponding primers. the cycling parameters for the sequencing rt-pcr were °c for min, followed by cycles of °c for s, °c for s, and °c for min. the analysis of labelled products was performed by the capillary electrophoresis abi prism genetic analyzer (applied biosystems, foster city, ca, usa), by the molecular genetics laboratory of the university hospital of caen. in this study, the term of molecular isolate was use to designate the sequence of hcov-oc detected directly from clinical sample. sequences were assembled in contigs corresponding to the entire nsp , s or n genes with codoncode aligner software, version . . (codoncode corporation, centerville, ma, usa). multiple sequence alignment and phylogenetic analysis were performed using mega software, version . (http://www.megasoftware.net). twenty-one sequences available in genbank were added to the alignment, including hcov-oc s hku _ and hku _ from hong-kong [ ] ; hcov-oc s be and be from belgium [ , , ] . the accession numbers corresponding to these sequences are given in table . phylogenetic trees were constructed using the method of neighbor joining, with the substitution model of kimura- , implemented in mega [ , ] . these trees were used to perform the comparative analysis of our results with those of lau et al in [ ] . in order to support neighbour joining trees with a probabilistic method and to estimate divergence time between groups and subgroups, trees of s and n genes were also inferred using the same sequences and a bayesian markov chain monte carlo method, implemented in the beast package, version . . [ , ] . inferences were calculated with the one parametric coalescent model with a constant size, under the tn +g substitution model according to bayesian information criterion (bic) and akaike information criterion (aic) values of a model test carried out on mega software. the tmrca was estimated using a relaxed molecular clock with an uncorrelated lognormal distribution. the length of mcmc was fixed at × − states for the both genes. the tracer software, version . , implemented in the beast package, was used to assess the fitness of the model used, focusing the effective sampling size (ess) data, after a burning of %. the target trees were obtained with the maximum credibility clades, with a posterior probability limit of . . the tmrca age for each genotype was compared for s and n genes. evolving the largest rna virus genome mesoniviridae: a proposed new family in the order nidovirales formed by a single species of mosquito-borne viruses ratification vote on taxonomic proposals to the international committee on taxonomy of viruses virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus bovine-like coronaviruses isolated from four species of captive wild ruminants are homologous to bovine coronaviruses, based on complete genomic sequences coronavirus diversity, phylogeny and interspecies jumping two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism comparison of genomic and predicted amino acid sequences of respiratory and enteric bovine coronaviruses isolated from the same animal with fatal shipping pneumonia genomic characterization of equine coronavirus 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findings from a retrospective investigation severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia human respiratory coronavirus oc : genetic stability and neuroinvasion complete genomic sequence of human coronavirus oc : molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event implications of altered replication fidelity on the evolution and pathogenesis of coronaviruses lack of evidence for proofreading mechanisms associated with an rna virus polymerase high-frequency rna recombination of murine coronaviruses inter-and intra-variant genetic heterogeneity of human coronavirus oc strains in france molecular epidemiology of human coronavirus oc reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination circulation of genetically distinct contemporary human coronavirus oc strains the neighbor-joining method: a new method for reconstructing phylogenetic trees molecular analysis of the s glycoprotein gene of bovine coronaviruses isolated in japan from to pathology of neonatal calf diarrhea induced by a coronavirus-like agent full-length genomic sequence of bovine coronavirus ( kb). completion of the open reading frame a/ b sequences evolutionary history of the closely related group coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc confidence limits on phylogenies: an approach using the bootstrap molecular evolutionary genetics analysis version . bayesian phylogenetics with beauti and the beast . the dominance of human coronavirus oc and nl infections in infants severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus in travelers progress in global surveillance and response capacity years after severe acute respiratory syndrome the first pandemic of the st century genomic analysis of colorado human nl coronaviruses identifies a new genotype, high sequence diversity in the n-terminal domain of the spike gene and evidence of recombination comparative analysis of coronavirus hku genomes reveals a novel genotype and evidence of natural recombination in coronavirus hku structural and functional analysis of the s proteins of two human coronavirus oc strains adapted to growth in different cells prevalence and genetic diversity analysis of human coronavirus oc among adult patients with acute respiratory infections in beijing a cis-acting function for the coronavirus leader in defective interfering rna replication a simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences prospects for inferring very large phylogenies by using the neighbor-joining method this work was supported by the french agence nationale de la recherche (anr) on the behalf of epicorem project (eco-epidemiology of coronaviruses, from wildlife to human: emergence prototype strains vr as reference and for validation of our method. this strain was obtained from the atcc and maintained in hrt- cells. threat assessment), grant anr- -bsv - . we thank paul brown for the english proofreading of the manuscript. a.v. and n.k. design the study. n.k. and w.l. performed the experiments. nk. and m.a.g. performed the analyses. n.k. conceived the manuscript with contributions from f.m., m.a.g. and a.v. all authors contributed toward the preparation, editing and proofreading of the manuscript. the authors declare no conflict of interest. key: cord- - ent fx authors: stoddard, shana v.; stoddard, serena d.; oelkers, benjamin k.; fitts, kennedi; whalum, kellen; whalum, kaylah; hemphill, alexander d.; manikonda, jithin; martinez, linda michelle; riley, elizabeth g.; roof, caroline m.; sarwar, nowreen; thomas, doni m.; ulmer, emily; wallace, felissa e.; pandey, pankaj; roy, sudeshna title: optimization rules for sars-cov- m(pro) antivirals: ensemble docking and exploration of the coronavirus protease active site date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ent fx coronaviruses are viral infections that have a significant ability to impact human health. coronaviruses have produced two pandemics and one epidemic in the last two decades. the current pandemic has created a worldwide catastrophe threatening the lives of over million as of july . current research efforts have been focused on producing a vaccine or repurposing current drug compounds to develop a therapeutic. there is, however, a need to study the active site preferences of relevant targets, such as the sars-cov- main protease (sars-cov- m(pro)), to determine ways to optimize these drug compounds. the ensemble docking and characterization work described in this article demonstrates the multifaceted features of the sars-cov- m(pro) active site, molecular guidelines to improving binding affinity, and ultimately the optimization of drug candidates. a total of compounds were docked into both the r z and lu sars-cov- m(pro) crystal structures. several key preferences for strong binding to the four subsites (s , s ′, s , and s ) were identified, such as accessing hydrogen binding hotspots, hydrophobic patches, and utilization of primarily aliphatic instead of aromatic substituents. after optimization efforts using the design guidelines developed from the molecular docking studies, the average docking score of the parent compounds was improved by . −log( )(kd) in binding affinity which represents an increase of greater than six orders of magnitude. using the optimization guidelines, the sars-cov- m(pro) inhibitor cinanserin was optimized resulting in an increase in binding affinity of . −log( )(kd) and increased protease inhibitor bioactivity. the results of molecular dynamic (md) simulation of cinanserin-optimized compounds cm , cm , and cm revealed that cm and cm fit well into the active site of sars-cov- m(pro) [protein data bank (pdb) accession number lu ] and formed strong and stable interactions with the key residues, ser- , his- , and glu- . the enhanced binding affinity produced demonstrates the utility of the design guidelines described. the work described herein will assist scientists in developing potent covid- antivirals. coronaviruses cause upper respiratory tract illnesses and can infect multiple species [ ] . the first severe coronaviruses to impact humans were the severe acute respiratory syndrome-coronavirus (sars-cov), which emerged in [ ] [ ] [ ] and the middle east respiratory syndrome (mers-cov), which emerged in [ ] . after the emergence of mers-cov, researchers expressed the importance of developing future therapeutic interventions for coronaviruses [ ] . the current coronavirus which emerged in is a result of a new strain of severe acute respiratory syndrome-coronavirus (sars-cov- ) [ ] . this new strain has resulted in a pandemic, termed covid- , and has infected almost million individuals worldwide, upending the way of life of individuals across the globe. this covid- pandemic has resulted in over , deaths between late and july [ ] . currently, there are no antiviral therapeutic options available for covid- . although vaccines are being pursued for covid- as a possible therapeutic route, vaccines can have limited effectiveness when strains continually change. coronaviruses appear to be on a path of continual reemergence with new strains developing, and, as a result, other therapeutic interventions in addition to vaccines must be pursued. the coronavirus main proteases are essential for the viral rna to be processed [ ] . currently over crystal structures are available of the specific covid- main protease (sars-cov- m pro ). drug compounds targeting the main protease are being pursued as a major therapeutic route for the treatment of covid- . many of these crystal structures have a compound from the zinc database [ ] bound in the active site or non-specifically bound to an allosteric site that could be further optimized as a drug candidate. additionally, several peptide or peptide-like inhibitors are being pursued as potential therapeutic agents against the coronavirus main protease [ , [ ] [ ] [ ] [ ] . the crystal structures lze, m k, wnp, lu , and bqyof the main protease demonstrates the molecular interactions between four of these peptide-based inhibitors and sars-cov- m pro . the peptide-based inhibitors reported in zhang, , however , have yet to be crystallized in the sars-cov- m pro active site [ ] . many scientists are also attempting to repurpose numerous approved drugs for coronaviruses as well [ ] [ ] [ ] [ ] [ ] . when mers emerged, this process was also pursued as a strategy to develop therapeutic options [ ] . the current inhibitors have been developed through virtual screening and further developed using rational drug design. some docking studies have been performed on already available drug compounds to rank their potential for development as antiviral agents [ , ] . studies using molecular docking have been utilized to analyze binding interactions of antiviral compounds targeting; the hiv protease [ , ] , topoisomerase ii dna gyrase enzymes [ , ] , and anti-cancer compounds which target histone deacetylases [ ] [ ] [ ] and a range of other diseases can assist in the optimization of molecular interactions for drug design. studies performed to explore the broad substrate binding preferences that sars-cov- m pro has would therefore significantly assist in the optimization of any lead drug compounds that are being pursued. furthermore, exploration of the range of binding interactions that can be utilized to strengthen binding would assist researchers in producing the best broad-spectrum options for the current coronavirus and any potential future coronavirus. to address these needs, in this study an ensemble molecular docking approach was performed to probe molecular considerations that can enhance the binding of potential drug candidates to the sars-cov- m pro . the design guidelines discovered were utilized to develop novel optimized inhibitor compounds and optimize the cinanserin drug compound currently being explored by other researchers [ ] . all of our novel optimized inhibitor compounds designed outcompeted the zinc database parent compounds and the cinanserin inhibitor. two of the main protease x-ray crystal structures for sars-cov- m pro , pdb ids lu [ ] and r z [ ] , were imported into the molecular visualization program ucsf chimera [ ] from the protein databank and saved as mol files. receptors were prepared according to default parameters in sybyl-x. protonation states of all residues were established at ph . during the receptor preparation. the protomol, which defines hydrophobic, hydrogen bond acceptor, and donor regions in the active site, was generated using the native ligand bound in each of the x-ray respective crystal structures. the flexible surflex-dock geom docking protocol was used for all docking analyses. a total of inhibitor structures were analyzed in this study. the ligands bound in the sars-cov- m pro receptors available at the time of the start of the study were pooled into one file to evaluate their initial binding affinity. the pdb ids of receptors utilized are lu [ ] , rf , ree, rec, ret, re , r z, rf , w , re , rfp, rfb, ref, rel, rfk, rex, re , rey, rfl, r , rfy, rfw, rfh, reg, rfs, r y, reo, rf , rfi, rfd, rfr, rez, rfx, rfq, rf , rew, r , rei, rev, res, rfa, rer, re , rej, red, rep, reu, rek, ren, rfj, rft, rfv, rf , rf , rfo, rea, rem, r , rfz, rff, re , rfu, r , rf , rf , rg , and rf . structures were either drawn in pubchem or chemdraw then converted to the simplified molecular line input entry specification (smiles) notation. structures were converted to d format in uscf chimera by pasting the smiles strings into the build structure module. compounds to be docked were combined into several mol files for docking. in sybyl-x the drug-like setting in the ligand structure preparation step was utilized to prepare the parent, derivative, and optimized compounds for docking, except where noted. a conformational sample was also developed in the ligand preparation step for all parent and derivative compounds. for the dataset containing the optimized compounds designed in this work, the option "generate preferred conformer/stereoisomer" was used. a minimum of conformers/stereoisomers were generated for each compound. the most diverse set of conformers/stereoisomers was selected for docking. additionally, compounds retrieved from crystal structures were also docked in the crystallized conformation. all compounds were docked into both the r z and lu sars-cov- m pro crystal structures. binding affinity for all compounds was predicted using the surflex-dock geom (sfxc) protocol for all docking simulations. the surflex-dock geom protocol default parameters were used for all docking runs. briefly, parameters were set to start docking with an additional four starting conformations to enable rotatable bonds and poses max and minimize compounds both pre-dock and post-docking. the binding affinities were calculated using the c-scoring method in sybyl-x and are reported as −log (kd) values. the empirical scoring function used accounts for hydrophobic contact, polar interactions, and entropic fixation cost in translational, torsional, and rotational degrees of freedom [ , ] . docking scores are outputted in −log (kd) values. thus, a docking score of correlates to a binding affinity of × − m. a larger binding score indicates a stronger binding to the protein receptor. docking poses were visualized in both sybyl-x and ucsf chimera programs. molecular interactions were also evaluated in both programs. all images were produced in ucsf chimera. structural characteristics of the coronavirus main protease protein were evaluated in ucsf chimera. characterization of the hydrophobicity and electrostatic potential map of sars-cov- m pro was performed to further understand binding preferences to docked substrates. all analysis and development of hydrophobicity and electrostatic potential were performed in ucsf chimera. the degree of conservation of residues in the main protease was determined with the program consurf [ , ] . the lu pdb file was submitted to the consurf server. default parameters were used to calculate conservation results. each receptor was recolored in chimera to indicate the degree of conservation using the consurf coloring scheme, and then the results were evaluated. logp, logs, topological polar surface area (tpsa), and bioactivity were predicted for optimized drug candidates. prediction of logp and logs values for compounds was performed using alogps . [ ] . smiles strings for compounds were uploaded into the online server and submitted for calculation of logp and logs values using the non-java interface. the topological polar surface area and the bioactivity of all optimized candidates were calculated using the program molinspiration [ , ] . smiles strings were uploaded to the molinspiration server and the calculate properties and predict bioactivity options were selected. the top three compounds (cm , cm , and cm ) that showed a stronger binding to the protein receptor (larger binding score −log (kd) values) were subjected to all-atom molecular dynamics ( ns) using desmond software, ver. . , . (schrödinger) [ ] . the selected ligand-protein complexes were first solvated with a tip p [ ] explicit water model. counter ions were added to neutralize the charges. the whole system was neutralized using sodium chloride (nacl) and set to an ionic strength of . m. an orthorhombic box was used with a buffer distance of Å for each dimension by the desmond software. the relaxation protocol was modified as specified in desmond release and as described in our previous publication [ ] . in brief, five steps were involved in the equilibrium stage. the first step involves brownian nvt dynamics (constant volume and temperature) with t = k and restraints on solute heavy atoms for ns; the second step includes t = k, h o barrier, brownian npt (constant pressure and temperature), membrane restrained in the z direction and protein restrained for ps; the third step consists of npgt, heating from → k, h o barrier and gradual release of restraints; the fourth step includes nvt production, t = k and no restraints for ps; and the final step involves npt production, t = k, and no restraints for ns. after the equilibration stage, the production run was performed in the npt ensemble using a langevin thermostat at a temperature of k and . bar pressure over ns (time-step fs) with recording intervals of . ps for energy and ps for trajectory. simulations were run with the opls- force field. plots and figures were generated with the desmond simulation interaction diagram (sid) implemented in the desmond software [ ] . coronaviruses typically have several binding pockets within their protease which makes up the active site. based on the crystal structure sars-cov- m pro (pdb id lu ) four binding pockets, s , s , s , and s , are present [ ] . the side chains of residues phe- , asn- , ser- , cys , his- , his- , and glu- , and the backbone of leu- , gly- , his- , and met- create the s binding site (figure ). the s site is created by the side chains of his- , val , asn- , thr- , cys- , gly- , and the backbone of thr- . subsite s is formed by the side chains of tyr- , asp- , met- , and his- , and the backbone of arg- . the last subsite s is created by the side chains of met- , leu- , pro- , , and the backbones of residues glu- , arg- , thr- . the surfaces and ribbon structure of the actives site for sars-cov- m pro lu and r z crystal structures are shown in figure a -e. overview of sars-cov- active site using pdb lu and r z: (a) surface of sars-cov- m pro active site in pdb lu , regions colored represent the binding subsites s , s , s , s , and the cleft s , which is accessible in the lu receptor; (b) surface of sars-cov- m pro active site in pdb r z, regions colored represent the binding subsites s , s , s , s . (c) residues comprise the key active site regions in sars-cov- m pro (pdb lu ); (d) residues which make of the key active site regions in sars-cov- m pro (pdb r z); (e) the ribbon structure of the lu crystal structure of sars-cov- m pro showing residues contributing to the s , s , s subsites. the n inhibitor is shown to assist in visualizing the subsite binding pockets. (f) overlay of the lu (cyan) and the r z (pink) active sites. the highlighted residues are the residues shown to have different side-chain orientations. proteins are flexible; thus, the shape of the active site can change as they move. proteins can be crystallized in multiple conformations resulting in variations of the side-chain orientations in the residues located in a protein's active site. a comparison of the crystal structures for sars-cov- m pro lu and r z was performed to determine structural variations that could contribute to differences in binding poses. when overlaying lu and r z, the crystal structure's residues thr- , ser- , met- , leu- , asn- , met- , glu- , and gln- were observed to have side chains in different orientations ( figure f ). the most significant variation to the active site can be seen when comparing met- . the change in orientation decreases the length of the s subsite by at least . Å. when the side chain of met- is up, as seen in lu , then the s active site extends all the way to the hydroxyl group of tyr- . when the met- side chain is down then the tyr- becomes buried ( figure ) . a second variation that can be seen when comparing these two receptors is the cleft on the surface that is created behind the s pocket in lu , which is significantly smaller in r z ( figure a ,b). this cleft represents another accessible site for binding. these observations taken together demonstrate the need for researchers to consider the impact of the multiple conformations on the s pocket and binding cleft. the use of multiple conformations when using docking will assist in the prediction of new antivirals agents targeting sars-cov- m pro as the diversity of accessible variations can produce distinct binding poses for an inhibitor compound. compounds not docked in multiple receptors may produce false negatives if not considered through a holistic methodology. as we will show in this study, the conformation of met- can contribute to drastic differences in binding poses and affinity. the properties of each of the substrate binding pockets were analyzed using conservation analysis, electrostatics, and hydrophobicity to understand the characteristics of each subsite ( figure ). this information can assist in better rational design of drug compounds. the s and s substrate binding sites were determined to be highly conserved, while the s and s did consist of residues with a lower degree of conservation (table ). in the s subsite, the residues thr- and ala- were slightly variable in sars-cov- m pro , while val- was highly variable. the residue met- of the s subsite was shown to be slightly variable while the leu- forming the cleft behind the s pocket was demonstrated to be highly variable. these variations are important to note as broad-spectrum inhibitors should attempt to bind to very conserved residues. thus use of the cleft, even though this can represent another binding site, for broad-spectrum design will need to be further studied. it is possible that these variable residues could confer specificity between coronaviruses, therefore inhibitors which can be created to selectively target these residues could be helpful in differentiating between the biological mechanisms utilized between different coronaviruses, if any. pocket was demonstrated to be highly variable. these variations are important to note as broadspectrum inhibitors should attempt to bind to very conserved residues. thus use of the cleft, even though this can represent another binding site, for broad-spectrum design will need to be further studied. it is possible that these variable residues could confer specificity between coronaviruses, therefore inhibitors which can be created to selectively target these residues could be helpful in differentiating between the biological mechanisms utilized between different coronaviruses, if any. an evaluation of the electrostatics showed the active site has several negatively charged regions. the most electronegative subsites were sites s and s . due to the electronegative character of the sars-cov- m pro active site, positively charge substituents represent a viable option to increase binding affinity, as will be demonstrated in this study. sars-cov- m pro also has several hydrophobic regions located in each subsite and in the cleft behind subsite s . these hydrophobic regions permit hydrophobic contacts in the active site and this study will also demonstrate the importance of accessing these patches to increase binding affinity. further implications of these structural insights will be discussed as the docking studies are explored. due to the variation, both receptors were used to evaluate the docking of all compounds. the highest binding score was accepted as the preferred mode of binding and further analyzed. the docking scores for the zinc database inhibitors that were successfully docked are listed in supplementary table s . five of the docked zinc database inhibitors, t j, sfy, t , k s, and t d ( figure and table ), produced docking scores of . , . , . , . , and . , respectively. these inhibitors were selected as templates to investigate the impact of structural variations of inhibitor compounds on the sars-cov- m pro receptor. in the crystal structures containing t j, sfy and k s are shown to bind to an allosteric site. the compounds t and t d are shown to be covalently bound to the active site serine in their crystal structures. for the purposes of evaluating binding preferences in sars-cov- m pro , all compounds were docked into the active site of sars-cov- m pro and used as template reference structures. it was observed that the compound t has a cl atom on the benzene ring, which is located at the s subsite of the crystal structure. the majority of the inhibitors published this year as new antiviral options for covid- have not incorporated halogens in the inhibitor structure [ ] [ ] [ ] . in order to explore the impact of the presence of halogens on inhibitor compounds designed to target sars-cov- m pro , ten compounds were created using t and t j as templates ( figure ). the impacts of f, cl, br, and i in addition to the impact of one, two, or three halogens were explored. the typical mode of binding for the halogenated compounds produced a pose with the halogen atoms in the s subsite or the opening of the active site gorge between the s or s subsites. the iodine atom of gm was shown to site in the s subsite. the t compound placed the cl atom in the s subsite. no halogens were placed in the s subsite. the docking scores for the parent compounds and the derivatives are shown in table . all ten of the derivative compounds produced docking scores that were similar or lower than the parent compound. these data suggest that halogens do not have a significant impact on binding affinity to the sars-cov- m pro receptor. the sars-cov- m pro active site has several hydrophobic regions. to determine optimal binding interactions using hydrophobic substituents, derivatives of both sfy and t were designed by adding hydrophobic substituents to each compound. the impact of the length of the aliphatic substituent on binding affinity was evaluated by adding a methyl, ethyl, propyl, or butyl substituent to sfy and t . tert-pentyl substituents and -butenyl groups were also used as substituents for sfy derivatives to evaluate the impact of bulky groups and more rigid alkyl groups. for sfy derivatives, the impact of the substituent location was also evaluated by creating ortho, meta, and para versions of each compound. in the t derivatives series, the impact of adding a benzene ring and the methylene linkers between the benzene ring and the amide was also interrogated. collectively, a total of derivatives ( figure ) were designed, successfully docked, and evaluated. docking scores for each derivative are shown in table . all derivatives were demonstrated to produce higher binding affinities than the parent compounds. compounds hs , sd , sd , sd , sd , and sd produced docking scores at least . orders of magnitude greater than the parent compounds. compounds bd , sd , sd , sd , and sd produced docking scores at least one order of magnitude greater than the parent compounds. the average binding scores for compounds having butyl, propyl, ethyl, and methyl substituents were . , . , . , and . −log (kd), respectively. this trend was also consistent with the derivative compounds db , db , and bd , which produced binding scores of . , . , and . −log (kd). bd , which has the most methylene linkers between the benzene ring and the carbonyl carbon, scored . and . −log (kd) better than bd and bd , which had one linker or no linker, respectively. when comparing docking scores of the derivatives against the parent compounds it was clearly demonstrated that increasing the length of the aliphatic substituent results in increased binding affinity. average binding scores for the meta, para, and ortho compounds were . , . , and . −log (kd), respectively. additionally, the data demonstrated a preference for meta conformers over para and ortho conformers. the modes of binding were evaluated further to understand the preference for longer aliphatic substituents and meta substituents. hs , bd , hs , bd , and bd all produced modes of binding which interacted with the s and the s subsites. hs , hs , and bd positioned the butyl substituent in the s pocket and the chlorinated benzene ring in the s subsite. this trend was shown to be reversed in bd and bd which placed the chlorinated benzene ring in the s subsite and the unsubstituted benzene ring in the s subsite. hs ( figure a ) was shown to produce a hydrogen bond with gln- and the piperazine ring while hs formed a hydrogen bond with the nitrogen in the piperazine ring and backbone of his- . no hydrogen bonds were produced between hs , bd , bd , or bd and the active site. the modes of binding for hs and hs were significantly different than the modes of binding for the other derivatives in this dataset. hs was shown to interact with the s and the s subsite forming a hydrogen bond with gln- . the chlorinated benzene ring was bound in s while the ethyl group was positioned in the s subsite. hs positioned the chlorinated benzene ring in the s subsite and the piperazine ring in the s subsite. the best binding pose for all t derivatives had piperazine rings that had the nitrogen not involved in the amide bond protonated, which result in a positively charged core on the inhibitor compound. the active site gorge between the s and s subsites is electronegative, therefore the positively charged ring would increase the attraction between the inhibitors and the receptor. several modes of binding were observed for the sfy derivatives in the active site ( figure ). the top five binders were sd , sd , sd , sd , and sd ( figure b -f). four of these compounds (sd , sd , sd , and sd ) are meta-substituted, while sd is para-substituted. the mode of binding for meta-substituted compounds sd , sd , sd shows interactions with the s and the s subsites. s has a hydrogen bond to the backbone of glu- , the alkyl substituent is bound in the s subsite, and the aniline ring is in the s subsite. the para-substituted compounds sd and sd also interact with the s and s subsites in a similar fashion as the meta-substituted compounds. sd and sd , however, bind differently, positioning the alkyl group in s and the aniline ring in the s subsite. the increase in binding affinity of sd over sd is due to the larger alkyl group positioned in the s subsite. the para-substituted compounds sd , sd , and sd bind in a unique mode of binding positioning the alkyl group in the s subsite, and the aniline ring is bound in s subsite. sd , which also binds to the s and s , binds in the reverse manner, positioning the aniline ring in the s and the alkyl substituent in the s subsite. the para-substituted compounds sd and sd interacted with the sars-cov- m pro active sites in a similar fashion as the sd . namely, binding to the s and the s subsites by positioning the alkyl substituent in s and the aniline ring in s . the ortho-substituted compounds sd , sd , sd also interact primarily with s and s . sd was demonstrated to have a reversed orientation compared to sd and sd , placing the aniline ring in s and the alkyl group in s . ortho-substituted compounds sd and sd position the alkyl groups near the s subsite, but do not seem to achieve the same depth of penetration that the meta-substituted compounds access. sd does penetrate significantly into the s subsite. this compound is bound such that it π-π stacks with his- . this stacking interaction rotates the compound such that the alkyl group is inserted directly into the s subsite, resulting in the slightly higher binding affinity for the propyl substituted sd over the butyl substituted sd . sd also creates a π-π stacking interaction with his- . these results taken together suggest that the s subsite plays a significant role in establishing binding affinity with hydrophobic groups. while the s subsite is also a major hydrophobic area, groups that could produce hydrophobic interactions in the s subsite yield more binding affinity than do groups that position the hydrophobic groups in the s subsite. the hydrophobicity plot indicates that the portion of the s subsite which is formed by met- is quite hydrophobic. consequently, inhibitors that could access this hydrophobic patch adjacent to met- in the s subsite would have increased binding affinities. the optimal bond angles are important to note as the angling of the ortho-substituted compounds typically did not produce gains in binding affinity that were as significant as the meta-or para-substituted compounds. the para orientation promoted binding to compounds to the active site in a linear conformation, while the meta substitution pattern promotes binding to s and s subsites efficiently. the ortho conformation decreased the ability of these compounds to access the s subsite with the alkyl groups, resulting in weaker binding affinities compared to the meta-and para-derivative compounds. this decrease in s subsite access can be explained in terms of the depth of penetration of the alkyl substituent into the s subsite. distances were measured from the tyr- oxygen atom and the methyl group of the butenyl substituent of compounds sd and sd . when comparing differences in depth of penetration into the s subsite, the meta-substituted sd is . Å from tyr- while the ortho-substituted sd is a distance of . Å from tyr- ( figure ). this is a difference of . Å in depth of penetration into the s subsite, increasing sd s binding affinity of . −log (kd) over sd s binding affinity of . −log (kd) by almost one order of magnitude. thus alkyl substituents in the s subsite improve binding affinity the most when they penetrate deep into the s subsite. the n inhibitor which has a leucine residue that sits in the s subsite [ ] is at a distance of . Å from the tyr- (measured in the lu pdb crystal structure). thus, this inhibitor could be further optimized if the residue was changed to a hydrophobic substituent which could penetrate deeper into the s subsite. while the angling of the ortho-substituted compounds decreases the ability of these compounds to access the s subsite with the alkyl groups, if these compounds are able to participate in π-π stacking interactions with his- they can be successfully directed into the s subsite which can serve to direct the alkyl groups into the s subsite. nitrogen atoms permit increased hydrogen bonding interactions and can have different effects on π-π stacking interactions in the active site [ ] . thus, in this study, we substituted the benzene rings with nitrogen-containing heterocycles to determine what impact inclusion of these heteroatoms would have on binding affinity. twenty derivates of sfy and t were designed and successfully docked into the sars-cov- m pro active site (figure ). the number of nitrogen atoms and the location of the nitrogen atoms in the ring structure were evaluated by creating derivatives which contained either pyridine, pyrimidine, pyridazine, or triazine rings. the docking scores for these compounds are listed in table . for the t derivatives, inclusion of a second nitrogen produced increases in binding affinity to sars-cov- m pro . comparing compound bd , which has no nitrogens atoms in the aromatic ring, to compounds db , db , and db , increases of . , . and . were observed, respectively. both db and db produced a hydrogen bond to tyr- , placing the nitrogen-containing pyridine ring in the s subsite. however, the pyridine ring of db , which has the nitrogen atom in the para position, is able to hydrogen bond by penetrating straight into the s subsite. because db is able to penetrate straight into the s subsite, it permits the formation of a hydrogen bond with the backbone of asp- in addition to being involved in a slipped stack π-π stacking with his- . the pyridine ring of db has the nitrogen in the ortho position, thus to hydrogen bond to tyr- it must bend the methylene linkers to position the nitrogen atom near the hydroxyl group on tyr- ( figure a,b) . the bending of db to facilitate hydrogen bonding to tyr- results in the loss of ability to hydrogen bond to asp- and produces a weaker edge-to-edge π-π stacking interaction with his- , subsequently decreasing its binding affinity compared to db . db has a pyrimidine ring where one of the nitrogen atoms is in the para position yet produces a binding affinity similar to db . while db also produces a hydrogen bond interaction between the nitrogen in the para position on the pyrimidine ring to tyr- , the second nitrogen in the ring is located in the ortho position. this results in this hydrophilic nitrogen being positioned against the hydrophobic met- residue. db has a carbon atom in this position on the ring and thus can participate in hydrophobic interactions with met- . the reduction in binding affinity between db and db is a result of the more hydrophilic nitrogen interacting with the hydrophobic met- compared to having a more hydrophobic carbon atom of db . db and db , which produced only small increases in binding affinity over t , both incorporated pyridazine rings. these pyridazine rings π-π stack against the his- . in other studies, it has been demonstrated that pyridazine rings are also less favorable when utilized in stacking interactions [ , ] , which may be why these compounds did not show significant gains in binding affinity. sd , which has a pyridine ring with a nitrogen in the para position, outcompeted the parent compound sfy and sd . both sfy and sd also contain pyridine rings but have nitrogens in the ortho and meta positions, respectively. the sfy derivatives, having a nitrogen atom in the para positions, showed improvement in binding affinity over sfy, except sd . all other sfy derivatives (sd , sd , sd , and sd ) produced similar or lower binding scores relative to sfy. incorporating two nitrogens in the ring showed improvement: strong improvement when comparing sd to sfy (increase of . −log(kd)), moderate improvement between sd to sd (increase of . −log(kd)), and weak improvement when comparing sd to sd and sd to sd ( . and . −log(kd) increases, respectively). sd , sd , sd , and sd all contain pyrimidine rings. sd was shown to access hydrogen bonds with tyr- with the para-substituted nitrogen, hydrogen bond with the backbone of his- with the sulfonamide amine, and his- and ser- with the amine group on the aniline ring. this network of hydrogen bonds aligns the compound in a position to also permit a parallel displaced π-π stacking interaction with his- . sd and sd have a different mode of binding compared to the other para-substituted nitrogen compounds ( figure c ). both sd and sd position the aniline ring in the s subsite by hydrogen bonding with thr- and thr- . the nitrogen heterocycle is placed in the s subsite where hydrogen bonding occurs with the backbone of cys- instead of the s subsite hydrogen bonding occurring with tyr- . the change in the mode of binding is due to the alkyl substituent on the nitrogen heterocycle. this bulky group would create more steric clashing if the nitrogen compound was positioned to optimize hydrogen bonding interactions with tyr- , thus the moiety is placed in the s subsite where the alkyl group can spread out and backbone hydrogen bonding can simultaneously be accessed. sd and sd showed the greatest improvement in binding affinity, which can be attributed to the presence of the butenyl and neopentyl substituents on the ring in addition to the inclusion of a second nitrogen atom in the para position. with met- . the reduction in binding affinity between db and db is a result of the more hydrophilic nitrogen interacting with the hydrophobic met- compared to having a more hydrophobic carbon atom of db . db and db , which produced only small increases in binding affinity over t , both incorporated pyridazine rings. these pyridazine rings π-π stack against the his- . in other studies, it has been demonstrated that pyridazine rings are also less favorable when utilized in stacking interactions [ , ] , which may be why these compounds did not show significant gains in binding affinity. the nitrogen atoms placed in the para position of a benzene ring proved to yield the greatest increases in binding affinity. this arrangement allows for optimized hydrogen bonding to tyr- and the backbone of asp- when substituents are not included in the nitrogen heterocycle. the tyr- interaction has, to our knowledge, not been a residue that has been targeted for increased binding affinity by other compounds being developed for covid- [ ] [ ] [ ] . this work demonstrates a feasible way to access this interaction in the s subsite while simultaneously π-π stacking with his- . while the inclusion of two nitrogens can increase binding affinity, placement is important, as demonstrated by the compounds sd , sd , sd , and sd which produced lower binding affinities than the sfy parent compound. additionally, the azide moiety seems to reduce binding affinity in the active-site gorge. it is notable that sd and sd produced the best binding scores in this dataset, suggesting that the inclusion of the hydrophobic substituent on the nitrogen heterocycle in addition to para placement of a nitrogen atom would optimize binding interactions to the sars-cov- m pro active site, albeit through a different mode of binding. the results of this dataset indicate that nitrogen on heterocycles should be at the para position to optimize h-bonding with the hydroxyl group of tyr- in the s subsite. aromatic versus aliphatic structures can make significant changes in binding affinity to protein receptors. in the design of glioblastoma therapeutics targeting histone deacetylase (hdac ) inhibitors, it was determined that aliphatic substituents increased the potency of these compounds to the hdac receptor [ ] . thus, in this study the impact of aromaticity versus aliphatic character was also evaluated. derivatives of sfy and ks which were evaluated in the study of aliphatic substituents and the study of nitrogen heterocycles were designed for analysis. the aromatic rings of the previously discussed sfy and ks derivatives were substituted with their aliphatic version (i.e., benzene was converted to cyclohexane ring). a total of compounds (figure ) were designed and successfully docked. the docking scores for each derivative are shown in table . using the sfy derivatives, both the impact of substituent length and the impact of nitrogen placement in the ring was evaluated. for the purposes of clarity and easier comparison between structures, the ortho, meta, para nomenclature system which is used for benzene rings was used for the cyclohexane ring instead of the , ; , ; , nomenclature system typically used for cyclohexane rings. a new dataset using the ks compound was generated, having both aromatic versions and aliphatic versions of the compound. additionally, the impact of various alkyl group substituents on the nitrogen atoms that were not in the ring structure was determined. the compounds developed in this dataset had the most significant gains in binding affinity compared to the parent compound. thirty-five of the compounds produced binding scores greater than the parent compounds. of those compounds which outcompeted the initial inhibitor, produced docking scores great than . orders of magnitude or better. compounds sd and lea produced docking scores of . and . , respectively. this shows an increase of . orders of magnitude. the docking scores are outputted in −log (kd) units, thus this unexpected result suggests that these compounds could have as much as times stronger binding affinity against sars-cov- m pro than the parent compounds. therefore, incorporation of these features in current antiviral drug candidates being optimized for covid- could yield highly potent inhibitors with low nanomolar inhibition values. compounds lea , kt , kth , kth , sd , kth , and lea produced docking scores at least . orders of magnitude stronger in binding to the sars-cov- m pro receptor than the sfy parent compound. the ten compounds sd , sd , sd , kt , lm , lea , l , kt , sd , sd , kt , and e produced binding affinities at least . orders of magnitude greater than the parent compound. these seventeen compounds are significantly better than any of the aromatic versions considered in this study. when considering the twelve monosubstituted compounds, the preferred placement of the alkyl substituent was in the s subsite (l , l , lm , sd , kt , kt ), then the s subsite (sd , sd , kt , kt ) and lastly, the s subsite (l and sd ). with the exception of l and sd , the amino group was placed in s and typically accessed in hydrogen bonds with the backbone of leu- and the side chains of ser- and his- . the amino group of both l and sd is bound to the s subsite through hydrogen bonding with the backbone of asp- . furthermore, sd produced a hydrogen bond with the backbone of met- . these results further support the trend that increasing the length of aliphatic substituents increases binding affinity to the sars-cov- m pro receptor and that the s subsite plays a strong role in the binding of hydrophobic substituents. while the aromatic versions of the sfy compounds preferred the meta, then para, then the ortho form of the inhibitor compound, this trend was not totally observed for the aliphatic versions. the average binding affinity for aliphatic sfy derivatives having substituents located in the para, meta, or ortho position was . , . , and . , respectively. the difference in preference between the meta and para can be explained by difference in the structure of the benzene ring versus the cyclohexane ring. the benzene ring is planar and aromatic which introduces more rigidity into the aromatic sfy derivatives. it was noticed that the para versions of the aromatic sfy derivative compounds are much more linear compared to the meta version, which is due to the aromatic planar benzene ring. the planarity decreases the potential to bind to the s and s subsites simultaneously. the aliphatic sfy derivatives, however, are not constrained by aromaticity and the para-substituted compounds are able to adopt a conformation allowing them to bind to s , forming hydrogen bonds and bending to also form hydrophobic contacts in s . after running the single substituted compounds, eleven additional hybrid compounds were designed that had both para-and meta-substituents or substituents located in ortho and para positions (lea , lea , lea , lea , kth , kth , kth , sd , sd , sd , sd ). the average docking score for the ortho/para versions was surprisingly larger than the average docking for the meta/para versions, . compared to . , respectively. the difference in average docking score, however, is not substantial, suggesting that both versions of the compound would be similar in binding affinity. the compounds sd and lea produced binding scores of . and . , respectively, that were significantly better than sfy which only produced a binding affinity of . . these compounds were shown to be able to place one hydrophobic substituent in the s , the second in s , and the amine group in s , accessing all the observed interactions of kt and sd . the binding modes for sd and lea are shown in figure a -d. compounds lea , lea , lea , kth , kth , and kth were also bound in the same orientation as sd and lea . however, an unexpected mode of binding was observed with sd , sd , and sd . these three compounds placed both aliphatic tails in the s subsite and the amine group in s . the ability for s to accommodate two aliphatic tails demonstrates that large substituents can be placed in this subsite. a key feature that permitted both aliphatic tails being inserted into s was that sd , sd , and sd all had meta/para placements of the substituents. the mode of binding for sd and sd demonstrating the interaction in the s subsite is shown in figure e ,f. all other compounds with the exception of sd had ortho/para placements of the two substituents, thus these chains were not able to pack closely enough to successfully be inserted into the s subsite. even though sd has the ortho/para placements of the substituents, each chain is a butyl group which might limit effective packing of the longer aliphatic chains. the results of this study indicate that inclusion of the aliphatic, not aromatic ring structures will yield sars-cov- m pro inhibitors with the greatest binding affinity to the active site. consideration should also be made to fully access the full depth of the s subsite. hydrogen bonding is prevalent in biological systems and can play a significant role in binding interactions. the sars-cov- m pro has a number of residues in the active site which can hydrogen bond. thus, regions of the active site which facilitate hydrogen bonding and compound structures that promote access to hydrogen bonding were evaluated. nineteen derivatives of the scaffolds t j, t , and t d were produced to develop a dataset to interrogate this question ( figure ). additionally, six compounds amm , amm , amm , ew , ew , and ew were created to determine the impact of distance in interacting with multiple hydrogen bonding regions in the active site. the compounds were designed with different variations of hydrogen bond donors and acceptors to identify the regions of sars-cov- m pro which could best accommodate hydrogen bonding interactions. the docking scores for each compound are listed in table . hydrogen bonding is prevalent in biological systems and can play a significant role in binding interactions. the sars-cov- m pro has a number of residues in the active site which can hydrogen bond. thus, regions of the active site which facilitate hydrogen bonding and compound structures that promote access to hydrogen bonding were evaluated. nineteen derivatives of the scaffolds t j, t , and t d were produced to develop a dataset to interrogate this question ( figure ). additionally, six compounds amm , amm , amm , ew , ew , and ew were created to determine the impact of distance in interacting with multiple hydrogen bonding regions in the active site. the compounds were designed with different variations of hydrogen bond donors and acceptors to identify the regions of sars-cov- m pro which could best accommodate hydrogen bonding interactions. the docking scores for each compound are listed in table . in panels (a) and (b) it can be seen that the aliphatic tails bind in the s and the s subsites. in panels (c) through (f) of the surface of the subsites, each of the bound aliphatic substituents is shown. the s subsite is colored blue, and the s subsite is green. grey regions are portions of the s subsite. hydrogen bonds are indicated by solid yellow lines. in general, inclusion of hydrogen bonding substituents produced improvement in binding affinities for the inhibitor compounds to the sars-cov- m pro . jn , which was demonstrated to improve the binding affinity of . orders of magnitude greater than t dm, was shown to hydrogen bond to the backbone of arg- and thr- by positioning the substituent containing the alcohol and the amine group in the s subsite ( figure a ). the nh in the linker region of gm and kf also produced hydrogen bonds with arg- . the benzimidazole ring nitrogen of gm , the benzimidazole ring nitrogen, the amine linker of kf and kf , the benzimidazole ring nitrogen, and the alcohol of kf , the amide nh of jn and jn , and the alcohol of jn all produced hydrogen bonds with residue glu- . compounds hs , hs , kf , and jcn were shown to produce modes of binding that formed hydrogen bonds with residue gln- , across from glu- . kf produced a mode of binding that allowed for hydrogen bonding interactions spanning in the center of the active site gorge between gln- and glu- ( figure b ). hydrogen bonds were formed with histidine rings in the active site; kf formed a hydrogen bond to his- ( figure c ), and compounds jn and jcn formed hydrogen bonds to his- . histidine rings, also being aromatic, formed several π-π stacking interactions with compounds in this dataset. his- formed stacking interactions with gm , jcn , jn , and jn , while his- formed π-π stacking interactions with jn . compound kf formed a hydrogen bond with gln- in the s subsite. one major area for hydrogen bonding was found in the s subsite. in the s subsite, the side chains of ser- and asn- , in addition to the backbone of residues leu- and gly- , were shown to form hydrogen bonds to a number of compounds. the amine group of compound jcn was observed to hydrogen bond to ser- and leu ; the hydroxyl of jcn produced a hydrogen bond to ser- ; the nh in the linker region of kf formed a hydrogen bond with ser- and leu- in addition to forming a hydrogen bond between the hydroxyl and asn- ( figure d ). the nh in the linker region of jn was observed to hydrogen bond with the backbone of residue gly- . (b) kf (pink) hydrogen bonding in hotspots located in the s subsite and the active site core; (c) kf (salmon) hydrogen bonding in hotspots located in the s subsite and the active site core; (d) kf (olive) hydrogen bonding s subsite hotspots; (e) ew (tan) hydrogen bonding in the hotspots located in the s subsite and the s ; (f) hs (purple) hydrogen bonding hotspots located in the s subsite and the active site core; (g) amm (lime) shown hydrogen bonding in s subsite hotspot and the active site core; and (h) amm compounds designed for aliphatic substituent dataset in sars-cov- m pro receptor. hydrogen bonds are indicated by solid yellow lines. several compounds were evaluated for modes of binding that promoted hydrogen bonding to multiple subsites. when analyzing ew it produced a mode of binding which permitted hydrogen bonding to both the s subsite and the s subsite ( figure e ). this hydrogen bonding pattern was also seen in several of the compounds in the aliphatic substituent and the aliphatic ring datasets ( figure d) . hs was also able to access hydrogen bonding hotspots in two regions of the active site ( figure f ). the first was the s hotspot and the second was hydrogen bonding in the core which formed a hydrogen bond to gln- , and similarly to kf . compound amm revealed an additional hydrogen bond that can be accessed in the s subsite with the backbone of met- ( figure g) . this compound was able to produce four hydrogen bonds in the s subsite using a simple amine group. the amine group, which in many cases will be protonated at physiological ph, would have three hydrogen atoms. based on our results, the amine group is an ideal substituent to access the hotspots in each of the subsites. while the amine group seems to be preferred, alcohol substituents are certainly still capable of forming several hydrogen bonds in the s subsite ( figure h) . when considering the hydrogen bonding patterns of compounds in this dataset and in previous datasets together, five accessible hydrogen bonding hotspots locations can be identified in the sars-cov- m pro active site. there is one hydrogen bond hotspot in each subsite and in the core of the active site spanning from gln- to glu- . the compounds jn and jn access the hydrogen bonding hotspot identified in the s subsite ( figure a ). compounds that can access the s hydrogen bonding hotspot will successfully hydrogen bond with the backbone of thr- and the backbone of arg- simultaneously. this can be done using either an amino group or a substituent having both an amine and an alcohol separated by a three-carbon linker. the s subsite has a hydrogen-bonding hotspot facilitated by tyr- , his- , and the backbone of asp- , which can be seen in the mode of binding of the compound kf (figure b) . we also observed this hydrogen bonding pattern in the previous dataset for the nitrogen-containing heterocycles. kf also formed a hydrogen bond pair across the core of the active site between gln- and glu- . this represents a third hotspot for hydrogen bonding. the fourth hydrogen bonding hotspot is located in the s subsite near residues thr- and thr- ( figures c and c ). both the backbone of thr- and the side chains of thr- and thr- can be involved in interacting with the inhibitor compound through hydrogen bonding, creating a forked-like position with the inhibitor in the active site. the s pocket has another very prominent hydrogen bonding hotspot created by the side chains of residues ser- and his- , the backbone of leu- and gly- , and sometimes the side chain of asn- . while it is possible to simultaneously form a hydrogen bond to all four residues, ser- , his- , leu- , and gly- , using an amino group ( figure d) , it was observed that ser- and the backbone of leu- were almost always hydrogen bound together ( figure d ). to determine the overall effectiveness of the guidelines developed in this study, a new dataset was created with twenty-six optimized compounds and given the name foundations-lab (fl) compounds series ( figure ). these compounds were designed and successfully docked into the sars-cov- receptors. because the conformational flexibility of the compounds was outside of the sybyl-x program capabilities, "generate favored tautomers/stereoisomers" selection was used for ligand preparation. five compounds (fl , fl , fl , fl , and fl ) had molecular weights (mws) ranging from to da, which is over the mw cutoff used for lipinksi's rules. thus, for the purposes of evaluating the effectiveness of the rules, the drug-like setting was not used to prepare the compounds in the final hybrid dataset. the docking scores of the fl compounds are listed in table . the fl compounds were produced by selecting the multiple features of the derivative compounds used in the datasets in this molecular docking study. compounds were designed to strategically interact with multiple hydrogen bonding hotspots in the s , s , s , s . some compounds were optimized to access the s subsite with hydrophobic substituents. the fl compounds produced binding scores ranging from . to . . the average binding scores of the zinc database parent compounds was . −log (kd), while the average docking score of the optimized compounds was . −log (kd). this represents an overall average increase in binding affinity by . orders of magnitude. thus, the fl compounds are predicted to significantly outperform the zinc database parent compounds. these data support the effectiveness of the optimization guidelines developed by the docking experiments. the fl compounds produced modes of binding which access all of the major hydrogen bonding hotspots and the hydrophobic patches (figure ), significantly increasing their binding affinity over the parent compounds. fl produced a total of ten hydrogen bonds (figure a) . the alcohol and amino portion taken from jn produced four hydrogen bonds between the amino group and residues tyr- , his- , and asp- in the s subsite; the cyclohexane ring with amino substituent portions taking the compounds in the sfy derivatives series formed four hydrogen bonds with residues ser- , his- , leu- , of the s subsite; and the sulfonamide and benzimidazole ring formed two hydrogen bonds spanning the core of the active site between gln- and glu- . the hydrophobic substituent which was intended to interact with the s subsite was determined to interact with the hydrophobic patch in the s subsite. a few of the fl compounds produced modes of binding that were unexpected, yet still accessed the intended molecular interactions albeit with a different substituent than expected. the compound fl formed hydrophobic interactions in the s subsite, accessed the hydrogen bonding hotspot in the s subsite using the alcohol and amino portion taken from jn , and accessed the s hydrogen bonding hotspot (figure b ). the molecular features identified in the fl series can be reliably used to design tight-binding inhibitor compounds targeting sars-cov- m pro . to further confirm the effectiveness of the selected inhibitors, the compound cinanserin from the work of jin et al., [ ] was selected for optimization of binding affinity to sars-cov- m pro . cinanserin has a micromolar ic inhibition against the sars-cov- m pro and produced a docking score of . . ten compounds were developed using defined guidelines. the docking run was also completed according to the same protocol as the fl inhibitors, wherein the conformational sample was created using the "generate favored tautomers/stereoisomers" selection for ligand preparation and drug-like setting disabled. the structures of cinanserin and the optimized inhibitors are shown in figure . the optimized compounds were then docked into sars-cov- m pro to determine if stronger binding was produced. the docking scores for cinanserin and the optimized compounds are shown in table . the docking pose of cinanserin [ ] demonstrated that the core benzene ring was placed in the s subsite, the terminal benzene was located in the s subsite and the amine group was located in the s subsite. thus, optimization of binding interactions in the s , s , and s subsites were prioritized. briefly, to improve the affinity of cinanserin, aliphatic rings were introduced in some derivatives, hydrogen bonding substituents were strategically placed to access the hotspots in the s , s , and s subsites, and larger hydrophobic groups were substituted for the amine group to increase hydrophobic interactions in the s subsite. an nh group was added to the core rings on each of the derivatives to anchor the derivative in the s hydrogen bonding subsite. all ten of the optimized compounds outperformed the cinanserin parent inhibitor. the greatest improvement was garnered by cm which produced a binding score of . , an increase of . -log (kd) over the cinanserin parent compound. the modes of binding produced mirrored the expected binding poses. the modes of binding for the compounds cm and cm produced the most significant increases in binding affinity and are shown in figure a ,b. when considered in conjunction, this demonstrates that current drug compounds that are being pursued as therapeutic options for covid- by targeting sars-cov- m pro can be optimized using the molecular guidelines described in this paper and yield significant gains in binding affinity. the bioactivity of compounds can be predicted to evaluate their potential against certain drug targets. the program molinspiration predicts the bioactivity of potential compounds to serve as g-protein coupled receptor (gpcr) ligands, ion channel modulators, kinase inhibitors, nuclear receptor ligands, enzyme inhibitors, and protease inhibitors. because sars-cov m pro is a protease, ideally compounds should be predicted to be protease inhibitors. surprisingly, cinanserin was not predicted to be a protease inhibitor, producing a bioactivity score of − . . all but one of the optimized cinanserin compounds, however, were predicted to be protease inhibitors. compounds cm , cm , cm , and co produced the strongest bioactivity scores for protease inhibitors. no correlation between binding affinity and bioactivity score could be produced as cm produced the highest bioactivity score, followed by c , the cm , and finally cm . based on the data in compound, cm might produce an effective low nanomolar sars-cov- m pro inhibitor. all compounds were predicted to have protease inhibitor activity and had the most significant bioactivity scores. taken together, this data suggests that not only do the optimization guidelines discovered in this work help to enhance binding affinity to sars-cov- , but they are useful in strengthening the bioactivity of drug candidates to be protease inhibitors overall. the compounds designed in this work could also be used as potential inhibitor compounds for the treatment of covid- . table ) were used for md studies with the opls- force field. the conformational dynamics and stability of the protein-ligand complexes, cm , cm , and cm , with sars-cov- m pro (pdb id: lu ), were analyzed using the root mean square deviation (rmsd), and by the root mean square fluctuation (rmsf). the rmsd graphs of the protein c-α atoms ( figure a ) of all three protein-ligand complexes, cm , cm , and cm , indicate that after ns, the proteins have equilibrated, and simulations are converged with respect to the reference frame at time ns. all the frames from ns trajectory were aligned on c-α atoms of the reference frame. in the case of ligand rmsd ( figure b ), the fluctuation was observed during the first ns of simulation, after that complexes did not undergo any significant conformational changes except for cm . the rmsf of sars-cov- m pro (pdb id: lu ) was calculated to understand the fluctuation of amino acids interacting with cm , cm , and cm during the ns simulations. the amino acids of lu that were involved in interactions with cm and cm did not show much fluctuation in their rmsf values (supporting information, figure s ), indicating the stability of the biomolecular system. however, amino acids of lu that were involved in the interactions with cm showed significant fluctuations in their rmsf values, indicating a less stable biomolecular system compared to the lu -cm and lu -cm complex. the residue interactions ( figure a ) and protein-ligand d contact contour map ( figure b ) of sars-cov- m pro (pdb id: lu )-cm complex shows that leu- (water bridges), ser- ( % contribution (h-bond) and water bridges), his- ( % contribution (h-bond) and water bridges), glu- ( % and % contributions (side-chain and backbone h-bonds)) and his- ( % contribution (hydrophobic)) were crucial amino acids that formed strong and stable interactions with cm . the interactions histogram ( figure a ) and protein-ligand d contact contour map ( figure b ) shows the residue interactions of viral protease sars-cov- m pro (pdb id: lu ) with cm . these graphs show that ser- ( % contribution (h-bond) and water bridges), glu- ( % and % contributions (side-chain and backbone h-bonds), and thr- ( % contribution (h-bond)) were crucial amino acids that formed strong and stable interactions with cm . similarly, the interaction histogram and protein-ligand contact contour map of sars-cov- m pro (pdb id: lu ) with cm (supporting information, figure s ) exhibited interactions with gln- (< % contribution, h-bond) thr- (water bridges), gln- (< % contribution, h-bond), asn- (< % contribution, h-bond) and glu- ( % contribution, h-bond, water bridges, and ionic interaction) and identified crucial amino acids that formed interactions with lu . the overall contributions of critical amino acid interactions with cm were significantly less during the entire ns of simulation, indicating that this compound diffused slightly with the initial binding pocket of lu and showed lower affinity towards crucial residues identified for cm and cm . taken together, the results of the docking study and the md simulations demonstrate several key interactions that facilitate improved binding affinity to the sars-cov- m pro receptor. in figure , a visual summary of the key molecular interactions is shown. figure . visual summary of key molecular interactions facilitating strong binding affinity to sars-cov- m pro . hydrogen bond hotspots are shown as green spheres, with hydrogen bonds shown as yellow lines. the hydrophobic core in s pocket is indicated by pink sphere. blue sphere indicates π-π stacking region. the ensemble molecular docking study revealed several tools which can be utilized to optimize current drug candidates for sars-cov- m pro and subsite binding preferences. binding in the s , s , and s subsites can all be facilitated by both hydrophobic interactions or hydrogen bonding interactions. the s subsite was one of the most prominent binding sites utilized in the inhibitor mode of binding. two methods can be successfully employed for drug compounds to access the s subsite: the inclusion of hydrophobic substituents that can interact with met- , or the inclusion of hydrogen bonding donor or acceptors atoms, which can access the hydrogen bonding hotspot formed by the backbone of residues asp- , arg- , gln- , and the side chain of tyr- . compounds can be developed to access the s subsite with high affinity by accessing the hydrogen bonding hotspot formed by leu- , ser- , his- , and asn- . the most facile way to achieve binding in this region is a protonated nitrogen atom. the hydrogen bonding hotspot in the s subsite can also be easily accessed with an amine group or an alcohol group. accessing the hydrogen bonding hotspot in the s subsite seems to be a molecular guideline that is not currently being employed in the design of covid- inhibitors. aliphatic ring systems outperform aromatic ring systems and can be an easy change in drug compound structures to produce substantial increases in binding affinity of drug compounds to sars-cov- m pro . however, when using compounds, having an aromatic ring structure inclusion of nitrogen heterocycles or aliphatic substituents can significantly improve binding affinity as long as care is taken to optimize the most effective angle, permitting access to the binding interactions of interest, whether that is hydrophobic interactions or hydrogen bonding interactions in a number of subsites. the optimization guidelines proved to be effective in increasing binding affinity specifically to sars-cov- m pro and in increasing drug compounds' potential to be protease inhibitors. the optimized compounds produced in this work used to test the design guidelines and the optimized cinanserin compounds are predicted to be viable drug candidates for covid- therapeutics. md simulations of optimized cinanserin derivatives cm , cm , and cm revealed that cm and cm adopt a very similar orientation as observed in docking pose and formed stable and strong interactions with sars-cov- m pro (pdb id: lu ) with the key residues, ser- , his- , and glu- . based on our studies, the optimized cinanserin compounds are predicted to be viable drug candidates that can be pursued to develop covid- therapeutics. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : docking scores of zinc database inhibitors in lu and r z actives sites. supporting information, figure s : a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia is the discovery of the novel human betacoronavirus c emc/ (hcov-emc) the beginning of another sars-like pandemic? a novel coronavirus from patients with pneumonia in china from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design zinc -ligand discovery for everyone structure of m pro from sars-cov- and discovery of its inhibitors structure-based design of antiviral drug candidates targeting the sars-cov- main protease crystal structure of sars-cov- main protease provides a basis for design of improved α-ketoamide inhibitors the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor rapid repurposing of drugs for covid- combating devastating covid- by drug repurposing in silico studies on therapeutic agents for covid- : drug repurposing approach targeting sars-cov- : a systematic drug repurposing approach to identify promising inhibitors against c-like proteinase and -o-ribose methyltransferase drug repurposing against sars-cov- using e-pharmacophore based virtual screening, molecular docking and molecular dynamics with main protease as the target repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection molecular docking and dynamics simulation of fda approved drugs with the main protease from novel coronavirus computational screening of antagonist against the sars-cov- (covid- ) coronavirus by molecular docking molecular docking studies of marine diterpenes as inhibitors of wild-type and mutants hiv- reverse transcriptase application of d-qsar, pharmacophore, and molecular docking in the molecular design of diarylpyrimidine derivatives as hiv- nonnucleoside reverse transcriptase inhibitors design, synthesis, molecular docking, and antibacterial evaluation of some novel flouroquinolone derivatives as potent antibacterial agent synthesis, antibacterial and molecular docking studies of new benzimidazole derivatives design of potent panobinostat histone deacetylase inhibitor derivatives: molecular considerations for enhanced isozyme selectivity between hdac and hdac design and synthesis of diazine-based panobinostat analogues for hdac inhibition in silico design of novel histone deacetylase inhibitors: design guidelines for improved binding affinity ucsf chimera a visualization system for exploratory research and analysis surflex: fully automatic flexible molecular docking usig a molecular similarity-based search engine surflex-dock . : robust performance from ligan energetic modeling, rig flexibility, and knowledge-based search consurf: identification of functional regions in proteins by surface-mapping of phylogenetic information the projection of evolutionary conservation scores of residues on protein structures virtual computational chemistry laboratory-design and description molinspiration cheminformatics free web services; molecular property and bioactivity score calculation toolkit fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties shaw research desmond molecular dynamics system; maestro-desmond interoperability tools quantum and statistical mechanical studies of liquids: . transferable intermolecular potential functions for water, alcohols, and ethers. application to liquid water selective inhibition of human monoamine oxidase b by acacetin -methyl ether isolated from turnera diffusa (damiana) heteroaromatic π-stacking energy landscapes this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to acknowledge the rhodes college chemistry department for support of the research project during the spring semester. the authors declare no conflict of interest. key: cord- - z org authors: tzou, philip l.; tao, kaiming; nouhin, janin; rhee, soo-yon; hu, benjamin d.; pai, shruti; parkin, neil; shafer, robert w. title: coronavirus antiviral research database (cov-rdb): an online database designed to facilitate comparisons between candidate anti-coronavirus compounds date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: z org background: to prioritize the development of antiviral compounds, it is necessary to compare their relative preclinical activity and clinical efficacy. methods: we reviewed in vitro, animal model, and clinical studies of candidate anti-coronavirus compounds and placed extracted data in an online relational database. results: as of august , the coronavirus antiviral research database (cov-rdb; covdb.stanford.edu) contained over cell culture, entry assay, and biochemical experiments, animal model studies, and clinical studies from over published papers. sars-cov- , sars-cov, and mers-cov account for % of the data. approximately % of experiments involved compounds with known or likely mechanisms of action, including monoclonal antibodies and receptor binding inhibitors ( %), viral protease inhibitors ( %), miscellaneous host-acting inhibitors ( %), polymerase inhibitors ( %), interferons ( %), fusion inhibitors ( %), and host protease inhibitors ( %). of compounds with known or likely mechanism, ( %) are licensed in the u.s. for other indications, ( %) are licensed outside the u.s. or are in human trials, and ( %) are pre-clinical investigational compounds. conclusion: cov-rdb facilitates comparisons between different candidate antiviral compounds, thereby helping scientists, clinical investigators, public health officials, and funding agencies prioritize the most promising compounds and repurposed drugs for further development. cov-rdb contains four main types of antiviral experimental data, six main lookup/explanation tables, and a registry of ongoing or planned clinical trials. the four main types of antiviral experimental data include (i) cell culture and entry assay experiments; (ii) biochemical experiments; (iii) animal model studies; and (iv) clinical studies. the six main lookup/explanation tables provide information on viruses, virus strains/isolates, tested compounds, compound targets, cell types, and animal models. cov-rdb data are stored in a postgresql relational database, but there is not necessarily a oneto-one relationship for the tables displayed on the web and their underlying database structure. indeed, several of the website tables contain information from more than one underlying database table. as of august , the cov-rdb contains data from more than virus cell culture experiments, entry assay experiments, biochemical experiments, animal model experiments, and clinical studies from more than peer-reviewed publications and preprints. research articles are identified through incremental daily searches of pubmed and biorxiv using the search term "coronavirus" and by citations identified through reading these papers. publications containing experimental data are imported into a staging zotero database and then annotated to extract the data described in the sections below. preprints that are subsequently published in a peer-reviewed journal are identfied using a computer script that parses datasets downloaded from the stephen b. thacker cdc (centers for disease control and prevention) library the cell culture experiments table contains fields, including four fields present in each of the experiment tables: reference, compound, virus category, and virus isolate/strain. the nine fields unique to the cell culture experiments table include six that describe experimental conditions and the three experiment results are the half-maximal effective concentration (ec ), percent inhibition, and the % cytotoxic concentration (cc ). the ec can only be determined using a series of compound dilutions. while the ec is usually reported as µm, inhibitory activity for interferons is also often reported as international units (iu)/ml and inhibitory activity for monoclonal antibodies is often reported as ng/ml. the ec is available for the vast majority of in vitro cell culture experiments. however, for a few experiments, the experimental setup involved a single compound concentration (rather than a dilution series). for these experiments, the percent virus inhibition with the single compound concentration is reported. there are two tables for entry assay experiments-one for pseudovirus entry assays and another for cell-cell fusion assays. the pseudovirus assay table contains the following six unique fields: (i) pseudovirus vector, (ii) pseudovirus number, (iii) target cell type, (iv) time to addition of drug, (v) indicator of virus replication, and (vi) ec . in the pseudovirus experiments, the virus strain is a virus construct composed of a virus that does not require a biosafety level (bsl- ) laboratory, such as vesicular stomatitis virus (vsv) or human immunodeficiency virus type (hiv- ), into which the coronavirus spike (s) gene has been cloned. this construct also has a reporter gene such as luciferase or gfp. the cell-cell fusion assay table contains the following seven unique fields: (i) effector cell type, (ii) effector cell number, (iii) target cell type, (iv) target cell number, (v) time to addition of drug, (vi) indicator of virus replication, and (vii) ec . the biochemical experiments table contains two unique fields: the biochemical target and the half maximal inhibitory concentration (ic ). the biochemical target is usually one of the virus enzymes including rna-dependent rna polymerase (rdrp), main protease (also called c-like protease; cl pro ), papain-like protease (pl pro ), and helicase. however, cell-free assays that test inhibitors of the spike (s) protein binding to angiotensin converting enzyme (ace ) are also included. the animal model experiments are characterized by comparisons between a group of animals receiving a treatment intervention either shortly before or after virus infection and a group of untreated virus-infected control animals. the animal model experiments table has two parts-experimental conditions and experimental results. the experimental conditions include the (i) animal model, (ii) size and route of virus challenge, (iii) treatment intervention, (iv) treatment dosage, (v) treatment timing in relation to the addition of virus, (vi) number of treated subjects, and (vii) number of control subjects. the experimental results, which often depend on the study, include endpoints such as mortality, weight loss, fever, respiratory rate, lung pathology, and virus load measurements. the reduction of endpoint severity is reported on an ordinal scale ranging from to . there are more than references containing more than animal model experiments, nearly all involving sars-cov- , sars-cov, or mers-cov. approximately % of the studies involve mice; % involve non-human primates (rhesus macaques, marmosets, and cynomolgus macaques); and % involve hamsters, ferrets, cats, dogs, or rabbits. the most commonly studied interventions have included monoclonal antibodies, fusion inhibitors, interferons, and the nucleoside analog polymerase inhibitors. the clinical studies are represented using several enumerated and free-text fields. the enumerated fields include the reference, virus category, and type of study (e.g., observational, randomized trial, randomized placebo-controlled trial). the free-text fields include descriptions of the interventions and regimen details, the study population and methods, and the study findings. cov-rdb does not randomized trial, randomized placebo-controlled trial). the free-text fields include descriptions of the interventions and regimen details, the study population and methods, and the study findings. cov-rdb does not provide an assessment of study quality such as validity and risk of bias as there are other research groups providing this type of assessment. antiviral data on coronaviruses other than sars-cov- provide insight into the robustness of an antiviral compound, in that, compounds that are active against multiple viral species will be more likely to inhibit future pandemic coronaviruses and will be less vulnerable to the development of drug-resistance mutations. indeed, for many drug targets, such as the virus rdrp and cl pro enzymes, and for host processes upon which coronaviruses depend, inhibitory compounds will likely have broad-spectrum activity. cov-rdb contains antiviral data for six categories of coronaviruses: sars-cov- , sars-cov, mers-cov, endemic human coronaviruses, bat coronaviruses, and non-bat mammalian coronaviruses. sars-cov- ( %), sars-cov ( %), and mers-cov ( %) account for % of the data. however, the proportion of data associated with sars-cov- is rapidly increasing. figure shows the distribution of study types for sars-cov- , sars-cov, and mers-cov. sars-cov and sars-cov- belong to the same betacoronavirus b (sarbecovirus) clade, and their amino acids are approximately % identical in the rdrp and cl pro enzymes and % identical in the spike protein. in contrast, mers, a clade c betacoronavirus, is approximately % identical to sars-cov and sars-cov- in the rdrp gene, % identical in the cl pro gene, and % identical in the s gene. within each of these three viruses, there is little diversity, with median pairwise distances ranging between % and . %. the four endemic human coronaviruses include two clade a betacoronaviruses and two alphacoronaviruses. bat coronaviruses are distributed widely among different clades [ , ] . indeed, of the betacoronavirus clades and of coronavirus clades are found only in bats. the mammalian coronaviruses include murine hepatitis virus (mhv), which is a longstanding experimental model for coronavirus infection, and several other coronaviruses that have been studied because they are important livestock diseases [ ] . although infectious bronchitis virus is an avian gammacoronavirus, we have included it in the non-bat mammalian coronavirus category. cov-rdb uses the terms isolate and strains to describe the different viruses used in antiviral studies, although "strains" is usually reserved for describing isolates that have distinct phenotypic properties [ ] . where possible, isolates are named according to the recommendations from the international committee on taxonomy of viruses [ ] , i.e., virus/host/location/isolate/date. most sars-cov- isolates are nearly identical to one another with the upper limit for the pairwise amino acid distance being about . %, although this number varies depending upon the gene [ ] . therefore, the biological significance of the isolate used for a particular study is not known. however, for some treatments such as monoclonal antibodies, changes in the sequence encoding the relevant epitope, specifically in the s protein receptor binding domain may prove to have biological and clinical significance [ , ] . although sars-cov resulted from at least two zoonotic introductions from civet cats [ ] , and although mers-cov resulted from multiple zoonotic introductions from dromedary camels, these viruses also demonstrate little genetic variability. several of the most commonly used isolates have been cloned, either as intact viruses (e.g., by plaque purification or limiting dilution) or by constructing a cdna copy representing a single sequence variant. modification of these clones, such as selection of a resistant variant in vitro [ ] or introduction of a reporter gene like gfp or luciferase, presumably retains the characteristics of the original parental virus isolate or strain [ ] . commonly used isolates that have been cloned and manipulated in the laboratory include mers-cov/human/amsterdam/emc/ [ ] , sars-cov/human/hanoi/urbani/ [ ] , sars-cov- /human/usa/wa / [ ] , and sars-cov- /human/munich/ / [ ] . the cell lines table provides descriptions for the cell lines used in cell culture and entry assay experiments. it contains four fields: (i) the cell line's commonly used name, (ii) the source of the cell line, (iii) closely related cell lines, and (iv) a description of the cell line and one or more of the closely related cell lines. the most commonly used cell lines for sars-cov and sars-cov- include a variety of different vero cell clones [ ] [ ] [ ] [ ] [ ] , huh [ , ] , caco- [ ] , calu- [ ] , and t/ace cells [ ] [ ] [ ] ( table ) . while each of these cell lines expresses ace , only calu- cells were originally derived from lung epithelial cells. the t cells are typically used for cell-cell fusion and pseudovirus entry experiments. several studies have also used human alveolar epithelial cells or a variety of different respiratory system or kidney organoids [ , ] . the cell lines used for mers-cov are similar, with the main exception that t/dpp (dipeptidyl peptidase ) cells are used instead of t/ace cells because dpp (aka cd ) is the mers-cov receptor [ ] . vero cells support the replication of many viruses often producing a visual cytopathic effect [ ] [ ] [ ] . they express ace , the receptor for sars-cov and sars-cov , and dpp , the receptor for mers-cov. although vero cells are ifn-deficient, they express the ifn-α/β receptor and thus retain the ability to respond to exogenous ifn [ ] . vero e cells engineered to express greater amounts of tmprss produce higher sars-cov- titers of sars-cov- [ ] . drugs that target tmprss are often inactive in vero cells. human lung epithelial cell line calu- cells form differentiated pseudostratified columnar epithelia highly permissible to coronavirus infection. they are polarized with an apical domain facing the airway lumen and a basolateral domain facing internally. they produce a visual cytopathic effect. the b clone has high ace expression. they are often used for the preclinical development of respiratory drugs [ ] . heterogeneous human epithelial colorectal adenocarcinoma caco- cells are considered to be more pharmacologically relevant than vero cells for some studies because of their human origin [ ] . human hepatoma huh- cells express ace and tmprss , yet do not support levels of replication as high as vero cells [ , ] . the t cells are derived from the human embryonic kidney cell line. t cells contain the sv large t-antigen, which facilitates replication of transfected plasmids containing the sv origin of replication. the t/ace cells are transfected to express ace and have been used for many sars-cov cell-cell fusion and pseudovirus entry inhibitor studies [ ] [ ] [ ] . human airway epithelial cells differentiated human airway cells have occasionally been used to study antiviral agents, although they are more commonly used to study viral pathogenesis [ , ] . tmprss -transmembrane serine protease ; ace -angiotensin converting enzyme ; ifn-interferon; dpp -dipeptidyl peptidase ; sv -simian virus . the over different animal models used in experiments described in the cov-rdb include three non-human primate models (rhesus macaques, cynomolgus macaque, and marmosets), multiple transgenic and non-transgenic mouse models, and several additional rodent models including hamsters and ferrets [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the transgenic mice have been modified in multiple ways, including to express hdpp so that they can be infected with mers-cov, to knock out the interferon (ifn)-α/β receptor to compromise innate immunity [ ] ; to knock out recombination activating gene (rag ) to compromise adaptive immunity [ ] ; to knock out carboxylesterase c, which causes poor plasma stability of remdesivir; and to express human rather than mouse ace [ ] [ ] [ ] . table describes the utility of the most common non-human primate and mammalian models for studies of the pandemic coronaviruses. pathological changes observed in the aged mouse model infected with sars-cov more closely resemble those observed in humans [ ] . rag −/− mice lack t and b cells and lack adaptive immunity and experience prolonged coronavirus shedding [ ] . ifnar −/− mice are vulnerable to greater coronavirus disease severity [ ] . there are many hace transgenic mouse models. these mice are more likely to experience weight loss, detectable virus loads, and interstitial pneumonia following challenge with sars-cov- than those with the murine ace receptor [ ] [ ] [ ] . rhesus macaque infection causes a self-limiting disease associated with virus replication. radiographic and pathologic examination of sars-cov- -infected animals display evidence of pneumonia [ , , , ] . cynomolgus macaque infection results in a productive infection in respiratory epithelial cells. symptoms are minimal but virus shedding can last up to weeks. chest radiographs reveal unifocal or multifocal pneumonia. autopsy reveals variable amounts of foci of diffuse alveolar damage [ , ] . rag-recombination activating gene; ifnar-interferon-α/β receptor; hace -human angiotensin-converting enzyme . the target table has two main fields: name and description. the target classification organizes drugs, treatments, and compounds according to the virus or host process targeted by a compound including virus enzymes, virus entry into cells, host immunological responses, and other host processes. there are three virus enzyme inhibitor classes: rdrp, protease (including cl pro , pl pro ), and helicase inhibitors. there are four inhibitor classes targeting virus entry: convalescent plasma and polyclonal antibody preparations, monoclonal antibodies, miscellaneous other compounds that inhibit virus receptor binding, and fusion inhibitors. there are two classes that target host immunological responses: interferons and other potential immunostimulatory compounds. although there are potentially many mechanisms by which targeting a host processes may interfere with virus replication, we have divided these into two broad categories: host protease enzymes used by coronaviruses to cleave the spike protein, thus, priming it for fusion and other host proteins or pathways utilized by coronaviruses. the classification of host-acting compounds is likely to continue to evolve as mechanistic pathways become better defined. table describes each of the targets and compound classes described above. figure shows the distribution of experimental data types according to target. the database contains experiments involving approximately compounds. more than of these compounds appear in the online compounds tables, which contain the following fields: (i) name, (ii) synonyms including abbreviations, (iii) closely related compounds, (iv) drug availability, (v) drug class, (vi) target, and (vii) description. for interferons, the drug class is the type of interferon (α, β, γ, or λ). for monoclonal antibodies, additional data are stored and displayed, including the antibody source and information on sequence and structure availability. the closely related compounds are subjectively defined as those that we intend to be returned by a query even if that compound was not entered by the user. there are five broad categories of closely related compounds: (i) monoclonal antibodies described in the same publication, (ii) interferons belonging to the same type (i.e., α, β, γ, or λ), (iii) a series of compounds derived from the same lead compound, (iv) prodrugs such as those for gs- (i.e., remdesivir) and β-nhydroxycytidine (i.e, eidd- ), (v) drug combinations such as lopinavir and ritonavir-boosted lopinavir (lopinavir/r), and (vi) compounds presumed to act by a highly similar mechanism of action (e.g., hydroxychloroquine and chloroquine). the drug availability category indicates whether the compound has been licensed in the u.s. or another country or has been studied in humans. of compounds with a known or likely mechanism of action, ( %) are u.s. fda (u.s. food and drug administration) -approved drugs (for indications other than covid- ), ( %) have been or are currently being evaluated in human clinical trials or are approved outside the u.s., and ( %) are preclinical investigational compounds. the database contains experiments involving approximately compounds. more than of these compounds appear in the online compounds tables, which contain the following fields: (i) name, (ii) synonyms including abbreviations, (iii) closely related compounds, (iv) drug availability, (v) drug class, (vi) target, and (vii) description. for interferons, the drug class is the type of interferon (α, β, γ, or λ). for monoclonal antibodies, additional data are stored and displayed, including the antibody source and information on sequence and structure availability. the closely related compounds are subjectively defined as those that we intend to be returned by a query even if that compound was not entered by the user. there are five broad categories of closely related compounds: (i) monoclonal antibodies described in the same publication, (ii) interferons belonging to the same type (i.e., α, β, γ, or λ), (iii) a series of compounds derived from the same lead compound, (iv) prodrugs such as those for gs- (i.e., remdesivir) and β-n-hydroxycytidine (i.e, eidd- ), (v) drug combinations such as lopinavir and ritonavir-boosted lopinavir (lopinavir/r), and (vi) compounds presumed to act by a highly similar mechanism of action (e.g., hydroxychloroquine and chloroquine). the drug availability category indicates whether the compound has been licensed in the u.s. or another country or has been studied in humans. of compounds with a known or likely mechanism of action, ( %) are u.s. fda (u.s. food and drug administration) -approved drugs (for indications other than covid- ), ( %) have been or are currently being evaluated in human clinical trials or are approved outside the u.s., and ( %) are preclinical investigational compounds. table . antiviral coronavirus therapy targets. inhibitors of the coronavirus rna-directed rna polymerase (rdrp) enzymes include nucleoside analogs that cause immediate chain termination, delayed chain termination, or viral mutagenesis. coronaviruses contain two protease enzymes: chymotrypsin-like cysteine protease ( cl pro or main (m)-pro) and papain-like (pl pro ). coronavirus helicases catalyze the unwinding of duplex rna molecules into single strands. entry convalescent plasma and polyclonal sera. convalescent plasma is one of the most widely studied treatments for covid- . polyclonal sera and immunoglobuline preparations have also entered clinical trials. recovery during sars-cov, mers-cov, and sars-cov- is usually associated with the development of neutralizing antibodies. most sars-cov- neutralizing mabs target the part of the receptor binding domain (rbd) that binds ace while most mers-cov neutralizing mabs target the part of the rbd that binds dpp . many highly potent neutralizing mabs targeting each of the pandemic coronaviruses have shown protection in vitro and in animal models. structural studies have defined specific rbd epitopes recognized by individual mabs and identified amino acid residues that are critical for mab binding. other receptor binding inhibitors sars-cov and sars-cov- spike s binds to the cellular angiotensin converting enzyme (ace ) receptor. mers-cov binds to dipeptidyl peptidase (dpp ). a variety of compounds including non-antibody proteins, peptides, and small molecules have been shown to prevent the binding of the coronavirus spike protein to its cellular receptor. following receptor binding and spike s /s cleavage and s priming, heptad region (hr ), which is close to the fusion peptide sequence, and hr , which is close to the virus membrane, collapse on to one another to bring virus and cell membranes together. nearly all fusion inhibitors are hr -mimicking peptides less than kda that bind hr , thus preventing hr −hr binding. interferons have been extensively studied for their ability to inhibit each of the pandemic coronaviruses in cell culture, animal models, and/or clinical studies [ ] . sars-cov- may be more susceptible to interferons than sars-cov is [ ] . there are several clinical trials using immunostimulatory cytokines and compounds purported to induce interferon. cleavage of coronavirus spike proteins is necessary for the virus to transition from receptor attachment to cell fusion. for sars-cov- , there is a poly-basic furin cleavage site at the s /s boundary and another cleavage site within s believed to be cleaved at the cell surface by host tmprss enzymes [ ] . multiple intracellular processes essential to virus replication are vulnerable to pharmacologic inhibitors including endosomal acidification, membrane formation, various signaling pathways, nucleotide biosynthesis, and autophagy [ ] . many compounds with uncertain mechanisms of action have been found to inhibit coronaviruses in vitro. several of these are also being studied in clinical trials. mabs-monoclonal antibodies. figure displays ec values for many of the directly acting antiviral compounds currently in clinical trials for the treatment of covid- including six polymerase inhibitors (remdesivir, eidd- , favipiravir, ribavirin, galidesivir, and sofosbuvir), three hiv- protease inhibitors (lopinavir, atazanavir, and darunavir), and three entry inhibitors (receptor binding monoclonal antibodies, soluble recombinant human ace , and umifenovir). figure displays ec values for many of the repurposed compounds that target host processes required for virus replication including two host pis that target the transmembrane serine protease (tmprss ) enzyme (camostat and nafamostat), three chloroquine analogs that interfere with endosomal acidification (chloroquine, hydroxychloroquine, and mefloquine), three other compounds believed to interfere with membrane trafficking (niclosamide, imatinib, and chlorpromazine), and four compounds acting by a variety of different cellular mechanisms (ivermectin, nitazoxanide, ciclesonide, and cyclosporin). trafficking (niclosamide, imatinib, and chlorpromazine), and four compounds acting by a variety of different cellular mechanisms (ivermectin, nitazoxanide, ciclesonide, and cyclosporin). figures and show that the potency of currently studied compounds extends over several orders of magnitude with monoclonal antibodies having ec s in the high picomolar to low nanomolar range and some compounds displaying no activity at concentrations above μm. however, there is also marked heterogeneity in the ec values for the same compound in different experiments. for several drugs, the heterogeneity can be explained by the type of cells used, inoculum size, drug timing, and culture duration. for example, the host tmprss inhibitors camostat and nafamostat are practically inactive against sars-cov- in vero cells but have ec s consistently below μm in caco- and calu- cells, because these cells require tmprss for virus replication whereas vero cells do not [ , [ ] [ ] [ ] [ ] [ ] [ ] . show that the potency of currently studied compounds extends over several orders of magnitude with monoclonal antibodies having ec s in the high picomolar to low nanomolar range and some compounds displaying no activity at concentrations above µm. however, there is also marked heterogeneity in the ec values for the same compound in different experiments. for several drugs, the heterogeneity can be explained by the type of cells used, inoculum size, drug timing, and culture duration. for example, the host tmprss inhibitors camostat and nafamostat are practically inactive against sars-cov- in vero cells but have ec s consistently below µm in caco- and calu- cells, because these cells require tmprss for virus replication whereas vero cells do not [ , [ ] [ ] [ ] [ ] [ ] [ ] . viruses , , x for peer review of table describes a set of the most promising compounds for the treatment of sars-cov- based on the following criteria: (i) act by a validated direct or indirect antiviral mechanism, (ii) display submicromolar activity in vitro and/or inhibitory activity in an animal model, and (iii) have a record of safety and favorable pharmacokinetics in human subjects. the majority of these compounds are being studied in clinical trials, although the numbers of these trials are far fewer than those for less promising compounds. table . promising sars-cov- antiviral compounds. remdesivir is a delayed chain terminator monophosphate prodrug of a ′cyano-substituted adenine c-nucleoside analog. it has high nanomolar inhibitory activity in vitro against sars-cov- particularly in cells other than vero cells [ , , [ ] [ ] [ ] . it reduces viral replication and lung pathology in mice and rhesus macaques when administered shortly after infection [ , ] . in a double-blind randomized clinical trial, its intravenous administration led to a significant reduction in time to recovery from to days (p < . ) and a non-statistically significant reduction in day mortality of . % vs. . % (p = . ) [ ] . based on this trial, the fda issued an emergency use authorization for remdesivir in patients with severe covid- . ongoing trials are examining its safety and efficacy when administered subcutaneously or via inhalation. β-d-n hydroxycytidine- ′isopropyl ester (eidd- ) eidd- is a nucleoside analog, which like remdesivir has high nanomolar inhibitory activity in vitro against sars-cov- [ ] . it reduces sars-cov and mers-cov replication and lung pathology in a mouse model [ ] . it is being evaluated in two phase ii clinical trials. table describes a set of the most promising compounds for the treatment of sars-cov- based on the following criteria: (i) act by a validated direct or indirect antiviral mechanism, (ii) display sub-micromolar activity in vitro and/or inhibitory activity in an animal model, and (iii) have a record of safety and favorable pharmacokinetics in human subjects. the majority of these compounds are being studied in clinical trials, although the numbers of these trials are far fewer than those for less promising compounds. table . promising sars-cov- antiviral compounds. remdesivir is a delayed chain terminator monophosphate prodrug of a -cyano-substituted adenine c-nucleoside analog. it has high nanomolar inhibitory activity in vitro against sars-cov- particularly in cells other than vero cells [ , , [ ] [ ] [ ] . it reduces viral replication and lung pathology in mice and rhesus macaques when administered shortly after infection [ , ] . in a double-blind randomized clinical trial, its intravenous administration led to a significant reduction in time to recovery from to days (p < . ) and a non-statistically significant reduction in day mortality of . % vs. . % (p = . ) [ ] . based on this trial, the fda issued an emergency use authorization for remdesivir in patients with severe covid- . ongoing trials are examining its safety and efficacy when administered subcutaneously or via inhalation. β-d-n hydroxycytidine- isopropyl ester (eidd- ) eidd- is a nucleoside analog, which like remdesivir has high nanomolar inhibitory activity in vitro against sars-cov- [ ] . it reduces sars-cov and mers-cov replication and lung pathology in a mouse model [ ] . it is being evaluated in two phase ii clinical trials. regn and regn are mabs with subnanomolar inhibitory activity that bind to non-overlapping ace -competing sars-cov- spike receptor binding domain epitopes [ , ] . this mab combination also reduces virus replication and lung pathology in syrian hamsters and rhesus macaques [ ] . the combination is being evaluated in phase iii trials for preventing and treating sars-cov- infection. ly is a sars-cov- mab in phase iii trials for preventing and treating covid- . as of august , there are no associated published preclinical data. mabs (phase i/ii trials) azd , brii- , js , scta , sti- , and ty are mabs in phase i/ii trials. as of august , there are no associated published preclinical data linked to mabs with these names. many research groups that have published preclinical data on one or more mabs (or mab variants such as nanobodies) including their in vitro inhibitory activity, genetic sequence data, three-dimensional structural data, and/or animal model data [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . interferons ifn-α, ifn-β, and ifn-λ ifn-α, ifn-β, and ifn-λ each inhibit sars-cov- by %- % at low concentrations of about international units (iu)/ml [ , [ ] [ ] [ ] [ ] . inhalational ifn-α and parenteral ifn-β were associated with modest reductions in disease severity and/or virus loads in two small open-label clinical trials [ , ] . an inhaled formulation of ifn-β was reported in the news to significantly reduce the odds of developing severe disease or death in a blinded randomized control trial (sng ) of patients that has not yet been published (https://www.synairgen.com/covid- /). there are currently four planned or ongoing placebo-controlled trials of parenteral or inhaled ifn-β (~ patients) and of parenteral ifn-λ (~ patients). camostat and nafamostat are tmprss inhibitors with nanomolar coronavirus inhibitory activity in biochemical and cell culture assays [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . both drugs are used in japan for the treatment of pancreatitis, while nafamostat is also used as an anticoagulant and for the treatment of disseminated intravascular coagulation. although nafamostat has approximately -fold greater inhibitory activity than camostat, it may be associated with greater toxicity. camostat is being studied in two blinded and two open-label randomized controlled studies totaling about patients. nafamostat is being studied in three small randomized open-label studies totaling about patients. apilimod was found to inhibit sars-cov- at two-digit nanomolar levels with high selectivity indexes in multiple drug screens [ ] [ ] [ ] [ ] . it inhibits the membrane protein pi ( , ) p by inhibiting the enzyme pi- p- -kinase (pikfyve) thus interfering with endosomal trafficking of sars-cov- and additional viruses utilizing the same endosomal pathway [ , ] . it has been studied in humans in multiple clinical trials and been found to be safe and well tolerated. it is being studied for the treatment of mild sars-cov- infections in one randomized placebo-controlled phase ii trial. ptc is an inhibitor of dihydroorotate dehydrogenase (dhodh), a rate limiting enzyme in the pyrimidine biosynthesis pathway [ ] . dhodh inhibitors are therapeutic targets for autoimmune disases and viral infections [ , ] . ptc has been found to be safe and have favorable pharmacokinetics in more than human subjects and has low nanomolar sars-cov- inhibitory activity and a high selectivity index [ ] . as both viral replication and cytokine overproduction depend on pyrmidine synthesis, dhodh inhibition may have a dual role in covid- treatment. dhodh inhibition is synergistic with viral polymerase inhibition [ ] . there is one phase ii/iii trial of ptc for patients with severe but not critical covid- . leflunomide, another repurposed dhodh inhibitor was found to reduce virus load in an open-label pilot study of patients [ ] . soluble recombinant human ace (rhace ) rhace protects mice for sars-cov- ards and has been studied as a treatment for ards in humans [ , ] . it inhibits sars-cov- spike binding at nanomolar concentrations in a wide variety of cell lines [ , ] . there are two ongoing phase ii trials of an intravenous commercial rhace preparation. notes: some compounds with sub-micromolar in vitro activity are not included in this table either because (i) they have not been used in humans, (ii) they have been identified only in high-throughput drug screens, or (iii) they have unfavorable pharmacokinetics. several compounds that inhibit sars-cov- less potently but are being studied as inhalational and/or intranasal therapies are also not shown. this last category includes (i) compounds that inhibit the interaction of sars-cov- spike and cell surface heparin sulfate proteoglycans such as lactoferrin and heparin; and (ii) ciclesonide, an inhaled corticosteroid that may interfere with membrane trafficking by binding directly to nsp- or nsp- or indirectly through a host protein. the clinical trials registry table is a regularly updated, annotated list of ongoing, planned, or completed clinical trials obtained from the clinicaltrials.gov, who ictrp, and chinese clinical trial websites. it contains trials of compounds with potential antiviral activity but not studies of non-antiviral interventions, such as those designed to optimize intensive-care management or reduce the inflammatory response associated with severe covid- . the clinical trials registry classifies trials according to the compound target, the type of trial (e.g., observational or randomized controlled study), the status of the trial (pending, active, or completed), and the population studied. as of august , it contains more than trials of which about % are listed on clinicaltrials.gov and % are listed only on the who international clinical trials platform. figure a displays the distribution of planned, ongoing, and published studies according to the compound targets of the drugs studied. figure b displays the same distribution for those drugs in three or more studies. it is notable that many of the most commonly studied compounds have either little or no activity against sars-cov- , including several drugs used for non-coronavirus infections such as the hiv protease inhibitors lopinavir and darunavir and the influenza inhibitors favipiravir, oseltamivir, and umifenovir. the chloroquine analogs, chloroquine and hydroxychloroquine, have weak in vitro activity but have failed to show clinical efficacy in multiple clinical trials [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the search function allows users to specify one or more of the following options from four drop-down lists: (i) compound target, (ii) compound, (iii) virus category, and (iv) study type. if the user selects "any" for one of these and leaves the others in their default position, the search function returns the database's complete set of cell culture experiments, biochemical experiments, entry assay experiments, animal model studies, and published clinical studies. by selecting one or more of the above options, the search function restricts the data returned to those meeting the search criteria. by using the "copy to clipboard" link, users can import the results of any query into a spreadsheet where they can further sort and filter query results. the search function also provides a link to the trials in the clinical trials registry for selected compounds and compound targets. the compound drop-down list displays of the most well recognized compounds. selecting a compound returns the data for that compound as well as for an additional closely related compounds (as described in the compound table section . . ). if the user selects the compound target from the dropdown menu, then the compound menu will list all compounds designed to inhibit the selected target. the compounds entry on the compounds page also links to all the data on that compound in the database. the compound drop-down list displays of the most well recognized compounds. selecting a compound returns the data for that compound as well as for an additional closely related compounds (as described in the compound table section . . ). if the user selects the compound target from the dropdown menu, then the compound menu will list all compounds designed to inhibit the selected target. the compounds entry on the compounds page also links to all the data on that compound in the database. to prioritize licensed drugs and investigational compounds for the treatment of covid- , it is necessary to compare their relative antiviral activities. compounds that are not active in vitro will almost certainly not be useful clinically. therefore, pre-clinical data are necessary to prioritize animal to prioritize licensed drugs and investigational compounds for the treatment of covid- , it is necessary to compare their relative antiviral activities. compounds that are not active in vitro will almost certainly not be useful clinically. therefore, pre-clinical data are necessary to prioritize animal model and clinical studies. compounds that are active in vitro, however, may also not be clinically useful if their associated in vitro data do not reflect physiologic conditions or if standard dosing with these compounds does not result in sufficient inhibitory concentrations at sites of infection. the creation of the cov-rdb was primarily motivated by the observation that many of the drugs being evaluated in covid- clinical trials demonstrate little or no in vitro anti-coronavirus activity. for example, as recently as july , four of the most commonly studied drugs-chloroquine analogs, azithromycin, lopinavir/r, and favipiravir-demonstrated little if any in vitro activity. the creation of the cov-rdb was secondarily motivated by the observation that results for the same compound often vary across different laboratories as a result of experimental design such as cell line, inoculum size, drug-addition timing, duration of culture, and method for measuring virus replication. given sufficient data, a database makes it possible to eventually identify the experimental features responsible for the heterogeneity in published results, thus improving the ability to compare the antiviral activity of different compounds. indeed, we have already noted in this manuscript several compound classes for which viral inhibition is influenced by the cells used for virus culture. the cov-rdb is also designed to be educational as it provides multiple lookup tables for the viruses, drugs, cell lines, and animal models used in reported experiments. these tables contain descriptions of viruses, virus isolate/strains, cell lines, animal models, and more than licensed and investigational compounds. work is underway to also add comprehensive, yet detailed, summaries of sars-cov- monoclonal antibodies and of pharmacokinetic data for those drugs with well-documented in vitro inhibitory activity. there are several additional web resources devoted to coronavirus drug development including sites devoted to high-throughput drug screening [ ] , the genetics of monoclonal antibodies [ ] , and meta-analyses of published clinical trials [ , ] . the national institutes of health (nih) recognizes the importance of such resources and has recently announced a notice of special interest: national institute of allergy and infectious diseases (niaid) priorities for biomedical knowledgebases and repositories (not-ai- - ). the cov-rdb database, user interface, and underlying computer code represent a framework for organizing a vast amount of data and for facilitating data curation. however, the value of this resource depends upon ongoing manual data curation and annotation. in conclusion, the cov-rdb provides a uniquely integrated interdisciplinary synthesis of in vitro, animal model, and clinical studies of compounds with proven or possible anti-coronavirus activity. it helps researchers place their findings in the context of previously published data and it facilitates comparisons between different candidate antiviral compounds, thereby helping scientists, clinical investigators, public health officials, and funding agencies to prioritize the most promising compounds and repurposed drugs for further development. the authors declare no conflict of interest. centers for disease control and prevention (cdc) the stepehn b. thacker cdc library research articles downloadable database origin and evolution of pathogenic coronaviruses viral determinants of interspecies transmission virus nomenclature below the species level: a standardized nomenclature for natural variants of viruses assigned to the family filoviridae coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- the establishment of reference sequence for sars-cov- and variation analysis antibody cocktail to sars-cov- spike protein prevents rapid mutational escape seen with individual antibodies escape from neutralizing antibodies by sars-cov- spike protein variants coronavirus susceptibility to the antiviral remdesivir (gs- ) is mediated by the viral polymerase and the proofreading exoribonuclease coronavirus reverse genetic systems: infectious clones and replicons reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus an infectious cdna clone of sars-cov- rapid reconstruction of sars-cov- using a synthetic genomics platform sars-coronavirus- replication in vero e cells: replication kinetics, rapid adaptation and cytopathology sars-associated coronavirus replication in cell lines differential cell line susceptibility to the emerging novel human betacoronavirus c emc/ : implications for disease pathogenesis and clinical manifestation regulation of the interferon system: evidence that vero cells have a genetic defect in interferon production enhanced isolation of sars-cov- by tmprss 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and countermeasure development passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection treatment with lopinavir/ritonavir or interferon-β b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset type i and type iii interferons-induction, signaling, evasion, and application to combat covid- sars-cov- sensitive to type i interferon pretreatment coronavirus membrane fusion mechanism offers a potential target for antiviral development coronaviruses -drug discovery and therapeutic options the anticoagulant nafamostat potently inhibits sars-cov- s protein-mediated fusion in a cell fusion assay system and viral infection in vitro in a cell-type-dependent manner. viruses identification of antiviral drug candidates against sars-cov- from fda-approved drugs sars-cov- and sars-cov differ in their cell tropism and drug sensitivity profiles comparative analysis of antiviral efficacy of fda-approved drugs against sars-cov- in human lung cells sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor nafamostat mesylate blocks activation of sars-cov- : new treatment option for covid- a large-scale drug repositioning survey for sars-cov- antivirals remdesivir inhibits sars-cov- in human lung cells and chimeric sars-cov expressing the sars-cov- rna polymerase in mice drug repurposing screens reveal fda approved drugs active against sars-cov- clinical benefit of remdesivir in rhesus macaques infected with sars-cov- remdesivir for the treatment of covid- -preliminary report an orally bioavailable broad-spectrum antiviral inhibits sars-cov- in human airway epithelial cell cultures and multiple coronaviruses in mice studies in humanized mice and convalescent humans yield a sars-cov- antibody cocktail regn-cov antibody cocktail prevents and treats sars-cov- infection in rhesus macaques and hamsters a human neutralizing antibody targets the receptor binding site of sars-cov- human neutralizing antibodies elicited by sars-cov- infection cross-neutralization of sars-cov- by a human monoclonal sars-cov antibody convergent antibody responses to sars-cov- in convalescent individuals isolation of potent sars-cov- neutralizing antibodies and protection from disease in a small animal model potent neutralizing antibodies from covid- patients define multiple targets of vulnerability potent neutralizing antibodies against sars-cov- identified by high-throughput single-cell sequencing of convalescent patients' b cells neutralization of sars-cov- by destruction of the prefusion spike neutralizing nanobodies bind sars-cov- spike rbd and block interaction with ace potently neutralizing and protective human antibodies against sars-cov- a cross-reactive human iga 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inhibitor with involvement of the host antiviral response enhancing the antiviral efficacy of rna-dependent rna polymerase inhibition by combination with modulators of pyrimidine metabolism the dhodh inhibitor ptc arrests sars-cov- replication and suppresses induction of inflammatory cytokines efficacy and safety of leflunomide for refractory covid- : an open-label controlled study angiotensin-converting enzyme protects from severe acute lung failure a pilot clinical trial of recombinant human angiotensin-converting enzyme in acute respiratory distress syndrome inhibition of sars-cov- infections in engineered human tissues using clinical-grade soluble human ace neutralization of sars-cov- spike pseudotyped virus by recombinant ace -ig effect of hydroxychloroquine in hospitalized patients with covid- : preliminary results from a multi-centre, randomized hydroxychloroquine in nonhospitalized adults with early covid- : a randomized trial hydroxychloroquine with or without azithromycin in mild-to-moderate covid- hydroxychloroquine in patients with mainly mild to moderate coronavirus disease : open label, randomised controlled trial a cluster-randomized trial of hydroxychloroquine as prevention of covid- transmission and disease hydroxychloroquine for early treatment of adults with mild covid- : a randomized-controlled trial a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- an opendata portal to share covid- drug repurposing data in real time the coronavirus antibody database. biorxiv characteristics of registered studies for coronavirus disease (covid- ): a systematic review ongoing clinical trials for the management of the covid- pandemic this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - bi edcw authors: lennemann, nicholas j.; evans, azia s.; coyne, carolyn b. title: imaging-based reporter systems to define cvb-induced membrane remodeling in living cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: bi edcw enteroviruses manipulate host membranes to form replication organelles, which concentrate viral and host factors to allow for efficient replication. however, this process has not been well-studied in living cells throughout the course of infection. to define the dynamic process of enterovirus membrane remodeling of major secretory pathway organelles, we have developed plasmid-based reporter systems that utilize viral protease-dependent release of a nuclear-localized fluorescent protein from the endoplasmic reticulum (er) membrane during infection, while retaining organelle-specific fluorescent protein markers such as the er and golgi. this system thus allows for the monitoring of organelle-specific changes induced by infection in real-time. using long-term time-lapse imaging of living cells infected with coxsackievirus b (cvb), we detected reporter translocation to the nucleus beginning ~ h post-infection, which correlated with a loss of golgi integrity and a collapse of the peripheral er. lastly, we applied our system to study the effects of a calcium channel inhibitor, apb, on virus-induced manipulation of host membranes. we found that apb treatment had no effect on the kinetics of infection or the percentage of infected cells. however, we observed aberrant er structures in cvb-infected cells treated with apb and a significant decrease in viral-dependent cell lysis, which corresponded with a decrease in extracellular virus titers. thus, our system provides a tractable platform to monitor the effects of inhibitors, gene silencing, and/or gene editing on viral manipulation of host membranes, which can help determine the mechanism of action for antivirals. positive-strand rna viruses represent a large group of viruses that are responsible for the development of severe disease manifestations worldwide. enteroviruses, including coxsackievirus b (cvb) and enterovirus (ev ), are small, non-enveloped, positive-strand rna viruses. infection by these viruses can lead to the development of severe disease, including acute flaccid paralysis, meningitis, and encephalitis [ ] [ ] [ ] [ ] [ ] [ ] . currently, there are no antivirals and vaccines are only available for enterovirus and poliovirus. thus, a better understanding of the interactions of these viruses with the host cell can aid in the development of anti-enterovirus small molecule therapeutics. all positive-strand rna viruses manipulate host cell membranes to form membranous structures, termed replication organelles (ros), that concentrate viral and host factors to allow for efficient genome replication [ ] [ ] [ ] [ ] . enteroviruses induce extensive remodeling of the host secretory pathway during infection, which results in the accumulation of large clusters of vesicular structures in the cytoplasm that harbor viral replication proteins [ ] [ ] [ ] [ ] . previous studies have shown that at early stages of enterovirus infection, ros are observed in contact with the endoplasmic reticulum (er) membrane [ , ] . as infection progresses, the accumulation of viral nonstructural proteins a and b leads to an inhibition of secretory trafficking, resulting in the dispersal of the golgi complex [ , [ ] [ ] [ ] [ ] [ ] . the infection eventually leads to the release of progeny virion through disruption of the plasma membrane or non-lytic release [ ] . while these events have been well-studied using traditional imaging techniques on fixed samples, the single-cell kinetics have not been well defined. imaging of virus-infected cells typically involves taking still images of fluorescently labeled fixed samples. while these methods are widely used and highly practical, there are a number of limitations. the choice of fixation and permeabilization reagents can have a significant impact on the outcome of immunofluorescence staining [ ] [ ] [ ] . additionally, imaging of fixed samples only allows for 'snapshots' of different cells at various time points, but infection is a dynamic process. thus, a large sample size is needed to accurately confirm observed phenotypes. to overcome these limitations, live-cell imaging has been used in combination with recombinant viruses encoding fluorescent reporter proteins, such as gfp. these recombinant viruses are highly beneficial for many assays, including high-throughput screens. however, the insertion of a fluorescent protein open reading frame in small positive-strand rna viruses, including enteroviruses and flaviviruses, can dramatically attenuate viral progeny [ , , ] , thereby limiting the scope of assays for which these viruses can provide accurate information. previous reports have overcome these limitations by developing plasmid-based reporters to detect hepaci-and flavivirus infections [ ] [ ] [ ] . these reporters rely on viral protease-dependent cleavage to allow for the translocation of a fluorescent protein to the nucleus from the mitochondria [ ] or the er [ , ] . however, these reporters only detect virus infection and do not allow for the characterization of virus-induced remodeling of intracellular host cell structures without the introduction of multiple fluorescent protein marker expression vectors. thus, we sought to develop plasmid-based multipartite reporters for live-cell imaging that allow for the visualization of host cell organelles and enterovirus infection. in this report, we describe the characterization of enterovirus reporter constructs that allow for the visualization of the er and golgi complex throughout infection. using long-term time-lapse imaging over the complete course of infection, we define the real-time remodeling of host secretory pathway organelles during cvb infection. hela cells were cultured in modified eagle's medium supplemented with % fetal bovine serum, × non-essential amino acids, and % penicillin/streptomycin. u os osteosarcoma cells were cultured in dulbecco's modified eagle's medium supplemented with % fetal bovine serum and % penicillin/streptomycin. u os cells were transfected using xtremegenehp (roche, mannheim, germany), and selected and maintained in µg/ml and . µg/ml blasticidin s hcl (research products international corp, mount prospect, il, usa), respectively. heterogeneous populations of stable cells were propagated and used for experiments. all enteroviruses were propagated in hela cells and stocks were prepared and titrated by plaque assay, as previously described [ ] . plasmids expressing myc-tagged wild-type and catalytically inactivated cvb c pro have previously been described [ , ] . all primer sequences for cloning are listed in supplementary materials table s . reper: a pcr fragment corresponding to amino acids - of the poliovirus receptor, which includes the type i transmembrane region, was amplified with flanking kpni and ecori restriction sites from cdna and cloned into pcdna . -v -his topo ta vector. the er-localized mcherry-kdel (a gift from gia voeltz, university of colorado), which includes the bip signal sequence and a flavivirus octapeptide host signal peptidase cleavage sequence (lvnslvta), was amplified and inserted upstream of the pvr fragment at hindiii and kpni sites. next, an enterovirus c pro cleavage sequence (leaefq↓gppk) flanked by ecori and bamhi sites was inserted with an amplified gfp with the c-myc nuclear localization sequence (paakrvkld) flanked by bamhi and xhoi sites into the vector. lastly, the neomycin resistance gene was replaced with a blasticidin resistance gene using gibson assembly (neb, ipswich, ma, usa). to generate the reper mutant that is unable to be cleaved by c pro , the protease recognition-gfp-nls fragment was amplified and ligated into reper at ecori-xbai sites. repor: the gfp-nls sequence in reper was replaced with an ebfp -nls sequence, ebfp -nucleus- was a gift from michael davidson (addgene plasmid # ). a fluorescent golgi marker, memerald-golgi- (a gift from michael davidson, addgene plasmid # ), flanked by hindiii and ecori was produced by pcr. next, the emcv ires sequence flanked by ecori and noti was amplified. the new reper cassette containing ebfp -nls flanked by noti and xbai was amplified and ligated with memerald-golgi- and emcv-ires amplicons into pcdna . reper plasmid at the hindiii and xhoi. rabbit polyclonal antibodies against c-myc (a- ) and gfp (fl) were purchased from santa cruz biotechnology. rabbit polyclonal antibodies against gapdh were purchased from proteintech. mouse monoclonal antibodies against vinculin, clone hvin- , were purchased from sigma aldrich (st. louis, mo, usa). mouse monoclonal antibodies against enterovirus vp (ncl-entero) were purchased from novocastra (buffalo grove, il, usa). xtremegene hp plasmid transfection reagent was purchased from sigma aldrich. brefeldin a (invitrogen, carlsbad, ca, usa) was dissolved in dmso and used at a final concentration of µg/ml. also, -aminoethoxydiphenylborane ( apb, sigma aldrich) was diluted in dmso and used at a final concentration of µm. cvb growth kinetics were determined using u os cells transfected with the indicated plasmids. u os cells were bound with cvb ( pfu/cell) for h at • c, washed × in pbs, and placed at • c. at the indicated time, samples were collected from infected cells and used for plaque assays to determine the titer of the extracellular virus. inhibition of infection using apb was performed in u os cells. cells were treated with apb ( µm) for h at • c, cvb ( pfu/cell) was absorbed for h in the presence of apb at room temperature with gentle rocking, washed three times, and replaced with growth media containing apb for h. supernatants were collected to determine the extracellular virus titer by plaque assay. u os cells or hela cells were transfected with the indicated plasmids using xtremegenehp (roche). when specified, hela and u os cells were infected with the indicated virus for h. cell lysates were prepared on ice in cold ripa buffer (millipore, mm tris-hcl, ph . , % np- , . % sodium deoxycholate, mm nacl, mm edta) supplemented with × pierce protease inhibitor cocktail (aebsf, aprotinin, bestatin, e , leupeptin, pepstatin a). lysates were clarified by centrifugation at , × g for min at • c. lysates ( - µg) were separated on - % tgx pre-cast gels (bio-rad laboratories, hercules, ca, usa) and transferred to nitrocellulose membranes. membranes were blocked in pbs + % nonfat milk for min prior to probing with primary and ir-dye conjugated secondary antibodies (li-cor biosciences, lincoln, ne, usa) in pbs-t + % nonfat milk. immunoblots were visualized using an odyssey clx infrared imaging system (li-cor biosciences). quantification was performed using imagestudio (li-cor biosciences). u os cells were transfected with reper plasmid using xtremegenehp (roche). cells were cultured in chamber slides (millipore, burlington, ma, usa) and infected with cvb ( pfu/cell) for h. cells were fixed in % paraformaldehyde and permeabilized with . % triton x- . monolayers were washed, incubated with anti-vp primary antibody, washed, incubated with secondary antibody conjugated to alexa fluor- (invitrogen), washed, and mounted with vectashield containing , -diamidino- -phenylindole (dapi; vector laboratories, burlingame, ca, usa). when indicated, samples were incubated with primary antibodies for h at room temperature, washed, incubated with alexa fluor conjugated secondary antibodies for min, and mounted using vectashield (vector laboratories). images were captured using a zeiss lsm confocal microscope (zeiss, oberkochen, germany). u os cells stably expressing reper or repor were plated into mm dishes with a mm glass no. . coverslip (mattek corporation, ashland, ma, usa). live-cell imaging was performed as previously described [ , ] . briefly, dishes were placed in a • c, co -controlled incubator positioned over a motorized, inverted fluorescent microscope to allow for long-term time-lapse imaging (vivaview fl; olympus, tokyo, japan). between and stage positions were selected for each sample. cvb ( pfu/cell) was added to cells and images were captured every - min for - h. image series were cropped using imagej software (nih, bethesda, ma, usa) and multi-panel movies were rendered using photoshop cc (adobe, san jose, ca, usa). the plot profile tool in imagej was used to perform line plot analyses on the red, green, and blue channels of confocal images. data was plotted and the area under the curve was calculated for the area corresponding to the nucleus using prism software (graphpad, san diego, ca, usa). imagej was used to determine the fluorescence intensity of the whole cell and nuclear, gfp and mcherry signals for individual cells. the cytoplasmic fluorescence intensity was calculated by subtracting the gfp value from the whole cell value. due to variable fluorescence intensity between cells, we normalized nuclear fluorescence intensity to the cytoplasmic intensity. the ratio of gfp to mcherry was then calculated for each cell to determine if infection changed nuclear gfp fluorescence compared to mcherry. in order to account for changes in fluorescent signal throughout live-cell imaging, due to debris moving into the field of view, the fluorescent signal was normalized to an internal control. imagej was used to measure the mean signal intensities of a circular area within the nucleus and in er-dense microtubule-organizing center region for individual cells from every image series frame. infection was then calculated as the ratio between the nuclear and perinuclear signals. nonlinear regression curve analysis in prism (graphpad) was used to determine the time at which % of the maximum infection signal was observed for individual infected cells. peripheral er integrity was determined by quantifying the ratio of peripheral er signal to dense er sheet-like signal. images were thresholded to exclude the fine er structures in the cell periphery. the area of the nucleus was subtracted from the area of the thresholded region to calculate the er sheet area. next, a boundary was drawn around the perimeter of the er network and the area was measured. the areas of the nucleus and er sheets were subtracted from this value to calculate the peripheral er area. the ratio of peripheral er to er sheet area was used to quantify peripheral er integrity. these measurements were performed on individual cells from every image series frame using the same thresholding values. golgi dispersal was measured by quantifying golgi area, which was performed similarly to previously described methods for imaging flow cytometry [ ] . the first frame of an image series was thresholded to include the high-intensity cluster of memerald signal, indicative of the golgi, for a single cell, and this area was measured. this measurement was taken for the same cell for every frame of an image series using the same thresholding value. this was performed for individual cells from every image series frame. image series were used to determine the percentage of infected cells at hpi by observing infection reporter translocation. infected cells with aberrant er structures at hpi in the image series were manually counted using imagej (nih). image series were used to calculate the percentage of lysed cells, which was determined by counting the number of cells showing infection at hpi, then tracking these cells up to hpi and looking for the loss of the cytoplasmic reporter signal due to loss of plasma membrane integrity. student's t-tests and one-way anova with bonferroni multiple comparisons tests were performed using prism software (graphpad). to monitor enterovirus infection in real-time, we adapted cell-based reporter methodologies previously used for flaviviruses and hepatitis c virus that rely on viral protease cleavage-dependent translocation of a membrane-anchored cytoplasmic fluorescent proteins to the nucleus [ ] [ ] [ ] . however, given that these systems do not allow for the visualization of virus-induced manipulation of host cell organelles, we sought to design reporter plasmids expressing a fluorescent protein to indicate infection in addition to fluorescent protein-targeted organelle markers. as a proof of principle, we developed a dual reporter construct (reper) that expresses an er lumen localized mcherry containing an er retention signal (kdel) and signal peptidase cleavage site fused to a type i transmembrane domain followed by an enterovirus c protease ( c pro ) target sequence and a cytoplasmic gfp containing a nuclear localization signal (gfp-nls) ( figure a ). upon infection, the expression of c pro is predicted to release the er-tethered gfp-nls, allowing for translocation to the nucleus ( figure a) . importantly, given that the mcherry-kdel remains localized in the er, we can monitor er dynamics in parallel. we first validated the cleavage of the reporter by expressing myc-tagged cvb c pro and c mut , a catalytically inactive protease mutant, in u os cells. immunoblot analysis of cell lysates indicated that expression of cvb c pro led to the efficient release of gfp-nls from the full reper protein, whereas cleavage was absent in c mut expressing cells (figure b ). to ensure that cleavage of reper is dependent on viral protease cleavage during infection, we generated a construct containing a double alanine mutation (aa) of the glutamine-glycine (qg) c pro cleavage site. we found that mutation of the viral protease cleavage site blocked cvb-induced cleavage of reper ( figure c ). next, we sought to determine if reper was cleaved by a panel of enteroviruses during infection. we found that infection of reper-expressing hela cells with cvb, poliovirus, echovirus , and enterovirus resulted in cleavage of reper to varying degrees ( figure d ) and efficiency of cleavage correlated with moi ( figure e ). lastly, using immunofluorescence microscopy, we verified that cvb infection resulted in translocation of gfp-nls to the nucleus, whereas mcherry was retained in the er (figure f ). notably, we did not observe complete gfp-nls translocation, which is consistent with previous reports demonstrating that enterovirus infection restricts nuclear transport [ ] ; additionally, there may be uncleaved reporter present on the er, which can account for some of the cytoplasmic gfp fluorescence. intensity profile analysis of confocal images showed that mock-infected cells had lower gfp-nls reporter and mcherry-er signal intensities in the nuclear region, defined by peaks of dapi signal (figure g) . importantly, cvb infection led to an increase in gfp-nls reporter signal intensity in the nuclear region, while mcherry-er maintained a lower signal intensity in this region. upon quantification of confocal images, we observed a significant increase in the ratio of gfp to mcherry signals in the nucleus relative to the cytoplasmic during cvb infection compared to mock (figure h ). together, these results indicate that the reper reporter can be used to efficiently detect infection by a broad range of enteroviruses and monitor virus-induced changes of the er. viruses , , x for peer review of cytoplasmic gfp fluorescence. intensity profile analysis of confocal images showed that mockinfected cells had lower gfp-nls reporter and mcherry-er signal intensities in the nuclear region, defined by peaks of dapi signal (figure g ). importantly, cvb infection led to an increase in gfp-nls reporter signal intensity in the nuclear region, while mcherry-er maintained a lower signal intensity in this region. upon quantification of confocal images, we observed a significant increase in the ratio of gfp to mcherry signals in the nucleus relative to the cytoplasmic during cvb infection compared to mock (figure h ). together, these results indicate that the reper reporter can be used to efficiently detect infection by a broad range of enteroviruses and monitor virus-induced changes of the er. we next utilized long-term time-lapse imaging to determine the kinetics of gfp-nls translocation during virus infection. we used u os cells, which are highly conducive to live-cell imaging. cvb infection of reper expressing u os cells resulted in a clear translocation of gfp-nls reporter to the nucleus and eventual dispersal due to cell lysis, while the mcherry-kdel was maintained in the membranous structure of the er (video s and figure a) . image series analysis revealed that infected cells exhibited a linear increase in nuclear translocation of gfp-nls starting around~ hpi on average (figure b) with % of the maximum infection signal occurring~ . hpi (figure c) , whereas mock-infected cells showed no increase in nuclear gfp signal. we next utilized long-term time-lapse imaging to determine the kinetics of gfp-nls translocation during virus infection. we used u os cells, which are highly conducive to live-cell imaging. cvb infection of reper expressing u os cells resulted in a clear translocation of gfp-nls reporter to the nucleus and eventual dispersal due to cell lysis, while the mcherry-kdel was maintained in the membranous structure of the er (video s and figure a) . image series analysis revealed that infected cells exhibited a linear increase in nuclear translocation of gfp-nls starting around ~ hpi on average (figure b ) with % of the maximum infection signal occurring ~ . hpi (figure c ), whereas mock-infected cells showed no increase in nuclear gfp signal. the er has recently been shown to be involved in the early stages of cvb ro formation [ ] . thus, we sought to determine virus-induced changes to the er throughout infection using long-term time-lapse imaging. we observed a dramatic loss of the structural integrity of the er, as shown by the collapse of the peripheral er network in cells infected with cvb (video s , video s , and figure a ). analysis of image series from individual cells indicated a decrease in the ratio of peripheral er to sheet areas, a measurement of peripheral er integrity, that coincided with an increase in signal for infection ( figure d) . furthermore, analysis of multiple image series showed a reverse correlation between the infection signal and the peripheral er integrity (figure e ). interestingly, we found that the collapse of the peripheral er network preceded cell lysis by ~ h (videos s and s ). the er has recently been shown to be involved in the early stages of cvb ro formation [ ] . thus, we sought to determine virus-induced changes to the er throughout infection using long-term time-lapse imaging. we observed a dramatic loss of the structural integrity of the er, as shown by the collapse of the peripheral er network in cells infected with cvb (video s , video s , and figure a ). analysis of image series from individual cells indicated a decrease in the ratio of peripheral er to sheet areas, a measurement of peripheral er integrity, that coincided with an increase in signal for infection ( figure d) . furthermore, analysis of multiple image series showed a reverse correlation between the infection signal and the peripheral er integrity (figure e) . interestingly, we found that the collapse of the peripheral er network preceded cell lysis by~ h (videos s and s ). enterovirus infection has been previously shown to restrict secretory trafficking, resulting in golgi dispersal [ , [ ] [ ] [ ] [ ] [ ] . thus, we developed a bicistronic, triple reporter to visualize the er and golgi as well as monitoring virus infection (repor). the repor construct encodes an memerald-golgi marker followed by an ires-driven reper cassette that has been modified to include a bfp-nls reporter protein (figure a) . time-lapse imaging of cells expressing repor showed correct localization of organelle reporters that were maintained throughout time-lapse imaging (video s and figure b ). to initially validate this construct, we performed time-lapse imaging of repor cells treated with brefeldin a (bfa), an inhibitor of er to golgi protein transport [ ] . as expected, bfa treatment resulted in a clear change in the localization of the golgi marker from a tight cluster to a dispersed signal that colocalized with the er marker, which indicated a disruption of golgi integrity and a block in protein transport out of the er (video s and figure b ). next, we sought to monitor virus-induced changes to the golgi during infection of repor expressing cells. to do this, we performed time-lapse imaging of cells infected with cvb. consistent with previous reports [ , [ ] [ ] [ ] [ ] [ ] ], cvb infection led to a clear dispersal of the golgi marker, as shown by a decrease in the high-intensity golgi signal area and presence of a diffuse low-intensity signal, indicating a disruption of golgi integrity (video s and figure b ). analysis of this image series indicated that dispersal of the golgi coincided with an increase in the signal for infection (figure c ) and analysis of multiple image series showed a reverse correlation between infection signal and golgi area (figure d ). together, these results indicated the repor construct can be used to monitor pharmacological and viral manipulation of the secretory pathway. previous studies have utilized recombinant enteroviruses that encode fluorescent protein reporters to visualize infection in real-time. however, these viruses display dramatic defects in replication kinetics [ , , ] . thus, we sought to determine if reporter plasmid expression attenuates virus replication. we performed single-step growth curves for cvb in u os cells transfected with each reporter plasmid. our results indicated there were no differences in growth of cvb in u os cells transfected with empty, reper, or repor plasmids (figure ) . these results suggest our reporter plasmids can be used to accurately monitor viral replication kinetics and virus-induced manipulation of host cells in real-time. previous studies have utilized recombinant enteroviruses that encode fluorescent protein reporters to visualize infection in real-time. however, these viruses display dramatic defects in replication kinetics [ , , ] . thus, we sought to determine if reporter plasmid expression attenuates virus replication. we performed single-step growth curves for cvb in u os cells transfected with each reporter plasmid. our results indicated there were no differences in growth of cvb in u os cells transfected with empty, reper, or repor plasmids (figure ) . these results suggest our reporter plasmids can be used to accurately monitor viral replication kinetics and virus-induced manipulation of host cells in real-time. lastly, we sought to demonstrate the utility of our reporter system by visualizing the effect of a pharmacological inhibitor on virus infection. we have previously reported that inhibition of inositol , , -triphosphate receptors (ip r), which function as calcium channels in the er, restricts the release of an infectious virus through modulating cell death pathways [ ] . long-term time-lapse imaging of cvb infected repor cells treated with dmso produced results similar to those described above (video s , video s , and figure a ). time-lapse imaging of repor cells in the presence of aminoethoxydiphenylborane ( apb), an ip r inhibitor, did not prevent the nuclear translocation of the virus reporter or dispersal of the golgi (video s , video s , and figure a ). furthermore, image series analysis showed no defect in the kinetics of nuclear translocation of the reporter, indicating this drug does not affect viral protein production and replication (figure b) . additionally, we did not observe a difference in the percentage of cells infected at hpi, indicated by reporter translocation to the nucleus (figure c ). interestingly, apb treatment of cells led to the formation of aberrant er structures that coincided with reporter translocation to the nucleus (video s and video s ). these structures resembled large membrane aggregates and inflated regions of er (figure a , asterisks and arrowheads, respectively), which were rarely observed in infected dmso-treated cells (figure c ) and not observed in uninfected cells. furthermore, treatment with apb led to a significant decrease in the number of infected cells that progressed to lysis (figure c ), which is consistent with a significant decrease in the titer of extracellular virus (figure d ). together, these results indicate that our plasmid-based reporters can be used to monitor virus-induced changes to host membranes upon inhibition of specific cellular functions, which can further our understanding of inhibitor mechanisms of action during infection. lastly, we sought to demonstrate the utility of our reporter system by visualizing the effect of a pharmacological inhibitor on virus infection. we have previously reported that inhibition of inositol , , -triphosphate receptors (ip r), which function as calcium channels in the er, restricts the release of an infectious virus through modulating cell death pathways [ ] . long-term time-lapse imaging of cvb infected repor cells treated with dmso produced results similar to those described above (video s , video s , and figure a ). time-lapse imaging of repor cells in the presence of -aminoethoxydiphenylborane ( apb), an ip r inhibitor, did not prevent the nuclear translocation of the virus reporter or dispersal of the golgi (video s , video s , and figure a ). furthermore, image series analysis showed no defect in the kinetics of nuclear translocation of the reporter, indicating this drug does not affect viral protein production and replication ( figure b ). additionally, we did not observe a difference in the percentage of cells infected at hpi, indicated by reporter translocation to the nucleus (figure c ). interestingly, apb treatment of cells led to the formation of aberrant er structures that coincided with reporter translocation to the nucleus (video s and video s ). these structures resembled large membrane aggregates and inflated regions of er (figure a , asterisks and arrowheads, respectively), which were rarely observed in infected dmso-treated cells (figure c ) and not observed in uninfected cells. furthermore, treatment with apb led to a significant decrease in the number of infected cells that progressed to lysis (figure c ), which is consistent with a significant decrease in the titer of extracellular virus (figure d ). together, these results indicate that our plasmid-based reporters can be used to monitor virus-induced changes to host membranes upon inhibition of specific cellular functions, which can further our understanding of inhibitor mechanisms of action during infection. the data was collected from two independent experiments, with four separate fields/experiment. significance was determined by one-way anova, *** p < . . (d) titer of extracellular virus from cvb infected u os cells treated with dmso or apb ( µm) at hpi. the data is shown as the average ± sd titer (pfu/ml) from three independent experiments. significance was determined by student's t-test, ** p < . . live-cell imaging is a powerful tool to study virus-host interactions in real-time. to facilitate this, we developed and characterized multipartite fluorescent-reporters to monitor enterovirusinduced remodeling of organelles in the host secretory pathway. these reporters rely on enterovirus c pro activity to release an nls-tagged fluorescent protein, which translocates to the nucleus while retaining er and golgi localized fluorescent markers. live-cell imaging of cvb infected cells the data was collected from two independent experiments, with four separate fields/experiment. significance was determined by one-way anova, *** p < . . (d) titer of extracellular virus from cvb infected u os cells treated with dmso or apb ( µm) at hpi. the data is shown as the average ± sd titer (pfu/ml) from three independent experiments. significance was determined by student's t-test, ** p < . . live-cell imaging is a powerful tool to study virus-host interactions in real-time. to facilitate this, we developed and characterized multipartite fluorescent-reporters to monitor enterovirus-induced remodeling of organelles in the host secretory pathway. these reporters rely on enterovirus c pro activity to release an nls-tagged fluorescent protein, which translocates to the nucleus while retaining er and golgi localized fluorescent markers. live-cell imaging of cvb infected cells expressing these reporters allowed for the real-time visualization of virus-induced changes to the host cell, including the collapse of the peripheral er network and loss of golgi integrity. furthermore, we used live-cell imaging with repor to show that a calcium channel inhibitor, apb, resulted in cvb-induced formation of aberrant er structures and decreased the lytic effects of the virus. overall, we show that our reporters present innovative opportunities for studying enterovirus-induced remodeling of host cell membranes during infection in real-time. previous studies have utilized plasmid-based reporters for live-cell imaging of other small positive-strand rna virus infections [ ] [ ] [ ] . plasmid-based reporters for dengue virus and zika virus utilized viral nonstructural protein b (ns b), a multi-pass er membrane-anchored protein, fused to gfp-nls [ , ] . however, expression of flavivirus ns b has previously been associated with the induction of autophagy and remodeling of the er, which can complicate interpretation of results and restrict the applications in which these reporters can be used [ , ] . additionally, these reporters are limited to the detection of virus infection. to monitor virus-induced changes to the host-cell they would need to be used in combination with other fluorescent-marker expression vectors or stable cells. thus, we expanded on these reporters to develop multipartite fluorescent reporters that anchor the virus reporter via a single-pass host-derived transmembrane domain. this design eliminates the need for transfection of multiple constructs to visualize host structures and virus infection and significantly simplifies the generation of stable cells or transgenic animals. whereas the flavivirus reporters are virus-specific [ ] , the reper and repor reporters can be used for a broad array of enteroviruses. while all enterovirus infections tested resulted in reporter cleavage, there were differences in efficiency with cvb infection consistently resulting in the highest reporter cleavage efficiency. one explanation is the engineered cleavage site may not be optimal for all enteroviruses and may need to be modified for specific viruses. alternatively, there may be slight differences in replication kinetics between these viruses, which results in lower levels of cleavage by the time point tested. thus, these reporters can be used to compare virus-specific manipulation of host membranes in a wide variety of target cells. all positive-strand rna viruses remodel host membranes to form ros that facilitate the sequestration of viral and host factors to promote efficient replication [ ] [ ] [ ] [ ] . recently, a recombinant cvb was developed to show in real-time that the accumulation of the viral a coincides with golgi disruption [ ] . this recombinant virus was also used to identify the er as an early source for ro membranes [ ] . however, the manipulation of the cvb genome to introduce fluorescent-based reporters resulted in dramatic attenuation, which is common in the development of most recombinant small positive-strand rna viruses [ , , ] . an important benefit of our reporters is their expression did not restrict cvb infection. thus, we utilized the reporters to evaluate the kinetics of cvb-induced membrane remodeling at a single-cell level. using live-cell imaging and image series analysis, we were able to demonstrate that golgi dispersal and loss of peripheral er integrity coincided with an increase in cvb infection. interestingly, the diffuse reporter signal in the cytoplasm indicated that the loss of the peripheral er network did not coincide with cell rounding, shrinkage, or result in a sudden progression to cell death, which occurred~ h post er collapse. interestingly, cmv infection leads to the collapse of the er due to the expression of the viral pul × protein [ ] . thus, future studies are warranted to determine if interactions between viral and host proteins are responsible for cvb-induced remodeling of the er. we applied our reporter systems to better understand the effects of calcium channel inhibition on infection, which we have previously shown decreases the release of infectious virus [ , ] . we found that apb inhibition of calcium channels did not block virus-induced nuclear translocation of the virus reporter, golgi dispersal, or er collapse. however, we did find that apb treated cells showing virus reporter translocation to the nucleus contained aberrant er structures and prevented cell lysis. previous studies have shown similar structures represent er whorls or stacked er, which have been proposed to regulate cargo exit from the er [ ] [ ] [ ] . in yeast, these structures have also been associated with induction of er stress and the unfolded protein response [ , ] , which could explain their presence in cvb infected cells that are unable to regulate ca + release. thus, our results demonstrate an important application for our reporters in understanding the real-time effects of pharmacological inhibitors on virus-induced manipulation of the host cell. overall, we have shown the benefits of using multipartite plasmid-based reporters to visualize virus-induced remodeling of the host cell using live-cell imaging. our system can be readily modified to include combinations of other fluorescent markers of host cell structures or viral host factors. additionally, these constructs can be modified to include recognition sites for proteases encoded by other positive-strand rna viruses, including flaviviruses, alphaviruses, and coronaviruses. furthermore, these reporters can be used for other applications, including diagnostics and high-throughput screens. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : primer and oligonucleotide sequences, video s : enterovirus infections of the central nervous system neonatal enterovirus infections reported to the national enterovirus surveillance system in the united states centers for disease control and prevention. enterovirus surveillance-united states pediatric group b coxsackievirus infections neurologic complications in children with enterovirus infection pathogenesis of enterovirus brainstem encephalitis in pediatric patients: roles of cytokines and cellular immune activation in patients with pulmonary edema architecture and biogenesis of plus-strand rna virus replication factories the transformation of enterovirus replication structures: a three-dimensional study of single and double-membrane compartments building viral replication organelles: close encounters of the membrane types remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles association of polioviral proteins of the p genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography electron microscopic study of the formation of poliovirus cellular origin and ultrastructure of membranes induced during poliovirus infection illuminating the sites of enterovirus replication in living cells by using a split-gfp-tagged viral protein origins of enterovirus replication organelles established by whole-cell electron microscopy viral reorganization of the secretory pathway generates distinct organelles for rna replication hijacking components of the cellular secretory pathway for replication of poliovirus rna inhibition of cellular protein secretion by poliovirus proteins b and a poliovirus infection blocks ergic-to-golgi trafficking and induces microtubule-dependent disruption of the golgi complex enterovirus protein b po(u)res out the calcium: a viral strategy to survive? nonlytic viral spread enhanced by autophagy components tissue preparation for immunocytochemistry pfa fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins immunolabeling artifacts and the need for live-cell imaging dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro development and characterization of a stable egfp enterovirus for antiviral screening real-time imaging of hepatitis c virus infection using a fluorescent cell-based reporter system a fluorescent cell-based system for imaging zika virus infection in real-time a plasmid-based reporter system for live cell imaging of dengue virus infected cells comparative rnai screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers the coxsackievirus b c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling rip regulates autophagy and promotes coxsackievirus b infection of intestinal epithelial cells adap is an interferon stimulated gene that restricts rna virus entry bpifb regulates autophagy and coxsackievirus b replication through a noncanonical pathway independent of the core initiation machinery effects of poliovirus infection on nucleo-cytoplasmic trafficking and nuclear pore complex composition dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells calcium signals and calpain-dependent necrosis are essential for release of coxsackievirus b from polarized intestinal epithelial cells induction of endoplasmic reticulum-derived replication-competent membrane structures by west nile virus non-structural protein b zika virus ns a and ns b proteins deregulate akt-mtor signaling in human fetal neural stem cells to inhibit neurogenesis and induce autophagy human cytomegalovirus pul x induces the release of endoplasmic reticulum calcium stores release of intracellular calcium stores facilitates coxsackievirus entry into polarized endothelial cells mouse stbd is n-myristoylated and affects er-mitochondria association and mitochondrial morphology yip a structures the mammalian endoplasmic reticulum membrane biogenesis during b cell differentiation: most endoplasmic reticulum proteins are expressed coordinately er-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response we thank kaman fan (tsinghua university school of medicine) for technical assistance and members of the coyne laboratory for discussion of the data presented in the manuscript. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -o atz c authors: xiao, li; sakagami, hiroshi; miwa, nobuhiko title: ace : the key molecule for understanding the pathophysiology of severe and critical conditions of covid- : demon or angel? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: o atz c recently, the sars-cov- induced disease covid- has spread all over the world. nearly % of the patients have severe or critical conditions. sars-cov- exploits ace for host cell entry. ace plays an essential role in the renin–angiotensin–aldosterone system (raas), which regulates blood pressure and fluid balance. ace also protects organs from inflammatory injuries and regulates intestinal functions. ace can be shed by two proteases, adam and tmprss . tmprss -cleaved ace allows sars-cov- cell entry, whereas adam -cleaved ace offers protection to organs. sars-cov- infection-caused ace dysfunction worsens covid- and could initiate multi-organ failure. here, we will explain the role of ace in the pathogenesis of severe and critical conditions of covid- and discuss auspicious strategies for controlling the disease. since december , the new coronavirus (severe acute respiratory syndrome coronavirus , sars-cov- ) induced disease, covid- , has spread rapidly all over the planet. as shown in covid- dashboard by the center for systems science and engineering (csse) at johns hopkins university (jhu), up to date , , people have been infected and , deaths have been confirmed worldwide [ ] .who officially declared covid- a pandemic on march th, . according to a report based on , cases (test confirmed cases: , ( %) from the chinese center for disease control and prevention, % of covid- patients have cold-like symptoms and mild pneumonia, % have severe respiratory inflammation, and % have critical conditions including respiratory failure, septic shock, and/or multiple organ dysfunction or failure. the mortality is . % ( . % in critical cases) [ ] . since the rates of severe and critical cases are much higher than seasonal influenza, it is essential to understand the pathophysiology and then devise strategies to fight the disease. sars-cov- is a positive-sense single-stranded rna virus with a crown-like appearance of spike proteins (s proteins) that project from their envelope. similar to sars (severe acute respiratory syndrome, [ ] [ ] coronavirus (sars-cov) [ ] , sars-cov- primarily uses the s protein to invade host cells through ace , an enzyme which is known to be important in the renin-angiotensin-aldosterone system (raas) [ , ] . cellular ace , adam , and tmprss are all expressed on the cell membrane. after being shed by adam , soluble ace is released from its full-length form to counteract the effects of ang ii signaling. alternatively, cellular ace is also shed by tmprss , which results in sars-cov- -cell membrane fusion. sars-cov- rna is then released into the cytoplasm and viral replication is efficiently processed. because soluble ace contains the virus binding site, it can also bind to the virus. however, without an intracellular environment, the virus cannot be duplicated. ace is a close homologue of human ace [ , ] . as is well-known, ace cleaves angiotensin i (ang i) to ang ii. ang ii binds to ang ii receptor (at ) and then mediates numerous systemic and local effects (such as promoting vasoconstriction, fibrosis, and salt retention) in the cardiovascular system ( figure ). in the raas, ace exerts the opposite role of ace. ace catalyzes the conversion of ang i to ang-( - ) and ang ii to ang-( - ). the converting efficiency of ace on substrate ang ii is -fold higher than that on ang i [ ] . ang-( - ) binds to the g protein-coupled receptor mas to mediate various effects including vasorelaxation, cardioprotection, anti-oxidative action [ ] , antiinflammation [ ] , and the inhibition of ang ii-induced signaling [ , ] . the ace -ang-( - ) axis is considered an important therapeutic target in cardiovascular disorders [ ] . a large cohort study showed that circulating ace was only detectable in the serum of among test subjects, and its concentration was approximately -fold lower than that of circulating ace [ ] . more evidence showed that circulating ace is increased in patients with type or type diabetes, hypertension, heart failure, and chronic kidney diseases [ ] [ ] [ ] . the reason for high levels of ace in these patients is that increased ace is a defensive response to counteract the adverse effect of ang ii. since ang ii-at receptor signaling also promotes autoimmune response, ace may control immune functions through the ang-( - )-mas axis [ , ] . cellular ace , adam , and tmprss are all expressed on the cell membrane. after being shed by adam , soluble ace is released from its full-length form to counteract the effects of ang ii signaling. alternatively, cellular ace is also shed by tmprss , which results in sars-cov- -cell membrane fusion. sars-cov- rna is then released into the cytoplasm and viral replication is efficiently processed. because soluble ace contains the virus binding site, it can also bind to the virus. however, without an intracellular environment, the virus cannot be duplicated. ace is a close homologue of human ace [ , ] . as is well-known, ace cleaves angiotensin i (ang i) to ang ii. ang ii binds to ang ii receptor (at ) and then mediates numerous systemic and local effects (such as promoting vasoconstriction, fibrosis, and salt retention) in the cardiovascular system ( figure ). in the raas, ace exerts the opposite role of ace. ace catalyzes the conversion of ang i to ang-( - ) and ang ii to ang-( - ). the converting efficiency of ace on substrate ang ii is -fold higher than that on ang i [ ] . ang-( - ) binds to the g protein-coupled receptor mas to mediate various effects including vasorelaxation, cardioprotection, anti-oxidative action [ ] , anti-inflammation [ ] , and the inhibition of ang ii-induced signaling [ , ] . the ace -ang-( - ) axis is considered an important therapeutic target in cardiovascular disorders [ ] . a large cohort study showed that circulating ace was only detectable in the serum of among test subjects, and its concentration was approximately -fold lower than that of circulating ace [ ] . more evidence showed that circulating ace is increased in patients with type or type diabetes, hypertension, heart failure, and chronic kidney diseases [ ] [ ] [ ] . the reason for high levels of ace in these patients is that increased ace is a defensive response to counteract the adverse effect of ang ii. since ang ii-at receptor signaling also promotes autoimmune response, ace may control immune functions through the ang-( - )-mas axis [ , ] . angiotensinogen is produced in the liver and released into the blood stream. the renal juxtaglomerular apparatus-secreted renin cleaves angiotensinogen to ang i. ang i is further converted to ang ii by ace which is mostly produced in the lungs. ang ii binds to both at and at receptors to regulate the blood pressure and inflammation. most actions of ang ii occur via the at receptor. meanwhile, cellular ace is cleaved by adam . after the active form of ace being released into the extracellular environment, ace converts ang i to ang-( - ) and ang ii to ang-( - ). ace also converts ang-( - ) to ang-( - ). ang-( - ) binds to mas receptor to mediate the opposite effects of ang ii. genetic ace deficiency is associated with upregulation of inflammatory mediators, elevated inflammatory responsiveness to proinflammatory stimuli, and enhanced ang ii-induced cardiac and aortic remodeling [ , ] . the anti-inflammatory effects of ace are exerted mostly through the ace -ang-( - ) axis against ang ii-at activities [ ] . ace also has a raas-independent function. in the intestine, cellular ace (not circulating ace ) regulates the absorption of amino acids and the intestinal bacterial balance to reduce intestinal inflammation [ ] . cellular ace is required for expression of the neutral amino acid transporter b at in intestinal epithelial cells [ ] . without ace -b at complex, serum levels of the neutral amino acids valine (val), threonine (thr), and tyrosine (tyr), and the essential amino acid tryptophan (trp) were markedly reduced and resulted in severe intestinal inflammation and microbial imbalance in ace knockout mice. administration with trp or nicotinamide (the metabolic product of trp) could increase the expression of antimicrobial peptide α-defensins and reverse the above-mentioned microbial imbalance and inflammation in the gut. structural modelling suggested that the ace -b at complex could bind to the s-protein of sars-cov- simultaneously [ ] . the intestinal cellular angiotensinogen is produced in the liver and released into the blood stream. the renal juxtaglomerular apparatus-secreted renin cleaves angiotensinogen to ang i. ang i is further converted to ang ii by ace which is mostly produced in the lungs. ang ii binds to both at and at receptors to regulate the blood pressure and inflammation. most actions of ang ii occur via the at receptor. meanwhile, cellular ace is cleaved by adam . after the active form of ace being released into the extracellular environment, ace converts ang i to ang-( - ) and ang ii to ang-( - ). ace also converts ang-( - ) to ang-( - ). ang-( - ) binds to mas receptor to mediate the opposite effects of ang ii. genetic ace deficiency is associated with upregulation of inflammatory mediators, elevated inflammatory responsiveness to proinflammatory stimuli, and enhanced ang ii-induced cardiac and aortic remodeling [ , ] . the anti-inflammatory effects of ace are exerted mostly through the ace -ang-( - ) axis against ang ii-at activities [ ] . ace also has a raas-independent function. in the intestine, cellular ace (not circulating ace ) regulates the absorption of amino acids and the intestinal bacterial balance to reduce intestinal inflammation [ ] . cellular ace is required for expression of the neutral amino acid transporter b at in intestinal epithelial cells [ ] . without ace -b at complex, serum levels of the neutral amino acids valine (val), threonine (thr), and tyrosine (tyr), and the essential amino acid tryptophan (trp) were markedly reduced and resulted in severe intestinal inflammation and microbial imbalance in ace knockout mice. administration with trp or nicotinamide (the metabolic product of trp) could increase the expression of antimicrobial peptide α-defensins and reverse the above-mentioned microbial imbalance and inflammation in the gut. structural modelling suggested that the ace -b at complex could bind to the s-protein of sars-cov- simultaneously [ ] . the intestinal cellular ace might be another viral entry point for sars-cov- as well as tmprss -sheded ace in the lungs. covid- is a lower respiratory tract disease. the first anatomic pathology report of a covid- death indicated that the most seriously injured organs were the lungs. exudative lesions and fibrosis were observed in the lungs. phlegm and exudates filled up the lower respiratory tract and pulmonary alveolus. compared to sars, the exudative lesions were far worse but the fibrosis was much lighter. notably, segmental dilatation and stenosis of the small intestine were observed in the cadaver suggesting that the small intestine was also severely injured by sars-cov- infection. lesions on other organs were not obvious [ ] . the pathological findings of another covid- victim showed that the bilateral diffuse alveolar damage with cellular fibromyxoid exudates, desquamation of pneumocytes, and hyaline membrane formation occurred in the lungs [ ] . this evidence suggests that sars-cov- invades the human body mostly through the respiration system, and possibly also through the intestine and other tissues. in , ding et al. detected organ distribution of sars-cov by immunohistochemistry and in situ hybridization. sars-cov was mainly found in the respiratory system, stomach, small intestine, kidneys, and sweat glands [ ] . the organ distribution of sars-cov- is probably similar to sars-cov. based on the evidence described above, we hypothesize that sars-cov- invades the lungs and intestine through tmprss -cleaved ace . if the immune system is not able to defeat the infection, sars-cov- will be massively replicated, occupy cellular ace , and destroy the host's cells. as a consequence, the ang ii-at signaling cannot be inactive. together, the intestinal function is ruined and the inflammation exacerbated. as a result, a cytokine storm occurs and eventually the respiration system, cardiovascular system and other organs lose function ( figure ) . clinical data showed that among covid- inpatients, about % have underlying diseases. these patients have an increased risk of death. hypertension was the most common, followed by diabetes and coronary heart disease [ , ] . since the ang ii-at axis is hyperactive in these diseases already [ , ] , sars-cov- further decreases the production of functional ace . therefore, in patients with those underlying diseases it is much easier to develop severe and critical conditions. cellular ace can be shed by both adam and tmprss . the adam -cleavage is a normal path which results in the production of circulating ace . circulating ace can prevent severe pathological conditions and protect organs during sars-cov- infection. in contrast, tmprss -shed ace allows the sars-cov- to invade cells in the lungs and intestine. tmprss -cleavage path might inhibit adam -cleavage path. if the immune system is not able to defeat the virus, sars-cov- will be massively reproduced, occupy cellular ace , and destroy the host's cells in the lungs and intestine. as sars-cov- reduces ace expression, there is not enough adam -shed circulating ace against the ang ii signaling-induced inflammatory injuries, and inflammation is accelerated until the immune system is overwhelmed. meanwhile, cellular ace /b at in the intestine is ruined by the virus. essential amino acids cannot be absorbed, the antimicrobial peptides are reduced, and the ecology of the gut microbiome is damaged. these intestinal changes will precipitate inflammation. as a result, a cytokine storm occurs and eventually induces multiple organ dysfunction or failure. cellular ace can be shed by both adam and tmprss . the adam -cleavage is a normal path which results in the production of circulating ace . circulating ace can prevent severe pathological conditions and protect organs during sars-cov- infection. in contrast, tmprss -shed ace allows the sars-cov- to invade cells in the lungs and intestine. tmprss -cleavage path might inhibit adam -cleavage path. if the immune system is not able to defeat the virus, sars-cov- will be massively reproduced, occupy cellular ace , and destroy the host's cells in the lungs and intestine. as sars-cov- reduces ace expression, there is not enough adam -shed circulating ace against the ang ii signaling-induced inflammatory injuries, and inflammation is accelerated until the immune system is overwhelmed. meanwhile, cellular ace /b at in the intestine is ruined by the virus. essential amino acids cannot be absorbed, the antimicrobial peptides are reduced, and the ecology of the gut microbiome is damaged. these intestinal changes will precipitate inflammation. as a result, a cytokine storm occurs and eventually induces multiple organ dysfunction or failure. as addressed above, the strategies for preventing and reversing severe and critical covid- should include: since tmprss plays a very important role in sars-cov- cell entry and ace dysfunction, blocking the activity of tmprss should be the primary strategy for preventing severe and critical conditions of covid- . hoffmann et al. discovered that a serine protease inhibitor, camostat mesylate, partially blocked tmprss -ace -mediated sars-cov- entry [ ] . similarly, nafamostat mesylate inhibited the tmprss -ace -mediated sars-cov- envelope-plasma membrane fusion, and then showed times higher efficiency than camostat mesylate in preventing sars-cov- cell entry [ ] . both camostat mesylate and nagamostat mesylate are clinically approved medicines with verified safety, and thus can be applied for covid- treatment in clinical practices immediately. nafamostat mesylate also has been used as a short-acting anticoagulant due to its ability to prevent the proteolysis of fibrinogen into fibrin. clinical reports of covid- showed that increased d-dimer (a fibrin degradation product) levels in severe and critical patients were often observed [ , , ] . high levels of d-dimer (> µg/ml) are related to higher risk of mortality [ ] . thus, nafamostat mesylate not only can block the viral entry, but also prevent thrombosis and disseminated intravascular coagulation (dic) in covid- patients. a clinical trial of covid- with nafamostat mesylate treatment in japan started in march . because sars-cov- exploits ace for entry, some scientists are concerned that the expression of ace might provide possible advantageous routes for virus entry. they suggest that patients with hypertension, diabetes, and cardiovascular diseases should reduce the use of ace inhibitors and ang ii-at blockers because these medicines can increase the expression of ace [ ] . ibuprofen and thiazolidinediones were also suspected of increasing the risk of covid- for the same reason [ ] . however, there is no evidence that those medicines can facilitate sars-cov or sars-cov- infection. on the contrary, sars-cov infection could reduce ace expression and worsen acute lung failure, which could be attenuated by blocking the raas pathway [ ] . ace expression was also shown to protect against severe acute lung failure in a mouse model [ ] . in addition, cell tropism of sars-cov and sars-cov- did not firmly associate with ace expression. ace is expressed on both type i and type ii pneumocytes, whereas sars-cov and sars-cov- only make use of type ii pneumocytes because the infection requires co-expression of ace and tmprss as we described before [ , , ] . therefore, increasing ace (especially circulating ace ) or ang-( - ) is a potential approach to reduce sars-cov- -induced severe damage and to protect organs. recently, an international research team showed that clinical-grade human soluble ace could combine with sars-cov- and decrease its infection rate to -times in engineered human blood vessel organoids and human kidney organoids [ ] . evidence showed that administration with trp or nicotinamide could reverse severe intestinal inflammation in ace knockout mice [ ] . as a nutrient enhancer, trp and its metabolites play crucial roles in gut microbiota maintenance, microbial metabolism, the host's immune system, the host-microbiome interface, and host immune system-intestinal microbiota interactions. [ ] . thus, supplementation with trp or nicotinamide may regulate the gut microbiome and increase antimicrobial peptides to convert sars-cov- -induced lesions in the intestine and further improve systemic conditions. in summary, ace plays essential roles in sars-cov- cell entry and makes an impact on the progress and prognosis of severe and critical conditions of covid- . regulating ace -related enzymes and amino acid intake would be desirable for disease control. however, more experimental and clinical studies are required. moreover, pathogenesis of severe and critical conditions of covid- is very complicated. other molecules, such as il- , also play important roles in the disease. therefore, therapeutic strategies should be flexibly changed according to the patient's clinical inspection results. funding: this research received no external funding. characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention angiotensin-converting enzyme is a functional receptor for the sars coronavirus sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor enhanced isolation of sars-cov- by tmprss -expressing cells a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase tumor necrosis factor-alpha convertase (adam ) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme- (ace ) receptor and viral determinants of sars-coronavirus adaptation to human ace tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis angiotensin-converting enzyme is an essential regulator of heart function angiotensin-converting enzyme overexpression in the subfornical organ prevents the angiotensin ii-mediated pressor and drinking responses and is associated with angiotensin ii type receptor downregulation tmprss and adam cleave ace differentially and only proteolysis by tmprss augments entry driven by the severe acute respiratory syndrome coronavirus spike protein efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry differential downregulation of ace by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection tmprss contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection cloning and characterization of the cdna and gene for human epitheliasin prostate-localized and androgen-regulated expression of the membrane-bound serine protease tmprss sars-cov- receptor ace is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - aceh/ace is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ace inhibitors. can hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase angiotensin-( - ) prevents activation of nadph oxidase and renal vascular dysfunction in diabetic hypertensive rats angiotensin-( - ) inhibits allergic inflammation, via the mas receptor, through suppression of erk / -and nf-κb-dependent pathways angiotensin-( - ) - ) is an endogenous ligand for the g protein-coupled receptor mas angiotensin-converting enzyme as a therapeutic target for heart failure circulating activities of angiotensin-converting enzyme, its homolog, angiotensin-converting enzyme , and neprilysin in a family study circulating angiotensin-converting enzyme activity in patients with chronic kidney disease without previous history of cardiovascular disease expression of ace and ace in patients with hypertensive nephrosclerosis a review of urinary angiotensin converting enzyme in diabetes and diabetic nephropathy angiotensin ii revisited: new roles in inflammation, immunology and aging identification of splenic reservoir monocytes and their deployment to inflammatory sites genetic ace deficiency accentuates vascular inflammation and atherosclerosis in the apoe knockout mouse enhanced angiotensin ii-induced cardiac and aortic remodeling in ace knockout mice the anti-inflammatory potential of ace /angiotensin-( - )/mas receptor axis: evidence from basic and clinical research ace links amino acid malnutrition to microbial ecology and intestinal inflammation expression and regulation of the neutral amino acid transporter b at in rat small intestine structure of dimeric full-length human ace in complex with b at . biorxiv gross examination report of a covid- death autopsy pathological findings of covid- associated with acute respiratory distress syndrome organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways renin-angiotensin-aldosterone system in diabetes and hypertension use of angiotensin receptor blockers in cardiovascular protection: current evidence and future directions , nejmoa . [crossref] . identification of an existing japanese pancreatitis drug, nafamostat, which is expected to prevent the transmission of new coronavirus infection (covid- ) diagnostic utility of clinical laboratory data determinations for patients with the severe covid- clinical features and treatment of covid- patients in northeast chongqing covid- and the cardiovascular system are patients with hypertension and diabetes mellitus at increased risk for covid- infection? a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury angiotensin-converting enzyme protects from severe acute lung failure sars-cov replicates in primary human alveolar type ii cell cultures but not in type i-like cells histopathologic changes and sars-cov- immunostaining in the lung of a patient with covid- inhibition of sars-cov- infections in engineered human tissues using clinical-grade soluble human ace this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to nathaniel green's proofreading. the authors declare no conflict of interest. key: cord- -uc i h m authors: izaguirre, gonzalo title: the proteolytic regulation of virus cell entry by furin and other proprotein convertases date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: uc i h m a wide variety of viruses exploit furin and other proprotein convertases (pcs) of the constitutive protein secretion pathway in order to regulate their cell entry mechanism and infectivity. surface proteins of enveloped, as well as non-enveloped, viruses become processed by these proteases intracellularly during morphogenesis or extracellularly after egress and during entry in order to produce mature virions activated for infection. although viruses also take advantage of other proteases, it is when some viruses become reactive with pcs that they may develop high pathogenicity. besides reacting with furin, some viruses may also react with the pcs of the other specificity group constituted by pc /pc /pace /pc . the targeting of pcs for inhibition may result in a useful strategy to treat infections with some highly pathogenic viruses. a wide variety of pc inhibitors have been developed and tested for their antiviral activity in cell-based assays. the regulation of viral cell entry by proteases is a control mechanism common among viruses ( table ). the proteolytic processing of viral proteins is often required for virus maturation and infectivity. a critical group of host-cell proteases exploited by a variety of viruses is the family of proprotein convertases (pcs), which includes furin, pc , pc , pace , and pc [ , ] . although other types of proteases besides pcs can also perform the proteolytic maturation of viruses, it has been observed that when pcs process viral proteins, some viruses become comparatively more infective and pathogenic. most of the research done on the maturation of viruses by pcs has focused on furin. however, there is evidence of the involvement of other pcs in the regulation of virus maturation [ ] . the scattered information about the role of pcs in the life cycle of a wide variety of viruses [ , ] , in addition to the new developments on pc activity regulation and reaction specificity [ ] [ ] [ ] [ ] , calls for an effort to integrate this knowledge, analyze the relevance of pcs in the pathogenicity of viruses, and evaluate the feasibility of inhibiting pcs as a sound strategy for antiviral therapy. this review will discuss the importance of differences of pc reactivity and selectivity, and the pc gene expression profile of infected cells, in determining virus infectivity and tropism. the proteolytic maturation of viruses by pcs generally involves the processing of proteins localized on the surface of viral particles, either of non-enveloped or enveloped viruses [ ] . the cleavage of the surface viral proteins mostly occurs inside the host cells during virus morphogenesis and before egress, although cleavage by the target-cell pcs can occur extracellularly or during cell entry with some viruses. the proteolytic processing by pcs promotes binding and fusion of viral particles to target cells. pcs are eukaryotic serine proteases classified in the merops peptidase database within the s b family. furin, pc , pc , pace , and pc are part of the kexin-like subfamily of pcs and localize to the organelles of the constitutive protein secretion pathway [ ] . these pcs perform the proteolytic post-translational modification of a large variety of peptides and proteins in the trans-golgi network, endosomes, and pericellular environment, and are critical regulators of central cellular processes, such as growth, proliferation, and differentiation [ ] [ ] [ ] . the gene expression profile of the kexin-like pcs is cell-type dependent, but most cells express some or all of them, except for pc whose expression is restricted to cell types in the testes, ovaries and the placenta. pcs are large-size multidomain proteins composed of conserved catalytic and regulatory p domains that share %- % amino acid sequence homology ( figure ). furin, pc , and pc are type i membrane-bound proteins, and furin pcs are eukaryotic serine proteases classified in the merops peptidase database within the s b family. furin, pc , pc , pace , and pc are part of the kexin-like subfamily of pcs and localize to the organelles of the constitutive protein secretion pathway [ ] . these pcs perform the proteolytic post-translational modification of a large variety of peptides and proteins in the trans-golgi network, endosomes, and pericellular environment, and are critical regulators of central cellular processes, such as growth, proliferation, and differentiation [ ] [ ] [ ] . the gene expression profile of the kexin-like pcs is cell-type dependent, but most cells express some or all of them, except for pc whose expression is restricted to cell types in the testes, ovaries and the placenta. pcs are large-size multidomain proteins composed of conserved catalytic and regulatory p domains that share - % amino acid sequence homology ( figure ). furin, pc , and pc are type i membrane-bound proteins, and furin and pc can be shed extracellularly; in contrast, pace is a secreted protein. kexin-like pcs cleave their substrates at sites specified by a motif composed of p arg-p x-p x-p arg-p x, where x is any amino acid residue, and cleavage occurs between the p arg and p x residues. this sequence motif is found in many viral surface proteins and determines cleavage by pcs [ ] . the kexin-like pcs are divided into two specificity groups, one represented by furin, and the other by pc /pc /pace /pc [ ] . furin is viruses , , of more reactive than the other pcs, and the differences in reaction specificity between the two groups are based on active-site and exosite determinants of reactivity. kexin-like pcs are considered potential pharmacological targets for the treatment of viral infections by blocking virus maturation and infectivity. other uses for the targeting of these pcs include the inhibition of the activation of bacterial toxins such as shiga, anthrax, clostridium, pseudomonas, and diphtheria; and also, for the treatment of degenerative diseases such as metastatic cancer, alzheimer's, and osteoarthritis [ , , ] . the only known natural inhibitors of pcs are serpins, which are slow-binding type inhibitors that form covalently-linked inhibitory complexes with their target proteases [ ] . serpin b , currently the only pc inhibitory serpin identified in vertebrates, has higher specificity for furin than for pc /pc /pace /pc [ , , ] . more pc inhibitory serpins have been characterized in other organisms as well [ ] [ ] [ ] . a variety of synthetic pc inhibitors have been developed based on small molecules, peptides and their mimetic derivatives, and larger proteins [ , ] . however, the main obstacle for their therapeutic use has been their toxicity, and their lack of pc selectivity. an important research tool is the pc inhibitor α pdx, which is a derivative of the serpin α -antitrypsin with an engineered pc cleavage site motif at its reactive loop [ ] . this engineered serpin inhibits all the kexin-like pcs with the same specificity. more recently, we developed two α pdx-serpin b chimeras that selectively target each of the two pc specificity groups. one is α ord that specifically inhibits furin, and the other is α mdw that specifically inhibits pc /pc /pace /pc [ , ] . the literature on the proteolytic processing of viral surface proteins by pcs and the role that pcs play on the maturation of viruses will be reviewed, and finally, the development of pc inhibitors and their antiviral properties will be discussed. the human papillomavirus (hpv) infects the basal cells of stratified epithelium, and virion replication depends on the infected basal cells progressing into differentiated squamous cells. hpv infects by reaching the lower layers of the stratified epithelium through micro-wounds in the tissue. there, the viral particles bind to heparin sulfate proteoglycan receptors localized either on the extracellular matrix of the basement membrane or the cell surface [ , ] . hpv particles are constituted by a naked nucleocapsid, and work done with pseudovirion particles has suggested that conformational changes in the nucleocapsid proteins l and l , that are induced upon binding of the virus to cell-surface proteoglycans, prime l for cleavage by extracellular or pericellular pcs at the arg residue [ ] . the cleavage of l modifies the conformation of the coat proteins and allows the virion to engage another receptor, and that leads to cell internalization and infection [ , ] . the inhibition of the target-cell pcs blocks hpv infection, but the treatment of the pseudovirion particles with furin beforehand bypasses the inhibition. in contrast, the cleavage of live native hpv virions by pcs occurs during virion morphogenesis, so infectivity becomes independent from the target-cell pcs [ ] [ ] [ ] . also, the proteolytic processing seems to be hpv-type dependent, as evidenced by the native hpv virions being poorly processed during morphogenesis, and their infectivity being mostly dependent on the pcs of the target cells [ ] . l and l in hpv and hpv contain more pc cleavage site motifs besides the commonly studied l -arg . two pc cleavage site motifs, one at l -arg and the other at l -arg , are conserved in many hpv types ( table ). mutagenesis of the l -arg site has been reported to affect pseudovirion morphogenesis [ ] . the l -arg site is located in a region of l involved in the regulation of retrograde trafficking of the l -viral genome complex from the trans-golgi network and into the nucleus [ , ] . surprisingly, low-risk hpv types have lys at the l - position instead of the arg of high-risk types ( table ). if cleavage at the l - site is required for virus morphogenesis, lowand high-risk types may use different proteases, unless their morphogenesis is different. if the cleavage at the l -arg and l -arg sites takes place during cell entry, the cleavage sites may be hidden inside the folded protein and protected from being accessed by the pcs in the intact virion. however, the virions undergo controlled unfolding during entry and trafficking, so that the pc cleavage sites may become exposed and cleaved along their cell internalization route. the potential diverse expression of the pc genes in keratinocytes at different anatomical sites of hpv infection may contribute to the restricted cell tropism by hpv types, which is especially different between the skin and mucosal types [ ] . hpv is commonly found in the stratified epithelium of the ectocervix and tonsilar crypts, and hpv is mainly found in the glandular epithelium of the endocervix. their differences in pc reactivity may play a role in determining their particular tropism. table . pc cleavage site motifs in the coat proteins l and l of hpv types. the cleavage site numbers correspond to those in the hpv sequences. the p arg and p arg residues are denoted in red. the envelope glycoprotein b (gb) is the most conserved protein among all herpes viruses, and its function is to regulate virus to cell membrane fusion. gb is synthesized as a precursor protein, and pcs cleave it at a loop, which is located in domain ii of the ectodomain and at a distance from the fusion loop at domain i. the cleavage site loop is highly variable in length and amino acid sequence among herpes viruses ( table ). the cleavage of gb by pcs has been demonstrated [ ] . most herpes viruses have at least one pc cleavage site motif in the cleavage loop, although, hhsv is an exception by having no pc motifs at all. in contrast, other viruses have more than one pc cleavage site, which may be cleaved sequentially [ ] . the experimental inactivation of the pc cleavage site of several herpes viruses did not severely affect viral cell entry into cells growing in vitro; however, the lack of pc cleavage reduced virus spread and replication in vivo [ , ] . the cleavage of gb promotes virus-to-cell and cell-to-cell fusion [ , ] . although much is still needed to consolidate our knowledge of the cleavage of gb by pcs, there is no doubt that the presence of pc cleavage site motifs in gb is the result of selective evolutionary pressure [ ] . two proteins predominate in the envelope of flaviviruses, prm, and glycoprotein e [ ] . the association between the two proteins (prm-e) in the immature virus changes upon cleavage of prm by pcs during egress. the pr segment is removed to render the mature virions (m-e) (m-e) [ ] . all flaviviruses contain a pc cleavage site motif at the pr-m junction (table ) . a peculiar case is the maturation of the dengue virus (denv). its proteolytic processing is known to be very inefficient, and virions are produced in the prm-e form in high proportion. it was initially suspected that maturation might not be necessary for infectivity but later demonstrated that it is indeed needed [ ] . the inefficient maturation of the denv agrees with studies that show that anti-prm antibodies represent a significant proportion of the immune response to denv and that these antibodies are responsible for the development of antibody-dependent enhancement (ade) of infection in individuals suffering from recurrent denv infections [ ] . these observations suggest that the denv pc reactivity is weaker compared to that of other flaviviruses, which seem to mature more efficiently. the pc site sequence alignment presented in table shows that the four denv types have asp or glu residues at the p position of the cleavage site, compared to ser or thr in most other flavivirus sequences, including that of the zika virus (zikv). acidic residues at this position in the substrate sequence are detrimental to reactivity with pcs [ ] . based on these differences, it is expected that denv reacts with a dramatically lower reactivity toward the pcs compared to other flaviviruses and that higher rates of pc reactivity align with the strong virulence and broad cell tropism observed with other flaviviruses such as [ ] . therefore, it is not surprising that zikv can even reach the fetus and remain in bodily fluids of asymptomatic patients for more extended periods when compared to denv [ ] . variations of the pc gene expression profile may be a key factor determining the difference of tropism to testes between zikv and denv. pc is the primary pc expressed in testes [ ] . a vigorous reactivity of zikv with pc would explain why the testes suffer the highest loads of zikv compared to other organs and the sexual transmission of the virus. the viral pc reactivity and the cell pc gene expression profile both probably play a role in determining the cell tropism differences observed with flaviviruses [ ] [ ] [ ] . viruses of the genus alphavirus like chikungunya (chikv), semliki forest (sfv), sindbis, and ross river, all are arboviruses structurally related to flaviviruses [ ] . their glycoprotein precursor e e is cleaved by pcs in order to regulate its interaction with the glycoprotein e , which promotes virus to cell fusion and infection [ , ] . the information available about the processing of togavirus proteins by pcs is scant, but it reveals the existence of amino acid sequence variability in the pc cleavage sites between the chikv asian and african strains, and that this variability probably determines the observed differences of pc selectivity [ ] . the family of coronaviruses includes viruses of relevance to human and veterinary health. like other enveloped viruses that rely on surface glycoproteins for binding and fusion, coronaviruses have the spike (s) protein, which is cleaved by proteases during virion biosynthesis, as well as during entry into target cells [ ] . the proteolytic regulation of coronaviruses is probably one of the best-studied systems, and a complete picture of the regulatory system mechanism has been developed compared to other families of viruses that are less well-studied. the general principles of the proteolytic regulatory mechanism of coronaviruses based on the accumulated evidence include: ( ) these viruses are regulated by a variety of proteases, ( ) the protein s is cleaved sequentially at two cleavage sites, ( ) viruses can quickly adapt to the proteolytic environment of the infected cells, and ( ) the compatibility between the cleavage site-specificity and cell protease expression profile determines the cell and tissue tropism and pathogenicity of the virus. furin is not the only protease that regulates the function of the coronavirus fusion protein. other proteases, such as the membrane-bound tmprss, the lysosomal cathepsins, elastase, and coagulation factor xa have also been implicated [ , ] . protein s is cleaved at the s -s junction during biosynthesis to separate the two major domains of the protein. the s domain is involved in receptor binding, and the s domain mediates the fusion step of the cell entry mechanism. during cell entry, the cleavage at s -s primes s for the second cleavage at the s site [ ] [ ] [ ] [ ] . in many coronaviruses, the s -s cleavage seems to be dispensable; however, the cleavage at s is not. the cleavage at s has been suggested to serve as a virulence marker [ ] . predictions of the furin/pc reactivity, based on the amino acid sequence surrounding the cleavage site, have been made based on computer algorithms [ ] ; however, the dependency of furin/pc reactivity on the conformation of the substrate and exosites lends uncertainty to those predictions. the highly virulent mers-cov (middle east respiratory syndrome coronavirus) is the only natural virus known to have pc cleavage site motifs at both the s -s and s sites. other viruses with two pc sites are the result of laboratory selection by their serial passage in cell lines in vitro, one such virus being the infectious bronchitis virus ibv-beaudette strain [ ] . mers-cov has an expanded tropism compared to other coronaviruses, so it is considered polytropic [ ] . only the s site in sars-cov (severe acute respiratory syndrome coronavirus) has a pc cleavage site motif [ , ] . the fact that mers-cov and sars-cov are highly pathogenic, and that ibv-beaudette is apathogenic is in line with these viruses reacting with proteases other than the pcs [ , ] . tmprss promotes sars-cov and mers-cov infection in vivo [ ] . the engineering of pc specificity at the cleavage sites of coronavirus s proteins can modify the virus tropism and virulence [ , ] . the conversion of a monobasic cleavage site into a polybasic site not only makes the virus susceptible to pc cleavage but also increases the chance of cleavage by other proteases that target single arginine residues, so it is not surprising that mers-cov is so pathogenic. because coronaviruses are adapted to the different proteolytic environments of the many cell types they infect, each virus may be activated by a specific set of proteases. it is crucial to define the protease cleavage specificity of viruses that impact human or animal health. the use of the pc inhibitor, dec-rvkr-cmk, has created some controversy as sometimes the inhibitor is used in excessive concentrations. the inhibitor binds pcs with a very high affinity, at low nm concentrations; it slowly forms covalent complexes with the enzymes, so it inhibits pcs in a stoichiometric manner. in our hands, treating cells with this inhibitor at a concentration of µm is enough to block hpv cell entry completely. concentrations up to µm reported in some studies should not be considered pc-specific; such high inhibitor concentrations most probably inhibit other proteases besides pcs [ , ] . medically relevant retroviruses of the retroviridae family have also been studied concerning their proteolytic regulation. the most studied viruses are the bovine and murine leukemia viruses, which are related to the human t-lymphotropic viruses. their envelope glycoproteins are cleaved by furin and other pcs [ ] . like the coronaviruses, leukemia virus glycoproteins are cleaved twice. pcs perform the first cleavage, which induces conformational changes and disulfide isomerizations that prime the protein for further proteolysis [ , ] . the second proteolytic event is performed by a viral protease that fully activates the glycoprotein [ , ] . the human immunodeficiency virus (hiv) env glycoprotein gp precursor is cleaved by furin during biosynthesis into gp and gp in the trans-golgi network (figure ). gp is further processed by furin into gp and gp after leaving the tgn [ , ] . the env glycoprotein is the only antigenic hiv protein, and furin cleavage-independent forms stabilized in the native form have been produced for vaccine development purposes [ ] . interestingly, a polybasic region located upstream from the pc cleavage site at the gp /gp junction was shown to bind heparin and promote cleavage [ ] . medically relevant retroviruses of the retroviridae family have also been studied concerning their proteolytic regulation. the most studied viruses are the bovine and murine leukemia viruses, which are related to the human t-lymphotropic viruses. their envelope glycoproteins are cleaved by furin and other pcs [ ] . like the coronaviruses, leukemia virus glycoproteins are cleaved twice. pcs perform the first cleavage, which induces conformational changes and disulfide isomerizations that prime the protein for further proteolysis [ , ] . the second proteolytic event is performed by a viral protease that fully activates the glycoprotein [ , ] . the human immunodeficiency virus (hiv) env glycoprotein gp precursor is cleaved by furin during biosynthesis into gp and gp in the trans-golgi network (figure ). gp is further processed by furin into gp and gp after leaving the tgn [ , ] . the env glycoprotein is the only antigenic hiv protein, and furin cleavage-independent forms stabilized in the native form have been produced for vaccine development purposes [ ] . interestingly, a polybasic region located upstream from the pc cleavage site at the gp /gp junction was shown to bind heparin and promote cleavage [ ] . figure . x-ray crystal structure of the hiv- envelope pg glycoprotein monomer. the fusion machine is composed of three gp monomers which are divided into n-terminal gp (green) and c-terminal pg (gray). residue in red denotes the end of gp and residue in blue the beginning of gp after pc cleavage. pdb id code mtj. the duck hepatitis b virus (dhbv) has been used as a model to study the hepatitis b virus (hbv). the proteolytic events that regulate the cell entry mechanism of this hepadnavirus have not attracted much attention, but there is evidence of the cleavage of the envelope proteins by pcs [ ] . single-stranded negative-sense rna viruses of the filoviridae and arenaviridae families and the new-order bunyavirales are the causative agents of lethal hemorrhagic fever diseases. despite the seriousness of the health threat these viruses represent, the information about the proteolytic regulation of their entry mechanism is scarce. the envelope glycoproteins of the ebola (ebov) and marburg (mbgv) viruses are processed by furin into two disulfide-linked subunits [ ] [ ] [ ] . except for the reston strain that has no pc cleavage site motifs, all other ebov strains have one; the reston strain is less pathogenic than the other ebov strains [ ] . the figure . x-ray crystal structure of the hiv- envelope pg glycoprotein monomer. the fusion machine is composed of three gp monomers which are divided into n-terminal gp (green) and c-terminal pg (gray). residue in red denotes the end of gp and residue in blue the beginning of gp after pc cleavage. pdb id code mtj. the duck hepatitis b virus (dhbv) has been used as a model to study the hepatitis b virus (hbv). the proteolytic events that regulate the cell entry mechanism of this hepadnavirus have not attracted much attention, but there is evidence of the cleavage of the envelope proteins by pcs [ ] . single-stranded negative-sense rna viruses of the filoviridae and arenaviridae families and the new-order bunyavirales are the causative agents of lethal hemorrhagic fever diseases. despite the seriousness of the health threat these viruses represent, the information about the proteolytic regulation of their entry mechanism is scarce. the envelope glycoproteins of the ebola (ebov) and marburg (mbgv) viruses are processed by furin into two disulfide-linked subunits [ ] [ ] [ ] . except for the reston strain that has no pc cleavage site motifs, all other ebov strains have one; the reston strain is less pathogenic than the other ebov strains [ ] . the glycoprotein of mbgv has two pc cleavage site motifs that do not agree in their amino acid sequence and position compared to the single pc site in the ebov protein [ ] . the cleavage by furin seems to be dispensable because the elimination of the pc site in the ebov protein does not affect the virus replication in cultured cells or the disease progression in experimental animals [ ] [ ] [ ] . ebov requires further proteolytic processing of the glycoprotein binding domain by endosomal cathepsins in order to gain binding activity [ ] [ ] [ ] . filoviruses are different from other viruses in that they require additional factors or modifications of the glycoprotein in order to gain infectivity [ , ] . the crimean-congo hemorrhagic fever bunyavirus (cchfv) glycoprotein is processed by furin and the proprotein convertase ski- , a pc of the pyrolysin-like type and also known as s p, which has a cleavage site specificity different from the polybasic specificity of the kexin-like pcs [ , ] . the cleavage by furin is not essential, but inactivating the cleavage site slows down virus replication [ , ] . the lymphocytic choriomeningitis (lcmv) and the lassa (lasv) arenaviruses are known to also require ski- activity for the cleavage of their envelope glycoproteins [ , ] . the paramyxoviridae is a diverse family of viruses, and a variety of proteases activate their fusion proteins. some paramyxoviruses are highly pathogenic. single proteolytic processing of the fusion protein occurs for most of these viruses. pcs perform the cleavage in the parainfluenza and the measles (mv) viruses [ ] [ ] [ ] . there are several serotypes of the avian paramyxoviruses (apmv). the glycoprotein of the highly pathogenic apmv- , or newcastle disease virus (ndv), is cleaved by furin, and the proteins of other serotypes are cleaved by undetermined trypsin-like proteases [ , ] . the mutation of the trypsin-like sites into pc site motifs made the viruses replicate faster in cell culture, but they remained non-virulent in vivo [ ] [ ] [ ] . conversely, the transformation of the pc cleavage site of the virulent apmv- strain into a trypsin-like site induced the virus to become highly attenuated [ ] . the pathogenic respiratory syncytial virus (rsv) is unique among paramyxoviruses in that its glycoprotein is cleaved at two sites by pcs [ ] . the first cleavage takes place before the virus enters the target cells, and the second occurs after entry into the endosomes [ ] . furin does not activate the lethal nipah (niv) and hendra (hev) viruses for entry; instead, the viruses depend on endosomal cathepsins [ , ] . these viruses produce systemic infections in several different hosts. the glycoprotein of the sendai virus (sev) requires the participation of the homologous attachment protein hemagglutinin-neuraminidase (hn), which binds the cell surface sialic acid receptors. sev glycoprotein has only one trypsin-like site, but by replacing it with the two rsv pc sites, the dependency on hn for infection is reduced [ ] . the influenza viruses cause respiratory disease and occasional pandemics. the virus envelope contains two glycoproteins, hemagglutinin (ha) and neuraminidase (na). both proteins contribute to the virus pathogenicity and the cleavage of ha precursor into ha and ha by the host cell pcs is a significant contributor of virulence for avian influenza (figure ). the extent and diversity of the cellular proteolytic activity is also an essential factor determining pathogenicity, spread, and tropism of the influenza virus [ , ] . there are ha types, but h and h are the most commonly present types in seasonal human infections, other types are found in birds. the pandemics of and were caused by the h n and h n strains, respectively. the proteolytic cleavage of ha occurs in a loop that varies in length and amino acid sequence depending on the strain ( table ). the loop usually contains one arg residue that determines cleavage by trypsin-like proteases. the cleavage can occur during synthesis, after release or before entry, and may depend on different proteases. the highly pathogenic virus strain responsible for the spanish influenza pandemic was of the h n type with only one arg residue in its cleavage loop. two proteases highly expressed in the respiratory tract, especially in the lungs, tmprss and tmprss , were shown to cleave the influenza ha [ ] . hat is a protease expressed in the airways, mostly in the larynx but not in lungs. it is also capable of activating influenza viruses [ ] . multibasic cleavage sites in ha arise by single substitution mutations like in the case of some h n types, or by insertions that result in longer loops, as observed with the highly pathogenic h and h types. viruses that acquire multibasic cleavage sites become independent of trypsin-like proteases. in low-pathogenic h n strains carrying the cleavage site motif, r-s-k-r, cleavage is not performed by pcs but by matriptase, which recognizes the same cleavage site motif of pcs and determines the nephrotropism of the virus [ ] . however, h n can become reactive with pcs by the removal of a glycosylation site near the cleavage site [ ] . the long and multibasic loops in some h and h strains are highly reactive with furin [ ] [ ] [ ] [ ] ; this reactivity leads to high pathogenicity that causes systemic infections [ , ] . an outbreak of a highly pathogenic avian h n strain that infected humans occurred in in hong kong. in some highly pathogenic h and h types that have the k-k-k-r motif, cleavage is carried out by the ubiquitous protease mspl and its splice variant mtprss , which are also capable of cleaving at the pc cleavage site motif [ ] . table . cleavage loop in the ha protein of influenza viruses. the variable region is blue with the arrow denoting the cleavage site. the p arg and p arg residues are colored red. figure . x-ray crystal structure of the ha trimer from the influenza virus a type h . each ha monomer is divided into ha (red, blue, and gray) and ha (oarange, cyan, and white, respectively) subunits after the pc cleavage denoted by residues at the end ha (yellow) and the beginning of ha (purple). pdb id code ibx. multibasic cleavage sites in ha arise by single substitution mutations like in the case of some h n types, or by insertions that result in longer loops, as observed with the highly pathogenic h and h types. viruses that acquire multibasic cleavage sites become independent of trypsin-like proteases. in low-pathogenic h n strains carrying the cleavage site motif, r-s-k-r, cleavage is not performed by pcs but by matriptase, which figure . x-ray crystal structure of the ha trimer from the influenza virus a type h . each ha monomer is divided into ha (red, blue, and gray) and ha (oarange, cyan, and white, respectively) subunits after the pc cleavage denoted by residues at the end ha (yellow) and the beginning of ha (purple). pdb id code ibx. the search for effective pc inhibitors centers into finding the inhibitor with the best characteristics of specificity, stability, and bioavailability [ ] . most pc inhibitors reported have been developed against furin. although these inhibitors are of high pc specificity, many of them still lack proper characterization of their pc selectivity. knowing the pc selectivity of an inhibitor is a critical issue as pcs differ in substrate specificity, and viruses can be pc-selective. synthetic pc inhibitors come in several forms, from small molecules identified by high-throughput screening [ ] [ ] [ ] [ ] ; to peptide substrates [ , ] , or viral cleavage sites [ ] ; peptide mimetic derivatives that add unnatural amino acids [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ; cyclic peptides [ ] ; polyarginine [ ] [ ] [ ] ; and larger engineered proteins like the leech eglin c [ ] , turkey ovomucoid [ ] , α -macroglobulin [ ] ; and the engineered serpin α -pdx [ ] . peptide derivatives seem more efficient at producing high-affinity pc inhibitors compared to small molecules [ ] . due to the high density of negative charges at the pc active site, highly basic peptides show strong specificity and bioavailability, but may also be highly toxic [ ] . larger proteins are poised to become the most effective pc inhibitors as they offer better opportunities to build specificity and selectivity compared to small molecules. they can also be made bioavailable through a variety of routes. several of these pc inhibitors have been confirmed to have antiviral activity in cell-based assays of viral propagation (table ). table . inhibitors of pcs tested for their antiviral activity in cell-based assays of viral propagation. ski- inhibitors lcmv, lasv [ , ] peptides and peptidomimetics chikv, sfv wnv, denv h and h influenza [ , ] the ubiquitous presence of furin and related pcs throughout the cells of the body makes these proteases vulnerable to being exploited by viruses. the location of furin and related pcs in the vesicles of the constitutive protein secretion pathway, where viruses are assembled during morphogenesis or disassembled during cell entry, explains why a diversity of virus types have evolutionarily converged to depend on pcs. viruses also use other types of proteases for the proteolytic regulation of the binding and fusion functions; however, proteases are restricted to specific cell types, which limits the range of the viral infection, so when some viruses mutate and acquire pc reactivity, they may expand their cell tropism and become more pathogenic. the targeting of pcs for inhibition as an antiviral strategy is a sound possibility. probably the major advantage of this approach is that by not targeting a viral component or function, it reduces the chance of producing resistance. the main drawback is the ubiquitous distribution of pcs and the potential toxicity and secondary effects that their inhibition may cause. in consequence, it is essential to know the virus pc selectivity and to have pc inhibitors that are selective for one of the two pc specificity groups, furin or pc /pc /pace /pc [ ] . funding: this research received no external funding. the author declares no conflict of interest. the 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serpin b reactive-site and exosite determinants of reactivity to the serpin α pdx processing of viral glycoproteins by the subtilisin-like endoprotease furin and its inhibition by specific peptidylchloroalkylketones substrate cleavage analysis of furin and related proprotein convertases. a comparative study high-resolution analysis and functional mapping of cleavage sites and substrate proteins of furin in the human proteome a residues motif delineates the furin cleavage site and its physical properties may influence viral fusion the serpins are an expanding superfamily of structurally similar but functionally diverse proteins. evolution, mechanism of inhibition, novel functions, and a revised nomenclature inhibition of soluble recombinant furin by human proteinase inhibitor the serpin proteinase inhibitor . an endogenous furin inhibitor released from human platelets drosophila serpin functions as a neuroserpin-like inhibitor of subtilisin-like proprotein convertases the spn gene of 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papillomaviral vectors maturation of papillomavirus capsids kallikrein- proteolytically processes human papillomaviruses in the extracellular space to facilitate entry into host cells the nuclear retention signal of hpv l protein is essential for incoming viral genome to transverse the trans-golgi network a central region in the minor capsid protein of papillomaviruses facilitates viral genome tethering and membrane penetration for mitotic nuclear entry microarray analysis of human keratinocytes from different anatomic sites reveals site-specific immune signaling and responses to human papillomavirus type transfection proteolytic processing of human cytomegalovirus glycoprotein b (gpul ) is mediated by the human endoprotease furin glycoprotein b of equine herpesvirus type has two recognition sites for subtilisin-like proteases that are cleaved by furin glycoprotein b cleavage is important for murid herpesvirus to infect myeloid cells mutagenesis of varicella-zoster virus glycoprotein b: putative fusion loop residues are essential for viral replication, and the furin cleavage motif contributes to pathogenesis in skin tissue in vivo cleavage of epstein-barr virus glycoprotein b is required for full function in cell-cell fusion with both epithelial and b cells positively selected sites at hcmv gb furin processing region and their effects in cleavage efficiency the . Å resolution cryo-em structure of zika virus a structural perspective of the flavivirus life cycle functional importance of dengue virus maturation: infectious properties of immature virions antibodies against the envelope glycoprotein promote infectivity of immature dengue virus serotype zika virus pathogenesis and tissue tropism persistence of zika virus in body fluids-preliminary report testicular expression of pc in the rat: molecular diversity of a novel germ cell-specific kex /subtilisin-like proprotein convertase changing the protease specificity for activation of a flavivirus, tick-borne encephalitis virus comparative analysis between flaviviruses reveals specific neural stem cell tropism for zika virus in the mouse developing neocortex zika virus impairs growth in human neurospheres and brain organoids glycoprotein organization of chikungunya virus particles revealed by x-ray crystallography furin processing and proteolytic activation of semliki forest virus inhibition of chikungunya virus infection in cultured human muscle cells by furin inhibitors: impairment of the maturation of the e surface glycoprotein host cell proteases: critical determinants of coronavirus tropism and pathogenesis cathepsin l functionally cleaves the severe acute respiratory syndrome coronavirus class i fusion protein upstream of rather than adjacent to the fusion peptide different host cell proteases activate the sars-coronavirus spike-protein for cell-cell and virus-cell fusion proteolytic activation of the spike protein at a novel rrrr/s motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein proteolytic processing of middle east respiratory syndrome 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cleavage potentiates the membrane fusion-controlling intersubunit disulfide bond isomerization activity of leukemia virus env furin cleavage of the moloney murine leukemia virus env precursor reorganizes the spike structure cooperative cleavage of the r peptide in the env trimer of moloney murine leukemia virus facilitates its maturation for fusion competence maturation cleavage of the murine leukemia virus env precursor separates the transmembrane subunits to prime it for receptor triggering comparative cellular processing of the human immunodeficiency virus (hiv- ) envelope glycoprotein gp by the mammalian subtilisin/kexin-like convertases the role of eukaryotic subtilisin-like endoproteases for the activation of human immunodeficiency virus glycoproteins in natural host cells structure of a cleavage-independent hiv env recapitulates the glycoprotein architecture of the native cleaved trimer heparin enhances the furin cleavage of hiv- gp peptides initiation of duck hepatitis b virus infection requires cleavage by a furin-like protease processing of the ebola virus glycoprotein by the proprotein convertase furin biochemical analysis of the secreted and virion glycoproteins of ebola virus proteolytic processing of marburg virus glycoprotein endoproteolytic processing of the ebola virus envelope glycoprotein: cleavage is not required for function reverse genetics demonstrates that proteolytic processing of the ebola virus glycoprotein is not essential for replication in cell culture proteolytic processing of the ebola virus glycoprotein is not critical for ebola virus replication in nonhuman primates endosomal proteolysis of the ebola virus glycoprotein is necessary for infection role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion crimean-congo hemorrhagic fever virus glycoprotein precursor is cleaved by furin-like and ski- proteases to generate a novel -kilodalton glycoprotein recovery of recombinant crimean-congo hemorrhagic fever virus reveals a function for non-structural glycoproteins cleavage by furin antiviral activity of a small-molecule inhibitor of arenavirus glycoprotein processing by the cellular site protease targeting the proteolytic processing of the viral glycoprotein precursor is a promising novel antiviral strategy against arenaviruses proteolytic cleavage of wild type and mutants of the f protein of human parainfluenza virus type by two subtilisin-like endoproteases, furin and kex engineered serine protease inhibitor prevents furin-catalyzed activation of the fusion glycoprotein and production of infectious measles virus the role of subtilisin-like proprotein convertases for cleavage of the measles virus fusion glycoprotein in different cell types molecular characterization and complete genome sequence of avian paramyxovirus type prototype strain duck/hong kong/d / complete sequence of the genome of avian paramyxovirus type and comparison with other paramyxoviruses mutations in the fusion protein cleavage site of avian paramyxovirus serotype increase cleavability and syncytium formation but do not increase viral virulence in chickens mutation of the f-protein cleavage site of avian paramyxovirus type results in furin cleavage, fusion promotion, and increased replication in vitro but not increased replication, tissue tropism, or virulence in chickens mutations in the fusion protein cleavage site of avian paramyxovirus serotype confer increased replication and syncytium formation in vitro but not increased replication and pathogenicity in chickens and ducks effect of fusion protein cleavage site sequence on generation of a genotype vii newcastle disease virus vaccine cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion host cell entry of respiratory syncytial virus involves macropinocytosis followed by proteolytic activation of the f protein ubiquitous activation of the nipah virus fusion protein does not require a basic amino acid at the cleavage site cathepsin l is involved in proteolytic processing of the hendra virus fusion protein recombinant sendai viruses expressing fusion proteins with two furin cleavage sites mimic the syncytial and receptor-independent infection properties of respiratory syncytial virus influenza virus activating host proteases: identification, localization and inhibitors as potential therapeutics role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure proteolytic activation of the influenza virus hemagglutinin proteolytic activation of influenza viruses by serine proteases tmprss and hat from human airway epithelium hat, and tmprss activate the hemagglutinin of h n influenza a viruses a novel activation mechanism of avian influenza virus h n by furin influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease proprotein-processing endoproteases pc and furin both activate hemagglutinin of virulent avian influenza viruses sequence specificity of furin, a proprotein-processing endoprotease, for the hemagglutinin of a virulent avian influenza virus activation of an influenza virus a/turkey/oregon/ ha insertion variant by the subtilisin-like endoprotease furin targeted infection of endothelial cells by avian influenza virus a/fpv/rostock/ (h n ) in chicken embryos molecular basis for high virulence of hong kong h n influenza a viruses novel type ii transmembrane serine proteases, mspl and tmprss , proteolytically activate membrane fusion activity of the hemagglutinin of highly pathogenic avian influenza viruses and induce their multicycle replication furin inhibitors: importance of the positive formal charge and beyond a small-molecule furin inhibitor inhibits cancer cell motility and invasiveness inhibition of furin/proprotein convertase-catalyzed surface and intracellular processing by small molecules synthetic small molecule furin inhibitors derived from , -dideoxystreptamine identification of potent and compartment-selective small molecule furin inhibitors using cell-based assays potent inhibitors of furin and furin-like proprotein convertases containing decarboxylated p arginine mimetics the multi-leu peptide inhibitor discriminates between pace and furin and exhibits antiproliferative effects on prostate cancer cells targeting host cell furin proprotein convertases as a therapeutic strategy against bacterial toxins and viral pathogens a novel enediynyl peptide inhibitor of furin that blocks processing of propdgf-a selective and potent furin inhibitors protect cells from anthrax without significant toxicity highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases optimization of furin inhibitors to protect against the activation of influenza hemagglutinin h and shiga toxin design, synthesis, and structure−activity relationship studies of a potent pace inhibitor peptidomimetic furin inhibitor mi- in combination with oseltamivir and ribavirin efficiently blocks propagation of highly pathogenic avian influenza viruses and delays high level oseltamivir resistance in mdck cells novel furin inhibitors with potent anti-infectious activity optimization of substrate-analogue furin inhibitors elongated and shortened peptidomimetic inhibitors of the proprotein convertase furin effects of ns b-ns protease and furin inhibition on west nile and dengue virus replication design, synthesis, and characterization of macrocyclic inhibitors of the proprotein convertase furin polyarginines are potent furin inhibitors short polybasic peptide sequences are potent inhibitors of pc / and pc : use of positional scanning-synthetic peptide combinatorial libraries as a tool for the optimization of inhibitory sequences lindberg, i. cationic cell-penetrating peptides are potent furin inhibitors engineered eglin c variants inhibit yeast and human proprotein processing proteases, kex and furin arg -lys -arg turkey ovomucoid third domain inhibits human furin inhibition of intracellular proteolytic processing of soluble proproteins by an engineered α -macroglobulin containing a furin recognition sequence in the bait region polyarginine inhibits gp processing by furin and suppresses productive human immunodeficiency virus type infection blockage of filoviral glycoprotein processing by use of a protein-based inhibitor a protein-based therapeutic for human cytomegalovirus infection key: cord- -mxjsp cm authors: denner, joachim title: transspecies transmission of gammaretroviruses and the origin of the gibbon ape leukaemia virus (galv) and the koala retrovirus (korv) date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: mxjsp cm transspecies transmission of retroviruses is a frequent event, and the human immunodeficiency virus- (hiv- ) is a well-known example. the gibbon ape leukaemia virus (galv) and koala retrovirus (korv), two gammaretroviruses, are also the result of a transspecies transmission, however from a still unknown host. related retroviruses have been found in southeast asian mice although the sequence similarity was limited. viruses with a higher sequence homology were isolated from melomys burtoni, the australian and indonesian grassland melomys. however, only the habitats of the koalas and the grassland melomys in australia are overlapping, indicating that the melomys virus may not be the precursor of the galv. viruses closely related to galv/korv were also detected in bats. therefore, given the fact that the habitats of the gibbons in thailand and the koalas in australia are far away, and that bats are able to fly over long distances, the hypothesis that retroviruses of bats are the origin of galv and korv deserves consideration. analysis of previous transspecies transmissions of retroviruses may help to evaluate the potential of transmission of related retroviruses in the future, e.g., that of porcine endogenous retroviruses (pervs) during xenotransplantation using pig cells, tissues or organs. whereas some recent transspecies transmission of retroviruses, for example, the transmission of a lentivirus from chimpanzees to humans resulting in the human immunodeficiency virus- (hiv- ) or the transmission of a simian immunodeficiency virus (siv) from sooty mangabeys to humans resulting in hiv- , have been well studied [ ] , other transspecies transmissions have still not been fully analysed. most interestingly, in the case of hiv- and hiv- , such transmissions happened several times, producing numerous clades of hiv- and hiv- . transspecies transmission has also been reported for gammaretroviruses, for example, the porcine endogenous retrovirus (perv) is thought to be the result of a transmission of a murine retrovirus to pigs [ ] . since similar sequences have been found in mice, but not in rats and hamsters, it was suggested that pervs originated from mouse endogenous retroviruses approximately three and million years ago [ ] . these data were later confirmed using molecular clock calibrations, revealing that the precise age of perv is at most . × years old [ ] . the transspecies transmission of the gibbon ape leukaemia virus (galv) and the koala retrovirus (korv) is a prime example of a transmission that is still not yet fully understood. some captive gibbons in thailand were found infected with galv, which is an exogenous gammaretrovirus that induces leukaemia in infected animals living exclusively in captivity, as wild gibbons in thailand are not infected [ ] . the koala retrovirus (korv) is a close relative of galv, and it infects koalas in australia [ ] . the fact that both viruses are closely related suggests that they may have a common origin and could be the result of a transspecies transmission of an ancestral retrovirus via one or more intermediate hosts. however, the geographical distance between thailand and australia, separated by the wallace line, makes it difficult to identify the precursor virus and the original host. the wallace line delineates australian and southeast asian fauna. west of the line is the asian ecozone, east of it is the so-called wallacea, which includes a mixture of asian and australian fauna. the eastern border of wallacea is the lydekker line, behind which starts the australian fauna. many birds do not cross the wallace line, but some animals do, such as bats and crab-eating macaques [ , ] . searching for the precursor virus, retroviruses related to korv and galv have been described in rodents such as south east asian mice (e.g., mus caroli [ , ] and mus dunni [ ] ), as well as in two subspecies of melomys burtoni in australia and indonesia [ , ] . although these viruses are close relatives, it is still difficult to explain how gibbons and koalas, living in such diverse habitats, could be infected with the precursor virus. furthermore, only the habitat of melomys burtoni in australia and that of the koalas overlap. since retroviruses related to korv/galv were also identified in bats [ , ] , which have the ability to fly long distances, this review will analyse the hypothesis of whether these bat viruses may be the origin of korv/galv. galv is associated with hematopoietic neoplasms in captive colonies of white-handed gibbons (hylobates lar). one of five known strains was isolated from gibbons suffering from granulocytic leukaemia at the southeast asia treaty organization (seato) medical research laboratory in bangkok, thailand (seato strain), and was shown to cause chronic myelogenous leukaemia after injection into juvenile gibbons [ ] . a second strain was isolated from an animal with lymphatic leukaemia at the san francisco medical center (strain sf), san francisco, usa [ , ] , and a third strain was isolated from a gibbon with lymphocytic leukaemia from a colony on hall's island near bermuda (strain galv-h), the only galv strain identified outside of thailand and the usa [ , ] . a fourth strain, galv-br (brain), was isolated from two healthy gibbons inoculated with brain extracts from human patients with kuru [ ] . to understand the origin of galv, it is important to analyse whether all infected animals came from the seato facility in thailand or at least had contact with galv-infected animals. according to recent investigations, most of the infected gibbons were directly from the seato facility [ ] . even in the case of the gibbons from which galv-br was isolated, it is thought that either the virus has its origin in animals sent from seato in , or that the cell lines the gibbon brain was co-cultured with were contaminated with galv [ ] . a closely related virus was isolated from a woolly monkey (lagothrix lagotricha) with multiple fibrosarcomas, called first simian sarcoma-associated virus (ssav), and then later woolly monkey virus (wmv) [ , ] . wmv is a mixture of a replication-defective transforming virus and a replication-competent helper virus [ ] , which has a high sequence homology and antigenic similarity with galv [ , , [ ] [ ] [ ] . it was shown that the infected woolly monkey had close contact with an infected gibbon [ ] . in addition to these strains, related galv were isolated from a galv-ssav infected marmoset tumor cell line, designated galv-mar (accession number u . ) and from an hiv- -infected human cell line (galv-x) [ , ] , revealing laboratory contaminations similar to the situation with the xenotropic murine leukaemia virus (mulv)-related virus (xmrv) in human tissues [ ] . when using immunological methods to investigate for galv infection in sera from captive gibbons in north american zoological institutions, the presence of antibodies against galv antigens was revealed in % of the animals, indicating previous exposure to galv [ ] . however, virus detection in gibbon blood or serum using polymerase chain reaction (pcr) or co-culture of gibbon peripheral blood mononuclear cells (pbmcs) with human cells was negative for all samples submitted and most of the animals were healthy. hence it can be assumed that these animals were infected with galv, reacted with an antibody response, and succeeded in eliminating the virus. recent investigations have demonstrated that all antibody positive gibbons were derived from the seato facility or had contact with infected animals [ ] , thus revealing that only captive gibbons, originating from the seato facility in thailand, or animals having contact with them, but not free living animals, were infected and that the virus is exogenous. the detection and characterization of the korv has been well described [ , , ]. an involvement of retroviruses in lymphoma and leukaemia in captive and free-living koalas (phascolarctos cinereus) has been recognized quite early [ ] . convincing evidence was obtained when virus particles were found in mitogen-stimulated pbmcs and lymphoma tissues of hundreds of koalas and an infectious virus was then isolated and cloned [ ] . soon, it became clear that this virus may be endogenous, being integrated in the genome of normal koalas, but it may also exist as an exogenous virus [ ] . despite the taxonomical distance between gibbons and koalas, sequencing of the virus has demonstrated that korv is closely related to galv [ ] . additionally, to a lesser extent, galv and korv are also related to pervs and mulvs. therefore, since a korv-like virus was not found in marsupials related to koalas, it seems most likely that the galv/korv grouping is the result of a relatively recent transspecies transmission of a related virus into gibbons and koalas. in this context, it is interesting to note that the first infections of koalas with the precursor virus took place in the north of australia and the infection moved like a wave to the south of the koala habitat [ ] . at present, two main subtypes of korv are known: korv-a, which uses pit- , a sodiumdependent phosphate transporter as a receptor; and korv-b and korv-j, both using the thiamine transport protein (thtr ) ( table ) . since korv-b and korv-j are the result of a recombination between korv-a sequences with still unknown endogenous sequences, a new receptor binding domain (rbd) in the envelope protein was created and thtr is used instead of pit- . additional, still unclassified korvs have also been described in previous reviews [ ] . it remains unclear whether only korv-b or also korv-a are involved in tumor induction [ , ] . like other retroviruses, including hiv- [ ] , korv induces severe immunodeficiencies in the infected animals, leading to serious infections, such as severe chlamydial infections [ ] . since there are effective vaccines protecting cats from infections with the feline leukemia virus (felv) [ ] , a virus related to korv, successful attempts have also been undertaken to generate an effective vaccine against korv infections [ ] . unfortunately, vaccination can be difficult in animals carrying an endogenous korv which produces a subsequent tolerance against the vaccine [ ] . the first evidence of related retroviruses was reported shortly after the description of galv when an endogenous retrovirus (mcerv) with sequence homology to galv was detected in the asian feral mouse mus caroli [ , ] . additionally, immunological assays showed that their antigens cross-reacted. despite the similarity of the genomic sequence with galv, mcerv has a different host range and uses a different receptor (plasmolipin), and therefore it is unlikely to be the precursor of galv/korv [ ] . endogenous retroviruses related to galv/korv were also detected in mus cervicolor and in the rodent vandeleuria oleacea [ , ] . this similarity was mainly evaluated using immunological and dna hybridization techniques, however more recent full genome data showed differences between the sequences. mcerv is most closely related to the mus dunni endogenous virus (mdev) [ ] and the mus musculus endogenous retrovirus (mmerv) [ ] . phylogenetically, these viruses form a sister clade to the galv/korv clade [ ] . however, mmerv and mdev are closer related to the galv/korv group than to the mulv, including the moloney mulv, the friend mulv, and the rauscher mulv [ , ] . when australian vertebrate species were screened using pcr, in order to detect proviral sequences closely related to galv/korv, a novel gammaretrovirus was detected in an australian rodent, the grassland melomys, melomys burtoni (mbrv) [ ] . mbrv shares % sequence homology with galv-seato and % identity with korv, a much higher homology when compared with the previously described related rodent viruses. since the geographic ranges of the grassland melomys and koalas partially overlap, a transspecies transmission of mbrv from melomys to koalas could be theoretically possible. however, since melomys do not live in south east asia, mbrv cannot be the origin of the galv, despite the fact that mbrv is closer related to galv than to korv [ ] . recently, a new gammaretrovirus, melomys woolly monkey virus (melwmv), was detected in another melomys burtoni subspecies living in the wallacea in indonesia [ ] . this virus was detected as a result of screening southeast asian rodent species and all other species were negative. melwmv has % homology with the wmv and is a subtype of wmv, whereas mbrv from the australian animals is a sister taxon (figure a ). stop codons in env and pol indicate that it is a defective, endogenous retrovirus [ ] . since melwmv und mbrv are the closest relatives to galv, they could be the origin of galv, although it is also possible that the two melomys subspecies were infected by a galv-like virus from an unknown species. the first evidence of related retroviruses was reported shortly after the description of galv when an endogenous retrovirus (mcerv) with sequence homology to galv was detected in the asian feral mouse mus caroli [ , ] . additionally, immunological assays showed that their antigens cross-reacted. despite the similarity of the genomic sequence with galv, mcerv has a different host range and uses a different receptor (plasmolipin), and therefore it is unlikely to be the precursor of galv/korv [ ] . endogenous retroviruses related to galv/korv were also detected in mus cervicolor and in the rodent vandeleuria oleacea [ , ] . this similarity was mainly evaluated using immunological and dna hybridization techniques, however more recent full genome data showed differences between the sequences. mcerv is most closely related to the mus dunni endogenous virus (mdev) [ ] and the mus musculus endogenous retrovirus (mmerv) [ ] . phylogenetically, these viruses form a sister clade to the galv/korv clade [ ] . however, mmerv and mdev are closer related to the galv/korv group than to the mulv, including the moloney mulv, the friend mulv, and the rauscher mulv [ , ] . when australian vertebrate species were screened using pcr, in order to detect proviral sequences closely related to galv/korv, a novel gammaretrovirus was detected in an australian rodent, the grassland melomys, melomys burtoni (mbrv) [ ] . mbrv shares % sequence homology with galv-seato and % identity with korv, a much higher homology when compared with the previously described related rodent viruses. since the geographic ranges of the grassland melomys and koalas partially overlap, a transspecies transmission of mbrv from melomys to koalas could be theoretically possible. however, since melomys do not live in south east asia, mbrv cannot be the origin of the galv, despite the fact that mbrv is closer related to galv than to korv [ ] . recently, a new gammaretrovirus, melomys woolly monkey virus (melwmv), was detected in another melomys burtoni subspecies living in the wallacea in indonesia [ ] . this virus was detected as a result of screening southeast asian rodent species and all other species were negative. melwmv has % homology with the wmv and is a subtype of wmv, whereas mbrv from the australian animals is a sister taxon (figure a ). stop codons in env and pol indicate that it is a defective, endogenous retrovirus [ ] . since melwmv und mbrv are the closest relatives to galv, they could be the origin of galv, although it is also possible that the two melomys subspecies were infected by a galv-like virus from an unknown species. bats are the second largest group of mammals with approximately different species. bats harbor more than distinct emerging and re-emerging human viral pathogens, including ebola viruses and the severe acute respiratory syndrome coronavirus (sars-cov) [ , ] . interestingly, bats carry many viruses and yet they do not usually show overt signs of a disease. in addition to these exogenous viruses, numerous endogenous retroviruses have also been described in bats, however most of them have major deletions or frameshift mutations in the pol gene coding for the polymerase, indicating that they are defective. among the endogenous retroviruses are the megaderma lyra retrovirus (mirv), which is from an insectivorous bat, and the rousettus leschenaultia retrovirus (rirv), from a frugivorous bat [ , ] . rirv clusters with the pervs and mirv clusters with mdev, korv and galv (figure b) . all galv are able to grow on ccl- bat lung fibroblasts [ ] , which serves as a strong indicator that mammalian retroviruses may have originated in bats. additionally, numerous betaretroviruses have also been described in bats [ ] . therefore, it can be assumed that rodents and bats serve as equally important intermediate hosts for endogenous retroviruses [ , ] . bats are the second largest group of mammals with approximately different species. bats harbor more than distinct emerging and re-emerging human viral pathogens, including ebola viruses and the severe acute respiratory syndrome coronavirus (sars-cov) [ , ] . interestingly, bats carry many viruses and yet they do not usually show overt signs of a disease. in addition to these exogenous viruses, numerous endogenous retroviruses have also been described in bats, however most of them have major deletions or frameshift mutations in the pol gene coding for the polymerase, indicating that they are defective. among the endogenous retroviruses are the megaderma lyra retrovirus (mirv), which is from an insectivorous bat, and the rousettus leschenaultia retrovirus (rirv), from a frugivorous bat [ , ] . rirv clusters with the pervs and mirv clusters with mdev, korv and galv (figure b) . all galv are able to grow on ccl- bat lung fibroblasts [ ] , which serves as a strong indicator that mammalian retroviruses may have originated in bats. additionally, numerous betaretroviruses have also been described in bats [ ] . therefore, it can be assumed that rodents and bats serve as equally important intermediate hosts for endogenous retroviruses [ , ] . although the speculation that bat retroviruses represent the precursor of both the galv and the korv is highly attractive, this theory is still not proven and some important questions are still unanswered. firstly, when was the introduction of these viruses into their host populations? galv was reported for the first time in the s [ ] , and the introduction of the korv precursor into koalas was first dated approximately years ago [ ] . meanwhile, a widespread distribution of korv in the late s was described [ ] . these findings would be consistent with a historical account that an epidemic with symptoms that may have been similar to those caused by chlamydia killed large numbers of koalas during the period - [ ] . secondly, why is wmv more closely related to mbrv than to galv, even though wmv is the result of an infection of a woolly monkey with galv, whereas mbrv is suggested to be the precursor of the galv? thirdly, the galv/korv-like viruses found until now in the melomys subspecies [ , ] and in bats [ ] have been integrated into the genome of these species a long time ago and they are now defective. based on the similarity of its and long terminal repeats (ltr), melwmv integrated into the genome of the indonesian melomys , years ago [ ] . therefore, it is unlikely that they are responsible for the recent infections in koalas and captive gibbons. melomys can then be excluded as the origin of the galv because all melomys species never crossed the wallace line [ ] . modern viruses related to galv/korv from a species able to cross the wallace line and able to infect koalas and gibbons in captivity (but not free-living gibbons) may be the best candidates for the galv/korv precursor. fourthly, why are the gibbons in the seato facility infected and not animals in the wild? one explanation may be that the bats carrying the virus were attracted to food in the facility, or that infected bats were kept there in captivity. this analysis of the transspecies transmission of galv/korv-related gammaretroviruses is of great interest for the evaluation of a potential transmission of the closely related pervs during xenotransplantation using pig cells, tissues and organs. xenotransplantation is under development due to the increasing shortage of human cells and organs for the treatment of organ failure [ ] . whereas most of the potentially zoonotic microorganisms in pigs can be eliminated by treatment, vaccination and designated pathogen-free breeding, pervs cannot be eliminated this way because they are integrated into the genome of all pigs. since perv-a, perv-b and recombinant perv-a/c can infect human cells in vitro, the question arises whether pervs represent a special risk for xenotransplantation [ ] . viruses related to perv such as galv [ ] , korv [ , ] and felv [ ] can also infect human cells in vitro, however no transmission to humans in vivo has yet been reported. for example, in veterinarians with a reported extensive duration of work with cats (mean, . years) and multiple high-risk exposures (e.g., cat bites, scratches, and injuries with sharp instruments), neither serologic nor molecular evidence of felv infection was detected [ ] . reports that another gammaretrovirus coming from mice, xmrv, infects humans and causes prostate cancer and chronic fatigue syndrome described an artifact [ ] . no transmission of perv was also observed in the first clinical xenotransplantations (more than patients), in numerous pig to non-human primate transplantations, as well as in perv infection experiments in rodents and non-human primates with and without strong pharmaceutical immunosuppression [ ] . recently, in two prospective clinical trials, transplanting pig islet cells to diabetic patients in new zealand [ ] and argentina [ ] , perv transmission was also not observed. this indicates that gammaretroviruses could potentially not be able to infect humans, despite the fact that human cells carry the corresponding receptor and can be infected in vitro. to increase the safety of xenotransplantation, additional measures can be undertaken, such as selection of perv-c negative pigs, in order to prevent recombination with perv-a which would result in highly replication-competent perv-a/c, inhibition of perv expression by rna interference in vitro and in vivo in transgenic pigs expressing the corresponding sirna, and vaccines based on the envelope proteins [ ] . recently, attempts were undertaken through genome editing to inactivate all pervs in the genome, either using a zinc finger nuclease (zfn) [ ] , or clustered regularly interspaced short palindromic repeats (crispr) acting with crispr-associated nuclease (cas ) [ ] . using crispr/cas, proviruses were inactivated in immortalized pig cells and the question is, whether this strategy can be used to generate perv-free animals suitable for xenotransplantation [ ] . transspecies transmission of retroviruses is a common event, and can be seen in various viruses, such as hiv- , which is the result of a transspecies transmission of a lentivirus from chimpanzees to humans and in hiv- , which is the result of a transspecies transmission of a siv from sooty mangabeys to humans [ ] . transspecies transmission is also common for gammaretroviruses; perv, for example, is the result of a transspecies transmission from mice to pigs [ , ] . galv and korv are also the result of a transspecies transmission of a retrovirus from a still unknown host. related viruses have been found in rodents and bats and it is of great interest to find the precursor virus and its hosts. to study the transmission of retroviruses from one species to another is of great importance in the context of transmissions, which may occur, for example, when pervs are transmitted to the human recipients during xenotransplantation. the origin and molecular 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sequences in the pig genome by zinc finger nucleases (zfn) genome-wide inactivation of porcine endogenous retroviruses (pervs) elimination of porcine endogenous retroviruses from pig cells acknowledgments: i would like to thank v. morozov for critical reading of the manuscript. the author declares no conflict of interest. viruses , , key: cord- - a uvyz authors: xu, jiabao; zhao, shizhe; teng, tieshan; abdalla, abualgasim elgaili; zhu, wan; xie, longxiang; wang, yunlong; guo, xiangqian title: systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov- and sars-cov date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: a uvyz after the outbreak of the severe acute respiratory syndrome (sars) in the world in , human coronaviruses (hcovs) have been reported as pathogens that cause severe symptoms in respiratory tract infections. recently, a new emerged hcov isolated from the respiratory epithelium of unexplained pneumonia patients in the wuhan seafood market caused a major disease outbreak and has been named the severe acute respiratory syndrome coronavirus (sars-cov- ). this virus causes acute lung symptoms, leading to a condition that has been named as “coronavirus disease ” (covid- ). the emergence of sars-cov- and of sars-cov caused widespread fear and concern and has threatened global health security. there are some similarities and differences in the epidemiology and clinical features between these two viruses and diseases that are caused by these viruses. the goal of this work is to systematically review and compare between sars-cov and sars-cov- in the context of their virus incubation, originations, diagnosis and treatment methods, genomic and proteomic sequences, and pathogenic mechanisms. coronaviruses (covs) are a group of viruses that co-infect humans and other vertebrate animals. cov infections affect the respiratory, gastrointestinal, liver, and central nervous systems of humans, livestock, birds, bats, mice, and many other wild animals [ ] [ ] [ ] . for example, severe acute respiratory syndrome (sars) in and the middle east respiratory syndrome (mers) in were both coronaviruses that transmitted from animals to humans [ , ] . the source of unexplained pneumonia was first discovered in wuhan in dec, , and sars-cov- , a new coronavirus, was isolated from the respiratory epithelium of patients. it belongs to a new evolutionary branch within the cov. on feb. th, , the new coronavirus was officially renamed "sars-cov- " from " -ncov" [ ] . the disease caused by sars-cov- was called "coronavirus disease " (covid- ) [ ] . according to on nov. th, , a respiratory illness erupted in guangdong province, china [ ] . in feb, , the chinese ministry of health announced that this acute respiratory syndrome had thus far resulted in cases and five deaths [ ] . the following month, there were clusters of atypical pneumonia reported in other parts of mainland china, hong kong [ ] , canada [ ] , and singapore [ ] . in jul, , sars-cov spread across countries in six continents, and caused a cumulative , cases and deaths ( . %) [ ] . in particular, a higher mortality ( %) was found in hospital personnel [ , ] . on dec. th, , the health departments of hubei province received a report that four employees of the south china seafood wholesale market were diagnosed with unknown-caused pneumonia in a local hospital, which was the first report of sars-cov- [ ] . on dec. st, , the national health commission of people republic of china and chinese center for disease control and prevention (china cdc) participated in the investigation and case-searching work [ ] . on the same day, the government of wuhan released information about the disease outbreaks to society [ ] . nowadays, the number of patients infected with sars-cov- continues to climb worldwide. by the date of this paper's submission, a cumulative , cases and , deaths ( . %) were reported worldwide. in wuhan, china, the number is , . the main timeline of sars and covid- epidemic development were shown in figure a ,b, respectively. glucocorticoid and interferon lopinavir/ritonavir (in testing) on nov. th, , a respiratory illness erupted in guangdong province, china [ ] . in feb, , the chinese ministry of health announced that this acute respiratory syndrome had thus far resulted in cases and five deaths [ ] . the following month, there were clusters of atypical pneumonia reported in other parts of mainland china, hong kong [ ] , canada [ ] , and singapore [ ] . in jul, , sars-cov spread across countries in six continents, and caused a cumulative , cases and deaths ( . %) [ ] . in particular, a higher mortality ( %) was found in hospital personnel [ , ] . on dec. th, , the health departments of hubei province received a report that four employees of the south china seafood wholesale market were diagnosed with unknown-caused pneumonia in a local hospital, which was the first report of sars-cov- [ ] . on dec. st, , the national health commission of people republic of china and chinese center for disease control and prevention (china cdc) participated in the investigation and case-searching work [ ] . on the same day, the government of wuhan released information about the disease outbreaks to society [ ] . nowadays, the number of patients infected with sars-cov- continues to climb worldwide. by the date of this paper's submission, a cumulative , cases and , deaths ( . %) were the initial symptoms of sars patients were fever ( %), cough ( . %), myalgia ( . %), dyspnea ( . %), and diarrhea ( . %) [ ] , and the prognosis of patients was associated with host characteristics (including age, gender, etc.) [ ] . during hospitalization, respiratory distress occurred in . % of sars patients [ ] . the duration from disease onset to severe respiratory distress was an average of . ± . days [ ] . during the disease course, some patients developed leukopenia, lymphopenia, and thrombocytopenia with an upregulation of aspartate transaminase (ast), alanine aminotransferase (alt), lactic dehydrogenase (ldh), and c-reactive protein (crp) [ ] . in comparison, covid- showed similar trends with sars patients [ ] . fever, fatigue, and dry cough are the main manifestations of the patients, while nasal congestion, runny nose, and other symptoms of the upper respiratory tract are rare. beijing centers for diseases control and prevention indicated that the typical case of covid- has a progressive aggravation process. covid- can be classified into light, normal, severe, and critical types based on the severity of the disease [ ] : ( ) mild cases-the clinical symptoms were mild, and no pneumonia was found on the chest computed tomography (ct); ( ) normal cases-fever, respiratory symptoms, and patients found to have imaging manifestations of pneumonia; ( ) severe cases-one of the following three conditions: respiratory distress, respiratory rate ≥ times/min (in resting state, refers to oxygen saturation ≤ %), partial arterial oxygen pressure (pao )/oxygen absorption concentration (fio ) ≤ mmhg ( mmhg = . kpa); ( ) critical cases-one of the following three conditions: respiratory failure and the need for mechanical ventilation, shock, or the associated failure of other organs requiring the intensive care unit [ ] . the current clinical data shows that the majority of the deaths occurred in the older patients. however, severe cases have been documented in young adults who have unique factors, particularly those with chronic diseases, such as diabetes or hepatitis b. those with a long-term use of hormones or immunosuppressants, and decreased immune function, are likely to get severely infected. the incubation period of sars is - days [ ] . however, in a small number of patients, the incubation period may be longer than days [ ] . it has been demonstrated that the latency of covid- varies from - days on average, for up to days [ ] . during this incubation period, patients are contagious, and it has been reported that each case infected on average . other people (uncertainty range . - . ) [ ] . by comparison, we found that the average latency of covid- is slightly longer than that of sars. according to the demographic information of sars patients, infection occurred in all age groups (the average age was dyspnea ( . %), and diarrhea ( . %) [ ] , and the prognosis of patients was associated with host characteristics (including age, gender, etc.) [ ] . during hospitalization, respiratory distress occurred in . % of sars patients [ ] . the duration from disease onset to severe respiratory distress was an average of . ± . days [ ] . during the disease course, some patients developed leukopenia, lymphopenia, and thrombocytopenia with an upregulation of aspartate transaminase (ast), alanine aminotransferase (alt), lactic dehydrogenase (ldh), and c-reactive protein (crp) [ ] . in comparison, covid- showed similar trends with sars patients [ ] . fever, fatigue, and dry cough are the main manifestations of the patients, while nasal congestion, runny nose, and other symptoms of the upper respiratory tract are rare. beijing centers for diseases control and prevention indicated that the typical case of covid- has a progressive aggravation process. covid- can be classified into light, normal, severe, and critical types based on the severity of the disease [ ] : ( ) mild cases-the clinical symptoms were mild, and no pneumonia was found on the chest computed tomography (ct); ( ) normal cases-fever, respiratory symptoms, and patients found to have imaging manifestations of pneumonia; ( ) severe cases-one of the following three conditions: respiratory distress, respiratory rate ≥ times / min (in resting state, refers to oxygen saturation ≤ %), partial arterial oxygen pressure (pao )/oxygen absorption concentration (fio ) ≤ mmhg ( mmhg = . kpa); ( ) critical cases-one of the following three conditions: respiratory failure and the need for mechanical ventilation, shock, or the associated failure of other organs requiring the intensive care unit [ ] . the current clinical data shows that the majority of the deaths occurred in the older patients. however, severe cases have been documented in young adults who have unique factors, particularly those with chronic diseases, such as diabetes or hepatitis b. those with a long-term use of hormones or immunosuppressants, and decreased immune function, are likely to get severely infected. the incubation period of sars is - days [ ] . however, in a small number of patients, the incubation period may be longer than days [ ] . it has been demonstrated that the latency of covid- varies from - days on average, for up to days [ ] . during this incubation period, patients are contagious, and it has been reported that each case infected on average . other people (uncertainty range . - . ) [ ] . by comparison, we found that the average latency of covid- is slightly longer than that of sars. according to the demographic information of sars patients, infection occurred in all age groups (the average age was ≦ ) [ ] . there was a proportional difference between male and female (female predominance) [ ] , with a male-to-female ratio of : . [ ] . in addition, hospital staff had a higher risk due to the proximal interactions with large numbers from the infected population. for example, hospital staff accounted for % of all cases in hong kong and . % in guangdong [ ] . the mortality caused by sars increased with age (> years) [ ] , and the overall mortality rate during the outbreak of sars was estimated at . % [ , ] . li et al. reported that people who have not been exposed to sars-cov- are all susceptible to covid- [ ] . among the , patients who have been confirmed with covid- , nearly half of the patients have been aged years or older ( . %) [ ] . the male-to-female ratio is about . : [ ] and the average incubation period is . days [ ] . however, severe covid- cases and deaths have mostly been in the middle-aged adults and the elderly with long smoking histories or other ) [ ] . there was a proportional difference between male and female (female predominance) [ ] , with a male-to-female ratio of : . [ ] . in addition, hospital staff had a higher risk due to the proximal interactions with large numbers from the infected population. for example, hospital staff accounted for % of all cases in hong kong and . % in guangdong [ ] . the mortality caused by sars increased with age (> years) [ ] , and the overall mortality rate during the outbreak of sars was estimated at . % [ , ] . li et al. reported that people who have not been exposed to sars-cov- are all susceptible to covid- [ ] . among the , patients who have been confirmed with covid- , nearly half of the patients have been aged years or older ( . %) [ ] . the male-to-female ratio is about . : [ ] and the average incubation period is . days [ ] . however, severe covid- cases and deaths have mostly been in the middle-aged adults and the elderly with long smoking histories or other basic diseases, such as heart disease and hypertension [ , ] . at the time that this paper was been submitted, covid- patients mortality rate was . % [ ] . in , shi et al. isolated a sars-like coronavirus which was highly homologous to the sars-cov, confirming that rhinolophus sinicus (chinese rufous horseshoe bat) was a natural host of the sars-cov [ ] . bats are known to be hosts for coronaviruses based on complete genomic sequences analysis [ ] . epidemiological investigations have shown that civet cats in the wildlife market were the direct source of sars-cov [ ] . among the four patients with sars discovered during the winter of - , two were waitresses at a restaurant in guangzhou, china, and one was a customer who viruses , , of ate in the restaurant a short distance from a civet cage. all six civets in this cage were tested positive for sars-cov [ ] . the new emerging sars-cov- shares about % of the gene sequence of sars-cov, released by the military medical research institute of nanjing military region in [ ] . recently, shi et al. reported that the sequence similarity of coronavirus between sars-cov- and the coronavirus isolated from rhinolophus affinis is . %, and suggested that bats may be the source of the virus [ ] . so far, the intermediate hosts of sars-cov- are elusive and have been reported to be snakes, minks, or variable others [ , ] . recently, a research group of south china agricultural university reported that pangolins may be one of the intermediate hosts for sars-cov- , by analyzing more than , metagenomic samples, because they found that % of pangolins are positive for the coronavirus. moreover, the virus isolate from pangolin shared % sequence similarity with the current infected human strain sars-cov- [ ] . taking this recent research into consideration, we agreed that pangolin is more likely to be one of intermediate hosts of sars-cov- . according to the who data on jul. th, [ ] , a total of , clinically diagnosed cases of sars were reported worldwide, with deaths and countries and regions affected (figure a ). most cases were in asia, europe, and america. the main countries in asia were china (including mainland, macao, hong kong, and taiwan), singapore, and so on. the total number of cases in mainland china was , , with deaths [ ] . the cases were mainly concentrated in beijing, guangdong, and shanxi ( figure b ) [ ] . in total, , patients were from hong kong, macao, and taiwan, with deaths [ ] . viruses , , x for peer review of basic diseases, such as heart disease and hypertension [ , ] . at the time that this paper was been submitted, covid- patients mortality rate was . % [ ] . in , shi et al. isolated a sars-like coronavirus which was highly homologous to the sars-cov, confirming that rhinolophus sinicus (chinese rufous horseshoe bat) was a natural host of the sars-cov [ ] . bats are known to be hosts for coronaviruses based on complete genomic sequences analysis [ ] . epidemiological investigations have shown that civet cats in the wildlife market were the direct source of sars-cov [ ] . among the four patients with sars discovered during the winter of - , two were waitresses at a restaurant in guangzhou, china, and one was a customer who ate in the restaurant a short distance from a civet cage. all six civets in this cage were tested positive for sars-cov [ ] . the new emerging sars-cov- shares about % of the gene sequence of sars-cov, released by the military medical research institute of nanjing military region in [ ] . recently, shi et al. reported that the sequence similarity of coronavirus between sars-cov- and the coronavirus isolated from rhinolophus affinis is . %, and suggested that bats may be the source of the virus [ ] . so far, the intermediate hosts of sars-cov- are elusive and have been reported to be snakes, minks, or variable others [ , ] . recently, a research group of south china agricultural university reported that pangolins may be one of the intermediate hosts for sars-cov- , by analyzing more than , metagenomic samples, because they found that % of pangolins are positive for the coronavirus. moreover, the virus isolate from pangolin shared % sequence similarity with the current infected human strain sars-cov- [ ] . taking this recent research into consideration, we agreed that pangolin is more likely to be one of intermediate hosts of sars-cov- . according to the latest data on feb. th, [ , ] , there have been a total of , clinically diagnosed cases of covid- in worldwide, with , deaths. a total of countries and regions have infected people. due to the spring festival transportation peak, the disease has been spread more rapidly across china (figure c ). as the origin area of covid- , hubei province has been the most severely infected area, with , cumulative diagnosis cases. wuhan city has , cases. guangdong, henan, and zhejiang province have , cases, , cases, and , cases, respectively (figure d ). at present, the covid- outbreak has been spread to all parts of china and around the world, including the united states, thailand, and japan. it has been noticed that most of these patients have ever been to wuhan or contacted with people who had been in wuhan. the distribution of covid- patients in china (including hong kong, macao and taiwan) and hubei province is shown in figure . sars were reported worldwide, with deaths and countries and regions affected (figure a) . most cases were in asia, europe, and america. the main countries in asia were china (including mainland, macao, hong kong, and taiwan), singapore, and so on. the total number of cases in mainland china was , , with deaths [ ] . the cases were mainly concentrated in beijing, guangdong, and shanxi (figure b ) [ ] . in total, , patients were from hong kong, macao, and taiwan, with deaths [ ] . according to the latest data on feb. th, [ , ] , there have been a total of , clinically diagnosed cases of covid- in worldwide, with , deaths. a total of countries and regions have infected people. due to the spring festival transportation peak, the disease has been spread more rapidly across china (figure c ). as the origin area of covid- , hubei province has been the most severely infected area, with , cumulative diagnosis cases. wuhan city has , cases. guangdong, henan, and zhejiang province have , cases, , cases, and , cases, respectively (figure d ). at present, the covid- outbreak has been spread to all parts of china and around the world, including the united states, thailand, and japan. it has been noticed that most of these patients have ever been to wuhan or contacted with people who had been in wuhan. the distribution of covid- patients in china (including hong kong, macao and taiwan) and hubei province is shown in figure . as the number of covid- patients in china has been growing rapidly, preventing the spread of sars-cov- is the most important and urgent task [ ] . it was shown that human-to-human transmission of sars-cov- has spread via droplets or close contacts [ , ] , but aerosol and fecal-oral transmission still need further study [ , ] . to reduce virus transmission, early detection and isolation are essential. in addition, close monitoring in crowded places is also important [ ] . the possible pathogens of sars and covid- are both derived from wild animals [ ] . therefore, hunting, selling, and eating wild animals not only seriously damage the ecosystem, but also lead to the spread of epidemic diseases [ ] . thus, banning all wildlife trade is an effective measure to prevent viral prevalence. wearing level-d protective clothing can protect medical staff from infection of respiratory viruses [ ] . a vaccine against sars-cov has not been described in any published articles [ ] . however, on jan. th, , the china cdc started to develop a new vaccine for sars-cov- . the virus has been successfully isolated and seed strains have been screened [ ] . the early symptoms of sars and covid- are very similar to winter influenza, and the most important way to distinguish flu and pneumonia is to take throat swabs for viral testing [ ] . current diagnostic tests for coronavirus include rt-pcr, real-time reverse transcription pcr (rrt-pcr), reverse transcription loop-mediated isothermal amplification, as well as real-time rt-lamp [ ] [ ] [ ] [ ] . national medical products administration has approved seven new nucleic acid test reagents for coronavirus, which were developed based on fluorescence pcr by feb. st, [ ] . suspected infections can be detected accurately and quickly for timely isolation and treatment to avoid infecting others by using these test reagents. both sars-cov and sars-cov- are covs; hence, the treatment strategies of sars could be relevant for covid- [ ] . in , sars was mainly treated by isolation of the patients, hormones treatment, antiviral and symptomatic treatments, and many drugs such as glucocorticoid [ ] and interferon [ ] . now, isolation, antiviral, and symptomatic treatments are still mainly adopted for covid- treatment. as effective drugs for sars, hormones and interferons can also be used to treat covid- [ ] . lopinavir is one kind of protease inhibitor used to treat hiv infection, with ritonavir as a booster. lopinavir and/or ritonavir has anti coronavirus activity in vitro. hong kong scholars found that, compared with ribavirin alone, patients treated with lopinavir/ritonavir and ribavirin had lower risk of acute respiratory distress syndrome (ards) or death caused by sars-cov [ , ] . lopinavir/ritonavir has also been clinically tested in treatment of covid- , and showed wonderfully effective treatment for some patients, but the general clinical effect has not been determined [ ] . more effective treatments are still under continuing exploration: on jan. th, , a joint research team from the shanghai institute of materia medica, chinese academy of sciences, and shanghai tech university screened and identified potential drugs that are reported to be effective against sars-cov- [ ] . a high-resolution crystal structure of sars-cov- coronavirus cl hydrolase (mpro) was announced after the outbreak of covid- in the world [ ] , and human coronaviruses (hcovs) have been treated as severe pathogens in respiratory tract infections. nelfinavir was predicted to be a potential inhibitor of sars-cov- main protease [ ] . the first patient in the us had been trial-treated with intravenous remdesivir (a novel nucleotide analogue prodrug in development) due to a severe infection [ , ] . no adverse reactions were observed during the administration, and the patient's condition was effectively improved [ ] . clinical trials of remdesivir for treatment of covid- just started on feb. th and th, in wuhan and beijing, respectively, and the experimental results remain unclear [ , ] . covs are rna viruses and contain the largest genomes of all rna viruses [ ] . covs belong to the subfamily coronavirinae in the family of coronaviridae of the order nidovirales, and this subfamily includes four genera: α-coronavirus, β-coronavirus, γ-coronavirus, and δ-coronavirus [ ] . both sars-cov- and sars-cov are in the coronavirus family, β-coronavirus genera [ ] . the genome of sars-cov- is more than % similar to the genome of the sars-like virus zc (bat-sl-covzc , mg . ), and together these types of viruses form a unique orthocoronavirinae subfamily with another sars-like virus zxc in the sarbecovirus subgenus [ ] . all the three viruses show typical β-coronavirus gene structure. human sars-cov and a genetically similar bat coronavirus (bat-sl-covzxc , mg ) from southwest of china have formed another clade within the sarbecovirus [ ] . we also performed comparative genomic analyses of sars-cov- and sars-cov by zpicture. the results showed that the genomic sequences of sars-cov- and sars-cov have extremely high homology at the nucleotide level (figure a,b) . there are six regions of difference (rd) in the genome sequence between sars-cov and sars-cov- , and the rds are named according to the order of discovery. rd , rd , and rd ( nt, nt, and nt, respectively) are partial coding sequences of the orf lab gene; rd and rd ( nt and nt, respectively) are partial coding sequences of the s gene; rd is nt in size and is part of the coding sequence of the orf b and orf genes. these rds may sequences of the orf lab gene; rd and rd ( nt and nt, respectively) are partial coding sequences of the s gene; rd is nt in size and is part of the coding sequence of the orf b and orf genes. these rds may provide new molecular markers for the identification of sars-cov- and sars-cov, and also help to develop new drugs against sars-cov- . to analyze the homogeneity of sars-cov, mers-cov, and sars-cov- , an evolutionary tree was constructed based on the genomes of sars-cov- strains from different data submission units, one sars-cov strain, and two mers-cov strains ( figure s ). phylogenetic analysis showed that the distance of sars-cov (ay ) is closer to sars-cov- strains than mers-cov (kc , jx ). to analyze the homogeneity of sars-cov, mers-cov, and sars-cov- , an evolutionary tree was constructed based on the genomes of sars-cov- strains from different data submission units, one sars-cov strain, and two mers-cov strains ( figure s ). phylogenetic analysis showed that the distance of sars-cov (ay ) is closer to sars-cov- strains than mers-cov (kc , jx ). to further explore whether all encoded proteins of sars-cov- are homologous to that of sars-cov, we performed a protein sequence alignment analysis using blastp. the results showed that most of sars-cov- proteins are highly homologous ( %- %) to the proteins of sars-cov virus, indicating the evolutionary similarity between sars-cov and sars-cov- (table ) . however, two proteins (orf and orf ) in sars-cov- have no homologous proteins in sars-cov. the amino acid sequence of orf in sars-cov- is different from sequences of conserved orf or orf b derived from human sars-cov [ ] . orf protein of sars-cov- does not contain known functional domain or motif. an aggregation motif vlvvl (amino acid - ) has been found in sars-cov orf b which was shown to trigger intracellular stress pathways and activate nod-like receptor family pyrin domain-containing- (nlrp ) inflammasomes [ ] . therefore, it will be clinically meaningful to analyze the biological function of these two specific proteins (orf and orf ) in sars-cov- . as the most abundant protein in covs, nucleocapsid (n) protein is highly conserved across covs [ ] . the n protein in sars-cov- shares~ % amino acid identity with that in sars-cov [ ] , which indicates that antibodies against the n protein of sars-cov would likely recognize and bind the n protein of sars-cov- as well. n antibodies do not provide immunity to sars-cov- infection, but the antibodies have a cross reactivity with sars-cov n protein viruses, which would allow a serum-based assay to identify the asymptomatic sars-cov- infected-cases [ ] . although previous studies have found serum reactivity to group sars-cov n proteins in chinese populations [ ] , exposure to sars-cov- should increase the dilution factor if the infection had occurred. this information has important implications for preventing the spread of asymptomatic infections. in addition, spike stalk s in sars-cov- is highly conserved and shares % identity with those of the two bat sars-like covs (bat-sl-covzxc and bat-sl-covzc ) and human sars-cov [ ] . thus, the broad spectrum antiviral peptides against s has the potential to be effective treatment [ ] . many studies have been performed to study the pathogenesis of sars-cov [ ] [ ] [ ] . the spike (s) protein and n protein confer stability to the viral particle [ ] . the n protein is a structural protein involved in virion assembly, and plays a pivotal role in virus transcription and assembly efficiency [ ] . s protein can bind to the cellular receptors of sensitive cells and mediate infection of their target cells, after which it begins to replicate in the cytoplasm [ ] . sars-cov mainly targets the lungs, immune organs, and small systemic blood vessels and causes systemic vasculitis and decrease of immune function [ ] . more seriously, the infection leads to extensive pulmonary consolidation, diffuse alveolar damage, and the formation of a transparent membrane, finally deteriorating to respiratory distress [ ] . cov can enter host cells through the interaction between cov s protein and its host receptor angiotensin-converting enzyme (ace ), which is isolated from sars-cov-permissive vero-e cells. the receptor-binding motif (rbm) of s protein can directly contact ace [ , , ] . aec have been identified to be key binding residues and functional receptor for sars-cov, and it can also protect alveolar cells [ ] . the binding of spike protein to ace and the subsequent downregulation of this receptor contribute to severe alveolar injury during sars [ ] . the downregulation of ace results in the excessive production of angiotensin ii by the related enzyme ace, and the stimulation of type a angiotensin ii receptor (agtr a) can lead to the increase of pulmonary vascular permeability, which potentially explains the increased lung pathology when the expression of ace is decreased [ ] . ace -transfected t cells form multinucleated syncytia with cells expressing s protein. the virus was shown to replicate effectively in ace -transfected, but not in mock-transfected t cells. antibodies targeting ace can block viral replication in vero-e cells [ ] . recently, ji et al. demonstrated that the receptor binding domain of sars-cov- was capable of binding ace in the context of the sars-cov spike protein [ ] . among sras-cov spike protein's fourteen residues predicted to interact directly with human ace as the receptor for sars-cov, eight amino acids are well conserved in homology sars-cov- spike protein [ , ] . at the same time, wan et al. showed that sars-cov- uses ace receptors to infect humans, bats, civets, monkeys, and swine, but not mice [ ] . compared to previously reported sars-cov strains, sars-cov- uses ace receptors more efficiently than human sars-cov (year ), but less efficiently than human sars-cov (year ) [ ] . the mutation of proteins determines two important characteristics of the sars-cov- : a higher ability to infect and enhanced pathogenicity than the bat-like sars-cov, but a lower pathogenicity than sars-cov [ ] . as a large number of people have left wuhan, the control of the epidemic situation is extremely urgent, and the treatments of covid- are imminent. on feb. th, , there were more than , confirmed patients in hubei province, china [ ] . due to the lack of effective antiviral drugs, the prognosis of patients solely depends on their age and physical condition [ ] . although it was reported that the clinically recovered patients exceed the number of dead, the majority of the patients are still not cured in hospital. in addition, the potential adaptive mutation of sars-cov- makes it difficult for vaccine development. therefore, it is urgent for us to develop more sensitive inspection methods and effective drugs. seven type of covs have been identified to cause human disease [ ] . the two highly pathogenic viruses, sars-cov and mers-cov, cause severe respiratory syndrome in humans. the other four human covs (hcov-nl , hcov- e, hcov-oc and hku ) induce only mild upper respiratory diseases, although some of them can cause severe infections in infants, young children, and elderly individuals [ , ] . the latest one is sars-cov- . it has been reported that sars-cov- shared almost % of the genome with sars-cov [ ] . our results also showed that almost all encoded proteins of sars-cov- are homologous to sars-cov proteins ( table ) . hence, clinical drugs and therapies for treating sars may be used as a reference for covid- treatment [ ] . in addition to the well-known sars-cov, mers-cov, as one merbecovirus subgenus of β-covs, is also extremely invasive. mers-cov is the pathogen of the middle east respiratory syndrome, which can infect both humans and animals, and can be transmitted through camels [ ] . it mainly occurs in saudi arabia and has a high mortality rate [ ] . studies had demonstrated that the clinical course of sars and mers was highly similar, and sars and mers may have similar pathogenesis [ ] . the genome sequence of sars-cov- also shows some similarities to that of mers-cov. it will be very interesting to study the relationship among sars-cov, mers-cov, and sars-cov- that may be exploited for future developing broad-spectrum antiviral therapies. although more and more studies for sars-cov- have sprung up since the outbreak of this epidemic covid- , based on our comparison, we propose some key questions to be clarified in future studies (table ). in-depth understanding the underlying pathogenic mechanisms of sars-cov- will reveal more targets for better therapy of covid- . table . proposed questions to study sars-cov- for future studies. what is the effect of the surface epitope and receptor binding domain of s protein of sars-cov- on the virus' infectivity? is there any effect of sars-cov vaccine designed according to s protein on sars-cov- ? does sars-cov- orf and orf proteins, which have no homology proteins in sars-cov, play roles in the infectivity and pathogenicity of sars-cov- ? can the susceptibility of asymptomatic carriers be judged by detecting the serum reactivity level of n protein? apart from droplet transmission and contact transmission, are there other methods to transmit sars-cov- ? what is the percentage of covid- patients have been infected with sars and produced antibodies? is there an effective specific anti-sars-cov- solution? does traditional chinese medicine have any effect on the treatment of covid- caused by sars-cov- ? do ethnic differences affect the transmissibility and pathogenicity of sars-cov- ? do any environmental factors, such as regional conditions or climate, affect sars-cov- transmission? supplementary materials: the following are available online at http://www.mdpi.com/ - / / / 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coronavirus-induced lung injury angiotensin-converting enzyme protects from severe acute lung failure angiotensin-converting enzyme is a functional receptor for the sars coronavirus receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars the novel chinese coronavirus ( -ncov) infections: challenges for fighting the storm epidemiology, genetic recombination, and pathogenesis of coronaviruses acknowledgments: all the genomic data of sars-cov- is from gisaid, ncbi/genbank, nmdc, cngb/cngbdb and ncovr (table s ). here, we would like to express our gratitude to all units and individuals who are responsible for the sample collection and data submission. the authors declare no conflict of interest. key: cord- -eo le v authors: qin, pan; du, en-zhong; luo, wen-ting; yang, yong-le; zhang, yu-qi; wang, bin; huang, yao-wei title: characteristics of the life cycle of porcine deltacoronavirus (pdcov) in vitro: replication kinetics, cellular ultrastructure and virion morphology, and evidence of inducing autophagy date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: eo le v porcine deltacoronavirus (pdcov) causes severe diarrhea and vomiting in affected piglets. the aim of this study was to establish the basic, in vitro characteristics of the life cycle such as replication kinetics, cellular ultrastructure, virion morphology, and induction of autophagy of pdcov. time-course analysis of viral subgenomic and genomic rna loads and infectious titers indicated that one replication cycle of pdcov takes to h. electron microscopy showed that pdcov infection induced the membrane rearrangements with double-membrane vesicles and large virion-containing vacuoles. the convoluted membranes structures described in alpha- and beta-coronavirus were not observed. pdcov infection also increased the number of autophagosome-like vesicles in the cytoplasm of cells, and the autophagy response was detected by lc i/ii and p western blot analysis. for the first time, this study presents the picture of the pdcov infection cycle, which is crucial to help elucidate the molecular mechanism of deltacoronavirus replication. porcine deltacoronavirus (pdcov), which belongs to the genus deltacoronavirus in the subfamily coronavirinae of the family coronaviridae, order nidovirales, was first reported in hong kong in [ ] and thereafter isolated from pigs in the united states [ ] and many asian countries including china [ ] [ ] [ ] . it causes acute diarrhea, vomiting, dehydration and mortality in nursing pigs [ , ] . the pdcov genome is a positive sense single-stranded rna of approximately . kb in size. the genome organization of pdcov is similar to those of other reported coronaviruses, with the typical gene order -orf a/ b-spike (s)-envelope (e)-membrane (m)-ns -nucleocapsid (n)/ns - . ultrastructural characterization of other covs in alpha-, beta-, or gamma-cov genus such as severe acute respiratory syndrome coronavirus (sars-cov) [ ] [ ] [ ] [ ] [ ] [ ] [ ] , human coronavirus nl (hcov-nl ) [ ] , infectious bronchitis virus (ibv) [ ] , transmissible gastroenteritis virus (tgev) [ ] and porcine epidemic diarrhea virus (pedv) [ ] has been performed. the studies on three cov genera demonstrated that the architectures of the organelle during cov infection are distinct between among alpha-/beta-coronaviruses and gammacoronavirus. alpha-and beta-coronaviruses formed clusters of the double-membrane vesicle (dmv), which is highly conserved among coronaviruses, sometimes linked by a convoluted membrane [ , , [ ] [ ] [ ] , whereas the gammacoronavirus ibv induced extensive paired membranes and smaller - nm spherules in addition to the dmvs [ ] . there is paucity of information on the ultrastructural picture of the newly discovered deltacoronavirus infection. the isolation, epidemiology, and pathogenicity of pdcov has been recently studied by researchers from different countries, but a better understanding of the pdcov life cycle is critically needed to help elucidate the mechanism of virus replication and antiviral activity of the host cells. in this study, some basic, in vitro characteristics of the life cycle, for example replication kinetics, cellular ultrastructure, virion morphology, and possible induction of autophagy of pdcov, were studied and elaborated. the pdcov chinese "hunan" strain was used in this study [ ] . the virus cultivation was done in a porcine kidney epithelial cell line, llc-pk (atcc cl- ), at • c in % co in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) and % (w/v) antibiotics (penicillin and streptomycin). llc-pk cells were seeded at % confluence in -well culture plates and incubated overnight. after the cells were washed with phosphate-buffered saline (pbs), viruses at a multiplicity of infection (moi) of were added to each plate. after h of incubation at • c, the cells were washed four times with pbs and then harvested along with supernatants at an hourly interval until h post-infection (hpi). at least three replicate experiments were performed. viral subgenomic rna (sgrna) load was monitored by one-step quantitative reverse-transcription polymerase chain reaction (qrt-pcr) targeting the membrane (m) gene as described previously (forward primer, -atcgaccacatggctccaa- ; reverse primer, -cagctcttgcccatgtagctt- ; and probe, -fam-cacaccagtcgttaagcatggcaagct-bhq- [ ] , as the m gene is expressed by the sgrna, and viral genomic rna (grna) load was detected by qrt-pcr targeting the non-structural protein (nsp) encoding region (forward primer, -gaaggtgaagatgatagtg- ; reverse primer, -gctctggtttaggataga- ; and probes, -fam-tacttcgtcgctgctggtctt-tamra- , as the nsp of orf a is expressed by the grna. standard curves were performed to allow absolute quantitation of pdcov rna copy numbers based upon the levels of in vitro transcribed rnas containing the targeting sequences [ ] . llc-pk cells were infected with pdcov at an moi of and cell supernatants were harvested hourly, to determine the onset of progeny virus release from the infected cells. the titers of the yield of progeny virus were determined by endpoint dilutions as % tissue culture infective dose (tcid ) on llc-pk cells. llc-pk cells were infected with pdcov at an moi of . the cells were harvested and fixed with . % glutaraldehyde in phosphate buffer ( . m, ph . ) and % osmium tetroxide (oso ) in phosphate at , , , , , , and hpi. specimens were dehydrated in a series of ethanol dilutions ( %, %, %, %, %, % and %) for - min at each step, then transferred to absolute acetone for min. the specimens were then placed in one of three mixtures of absolute acetone and spurr resin ( : , : , and pure spurr resin) for h, h, and overnight, respectively. finally, ultrathin sections were stained by uranyl acetate and alkaline lead citrate for - min and observed using a hitachi model h- transmission em [ ] . pdcov infected llc-pk cells were lysed in cellytic m lysis buffer (sigma, st. louis, mo, usa). the protein concentration was quantified by the bca (bicinchoninic acid) protein assay kit (beyotime biotechnology, shanghai, china). samples were separated by % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page). the proteins were transferred onto a polyvinylidene difluoride (pvdf) membrane that was subsequently blocked with tris-buffered saline (tbs) containing % bovine serum albumin (bsa) at room temperature for h and then incubated overnight at • c with primary antibodies. rabbit anti-lc (a , abclonal, wuhan, china) and rabbit anti-p /sqstm (p , sigma, st. louis, mo, usa) polyclonal antibodies were used in this study. the blots were then incubated with corresponding horseradish peroxidase (hrp) conjugated secondary antibody (thermo fisher scientific, waltham, ma, usa). viral grna was first detected in the supernatants at hpi, which increased significantly at hpi (p < . ) and reached a maximum of . × copies/ml at hpi ( figure a , left panel). intracellular grna load remained low until hpi, followed by a sharp increase at hpi from . × to . × copies/ cells, reaching a final titer of . × copies/ cells at hpi ( figure b , left panel). similarly, extracellular or intracellular viral sgrna amount gradually increased from hpi, with significant changes at or hpi ( figure a ,b, right panels). infectious progeny virus could be detected in the culture supernatants at hpi at a low titer ( . × tcid /ml), and then the amounts of progeny virus increased. by h post infection, infected llc-pk cells produced an infectious titer of . × tcid /ml ( figure c ). these results indicated that the first generation of progeny was assembled and released from infected cells at about hpi, suggesting that one replication cycle of pdcov takes approximately to h to complete. data represent mean values ± standard deviation (sd) of at least replicates. differences in the rna copies or virus titer were evaluated by t-test, asterisks indicate significant differences between the two groups, *p < . ; **p < . . . data represent mean values ± standard deviation (sd) of at least replicates. differences in the rna copies or virus titer were evaluated by t-test, asterisks indicate significant differences between the two groups, * p < . ; ** p < . . the diameter of known cov particles ranges from - nm. the size of the purified pdcov virion by ultracentrifugation (the approach described in [ ] ) was first assessed. electron microscopy (em) of a negatively stained sample demonstrated that the virus particle was to nm in diameter, and had surface projections typical of cov (supplemental figure s ). pdcov infected cells exhibited significant morphologic changes (figure a ,b) compared to uninfected controls ( figure c ,d) at hpi, such as the presence of many cytoplasmic vesicles including dilated rough endoplasmic reticulum, dmvs and injured mitochondria. the cytoplasmic vesicles surrounding the nucleus increased along with infection ( figure a ,b). the size of pdcov virions observed with infected cells, approximately - nm in diameter ( figure e ,f), was in line with the purified viral particle (supplemental figure s ). cov infection is initiated by the interaction of the viral s protein with specific host cellular receptors followed by membrane fusion, and the cellular receptor for pdcov entry has just been identified as porcine aminopeptidase n [ ] . at hpi, pdcov virions were seen attached to the cell surface ( figure e ). the process of likely endocytosis of pdcov was also observed ( figure f ), which needs further confirmation. following internalization, cov particles are transported and scattered into the cytoplasm. it is well known that viral grna is recognized directly by the host cell machinery and translated into non-structural proteins, which assemble into viral replication-transcription complexes (rtcs) on dmvs or other membrane structures [ , , ] , coordinating the enzymes required for viral rna transcription, proof-reading and capping of new viral transcripts. similar events should apply to pdcov infection as a large number of dmvs were seen at hpi and thereafter. after completion of genome replication and rna transcription, the virions of pdcov start to assemble in the expanded rough endoplasmic reticulum (rer) with irregular shape, as the surface was decorated with ribosomes ( figure g ,h). immature pdcov precursors with an electron-dense periphery and clear core were present in the rer ( figure h ) or in the lumen of a stack of adjacent cisternae, very likely the golgi apparatus ( figure i ). the precursors likely trafficked through the endoplasmic reticulum-golgi intermediate compartment (ergic), completing structural transformation into small, infectious virions in the golgi complex. the virions were also observed in large circular organelles ( figure j ). these structures, named as large virion-containing vacuoles (lvcvs), have earlier been described for other covs-infected cells, which are thought to originate from golgi compartments that expand to accommodate numerous virions [ ] . mature virions accumulate inside secretory vesicles, which tend to merge into bigger vesicles ( figure j ). after that, vesicles containing mature virions moved to the cell periphery and eventually fuse with the cell membrane. in the final phase of infection, mature virions must escape the cell. pdcov release was observed to occur via two pathways: exocytosis and cytolysis. early virions, those generated within the first few replicative cycles, may be released by exocytotic fusion of virus-containing vesicles with plasma membranes without damage to the cell ( figure k ). however, as viral infection progressed and the quantity of virus in the cells increased, large amounts of virus particles were released by cytolysis resulting in necrosis of the host cell ( figure l ). cov replication is closely tied to the formation of membrane-bound rtcs that alter cell membrane structures, as previously reviewed [ , ] . many such alterations were observed directly in an electron microscope (em) of pdcov-infected llc-pk cells, including dmvs ( figure. g, h, a and b), lvcvs ( figure. i and j) , zippered er ( figure c ) and damaged mitochondria ( figure. d ). the dmv architecture is a typical and highly conserved feature of cov infections, with a diameter ranging from - nm with electron-dense cores [ , , , [ ] [ ] [ ] [ ] , ] . dmvs play a role cov replication is closely tied to the formation of membrane-bound rtcs that alter cell membrane structures, as previously reviewed [ , ] . many such alterations were observed directly in an electron microscope (em) of pdcov-infected llc-pk cells, including dmvs ( figure g ,h and figure a ,b), lvcvs ( figure i ,j), zippered er ( figure c ) and damaged mitochondria ( figure d ). the dmv architecture is a typical and highly conserved feature of cov infections, with a diameter ranging from - nm with electron-dense cores [ , , , [ ] [ ] [ ] [ ] , ] . dmvs play a role not only in the synthesis of viral rna [ ] but also in helping to concentrate on viral proteins and offer protection from cellular antiviral detection and elimination machinery [ ] . dmvs could be detected at hpi in sars-cov or mouse hepatitis virus (mhv) infected cells and then the number of dmvs increased dramatically [ , ] . the appearance of dmvs in pdcov-infected cells was seen at hpi ( figure g ). the sizes of dmvs ranged from to nm. pdcov induced a low number of dmvs and most of the dmvs were separate entities or in small clusters in the cytoplasm. initially, the dmv inner and outer membranes were generally tightly apposed ( figure g ,h) but, occasionally, some luminal space between the two lipid bilayers could be discerned ( figure a ). similar observations were previously made for sars-cov and ibv [ ] . most of the dmvs contained few or no fibrous material in the inner vesicles ( figure g ,h and figure a ). interestingly, dmvs containing an electron-dense core could also be observed ( figure b ). previous researches confirmed that the detection of this core depended on the protocol used for the preparing of resin-embedded specimens. in chemically fixed samples, the interior of dmv appeared mainly electron translucent. however, the core can be preserved by using high-pressure freezing followed by freeze substitution. a similar phenomenon can be observed in the ibv infected cells and chemically fixed tracheal organ cultures. two types of dmvs could well represent early and later stages in the existence of a dmv [ ] . previous research suggested that cov dmv structures derive from er membranes [ ] [ ] [ ] , though the precise mechanism of membrane rearrangement remains controversial. in the context of nidovirus-induced dmv formation, two alternatives have been proposed: enwrapping and double budding [ ] . the multiple em images of zippered er and small vesicles close to the zippered er in pdcov infected cells ( figure c ) suggest that these structures were likely dmv precursors. interestingly, convoluted membranes structures or spherules, which can be observed in proximity to the dmv cluster in alpha-or gamma coronavirus-infected cells, were not detected in the pdcov infected cells, despite analysis at several time points post-infection (figures and ) . the large evolutionary distance between the coronavirus genera might be attributed to such differences. previous studies proposed that these structures were the site of viral genome translation and polyprotein processing. further work will be needed to determine the details on membrane structures that participate in pdcov rna synthesis. pdcov infection was shown to induce mitochondrion damage. irregular mitochondria and mitochondria lacking cristae were observed after pdcov infection, indicative of injured mitochondria ( figure d ). we hypothesize that these structures may donate their membrane material to form autophagosomes [ ] , as described below. autophagy is a dynamic and continuous cellular defence pathway, resisting viral infection by degrading both virus and necrotic organelles. regions of the cytoplasm become engulfed into large double membrane-bound vesicles termed autophagosomes, which are another type of distinct structures seen during cov infection [ , ] . autophagosomes are distinguished from dmvs by the size and specific markers of autophagic activity. double-membraned autophagosomes-like vesicles were detected in pdcov-infected cells at hpi and increased thereafter. the initial autophagic vacuoles contain cytosol and/or organelles that in appearance are morphologically intact, and similar to the cytosol and organelles elsewhere in the cell. the membrane of the vacuoles is partially visible as two bilayers separated by a narrow electron-lucent cleft [ ] (figure e ,f). it should be noted that these are characteristic features of coronavirus infection, and expression of sars-cov nsp alone induced similar structures. upon induction of autophagy, a series of conjugation reactions converts cytosolic microtubule-associated protein light chain (lc -i) to a lipidated form (lc -ii); the ratio of lc -ii/lc -i is regarded as an accurate indicator of autophagic activity [ ] . in order to further confirm that pdcov induced autophagy, lc conversion in pdcov-infected llc-pk cells was determined by western blot using an antibody recognizing both forms of lc . the lc -ii/lc -i ratio increased significantly from - hpi in pdcov-infected cells relative to mock-infected cells ( figure a,b) . this was consistent with the increase in autophagosomes observed in infected cells from - hpi under em ( figure e ,f). we also monitored autophagic flux, using western blot assay to measure changes in p protein levels upon infection. because p -bound polyubiquitinated proteins become incorporated into the completed autophagosome and are degraded in autolysosomes, they serve as an index of autophagic degradation [ ] . pdcov infection increased degradation of p in llc-pk cells starting at hpi ( figure c,d) , indicating that accumulation of autophagosome-like vesicles is linked to autophagic activation. these results provided evidence that pdcov infection may induce autophagy just like other covs [ , [ ] [ ] [ ] . a general response to viral infections is the activation of autophagy, which either has a positive or negative outcome depending on the nature of the virus. initial work with mhv showed that although capable of inducing autophagy, it was replication-deficiet in atg −/− embryonic stem cell lines (lacking atg , the e ubiquitin ligase essential for autophagosome elongation). this replication deficiency reversed in the presence of an atg -expressing plasmid [ ] . however, other studies showed that mhv and sars-cov replication does not require an intact autophagy pathway [ , ] . similar results were also observed in ibv infection, where induction or inhibition of autophagy did not affect replication, suggesting that classical autophagy may not be important for its life cycle [ ] . however, a recent study shows that tgev infection activates autophagy, which subsequently inhibits further tgev infection [ ] . as it is possible that replication mechanisms differ among viruses of the same family, further in-depth study is needed to confirm pdcov infection-associated autophagy and identify the impact of autophagy on viral replication. the protein blots were quantified by the image j software. the ratio of lc -ii to β-actin and the ratio of p to β-actin from three independent experiments were expressed as mean ± standard deviation (sd); **p < . , calculated using student's t-test. in summary, we demonstrate and elaborate some novel, in vitro characteristics of the life cycle of pdcov, including replication kinetics in cultured cells, cellular ultrastructure, virion morphology, and possible induction of autophagy, mainly by means of em observation. pdcov infection induced membrane rearrangements. however, convoluted membrane structures, caused by α-cov, were not observed in the pdcov-infected cells. this study gives the first detailed picture of the pdcov infection cycle, which will help elucidate the molecular mechanism of deltacoronavirus replication. supplementary materials: the following are available online at xxx. figure s . electron microscope image of a purified pdcov particle using phosphotungstic acid negative staining. . llc-pk cells were mock-treated or infected with pdcov (multiplicity of infection (moi) = ). at , , , and hpi, cells were lysed and subjected to western blot with antibodies against lc , p and β-actin (as the loading control). the protein blots were quantified by the image j software. the ratio of lc -ii to β-actin and the ratio of p to β-actin from three independent experiments were expressed as mean ± standard deviation (sd); ** p < . , calculated using student's t-test. in summary, we demonstrate and elaborate some novel, in vitro characteristics of the life cycle of pdcov, including replication kinetics in cultured cells, cellular ultrastructure, virion morphology, and possible induction of autophagy, mainly by means of em observation. pdcov infection induced membrane rearrangements. however, convoluted membrane structures, caused by α-cov, were not observed in the pdcov-infected cells. this study gives the first detailed picture of the pdcov infection cycle, which will help elucidate the molecular mechanism of deltacoronavirus replication. the following are available online at http://www.mdpi.com/ - / / / /s . figure s . electron microscope image of a purified pdcov particle using phosphotungstic acid negative staining. discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus detection and 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aminopeptidase n for infectious cellular entry genetic and pathogenic characterization of a novel reassortant mammalian orthoreovirus (mrv ) from a diarrheic piglet and seroepidemiological survey of mrv in diarrheic pigs from east china ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus does form meet function in the coronavirus replicative organelle coronavirus replication complex formation utilizes components of cellular autophagy biogenesis and architecture of arterivirus replication organelles mitochondria directly donate their membrane to form autophagosomes during a novel mechanism of parkin-associated mitophagy guidelines for the use and interpretation of assays for monitoring autophagy how to interpret lc immunoblotting johansen, t. p /sqstm forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death involvement of autophagy in coronavirus replication coronavirus nsp proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate coronavirus replication does not require the autophagy gene atg severe acute respiratory syndrome coronavirus replication is severely impaired by mg due to proteasome-independent inhibition of m-calpain visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we are grateful to the analysis center of agrobiology and environmental sciences, faculty of agriculture, life and environmental sciences, zhejiang university. we are grateful to the analysis center of agrobiology and environmental sciences, faculty of agriculture, life and environmental sciences, zhejiang university. the authors declare no conflict of interest.viruses , , key: cord- -iy vjpuh authors: schwartz, david a.; graham, ashley l. title: potential maternal and infant outcomes from coronavirus -ncov (sars-cov- ) infecting pregnant women: lessons from sars, mers, and other human coronavirus infections date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: iy vjpuh in early december a cluster of cases of pneumonia of unknown cause was identified in wuhan, a city of million persons in the people’s republic of china. further investigation revealed these cases to result from infection with a newly identified coronavirus, initially termed -ncov and subsequently sars-cov- . the infection moved rapidly through china, spread to thailand and japan, extended into adjacent countries through infected persons travelling by air, eventually reaching multiple countries and continents. similar to such other coronaviruses as those causing the middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars), the new coronavirus was reported to spread via natural aerosols from human-to-human. in the early stages of this epidemic the case fatality rate is estimated to be approximately %, with the majority of deaths occurring in special populations. unfortunately, there is limited experience with coronavirus infections during pregnancy, and it now appears certain that pregnant women have become infected during the present -ncov epidemic. in order to assess the potential of the wuhan -ncov to cause maternal, fetal and neonatal morbidity and other poor obstetrical outcomes, this communication reviews the published data addressing the epidemiological and clinical effects of sars, mers, and other coronavirus infections on pregnant women and their infants. recommendations are also made for the consideration of pregnant women in the design, clinical trials, and implementation of future -ncov vaccines. coronaviruses are spherical, enveloped, and the largest of positive-strand rna viruses. they have a wide host range, including birds, farm animals, pets, camels, and bats, in which they primarily cause respiratory and gastrointestinal disease. belonging to the order nidovirales, family coronaviridae, and the subfamily orthocoronaviridae there are four genera of coronaviruses-alphacoronavirus, betacoronavirus, deltacorona virus, and gammacoronavirus [ ] [ ] [ ] [ ] . in humans, they are a cause of mild illnesses including the common colds occurring in children and adults, and were believed to be of modest medical importance. however, two zoonotic coronaviruses-including the severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov)-can produce severe lower respiratory in the beginning of december , a cluster of persons with a pneumonia of unknown cause was identified in wuhan, the capital of hubei province and a large city of approximately million persons located in the central region of the people's republic of china [ , ] . between and december there were cases of pneumonia identified whose clinical features resembled that of a viral pneumonia. the outbreak was initially believed to be linked to the wuhan huanan (south china) seafood wholesale market. this market, termed a "wet" market, sells a variety of seafood, cuts of meat, and both live and dead animals in over one thousand stalls in constant close contact; however, whether this market was the origin of the outbreak remains unknown [ ] . on december , the chinese center for disease control and prevention (china cdc) sent a rapid response team to hubei to work alongside health personnel from the provincial and wuhan city health departments to conduct an epidemiologic investigation. as the disease was spreading through secondary and tertiary cases, the world health organization (who) china country office was informed on december of the occurrence of these cases of pneumonia of unknown etiology. during the period from december to january , patients with pneumonia of unknown etiology were reported by the chinese authorities to the who. on january investigators in china identified the etiological agent of the epidemic as a previously unknown coronavirus, and it was given the designation -ncov (for novel coronavirus) [ ] . analysis of the clinical features of hospitalized patients with laboratory-confirmed -ncov infection revealed that were men ( %); less than one-half had underlying co-morbid conditions ( ; %) which included diabetes ( , %) , hypertension ( , %), and cardiovascular disease ( ; %); and the average age was . years old. the most common symptoms at the beginning of their illness included fever ( , %) , cough ( , %) , and fatigue or myalgia ( , %) , sputum production ( , %) , and headache ( , %) [ ] . among these initial cases of -ncov infection there were patients ( %) who developed acute respiratory distress syndrome (ards), ( %) required intensive care and ( %) died. during the first weeks of january the infection spread rapidly through china and extended to adjacent countries where cases began to appear- january in thailand, january in japan, january in the republic of korea, and taiwan and the united states on january [ ] . infected travelers, mostly via commercial air travel, are known to have been responsible for introducing the virus outside of wuhan. the new coronavirus continued to spread throughout multiple countries and continents, and by february the who reported , confirmed cases in china that resulted in deaths, surpassing the number of deaths that occurred during the - sars epidemic. an additional cases of -ncov infection have occurred among other countries outside of china [ ] . (figure ) at the meeting of the emergency committee of the who on january, the novel coronavirus epidemic was declared a public health emergency of international concern (pheic) [ , ] . viruses , , of epidemic. an additional cases of -ncov infection have occurred among other countries outside of china [ ] . (figure ) at the meeting of the emergency committee of the who on january, the novel coronavirus epidemic was declared a public health emergency of international concern (pheic) [ , ] . this newly recognized coronavirus, producing a disease that has been termed covid- , is rapidly spreading throughout china, has crossed international borders to infect persons in neighboring countries, and humans infected by the virus are travelling via commercial airlines to other continents. it is certain that -ncov will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with sars-cov and mers-cov. in order to address the potential obstetrical outcomes of infection to both mother and infant, the present communication describes the current state of knowledge regarding the effects of other coronavirus infections in pregnancy. pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women. it is the most prevalent non-obstetric infectious condition that occurs during pregnancy [ ] [ ] [ ] . in one study pneumonia was the rd most common cause of indirect maternal death [ ] . approximately percent of pregnant women who develop pneumonia will need to be hospitalized in critical care units and require ventilatory support [ ] . although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [ ] . as with other infectious diseases, the normal maternal physiologic changes that accompany pregnancy-including altered cell-mediated immunity [ ] and changes in pulmonary function-have been hypothesized to affect both susceptibility to and clinical severity of pneumonia [ ] [ ] [ ] . this has been evident historically during previous epidemics. the case fatality rate (cfr) for pregnant women infected with influenza during the - pandemic was %-even higher when exposure occurred during the rd trimester and upwards of % if pneumonia supervened [ ] . during the - asian flu epidemic, % of all deaths occurred in pregnant women, and their cfr was twice as high as that of infected women who were not pregnant [ ] . the most common adverse obstetrical outcomes associated with maternal pneumonias from all causes include this newly recognized coronavirus, producing a disease that has been termed covid- , is rapidly spreading throughout china, has crossed international borders to infect persons in neighboring countries, and humans infected by the virus are travelling via commercial airlines to other continents. it is certain that -ncov will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with sars-cov and mers-cov. in order to address the potential obstetrical outcomes of infection to both mother and infant, the present communication describes the current state of knowledge regarding the effects of other coronavirus infections in pregnancy. pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women. it is the most prevalent non-obstetric infectious condition that occurs during pregnancy [ ] [ ] [ ] . in one study pneumonia was the rd most common cause of indirect maternal death [ ] . approximately percent of pregnant women who develop pneumonia will need to be hospitalized in critical care units and require ventilatory support [ ] . although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [ ] . as with other infectious diseases, the normal maternal physiologic changes that accompany pregnancy-including altered cell-mediated immunity [ ] and changes in pulmonary function-have been hypothesized to affect both susceptibility to and clinical severity of pneumonia [ ] [ ] [ ] . this has been evident historically during previous epidemics. the case fatality rate (cfr) for pregnant women infected with influenza during the - pandemic was %-even higher when exposure occurred during the rd trimester and upwards of % if pneumonia supervened [ ] . during the - asian flu epidemic, % of all deaths occurred in pregnant women, and their cfr was twice as high as that of infected women who were not pregnant [ ] . the most common adverse obstetrical outcomes associated with maternal pneumonias from all causes include premature rupture of membranes (prom) and preterm labor (ptl), intrauterine fetal demise (iufd), intrauterine growth restriction (iugr), and neonatal death [ ] [ ] [ ] . the sars epidemic began quietly at the turn of the st century. in november , a cook in guangdong province, china, died from an unidentified illness. he had worked at a restaurant in which meat from wild animals was served. on november chinese-language media and internet reports were picked up by canada's global public health intelligence network (gphin) that indicated a flu-like illness was occurring in china [ , ] . unfortunately, the reports were not translated, and china failed to report the occurrence of this illness to the world health organization (who) until february . the disease spread to other countries where it primarily infected healthcare workers. one of these was dr. carlo urbani, a who physician investigating a patient with the new disease in hanoi. he recognized that the pneumonia was probably caused by a new, highly infectious agent, and rapidly notified the who. he contracted the sars-cov while there, became febrile and later died after traveling to thailand to attend a conference. on march , who issued a global alert regarding the disease that was occurring primarily among health care workers in hanoi, vietnam and hong kong. the disease continued to spread, and by july there were probable cases, leading to deaths in countries, with the majority of cases occurring in mainland china and hong kong. approximately % of infections occurred in healthcare workers. by the termination of the epidemic the global cfr was % [ ] . although there were relatively few documented cases of sars occurring during pregnancy, several case reports and small clinical studies have described the clinical effects in pregnant women and their infants. in reviewing these reports describing pregnant women with sars in china it is possible, and perhaps even probable, that some of the same patients were included in more than one publication. however, even if this is the case, there is no doubt that sars coronavirus infection was found to be associated with severe maternal illness, maternal death, and spontaneous abortion [ , [ ] [ ] [ ] [ ] . martha anker, an expert in statistics formerly with the who and the university of massachusetts, estimated that more than cases of sars-cov infection occurred in pregnant women, which warrants closer inspection [ ] . the clinical outcomes among pregnant women with sars in hong kong were worse than those occurring in infected women who were not pregnant [ ] . wong et al. [ ] evaluated the obstetrical outcomes from a cohort of pregnant women who developed sars in hong kong during the period of february to july . four of the women ( %) that presented during the st trimester sustained spontaneous miscarriages, likely a result of the hypoxia that was caused by sars-related acute respiratory distress. among the women who presented after weeks gestation, had preterm deliveries ( %). a case-control study to determine the effects of sars on pregnancy compared pregnant and non-pregnant women with the infection at the princess margaret hospital in hong kong [ , ] . there were deaths among the pregnant women with sars (maternal mortality rate of %) and no deaths in the non-pregnant group of infected women (p = . ). renal failure (p = . ) and disseminated intravascular coagulopathy (p = . ) developed more frequently in pregnant sars patients when compared with the non-pregnant sars group. six pregnant women with sars required admission to the intensive care unit (icu) ( %) and required endotracheal intubation ( %), compared with a . % intubation rate (p = . ) and . % icu admission rate (p = . ) in the non-pregnant group. maxwell et al. [ ] reported pregnant women infected with sars-cov who were followed at a designated sars unit- of the died (cfr of %), and ( %) required icu hospitalization and mechanical ventilation. in contrast, the mortality rate was less than % and mechanical ventilation rate less than % among non-pregnant, age-matched counterparts who were not infected with sars-cov. two women with sars recovered and maintained their pregnancy but had infants with iugr. among the live newborn infants, none had clinical or laboratory evidence for sars-cov infection. the new mothers who had developed sars were advised not to breastfeed to prevent possible vertical transmission of the virus. zhang et al. [ ] described sars-cov infections in primagravidas from guangzhou, china at the height of the sars epidemic. two of the mothers became infected in the nd trimester, and developed infection in the rd trimester. two of the pregnant women had hospital-acquired sars infections, and the other were community-acquired. all pregnant women had fever and abnormal chest radiographs; had cough; developed hypoalbuminemia; had elevated alanine aminotransferase levels (alt), had chills or rigor, had decreased lymphocytes, and had decreased platelets. one pregnant woman required intensive care, but all recovered and there were no maternal deaths. the infants were clinically evaluated, and none had evidence of sars. two pregnant women with sars were reported from the united states. in a detailed case report, robertson et al. [ ] described a -year-old pregnant woman with an intermittent cough of approximately days duration and no fever. while travelling in hong kong during the epidemic, she was exposed at her hotel to a person subsequently known to be infected with sars-cov. at weeks gestation she developed fever, anorexia, headache, increasing cough, weakness, and shortness of breath. upon returning to the united states she was hospitalized with pneumonia. obstetrical ultrasounds revealed a low-lying placenta (placenta previa) but were otherwise normal. following her discharge home and clinical recovery, she was found to have antibodies to sars-cov. she underwent cesarean section at weeks gestation because of the placenta previa and a healthy baby girl was delivered [ , ] . the placenta was interpreted as being normal. at days post-maternal illness, maternal serum and whole blood, swabs from maternal nasopharynx and rectum, post-delivery placenta, umbilical cord blood, amniotic fluid, and breast milk were collected for analysis-no viral rna was detected in specimens tested by reverse transcriptase polymerase chain reaction (rt-pcr). antibodies to sars-cov were detected from maternal serum, umbilical cord blood, and breast milk by enzyme immunoassay (eia) and indirect immunofluorescence assay. no clinical specimens (except for cord blood) were available for testing from the infant. the second case in the usa occurred in a -year-old woman who had travelled to hong kong at weeks gestation where she was exposed to sars-cov in the same hotel as the aforementioned american woman [ ] . following her return to the united states, her husband developed the clinical onset of sars, and days later she became ill with fever, myalgia, chills, headache, coryza, and a productive cough with shortness of breath and wheezing. following her hospitalization for sars she recovered, serum samples taken on days and post-onset of illness were positive for antibodies to sars-cov by enzyme immunoassay and immunofluorescent assays. her pregnancy continued and was unremarkable except for developing elevated glucose levels. a cesarean section that was performed at weeks gestation due to preterm rupture of membranes and fetal distress resulted in a healthy baby boy. at the time of delivery, the mother's serum samples were positive for antibodies to sars-cov, but samples taken of umbilical cord blood and placenta were negative. breast milk sampled and days after delivery were also negative for sars-cov antibodies. specimens evaluated from maternal blood, stool, and nasopharynx samples, as well as umbilical cord blood of the infant, were all negative for coronavirus rna by rt-pcr. neonatal stool samples obtained on days-of-life and were also negative for viral rna. from canada, yudin et al. [ ] reported a -year-old pregnant woman who was admitted to the hospital at weeks gestation with a fever, dry cough, and abnormal chest radiograph demonstrating patchy infiltrates. she had acquired sars from contact with an infected family member. following a -day stay in the hospital, during which she did not require ventilatory support, her convalescent antibody titers were positive for coronavirus infection. she had a normal labor and delivery and her newborn girl had no evidence of infection. in a study of liveborn neonates who were delivered to women infected with sars-cov during the hong kong epidemic, results from multiple tests-including serial rt-pcr assays, viral culture, and paired neonatal serological titers-were negative for sars-cov [ ] . none of the neonates developed any clinical signs or symptoms of respiratory infection or compromise. fortunately, there were no cases of vertical transmission identified among pregnant women infected with sars-cov during the - asian epidemic [ , , , , ] , and with the exception of a small cluster of cases that recurred in late , no new cases of sars have occurred. in the only reported study of the placental pathology of mothers with sars, ng et al. [ ] reported the findings from pregnant women infected with sars-cov. in the case of women who were convalescing from sars-cov infection during the st trimester of pregnancy, the placentas were found to be normal. three placentas were delivered from pregnancies in which the mothers had acute sars-cov infection-these were abnormal and demonstrated increased subchorionic and intervillous fibrin, a finding that can be associated with abnormal maternal blood flow to the placenta. in the placentas of women who were convalescing from sars-cov infection in the rd trimester of pregnancy the placentas were highly abnormal. they showed extensive fetal thrombotic vasculopathy with areas of avascular chorionic villi-chronic findings of fetal vascular malperfusion. these pregnancies also were complicated by oligohydramnios and had poor obstetrical outcomes-both infants had developed iugr. it is interesting that villitis, the microscopic finding of inflammation of the chorionic villi that is the histologic hallmark of many maternal hematogenous infections that are transmitted through the placenta to the fetus, was not identified in any of these placentas. similar to other coronavirus infections, sars-cov is easily spread from person-to-person via respiratory droplets and secretions as well as through nosocomial contacts [ , ] . in addition to transmission of sars-cov through natural aerosols from infected patients, it was found that in hong kong the sars-cov could also be transmitted by mechanical aerosols [ ] . environmental factors had an important role when it was discovered that during the amoy gardens housing estate outbreak as many as two-thirds of infected persons had diarrhea, sars-cov was excreted in their stools, and that aerosols arising from the flushing of toilets could transmit the virus [ ] . healthcare facilities were also an important source of new sars infections during the - epidemic, and healthcare workers were also at high risk for acquiring the infection. in order to address the safety issues for the obstetrical management and delivery of pregnant women with sars, guidelines were prepared by the canadian task force on preventive health care and the society of obstetricians and gynaecologists of canada [ ] . these recommendations include: . "all hospitals should have infection control systems in place to ensure that alerts regarding changes in exposure risk factors for sars or other potentially serious communicable diseases are conveyed promptly to clinical units, including the labour and delivery unit. at times of sars outbreaks, all pregnant patients being assessed or admitted to the hospital should be screened for symptoms of and risk factors for sars. upon arrival in the labour triage unit, pregnant patients with suspected and probable sars should be placed in a negative pressure isolation room with at least air exchanges per hour. all labour and delivery units caring for suspected and probable sars should have available at least one room in which patients can safely labour and deliver while in need of airborne isolation. if possible, labour and delivery (including operative delivery or caesarean section) should be managed in a designated negative pressure isolation room, by designated personnel with specialized infection control preparation and protective gear. . either regional or general anaesthesia may be appropriate for delivery of patients with sars. neonates of mothers with sars should be isolated in a designated unit until the infant has been well for days, or until the mother's period of isolation is complete. the mother should not breastfeed during this period. . a multidisciplinary team, consisting of obstetricians, nurses, pediatricians, infection control specialists, respiratory therapists, and anaesthesiologists, should be identified in each unit and be responsible for the unit organization and implementation of sars management protocols. . staff caring for pregnant sars patients should not care for other pregnant patients. staff caring for pregnant sars patients should be actively monitored for fever and other symptoms of sars. such individuals should not work in the presence of any sars symptoms within days of exposure to a sars patient. . all health care personnel, trainees, and support staff should be trained in infection control management and containment to prevent spread of the sars virus. . regional health authorities in conjunction with hospital staff should consider designating specific facilities or health care units, including primary, secondary, or tertiary health care centers, to care for patients with sars or similar illnesses." middle east respiratory syndrome (mers) was first reported in september in saudi arabia, following isolation of mers-cov from a male patient who died months earlier from severe pneumonia and multiple organ failure [ ] . in the years since then, there have been more than confirmed cases of mers resulting in upwards of deaths globally [ ] . while countries have reported cases of mers, approximately % of confirmed cases originated in saudi arabia [ ] . to date, all known cases of mers can be linked to travel or residence in countries along the arabian peninsula-that is, bahrain; iraq; iran; israel, the west bank, and gaza; jordan; kuwait; lebanon; oman; qatar, saudi arabia; syria; the united arab emirates (uae); and yemen [ ] . the largest documented outbreak outside of this region occurred in in the republic of korea, in which infections occurred, resulting in deaths [ ] . the index case in this outbreak reportedly returned from the arabian peninsula just prior to onset of illness [ ] . mers-cov is characterized by sporadic zoonotic transmission events as well as spread between infected patients and close contacts (i.e., intra-familial transmission) [ ] . nosocomial outbreaks in health care settings-the result of poor infection control and prevention-are widely recognized as the hallmark of mers [ ] . superspreading events have been recorded in healthcare settings in jordan, al hasa, jeddah, abu dhabi and south korea [ , [ ] [ ] [ ] [ ] . like other coronaviruses, mers-cov can be spread through person-to-person contact, likely via infected respiratory secretions [ ] . transmission dynamics, however, are otherwise poorly understood [ ] . bats are believed to be the natural reservoir of mers-cov, and dromedary camels can have the virus and have been suggested as possible intermediary hosts as well as a source of infection to humans [ , , ] . there are no clinical or serological reports of perinatal transmission of mers, though vertical transmission has been reported for non-coronavirus respiratory viruses including influenza and respiratory syncytial virus (rsv) [ ] . researchers have not yet discovered ongoing transmission of mers-cov within communities outside of health care settings. the clinical presentation of mers varies from asymptomatic to severe pneumonia with acute respiratory distress syndrome (ards), septic shock, and multiple organ failure, often resulting in death. most patients with mers develop severe acute respiratory illness accompanied by fever, cough, and shortness of breath [ ] . progression to pneumonia is swift-usually within the first week -and at least one-third of patients also present with gastrointestinal symptoms [ ] . mers progresses much more rapidly to respiratory failure and has a higher case fatality rate than sars [ ] . unlike sars, however, infection with mers-cov is generally mild in healthy individuals but more severe in immunocompromised patients and people with underlying comorbidities [ ] . the overall cfr of mers is approximately . % [ ] . most fatalities have been associated with pre-existing medical conditions like chronic lung disease, diabetes, and renal failure, as well as weakened immune systems [ ] , making such individuals high risk. as a result of the immunological changes that occur during pregnancy, women who are pregnant are included in this high-risk group. pregnant women may develop severe disease and fatal maternal and/or fetal outcomes as a result of mers-cov infection; however, little is known of the pathophysiology of this infection during pregnancy. limited data exists on the prevalence and clinical features of mers during pregnancy, birth, and the postnatal period. it is likely, however, that the immunological changes that normally occur in pregnancy may alter susceptibility to the mers-cov and the severity of clinical illness [ ] . pregnant women infected with sars-cov, a related coronavirus, appear to have increased morbidity and mortality when compared to non-pregnant women, suggesting that mers-cov could also lead to severe clinical outcomes in pregnancy. to date, however, very few pregnancy-associated cases (n = ) have been documented, with % having adverse clinical outcomes. between november and february , there were cases of mers reported by the saudi arabia ministry of health (moh). of these, patients were pregnant, according to a retrospective study by assiri et al. [ ] , and all resulted in adverse outcomes. patient ages ranged from to years, with occurrence of exposure in either the nd or rd trimester. all cases received intensive care. two women died and there were cases of perinatal death- stillbirth and neonatal death shortly after emergency cesarean section. these instances of severe maternal and perinatal outcomes are consistent with other reports of mers-cov infection in pregnant women, as well as outcomes associated with sars-cov infection. the authors of the retrospectives study concede that unreported cases of mers in pregnancy are likely due to lack of routine pregnancy testing [ ] . they conclude that pregnancy testing for women of reproductive age should be considered for those who test positive for mers-cov, to contribute to overall understanding of pathogenesis and epidemiological risk. additionally, of the patients were healthcare workers, which corresponds with existing knowledge of higher risk of exposure to mers-cov in healthcare settings. in a separate case report of mers occurring in pregnancy, alserehi et al. [ ] described a -year-old critical care nurse who became infected during the rd trimester in the midst of a large hospital outbreak. in the days following hospital admission, she developed respiratory failure necessitating mechanical ventilation and administration of dexamethasone as prophylaxis for the fetus. following an emergency cesarean section at weeks gestation, she was transferred to the intensive care unit (icu) and later recovered. the preterm but otherwise healthy infant was kept in the neonatal unit for observation and later released along with his mother. in contrast to other reported cases, this patient had a successful outcome, perhaps due to the timing of mers-cov exposure, her young age, the use of steroids, and differences in immune response. alfaraj et al. [ ] described cases of maternal infection with mers-cov at the prince mohammed bin abdulaziz hospital (pmah) in saudi arabia. maternal infection in both cases was confirmed by nasopharyngeal swab testing by rt-pcr. one patient was a -year-old woman at weeks gestation with no underlying medical conditions. the second patient, a -year-old at weeks gestation, had several comorbidities, including end stage renal disease, hypertension, and hemodialysis. this woman presented to the hospital after contact with a mers-cov-infected person during an active outbreak. both patients later tested negative for mers-cov and were subsequently discharged. the younger patient delivered a healthy, full-term infant. the status of the other delivery is unknown. neither fetus was tested for mers-cov. according to payne et al. [ ] , epidemiologic investigation of the mers outbreak in zarqa, jordan, revealed that a nd trimester stillbirth ( months gestational age) had occurred as a result of maternal exposure to mers-cov. the mother experienced fever, fatigue, headache and cough, concurrently with vaginal bleeding and abdominal pain. on the th day of symptoms, she had a fetal death. the mother was confirmed to have antibody to mers-cov, and she self-reported having had unprotected contact with family members who later tested positive for the virus. this was the first documented occurrence of stillbirth during maternal infection with mers-cov. on november , a -year-old pregnant woman in the united arab emirates (uae) developed ards following admission to the icu after suspected community-acquired pneumonia advanced to respiratory failure and hypotension [ ] . later that day, her baby was delivered by caesarean section and subsequent apgar scores were within healthy range. the next day, rt-pcr evaluation revealed that the mother was positive for mers-cov. despite rigorous intervention, including oral ribavirin-peginterferon-α therapy and ventilator support, the woman continued to deteriorate, developed septic shock, and died. while the outcome for this mother was fatal, malik et al. noted that virus shedding ceased during therapy with ribavirin and peginterferon-α and radiographic evidence indicated clinical improvement before her death [ ] . more research is needed to determine safety, efficacy, and dosage of these therapies in the general population but also in pregnant women. while few data exist on the effects of these treatments in pregnant humans, ribavirin is generally contraindicated during pregnancy [ ] . outside of the middle east the only confirmed case of mers in pregnancy occurred in in south korea. jeong et al. [ ] reported that a -year-old patient was exposed during the rd trimester following contact with a patient having mers. despite abrupt vaginal bleeding and rupture of membranes, the patient recovered fully and delivered a healthy infant at weeks and days gestation. subsequent testing of the infant's blood did not detect any igg, igm, or iga antibodies to mers-cov. the mean maternal age of the confirmed maternal sars cases described above was . years, with a mean gestational age of . weeks. the source of infection in of the cases was attributed to contact with family members who tested positive for mers-cov, unknown in cases, likely due to animal exposure in case, and were healthcare-associated ( of these patients were healthcare workers). six patients required intensive care and died. of those who died, were exposed to mers-cov in the rd trimester, and was exposed during the nd trimester. the infant death rate for all cases was %. fetal survival did not appear to correlate with the timing of maternal infection and gestational age; however, more data are needed to draw conclusions about this relationship. according to alfaraj et al. [ ] , the cfr for the infected women-also %-was not statistically different from the overall cfr of mers in the general population ( %) (p = . ). only case resulted in both maternal and fetal death. similar to sars in pregnancy, more research is needed to understand the pathogenesis and epidemiology of mers in pregnancy including the relationship between the timing of maternal infection, gestational age of the fetus, the effects of comorbid factors, and the occurrence of adverse outcomes. few studies documented the presence of mers-cov antibodies in the umbilical cord or neonatal blood, making it difficult to assess perinatal transmission. as such, future studies should involve the collection of samples from relevant specimens including amniotic fluid, placenta, and umbilical cord [ ] . mers prevention should be high priority for high-risk exposures such as healthcare workers, pregnant women and individuals working with camels, camel meat-milk processors and in abattoirs [ ] . since , the saudi arabia moh has recommended that pregnant women postpone travel to saudi arabia for the hajj and umrah [ ] . to further reduce risk of exposure among pregnant women, additional measures such as avoiding contact with camels and sick persons-particularly in healthcare settings-are also recommended. pregnant women who present with symptoms of pneumonia, influenza-like illness (ili), or sepsis on the arabian peninsula may also benefit from mers-cov screening to expedite early diagnosis and improve disease management [ ] . while multiple agents have been used to treat mers, none have been tested in large clinical studies. available data are limited to the use of combination therapies of interferon and other agents in case reports and case series [ ] . a prospective or randomized study may prove difficult given the sporadic nature of mers-cov outbreaks. due to a gap in research on the treatment of mers in pregnancy, there are no therapeutic options currently recommended for pregnant women [ ] . therapies under development and testing may be considered inappropriate for pregnant women due to the unknown potential for teratogenic effects. for example, during the sars outbreak, ribavirin was administered to pregnant women with severe cases of the disease, but ribavirin therapy has been documented to increase the risk of teratogenic effects in newborns [ ] . the alphacoronaviruses hcov e and nl , as well as the betacoronaviruses hku and oc , can infect humans and cause the common cold. in order to investigate the potential maternal-fetal transmission of human coronaviruses during pregnancy, gagneur et al. [ , ] evaluated types of maternal-infant paired specimens that included maternal vaginal and respiratory specimens that were obtained during labor, as well as gastric samples from the newborn infants. these specimens were evaluated for the presence of hcov e, oc- , nl and hku using rt-pcr methodology. between the period from july to august the authors examined mother-infant dyads. human coronaviruses were identified in samples (hcov e: ; hku : ) from mother-child pairs. in mother-infant dyads only maternal respiratory samples were positive; in other pairs all of the samples tested positive for human coronavirus; in case only the maternal vaginal and newborn gastric samples were positive; and in another case the maternal vaginal sample alone was positive. there were no signs of clinical infection in any of the neonates that had positive gastric samples for human coronavirus. it is beyond the scope of this communication to discuss the various technical challenges inherent in developing a safe and efficacious vaccine for coronavirus infections in humans. there are clearly challenges to this endeavor-protective antibodies to coronaviruses are not long-lasting, tissue damage has been reported to occur as a result of exposure to sars-cov, development of animal models that closely resemble human infection are limited, and the extensive time and expense necessary to perform clinical trials in humans, to name a few [ ] [ ] [ ] . it is vitally important that pregnant women be considered in the design, clinical trial, and implementation of vaccine candidates for -ncov. in examining the history of vaccine design, it is clear that the needs of pregnant women have rarely been prioritized in either the preclinical development or the clinical trial phases of production. today, pregnant women are usually excluded from experimental trial of drugs and vaccines that do not target obstetric conditions [ ] . excluding pregnant women and their infants from participation in vaccine development and implementation undermines ethical principles of justice-fairness, equity, and maximization of benefit-and potentially places their health at risk during outbreaks and other health emergencies [ ] [ ] [ ] . on january the coalition for epidemic preparedness innovations (cepi) announced three programs to develop a vaccine against the novel wuhan coronavirus. the chief executive officer of cepi, richard hatchett, said [ ] : "given the rapid global spread of the ncov- virus the world needs to act quickly and in unity to tackle this disease. our intention with this work is to leverage our work on the mers coronavirus and rapid response platforms to speed up vaccine development." the novel coronavirus is the first epidemic disease to emerge since the formation of cepi in davos in . cepi was created with the express intent to enable speedy research and development of vaccines against emerging pathogens. in may , who released the target product profile (tpp) for mers-cov vaccines, following the prioritization of mers-cov as one of eight priority pathogens for prevention of epidemics [ ] . cepi and partners aim to use existing platforms-that is, the existing "backbone" that can be adapted for use against new pathogens-that are currently in preclinical development for mers-cov vaccine candidates. following the who declaration on january that the current -ncov outbreak is a public health emergency of international concern (pheic), global health organizations and researchers will be further mobilized-bolstered by new mechanisms for action and greater resources-to stop the spread of disease. a critical question that must be answered at this stage-with a clear view of the potential deleterious effects of a new coronavirus in pregnancy-is will maternal immunization be a priority in research and development? as of the pheic declaration, groups have announced that they are developing new vaccines against -ncov and seven others announced initiatives to develop new therapies [ ] . safe testing of experimental vaccines in a pregnant population is difficult and, as a result, vaccines are not typically developed with pregnant women in mind. to date, very few clinical trials for vaccines have proactively included pregnant women [ ] , and the exclusion of pregnant and lactating women from receiving the rvsv-zebov vaccine through ebola virus epidemics serves as a recent example [ ] [ ] [ ] . given the potential severity in pregnancy, as demonstrated by this review of maternal infections of sars and mers, women who are pregnant should be considered a priority population in all efforts to prepare for and prevent infection by novel coronaviruses. on february it was reported by multiple media outlets that a newborn infant delivered during the epidemic in wuhan had tested positive for -ncov at the wuhan children's hospital in hubei province hours following its birth. according to the official xinhua news agency, the infant was delivered on february to a mother who had tested positive for the virus. reports have stated that the infant had stable vital signs, no fever or cough, but had shortness of breath together with abnormal chest radiographs and abnormalities of liver function [ ] [ ] [ ] . dr. zeng lingkong, chief physician at the neonatal medicine department of the hospital, said [ ] , "this reminds us to pay attention to mother-to-child being a possible route of coronavirus transmission" the hospital also provided information about a previous case of a baby that had been delivered on january . following its birth, the infant's nanny was diagnosed with -ncov, and the mother was diagnosed days later [ ] . on january the baby began to develop symptoms. according to dr. zeng lingkong [ ] , "whether it was the baby's nanny who passed the virus to the mother who passed it to the baby, we cannot be sure at the moment. but we can confirm that the baby was in close contact with patients infected with the new coronavirus, which says newborns can also be infected" in considering whether these and future cases of neonatal infection are acquired prior to delivery, it is important to remember that newborn infants can acquire an infection in other ways beyond intrauterine maternal-fetal transmission. in some cases, viral infection can be acquired when the infant passes through the birth canal during a vaginal delivery or through post-partum breast feeding, although these mechanisms would be highly unusual for a respiratory virus. neonatal infection from respiratory viruses can occur after delivery through such mechanisms as inhalation of the agent through aerosols produced by coughing from the mother, relatives or healthcare workers or other sources in the hospital environment. based upon past experience with pregnant women who developed mers and sars, and realizing that the numbers are limited, there has never been confirmed intrauterine coronavirus transmission from mother to fetus. discussing the most recent baby to be diagnosed with the -ncov infection, dr. stephen morse, an epidemiologist at the mailman school of public health at columbia university stated [ ] , "it's more likely that the baby contracted the virus from the hospital environment, the same way healthcare workers get infected by the patients they treat," "it's quite possible that the baby picked it up very conventionally-by inhaling virus droplets that came from the mother coughing." and according to dr. paul hunter, professor of medicine at the university of east anglia [ ] , "as far as i am aware there is currently no evidence that the novel coronavirus can be transmitted in the womb. when a baby is born vaginally it is exposed to the mother's gut microbiome, therefore if a baby does get infected with coronavirus a few days after birth we currently cannot tell if the baby was infected in the womb or during birth." there is limited knowledge regarding coronavirus infections that occur during pregnancy-what is known has, for the most part, been the result of epidemics resulting from two different diseases, sars and mers. these previous experiences with coronavirus infections in pregnancy indicates that these agents are capable of causing adverse clinical outcomes including life-threatening maternal disease that in some cases requires hospitalization, intensive care and ventilatory support. both of these coronaviruses can result in maternal death in a small but significant number of cases, but the specific risk factors for a fatal outcome during pregnancy have not been clarified. coronaviruses can also result in adverse outcomes for the fetus and infant including intrauterine growth restriction, preterm delivery, admission to the icu, spontaneous abortion and perinatal death. unlike some viral infections, notably ebola virus [ ] and zika virus [ ] , the likelihood of intrauterine maternal-fetal transmission of coronaviruses is low-there have been no documented cases of vertical transmission occurring with either sars or mers. it remains to be seen during the current wuhan -ncov epidemic how this newly-emergent coronavirus affects pregnant women and their infants, as well as which factors may modulate obstetrical disease and outcomes including the timing of maternal coronavirus exposure by gestational age, the effects of medications or other treatment regimens, differences in host immune responses, occurrence of coexisting medical and obstetrical conditions, and other covariables. however, pregnant women should be considered to be at high risk for developing severe infection during this current outbreak of -ncov. additional clinical research on the treatment of sars, mers, and the new coronavirus -ncov is necessary if we are to understand the potential risks and benefits of novel therapies and new vaccines in pregnancy. this research will be critical in improving the care, and even saving the lives, of pregnant women in the current as well as future outbreaks. epidemic and emerging 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pregnancy and lactation being pregnant during the kivu ebola virus outbreak in dr congo: the rvsv-zebov vaccine and its accessibility by mothers and infants during humanitarian crises and in conflict areas maternal and infant death and the rvsv-zebov vaccine through three recent ebola virus epidemics-west africa, drc Équateur and drc kivu: years of excluding pregnant and lactating women and their infants from immunization coalition for epidemic preparedness innovations. cepi to fund three programmes to develop vaccines against the novel coronavirus a dozen vaccine programs underway as who declares coronavirus public health emergency. biocentury who. who target product profiles for mers-cov vaccines when is it acceptable to vaccinate pregnant women? risk, ethics, and politics of governance in epidemic crises chinese baby tests positive for coronavirus hours after birth a pregnant mother infected with the coronavirus gave birth, and her baby tested positive hours later coronavirus: doctors fear pregnant women can pass on illness after newborn baby is diagnosed expert reaction to newborn baby testing positive for coronavirus in wuhan zika virus infection in pregnancy, microcephaly and maternal and fetal health-what we think, what we know, and what we think we know this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -prgorr d authors: li, zhonghua; zeng, wei; ye, shiyi; lv, jian; nie, axiu; zhang, bingzhou; sun, yumei; han, heyou; he, qigai title: cellular hnrnp a interacts with nucleocapsid protein of porcine epidemic diarrhea virus and impairs viral replication date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: prgorr d the nucleocapsid (n) protein is a major structural component of porcine epidemic diarrhea virus (pedv), which is predicted to be a multifunctional protein in viral replication. heterogeneous nuclear ribonucleoprotein a (hnrnp a ) is a cellular protein participating in the splicing of pre-mrna in the nucleus and translation regulation in the cytoplasm. according to our previous proteomic study about pedv infection in vivo, hnrnp a was thought to be a cellular factor influencing pedv replication. in this report, pedv n protein was discovered to colocalize with cellular hnrnp a in perinuclear region of pedv infected cells. co-immunoprecipitation (co-ip) results clearly demonstrated that pedv n protein could bind to human hnrnp a . replication of pedv was inhibited by silencing the expression of hnrnp a in ccl- cells, suggesting the positive effect of hnrnp a on pedv infection. porcine epidemic diarrhea virus is the most important viral agent which can cause diarrhea in pigs. serious threat has been posed on the world pig industry as a result of the high morbidity and mortality caused by pedv in piglets [ ] [ ] [ ] [ ] [ ] [ ] [ ] . pedv, a single-stranded positive sense rna virus, is a member of coronavirus. four structural proteins, including spike protein (s), membrane protein (m), envelope protein (e) and nucleocapsid protein (n), and the genome constitute the virion. pedv n protein has multiple functions. firstly, as a structural protein, n protein along with the genomic rna forms the nucleocapsid of pedv. furthermore, n protein plays an important role in pedv rna synthesis and enhancing the pedv transcription and virion assembly [ ] [ ] [ ] . however, the effects of n protein on pedv infection usually depend on its interaction with some host factors. for example, pedv n protein binds with phosphoprotein nucleophosmin (npm ) and then protects it from proteolytic degradation by caspase- , enhancing cell survival and pedv growth [ ] . in addition, it has been proved that interaction between pedv n protein and tbk could inhibit irf activation and type i ifn production, further to causing the circumvention of the host's antiviral immunity [ ] . more than heterogeneous nuclear ribonucleoproteins (hnrnps) have been discovered and among them hnrnp a is the best-characterized one [ , ] . hnrnp a is a rna-binding protein which functions as binding to pre-mrna to form hnrnp particles in eukaryotic cells [ ] . this protein contains two rna-binding domains (rbds) and a glycine-rich domain responsible for protein-protein interaction [ , ] . it has been reported that hnrnp a selectively interacts with different rna-binding proteins through its glycine-rich domain [ ] . previous studies have demonstrated hnrnp a could interact with n proteins of sars coronavirus and mouse hepatitis virus (mhv) [ , ] . since pedv is also a member of coronavirus and pedv n protein is a rna binding protein, it is hypothesized that pedv n protein might also be able to interact with hnrnp a during pedv infection. our previous work has proved that hnrnp a underwent different regulations in jejunum tissues of piglets infected with pedv virulent strain and its attenuated strain [ ] . therefore, we assume that hnrnp a may play a role in the life cycle of pedv. in this study, it is found that hnrnp a could interact with pedv n protein and pedv replication could be inhibited by silencing the hnrnp a . the pedv yn (genbank accession no. kt ), yn (genbank accession no. kt ), and cv (genbank accession no. af . ) strain were used throughout this study. yn and yn strains were obtained by passaging the yn strain, a variant strain isolated from the intestine of a piglet with diarrhea, for and generations, respectively. our previous study has demonstrated that yn was a virulent strain and yn was an attenuation strain [ ] . cv strain, a classical pedv strain, was provided by chengdu tecbond biological product co., ltd. (chengdu, china). the ccl- cell line and hek t cell line were purchased from american type culture collection (atcc) and cultured in dulbecco's modified eagle's medium (dmem), supplemented with % fetal bovine serum (invitrogen, carlsbad, ca, usa) at • c with % co . the ccl- cell line was used for virus growth, infection, and cell lysate preparation. hek t cell line was used for co-ip analysis. the rabbit anti-hnrnp a , anti-flag polyclonal antibody (pab) and mouse anti-β-actin, anti-flag mono-antibody (mab) were purchased from abclonal (wuhan, china). mouse mab against pedv n protein was purchased from youlong biotech (shanghai, china). mouse mab against pedv spike protein was established by our laboratory. application of this mab has been described in some previous studies [ , ] . alexa fluor -conjugated anti-rabbit, anti-mouse and alexa fluor -conjugated anti-mouse antibodies were purchased from antgene biological (wuhan, china). the cdna expression construct encoding yn n protein was pcr amplified and cloned into pcaggs-flagc, which encode a c-terminal flag. details of this part have been described in our previous study [ ] . briefly, twelve piglets were randomly divided into three groups of four. the piglets in different groups were orally administrated with . ml yn and . ml yn with the same titer of . median tissue culture infective dose (tcid ) ml − and . ml dmem, respectively. all piglets were euthanized and necropsied when diarrhea was observed in the piglets in yn -infected group. jejunum tissues were separated rapidly, washed with ice-cold pbs buffer, snap-frozen in liquid nitrogen, and kept at − • c for subsequent proteome study. for the proteome study, itraq labeling coupled with lc-ms/ms was chose to analyze whole cell changes of jejunum of piglets, infected with pedv yn strain and yn strain. were precleared with protein a/g agarose (beyotime, shanghai, china) for h then centrifuged at rpm for min at • c. supernatants were incubated with mono-antibody against flag for h, then protein a/g beads were added and incubated at • c for h. the beads were then washed with ip lysis buffer five times and boiled in sample buffer, and the proteins were subjected to sds-page, followed by immunoblotting analysis with anti-flag pab or anti-hnrnp a pab. ccl- cells grown on coverslips were infected with pedv yn strain, yn strain and cv strain, respectively, at a multiplicity of infection (moi) . . at h post infection (hpi), the cells were fixed with % paraformaldehyde for min followed by being treated with methanol. fixed cells were blocked with % bovine serum albumin and incubated with anti-hnrnp a pab and mab against pedv n protein. alexa fluor -conjugated anti-rabbit and alexa fluor -conjugated anti-mouse antibodies were served as the secondary antibody. cell nucleus were stained with , -diamidino- -phenylindole (dapi). the localization of pedv n protein, hnrnp a and cell nucleus were then observed on a zeiss confocal microscope (zeiss, oberkochen, germany). ccl- cells were transfected with sirnas targeting to hnrnp a with lipofectamine reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. the sirnas targeting hnrnp a were synthesized by gene pharma (shanghai, china). effect of rna interference was tested by western blot at hpt. ccl- cells seeded in a -well plate were transfected without sirna or with nm nonspecial control sirna or sirna targeting to hnrnp a . at hpt, cells were infected with or without pedv. at hpi (yn ) or hpi (yn and cv ), the cells were fixed with % paraformaldehyde followed by treating with methanol. then the fixed cell was blocked with % bovine serum albumin and then incubated with mab against pedv s protein. alexa fluor -conjugated anti-mouse antibody was applied to detect the primary antibodies. dapi was selected to stain the cell nucleus. rna was extracted by using tripure isolation reagent (roche, in, usa) following the manufacturer's instructions. the cdna was obtained by rt-pcr using the primescript™ rt master mix (takara, tokyo, japan). the real-time rt-pcr assay for quantifying pedv genome used the following primer and probe sequences: pedv forward primer: -cgtacaggtaagtcaattac- , pedv reverse primer: -gatgaagcattgactgaa- , pedv taq-man ® probe: fam-ttcgtcaca gtcgccaagg-tamra. cells were lysed with lysis buffer (beyotime, shanghai, china) containing mm pmsf. these proteins were subjected to sds-page and then the separated protein bands were transferred onto pvdf membrane using a trans-blot (bio-rad, berkeley, ca, usa). the membrane was incubated in blocking buffer (tris-buffered saline (tbs), containing . % tween- (tbst) and % skim milk) for h at room temperature followed by washing three times by pbst. then the membrane was incubated with the corresponding primary antibodies for h at room temperature. after being washed three times by pbst, the membrane was incubated with (hrp)-conjugated goat-anti mouse/rabbit igg at room temperature for . h. finally, the protein bands were visualized using the clarity™ western ecl blotting substrate (bio-rad, hercules, ca, usa). according to the result of lc-ms/ms, hnrnp a was down-regulated in an attenuated pedv strain (yn ) infected jejunum tissues, while showed no apparent change in a virulent pedv strain (yn ) infected jejunum tissues [ ] . western blot assay was applied to confirm the changes of hnrnp a . as shown in figure , regulations of hnrnp a were consistent with the result of lc-ms/ms. hnrnp a has been proved to be involved in the replication process of other coronaviruses [ , ] . therefore, we assumed that hnrnp a may play a role in pedv replication and influence the pathogenicity of pedv in vivo. according to the result of lc-ms/ms, hnrnp a was down-regulated in an attenuated ped rain (yn ) infected jejunum tissues, while showed no apparent change in a virulent pedv stra n ) infected jejunum tissues [ ] . western blot assay was applied to confirm the changes rnp a . as shown in figure , regulations of hnrnp a were consistent with the result of l s/ms. hnrnp a has been proved to be involved in the replication process of other coronavirus , ] . therefore, we assumed that hnrnp a may play a role in pedv replication and influen e pathogenicity of pedv in vivo. previous studies have demonstrated that n protein of some coronaviruses, such as mhv an rs-cov, could interact with hnrnp a . in this study, we determined to investigate whether otein can be colocalized with hnrnp a during pedv infection. ccl- cells were infected wi e yn , yn , and cv strain of pedv, respectively, and the localization of np and hnrnp a as observed by confocal microscopy at hpi. as shown in figure , hnrnp a was main calized in the nucleus without pedv infection. however, some hnrnp a was transported fro e nucleus to the cytoplasm and colocalized with pedv n protein during pedv infectio rthermore, pedv n protein was localized in cytoplasm and no differences were found in t calization of n protein of these pedv strains. the immunofluorescence assay demonstrated th dv n protein and hnrnp a colocalized in cytoplasm. previous studies have demonstrated that n protein of some coronaviruses, such as mhv and sars-cov, could interact with hnrnp a . in this study, we determined to investigate whether n protein can be colocalized with hnrnp a during pedv infection. ccl- cells were infected with the yn , yn , and cv strain of pedv, respectively, and the localization of np and hnrnp a was observed by confocal microscopy at hpi. as shown in figure , hnrnp a was mainly localized in the nucleus without pedv infection. however, some hnrnp a was transported from the nucleus to the cytoplasm and colocalized with pedv n protein during pedv infection. furthermore, pedv n protein was localized in cytoplasm and no differences were found in the localization of n protein of these pedv strains. the immunofluorescence assay demonstrated that pedv n protein and hnrnp a colocalized in cytoplasm. the colocalization of pedv n protein and hnrnp a demonstrated that a certain interaction may exist between these two proteins. thus, co-immunoprecipitation (co-ip) was performed to testify this phenomenon. t cells transfected with plasmids expressing the flag-tagged pedv n protein were subjected to immunoprecipitation using anti-flag antibody. interaction of pedv n protein with host protein hnrnp a was analyzed by immunoblotting with anti-flag antibody and anti-hnrnp a antibody. as shown in figure , cellular hnrnp a protein was only detected in the presence of flag tagged pedv n by co-ip. these results indicate that n protein interacted with hnrnp a . the colocalization of pedv n protein and hnrnp a demonstrated that a certain interaction may exist between these two proteins. thus, co-immunoprecipitation (co-ip) was performed to testify this phenomenon. t cells transfected with plasmids expressing the flag-tagged pedv n protein were subjected to immunoprecipitation using anti-flag antibody. interaction of pedv n protein with host protein hnrnp a was analyzed by immunoblotting with anti-flag antibody and anti-hnrnp a antibody. as shown in figure , cellular hnrnp a protein was only detected in the presence of flag tagged pedv n by co-ip. these results indicate that n protein interacted with hnrnp a . cell lysates were also applied to confirm the expression of proteins (input). in order to investigate whether hnrnp a participates in the replication of pedv, three sirnas targeting to hnrnp a were synthesized to silence the expression of this protein. sequences of the sirnas were shown in table . ccl- cells were transfected with sirnas against hnrnp a expression, cells were collected at hpt to analyze the silencing efficiency of hnrnp a at the protein level. as shown in figure , sirna- showed the best performance for silencing hnrnp a and was chosen for the following research. in order to investigate whether hnrnp a participates in the replication of pedv, three sirnas targeting to hnrnp a were synthesized to silence the expression of this protein. sequences of the sirnas were shown in table . ccl- cells were transfected with sirnas against hnrnp a expression, cells were collected at hpt to analyze the silencing efficiency of hnrnp a at the protein level. as shown in figure , sirna- showed the best performance for silencing hnrnp a and was chosen for the following research. ccl- cells were transfected with sirna- for h followed by infection with pedv yn strain. these cells were subjected to western blot, indirect immunofluorescence assay (ifa) or real-time pcr at hpi figure . according to the results, a conclusion was reached that knockdown of hnrnp a reduced the replication of yn . hek t cells were transfected with a vector expressing pedv n protein with a flag tag (flag-pedv-n) or empty vectors (flag-pcaggs) and the whole-cell lysates obtained at hpt were immunoprecipitated with anti-flag mab. after separation by sds-page, proteins were detected by immunoblotting with the indicated antibodies (ip: flag). cell lysates were also applied to confirm the expression of proteins (input). in order to investigate whether hnrnp a participates in the replication of pedv, three sirnas targeting to hnrnp a were synthesized to silence the expression of this protein. sequences of the sirnas were shown in table . ccl- cells were transfected with sirnas against hnrnp a expression, cells were collected at hpt to analyze the silencing efficiency of hnrnp a at the protein level. as shown in figure , sirna- showed the best performance for silencing hnrnp a and was chosen for the following research. ccl- cells were transfected with sirna- for h followed by infection with pedv yn strain. these cells were subjected to western blot, indirect immunofluorescence assay (ifa) or realtime pcr at hpi figure . according to the results, a conclusion was reached that knockdown of hnrnp a reduced the replication of yn . sequences ( ′- ′) sirna- ggaagaguuguggaaccaatt sirna- ggauuugguaaugauggaatt sirna- gcgguggaggucaauacuutt negative control sirna (nc) uucuccgaacgugucacgutt the pedv virulent strain yn and pedv classical strain cv were further applied to test the positive effects of hnrnp a on pedv replication. as shown in figures and , similar results were obtained. these results demonstrated that hnrnp a is a positive regulator of pedv replication. sirna- ggaagaguuguggaaccaatt sirna- ggauuugguaaugauggaatt sirna- gcgguggaggucaauacuutt negative control sirna (nc) uucuccgaacgugucacgutt the pedv virulent strain yn and pedv classical strain cv were further applied to test the positive effects of hnrnp a on pedv replication. as shown in figures and , similar results were obtained. these results demonstrated that hnrnp a is a positive regulator of pedv replication. figure . knockdown of hnrnp a expression inhibits yn replication. ccl- cells were transfected without sirna (a) or with nc (b) or sirna- (c). at hpt, cells were infected with yn strain at a moi of . or mock infected (d). virus suspensions were harvested at hpi to calculate the virus titer by real-time rt-pcr. the virus copies per ml suspension was calculated (a). cells were harvested at hpi to analyze the expression of pedv n protein by western blot (b) and the numbers of cells infected with pedv by ifa (original magnification ×) (c). pedv n protein levels were quantified by measuring band intensities and normalized with respect to the amount of β-actin. data are shown as means ± the standard errors of the mean (sem) of at least three independent experiments, with the error bars representing the standard deviations. the pedv virulent strain yn and pedv classical strain cv were further applied to test the positive effects of hnrnp a on pedv replication. as shown in figures and , similar results were obtained. these results demonstrated that hnrnp a is a positive regulator of pedv replication. the virus titer by real-time rt-pcr. the virus copies per ml suspension was calculated (a). cells were harvested at hpi to analyze the expression of pedv n protein by western blot (b) and the numbers of cells infected with pedv by ifa (original magnification ×) (c). pedv n protein levels were quantified by measuring band intensities and normalized with respect to the amount of β-actin. data are shown as means ± sem of at least three independent experiments, with the error bars representing the standard deviations. in this study, interaction between hnrnp a and pedv n protein was verified by co-ip and immunofluorescence assay. our results established that n protein interacts with hnrnp a in pedv infected cells and t cells expressing pedv n protein. hnrnp a not only mainly localizes in the cell nucleus, but also shuttles between the nucleus and the cytoplasm [ , ] . however, this protein underwent a relocalization to cytoplasm during pedv infection, suggesting a possible functional link between hnrnp a and pedv infection. this phenomenon is very similar to a previous study about mhv [ ] . furthermore, we found that the pedv n protein and hnrnp a co-localized predominantly in the perinuclear region of pedv infected cells, in which active coronavirus replication/transcription complexes reside. this indicates both pedv n protein and hnrnpa might participate in constituting the pedv replication/transcription complex and their interaction may be involved in regulation of pedv replication. it is well known that hnrnp a is one part of replication/transcription complex of both sars-cov and mhv [ , , , , ] . overexpression of hnrnpa facilitates mhv replication while inhibition of hnrnp a expression results in the reduction of mhv replication [ ] . since pedv is in this study, interaction between hnrnp a and pedv n protein was verified by co-ip and immunofluorescence assay. our results established that n protein interacts with hnrnp a in pedv infected cells and t cells expressing pedv n protein. hnrnp a not only mainly localizes in the cell nucleus, but also shuttles between the nucleus and the cytoplasm [ , ] . however, this protein underwent a relocalization to cytoplasm during pedv infection, suggesting a possible functional link between hnrnp a and pedv infection. this phenomenon is very similar to a previous study about mhv [ ] . furthermore, we found that the pedv n protein and hnrnp a co-localized predominantly in the perinuclear region of pedv infected cells, in which active coronavirus replication/transcription complexes reside. this indicates both pedv n protein and hnrnpa might participate in constituting the pedv replication/transcription complex and their interaction may be involved in regulation of pedv replication. it is well known that hnrnp a is one part of replication/transcription complex of both sars-cov and mhv [ , , , , ] . overexpression of hnrnpa facilitates mhv replication while inhibition of hnrnp a expression results in the reduction of mhv replication [ ] . since pedv is also a member of coronavirus, hnrnp a may be involved in the process of pedv infection. in order to test this hypothesis, we silenced the hnrnp a and then studied its effect on pedv infection. similar to the result of mhv following silencing hnrnp a , replication of three pedv strains were all inhibited, suggesting a positive effect of hnrnp a on pedv infection. replication of coronavirus depend on the generation of nested subgenomic mrnas (sgmrnas) with a common capped leader sequence [ ] . optical transcription of sgmrnas requires the interaction between its leader sequence and the intergenic (ig) sequences of each orf. it has been reported that hnrnp a can bind to both the terminal leader sequences and ig sequences [ ] . so, inhibition of pedv by silencing hnrnp a is likely due to the break of the interaction between terminal leader sequences and ig sequences. however, till now we have not got evidence to support it. in addition, silencing of hnrnp a might also be able to reduce its interaction with pedv n protein. the function of their interaction on pedv infection is under the investigation of our laboratory. our previous study has demonstrated that hnrnp a was downregulated in the jejunum of pedv strain yn infected group, but no apparent change in group infected with yn [ ] . since yn caused diarrhea in piglets while yn could not, therefore, downregulation of hnrnp a was one of the reasons responsible for the lower pathogenicity of yn than yn in vivo. from the field to the lab-an european view on the global spread of pedv porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis new variants of porcine epidemic diarrhea virus, china porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines outbreak-related porcine epidemic diarrhea virus strains similar to us strains distinct characteristics and complex evolution of pedv strains first detection, clinical 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insights to post-transcriptional regulatory roles isolation of an active gene encoding human hnrnp protein a : evidence for alternative splicing the swiss army knife of gene expression viral and cellular mrna translation in coronavirus-infected cells comparative proteome analysis of porcine jejunum tissues in response to a virulent strain of porcine epidemic diarrhea virus and its attenuated strain he, q. itraq-based comparative proteomic analysis of vero cells infected with virulent and cv vaccine strain-like strains of porcine epidemic diarrhea virus comparative genomic analysis of classical and variant virulent parental/attenuated strains of porcine epidemic diarrhea virus the nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein a in vitro and in vivo a -mediated translational regulation of the g quadruplexcontaining ron receptor tyrosine kinase mrna linked to tumor progression cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein a controls translation initiation of specific mrnas heterogeneous nuclear ribonucleoprotein a regulates rna synthesis of a cytoplasmic virus multiple type a/b heterogeneous nuclear ribonucleoproteins (hnrnps) can replace hnrnp a in mouse hepatitis virus rna synthesis blocking eif e-eif g interaction as a strategy to impair coronavirus replication nuclear proteins hijacked by mammalian cytoplasmic plus strand rna viruses the authors have declared no conflict of interest. key: cord- -rafvxgx authors: hartmann, katrin title: clinical aspects of feline retroviruses: a review date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: rafvxgx feline leukemia virus (felv) and feline immunodeficiency virus (fiv) are retroviruses with global impact on the health of domestic cats. the two viruses differ in their potential to cause disease. felv is more pathogenic, and was long considered to be responsible for more clinical syndromes than any other agent in cats. felv can cause tumors (mainly lymphoma), bone marrow suppression syndromes (mainly anemia), and lead to secondary infectious diseases caused by suppressive effects of the virus on bone marrow and the immune system. today, felv is less commonly diagnosed than in the previous years; prevalence has been decreasing in most countries. however, felv importance may be underestimated as it has been shown that regressively infected cats (that are negative in routinely used felv tests) also can develop clinical signs. fiv can cause an acquired immunodeficiency syndrome that increases the risk of opportunistic infections, neurological diseases, and tumors. in most naturally infected cats, however, fiv itself does not cause severe clinical signs, and fiv-infected cats may live many years without any health problems. this article provides a review of clinical syndromes in progressively and regressively felv-infected cats as well as in fiv-infected cats. feline leukemia virus (felv) and feline immunodeficiency virus (fiv) belong to the most common infectious diseases in cats. both are retroviruses, but felv is a γ-retrovirus, while fiv is classified as open access a lentivirus. although felv and fiv are closely related, they differ in their potential to cause disease. in the united states, prevalence of both infections is about % in healthy cats and up to about % in high-risk or sick cats [ , ] . risk factors for infection include male gender, adulthood, and outdoor access [ , ] . retroviral tests can diagnose only infection, not clinical disease. felv is more pathogenic than fiv. for a long time, felv was considered to account for most disease-related deaths, and to be responsible for more clinical syndromes than any other single agent in cats. it was proposed that approximately one third of all tumor-related deaths in cats were caused by felv, and an even greater number of cats died of felv-related anemia and secondary infections caused by suppressive effects of the virus on bone marrow and the immune system. today, these statements have to be revised, as in recent years the prevalence and consequently the importance of felv as a pathogen in cats have been decreasing. still, if present in closed households with other viruses, such as feline coronavirus (fcov), or fiv, felv infection has the greatest impact on survival [ ] . the death rate of progressively felv-infected cats in multi-cat households has been estimated at approximately % in two years and % in three years [ , ] , but is much lower today, at least for cats that are well taken care of and that are kept strictly indoors in single-cat households. a survey in the united states compared the survival of more than felv-infected cats to more than age-and sex-matched uninfected control cats and found that in felv-infected cats median survival was . years compared to . years for control cats [ ] . despite the fact that progressive felv infection is associated with a decrease in life expectancy, many owners elect to provide treatment for their cats, and with proper care, felv-infected cats might live for many years with good quality of life. although fiv can cause an acquired immunodeficiency syndrome in cats ("feline aids") comparable to human immunodeficiency virus (hiv) infection in humans, with increased risk for opportunistic infections, neurologic diseases, and tumors, in most naturally infected cats, fiv does not cause a severe clinical syndrome. with proper care, fiv-infected cats can live many years and, in fact, can die at older age from causes unrelated to their fiv infection. in a follow-up study in naturally fivinfected cats, the rate of progression was variable, with death occurring in about % of infected cats within the first two years of observation (about five years after the estimated time of infection). an additional % developed increasingly severe disease, but more than % remained clinically asymptomatic during the two years [ ] . fiv infection has little impact on a cat population and does not reduce the number of cats in a household [ ] . thus, overall survival time is not shorter than in uninfected cats, and quality of life is usually fairly high over an extended period of time. both infections are chronic in nature and develop through different disease stages. characteristically for both infections, there is a long asymptomatic phase, in which cats do not show clinical signs. felv infection has different stages. recently, novel diagnostic tools, including very sensitive pcr methods providing new data on the course of felv infection, have questioned the traditional understanding of felv pathogenesis. cats believed to be immune to felv after infection were found to remain provirus-positive. antigen-negative, provirus-positive cats are frequently detected and their clinical relevance and role in felv epidemiology is still not fully understood. antigen-negative, provirus-positive cats are considered felv carriers. following reactivation, they can act as an infection source. as felv provirus is integrated into the cat's genome, it is unlikely to be fully cleared over time. antigen-negative, provirus-positive cats do not shed the virus, but reactivation with reoccurring virus shedding is possible [ , ] . based on this information, a new classification has been proposed, in which the stages of felv infection are defined as abortive infection (comparable to the former "regressor cats"), regressive infection (comparable to the former "transient viremia" followed by "latent infection"), progressive infection (comparable to the former "persistent viremia"), and focal or atypical infection (table ) [ ] [ ] [ ] [ ] . abortive infection. after infection, the virus starts initially to replicate in the local lymphoid tissue in the oropharyngeal area. in some immunocompetent cats (formerly called "regressor cats)", viral replication may be terminated by an effective humoral and cell-mediated immune response; these cats never become viremic. they have high levels of neutralizing antibodies. neither felv antigen nor viral rna or proviral dna can be detected in the blood at any time. abortive infection is likely caused when a cat is exposed to low doses of felv [ ] . it is still unknown, how often this situation really occurs in nature, because studies using very sensitive pcr methods have found that in many of the formerly considered "regressor cats", the virus actually can still be found later in these cats when investigating tissue samples. thus, it appears likely that none or only very few cats can completely clear felv infection from all cells. regressive infection develops following an effective immune response. in regressive infection, virus replication and viremia are contained prior to or shortly after bone marrow infection. after initial infection, replicating felv spreads systemically through infected mononuclear cells (lymphocytes and monocytes). during this stage, cats have positive results on tests that detect free antigen in plasma (e.g., elisa). they shed virus, mainly with saliva. in cats with regressive infection, this viremia, however, is terminated within weeks or months (therefore formerly called "transient viremia"). in some cats, viremia may persist longer than three weeks. after about three weeks of viremia, bone marrow cells become infected, and infected hematopoietic precursor cells develop into infected granulocytes and platelets that circulate in the body. even if bone marrow cells become infected, a certain percentage of cats is able to clear viremia. however, they cannot completely eliminate the virus from the body, even if they terminate viremia because the information for virus replication (proviral dna) is present in bone marrow stem cells. this condition has been called "latent infection" (and is now a part of the regressive infection). the molecular basis of latency is the integration of a copy of the viral genome (provirus) into cellular chromosomal dna. although proviral dna remains present within the cellular genome, no virus is actively produced. thus, cats with regressive infection have negative results in all tests that detect felv antigen. during cell division, proviral dna is replicated and the information given to the daughter cells. thus, complete cell lineages may contain felv proviral dna. however, proviral dna is not translated into proteins, and no infectious virus particles are produced. therefore, regressively infected cats do not shed felv and are not infectious to others. sensitive pcr methods can detect provirus in the blood of cats with regressive infection that are antigen-negative. in a study in switzerland it was shown that in addition to the antigen-positive, provirus-positive cats, about % of the cat population that were negative for antigen were positive for proviral dna in the blood [ ] . regressive infection can be reactivated because the information for producing complete viral particles is present and can potentially be reused when antibody production decreases (e.g., after immunosuppression). in cats with progressive infection, felv infection is not contained early in the infection. thus, extensive virus replication occurs, first in the lymphoid tissues, followed by the bone marrow and mucosal and glandular epithelial tissues. progressively infected cats remain persistently viremic. they are infectious to other cats for the remainder of their life. this condition has been called "persistent viremia" and is now classified as progressive infection. cats with progressive infection develop felv-associated diseases, and most of them will die within a few years. regressive and progressive infections can be distinguished by repeated testing for viral antigen in peripheral blood; regressively infected cats will turn negative at latest weeks after infection, while progressively infected cats will remain positive. initially both, regressive and progressive infections are accompanied by persistence of felv proviral dna in the blood detected by pcr but later are associated with different felv loads when measured by quantitative pcr; regressive infection is associated with low, progressive infection with high virus load [ , ] . focal infections or atypical infections have been reported in up to % of experimentally infected cats. focal infections or atypical infections may also be observed in natural infections, but are probably rare in the field. focal infections are characterized by a persistent atypical local viral replication (e.g., in mammary glands, bladder, eyes). this replication can lead to intermittent or low-grade production of antigen, and therefore, these cats can have weakly positive or discordant results in antigen tests, or positive and negative results may alternate [ ] . experimental fiv infection also progresses through several stages, similar to hiv infection in people, including an acute phase, a clinically asymptomatic phase of variable duration, and a terminal phase sometimes called "feline acquired immunodeficiency syndrome" ("aids") [ , ] . however, there is no clear distinction between these stages in naturally fiv-infected cats, and not all stages are apparent; therefore, the usefulness of this staging in natural fiv infection has been questioned. moreover, even cats in moribund condition with severe immunosuppression and secondary infections may fully recover with appropriate care and return to an asymptomatic stage. thus, different from hiv-infected people, cats classified as being in the "aids phase" (high virus load, severe clinical signs due to secondary infection) can recover and be asymptomatic again, and their virus loads can even decrease dramatically. clinical signs in both retrovirus infections are variable. after a long asymptomatic phase, cats can develop tumors, hematopoietic disorders, neurologic disorders, immunodeficiency, immune-mediated diseases, and stomatitis. the pathomechanism of these disorders is different in both retrovirus infections (table ) . although felv was named after a tumor that first garnered its attention, most infected cats are presented to the veterinarian not for tumors but for anemia or immunosuppression. clinical signs associated with felv infection can be classified as tumors, immunosuppression, hematologic disorders, immune-mediated diseases, and other syndromes (including neuropathy, reproductive disorders, fading kitten syndrome). of felv-infected cats presented to north american veterinary teaching hospitals, various co-infections (including fiv infection, feline infectious peritonitis (fip), upper respiratory infection, hemotropic mycoplasmosis, and stomatitis) were the most frequent findings ( %), followed by anemia ( %), lymphoma ( %), leukopenia or thrombocytopenia ( %), and leukemia or myeloproliferative diseases ( %) [ ] . the outcome of felv infection and the clinical course are determined by a combination of viral and host factors. some of the differences in outcome can be traced to properties of the virus itself, such as the subgroup that determines differences in the clinical picture (e.g., felv-b is primarily associated with tumors, felv-c is primarily associated with non-regenerative anemia). a study aiming to define dominant host immune effects or mechanisms responsible for the outcome of infection by using longitudinal changes in felv-specific cytotoxic t-lymphocytes (ctl) found that high levels of circulating felv-specific effector ctls appear before virus-neutralizing antibodies in cats that have recovered from exposure to felv. in contrast, progressive infection with persistent viremia has been associated with a silencing of virusspecific humoral and cell-mediated immunity host effector mechanisms [ ] . probably the most important host factor that determines the clinical outcome of cats infected with felv is the age of the cat at the time of infection [ ] . neonatal kittens develop marked thymic atrophy after infection ("fading kitten syndrome"), resulting in severe immunosuppression, wasting, and early death. as cats mature, they acquire a progressive resistance. when older cats become infected, they tend to have abortive or regressive infections or, if developing progressive infection, have at least milder signs and a more protracted period of apparent good health [ ] . clinical signs in naturally fiv-infected cats usually reflect secondary diseases, such as infections and neoplasia, to which fiv-infected cats are considered more susceptible. fiv itself may cause some clinical features (e.g., neurologic signs) resulting from abnormal function or inflammation of affected organs. in experimental infection, an initial stage is sometimes noticed usually with transient and mild clinical signs, including fever, lethargy, signs of enteritis, stomatitis, dermatitis, conjunctivitis, respiratory tract disease, and generalized lymph node enlargement [ ] . the acute phase may last several days to a few weeks, after which cats will enter a period in which they appear clinically healthy. this phase is usually not noticed by the owners in naturally infected cats. the duration of the following asymptomatic phase varies, but usually lasts many years. factors that influence the duration of the asymptomatic phase include the pathogenicity of the infecting isolate (also depending on the fiv subtype), exposure to secondary pathogens, and the age of the cat at the time of infection [ , ] . in the last, symptomatic phase ("aids phase") of infection, the clinical signs are a reflection of opportunistic infections, neoplasia, myelosuppression, and neurologic disease. while felv-infected cats are -times more likely to develop lymphoma or leukemia than noninfected cats and felv plays a direct role in tumorgenesis, fiv-infected cats have about a five-fold increased risk of tumor development, and the role of fiv is usually indirect. lymphomas are the most common tumors in felv-and fiv-infected cats. while felv-infected cats have most commonly t-cell lymphomas, lymphomas in fiv are mostly of b-cell origin [ , ] . felv is a major oncogene that causes different tumors in cats, most commonly lymphoma and leukemia, less often other hematopoietic tumors and rarely other malignancies (including neuroblastoma, osteochondroma, and others). the association between felv and lymphomas has been clearly established in several ways. first, these malignancies can be induced in kittens by experimental felv infection [ ] [ ] [ ] . second, cats naturally infected with felv have a higher risk of developing lymphoma than uninfected cats [ , ] . third, most cats with lymphoma were-at least in earlier times when prevalence of felv was still higher-felv-positive in tests that detected infectious virus or felv antigens. previously, up to % of feline lymphomas and leukemias were reported to be felv-related [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, since the s a reduction in the prevalence of viremia has been noted in cats with lymphoma [ ] [ ] [ ] . the decrease in prevalence of felv infection in cats with lymphoma or leukemia also indicates a shift in tumor causation in recent years. whereas % of all cats with lymphoma or leukemia were felv antigen-positive in one german study from to , only % of the cats were felv antigen-positive in the years to in the same university teaching hospital [ ] . in a recent study in the netherlands, only four of cats with lymphoma were felv-positive, although of these cats had mediastinal lymphoma, which previously was strongly associated with felv infection [ ] . a greater prevalence of lymphoma in older-age cats in now observed. the major reason for the decreasing association of felv with lymphoma is the decreasing prevalence of felv infection in the overall cat population as a result of felv vaccination as well as testing and elimination programs. however, prevalence of lymphomas caused by felv may be higher than indicated by conventional antigen testing of blood [ ] . cats from felv cluster households had a -fold higher rate of development of felv-negative lymphoma than did those from the general population. felv-negative lymphomas have also occurred in laboratory cats known to have been infected previously with felv [ ] . felv proviral dna was detected in lymphomas of cats that tested negative for felv antigen [ ] , also suggesting that the virus may be associated with a larger proportion of lymphomas than previously thought. felv has been shown to incorporate cellular genes; several such transducted genes also present in regressively infected cells have been implicated in viral oncogenesis [ ] [ ] [ ] . it is still unclear, how common regressive felv infection is responsible for felv-associated tumors in the field as study results have been controversial. proviral dna was detected in formalin-fixed, paraffin-embedded tumor tissue in / felv-negative cats with lymphoma [ ] . however, other groups found evidence of provirus in only / [ ] and in / felv antigen-negative lymphomas [ ] . the most important mechanism by which felv causes malignancy is by insertion of the felv genome into the cellular genome near a cellular oncogene (most commonly myc), resulting in activation and over-expression of that gene. these effects lead to uncontrolled proliferation of these cells (clone). a malignancy results in the absence of an appropriate immune response. felv may also incorporate the oncogene to form a recombinant virus (e.g., felv-b, fesv) containing cellular oncogene sequences that are then rearranged and activated. when they enter a new cell, these recombinant viruses are oncogenic. in a study of cats with lymphomas, transduction or insertion of the myc locus had occurred in cats ( %) [ ] . thus, felv-induced neoplasms are caused, at least in part, by somatically acquired insertional mutagenesis in which the integrated provirus may activate a proto-oncogene or disrupt a tumor suppressor gene. a recent study suggested that the u -ltr region of felv transactivates cancer-related signaling pathways through production of a non-coding base rna transcript that activates nf kappab [ ] . twelve common integration sites for felv associated with lymphoma development have been identified in six loci: c-myc, flvi- , flvi- (contains bmi- ), fit- , pim- , and flit- . oncogenic association of the loci is based on the fact that c-myc is known as a proto-oncogene, bmi- and pim- have been recognized as myc-collaborators, fit- appears to be closely linked to myb, and flit- insertion was shown to be associated with over-expression of cellular genes, e.g., activin-a receptor type ii-like (acvrl ). [ ] . flit- seems to have an important role in the development of lymphomas and appears to represent a common novel felv proviral integration domain that may influence lymphomagenesis by insertional mutagenesis. among felv-related tumors, / thymic lymphomas demonstrated proviral insertion within flit- locus, whereas / alimentary lymphomas, / multicentric lymphomas, and / t-lymphoid leukemia examined had rearrangements in this region. expression of acvrl mrna was detected in the two thymic lymphomas with flit- rearrangement, whereas normal thymuses and seven lymphoid tumors without flit- rearrangement had no detectable acvrl mrna expression [ ] . fibrosarcomas that are associated with felv are caused by fesv, a recombinant virus that develops de novo in felv-a-infected cats by recombination of the felv-a genome with cellular oncogenes. through a process of genetic recombination, fesv acquires one of several oncogenes, such as fes, fms, or fgr. as a result, fesv is an acutely transforming (tumor-causing) virus, leading to a polyclonal malignancy with multifocal tumors arising simultaneously after a short incubation period. with the decrease in felv prevalence, fesv also has become less common. fesv-induced fibrosarcomas are multicentric and usually occur in young cats. strains of fesv identified from naturally occurring tumors are defective and unable to replicate without the presence of felv-a as a helper virus that supplies proteins (such as those coded by the env gene) to fesv. fibrosarcomas caused by fesv tend to grow rapidly, often with multiple cutaneous or subcutaneous nodules that are locally invasive and metastasize to the lung and other sites. solitary fibrosarcomas in older cats are not caused by fesv. these tumors are slower growing, locally invasive, slower metastasizing, and only occasionally curable by excision combined with radiation and/or gene therapy. they usually are classified as feline injection site sarcomas (fiss) caused by the granulomatous inflammatory reaction at the injection site, commonly occurring after inoculation of adjuvant-containing vaccines. it has been demonstrated that neither fesv nor felv play any role in the development of fiss [ ] . a few other tumors have been found in felv-infected cats; some of them might have an association with felv, others likely have just been observed by chance simultaneously in an infected cat. iris melanomas, for example, are not associated with felv infections, although in one study three of eyes tested positive for felv/fesv proviral dna [ ] . in a more recent study, however, immunohistochemical staining and pcr did not find felv or fesv in the ocular tissues of any cat with this disorder [ ] . multiple osteochondromas (cartilaginous exostoses on flat bones of unknown pathogenesis) have been described in felv-infected cats. although histologically benign, they may cause significant morbidity if they occur in an area such as a vertebra and put pressure on the spinal cord or nerve roots [ , ] . in spontaneous feline olfactory neuroblastomas (aggressive, histologically inhomogenous tumors of the tasting and smelling epithelium of nose and pharynx with high metastasis rates), budding felv particles were found in the tumors and lymph node metastases, and felv dna was detected in tumor tissue [ ] . the exact role of felv in the genesis of these tumors is uncertain. cutaneous horns are a benign hyperplasia of keratinocytes that have been described in felv-infected cats [ ] , but the role of felv is also unclear. fiv-infected cats are about five times more likely to develop lymphoma or leukemia than noninfected cats [ , ] . lymphomas (mostly b-cell lymphomas) [ , , , ] , leukemias, but also several other tumors have been described in association with fiv infection [ , [ ] [ ] [ ] [ ] [ ] [ ] , including squamous cell carcinoma, fibrosarcoma, and mast cell tumor. fiv provirus, however, is only occasionally detected in tumor cells [ ] [ ] [ ] [ ] , suggesting a more indirect role in lymphoma formation, such as decreased cell-mediated immune surveillance or chronic b-cell hyperplasia [ , ] . however, clonally integrated fiv dna was found in lymphoma cells from one cat that had been experimentally infected six years earlier, indicating the possibility of an occasional direct oncogenic role of fiv [ , , ] . the prevalence of fiv infection in one cohort of cats with lymphoma was % [ ] , much higher than the fiv prevalence in the population of cats without lymphomas, which is also supportive of a cause and effect relationship. fiv could alternatively increase tumor incidence by decreasing tumor immunosurveillance mechanisms. it also could promote tumor development through the immunostimulatory effects of replicating in lymphocytes. myelosupression and other hematopoietic disorders can occur in both, felv and fiv infection. it is, however, much more common and more severe in felv-infected cats. hematologic changes described in association with felv include anemia (non-regenerative or regenerative), persistent, transient, or cyclic neutropenia, platelet abnormalities (thrombocytopenia and platelet function abnormalities), aplastic anemia (pancytopenia), and panleukopenia-like syndrome. for the majority of pathogenic mechanisms in which felv causes bone marrow suppression, active virus replication is required. however, it has been demonstrated that in some felv antigen-negative cats, regressive felv infection without viremia may be responsible for bone marrow suppression. in a recent study including cats with myelosuppression that tested felv antigen-negative in peripheral blood, / cats ( %) were found regressively infected with felv by bone marrow pcr (both had non-regenerative anemia) [ ] . in these regressively infected cats, felv provirus may interrupt or inactivate cellular genes in the infected cells, or regulatory features of viral dna may alter expression of neighboring genes. additionally, cell function of provirus-containing myelomonocytic progenitor and stromal fibroblasts that provide bone marrow microenvironment may be altered. alternatively, felv provirus may cause bone marrow disorders by inducing the expression of antigens on the cell surface, resulting in an immune-mediated destruction of the cell. anemia is a major nonneoplastic complication that occurs in a majority of felv-infected cats [ ] . anemia in felv-infected cats may have various causes. approximately % of felv-associated anemias are regenerative [ ] , most felv-associated anemias, however, are non-regenerative and are caused by the bone marrow suppressive effect of the virus resulting from primary infection of hematopoietic stem cells and infection of stroma cells that constitute the supporting environment for hematopoietic cells. in vitro exposure of normal feline bone marrow to some strains of felv caused suppression of erythrogenesis [ ] . in addition to the direct effect of the virus on erythropoiesis, other factors can cause nonregenerative anemia in felv-infected cats (e.g., anemia of chronic inflammation promoted by high concentration of cytokines). felv infection can cause decreased platelet counts. it also can be responsible for platelet function deficits, and the lifespan of platelets is shortened in some felvinfected cats. thrombocytopenia (resulting in bleeding disorders) can occur secondary to decreased platelet production from felv-induced bone marrow suppression or leukemic infiltration. platelets harbor felv, and megakaryocytes are frequent targets of progressive felv infection. immunemediated thrombocytopenia, which rarely occurs as a single disease entity in cats, often accompanies immune-mediated hemolytic anemia (imha) in cats with underlying felv infection. felv infection also can cause decreased neutrophil or lymphocyte counts. neutropenia is common in felv-infected cats [ ] and generally occurs alone or in conjunction with other cytopenias. in some cases, myeloid hypoplasia of all granulocytic stages is observed, suggesting infection on neutrophil precursors. in some neutropenic felv-infected cats, an arrest in bone marrow maturation can occur at the myelocyte and metamyelocyte stages. it has been hypothesized that an immune-mediated mechanism is responsible in cases in which neutrophil counts recover with glucocorticoid treatment ("glucocorticoidresponsive neutropenia"). hematopoietic neoplasia ("myeloprolifertaive disorders"), including leukemia, can also cause bone marrow suppression syndromes by crowding out. myelodysplastic syndrome (mds), characterized by peripheral blood cytopenias and dysplastic changes in the bone marrow, is a pre-stage of acute myeloic leukemia. it was found that changes of the ltr region of the felv genome (presence of three tandem direct -bp repeats in the upstream region of the enhancer (ure)) are strongly associated with the induction of mds [ ] . myelofibrosis, another cause of bone marrow suppression, is a condition characterized by abnormal proliferation of fibroblasts resulting from chronic stimulation of the bone marrow, such as chronic bone marrow activity from hyperplastic or neoplastic regeneration caused by felv. in severe cases, the entire endosteum within the medullary cavity can be obliterated. feline panleukopenia-like syndrome (fpls), also known as felv-associated enteritis (fae) or myeloblastopenia, consists of severe leukopenia (< cells/μl) with enteritis and destruction of intestinal crypt epithelium that mimics feline panleukopenia caused by feline panleukopenia virus (fpv) infection. however, fpv antigen has been demonstrated by ifa in intestinal sections of cats that died from this syndrome after being experimentally infected with felv [ ] . fpv was also demonstrated by electron microscopy despite negative fpv antigen tests. it appears that this syndrome might actually not be caused by felv itself, as previously thought, but by co-infection with fpv. the syndrome also has been referred to as fae in cats with progressive felv infection because the clinical signs observed are usually gastrointestinal, including hemorrhagic diarrhea, vomiting, oral ulceration or gingivitis, anorexia, and weight loss [ , ] . it is still unclear whether all theses syndromes have the same origin and are simply caused by co-infection with fpv (and even modified life fpv vaccines have been discussed) or if they are caused by felv itself [ ] . although cytopenias caused by bone marrow suppression are a common finding in felv infection, these are rather uncommon in fiv-infected cats. during the acute phase of infection, fiv-infected cats can exhibit mild neutropenia, which resolves as the cat progresses to the asymptomatic phase of infection. clinically ill fiv-infected cats in a later phase of infection may have a variety of cytopenias, with lymphopenia being most common. lymphopenia is caused by direct replication of the virus in cd + lymphocytes. anemia and neutropenia (usually mild) may also be seen [ , ] , although these abnormalities may be as much a reflection of concurrent disease as direct effects of fiv itself. a recent study in a high number ( ) of client-owned field cats compared hematologic parameters in fivinfected, felv-infected and uninfected cats [ ] . anemia and thrombocytopenia were not significantly more common in fiv-infected versus uninfected cats. only neutropenia was significantly more often present, in about % of fiv-infected cats. soluble factors have been shown to inhibit bone marrow function in fiv-infected cats, and bone marrow infection has been associated with decreased ability to support hematopoietic potential in vitro or has been proposed as a mechanism underlying the development of cytopenias [ ] . neurologic dysfunction may be present in felv-and in fiv-infected cats and is one of the few syndromes directly caused by the retrovirus. however, mechanisms of neurologic dysfunction are different with both viruses. in felv-infected cats, most neurologic signs are caused by lymphoma and lymphocytic infiltrations in brain or spinal cord leading to compression, but in some cases, no tumor is detectable with diagnostic imaging methods or at necropsy. in these cats, felv-induced neurotoxicity is suspected. anisocoria, mydriasis, central blindness, or horner's syndrome have been described in felv-infected cats without morphologic changes. in some regions (such as the southeastern united states), urinary incontinence caused by neuropathies in felv-infected cats has been described [ ] . direct neurotoxic effects of felv have been discussed as pathogenetic mechanisms. felv envelope glycoproteins may be able to produce increased free intracellular calcium leading to neuronal death (this has also been described in hiv-infected humans). a polypeptide of the felv envelope was found to cause dosedependent neurotoxicity associated with alterations in intracellular calcium ion concentration, neuronal survival, and neurite outgrowth. the polypeptide from a felv-c strain was significantly more neurotoxic than the same peptide derived from a felv-a strain [ , ] . neurologic signs in cats with progressive felv infection consisted of abnormal vocalization, hyperesthesia, and paresis progressing to paralysis. some cats developed anisocoria or urinary incontinence during the course of their illness. others had concurrent felv-related problems such as myelodysplastic disease. the clinical course of affected cats involved gradually progressive neurologic dysfunction. microscopically, white-matter degeneration with dilation of myelin sheaths and swollen axons was identified in the spinal cord and brain stem of affected animals [ ] . immunohistochemical staining of affected tissues revealed consistent expression of felv p antigens in neurons, endothelial cells, and glial cells, and proviral dna was amplified from multiple sections of the spinal cord [ ] . these findings suggest that in some felv-infected cats, the virus may directly affect cns cells cytopathically. neurologic signs also have been described in both natural and experimental fiv infections [ ] [ ] [ ] [ ] [ ] [ ] . about % of symptomatic fiv-infected cats have a neurological disease as a predominant clinical feature. neurologic disorders in fiv infection seem to be strain-dependent [ ] . both central and peripheral neurologic manifestations have been described, comparable to the changes in hiv-infected human beings. dementia in human patients with aids is often characterized by a slight decline in cognitive ability or behavior, changes that may be too subtle to be recognized in cats. neurological abnormalities seen in naturally infected cats tend to be more behavioral than motor. psychotic behavior, twitching movements of the face and tongue, compulsive roaming, dementia, loss of bladder and rectal control, and disturbed sleep patterns have been observed. other signs described include nystagmus, ataxia, seizures, and intention tremors [ ] [ ] [ ] . abnormal forebrain electrical activity and abnormal visual and auditory-evoked potentials have also been documented in cats that appeared otherwise normal [ , , , ] . although the majority of fiv-infected cats do not show clinically overt neurologic signs, a much higher proportion of infected cats have microscopic cns lesions. brain lesions may occur in the absence of massive infection, and abnormal neurologic function has been documented in fiv-infected cats with only mild to moderate histologic evidence of inflammation [ ] . pathologic findings include the presence of perivascular infiltrates of mononuclear cells, diffuse gliosis, glial nodules, and white matter pallor. these lesions are usually located in the caudate nucleus, midbrain, and rostral brain stem [ ] . mostly, abnormal neurologic function is the result of a direct effect of the virus on cns cells. neurologic signs upon fiv infection are highly strain-dependent. the virus infects the brain early, with virus-induced cns lesions sometimes developing within two months of experimental infection [ ] . microglia and astrocytes are infected by fiv, but the virus does not infect neurons. however, neuronal death has been associated with fiv infection; in particular, forebrain signs are often a result of direct neuronal injury from the virus. the exact mechanism of neuronal damage by fiv is unclear but may include neuronal apoptosis, effects on the neuron supportive functions of astrocytes, toxic products released from infected microglia, or cytokines produced in response to viral infection. in vitro studies support the hypothesis that fiv infection may impair normal metabolism in cns cells, particularly astrocytes [ ] . documented abnormalities of astrocyte function include altered intercellular communication, abnormal glutathione reductase activity that could render cells more susceptible to oxidative injury, and alterations in mitochondrial membrane potential that disrupt the energy-producing capacities of the cell [ ] . astrocytes are by far the most common cell type of the brain and are important in maintaining cns neuronal vascular microenvironment. one of the most important functions of astrocytes is to regulate the level of extracellular glutamate, a major excitatory neurotransmitter that accumulates as a consequence of neuronal activity. excessive extracellular glutamate often results in neuronal toxicity and death. fiv infection of feline astrocytes can significantly inhibit their glutamate-scavenging ability, potentially resulting in neuronal damage [ , ] . sometimes, neurologic signs may also be caused by opportunistic infections such as toxoplasmosis, cryptococcosis, or fip. the most clinically important consequence of both retrovirus infections is immunosuppression. immunosuppression can lead to secondary infectious diseases accounting for most clinical signs, but also can lead to decreased tumor surveillance mechanisms causing an increased risk of tumor development. it is important to realize that many of these secondary diseases in felv-and fivinfected cats are treatable. the mechanisms that cause the immunosuppression are different for the two infections. many felv-infected cats have concurrent bacterial, viral, protozoal, and fungal infections, but few controlled studies exist proving that these cats have a higher rate of infection than felv-negative cats. thus, although felv certainly can suppress immune function, it should not be assumed that all concurrent infections are a direct consequence of felv infection. progressively felv-infected cats develop immunosuppression similar to that in hiv-infected people. the exact mechanisms of how the virus destroys the immune system are poorly understood, as is why different animals have such varying degrees of immunosuppression. immunosuppression has been associated with non-integrated viral dna from replication-defective viral variants [ ] . these pathogenic immunosuppressive variants, such as felv-t, require a membrane-spanning receptor molecule (pit ) and a second co-receptor protein (felix) to infect t lymphocytes [ ] . the latter protein is an endogenously expressed protein encoded by an endogenous provirus arising from felv-a, which is similar to the felv receptor-binding protein of felv-b [ ] . felv-infected cats may develop thymic atrophy and depletion of lymph node paracortical zones following infection. lymphopenia and neutropenia are common. in addition, neutrophils of viremic cats have decreased chemotactic and phagocytic function compared with those of normal cats. in some cats, lymphopenia may be characterized by preferential loss of cd + helper t cells, resulting in an inverted cd /cd ratio (as typically seen in fiv infection) [ , ] , but more commonly, substantial losses of helper cells and cytotoxic suppressor cells (cd + cells) occur [ ] . many immune function tests of naturally felv-infected cats are abnormal, including decreased response to t-cell mitogens, prolonged allograft reaction, reduced immunoglobulin production, depressed neutrophil function, and complement depletion. il- and il- are decreased in some cats [ , ] , but felv does not appear to suppress il- production from infected macrophages. ifn-γ may be deficient or increased. increased tnf-α has been observed in serum of infected cats and in infected cells in culture. each cytokine plays a vital role in the generation of a normal immune response, and the excess production of certain cytokines, such as tnf-α, can also cause illness. t-cells of felvinfected produce significantly lower levels of b-cell stimulatory factors than do those of normal cats (this defect becomes progressively more severe over time) [ ] , but when b-cells of felv-infected cats are stimulated in vitro by uninfected t-cells, their function remains normal. primary and secondary humoral antibody responses to specific antigens are decreased and may occur delayed in felv-infected cats. in vaccination studies, felv-infected cats were not able to mount an adequate immune response to vaccines, such as rabies. therefore, protection in a felv-infected cat after vaccination is not complete and not comparable to that in a healthy cat;thus, more frequent vaccinations (e.g., every six months) have to be considered. in fiv-infected cats, immunosuppression usually occurs in later stages of the infection, and leads to predisposition for secondary infections. in a survey study of naturally fiv-infected cats examined at north american veterinary teaching hospitals, the most common disease syndromes were stomatitis, neoplasia (especially lymphoma and cutaneous squamous cell carcinoma), ocular disease (uveitis and chorioretinitis), anemia and leukopenia, opportunistic infections, renal insufficiency, lower urinary tract disease, and endocrinopathies, such as hyperthyroidism and diabetes mellitus [ ] . some of these problems, however, are most likely associated rather with the older age at which these cats presented (e.g., endocrinopathies, renal insufficiency) than with their fiv infection. infections with many different "opportunistic" pathogens of viral, bacterial, protozoal, and fungal origin have been reported in fiv-infected cats. few studies, however, have compared the prevalence of most of these infections in fiv-infected and non-infected cats, and thus, their relevancy as true secondary invaders is unclear. the most important immunologic abnormality shown in experimental [ ] [ ] [ ] as well as in natural [ , ] infection is a decrease in the number and relative proportion of cd + cells in the peripheral blood as well as in most primary lymphoid tissues [ ] . loss of cd + cells leads to inversion of the cd /cd ratio. in addition, an increase in the proportion of cd + cells also contributes to the inversion [ , , ] , in particular a population referred to as "cd + alpha-hi, beta-low cells" [ ] [ ] [ ] , a subset of cd + cells that may contribute to suppression of viremia in fivinfected cats. causes of cd + cell loss include decreased production secondary to bone marrow or thymic infection, lysis of infected cells induced by fiv itself (cytopathic effects), destruction of virusinfected cells by the immune system, or death by apoptosis (cell death that follows receipt of a membrane signal initiating a series of programmed intracellular events) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the degree of apoptosis correlates inversely with the cd + numbers and the cd /cd ratio [ ] . fiv env proteins are capable of inducing apoptosis in mononuclear cells by a mechanism that requires cxcr binding [ ] . ultimately, loss of cd + cells impairs immune responses, because cd + cells have critical roles in promoting and maintaining both humoral and cell-mediated immunity. a certain subset of cd + cells, the "treg" (for t-regulatory cells), also seems to play an important role, and treg cells with suppressive activity have been documented during early [ ] and chronic fiv infection [ ] . in fiv-infected cats, increased activity of treg cells could thus play a role in suppressing immune responses to foreign antigens or pathogens. in addition, treg cells are themselves targets for fiv infection [ , ] , and may serve as a fiv reservoir during the latent stage of infection and be capable of stimulating virus production [ ] . in addition, other immunologic abnormalities can be found. lymphocytes may lose the ability to proliferate in response to stimulation with mitogens or antigens, and priming of lymphocytes by immunogens may be impaired [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . lymphocyte function may be reduced by altered expression of cell surface molecules, such as cd , major histocompatibility complex ii antigens, or cytokines and cytokine receptors [ ] [ ] [ ] [ ] [ ] , or through over-expression of abnormal molecules, such as receptors [ ] , leading to disrupted production of cytokines or receptor function. impaired neutrophil adhesion and emigration in response to bacterial products have been described in fiv-infected cats [ ] [ ] [ ] . natural killer cell activity may be diminished [ ] or increased [ ] , in acutely or asymptomatically infected cats, respectively. changes in cytokine pattern include increased production of ifn-γ, tnf-α, il- , il- , il- , and il- [ ] [ ] [ ] [ ] , but also differences in cytokine ratios (e.g., il- /il- ratio) [ , ] . in addition to a dysregulation of the immune system leading to immunosuppression, retrovirusinfected cats can also develop immune-mediated diseases caused by an overactive immune response. the most commonly seen immune-mediated response is a hypergammaglobulinemia which is caused by an excessive antibody response against the chronic persistent infection. the produced antibodies are not neutralizing and thus, may lead to antigen antibody complex formation. these immune complexes can deposit, usually in narrow capillary beds, leading to glomerulonephritis, polyarthritis, uveitis, and vasculitis. secondary immune-mediated diseases are more commonly seen in fiv-than in felv-infected cats. when comparing plasma electrophoretograms, felv-infected cats do not show hypergammaglobulinemia and hyperproteinemia significantly more often than non-infected cats, whereas in fiv-infection, hypergammaglobulinemia and hyperproteinemia occur significantly more commonly [ , ] . nevertheless, immune-mediated diseases have been described in felv-infected cats as well. while humoral immunity to specific stimulation decreases during the course of felv infection, nonspecific increases of igg and igm have been noted. the loss of t-cell activity in combination with the formation of antigen antibody complexes promotes immune dysregulation [ ] . immunemediated diseases described in felv-infected cats include imha [ ] , glomerulonephritis [ ] , uveitis with immune complex deposition in iris and ciliary body [ ] , as well as polyarthritis [ ] . chronic progressive polyarthritis can be triggered by felv; in about % of cats with polyarthritis, felv seems to be an associated agent [ ] . measurement of felv antigen has shown that cats with glomerulonephritis have more circulating viral proteins than do other felv-infected cats. antigens that can lead to antigen antibody complex formation include not only whole virus particles, but also free gp , p , or p e proteins [ , ] . immune-mediated diseases observed in fiv-infected cats are caused by an excessive immune response leading to hypergammaglobulinemia [ , , ] . hypergammaglobulinemia reflects polyclonal b-cell stimulation and is a direct consequence of fiv infection, because experimentally fiv-infected specific pathogen-free (spf) healthy cats also develop hypergammaglobulinemia [ ] . increased igg as well as circulating immune complexes have been detected in fiv-infected cats [ ] . chronic ulcero-proliferative gingivostomatitis is very common in retrovirus-infected cats, especially in those with fiv infection. in cats naturally infected with fiv, it is the most common syndrome (affecting up to %). it characteristically originates in the fauces and spreads rostrally, especially along the maxillary teeth. histologically, the mucosa is invaded by plasma cells and lymphocytes, accompanied by variable degrees of neutrophilic and eosinophilic inflammation. lesions are often painful, and tooth loss is common. severe stomatitis can lead to anorexia and emaciation. the cause of this syndrome is unclear, but the histologic findings suggest an immune response to chronic antigenic stimulation or immune dysregulation. circulating lymphocytes of cats with stomatitis have increased expression of inflammatory cytokines [ ] , further implicating immune activation in the pathogenesis of this condition. this type of stomatitis is not always correlated with felv or fiv infection [ ] , and is usually not seen in spf cats experimentally infected with felv or fiv, suggesting that exposure to other infectious agents also plays a role [ ] . concurrent feline calicivirus (fcv) infection is often identified in the oral cavity of these cats, and experimental and naturally occurring co-infection of fiv and fcv infection results in more severe disease [ , ] . felv can cause severe clinical syndromes, and progressive felv infection is associated with a decrease in life expectancy. still, many owners still elect to provide therapy for their felv-infected cats, and with proper treatment, felv-infected cats, especially in indoor-only households, may live for many years with good quality of life. diseases secondary to immunosuppression account for a large portion of the syndromes seen in felv-infected cats, and it is important to realize that many of these secondary diseases are treatable. in most naturally infected cats, fiv does not cause a severe clinical syndrome. most clinical signs in fiv-infected cats reflect secondary diseases, such as infections and neoplasia, to which fiv-infected cats are more susceptible. with proper care, fiv-infected cats can live many years and, in fact, commonly die at an old age from causes unrelated to their fiv infection. while long-term studies describing clinical outcomes of naturally occurring felv and fiv infection are lacking, modalities for treatment of secondary infections or other co-incident diseases are available, and by treating these symptomatically, the life expectancy and quality of life of felv-and fivinfected animals can be significantly improved. rare, direct influence of the virus (specific fiv strains), impairment of astrocyte function immunodeficiency common, several mechanisms, e.g., replication of virus in all bone marrow cells (including neutrophils), changes in cytokine pattern common, several mechanisms, e.g., decrease in cd + cells, changes in cytokine pattern immune-mediated diseases rare, e.g., immune-mediated hemolytic anemia sometimes, hyperglobulinemia common with immune complex deposition leading to e.g., 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perinatally infected with the jsy molecular clone of feline immunodeficiency virus constitutive expression of types and cytokines by alveolar macrophages from feline immunodeficiency virus-infected cats effect of feline immunodeficiency virus on cytokine response to listeria monocytogenes in vivo altered bone marrow dendritic cell cytokine production to toll-like receptor and cd ligation during chronic feline immunodeficiency virus infection plasma electrophoretogram in feline immunodeficiency virus (fiv) and/or feline leukaemia virus (felv) infections feline leukemia virus infection primary immune-mediated hemolytic anemia in cats: diagnosis, therapy, and outcome membranous glomerulonephritis associated with leukaemia in cats ocular disease in felv-positive cats: cases ( - ) circulating immune complexes associated with naturally occurring lymphosarcoma in pet cats detection of circulating immune complexes by a clq/protein a-elisa during the preneoplastic stages of feline leukemia virus infection polyclonal b-cell activation in cats infected with feline immunodeficiency virus serum concentration of circulating immune complexes in cats infected with feline immunodeficiency virus detected by immune adherence hemagglutination method evaluation of the association of bartonella species, feline herpesvirus , feline calicivirus, feline leukemia virus and feline immunodeficiency virus with chronic feline gingivostomatitis feline immunodeficiencyvirus chronic oral infections of cats and their relationship to persistent oral carriage of feline calici-, immunodeficiency, or leukemia viruses effect of chronic feline immunodeficiency virus infection on experimental feline calicivirus-induced disease many doctoral students have contributed to various parts of the author's publications cited in this paper and are acknowledged for all their hard work. the authors declare no conflict of interest. key: cord- -qrddwebe authors: sebina, ismail; phipps, simon title: the contribution of neutrophils to the pathogenesis of rsv bronchiolitis date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qrddwebe acute viral bronchiolitis causes significant mortality in the developing world, is the number one cause of infant hospitalisation in the developed world, and is associated with the later development of chronic lung diseases such as asthma. a vaccine against respiratory syncytial virus (rsv), the leading cause of viral bronchiolitis in infancy, remains elusive, and hence new therapeutic modalities are needed to limit disease severity. however, much remains unknown about the underlying pathogenic mechanisms. neutrophilic inflammation is the predominant phenotype observed in infants with both mild and severe disease, however, a clear understanding of the beneficial and deleterious effects of neutrophils is lacking. in this review, we describe the multifaceted roles of neutrophils in host defence and antiviral immunity, consider their contribution to bronchiolitis pathogenesis, and discuss whether new approaches that target neutrophil effector functions will be suitable for treating severe rsv bronchiolitis. respiratory syncytial virus (rsv) is the leading viral cause of lower respiratory infections (lri) among young infants [ , ] . in , an estimated million cases and , deaths due to rsv infection were reported worldwide [ ] . the estimated cost of treating severe cases of rsv-induced bronchiolitis is over $ million per year [ ] , and an fda-approved vaccine against rsv remains elusive [ , ] . a greater understanding of the molecular mechanisms underlying the immunopathogenesis of rsv-bronchiolitis may reveal novel pathways that can be targeted for improving treatment therapies for severe rsv patients. severe viral bronchiolitis in infancy is a major independent risk factor for the development of chronic asthma in later life [ ] . therefore, ameliorating severe bronchiolitis in infancy may also act as a preventative strategy for later asthma in susceptible individuals. the prevailing view is that severe bronchiolitis results from excessive inflammation and consequent immunopathology, rather than virus-induced cytology. neutrophils are the dominant inflammatory cell in the airways of paediatric patients with rsv bronchiolitis [ ] [ ] [ ] , accounting for up to % of the cellular infiltrate at peak symptomatology [ ] , and yet the precise contribution of neutrophils to antiviral immunity remains ill-defined. in the context of anti-bacterial or anti-fungal immunity, neutrophils mediate protection through the production of antimicrobial peptides, respiratory oxygen species (ros), cytokines and chemokines, and the formation of neutrophil extracellular traps (nets). new information from omics-based technologies and advances in multi-parameter flow cytometry are shedding new light on neutrophil diversity and function, suggesting a greater complexity to a cell that is often unfavourably viewed as a blunt tool on account of its spectacular prowess at microbial killing and short life-span. here, we review the multifaceted roles of the neutrophil in host defence, antiviral immunity, and immunopathogenesis in the context of viral lris. we find that neutrophils make an important contribution to innate antiviral immunity and help to optimise the induction of an effective adaptive immune response. however, as a consequence of their ability to initiate and amplify the magnitude of an inflammatory reaction, it is clear that a failure to adequately regulate this response can lead to excessive collateral damage and a loss of tissue function, leading to significant morbidity from an rsv infection. we consider a number of novel approaches that might be employed to either harness or suppress the actions of neutrophils to promote antiviral immunity and/or ameliorate the immunopathology associated with severe rsv infection. an estimated four billion neutrophils are produced in the bone marrow every hour [ , ] , and accordingly, neutrophils are by far the most abundant leukocyte in blood, representing approximately % of the total white blood cell count. as well as circulating in large numbers, neutrophils enter and surveil tissues in the steady state [ ] . neutrophils are one of the host's first defensive measures against a sterile or microbial insult. neutrophils employ three major defensive mechanisms: phagocytosis, degranulation (secreting an array of anti-microbial peptides), and netosis [ , ] . during phagocytosis, neutrophils utilise a combination of surface-expressed opsonic receptors, intracellular signalling cascades, and cytoskeletal rearrangements to engulf and internalise microbes [ , ] . neutrophils contain cytoplasmic granules, which store antimicrobial molecules and facilitate cross-talk with other immune cells [ ] . these granules (summarised in table ) are subdivided into primary or azurophilic granules (containing, e.g., myeloperoxidase, cd ), secondary or specific granules (containing, e.g., lipocalin- and lactoferrin), and tertiary or gelatinase granules (containing, e.g., cathelicidins and neutrophil collagenase) [ ] . this classification is based on their production during granulopoiesis and expression of distinct protein markers [ ] . as a consequence of phagocytosis, the internalised microbe is trapped in 'bag-like' structures known as phagosomes, which fuse with neutrophil granules for the safe destruction of microbes [ ] . upon fusion, antimicrobial molecules (summarised in table ) are released into the phagosome, whereupon they kill the trapped microbe [ , ] . table . neutrophil granules and their anti-microbial properties. neutrophils can also eliminate microbes by releasing extracellular dna traps composed of decondensed nuclear chromatin, histones, and protein granules, in a process termed netosis [ , ] . classical activation of netosis depends on nadph oxidase-mediated production of ros [ ] , which promotes chromatin decondensation via histone deamination or citrullination. this process is aided by the downstream activation of the protein-arginine deiminase type (pad ), a nuclear enzyme that citrullinates arginine residues, converting amine groups to ketones [ ] [ ] [ ] . ros production can also activate azurophilic granules to release myeloperoxidase (mpo) and neutrophil elastase (ne). if these enzymes translocate to the nucleus, they can disrupt chromatin packaging [ , ] , and promote the release of chromatin, which then interacts with other granular and cytosolic proteins to form 'nets' [ ] . these nets are important for immunity against fungi and some bacterial species [ , , ] . for example, people who are unable to produce nets develop chronic granulomatous disease and are highly susceptible to invasive aspergillosis [ , ] . similarly, individuals lacking mpo are susceptible to recurrent fungal infections [ ] . in pad -deficient mice, neutrophil killing of shigella flexneri or group a streptococcus is diminished [ ] . collectively, these studies demonstrate that netosis is an important weapon in the host's defensive armoury. however, if left unchecked, net-induced responses may also mediate severe pathology. excessive netosis can damage the epithelium in pulmonary aspergillosis [ ] , damage the endothelium in transfusion-related acute lung diseases [ ] , and exacerbate rhinovirus infection-induced allergic asthma [ ] . therapeutic approaches to block netosis are now being assessed for the treatment of infectious diseases (including bacterial sepsis), autoimmune diseases (including systemic lupus erythematosus [ ] , rheumatoid arthritis [ ] ) and chronic lung disorders (such as cystic fibrosis [ ] ). neutrophils are a vital component of the innate immune system, with key roles in pathogen recognition, the killing of invading pathogens, the presentation of antigens to t-cells, the recruitment of other inflammatory cells, and the production of cytokines [ , ] . for sensing invading pathogens, neutrophils employ a vast array of pattern recognition receptors (prrs), including toll-like receptors (tlrs), c-type lectin receptors (e.g., dectin- ) and cytoplasmic sensors of ribonucleic acids (rig-i and mda ) [ , , ] . neutrophils also express nucleotide-binding oligomerisation domain (nod)-like receptors, important for inflammasome formation [ ] . the sensing of pathogens through these prrs activates the effector functions of neutrophils, including ros production, net formation, and degranulation (as highlighted in section ). activated neutrophils may also influence the quality of innate and adaptive immune responses by affecting the trafficking and function of other innate cells, such as dendritic cells (dcs) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, the neutrophil-derived chemokine ccl supports the rapid recruitment of dcs to the inoculation site during leishmania major infection [ ] . dc function can also be affected: in the context of an infection with toxoplasma gondii, neutrophil-depletion attenuated interleukin (il)- and tnf production by splenic dcs [ ] . neutrophils also affect the recruitment of other cells too, which they accomplish via the secretion of pro-inflammatory mediators such as danger-associated molecular patterns (damps; e.g., high mobility group box (hmgb ), double stranded dna, and s complex proteins), pro-inflammatory cytokines (e.g., il- α, il- β, il- , il- , and tnf) and chemokines (e.g., cxcl , cxcl , cxcl , ccl , and ccl ) [ , , ] . neutrophils can also initiate adaptive immune responses by directly presenting antigens themselves, or by acting as accessory cells, to support t-cell responses. for instance, human neutrophils upregulate mhc-ii and co-stimulatory molecule (cd and cd ) expression and can present antigens to cd + t-cells following phagocytosis [ ] . neutrophil acquisition of these antigen-presenting properties (mhc-ii, cd , and cd expression) is associated with increased activation and proliferation of cd + t-cells in response to tetanus toxoid. neutrophils can also direct t-cell recruitment to the site of infection. for example, in response to influenza virus infection, lung-infiltrating neutrophils were found to deposit a long-lasting chemoattracting trail (expressing the chemokine cxcl ) in the lung to guide antigen-specific cd + t-cells into specific niches [ ] . in the absence of neutrophils, influenza-specific cd + t-cells were lower in the lung, leading to increased viral load and delayed viral clearance. neutrophils can also influence cd + t cell helper (th) responses, in particular, th immune responses [ , , ] . in a mouse model of allergic asthma, neutrophil cytoplasts (enucleated cell bodies) augmented dc-mediated th responses in the lymph nodes, which subsequently increased asthma-like pathology in the lung [ ] . collectively, these studies demonstrate that neutrophils are important participants in innate immunity and contribute to effective adaptive immune responses. the vast majority of human respiratory viruses, including rsv, rhinovirus, influenza, coronavirus, adenovirus, and parainfluenza virus, can cause bronchiolitis [ ]. however, rsv-induced bronchiolitis is the leading cause of hospitalisation and death among infants within the first two years of life [ , ] . rsv is highly contagious and persists outside of the host for almost six hours [ ] . this prolonged survival facilitates its spread to susceptible individuals, mainly via inoculation of open mucous membranes lining the eyes and buccal cavity. upon inoculation, rsv infects the nasopharyngeal epithelium of the upper respiratory tract, replicates in epithelial cells, and then spreads to the lrt via the bronchiolar epithelium [ , ] . this occurs within - days post infection, with peak infectivity occurring at - days post-inoculation [ , ] . during this period, rsv triggers extensive inflammation (characterised by increased neutrophilia and levels of inflammatory cytokines), mucus hypersecretion, and oedema in the airways [ , , ] . in severe cases, increased mucus production and deposition of cellular debris can occlude the bronchiole lumen, contributing to bronchiolar obstruction, air trapping, and lobar collapse [ ] . patients with severe disease experience dyspnea, wheezing, and cough, the latter often persisting for three or more weeks. mild cases of bronchiolitis are manageable in outpatient departments. however, severe disease can be life threatening, necessitating urgent mechanical ventilation and admission to intensive care units for some patients. individuals with severe rsv-induced bronchiolitis during infancy are more likely to develop impaired lung function, recurrent wheezing, and asthma in adulthood [ ] . whether neutrophils play a beneficial or detrimental role during rsv bronchiolitis remains difficult to ascertain clinically due to difficulties in sampling young infants. however, the predominance of neutrophils in the airway wall and alveoli of lung autopsy samples from fatal cases of rsv-related lri [ , ] or in the airways of paediatric patients (~ % of the total cell infiltrate) with severe rsv-induced bronchiolitis [ ] would suggest that they are not innocent bystanders. we postulate that neutrophils promote lung pathophysiology during rsv bronchiolitis through the production of pro-inflammatory cytokines and the releasing of cytotoxic molecules (illustrated in figure ). these products, released through distinct cellular processes (e.g., netosis, degranulation) contribute to mucus hyperproduction, airway epithelial cell (aec) death and sloughing, and oedema ( figure ). in rsv-infected mice, the depletion of neutrophils decreases ne, mpo, and mmp levels in the airways [ ] . depletion of neutrophils has also been shown to lower tnfα levels and attenuate airway mucin production in the lungs of rsv infected mice [ ] . in addition, in vivo antagonism of cxcr (a chemokine receptor important for neutrophil recruitment into the lung) reduces mucus production in the airways of rsv-infected mice [ ] . in vitro, co-culture of rsv-infected aecs with neutrophils increases aec damage compared with rsv-infected aecs cultured without neutrophils [ ] . collectively, these findings suggest that neutrophils can contribute to pathogenesis; however, a limitation of modelling rsv in mice is that the virus replicates poorly, and hence this is not a particularly tractable system to assess the beneficial properties of neutrophils, such as their antiviral activities. ( ), degranulation ( ), respiratory oxygen species (ros) production ( ), and the release of neutrophil extracellular traps (netosis) ( ) are associated with increased lung inflammation, systemic fever, mucus hypersecretion, airway obstruction, and epithelial cell death. together, these factors contribute to increased lung damage during severe rsv bronchiolitis. infantile severe viral bronchiolitis remains a major risk factor for persistent wheezing and chronic asthma in childhood [ , ] . therefore, excessive neutrophilic inflammation in infants with severe rsv bronchiolitis may contribute to the onset of type inflammation and cause long-term defects in lung development that predispose susceptible infants to the development of asthma in later childhood. thus, regulating excessive neutrophilic responses during severe rsv-bronchiolitis might protect against the loss of lung function, curtail the development of allergic sensitisation, and reduce the prevalence of asthma. in light of this, it is important to understand ( ) the molecular events governing neutrophil recruitment to the lung, ( ) the type of neutrophils recruited to the lung, ( ) the molecular signals that control optimal neutrophil activation and function, and ( ) whether specific neutrophil subtypes can be targeted therapeutically to improve the management of severe rsv bronchiolitis. we discuss these questions in the sections below. the earliest stages of neutrophil recruitment are initiated by damps (table ) , including hmgb , dna, and histones, which are released by damaged or dying cells [ ] . indeed, hmgb is elevated in the nasopharynx of rsv-infected children compared with those infected with other viruses [ ] . animal models of rsv-bronchiolitis have improved our knowledge on the mechanisms of disease development and progression (reviewed extensively in [ ] [ ] [ ] ); however, as figure . potential roles of neutrophils in the pathophysiology of severe respiratory syncytial virus (rsv) bronchiolitis. excessive neutrophil-derived inflammatory cytokine production ( ), degranulation ( ), respiratory oxygen species (ros) production ( ), and the release of neutrophil extracellular traps (netosis) ( ) are associated with increased lung inflammation, systemic fever, mucus hypersecretion, airway obstruction, and epithelial cell death. together, these factors contribute to increased lung damage during severe rsv bronchiolitis. infantile severe viral bronchiolitis remains a major risk factor for persistent wheezing and chronic asthma in childhood [ , ] . therefore, excessive neutrophilic inflammation in infants with severe rsv bronchiolitis may contribute to the onset of type inflammation and cause long-term defects in lung development that predispose susceptible infants to the development of asthma in later childhood. thus, regulating excessive neutrophilic responses during severe rsv-bronchiolitis might protect against the loss of lung function, curtail the development of allergic sensitisation, and reduce the prevalence of asthma. in light of this, it is important to understand ( ) the molecular events governing neutrophil recruitment to the lung, ( ) the type of neutrophils recruited to the lung, ( ) the molecular signals that control optimal neutrophil activation and function, and ( ) whether specific neutrophil subtypes can be targeted therapeutically to improve the management of severe rsv bronchiolitis. we discuss these questions in the sections below. the earliest stages of neutrophil recruitment are initiated by damps (table ) , including hmgb , dna, and histones, which are released by damaged or dying cells [ ] . indeed, hmgb is elevated in the nasopharynx of rsv-infected children compared with those infected with other viruses [ ] . animal models of rsv-bronchiolitis have improved our knowledge on the mechanisms of disease development and progression (reviewed extensively in [ ] [ ] [ ] ); however, as pneumoviruses are host-specific, a major limitation with using mice to study human rsv stems from its inability to replicate efficiently in this setting. to better understand the pathogenic processes that underlie viral bronchiolitis, we and others have preferred to inoculate mice with pneumonia virus of mice (pvm), which is orthologous to human rsv [ , ] . because pvm is a natural pathogen in rodents, a low inoculum dose (e.g., pfu) can be employed, and this allows for greater insights into host-pathogen interactions over time as the virus replicates in the airway epithelium. importantly, inoculation with a low dose of pvm can induce the more severe pathologies of infant bronchiolitis, such as alveolar epithelial cell apoptosis, bronchial epithelial necrosis, multifocal acute alveolitis, intra-alveolar oedema, haemorrhage, and increased neutrophilia [ , , ] . these pathological features are similar to those observed in autopsy samples obtained from fatal cases of rsv bronchiolitis [ ] . although there are limitations with the use of pvm (highlighted in [ ] ), it is proving a useful model to understand the cellular and molecular processes that underlie pathogenesis. unlike wild-type (wt) control mice, plasmacytoid dendritic cell (pdc)-depleted, toll-like receptor (tlr) -deficient, or interferon regulatory factor (irf) -deficient neonatal mice develop severe pathology, characterised by increased neutrophilia and lung inflammation in response to acute pvm infection [ ] [ ] [ ] . in the absence of the tlr -irf signalling pathway, massive amounts of preformed airway epithelial cell hmgb is released into the extracellular environment, increasing the level of neutrophilic inflammation and amplifying tissue pathology, most notably tissue oedema, epithelial sloughing, and cell death [ , , , ] . the cell death occurs through programmed necroptosis and further increases the levels of hmgb , perpetuating the inflammatory response [ ] . rsv infection of primary human airway epithelial cells also induces necroptosis-dependent hmgb release [ ] . intriguingly, therapeutic inhibition of necroptosis during severe viral bronchiolitis protected mice against the subsequent development of experimental asthma in later-life [ ] , indicating that severe bronchiolitis is causally associated with subsequent asthma. examination of the infant bronchiolitis revealed that pharmacological inhibition of necroptosis or immunoneutralisation of hmgb decreases the expansion of il- -producing type- innate lymphoid cells (ilc s), and prevents alterations to the airway wall such as thickening of the airway smooth muscle (asm) layer [ , ] . in vitro, asm cells cultured with il- /il- -activated ilc s isolated from the lungs of pvm-infected mice undergo increased cell division, elucidating a cellular and molecular pathway by which excessive inflammation and immunopathology promotes both type inflammation and an aberrant repair response that alters the architecture of the developing lung [ ] . therefore, therapeutic targeting of the hmgb signalling axis may act as a novel asthma preventative by dampening ilc -mediated type- inflammation and associated asm remodelling. low circulating pdc numbers in infancy are associated with acute lris [ ] . interestingly, the temporal depletion of pdc in neonatal mice predisposes to severe pvm bronchiolitis due to a lack of immunoregulation by regulatory t-cells [ ] . the adoptive transfer of splenic regulatory t-cells to infected pdc-depleted neonatal mice decreased hmgb and il- levels, epithelial sloughing, and lung neutrophil infiltration. despite the lack of effect on viral load, the attenuated inflammatory response improved the clinical score. additionally, the adoptive transfer of regulatory t-cells in early life was sufficient to protect against the later development of experimental asthma [ ] . these data suggest that therapies that enhance immunoregulation will both ameliorate the severity of bronchiolitis and break its nexus with the development of asthma. damps can indirectly promote neutrophilic inflammation by inducing the production of neutrophil-active chemoattractants (table ) , such as il- [ ] and leukotriene b (ltb ) [ ] . il- levels correlate with neutrophil numbers in the airways and are elevated in nasal washes of rsv-infected children [ ] . moreover, genetic polymorphisms in the il- -encoding gene that increase il- production are associated with increased severity of rsv bronchiolitis [ ] , and pharmacological inhibition of il- in humans reduces neutrophilia and improves clinical outcomes [ ] . levels of the eicosanoid ltb are elevated in endotracheal aspirates of infants with rsv bronchiolitis compared with healthy controls [ ] , and are associated with increased neutrophilic responses [ ] . treatment of rsv-infected mice with the leukotriene inhibitor zileuton reduces cellular inflammation (including neutrophils) in the lung, prevents rsv-induced weight loss and decreases airway pathology compared with untreated control mice [ ] . mice lacking ccl or its receptor ccr display significantly reduced numbers of airway neutrophils in response to infection with pvm [ , ] . moreover, the blockade of ccl /ccr in combination with antiviral therapy improves the survival of mice infected with a lethal pvm dose [ ] . damps can also promote γδ-t cells [ ] and cd + th cells [ ] to produce il- a [ ] [ ] [ ] , which is elevated in nasal aspirates from rsv-infected infants in comparison with uninfected controls [ ] . il- a augments neutrophil recruitment into the lung by stimulating the production of il- and leukotrienes by microvascular endothelial and lung epithelial cells [ , ] . in addition, il- activates p mapk-induced expression of endothelial adhesion markers (e-selectin, vcam- , and icam- ) on lung endothelial cells, promoting trans-endothelial migration of neutrophils. lack of il- in rsv-infected mice reduces neutrophil numbers in the lung, limits mucus production, and improves the cytotoxic t-cell (ctl) responses against rsv [ ] . however, the mechanism by which il- deficiency enhances ctl responses to rsv remains unknown. nonetheless, inhibiting il- signalling may prevent neutrophil-induced inflammation and pathology during rsv infection. induce secretion of pro-inflammatory cytokines, drive ilc responses, induce necroptosis and aec death [ , ] neutrophils have traditionally been viewed as a homogenous cell population. however, it is now appreciated that diversity exists at the molecular, phenotypic, and functional level [ , , , ] . this heterogeneity is a consequence of differences in proliferative capacity, maturation status, transcriptional and epigenetic properties, and environmental cues in tissues [ ] . in one study that employed fucci- reporter mice to identify cells at different stages of the cell cycle, subsequent mass cytometry and transcriptomic analysis revealed the existence of three distinct neutrophil subsets in the bone marrow [ ] . these subsets consisted of a highly proliferative precursor that differentiated into two non-proliferating subsets, one immature and one mature. the precursor neutrophils were ly gand cxcr -negative, but expressed high levels of ckit and cxcr . the immature subset displayed low levels of surface ly g and cxcr and was positive for cxcr . the mature subset displayed higher levels of ly g, cxcr , and cd expression and lacked surface cxcr . immature and mature neutrophil subsets were subsequently identified in human blood, with immature neutrophils associating with increased tumour burden [ ] , suggesting that neutrophil heterogeneity influences disease outcomes. importantly, different neutrophil subsets have been identified in circulation and in the lung during acute rsv-bronchiolitis [ , ] . flow cytometric analysis revealed four distinct neutrophil subsets according to their differential expression of cd and cd l [ ] . these included a mature subset (defined as cd hi cd l hi ), an immature one (cd lo cd l hi ), a suppressive subset (cd hi cd l lo ), and a progenitor subset (cd lo cd l lo ). the relative frequency of these subsets changed over the course of infection, suggesting that they may play different roles, although functional differences between these subsets remain to be shown. in a separate study in rsv-infected patients, geerdink and colleagues identified that neutrophils in balf, compared to those in blood, displayed heightened expression of the inhibitory immune receptor leukocyte-associated immunoglobulin-like receptor- (lair- ) [ ] . flow cytometry analysis also revealed increased sirp-α, siglec- , cd b, and reduced cd l and cd expression on activated lair- -expressing neutrophils. agonistic ligation of lair- was shown to limit net formation by balf neutrophils in vitro. of note, rsv-infected lair- -deficient mice present with greater neutrophilic inflammation upon inoculation with rsv [ ] , supporting the notion that agonism of lair- is a logical strategy to ameliorate the severity of viral bronchiolitis. in a separate study performed in infants with an rsv infection, the neutrophils in the nasopharyngeal aspirates were shown to exhibit lower cd l and cd (pecam- ) and higher cd (icam-i), cd b, and cd expression compared to those in peripheral blood [ ] . cd and icam- expression on neutrophils are also increased in rsv-infected infants compared with uninfected control infants [ ] . in a more recent study, the addition of physiological concentrations of neutrophils to human rsv-infected nasal cultures in vitro increased epithelial cell damage, characterised by lower ciliary activity, cilium loss, less tight junction expression, and greater detachment of epithelial cells, compared to cultures with rsv-infected epithelial cells alone [ ] . high-throughput analysis of lung-infiltrating neutrophils using the latest transcriptomic and high-dimensional flow cytometry is now needed to better identify different neutrophil subsets and associate these with beneficial and deleterious outcomes. such knowledge, together with an understanding of the molecular signals controlling neutrophil recruitment and function, is likely to reveal novel prognostic biomarkers and targets for therapeutic intervention. neutropenic individuals are highly susceptible to bacterial and fungal infections. the evidence with regard to viral infections is less clear [ ] , suggesting that neutrophils are not critical participants. however, neutrophils do appear to be required for optimal host immunity against an rsv infection. to accomplish this, neutrophils detect virus-associated molecular patterns through pattern recognition receptors (such as tlrs), produce an array of antimicrobial products, and assist the adaptive immune responses. for example, tlr expression on neutrophils is important for generating optimal immunity against rsv infection [ , ] . the expression of tlr is lower on blood and airway neutrophils of infants with severe rsv infection compared with uninfected controls [ ] . in humans, two tlr gene mutations are associated with an increased risk of developing severe rsv bronchiolitis [ ] . of note, the beneficial or detrimental roles of tlr-signalling in neutrophils during respiratory viral infections may be context-dependent. for instance, tlr -signalling may be detrimental in rsv infection but protective against influenza. therapeutic tlr antagonism lowers tnf-α, il- α, ptgs , and cxcl gene expression and improves survival of mice infected with a lethal dose of influenza a [ ] . some neutrophil anti-microbial products may be exploited for boosting anti-viral immunity during rsv infection. for instance, inflammatory neutrophils produce cathelicidins; cationic proteins important for host defence. intriguingly, in infants hospitalised with viral bronchiolitis, those with the lowest levels of ll- were more likely to be infected with rsv, suggesting that ll- is beneficial [ ] . human neutrophils stimulated in vitro with rsv virions secrete ll- , inhibiting the formation of new viral particles and reducing the apoptosis of infected epithelial cells [ , ] . neutrophil-derived ll- may support the production of interferons, which are required for viral control. in mice, treatment with an exogenous ll- peptide protected against body weight loss, increased interferon-lambda production, and lowered rsv load [ ] . neutrophils may also influence the quality of adaptive immune responses that develop during rsv infection. for example, neutrophils may aid in the rapid recruitment of virus-specific cd + t-cells. in infants with rsv infection, a systemic influx of bone marrow-derived neutrophil precursors in blood precedes robust cd + t-cell activation and a reduction in severe disease symptoms [ ] . however, the precise mechanisms through which neutrophils influence cd + t-cells remain unclear. collectively, these studies suggest that neutrophils can act beneficially in mediating immune protection against rsv infection. however, perturbation of optimal neutrophil function during infection may amplify their effector responses and thus contribute to the pathology that occurs in severe rsv patients. excessive neutrophilia is associated with increased tissue damage during severe rsv bronchiolitis [ ] . in part, this is attributed to molecular dysregulation of neutrophil-effector pathways, including ros production, netosis, degranulation, and over-secretion of proteolytic enzymes [ , , [ ] [ ] [ ] [ ] , (highlighted in figure ). excessive ros production activates indiscriminate oxidative stress in the lung, contributing to lung damage [ ] [ ] [ ] . indeed, markers of oxidative stress are elevated in the lungs and blood of patients with severe rsv [ ] . in vivo treatment of mice with antioxidants significantly reduces rsv-induced inflammation and pathology [ ] . ne levels are heightened in nasal aspirates and serum from paediatric patients with acute rsv infection compared with uninfected controls [ , ] , and nets are readily detected in balf samples obtained from patients with an rsv infection [ ] . in another study that sampled patients with rsv bronchiolitis, blood neutrophils showed little net formation, whereas neutrophils from the airways underwent netosis [ ] . intriguingly, in an ex vivo model, this process was diminished, with an agonistic antibody directed against lair- , which was shown to be elevated on airway neutrophils but not on circulating neutrophils [ ] . in vitro stimulation of human neutrophils with rsv virions induces ros-dependent netosis via pad- citrullination and downstream activation of the pi k/akt, erk, and p mapk pathways [ ] . the deposition of net products in the culture was shown to trap rsv virions in dna lattices coated with ne and mpo, implicating a beneficial antiviral effect of nets [ ] . however, in calves with a severe bovine rsv infection, the widespread airway obstruction is caused by luminal plugs composed of mucins, cellular debris, and nets [ ] . as dna-rich mucus is associated with airway obstruction [ , ] , excessive net release likely contributes to pathogenesis, although this has yet to be demonstrated clinically or in an experimental animal model of bronchiolitis. findings from other experimental systems such as allergic asthma have demonstrated that nets can exacerbate immunopathology [ ] . virus-associated exacerbations of asthma are attenuated following inhibition of netosis, either through blockade of ne or degradation of nets with dnase [ ] . mechanistically, netosis inhibition reduced type -mediated allergic inflammation and lowered airway hyper-reactivity [ ] . clearly, as with most inflammatory processes, netosis is a double-edged sword. a greater understanding of netosis in the context of acute lris is needed; however, on balance it appears that limiting netosis in severe rsv bronchiolitis would be advantageous. neutrophil-derived toxic granules and proteolytic enzymes also contribute to the pathogenesis of rsv bronchiolitis. rsv increases the expression of matrix metalloproteinase- (mmp- ), which augments the influx of neutrophils to the site of infection [ ] . elevated levels of mmp- are detected in the respiratory secretions of rsv-infected paediatric patients and are associated with increased risk of requiring mechanical ventilation [ ] [ ] [ ] . hence, mmp- activity is considered a useful predictor of disease severity in children with rsv-induced respiratory failure [ ] . genetic mmp- deficiency in mice decreases viral burden and lung inflammation [ ] , although it is still unclear whether neutrophil-derived mmp- plays a dominant role in the mmp- -induced pathology during rsv infection. specific ablation of mmp- in neutrophils using cre-recombinase-loxp systems would help address this question. in summary, severe neutrophilia and excessive production of powerful antimicrobial mediators appears to cause significant collateral damage, resulting in airway obstruction and the clinical symptoms that characterise severe rsv bronchiolitis. pharmacological tools to inhibit the excessive neutrophilic response in the lung are likely to ameliorate the severity of rsv bronchiolitis. to achieve this goal, therapeutic strategies should seek to modify the expression of cytokines and/or chemokines, which regulate neutrophil trafficking/recruitment into the lung, and neutrophil activation and function in response to infection (as proposed in figure ). macrolide antibiotics, in particular azithromycin, which is efficacious against il- and neutrophil-induced inflammation, decrease the severity of symptoms during viral bronchiolitis [ , , ] . a randomised trial in infants hospitalised with rsv bronchiolitis demonstrated that, compared with the placebo-control group, a -day treatment with azithromycin significantly decreased wheezing episodes, time to recovery, and overall respiratory morbidity over the subsequent year in comparison [ ] . notably, this was associated with lower il- levels in nasal lavage fluid, suggesting that neutrophil numbers were attenuated, although this was not specifically assessed [ ] . preclinical studies in mice have also demonstrated that azithromycin decreases virus-induced neutrophil accumulation in the lung by abrogating the expression of neutrophil inflammatory mediators such as cxcl [ ] . in a follow-up study to the clinical trial, azithromycin treatment modified the composition of the upper airway microbiome, reducing the abundance of moraxella [ ] . this phenotype was associated with lower odds of developing recurrent wheezing episodes over the next months [ ] . ongoing phase ii clinical trials are evaluating whether the addition of azithromycin to routine bronchiolitis care in hospitalised infants reduces the frequency of recurrent wheeze episodes during preschool years (clinicaltrials.gov identifier: nct ). viruses , , x for peer review of would help address this question. in summary, severe neutrophilia and excessive production of powerful antimicrobial mediators appears to cause significant collateral damage, resulting in airway obstruction and the clinical symptoms that characterise severe rsv bronchiolitis. pharmacological tools to inhibit the excessive neutrophilic response in the lung are likely to ameliorate the severity of rsv bronchiolitis. to achieve this goal, therapeutic strategies should seek to modify the expression of cytokines and/or chemokines, which regulate neutrophil trafficking/recruitment into the lung, and neutrophil activation and function in response to infection (as proposed in figure ). macrolide antibiotics, in particular azithromycin, which is efficacious against il- and neutrophil-induced inflammation, decrease the severity of symptoms during viral bronchiolitis [ , , ] . a randomised trial in infants hospitalised with rsv bronchiolitis demonstrated that, compared with the placebo-control group, a -day treatment with azithromycin significantly decreased wheezing episodes, time to recovery, and overall respiratory morbidity over the subsequent year in comparison [ ] . notably, this was associated with lower il- levels in nasal lavage fluid, suggesting that neutrophil numbers were attenuated, although this was not specifically assessed [ ] . preclinical studies in mice have also demonstrated that azithromycin decreases virusinduced neutrophil accumulation in the lung by abrogating the expression of neutrophil inflammatory mediators such as cxcl [ ] . in a follow-up study to the clinical trial, azithromycin treatment modified the composition of the upper airway microbiome, reducing the abundance of moraxella [ ] . this phenotype was associated with lower odds of developing recurrent wheezing episodes over the next months [ ] . ongoing phase ii clinical trials are evaluating whether the addition of azithromycin to routine bronchiolitis care in hospitalised infants reduces the frequency of recurrent wheeze episodes during preschool years (clinicaltrials.gov identifier: nct ). therapeutic inhibition strategies may aim to: ( ) block excessive neutrophil granulopoiesis in the bone marrow (e.g., neutralising g-csf production and function); ( ) antagonise chemokine-mediated neutrophil activation and chemotaxis to the lung microenvironment (e.g., using cxcr small molecule inhibitors); ( ) ( ) ( ) ( ) regulate neutrophil function in the lung (e.g., blocking the production of antimicrobial products such as mmps ( ), ros ( ), inflammatory cytokines ( ) , and deposition of nets ( )). therapeutic anti-viral products ( ) may also limit rsv-induced recruitment of neutrophils in the lung during infection. targeting chemokine receptors, for instance cxcr , which is important for neutrophil migration, may also be a therapeutic option (figure ). danirixin, a selective and reversible antagonist of cxcr , decreases the activation and transmigration of neutrophils to the lungs of rats treated with aerosolised lipopolysaccharide [ ] . a clinical trial to evaluate the efficacy of danirixin in rsv-infected infants < years of age (clinicaltrials.gov identifier: nct ) is ongoing. pharmacological inhibition of mmp activity has also been employed as a therapeutic strategy for treating chronic lung diseases [ , , ] . for example, marimastat, a broad-spectrum mmp inhibitor, reduces bronchial hyperresponsiveness to inhaled allergens in atopic asthmatic individuals [ ] . similarly, mice treated with another broad-spectrum mmp inhibitor, r- , also exhibited reduced airway inflammation, characterised by reduced eosinophils and lymphocytes recruited to the lung airways during experimental allergic asthma [ ] . as mmp- levels are significantly elevated in rsv-infected children [ ] [ ] [ ] , selective inhibition of mmp- activity may be of significant benefit for the treatment of rsv bronchiolitis (figure ). inhibition of netosis may also be a therapeutic option. strategies to achieve this might involve the targeting of neutrophil granules (including elastases or mpo), neutrophil-activating pattern recognition receptors (e.g., tlrs) or enzymes (nadph-oxidase and pad ). the ne inhibitor azd improves lung function in patients with bronchiectasis [ ] ; however, it is yet to be tested for efficacy in infants with a severe rsv infection. similarly, selective inhibition of mpo with pf- , which attenuates tissue injury in experimental immune complex-mediated alveolitis [ ] , may offer a therapeutic benefit against severe rsv bronchiolitis ( figure ). the detection of pathogen-associated molecular patterns by pattern recognition receptors on neutrophils can induce netosis. therefore, pharmacological disruption of these interactions may attenuate netosis. for instance, tak- , a small molecule tlr inhibitor [ ] that reduces inflammation, may also be used to regulate netosis. considering that the formation of nets is largely dependent on the activity of nadph oxidase or pad , the inhibition of either enzyme is likely to decrease the magnitude of neutrophil-associated tissue damage. genetic deletion of pad or pad inhibition by cl-amidine or bb-cl-amidine improves clinical outcomes in several different mouse models of inflammatory disease [ , , ] , although whether pad inhibitors are beneficial against a severe rsv infection remains unknown. the modulation of neutrophil numbers or function is evidently a promising target to ameliorate the severity of rsv bronchiolitis. however, several feasibility issues remain, particularly in relation to safety and specificity. selective targeting of neutrophils is challenging due to their close relationship to other myeloid lineages, such as monocytes and macrophages. this may alter tissue homeostasis and immune defence against other intracellular pathogens such as mycobacterium tuberculosis or listeria monocytogenes. moreover, since neutrophils are important for robust antimicrobial host defence, their therapeutic inhibition may predispose the host to other infectious agents or effect colonisation by commensals, which can become pathobionts if left unchecked. to overcome this, one strategy would be to target specific neutrophil subsets, and thus ablate the pathogenic population without compromising the beneficial effect of neutrophils in mediating antimicrobial host defence. new insights are needed to identify approaches that can support this possibility. most therapeutic approaches in the pipeline have focused on inhibiting excessive neutrophil function in treating disease pathology. however, an alternative approach is to modify neutrophil function to boost host immunity against pathogenic microbes, particularly in the elderly. for instance, the cholesterol-lowering medication simvastatin has been associated with improved neutrophil function and immunity against bacterial infection in humans [ ] . in this study, adding simvastatin to the normal treatment regimen significantly reduced the severity of bacterial pneumonia and sepsis in patients older than years [ ] . mechanistically, simvastatin acted by augmenting the migratory accuracy of neutrophils into tissues, albeit with reduced netosis and ne release, which promoted optimal non-inflammatory bacterial control. therefore, pharmacological approaches to rejuvenate neutrophils in the elderly might be beneficial in boosting immunity against rsv. there remains a pressing need to develop new treatments to reduce the significant morbidity and mortality associated with severe rsv bronchiolitis. neutrophils contribute to host immunity against rsv; however, they are strongly associated with the development of severe disease and are thus a logical target for therapeutic intervention. new technological approaches, such as single cell rna sequencing, combined with proteomics, metabolomics, and advances in flow cytometry that allow for multi-parameter analyses, must now be applied to clinical samples from infants with mild and severe disease to gain a greater understanding 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myeloperoxidase inhibitor, prevents immune complex vasculitis and anti-glomerular basement membrane glomerulonephritis tak- , a small-molecule inhibitor of toll-like receptor signalling, unveils similarities and differences in lipopolysaccharide-and lipid-induced inflammation and insulin resistance in muscle cells neutrophil-mediated ifn activation in the bone marrow alters b cell development in human and murine systemic lupus erythematosus locally instructed cxcr (hi) neutrophils trigger environment-driven allergic asthma through the release of neutrophil extracellular traps simvastatin improves neutrophil function and clinical outcomes in pneumonia. a pilot randomized controlled clinical trial this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -admlzeu authors: wang, gang; liang, rui; liu, ziwei; shen, zhou; shi, jiale; shi, yuejun; deng, feng; xiao, shaobo; fu, zhen f.; peng, guiqing title: the n-terminal domain of spike protein is not the enteric tropism determinant for transmissible gastroenteritis virus in piglets date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: admlzeu transmissible gastroenteritis virus (tgev) is the etiologic agent of transmissible gastroenteritis in pigs, and the n-terminal domain of tgev spike protein is generally recognized as both the virulence determinant and enteric tropism determinant. here, we assembled a full-length infectious cdna clone of tgev in a bacterial artificial chromosome. using a novel approach, the clustered regularly interspaced short palindromic repeat (crispr)/crispr-associated protein (cas ) systems efficiently and rapidly rescued another recombinant virus with a -amino-acid deletion in the n-terminal domain of the tgev spike gene (s_ntd ), which is analogous to the n-terminal domain of porcine respiratory coronavirus. s_ntd notably affected the tgev growth kinetics in pk- cells but was not essential for recombinant virus survival. in animal experiments with two-day-old piglets, the tgev recombinant viruses with/without s_ntd deletion induced obvious clinical signs and mortality. together, our results directly demonstrated that s_ntd of tgev mildly influenced tgev virulence but was not the enteric tropism determinant and provide new insights for the development of a new attenuated vaccine against tgev. importantly, the optimized reverse genetics platform used in this study will simplify the construction of mutant infectious clones and help accelerate progress in coronavirus research. coronaviruses (covs) are single-stranded, positive-sense rna viruses closely related to animal and human health [ ] [ ] [ ] . covs belong to the coronaviridae family, which consists of the alpha-, beta-, gamma-and deltacoronavirus genera [ ] . since , covs, including severe acute respiratory syndrome cov (sars-cov), middle east respiratory syndrome cov (mers-cov), and porcine epidemic diarrhea virus (pedv), have swept across the world and caused considerable global economic losses [ ] [ ] [ ] [ ] [ ] . as the largest rna genome viruses, covs have at least six typical overlapping open reading frames pk- cells were cultured in dulbecco's modified eagle's medium (gibco, waltham, ma, usa) supplemented with % fetal bovine serum at • c with % co . tgev strain wh- (genbank accession number hq ) was propagated at • c in a % co incubator in dulbecco's modified eagle's medium (gibco, waltham, ma, usa) supplemented with % fetal bovine serum (gibco, waltham, ma, usa). all the experiments using live viruses were performed under biosafety level (bsl) conditions. total rna was extracted from virus-infected cultures using trizol reagent (invitrogen, carlsbad, ca, usa), and cdna was reverse transcribed with reverse transcriptase (takara, amv, kusatsu, japan) using random primers (takara, kusatsu, japan, mer). all the fragments were amplified by polymerase chain reaction (pcr) with phanta super fidelity dna polymerase (vazyme, nanjing, jiangsu, china). the natural complete genome of tgev wh- was determined by sequencing (genscript, nanjing, china) the overlapping pcr products cloned into the corresponding vectors in triplicate. compared with the parental tgev wh- from ncbi (bethesda, md, usa), several site mutations, including t c, g t, g c, c t, and c g, were observed, and these mutations were maintained during the cloning of the tgev full-length genome. a point mutation, a t, was introduced by overlap extension pcr to remove the natural van i site and maintained as the rescue marker. as described in previous research, the egfp gene was inserted into the tgev genome [ , ] to replace the original sequence positioned from , to , . the virus genome was divided into six continuous fragments (a to f), and each fragment was amplified from the total cdna using specific primers (available upon request). fragments a, b, d, and e were cloned into the pmd -t vector (takara, kusatsu, japan). fragment a was cloned with the saci and van i sites, and fragment e was cloned with the van i and kasi sites. notably, fragment a was cloned to contain a saci site (gagctcgtttagtgaaccgt) [ ] located in the terminal of the tgev genome sequence. fragments b and d were cloned with van i sites. fragment c was cloned into a bac plasmid (kindly provided by prof. cao gang) that was modified from pbelobac to include a van site [ ] . fragment f was also cloned into the bac plasmid with saci and kasi sites introduced at the and termini of the tgev genome, respectively. as the final recipient bac vector, subclone f also contained the synthesized essential element sequences, such as the cmv promoter, the poly(a) tail sequence ( a), the hdv rz sequence (hepatitis delta virus self-cleaving ribozyme sequence, rz), and the bovine growth hormone (bgh) transcription terminal signal (genscript, nanjing, china) [ ] . after the six subclones were sequenced in their corresponding vectors, subclones a and e were first digested with saci and kasi, respectively, and then treated with calf intestinal alkaline phosphatase (ciap, scientific). all the subclones were then digested with van i except subclone f, which was digested with saci and kasi. subsequently, all the digested products were purified with a gel extraction kit (omega, norcross, ga, usa), and fragments a to f were ligated for more than h at • c and transformed into chemically competent dh b cells (biomed, beijing, china). after determination of all the fragments by bacterial pcr, the positive clones were further determined by restriction fragment length polymorphism with kpni, and the correct clone was designated ptgev-gfp bac after sequencing (genscript, nanjing, china). pk- cells were seeded in a six-well plate and incubated for h, and the recovery of tgev-gfp or tgev-gfp-∆s_ntd was then performed by transfecting µg of the corresponding bac into pk- cells with µl of lipofectamine (invitrogen, carlsbad, ca, usa). at h post-transfection, the collected virus progenies were purified once by fluorescent plaques. subsequently, the purified virus clone was amplified and stored until use at − • c. for the design of a sgrna to mediate cleavage of the targeted site, a constant reverse primer (ssdna-r) and two forward primers (ssdnaa-f and ssdnab-f) specific for sites a and b were synthesized as shown in table , similar to the protocol described in a previous report [ ] . to anneal the primers ssdnaa-f and ssdnab-f with the reverse primer ssdna-r, pcr was conducted for cycles at • c for s, • c for s, and • c for s using × utaq mastermix (zoman). the pcr products were then purified with cp buffer (omega, norcross, ga, usa) and transcribed using a t transcription kit (neb, ipswich, ma, usa) according to the manufacturer's instructions to produce the targeted sgrna a and sgrna b. the purity of the sgrna products was analyzed by electrophoresis on agarose gels using . µg of each sgrna product. to modify the sequence of tgev s_ntd , ptgev-gfp bac was digested using the targeted sgrnas. specifically, ptgev-gfp bac was digested in a µl reaction mixture with µg of ptgev-gfp bac, µl of cas (neb, ipswich, ma, usa), µg of sgrna, and µl of nuclease reaction buffer incubated at • c for more than h or, preferably, overnight. for purification of the digested ptgev-gfp bac, an equivalent volume of solution i (plus rnase; omega, norcross, ga, usa) was first added to digest the sgrna at room temperature for min, and the cp buffer (omega, norcross, ga, usa) was then applied to recycle the digested bac according to the manufacturer's instructions. the pcr products with the -bp deletion were constructed by two-cycle pcr. first, the primers rec- sf and δs-ntdr or the primers rec- sr and δs-ntdf (table ) were used to amplify the primary pcr products from the template of the ptgev-gfp bac. second, the two primary pcr products were annealed to produce pcr products of the -bp deletion using the primers rec- sf and rec- sr (table ). homologous recombination was then performed using the clonexpress ii one step cloning kit (vazyme, nanjing, jiangsu, china) according to the manufacturer's instructions using ng of recycled linearized ptgev-gfp bac, ng of pcr products of s_ntd and two pairs of primers (rec- sf/δs-ntdr and rec- sr/δs-ntdf) ( table ) . subsequently, a pair of primers (primerf/primerr) ( table ) was designed to amplify the sequence of the modified s_ntd area for sequencing (genscript, nanjing, china). the recombinant virus corresponding to the correct ptgev-gfp-∆s_ntd bac was then recovered as described above. pk- cells in six-well plates were inoculated with -fold serially diluted recombinant virus. after virus adsorption for min, monolayer cells were washed three times with pbs and overlaid with a mixture of % low-melt agarose and times the concentration of dmem (invitrogen, carlsbad, ca, usa) supplemented with % fetal bovine serum (gibco). the overlay was then solidified at • c for min. subsequently, the plates were cultured at • c in a % co incubator, and days post-infection, the fluorescent plaques were visualized by fluorescence microscopy. thirteen -day-old piglets from a tgev-free sow were randomly divided into three groups and fed fresh liquid milk diluted in warm water every h. all piglets were confirmed to be free of tgev, pedv, porcine delta coronavirus (pdcov), and rotavirus (rv) through a rt-pcr assay of piglet feces before viral challenge. the piglet weights were measured and recorded at the beginning of the challenge. the piglet challenge group was intranasally and orally inoculated with µl ( × tcid ) of chimeric virus, and the mock-infected control group was intranasally and orally inoculated with µl of dmem. the piglets were monitored for their clinical status every h. any piglet exhibiting moribund signs were euthanized. at days post-inoculation, all surviving piglets were euthanized consecutively to reduce the stress of the other piglets. before necropsy, the weight of each piglet was recorded. at necropsy, five sections of the duodenum, jejunum, ileum, colon and stomach were collected, fixed in % formalin for histopathological examination and stained with hematoxylin and eosin (he). after necropsy, samples of jejunal contents and lung tissue were collected for virus detection by nested rt-pcr using the specific primers f /r and f /r (table ) [ ]. the animal experiments were performed according to the protocols approved by the scientific ethics committee of huazhong agricultural university (permit number: hzausw- - ). the animal care and maintenance protocols complied with the recommendations detailed in the regulations for the administration of affairs concerning experimental animals made by the ministry of science and technology of china. to construct an infectious clone of tgev, six overlapping cdna fragments designated a to f were generated by reverse transcriptase pcr (rt-pcr) using total rna extracted from pk- cells infected with tgev wh- ( figure a ,b). fragments a, b, d, and e were cloned into the pmd -t vector, and fragments c and f were cloned into the bacterial artificial chromosome (bac) to produce the corresponding subclones. subclone f was also constructed as the final recipient bac vector by inserting the cytomegalovirus (cmv) promoter at the terminus of fragment f and a -bp poly(a) tail ( a) followed by the hepatitis delta virus ribozyme (rz) and bovine growth hormone (bgh) transcription terminal signal sequences at the terminus of fragment f ( figure b ). to more conveniently observe the chimeric virus and exclude the influence of the accessory gene orf on tgev enteric tropism and virulence, the gene encoding orf at the genome position from , to , was replaced by the egfp gene ( figure a ,b). through the one-step assembly of fragments a to f, we successfully obtained a full-length cdna infectious clone of tgev, designated ptgev-gfp bac ( figure b ,c). the full-length ptgev-gfp bac was verified by sequencing. after propagation for more than generations in e. coli dh b cells, ptgev-gfp bac digestion with the kpni enzyme, which produced six different fragment products, also confirmed the correct ptgev-gfp bac clone ( figure c ,d). in other words, the cloning of fragment c into the bac yielded no toxic sequences in any of the experiments. produce the corresponding subclones. subclone f was also constructed as the final recipient bac vector by inserting the cytomegalovirus (cmv) promoter at the ′ terminus of fragment f and a -bp poly(a) tail ( a) followed by the hepatitis delta virus ribozyme (rz) and bovine growth hormone (bgh) transcription terminal signal sequences at the ′ terminus of fragment f ( figure b) . to more conveniently observe the chimeric virus and exclude the influence of the accessory gene orf on tgev enteric tropism and virulence, the gene encoding orf at the genome position from , to , was replaced by the egfp gene ( figure a ,b). through the one-step assembly of fragments a to f, we successfully obtained a full-length cdna infectious clone of tgev, designated ptgev-gfp bac ( figure b ,c). the full-length ptgev-gfp bac was verified by sequencing. after propagation for more than generations in e. coli dh b cells, ptgev-gfp bac digestion with the kpni enzyme, which produced six different fragment products, also confirmed the correct ptgev-gfp bac clone ( figure c,d) . in other words, the cloning of fragment c into the bac yielded no toxic sequences in any of the experiments. to rescue the recombinant cov of tgev-gfp (corresponding to ptgev-gfp bac), the ptgev-gfp bac was transfected into pk- cells using lipofectamine . sporadic green fluorescence was observed h post-transfection, as depicted in figure a , but the infected pk- cells grew and showed a normal morphology. however, compared with the mock-infected group, obvious green fluorescence and the cytopathic effect (cpe) could be observed h post-transfection ( figure b ). western blot and rt-pcr assays were performed to further confirm the recombinant virus tgev-gfp. the . -kilodalton band of tgev n protein ( figure c ) and the marker mutation at position in the tgev genome ( figures b and d ) confirmed the recovery of the tgev-gfp recombinant virus. to rescue the recombinant cov of tgev-gfp (corresponding to ptgev-gfp bac), the ptgev-gfp bac was transfected into pk- cells using lipofectamine . sporadic green fluorescence was observed h post-transfection, as depicted in figure a , but the infected pk- cells grew and showed a normal morphology. however, compared with the mock-infected group, obvious green fluorescence and the cytopathic effect (cpe) could be observed h post-transfection ( figure b ). western blot and rt-pcr assays were performed to further confirm the recombinant virus tgev-gfp. the . -kilodalton band of tgev n protein ( figure c ) and the marker mutation at position in the tgev genome ( figures b and d ) confirmed the recovery of the tgev-gfp recombinant virus. spike with n-terminal aa deletion in tgev wh is analogous to the spike of a reported natural prcv strain ( figure a ). to verify whether s_ntd is the enteric tropism determinant for tgev, we used the crispr-cas system to efficiently manipulate the tgev gene. briefly, two specific enzyme sites encompassing the sequence of s_ntd were selected. we then synthesized two types of single-stranded dna forward primers (ssdnaa-f or ssdnab-f) and a constant reverse primer (ssdna-r) ( table ) corresponding to the two enzyme cutting sites, designated sites a and b ( figure c ). after annealing pcr using the forward and reverse primers, the purified pcr products of short dna fragments were transcribed by t rna polymerase ( figure b ). the transcribed products corresponding to sites a and b (designated sgrna a and sgrna b) ( figure c ) were incubated with the nuclease cas to digest the ptgev-gfp bac in vitro, and the digestion yielded a linearized bac and a~ . -kb dna fragment that included the sequence of s_ntd ( figure a,d) . tgev, we used the crispr-cas system to efficiently manipulate the tgev gene. briefly, two specific enzyme sites encompassing the sequence of s_ntd were selected. we then synthesized two types of single-stranded dna forward primers (ssdnaa-f or ssdnab-f) and a constant reverse primer (ssdna-r) ( table ) corresponding to the two enzyme cutting sites, designated sites a and b ( figure c ). after annealing pcr using the forward and reverse primers, the purified pcr products of short dna fragments were transcribed by t rna polymerase ( figure b ). the transcribed products corresponding to sites a and b (designated sgrna a and sgrna b) ( figure c ) were incubated with the nuclease cas to digest the ptgev-gfp bac in vitro, and the digestion yielded a linearized bac and a ~ . -kb dna fragment that included the sequence of s_ntd ( figure a,d) . to construct the mutant infectious clone of the s_ntd deletion (designated ptgev-gfp-Δs_ntd bac) from the ptgev-gfp bac, we produced a -nucleotide deletion-specific mutation pcr product using two pairs of primers (rec- sf/δs-ntdr and rec- sr/δs-ntdf) ( table ). the mutation pcr products were then recombined into the linearized bac vector cleaved from the full-length ptgev-gfp bac ( figure d ). after the recombination products were transformed into dh b competent cells, all monoclonal colonies were identified as positive clones by pcr using the primer pair rec- sf/rec- sr ( figure e ). the sequencing of three randomly selected monoclonal colonies also confirmed the positive ptgev-gfp-Δs_ntd bac, and we then constructed an infectious clone with the corresponding -aa deletion in the n-terminus of the tgev-gfp s protein by sequencing one entire genome of the three positive clone ( figure e ). to construct the mutant infectious clone of the s_ntd deletion (designated ptgev-gfp-∆s_ntd bac) from the ptgev-gfp bac, we produced a -nucleotide deletion-specific mutation pcr product using two pairs of primers (rec- sf/δs-ntdr and rec- sr/δs-ntdf) ( table ). the mutation pcr products were then recombined into the linearized bac vector cleaved from the full-length ptgev-gfp bac ( figure d ). after the recombination products were transformed into dh b competent cells, all monoclonal colonies were identified as positive clones by pcr using the primer pair rec- sf/rec- sr ( figure e ). the sequencing of three randomly selected monoclonal colonies also confirmed the positive ptgev-gfp-∆s_ntd bac, and we then constructed an infectious clone with the corresponding -aa deletion in the n-terminus of the tgev-gfp s protein by sequencing one entire genome of the three positive clone ( figure e ). we rescued the recombinant virus tgev-gfp-∆s_ntd from pk- cells as previously described, and sporadic and more obvious green fluorescence was observed at and h post-transfection, respectively ( figure a ). we then verified the modified virus tgev-gfp-∆s_ntd by rt-pcr using the primers primerf and primerr (table ). an obvious deletion of approximately bp was observed in tgev-gfp-∆s_ntd in comparison with tgev-gfp ( figure b ). subsequently, the rt-pcr product was sequenced using the primers primerf and primerr ( figure c) . comparison with the ptgev-gfp bac showed that all the modified nucleotides were correct, as depicted by the model shown in figure d . to validate whether s_ntd determines the enteric tropism of tgev, two-day-old piglets were randomly divided into three groups, with five piglets in each virus-infected group and three piglets in the mock-infected control group. the piglets in the two virus-infected groups were to further evaluate the role of s_ntd in tgev, we measured the growth kinetics of the wild-type virus, tgev-gfp and tgev-gfp-∆s_ntd. the replication kinetics of the tgev-gfp and wild-type viruses were comparable to each other and considerably different from those of tgev-gfp-∆s_ntd ( figure e ). twelve hours after inoculation, the titer of tgev-gfp was more than -fold greater than that of tgev-gfp-∆s_ntd ( figure e ). to further identify the effect of s_ntd on tgev, we also analyzed the fluorescent viral plaques. the plaque size of tgev-gfp-∆s_ntd was notably different from that of tgev-gfp ( figure f ), which also indicated that tgev-gfp infected cells more effectively than tgev-gfp-∆s_ntd. to validate whether s_ntd determines the enteric tropism of tgev, two-day-old piglets were randomly divided into three groups, with five piglets in each virus-infected group and three piglets in the mock-infected control group. the piglets in the two virus-infected groups were inoculated intranasally and orally at a dose of × % tissue culture infective dose (tcid ) with the respective chimeric virus, and the mock-infected control piglets were inoculated with dulbecco's modified eagle's medium (dmem). all the piglets in the tgev-gfp group exhibited severe clinical symptoms and weight loss, and those in the tgev-gfp-∆s_ntd group showed ameliorated but still obvious clinical symptoms ( figure a,b) . in addition, the piglets in the tgev-gfp and tgev-gfp-∆s_ntd groups appeared moribund within days postinoculation, whereas the piglets in the mock-infected control group remained healthy ( figure c ). in addition, the piglets in the tgev-gfp group showed a higher mortality rate (as high as %) and presented earlier symptoms compared with those in the tgev-gfp-∆s_ntd group, which showed % mortality at days postinoculation ( figure a,c) . to better detect the presence of the inoculated virus in the euthanized piglet intestine, the presence of both tgev-gfp and tgev-gfp-∆s_ntd in intestinal tissue was detected by nested pcr using the primer pairs f /r and f /r (table ) . tgev-gfp and tgev-gfp-∆s_ntd were detected in intestinal tissue from the moribund piglets ( figure d ), but no chimeric virus was detected in the two piglets in the tgev-gfp-∆s_ntd group that were euthanized at days post-inoculation ( figure d ). tgev-gfp-Δs_ntd groups appeared moribund within days postinoculation, whereas the piglets in the mock-infected control group remained healthy ( figure c ). in addition, the piglets in the tgev-gfp group showed a higher mortality rate (as high as %) and presented earlier symptoms compared with those in the tgev-gfp-Δs_ntd group, which showed % mortality at days postinoculation ( figure a ,c). to better detect the presence of the inoculated virus in the euthanized piglet intestine, the presence of both tgev-gfp and tgev-gfp-Δs_ntd in intestinal tissue was detected by nested pcr using the primer pairs f /r and f /r (table ) . tgev-gfp and tgev-gfp-Δs_ntd were detected in intestinal tissue from the moribund piglets ( figure d ), but no chimeric virus was detected in the two piglets in the tgev-gfp-Δs_ntd group that were euthanized at days post-inoculation ( figure d ). the postmortem of the moribund piglets in the tgev-gfp and tgev-gfp-Δs_ntd groups revealed that the small intestines were filled with watery contents. in particular, the intestinal walls in the jejunal section of the intestines of these piglets were clearly thinner and more transparent compared with those of the mock group ( figure a) . he staining compared with the normal mock group also revealed that the tgev-gfp chimeric viruses caused more severe intestinal tissue the postmortem of the moribund piglets in the tgev-gfp and tgev-gfp-∆s_ntd groups revealed that the small intestines were filled with watery contents. in particular, the intestinal walls in the jejunal section of the intestines of these piglets were clearly thinner and more transparent compared with those of the mock group ( figure a ). he staining compared with the normal mock group also revealed that the tgev-gfp chimeric viruses caused more severe intestinal tissue damage than tgev-gfp-∆s_ntd ( figure b ). more severe villous atrophy was observed in the small intestine, particularly the jejunum and ileum, of the piglets in the tgev-gfp and tgev-gfp-∆s_ntd groups compared with those of the mock group ( figure b ). collectively, these results suggested that s_ntd has not altered the enteric tropism for tgev but exerts a mild influence on tgev virulence. the n-terminal domain of spike protein is recognized as the tgev enteric tropism determinant in piglets, as demonstrated through comparisons of the sequences of natural tgev isolates or those obtained after continuous passage in cell culture [ , , ] , but more direct evidence is still needed. in this study, based on a dna-launched infectious clone, we used a novel cov gene editing method to efficiently perform cov targeted gene editing. using reverse genetics, we found that s_ntd was not the enteric tropism determinant for tgev. the relevant insights regarding the novel cov targeted gene editing method and tgev s_ntd are discussed below. because covs are the viruses with the largest rna genomes, the construction of a cov infectious clone is hampered by two main challenges: large full-length cdna and toxic sequences in the bacterial clone [ , ] . although the problem of cov cdna sequence instability has been overcome by several methods, the manipulation of the large full-length cov genome remains a considerable challenge. until now, the direct editing of the full-length cdna of covs has not been reported. in this study, we constructed a tgev-gfp infectious clone by ligating six fragments in one the n-terminal domain of spike protein is recognized as the tgev enteric tropism determinant in piglets, as demonstrated through comparisons of the sequences of natural tgev isolates or those obtained after continuous passage in cell culture [ , , ] , but more direct evidence is still needed. in this study, based on a dna-launched infectious clone, we used a novel cov gene editing method to efficiently perform cov targeted gene editing. using reverse genetics, we found that s_ntd was not the enteric tropism determinant for tgev. the relevant insights regarding the novel cov targeted gene editing method and tgev s_ntd are discussed below. because covs are the viruses with the largest rna genomes, the construction of a cov infectious clone is hampered by two main challenges: large full-length cdna and toxic sequences in the bacterial clone [ , ] . although the problem of cov cdna sequence instability has been overcome by several methods, the manipulation of the large full-length cov genome remains a considerable challenge. until now, the direct editing of the full-length cdna of covs has not been reported. in this study, we constructed a tgev-gfp infectious clone by ligating six fragments in one step [ ] . the crispr/cas system was then used to finish the construction of ptgev-gfp-∆s_ntd. to our knowledge, this study provides the first demonstration of the direct in vitro manipulation of full-length coronavirus cdna. to edit specific cov genes, targeted cleavage of the bac was completed by cas protein through a reaction mediated by two types of sgrna transcribed together or separately ( figure b,d) . sgrna can be easily obtained by annealing pcr and transcription using an available kit. moreover, regardless of the exonuclease trimming activities of cas [ ] , in the experiment, we were able to insert the mutated fragments in the linearized bac with -bp overlapping sequences through homologous recombination. notably, numerous mutation fragments can be inserted efficiently into the linearized bac at the same time ( figure d ), which is perfect for the construction of a viral mutant library [ ] [ ] [ ] . similar to traditional plasmid manipulation, we edited the targeted gene by recombination in vitro with overlapping pcr products (e.g., mutations, deletions, or insertions). moreover, as little as ng of linearized, digested bac was sufficient to complete the recombination reaction. to determine the mutation of the targeted bac, we only needed to amplify the fragments by bacterial pcr using a pair of primers in duplicate, and this assay can be used to sequence the site of recombination and the modified fragment area. furthermore, almost any area of the targeted bac can be simply cleaved by the crispr/cas system with two types of specific sgrna. throughout the process, we accomplished recombinant virus recovery using only one plasmid of bac in a single week ( figure ) . namely, once an infectious clone was constructed, the recombinant coronaviruses was more efficiently and conveniently rescued in this study than in previous research, and the proposed approach thus greatly accelerates the gene editing speed of large rna virus rescue. moreover, the simple manipulation of a bac vector and modification of the specific small region throughout the procedure would theoretically lower the mutation probability of the full-length cov cdna. thus, this method is not only cost-effective but also reduces the probability of introducing additional mutations during the bac modification procedure. the spike gene of tgev has been shown to alter tgev virulence or enteric tropism. however, recombinant tgev with s protein n-terminal amino-acid deletion was constructed through targeted recombination and passaged several times, which might cause other locus mutations in addition to the s protein deletion [ ] . in particular, an early study reported that a -residue deletion in prcv corresponding to the n-terminal domain of the tgev s protein, as depicted in figure a , is likely responsible for the loss of replication observed in the enteric tract [ ] . no other studies have provided direct evidence demonstrating that only the n-terminal region of the spike gene changes the tgev virulence or enteric tropism [ ] . here, we emphasize the importance of the n-terminus of the tgev s protein for the enteric tropism of the virus. to that end, we constructed a recombinant virus with an s protein analogous to that of prcv ( figure a) , tgev-gfp-∆s_ntd, and this recombinant virus showed titers and fluorescent plaque sizes that greatly differed from those of tgev-gfp ( figure e,f) . these results indicated that the amino acids of the n-terminal of the tgev spike protein are not essential for viral survival but important for viral replication or infection, which was analogous to the findings obtained for pedv and other covs [ ] [ ] [ ] [ ] . our animal experiments revealed that tgev-gfp-∆s_ntd caused % mortality in piglets and obvious intestinal tissue damage, which indicates that s_ntd has a mild influence on virulence but does not alter the enteric tropism of tgev [ ] . using reverse genetics, we confirmed that changes in s_ntd alone altered, albeit not completely, the virulence of tgev. one reason explaining no detection of tgev-gfp-∆s_ntd in the two piglets euthanized at days post-inoculation might be related to immunity of recovering piglets. the role of s_ntd might be analogous to that of the -amino-acid region in the n-terminus of the pedv s gene when used as a viral virulence marker. consistent with previous reports, our experiments also detected tgev-gfp and tgev-gfp-∆s_ntd in the jejunal contents tissue by nested rt-pcr [ , ] , which indicates that changes in s_ntd alone do not alter tgev enteric tropism in vivo. and it is also possible that other genes in addition to the amino acids of the n-terminal of the tgev spike protein might regulate changes in tgev tissue tropism [ , ] . additional research is needed to determine the detailed mechanism of tgev enteric tropism in vivo. in summary, using the reverse genetics method, we have provided direct evidence showing that the n-terminal domain of spike protein is not the determinant of tgev enteric tropism in piglets, although s_ntd exerts a mild influence on tgev virulence. these results provide new insights into the development of a new attenuated vaccine against tgev. furthermore, the method developed in this study allows the efficient and rapid editing of the full-length cov genome in vitro and 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bronchus-associated lymphoid tissues of suckling pigs the authors declare no conflict of interest. key: cord- - jyv q e authors: ikegami, tetsuro; makino, shinji title: the pathogenesis of rift valley fever date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jyv q e rift valley fever (rvf) is an emerging zoonotic disease distributed in sub-saharan african countries and the arabian peninsula. the disease is caused by the rift valley fever virus (rvfv) of the family bunyaviridae and the genus phlebovirus. the virus is transmitted by mosquitoes, and virus replication in domestic ruminant results in high rates of mortality and abortion. rvfv infection in humans usually causes a self-limiting, acute and febrile illness; however, a small number of cases progress to neurological disorders, partial or complete blindness, hemorrhagic fever, or thrombosis. this review describes the pathology of rvf in human patients and several animal models, and summarizes the role of viral virulence factors and host factors that affect rvfv pathogenesis. rift valley fever (rvf), a mosquito-borne zoonotic disease among humans and ruminants, is caused by rift valley fever virus (rvfv) belonging to family bunyaviridae, genus phlebovirus [ , ] . rvf is endemic to sub-saharan african countries and has caused major outbreaks in several countries including kenya, tanzania, somalia, south africa, madagascar, egypt, sudan, mauritania, senegal, saudi arabia, and yemen [ ] . pregnant ruminants infected with rvfv typically are subject to highrate abortions, fetal malformation, and subclinical-to-fatal febrile illness, while newborn lambs usually die by acute hepatitis [ ] [ ] [ ] . rvfv infection in humans primarily causes a self-limiting febrile illness; however, some patients develop hemorrhagic fever, neurological disorders, or blindness after the febrile period [ , , ] . in endemic area, floodwater aedes mosquitoes, such as ae.mcintoshi or ae.vexans, serve as vectors, and the virus could be transmitted into offspring transovarially [ , ] . heavy rainfall or flooding of river banks due to construction of dams increases the number of permanent fresh water species of mosquitoes such as culex pipens, which play a role in amplifying rvfv among mosquitoes, ruminants and humans [ ] [ ] [ ] [ ] [ ] [ ] . an outbreak of rvf in developed countries, e.g., the u.s. or europe, could force a curtailing of livestock movement to prevent rvfv spread, causing massive economic loss, and a substantial degree of panic in our society, because the body fluids of infected animals contain infectious rvfv [ , ] , and mosquitoes such as culex spp. aedes spp. or anopheles spp. might further spread rvfv into other mosquitoes, humans and animals [ ] [ ] [ ] . effective vaccines and antiviral drugs are necessary for the containment of outbreaks and treatment of rvf patients, respectively. however, neither safe and effective vaccines nor efficient treatment is available. a correct understanding of rvf pathogenesis is essential for the development of effective vaccines and antiviral drugs against rvf. in this review, we will describe clinical and pathological findings of rvf in humans and animals and discuss viral and host factors that affect rvf pathogenesis. most rvf patients suffer from a self-limiting, febrile illness. however, some patients develop neurological disorders, vision loss, hemorrhagic fever, or thrombosis as shown in figure . in the s- s, many rvfv laboratory infections occurred due to a lack of appropriate biosafety procedures [ ] [ ] [ ] [ ] [ ] . however, the patients in most of these and later outbreaks suffered from self-limiting and nonfatal illness [ , , , , [ ] [ ] [ ] [ ] . typically, the incubation period for rvf is to days. symptoms start abruptly with severe chills, malaise, dizziness, weakness, severe headache, nausea and/or sensation of fullness over the liver region [ , , ] . these symptoms are followed by an elevated body temperature ( . °c to . °c); decreased blood pressure; pain in the back, shoulders, neck or legs; rigor; shivering; flushed face; red eye with sore; constipation; insomnia and/or photophobia. occasionally, other symptoms are seen which include epistaxis, abdominal pain, lack of gustatory discrimination, vomiting and/or diarrhea [ , , , , , ] . some lessening of symptoms can be observed on the rd day, and the body temperature often decreases to a normal level by the th day after the onset of symptoms. however, within to days after the recovery of body temperature, some patients again experience a temporal recurrence of high fever with a severe headache for a few days [ , , ] . moreover, patients may have a long-lasting high fever for as much as days [ ] . after body temperature becomes normal, some patients may develop a massive coronary thrombosis [ ] , persistent aching of legs for two weeks [ , ] , or persistent abdominal discomfort for weeks [ ] . the palpable enlargement of the liver and spleen is not common. in the convalescence phase, patients often experience weakness, malaise, a tendency to sweat, frequent headaches, pain on motion of the eye, and a sense of imbalance. virus has been demonstrated in the blood during the febrile period ( - days) , whereas neutralizing antibody also starts appearing around the th day of the onset of symptoms [ , , , , ] . maar et al. described a case of encephalitis in a rvf patient [ ] . the patient exhibited symptoms of sudden fever, rigor, and retro-orbital headache for two days. he had fever again at the nd day after the onset of illness and experienced neck rigidity lasting for five days from the th day. subsequently, he was sometimes confused and otherwise mentally affected, and experienced temporal vision loss without detectable retinopathy. he also exhibited convulsive attacks, hyperflexia and fever until the th day. his serum contained anti-rvfv hemagglutination (hai) antibodies of : at the th day and : at the th day, while his cerebrospinal fluid (csf) contained : of hai antibody at the th day and : at the th day. the csf also contained an increased number of white blood cells consisting mainly of lymphocytes at the th day, indicative of the possible occurrence of viral meningitis or meningoencephalitis. the patient recovered after treatment with amantadine, rifampicin, and dexamethasone for two weeks, although the effect of therapy could not be evaluated precisely. another case with encephalitis and retinitis was described by alrajhi et al. [ ] . the patient had a fever, ataxic gait, and bilateral retinal hemorrhage. she could not count fingers, and the csf contained many leukocytes, including lymphocytes. her consciousness level was decreased. she was discharged on day of the illness to her home, at which time she was awake, blind, quadreparetic, and incontinent. moreover, her neurologic conditions did not improve for the next year. an additional report described a patient who had persistent hemiparesis for four months after the onset of illness [ ] , and another paper reported rvf patients, who developed neurological signs and symptoms, including meningeal irritation, confusion, stupor and coma, hypersalivation, teeth-grinding, visual hallucinations, locked-in syndrome, and choreiform movement of upper limbs [ ] ; in these patients, the histopathological lesions in brains were characterized by focal necroses associated with an infiltration of round cells, mostly lymphocytes and macrophages, and perivascular cuffing [ ] . some patients suffer from maculopathy or retinopathy. patients noticed the loss of central vision or blurred eye occurring at various times after infection; e.g., from immediately after the disease onset to several weeks or months later. one or both eyes could be affected [ ] [ ] [ ] , and the affected eyes had macular edema with exudates containing a white mass covering the macular area with or without retinal hemorrhage, vasculitis, infarction or vitreous haze [ ] [ ] [ ] [ ] [ ] [ ] . in addition, retinal detachment [ , ] , uveitis [ , ] , or arterial occlusion [ , , [ ] [ ] [ ] was reported in some patients. in many cases, a complete recovery of vision does not occur, and chorioretinal scarring can remain in macular and paramacular areas, in spite of the resorption of exudates [ , , [ ] [ ] [ ] [ ] [ ] , while some patients show partial improvement in vision after several months of rvfv infection [ , , , ]. fatal rvf cases often involve hemorrhagic manifestations but the time to death varies among cases [ ] [ ] [ ] . most typically, the illness starts suddenly, and the patients experience fever, rigor, nausea, vomiting, headache, injected conjunctives, drowsiness, and/or body pains. the patients may also have such symptoms as macular rash over the entire trunk, ecchymoses on the arms, limbs, and/or eyelids, bleeding from the gums and/or gastrointestinal mucosal membrane, low blood pressure, hematemesis, melena, diarrhea, throat pain, pneumonitis, jaundice, and/or hepatosplenomegaly [ , ] . typically, elevation of alanine aminotransferase (alt), aspartate aminotransferase (ast), lactate dehydrogenase (ldh), and reduction of platelet count and hemoglobin are seen in these patients [ , ] . in many cases, death occurs in to days after patients become symptomatic; however, in some cases, death occurs in to days after the onset of symptoms. postmortem examination shows diffuse necrosis of hepatocytes which more greatly affect the centrilobular area than the portal area, which may indicate association with acute hepatic injury in this type of pathogenesis [ , ] . it should be noted that some patients who do not exhibit jaundice or hemorrhage, die from renal failure or disseminated intravascular coagulation (dic) accompanied by an elevation of alt, ast, ldh or d-dimer, or a decrease in platelet count [ , ] . a group of rvf patients who died from typical hemorrhagic fever also had encephalitis in addition to hepatic and gastro-intestinal necroses [ ] , which demonstrates the neuroinvasiveness of rvfv in hemorrhagic patients. another type of fatal case of rvfv infection was described by schwentker et al. [ ] . they reported that the temperature of the patient fell to normal on the th day after the onset of symptoms, whereas two papular areas of several centimeter in diameter were found on the patient's thigh and leg on the th day and remained until day . after temporal recovery by the th day, the patient experienced phlebitis of the popliteal vein, which was followed by infarcts in the lungs on the th, th and th days at multiple locations; these eventually caused a fatal embolus in the pulmonary vessels on the th day of illness. the liver of the patient was normal, did not contain infectious rvfv, and no typical rvf lesions were confirmed at the postmortem histopathological examination. however, there was a large thrombus in the inferior vena cava, a part of which might have detached and caused an embolus in the pulmonary artery. the neutralizing antibody showed up on the th day of illness, and its titer increased toward the th day. in a retrospective study in egypt, no increases in the total number of abortions were seen during an rvf outbreak, and the serological conversion rate of aborted women before and after outbreak was . % and . %, respectively [ ] . a report describing a potential vertical infection of rvfv concerned a pregnant woman, who experienced fever, headache, dizziness and generalized muscle ache four days before delivery during the rvf outbreak in saudi arabia in and developed igg specific to rvfv [ ] . her newborn baby presented with an anti-rvfv igm antibody, as well as alt/ast elevation, jaundice, extension of the activated partial thromboplastin time (aptt: test for the deficiency of intrinsic pathway factors) and the prothrombin time (pt: test for the deficiency of extrinsic pathway factors), and died on the th day after birth [ ] . although it is unknown whether the newborn baby died from rvf, it is possible that a vertical transmission in utero might have occurred in this case. the clinical symptoms of rvf vary among patients. the determinant of host susceptibility to induce hemorrhagic fever in humans has not been characterized. it is also unknown how rvfv causes diseases such as neurological disorders, vision loss or thrombosis in the presence of protective antibodies. several animal models have been used to understand the pathology of rvf, and the advantages and disadvantages of different animal models are summarized in table . we discuss the pathological findings in various different animal models in the next chapter. mice are one of the most susceptible animal species to rvfv infection [ , ] , and rvf pathology in infected mice mimics the pathological findings in newborn lambs [ ] . most of the mice infected with wild-type (wt) rvfv zh or zh strains die in to days [ ] [ ] [ ] , whereas they die faster by infection with other wt isolates [ , ] . infected mice show ruffed fur with decreased activity in to days, and then become more lethargic while lying with their back legs wide apart [ , ] . the symptom is often followed by death within one hour [ ] . occasionally, mice survive this stage, yet have hind limb paralysis at days - post infection (p.i.) and die from encephalitis [ ] . the rectal temperature of infected mice is often normal or decreased to below normal [ ] . also, the clotting time of blood derived from rvfv-infected mice is significantly extended, and it clots normally by mixing with normal sera, a finding that may indicate the shortage of coagulation factors is important for the extension of clotting time [ ] . the liver is the major target organ of rvfv, while the enlargement of liver is not common [ , ] . liver lesions are characterized by fulminant hepatitis, with coagulative necroses leaving the portal space intact [ , , , ] . depletion of glycogen in hepatocytes is also common at an early stage [ , , ] . infected hepatocytes in mice contain eosinophilic intranuclear inclusion bodies [ , ] , which are not reactive to the feulgen reaction, a nucleic acid stain [ ] . the intranuclear inclusion bodies in rvfv-infected cultured cells were visualized by an indirect immunofluorescent assay with antisera against rvfv and found to have a filamentary shape [ ] . inclusion bodies are formed by the nss protein [ ] , a viral nonstructural protein, and the -to- amino acids at the carboxyl terminus of nss are responsible for the formation of the filamentous structures via self-association [ ] . viral antigens start accumulating in hepatocytes at day , and their abundance increases extensively at day p.i. [ ] . the infected hepatocytes are stained by using a terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay, indicating that rvfv replication induces apoptosis in hepatocytes [ ] . mice that survived the early hepatitis phase often are able to regenerate hepatocytes [ , ] . although swollen endothelial cells can be observed in the liver [ ] , antigens are not easily detectable in endothelial cells or kuppfer cells, which indicate that these cells are not the primary targets of rvfv [ , ] . in addition to hepatocytes, viral antigens have been detected in, odontogenic and gingival epithelium; lipocytes; pituicytes; olfactory neurons and multiple types of neurons in the brain; mononuclear phagocytes; cardiac myofibers; and in perineural, periosteal, adrenocortical, endosteal, perivascular, bone marrow stromal, fibroblastic reticular, and vascular smooth muscle cells, as well as in cells morphologically consistent with dendritic, pancreatic islet, and adrenal medullary cells; however, no viral antigens were reported in any ocular structure, including the retina [ ] . apoptosis of lymphocytes were found in the thymus, spleen, lymph nodes and mucosa-associated lymphoid tissues [ ] . congestion and hemorrhage are common to the liver, spleen, lymph nodes, large intestine, kidneys and brain [ , ] , but are uncommon in the jejunum [ ] . in some mice that survived acute viral hepatitis, a sharp decrease in viral antigens occurred at days p.i., and no virus could be detected in the sera, liver, lung, pancreas, large intestine and ovaries [ ] . in the late stage of infection, however, lethal meningoencephalitis characterized by neuronal necrosis, microhemorrhages, and perivascular cuffs occurs in mice that survived the acute hepatitis [ ] . the susceptibility of rats to rvfv differs among rat strains [ ] . peters et al. demonstrated that -to- -week-old inbred rats from u.s. breeders exhibited three different responses to subcutaneous (s.c.) rvfv inoculation [ , ] . wistar-furth (wf) and brown norway strains were highly susceptible to rvfv and died within four days p.i. by liver necrosis, while the fisher , buffalo, da and lewis strains were largely resistant to rvfv infection [ ] . aci and maax strains proved to be moderately susceptible and showed ascending paralysis; lesions were mainly in the brain and spinal cord and characterized as mild-to-severe necrotizing encephalitis and encephalomyelitis with focal necrosis with neutrophilic infiltrate and perivascular cuffing primarily with lymphocytes. in the aci and maax strains, viruses were undetectable in the liver and blood, whereas -to- log pfu/g of viruses could be detected from brain tissue, even in the presence of neutralizing antibody in the serum. the intracranial injection of rvfv uniformly caused encephalitis in these rats, including the resistant lewis strain. the immunosuppression of the resistant lewis rats by treating animals with cyclophosphamide day prior to s.c. rvfv infection resulted in death at around -to- days p.i. with increased viral titers in the serum, liver, spleen, brain, kidneys and adrenal gland, although the virus titers in these organs in the lewis rat were still lower than those in the corresponding organs of the wf strain [ ] . these data suggest that the lewis rat encodes a gene(s) important for the resistant phenotype. interestingly, wf (wf/mol) and lewis rats (lewis/mol) obtained from a european breeding colony are resistant and susceptible to rvfv, respectively; hence, these rats showed the opposite susceptibilities to rvfv infection to those of the same strains from u.s. breeders. furthermore, both wf and lewis rats obtained from another european breeder were resistant to rvfv infection; taken together, these findings indicate the possible genetic variability of inbred rats among different breeders [ ] . cross-breeding experiments culminated in findings that indicated the resistance of wf/mol rat was segregated as a single mendelian dominant locus [ ] . findlay et al. also showed that the albino rat of the glaxo strain had an age-dependent susceptibility to rvfv via the i.p. route infection [ ] ; rats younger than days died in to days with extensive liver necrosis, whereas -day-old rats survived rvfv infection [ ] . the syrian hamster is one of the most susceptible rodents to rvfv. death occurs in to days p.i. after intraperitoneal inoculation with massive liver necrosis [ , ] . the pathological changes are similar to those seen in mice [ ] . administration of low titers of neutralizing antibodies protects hamsters from fatal liver necrosis, yet infected hamsters die from encephalitis by day [ ] . the gerbil, meriones unguiculatus, represents a unique rvfv animal model, which produces fatal encephalitis with minimal liver involvement after infection of non-neuroadapted wt rvfv. the gerbil has proven moderately susceptible to rvfv, and the survival rate of -week-old gerbils after s.c. inoculation is reported to range from to %, dependent on the strain and inoculation dose [ ] . death was reported to occur around to weeks in a dose-independent manner; s.c. inoculation of , , , pfu of zh resulted in %, %, % and % survival of outbred tum:(mon) gerbils, respectively, and %, %, % and % survival of inbred mon/tum gerbils, respectively [ ] . the infected gerbils exhibited hind-limb paralysis, generalized weakness and wasting. gerbils also showed an age-dependent resistance to rvfv infection. most of the -to -week-old tum:(mon) gerbils died after pfu s.c. inoculation of zh from encephalitis, whereas % of -week-old gerbils were able to survive the infection. after s.c. inoculation, rvfv replicated temporally in the livers of both -week-old and -week-old gerbils on days and (~ pfu/g), while subsequent efficient virus replication in the brain occurred in -week-old gerbil (from day to day up to pfu/g), but not in -week-old gerbil (temporal increase up to at day ) [ ] . intracerebral wt rvfv inoculation of pfu into -week-old gerbils resulted in an efficient viral replication (~ pfu/g) in the brain and the mean time to death was six days, which is not statistically different from that of -week-old gerbils, which may indicate the presence of host factors influencing the neuroinvasiveness in an age-dependent manner [ ] . histopathologically, minimal multifocal necroses of hepatocytes are seen at days or after the s.c. inoculation of wt rvfv, while focal necrotizing encephalitis with neuronal necrosis, a neutrophilic infiltrate, and perivascular cuffing are seen in the brain at later time points [ ] . mild, necrotizing encephalitis without detectable infectious rvfv could be observed even in clinically normal rvf-infected gerbils [ ] . rhesus macaques are moderately susceptible to rvfv infection [ ] . after i.p or intranasal inoculations, body temperatures of infected macaques increased to - °c at to days p.i. and the febrile period lasted for to h, whereas some infected animals did not show any febrile reactions [ ] . peters et al. first described hemorrhagic fever-like illness in rhesus macaques that were experimentally infected with a wt rvfv zh strain [ ] . three out of fifteen rhesus macaques intravenously inoculated with rvfv zh became ill; two became moribund and were euthanized on days and , and one recovered from illness, whereas the others showed temporal viremia, the maximum viral titer of which occurred around day , and were clinically normal. all three monkeys exhibited lassitude, weakness, the cessation of food intake, petechiae, ecchymoses and bleeding from nares, gums or venipuncture sites. clear extensions of aptt, slight extension of pt, and a decrease in the number of platelets were observed in the two dead monkeys, possibly indicating a deficiency of coagulation factors and platelets. histopathologically, the dead monkeys showed moderate focal or midzonal coagulative necrosis of the liver involving approximately / to / of hepatocytes, necrosis in the ventricular myocardium, fibrin thrombi in the glomeruli and small intertubular vessels of renal medulla in the kidneys, and mild depletion of lymphocytes from white pulp and the deposition of eosinophilic amorphous fibrin-like material in red pulp cords in the spleen [ ] . morrill et al. reported that after intravenous inoculation of × pfu zh strain into rhesus macaques, three developed signs of hemorrhagic fever, seven were clinically ill but survived, and the other seven survived without clinical signs [ ] . serum interferon (ifn)- was detected from - h p.i. and from - h p.i. in the surviving monkeys and in those dying, respectively, and the delayed ifn response was preceded by viremia in two of the three lethally-infected monkeys [ ] . no surviving monkeys developed signs of encephalitis or retinal complications in follow-up observations at two months to two years [ ] . morrill et al. also demonstrated that the administration of recombinant leukocyte a ifn ( × u, i.m) at h after rvfv intravenous inoculation reduced the peak viremia titer by -times and cleared viruses by h p.i. [ ] . these studies suggested the importance of ifn- in limiting viral replication. findlay et al. reported that three species of african monkey, i.e., the green guenon (cercopithecus callitrichus), the sooty mangabey (cercocebus fuliginosus) and the patas guenon (erythrocebus patas), did not exhibit any febrile reaction after inoculation of rvfv, whereas virus was detected in the blood [ ] . in contrast, four species of south american monkeys, two brown capuchin monkeys (cebus fatuellus and cebus chrysopus) and two common marmosets (callithrix jacchus and callithrix penicillata) exhibited febrile reactions for to days upon rvfv infection, which may indicate that south american monkeys are more susceptible to rvfv infection than african monkeys [ ] . davies et al. reported that rvfv-infected baboons (papio anubis) had viremia for to days without developing significant clinical signs [ ] . daubney et al. originally reported an outbreak of rvf in a herd of sheep in kenya in , which was characterized as a high rate of abortion in pregnant ewes and high mortality of newborn lambs [ ] . a later study by easterday et al. described that the mortality of adult sheep following experimental rvfv infection was approximately % [ ] . typically, sheep with more than one week old were relatively resistant to rvfv infection, yet did exhibit fever ( to °c), viremia, diarrhea, nasal discharge, and decreased activity [ , ] . nine-to ten-week-old young adult sheep (ripollesa breed) that were subcutaneously inoculated with rvfv had corneal and choroidal edema with inflammatory infiltrate, which could be associated with drainage failure or inadequate corneal dehydration after transient viremia [ ] . on the other hand, -to -month-old yansaka sheep subcutaneously inoculated with rvfv died during the viremic febrile phase and displayed symptoms of epistaxis ( days p.i.~), severe and bloody diarrhea, conjunctival hemorrhage, widespread petechiae and ecchymoses in hairless areas, pulmonary edema/hemorrhage, and thrombi formation in the blood vessels of the heart, kidneys and brain. rvfv-infected west african dwarf or the ouda breed did not exhibit such rapid hemorrhagic symptoms and rather exhibited marked coagulative hepatic necrosis, and brain lesions, including mild gliosis, neural degeneration, neurophagia, and satellitosis [ ] . interestingly, yansaka, west african dwarf and ouda also had increased prothrombin time, which may have indicated that hemorrhage was induced by a combination of vascular endothelial damage and an inability to clot blood in response to the damage [ ] . the inconsistency of symptoms and mortality in adult sheep described in these publications suggest the divergence of host genetic background, even within the same breed of sheep, affects susceptibility to rvfv infection. several studies examined the effects of rvfv vaccine candidates for protecting pregnant ewes from wt rvfv infection. however, insufficient immunogenicity of inactivated vaccines or residual virulence of live-attenuated vaccines induced fetal malformations. pregnant ewes were untreated (n = ) or vaccinated (n = ) once with formalin-inactivated rvfv and then challenged with wt rvfv zh at days post vaccination [ ] . abortion occurred in both unvaccinated and vaccinated ewes, between to days p.i., and % of the ewes aborted their fetuses. one out of vaccinated ewes and one out of eight unvaccinated ewes died. these data may indicate that the inactivated rvfv vaccine induced an insufficient immunity for sheep. necropsy at days p.i. revealed that % of the ewes had either aborted or dead fetuses, while the dead or aborted lambs showed extensive liver necrosis typical of rvf [ ] . coetzer et al. reported that immunization of pregnant ewes with a live-attenuated smithburn vaccine strain at to days of pregnancy could cause a teratogenic effect in the fetus, including arthrogryposis, hydranencephaly, or mineralization of brain with or without hydrop amnii, and two out of six lambs from the nine vaccinated ewes showed such effects [ ] . hunter et al. described that pregnant ewes inoculated with live-attenuated mp- vaccine strain at to days of gestation either miscarried or produced lambs showing teratogenic effects ( out of lambs from vaccinated ewes), such as cerebellar hypoplasia, spinal hypoplasia, hydranencephaly, prognathia inferior, brachygnathia inferior, arthrogryposis, scoiliosis, lordosis, kyphosis, or dormed head [ ] . the abortion and teratogenous effects did not occur when pregnant ewes were vaccinated with mp- at the third trimester of pregnancy, i.e., at - days of gestation [ , ] . on the other hand, pregnant ewes vaccinated at days of gestation with a rvfv clone (c ) strain, which has a % in-frame deletion of nss [ ] , did not cause abortion or fetal malformations and were protective against wt rvfv challenge [ ] . these data may imply the involvement of mp- nss in the abortion of ewes and the teratogenic effects in lambs. rvfv infection causes an acute and fatal disease in newborn lambs [ ] . rvfv-infected newborn lambs usually exhibit obvious illness, including elevated body temperature ( to °c), loss of appetite, decreased activity, and prostration, about to h prior to death [ ] . the mortality rate in rvfv-infected newborn lambs is to % [ ] . studies of experimental infection of - day-old lambs with rvfv via s.c. resulted in necrosis of isolated hepatocytes ( - h p.i.), focal coagulative necrosis of hepatocytes ( - h p.i.), and extensive hepatocyte necrosis ( - h p.i.) with a progressive increase in viral antigens, whereas no viral antigens could be detected in the endothelial or kupffer cells in the liver, suggesting that hepatocytes are the primary target of rvfv [ ] . the necrosis is predominantly centrilobular or midzonal, and yet there is no definite distribution pattern in liver necrosis [ , ] . some infected lambs also exhibited necrosis in the villi at the distal jejunum and ileum and depletion of lymphocytes in the spleen, whereas the brain and eyes had no lesions [ ] . overall, the liver pathology of newborn lambs resembles that of mice or hamsters, which are extremely susceptible to rvfv. however, the rvfv neurovirulence in lambs is less prominent when compared with that in rodents. rvfv also causes diseases in other animals including goats, cattle, camels, dogs, cats, and ferrets, but does not cause any symptomatic diseases in rabbits, guinea pigs, birds, horses, pigs and other animals, as reviewed in detail previously [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] [ ] [ ] . as of the present, the mechanism of species-specific susceptibility to rvfv infection is unknown. rvfv infection shows unique pathogenesis in each animal model. because viral replication and host antiviral responses most probably contribute to viral pathogenecity, an understanding of rvf pathogenesis requires identification and characterization of the viral virulence factors and host antiviral factors. the next chapter describes the viral determinant of virulence. the rvfv genome is comprised of three rna segments named the s-, m-and l-segments ( figure ) [ , ] . the s-segment encodes n and nss genes in an ambisense manner, the m-segment. nsm (nsm ), kd (nsm ), gn and gc genes, and the l-segment, the rna-dependent rna polymerase (l) gene [ ] . rvfv virions bind to an unidentified cellular receptor, and enter the cells in a ph-dependent manner [ ] , probably through a clathrin-mediated endocytic pathway, as described for another phlebovirus [ ] . after viral uncoating, viral ribonucleocapsid (rnp) composed of viral genomic rna segments and n protein [ ] is released into the cytoplasm, and the viral polymerase, which probably is attached to the rnp exerts primary transcription to synthesize viral mrna [ ] . both n mrna and nss mrna are transcribed during primary transcription as early as min after infection from an efficiently packaged, viral-sense (negative-sense) s-segment and anti-viral-sense (positive-sense) s-segment, respectively; the packaging mechanism of rvfv rnp and the presence of specific cis-signals in the genomic rna are not known [ ] . viral rna replication starts around to h after infection [ ] and an increase in the amount of viral genomic rna results in increases in viral mrnas and proteins. the rnp is packaged into viral virions probably by its interaction with the cytoplasmic domains of gn/gc at the golgi apparatus, as reported for other bunyaviruses [ , ] . the three different rna segments could be co-packaged in a coordinated manner, in which the co-packaging of m and s-segments could support the packaging of the l-segment [ ] . the rvfv virion surface is highly symmetric t = icosahedral lattice [ ] , which is formed by a shell of glycoprotein capsomers most probably composed of gn-gc heterodimers [ ] . the rvfv m-segment encodes kd, nsm, gn and gc proteins in m mrna (figure ). those proteins are synthesized from a single open reading frame of m mrna at different augs present at the ' region of m mrna by leaky scanning of ribosomes, while the n-terminus of gn and gc is co-translationally cleaved by host proteins [ ] [ ] [ ] . nsm is synthesized from the nd aug, and the c-terminus is generated by cleavage at the n-terminus of gn, while the kd protein is synthesized from the st aug, and the c-terminus is identical to that of gn. among seven viral proteins, nss and nsm are nonstructural proteins which are not incorporated into virions [ , ] . the kd protein has not been studied in detail, while an " kd" protein induced by rvfv infection (most probably corresponding to the current kd protein) is known to be incorporated into virions [ ] , which may indicate that the kd protein is a structural protein. nss, kd protein and nsm are dispensable for viral replication in cell cultures [ , [ ] [ ] [ ] . infection of -week-old female wf rats with a recombinant rvfv zh strain lacking both kd protein and nsm induced acute fatal hepatic disease, causing the deaths of some infected rats around four days p.i., or a delayed fatal neurologic disease, resulting in death of some of infected animals around days p.i. (mortality rate: to %), while wt zh -infected rats developed acute hepatitis and % died. these data suggest that nsm is not essential for virulence and lethality [ ] . on the other hand, a recombinant rvfv mp- lacking the expression of both kd and nsm induced more extensive apoptosis than did mp- in cultured cells and the expression of nsm significantly inhibited the cleavage of caspase- and - induced by staurosporine [ ] , demonstrating that nsm protein suppresses apoptosis. thus, nsm probably suppresses apoptosis in infected hosts and affects viral pathogenicity. nss is known to be a major virulence factor of rvfv; a rvfv c strain, which has a % in-frame deletion of nss [ ] , and is completely attenuated in mice or sheep [ , ] . further studies showed that nss inhibits the synthesis of ifn- mrna under the conditions that transcription factors, such as ifn regulatory factor (irf- ), nf-b and activator protein (ap)- , are activated in wt rvfv zh -infected cells [ ] . it was found that nss can bind and sequester the p subunit of tfiih, an essential transcription factor for rna polymerase i and ii; and hence nss was reported to prevent the assembly process of the tfiih complex, resulting in the suppression of host mrna transcription [ ] . le may et al. also described studies demonstrating that a region of nss, corresponding to amino acid to , specifically binds to sin a-associated protein (sap ) and forms a complex that represses the histone acetylation required for the transcriptional activation of ifn- promoter; this repression worked even after the binding of irf- to the ifn- promoter [ ] ; hence, recombinant zh carrying a deletion at amino acid to of nss cannot suppress the ifn- mrna synthesis. it should be noted that c nss bound to sap , yet it did not suppress ifn- expression [ ] . although the inability of c nss to suppress ifn- expression may have been due to its poor accumulation in infected cells, further studies are required to know how the binding of nss to sap can lead to the suppression of ifn- mrna synthesis. it is also uncertain whether the general host transcriptional suppression is required for the inhibition of ifn- mrna synthesis. because nss induces host general transcription suppression [ ] , rvfv might have the ability to replicate under host transcription suppression. actinomycin d (actd) is a general inhibitor of host rna synthesis. viral titers of several cytoplasmic rna viruses including arenaviruses [ ] [ ] [ ] , measles virus [ ] , sindbis virus [ ] , rubella virus [ ] , polio virus [ , ] , coronaviruses [ , ] , and lactic dehydrogenase elevating virus [ ] could be reduced in the presence of actd, while the viral rna synthesis is often unaffected [ , , ] . we found that expression of nss protein is essential for the rvfv mp- strain to actively synthesize viral proteins and produce high titers of infectious viruses in the presence of actd [ ] ; cells infected with recombinant rvfv mp- lacking nss failed to synthesize viral proteins; and while cells infected with mp- lacking nss accumulated phosphorylated eif . the latter made cellular translation initiation inactive through the activation of dsrna-dependent protein kinase (pkr) at around h p.i. [ ] , resulting in the suppression of viral protein synthesis. further studies revealed that mp- nss promoted the degradation of pkr through the proteasome pathway and prevented an accumulation of phosphorylated eif , thereby securing efficient viral protein synthesis under host transcription shut-off induced by actd [ ] . habjan et al. reported that wt rvfv nss also induced pkr degradation and demonstrated that an rvfv c strain carrying biologically inactive nss induced fatal hepatic disease in c bl/ mice lacking pkr [ ] ; these mice are competent for inducing type-i ifns in response to viral rna replication or poly (i):poly (c) [ ] . pkr is one of several ifn-stimulated genes (isgs) and plays an important role in inhibiting viral replication in vivo; other rna viruses, including vesicular stomatitis virus (vsv) and influenza virus, also replicate more efficiently in mice lacking pkr than in those with an intact pkr [ ] . in addition to pkr, ifn-induced mxa proteins is also known to inhibit rvfv replication [ ] . although the genetic diversity of rvfv strains is relatively low (approximately % in primary sequences [ ] ), the susceptibilities to rat ifn-/ differed among rvfv strains. most of the sub-saharan rvfv strains are very sensitive to rat ifn-/ (ed : . - . units), whereas egyptian strains, including zh and zh , and a zimbabwean isolate ( / ) are relatively resistant to rat ifn-/ (ed : - units) [ ] . all of those rvfv isolates show a similar sensitivity to human ifn- (ed : - units). in summary, rvfv nss induces the shut-down of host transcription, including transcription of both type-i ifn and isgs mrnas, to prevent antiviral responses. nss also induces the degradation of pkr to prevent eif -mediated host and viral translational shut-off and promote an efficient viral protein synthesis. although nss is a major virulence factor to escape host innate immune responses, the virulence of rvfv could be controlled in a polygenic manner. the rvfv mp- strain is a highly attenuated strain derived from wt rvfv zh [ ] and encodes a functional nss gene. mutations in the m-and l-segments are major determinants of mp- attenuation [ , ] . in contrast, the c strain encodes an s-segment lacking a functional nss gene, while the m-and l-segments of c strain are still virulent phenotypes [ , ] . mice lacking ifn-ar (ifn-ar -/mice) are susceptible to both mp- and c , while the viral replication kinetics of mp- and c differ in those mice; the highest titer of viremia was reached within h p.i. and around h p.i. in c -infected mice and in mp- -infected mice, respectively [ ] . thus, there is a possibility that m-and l-segments of c may facilitate a rapid replication of c in these mice, reaching the highest virus titer substantially earlier compared to mp- -infected mice. recently, we found that wt rvfv zh virus stock contains two major viral populations, rzh -m -a (glu at aa. of gn protein) and rzh -m -g (gly at the corresponding site); the difference in the amino acid is mapped within one of the neutralizing epitopes in gn protein [ , ] . although it is not known how the two different populations have emerged in the zh virus stock, which was amplified once in the mouse brain and passaged twice in frhl cells and twice in vero e cells, the rzh -m g replicated less efficiently than rzh -m a in infected mice, whereas both of them replicated efficiently in tissue cultures such as mrc- , veroe , j . , and nih t cells. our study showed that one amino acid change in the gn can substantially alter replication and pathogenesis of zh in vivo, and yet the mechanism of the gn mutation in the pathogenesis remains unknown. clearly further studies will be needed for understanding the roles of viral proteins in rvfv pathogenesis. rvfv encodes several virulence factors, and the major virulence factor nss plays an important role in evading host innate immune responses. in the next chapter, we discuss how humans or host animals develop protective immune responses against highly virulent wt rvfv. adequate immunity against rvfv can attenuate the virulence of rvfv in animals and vaccination against rvfv can save animals from lethal rvfv challenge [ , , [ ] [ ] [ ] [ ] . the passive transfer of neutralizing antibodies is sufficient for protection from lethal rvf [ , [ ] [ ] [ ] [ ] , whereas the role of the cellular immune response for the protection is not sufficiently evaluated. yet, mandell et al. demonstrated that mice immunized with virus-like particles containing n have a higher survival rate (survival: / ) than those not containing n after lethal rvfv challenges (survival: / ). because anti-n protein antibody is unlikely to neutralize rvfv, these data may point to the involvement of cellular immune responses against n protein for rvfv protection [ ] . alternatively, antiviral immune responses might be induced by forming a complex between anti-n antibody and n proteins released from dead cells or infected cells in vivo [ ] . if n proteins are present in cell surface as reported in influenza virus-infected cells [ , ] , complement-mediated cell lysis could be another mechanism to support the elimination of infected cells [ ] . aerosol exposure is one of the most likely routes for both laboratory infections and bioterrorism attack. immunization of rats by s.c. inoculation of a formalin-inactivated rvfv vaccine (tsi-gsd- ) partially protected lethal rvfv aerosol exposure at days post immunization (survival: / : %), whereas out of surviving rats developed encephalitis without clinical signs at - days post-challenge, which was revealed during necropsy [ ] . another study showed that three vaccinated rats challenged with rvfv aerosol developed uveitis, although no histopathological analysis was presented [ ] . mice are highly susceptible to wt rvfv aerosol exposure (ld : to pfu), which may cause lethal hepatitis, but not pneumonia [ ] . a study in mice vaccinated at several different routes with formalin-inactivated rvfv vaccine (ndbr- ) [ ] and challenged with rvfv subcutaneously showed that the immunization route affected survival rates; s.c. immunization, intraperitoneal (i.p.) immunization, and intraduodenal (i.d.) immunization resulted in survival rates of . %, % and less than %, respectively. another study reported that fewer than % of mice immunized via s.c. or i.d. and about % of mice immunized via i.p. survived after aerosol route challenge of rvfv in the -week observation period [ ] . findings from this study using aerosol rvfv challenge indicated that the mucosal immunity elicited by i.d. immunization successfully lowered the occurrence of olfactory bulb encephalitis, but failed to prevent necrotic hepatitis, and immunity induced by s.c. or i.d. did not prevent hepatitis, olfactory bulb encephalitis and multifocal encephalitis, while i.p. immunization completely prevented the occurrence of hepatitis, but not multifocal encephalitis [ ] . these reports seem to indicate that immunization with inactivated vaccines via s.c. cannot prevent rvfv-induced diseases after aerosol challenge. it will be important to establish a reliable countermeasure against bioterrorism by use of rvfv through further detailed characterization of rvfv replication in various organs after aerosol challenge and examination of the efficacy of immunization of current live-attenuated rvfv vaccine candidates, such as mp- or c , for preventing rvf-induced diseased after rvfv aerosol challenge. exposure of animals to aerosol containing rvfv probably results in initial rvfv infection in lung epithelial cells, such as type i alveolar epithelial cells. both infection and release of rvfv occur in polarized epithelial cells, such as caco- cells (human colorectal adenocarcinoma cells), at apical and basolateral membranes [ ] . infection by punta toro virus (ptv), which belongs to the phlebovirus genus and causes a lethal necrotic hepatitis in hamsters [ , ] and mice [ ] , results in virus being released into the basolateral membrane [ ] , which may contribute to systemic viral spread in infected animals. as described in section . , several studies point to the possibility that unidentified host genetic factors influence the susceptibility of inbred rat species to rvfv. the primary rat hepatocytes derived from resistant american lewis rats or wf/mol rats are less permissive to rvfv infection than those derived from susceptible american wf rat or lewis/mol rats [ , ] , whereas rvfv replicates efficiently in primary cortical glial cells derived from wf/mol rats [ ] or spontaneously transformed cell lines generated from embryonic thymus cells of either resistant lew rats or susceptible wf rats [ ] , suggesting that hepatocytes in those resistant inbred rats are under the control of a host factor(s) that restricts efficient rvfv replication. peritoneal macrophages (pm) derived from resistant lewis rat or susceptible wf rat were treated with . or u of ifn, which resulted in a -or -fold reduction of zh replication in lewis pm, and a -or -fold reduction in wf pm, respectively, possibly indicative of the role of ifn to increase resistance in lewis rats [ ] . on the other hand, primary hepatocytes derived from susceptible lewis/mol rats and resistant wf/mol rats are resistant to rvfv after treatment of the cells with rat type-i ifn, suggesting the resistance induced by type-i ifn is not necessarily important for the host specific resistance seen in wf/mol rats [ ] . a recent study showed that mbt/pas mice, but not balb/cbyi mice, are highly susceptible to rvfv zh infection [ ] . experiments using microarray and quantitative real-time pcr showed that rvfv zh replication in mouse embryonic fibroblast (mef) cells derived from susceptible mbt/pas mice induced higher levels of ifnb and ifna mrnas than did those derived from resistant balb/cbyj mice, while mef cells derived from mbt/pas mice failed to induce several isgs, including the ifn regulatory factor (irf ) mrna, the '- ' oligoadenylate synthetase-like (oasl ) mrna, and the ifn-induced kda protein (isg ) mrna [ ] . the knockout of isg or oasl mrna expressions increased viral replication in mef cells derived from resistant balb/cbyj mice, leading the authors to suggest that mbt/pas mice have a defect in their ifn responses which controls rvfv spread [ ] . it is of interest to know whether the mice lacking isg or oasl genes are susceptible to rvfv. it is unknown whether the susceptibility of inbred rat to rvfv is due to a deficit in some isgs. the host factors determining the host susceptibility to ptv infection have been explored; ptv infection in mice mimics rvfv-induced lethal hepatitis. c bl/ j mice show an age-dependent susceptibility to ptv infection; the mice gradually become resistant to ptv from to weeks, and they are resistant at eight weeks of age [ ] . the -week-old c bl/ j mice show a delayed viremia, when compared to -week-old mice, and the peak viremia titers in the -week-old mice are -to -times lower than those in the -week-old mice [ ] . likewise, ptv replication was lower in primary cultured hepatocytes, kupffer cells and peripheral blood monocytes isolated from -week-old c bl/ mice than in those cells obtained from -week-old c bl/ mice [ , ] . adding stress to the -week-old mice by daily handling and observation increased their susceptibility to ptv replication [ ] . the role of toll-like receptor (tlr) , which is a pathogen recognition receptor recognizing double-stranded rna, in the susceptibility of the mice to ptv was studied by using -week-old tlr -/mice of the c bl/ background [ ] . the wt mice infected with ptv had a % mortality and high levels of il- induction (yet no remarkable increase in tnf-), whereas mice lacking tlr infected with ptv had increased survival rates, slightly earlier reduction of serum virus titers, and decreased levels of serum il- [ ] . on the other hand, il- -/mice were more susceptible to ptv infection and supported higher levels of viremia than did wt mice, which possibly means that il- is indispensable for protective immunity. a similar attenuation of virulence has been also reported for west nile virus infection in tlr -/mice [ ] . the authors hypothesized that over-production of il- in wt mice would be detrimental to the outcome of ptv infection [ ] , pointing out that the balance of cytokines might alter the pathogenesis of ptv. stat- is a key molecule in the ifn signaling pathway [ ] . it was reported that ptv replicated substantially better in the brains of stat- -/mice than in wt mice. also ptv titers in sera, spleens and livers of the stat- -/mice were high, which may emphasize the importance of ifn responses for limiting viral replication in the liver, spleen and brain [ ] . since the first recognition of rvf in an outbreak in , more than years has passed. although there has been good progress in characterizing clinical, pathological, and virological features of rvfv infection, rvfv still causes outbreaks in african countries or the arabian peninsula [ , ] . the development of effective, safe, highly immunogenic and economic vaccines for animals and humans will prevent rvf in the endemic countries [ ] . there are several important questions to address in controlling rvfv and in further understanding rvf pathogenesis. some of them include determining the: ( ) mechanism that triggers hemorrhagic fever, ( ) route of viral entry to the brain, ( ) mechanism of prolonged diseases in rvf patients in the presence of neutralizing antibodies, and ( ) significance of vaccination to prevent rvf after aerosol exposure. addressing these questions will have a substantial impact on understanding of rvf pathogenesis and development of anti-rvfv reagents. fields virology rift valley fever virus enzootic hepatitis or rift valley fever: an undescribed virus disease of sheep cattle and man from east rift valley fever the virus of rift valley fever or enzootic hepatitis rift valley fever rift valley fever-a review rift valley fever virus (family bunyaviridae, genus phlebovirus). isolations from diptera collected during an inter-epizootic period in kenya rift valley fever virus (bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention rift valley fever surveillance in the lower senegal river basin: update years after the epidemic risk factors for severe rift valley fever infection in kenya rift valley fever during rainy seasons, madagascar climate and satellite indicators to forecast rift valley fever epidemics in kenya prediction of a rift valley fever outbreak prevalence of anti-rift-valley-fever igm antibody in abattoir workers in the nile delta during the outbreak in egypt prevalence of rift valley fever immunoglobulin g antibody in various occupational groups before the outbreak in tanzania. vector borne zoonotic dis replication and dissemination of rift valley fever virus in culex pipiens vector potential of selected north american mosquito species for rift valley fever virus vector competence of a houston, texas strain of aedes albopictus for rift valley fever virus report of a fatal laboratory infection complicated by thrombophlebitis laboratory infections with the virus of rift valley fever am rift valley fever : a report of three cases of laboratory infection and the experimental transmission of the disease to ferrets human infection with rift valley fever virus and immunity twelve years after single attack rift valley fever; accidental infections among laboratory workers rift valley fever; i. the occurrence of human cases in johannesburg rift valley fever in south africa. . the occurrence of human cases in the orange free state, the north-western cape province, the western and southern transvaal. b. field and laboratory investigation rift valley fever in south africa: . the occurrence of human cases in the orange free state, the north-western cape province, the western and southern transvaal. a epidemiological and clinical findings rift valley fever encephalitis. a description of a case rift valley fever encephalitis clinical studies on rift valley fever. part : ophthalmologic and central nervous system complications rift valley fever affecting humans in south africa: a clinicopathological study rift valley fever ocular manifestations: observations during the epidemic in egypt epidemic maculopathy rift valley fever retinitis macular changes in rift valley fever rift valley fever in man, complicated by retinal changes and loss of vision ocular disease resulting from infection with rift valley fever virus ocular complications of rift valley fever outbreak in saudi arabia ocular complications of rift valley fever ocular manifestations of rift valley fever fatal rift valley fever of man in rhodesia clinico-pathological picture in five human cases died with rift valley fever rift valley fever virus infections in egypt: pathological and virological findings in man epidemic rift valley fever in saudi arabia: a clinical study of severe illness in humans rift valley fever hepatitis complicated by disseminated intravascular coagulation and hepatorenal syndrome acute renal failure associated with the rift valley fever: a single center study rift valley fever as a possible cause of human abortions vertical transmission of fatal rift valley fever in a newborn the s segment of rift valley fever phlebovirus (bunyaviridae) carries determinants for attenuation and virulence in mice genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein nss the pathogenesis of rift valley fever virus in the mouse model rift valley fever virus in mice. iii. further quantitative features of the infective process rift valley fever virus in mice. i. general features of the infection rift valley fever virus hepatitis: light and electron microscopic studies in the mouse pathogenicity of different strains of rift valley fever virus in swiss albino mice the feulgen reaction years on demonstration of nuclear immunofluorescence in rift valley fever infected cells identification of a major non-structural protein in the nuclei of rift valley fever virus-infected cells the carboxy-terminal acidic domain of rift valley fever virus nss protein is essential for the formation of filamentous structures but not for the nuclear localization of the protein rapid accumulation of virulent rift valley fever virus in mice from an attenuated virus carrying a single nucleotide substitution in the m rna the susceptibility of rats to rift valley fever in relation to age pathogenesis of rift valley fever inbred rat strains mimic the disparate human response to rift valley fever virus infection pathogenesis of rift valley fever virus (rvfv) in inbred rats resistance to rift valley fever virus in rattus norvegicus: genetic variability within certain 'inbred' strains active and passive immunization against rift valley fever virus infection in syrian hamsters the gerbil, meriones unguiculatus, a model for rift valley fever viral encephalitis experimental rift valley fever in rhesus macaques pathogenesis of rift valley fever in rhesus monkeys: role of interferon response prevention of rift valley fever in rhesus monkeys with interferon-alpha the infectivity of rift valley fever for monkeys the pathogenicity of rift valley fever virus for the baboon experimental rift valley fever in lambs and sheeps clinical, virological and serological response of the west african dwarf sheep to experimental infection with different strains of rift valley fever virus experimental infection of young adult european breed sheep with rift valley fever virus field isolates. vector borne zoonotic dis experimental infection of three nigerian breeds of sheep with the zinga strain of the rift valley fever virus abortion in vaccinated sheep and cattle after challenge with rift valley fever virus hydrops amnii in sheep associated with hydranencephaly and arthrogryposis with wesselsbron disease and rift valley fever viruses as aetiological agents teratogenicity of a mutagenised rift valley fever virus (mvp ) in sheep further evaluation of a mutagen-attenuated rift valley fever vaccine in sheep pathogenicity and immunogenicity of a mutagen-attenuated rift valley fever virus immunogen in pregnant ewes characterization of clone , a naturally attenuated avirulent isolate of rift valley fever virus, which is altered in the small segment evaluation of the efficacy and safety of the rift valley fever clone vaccine in sheep the pathogenesis of rift valley fever in lambs distribution of viral antigen in tissues of new-born lambs infected with rift valley fever virus the pathology of rift valley fever. i. lesions occurring in natural cases in new-born lambs the clinical aspects of rift valley fever virus in household pets. i. susceptibility of the dog the clinical aspects of rift valley fever virus in household pets. ii. susceptibility of the cat susceptibility of dogs and cats to rift valley fever by inhalation or ingestion of virus pigs and rift valley fever rift valley fever in camels rift valley fever development and characterization of a rift valley fever virus cell-cell fusion assay using alphavirus replicon vectors helenius, a. entry of bunyaviruses into mammalian cells structure of the rift valley fever virus nucleocapsid protein reveals another architecture for rna encapsidation rift valley fever virus nss mrna is transcribed from an incoming anti-viral-sense s rna segment role of the cytoplasmic tail domains of bunyamwera orthobunyavirus glycoproteins gn and gc in virus assembly and morphogenesis the glycoprotein cytoplasmic tail of uukuniemi virus (bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging mechanism of tripartite rna genome packaging in rift valley fever virus threedimensional organization of rift valley fever virus revealed by cryoelectron tomography electron cryo-microscopy and singleparticle averaging of rift valley fever virus: evidence for gn-gc glycoprotein heterodimers rift valley fever virus m segment: phlebovirus expression strategy and protein glycosylation rift valley fever virus m segment: use of recombinant vaccinia viruses to study phlebovirus gene expression synthesis, proteolytic processing and complex formation of nterminally nested precursor proteins of the rift valley fever virus glycoproteins protein synthesis in rift valley fever virus-infected cells nsm and -kilodalton proteins of rift valley fever virus are nonessential for viral replication in cell culture the nsm proteins of rift valley fever virus are dispensable for maturation, replication and infection rescue of infectious rift valley fever virus entirely from cdna, analysis of virus lacking the nss gene, and expression of a foreign gene rift valley fever virus lacking nsm proteins retains high virulence in vivo and may provide a model of human delayed onset neurologic disease nsm protein of rift valley fever virus suppresses virus-induced apoptosis nss protein of rift valley fever virus blocks interferon production by inhibiting host gene transcription tfiih transcription factor, a target for the rift valley hemorrhagic fever virus a sap complex inhibits ifn-beta expression in rift valley fever virus infected cells replication and physical parameters important for preparing purified junin virus inhibition of pichinde virus replication by actinomycin d inhibition of lymphocytic choriomeningitis virus replication by actinomycin d and -azauridine the effects of actinomycin d on rna synthesis in measles virus-infected cells requirement for host transcription in the replication of sindbis virus rubella virus replication: effect of interferons and actinomycin d the effect of rifampicin, actinomycin d and mitomycin c on poliovirus and foot-and-mouth disease virus replication the inhibition by actinomycin d of poliovirus multiplication hep cells inhibition of coronavirus e replication by actinomycin d differential in vitro inhibition of feline enteric coronavirus and feline infectious peritonitis virus by actinomycin d inhibition of replication of lactic dehydrogenase virus by actinomycin rift valley fever virus nss protein promotes post-transcriptional downregulation of protein kinase pkr and inhibits eif alpha phosphorylation nss protein of rift valley fever virus induces the specific degradation of the double-stranded rna-dependent protein kinase deficient signaling in mice devoid of double-stranded rna-dependent protein kinase essential role for the dsrna-dependent protein kinase pkr in innate immunity to viral infection inhibition of bunyaviruses, phleboviruses, and hantaviruses by human mxa protein complete genome analysis of ecologically and biologically diverse rift valley fever virus strains reveals widespread virus movement and low genetic diversity due to recent common ancestry viral determinants of virulence for rift valley fever (rvf) in rats mutagen-directed attenuation of rift valley fever virus as a method for vaccine development rna polymerase i-mediated expression of viral rna for the rescue of infectious virulent and avirulent rift valley fever viruses reassortant rvfv between mp- and zh by reverese genetics. the university of texas medical branch use of bacterial expression cloning to define the amino acid sequences of antigenic determinants on the g glycoprotein of rift valley fever virus rift valley fever vaccines immunization against rift valley fever virus. studies onthe immunogenicity of lyophilized formalin-inactivated vaccine the development of a formalin-killed rift valley fever virus vaccine for use in man rift valley fever; the neurotropic adaptation of the virus and the experimental use of this modified virus as a vaccine efficacy of a rift valley fever virus vaccine against an aerosol infection in rats baculovirus expression of the m genome segment of rift valley fever virus and examination of antigenic and immunogenic properties of the expressed proteins evaluation of a formalin-inactivated rift valley fever vaccine in sheep long term existence of rift valley fever virus in immune mice flick, r. a replication-incompetent rift valley fever vaccine: chimeric virus-like particles protect mice and rats against lethal challenge contributions of antinucleoprotein igg to heterosubtypic immunity against influenza virus early presence of ribonucleoprotein antigen on surface of influenza virus-infected cells expression of influenza a virus internal antigens on the surface of infected p cells rift valley fever vaccine for humans respiratory infectivity of a recently isolated egyptian strain of rift valley fever virus mucosal priming alters pathogenesis of rift valley fever bidirectional infection and release of rift valley fever virus in polarized epithelial cells pathogenesis of a phleboviral infection (punta toro virus) in golden syrian hamsters induction of severe disease in hamsters by two sandfly fever group viruses, punta toro and gabek forest (phlebovirus, bunyaviridae), similar to that caused by rift valley fever virus punta toro virus infection of c bl/ j mice: a model for phlebovirusinduced disease assembly and polarized release of punta toro virus and effects of brefeldin a immunoelectron microscopy of rift valley fever viral morphogenesis in primary rat hepatocytes the effects of aging in vitro and interferon on the resistance of rat macrophages to rift valley fever virus (rvfv) a new mouse model reveals a critical role for host innate immunity in resistance to rift valley fever role of hepatocytes and kupffer cells in agedependent murine hepatitis caused by a phlebovirus, punta toro effect of macrophage source and activation on susceptibility in an age-dependent model of murine hepatitis caused by a phlebovirus tlr deletion limits mortality and disease severity due to phlebovirus infection toll-like receptor mediates west nile virus entry into the brain causing lethal encephalitis fagard, r. stat and pathogens, not a friendly relationship punta toro virus (bunyaviridae, phlebovirus) infection in mice: strain differences in pathogenesis and host interferon response the authors (ti and sm) were supported by grant number u ai through the western regional center of excellence. ti was also supported by r ai from the national institute of allergy and infectious diseases and internal funding from the sealy center for vaccine development at the university of texas medical branch. sm was also supported by a grant from department of homeland security. key: cord- -m m zgfy authors: pharo, elizabeth a.; williams, sinéad m.; boyd, victoria; sundaramoorthy, vinod; durr, peter a.; baker, michelle l. title: host–pathogen responses to pandemic influenza h n pdm in a human respiratory airway model date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: m m zgfy the respiratory influenza a viruses (iavs) and emerging zoonotic viruses such as severe acute respiratory syndrome-coronavirus- (sars-cov- ) pose a significant threat to human health. to accelerate our understanding of the host–pathogen response to respiratory viruses, the use of more complex in vitro systems such as normal human bronchial epithelial (nhbe) cell culture models has gained prominence as an alternative to animal models. nhbe cells were differentiated under air-liquid interface (ali) conditions to form an in vitro pseudostratified epithelium. the responses of well-differentiated (wd) nhbe cells were examined following infection with the pandemic influenza a/h n pdm strain or following challenge with the dsrna mimic, poly(i:c). at h postinfection with h n pdm , the integrity of the airway epithelium was severely impaired and apical junction complex damage was exhibited by the disassembly of zona occludens- (zo- ) from the cell cytoskeleton. wdnhbe cells produced an innate immune response to iav-infection with increased transcription of pro- and anti-inflammatory cytokines and chemokines and the antiviral viperin but reduced expression of the mucin-encoding muc b, which may impair mucociliary clearance. poly(i:c) produced similar responses to iav, with the exception of muc b expression which was more than -fold higher than for control cells. this study demonstrates that wdnhbe cells are an appropriate ex-vivo model system to investigate the pathogenesis of respiratory viruses. over the past years, there has been a rapid rise in emerging infectious diseases both of zoonotic and human origin [ ] . the recent emergence and worldwide spread of the severe acute respiratory syndrome-coronavirus- (sars-cov- ) virus which produces the coronavirus disease (covid- ) respiratory illness demonstrates the risk to human health and the economy posed by respiratory pathogens [ ] . for novel viruses, often no diagnostics or therapeutics exist, hence they present an enormous risk of a catastrophic global pandemic [ , ] . respiratory rna viruses, particularly influenza a viruses (iav) of the orthomyxoviridae family are a major pandemic risk as they replicate rapidly, lack a proof-reading mechanism, have a high mutation rate and are readily transmissible [ , [ ] [ ] [ ] . over years ago, the h n "spanish flu" pandemic killed up to million people worldwide [ ] [ ] [ ] . three global influenza pandemics have occurred since, "asian flu" h n undifferentiated nhbe and wdnhbe cells were fixed in % neutral buffered formalin and embedded in paraffin, sectioned ( µm; microtome), mounted and stained with hematoxylin and eosin. for immunofluorescence assays, airlifted nhbe cells were fixed in % w/v paraformaldehyde for at least min, rinsed with and then stored in dpbs at • c. fixed cells were permeabilized for min at rt with % v/v triton x- /dpbs (sigma-aldrich, st. louis, mo, usa), blocked for h in % w/v bovine serum albumin (bsa; sigma-aldrich, st. louis, mo, usa)/ . % v/v triton x- /dpbs. apoptotic dna fragmentation which occurs in the last phase of apoptosis (programmed cell death) was detected by terminal deoxynucleotidyl transferase dutp nick end labelling (tunel) staining using the in situ cell death detection kit, tmr red (cat# ; roche, mannheim, germany) according to the manufacturer's instructions. cells were then stained with primary antibody overnight at • c, washed three times with dpbs and then incubated with the appropriate species-specific secondary antibody diluted at : in % w/v bsa/ . % v/v triton x- /dpbs for at least h at rt. transwell membranes were rinsed three times with dpbs, stained with phalloidin (a , alexa fluor phalloidin, thermofisher scientific, usa) which stains f-actin filaments for min at rt, washed three times with dpbs, stained with the nuclear stain ', -diamidino- -phenylindole (dapi, d ; thermofisher scientific, usa) for min at rt. transwells were washed twice with sterile water, excised and mounted on glass slides with prolong gold antifade mountant (p , thermofisher scientific, usa). primary antibodies were used at the following dilutions : mucin ac (muc ac, ma - , thermofisher scientific, usa), : mucin b (muc b, hpa , sigma-aldrich, usa), : zo- (zo- - a ; - , thermofisher scientific, usa), : acetylated tubulin (tubac, t , sigma-aldrich, usa) and : claudin (cldn , ab , abcam, cambridge, uk). a monoclonal antibody specific to the nucleoprotein (np) of type a specific influenza (iav np) was produced using hybridoma technology in mice, as previously described [ ] . the hybridoma iav np monoclonal antibody h -l - used at : dilution was kindly provided by paul selleck (australian centre for disease preparedness, csiro, geelong, australia). secondary antibodies were purchased from thermofisher scientific, usa and included goat anti-mouse igg (h+l), alexa fluor and alexa fluor (a and a respectively), goat anti-rabbit igg (h+l), alexa fluor (a ) and donkey anti-rabbit igg (h+l) alexa fluor (a ). cells were imaged using the zeiss lsm confocal microscope (zeiss, oberkochen, bw, germany) using a × oil immersion objective unless specified otherwise. images were captured as z-stacks and maximum intensity projections generated. images were captured and processed using zen . blue software (zeiss, germany). the presence of α - and α - -linked sialic acids (sas) on the surface of wdnhbe cells was analyzed by fluorescence activated cell sorting (facs). wdnhbe cells were detached from transwell membranes by two the pandemic strain of influenza virus a/california/ / (h n )pdm , kindly provided by the who collaborating centre for reference and research, melbourne, vic, australia was propagated by allantoic cavity inoculation of day old embryonated chicken eggs at • c for h. the virus was passaged once in mdck cells in the presence of µg/ml l- -tosylamide- -phenylethyl chloromethyl ketone (tpck)-treated trypsin (sigma-aldrich, usa) at • c for h or until cytopathic effect was observed. virus was aliquoted and titrated to determine the median tissue culture infectious dose (tcid ) on mdck cells in the presence of µg/ml tpck-treated trypsin. briefly, virus was serially diluted -fold and applied in quadruplicate to cell monolayers. cytopathic effect was assessed five days post infection. viral titres were calculated according to the method of reed-muench as tcid /ml [ ] . . . immune challenge of wdnhbe cells with influenza a virus and poly (i:c) wdnhbe cells were washed three times with ali media to remove excess mucus from the upper surface and inoculated with influenza a/h n pdm ( µl) at a multiplicity of infection (moi) of . for h at • c. the virus inoculum was removed, the cells washed three times with ali media to remove unbound virus and the cells incubated at • c for h postinfection (experiment endpoint). at each time point ( , , and h postinfection), the virus was harvested by the addition of ali media to the upper surface for min at • c, with both apical and basolateral media collected and stored at − • c for endpoint analyses. in addition to iav challenge, wdnhbe cells were inoculated with varying concentrations of poly(i:c) dsrna viral mimic [poly(i:c) high molecular weight, cat: tlrl-pic, invivogen, san diego, ca, usa]. briefly, airlifted cells were washed in prewarmed dpbs for min at • c to remove excess mucus. poly(i:c) ( µg and µg) in ali media was added to the apical surface of wdnhbe cells and ali media added to the basolateral compartment. cells were incubated for h at • c, then washed three times with ali media to remove excess poly (i:c). basolateral media was replaced and the cells incubated for an additional h at • c. transepithelial electrical resistance (teer) across the epithelium of undifferentiated and wdnhbe cells was measured using an epithelial voltohmmeter (evom ) with an stx chopstick electrode (world precision instruments, sarasota, fl, usa) [ ] . ali media was added to the apical and basolateral compartments, equilibrated for at least - min at rt and the teer measured. teer readings were membrane-corrected by subtraction of measurements from control transwells (no cells) and expressed in units of Ω or Ω × cm . dextran conjugated to fluorescein isothiocyanate (fitc-dextran, kda; sigma-aldrich, usa) was used to determine the paracellular permeability of wdnhbe cells, i.e., the transport of fitc-dextran from the apical to the basolateral compartment of the transwell via extracellular space in the pseudostratified airway epithelium [ ] . fitc-dextran diluted in ali media ( . mg/ml) was added to the upper transwell compartment and ali media alone added to the basolateral compartment and the cells incubated for h at • c. basolateral media was mixed, sampled in triplicate in a microtiter plate and fitc-dextran analyzed on a biotek synergy ht microtitre plate reader. assay standards and sample solutions were prepared concurrently. lactate dehydrogenase (ldh) release was quantified using the cytotox non-radioactive cytotoxicity assay (g , promega corporation, madison, wi, usa) according to the manufacturer's instructions. controls including no cells, untreated cells and maximum ldh release (triton x- -treated cells, % cytotoxicity) were analyzed together with the apical samples using a biotek synergy ht microtitre plate reader. cytotoxicity percentage was expressed as experimental ldh release compared to maximum ldh release for triton x- treated cells. quantitative reverse transcription pcr (rt-qpcr) was performed on total rna extracted from cells. total rna was isolated from wdnhbe cells using the rneasy mini kit (qiagen, hilden, nw, germany) with on-column dnase i digestion. total rna was quantified using the ds- fx spectrophotometer (denovix, wilmington, de, usa) and first strand cdna made using the superscript iii first-strand synthesis system (thermofisher scientific, usa), total rna ( ng) and oligo(dt) . gene expression was determined by rt-qpcr using first strand cdna ( : dilution), taqman gene expression master mix and taqman inventoried probes (applied biosystems, foster city, ca, usa), all of which were exon-spanning apart from the single exon interferon beta (ifnb ) probe (table s ). rt-qpcr reactions for the iav h n pdm and poly(i:c) experiments were performed on the quantstudio system and quantstudio flex real-time pcr system (applied biosystems, usa) respectively with the appropriate no template controls included on each plate. samples were assayed in triplicate. conditions used were • c for min, denaturation at • c for min followed by cycles of • c for s, • c for min. a cycle threshold (ct) of . was applied to all gene probes. gene expression levels were normalized to the human glyceraldehyde -phosphate dehydrogenase viruses , , of (gapdh) housekeeping gene and expressed as fold over detectable (fod), as described [ ] . minimum detectable ct was set at cycles. to assess the effect of iav h n pdm infection in wdnhbe cells on the transcription of twelve innate immune genes and two mucin-encoding genes quantified by rt-qpcr, we constructed a gene coexpression network [ ] . we used the normalized gene copy number in n = transwells at h postinfection with iav h n pdm , i.e., the number of gene copies per copies of gapdh (see above) to produce the network. for each pair of genes, the pearson correlation coefficient (pcc) was calculated to estimate the strength of the linear relationship between gene expression levels. a pcc value exceeding ± . was used as the coexpression threshold. for each gene-pair, a network was constructed with the expressed genes forming the network's nodes and the pcc values exceeding the . threshold forming an edge. the pcc values were estimated using the "corr" function in the r stats package, and the network was constructed and visualized using the igraph package [ ] . statistical analyses for most experiments were performed in graphpad prizm . . (graphpad software, inc., ca, usa). data from three independent experiments for each of the iav and poly(i:c) challenges was pooled and analyzed as described below. for iav h n pdm experiments, growth kinetics of iav h n pdm (tcid /ml) were analyzed by one-way anova using tukey's multiple comparison test; percentage of preinfection teer, fitc-dextran transported across the epithelium and percentage cytotoxicity were analyzed by multiple t-tests using the two-stage linear step-up procedure of benjamini, krieger and yekutieli, with a false discovery rate (fdr) of %. teer resistance in iav h n pdm -inoculated and mock-inoculated wells at h postinfection was also analyzed with r using a mixed effects model using the "aov" and the "lme" functions in the r stats and nlme packages respectively [ ] . poly(i:c) teers were analyzed by one-way anova with brown-forsythe and welch anova tests and dunnett's t multiple comparison test. poly(i:c) fitc-dextran transported and cell cytotoxicity were analyzed by one-way anova and tukey's multiple comparisons test. iav muc ac and muc b gene expression was analyzed by unpaired t-tests and for poly(i:c), by one-way anova using tukey's multiple comparison test. cytokines secreted apically form iav-infected cells were analyzed by unpaired t-tests. the use of human cells in all experiments was approved by the csiro health and medical human research ethics committee (ethics approval # _ _lr, th may, ). prior to innate immune system challenge studies, we characterized our commercially sourced nhbe cells. cells cultured at ali on collagen i-coated transwells formed a polarized, pseudostratified epithelium around four weeks postairlift. wdnhbe cells exhibited a mucociliary phenotype, characterized by the secretion of mucus on the apical surface and the coordinated beating of cilia on the surface of ciliated cells (video s ). histological staining confirmed that wdnhbe cells formed a columnar epithelium - cells thick, consisting of basal, goblet, club and ciliated cells, representative of the human airway epithelium in vivo (figure a ). immunofluorescence assays confirmed the expression of muc ac, a goblet cell marker and muc b, produced in secretory cells and the partial colocalization of muc ac and muc b (figure b) . immunostaining of wdnhbe cells also confirmed the presence of ciliated cells, characterized by the expression of acetylated tubulin on cell-surface cilia ( figure c ). in order to confirm that airlifted cells formed an intact epithelial barrier, we measured the teer of undifferentiated and differentiated nhbe cells. the teer indicates the degree of polarization and differentiation of nhbe cells and hence the formation of an intact airway epithelial barrier [ , ] . for undifferentiated nhbe cells, the average teer was < Ω × cm . in contrast, teer values ≥ Ω × cm were recorded for a majority of wdnhbe cells with average values consistently ≥ - Ω × cm ( figure s ; table s ; table s ). therefore, our cells provide an excellent model to study the integrity of the airway epithelium in vitro before, during and after immune system challenges. iav infects cells by binding to cell-surface sialic acid receptors. therefore, we characterized the iav receptors present on the exterior of wdnhbe cells by facs using the lectins sna i and maa ii which bind to α - -linked sialic acids (sa) and α - -linked sas respectively. while hemagglutinins of human iavs preferentially bind α - -sa, those from avian iavs recognize α - sa [ , ] . almost all ( . %) wdnhbe cells had α - -linked sialic acids on the cell surface, while . % had cell-surface α - -linked sialic acids ( figure s ), demonstrating the susceptibility of wdnhbe cells to iav infection in this study. wdnhbe cells were infected with h n pdm at moi of and apical virus titre monitored over a h time period postinfection for three independent replicate experiments ( figure a ). h n pdm replicated efficiently in wdnhbe cells exhibiting peak viral titre of tcid /ml at h postinfection. the titre decreased by . logs at h postinfection ( . × ; p < . ). the virus titre at h and h postinfection ( tcid /ml) when the virus binds and penetrates the cells is slightly higher than expected, which may be due to incomplete removal of unbound virus, despite numerous washes, and also the binding of the virus to mucus on the transwell surface. this was consistent across all three experiments. in preliminary experiments, no virus was detected in basolateral media at h postinfection of wdnhbe cells with h n pdm . this indicated that the virus is shed on the apical surface of polarized epithelial cells, consistent with previous studies [ , ] . immunofluorescence assays confirmed that at h postinfection, the majority of wdnhbe cells were infected with h n pdm , as indicated by staining with the influenza a nucleoprotein (iav np, figure b ). teer indicates the degree of polarization and differentiation of nhbe cells and hence the formation of an intact airway epithelial barrier [ , ] . for undifferentiated nhbe cells, the average teer was < Ω x cm . in contrast, teer values ≥ Ω x cm were recorded for a majority of wdnhbe cells with average values consistently ≥ - Ω x cm ( figure s ; table s ; table s ). therefore, our cells provide an excellent model to study the integrity of the airway epithelium in vitro before, during and after immune system challenges. to understand the effect of h n pdm on the integrity of the barrier formed by wdnhbe cells, the effect of the virus on the airway epithelium was determined by the change in teer, paracellular permeability and ldh release (percent cytotoxicity) (figure ). at - h postinfection, there was no difference in percentage teer values. during this period, the virus is undergoing virus assembly and early release of virus particles. however, at h and h postinfection, dramatic reductions in teer values of . and . % respectively occurred, significantly lower values than the teers of mock-infected cells (p < . ; refer to figure s for a graph of the individual teer values (Ω x cm ) for all three independent h n pdm experiments). this significant decrease in teer resistance was also confirmed by mixed effects modelling (p < . ). from - h postinfection, higher viral replication (observed in figure a ) correlates with a reduction in barrier integrity of wdnhbe cells. in contrast to virus-infected cells, teer values for to understand the effect of h n pdm on the integrity of the barrier formed by wdnhbe cells, the effect of the virus on the airway epithelium was determined by the change in teer, paracellular permeability and ldh release (percent cytotoxicity) (figure ). at - h postinfection, there was no difference in percentage teer values. during this period, the virus is undergoing virus assembly and early release of virus particles. however, at h and h postinfection, dramatic reductions in teer values of . and . % respectively occurred, significantly lower values than the teers of mock-infected cells (p < . ; refer to figure s for a graph of the individual teer values (Ω × cm ) for all three independent h n pdm experiments). this significant decrease in teer resistance was also confirmed by mixed effects modelling (p < . ). influenza a viruses damage apical junctional complexes and alter the cell cytoskeleton and morphology of airway epithelial cells [ , , ] . to investigate these characteristics in our model we performed immunofluorescence assays on mock and h n pdm -infected wdnhbe cells h postinfection ( figure ) . we characterized the distribution of the zo- adapter protein that links tight junctions and adherens junctions to the cell cytoskeleton [ ] . phalloidin was used to stain f-actin filaments of the cytoskeleton. in mock infected wdnhbe cells, zo- forms an intact apical to further characterize airway barrier integrity in response to iav infection, the paracellular transport of fitc-dextran ( kda) across the epithelium was measured. paracellular transport is regulated by ajcs, hence increased fitc-dextran transport from the apical to basolateral compartment of the transwell is an indicator of "leaky" or damaged junctions [ ] . at h postinfection, fitc-dextran transported across iav-infected cells was . -fold higher than for mock-treated cells (p < . ; figure b ), indicative of increased epithelium permeability. influenza a virus induces cell death [ , ] . hence, we determined the cytotoxicity of h n pdm to wdnhbe cells by measuring the apical release of ldh h postinfection. ldh is a soluble cytoplasmic enzyme that is released from the cytoplasm upon damage to the plasma membrane and is directly proportional to the number of cells undergoing apoptosis or necrosis [ , ] . at h postinfection, cell death due to h n pdm was . -fold greater for iav-infected cells compared to cells treated with media alone (p < . ; figure c ). tunel staining also showed a greater number of apoptotic cells in wdnhbe cells infected with pandemic influenza compared to mock-infected cells ( figure s ). therefore, h n pdm damage to the in vitro airway epithelium is characterized by a reduced teer, increased paracellular flux and increased cell death. in order to compare the immune response of wdnhbe cells to different immunostimulants, we challenged our cells with poly(i:c) at µg and µg and evaluated barrier integrity as for iav-infected cells. at h poststimulation, teer values were significantly reduced in a dose-dependent manner in comparison to mock-treated cells (p < . ; figure d ). similar to the h n pdm treatment, a . % reduction in teer was recorded for ug poly(i:c) and a . % reduction for the µg treatment. in contrast, the teer of mock-treated cells increased by . %. a graph of the individual teer values (Ω × cm ) for all three, independent poly(i:c) experiments is also provided ( figure s ). the effect of poly(i:c) on paracellular flux was not as great as for h n pdm . at µg and µg poly (i:c), fitc-dextran transported across the airway epithelium was -fold and -fold greater respectively compared to control cells (p < . and p < . respectively; figure e ). similarly, poly(i:c) was not as cytotoxic as h n pdm . at the higher, µg poly(i:c) dose, cell death was -fold higher than for control cells (p < . ; figure f ). however, there was no difference in cytotoxicity between the µg poly(i:c) and mock treatments. in summary, both h n pdm and poly(i:c) had a detrimental effect on the integrity of the in vitro pseudostratified airway epithelium. influenza a viruses damage apical junctional complexes and alter the cell cytoskeleton and morphology of airway epithelial cells [ , , ] . to investigate these characteristics in our model we performed immunofluorescence assays on mock and h n pdm -infected wdnhbe cells h postinfection ( figure ) . we characterized the distribution of the zo- adapter protein that links tight junctions and adherens junctions to the cell cytoskeleton [ ] . phalloidin was used to stain f-actin filaments of the cytoskeleton. in mock infected wdnhbe cells, zo- forms an intact apical circumferential belt (also known as the perijunctional actomyosin ring) around the plasma membrane of each cell (figure a ). in sharp contrast, zo- was disrupted and discontinuous in many iav h n pdm -infected cells, suggestive of damage to ajcs and the epithelium (figure b) . these results were consistent with phalloidin staining of f-actin filaments of the cell cytoskeleton. f-actin filaments are intact in mock-infected cells (figure a ) but disrupted in iav-infected cells (figure b) . merged images show that the disruption of zo- and f-actin of the cytoskeleton occurs in the same cells in the in vitro airway epithelium (merge, figure b) . furthermore, iav-infected cells show evidence of cell distension. while h n pdm disrupted zo- and f-actin in wdnhbe cells, this was not observed in cells treated with µg poly(i:c) ( figure s ). results were consistent with phalloidin staining of f-actin filaments of the cell cytoskeleton. f-actin filaments are intact in mock-infected cells (figure a ) but disrupted in iav-infected cells (figure b) . merged images show that the disruption of zo- and f-actin of the cytoskeleton occurs in the same cells in the in vitro airway epithelium (merge, figure b) . furthermore, iav-infected cells show evidence of cell distension. while h n pdm disrupted zo- and f-actin in wdnhbe cells, this was not observed in cells treated with µg poly(i:c) ( figure s ). the mucosal barrier is a major component of the innate immune system in the lungs [ , ] . muc ac and muc b are the major secreted mucins and play a critical, though poorly understood role in airway defense and mucociliary clearance [ , ] . therefore, we analyzed the effect of h n pdm on the expression of these genes by rt-qpcr (figure a) . iav had no effect on the expression of the muc ac goblet cell marker in wdnhbe cells (figure a ). in contrast, muc b was suppressed more than -fold in h n pdm -inoculated cells (p < . ; figure a ). poly(i:c) treatment also had no effect on muc ac expression when compared to mock-treated cells (ns, figure b) , consistent with pandemic iav. in contrast, poly(i:c) had a dose-dependent effect the mucosal barrier is a major component of the innate immune system in the lungs [ , ] . muc ac and muc b are the major secreted mucins and play a critical, though poorly understood role in airway defense and mucociliary clearance [ , ] . therefore, we analyzed the effect of h n pdm on the expression of these genes by rt-qpcr (figure a) . iav had no effect on the expression of the muc ac goblet cell marker in wdnhbe cells (figure a ). in contrast, muc b was suppressed more than -fold in h n pdm -inoculated cells (p < . ; figure a ). mock, n = ; iav, n = for muc b; poly(i:c) data n = biological replicates per treatment. statistical significance: ns, not significant; *, p < . ; **, p < . ; ***, p < . ; ****, p < . . we used rt-qpcr to determine whether wdnhbe cells produced an innate immune response to iav h n pdm in vitro. genes were selected based on their response to iav infection with poly(i:c) treatment also had no effect on muc ac expression when compared to mock-treated cells (ns, figure b) , consistent with pandemic iav. in contrast, poly(i:c) had a dose-dependent effect on muc b expression with -fold and -fold higher expression in wdnhbe cells treated with µg and µg poly(i:c) respectively compared to mock-treated cells ( µg poly(i:c): p < . ; µg: p < . ; figure b ). we used rt-qpcr to determine whether wdnhbe cells produced an innate immune response to iav h n pdm in vitro. genes were selected based on their response to iav infection with h n pdm and/or h n subtypes as reported for in vitro and/or in vivo studies [ , , [ ] [ ] [ ] [ ] [ ] [ ] . genes assayed included: the chemokine-encoding c-x-c motif chemokine ligand (cxcl ), also known as interferon γ-induced protein (ip ), c-c motif chemokine ligand (ccl ), alias regulated on activation, normal t cell expressed and secreted (rantes), c-c motif chemokine ligand (ccl ), also known as monocyte chemoattractant protein- (mcp ), c-c motif chemokine ligand (ccl ), alias macrophage inflammatory protein -α (mip α), and c-x-c motif chemokine ligand (cxcl ), also known as interleukin (il ). proinflammatory cytokine-encoding genes characterized included: tumor necrosis factor (tnf), known as tnf-α, interleukin β (il b), colony stimulating factor (csf ), also referred to as granulocyte-macrophage colony stimulating factor (gmcsf) and the potent proinflammatory interleukin (il ). expression of the anti-inflammatory-encoding interleukin (il ) and antiviral-encoding interferon β (ifnb ) and radical s-adenosyl methionine domain containing (rsad ), commonly known as virus inhibitory protein, endoplasmic reticulum-associated, ifn-inducible; viperin [ ] was also characterized. all genes assayed were upregulated in h n pdm -infected versus mock-inoculated cells (table ; figure s ). muc ac and muc b values ( figure ) have also been included in table . notably, there was a difference in the magnitude of the response to pandemic influenza with four different levels of fold-change observed. the highest fold-increases in expression were recorded for cxcl (> , -fold) and the antiviral-encoding ifnb (> -fold). ccl and rsad expression were also significantly upregulated in response to iav (> and > fold respectively). il , tnf, mip α and il exhibited a - -fold increase in expression compared to mock-inoculated cells. in contrast, the magnitude of response to iav-infection was lower for the ccl , cxcl , csf and il b genes ( - -fold increases). the increased expression of these cytokine, chemokine and antiviral genes suggests that innate immune responses are activated in our wdnhbe cells in response to pandemic influenza. this relationship was explored by the generation of a gene coexpression network ( figure ) based on gene copy numbers (table s ). the relationship between pro-and anti-inflammatory cytokines and chemokines genes is highlighted by the network connectivity. similarly, association between mucin gene expression is shown. in contrast, the expression of the antiviral ifnb and rsad were not linked to other genes. the induction of innate immune response genes in wdnhbe cells was also investigated by the stimulation of wdnhbe cells with µg or µg poly(i:c) ( figure s ). in contrast to iav, poly(i:c) had no effect on csf expression. while the lower, µg dose of poly(i:c) was sufficient to upregulate il and cxcl expression, tnf and il b were only induced by µg of poly(i:c) compared to mock-treated cells. to determine whether gene expression was correlated with protein secretion, multiplex immunoassays for il- , tnf-α, cxcl (il- ) and csf (gm-csf) were performed on media harvested from the apical (figure ) and basolateral compartments ( figure s ) at h postinfection of wdnhbe cells with h n pdm . apical secretion levels of il- and tnf-α after a min period were > - -fold higher in iav-infected versus mock cells (figure a, b) , with il- more than -fold higher (figure c ). in contrast, while csf trended lower in iav-infected cells, it was not significantly different to mock levels (figure d ). il- β was secreted at the minimum limit of detection (data not shown). cytokine and chemokine secretion into the lower compartment of transwells over a h period from to h postinfection exhibited similar trends to apical supernatants, although protein levels were generally reduced apart from il- ( figure s ). multiplex immunoassays for il- , tnf-α, cxcl , csf and il- β were also performed for apical and basolateral media from the µg and µg poly(i:c) treatments ( figure s ). trends were analogous to the response to iav, apart from csf , which was stimulated, rather than suppressed by poly(i:c). the apical secretion of il- β, il- and csf was only higher for µg poly(i:c) compared to mock-treated cells. in contrast, tnf-α and cxcl levels were greater than mock cells for both poly(i:c) doses. cytokine and chemokine secretion in basolateral media over a h period the induction of innate immune response genes in wdnhbe cells was also investigated by the stimulation of wdnhbe cells with µg or µg poly(i:c) ( figure s ). in contrast to iav, poly(i:c) had no effect on csf expression. while the lower, µg dose of poly(i:c) was sufficient to upregulate il and cxcl expression, tnf and il b were only induced by µg of poly(i:c) compared to mock-treated cells. to determine whether gene expression was correlated with protein secretion, multiplex immunoassays for il- , tnf-α, cxcl (il- ) and csf (gm-csf) were performed on media harvested from the apical (figure ) and basolateral compartments ( figure s ) at h postinfection of wdnhbe cells with h n pdm . apical secretion levels of il- and tnf-α after a min period were > - -fold higher in iav-infected versus mock cells (figure a,b) , with il- more than -fold higher (figure c ). in contrast, while csf trended lower in iav-infected cells, it was not significantly different to mock levels ( figure d ). il- β was secreted at the minimum limit of detection (data not shown). cytokine and chemokine secretion into the lower compartment of transwells over a h period from to h postinfection exhibited similar trends to apical supernatants, although protein levels were generally reduced apart from il- ( figure s ). multiplex immunoassays for il- , tnf-α, cxcl , csf and il- β were also performed for apical and basolateral media from the µg and µg poly(i:c) treatments ( figure s ). trends were analogous to the response to iav, apart from csf , which was stimulated, rather than suppressed by poly(i:c). the apical secretion of il- β, il- and csf was only higher for µg poly(i:c) compared to mock-treated cells. in contrast, tnf-α and cxcl levels were greater than mock cells for both poly(i:c) doses. cytokine and chemokine secretion in basolateral media over a h period exhibited similar trends to apical media. therefore, airlifted nhbe cells secrete cytokines and chemokines in response to both h n pdm and poly(i:c) immune system challenges. however, under experimental conditions used and properties measured, iav elicits a much stronger inflammatory response than poly(i:c) in wdnhbe cells. exhibited similar trends to apical media. therefore, airlifted nhbe cells secrete cytokines and chemokines in response to both h n pdm and poly(i:c) immune system challenges. however, under experimental conditions used and properties measured, iav elicits a much stronger inflammatory response than poly(i:c) in wdnhbe cells. and mock csf (n = ); ns, not significant; *, p < . ; **, p < . . we show that well-differentiated nhbe cells grown at the air-liquid interface are an anatomically and physiologically relevant in vitro model to investigate changes in the airway epithelium in response to h n pdm and the dsrna viral analogue, poly(i:c). our model we show that well-differentiated nhbe cells grown at the air-liquid interface are an anatomically and physiologically relevant in vitro model to investigate changes in the airway epithelium in response to h n pdm and the dsrna viral analogue, poly(i:c). our model recapitulated the in vivo response of the human airway epithelium to influenza in vitro. the cytopathic effect of h n pdm included damage to the airway epithelium, the induction of innate immune responses including the expression of pro-and anti-inflammatory cytokines and chemokines and antiviral genes and proteins, consistent with pulmonary host defense. to our knowledge, this is the first study that has shown zo- damage in wdnhbe cells treated with h n pdm . additionally, the downregulation of muc b, which plays an important role in mucociliary clearance suggests another mechanism of pathogenesis induced by the virus. these results form the basis for using the ali model for studying host-pathogen interactions and for testing therapeutics against newly emerged and re-emerging respiratory pathogens. the striking effect of h n pdm on zo- and the disruption of the apical perijunctional actomyosin ring of the cytoskeleton highlights iav damage to the d architecture of cells within the pseudostratified epithelium and is consistent with reduced teer, increased paracellular flux across the epithelium and increased apoptosis. the pattern of zo- damage in our wdnhbe cells infected with pandemic iav is similar to that of the immortalized human bronchial epithelial cell line ( hbe o-) infected with tissue culture adapted influenza virus a (h n ) [ ] . it also mimics zo- dissociation from tjs caused by rhinovirus infection of hbe o-cells, human airway epithelial cells grown at ali and in mice in vivo [ , ] . zo- dissociation is also consistent with the in vivo airway epithelium of asthmatic patients [ ] . while poly(i:c) disrupted zo- in cell membranes of immortalized hbe ocells [ ] , this was not observed in our primary wdnhbe cells. this may reflect differences between primary and immortalized cells and/or different concentrations and preparations of poly(i:c). airway protection is provided by a broad-spectrum of antimicrobial factors in mucus, e.g., lysozyme, β-defensins, cathelicidin, lactoferrin, elafin, secretory leukocyte peptidase inhibitor (slpi) and mucins [ ] [ ] [ ] . however, the precise roles of mucins, in particular, muc ac and muc b, the major secretory mucins in the human airway epithelium are still to be determined. optimal airway defense requires a balance between mucus production and clearance [ ] . in our wdnhbe model, mucus removal does not occur due to the closed nature of the transwell system. however, differences in mucin gene expression for h n pdm and poly(i:c) compared to mock-inoculated cells demonstrated that wdnhbe cells respond to these immune system challenges. differences in mucus composition and the relative abundance of muc ac and muc b can alter mucus gel viscosity, promoting pathogen growth, rather than their removal [ , ] . hence, the significant increase in muc b expression in response to poly(i:c) but decrease in response to h n pdm and unchanged muc ac for both immune system stimulants was intriguing. whilst the increase in muc b in response to poly(i:c) in our study was consistent with lever and colleagues, we did not observe an increase in muc ac expression [ ] . additionally, different iav strains (seasonal and pandemic) and different subtypes (h n and h n ) induced different levels of muc ac expression in an nci-h human pulmonary mucoepidermoid carcinoma cell line [ ] . seasonal strains were stronger inducers of muc ac expression than pandemic strains and h n generally had a greater effect on expression than h n [ ] . the differences observed in muc ac expression in our study may reflect the use of normal primary cells versus immortalized cancer lines, the use of different virus preparations and mois, different concentrations and/or preparations of poly(i:c) and a difference between cell donors. once the mucus layer of the airway epithelium has been penetrated, pathogens such as iav attach to cell-surface receptors (α , -and/or α , sialic acids), enter and replicate in epithelial cells. the virus egresses and spreads to other nonimmune and immune cells [ ] . although our model lacks immune cells, wdnhbe cells mount an innate immune response to both iav and poly(i:c), consistent with separate studies of these stimulants [ , ] . the body's natural defense mechanism of inflammation which promotes cell repair and healing [ ] was mimicked in our wdnhbe model, with the increased expression of proinflammatory cytokines and chemokines such as tnf, il b, il , cxcl and cxcl . this suggests that wdnhbe cells recognize iav and poly(i:c) through binding to pattern recognition receptors (prrs) such as the toll-like receptors (tlr , tlr , and tlr ), retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated protein- (mda- ), triggering innate immune response signaling cascades as occurs in vivo [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . antiviral, pro-and anti-inflammatory cytokines and chemokines are then upregulated in the host [ , , ] . key genes upregulated in our h n pdm in vitro challenge model, mimic the innate immune and inflammatory response in human patients in vivo infected with the pandemic iav. these include ifnb , cxcl , il , tnf and rsad . the more than -fold increase in ifnb , which encodes the antiviral type i interferon, ifn-β, confirms the host recognition of iav and the host-defense response to the virus in the airway epithelium in vitro, as observed by chan et al., [ ] . interestingly, the kinetics and magnitude of ifn-β secretion and hence the early innate and antiviral responses can vary for different influenza a subtypes [ ] . ifn-α/β stimulates the activation of hundreds of ifn-stimulated genes (isgs, e.g., cxcl , rsad , etc.), the first line of defence against viral infection [ ] [ ] [ ] [ ] [ ] . this is observed in our model with the dramatic, , -fold increase in cxcl expression in response to h n pdm infection, consistent with differentiated human airway epithelial cells infected with a hong kong isolate of the a/h n pandemic strain [ ] , wdnhbe cells infected with pandemic iav strains from a fatal (a/ky/ / ) and nonfatal (a/ky/ / ) case [ ] and in patients infected with pandemic iav [ , [ ] [ ] [ ] . upregulation of cxcl also occurs in vitro in the a human lung alveolar adenocarcinoma response to iavs [ ] . cxcl is a key chemokine responsible for early response to viral infection, is associated inflammation, e.g., asthma [ ] and is a biomarker that predicts disease severity and pathogenesis [ ] [ ] [ ] . the impact of cxcl is reflected in the gene coexpression network, with the increased expression of a number of other chemokines, cytokines and the anti-inflammatory il gene. cxcl is upregulated in response to other respiratory viruses, including rsv, rhinovirus and h n [ , , ] . however, while cxcl is protective in sars-cov infection [ ] , its precise role in h n pdm infection is unclear. increased levels of cytokines and chemokines are detected in the sera of patients with pandemic influenza [ ] , consistent with our model. in particular, increased levels of the proinflammatory il- and the anti-inflammatory il- which protects host tissue from damage during acute inflammatory responses are observed [ , , , [ ] [ ] [ ] . elevated levels of tnf and il b [ ] are also detected [ , , ] . tnf-α stimulates il- β which exacerbates lung injury during severe influenza but may also have a vital role in lung repair after infection [ ] . other cytokines and chemokines elevated in sera of patients include the chemoattractants cxcl [ , ] and ccl [ ] [ ] [ ] . increased expression and/or secretion of these and other chemoattractants such as ccl and ccl were observed in our wdnhbe model, consistent with other in vitro lung cell culture studies [ , , ] . hence, cell-mediated immunity is triggered in wdnhbe cells, as occurs in vivo [ , ] . the more than -fold induction of the antiviral-encoding rsad (viperin) gene in our pandemic iav-infected wdnhbe cells confirms the antiviral response of the airway epithelium in vitro. it is consistent with increased rsad expression in wdnhbe cells infected with the pandemic influenza strains a/ky/ / and a/ky/ / [ ] and in immortalized nci-h [ ] and a cells [ ] . rsad has multiple modes of antiviral activity. it catalyzes the conversion of cytidine triphosphate (ctp) to -deoxy- , -didehydro-ctp (ddhctp) which prematurely terminates rna-dependent rna polymerase (rdrp) of selected viruses but does not interfere with host rna and dna polymerases [ ] . hence, it reduces the replication of a range of rna and dna viruses including iavs, rabies virus, hiv, west nile virus, zika virus, dengue virus and hepatitis c virus [ ] [ ] [ ] . rsad also restricts the release (budding) of iav h n and other viruses by disrupting lipid raft microdomains on the plasma membrane of host cells [ , , ] . rsad also plays a role in the activation of t-cells and t-cell-receptor-mediated activation of nf-κb and activating protein (ap- ), key transcription factors in the expression of proinflammatory cytokines [ ] . hence, rsad is a multifunction antiviral that has a vital role in combatting viruses such as iav. airlifted primary nhbe cells grown on transwells at the air-liquid interface are the gold standard for bronchial epithelial cell culture [ ] . this model provided an excellent system to investigate innate immune responses to h n pdm and poly(i:c) in the airway epithelium in vitro. future studies could investigate the addition of other cell types including fibroblasts, endothelial smooth muscle cells and immune cells, e.g., neutrophils, to better reproduce the human airway in vitro and will undoubtably provide further information on host-pathogen interactions in the lung. airlifted nhbe cells recapitulated the pseudostratified airway epithelium in vivo, with the formation of ajcs, mucus secretion and the coordinated beating of ciliated cells. the disruption of the airway epithelium by iav h n pdm and poly(i:c), plus the induction of the innate immune response and antiviral, and pro-and anti-inflammatory genes demonstrated the viability of this model to investigate pandemic influenza. the disassembly of the ajc by h n pdm as shown by damage to zo- suggests that the virus can penetrate the epithelium and hence produce systemic infection. the use of multiple immune system challenges enabled the identification of differential responses of wdnhbe cells to h n pdm and poly(i:c). the reduction of muc b in response to iav is indicative of impaired mucociliary clearance of iav which may contribute to the severity of pandemic influenza in vivo. future studies will focus of the use of this model to investigate zoonotic, emerging infectious viruses with pandemic potential including the sars-cov- coronavirus currently sweeping the world today. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : taqman probes used for rt-qpcr analyses, video s : beating of cilia on the surface of ciliated cells of the wdnhbe epithelium, figure s :'transepithelial electrical resistance (teer) readings of wdnhbe cells, table s : teer readings (Ω & Ω × cm ) wdnhbe cells -iav h n pdm , table s : teer readings (Ω & Ω × cm ) wdnhbe cells -poly(ic), figure s : facs analysis of α- - and α- - -linked sialic acids on the surface of wdnhbe cells, figure s : transepithelial electrical resistance (teer) readings for three independent experiments of mock vs iav h n pdm inoculated wdnhbe cells, figure s : apoptosis in wdnhbe cells infected with pandemic iav h n pdm , figure s : transepithelial electrical resistance (teer) readings for three independent experiments of wdnhbe cells treated with µg and µg poly(i:c) 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cytokine response patterns in severe pandemic h n and seasonal influenza among hospitalized adults immunologic changes during pandemic (h n ) il- b and il- upregulation in children with h n influenza virus infection avian influenza virus a/hk/ / (h n ) ns protein induces apoptosis in human airway epithelial cells a naturally occurring antiviral ribonucleotide encoded by the human genome the role of viperin in the innate antiviral response viperin is an important host restriction factor in control of zika virus infection the interferon inducible gene: viperin in vivo and in vitro studies on the antiviral activities of viperin against influenza h n virus infection engineering airway epithelium this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors gratefully acknowledge the assistance of dianne green and jean payne for histology, john bingham and jemma bergfeld for pathology expertise, jenni harper for assistance with immunofluorescence assays, matt bruce for facs analysis, leanne davis for assistance with rt-qpcr and ryan farr for advice on rt-qpcr analysis. we acknowledge the insightful advice and assistance from dr kirsty short in establishing the transwell models at acdp. we thank microscopy australia for supporting the confocal microscopy capability utilized in this study through national collaborative research infrastructure strategy (ncris) funding. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -e paw n authors: klein-richers, ute; hartmann, katrin; hofmann-lehmann, regina; unterer, stefan; bergmann, michèle; rieger, anna; leutenegger, christian; pantchev, nikola; balzer, jörg; felten, sandra title: prevalence of feline coronavirus shedding in german catteries and associated risk factors date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: e paw n the aim of this prospective study was to determine prevalence and potential risk factors of feline coronavirus (fcov) shedding. four consecutive fecal samples of cats from german breeding catteries were analyzed for fcov ribonucleic acid (rna) by real-time reverse transcriptase polymerase chain reaction (rt-qpcr). prevalence of shedding was calculated using different numbers of fecal samples per cat ( – ) and different sampling intervals ( – days). information on potential risk factors for fcov shedding was obtained by a questionnaire. risk factor analysis was performed using a generalized linear mixed model (glmm). most cats ( / , . %, % confidence interval (ci) . – . ) shed fcov at least at once. none of the tested catteries was free of fcov. prevalence calculated including all four ( . %, % ci . – . ) or the last three ( . %, % ci . – . ) samples per cat was significantly higher than the prevalence calculated with only the last sample ( . %, % ci . – . ; p = . and . , respectively). young age was significantly associated with fcov shedding while the other factors were not. for identification of fcov shedders in multi-cat households, at least three fecal samples per cat should be analyzed. young age is the most important risk factor for fcov shedding. feline coronaviruses (fcov) are single-stranded, positive-sense ribonucleic acid (rna) viruses of the family coronaviridae [ , ] , that exist as two pathotypes. cats become infected with the avirulent pathotype, which usually causes no clinical signs or only mild enteritis. however, in up to % of the infected cats, a highly virulent mutant of fcov will lead to the fatal syndrome of feline infectious peritonitis (fip) [ ] [ ] [ ] . fcov is ubiquitous in most multi-cat environments, and it is important to detect fcov shedders in these situations [ ] [ ] [ ] [ ] . the prevalence of fcov shedding has been investigated in several countries by testing fecal samples or rectal swabs for fcov rna by reverse transcriptase polymerase chain reaction (rt-pcr), and the results range from . % to . % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . crowded living conditions and sharing litter boxes have been discussed as predisposing factors, but there are only a limited number of studies prospectively evaluating risk factors for fcov shedding. as of today, preventing fcov infection is the only method of preventing fip. once a cat is infected, development of the fatal disease cannot be prevented. an inherited susceptibility to fip has been shown in pedigree cats [ ] but attempts to selectively breed resistant cats have failed [ ] . variants of the feline interferon-gamma gene (fifng) are thought to be associated with the risk of disease, but a study investigating the clinical use of this association to select cats for breeding could not show reliable results [ ] . another study evaluated the use of a novel feline infectious peritonitis virus (fipv)-targeted rt-pcr to distinguish the avirulent pathotype from the virulent mutant, but the differentiation was not accurate [ ] . fcov-infected cats can shed the virus persistently, intermittently, or not at all [ , , , ] . thus, for detection of fcov shedders in multi-cat households, testing of several fecal samples has been recommended [ ] [ ] [ ] [ ] [ ] [ ] . the optimal time interval between sampling, however, has not been determined prospectively [ , [ ] [ ] [ ] . the current prevalence of fcov shedding in catteries in germany is unknown, as are factors influencing this prevalence. therefore, the aim of this study was to determine the prevalence of fcov shedding in german breeding catteries and to evaluate associated risk factors. additionally, serial fecal sampling at different time intervals was compared to single sampling in terms of efficacy to detect fcov shedders within a multi-cat environment. the prospective study included cats from catteries from all over germany. catteries were defined as private breeding establishments with at least one intact female cat and were included if they kept five or more cats. the study protocol was approved by the responsible veterinary authority (reference number . - - - . - - ). owners gave their informed consent prior to participation. breeders were contacted via phone, email, or personally at cat shows. those who were willing to participate were instructed to collect four consecutive fecal samples from an unlimited number of cats in their catteries; the samples were to be taken at intervals of five to days. the first three samples were stored at − • c until all four samples were collected. following collection of the fourth sample, which was kept unfrozen, all samples were immediately shipped refrigerated to the investigators. all four samples of each cat were analyzed for fcov by real-time rt-pcr (rt-qpcr) using forward primer, reverse primer and probe as described previously [ ] . total nucleic acid was extracted using the magvet™ universal purification kit (thermofisher scientific, darmstadt, germany) on an automated platform (kingfisher flex ; thermofisher scientific, darmstadt, germany) according to the manufacturer's instructions. rt-qpcr was performed using the lightcycler system (roche, mannheim, germany). the target gene was fcov b gene (dq . ). rt-qpcr was run with six quality controls, including rt-qpcr-positive controls (synthetic desoxyribonucleic acid (dna) covering the rt-qpcr target region), rt-qpcr-negative controls (pcr-grade nuclease-free water), negative extraction controls (extraction positions filled with lysis solution and pcr-grade nuclease free water only), an internal positive control spiked into the lysis solution to monitor the nucleic acid extraction efficiency, and presence or absence of inhibitory substances (using lambda phage dna), rna pre-analytical quality control targeting feline ssr rrna ( s rrna) gene complex, and a swab-based environmental contamination monitoring control [ , , ] . samples with a cp value below were considered positive. overall prevalence of fcov shedding was defined as the proportion of cats that tested positive for fcov in at least one of the four samples. in order to evaluate if the number of analyzed samples per cat had a significant influence on the prevalence, prevalence was also calculated for one, two, or three samples per cat. this analysis always included the last samples of each cat; for example, sample number was used for analysis of one sample per cat and samples number and were used for analysis of two samples per cat. comparison of prevalence was performed using fisher's exact test. the time intervals between the collection of each individual fecal sample ranged from five to days, and each cat was assigned to one of four groups according to the longest interval between their four samplings ((group ): longest interval - days; (group ): longest interval - days; (group ): longest interval - days; (group ): longest interval - days). prevalence was calculated separately for each of these groups and compared using fisher's exact test. evaluated risk factors included signalment (breed, gender, age, reproductive status) and anamnestic data (number of partner cats, hygiene management, outdoor access, feeding routine). cat owners were asked to fill in a questionnaire (provided as supplementary material) for each cat including age, gender, breed, data on hygiene management (contact with cats from other households, litter box cleaning, disinfection routine), and general husbandry conditions (number of cats in the household, outdoor access, feeding, available space in general and per cat). univariate analysis was carried out using fisher's exact test for categorical variables and mann-whitney u test for continuous variables. a multivariate analysis was performed using statistical software (r foundation for statistical computing, vienna, austria, version . . ), and in order to control for multiple observations of individual breeders, a generalized linear mixed model (glmm) with logit link function and a random intercept per breeder was selected. this served as "breeder or cattery effect" to capture the impact of yet unknown risk factors for fcov infection not considered in the questionnaire, such as environment ventilation, feeding interval, use of different disinfection agents and other management differences. selection of variables was done with glmm lasso (r package glmmlasso, andreas groll ( )). with lasso, some variable coefficients are eliminated by the variable selection process in order to achieve a simple model containing only relevant risk factors. the optimal shrinking parameter was determined using akaike information criterion (aic). none of the tested catteries was entirely free of fcov. the number of cats in the individual catteries ranged from five to with a median number of cats per cattery. the majority of catteries ( / , . %) kept more than ten cats in the household. the proportion of cats shedding fcov within the individual catteries ranged from . % to . % of the sampled cats. of tested cats, ( . %, % confidence interval (ci) . - . ) tested positive for fcov rna in at least one of the four samples (table ) . prevalence calculated with different numbers of samples per cat are shown in table . prevalence was significantly lower when calculated from only one sample per cat compared to three or four samples per cat (p = . and . , respectively). there was no significant difference when comparing prevalence calculated with different sampling intervals (table ) . univariate analysis suggested that breed and the number of cats in the household had a significant influence on the prevalence of fcov shedding. however, multivariate analysis (glmm), which captured the breeders' influence, determined that the age of the cats was the only parameter significantly and independently associated with fcov shedding. cats under one year of age had a . -times higher risk of shedding fcov than cats between one and five years of age (p = . , odds ratio (or) . , % ci . - . ). the number of cats per cattery, breed, hygiene management, husbandry conditions and outdoor access were not significantly associated with fcov shedding in this population (tables and ). outdoor access in this population referred to a fenced enclosure on the owner's property, preventing cats from leaving the premises. overall prevalence of fcov shedding in breeding catteries with more than five cats was . % ( % ci . - . ). other studies investigating fcov shedding prevalence showed varying results depending on the examined cat population. in canada, of ( . %) healthy cats from shelters and private households were positive for fcov rna in the feces [ ] . in florida, usa, prevalence of fcov shedding among cats entering an animal shelter was . % in cats with diarrhea and . % in cats with normal feces [ ] . in california, usa, the overall prevalence of shedding upon admission to a shelter was %, with a prevalence of fcov shedding in kittens and young cats under weeks of age as high as % [ ] . these studies examined either cats entering a hospital, or cats that were newly relinquished to a shelter, so a mixed population (consisting of cats originating from single-and multi-cat environments) can be assumed. this could explain the lower prevalence among adult cats when compared to the results of the present study. prevalence is expected to be higher when evaluating a population of cats from multi-cat households only. a few studies investigating fcov shedding in a single cattery or shelter reported prevalence ranging from . % to % [ , , ] , but these are not comparable to the present study, that investigated the prevalence of fcov shedding in a population of cats from different breeding catteries. studies investigating fcov antibody prevalence in catteries in the united kingdom and in california, usa, found antibody prevalence of % [ ] and % [ ] , respectively, but this is not comparable either, because antibody presence does not equal shedding. every cattery examined in the present study had at least one cat that was shedding fcov; no cattery was free of the infection. there are several possible reasons. first, as shown in previous studies, multi-cat environments facilitate the spread of this highly contagious virus [ , , [ ] [ ] [ ] [ ] [ ] , and the fecal-oral route of transmission results in very effective propagation of fcov through shared litter boxes, which is common practice in catteries [ , , , , , , , ] . after natural infection, cats start to shed high amounts of virus within seven days and continue to do so for several weeks or up to months [ , , , ] . in most cats, shedding will gradually decrease after this initial phase and can even stop entirely, but cats remain susceptible to reinfection and will then start shedding again [ , , , , , ] . some cats become lifelong shedders and only very few cats seem to be resistant and never shed the virus [ , , , , , [ ] [ ] [ ] . second, catteries are usually home to kittens, which are known to shed the virus in particularly high amounts [ , , , ] , and third, most cats in catteries are purebreds, which are discussed to be more susceptible to the infection [ , ] . according to previous studies, - % of infected cats will become intermittent shedders [ , , , ] . thus, these shedders could be missed when only a single fecal sample is analyzed [ , , , ] . intermittent shedding can be caused either by reinfection or by intermittent virus excretion in persistently infected cats, and in both cases multiple phases without virus shedding can occur [ , , , , ] . in order not to miss intermittent shedders, four samples from each cat (collected every - days) were analyzed in the present study. the proportion of shedding cats identified was significantly higher when all four samples of each cat were taken into account compared to only one sample per cat ( . % and . %, respectively; table ). these results support the recommendation that, for identification of fcov shedders in a given population, serial fecal rt-qpcr tests should be performed [ ] [ ] [ ] [ ] [ ] [ ] . the most suitable time interval for serial fecal sampling of an individual cat has not been clearly defined and the recommendations made by different authors vary from a few days to one month [ , , [ ] [ ] [ ] ] . in the present study, no significant difference could be found when comparing separately calculated prevalence for different sampling intervals ( - , - , - , or - days; table ). thus, for detection of fcov shedders, sampling intervals of one week to one month can be recommended. univariate risk factor analysis in the present study suggested that breed and the number of cats in the household had a significant influence on the prevalence of fcov shedding, but these results have been distorted by the effect each breeder has on hygienic conditions and the risk of infection within their cattery. additionally, most breeders keep only one, rarely two breeds within their cattery, so that the influence of the breed cannot easily be separated from the influence of the breeder. multivariate risk factor analysis (glmm) found no association between breed and fcov shedding, suggesting that in this population, the breeders and their specific husbandry routine and hygiene management were truly influencing the prevalence of fcov shedding, not the breed. however, if analysis had been performed with a greater number of cats from each breed, a significant influence of breed on fcov shedding might have been found. moreover, multivariate analysis revealed that the age of the cats was significantly associated with fcov shedding in this population, while univariate analysis did not find a significant association between age and fcov shedding. thus, when considering the breeders' effect, the influence of age on shedding becomes obvious. the higher risk of fcov shedding in young cats is consistent with previous studies [ , , ] . moreover, it was shown that kittens (under six months of age) also shed significantly more virus as determined by rt-qpcr than older cats [ ] . kittens in multi-cat households will usually acquire infection between the th and th week of age, when maternal antibodies wane [ , , , , ] . most previous studies could not demonstrate virus shedding before nine weeks [ , , , ] , while harpold and others demonstrated fcov infection of kittens as early as four weeks of age [ ] . virus shedding starts a few days after the primary infection and is especially high and consistent in this early phase [ , , ] . the higher frequency of shedding in kittens is likely due to the fact that the immune system is not fully developed and allows the virus to replicate efficiently [ , , , ] . the number of cats living together in one household was not significantly associated with fcov shedding, once the breeders' effect was controlled for. living in a multi-cat household has already been confirmed as a risk factor for fcov infection [ , , , [ ] [ ] [ ] ] and every cat tested in the present study came from a household with five or more cats. it can be concluded that for households with more than five cats, additional cats do not additionally increase the risk of fcov shedding. hygiene management could have been expected to play a role in the distribution of fcov, as the virus is transmitted via the fecal-oral route. in the present population, however, no significant association of hygiene management and fcov shedding could be shown. neither the number of litter boxes nor the cleaning and disinfection frequencies were associated with the prevalence of fcov shedding. there are several possible explanations for this. first, the individual effect of the breeder on certain management-related risk factors was not assessed in the questionnaire, e.g., thoroughness of cleaning or the use of different cleaning agents. second, it has been shown in previous studies that keeping cat populations with more than five cats free of fcov is extremely difficult, due to the ubiquitous nature of the virus and the ease of transmission [ , , ] . in order to prevent endless reinfection within such a cat population, shedders must be isolated, and the level of quarantine required to prevent contamination is extremely high and costly [ , ] . it is possible that hygiene-related factors, such as frequency of litter box cleaning and disinfection, do not have an influence on fcov prevalence in households with more than five cats, because the virus is distributed so efficiently that normal sanitary measures are simply not enough to slow the spread. outdoor access has been suggested to reduce the risk of fcov infection, because cats with outdoor access have the opportunity to bury their feces in distinct places and therefore have little to no contact with contaminated feces from other cats [ , , , , ] . the results of previous studies are inconsistent regarding this topic. in an australian study, cats living exclusively indoors had a higher prevalence of fcov antibodies than cats with outdoor access, but the difference was not significant [ ] . in a british study, feral and semi-feral cats were -times as likely as tame cats to have fcov antibodies [ ] and in another british study investigating risk factors for the presence of fcov antibodies, feral cats did not have a reduced risk of having fcov antibodies [ ] . in the present study, there was no significant difference in fcov shedding between cats with outdoor access and cats living indoors only. however, outdoor access in the present population did not refer to free-roaming, but to access to open-air enclosures on the breeders' properties. the latter obviously did not differ much from keeping cats strictly indoors, because the cats will still use litter boxes or share places for defecation within the open-air enclosures. stress has been suggested to increase the risk of fcov shedding [ ] and the development of fip [ ] . a stress-related increase in glucocorticoid release is believed to be responsible for the suppression of cell-mediated immunity, resulting in higher fcov replication [ , ] . one study could show an association between immunosuppression and an increased risk of fip in feline immunodeficiency virus (fiv)-positive cats [ ] . another study examined the effect of stress on fcov shedding in experimentally infected cats by administering methylprednisolone acetate to ten of these cats. seven additional cats became pregnant and had kittens during the experiment. no increase in virus shedding could be found in any of these cats, indicating that stress had no influence on fcov shedding in the examined population [ ] . although a shelter environment is assumed to be more stressful for cats than a breeding cattery environment, the prevalence of fcov shedding in this population was higher than many of the prevalence previously reported from shelters [ , , , ] . thus, also a breeding cattery environment with more than five cats might represent a stressful situation for the individual animal. this study had some limitations. first, the breeders' statements in the epidemiological surveys were subjective and could not be verified. second, not every cat from every participating cattery was tested, which impedes a direct comparison of catteries concerning management-related risk factors. another limitation is that we cannot exclude that owners were inclined to specifically sample cats that were not healthy. however, as owners did not receive the results of this study immediately, there was no benefit of specifically picking those cats with disease signs. the limited number of cats from each breed is also a limitation which should be improved in future studies. nevertheless, the fact that no cattery was fcov-free confirms once more that large groups of cats, especially in breeding catteries, are at a high risk for fcov infection. additionally, keeping a large number of cats increases the risk of infection in such a manner that normal hygiene measures will not prevent the spread of the infection. prevention strategies for fcov infection and in general for virus infections are less likely to be successful when large groups of cats are kept together; smaller groups of cats should be preferred. the development of fip currently can neither be predicted nor prevented once a cat is infected with fcov, so the main focus should rather be on prevention of fcov infection to avoid the development of this severe disease. the overall prevalence of fcov shedding in german catteries was . % ( % ci . - . ), and no cattery was identified to be free of fcov. for identification of fcov shedders in a multi-cat environment, at least three samples collected at intervals between one week and one month should be analyzed. in this population of cats from private multi-cat households housing more than five cats, only age 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title: assessment of metagenomic sequencing and qpcr for detection of influenza d virus in bovine respiratory tract samples date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: yoq geu high throughput sequencing is currently revolutionizing the genomics field and providing new approaches to the detection and characterization of microorganisms. the objective of this study was to assess the detection of influenza d virus (idv) in bovine respiratory tract samples using two sequencing platforms (miseq and nanopore (gridion)), and species-specific qpcr. an idv-specific qpcr was performed on samples ( nasal swabs and tracheal washes) that had been previously subject to virome sequencing using miseq. nanopore sequencing was performed on samples positive for idv by either miseq or qpcr. nanopore sequence data was analyzed by two bioinformatics methods: what’s in my pot (wimp, on the epi me platform), and an in-house developed analysis pipeline. the agreement of idv detection between qpcr and miseq was . %, between qpcr and nanopore was . % (in-house) and . % (wimp), and between miseq and nanopore was . % (in-house) and . % (wimp). idv was detected by miseq in of idv qpcr-positive samples with cq (cycle quantification) values below , despite multiplexing samples for sequencing. when qpcr was regarded as the gold standard, the sensitivity and specificity of miseq sequence detection were . % and . %, respectively. we conclude that both miseq and nanopore sequencing are capable of detecting idv in clinical specimens with a range of cq values. sensitivity may be further improved by optimizing sequence data analysis, improving virus enrichment, or reducing the degree of multiplexing. high throughput sequencing is currently revolutionizing the genomics field and providing new approaches to the detection and characterization of viruses. the utilization of metagenomic sequencing to elucidate genome sequences of viruses, particularly rna viruses, directly from clinical samples offers several benefits. first, metagenomics enables identification and genomic characterization of unexpected viruses or even novel viruses either as primary pathogens or as co-infectants, without prior knowledge of their clinical significance [ ] [ ] [ ] [ ] . second, it eliminates the need for ongoing optimization of primers and/or probes for rapidly evolving or highly diverse rna viruses [ ] . third, it facilitates routine surveillance and early detection of outbreaks of novel virus strains that are distinct from currently circulating strains. finally, the development of portable sequencing devices creates the potential for timely identification of routine cases or outbreaks in the field [ , ] . sequencing technology continues to evolve rapidly. with the capability of generating long reads, relatively lower set-up cost and portability, oxford nanopore sequencing has attracted increasing attention for its potential advantages in some circumstances over short-read sequencing technologies [ , ] . metagenomic sequencing has been widely used for non-targeted detection of viruses and has been applied to identify several "new" viruses associated with bovine respiratory disease (brd) [ ] [ ] [ ] [ ] . in dairy cattle, bovine adenovirus (badv ), bovine rhinitis a virus (brav), and influenza d virus (idv) showed significant association with brd [ ] . in beef cattle, bovine rhinitis b virus (brbv), brav, and idv showed statistical association with brd [ , ] . among all of the viruses detected by sequencing of the bovine respiratory tract metagenome, influenza d virus (idv) has been identified as a common virus associated with brd in both beef and dairy cattle, suggesting the potential contribution of idv to brd [ ] [ ] [ ] . influenza d virus (idv) belongs to the orthomyxoviridae, and is a single-stranded, enveloped, segmented and negative-sense rna virus [ ] . since its discovery in swine in usa in , idv has been reported all over the world, and cattle are thought to be the natural host reservoir [ , [ ] [ ] [ ] [ ] . in addition to cattle and swine, idv has been reported in sheep, goats, laboratory animals (ferrets and guinea pigs), and seropositivity has been detected in humans [ , [ ] [ ] [ ] . concerns about interspecies transmission and potential zoonosis have been raised due to the high seroprevalence of idv antibodies in people exposed to cattle [ ] . given the diversity of viruses now known to be associated with brd and the potential for discovery of novel viruses like idv, there is increasing interest in application of metagenomic sequencing for diagnostics, since screening for many individual viruses using targeted pcr assays quickly becomes logistically complex, expensive, and time-consuming. the relative performance of metagenomic sequencing compared to pcr in terms of analytical sensitivity, however, has not been widely explored. at the time of writing, there is only one published study focused on the assessment of performance of miseq and nanopore sequencing relative to gold standard qpcr, which included a limited number of samples and a narrow range of viral loads [ ] . in this study, idv was used as a representative brd-associated virus to examine the feasibility of using metagenomic sequencing for detection of viruses in clinical bovine respiratory samples. we compared results of long-read sequencing on the oxford nanopore gridion platform and previously generated illumina miseq data [ ] to the results of an idv-specific qpcr. the objective was to assess idv detection using all three approaches applied to a set of bovine respiratory tract samples containing a wide range of viral loads. the samples used in this study were collected as part of a previous study [ ] . collection of the samples was approved by the university of calgary veterinary sciences animal care committee (ac - ). the overall sample preparation workflow is shown in figure a . sample collection and preparation were described previously [ , ] . briefly, paired nasal swabs (n = ) and tracheal washes (n = ) were collected from cattle with brd and healthy controls from four different feedlots in alberta, canada between november and january [ ] . the samples were centrifuged and supernatants were treated with dnase (life technologies, carlsbad, ca, usa) and rnase (promega, madison, wi, usa), followed by extraction of viral nucleic acids using a commercial kit (qiaamp minelute virus spin kit, qiagen, venlo, netherlands). a portion ( . µl) of extracted total nucleic acids was used directly as a template for idv qpcr and another portion ( µl) used to generate cdna for sequencing. the first strand was reverse transcribed with primer fr rv-n ( -gcc gga gct ctg cag ata tcn nnn nn- ) using superscript iii enzyme (life technologies, carlsbad, ca, usa) , followed by complementary strand synthesis using sequenase polymerase (affymetrix, santa clara, ca, usa) as per manufacturer's instructions [ ] . double-stranded dna was purified using nucleomag beads (macherey-nagel inc., bethlehem, pa, usa) and subsequently subjected to random amplification with primer fr rv ( -gcc gga gct ctg cag ata tc- ) prior to sequencing library preparation [ ] . quantitative real-time pcr for idv was performed on the extracted total nucleic acids for each sample (total number of samples = ) using previously described primers and probe specific for idv [ ] . the qpcr was carried out using agpath-id one-step rt-pcr reagents (thermofisher scientific, waltham, usa) in a total volume of . µl, which included . µl template, pm forward/reverse primers, pm probe and . µl amplitaq gold dna polymerase in a bio-rad cfx real-time detection system (bio-rad, hercules, ca). the following cycling conditions were used: reverse transcription phase at • c for min; initial activation phase at • c for min; two-step cycles of denaturation at • c for s; and annealing and extension at • c for min. to obtain a positive control template, a dna fragment corresponding to a bp portion of the pb gene of idv (accession number: jq ) was synthesized and inserted into puc -amp vector (bio basic, markham, on, canada). a -fold dilution series of the positive control plasmid was used to construct a standard curve to determine the efficiency of the pcr. all samples were tested in duplicate along with the standard curve and no template controls. samples, for which both of the duplicates gave a sigmoid amplification curve with a cq (cycle quantification) value, were considered positive. nineteen samples that were idv positive by either miseq virome sequencing or qpcr were selected for nanopore sequencing. samples were selected to represent the full range of cq values observed in the qpcr results. three batches of six samples were multiplexed and run on individual flow cells, while one sample (sample ) was run individually. the dna used for gridion nanopore library preparation was from the same randomly amplified dna that was used for miseq sequencing ( figure a ). ligation d sequencing kit sqk-lsk was used for library preparation. end-repair and da-tailing were performed on randomly amplified dna for each sample using nebnext ffpe dna repair mix and ultra ii end-prep enzyme mix (new england biolabs, ipswich, ma, usa). after purification with ampure xp beads (beckman coulter, brea, ca, usa), native barcode ligation using the exp-nbd barcode kit (oxford nanopore technologies, oxford, uk) and blunt/ta ligase master mix (new england biolabs, ipswich, ma, usa) was performed as per manufacturer's instructions. the concentration of barcoded libraries was determined using a qubit fluorometer (thermofisher scientific, waltham, ma, usa) and subsequently equimolar amounts of each barcoded library (total amount = µg) were pooled, and adaptors were added using quick t dna ligase (new england biolabs, ipswich, ma, usa). each pooled library ( . µl) was mixed with µl priming buffer and . µl loading beads and loaded dropwise through the sample port into the flow cell (flo-min ) as per manufacturer's instructions. the minknow platform qc check confirmed at least available pores, and the high accuracy basecalling (hac) flip-flop model was applied. the workflow of bioinformatic analysis is illustrated in figure b . once nanopore raw data were demultiplexed and trimmed using porechop (https://github.com/rrwick/porechop) and passed the quality score (qscore) , high quality reads were aligned to the bovine genome (bioproject accessions prjna , prjna ) using minimap [ ] , and unmapped reads (i.e., non-host derived reads) from each sample were de novo assembled using trinity [ , ] . assembled contigs were mapped to the virus reference sequence (refseq) database using blastn and virus-like contigs with a minimum alignment length of bp and an expectation (e) value < − were further examined by blastx alignment to the genbank non-redundant protein sequence database to confirm the nucleotide sequence-based identification and to remove any spurious matches [ ] . the total number of viral reads was determined as previously described [ ] . quality filtered reads from the nanopore sequencing were also uploaded to the epi me (https: //epi me.nanoporetech.com/) platform for analysis with the wimp (what's in my pot, version . . ) application for taxonomic classification of reads. viruses , , x for peer review of from each sample were de novo assembled using trinity [ , ] . assembled contigs were mapped to the virus reference sequence (refseq) database using blastn and virus-like contigs with a minimum alignment length of bp and an expectation (e) value < − were further examined by blastx alignment to the genbank non-redundant protein sequence database to confirm the nucleotide sequence-based identification and to remove any spurious matches [ ] . the total number of viral reads was determined as previously described [ ] . quality filtered reads from the nanopore sequencing were also uploaded to the epi me (https://epi me.nanoporetech.com/) platform for analysis with the wimp (what's in my pot, version . . ) application for taxonomic classification of reads. extracted rna was used directly for qpcr, while dna randomly amplified from the same extracts was used for miseq sequencing and gridion nanopore sequencing; (b) bioinformatic workflow to identify viruses in brd samples. wimp (what's in my pot) analysis was used only for nanopore data. the remaining analysis was the same for data from both miseq and gridion nanopore sequencing except minimap was used instead of bowtie for host sequence subtraction. viruses , , of samples totaling ( nasal swabs and tracheal washes) that had been sequenced using miseq previously ( cycle v chemistry, libraries of multiplexed samples) were tested by an idv-specific qpcr (figure a ) [ ] . the detection limit of the pcr was demonstrated to be . copies per reaction (data not shown). there were idv positive samples based on the qpcr and the range of cq values was from . ( . × copies per reaction) to . ( . copies per reaction) with median cq value being . ( . × copies per reaction). the agreement of idv detection between qpcr and miseq was . %. when qpcr was regarded as the gold standard, the sensitivity and specificity of miseq detection were . % and . %, respectively. idv was detected by miseq in of idv qpcr-positive samples with cq values below (figure ), when multiplexing samples in the miseq flow cell. only of idv qpcr-positive samples with a cq value above was detected by miseq ( figure ). nineteen samples that were positive for idv by qpcr or miseq, and that represented the full range of qpcr cq values, were selected for further analysis by nanopore sequencing. extracted rna was used directly for qpcr, while dna randomly amplified from the same extracts was used for miseq sequencing and gridion nanopore sequencing; (b) bioinformatic workflow to identify viruses in brd samples. wimp (what's in my pot) analysis was used only for nanopore data. the remaining analysis was the same for data from both miseq and gridion nanopore sequencing except minimap was used instead of bowtie for host sequence subtraction. samples totaling ( nasal swabs and tracheal washes) that had been sequenced using miseq previously ( cycle v chemistry, libraries of multiplexed samples) were tested by an idv-specific qpcr (figure a ) [ ] . the detection limit of the pcr was demonstrated to be . copies per reaction (data not shown). there were idv positive samples based on the qpcr and the range of cq values was from . ( . × copies per reaction) to . ( . copies per reaction) with median cq value being . ( . × copies per reaction). the agreement of idv detection between qpcr and miseq was . %. when qpcr was regarded as the gold standard, the sensitivity and specificity of miseq detection were . % and . %, respectively. idv was detected by miseq in of idv qpcr-positive samples with cq values below (figure ), when multiplexing samples in the miseq flow cell. only of idv qpcr-positive samples with a cq value above was detected by miseq ( figure ). nineteen samples that were positive for idv by qpcr or miseq, and that represented the full range of qpcr cq values, were selected for further analysis by nanopore sequencing. a total of . million reads were obtained from miseq. after removing low-quality reads and host-derived reads, . million reads remained. a total of . million high-quality viral reads was generated, accounting for . % of the total reads obtained from miseq [ ] . a total of . million raw nanopore reads was generated, . % of which passed the quality filter (qscore ) and were classified taxonomically when the data was uploaded to the epi me platform. these classified reads included a total of . million viral reads, accounting for . % of filter passed, classified reads ( figure ). the proportion of viral reads per sample was . % to . % (nanopore, wimp analysis) compared to a total of . million reads were obtained from miseq. after removing low-quality reads and host-derived reads, . million reads remained. a total of . million high-quality viral reads was generated, accounting for . % of the total reads obtained from miseq [ ] . a total of . million raw nanopore reads was generated, . % of which passed the quality filter (qscore ) and were classified taxonomically when the data was uploaded to the epi me platform. these classified reads included a total of . million viral reads, accounting for . % of filter passed, classified reads ( figure ). the proportion of viral reads per sample was . % to . % (nanopore, wimp analysis) compared to . % to . % for the previously generated miseq data; however, with both sequencing approaches, the majority of reads obtained were identified as host-derived or other (bacteria, fungi, unclassified) ( figure ). the samples selected for sequencing on the nanopore gridion platform represented a range of idv concentrations based on qpcr of . to . × genome copies per reaction, corresponding to cq values ranging from . to as low as . (table ). the agreement of idv detection between qpcr and nanopore was . % (in-house) and . % (wimp), and that between miseq and nanopore was . % (in-house) and . % (wimp). idv was detected in the nanopore data from all but one ( / ) of the idv-positive samples when reads were classified using the wimp application, but this proportion dropped to / when the in-house read assembly workflow was used. for most ( / ) of the samples with disparate results, or fewer idv reads were identified in the wimp analysis. the exception was sample t with idv reads. in order to explore qualitatively whether detection of other viruses in addition to idv was comparable between the two metagenomic sequencing platforms, we compared the complete lists of viruses detected by miseq or nanopore in the idv-positive samples. the number of different viruses detected in each sample varied from none to a maximum of four. the proportion of samples with perfect agreement between miseq and nanopore (in-house) was . %, by miseq and nanopore (wimp) it was . %; and by nanopore (in-house) and nanopore (wimp) it was . % (supplementary table s ). metagenomic sequencing is transforming routine detection of viruses from traditional cell culture, antibody-antigen techniques and qpcr to detection of viruses in a target-independent manner. sequencing approaches have now been widely applied for detection of known and novel agents in various types of clinical specimens in both human and veterinary medicine [ , , ] . the potential usefulness of viral metagenomics for virus surveillance and diagnostics is still in debate due to its performance relative to the gold-standard method of real-time qpcr routinely employed in diagnostic laboratories [ ] . a recent assessment of the performance of nanopore, miseq and qpcr for detection of chikungunya and dengue viruses in serum or plasma samples with relatively high viral loads (cq values from to ) demonstrated % agreement between these methods [ ] . in this investigation, however, a maximum of samples was multiplexed and sequenced using miseq, and each sample was sequenced individually on nanopore [ ] . this low degree of multiplexing translates to high analytical sensitivity, but correspondingly makes these technologies relatively more expensive per sample and decreases the potential application for routine diagnostics. in our current in addition to wimp classification of quality-filtered nanopore reads, we also performed a de novo assembly of the nanopore reads. the largest idv contigs assembled for each sample from nanopore data (using the in-house bioinformatics workflow, figure b) were generally longer than those from miseq data and ranged from to bp (nanopore), and to bp (miseq) ( table ) . the genome segment coverage of each largest contig from each sample was from . % to . %. the proportion of nanopore reads mapped to idv for each sample by in-house analysis was higher than that from miseq except for sample t and t . the proportion of idv reads identified in the wimp analysis of the nanopore data, however, was generally comparable to that from the nanopore (in-house) workflow (table ). the proportion of reads identified as idv in nanopore (wimp), nanopore (in-house) and miseq sequencing was generally extremely low (average . %, . %, and . %, respectively). as expected, approximately six times more reads were obtained for the individually sequenced sample than for those from the multiplexed samples (table ). sample also had the lowest cq value in the idv qpcr ( . , corresponding to . × copies per reaction) and the highest proportion of idv reads in the metagenomic sequencing results (nanopore-wimp . %, nanopore-in-house . %, miseq . %) ( table ) . the samples selected for sequencing on the nanopore gridion platform represented a range of idv concentrations based on qpcr of . to . × genome copies per reaction, corresponding to cq values ranging from . to as low as . (table ). the agreement of idv detection between qpcr and nanopore was . % (in-house) and . % (wimp), and that between miseq and nanopore was . % (in-house) and . % (wimp). idv was detected in the nanopore data from all but one ( / ) of the idv-positive samples when reads were classified using the wimp application, but this proportion dropped to / when the in-house read assembly workflow was used. for most ( / ) of the samples with disparate results, or fewer idv reads were identified in the wimp analysis. the exception was sample t with idv reads. in order to explore qualitatively whether detection of other viruses in addition to idv was comparable between the two metagenomic sequencing platforms, we compared the complete lists of viruses detected by miseq or nanopore in the idv-positive samples. the number of different viruses detected in each sample varied from none to a maximum of four. the proportion of samples with perfect agreement between miseq and nanopore (in-house) was . %, by miseq and nanopore (wimp) it was . %; and by nanopore (in-house) and nanopore (wimp) it was . % (supplementary table s ). metagenomic sequencing is transforming routine detection of viruses from traditional cell culture, antibody-antigen techniques and qpcr to detection of viruses in a target-independent manner. sequencing approaches have now been widely applied for detection of known and novel agents in various types of clinical specimens in both human and veterinary medicine [ , , ] . the potential usefulness of viral metagenomics for virus surveillance and diagnostics is still in debate due to its performance relative to the gold-standard method of real-time qpcr routinely employed in diagnostic laboratories [ ] . a recent assessment of the performance of nanopore, miseq and qpcr for detection of chikungunya and dengue viruses in serum or plasma samples with relatively high viral loads (cq values from to ) demonstrated % agreement between these methods [ ] . in this investigation, however, a maximum of samples was multiplexed and sequenced using miseq, and each sample was sequenced individually on nanopore [ ] . this low degree of multiplexing translates to high analytical sensitivity, but correspondingly makes these technologies relatively more expensive per sample and decreases the potential application for routine diagnostics. in our current study, we performed further exploration to assess the performance of metagenomic sequencing approaches with a higher degree of multiplexing of clinical samples in both miseq and nanopore sequencing. idv presented an excellent target for this comparison given its association with brd in beef and dairy cattle, and the availability of specimens with a wider range of viral loads than has been included in previous investigations [ , , ] . different viral extraction kits have been demonstrated to have variable extraction efficiencies for different viruses in respiratory clinical samples [ ] . the qiaamp minelute virus spin kit (mvsk) has been found to be generally applicable for isolating nucleic acid for qpcr or metagenomic virus identification of adenovirus, influenza virus a, human parainfluenza virus , human coronavirus oc , and human metapneumovirus in respiratory clinical samples [ ] . in our current study, the original nucleic acids extracted with the mvsk were used for both qpcr and metagenomic sequencing, eliminating the influence of different extraction methods and kits on our results (figure a) . the idv-specific qpcr assay detected its target in . % ( / ) of specimens. while the majority of idv positive samples with cq value below were detected by miseq, only / samples with a cq above this threshold were positive by sequencing (figure ). these results demonstrate that for samples where the viral load exceeds . × per reaction even a relatively modest miseq sequencing effort ( samples multiplexed in a single flow cell) is sufficient to detect the virus. the agreement between qpcr and nanopore of . % (in-house) and . % (wimp) demonstrated that relatively modest nanopore sequencing effort (six samples multiplexed) is also sufficient to detect the virus. the results from nanopore sequencing (in-house), however, showed no consistent relationship between viral load and detection by sequencing; furthermore, no consistent relationship between viral load and proportion of viral reads was observed in either miseq or nanopore sequencing (table ) . for example, the two idv positive samples and had cq values of . and . , respectively; however, sample had a higher proportion of idv sequence reads in nanopore and miseq than sample ( table ) . there are several possible explanations for differences in both the proportion of idv reads and total viral reads detected in each sample by miseq and nanopore. first, variation in the amounts of dna used for sequencing library preparation for the two sequencing platforms may play an important role. second, the abundance of virus relative to host or bacterial genetic material is a critical determinant of the detection threshold of metagenomic sequencing. a greater proportional abundance of a virus increases the chance that it will be detected by sequencing and improves the genome coverage obtained. therefore, virus enrichment is commonly applied to clinical samples and enrichment methods such as those used in this study (a combination of centrifugation and nuclease-treatment) should lead to removal of bacteria and host cells, thus improving virus detection [ ] . virus propagation in cell culture is a less appealing method for virus enrichment since it is time-consuming, requires specific expertise and creates the potential for introduction of mutations [ ] . reduction of the degree of multiplexing of samples is an alternative way to improve virus detection, but there is a corresponding increase in cost per sample and a corresponding reduction in throughput that are undesirable in research or clinical diagnostic settings. reduction of the degree of multiplexing of samples also reduces the chances of cross-barcode contamination because barcode reagents are susceptible to cross-contamination [ ] . bioinformatic analysis in metagenomic sequencing remains challenging but is crucial for accurate identification of diagnostic targets. we used the comparable pipeline to analyze both data from miseq and nanopore sequencing (in-house), which showed the feasibility of metagenomic viral whole-genome-sequencing using both nanopore and miseq technology with the assembled contigs covering from to % of each idv genome segment. although de novo assembly was performed on nanopore sequencing data, for the majority of the samples, the length of the largest contig was that of one single read. skipping the assembly step in bioinformatic analysis of nanopore data could provide an advantage for timely identification of potential pathogens. the long reads of nanopore sequencing are thought to provide good confidence for species level identification, but the low coverage combined with the error rates of this platform preclude its use for strain-level resolution [ ] . the detection rates of idv, the number or proportion of idv reads (table ) , and other viruses detected (supplementary table s ) from nanopore (wimp) and nanopore (in-house) were different, which demonstrates that bioinformatic analysis affects the results of virus detection. taxonomic classification in wimp is based on centrifuge [ ] , which compares query sequences to a (undescribed) reference database with a high speed and space-optimized k-mer-based algorithm. for nanopore (in-house) and miseq analysis, blast [ ] was used to compare assembled contig sequences to the ncbi refseq virus database, which is a more computationally intense process that produces more detailed results. the identification of a match using centrifuge is based on probabilities of particular k-mer combinations occurring in the query and reference and not a consideration of the entire query sequence, thus increasing the possibility of false positives [ ] . in contrast, trinity assembly and then blast search against a reference database could lead to false negatives if the particular target sequence is very rare [ ] . if there are very few reads derived from some component of the metagenome, these reads may not be included in the assembly since there is insufficient "evidence" to support building contigs from them [ , ] . the current lack of definition of the reference database or the ability to use custom databases with wimp make this approach inappropriate for clinical diagnostic applications due to the difficulty of validating such approaches. our results provide an illustration of the profound effects that post-sequencing analysis can have on results, and the trade-offs associated with each choice. selection of the most appropriate analysis pipeline must consider the sequencing platform, as well as tolerance for false negatives and false positives, logistical considerations, and the required taxonomic resolution. analytical sensitivity is currently one of the main limitations of metagenomics. in this study, idv was detected by miseq sequencing in specimens with qpcr cq value as high as . when samples were multiplexed in comparison to a maximum cq value of . using nanopore with multiplexing of six samples. for the idv positive samples with low virus loads (e.g., sample ), targeted qpcr may be preferable given its higher analytical sensitivity. interestingly, we observed two samples that were idv positive by both nanopore (wimp) and miseq but negative by qpcr (samples t and t , table ). these cases illustrate a potential advantage of metagenomic sequencing compared to qpcr since a likely explanation for this observation is that these specimens contained strain variants of idv that were not detected by the qpcr assay. we were unable to determine if this was the case since the idv sequence reads did not cover the region of the genome targeted by the species-specific qpcr assay. targeted pcr assays for rapidly evolving rna viruses require ongoing performance monitoring, and optimization of primers and probes [ ] . no single method is suitable for application for all pathogens or specimen types, and each one has advantages in different circumstances. taken together our results demonstrate the potential of metagenomic sequencing on the illumina miseq and oxford nanopore platforms for detection of viruses, including idv, in clinical samples from naturally infected animals with a wide range of viral loads. while application of these approaches to screening animal populations or infectious disease research is feasible, their deployment for routine virology diagnostics in clinical settings will require additional research, laboratory and bioinformatic method development, and performance evaluation. our exploration of two sequencing platforms, different degrees of multiplexing, including samples 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flora of california sea lions the fecal virome of pigs on a high-density farm metagenomic analysis of viral nucleic acid extraction methods in respiratory clinical samples evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples accurate multiplexing and filtering for high-throughput amplicon-sequencing centrifuge: rapid and sensitive classification of metagenomic sequences basic local alignment search tool trimmomatic: a flexible trimmer for illumina sequence data this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank anju tumber (prairie diagnostic services, inc.) for reagent purchasing and other logistics. we are grateful for kara toews, anatoliy trokhymchuk, kazal krishna ghosh (pds) for technical support. the authors declare no conflict of interest. key: cord- -kxhpdvri authors: grandvaux, nathalie; mccormick, craig title: csv : the nd symposium of the canadian society for virology date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: kxhpdvri the nd symposium of the canadian society for virology (csv ) was held in june in halifax, nova scotia, canada, as a featured event marking the th anniversary of dalhousie university. csv attracted attendees from across canada and around the world, more than double the number that attended the first csv symposium two years earlier. csv provided a forum to discuss a wide range of topics in virology including human, veterinary, plant, and microbial pathogens. invited keynote speakers included david kelvin (dalhousie university and shantou university medical college) who provided a historical perspective on influenza on the th anniversary of the pandemic; sylvain moineau (université laval) who described crispr-cas systems and anti-crispr proteins in warfare between bacteriophages and their host microbes; and kate o’brien (then from johns hopkins university, now relocated to the world health organization where she is director of immunization, vaccines and biologicals), who discussed the underlying viral etiology for pneumonia in the developing world, and the evidence for respiratory syncytial virus (rsv) as a primary cause. reflecting a strong commitment of canadian virologists to science communication, csv featured the launch of halifax’s first annual soapbox science event to enable public engagement with female scientists, and the live-taping of the th episode of the this week in virology (twiv) podcast, hosted by vincent racaniello (columbia university) and science writer alan dove. twiv featured interviews of csv co-founders nathalie grandvaux (université de montréal) and craig mccormick (dalhousie university), who discussed the origins and objectives of the new society; ryan noyce (university of alberta), who discussed technical and ethical considerations of synthetic virology; and kate o’brien, who discussed vaccines and global health. finally, because csv seeks to provide a better future for the next generation of canadian virologists, the symposium featured a large number of oral and poster presentations from trainees and closed with the awarding of presentation prizes to trainees, followed by a tour of the halifax citadel national historic site and an evening of entertainment at the historic alexander keith’s brewery. the canadian society for virology (csv) was founded in to provide support for the canadian virology research community, including basic, clinical, government, and industry researchers working on a broad range of viruses. to launch the new society, csv planned a satellite workshop in conjunction with the annual american society for virology (asv) meeting in blacksburg, virginia in june , which was supported by then-asv president and ex-pat canadian dr. grant mcfadden [ ] . the symposium opened with a keynote lecture from dr. david kelvin (dalhousie university and shantou university medical college) who provided a historical perspective on influenza on the th anniversary of the pandemic. dr. kelvin shared anecdotes from the pandemic and described how factors such as the impact of exposure to past circulating viruses may have influenced pathogenic outcome to the pandemic virus. he discussed the circumstances of other influenza pandemics of the past two centuries, and the unpredictable evolution of pandemic viruses, whereby some pandemic viruses can enter circulation as an endemic virus, whereas others, such as the h n virus, can disappear entirely. lessons learned from the study of these viruses could be applied to prevent or mitigate the impact of another influenza pandemic. the nd keynote address of the evening was delivered by dr. sylvain moineau (université laval) who transitioned from human hosts to bacterial hosts in a fascinating talk about crispr-cas antiviral systems. bacteria thrive in virus-rich ecosystems by using a combination of antiviral defenses including restriction endonucleases and crispr-cas enzymes that effectively act as bacterial intrinsic and adaptive immune systems, respectively. dr. moineau focused on bacterial crispr-cas type ii systems that function by archiving short dna "spacers" derived from invading phage genomes into a crispr array in their genome, providing a chronological record of past infections. the crispr array is then transcribed into a "guide" rna that associates with a cas the symposium opened with a keynote lecture from dr. david kelvin (dalhousie university and shantou university medical college) who provided a historical perspective on influenza on the th anniversary of the pandemic. dr. kelvin shared anecdotes from the pandemic and described how factors such as the impact of exposure to past circulating viruses may have influenced pathogenic outcome to the pandemic virus. he discussed the circumstances of other influenza pandemics of the past two centuries, and the unpredictable evolution of pandemic viruses, whereby some pandemic viruses can enter circulation as an endemic virus, whereas others, such as the h n virus, can disappear entirely. lessons learned from the study of these viruses could be applied to prevent or mitigate the impact of another influenza pandemic. the nd keynote address of the evening was delivered by dr. sylvain moineau (université laval) who transitioned from human hosts to bacterial hosts in a fascinating talk about crispr-cas antiviral systems. bacteria thrive in virus-rich ecosystems by using a combination of antiviral defenses including restriction endonucleases and crispr-cas enzymes that effectively act as bacterial intrinsic and adaptive immune systems, respectively. dr. moineau focused on bacterial crispr-cas type ii systems that function by archiving short dna "spacers" derived from invading phage genomes into a crispr array in their genome, providing a chronological record of past infections. the crispr array is then transcribed into a "guide" rna that associates with a cas endonuclease and allows it to cleave incoming foreign viral dna. bacterial viruses, known as phage, are not defenseless; they can bypass crispr-cas targeting by mutation or deletion of the crispr target in their genome, or by production of anti-crispr proteins that subvert the crispr-cas machinery. dr. moineau discussed current and potential applications of crispr technology and introduced the audience to a wide variety of newly discovered anti-crispr proteins that will undoubtedly be the subject of intense investigation for years to come [ , ] . he also described his efforts to bring crispr-cas technology to the undergraduate laboratory curriculum at université laval [ ] . the second day of csv started early for some, with a student-led fun run through south end halifax and point pleasant park. when fun run survivors arrived at breakfast, they were greeted by volunteers who gave them seating assignments that maximized potential interactions between pis and students. following this, the first scientific session of the day commenced, chaired by dr. hugo soudeyns (université de montréal), on "emerging viruses". to lead off, dr. vikram misra (university of saskatchewan, saskatoon, sk) presented his studies of bat viruses. in recent years several viruses that appear to cause no overt disease in their natural bat hosts have been transmitted to new mammalian hosts, causing serious and sometimes fatal disease. dr. misra's research program is focused on examining the apparently benign relationship between these viruses and their natural bat hosts, as well as factors that upset this relationship leading to increased viral replication and transmission to new mammalian hosts (recently reviewed in [ ] ). his team, in a comparison of bat and human host responses to viral infection, demonstrated that while both animals mount antiviral responses, inflammatory responses are diminished in bat cells [ ] potentially decreasing viral pathology. moreover, antiviral responses in bat cells were resistant to viral subversion mechanisms that normally operate in human host cells. the misra laboratory has identified a new bat coronavirus [ ] and a new bat herpesvirus [ ] that persistently infect two canadian bat species with no overt signs of disease. both viruses infect many, if not most, bats in long-term associations without causing overt disease. the stress of white-nose syndrome, a fungal infection that has decimated some north american bat species, leads to a dramatic increase in corona virus replication in little brown bats, potentially increasing the chances of transmission to other species [ ] . long-term viral persistence followed by stress-induced reactivation could be modeled in tissue-culture where the mers-coronavirus persistently infected bat cells and the suppression of innate antiviral responses led to increased viral replication [ ] . studying interactions between bat viruses and their natural host may provide key information to prevent future zoonoses. national microbiology laboratory (nml) researchers developed an ebola vaccine by pseudotyping vesicular stomatitis virus (vsv) with ebolavirus (ebov) glycoprotein precursor (gpc), yielding the rvsv-ebov-gpc vaccine. this vaccine was safe and efficacious in early clinical trials and was deployed in west africa in as an emergency measure to quell an ebola epidemic by ring vaccination (effectively, a phase iii clinical trial) [ ] . lassa virus is an arenavirus endemic across much of west africa. rodents are the natural host, and zoonotic transmission to humans can cause severe hemorrhagic fevers in % of infected individuals. no lassa virus vaccine is currently available because even though many candidates have performed well in clinical trials, none have yet shown efficacy and safety in humans. nml researchers developed a lassa fever vaccine using the same vsv platform previously used to create the successful ebola vaccine by inserting lassa virus gpc gene into the vsv platform [ ] . dr. michael drebot from the zoonotic diseases and special pathogens program at the nml summarized the pre-clinical studies conducted to date on the "made-in-canada" lassa fever vaccine, which performed well in guinea pig, rhesus and cynomolgus macaque models [ ] [ ] [ ] . dr. drebot also described work from dr. david safronetz and other colleagues at the nml who are working on the next steps required to bring this vaccine to clinical trials. the "emerging viruses" session also included presentations from the following trainees: md niaz rahim (university of manitoba) who discussed a pan-filovirus t-cell vaccine for ebola and marburg viruses, corina warkentin (university of ottawa) who discussed roles for kinases in filovirus entry pathways, and neda barjesteh (mcmaster university) who discussed links between viral infection and amyotrophic lateral sclerosis. the next session on "viral subversion of host cell processes" opened with a talk on herpesvirus assembly from dr. bruce banfield (queens university). herpesviruses replicate in the cell nucleus, where the viral genome is copied into branched concatemers that are trimmed and packaged into newly assembled procapsids. because these dna-containing capsids are too large to exit the nucleus via nuclear pores, herpesviruses must transit the nuclear envelope to access the cytoplasm where the final stages of virion maturation occur. this process, called nuclear egress, is achieved by budding of capsids into the inner nuclear membrane yielding an enveloped particle in the perinuclear space. the envelopes of these perinuclear virions then fuse with the outer nuclear membrane releasing dna-containing capsids into the cytoplasm. although this fusion mechanism is poorly understood, several groups have partially described the remarkable mechanism of budding, whereby two conserved viral proteins, pul and pul , assemble into an oligomeric nuclear egress complex (nec) on the inner nuclear membrane and facilitate capsid recruitment and budding into the perinuclear space [ , ] . dr. banfield described the herpes simplex virus type- (hsv- ) nec complex, which is sufficient to promote nuclear envelope vesiculation and drive nucleoplasmic invagination of the inner nuclear membrane in transfected cells. however, these nuclear envelope perturbations are not normally seen in cells infected with wild-type hsv- strains suggesting that these nec activities are negatively regulated during infection. the capacity of the nec to drive the formation of long inner nuclear membrane-derived tubules that extend deep into the nucleus suggest that capsid envelopment could, paradoxically, occur deeper in the nucleus rather than at the nuclear periphery where marginalized cellular chromatin and the nuclear lamina create formidable barriers to capsid envelopment. pul is a tegument protein that is required for efficient nuclear egress of hsv- capsids [ ] . new data from the banfield lab demonstrated that enveloped capsids accumulate in inner nuclear membrane bound structures deep within the nucleus of cells infected with ul mutant viruses suggesting that: ) pul negatively regulates the inner nuclear membrane invagination activity of the hsv- nec; and ) that inner nuclear membrane invaginations can serve as sites of capsid envelopment. mammalian orthoreoviruses selectively replicate in cancer cells and have been extensively studied as oncolytic agents in animal models and human clinical trials [ , ] ). dr. maya shmulevitz (university of alberta) described how knowledge of reovirus molecular genetics can be exploited to create mutant viruses that enter and kill cancer cells more effectively without compromising their selectivity towards transformed cells [ ] . for example, while virions bearing the full complement of twelve trimeric σ attachment protein complexes on their surfaces are relatively stable in the natural enteric environment, experimentally reducing the number of these attachment proteins on the virion accelerated uncoating and replication in cancer cells and increased oncolytic potency in a mouse melanoma model [ , ] . dr. shmulevitz also described how σ attachment protein cleavage by breast cancer metalloproteases dramatically lowered reovirus infectivity. mutagenesis of defined protease cleavage sites on σ prevented cleavage and inactivation of reoviruses by breast tumor metalloproteases. these studies suggest that reoviruses are quite amenable to repurposing for superior oncolytic properties. the "viral subversion of host cell processes" session also included presentations from the following trainees: nichole mcmullen (dalhousie university) who reported the unconventional egress mechanisms of non-enveloped reoviruses, justine sitz (université laval) who described interactions between a human papillomavirus protein and a host dna repair-specific e ubiquitin ligase, and quentin osseman (université de montréal) who described interactions between respiratory syncytial virus (rsv) and the host autophagy pathway. following this session, the csv conducted its first face-to-face business meeting, where members learned about the state of the society budget and adopted a set of by-laws. csv symposia and trainee-focused opportunities were discussed, as well as imminent plans for the first open election of a csv executive from the membership (which was completed in late ). meanwhile, in another theatre, many of the trainee attendees were treated to a writing workshop designed by science writer alan dove, plos pathogens editor karen mossman and plos one senior editor eileen clancy, who presented strategies to communicate scientific discoveries effectively in writing. particular attention was paid to making a compelling case for the importance of your research, articulation of a clear take-home message, and considering the audience for your research. due attention was paid to ethical issues in publishing, and open-access publishing was promoted as a mechanism to maximize the impact of research. the first afternoon session focused on "viruses of microbes" was chaired by dr. karen maxwell (university of toronto). in the first talk of the session, dr. maxwell described a new host defense mechanism against viral infection. bacteria have long been known to defend against bacteriophage infection using restriction endonucleases, cell surface modifications and abortive infection systems, and the past years have seen a rapid expansion of our understanding of diverse crispr-cas systems that provide a form of adaptive immune memory for bacteria [ ] . all these systems rely on bacterial proteins or protein/rna complexes to execute antiviral defense. dr. maxwell described the discovery of a new chemical defense against phage infection of the soil-dwelling bacteria streptomyces. these molecules include anthracyclines that act as dna-intercalating agents that disrupt the early stages of infection by phages with dna genomes, perhaps by blocking circularization of the incoming linear dna [ ] . dr. maxwell showed that streptomyces bacteria can secrete anthracyclines into the extracellular environment, which are then taken up by neighboring cells to block viral infection. in this way, these chemicals act as broad-spectrum antivirals that can protect and influence the evolution of bacterial communities. fecal microbiota transplants (fmts) are increasingly used to treat human intestinal diseases including ulcerative colitis [ ] and antibiotic-resistant clostridium difficile infections [ ] . however, documenting successful transplant of gut microbiota in fmt recipients remains challenging. fmt inevitably transfer bacteriophages as well [ ] , but little is known about how transferred phage affect the recipient microbiome. dr. alexander hynes (mcmaster university) described studies designed to address this directly by studying crassphage, the most abundant phage in humans. crassphage was recently discovered by metagenomic sequencing [ ] but its natural bacterial host had not yet been identified. using pcr and metagenomic analysis, dr. hynes's group tracked crassphage transfer from phage-positive human donors to human fmt recipients or germ-free mice. studying the bacteria transferred along with the crassphage into naïve recipients may provide opportunities to discover the elusive crassphage host. the "viruses of microbes" session also included presentations from the following trainees: casey jones (dalhousie university) who reported on the state of the gut virome in pediatric crohn's disease [ ] , jaclyn mccutcheon (university of alberta) who presented her discovery of type iv pili as receptors for bacteriophages on stenotrophomonas maltophilia [ ] , and nikhil george (university of waterloo) who presented his studies of crispr-cas-based warfare between phage and bacteria in a municipal landfill site. following an afternoon break, dr. matthias götte (university of alberta) chaired a session on "antivirals and vaccines". this session was kicked off in an electric fashion by four -minute thesis-style presentations from trainees. using only a single slide, ali zhang (mcmaster university), briti saha (university of ottawa) and mariel kleer and andrea monjo from dalhousie university clearly and concisely presented their respective research projects. the general consensus was that future csv symposia should feature more of these "flash" talks from trainees (and maybe the pis should be challenged to do the same!). this was followed by a panel discussion about the future of antiviral and vaccine discovery and development, chaired by dr. götte and featuring panel members following the "antivirals and vaccines" session, everyone had an opportunity to get some fresh air and participate in a preview of halifax's first soapbox science event, spearheaded by dalhousie university graduate student emma finlayson-trick. soapbox science is an international program that promotes the creation of events that showcase female scientists and provides opportunities for public outreach and science communication. for this preview, dr. maya shmulevitz from the university of alberta and drs. alyson kelvin and sarah wells and phd student krysta coyle from dalhousie university put on lab coats embossed with the purple soapbox science logo and stood on "soapboxes" built by volunteer engineers (figure ). they explained their research using simple props to engage and interact with the audience. of course, csv attendees were exactly the opposite of the laypeople that soapbox science is designed to target, but nevertheless this preview event provided a good example of how scientists can reach out and engage the public. on the saturday following the symposium, the actual soapbox science event launch at the halifax seaport farmers' market featured twelve scientists and attracted members of public over h. figure . soapbox science preview event. soapbox science is a science communication event in which female scientists stand on "soapboxes" (see above) and discuss their research with the lay public using simple props. these events are intended to boost the visibility of female scientists and inspire the next generation. in this photo, dr. maya shmulevitz (university of alberta) is explaining her research program using art supplies and candy; she is illustrating how scientists can transform a virus that causes disease, into a virus that treats disease. volunteer lucas jarche (dalhousie university) is seen here holding a "typical healthy human", which is composed of many human organs that each have their own unique features (e.g., bones are licorice, heart is a cherry gummy). the audience was asked to make a virus that targets a specific organ, using the corresponding candy. for example, a virus that homes to the heart would have evolved to recognize specific features of the heart/cherry gummy. additional features of viruses were then added; for example, viruses that cause disease often interfere with immune responses, so candies were added to represent these attributes. following a discussion of the features of cancer, the audience were instructed to modify their candy viruses so that they will target cancer cells rather than their natural host cells. following the soapbox science preview, the audience returned to the lecture hall for a keynote lecture from dr. kate o'brien, then from johns hopkins bloomberg school of public health, who had recently been awarded a prestigious canada research chair to support her recruitment to dalhousie university (dr. o'brien was subsequently recruited by world health organization in geneva, switzerland, where she is director of immunization, vaccines and biologicals). dr. o'brien's scientific and policy work is focused on vaccine-preventable illnesses in children and adults. she reported on results from the pneumonia etiology research for child health (perch) study, which is a collaborative study of populations in seven countries in africa and asia designed to determine the etiology of severe pediatric pneumonia in the context of widespread conjugate hemophilus influenzae type b (hib) and pneumococcal vaccination [ ] . the study included over hospitalized soapbox science is a science communication event in which female scientists stand on "soapboxes" (see above) and discuss their research with the lay public using simple props. these events are intended to boost the visibility of female scientists and inspire the next generation. in this photo, dr. maya shmulevitz (university of alberta) is explaining her research program using art supplies and candy; she is illustrating how scientists can transform a virus that causes disease, into a virus that treats disease. volunteer lucas jarche (dalhousie university) is seen here holding a "typical healthy human", which is composed of many human organs that each have their own unique features (e.g., bones are licorice, heart is a cherry gummy). the audience was asked to make a virus that targets a specific organ, using the corresponding candy. for example, a virus that homes to the heart would have evolved to recognize specific features of the heart/cherry gummy. additional features of viruses were then added; for example, viruses that cause disease often interfere with immune responses, so candies were added to represent these attributes. following a discussion of the features of cancer, the audience were instructed to modify their candy viruses so that they will target cancer cells rather than their natural host cells. following the soapbox science preview, the audience returned to the lecture hall for a keynote lecture from dr. kate o'brien, then from johns hopkins bloomberg school of public health, who had recently been awarded a prestigious canada research chair to support her recruitment to dalhousie university (dr. o'brien was subsequently recruited by world health organization in geneva, switzerland, where she is director of immunization, vaccines and biologicals). dr. o'brien's scientific and policy work is focused on vaccine-preventable illnesses in children and adults. she reported on results from the pneumonia etiology research for child health (perch) study, which is a collaborative study of populations in seven countries in africa and asia designed to determine the etiology of severe pediatric pneumonia in the context of widespread conjugate hemophilus influenzae type b (hib) and pneumococcal vaccination [ ] . the study included over hospitalized children to months of age, and analysis including molecular and culture methods, chest x-rays and descriptive etiology analysis. the perch study identified the frequency of etiologic association of different viruses with severe childhood pneumonia, which notably included rsv, widely recognized as a major cause of childhood mortality worldwide [ ] . after the lecture, the audience adjourned to an evening at second poster session, after which, many reunited at a beer garden on spring garden road. on friday morning, the first scientific session focused on "emerging methods in virology" was chaired by dr. marceline côté (university of ottawa) and was led off by dr. roger lippé (université de montréal) who described a powerful new technique called flow virometry. traditionally, flow cytometry has been a very useful approach to study virus infection on a single-cell basis, but viruses themselves have been too small to analyze due to the . µm resolution of most instruments. dr. lippé described how his research group prepares herpesvirus particles for analysis on these instruments by labelling with nucleic acid dyes that can penetrate viral particles or engineering viral particles with fluorescent fusion proteins (including capsid proteins, tegument proteins or glycoproteins) [ ] [ ] [ ] . this approach enabled characterization of individual hsv- particles harvested from different cellular compartments, as well as sorting to > % purity. flow virometry also enables the study of the impact of heterogeneity of viral populations on viral fitness and facilitates the analysis by mass spectrometry of highly enriched viral particles isolated along the egress pathway to monitor their maturation. because flow virometry can be performed on standard flow cytometers and flow sorters available at most institutions, these approaches can be rapidly adopted by others and applied to the study of other large viruses. low-cost dna synthesis has catalyzed a new revolution in synthetic biology, enabling efficient manipulation and re-coding of microbial and eukaryotic genomes. ryan noyce (university of alberta) described his efforts to use the principles of synthetic biology to create a synthetic poxvirus vector that could be used for a variety of therapeutic applications. edward jenner believed that his variolae vaccinae originated in horses, which has been corroborated by molecular analyses of modern vaccinia virus (vacv) isolates that share common ancestry with horsepox virus (hpxv) [ ] [ ] [ ] (incidentally, dr. noyce told us, this means that the term "vaccination" could be a misnomer; cows may have had nothing to do with it, and the term "equination" may be more accurate [ ] ). to improve on the safety and efficacy of vacv, dr. noyce and colleagues chose to reactivate the ancestral hpxv and use it as a template for safer and more efficacious vaccines and other therapeutics including oncolytic viruses. dr. noyce synthesized ten large ( - kilobase pair) fragments of dna based on the published hpxv sequence [ ] , along with vacv-derived terminal sequences [ ] . these sequences were recombined into a kilobase pair live synthetic chimeric (schpxv) virus in cells infected with shope fibroma virus (sfv), which served as a helper virus. schpxv was safe and effective in pre-clinical studies; it produced smaller plaques in cell culture and was less virulent in mice than vacv, but still provided protection against a lethal vacv challenge. dr. noyce discussed the technical challenges associated with using synthetic biology as a tool to build new viruses, as well as biosafety and ethical considerations. the "emerging methods in virology" session also included presentations from the following trainees: vanessa meier-stephenson (university of calgary and university of lethbridge) who presented her findings of complex dna structures in the hepatitis b virus (hbv) genome, yumiko komatsu (kyoto university) who presented her research on borna disease virus vectors, and brennan dirk (western university) who discussed his efforts to trace multiprotein complexes involved in hiv- immune evasion [ , ] . dr. andrew white (york university) chaired the next session on "rna in virus infection", which opened with a talk from dr. selena sagan (mcgill university). the liver-specific microrna- (mir- ) directly binds to the '-end of the hepatitis c virus (hcv) genome and promotes the accumulation of viral rna in cell culture and animal models, at least in part by protecting the genome from degradation by the host '- ' exonucleases xrn and xrn . because the hcv '-triphosphate structure is a molecular pattern expected to be recognized by pattern recognition receptors (prrs), the sagan (mcgill university) and wilson (university of saskatchewan) groups investigated whether mir- binding to the hcv genome affects prr binding and activation of innate antiviral signaling pathways. dr. sagan reported that mir- does not affect recognition of the hcv genome by protein kinase r (pkr), retinoic acid inducible protein i (rig-i)-like receptors, or interferon-induced protein with tetratricopeptide repeats (ifits) and [ ] . '-triphophate-containing rnas can be converted to '-monophosphate rna by the action of the cytoplasmic pyrophosphatases dom z and dusp ; they found that rna silencing of dom z or dusp rescued rna accumulation of hcv subgenomic replicons in the absence of mir- , suggesting that mir- normally protects the '-triphosphate structure on the viral rna and prevents pyrophosphatase activity and subsequent degradation by xrn / . using selective ' hydroxyl acylation analyzed by primer extension (shape), the sagan lab is exploring how mutations in the ' non-coding region of hcv genomes from patient isolates may help to promote genome stability and relieve hcv's dependence on mir- . however, there is evidence to support an additional role for mir- in the hcv replication cycle beyond genome stabilization. influenza a virus infection delivers eight (−)-sense single-stranded viral rna (vrna) genome segments to the cell nucleus, each bound to the tripartite viral rna-dependent rna polymerase (rdrp) complex. to initiate transcription, the rdrp complex simultaneously binds to host rna polymerase ii (pol ii) and '-cap structures on nascent host pre-mrnas. once in position, the polymerase acidic (pa) subunit of the rdrp complex catalyzes endoribonucleolytic cleavage of host pre-mrnas at a position - nucleotides downstream of the '-cap structure. this process, known as "cap-snatching", enables priming of viral mrna synthesis. it remains unclear whether there is some selectivity in this process, or the rdrp simply "snatches" these primers from the most abundant nearby host pre-mrnas. dr. martin pelchat (university of ottawa) described deep sequencing studies that support a more nuanced model of cap-snatching that integrates host pre-mrna abundance with selectivity, whereby each of the eight rdrp:vrna complexes acquire distinct subsets of host rnas due to differences in vrna untranslated region (utr) sequence [ , ] . importantly, he reported that host mrnas associated with antiviral responses are over-represented in the cap-snatched population. this suggests that cap-snatching may indeed play an important role in influenza a virus-host shutoff. the "rna in virus infection" session also included presentations from the following trainees: carolyn robinson (dalhousie university) who discussed kaposi's sarcoma-associated herpesvirus (kshv) modulation of rna granules, eric pringle (dalhousie university) who discussed how kshv mrnas are translated [ ] , and tyler mrozowich (university of lethbridge) who presented structural analysis of the japanese encephalitis virus rna genome. the "this week in virology" or "twiv" podcast was created in september by columbia university professors vincent racaniello and dickson despommier to discuss science in an informal and informative manner. ten years later, dr. racaniello and twiv co-host dr. alan dove accepted our invitation to record their th podcast at csv . after discussing the weather (as is their custom), the twiv hosts got down to business with an interview of drs. grandvaux and mccormick, who discussed their motivation in founding the csv, the current research funding climate in canada, and plans for the new society ( figure ). they also interviewed dr. ryan noyce from the university of alberta, who discussed his high-profile synthetic virology (or "synviro") research project, in which he used principles of synthetic biology to create a horsepox virus vector that could be used as the basis for new poxvirus vaccines and oncolytic viruses [ , ] . this project had been previously discussed on twiv , entitled "a pox on your horse" [ ] , and received scrutiny in the scientific and mainstream media due to concerns about ethics and potential "dual-use" of these new poxviruses. finally, the twiv team interviewed dr. kate o'brien from johns hopkins university, who discussed her global health research in vaccines, and in particular how certain viruses are etiologically linked to severe pneumonia in the developing world. the podcast was entitled "good virologists go to halifax" [ ] . viruses , , x for peer review of which he used principles of synthetic biology to create a horsepox virus vector that could be used as the basis for new poxvirus vaccines and oncolytic viruses [ , ] . this project had been previously discussed on twiv , entitled "a pox on your horse" [ ] , and received scrutiny in the scientific and mainstream media due to concerns about ethics and potential "dual-use" of these new poxviruses. finally, the twiv team interviewed dr. kate o'brien from johns hopkins university, who discussed her global health research in vaccines, and in particular how certain viruses are etiologically linked to severe pneumonia in the developing world. the podcast was entitled "good virologists go to halifax" [ ] . after the twiv live-taping, our program resumed on the topic of "viruses of flora and fauna", with dr. eric jan (university of british columbia) chairing and giving the first talk. internal ribosome entry sites (ires) can start translation in alternative reading frames. for example, dr. jan's lab previously showed that the ires of the honey bee dicistrovirus not only directs translation in the reading frame yielding a polyprotein, but also in the + reading frame [ , ] . dr. jan reported that the ires of the related dicistrovirus cricket paralysis virus (crpv) also promotes translation in the + reading frame, resulting in the translation of a new protein called orfx [ ] . however, unlike the honey bee ires, a subset of ribosomes recruited to the crpv ires can undergo a bypass mechanism to initiate translation downstream at the + frame th non-aug codon. dr. jan showed that mutagenesis of this downstream + frame orfx attenuates the virus in a drosophila infection model, suggesting that orfx plays a role in viral pathogenesis. these studies highlight the diversity of viral translation recoding mechanisms to increase coding capacity. in the next talk, hélène sanfaçon (summerland research and development centre, agriculture and agri-food canada) presented evidence for non-canonical proteases encoded by plant viruses. previously described plant virus proteases are grouped according to conserved catalytic active site residues: cysteine, serine or aspartic acid [ , ] . dr. sanfaçon described investigations of viruses of the family secoviridae that constitute a large group of picorna-like viruses [ ] and include strawberry mottle virus (smov). smov has a bipartite genome, with each rna encoding a large polyprotein. rna encodes a cysteine protease that is related to the canonical c protease of picornaviruses and cleaves at multiple locations on polyproteins generated from rna and rna [ ] . dr. sanfaçon and her team detected an additional proteolytic activity associated with the carboxy-terminal domain of the rna polyprotein [ ] . surprisingly, mutation of conserved serine, cysteine, histidine or aspartic acid residues did not affect this activity. instead, two glutamic acid residues were shown to be after the twiv live-taping, our program resumed on the topic of "viruses of flora and fauna", with dr. eric jan (university of british columbia) chairing and giving the first talk. internal ribosome entry sites (ires) can start translation in alternative reading frames. for example, dr. jan's lab previously showed that the ires of the honey bee dicistrovirus not only directs translation in the reading frame yielding a polyprotein, but also in the + reading frame [ , ] . dr. jan reported that the ires of the related dicistrovirus cricket paralysis virus (crpv) also promotes translation in the + reading frame, resulting in the translation of a new protein called orfx [ ] . however, unlike the honey bee ires, a subset of ribosomes recruited to the crpv ires can undergo a bypass mechanism to initiate translation downstream at the + frame th non-aug codon. dr. jan showed that mutagenesis of this downstream + frame orfx attenuates the virus in a drosophila infection model, suggesting that orfx plays a role in viral pathogenesis. these studies highlight the diversity of viral translation recoding mechanisms to increase coding capacity. in the next talk, hélène sanfaçon (summerland research and development centre, agriculture and agri-food canada) presented evidence for non-canonical proteases encoded by plant viruses. previously described plant virus proteases are grouped according to conserved catalytic active site residues: cysteine, serine or aspartic acid [ , ] . dr. sanfaçon described investigations of viruses of the family secoviridae that constitute a large group of picorna-like viruses [ ] and include strawberry mottle virus (smov). smov has a bipartite genome, with each rna encoding a large polyprotein. rna encodes a cysteine protease that is related to the canonical c protease of picornaviruses and cleaves at multiple locations on polyproteins generated from rna and rna [ ] . dr. sanfaçon and her team detected an additional proteolytic activity associated with the carboxy-terminal domain of the rna polyprotein [ ] . surprisingly, mutation of conserved serine, cysteine, histidine or aspartic acid residues did not affect this activity. instead, two glutamic acid residues were shown to be required for polyprotein processing. n-terminal sequencing by edman degradation method enabled precise mapping of two protease cleavage sites with the consensus sequence p↓xfp. together, these findings suggest that smov polyprotein processing requires two viral proteases, the c-like cysteine protease encoded by rna , and a novel protease encoded by rna that features two putative glutamic acid residues in the catalytic site. the novel protease domain is only found in the rna polyproteins of two other definite or putative members of the family secoviridae, suggesting that it was recently acquired. the "viruses of flora and fauna" session also included presentations from the following trainees: shuang yang (university of british columbia) who described a unique nuclear entry pathway for parvoviruses, gerard gaspard (dalhousie university) who presented mechanistic studies of the reptilian reovirus p fusion protein, and jared rowell (university of calgary) who presented studies of cell fate during porcine circovirus- infection. during the break following this session, everyone assembled in the courtyard for a group photo (figure ). viruses , , x for peer review of required for polyprotein processing. n-terminal sequencing by edman degradation method enabled precise mapping of two protease cleavage sites with the consensus sequence p↓xfp. together, these findings suggest that smov polyprotein processing requires two viral proteases, the c-like cysteine protease encoded by rna , and a novel protease encoded by rna that features two putative glutamic acid residues in the catalytic site. the novel protease domain is only found in the rna polyproteins of two other definite or putative members of the family secoviridae, suggesting that it was recently acquired. the "viruses of flora and fauna" session also included presentations from the following trainees: shuang yang (university of british columbia) who described a unique nuclear entry pathway for parvoviruses, gerard gaspard (dalhousie university) who presented mechanistic studies of the reptilian reovirus p fusion protein, and jared rowell (university of calgary) who presented studies of cell fate during porcine circovirus- infection. during the break following this session, everyone assembled in the courtyard for a group photo (figure ) . dr. karen mossman (mcmaster university) chaired the final symposium session on "antiviral innate immunity", which was led off by her former post-doctoral fellow dr. stephanie dewitte-orr (wilfrid laurier university) who discussed roles for class a scavenger receptors (sr-as) in innate antiviral immunity in lower vertebrates. in mammals the sr-as include five family members (sr-ai, marco, scara , scara and scara ) that play diverse roles in immunity, aiding macrophage clearance of pathogens and apoptotic cells, and acting as receptors for immune-stimulating molecules. they have also been shown to serve as virus receptors. notwithstanding how important these receptors are to host immunity, little is known regarding sr-a function in lower vertebrates. dr. dewitte-orr presented her findings to date regarding sr-as and their functions in fish and frogs. dr. dewitte-orr has identified marco, scara , scara and scara sequences from rainbow trout [ , ] . these sr-as appear to be functional in rainbow trout, and act as surface receptors for double-stranded rna (dsrna), a virus-derived innate immune stimulant [ ] . interestingly, marco does not appear to bind dsrna, a unique finding in vertebrates [ ] . sr-as appear to be surface receptors in amphibian cells as well, as dr. dewitte-orr presented her findings regarding sr-a mediated dsrna responses in the american toad cell line, bufotad [ ] . in addition to function studies, the dewitte-orr group has identified a cell line derived from chinook salmon (chse- ) that appears to lack functional sr-as and can be used as a model for sr-a/dsrna binding [ ] . finally, she presented evidence that sr-as could serve as surface receptors for the ranavirus, frog dr. karen mossman (mcmaster university) chaired the final symposium session on "antiviral innate immunity", which was led off by her former post-doctoral fellow dr. stephanie dewitte-orr (wilfrid laurier university) who discussed roles for class a scavenger receptors (sr-as) in innate antiviral immunity in lower vertebrates. in mammals the sr-as include five family members (sr-ai, marco, scara , scara and scara ) that play diverse roles in immunity, aiding macrophage clearance of pathogens and apoptotic cells, and acting as receptors for immune-stimulating molecules. they have also been shown to serve as virus receptors. notwithstanding how important these receptors are to host immunity, little is known regarding sr-a function in lower vertebrates. dr. dewitte-orr presented her findings to date regarding sr-as and their functions in fish and frogs. dr. dewitte-orr has identified marco, scara , scara and scara sequences from rainbow trout [ , ] . these sr-as appear to be functional in rainbow trout, and act as surface receptors for double-stranded rna (dsrna), a virus-derived innate immune stimulant [ ] . interestingly, marco does not appear to bind dsrna, a unique finding in vertebrates [ ] . sr-as appear to be surface receptors in amphibian cells as well, as dr. dewitte-orr presented her findings regarding sr-a mediated dsrna responses in the american toad cell line, bufotad [ ] . in addition to function studies, the dewitte-orr group has identified a cell line derived from chinook salmon (chse- ) that appears to lack functional sr-as and can be used as a model for sr-a/dsrna binding [ ] . finally, she presented evidence that sr-as could serve as surface receptors for the ranavirus, frog virus- (work described in this special issue). together, these findings provide the first glimpse into the role of sr-as in innate antiviral immunity in lower vertebrates. continuing with the theme on innate immunity in non-human hosts, dr. katharine magor (university of alberta) presented her studies of non-pathogenic and pathogenic influenza a virus (iav) infection of ducks. dr. magor shared a photo of herself as a child cradling her pet duck in yarmouth, nova scotia, and explained how this early love of animals influenced her career path. ducks are the natural iav reservoir host; the virus infects the gastrointestinal tract and typically does not cause overt disease. however, the emergence of avian h n iav strains with pandemic potential have disrupted this balance and can cause lethal infection in ducks. to better understand the pathogenic effects of lethal h n infection in ducks, dr. magor established an infection model of white pekin ducks with three similar h n viruses with known differences in pathogenicity and replication rate [ ] . viral replication and host responses were measured by rt-qpcr at different times post-infection. viral replication peaked and viral load declined in the spleen over time. the ducks rapidly mounted interferon and cytokine responses that peaked on the first day of infection and declined thereafter, with the strongest responses coming from the high-pathogenic viruses. further characterization of the duck immune responses to lethal and sub-lethal infections could advance our understanding of virus-host interactions in the reservoir host. the "antiviral innate immunity" session also included presentations from the following trainees: jiangyi he (memorial university) who described mechanisms of murine cytomegalovirus immune evasion, dacquin kasumba (université de montréal) who described roles for duox enzymes in host responses to respiratory virus infections, and hannah stacey (mcmaster university) who presented her work on iga immune complexes in infection and autoimmunity. csv closed with comments from dr. grandvaux thanking sponsors, the scientific advisory committee, and the local organizing committee. following an ovation for the small army of local volunteers who were instrumental to the success of the symposium, dr. grandvaux announced the travel awards ( figure ). we then gathered outside the lecture theatre where we were met by two members of the th highlanders pipe band, who marched us through the streets of halifax and ascended the slopes of the halifax citadel national historic site ( figure ). inside the walls of the star-shaped fortress, highlanders conducted historic tours in english and french. then pipe and drum sounded once again, and we marched out of the citadel and down sackville street to the historic alexander keith's brewery for an evening featuring local food, drink, and music. a good time was had by all, including drs. grandvaux and mccormick, who could be found relaxing in mr. keith's leather-bound office following a very successful symposium (figure ) , and perhaps twisting a few arms to encourage colleagues to assume leadership positions and host future csv symposia. pipe and drum sounded once again, and we marched out of the citadel and down sackville street to the historic alexander keith's brewery for an evening featuring local food, drink, and music. a good time was had by all, including drs. grandvaux and mccormick, who could be found relaxing in mr. keith's leather-bound office following a very successful symposium (figure ) , and perhaps twisting a few arms to encourage colleagues to assume leadership positions and host future csv symposia. pipe and drum sounded once again, and we marched out of the citadel and down sackville street to the historic alexander keith's brewery for an evening featuring local food, drink, and music. a good time was had by all, including drs. grandvaux and mccormick, who could be found relaxing in mr. keith's leather-bound office following a very successful symposium (figure ) , and perhaps twisting a few arms to encourage colleagues to assume leadership positions and host future csv symposia. author contributions: all authors contributed equally to the conception and writing of this paper. workshop of the canadian society for virology an anti-crispr from a virulent streptococcal phage inhibits streptococcus pyogenes cas widespread anti-crispr proteins in virulent bacteriophages inhibit a range of cas proteins crispr-cas in the laboratory classroom workshop of the canadian society for virology an anti-crispr from a virulent streptococcal phage inhibits streptococcus pyogenes cas widespread anti-crispr proteins in virulent bacteriophages inhibit a range of cas proteins crispr-cas in the laboratory classroom lack of inflammatory gene expression in bats: a unique role for a transcription repressor a persistently infecting coronavirus in hibernating myotis lucifugus, the north american little brown bat isolation, characterization and prevalence of a novel gammaherpesvirus in eptesicus fuscus, the north american big brown bat environmentally persistent pathogens present unique challenges for studies of host-pathogen interactions: reply to field generation and characterization of eptesicus fuscus (big brown bat) kidney cell lines immortalized using the myotis polyomavirus large t-antigen efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses development of a new vaccine for the prevention of lassa fever vesicular stomatitis virus-based vaccines against lassa and ebola viruses a recombinant vesicular stomatitis virus-based lassa fever vaccine protects guinea pigs and macaques against challenge with geographically and genetically distinct lassa viruses will travel: structural basis of membrane budding during nuclear egress in herpesviruses. adv. virus res nuclear egress of herpesviruses: the prototypic vesicular nucleocytoplasmic transport the herpes simplex virus ul protein is essential for virus propagation reovirus therapy of tumors with activated ras pathway reovirus in cancer therapy: an evidence-based review potential for improving potency and specificity of reovirus oncolysis with next-generation reovirus variants reovirus variants with mutations in genome segments s and l exhibit enhanced virion infectivity and superior oncolysis reduction of virion-associated σ fibers on oncolytic reovirus variants promotes adaptation toward tumorigenic cells an updated evolutionary classification of crispr-cas systems a chemical defence against phage infection systematic review and meta-analysis: fecal microbiota transplantation for treatment of active ulcerative colitis clinical inquiries: how effective and safe is fecal microbial transplant in preventing c difficile recurrence bacteriophage transfer during faecal microbiota transplantation in clostridium difficile infection is associated with treatment outcome a highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes multi-omics differentially classify disease state and treatment outcome in pediatric crohn's disease identification and characterization of type iv pili as the cellular receptor of broad host range stenotrophomonas maltophilia bacteriophages dlp and dlp the pneumonia etiology research for child health project: a st century childhood pneumonia etiology study global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis quantitative evaluation of protein heterogeneity within herpes simplex virus particles flow virometry: a powerful tool to functionally characterize viruses analysis of herpes simplex virus type i nuclear particles by flow cytometry genomic analysis, phenotype, and virulence of the historical brazilian smallpox vaccine strain ioc: implications for the origins and evolutionary relationships of vaccinia virus genome of horsepox virus an early american smallpox vaccine based on horsepox equination (inoculation of horsepox): an early alternative to vaccination (inoculation of cowpox) and the potential role of horsepox virus in the origin of the smallpox vaccine construction of an infectious horsepox virus vaccine from chemically synthesized dna fragments hiv- nef sequesters mhc-i intracellularly by targeting early stages of endocytosis and recycling where in the cell are you? probing hiv- host interactions through advanced imaging techniques mir- does not impact recognition of the hcv genome by innate sensors of rna but rather protects the end from the cellular pyrophosphatases, dom z and dusp influenza a virus cap-snatches host rnas based on their abundance early after infection insight into influenza: a virus cap-snatching. viruses mccormick, c. mtorc activity is dispensable for synthesis of kshv lytic proteins synthetic horsepox viruses and the continuing debate about dual use research a pox on your horse good virologists go to halifax global shape mimicry of trna within a viral internal ribosome entry site mediates translational reading frame selection functional insights into the adjacent stem-loop in honey bee dicistroviruses that promotes internal ribosome entry site-mediated translation and viral infection ires-dependent ribosome repositioning directs translation of a + overlapping orf that enhances viral infection plant viral proteases: beyond the role of peptide cutters. front expanding repertoire of plant positive-strand rna virus proteases identification of cleavage sites recognized by the c-like cysteine protease within the two polyproteins of strawberry mottle virus strawberry mottle virus (family secoviridae, order picornavirales) encodes a novel glutamic protease to process the rna polyprotein at two cleavage sites scavengers for bacteria: rainbow trout have two functional variants of marco that bind to gram-negative and -positive bacteria in vitro transcribed dsrna limits viral hemorrhagic septicemia virus (vhsv)-ivb infection in a novel fathead minnow (pimephales promelas) skin cell line class-a scavenger receptor function and expression in the rainbow trout (oncorhynchus mykiss) epithelial cell lines rtgutgc and rtgill-w class a scavenger receptors mediate extracellular dsrna sensing, leading to downstream antiviral gene expression in a novel american toad cell line chse- : a model for studying extracellular dsrna sensing in vitro duck innate immune responses to high and low pathogenicity h avian influenza viruses we are grateful to our colleagues who allowed us to communicate elements of their acknowledgments: we are grateful to our colleagues who allowed us to communicate elements of their workshop presentations in this report. we thank members of the csv scientific advisory committee (drs. marceline côté, darryl falzarano, matthias götte, karen maxwell, nelly pante and andrew white) who planned scientific sessions and reviewed travel award applications. we thank the csv local organizing committee (drs. lisa barrett, jeanette boudreau, jenn corcoran, roy duncan, may el-sherif, todd hatchette, alyson kelvin, denys khaperskyy, jason leblanc, chris richardson and rod russell) who raised funds and planned meeting logistics. we thank the csv student committee, led by dr. denys khaperskyy and including quinelle boudreau, katrina bouzanis, beth castle, becca chilvers, brett duguay, lucas jarche, mariel kleer, grant macneil, andrea monjo, rory mulloy, jacob nearing, carolyn robinson, eric pringle, jacob sicheri, gill singh, patrick slaine, iona stylianides and mackenzie thornbury, who provided essential on-the-ground logistical support for the symposium. we thank emma finlayson-trick for organizing the soapbox science preview and launch events. we thank drs. maya shmulevitz, alyson kelvin and sarah wells and phd student krysta coyle for their soapbox science presentations. we thank quentin osseman, gillian singh and denys khaperskyy for photography. we thank manon livernois (crchum) for administrative assistance. we thank lucas jarche for csv logo artwork. csv was supported by grants from the canadian institutes of health research (cihr-mpl- ) and the dalhousie medical research foundation. additional support was provided by dalhousie university, the li ka shing institute of virology, vido-intervac, plos pathogens, plos one, viruses, canadian journal of microbiology, imv™, zeiss, nikon and sanofi. the authors declare no conflict of interest. key: cord- - n rgfz authors: kleines, michael; häusler, martin; krüttgen, alexander; scheithauer, simone title: wu polyomavirus (wupyv): a recently detected virus causing respiratory disease? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: n rgfz the wu polyomavirus (wupyv) is a novel member of the family polyomaviridae recently detected in respiratory tract specimens by shotgun sequencing. intriguingly, viral genome has been detected in . % to . % of respiratory tract specimens from children with respiratory disease. the levels of co-infection with established respiratory viruses were in the range between . % and . %. moreover, some studies report detection of wupyv in stool or serum. so far, wupyv infections can not be distinguished from other viral infections by means of clinical symptoms. respiratory tract disease like pneumonia or bronchitis is frequently observed in patients harbouring wupyv. detection of viremia suggests systemic infections. however, the available data do not prove wupyv to be a human pathogen. further investigations are necessary. respiratory tract infections are a major cause of hospital admission in infants and young children. they are caused by a broad spectrum of established respiratory tract pathogens, particularly by viruses including the respiratory syncytial virus (rsv), the most important cause of severe respiratory tract infections in young children, influenza viruses, parainfluenza viruses, rhinoviruses, coronaviruses ( e, oc ), and adenoviruses. studies investigating the entire spectrum of established respiratory tract pathogens are rare and mostly based on the detection of pathogen specific antibodies. only in recent years, a few studies became available presenting data based on the direct detection of relevant infectious agents. creer and co-workers [ ] identified at least one potential pathogen in % of specimens from adults suffering from lower respiratory tract infections (lrti, % contained viruses, % contained bacteria) still leaving a significant diagnostic gap to be filled with so far unidentified pathogenic microorganisms. additional agents associated with respiratory tract infections have been discovered recently, e.g. the human metapneumovirus (hmpv) [ ] , coronaviruses (nl , hku ) [ , ] , and the human bocavirus (hbov) [ ] . furthermore, herpes viruses may contribute to fill this gap as they are assumed to trigger respiratory tract infections [ , ] . unfortunately, relevant studies did not often include them. by now, the pathogenic impact of the human metapneumovirus and its relevance on respiratory tract infections has been well established. it is a common cause of upper (urti) and lower respiratory tract infections (lrti) in children, elderly, and immunocompromised persons; asymptomatic infections seem to be uncommon [ ] . hmpv has been identified to be an important cause of bronchiolitis in young children. the novel human coronavirus hcov-nl is also widely accepted to be a significant respiratory tract pathogen. a strong association with croup has been shown [ ] . human coronavirus hcov-hku infections are observed rather infrequent, often in children with underlying diseases [ ] . several reports link the virus with pneumonia and bronchiolitis. for the human bocavirus (hbov) an increasing number of reports present evidence for an association with respiratory tract infections; however, co-infections with established respiratory tract pathogens are frequent. recently, the wu polyomavirus was detected in respiratory tract specimens [ ] . the first polyomaviruses were isolated in and named jc virus and bk virus. in and further viruses of this particular family were described for the first time: wu polyomavirus, ki polyomavirus, and merkel cell polyomavirus [ ] . wupyv was discovered by shot gun sequencing of nasopharyngeal aspirate of a three-years-old child from australia suffering from pneumonia [ ] . the investigators isolated total nucleic acids from the nasopharyngeal aspirate, randomly amplified fragments, and cloned them. sequences of the obtained clones gained by shot gun sequencing were subjected to automated editing and database searches. the combination of these technologies led to the identification of a dna-sequence distant to all sequences included in the sequence database, but related to viruses of the family polyomaviridae. thus, the identification of a novel virus belonging to this viral family has been proposed, exclusively based on molecular data. so far, no system for in vitro or in vivo propagation has been discovered, neither a cell line nor an animal model. thus, until now koch's postulates could not be fulfilled. however, the molecular data form reasonable evidence to propose the existence of the novel virus wupyv. large-scale screening based technologies as the one used for the discovery of wupyv may be a powerful tool in discovering new infectious agents causing respiratory tract diseases particularly with regard to the etiological gap of up to %. the novel virus has a bp dsdna genome with a gc-content of %. the genome contains an early coding region encoding the t-antigens, a late coding region encoding structural proteins, and a non-coding control region [ ] . all features are typical for members of the polyomavirus family. the sequence identity of wupyv at the amino acid level to all of the well-known polyomaviruses ranged from % - %. the closest relative within the virus family is the ki polyomavirus (kipyv) with a sequence identity of %, which was described briefly before wupyv. thus, both viruses were grouped into a new subclass of the polyomavirus family. however, the international committee on taxonomy of viruses (ictv) has not recognised wupyv or kipyv yet. typical members of the family polyomaviridae form small, non-enveloped, icosahedral particles with - nm in diameter containing a supercoiled dsdna genome [ ] . the virion is made up by pentamers of the structural protein vp , each pentamer binds to a single copy of vp or vp . the genome is attached to cellular histones. there is no evidence hinting at a virus structure for wupyv differing from other polyomaviruses. however, wupyv virions have not been displayed by electron microscopy, thus there is no evidence for the precise virion structure at this stage. based on the genome organisation, two subclasses of the polyomavirus family have been postulated: the primate-like group and the mouse polyoma-like group [ ] . the genome of members of both subclasses is divided into three regions. the early coding region encodes at least the small tantigen and the large t-antigen. both are binding partners of the rb-protein and p . the late coding region encodes at least the structural proteins vp -vp , and the non coding control region contains viral promoters and the origin of replication. the early coding region of members of the mouse polyoma-like group additionally encodes the medium t-antigen, whereas the late coding region of members of the primate-like group additionally encodes the structural protein agnoprotein. apart from kipyv, the jc virus and the bk virus are the closest relatives of wupyv based on genome organisation and amino acid sequence. the early coding region contains a splice site for the large tantigen and binding domains for the rb-protein and p . however, wupyv neither encodes open reading frames for the middle t-antigen nor the agnoprotein. thus, wupyv and kipyv were provisionally grouped into a third subclass of the polyomavirus family. so far, no expression studies have been conducted on wupyv to clarify whether the predicted open reading frames are in fact expressed during the viral infection of the target. interestingly, as shown for hbov the predicted transcripts of a virus are not necessarily identical to the transcripts detectable in an infected cell [ ] . there is the need for an appropriate experimental system to carry out expression studies on wupyv for further characterization. polyomaviruses have a restricted host range. the bk virus and the jc virus, the closest relatives of wupyv within the well established members of the virus family, are human-specific. this suggests a host range restricted to humans for wupyv, too. infection with bkv takes place in early childhood; % of - years old children are seropositive. wupyv infections have been reported predominantly in children < years of age [ ] suggesting a strong increase of seropositivity during early childhood for this virus, too. the incubation period of wupyv infections is unknown. respiratory transmission has been suggested for jc virus and bk virus although the diseases related to these viruses are not involving the respiratory tract. both viruses can be extracted from tonsil tissue [ , ] . the viral entry route for wupyv is not completely defined. it could be defined by uptake through dividing cells of the upper respiratory tract and the oropharynx. in this case it would be likely that mostly children are at risk of infection as, due to their growing up, their anabolism is enhanced involving increased cell division. alternatively transmission may occur occasionally by blood transfusion and organ transplantation. jc virus and bk virus primary infections are typically subclinical or linked to mild respiratory illness [ , ] . viral dissemination to the target cells occurs via blood [ ] . sites of lifelong persistence are epithelial cells of the kidney for bk virus [ ] and lymphocytes, oligodendrocytes as well as epithelial cells of kidney for jc virus [ ] . % of immunocompetent bk virus carriers show viruria [ ] , - % show shedding of jc virus [ ] . this indicates that viral persistence can be terminated and viral reactivation can take place even in immunocompetent persons. reactivation can be assumed to an obviously higher extent in immunocompromised individuals. concordantly the related diseases, hemorrhagic cystitis and polyomavirus nephropathy for bk virus and progressive multifocal leukencephalopathy (pml) for jc virus, predominantly occur in persons with reduced immunocompetence. as wupyv was detected in a respiratory tract specimen the respiratory tract can be assumed to be the entry site for this virus, too. the virus was detected in serum indicating that the dissemination occurs via blood. so far, sites of persistence have not been identified. the virus has not been detected in urine [ ] . thus, epithelial cells of kidney may not be the site of persistence for wupyv. wupyv is made responsible for upper and lower respiratory tract diseases, e.g. bronchiolitis, croup, and pneumonia [ ] . besides nasopharyngeal samples, wupyv could also be detected in stool samples [ ] , even in patients without any apparent clinical respiratory symptoms [ ] . however, it has to be determined, whether wupyv is able to replicate in the intestine or whether it accumulates there by oral ingestion. for detection of wupyv-infections several conventional pcr formats as well as real-time pcr protocols were developed reporting sensitivities up to % and specificities up to . % [ , [ ] [ ] [ ] . antigen or antibody detection formats have not been reported, so far. no culture system is known at this time. the diagnostics of established respiratory viruses is based on a broad spectrum of available assays, aiming at the detection of virus-specific antibodies, viral protein, or viral nucleic acids. acute respiratory tract infections commonly have a short incubation period. thus, antibodies are usually not present at the beginning of the acute phase of the disease. for this reason, detection of virus-specific antibodies by complement fixation assays, enzyme linked immunosorbent assays (elisa), or immunofluorescence assays (ifa), all of which are available for established respiratory viruses, is inappropriate for the identification of the etiologic agent causing an acute respiratory tract infection, but useful for retrospective or epidemiological studies. direct detection of viral protein or viral nucleic acids is the mode of choice for respiratory virus diagnostics. viral cell culture represents the gold standard for the well established respiratory viruses, but is too slow for in time diagnostics. less time consuming methods have been developed. these comprise polymerase chain reaction (pcr), antigenspecific elisa, antigen-specific ifa, and, available only for a few respiratory viruses, very fast immunochromatographic assays. sensitivity and specificity of these assays vary considerably. the pcr displays the highest sensitivity. thus, being restricted to pcr formats for detection of wupyv is no bias to sensitivity. particularly real time pcr formats have an established track record in both, very sensitive detection and differentiation between colonisation and acute infection based on the quantitative data [ , ] . availability of automated extraction of nucleic acids permits short processing times and, by this, handling of large specimen cohorts [ ] . following the discovery of wupyv in australia, the virus was detected in specimens from patients with respiratory tract disease on all continents suggesting a worldwide distribution [ , [ ] [ ] [ ] . so far, wupyv-dna was reported to be found in respiratory tract specimens (e.g. nasopharyngeal washes, tracheal secretion, bal), serum, and faeces. the virus could not be detected in urine or from uv lightassociated primary malignant lymphomas [ , ] . specimens from other malignant diseases have not been investigated. the use of tracheal secretion for diagnostics has been shown to lead to an underestimation of the rate of positive specimens compared to other respiratory materials for hbov [ ] . this may be true for wupyv, too. the data available are mainly based on retrospective studies exclusively including symptomatic patients. the detection rate in respiratory samples from children with respiratory disease varies from . % to . % [ , ] . the age of wupyv infected patients ranged from a few weeks to years, children < years of age were dominating. infections were predominantly detected in late winter, spring, and early summer [ ] . high infection rates were reported for study populations preselected for lack of immunocompetence. hiv positive patients had detection rates of up to . % in respiratory tract specimens and . % in blood [ , ] . the rates of co-infection with established respiratory viruses lay between . % and . % [ , ] , commonly exceeding %. wupyv was detectable in blood, possibly indicating its potential for systemic infections. le and co-workers [ ] presented evidence for viral persistence, wattier et al. [ ] for nosocomial infections with wupyv. the real time protocols available allow the quantification of wupyv. quantification of viral loads in respiratory tract specimens revealed viral titers up to copies/ml, but low and medium viral loads were dominating [ , ] . no correlation between viral load and the rate of co-infection or clinical diagnoses was observed. few studies included asymptomatic control groups [ , , [ ] [ ] [ ] . the results were not concordant and reached from higher detection rates in the control group to higher detection rates among the group of patients with respiratory tract diseases. prospective studies have been published only recently. van der zalm and co-workers [ ] reported the detection of wupyv in a cohort of children. their parents were contacted twice a week over a -months period (november to april) and asked for symptoms of respiratory tract disease in their children. every two weeks respiratory tract specimens were collected, regardless of respiratory symptoms. . % of the specimens of children with symptoms were wupyv positive, but only . % of specimens of healthy children, indicating at least an association of wupyv with disease. to this time there are no studies available concerning therapeutic interventions. this is mainly due to the retrospective nature of most investigations. further, no causal association between wupyv and respiratory disease has been shown yet [ ] . as the need for therapeutics is driven by associating infections with disease, studies on therapeutic interventions are not to be expected in the near future. for bk virus associated nephropathy in renal transplant patients, reduction of immunosuppressive therapy is recommended if possible. in the case of progressive renal dysfunction fluoroquinolones (antibacterials), intravenous immunoglobulines, leflunomide, and -in otherwise refractory casescidofovir could be administered. for jc virus caused progressive multifocal leukoencephalopathy (pml) no specific therapy exists. in hiv-positive patients haart induced improvement of cellular function may lead to an at least temporary improvement. failure of treatment approaches with interferon alfa- b, cytarabine, cidofovir, and topotecan has been documented [ ] . the discovery of novel respiratory viruses has the potential to diminish the diagnostic gap for respiratory tract infections. creer and co-workers [ ] , who omitted the recently identified respiratory viruses in their study on adults, reported a diagnostic gap of %. the share of specimens from children negative for any respiratory pathogen investigated was % when hmpv, hbov, and hcov-nl were included [ ] . no study included the novel polyomaviruses or herpes viruses until now. thus, a further reduction of the respiratory tract infections of unknown origin seems reasonable. however, a substantial gap is remaining leaving sufficient room for additional respiratory viruses to be discovered in the future. so far it has not been finally proven, if wupyv is a real causative agent for respiratory diseases. association of virus detection with previously unexplained respiratory disease led to the tempting idea of wupyv representing a new etiologic agent, particularly in cases where no other respiratory tract pathogen could be identified. furthermore, the association of mouse pneumotropic polyomavirus with intestinal pneumonia and significant mortality [ ] indicates that polyomaviruses have the capacity to be respiratory tract pathogens. many studies correlating the detection of wupyv with the incidence of respiratory symptoms argue for this hypothesis, but it remains difficult to prove as asymptomatic control groups have not been investigated sufficiently. additionally, the studies including control groups report dissenting findings. however, in two studies asymptomatic individuals display a lower frequency of virus detection compared to symptomatic patients. one of these is the only prospective study available. the difficulty to prove wupyv to be a respiratory pathogen may be due to the fact that most studies available so far try to correlate the virus with respiratory tract disease in general. in case of wupyv being the causative agent of a particular entity of respiratory tract disease, inclusion of patients displaying any kind of respiratory disease may bias the investigation. focussing on a single entity of respiratory disease proved successful for hcov-nl , which was shown to cause croup. no association of wupyv viral loads and clinical symptoms could be observed so far, and coinfections with other viruses were described frequently. this weakens the hypothesis of wupyv being a respiratory tract pathogen. however, collection of samples was not performed by means of a standardized protocol among the various groups or at a defined time point, after onset of symptoms. as viral loads decrease during the acute phase of infection, the combination of longitudinal data from different time points in one analysis may bias the correlation of viral loads and symptoms. furthermore, detection of co-infection does not exclude pathogenic potential for wupyv, as early childhood is characterized by subsequent episodes of respiratory infections. co-detection of the declining pathogen responsible for the last episode of respiratory disease and the pathogen responsible for the present, acute disease has to be expected. only determination of the viral load kinetics would allow defining the clinical impact of a detectable microorganism. the large t-antigen of wupyv contains binding sites for the rb protein and p . thus, tumourigenic potential cannot be excluded. however, the association of wupyv with other diseases, particularly tumour diseases has not been sufficiently investigated yet. taken together, the currently available data neither prove nor deny wupyv to be a respiratory tract pathogen. several scenarios may describe the role of the virus best. wupyv may be ( ) part of the endogenous viral flora without pathogenic potential; ( ) an opportunistic pathogen with pathogenic potential in the respiratory tract under conditions still to be defined; ( ) a pacemaker for secondary infections; ( ) a viral pathogen using the respiratory tract only as an entry route to reach the final target cell; infection of this cell type is the basis of a disease not related to the respiratory tract. to reach a final conclusion more powerful, controlled studies need to be performed prospectively. however, retrospective analysis of age-and time-matched control populations also permits conclusions if the data collected at the time are related to respiratory illness. ideally, prospective studies would be carried out in a multicenter-design including control groups concentrating on the correlation of wupyv with single entities of respiratory tract diseases. criteria for inclusion of specimens have to be comparable, as well as the time point and the protocol for extraction of specimens. all known or suspected respiratory tract pathogens should be included and follow-up investigations should be performed. the detection method of choice would be quantitative pcr to allow discrimination between colonisation and productive infection. additionally, serological investigations should be included to show that the patients seroconvert in response to an infection. the study participants should be evaluated before enrolment. beside investigation of respiratory tract disease, the cell type hosting the persisting virus has to be identified and a possible association with tumour diseases has to be investigated. aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (lrti) in primary care a newly discovered human pneumovirus isolated from young children with respiratory tract disease identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia cloning of a human parvovirus by molecular screening of respiratory tract samples herpes simplex virus (hsv) pneumonia in a heart transplant: diagnosis and therapy. heart lung epstein-barr-virus-induced interstitial lung disease human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children croup is associated with the novel coronavirus nl identification of a novel polyomavirus from patients with acute respiratory tract infections the role of polyomaviruses in human disease human bocavirus can be cultured in differentiated human airway epithelial cells the role of bk virus in acute respiratory tract disease and the presence of bkv dna in tonsils detection of jc virus dna in human tonsil tissue: evidence for site of initial viral infection human papovavirus isolated from urine of a child with acute tonsillitis human endothelial cells allow passage of an archetypal bk virus (bkv) strain-a tool for cultivation and functional studies of natural bkv strains bk virus infection of the human urinary tract cultivation of papovalike virus from human brain with progressive multifocal leucoencephalopathy human polyomavirus jc and bk persistent infection discovery and epidemiology of the human polyomaviruses bk virus (bkv) and jc virus (jcv) wu polyomavirus in fecal specimens of children with acute gastroenteritis detection of wu polyomavirus dna by real-time pcr in nasopharyngeal aspirates, serum, and stool samples a single-tube real-time pcr assay for detection of the two newly characterized human ki and wu polyomaviruses low to medium wu-virus titers in young children with lower respiratory tract infections development and evaluation of real-time pcr assays for the detection of the newly identified ki and wu polyomaviruses detection of cytomegalovirus dna in human specimens by lightcycler pcr detection of cytomegalovirus dna in human specimens by lightcycler pcr: melting point analysis is mandatory to detect virus strains with point mutation in the target sequence of the hybrdization probes efficient extraction of viral dna and viral rna by the chemagic viral dna/rna kit allows sensitive detection of cytomegalovirus, hepatitis b virus, and hepatitis g virus by pcr wu polyomavirus infection in children human polyomaviruses, wu and ki in hiv exposed children with acute lower respiratory tract infections in hospitals in south africa wu polyomavirus in children with acute lower respiratory tract infections dna from bk virus and jc virus and from ki, wu, and mc polyomaviruses as well as from simian virus is not detected in non-uv-light-associated primary malignant melanomas of mucous membranes high prevalence of human bocavirus detected in young children with severe acute lower respiratory tract disease by use of a standard pcr protocol and a novel real-time pcr protocol prevalence and pathogenicity of wu and ki polyomaviruses in children; the netherlands discovery and identification of wu polyomavirus in children from zhejing region role of human polyomaviruses in respiratory tract disease in young children wu polyomavirus in patients infected with hiv or hepatitis c virus reactivation and mutation of newly discovered wu, ki, and merkel cell carcinoma polyomaviruses in immunosuppressed individuals wu and ki polyomavirus present in the respiratory tract of children, but not in immunocompetent adults clinical and epidemiologic characterization of wu polyomavirus infection human bocavirus and ki/wu polyomaviruses in pediatric intensive care patients wu polyomavirus in children with acute lower respiratory tract infections wu polyomavirus in children no evidence for an association between infection with wu and ki polyomaviruses and respiratory disease treatment options for aids patients with progressive multifocal leukencephalopathy molecular epidemiology and disease severity of respiratory syncytial virus in relation to other potential pathogens in children hospitalized with acute respiratory infection in jordan effect of host age on experimental k virus infection in mice this study was supported by an eu-grant to m.k. (contract no. ). key: cord- -xfir m p authors: hyseni, inesa; molesti, eleonora; benincasa, linda; piu, pietro; casa, elisa; temperton, nigel j; manenti, alessandro; montomoli, emanuele title: characterisation of sars-cov- lentiviral pseudotypes and correlation between pseudotype-based neutralisation assays and live virus-based micro neutralisation assays date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: xfir m p the recent outbreak of a novel coronavirus (sars-cov- ) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against sars-cov- spike protein and to evaluate the serological immunity in humans. since the accessibility of live virus microneutralisation (mn) assays with sars-cov- is limited and requires enhanced bio-containment, the approach based on “pseudotyping” can be considered a useful complement to other serological assays. after fully characterising lentiviral pseudotypes bearing the sars-cov- spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an italian sero-epidemiological study. the results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and mn results. the overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context. in early december , cases of severe pneumonia of unknown aetiology were reported by the china health authority. in january , a novel coronavirus was identified as -ncov (subsequently renamed as sars-cov- ). an initial site of infections was the huanan seafood market, where live animals are sold. progressively, human-to-human transmission occurred [ ] , causing a disease named coronavirus disease . on th july , the world health organization (who) estimated the global incidence of covid- as , , cases and the number of the deaths as , [ ] . hep g cells were cultured in rpmi (gibco) supplemented with mm l-glutamine (lonza, milan, italy), units/ml penicillin-streptomycin (lonza, milano, italy) and % foetal bovine serum (fbs) (euroclone, pero, italy). hek t/ , mdck and huh- cell lines were cultured in dulbecco's modified eagle's medium (dmem) high glucose (euroclone, pero, italy) supplemented with mm l-glutamine (lonza, milan, italy), units/ml penicillin-streptomycin (lonza, milan, italy) and % of fbs (fbs euroclone, pero, italy). vero e and caco cells were cultured in eagle's minimum essential medium (emem) (lonza, milan, italy) supplemented with mm l-glutamine (lonza, milan, italy), units/ml penicillin-streptomycin (lonza, milan, italy) and fbs (euroclone, pero, italy) to a final concentration of % for vero e and % for caco . all the cell lines used were incubated at • c, % co in humidified atmosphere and were sub-cultured twice a week until passage . a total of samples from an italian sero-epidemiological study, anonymously collected in compliance with italian ethics law, were provided by the laboratory of molecular epidemiology of the university of siena, italy. the human sera, derived from a sero-epidemiological study, had previously been tested in an elisa assay as pre-screening, and positive, borderline and negative elisa samples were tested in a micro-neutralisation assay, as previously described [ ] . this panel of sera was subsequently tested with sars-cov- pseudotypes in order to compare the neutralisation profiles when they were tested against the live virus and the surrogate virus. as an internal positive control, a panel of samples collected from health care workers (confirmed positive for sars-cov- by reverse real-time pcr) were kindly provided by prof. valentina bollati, university of milan. in addition, three monoclonal antibodies (mabs) were included in the serological assay: human igg anti-s cr (native antigen, oxford, uk), human igg anti-rbd (eenzyme, gaithersburg, md, usa) and human anti-igm sars-cov- spike s cr (absolute antibody, drydock avenue, th floor boston, ma , usa ( : starting dilutions). the full-length s protein (genbank accession number: yp_ . ) was codon-optimised and synthesised (genscript, cina), and the s fragment was cloned into the expression vector as described previously [ ] . the hiv gag-pol plasmid (p . ) [ ] , the firefly luciferase-expressing plasmid (pcsflw) [ ] , the pcaggs-tmprss plasmid and the plasmid encoding for the spike's human ace receptor were kindly provided by dr. nigel temperton and have previously been described [ ] [ ] [ ] . as a control, a vesicular stomatitis virus (vsv-g) plasmid was used (pcmv-vsv-g) (addgene plasmid ; http://n t.net/addgene: ) [ ] . the day before transfection, confluent plates of hek t/ cells were split : and seeded into cm plates in dmem % fbs. cells (at the % of confluence) were co-transfected with the s plasmid from sars-cov- ( ug/ul), hiv gag-pol ( ug/ul) and the pcsflw ( . ug/ul) using endofectin™ lenti transfection reagent (tebu bio- ef , via pretorio -c.p. . i- magenta, milano) in accordance with the manufacturer's instructions. the following day, the supernatants were replaced with dmem without phenol red (thermo fisher scientific, rd ave, waltham, ma , united states containing % fbs, and the plates were incubated for h at • c in an atmosphere of % co . after h, the supernatants of transfected cells were harvested and filtered by millex-ha . um filter. concurrently, hek t/ cells were also transfected with vsv-g plasmid ( µg/µl), and a no-spike control (∆ envelope) was generated by co-transfection with hiv gag-pol and pcsflw plasmids only. for sars-cov- titration, a further transfection is required in order to allow pseudotypes to enter target cells. the day before titration, hek t/ cells were co-transfected with two plasmids encoding for the ace receptor gene and tmprss , by means of endofectin™ lenti transfection reagent; after overnight incubation at • c, the supernatant was replaced by dmem containing % fbs. the following day, supernatants were serially two-fold diluted in a fresh cell culture medium in -well, flat-bottomed culture plates, and × hek t/ target cells were added to each well. as controls, vsv-g and ∆-envelope pseudotypes were also included. after h, the luminescence of cell cultures (in relative luminescence units or rlus) was evaluated by luminometry (tecan infinite m pro multi-detection plate reader) using the bright-glo assay system (bright-glo™ luciferase assay system, promega, viale piero e alberto pirelli, , milano mi). serial dilutions of sars-cov- pseudotype-containing media were tested by means of the lenti-x p rapid titre kit (cat. no. ) (takara, japan) in accordance with the manufacturer's instructions. in order to verify the incorporation of the s glycoprotein of sars-cov- , the s protein expressed on the lentiviral pseudotypes was detected by western blot analysis. western blot analysis of spike protein was performed on the supernatant from sub-confluent hek t/ cells co-transfected with hiv gag-pol plasmid, csflw plasmid and the s plasmid from sars-cov- . western blot analysis was also performed on lv in a bsl facility. ∆-envelope (no-spike control) pseudotypes prepared with the same procedure were run as a negative control. sars-cov- pseudotypes, sars-cov- live virus and ∆-envelope pseudotypes were mixed with sds sample buffer. the mixture was heated for min at • c and electrophoresis ( µg of protein/sample) was carried out in - % bis-tris gels (life technologies, carlsbad, ca, usa). proteins were then blotted onto nitrocellulose membranes and incubated overnight with -fold diluted sera from convalescent sars-cov- patients. a goat anti-human igg (bethyl, fm , montgomery, tx , united states) was used as a secondary antibody ( : dilution). the chemiluminescent signals from the nitrocellulose membranes were captured by a camera system (imagequant las instrument). in this study, different cell lines have been tested in order to study their susceptibility to sars-cov- s protein driven entry, the role of the ace receptor and tmpssrs for s protein priming. one day before pseudotypes titration, mdck, vero e , caco , hep g and huh cells were transfected with the pcaggs-tmprss plasmid, while hek t/ cells were co-transfected with ace and pcaggs-tmprss plasmids. after h, the cells were removed by trypsinisation, counted and used for the subsequent titration. in parallel with sars-cov- pseudotypes, vsv-g and ∆-envelope pseudotypes were titrated as controls. plates were incubated for - h with the pseudotypes, at • c in an atmosphere of % co , and the efficiency of pseudotypes entry was characterised on the basis of luciferase activity (relative luminescence units or rlus). to measure the neutralisation activity of this panel of sera, neutralising antibody titres were defined as endpoint two-fold seral dilutions of test samples, and the % inhibitory concentration (ic ) was determined as the serum dilution resulting in a % reduction of a single round of infection (reporter gene-mediated signal). values were expressed as a percentage in comparison with the signal from the cell-only control (equivalent to % neutralisation and/or no infection) and the signal from a pseudotype-only control (equivalent to % neutralisation or % infection). in brief, two-fold serial dilution of serum samples, starting from : , was performed in a culture medium (dmem, % fbs, % pen-strep, % l-glutamine). the serum was mixed with sars-cov- pseudotypes in a : vol/vol ratio in a -well culture plate. the virus input used was × rlu/well (based on the previous titration). the serum-pseudotypes mixture was then incubated for h at • c in a humidified atmosphere with % co . after h, hek /ace transfected cell suspensions ( . × cell/ml) were seeded into each well of flat-bottomed tissue culture plates. the plates were incubated at • c for - h, and the neutralising antibodies were characterised on the basis of luciferase activity. the sars-cov- strain -ncov/italy-inmi -wild-type virus was purchased from the european virus archive goes global (evag, spallanzani institute, via portuense, , - , rome) and propagated in vero e cells. the virus was titrated in a tissue culture infective dose % assay (tcid ) on -well culture plates with -log serial dilution. the plates were observed daily for a total of four days for the presence of cytopathic effect (cpe). the end-point titre was calculated according to the spearman-karber formula [ ] . the mn assay was performed as previously reported by manenti et al. [ ] . briefly, two-fold serial dilutions of serum samples were mixed with an equal volume of viral solution containing tcid of sars-cov- . the serum-virus mixture was incubated for h at • c in a humidified atmosphere with % co , then passed to a vero e culture plate. the plates were incubated for four days at • c in a humidified atmosphere with % co . after the incubation time, each well of a -well plate was inspected by means of an inverted optical microscope to evaluate the percentage of cpe. pseudotype transduction titres were estimated by means of excel tm software; the pseudotype titres obtained at each point in a range of dilution points were expressed as rlu/ml, and the arithmetic mean was calculated. for the analyses of pseudotype-based neutralisation assays, titres were firstly normalised, and ic values were calculated by a non-linear regression model (log (inhibitor) vs. normalised response-variable slope) analysis. titres were subsequently expressed as the range of dilution in which the ic value lay. in order to evaluate cell infectivity, two-way analysis of variance (anova) with dunnett posttest was used to test for statistical significance (p > . (ns, not significant), p ≤ . (*), p ≤ . (**), p ≤ . (***), and p ≤ . (****)). for all statistical analyses, the graphpad prism version . package was used (graphpad software, graphpad, northside dr., suite , san diego, ca , usa). the statistical analyses have been undertaken with the r software (version . . ). different approaches drove our statistical analyses, as shown in figure . all the titres underwent preliminary base- logarithmic transformation. in accordance with the classification approach, the titres greater than log ( ) were labelled as "positive," and otherwise as "negative." with mnt taken as the reference ("true") results, the misclassifications were counted and displayed in an error matrix table. a linear regression model provided a measure of the strength of the relationship between pnt and mnt. as the dependent variable, we considered the log of pnt, and as the independent variable, the log of mnt. for the evaluation of the agreement between pnt and mnt, we used the intra-class correlation coefficient (icc). in addition, the bland-altman method (ba) enabled us to search for possible systematic difference (bias) between the pnt and mnt, as well as to identify the presence of outliers. the ba evaluation mainly consists in a scatter plot of the differences between the measurements vs. their means. the % limits of agreement (loas) were calculated around the mean of the differences. we set a maximum acceptable difference (mad) of . times the mnt, below which the observed pnt-mnt differences were considered as not having a significant biological effect. we interpreted the differences below the mad and within the % limits of agreement as interchangeable. by comparing the distributions of pnt and mnt, we got further insight into their similarity degree. thus, we applied the kolmogorov-smirnov test and measured the kullbac-leibler divergence (kld). the former considers the largest difference between the empirical distribution functions of the pnt and mnt tests, and the latter is an information theoretic-based value, which indicates how much information is lost when taking pnt as an approximation of mnt. the kld was calculated as the "true" reference the mnt distribution, and normalised as follows: where nkld is the normalised divergence, and pmnt and ppnt are the distributions of mnt and pnt, respectively. this normalisation restricts the divergence values in the range [− . , + . ], such that nkld = if the pnt distribution perfectly reproduces the mnt distribution, while the extremes nkld = − . or nkld = . are attained if kld tends towards infinite, (positive or negative infinite, respectively). lastly, we conducted a bootstrap test ( , resamples with replacement from the mnt and pnt data) on the kld statistic under the null hypothesis of nkld = . all the titres underwent preliminary base- logarithmic transformation. in accordance with the classification approach, the titres greater than log ( ) were labelled as "positive," and otherwise as "negative." with mnt taken as the reference ("true") results, the misclassifications were counted and displayed in an error matrix table. a linear regression model provided a measure of the strength of the relationship between pnt and mnt. as the dependent variable, we considered the log of pnt, and as the independent variable, the log of mnt. for the evaluation of the agreement between pnt and mnt, we used the intra-class correlation coefficient (icc). in addition, the bland-altman method (ba) enabled us to search for possible systematic difference (bias) between the pnt and mnt, as well as to identify the presence of outliers. the ba evaluation mainly consists in a scatter plot of the differences between the measurements vs. their means. the % limits of agreement (loas) were calculated around the mean of the differences. we set a maximum acceptable difference (mad) of . times the mnt, below which the observed pnt-mnt differences were considered as not having a significant biological effect. we interpreted the differences below the mad and within the % limits of agreement as interchangeable. by comparing the distributions of pnt and mnt, we got further insight into their similarity degree. thus, we applied the kolmogorov-smirnov test and measured the kullbac-leibler divergence (kld). the former considers the largest difference between the empirical distribution functions of the pnt and mnt tests, and the latter is an information theoretic-based value, which indicates how much information is lost when taking pnt as an approximation of mnt. the kld was calculated as the "true" reference the mnt distribution, and normalised as follows: where nkld is the normalised divergence, and pmnt and ppnt are the distributions of mnt and pnt, respectively. this normalisation restricts the divergence values in the range [− . , + . ], such that nkld = if the pnt distribution perfectly reproduces the mnt distribution, while the extremes nkld = − . or nkld = . are attained if kld tends towards infinite, (positive or negative infinite, respectively). lastly, we conducted a bootstrap test ( , resamples with replacement from the mnt and pnt data) on the kld statistic under the null hypothesis of nkld = . sars-cov- s glycoprotein and gag-p in pseudotypes were characterised by immunoblot analysis and p elisa, respectively. to verify the expression of the spike protein in the sars-cov- pseudotypes, the spike was detected by western blot; sera from convalescent sars-cov- patients, which have been shown to have a high neutralising titre in microneutralisation with a live virus, were used as the primary antibody, and goat anti-human igg as the secondary antibody. sars cov- strain -ncov/italy wild-type virus (lv), which was handled in a level bio-containment facility (bsl ), was used as positive control in order to evaluate the spike glycoprotein expression, while a ∆-envelope pseudotype, prepared with the same procedure, was used as a negative control. three different batches of pseudotypes were tested; specific bands were found for sars-cov- pseudotypes and for sars-cov- live virus, but not for the ∆-envelope control ( figure ). sars-cov- s glycoprotein and gag-p in pseudotypes were characterised by immunoblot analysis and p elisa, respectively. to verify the expression of the spike protein in the sars-cov- pseudotypes, the spike was detected by western blot; sera from convalescent sars-cov- patients, which have been shown to have a high neutralising titre in microneutralisation with a live virus, were used as the primary antibody, and goat anti-human igg as the secondary antibody. sars cov- strain -ncov/italy wild-type virus (lv), which was handled in a level biocontainment facility (bsl ), was used as positive control in order to evaluate the spike glycoprotein expression, while a Δ-envelope pseudotype, prepared with the same procedure, was used as a negative control. three different batches of pseudotypes were tested; specific bands were found for sars-cov- pseudotypes and for sars-cov- live virus, but not for the Δ-envelope control (figure ). . the lv was used as a positive control and the Δ-envelope (particles bearing no envelope protein), prepared with the same procedure, was used as a negative control. uncleaved s protein, about kda; cleaved s protein, about kda; dimeric-trimeric s protein, above kda; nucleocapsid protein, about kda. experiments were done twice and one is shown. regarding the pseudotypes, we observed three main bands: one just below kda, and the remaining two bands at kda and kda, corresponding to the full-length and cleaved s protein, as shown in previous studies [ ] . these two bands ( kda and kda) were barely detectable in the live virus, in which was observed one just above kda, possibly reflecting the dimeric-trimeric s protein (detected in the pseudotypes below kda), and the other band was around kda, possibly corresponding to the nucleocapsid protein (np). the high glycosylation potential to which the spike is subjected during the infection differs from the spike expressed on the pseudotyped particles that do not undergo the same post-translational modifications [ ] . this would explain the presence of a protein with a weight greater than kda in the wild type virus, compared to the three isoforms with detectable molecular weight between kda and below kda related to the pseudotypes [ ] . moreover, the quantitative data can slightly differ when a convalescence serum sample is used instead of an antibody (e.g., monoclonal antibody), specifically directed against a defined protein's portion. although, in this study, the evaluation of sars-cov- pseudotype titres is based on the reporter gene expression (rlus/ml), a number of eight different batches of sars-cov- lentiviral pseudotypes were also compared for the hiv- viral core protein p amount (directly correlating to the number of particles) by elisa (values reported as pg of p for ml). regarding the pseudotypes, we observed three main bands: one just below kda, and the remaining two bands at kda and kda, corresponding to the full-length and cleaved s protein, as shown in previous studies [ ] . these two bands ( kda and kda) were barely detectable in the live virus, in which was observed one just above kda, possibly reflecting the dimeric-trimeric s protein (detected in the pseudotypes below kda), and the other band was around kda, possibly corresponding to the nucleocapsid protein (np). the high glycosylation potential to which the spike is subjected during the infection differs from the spike expressed on the pseudotyped particles that do not undergo the same post-translational modifications [ ] . this would explain the presence of a protein with a weight greater than kda in the wild type virus, compared to the three isoforms with detectable molecular weight between kda and below kda related to the pseudotypes [ ] . moreover, the quantitative data can slightly differ when a convalescence serum sample is used instead of an antibody (e.g., monoclonal antibody), specifically directed against a defined protein's portion. although, in this study, the evaluation of sars-cov- pseudotype titres is based on the reporter gene expression (rlus/ml), a number of eight different batches of sars-cov- lentiviral pseudotypes were also compared for the hiv- viral core protein p amount (directly correlating to the number of particles) by elisa (values reported as pg of p for ml). the results showed that all batches tested consistently contained around - pg/ml of p gag capsid protein, corresponding approximately to a titre of . e + rlus/ml. similar vector infectivity was also identified for vsv-g pseudotyped vectors, around - pg/ml of p gag capsid protein corresponding to an approximate titre of . e + rlus/ml, while a value of - pg/ml, corresponding to a titre of . e + , was obtained for ∆-envelope pseudotypes, as shown in figure . the values obtained for the delta envelope are slightly higher for the p amount compared to the rlus. a possible explanation, as showed by geraerts [ ] , is that p quantification by elisa will detect cores lacking envelope glycoproteins (non-functional) as well as cores belonging to transduction competent (functional) pseudotypes, and this technique usually overestimates the functional vector titre. in fact, it also been shown that omission of the envelope plasmid during the vector production resulted in p being comparable with those of a normal production although with a non-detectable functional titre. the results showed that all batches tested consistently contained around - pg/ml of p gag capsid protein, corresponding approximately to a titre of . e + rlus/ml. similar vector infectivity was also identified for vsv-g pseudotyped vectors, around - pg/ml of p gag capsid protein corresponding to an approximate titre of . e + rlus/ml, while a value of - pg/ml, corresponding to a titre of . e + , was obtained for Δ-envelope pseudotypes, as shown in figure . the values obtained for the delta envelope are slightly higher for the p amount compared to the rlus. a possible explanation, as showed by geraerts [ ] , is that p quantification by elisa will detect cores lacking envelope glycoproteins (non-functional) as well as cores belonging to transduction competent (functional) pseudotypes, and this technique usually overestimates the functional vector titre. in fact, it also been shown that omission of the envelope plasmid during the vector production resulted in p being comparable with those of a normal production although with a non-detectable functional titre. figure . p quantification. vector particle concentration (pg p protein per ml) determined by p elisa and corresponding lentiviral vector infectivity (rlus/ml). values expressed as the means ± sd of independent measurements (n = ). after the production of the sars-cov- pseudotypes, we asked which cell lines were susceptible to pseudotype-driven entry in order to have a panel of cell lines that can be used in downstream pseudotype-neutralisation assays. for this purpose, we used a panel of cell lines of human and animal origin. since sars-cov- live virus has been successfully isolated in vero (african green monkey kidney cell line), vero e and huh cell lines at high titres (as shown previously [ ] ), these were chosen for the cell tropism study. the hep g , mdck and caco cells were also included in this panel because a previous study evidenced ace receptor expression, except for hek t/ [ ] . however, hek / cells have also been included as control cell line due to their high transfectability, and they were firstly optimised using different ace -expressing plasmid figure . p quantification. vector particle concentration (pg p protein per ml) determined by p elisa and corresponding lentiviral vector infectivity (rlus/ml). values expressed as the means ± sd of independent measurements (n = ). after the production of the sars-cov- pseudotypes, we asked which cell lines were susceptible to pseudotype-driven entry in order to have a panel of cell lines that can be used in downstream pseudotype-neutralisation assays. for this purpose, we used a panel of cell lines of human and animal origin. since sars-cov- live virus has been successfully isolated in vero (african green monkey kidney cell line), vero e and huh cell lines at high titres (as shown previously [ ] ), these were chosen for the cell tropism study. the hep g , mdck and caco cells were also included in this panel because a previous study evidenced ace receptor expression, except for hek t/ [ ] . however, hek / cells have also been included as control cell line due to their high transfectability, viruses , , of and they were firstly optimised using different ace -expressing plasmid concentrations. based on these preliminary results, this panel of cell lines was tested against sars-cov- , vsv-g and delta envelope pseudotypes. as also seen in previous studies [ , ] , all the cell lines tested were highly susceptible to entry driven by vsv-g pseudotypes as demonstrated by titres of - rlus/ml (figure ) . all the cell lines tested were also susceptible to entry by sars-cov- pseudotypes, in particular, hek ace /tmprss -transfected cells, as demonstrated by titres of - rlus/ml. however, no statistical differences have been observed when sars-cov- pseudotypes have been tested against different cell lines. this comparable susceptibility can be due to the similar ace expression (except for mdck cell line as also showed by nie et al. [ ] ). moreover, co-transfection with tmprss protease can potentially level the titres obtained with different cell lines except for hek / . concentrations. based on these preliminary results, this panel of cell lines was tested against sars-cov- , vsv-g and delta envelope pseudotypes. as also seen in previous studies [ , ] , all the cell lines tested were highly susceptible to entry driven by vsv-g pseudotypes as demonstrated by titres of - rlus/ml (figure ) . all the cell lines tested were also susceptible to entry by sars-cov- pseudotypes, in particular, hek ace /tmprss -transfected cells, as demonstrated by titres of - rlus/ml. however, no statistical differences have been observed when sars-cov- pseudotypes have been tested against different cell lines. this comparable susceptibility can be due to the similar ace expression (except for mdck cell line as also showed by nie et al. [ ] ). moreover, co-transfection with tmprss protease can potentially level the titres obtained with different cell lines except for hek / . as expected, when cell lines were tested with Δ-envelope control (particles without envelope proteins) the transduction activity dropped drastically ( -log), corresponding to - rlus/ml (figure ). as shown in table , of human serum samples tested, proved positive for pnt, with titres ranging between - and > , and positives for mnt, with titres ranging between and (table ) . forty-one human samples were found negative for pnt and negative for mnt. therefore, only titres obtained for sera were found to be discordant. when mabs have been tested against pseudotypes and the live virus, no neutralisation activity was observed on mn, while an evident neutralisation profile was seen for pnt against human anti-igm [ ] sars-cov- spike s (ic > ). as expected, when cell lines were tested with ∆-envelope control (particles without envelope proteins) the transduction activity dropped drastically ( -log), corresponding to - rlus/ml ( figure ). as shown in table , of human serum samples tested, proved positive for pnt, with titres ranging between - and > , and positives for mnt, with titres ranging between and ( table ) . forty-one human samples were found negative for pnt and negative for mnt. therefore, only titres obtained for sera were found to be discordant. table . panel of human sera tested by live virus mn and pseudotype-based neutralisation assays. responses against sars-cov- are expressed as antibody titres for both assays (end-point dilution). titres (pnt) when mabs have been tested against pseudotypes and the live virus, no neutralisation activity was observed on mn, while an evident neutralisation profile was seen for pnt against human anti-igm [ ] sars-cov- spike s (ic > ). in addition, we defined the parameters of sensitivity, specificity and accuracy. from the error matrix (table ) , we obtained the following results: accuracy = . % ( % ci ( . %- . %)), which was significantly higher (p < . ) than the no-information rate ( . %), sensitivity = . % ( % ci ( . %- . %)) and specificity = % ( % ci ( . %- %)). a simple linear regression was conducted to predict the log pnt based on the log mnt data ( figure ). the linear regression was found to be significant, f ( , ) = . , p < . , with an r = . . the pnt increased . for each log of the mnt. viruses , , x for peer review of < < < < < < < < < < < < < < < < < < < in addition, we defined the parameters of sensitivity, specificity and accuracy. from the error matrix (table ) , we obtained the following results: accuracy = . % ( % ci ( . %- . %)), which was significantly higher (p < . ) than the no-information rate ( . %), sensitivity = . % ( % ci ( . %- . %)) and specificity = % ( % ci ( . %- %)). a simple linear regression was conducted to predict the log pnt based on the log mnt data ( figure ). the linear regression was found to be significant, f ( , ) = . , p < . , with an r = . . the pnt increased . for each log of the mnt. to evaluate the agreement between pnt and mnt, we used the intra-class correlation coefficient (icc). the one-way random single score icc (table ) calculated between the neutralisation titres pnt and mnt was . ( % ci ( . - . )), p < . . this indicated excellent agreement [ ] . to evaluate the agreement between pnt and mnt, we used the intra-class correlation coefficient (icc). the one-way random single score icc (table ) calculated between the neutralisation titres pnt and mnt was . ( % ci ( . - . )), p < . . this indicated excellent agreement [ ] . table . summary of the intra-class correlation analysis. the one-way random single score icc calculated between the neutralisation titres pnt and mnt was . ( % ci ( . - . )), p < . . the bland-altman (ba) analysis ( figure ) revealed the presence of a significant relationship between differences and means, which made applying a regression approach consistent [ ] . calculated between the neutralisation titres pnt and mnt was . ( % ci ( . - . )), p < . . the bland-altman (ba) analysis ( figure ) revealed the presence of a significant relationship between differences and means, which made applying a regression approach consistent [ ] . we found that most of the data-points were within the % limit of agreements (loas), while one outlier, corresponding to a means of titres equal to . (log -units), was detected. these findings suggested that there was substantial agreement and interchangeability between pnt and mnt. in addition, the ba method enabled us to search for possible systematic difference (bias) between the pnt and mnt, and to identify the presence of outliers. the maximum acceptable differences (blue lines) embrace the loas (red lines), which makes the interpretation of the statistical results biologically plausible. we found a significant linear relationship between the differences and the means of the titres. moreover, except for one outlier, all the differences were both biologically and statistically acceptable. in other words, there was substantial agreement between pnt and mnt. we also concluded, on the basis of the kolmogorov-smirnov test, that there was not enough evidence to reject the null hypothesis of equal distributions of pnt and mnt, (ks test = . , p = . ). the normalised kullback-leibler divergences (nkld) between the original pnt and mnt data was equal to − . . the result of the bootstrap testing evidenced that the hypothesis of a zero nkld between pnt and mnt was consistent with the data, p = . (figure ). the maximum acceptable differences (blue lines) embrace the loas (red lines), which makes the interpretation of the statistical results biologically plausible. we found a significant linear relationship between the differences and the means of the titres. moreover, except for one outlier, all the differences were both biologically and statistically acceptable. in other words, there was substantial agreement between pnt and mnt. viruses , , x for peer review of since the observed nkld was lower (in absolute value) than the % lower-tail quantile, we concluded that the divergence was not significantly different from zero (p-value = . ). the recent emergence of the novel pathogenic sars-coronavirus- constitutes a global health emergency. as previously shown [ , ] , infection with sars-cov- elicits antibodies that bind to the virus. although several studies are still ongoing regarding the virus and the complexity of the human immune responses, neutralising antibodies are known to be strongly correlated with protection [ ] . currently, polyclonal antibodies from recovered sars-cov- -infected patients have been used to treat the sars-cov- infection, but identification of sars-cov- -specific neutralising mabs is still ongoing. once such antibodies are selected and produced, the subsequent steps will involve testing for neutralising and/or cross-neutralising activity [ ] , which should simplify the analysis of functional humoral immune responses. the demand for serological testing for sars-cov- is high, as there is a need to better quantify the number of cases of covid- , including asymptomatic carriers [ ] and patients who have recovered. as we know, sars-cov- have a strong pathogenicity and working with the wild-type virus implies the need for level bio-containment facility, according to the who guidelines. however, serological assays for the evaluation of neutralising activity against the sars-cov- currently require the use of isolated-live virus. indeed, high-throughput methods (such as elisa) that do not require the live virus are limited by the fact that they detect total antibody binding to sars-cov- or to some of its key constituent proteins [ , ] . for this reason, we produced and characterised a lentiviral pseudotype system that expresses the s protein of sars-cov- with the same approach used by for other pathogenic coronaviruses including sars-cov and mers [ , ] . this pseudotype system can be a useful tool because of its safety and versatility. its versatility lies in the fact that the virus can be pseudotyped with different envelope proteins [ , , ] . moreover, this approach does not necessitate handling the live virus, and does not require high-level the nkld between the original mnt and pnt was − . (blue dotted line). the mnt and pnt data were re-sampled with replacement , times. the red dotted line shows the % lower-tail quantile (− . ) of the bootstrap distribution. since the observed nkld was lower (in absolute value) than the % lower-tail quantile, we concluded that the divergence was not significantly different from zero (p-value = . ). the recent emergence of the novel pathogenic sars-coronavirus- constitutes a global health emergency. as previously shown [ , ] , infection with sars-cov- elicits antibodies that bind to the virus. although several studies are still ongoing regarding the virus and the complexity of the human immune responses, neutralising antibodies are known to be strongly correlated with protection [ ] . currently, polyclonal antibodies from recovered sars-cov- -infected patients have been used to treat the sars-cov- infection, but identification of sars-cov- -specific neutralising mabs is still ongoing. once such antibodies are selected and produced, the subsequent steps will involve testing for neutralising and/or cross-neutralising activity [ ] , which should simplify the analysis of functional humoral immune responses. the demand for serological testing for sars-cov- is high, as there is a need to better quantify the number of cases of covid- , including asymptomatic carriers [ ] and patients who have recovered. as we know, sars-cov- have a strong pathogenicity and working with the wild-type virus implies the need for level bio-containment facility, according to the who guidelines. however, serological assays for the evaluation of neutralising activity against the sars-cov- currently require the use of isolated-live virus. indeed, high-throughput methods (such as elisa) that do not require the live virus are limited by the fact that they detect total antibody binding to sars-cov- or to some of its key constituent proteins [ , ] . for this reason, we produced and characterised a lentiviral pseudotype system that expresses the s protein of sars-cov- with the same approach used by for other pathogenic coronaviruses including sars-cov and mers [ , ] . this pseudotype system can be a useful tool because of its safety and versatility. its versatility lies in the fact that the virus can be pseudotyped with different envelope proteins [ , , ] . moreover, this approach does not necessitate handling the live virus, and does not require high-level bio-containment facilities, as the pseudotype is devoid of virulent viral components and it is involved in a single round of replication [ ] . the production of pseudotypes harbouring novel glycoproteins could permit elucidation of viral biological characteristics and a better understanding if they have potential to cause pandemics by the generation of mutants (via mutation of glycoprotein) without the risk of creating potentially dangerous viruses. it can also reflect key aspects of host cell entry and receptor binding specificity [ , ] . however, the need of additional reagents seems a requirement due to cellular receptor specificity and tmprss priming for sars-cov- pseudotypes; thus, necessitating us to optimise the batch-to-batch variations in order to reduce the variability in terms of pseudotype titres and stability. since previous studies have evidenced that most amino acid residues for ace binding by sars-cov were also conserved in sars-cov- [ ] , we have studied different cell susceptibility to sars-cov- pseudotype entry. understanding the entry mechanisms determined by the s glycoprotein and the susceptibility of different cells (based on specific cellular receptor expression) can also provide important information to study critical process in which the s spike protein of sars-cov- is involved. moreover, the use of multiple target cell lines is particularly valid since one of the advantages of the pseudotype-based assays is that they are deployable across different stakeholder laboratories who have access to different cell lines. it also represents a determinant for the development and optimisation of cell-based assays and for the screening of potential entry inhibitors. once the sars-cov- pseudotypes have been efficiently produced, we have also developed a lentiviral pseudotype-based assay that facilitates the accurate determination of neutralising antibody responses to sars-cov- that can be paired to the more intensive and laborious mn. although it is unclear as to how the display of s protein on heterologous virus impacts viral entry, antibody recognition and antibody neutralisation [ ] , our results suggest that pseudotype-based neutralisation assays correlate well with the mn assays when testing human sera, and our findings show significant accuracy, sensitivity and specificity when both assays are compared. when employed in the screening of vaccinated human sera and other influenza serological studies [ ] [ ] [ ] [ ] , pseudotype-based neutralisation assays have been described to be more sensitive than classical mn. the sensitivity of pseudotypes can also detect particularly low antibody responses or facilitate the antibody's recognition that cannot always be determined by conventional assays, possibly due to lower density/quantity of glycoprotein expressed on the pseudotypes [ , ] . this could explain the ability of human mab igm to recognise certain epitopes of s protein, as it has been seen for pseudotype-based neutralisation but not for live virus mn. although the model (pnt instead of mnt) returned a relatively high percentage of false-negatives, in large-scale serological testing, having a highly specific test is advantageous when a false-positive result has a great clinical impact. indeed, it guarantees against the risk of misclassification of the true-negative samples. undoubtedly, for a wider use of pseudotypes, it will be necessary to take into consideration additional aspects required for standardisation such as comparison between viral vectors/expression plasmids used, optimisation of particle titres and establishment of adequate positive, negative controls and reference standards (making it more difficult to evaluate the reproducibility of different serological assays). moreover, as in all the cell-based assays, cell input [ , ] is an important aspect in standardisation: the use of an automatic cell counter, cell viability and requirement of different proteases should be taken into account and it could be important for the consistency of the assay during different analytical sessions. undoubtedly, a permanent cell line could be a valid alternative [ ] (it would require only tmprss priming), and the generation of a cell line expressing proteases as a pseudotype producer could be investigated. our findings suggest that sars-cov- pseudotype-based assays and the live-virus microneutralisation correlate well when employed in testing antibody responses against the novel sars-cov- virus. undoubtedly, they would help to dissect out these diversities of immunological 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and h subtypes for sensitive and specific detection of neutralizing antibodies optimization and evaluation of an influenza a (h ) pseudotyped lentiviral particle-based serological assay investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison efficient generation of vesicular stomatitis virus (vsv)-pseudotypes bearing morbilliviral glycoproteins and their use in quantifying virus neutralising antibodies the united states pharmacopeial convention. design and development of biological assays; the united states pharmacopeial convention biological assay validation; the united states pharmacopeial convention this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we found that most of the data-points were within the % limit of agreements (loas), while one outlier, corresponding to a means of titres equal to . (log -units), was detected. these findings suggested that there was substantial agreement and interchangeability between pnt and mnt.in addition, the ba method enabled us to search for possible systematic difference (bias) between the pnt and mnt, and to identify the presence of outliers.we also concluded, on the basis of the kolmogorov-smirnov test, that there was not enough evidence to reject the null hypothesis of equal distributions of pnt and mnt, (ks test = . , p = . ). the normalised kullback-leibler divergences (nkld) between the original pnt and mnt data was equal to − . . the result of the bootstrap testing evidenced that the hypothesis of a zero nkld between pnt and mnt was consistent with the data, p = . (figure ) . developmental medicine, university of siena, italy, for providing us the human serum samples from the sero-epidemiological study on sars-cov- . thanks to prof. valentina bollati, from the university of milan, for providing the sera sample from covid- -positive healthcare workers. thanks to nigel temperton, and the pseudotypes unit, university of kent, for providing the panel of plasmids used for sars-cov- pseudotype production. we thank also: virginia cianchi and donata martinuzzi for their lab support in vismederi research. the authors declare no conflict of interest. key: cord- - jtfc authors: van nguyen, dung; van nguyen, cuong; bonsall, david; ngo, tue tri; carrique-mas, juan; pham, anh hong; bryant, juliet e.; thwaites, guy; baker, stephen; woolhouse, mark; simmonds, peter title: detection and characterization of homologues of human hepatitis viruses and pegiviruses in rodents and bats in vietnam date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jtfc rodents and bats are now widely recognised as important sources of zoonotic virus infections in other mammals, including humans. numerous surveys have expanded our knowledge of diverse viruses in a range of rodent and bat species, including their origins, evolution, and range of hosts. in this study of pegivirus and human hepatitis-related viruses, liver and serum samples from vietnamese rodents and bats were examined by pcr and sequencing. nucleic acids homologous to human hepatitis b, c, e viruses were detected in liver samples of ( . %) of bats, ( . %), and ( %) of rodents, respectively. hepacivirus-like viruses were frequently detected ( . %) in the bamboo rat, rhizomys pruinosus, while pegivirus rna was only evident in ( . %) of rodent serum samples. complete or near-complete genome sequences of hbv, hev and pegivirus homologues closely resembled those previously reported from rodents and bats. however, complete coding region sequences of the rodent hepacivirus-like viruses substantially diverged from all of the currently classified variants and potentially represent a new species in the hepacivirus genus. of the viruses identified, their routes of transmission and potential to establish zoonoses remain to be determined. unlike many other communicable diseases, the burden of viral hepatitis has substantially increased over the last two decades to recently become the seventh leading cause of mortality worldwide. viral hepatitis now causes more deaths than tuberculosis, aids or malaria each year. hepatitis c virus (hcv) and hepatitis b virus (hbv) are responsible for > % ( % in ) of viral viruses , , of hepatitis-related mortality and disability. as such, these hepatitis viruses are the targets of efforts to combat viral hepatitis [ ] , including hbv vaccination, development of hcv vaccines, and highly effective drugs. in contrast, hepatitis e virus (hev) is endemic in many low-income countries [ ] but usually causes self-limiting hepatitis. infection with hev occasionally results in liver failure [ ] . hbv, hcv, and hev are members of virus families hepadnaviridae, flaviviridae, and hepeviridae, respectively. hbv has a partially double-stranded dna genome with overlapping open reading frames (orfs) [ ] , whereas hcv and hev have a single-stranded rna genome [ , ] . while the origins of these human viruses are unknown, rodents and bats are putative reservoir hosts because they host a diverse array of hepadnaviridae [ ] [ ] [ ] , hepeviridae [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and genera pegivirus [ ] [ ] [ ] and hepacivirus [ , , ] of the flaviviridae family including homologues of the human hepatitis viruses under question. among these, it is of concern that a bat hepadnavirus may possess the ability to infect human liver cells [ ] . several factors may contribute to the risk of zoonotic rodent or bat virus transmission. rodent meat is a popular source of protein for human consumption in vietnam, particularly in the mekong delta, where rats (rattus spp. and bandicota indica) are commonly trapped and sold live in wet markets [ ] . the total annual consumption of rat meat in vietnam is estimated at - tonnes [ ] . hoary bamboo rats (rhizomys pruinosus) are additionally farmed for human consumption. moreover, bat faeces collected from bat caves or farms is used as natural fertilizer ("guano") in vietnam. as rodents and bats are reservoirs or carriers of a significant number of zoonotic pathogens [ ] and viruses with unknown zoonotic potential, there are health risks that are associated with exposure to these animals. however, a previous study [ ] showed none of the surveyed rat catchers or processors were aware of infection risks from contact with live rats. consequently, no precautions were taken for the handling of rodents. in the search for viral diversity and zoonotic viruses, novel paramyxovirus and coronavirus in vietnamese bats and rats were detected in a previous study [ ] . here, we report the detection pegivirus and human hepatitis-related viruses in these mammals. rodent and bat samples were collected within the vizions (vietnam initiative on zoonotic infections) framework [ ] for the screening of zoonotic microorganisms [ , [ ] [ ] [ ] . rodent samples. as it is important and essential to understand the risk associated with rodents, including those sold in the markets, a total of rats purchased from markets in five of twelve provinces in the mekong delta during - and farmed bamboo rats purchased from a market in dak lak in - were enrolled. in addition, trapped rats were also included. rat trapping was carried out in different locations (pig and poultry farms, rice fields, fruit groves, tropical forests, markets, slaughter-house) in the provinces of dong thap during march and dak lak in april , as previously described [ ] . serum and liver samples were collected post-mortem. species identification of rats was carried out on the basis of morphological characteristics and sequencing of a conserved housekeeping gene [ ] . all of the samples were stored in sterile tubes with rna later at − • c until nucleic acid extraction. special precautions were taken to avoid cross-contamination. bat samples. a total of bats were trapped at six sites in the provinces of dong nai (in cat tien national park) and quang ngai in may using mist nets and harp traps as described [ ] . trapped bats were speciated according to morphology [ ] , and liver samples were collected and stored as described above for rats. rna was manually extracted from rodent serum samples using qiaamp viral rna mini kit (qiagen, manchester, uk) and following instructions from the manufacturer. liver samples from bats and rodents were subjected to nucleic acid extraction using allprep dna/rna mini kit (qiagen, manchester, uk). briefly, about mg of liver per sample was first lysed and homogenised using tissuelyser (qiagen, manchester, uk). the lysate was applied to an allprep dna spin column for dna to bind onto the column. ethanol was added to the flow-through and rna and bound to the membrane when the sample was passed through an rneasy spin column. after washing steps, dna and rna was eluted separately in µl of nuclease-free water. extracted nucleic acid was used in screening for the targeted hepatitis viruses. in order to minimize contamination, pcr mastermix preparation, and the addition of templates were carried under separated laminar flow hoods and lab spaces. all of the surfaces, tubes, and equipment were uv irradiated between each pcr. reverse transcription using superscript iii reverse transcriptase (invitrogen, paisley, uk) was performed according to the manufacturer's instruction. synthesized cdna was screened for hepaciviruses and pegiviruses using a semi-nested pcr protocol with broad spectrum degenerate primers, which can detect all known hepaciviruses and pegiviruses. amplification conditions (using gotaq from promega, southampton, uk) included • c for min, and cycles of denaturation ( • c, s), annealing ( • c, s) and elongation ( • c, s). similarly, hev was screened using broadly reactive primers targeting viral rna-dependent rna polymerase as described in drexler et al., [ ] . dna extracted from liver samples was used for screening of hbv. degenerate primers targeting a highly conserved region of the polymerase gene of sequences from all known hbv hosts were designed for a nested pcr protocol using the above amplification conditions. primers for screening are listed in table s . the length of the sequenced screening fragments (excluding primer sequences) of homologues of hbv, hev, hcv, and pegivirus was , and nucleotides, respectively. for rodent hepacivirus, hev and pegivirus genomes, extracted rna from representative positive samples was subjected to deep sequencing using an illumina platform. libraries were prepared from total extracted rna using the nebnext ultra directional sequencing kit (new england biolabs, hitchin, uk) with omission of actinomycin d, then pooled and sequenced on a hiseq instrument (illumina, nr saffron walden, uk). quality control and trimming of reads were performed before genome mapping using clc genomics workbench (qiagen bioinformatics, redwood city, ca, usa) with the default affine gap cost parameters. the closest related virus genomes (genbank numbers kc , jx and kj for hepacivirus, hev and pegivirus, respectively) were used as templates for genome mapping. additional primers were designed using the obtained contigs for gap fillings. for bat hbv, primers were designed using sequences available from genbank and the obtained sequences from screening. these primers amplified amplicons, with overlapping regions as presented in table s . all of these nested or hemi-nested pcr protocols used superscript iii one-step rt-pcr system with platinum taq dna polymerase (invitrogen, paisley, uk) for rna viruses or q high-fidelity dna polymerase (new england biolabs, hitchin, uk) for hbv in the first round pcr, according to instructions from the manufacturers. q high-fidelity dna polymerase was also used in the second round pcr. two real-time pcr primer sets (table s ) in the untranslated region of bamboo rat hepaciviruses and the screening fragment of other rat hepaciviruses were designed using sequences available from this study. the targeted regions were amplified from positive samples and cloned into pgem-t easy vector (promega, southampton, uk) for in vitro transcription, as previously described [ ] . the obtained rna transcripts were used to generate standard curves of the real-time pcr assays for measurement of rodent hepacivirus rna titers using superscript iii reverse transcriptase (invitrogen, paisley, uk) and powerup sybr green master mix (thermo fisher scientific, northumberland, uk). sequences were imported into sse (simmonic sequence editor) [ ] for the alignment and calculation of sequence distance values from reference sequences of known viruses from which sequence identities were inferred. sequence distances instead of sequence identities in the regions used for classification of hepaciviruses and pegiviruses were presented to easily compare with the species p-distance thresholds set in the proposed update to the taxonomy of the genera hepacivirus and pegivirus [ ] . maximum-likelihood phylogenetic trees were reconstructed using the mega . software package [ ] with bootstrap resamples. the best-fitting model for each sequence dataset (as shown in figure captions) was first determined and used for phylogenetic reconstruction. sequences obtained in this study have been assigned the following genbank accession numbers mg -mg . nucleic acid that was extracted from liver samples of bats ( species; table s ) and rodents (six species) was screened for pegivirus and human hepatitis b, c, e viruses and their homologues ( table ) by nested and semi-nested pcr assays with degenerate primers. hepaciviruses were the most commonly detected ( . % of rodent samples, from three species), followed by hepatitis e related virus ( % of rodent samples, from four species) while hepatitis b related viral dna was only detectable in three bats ( species). most of the hepacivirus positive samples were from farmed hoary bamboo rats in dak lak province although the predominantly sampled rat species was rattus argentiventer. coinfection with hepacivirus and hev was observed in a sample from rattus losea. all liver samples from bats were negative for hepacivirus and hepatitis e related virus and no sample was positive for pegivirus. and other rats whose liver samples were screened for hepacivirus as above. hepacivirus rna was only detected in serum samples of bamboo rats with positive liver samples. pegivirus rna was detected in two samples from rattus tanezumi. two real-time pcr assays specific for bamboo rat hepaciviruses, and other hepaciviruses were used to determine viral rna concentration in the positive samples. the concentration of rna ( figure ) was high in both liver tissue (median, . × copies/gram; range, . × - . × ) and sera (median, . × copies/ml; range, . × - × ). viruses , , x of ( figure ) was high in both liver tissue (median, . × copies/gram; range, . × - . × ) and sera (median, . × copies/ml; range, . × - × ). amplicons derived from pcr screening experiments were all successfully sequenced and complete or near complete genome sequences were determined for representatives of the viruses by pcr or deep sequencing. after exclusion of primers, the obtained screening sequences from each targeted virus clustered in one or two clades of those with high identity and were most closely related to sequences previously reported from rodents or bats from the us [ , ] , china [ ] and vietnam [ ] . rodent hepacivirus sequences ( nucleotides) formed two well supported clades (figure a) . clade included all of sequences from rhizomys pruinosus which shared . - % pairwise nucleotide identity while three sequences (nucleotide identity of - %) from rattus losea and rattus argentiventer grouped in clade . these clades differed from each other by mean distances of . % and . % at nucleotide and amino acid levels, respectively. while the four vietnamese bamboo rat hepacivirus genomes were highly similar to each other (< % nucleotide and < % amino acid distances in the complete coding region (cds)), they were remarkably different from the closest sequence (kc ) with the corresponding distances of % and %, respectively ( table ). the amino acid p-distances of the obtained hepacivirus sequences and kc in the regions - and - (numbered relative to m ) were % and %, respectively. the newly identified hepaciviruses therefore could be provisionally classified as members of a new hepacivirus species (figure b ) [ ] . the other bamboo rat hepaciviruses in clade may also belong to this new virus species on the basis of the high sequence identity in this clade. similarly, although complete genome sequences were not determined for hepaciviruses in clade , they likely belong to species hepacivirus g due to their low amino acid p-distances ( . - . %) in the screening region to kj . the ' untranslated region sequences of these hepaciviruses contain two mir- seed sites (cacucc), which were located nucleotides from each other, as consistent with the suspected hepatotropism of the viruses. in contrast to host specificity of rodent hepaciviruses, the hev sequences ( nucleotides) from four rodent species were . - . % identical to each other and phylogenetically interspersed with each other and with reference sequences from other rat species (figure a) . the obtained complete genome of rat hev comprised of nucleotides excluding the poly a tail. its concatenated orf and orf differed by . % (table ) at amino acid level to the closest match (jx ) and these two sequences therefore belong to a same genotype in the species orthohepevirus c (figure b ), according to the latest proposal for classification of hepeviruses [ ] . amplicons derived from pcr screening experiments were all successfully sequenced and complete or near complete genome sequences were determined for representatives of the viruses by pcr or deep sequencing. after exclusion of primers, the obtained screening sequences from each targeted virus clustered in one or two clades of those with high identity and were most closely related to sequences previously reported from rodents or bats from the us [ , ] , china [ ] and vietnam [ ] . rodent hepacivirus sequences ( nucleotides) formed two well supported clades (figure a) . clade included all of sequences from rhizomys pruinosus which shared . - % pairwise nucleotide identity while three sequences (nucleotide identity of - %) from rattus losea and rattus argentiventer grouped in clade . these clades differed from each other by mean distances of . % and . % at nucleotide and amino acid levels, respectively. while the four vietnamese bamboo rat hepacivirus genomes were highly similar to each other (< % nucleotide and < % amino acid distances in the complete coding region (cds)), they were remarkably different from the closest sequence (kc ) with the corresponding distances of % and %, respectively ( table ). the amino acid p-distances of the obtained hepacivirus sequences and kc in the regions - and - (numbered relative to m ) were % and %, respectively. the newly identified hepaciviruses therefore could be provisionally classified as members of a new hepacivirus species (figure b ) [ ] . the other bamboo rat hepaciviruses in clade may also belong to this new virus species on the basis of the high sequence identity in this clade. similarly, although complete genome sequences were not determined for hepaciviruses in clade , they likely belong to species hepacivirus g due to their low amino acid p-distances ( . - . %) in the screening region to kj . the ' untranslated region sequences of these hepaciviruses contain two mir- seed sites (cacucc), which were located nucleotides from each other, as consistent with the suspected hepatotropism of the viruses. in contrast to host specificity of rodent hepaciviruses, the hev sequences ( nucleotides) from four rodent species were . - . % identical to each other and phylogenetically interspersed with each other and with reference sequences from other rat species (figure a) . the obtained complete genome of rat hev comprised of nucleotides excluding the poly a tail. its concatenated orf and orf differed by . % (table ) at amino acid level to the closest match (jx ) and these two sequences therefore belong to a same genotype in the species orthohepevirus c (figure b) , according to the latest proposal for classification of hepeviruses [ ] . the three hbv variants (from bat species hipposideros pomona and hipposideros larvatus) clustered with known bat hbv sequences (figure ). the two bat hbv complete genome sequences comprised and nucleotides. as with other hepadnaviruses, the bat hbv genomes have four open reading frames (orfs) encoding the surface (s), polymerase (p), core (c), and x proteins. a detailed comparison of the orfs of these viruses with their closest sequences is shown in table . bat consistently showed greatest sequence identity to ky in all orfs. in contrast, bat shared highest identity to ky in the p and s orfs, but was more similar to ky and ky in the orfs encoding for x and c, respectively. the two vietnamese pegivirus sequences from rattus tanezumi grouped with a sequence previously reported from rattus norvegicus (figure a ). the amino acid p-distance between the obtained rodent pegivirus sequence and kj in the region - (numbered relative to u ) was . % and the two viruses could be classified as members of species pegivirus j (figure b) , according in the update to the taxonomy of the pegivirus genus [ ] . the three hbv variants (from bat species hipposideros pomona and hipposideros larvatus) clustered with known bat hbv sequences (figure ) . the two bat hbv complete genome sequences comprised and nucleotides. as with other hepadnaviruses, the bat hbv genomes have four open reading frames (orfs) encoding the surface (s), polymerase (p), core (c), and x proteins. a detailed comparison of the orfs of these viruses with their closest sequences is shown in table . bat consistently showed greatest sequence identity to ky in all orfs. in contrast, bat shared highest identity to ky in the p and s orfs, but was more similar to ky and ky in the orfs encoding for x and c, respectively. the two vietnamese pegivirus sequences from rattus tanezumi grouped with a sequence previously reported from rattus norvegicus (figure a ). the amino acid p-distance between the obtained rodent pegivirus sequence and kj in the region - (numbered relative to u ) was . % and the two viruses could be classified as members of species pegivirus j (figure b) , according in the update to the taxonomy of the pegivirus genus [ ] . the present study reports the findings of pegivirus and human hepatitis-related viruses in vietnamese rodents and bats. the detection of hepacivirus, hev homologue and pegivirus in a number of rodent species, and hbv homologue in hipposideros larvatus indicates wider host ranges of these viruses. whereas, the identified rat hev and bat hbv showed relatively high sequence identity to previously characterized viruses infecting rattus rattus, rattus tanezumi and hipposideros pomona, the rodent pegivirus and hepacivirus showed substantial sequence distances to their closest sequences and represent new pegivirus variants and a new hepacivirus species. this highlights the incomplete genetic characterization of these viruses. thus far, only one complete genome and two complete coding sequences (including the one from this study) of rodent pegivirus are available on genbank. more highly divergent hepacivirus and pegivirus sequences are expected to be discovered from rodents in future studies. the absence of bat hev, hepacivirus, pegivirus, and low detection frequency of bat hbv are consistent with their low prevalence ( - %) reported in previous studies [ , , , ] . contrastingly, the prevalence of hepacivirus in the farmed bamboo rats was unprecedentedly high ( . %). the inbred nature of farmed bamboo rats that were investigated in this study may have contributed to susceptibility to infection and likelihood of persistence [ , ] . the existence of relatively homozygous individuals may play a key role in the maintenance of pathogens in a population [ ] . the high prevalence of hepacivirus in bamboo rats also indicates the need to reconsider transmission routes of hepaciviruses. among hepaciviruses, the transmission route of hcv has been relatively well studied, while those of other hepaciviruses are mostly speculative [ , ] . as a bloodborne virus, hcv is thought to be mainly transmitted through injections or blood transfusion. however, this does not explain how a range of divergent hcv strains have been maintained for centuries in some rural populations in central africa and southeast asia [ ] . equine hepacivirus has been shown to be transmittable via direct inoculation [ ] and via vertical transmission [ ] . rodent hepacivirus may utilize a similar transmission route as experimental intravenous injection of the supernatants of homogenised liver tissues from infected rats lead to viraemia [ ] . the high prevalence of hepacivirus in bamboo rats (in this study) and commensal rattus norvegicus ( . % in firth et al. [ ] ) indicates other more efficient transmission routes may exist such as via saliva the present study reports the findings of pegivirus and human hepatitis-related viruses in vietnamese rodents and bats. the detection of hepacivirus, hev homologue and pegivirus in a number of rodent species, and hbv homologue in hipposideros larvatus indicates wider host ranges of these viruses. whereas, the identified rat hev and bat hbv showed relatively high sequence identity to previously characterized viruses infecting rattus rattus, rattus tanezumi and hipposideros pomona, the rodent pegivirus and hepacivirus showed substantial sequence distances to their closest sequences and represent new pegivirus variants and a new hepacivirus species. this highlights the incomplete genetic characterization of these viruses. thus far, only one complete genome and two complete coding sequences (including the one from this study) of rodent pegivirus are available on genbank. more highly divergent hepacivirus and pegivirus sequences are expected to be discovered from rodents in future studies. the absence of bat hev, hepacivirus, pegivirus, and low detection frequency of bat hbv are consistent with their low prevalence ( - %) reported in previous studies [ , , , ] . contrastingly, the prevalence of hepacivirus in the farmed bamboo rats was unprecedentedly high ( . %). the inbred nature of farmed bamboo rats that were investigated in this study may have contributed to susceptibility to infection and likelihood of persistence [ , ] . the existence of relatively homozygous individuals may play a key role in the maintenance of pathogens in a population [ ] . the high prevalence of hepacivirus in bamboo rats also indicates the need to reconsider transmission routes of hepaciviruses. among hepaciviruses, the transmission route of hcv has been relatively well studied, while those of other hepaciviruses are mostly speculative [ , ] . as a bloodborne virus, hcv is thought to be mainly transmitted through injections or blood transfusion. however, this does not explain how a range of divergent hcv strains have been maintained for centuries in some rural populations in central africa and southeast asia [ ] . equine hepacivirus has been shown to be transmittable via direct inoculation [ ] and via vertical transmission [ ] . rodent hepacivirus may utilize a similar transmission route as experimental intravenous injection of the supernatants of homogenised liver tissues from infected rats lead to viraemia [ ] . the high prevalence of hepacivirus in bamboo rats (in this study) and commensal rattus norvegicus ( . % in firth et al. [ ] ) indicates other more efficient transmission routes may exist such as via saliva and bites, the zoonotic potential of the detected viruses is unknown and requires further investigation. while the identified rodent hepaciviruses appear host species specific, four rodent species were infected with highly similar hev homologues, which were phylogenetically interspersed, indicative of low host species specificity. this is a characteristic that may lead to their establishment and emergence in new hosts. understanding the receptor usage for cell entry of hev in rodents and other host species would potentially help predict the host range of the virus. furthermore, a surrogate assay with pseudotyped viruses carrying surface/envelope proteins of the identified viruses may be useful in assessing their potential to infect human liver cells. such an assay was used to show that a bat hbv could infect primary human hepatocytes [ ] . in summary, this study demonstrated the wide circulation of diverse pegivirus and human hepatitis-related viruses in new rodent and bat species. the presented findings form a framework for future investigations of human transmission risk, now that the rodent and bat species infected with these viruses have been identified and the human contact groups are better defined (e.g., bamboo rat farmers, rat catchers, rat sellers, and bat farmers). the transmission routes of the identified viruses are to be determined. the following are available online at http://www.mdpi.com/ - / / / / s . the global burden of viral hepatitis from to : findings from the global burden of disease study the global burden of hepatitis e virus genotypes and in hepatitis b: the virus and disease genetic organization and diversity of the hepatitis c virus hepatitis e virus: molecular virology, clinical features, diagnosis, transmission, epidemiology, and prevention bats carry pathogenic hepadnaviruses antigenically related to hepatitis b virus and capable of infecting human hepatocytes detection and genome characterization of four novel bat hepadnaviruses and a hepevirus in china hepatitis virus in long-fingered bats hepatitis e virus in rats hepatitis e virus genotype in wild rats, united states bats worldwide carry hepatitis e virus-related viruses that form a putative novel genus within the family hepeviridae complete genome sequence of a rat hepatitis e virus strain isolated in the united states hepatitis e virus in norway rats (rattus norvegicus) captured around a pig farm novel hepatitis e virus genotype in norway rats detection of a novel hepatitis e-like virus in faeces of wild rats using a nested broad-spectrum rt-pcr characterization of full genome of rat hepatitis e virus strain from vietnam identification of rodent homologs of hepatitis c virus and pegiviruses detection of zoonotic pathogens and characterization of novel viruses carried by commensal rattus norvegicus bats are a major natural reservoir for hepaciviruses and pegiviruses evidence for novel hepaciviruses in rodents rodents and risk in the mekong delta of vietnam: seroprevalence of selected zoonotic viruses in rodents and humans. vector-borne zoonotic dis market study of meat from field rats in the mekong delta aciar monograph rodent-borne diseases and their risks for public health detection of potentially novel paramyxovirus and coronavirus viral rna in bats and rats in the mekong delta region of southern viet nam the vietnam initiative on zoonotic infections (vizions): a strategic approach to studying emerging zoonotic infectious diseases bartonella species and trombiculid mites of rats from the mekong delta of vietnam. vector borne zoonotic dis how important are rats as vectors of leptospirosis in the mekong delta of vietnam? vector borne zoonotic dis a guide to the mammals of southeast asia large-scale screening and characterization of enteroviruses and kobuviruses infecting pigs in vietnam sse: a nucleotide and amino acid sequence analysis platform proposed update to the taxonomy of the genera hepacivirus and pegivirus within the flaviviridae family mega : molecular evolutionary genetics analysis version . for bigger datasets consensus proposals for classification of the family hepeviridae parasite-mediated selection against inbred soay sheep in a free-living, island population disease susceptibility in california sea lions consanguinity and susceptibility to infectious diseases in humans hepacivirus cross-species transmission and the origins of the hepatitis c virus viraemic frequencies and seroprevalence of non-primate hepacivirus and equine pegiviruses in horses and other mammalian species the virus whose family expanded experimental transmission of equine hepacivirus in horses as a model for hepatitis c virus vertical transmission of hepatitis c virus-like non-primate hepacivirus in horses mouse models of acute and chronic hepacivirus infection key: cord- -hpo bboi authors: wasilenko, shawn t.; montano-loza, aldo j.; mason, andrew l. title: is there a role for cyclophilin inhibitors in the management of primary biliary cirrhosis? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: hpo bboi autoimmune hepatitis (aih) and primary biliary cirrhosis (pbc) are poorly understood autoimmune liver diseases. immunosuppression is used to treat aih and ursodeoxycholic acid is used to slow the progression of pbc. nevertheless, a proportion of patients with both disorders progress to liver failure. following liver transplantation, up to a third of patients with pbc experience recurrent disease. moreover a syndrome referred to as “de novo aih” occurs in a proportion of patients regardless of maintenance immunosuppression, who have been transplanted for disorders unrelated to aih. of note, the use of cyclosporine a appears to protect against the development of recurrent pbc and de novo aih even though it is a less potent immunosuppressive compared to tacrolimus. the reason why cyclosporine a is protective has not been determined. however, a virus resembling mouse mammary tumor virus (mmtv) has been characterized in patients with pbc and aih. accordingly, we hypothesized that the protective effect of cyclosporine a in liver transplant recipients may be mediated by the antiviral activity of this cyclophilin inhibitor. treatment of the mmtv producing mm mt cells with different antivirals and immunosuppressive agents showed that both cyclosporine a and the analogue nim inhibited mmtv production from the producer cells. herein, we discuss the evidence supporting the role of mmtv-like human betaretrovirus in the development of pbc and de novo aih and speculate on the possibility that the agent may be associated with disease following transplantation. we also review the mechanisms of how both cyclosporine a and nim may inhibit betaretrovirus production in vitro. compared to tacrolimus was associated with eight-fold reduction in the risk of pbc recurrence, in agreement with other liver transplant centers [ , , ] . the -year probability of recurrence was % and %, respectively (p = . , log-rank test). the -year probability of recurrence was % and % in these same groups, but fewer patients were followed. (with permission of john wiley and american journal of transplantation). the immunosuppression regimens used in the s probably contributed to an era effect, when pbc patients undergoing liver transplantation experienced a lower incidence of disease recurrence. others liver transplant centers have reported a similar protective era effect that has been linked with several factors including specific immunosuppression regimens, the use of younger donors and decreased cold ischemic time [ , ] . nevertheless, a major conclusion of most series documenting outcomes in patients with pbc following liver transplantation is that csa is associated with a lower incidence of recurrent disease [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] . also, of interest, the primary use of csa has been shown to confer a protective effect against other autoimmune diseases, such as de novo aih in liver transplant recipients in general [ ] as well as a decreased risk of recurrent or de novo inflammatory bowel disease [ ] after liver transplantation for primary sclerosing cholangitis (psc). aih is a heterogeneous disorder observed in pediatric and adult populations with a variable presentation and prognosis. the diagnosis is usually established by an exclusion of other causes of liver disease, liver histology and the presence of a variety of autoantibodies. also included in the spectrum of aih are the poorly understood overlap syndromes with both pbc and psc; however, the overlap syndromes are considered contentious because both pbc and psc are exclusion criteria for making a diagnosis of aih [ , , ] . de novo aih has been recognized for over a decade as a condition that affects patients transplanted for hepatic disorders other than aih [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . comparable to classical aih, the diagnosis of de novo aih is essentially based on the presence of autoantibodies, distinctive histological findings and the exclusion of other conditions, such as viral hepatitis, acute or chronic rejection and immune mediated biliary disease [ ] . similar to the diagnosis of aih in the general population, it has to be acknowledged that the diagnosis of de novo aih following liver transplantation is not clear-cut. while we lack evidence-based diagnostic criteria to distinguish the differing entities, there are strong similarities between de novo aih, the recurrence of aih in liver transplant recipients, and the classical aih in the non-transplant setting. in a study based at our center we found that the probability of de novo aih was approximately % at years with an overall incidence of cases per patient-years. it is notable that the frequency of de novo aih may be higher than the prevalence of aih in the general population, probably because transplant patients are exposed to more potential risk factors [ ] . with regard to immunosuppression usage, liver transplant recipients maintained on csa had a -fold lower risk of de novo aih, whereas those receiving tacrolimus or mycophenolate mofetil had a -and -fold higher risk of de novo aih, respectively ( figure ) [ ] . intriguingly, we found that patients who had donors aged years or older or female donors had a -fold and -fold higher risk of developing de novo aih, respectively. moreover, female recipients with gender mismatch were protected against de novo aih, reducing the risk by -fold [ ] . in other words, having a younger male donor and primary use of csa is protective against the development of de novo aih. similar to patients with pbc, the immunosuppression regimens used in the s probably contributed to the cohort effect observed in this study. patients undergoing liver transplantation in this period experienced a -fold lower risk of de novo aih compared with patients transplanted in the decade following . it can be argued that the protective effect of csa could also be attributable to the concomitant use of steroids, as previous studies have shown a role for steroid use in preventing development of de novo aih [ ] . the -and -year probability of development of de novo autoimmune hepatitis with cyclosporine a was and . % respectively; the -and -year probability of development with tacrolimus was . % and . % respectively; and the -and -year probability of development with mycophenolate mofetil was . and . % respectively. (with permission of john wiley and sons and liver international). while the genetic and environmental factors that trigger pbc are poorly understood, one could argue that the same mechanisms likely mediate recurrent disease in the allograft. there is a strong heritable component to disease that is difficult to quantify. the risk of developing pbc in family members ranges from % to % and pbc is more prevalent in monozygotic as compared to dizygotic twins [ , ] . several case control and genome wide association studies have linked pbc with the hla dr and dq class ii region, the il- /interferon  cytokine axis and other genes associated with host pathogen interaction [ ] [ ] [ ] [ ] [ ] [ ] [ ] . collectively, the susceptibility data suggest a hypothesis that patients with pbc have a global disturbance in sensing and response to environmental agents [ ] . it is tempting to speculate, therefore, that the penetrance of recurrent disease maybe related in part to the acquisition of protective alleles in the new allograft that prevents establishment of an infectious process but data are lacking to support this conjecture. the recurrence of pbc following transplantation strongly suggests an infectious process and epidemiological data are suggestive of a transmissible etiology as well. spouses, other unrelated family members, and even care providers have been reported to develop pbc suggesting a role for environmental factors in disease [ , ] . also, pbc has been reported to cluster in specific geographical regions [ , ] and migration studies show that the children develop the relative incidence of their adopted host country, whereas their parents do not [ , ] . with regard to environmental agents, xenobiotics, specific bacteria and a betaretrovirus have been linked with the development of pbc, but none has been confirmed to cause the disease to date [ ] . our group first characterized a human betaretrovirus resembling the mouse mammary tumor virus (mmtv) in patients with pbc nearly ten years ago [ , ] . the initial finding was important because the betaretrovirus was shown to trigger the mitochondrial phenotype of pbc in vitro and was found in cells displaying the mitochondrial phenotype in vivo [ ] . while % of patients with pbc had evidence of the hbrv in their peri-hepatic lymph nodes, the virus was difficult to detect in the liver and questions were raised concerning the validity of the original findings [ ] . accordingly, studies are ongoing to demonstrate proviral integrations in patients with liver disease to provide more substantial evidence that hbrv is a potential human pathogen [ ] . it is understood that multiple layers of proof will be required to link hbrv with the pathogenesis of pbc and there are several inter-related ongoing studies that aim to address this issue. on and above demonstrating proviral integration in the bile ducts of patients with pbc, an elisa is being constructed to determine the seroprevalence of hbrv infection in a large cohort of patients with liver disease. also, mouse models are being used to demonstrate how infection with the closely related mmtv triggers autoimmune biliary disease [ ] . these animal models are being used to validate combination antiviral treatments to inhibit betaretrovirus infection and ongoing clinical trials are investigating whether anti-retroviral therapy can improve histological, biochemical and clinical endpoints in patients with pbc [ , , ] . accordingly, we are working on the model that a betaretrovirus may trigger viral cholangitis in susceptible individuals with specific hla dr and dq class ii alleles and genetic polymorphisms associated with the il- cytokine axis. a similar model could be used to propose that factors that trigger the original disease also persist in the allograft. while more than two thirds of patients develop both ama and the mitochondrial phenotype in bile ducts, only one third of patients develop biochemical and histological disease recurrence years following transplantation. these data suggest that approximately one third of the patients with pbc who receive a liver transplant are protected against recurrent disease. the evidence supports the hypothesis that the environmental trigger(s) may persist in about two thirds of patients, whereas only a third develop progressive disease. whether the factors modulating disease recurrence are protective polymorphisms such as those involving the hla or il- alleles in the allograft or the use of younger donors with shorter cold ischemic times remains to be resolved. however, a major factor that has reproducibly been shown to be protective at this juncture is the primary use of csa. little is known about the etiology and pathogenesis of aih in the non-transplant setting and the development of de novo aih remains a total mystery. indeed, three diverse processes could be involved with the pathogenesis of de novo aih including autoimmunity, a forme fruste of allograft rejection and the possibility of a viral infection. in fact, it has been suggested that aih may represent serologically unidentified viral infection(s) [ ] and we have found evidence of hbrv infection in patients with aih. in one case with acute onset aih, the viral load was highest with acute presentation and diminished by corticosteroid treatment [ ] . this observation lead to the proposal of a model that the use of corticosteroids may have the dual purpose of limiting viral spread and diminishing inflammation [ ] . indeed the related agent, mmtv, has an obligate prerequisite for replicating in dividing lymphocytes. in the setting of liver transplantation, this model would also be consistent with the antiviral and anti-lymphocytic activity of csa in protecting against the development of de novo aih. the observation that the more potent immunosuppressant, tacrolimus is associated with earlier and more severe recurrence of pbc suggests two potentially compatible hypotheses. the first is that tacrolimus inhibits the immune system to a greater degree permitting the emergence of an infectious agent. the second is that csa may be acting in part as an antiviral in patients with pbc following liver transplantation. it has to be acknowledged, however, that data demonstrating recurrent hbrv in the allograft are lacking to support these hypotheses. nevertheless, we have found that csa and the analogue nim can inhibit betaretrovirus production suggesting a critical role for cyclophilins in the replication and infectivity of betaretroviruses [ ] . for our in vitro studies, we used the mm mt mouse breast cancer cells containing integrated mmtv provirus for testing the sensitivity of antiviral and immunosuppressive agents to diminish mmtv production. by design, this model cannot be used to test early events in the retrovirus life cycle, such as viral internalization, uncoating, disassembly, reverse transcription, nuclear import of the preintegration complex and proviral integration ( figure ) . nevertheless, the downstream production of infectious particles from integrated provirus can be studied. in these in vitro studies, we observed a reduction in reverse transcriptase activity and viral genome levels in supernatants from mm mt cells incubated with the cyclophilin inhibitors, csa and nim , whereas tacrolimus and reverse transcriptase inhibitors had significantly less effect [ ] . steady state viral protein levels were unaffected by the presence of csa or nim , however. we observed change in protease processing of mmtv gag proteins with either csa or nim , whereas the combination hiv protease inhibitor, lopinavir and ritonavir (kaletra tm ) partially inhibited the production of the mmtv p capsid protein [ ] . it is clear however that the likely block in viral replication is independent of the immunosuppressive nature of csa as incubation with nim resulted in similar levels of reverse transcriptase and genome equivalents. at this point however, the mechanism(s) of how either csa or nim block viral production has yet to be resolved. the early events of retroviral infection include (i) internalization following receptor engagement, (ii) disassembly and (iii) uncoating during reverse transcription, (iv) nuclear import of the preintegration complex and proviral integration. in the mmtv producing mm mt cells, antiviral blockade with cyclophilins can impact on the more downstream events such as (v) rna transcription, (vi) pre-translational and post-translational protein processing as well as (vii) viral budding and maturation. reviewing the betaretroviral life cycle helps us to understand the critical steps that csa and nim may interfere with viral replication (figure ). as discussed, the use of mm mt cells with a stably integrated mmtv provirus prevented the study the early events of infection. nevertheless, cyclophilin antagonists may function to prevent internalization of virions as observed with several agents including human papilloma virus (hpv), measles virus and hiv. for example, both csa and nim counteract cyclophilin b enabling the necessary conformational changes of the hpv minor capsid protein required for viral internalization [ , ] . it is also known that both cyclophilin a and cyclophilin b assist the cellular entry of hiv and measles virus, respectively, through a critical interaction with cd , a transmembrane glycoprotein belonging to the immunoglobulin superfamily [ , ] . however, no one has demonstrated to date that csa or its analogues specifically function by blocking cd mediated viral entry. cyclophilins also play an important role in viral uncoating and disassembly. for hpv this process occurs in endosomal compartments with the assistance of cyclophilin b, for example, and this activity inhibited by csa [ ] . another example of cyclophilin mediated viral replication includes both the disassembly of mature cores and the nuclear import of the hiv preintegration complex (figure iii ). cyclophilins play a critical role in assisting the interaction of the viral capsid protein with the major cytoplasmic component of the nuclear pore complex, nup necessary for uncoating the capsid and shuttling of the preintegration complex into the nucleus [ ] [ ] [ ] . in this case, inhibition of the cyclophilin a interaction with hiv capsid by csa impairs the passage of the mature viral core to the nuclear pore complex. following proviral integration, cyclophilins have been shown to play a role in several processes in the viral life cycle comprising viral rna and protein production, assembly and maturation (figure ). for example, csa has been shown to block the b activation of the hiv enhancer region to inhibit viral rna transcription [ ] . other agents, such as hepatitis c virus (hcv) rely on cyclophilin a interactions with viral components of the replication complex to produce rna transcripts, a process also blocked by csa [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in a similar vein, csa blocks coronavirus replication via an unknown mechanism at a step prior to rna and protein synthesis [ ] . while there is significant anecdotal evidence to favor the inhibition of rna transcription, the presence of csa and tacrolimus can potentiate, rather than block, transcriptional activity of the mmtv promoter [ ] . however, this process is modulated by dexamethasone activation of the glucocorticoid responsive elements in the long terminal repeat. it is also possible cyclophilin inhibitors negatively regulate viral protein steady state levels at any one of the many steps involved in viral protein production and turnover. for example, csa blocks nuclear export of influenza rna transcripts [ ] [ ] [ ] . furthermore, processing of the hcv polyprotein into functional components requires the support of cyclophilin a interacting with ns that can be blocked by csa and debio- [ ] [ ] [ ] . the presence of csa also accelerates proteolytic degradation of the influenza matrix protein m by augmenting its interaction with cyclophilin a. in contrast, cyclophilin inhibitors trigger loss of the hiv vpr protein-a known partner of cyclophilin a-independent of proteosome activity [ ] . as discussed for our in vitro experiments with mmtv producing cells, it is unlikely that the cyclophilin inhibitors impact on any of the steps from viral entry to viral transcription and protein production given that csa and nim treatment did not impact on mmtv rna or protein levels in cells [ , ] . these data suggest that viral transcription and protein production likely proceeds appropriately in producer cells. therefore, additional studies should be directed towards investigating viral assembly, maturation and the infective stages of the viral life cycle as numerous viruses utilize cyclophilins during target cell infection. specifically, we know that hiv incorporates cyclophilins into budding virions and interference of this process by csa or nim reduces infectivity [ , , ] . these data suggest a model that virion incorporated cyclophilin, or target cell cyclophilin may be necessary for effective internalization, uncoating and/or nuclear import of the mmtv genome. at present, patients with pbc have limited options for therapeutic intervention. clearly, better treatments are required for the ~ % of patients that progress to liver failure and to treat those with recurrent disease following liver transplantation. while anti-retroviral therapy for pbc has shown promise with improvements in hepatic biochemistry and histology [ , , , ] , the specific drugs employed in clinical trials to date have been manufactured for treating hiv rather than hbrv. our preliminary data support the hypothesis that csa and other cyclophilin inhibitors may inhibit betaretrovirus activity during maturation or infective stages of target cells. as such, further characterization of the role of cyclophilins in the betaretrovirus life cycle would be beneficial both in vitro and in mouse models of pbc with mmtv infection. [ ] such studies are encouraged as they would serve the dual purpose of characterizing the mechanism of cyclophilin inhibitors in blocking hbrv production and for identifying novel management strategies for patients with pbc. primary biliary cirrhosis: a update effects of ursodeoxycholic acid on survival in patients with primary biliary cirrhosis controlled trial of ursodiol for the treatment of primary biliary cirrhosis. udca-pbc study group liver transplantation for primary biliary cirrhosis autoimmune liver disease. current standards, future directions other potential medical therapies: the use of antiviral agents to investigate and treat primary ciliary cirrhosis cyclosporin a treatment in primary biliary cirrhosis: results of a long-term placebo controlled trial hepatitis and cholestasis in a middle-aged woman recurrent primary biliary cirrhosis recurrence of primary biliary cirrhosis after liver transplantation long-term survival and impact of ursodeoxycholic acid treatment for recurrent primary biliary cirrhosis after liver transplantation recurrent primary biliary cirrhosis: peritransplant factors and ursodeoxycholic acid treatment post-liver transplant cyclosporine a protects against primary biliary cirrhosis recurrence after liver transplantation long-term follow-up after recurrence of primary biliary cirrhosis after liver transplantation in patients the effect of immunosuppressive regimens on the recurrence of primary biliary cirrhosis after liver transplantation transplantation for primary biliary cirrhosis: retrospective analysis of patients in a single center immunosuppression affects the rate of recurrent primary biliary cirrhosis after liver transplantation the changing clinical presentation of recurrent primary biliary cirrhosis after liver transplantation cyclosporine a inhibits in vitro replication of betaretrovirus associated with primary biliary cirrhosis incidence and risk factors associated with de novo autoimmune hepatitis after liver transplantation inflammatory bowel disease after liver transplantation: risk factors for recurrence and de novo disease de-novo autoimmune hepatitis after liver transplantation current topics in autoimmune hepatitis de novo autoimmune hepatitis following liver transplantation for primary biliary cirrhosis successful treatment of de novo autoimmune hepatitis and cirrhosis after pediatric liver transplantation posttransplant immune hepatitis in pediatric liver transplant recipients: incidence and maintenance therapy with azathioprine de novo hepatitis with autoimmune antibodies and atypical histology: a rare cause of late graft dysfunction after pediatric liver transplantation late cellular rejection in paediatric liver transplantation: aetiology and outcome late graft dysfunction and autoantibodies after liver transplantation in children: preliminary results of an italian experience antibodies against cytokeratin / in a patient with de novo autoimmune hepatitis after living-donor liver transplantation response to steroids in de novo autoimmune hepatitis after liver transplantation de novo autoimmune hepatitis following liver transplantation: a case report diagnosis and management of autoimmune hepatitis primary biliary cirrhosis in monozygotic and dizygotic twins: genetics, epigenetics, and environment. gastroenterology genetics and geoepidemiology of primary biliary cirrhosis: following the footprints to disease etiology hla and interleukin gene polymorphisms in primary biliary cirrhosis: associations with disease progression and disease susceptibility the genetics of complex cholestatic disorders variants at irf -tnpo , q - and mmel are associated with primary biliary cirrhosis primary biliary cirrhosis associated with hla, il a, and il rb variants association of primary biliary cirrhosis with variants in the clec a, socs , spib and siae immunomodulatory genes genome-wide meta-analyses identify three loci associated with primary biliary cirrhosis hla drw and complement c deficiency as risk factors in primary biliary cirrhosis primary biliary cirrhosis: definition and epidemiological features primary biliary cirrhosis: an epidemiological study the epidemiology of primary biliary cirrhosis the geoepidemiology of primary biliary cirrhosis linking human betaretrovirus infection with primary biliary cirrhosis cloning the human betaretrovirus proviral genome from patients with primary biliary cirrhosis does a betaretrovirus infection trigger primary biliary cirrhosis? lack of immunological or molecular evidence for a role of mouse mammary tumor retrovirus in primary biliary cirrhosis mouse mammary tumor virus in anti-mitochondrial antibody producing mouse models pilot studies of single and combination antiretroviral therapy in patients with primary biliary cirrhosis clinical trial: randomized controlled trial of zidovudine and lamivudine for patients with primary biliary cirrhosis stabilized on ursodiol reverse transcriptase activity in patients with primary biliary cirrhosis and other autoimmune liver disorders target cell cyclophilins facilitate human papillomavirus type infection cyclophilins facilitate dissociation of the human papillomavirus type capsid protein l from the l /dna complex following virus entry cd /emmprin acts as a functional entry receptor for measles virus on epithelial cells cd facilitates hiv- infection by interacting with virus-associated cyclophilin a cyclophilin interactions with incoming human immunodeficiency virus type capsids with opposing effects on infectivity in human cells target cell cyclophilin a modulates human immunodeficiency virus type infectivity cyclophilin a is required for the replication of group m human immunodeficiency virus type (hiv- ) and simian immunodeficiency virus siv(cpz)gab but not group o hiv- or other primate immunodeficiency viruses inducible nuclear factor binding to the kappa b elements of the human immunodeficiency virus enhancer in t cells can be blocked by cyclosporin a in a signal-dependent manner cyclophilin a is an essential cofactor for hepatitis c virus infection and the principal mediator of cyclosporine resistance in vitro a major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis c virus identified by a genetic approach cyclophilin b is a functional regulator of hepatitis c virus rna polymerase cyclophilin b stimulates rna synthesis by the hcv rna dependent rna polymerase cyclosporine inhibits a direct interaction between cyclophilins and hepatitis c ns a cyclophilin a interacts with domain ii of hepatitis c virus ns a and stimulates rna binding in an isomerase-dependent manner mechanism of resistance of hepatitis c virus replicons to structurally distinct cyclophilin inhibitors cyclosporine inhibits flavivirus replication through blocking the interaction between host cyclophilins and viral ns protein cyclosporin a inhibits the replication of diverse coronaviruses cyclosporin a potentiates the dexamethasone-induced mouse mammary tumor virus-chloramphenicol acetyltransferase activity in lmcat cells: a possible role for different heat shock protein-binding immunophilins in glucocorticosteroid receptor-mediated gene expression in vivo effects of cyclosporine on influenza a virus-infected mice the hepatitis c virus ns / protease cyclosporin a inhibits the influenza virus replication through cyclophilin a-dependent and -independent pathways construction and characterization of infectious intragenotypic and intergenotypic hepatitis c virus chimeras essential role of cyclophilin a for hepatitis c virus replication and virus production and possible link to polyprotein cleavage kinetics cyclosporine a inhibits hepatitis c virus nonstructural protein through cyclophilin a cyclophilin a interacts with hiv- vpr and is required for its functional expression specific incorporation of cyclophilin a into hiv- virions functional association of cyclophilin a with hiv- virions killing two birds with one stone key: cord- -dc t vh authors: todt, daniel; walter, stephanie; brown, richard j. p.; steinmann, eike title: mutagenic effects of ribavirin on hepatitis e virus—viral extinction versus selection of fitness-enhancing mutations date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: dc t vh hepatitis e virus (hev), an important agent of viral hepatitis worldwide, can cause severe courses of infection in pregnant women and immunosuppressed patients. to date, hev infections can only be treated with ribavirin (rbv). major drawbacks of this therapy are that rbv is not approved for administration to pregnant women and that the virus can acquire mutations, which render the intra-host population less sensitive or even resistant to rbv. one of the proposed modes of action of rbv is a direct mutagenic effect on viral genomes, inducing mismatches and subsequent nucleotide substitutions. these transition events can drive the already error-prone viral replication beyond an error threshold, causing viral population extinction. in contrast, the expanded heterogeneous viral population can facilitate selection of mutant viruses with enhanced replication fitness. emergence of these mutant viruses can lead to therapeutic failure. consequently, the onset of rbv treatment in chronically hev-infected individuals can result in two divergent outcomes: viral extinction versus selection of fitness-enhanced viruses. following an overview of rna viruses treated with rbv in clinics and a summary of the different antiviral modes of action of this drug, we focus on the mutagenic effect of rbv on hev intrahost populations, and how hev is able to overcome lethal mutagenesis. hepatitis e virus (hev) was first described as novel agent responsible for enterically transmitted non-a, non-b hepatitis by reyes and colleagues in [ ] . this was years after the first documented epidemic outbreak ( ) ( ) ) of a retrospectively identified hev infection-transmitted via the fecal-oral route-in new delhi, india [ ] . hev is a nonenveloped single-stranded rna virus with a . kb genome of positive orientation. three open reading frames (orfs) encode for: ( ) the nonstructural proteins (orf ), comprising a methyltransferase, a papain-like cysteine protease, a helicase, and an rna-dependent rna polymerase (rdrp), connected by a y-domain and a hypervariable region (hvr); ( ) the capsid protein (orf ); and ( ) small proteins whose functions are not yet completely understood (orf ) [ ] . the viral subgenomic rna is comparable to mammalian mrnas, flanked by a -methylguanine cap and a -poly(a) tail. hev has recently been taxonomically reassigned to the genus orthohepevirus in the family of hepeviridae [ ] . differences in the sequences of isolates led to the current classification into seven genotypes, four of which infect humans. hev- and hev- (i.e., genotypes and ) are solely human pathogens and are mainly transmitted orally by feces-contaminated drinking water. in , rbv was described as a broad-spectrum antiviral against several dna and rna viruses [ ] . since then, numerous studies have reported on the in vitro antiviral properties of rbv. figure provides an overview of a selection of rna viruses against which rbv was shown to be active: hepatitis c virus (hcv, flaviviridae), dengue virus (denv, flaviviridae), respiratory syncytial virus (rsv, paramyxoviridae), influenza a and b virus (orthomyxoviridae), chikungunya virus (chikv, togaviridae), poliovirus (picornaviridae), hantaan virus (bunyaviridae), and lassa virus (arenaviridae) [ , ] ( figure ). for further reading we would like to refer to other reviews like [ ] [ ] [ ] . studying multiple viruses from the family flaviviridae, crance et al. investigated the in vitro antiviral properties of rbv against flaviviruses including denv, japanese encephalitis virus (jev), and yellow fever virus (yfv). inhibition of virus replication was observed for all tested flaviviruses [ ] . furthermore, effectiveness of rbv could be confirmed in vivo for yfv using a hamster model by administering early upon infection [ , ] . however, these effects could not be confirmed in a nonhuman primate model [ ] . therefore, further studies are required to evaluate the possible application of rbv as a treatment option for yfv. here, the dosage as well as the time points of treatment represent the major hurdles, which need to be overcome [ ] . additionally, hemorrhagic fever-causing viruses, which are categorized into the families of arena-, bunya-, and togaviridae, were demonstrated to be susceptible to inhibition by rbv ( figure ). for example, for lassa virus the antiviral efficiency of rbv was proven both in vitro and in vivo in guinea pigs and monkeys [ , ] . hantaviruses (i.e., hantaan virus) and phleboviruses (i.e., rift valley fever virus, rvf) were also shown to be susceptible to rbv treatment [ ] . in a mouse model for hantaan virus, an increase of survival and milder signs of disease were described [ ] . in experimental rvf infections of mice and hamsters, rbv led to a prevention of death, delay of death, or the onset of milder symptoms, depending on the time point of administration [ ] . in general, higher doses of rbv were needed to inhibit flaviviruses compared to arena-, bunya-, and hantaviruses [ ] . for chikv, rbv also demonstrated antiviral effects, although to a lesser extent when compared to interferon-α (ifn-α). nevertheless, a synergistic effect of rbv and ifn-α could be demonstrated in vitro [ , ] . moreover, the antiviral properties of rbv could be shown for members of the picornaviridae: both foot-and-mouth disease virus (fmdv) [ , ] and poliovirus (pv) [ ] were inhibited by rbv (figure ). demonstrated antiviral effects, although to a lesser extent when compared to interferon-α (ifn-α). nevertheless, a synergistic effect of rbv and ifn-α could be demonstrated in vitro [ , ] . moreover, the antiviral properties of rbv could be shown for members of the picornaviridae: both foot-and-mouth disease virus (fmdv) [ , ] and poliovirus (pv) [ ] were inhibited by rbv ( figure ). figure . antiviral properties of ribavirin (rbv) against rna viruses. the broad-spectrum antiviral properties of rbv have been reported for several rna viruses. depicted is a selection of the different viral families and the respective genus and species. viruses for which rbv was clinically approved are highlighted with an orange box. viruses for which lethal mutagenesis or increased mutation rate was proposed as a possible rbv mechanism are indicated in blue. while displaying broad antiviral activity against a wide range of rna viruses, clinical data on the application of rbv are still limited and restricted to only a few viruses. initially, rbv was considered as a treatment option for influenza a and b virus infections. however, clinical trials showed inconclusive data; although some studies reported an improvement of symptoms of influenza virus infection, results were generally inconsistent [ , ] . due to lack of conclusive data from clinical trials, coupled with the development of alternative antiviral therapies, rbv has never been approved for the treatment of influenza virus. nonetheless, lassa virus, hcv, and rsv are prominent examples of viruses for which rbv has received approval as an antiviral compound for clinical application [ ] . rbv was shown to be effective in treating patients suffering from lassa fever [ ] and can be administered orally, intravenously, or as pre-or post-exposure prophylaxis [ ] ., in , rbv was approved by the food and drug administration (fda) as a treatment option for hcv [ ] and was, in combination with pegylated ifn-α, the standard treatment for chronic hcv infection for over two decades [ ] . it has been shown that after failure of a monotherapy with ifn-α alone, a combination therapy with rbv is more effective than subsequent repetition of ifn-α monotherapy [ ] . however, the sustained virological response (svr) rates varied among genotypes, and dual therapy was associated with severe side effects [ ] . nowadays, rbv is no longer the standard-ofcare anti-hcv therapy, and has been replaced by direct-acting antivirals (daas). initial trials for the treatment of rsv infection showed a reduced duration of hospitalization and requirement of mechanical ventilation [ , ] . a routine use of rbv in rsv-infected children is not recommended; however, treatment can be considered for individual cases [ ] . while displaying broad antiviral activity against a wide range of rna viruses, clinical data on the application of rbv are still limited and restricted to only a few viruses. initially, rbv was considered as a treatment option for influenza a and b virus infections. however, clinical trials showed inconclusive data; although some studies reported an improvement of symptoms of influenza virus infection, results were generally inconsistent [ , ] . due to lack of conclusive data from clinical trials, coupled with the development of alternative antiviral therapies, rbv has never been approved for the treatment of influenza virus. nonetheless, lassa virus, hcv, and rsv are prominent examples of viruses for which rbv has received approval as an antiviral compound for clinical application [ ] . rbv was shown to be effective in treating patients suffering from lassa fever [ ] and can be administered orally, intravenously, or as pre-or post-exposure prophylaxis [ ] . in , rbv was approved by the food and drug administration (fda) as a treatment option for hcv [ ] and was, in combination with pegylated ifn-α, the standard treatment for chronic hcv infection for over two decades [ ] . it has been shown that after failure of a monotherapy with ifn-α alone, a combination therapy with rbv is more effective than subsequent repetition of ifn-α monotherapy [ ] . however, the sustained virological response (svr) rates varied among genotypes, and dual therapy was associated with severe side effects [ ] . nowadays, rbv is no longer the standard-of-care anti-hcv therapy, and has been replaced by direct-acting antivirals (daas). initial trials for the treatment of rsv infection showed a reduced duration of hospitalization and requirement of mechanical ventilation [ , ] . a routine use of rbv in rsv-infected children is not recommended; however, treatment can be considered for individual cases [ ] . taken together, since its first description as an antiviral in , rbv has been shown to be active against a broad range of rna viruses. however, due to limited clinical trial data supporting its in vivo efficacy, clinical applications are currently limited to a minority of viruses. the broad antiviral effect of rbv against numerous rna viruses suggests different modes of action for the molecule; indeed, several antiviral mechanisms have been described in the past [ , ] and are summarized in figure a . among the indirect mechanisms, a t-cell-mediated effect was described for hcv ( figure a) . here, the balance of t helper cells was changed by switching from a t helper type phenotype to a t helper type [ ] . in a study by hultgren et al., an inhibition of in vitro t-cell proliferation as well as a change in secreted cytokines was observed [ ] . simultaneously, alanine transaminase (alt) levels in serum were reduced with no change in hcv titers [ ] . furthermore, an early switch of a t helper type immune response to a t helper type immune response was associated with disease progression and the development of chronicity [ ] . thus, rbv restored the t helper phenotype needed for balanced expression and secretion of cytokines produced from type and t helper cells [ ] . another example where an immunomodulatory effect was described for rbv is in rsv infection. it was proposed that a t helper type cytokine response initiated the cascade leading to airway hyper-reactivity, which in turn can be blocked by rbv treatment [ ] (figure a) . another indirect mode of action for rbv is the inhibition of the cellular inosine monophosphate dehydrogenase (impdh), which was already proposed in [ ] (figure a ). after uptake into the cell, rbv is phosphorylated to rbv mono-, di-, and triphosphate (rmp, rdp, and rtp, respectively). rmp represents a good mimic of inosine monophosphate (imp) and thereby inhibits the synthesis of imp to xanthosine monophosphate (xmp) by impdh. consequently, no guanosine monophosphate (gmp), and subsequently guanosine triphosphate (gtp), can be synthesized. in vitro, replication of measles virus in vero cells could be blocked by the addition of xmp, gmp, and to a lesser extent, also imp [ ] , which underlines the mode of action of rmp. a linear correlation of the depletion of gtp pools and in vitro antiviral activity of rbv against human parainfluenza virus and yfv was confirmed [ ] . furthermore, the addition of guanosine to cell cultures restored the antiviral activity of rbv against gb virus b (gbv-b) [ ] . in contrast, in vitro experiments with lassa virus and hantaan virus indicated that rbv did not primarily act via depletion of gtp pools for these two viruses [ , ] . moreover, experiments with influenza a virus showed no linear correlation of intracellular gtp pools and viral replication with increasing concentrations of rbv [ ] . additionally, the authors did not observe a complete restoration of influenza a virus replication after addition of guanosine [ ] . no effect of guanosine or gmp on the antiviral effect of rbv against influenza a virus in mice could be demonstrated [ ] . overall, these data suggest that other mechanisms for the mode of action of rbv exist. the influence of rbv on the expression of ifn-stimulated genes (isg) is controversial in the literature. most studies, both in vivo and in vitro, come from the hcv and rsv fields. rbv is able to increase the antiviral effects of an ifn-based therapy and restore ifn-responsiveness in hcv-infected livers [ ] [ ] [ ] [ ] . also, a direct, ifn-independent upregulation of isgs has been proposed [ , ] . however, a recent study with hcv patients receiving rbv monotherapy showed a downregulation of abnormally preactivated isgs through chromatin remodeling and modulation of histone methylation, resulting in a higher liver susceptibility to ifn by lowering the baseline expression of certain isgs [ ] . some rna viruses, as well as cellular mrnas, harbor a -methylguanosine cap structure at the end [ ] . the rbv-induced reduction of gtp pools within the cell was proposed to also have an effect on the capping efficiency of rna viruses (figure a ). for example, denv encodes for a -o-methyltranferase at the n-terminus of the ns polymerase, termed ns mtase dv . ns mtase dv binds gtp and catalyzes the formation of a cap structure [ ] . after rbv treatment, less gtp is present and rtp was shown to compete for binding to the ns mtase dv , thereby blocking the synthesis of the cap [ ] . likewise, rbv directly and strongly inhibited the viral mrna guanylyltransferase of vaccinia virus and thus prevented capping of nascent viral rna [ , ] . however, this mechanism is controversially discussed in literature [ ] [ ] [ ] [ ] , and not all rna viruses display a -methylguanosine cap structure at the end. therefore, this mode of action cannot account exclusively for the observed effects of rbv. another suggested mechanism is the direct impact of rbv treatment on the function of viral polymerases (figure a,b) . here, rtp is thought to directly inhibit viral rna replication by being recognized by the viral polymerase and thereby leading to chain termination or preventing the binding of other nucleotides important for elongation [ ] . in a cell-free system, rtp was shown to inhibit the rna polymerase of influenza a virus [ ] . moreover, inhibition of viral rna synthesis of vesicular stomatitis virus (vsv) in the presence of rmp, rdp, and rtp was described with the triphosphorylated form being the least active [ ] . this would argue against a mode of action that is based on the incorporation of rtp in the nascent viral rna in vsv. in the same study, an inhibitory effect of rdp on la crosse virus rna synthesis was also reported [ ] . interestingly, crotty et al. could demonstrate that rtp is indeed employed by pv rdrp, and that integrated rbv acts as mutagen [ ] . another example of an effect of rtp on the viral polymerase is the case of reovirus. rankin et al. proposed that rtp binds close to the catalytic site of the transcriptase, thereby affecting the helicase function and subsequently lowering the binding affinity of viral rna [ ] . as a consequence, elongation of the viral rna is inhibited. interestingly, no effect on the capping activity was demonstrated [ ] . the nucleotide binding site of the polymerase is highly conserved among hcv genotypes, supporting this proposed mechanism [ ] . indeed, in vitro analysis showed a minor decrease of hcv replication [ , , ] . however, in clinical trials with rbv monotherapy, only a mild decrease of hcv replication was noticed [ , ] . in recent years, a mutagenic effect of rbv via its incorporation into newly synthesized rna genomes, leading to viral extinction was described for several rna viruses ( figure b ). in contrast to dna viruses, the major characteristic of rna viruses is the occurrence of a cloud of related but genetically distinct variants in infected patients, often referred to as a quasispecies. however, the term "quasispecies" refers to a particular mutation-selection balance, with natural selection acting on the group rather than on the individual [ , ] . it is not simply a surrogate for genetic heterogeneity [ ] . while quasispecies behavior has been demonstrated experimentally in artificially expanded poliovirus populations in infected mice [ ] , evidence is lacking for quasispecies' behavior in many viruses, including hev. these diverse intra-host viral populations are the result of the lack of proofreading activity of rdrp. however, due to this high variation, viral isolates are close to the error threshold, which would lead to reduction in viral fitness [ ] . incorporation of rbv into newly synthesized rna genomes thereby increases the frequency of mutations in the population, pushing the virus over an error threshold and resulting in viral extinction. this mechanism of action for rbv has been described, at least in vitro, for fmdv [ ] , poliovirus [ ] , hcv [ ] , gbv-b [ ] , hantaan virus [ ] , and hev [ , ] . ever since the first reports by sidwell et al. describing rbv as a broad-spectrum antiviral [ ] , there have been multiple discussions about its mechanisms of action. of course one has to always keep in mind that in vitro data, where most of the proposed models arose from, cannot just be translated into in vivo situations. remarkably enough, monotherapy with rbv is only potently effective against lassa virus [ ] and hev [ , ] . future studies should address questions regarding the biocompatibility of rbv and its availability in the targeted liver to investigate if intracellular concentrations can account for the different proposed mechanisms-for example, to outcompete cellular nucleoside triphosphates (ntps) for misincorporation. in summary, several mechanisms have been postulated for rbv activity. among these, there are indirect, immunomodulatory mechanisms and effects on impdh. furthermore, mechanisms on the virus itself were described by inhibition of the capping efficiency, the viral polymerase, and a mutagenic effect on newly synthesized rna genomes. rna viruses do not exist as a clonal population of genomes within the infected host, but rather diversify into a swarm of related but non-identical genome sequences [ ] . this heterogeneous viral population-also referred to as mutant cloud, mutant swarm, or mutant spectra-is capable of better adapting to changing environmental conditions and rapidly evolving, during passage from host to host, due to its high heterogeneity. the concept of quasispecies was mainly developed by manfred eigen and peter schuster [ ] . by demonstrating viral heterogeneity for fmdv [ , ] and vsv [ , ] domingo and colleagues and holland and colleagues were the first to extrapolate this concept to virology [ , ] . these viral populations are the product of very high replication rates found in rna viruses, coupled with a lack of an rdrp proofreading function. for hcv, it is estimated that between and new virions are produced in one infected individual per day [ , ] . estimates for hev do not currently exist, although comparably high replication rates can be assumed. there is data on -end repair mechanisms identified in small rna viral polymerases [ ] . in coronaviruses, for example, a -to- exoribonuclease (exon) domain within the nonstructural protein was identified as being essential for high-fidelity replication [ , ] ; for hcv, pyrophosphorolytic and ntp-mediated nucleotide excision activity of the ns b rdrp have been described as viral mechanisms for removing misincorporated bases [ , ] . despite these reports, most rna viruses, and most likely also hev, do not have any real proofreading capability, causing an error-prone replication of viral genomes. together with the short generation times, this results in highly diverse intra-host populations [ ] . as expected, hev also exists as a heterogeneous population within infected individuals [ , , [ ] [ ] [ ] . early publications relied on the classical tools for detecting diversification of viral genomes, including restriction fragment length polymorphism (rflp) and haplotype profiling [ , ] or clonal sequencing [ ] to characterize hev intra-host diversity. recently, next-generation sequencing (ngs) methods have been utilized to study the distribution of snvs in hev genomes over time [ , ] . hev and most other rna virus populations exist in close proximity to the so-called genomic error threshold, which defines a maximum error rate that still guarantees the maintenance and transmission of the genetic information of the master sequence [ , ] . a replication and, most importantly, mutation rate beyond this extinction threshold causes a sharp reduction in the efficiency of transmission of the genetic information contained in the population master sequence to the next generation of viral progeny, a phenomenon sometimes referred to as error catastrophe [ ] : the majority of genomes in the population are nonfunctional. broad-spectrum antiviral agents like rbv can cause increased mutation rates, and potentially can result in the extinction of the virus population in a process called lethal mutagenesis [ ] . however, the mutated viral intra-host populations can acquire mutations accounting for drug resistance or decreased sensitivity to rbv as a direct consequence of the boosted complexity of the mutational spectra. this has been shown for several viruses like hcv [ ] , fmdv [ ] , pv [ ] , sindbis virus [ ] , and also for hev [ ] [ ] [ ] . the dynamics of hev populations in patients under rbv therapy is not fully understood, but recent studies and reports from other rna viruses point to a dichotomy of opposing outcomes resulting from rbv therapy: rbv-induced lethal mutagenesis resulting in viral extinction versus the accumulation of mutations beneficial to the virus in the population, which can lead to therapeutic failure [ , ] . as a consequence of the emergence of rbv-resistant mutations and subsequent treatment failure, clinicians could draw back on combination therapies to overcome or avoid this phenomenon. possible combinations are one mutagen and a conventional antiviral drug or using several rna mutagens in combination or sequence as proposed by perales and domingo [ ] [ ] [ ] . hev is one of the pathogenic viruses that can currently only be treated with rbv as an off-label drug. ifn-α as an alternative therapy has been evaluated in small patient cohorts with limited success and considerable side effects [ , ] . in addition, in vitro data suggests careful assessment of ifns when treating hev [ , ] . considering high mortality rates of over % for genotype- -infected pregnant women [ , ] , the urgent need for extensive research in the field of novel anti-hev treatment regimens is required. patients who fail to achieve sustained virological responses after rbv therapy for hev have no further treatment options: this is particularly of importance in a solid organ transplant setting, as a reduction of immunosuppression beyond a certain level will lead to the rejection of the allograft [ , ] , and hepatitis caused by hev cannot be impeded. recently, two independent studies were able to correlate rbv treatment failure with the emergence of novel single-nucleotide variations in the viral genome during treatment [ , ] . both research groups identified a variant previously described, g r [ ] , as well as other new variants, k n, d g, k r, v i, and y f, all in the polymerase region of orf . in addition, todt et al. also determined nine additional snvs in orfs and [ ] . in both studies, k n mutations emerged in several patients; additionally, an overall increase in viral intra-host heterogeneity could be shown [ , ] . the authors demonstrated significant increases in the number of sites exhibiting snvs, synonymous as well as nonsynonymous, in viral populations after the first administration of rbv in nine patients. this phenomenon was observed for all orfs of the hev genome. interestingly, this increase in heterogeneity was reversible with a decline in the number of snv sites when rbv treatment was stopped. strikingly, none of the described variants that became dominant in the viral populations under treatment resulted in a decreased sensitivity to rbv when cloned into an hev subgenomic reporter replicon in tissue culture. only g r mutations altered the viral replication efficacy, increasing replication rates [ , ] , while rbv sensitivity was unmodified [ , , ] . why rbv treatment fails in some patients, while others are able to clear the virus under rbv monotherapy, remains an open question. rbv has been shown to block hev replication through a depletion of cellular gtp pools in cell culture model systems [ ] , in addition to the strong mutagenic effect of rbv on the hev genome in vivo described above. rbv inhibits the impdh, thus causing a two-fold reduction of the intracellular gtp pools and increasing ctp and utp concentrations at the same time [ , ] . hev genome replication is a cyclic process of alternating synthesis of negative-strand rna and positive-strand rna [ ] . during the replication process, the extrinsically administered, rtp is randomly incorporated into the nascent negative-stranded rna as a result of pairing with either of the pyrimidine bases cytidine or uracil ( figure b, upper panel) . this negative-stranded antigenome rna then serves as a template for subsequent production of positive-stranded genomic rna. the rdrp subsequently incorporates, again randomly, a cytidine or uracil at rbv residues located in the antigenome template ( figure b, middle panel) . these stochastic incorporations lead to nucleotide substitutions in the newly synthesized viral genomes. additionally, rbv will also be incorporated in the positive-stranded rna genome, leading to increased amounts of replication-defective viral genomes packaged into the capsid, ultimately leading to an increase in frequency of replication-defective virions. additionally, new antigenome templates can be produced from defective positive-stranded genomes, so misincorporations are amplified in the replication process ( figure b, lower panel) . this results in the fixation of transitional substitutions in nascent rnas. transitional purine-to-purine (g<>a) or pyrimidine-to-pyrimidine (c<>t) nucleotide substitutions are preferentially enriched during rbv monotherapy, leading to the observed synonymous exchanges as well as to the amino acid replacements favorable for the survival of the viral population [ , ] . whether rbv also inhibits the hev methyltransferase comparably to the direct inhibition of the vaccinia virus guanylyltransferase (see above), or if the rtp is incorporated as a cap analog [ , ] (thus impacting correct translation) has not been investigated yet. the mutagenic effect of rbv-based therapy can have divergent effects on hev populations, which may impact the therapy success. on the one hand, rbv increases the mutation rate in the viral genome, driving the population towards its extinction threshold. in contrast, the increased variability in the viral population can result in selection of variants with improved replication fitness which become dominant in the viral population and are associated with therapeutic failure. these advantageous variants could be (i) a downregulation of the replication machinery, thus preventing the accumulation of more mutations, as shown from in vitro data when reverse engineering the k n variant into hev cell culture systems [ ] , and (ii) an increase in viral polymerase fidelity as hypothesized by debing et al. for the k n variant-a mutant with a substitution in the f -motif of the rdrp-which could hinder the incorporation of rbv into the viral genome. in fact, the lab of esteban domingo was able to dissect a multistep process of viral adaption to a mutagenic nucleoside analog in fmdv that led to an extinction escape by changing the fidelity of the polymerase [ ] . hev is a life-threatening infection when immunosuppressed individuals fail to achieve an svr during rbv treatment. currently, clinicians do not have alternative therapy regimens available. recent studies have suggested that the heterogeneous viral population is able to acquire snvs that decrease rbv sensitivity [ , ] . their data supports a conclusion whereupon the mutagenic effect of the broad-spectrum antiviral agent leads to increased heterogeneity in the intra-host viral population introducing a race between the virus trying to gain 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viral adaptation to a mutagenic nucleoside analogue by modulation of transition types leads to extinction-escape acknowledgments: this work was supported by the german ministry for education and research (bmbf) through a ginaico grant gw . e.s. was further supported by the helmholtz centre for infection research. the authors declare no conflict of interest. key: cord- -tzmev c authors: yuan, shuofeng; chan, chris chun-yiu; chik, kenn ka-heng; tsang, jessica oi-ling; liang, ronghui; cao, jianli; tang, kaiming; cai, jian-piao; ye, zi-wei; yin, feifei; to, kelvin kai-wang; chu, hin; jin, dong-yan; hung, ivan fan-ngai; yuen, kwok-yung; chan, jasper fuk-woo title: broad-spectrum host-based antivirals targeting the interferon and lipogenesis pathways as potential treatment options for the pandemic coronavirus disease (covid- ) date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: tzmev c the ongoing coronavirus disease (covid- ) pandemic caused by severe acute respiratory syndrome coronavirus (sars-cov- ) signals an urgent need for an expansion in treatment options. in this study, we investigated the anti-sars-cov- activities of antiviral agents with known broad-spectrum antiviral activities against coronaviruses and/or other viruses. they were first evaluated in our primary screening in veroe cells and then the most potent anti-sars-cov- antiviral agents were further evaluated using viral antigen expression, viral load reduction, and plaque reduction assays. in addition to remdesivir, lopinavir, and chloroquine, our primary screening additionally identified types i and ii recombinant interferons, -hydroxycholesterol, and am as the most potent anti-sars-cov- agents among the antiviral agents. betaferon (interferon-β b) exhibited the most potent anti-sars-cov- activity in viral antigen expression, viral load reduction, and plaque reduction assays among the recombinant interferons. the lipogenesis modulators -hydroxycholesterol and am exhibited ec( ) at low micromolar levels and selectivity indices of > . . combinational use of these host-based antiviral agents with virus-based antivirals to target different processes of the sars-cov- replication cycle should be evaluated in animal models and/or clinical trials. severe acute respiratory syndrome coronavirus (sars-cov- ) is a novel betacoronavirus that was first identified in patients with unexplained pneumonia in wuhan, hubei, china in december sars-cov- hku- a (genbank accession number: mt ) was isolated from the nasopharyngeal aspirate specimen of a laboratory-confirmed covid- patient in hong kong [ ] . the virus was propagated in veroe cells and kept at − • c in aliquots. plaque forming unit (pfu) and tcid assays were performed to titrate the cultured virus as we described previously [ ] . veroe (atcc ® crl- ™) cells were purchased from atcc (manassas, va, usa) and maintained in dulbecco's modified eagle medium (dmem, gibco, carlsbad, ca, usa) culture medium supplemented with % heat-inactivated fbs (fetal bovine serum, gibco), u/ml penicillin, and µg/ml streptomycin as previously described [ ] . all experiments involving live sars-cov- followed the approved standard operating procedures of the biosafety level facility at the department of microbiology, the university of hong kong [ ] . the recombinant ifns were obtained from the following sources: pegasys (roche, basel, switzerland), avonex (ucb, brussels, belgium), rebif (emd serono, inc. rockland, ma, usa), betaferon (bayer schering pharma, berlin, germany), and immukin (boehringer ingelheim, ingelheim am rhein, germany). all other antiviral agents were purchased from medchemexpress (monmouth junction, nj, usa). twenty-two antiviral agents with reported activities against coronaviruses and/or other viruses were included in the study. in the primary screening, , iu/ml of each recombinant ifn or µm of each of the other antiviral agents was used to treat sars-cov- -infected (moi = . ) veroe cells for h. the viral loads in the cell culture supernatants were then determined by quantitative reverse transcription-polymerase chain reaction (qrt-pcr) as previously described [ ] . the cut-off value of ≥ % inhibition (i.e., ≥ log copies/ml reduction when compared with the dimethyl sulfoxide (dmso) control) was used to select the antiviral agents for further evaluation. the celltiterglo luminescent assay (promega corporation, madison, wi, usa) was performed to detect the cytotoxicity of the selected antiviral agents as previously described [ ] . briefly, veroe cells ( × cells/well) were incubated with different concentrations of the individual compound for h, followed by the addition of substrate and measurement of luminance min later. the % cytotoxic concentrations (cc ) of the antiviral agents were calculated by sigmaplot (systat software inc., san jose, ca, usa) in an excel add-in ed v . viral load reduction assay was performed as described previously with modifications [ ] . briefly, the culture supernatants of the sars-cov- -infected veroe cells were harvested at h post-inoculation (hpi) for qrt-pcr analysis of viral rna load. a total of µl of culture supernatant was lysed with µl of avl buffer, which was subsequently extracted for total rna with the qiaamp viral rna mini kit (qiagen, hilden, germany). qrt-pcr was used for quantitation of sars-cov- replication using the quantinova probe rt-pcr kit (qiagen) with a lightcycler real-time pcr system (roche) as we described previously [ ] . each µl reaction mixture contained µl of × quantinova probe rt-pcr master mix, . µl of rnase-free water, . µl of quantinova probe rt-mix, . µl each of µm forward and reverse primer, . µl of µm probe, and µl of extracted rna as the template. reactions were incubated at • c for min for reverse transcription, • c for min for denaturation, followed by cycles of • c for s and • c for s. signal detection and measurement were taken in each cycle after the annealing step. the cycling profile ended with a cooling step at • c for s. the primers and probe sequences were against the rna-dependent rna polymerase/helicase (rdrp/hel) gene region of sars-cov- : forward primer: -cgcatacagtcttrcaggct- ; reverse primer: -gtgtgatgttgawatgacatggtc- ; specific probe: -fam ttaagatgtggtgcttgcatacgtagac-iabkfq- . viral n antigen expression in sars-cov- -infected veroe cells was performed as we described previously using immunofluorescent staining with an in-house rabbit antiserum against sars-cov- -n protein as previously described [ ] . cell nuclei were labelled with , -diamidino- -phenylindole (dapi) nucleic acid stain from thermo fisher scientific (waltham, ma, usa). the alexa fluor secondary antibody was obtained from thermo fisher scientific. mounting was performed with the diamond prolong antifade mountant from thermo fisher scientific. plaque reduction assay was performed to plot the % maximal effective concentration (ec ) as we previously described with slight modifications [ ] . briefly, veroe cells were seeded at × cells/well in -well tissue culture plates on the day before carrying out the assay. after h of incubation, plaque-forming units (pfu) of sars-cov- were added to the cell monolayer with or without the addition of antiviral agents and the plates were further incubated for h at • c in % co before removal of unbound viral particles by aspiration of the media and washing once with dmem. monolayers were then overlaid with media containing % low melting agarose (cambrex corporation, east rutherford, nj, usa) in dmem and appropriate concentrations of individual compound, inverted and incubated as above for another h. the wells were then fixed with % formaldehyde (bdh, merck, darmstadt, germany) overnight. after removal of the agarose plugs, the monolayers were stained with . % crystal violet (bdh, merck) and the plaques counted. the percentage of plaque inhibition relative to the control (i.e., without the addition of compound) wells were determined for each antiviral agent concentration. ec was calculated using a sigma plot (spss) in an excel add-in ed v . the plaque reduction assay experiments were performed in triplicate and repeated twice for confirmation. time-of-drug-addition assay was performed for selected compounds as previously described with slight modifications [ ] . briefly, veroe cells were seeded in -well plates ( × cells/well). the cells were inoculated with sars-cov- (multiplicity of infection, moi = . ) and then incubated for h for virus internalization at • c. dmso ( . %) was included as a negative control. the viral loads in the culture supernatants normalized by dmso at the different phases of the assay were determined by qrt-pcr. a total of antiviral agents with reported activity against coronaviruses and/or other viruses were included in the primary screening. these included types i (pegasys, avonex, rebif, and betaferon) and ii (immukin) ifns, nucleoside analogues (favipiravir, galidesivir, remdesivir, and ribavirin), -aminoquinoline (chloroquine), anthracycline (idarubicin), antihistamine (chlorcyclizine), calcineurin inhibitor (cyclosporine), flavonoid (silibinin), kinase inhibitors (erlotinib and everolimus), macrolide (azithromycin), oxysterol ( -hydroxycholesterol), polymerase inhibitor (filibuvir), protease inhibitors (lopinavir and rupintrivir), and retinoic acid receptor agonist (am ) ( table ). in this primary screening, chloroquine, lopinavir, and remdesivir which were recently reported to have anti-sars-cov- activity, exhibited about . - . log copies/ml reduction in viral rna load ( figure ). in comparison, recombinant ifn-β demonstrated the most potent anti-sars-cov- activity, with avonex (ifn-β a), rebif (ifn-β a), and betaferon (ifn-β b) each achieving about log copies/ml reduction in viral load. pegasys (pegylated ifn-α a) and immukin (ifn-γ b) also showed anti-sars-cov- activity with viral load reduction of . log copies/ml and . log copies/ml, respectively. additionally, two other antiviral agents, namely, am and -hydroxycholesterol, exhibited anti-sars-cov- activity. they each achieved about -log reduction in viral load. the other antiviral agents demonstrated < log reduction in sars-cov- load and were therefore not investigated further in this study. based on the results from the primary screening, avonex (ifn-β a), rebif (ifn-β a), betaferon (ifn-β b), pegasys (pegylated ifn-α a), immukin (ifn-γ b), am , and -hydroxycholesterol were further evaluated in the sars-cov- n antigen expression assay. as shown in figure , at the fixed concentrations of iu/ml of each of the recombinant ifns and µm of am and -hydroxycholesterol, all seven antiviral agents reduced viral n antigen expression in the immunofluorescent staining assay. the most prominent reduction in viral n antigen expression was observed in cells treated with avonex, rebif, betaferon, and am , which achieved similar degree of viral n antigen expression reduction as redemsivir. pegasys, immukin, and -hydroxycholesterol also moderately reduced viral n antigen expression to a similar degree as lopinavir. viruses , , x; doi: for peer review of based on the results from the primary screening, avonex (ifn-β a), rebif (ifn-β a), betaferon (ifn-β b), pegasys (pegylated ifn-α a), immukin (ifn-γ b), am , and -hydroxycholesterol were further evaluated in the sars-cov- n antigen expression assay. as shown in figure , at the fixed concentrations of iu/ml of each of the recombinant ifns and µm of am and -hydroxycholesterol, all seven antiviral agents reduced viral n antigen expression in the immunofluorescent staining assay. the most prominent reduction in viral n antigen expression was observed in cells treated with avonex, rebif, betaferon, and am , which achieved similar degree to quantify the anti-sars-cov- activity of the identified antiviral agents in the primary screening and the viral n antigen expression assay, sars-cov- viral load reduction assay by qrt-pcr was conducted to determine the sars-cov- rna copies released in the cell culture supernatant with or without antiviral agent treatment. as show in figure , the mean baseline viral load in the cell culture supernatants without any antiviral agent was about . log copies/ml. there was dose-dependent and significant (p < . ) reduction in viral load of > % as compared to the baseline in cell culture supernatants inoculated with each of the eight antiviral agents. as to quantify the anti-sars-cov- activity of the identified antiviral agents in the primary screening and the viral n antigen expression assay, sars-cov- viral load reduction assay by qrt-pcr was conducted to determine the sars-cov- rna copies released in the cell culture supernatant with or without antiviral agent treatment. as show in figure , the mean baseline viral load in the cell culture supernatants without any antiviral agent was about . log copies/ml. there was dose-dependent and significant (p < . ) reduction in viral load of > % as compared to the baseline in cell culture supernatants inoculated with each of the eight antiviral agents. as controls, remdesivir and lopinavir achieved . log copies/ml and . log copies/ml reduction in viral rna load, respectively, at a concentration of µm. in comparison, -hydroxychloestero and am achieved . log copies/ml and . log copies/ml reduction in viral rna load, respectively, at the same antiviral agent concentration. consistent with the primary screening results, ifn-β a ifn-β b demonstrated more potent anti-sars-cov- activity than ifn-α a and ifn-γ b in the viral load reduction assay. at a concentration of iu/ml, avonex (ifn-β a), rebif (ifn-β a), and betaferon (ifn-β b) respectively achieved . log copies/ml, . log copies/ml, and . log copies/ml reduction in viral rna load, whereas pegasys (pegylated ifn-α a) and immukin (ifn-γ b) achieved only . log copies/ml and . log copies/ml reduction in viral rna load. at a higher concentration of , iu/ml, pegasys and immukin achieved modestly higher reduction in viral rna load ( . log copies/ml and . log copies/ml, respectively). in addition to reduction in viral n antigen expression and rna load, inhibition of infectious sars-cov- particles was evaluated using plaque reduction assay (figure ) in addition to reduction in viral n antigen expression and rna load, inhibition of infectious sars-cov- particles was evaluated using plaque reduction assay (figure ). among the recombinant ifns, betaferon (ifn-β b) demonstrated the most potent anti-sars-cov- effect with % inhibition of virus plaque formation at a concentration of iu/ml. two different brands of ifn-β a (avonex and rebif) exhibited similar anti-sars-cov- activity with % inhibition of virus formation at a concentration of iu/ml. pegasys (pegylated ifn-α a) and immukin (ifn-γ b) again demonstrated less potent anti-sars-cov- activity than the recombinant ifn-β's, and demonstrated % inhibition of virus plaque formation at higher concentrations of , and iu/ml, respectively. -hydroxycholesterol demonstrated marked virus plaque formation at µm. am showed dose-dependent plaque reduction effect with partial inhibition of virus plaque formation at the highest tested concentration of µm. remdesivir and lopinavir achieved nearly complete inhibition of virus plaque reduction at µm and µm, respectively, but demonstrated cytotoxicity in veroe cells at hpi at higher concentrations. based on these plaque reduction assay results, the ec of the antiviral agents were determined ( µm. am showed dose-dependent plaque reduction effect with partial inhibition of virus plaque formation at the highest tested concentration of µm. remdesivir and lopinavir achieved nearly complete inhibition of virus plaque reduction at µm and µm, respectively, but demonstrated cytotoxicity in veroe cells at hpi at higher concentrations. based on these plaque reduction assay results, the ec of the antiviral agents were determined ( to explore the mode of action of the two non-ifn antiviral agents, a time-of-drug-addition assay was performed. as shown in figure , -hydroxycholesterol and am both exhibited anti-sars-cov- effect only when they were added to the sars-cov- -infected veroe cells at or after one hour post-inoculation. these results suggested that -hydroxycholesterol and am both targeted the post-entry steps of the sars-cov- replication cycle. viruses , , x; doi: for peer review of a > , iu/ml, > µm, and > µm indicate the highest antiviral agent concentrations tested in the cytotoxicity assay was , iu/ml (ifns), µm ( -hydroxycholesterol), and µm (remdesivir), respectively. abbreviations: cc , % cytotoxic concentration; ec , % maximal effective concentration; ifn, interferon. to explore the mode of action of the two non-ifn antiviral agents, a time-of-drug-addition assay was performed. as shown in figure , -hydroxycholesterol and am both exhibited anti-sars-cov- effect only when they were added to the sars-cov- -infected veroe cells at or after one hour post-inoculation. these results suggested that -hydroxycholesterol and am both targeted the post-entry steps of the sars-cov- replication cycle. in this study, we evaluated the anti-sars-cov- activity of antiviral agents with broad-spectrum antiviral activities against coronaviruses and/or other viruses. in our primary in this study, we evaluated the anti-sars-cov- activity of antiviral agents with broad-spectrum antiviral activities against coronaviruses and/or other viruses. in our primary screening using a fixed antiviral agent concentration and virus inoculum, we identified recombinant ifns and lipogenesis modulators to be the most potent anti-sars-cov- agents among broad-spectrum antivirals. these findings have important implications for the choice of clinically available recombinant ifns to be used in covid- patients and development of lipogenesis modulators as potential anti-sars-cov- therapeutics. ifns are glycoproteins with strong antiviral activities that represent one of the first lines of host immune response against invading pathogens [ ] . these proteins are classified into three groups, types i, ii, and iii ifns, based on the structure of their receptors on the cell surface [ ] . ifns are known for their broad-spectrum antiviral activities against a wide range of dna and rna viruses, through inducing the expressions of interferon-stimulated genes in host cells, such as protein kinase r, oligoadenylate synthetase, and rnase l [ ] . these interferon-stimulated genes suppress viral replication by inhibiting multiple steps in a viral life cycle, including viral rna transcription and viral protein translation. recombinant ifn-α and ifn-β exhibited potent antiviral activity against sars-cov and mers-cov in vitro and in animal models [ , [ ] [ ] [ ] [ ] . recombinant ifn-γ exhibited limited anti-coronaviral activity in vitro, but might be synergistic with type i ifns [ , , ] . in this study, we demonstrated the anti-sars-cov- activity of five clinically-approved preparations of recombinant ifns, including pegasys (pegylated ifn-α a), avonex (ifn-β a), rebif (ifn-β a), betaferon (ifn-β b), and immukin (ifn-γ b). among them, betaferon exhibited the most potent anti-sars-cov- effect with the lowest ec of . iu/ml and the highest selectivity index of > . . importantly, the ec of betaferon against sars-cov is below its achievable peak serum concentration (cmax) with standard subcutaneous dosing of million units of betaferon ( iu/ml). notably, betaferon was similarly found to be the most potent recombinant ifn for the highly virulent mers-cov and significantly improved the clinical, virological, and histopathological parameters of mers-cov-infected common marmosets [ , ] . in sars and mers patients, the use of recombinant ifn-α and/or ifn-β treatment was generally well tolerated with minimal adverse effects [ , , ] . although the clinical benefits of recombinant ifn treatment in sars and mers patients remain inconclusive, the apparent discrepancy between the in vitro and in vivo antiviral effects might be related to the delay in treatment commencement after symptom onset. because sars-cov- is able to achieve more than folds higher viral load than sars-cov within h in human lung tissues by minimally eliciting the host ifn response, it would be important to supplement covid- patients with recombinant ifns, especially ifn-β b, before cytokine storm develops, with other effective virus-targeting antivirals [ ] . importantly, inhaled ifn-β is well tolerated and enhances both systemic and local innate immunity with upregulated antiviral gene expression and reduced proinflammatory cytokines in sputum [ ] . this treatment strategy might be especially useful when given early to covid- patients who usually have the peak respiratory tract viral loads within the first week of symptom onset [ ] . in addition to the recombinant ifns, we also identified antiviral agents that target the host lipogenesis pathways as potential anti-sars-cov- agents. am is a selective retinoic acid receptor-α agonist which was recently identified to have broad-spectrum antiviral activities against various families of dna and rna viruses, including coronaviridae, flaviviridae, orthomyxoviridae, picornaviridae, and adenoviridae [ ] . am inhibits virus replication through interaction with sterol regulatory element-binding protein (srebp) and downregulation of multiple srebp proteolytic processes and srebp-regulated lipid biosynthesis pathways, such as double-membrane vesicle formation by mers-cov [ ] . our time-of-drug-addition assay showed that am inhibited the post-entry events of the sars-cov- replication cycle, which corroborated with the hypothesized restrictive effects of am on lipid biosynthesis in sars-cov- infection. the oxysterol -hydroxycholesterol is a metabolite of cholesterol that is produced and secreted by macrophages and has multiple effects on lipid metabolism, especially lipid biosynthesis and immunity [ ] . -hydroxycholetserol has been shown to inhibit feline coronavirus, porcine epidemic diarrhea virus, and porcine transmissible gastroenteritis virus possibly through induction of intracellular cholesterol accumulation [ , ] . moreover, -hydroxycholesterol is active against various emerging rna viruses, including ebola virus, nipah virus, rift valley fever virus, and zika virus, and dna and rna viruses that cause chronic infections, such as human immunodeficiency virus, herpes simplex virus, and varicella zoster virus [ , ] . mechanistically, -hydroxycholesterol and its downstream metabolite -hydroxycholesterol- -sulfate ( hc s) block membrane fusion between virions and host cells through diametrical regulation of lipid metabolism and inflammatory response via lxr/srebp- and ikappabalpha/nf-kappab signaling [ , ] . addition of hc s to primary rat hepatocytes decreased nuclear lxr and srebp- protein levels, downregulated their target genes, acetyl coa carboxylase , fatty acid synthase, and srebp- target gene hmg reductase, which are key enzymes involved in fatty acid and cholesterol biosynthesis [ ] . interestingly, the expression of the interferon stimulating gene-encoded cholesterol- -hydroxylase (ch h) is upregulated by ifns and toll-like receptors to convert cholesterol into -hydroxycholesterol [ ] . thus, combination treatment with ifns may further enhance the antiviral effects of -hydroxycholesterol and should be further investigated. our study had limitations. first, we used a fixed antiviral agent concentration in our primary screening in order to identify the antiviral agents with the lowest ec among the broad-spectrum antivirals. this might have overlooked antiviral agents that can inhibit sars-cov- at higher concentrations. for example, favipiravir has been shown to inhibit sars-cov- replication with an ec of µm [ ] . similarly, galidesivir, a broad-spectrum rna-dependent rna polymerase inhibitor, inhibited the sars-cov with an ec of . µm [ ] . second, antiviral evaluation of the selected reagents should be performed in additional primary cells to comprehensively document their antiviral activities. third, the combination effects of the host-based ifn-β b, am , and -hydroxycholesterol with virus-based antivirals, such as remdesivir and lopinavir, should be further evaluated in vitro and/or in vivo. targeting multiple steps in the viral replication cycle might help to enhance the therapeutic effects of these virus-based antivirals in covid- patients. indeed, during the revision of this manuscript, a multi-center, open-label, randomized phase clinical trial comparing adult covid- patients treated with triple combination antiviral therapy (ifn-β b, lopinavir-ritonavir, and ribavirin) with those treated with lopinavir-ritonavir monotherapy was reported. the results showed that the combination therapy group had a significantly shorter median time from commencement of treatment to negative nasopharyngeal swab than the control monotherapy group ( vs. days) [ ] . additional studies to evaluate the effects of combination therapies using the other antiviral agents identified in this study should be considered. hong kong corporation limited, and was an invited speaker for gilead sciences hong kong limited and luminex corporation. the other authors declared no conflict of interests. the funding sources had no role in study design, data collection, analysis or interpretation or writing of the report. the corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan world health organization. coronavirus disease (covid- ) situation report - a familial cluster of pneumonia associated with the novel coronavirus indicating 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norovirus replicon silibinin is a potent antiviral agent in patients with chronic hepatitis c not responding to pegylated interferon/ribavirin therapy treatment of sars with human interferons inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques treatment with interferon-alpha b and ribavirin improves outcome in mers-cov-infected rhesus macaques interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (sars-cov) potent inhibition of sars-associated coronavirus (scov) infection and replication by type i interferons (ifn-alpha/beta) but not by type ii interferon (ifn-gamma) ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ifn-alpha a or ifn-beta a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study comparative replication and immune activation profiles of sars-cov- and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid- the effect of inhaled ifn-beta on worsening of asthma symptoms caused by viral infections. a randomized trial temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study -hydroxycholesterol acts as an amplifier of inflammatory signaling endocytic pathway of feline coronavirus for cell entry: differences in serotype-dependent viral entry pathway cholesterol -hydroxylase negatively regulates porcine intestinal coronavirus replication by the production of -hydroxycholesterol -hydroxycholesterol protects host against zika virus infection and its associated microcephaly in a mouse model lipids in innate antiviral defense regulation of hepatocyte lipid metabolism and inflammatory response by -hydroxycholesterol and -hydroxycholesterol- -sulfate -hydroxycholesterol production by the cholesterol- -hydroxylase interferon-stimulated gene restricts mammalian reovirus infection triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial key: cord- -xorj tnz authors: kao, chi-fei; chiou, hue-ying; chang, yen-chen; hsueh, cheng-shun; jeng, chian-ren; tsai, pei-shiue; cheng, ivan-chen; pang, victor fei; chang, hui-wen title: the characterization of immunoprotection induced by a cdna clone derived from the attenuated taiwan porcine epidemic diarrhea virus pintung strain date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: xorj tnz the porcine epidemic diarrhea virus (pedv) poses a great threat to the global swine industries and the unreliable protection induced by the currently available vaccines remains a major challenge. we previously generated a genogroup b (g b) pedv taiwan pintung (pedvpt) strain, pedvpt-p , and determined its promising host immune response against the virulent pedvpt-p strain. to study the attenuation determinants of pedvpt-p and establish a pedvpt-p -based recombinant vector as a vaccine platform for further antigenicity modification, ipedvpt-p , a full-length cdna clone of pedvpt-p , was established. comparing to the parental pedvpt-p virus, the ipedvpt-p virus showed efficient replication kinetics with a delayed decline of viral load and similar but much more uniform plaque sizes in vero cells. in the -week-old piglet model, fecal viral shedding was observed in the pedvpt-p -inoculated piglets, whereas those inoculated with ipedvpt-p showed neither detectable fecal viral shedding nor pedv-associated clinical signs. moreover, inoculation with ipedvpt-p elicited comparable levels of anti-pedv specific plasma igg and fecal/salivary iga, neutralizing antibody titers, and similar but less effective immunoprotection against the virulent pedvpt-p challenge compared to the parental pedvpt-p . in the present study, an infectious cdna clone of an attenuated g b pedv strain was successfully generated for the first time, and the in vitro and in vivo data indicate that ipedvpt-p is further attenuated but remains immunogenic compared to its parental pedvpt-p viral stock. the successful development of the ipedvpt-p cdna clone could allow for the manipulation of the viral genome to study viral pathogenesis and facilitate the rapid development of effective vaccines. the porcine epidemic diarrhea virus (pedv) is an enveloped, positive-sense and single-stranded rna virus, belonging to the order nidovirales, family coronaviridae, and genus alphacoronavirus [ ] . the genome of pedv is about kilobase pairs in length and comprises of seven open reading frames (orf), including orf a and b genes that constitute the two thirds of the genome and encode the replication complex; the spike (s) gene that governs viral entry; the envelop (e), membrane (m), and nucleocapsid (n) genes that are responsible for virion assembly; and the accessory orf gene with an undetermined function [ ] . the porcine epidemic diarrhea virus is the causative agent of porcine epidemic diarrhea (ped), a historic, highly contagious enteric swine disease characterized by diarrhea, dehydration, and the growth retardation in pigs of all ages [ ] . in late , new and highly virulent pedv strains arose in china and spread rapidly worldwide by late , resulting in nearly % mortality in the affected nursing piglets [ ] [ ] [ ] . to date, there are still indelible endemics and considerable economic losses in the global swine market [ ] . besides, the protection conferred by currently available vaccines is, unfortunately, unsatisfactory [ , ] . based on the nucleotide identity of the s gene, pedvs are categorized into four genogroups (gs), namely g a, g b, g a, and g b [ ] . among these, the g b pedv strains that predominate the field in asia and north america, show higher pathogenicity [ ] and appear to elicit broader protection across different genogroups [ ] [ ] [ ] . although the increased virulence of new pedv strains was ascribed to several mutations in the s gene through viral escape from antibody neutralization induced by traditional vaccines [ , ] , the detailed mechanism remains elusive. previously, we generated an attenuated g b taiwan pedv strain, pedv pintung passage (pedvpt-p ) virus, by serial passage of the parental pedvpt strain in vero cells [ ] . despite the high potential of pedvpt-p as a future vaccine candidate against pedv as indicated by its reduced pathogenicity and robust host immune response in our -week-old piglet model [ ] , the difficulty in pedv isolation and subsequent lengthy passage process rendered the pedvpt-p unable to promptly respond to the vast outbreak in late . this year, zhou et al. [ ] reported a new disastrous swine disease outbreak in china in caused by an hku -related coronavirus of bat origin, again highlighting the potential burden of the interspecies jumping of coronavirus, and a pressing need for a readily applicable vaccine platform for new emergences. the reverse genetics system has been widely used to study viral pathogenesis and novel vaccine design. at present, the reported pedv infectious cdna clones were exclusively constructed based on sequences of the representative wild-type pedv isolates [ ] [ ] [ ] [ ] [ ] . consequently, they are highly pathogenic and fatal in suckling piglets, comparable to their parental viruses. with regard to vaccine use, further genetic editing is necessary to attenuate these recombinant viruses. alternatively, a complementary approach exploiting the attenuated phenotype to address the safety concerns has not been described. in the present study, a full-length cdna clone of the attenuated pedvpt-p , ipedvpt-p , was generated. in addition, the pathogenicity, immunogenicity, and protection against virulent pedvpt-p challenge by ipedvpt-p were evaluated in the -week-old piglet model. our data suggest that the ipedvpt-p virus was more attenuated but able to elicit similar immunogenicity and immunoprotection against the autologous virulent pedvpt-p challenge compared to the parental pedvpt-p virus. this ipedvpt-p cdna clone is expected to allow for the manipulation of the viral genome to study viral pathogenesis. on the other hand, it can serve as a vaccine platform, for example, by directly replacing the s gene with that of the (re)emerging swine coronaviruses. vero c cells (atcc no. crl- ) were maintained in dulbecco's modified eagle's medium (dmem, gibco, grand island, ny, usa) supplemented with % fetal bovine protein (fbs), ng/ml amphotericin b, u/ml penicillin, and µg/ml streptomycin. viral stock of pedvpt-p in post-inoculation medium (pi medium) containing dmem supplemented with . % tryptose phosphate broth (tbp), . % yeast extract ( . %), and µg/ml trypsin, as prepared in our previous study [ ] , was used for generating the infectious cdna clone and as the control for in vivo and in vitro studies, whereas the virulent pedvpt-p virus was used for animal challenge. the strategy used to engineer the full-length cdna clone of pedvpt-p , namely ipedvpt-p , was modified according to a previously published method [ ] . briefly, the complete genome of pedvpt-p (genbank accession no. ky ) was divided into six fragments by pcr amplification using primer pairs (table ) incorporated with specific type-iis restriction enzyme sites for seamless ligation. fragment a contained a t promoter sequence at its end to allow in vitro transcription and the sequence was designed following the article published previously [ ] ; a -adenosine sequence was added to the end of fragment f to simulate polyadenylation. a naturally occurring bsai site in fragment e was removed by introducing a silent mutation (c t) by site-directed mutagenesis according to the previously described protocol [ ] . an additional synonymous mutation (t c) was generated near the junction of fragments e and f, to create a novel bsmbi recognition site. pcr amplicons of all fragments were subcloned into plasmid vectors (pjet . ; thermo fisher scientific, waltham, ma, usa). each subclone was digested with the corresponding type-iis restriction enzymes as indicated in figure and gel-purified using a qiaquick gel extraction kit (qiagen, hilden, germany). assembly of the full-length cdna was conducted by employing t ligase (neb, ipswich, ma, usa) overnight at • c. the ligated full-length cdna was phenol-chloroform extracted and in vitro transcribed to full-length rna transcripts using a mmessage mmachine t transcription kit (ambion, austin, ca, usa) following the manufacturer's instructions. the cap analog to gtp ratio was adjusted to : to increase the yield of full-length transcripts. to facilitate viral recovery, nucleocapsid (n) transcripts were also generated from amplicons flanking the entire n gene with the addition of the t promoter sequence and poly-a tail at the and ends, respectively. the n transcripts were precipitated using lithium chloride (ambion, austin, ca, usa) and purified with ethanol. the reaction mixture of full-length transcripts ( µl) and µg of n transcripts were mixed thoroughly and electroporated into vero cells in a suspension of µl with cells/ml in rnase-free phosphate buffered saline (pbs) using a gene pulser xcell™ electroporation system (bio-rad, hercules, ca, usa), with four pulses at v, µf and - s rest between each pulse. after electroporation, the cells were initially incubated at room temperature for min and then resuspended in dmem supplemented with % fbs in a -cm flask overnight at • c to allow for recovery. on the next day, the cells were washed twice with dulbecco's phosphate-buffered saline (dpbs) and maintained in pi medium for an additional - days until cytopathic effects (cpe) characterized by cell fusion, syncytial cell formation, and cell detachment were observed. the whole flask was subjected to one freeze-and-thaw cycle and the rescued virus was passaged once to generate a viral stock of ipedvpt-p for further use. the viral stock was titrated in vero cells in a -well plate to determine viral titer. to detect the pedv antigen, immunocytochemistry (icc) was performed as described previously [ ] . briefly, vero cells infected with ipedvpt-p showing typical cpe were fixed with % ice-cold acetone, air-dried, and incubated with an in-house anti-pedv n antibody at room temperature (rt) for h. after washing three times with pbs, a polyclonal anti-rabbit/mouse immunoglobulin, envision-dab + system (dako, carpinteria, ca, usa), was applied for h at rt. following three washes with pbs, the cells were incubated with , -diaminobenzidine (dab) chromogen from a peroxidase dab substrate kit (dako, carpinteria, ca, usa) according to the manufacturer's instructions. positive signals were visualized under an inverted light microscope (nikon, tokyo, japan). sequence analysis was conducted as described previously [ ] and a primer pair (sf and n- listed in table ) targeting the genome between nucleotides to was used to identify the presence of marker mutations, c t and t c, in the ipedvpt-p viral stock. the growth characteristics of ipedvpt-p and pedvpt-p in vero cells were evaluated and compared by examining the growth kinetics and plaque morphologies. confluent monolayers of vero cells were prepared on -well plates and infected with each virus at the multiplicity of infection (moi) . for h at • c in triplicate. to determine the growth kinetics, cells were washed twice with dpbs and then maintained in the pi medium. the supernatants at indicated time points, , , , and h post-inoculation (hpi), were collected and subjected to titration in vero cells seeded in -well plates. plaque assays were performed to characterize the plaque morphologies. after the adsorption of pedvs at moi . , vero cells were washed twice with dpbs and overlaid with pi medium containing % agarose (invitrogen, carlsbad, usa) pre-warmed to • c. upon solidification of the overlays, the plates were incubated for days at • c to allow pedv-infected vero cells to produce distinct plaques. the cells were fixed in . % neutral formalin for h at rt. the semisolid overlays were then removed manually and the cells were stained with % crystal violet in % ethanol and distilled water for min. the viral plaques were inspected after washing off the crystal violet, rinsing the plates with water, and air-drying at rt. the diameters of representative plaques for each virus were measured and compared. fifteen, -week-old, large white × duroc, crossbred piglets that were pedv-seronegative and pedv-shedding negative were selected from a conventional pig farm with no history of a g b pedv strain infection. these piglets were randomly assigned to three groups, including the pedvpt-p group (n = ), ipedvpt-p group (n = ), and mock group (n = ), and acclimated for one week prior to inoculation. at weeks of age, the piglets in each group were orally inoculated with ml of × tcid /ml of pedvpt-p , × tcid /ml of the pedvpt-p virus, or pi medium, respectively. to evaluate the protective efficacy induced by ipedvpt-p , piglets at weeks of age in all groups were orally challenged with ml tcid /ml of pedvpt-p . the animal experimental procedure was reviewed and approved by the institutional animal care and use committee (iacuc) of the national taiwan university (taiwan, republic of china) with approval no.: ntu el- . fecal consistency was monitored daily and scored visually as = normal, = loose, = semi-fluid, and = watery, as described previously [ ] . the body weight of each piglet was measured weekly. to quantitate the viral rna in stools, fecal samples collected from rectal swabs were resuspended in µl dpbs, pulse-vortexed for s and precipitated by centrifugation at , rpm for min. rna was extracted automatically from µl of stool suspension on a qiacube using the cador pathogen qiacube ht kit (qiagen, hilden, germany) according to the manufacturer's instructions. cdna was reverse-transcribed using a quantinova reverse transcription kit (qiagen, hilden, germany) following the manufacturer's protocols and it was used for quantitative real-time pcr analysis (qpcr). qpcr was conducted using the previously published primer-probe set [ ] and a quantinova ® probe pcr kit (qiagen, hilden, germany) on a cfx thermal cycler (bio-rad, hercules, ca, usa). the thermal profile comprised an initial denaturation at • c for min followed by cycles of • c for s followed by • c for s. the detection limit of the assay was determined by generating standard curves from serial -fold dilutions of known amounts of in vitro transcribed rna followed by reverse transcription and qpcr quantification as described above. the detection limit was calculated as rna copies per ml. to detect pedv specific plasma igg and fecal and salivary iga, an in-house, pedv s protein based indirect enzyme-linked immunosorbent assay (elisa) was conducted as described in the previous study [ ] . in brief, -well, flat-bottom microtiter plates (nunc, roskilde, denmark) were coated with µg/ml purified recombinant pedvpt s protein ( ng/well) and incubated overnight at • c. the plates were washed six times with µl of pbst (pbs containing . % tween ) and then blocked with µl of blocking buffer ( % bovine serum albumin in pbs) at rt for h. for the detection of plasma igg, µl of -fold diluted plasma samples in blocking buffer were added following six washes and incubated at rt for h. for fecal and salivary iga, µl of eluted fecal suspension and saliva at : dilution in blocking buffer were added following six washes and kept overnight at • c. after incubation, the samples were discarded and the plates were washed six times. to detect plasma igg, and the fecal and salivary iga, µl of either horseradish peroxidase (hrp) conjugated goat anti-pig igg (kirkegaard & perry laboratories, milford, ma, usa) at : dilution, or goat-anti-pig iga (abcam, cambridge, uk) diluted : , were added, respectively, and incubated at rt for h. following a wash step, µl of tetramethylbenzidine (tmb) substrate solution (kirkegaard and perry laboratories) was added to allow color development at rt for min. the reactions were terminated by adding µl of tmb stop solution (kirkegaard and perry laboratories) to each well. the optical density (od) at nm was measured on an elisa reader (molecular devices, sunnyvale, ca, usa). the antibody titers were expressed as sample-to-positive control ratio (s/p ratio) values. plasma samples of piglets were heated at • c for min to inactivate complement prior to use. for each well, mixtures containing µl of pedvpt-p virus ( viral particles) and µl of -fold diluted plasma samples in pi medium were incubated at • c for h before applying to vero cells ( × /well). after incubation with the virus-plasma mixture for h, vero cells were washed twice and maintained in pi medium for h. cytopathic effects were detected using inverted light microscopy (nikon, tokyo, japan). the neutralizing titer was defined as the highest dilution without cpe. the results of body weight, antibody titers, viral titer of growth kinetics at each time point, and fecal viral shedding were analyzed statistically on graphpad prism . (graphpad software, san diego, ca, usa) with two-way anova by time. a p value less than . was considered statistically significant. to generate the full-length cdna clone of ipedvpt-p , the complete genome sequence of pedvpt-p was split into six fragments by designing primer pairs fused with specific type-iis restriction enzymes. after the restriction enzyme digestion and dna ligation, transcripts containing the full-length ipedvpt-p sequence were successfully prepared. typical pedv-associated cytopathic effects in vero cells, characterized by multinucleated giant syncytia, were observed at about one day post-electroporation ( figure a) . the presence of ipedvpt-p was confirmed by the detection of the pedv n protein by immunocytochemistry ( figure a ) and sequence analyses for identification of the introduced substitutions, c t and t c ( figure b ). after an additional day, recombinant viral supernatants were harvested when the cpe reached % of the confluent monolayer. an ipedvpt-p viral stock with a titer of . × tcid /ml was prepared by an additional passage of the viral supernatants in the vero cells. to characterize the recombinant ipedvpt-p , plaque morphologies and growth kinetics of the recombinant ipedvpt-p in vero cells were compared to those of the parental pedvpt-p virus. interestingly, aside from the constantly short diameter, the plaque size of ipedvpt-p appeared to be more uniform than that of pedvpt-p ( figure c ). as to the replicative capacity, the multistep growth kinetics of both the ipedvpt-p and pedvpt-p viruses in vero cells were examined. at moi of . , while pedvpt-p replicated rapidly to the titer of . ± . tcid /ml at hpi, reached the peak viral titer of . ± . tcid /ml at hpi, and showed a declined viral titer of . ± . tcid /ml at hpi. ipedvpt-p showed a steady increase in viral progenies and a significantly decreased viral titer of . ± . tcid /ml at hpi, a comparable viral titer of . ± . tcid /ml at hpi, and a significantly high viral titer of . ± . tcid /ml at hpi ( figure d ). at mois of . and . , similar to those observed at . moi, ipedvpt-p replicated slower in the beginning, reached a similar peak viral titer as compared to pedvpt-p and showed a relatively slower reduction of viral titer after reaching the peak titer (see figure s ). to evaluate the pathogenicity of the ipedvpt-p virus, -week-old crossbred piglets were orally inoculated with ml of × tcid /ml of the ipedvpt-p virus, pedvpt-p virus, or pi medium. the clinical impact of viral inoculation in each piglet was evaluated by daily clinical fecal scoring, fecal viral shedding, and weekly body weight changes. during the study, no significant difference in body weight was revealed among the different groups at any indicated time points (figure ). while one piglet in the pedvpt-p group showed intermittent loose diarrhea (score = ) and viral shedding at to days post-inoculation (dpi) with the peak viral titer of . ± . log rna copies/ml at dpi (figure ) , no evidence of pedv-associated clinical signs and fecal viral shedding were demonstrated in both ipedvpt-p and mock groups. after the oral inoculation of piglets with the ipedvpt-p and pedvpt-p viruses, systemic pedv-specific igg in blood and mucosal specific iga in feces and saliva were measured at the indicated time points. compared to the mock group, an elevated mean s/p ratio of blood pedv-specific igg was detected at dpi and sustained until dpi in piglets from both the ipedvpt-p and pedvpt-p groups ( figure ) . moreover, subtle increases in the mean s/p ratios of fecal and salivary pedv-specific iga were also observed in both the ipedvpt-p and pedvpt-p groups compared to those of the mock group at dpi ( figure ). after oral inoculation with the virulent pedvpt-p virus, the daily fecal viral sheddings and fecal consistencies in all groups were evaluated. similar to our previous data [ ] , all piglets in the mock group showed the differing severity of clinical signs for diarrhea at - days post-challenge (dpc) (figure ) . the mean value of the pedv rna copies in feces of the mock group became detectable at dpc, shot up to a peak titer of . ± . log rna copies/ml at dpc, and then declined to a constantly low viral load of approximately log rna copies/ml at - dpc. in the pedvpt-p group, two piglets showed loose diarrhea (score = ) for one day at and dpc, respectively. the onset of fecal viral shedding in the pedvpt-p group after the challenge was further delayed to dpc compared to the other two groups. the highest viral titer of the pedvpt-p group also appeared at dpc, calculated as . ± . log rna copies/ml. in the ipedvpt-p group, all pigs remained clinically normal without an observation of diarrhea. compared to the mock group, only three of the five pigs in the ipedvpt-p group established fecal viral shedding through the experiment, with a delayed onset and a steadily fluctuated low viral shedding, ranging from the highest titer of . ± . to log rna copies/ml to undetectable levels ( figure ). in the mock group, the mean value of the s/p ratio of the systemic pedv-specific igg titers in the blood of piglets remained unchanged prior to the pedvpt-p challenge, after which the mean s/p value rose to . ± . at dpc ( figure ). on the other hand, the mean values of systemic pedv-specific igg in blood from the ipedvpt-p and pedvpt-p groups increased sharply to . ± . and . ± . at dpc, respectively, which was significantly higher than that of the mock group ( figure ). as for mucosal immunity, similar to the trend noticed in the systemic pedv-specific igg in blood, significant increases in the mean s/p values of both fecal and salivary anti-pedv specific iga were noted in the ipedvpt-p and pedvpt-p groups after challenge ( figure ). among them, the s/p values of salivary anti-pedv specific iga, . ± . and . ± . , in the ipedvpt-p and pedvpt-p groups, respectively, were significantly higher than those of the mock group ( . ± . ) at dpc (figure ) . the vn antibody titers of piglets in each group are depicted in figure . similar to the trend observed in systemic plasma igg titers, the ipedvpt-p group showed a comparable vn antibody titer against pedvpt-p to that of the pedvpt-p group through the study. the mean vn antibody titers of both groups increased mildly at dpc and spectacularly at dpc and were higher than those of the mock group during the study (figure ). data were expressed as the mean ± standard deviation. statistically significant differences between each group were examined using a two-way anova by time. in the present study, we described the first development of an infectious cdna clone of ipedvpt-p , and evaluated it's in vitro and in vivo characteristics. compared to the parental pedvpt-p virus, ipedvpt-p replicated more slowly in the beginning and reached a similar peak viral titer with similar but more uniform plaque sizes, suggesting that the composition of viral quasispecies in ipedvpt-p is less complex than that in the original pedvpt-p stock. moreover, neither fecal pedv rna shedding nor a pedv-associated clinical illness was detected in conventional -week-old piglets inoculated with the ipedvpt-p virus, indicating a further attenuated phenotype in vivo. importantly, piglets in the ipedvpt-p -inoculated group showed comparable levels of anti-pedv specific plasma igg, fecal/salivary iga, plasma neutralizing antibody titers, and a weakened but modest immunoprotection against the virulent pedvpt-p challenge compared to the parental pedvpt-p -inoculated piglets. taken together, our results suggest that ipedvpt-p is immunogenic in piglets and could be a potential safe viral vector candidate for vaccine development. while inoculation with ipedvpt-p was demonstrated to completely protect the piglets from developing diarrhea after challenge with the virulent pedvpt-p , the ipedvpt-p -inoculated group showed an earlier onset and longer duration of fecal pedv rna shedding with a higher peak value than that of the parental pedvpt-p -inoculated group. these data suggested that ipedvpt-p conferred a relatively weakened protection than that of the parental pedvpt-p . considering that the major variation between the pedvpt-p and ipedvpt-p viruses should be the heterogeneity of viral population as noted in plaque assays wherein the ipedvpt-p virus produced more uniform plaques than those of the parental pedvpt-p virus in vero cells, we speculate that the decrease in quasispecies diversity in ipedvpt-p may partly contribute to its further attenuation in vivo. to investigate the speculation, we conducted the next generation sequencing (ngs) to determine the quasispecies diversity of both viruses by exploring the recovered variants against our previous published sequence of pedvpt-p generated by sanger sequence (see table s ). the results clearly demonstrated that the pedvpt-p carried a greater sequence diversity than that of the ipedvpt-p , including single nucleotide variants (snv) and resultant amino acid substitutions; of interest, seven snv were found in the spike gene and of them were the same as the virulent pedvpt-p . for ipedvpt-p , excluding the artificially introduced marker mutations and those derived from the snv of pedvpt-p upon the initial construction of subclones of ipedvpt-p , only snv were uncovered. indeed, generating viruses with high-fidelity replication has been proposed as a rational strategy to develop genetically stable and safe attenuated vaccines [ , ] . however, for attenuated viruses, the in vivo fitness and antigenicity are already abated after serial cell culture passage. on the basis of quasispecies theory that the cooperative interplay between different variants determines viral characteristics including virulence [ , ] , it is possible that the consensus sequence used for constructing ipedvpt-p no longer sustained the original affinity of virus-host interaction or viral replication in enterocytes, which therefore impaired viral entry and/or limited the viral infection. furthermore, the limited diversity of the s gene in the ipedvpt-p viral stock might also diminish the potency and protective broadness of the induced antibody responses explaining the comparable level of systemic and mucosal antibody response but weakened protection in the ipedvpt-p -inoculated group. this speculation could be tested by comparing the tissue tropism and the quantity of pedv antigens of both pedvpt-p and ipedvpt-p in enterocytes using a -day-old piglet model. other explanation for the attenuation of ipedvpt-p might be attributed to the possible addition of the five nucleotides "ggaga" at the very extreme of the utr based on our primer design (see table ). considering the location that these additional nucleotides sequence should neither alter the secondary structures of the sl stem-loop, that serve as cis-acting elements required for driving subgenomic rna synthesis nor change any consensus transcription regulatory sequences -xua(a/g)ac- , we assumed that the effect of these additional nucleotides in the rescued ipedvpt-p virus on replication might be minimal. nonetheless, further studies are required to determine the potential effect of these five additional nucleotides on ipedvpt-p replication. in the present study, the profile of fecal pedv rna shedding and the pattern of antibody responses after inoculation with pedvpt-p appeared milder than those observed in our previous findings despite the fact that the same viral stock was used and that the conventional piglets used in this study were purchased from the same pig farm and housed in the same animal facility as described previously [ ] . that is, it seemed that the conventional piglets used in the current study were more resistant to the pedvpt-p infection. several host factors such as genetic variation, type of feed, gut microflora, and immune status among different litters at the time of inoculation may play an important role in the varying severity of clinical outcomes and immune responses [ ] . ideally, the discrepancy between the previous experiment might be minimized by expanding the size of groups, particularly if we chose conventional piglets as our target animals. nevertheless, due to the difficulty in collecting large numbers of pedv-negative piglets in taiwan where ped has become endemic, we decided to use five piglets per treatment to reach a statistical effect practice. although the use of conventional pigs often magnifies those variables and results in higher variation in the experimental results, to mimic the pathogenicity and immunoprotection of pedv in field conditions, the conventional pig model is still a preferred as a preclinical trial model. since the sudden appearance of the severe acute respiratory syndrome-coronavirus (sars-cov) in the early st century, the emergence and re-emergence of coronaviruses continually threatens the global public and animal health by causing severe illnesses, by having a high potential of zoonosis, and by causing great economic losses, indicating a desperate need for an effective and readily responsive vaccine platform. the large genome size of coronaviruses warrants a great tolerance for foreign genes and subsequent expression of heterologous antigens [ , ] . in addition, the insertion of other antigens by replacing the accessory orf gene or creating a novel expression cassette is also an attractive approach to design multivalent vaccines [ , , ] . under this concept, ipedvpt-p could be a potential safe vaccine backbone that facilitates the prompt generation of chimeric vaccines with the induction of mucosal immunity. for instance, viral antigenicity can be manipulated by replacing the ipedvpt-p s gene with that of other pedv or emerging swine coronaviruses although the potential loss of attenuation due to the s substitution requires further clarification. however, it is noteworthy that the inherent genetic instability of coronaviruses due to high recombination and mutation frequency is also a matter of concern as it raises the possibility of virulence reversion and acquisition of new tropism [ ] . besides, the intrinsic characteristics of the gene of interest, the targeted locus within the coronavirus genome, may also affect the expression level and stability of the recombinant viruses [ ] . accordingly, further characterization of the virulence variation and genetic stability of ipedvpt-p after different genetic modifications needs to be conducted. in this article, we described the first successful construction of an attenuated g b pedv infectious cdna clone of the ipedvpt-p virus and demonstrated the maintenance of fitness in vitro along with further attenuation in vivo. we also proposed that the initial low quasispecies diversity and the additional nucleotides at the end of ipedvpt-p may contribute to a further attenuated phenotype and potentially less effective immunoprotection against the virulent strain challenge. based on the results of antibody response, a prime-boost strategy or further optimization of ipedvpt-p antigenicity is essential to induce sufficient protective immunity in all recipients. together, the full-length cdna clone of ipedvpt-p generated herein is expected to provide an access to study the attenuation determinants of pedvpt-p and establish a pedvpt-p -based recombinant vector as a vaccine platform for developing multivalent vaccines for pedv and other porcine pathogens. a new coronavirus-like particle associated with diarrhea in swine genome organization of porcine epidemic diarrhoea virus new variants of porcine epidemic diarrhea virus, china pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs phylogenetic analysis of the spike (s) gene of the new variants of porcine epidemic diarrhoea virus in taiwan swine enteric coronavirus disease: a review of years with porcine epidemic diarrhoea virus and porcine deltacoronavirus in the united states and canada 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genomes: transmissible gastroenteritis virus model an efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol efficacy of heat-labile enterotoxin b subunit-adjuvanted parenteral porcine epidemic diarrhea virus trimeric spike subunit vaccine in piglets ribavirin-resistant variants of foot-and-mouth disease virus: the effect of restricted quasispecies diversity on viral virulence quasispecies diversity determines pathogenesis through cooperative interactions in a viral population viral quasispecies quasispecies theory in virology virus-based vectors for gene expression in mammalian cells coronaviruses as vectors: stability of foreign gene expression recombination in large rna viruses: coronaviruses. seminars in virology the authors would like to thank center for biotechnology, national taiwan university for next generation sequencing and bioinformatics and biostatistics core lab, center of genomic and precision medicine, national taiwan university for ngs data analysis. the authors declare no conflict of interest with respect to the research, authorship, and/or the publication of the article. key: cord- - voi y authors: han, hui-ju; liu, jian-wei; yu, hao; yu, xue-jie title: neutralizing monoclonal antibodies as promising therapeutics against middle east respiratory syndrome coronavirus infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: voi y since emerging in , middle east respiratory syndrome coronavirus (mers-cov) has been a global public health threat with a high fatality rate and worldwide distribution. there are no approved vaccines or therapies for mers until now. passive immunotherapy with neutralizing monoclonal antibodies (mabs) is an effective prophylactic and therapeutic reagent against emerging viruses. in this article, we review current advances in neutralizing mabs against mers-cov. the receptor-binding domain (rbd) in the spike protein of mers-cov is a major target, and mouse, camel, or human-derived neutralizing mabs targeting rbd have been developed. a major problem with neutralizing mab therapy is mutant escape under selective pressure, which can be solved by combination of neutralizing mabs targeting different epitopes. neutralizing mabs are currently under preclinical evaluation, and they are promising candidate therapeutic agents against mers-cov infection. middle east respiratory syndrome (mers) emerged in in saudi arabia with the death of a man with pneumonia; the causative agent was subsequently identified as mers-cov, which belonged to lineage c betacoronaviruses [ ] . with dromedary camels (camelus dromedarius, also known as arabian camel) as direct sources and bats as potential reservoirs [ ] , mers-cov has been frequently introduced into human populations. once mers-cov is introduced into a person, person-to-person transmission might occur, and is responsible for approximately % of mers cases globally [ ] . mers-cov has been a consistent threat to humans. as of october , mers-cov has caused laboratory-confirmed human cases, including deaths in countries, with the fatality rate as high as % (http://www.who.int/emergencies/mers-cov/en/). although mers cases are primarily reported in the middle east, facilitated by international travelling, mers-cov can also be a worldwide threat, which is well illustrated by the mers outbreak in south korea in [ ] . given the potential risk of causing worldwide public health emergencies and the absence of licensed vaccines and antiviral therapeutics, the world health organization has listed mers-cov in the "list of blueprint priority diseases" (http://www.who.int/blueprint/priority-diseases/en/). vaccines are the most important approach against viral infections, but usually take a long time to develop. they are also unable to provide either immediate prophylactic protection or treat ongoing viral infections. neutralizing monoclonal antibodies (mabs) have recently emerged as a powerful tool to provide prophylactic and therapeutic protection against emerging viruses [ ] . potent neutralizing mabs can be achieved by various technologies, such as hybridoma technology, humanized mouse, phage or yeast display, and single b cell isolation [ ] . mers-cov is a single, positive-stranded rna virus of about kb, which encodes four major viral structural proteins-including spike (s), envelope (e), membrane (m) and nucleocapsid (n)-as well as several accessory proteins [ ] . the s protein ( aa) plays an important role in virus infection and consists of a receptor-binding subunit s (aa - ) and a membrane-fusion subunit s (aa - ). s mediates viral attachment to host cells and s mediates virus-cell membrane fusion [ ] . the s subunit contains a receptor-binding domain (rbd) (aa - ) [ ] that can bind to cell receptor dipeptidyl peptidase (dpp , also known as cd ), and mediates viral attachment target cells [ ] . the rbd consists of a core subdomain and a receptor-binding motif (rbm) (aa - ). the schematic representation of mers-cov s protein is shown in figure a . neutralizing mabs binding to the s protein of mers-cov can prevent viral attachment to the cell receptor and inhibit viral entry [ ] . the s protein of mers-cov is a key target for antivirals, and rbd is the most popular focus. in this study, we review the current knowledge on neutralizing mabs targeting the rbd of mers-cov. stable hybridoma cell lines were generated by fusing myeloma cells with splenocytes of mice that were immunized with mers-rbd protein. two neutralizing mabs, c and e , had high affinity for the rbd of mers-cov and blocked both pseudovirus and live mers-cov entry into cells with high efficacy [ ] . humanized c showed similar neutralizing activity in cell entry tests. in vivo tests indicated that c could significantly reduce the virus titers in the lungs of ad -hcd -transduced mice which were infected with mers-cov, highlighting its potential application in humans not only for preventing but also treating mers-cov infection. crystallization of the c fab/mers-rbd complex showed that the c recognized conformational epitopes (y -n , k , l -k , p , v -s , w -e , and d -q ), which were partially overlapped the receptor-binding footprint in the rbd of mers-cov. the c complex interfered with mers-cov binding to dpp by both steric hindrance and interface-residue competition. e competed with c to bind to mers-rbd, indicating that they recognized proximate or overlapping epitopes [ ] . neutralizing mab mersmab was obtained by fusing myeloma cells with splenocytes of a mouse that was immunized with recombinant mers-cov s [ ] . mersmab effectively blocked the entry of pseudovirus and live mers-cov into cells. structural analysis showed that mersmab bound to the rbd of mers-cov through recognizing conformational epitopes, and all of the residues critical for mersmab binding were located on the left ridge of rbm. mersmab neutralized mers-cov by competitively blocking the binding of mers-cov rbd to dpp . based on escape mutant analysis of the key residues on the rbd, it was found that residue l , d , r , e , and w were critical for mersmab binding to the rbd, while mutation of e , d , or e did not affect the interaction of mersmab and the rbd at all [ ] . an ultra-large nonimmune human antibody-phage display library was constructed with b cells of unimmunized donors. with a unique spanning strategy, seven human neutralizing mabs with varying neutralization efficacy to mers-cov were identified [ ] . binding detection demonstrated that the epitopes of these mabs lay within aa - of the s protein, which overlapped a large part of the rbd of mers-cov. binding competition assays showed that these mabs recognized at least three distinct epitope groups, which was further confirmed by escape studies. with no cross-epitope resistance, these mabs neutralized mers-cov by competitively blocking the binding of the rbd of mers-cov to dpp . escape mutant assays showed that five residues were critical for neutralization of these mabs, namely l , t , y , r , and p . of the seven mabs, b exhibited the best neutralization activity against both pseudovirus and live mers-cov infectivity in cells. moreover, under the selective pressure of these mabs, the igg form of b was superior, since it did not induce neutralization escape [ ] . in vivo tests demonstrated that b reduced lung pathology in rhesus monkeys infected with mers-cov [ ] . with its high neutralizing activity and suppression of mutant escape, b in the igg form is a promising therapeutic mab against mers-cov. three human mabs-m , m , and m -were identified from a large naïve human phage display antibody library, which was constructed with peripheral blood mononuclear cells from healthy volunteers [ ] . the binding sties of the three mabs were within the rbd of mers-cov (aa - ), therefore they neutralized mers-cov by competing with dpp binding to the rbd. the three mabs also competed with each other to bind to the rbd of mers-cov, and mutant analysis showed that the three mabs possessed overlapping but distinct epitopes. of the three mabs, m neutralized both pseudovirus and live mers-cov infectivity in cells with exceptional potency (m inhibited % mers-cov pseudovirus infection at a concentration of . g/ml, and neutralized live mers-cov with ic of g/ml and ic of . g/ml). residues in the rbd crucial for m binding were l , d , e , d , w , and v [ ] . in vivo study demonstrated that prophylaxis with m reduced virus titers in the lung of rabbits infected with mers-cov [ ] , and m also provided transgenic mice expressing human dpp with full prophylactic and therapeutic protection from mers-cov [ ] . however, another study with a non-human primate, the common marmoset showed that m could only alleviate the severity of the disease, and did not provide complete protection against mers-cov [ ] . igg+ memory b cells were isolated from a mers patient, and were subsequently immortalized with epstein-barr virus. a neutralizing mabs, lca , was identified, and was the first fully human neutralizing mab with naïve heavy and light chain pairs [ ] . lca efficiently neutralize mers-cov infectivity in cells. in vivo study showed that lca provided balb/c mice transduced with adenoviral vectors expressing human dpp (hdpp ) with both prophylactic and postexposure protection against mers-cov. furthermore, the neutralizing efficacy of lca was evaluated in ifn-α/β receptor-knockout mice that were more stringent models of mers-cov infection. after transducing with hdpp , these mice showed more profound clinical symptoms when challenged with mers-cov [ ] ; administration of lca reduced mers-cov titer in the lungs of these mice more effectively (lung viral titer reduced by three logs in one day for ifn-α/β receptor-knockout mice vs. three days for balb/c mice) [ ] . with naïve heavy and light chain pairs, lca was more potent than b and comparable to m . cross-competition experiment demonstrated that lca competed with b to bind to the rbd. lca interacted with rbd residues around k , and the lca footprint on the rbd was partially overlapped with that of dpp . four residues in the rbd affected the binding of lca -namely t , k , e , and e -which were conserved in all mers-cov isolates. moreover, compared with dpp , the binding affinity of lca to rbd was significantly higher (~ -fold). therefore, one major neutralization mechanism of lca was to competitively inhibit the interaction of the rbd with dpp . interestingly, virus escape studies demonstrated that under the selective pressure of lca , a mutant variant (v a) in the n-terminal domain (ntd) of mers-cov s subunit was also generated [ ] . a gmp-approved cell line (lca . . ) that expresses lca in high concentrations has been established, highlighting its application as promising therapeutics against mers-cov infection [ ] . hybridoma b cells producing neutralizing mabs against the s protein of mers-cov were generated by immunizing humanized transgenic mice (velocimmune mice) with dna encoding the mers-cov s protein. two fully human neutralizing mabs, regn and regn , were obtained [ ] . the two mabs bound with high affinity to distinct epitopes on the rbd of mers-cov, which were conserved during the natural evolution of mers-cov. mutation as a result of selective pressure by one mab should not affect the binding of the other mab. regn neutralized a broad range of mers-cov isolates, the prototype emc/ strain and all clinical mutants including a p, s g, s f, a v, l f, d g, and v a. with the exception of v a variant, regn achieved similar neutralizing activity. in vivo study demonstrated that regn and regn reduced mers-cov replication in humanized dpp mice in both prophylactic and therapeutic settings [ ] . when evaluated in the common marmoset, both mabs seemed to be more effective for prophylaxis rather than for treatment of mers-cov infection [ ] . an anti-mers-cov phage display antibody library was constructed with the peripheral b cells of a mers survivor, and a human neutralizing mab against mers-cov, mca , was identified [ ] . mca showed potent neutralizing activity against mers-cov in cell entry tests. in vivo, mca completely inhibited the replication of mers-cov in common marmosets when administrated prophylactically or therapeutically. structure analysis of the mca fab-rbd complex showed that mca formed direct contacts with the receptor-binding site (rbs) subdomain on the rbd. epitopes on the rbs critical for mca binding were d , w , e , d , y , r , and q . superimposed structure analysis of mca -rbd and hdpp -rbd complexes showed that the binding interface of mca was largely overlapped with that of hdpp . therefore, the neutralizing mechanism of mca was achieved by competing with dpp for binding to the rbd [ ] . two potent human neutralizing mabs, mers- and mers- , were derived from a nonimmune human yeast display antibody library, which was constructed with spleen and lymph node polyadenylated rna from normal humans [ ] . [ ] . further structural analysis showed that mers- bound to unique epitopes and caused conformational changes in the rbd interface critical for accommodating dpp , therefore indirectly disrupting the interaction between the two. moreover, mers- also demonstrated synergistic effects with m and f (a ntd-specific mab). the special neutralizing mechanism made mers- a valuable addition for the combined use of mabs against mers-cov infection [ ] . thirteen ultrapotent neutralizing mabs, which all targeted the rbd of mers-cov were generated following a protocol for the rapid production of antigen-specific human mabs [ ] . briefly, antibody-secreting b cells were isolated from the whole blood of a mers patient, and the antibody genes were amplified and cloned into vectors to transfect human cell lines for mab production. of the mabs, mers-gd and mers-gd exhibited the strongest neutralizing activity against both pseudovirus and live mers-cov in cell infection tests. mers-gd directly competed with dpp to bind to the rbd to dpp , and the crystal structure of mers-gd showed that its epitopes were almost completely overlapped with dpp -binding sites. mers-gd and mers-gd recognized distinct epitopes on the rbd, and had a low level of competing activity. the combined use of the two mabs demonstrated synergistic effects in neutralization against pseudotyped mers-cov. mutant analysis demonstrated that residues l , d , v , e , and a on rbd were important for the neutralizing activity of mers-gd , and residue r was critical for mers-gd [ ] . moreover, in vivo study found that mers-gd could provide both prophylactic and therapeutic protection for hdpp -trangenic mice against mers-cov infection [ ] . dromedary camels exposed to mers-cov showed mild clinical signs but developed exceptionally potent neutralizing antibodies. camelid species naturally produced heavy chain-only antibodies (hcabs) [ ] , which are dimeric and devoid of light chains, and their antigen recognition region is solely formed by the variable heavy chains (vhhs) (also called nanobodies, nbs). vhhs or nbs have long complementarity-determining region (cdr ) loops and are capable of binding to unique epitopes not accessible to conventional antibodies [ ] . notably, camelid vhhs are relatively stable and can be produced with high yields in prokaryotic systems [ ] . because of their small size; good tissue permeability; and cost-effective production, storage, and transportation [ ] [ ] [ ] , vhhs or nbs have been gaining acceptance as antiviral agents. a vhh complementary dna library was constructed with the bone marrow of dromedary camels infected with mers-cov. four vhhs (vhh- , vhh- , vhh- , and vhh- ) with high neutralizing activity were identified by direct cloning and screening of the phage display antibody library [ ] . the four vhhs competed for a single epitope that partially overlapped with the rbd-dpp interface. mutant analysis showed that the four vhhs did not bind to the d n variant, which was a critical residue on the rbd for dpp binding [ , ] . therefore, these vhhs most likely neutralized mers-cov by blocking its binding to dpp . of the vhhs, vhh- showed the best neutralizing activity and epitope recognition. vhh- efficiently blocked the entry of mers-cov into cells, and it also prophylactically protected k transgenic mouse expressing hdpp from mers-cov infection. to extend the half-life of vhh- in serum, it was linked to a human fc domain lacking the ch exon to construct the chimeric camel/human hcab- , which showed similar neutralizing activity as vhh- . the chimeric camel/human hcab- was highly stable in mice and provided k mice with fully prophylactic protection against mers-cov infection [ ] . alpacas were immunized with recombinant mers-cov rbd-containing a c-terminal human igg fc tag, and vhhs were amplified from their peripheral blood mononuclear cells to construct a vhh phage display library. a neutralizing nb, nbms , which bound with high affinity to the rbd of mers-cov and blocked the binding of rbd to dpp , was identified [ ] . to extend its in vivo half-life, the human-fc-fused version, nbms -fc, was constructed. nbms competed with dpp to bind to rbd, indicating that the binding site of nbms on rbd overlapped with that of dpp . the binding site of the nbms on the rbd was mapped to be around residue d , which is part of a highly conserved conformational epitope at the receptor-binding interface in almost all the natural mers-cov published to date. nbms did not neutralize psuedotyped mers-cov bearing a mutation in d , confirming that residue d was critical for nbms binding. nbms efficiently neutralized the cell entry of live mers-cov. moreover, nbms showed potent prophylactic and therapeutic efficacy in protecting hdpp -transgenic mice against mers-cov infection [ ] . for their exceptionally high neutralization activity in vitro and in vivo, these newly identified neutralizing mabs are promising candidate therapeutics against the infection of mers-cov. however, the use of a single neutralizing antibody bears the risk of selecting escape mutants, a fact that has been observed for lca and other described antibodies [ , , ] . notably, the majority of these escape mutations had little impact on viral fitness and the interaction of dpp with the rbd [ ] . moreover, mutants of mers-cov during natural infection have also been reported [ ] . escape from neutralization is a major concern with therapeutic neutralizing mabs, however, this potential problem can be solved by combining mabs that target distinct epitopes and show different neutralizing mechanisms [ ] . this strategy can take advantage of the synergistic effects while decreasing the possibility of viral escape. currently, most of the mers-cov neutralizing mabs compete with dpp binding to the rbd, and residues on the rbd critical for mab neutralization are identified by mutant analysis. almost all of the residues identified critical for mab neutralization are located in rbm, and overlap with those critical for dpp binding ( figure b) . with the availability of crystal structure of mab fab-rbd complex, the neutralization mechanism of these mabs will be better illustrated. based on the crystal structure of rbd-dpp , it was found that several conserved residues in the rbd are critical for the interaction of the rbd with dpp (y , l , d , e , e , d , d , y , r , w , and v ) [ , ] . development of therapeutic neutralizing mabs targeting those critically conserved residues might be important for combating mers-cov. moreover, a study found a mouse-derived neutralizing mab, f , which bound to a possible linear epitope in the ntd of the mers-cov s subunit, exhibited efficient neutralizing activity against pseudovirus and live mers-cov in cell entry tests. this study highlighted the important role of ntd during the infection process of mers-cov. ntd might have significant implications for the development of prophylactic and therapeutic mabs against mers-cov infection [ ] . although the in vitro neutralizing potency of f was approximately -fold lower than that of the rbd-targeting neutralizing mabs [ ] , it may provide an alternative for the immunotherapy against mers-cov, once the virus mutates and is no longer susceptible to rbd-specific mabs. so far, there is a lack of appropriate animal models to mimic the pathology of merd-cov in humans. commonly-used laboratory animals-such as wild-type mouse, ferret, hamster, and guinea pig-are not susceptible to mers-cov infection due to differences in critical amino acids in the s-binding domain of their dpp [ ] [ ] [ ] . new zealand rabbits, hdpp -transduced/transgenic mice, camelids and non-human primates (rhesus macaque and common marmoset) are susceptible to mers-cov infection, however, rabbits showed asymptomatic infection [ ] ; dromedary camels displayed different clinical manifestations to that of humans [ ] ; rhesus macaque only showed transient lower respiratory infection [ ] , while common marmoset developed progressive pneumonia [ ] ; hdpp -trangenic mouse expressed hdpp extensively, and resulted in multiple organ damage [ ] ; hdpp -transduced mouse only exhibited mild transient clinical diseases [ ] . with robust animal models, the protective effects of these neutralizing mabs will be better evaluated. furthermore, ongoing efforts on developing therapeutic neutralizing mabs against mers-cov should also consider the different target populations (dromedary camels and humans) and their protective efficacy. isolation of a novel coronavirus from a man with pneumonia in saudi arabia evidence for zoonotic origins of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea human monoclonal antibodies as candidate therapeutics against emerging viruses. front genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans mers-cov spike protein: a key target for antivirals structure of mers-cov spike receptor-binding domain complexed with human receptor dpp dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution b -n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n / . virology exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection efficacy of antibody-based therapies against middle east respiratory syndrome coronavirus (mers-cov) in common marmosets prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus rapid generation of a mouse model for middle east respiratory syndrome rapid generation of a human monoclonal antibody to combat middle east respiratory syndrome pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection prophylactic and therapeutic efficacy of mab treatment against mers-cov in common marmosets human neutralizing monoclonal antibody inhibition of middle east respiratory syndrome coronavirus replication in the common marmoset potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein structural definition of a unique neutralization epitope on the receptor-binding domain of mers-cov spike glycoprotein ultrapotent human neutralizing antibody repertoires against middle east respiratory syndrome coronavirus from a recovered patient a novel human mab (mers-gd ) provides prophylactic and postexposure efficacy in mers-cov susceptible mice naturally-occurring antibodies devoid of light-chains molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies nanobodies: natural single-domain antibodies application of camelid heavy-chain variable domains (vhhs) in prevention and treatment of bacterial and viral infections nanobodies(r) as inhaled biotherapeutics for lung diseases generation and characterization of alx- , a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection chimeric camel/human heavy-chain antibodies protect against mers-cov infection molecular basis of binding between novel human coronavirus mers-cov and its receptor cd a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on mers-cov spike to avoid neutralization escape severe respiratory illness caused by a novel coronavirus identification of residues on human receptor dpp critical for mers-cov binding and entry a novel neutralizing monoclonal antibody targeting the n-terminal domain of the mers-cov spike protein wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase -mediated entry of the middle east respiratory syndrome coronavirus asymptomatic middle east respiratory syndrome coronavirus infection in rabbits experimental infection of dromedaries with middle east respiratory syndrome-coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase an animal model of mers produced by infection of rhesus macaques with mers coronavirus infection with mers-cov causes lethal pneumonia in the common marmoset multi-organ damage in human dipeptidyl peptidase transgenic mice infected with middle east respiratory syndrome-coronavirus funding: this study was supported by a grant from national natural science funds of china (nos. ). the authors declare that they have no conflict of interest. key: cord- - l lb authors: pedersen, niels c.; liu, hongwei; dodd, kimberly a.; pesavento, patricia a. title: significance of coronavirus mutants in feces and diseased tissues of cats suffering from feline infectious peritonitis date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: l lb the internal fecv→fipv mutation theory and three of its correlates were tested in four sibs/half-sib kittens, a healthy contact cat, and in four unrelated cats that died of fip at geographically disparate regions. coronavirus from feces and extraintestinal fip lesions from the same cat were always > % related in accessory and structural gene sequences. snps and deletions causing a truncation of the c gene product were found in almost all isolates from the diseased tissues of the eight cats suffering from fip, whereas most, but not all fecal isolates from these same cats had intact c genes. other accessory and structural genes appeared normal in both fecal and lesional viruses. deliterious mutations in the c gene were unique to each cat, indicating that they did not originate in one cat and were subsequently passed horizontally to the others. compartmentalization of the parental and mutant forms was not absolute; virus of lesional type was sometimes found in feces of affected cats and virus identical to fecal type was occasionally identified in diseased tissues. although c gene mutants in this study were not horizontally transmitted, the parental fecal virus was readily transmitted by contact from a cat that died of fip to its housemate. there was a high rate of mutability in all structural and accessory genes both within and between cats, leading to minor genetic variants. more than one variant could be identified in both diseased tissues and feces of the same cat. laboratory cats inoculated with a mixture of two closely related variants from the same fip cat developed disease from one or the other variant, but not both. significant genetic drift existed between isolates from geographically distinct regions of the western us. feline infectious peritonitis (fip) was first introduced as an "important disorder of cats" by holzworth [ ] and a clinico-pathologic conference on the disease was published the following year [ ] . the incidence of fip rose progressively over the next two decades. the occurrence of fip among all cats seen at veterinary medical teaching hospitals in the usa from - was : among new feline visits, : among total cat accessions, and % of accessions at diagnostic laboratories [ ] . the incidence is several times higher among kittens and young cats originating from catteries or shelters. the disease was thought to be viral when first described but no specific etiologic agent was identified at the time [ ] . zook et al. [ ] observed virus particles in the tissues of experimentally infected cats, however, the close similarities of fip virus (fipv) in tissues to members of the family coronaviridae was noted by ward [ ] . the ability of fipv to cause either a non-effusive (dry, parenchymatous) or effusive (wet, non-parenchymatous) form of the disease was first reported by montali and strandberg [ ] . the close genetic relationship of fipv to coronaviruses of dogs and swine was first recognized by pedersen et al. [ ] . the existence of two serotypes, feline-or canine-coronavirus like, was described in [ ] . fip was originally believed to be an uncommon clinical manifestation of a ubiquitous and largely nonpathogenic agent named feline enteric coronavirus (fecv) [ ] . subsequent studies demonstrated that the agent of fip was distinct from fecv in disease potential but that both viruses co-existed in the same population and were antigenically identical [reviewed in , ] . it was subsequently hypothesized that fipv might be a simple mutant of fecv [ ] , and the two viruses were later described as biotypes of each other [ ] . animal studies, with both natural [ ] and experimental [ ] infection, also suggest that fipvs arise spontaneously during the course of fecv infection. vennema et al. [ ] demonstrated that all major structural and accessory genes of wild type fecvs were virtually identical to fipvs from the same or closely related cats. however, % of fipvs studied had deleterious mutations in a small accessory gene called c. these mutations, which were either deletions or introduced stop codons, were also found to be unique to each cat. in spite of indirect and direct supporting evidence for internal fecv→fipv mutation, the role of fecv mutation in fip, and especially in the c gene, has not been given much attention in the literature of fip [reviewed ] . in fact, there is a general feeling that fipv and fecv are either the same virus, with disease being dependent on the nature of the host's immune response [reviewed ] , or that the causative mutation is in other genes [ ] . although the precise origin of fipvs is debated, there appears to be agreement regarding the relative cell tropisms of fecvs and fipvs. fecvs are thought to have greater tropism for the mature apical intestinal epithelium, while fipvs are believed to have a greater tropism for macrophages [reviewed ]. this has led to the a strongly held belief that coronaviruses found in the feces are fecv-like, while viruses found in extra-intestinal (usually lesional) tissues are fipv-like [ ] . the purpose of this study was to repeat the original work of vennema et al. [ ] with a new and geographically diverse group of cats and to test the major tenant of the fecv→fipv theory and three of its possible correlates. the major tenant of the theory assumes that functional mutations in the c gene are somehow related to the fip biotype. the first correlate of this theory supposes that each fip cat will have its own unique c mutant which is not transmitted cat-to-cat. the second correlate assumes compartmentalization of enteric and fip biotypes to gut and internal tissues, respectively. the third correlate, if correct, should show fipvs to be as geographically diverse as the fecvs from which they arise complete structural (s, e, m, n) and accessory ( a-c and a, b) gene sequences were obtained from diseased omentum of the four related cats that died of fip and the isolates designated were fipv-ucd , , and ( table ). the numbers of nucleotides sequenced for isolates fipv-ucd to ucd are shown in table , while the relationship of the fipvs isolated from the related scottish fold cats is shown in figure . the overall sequence identity for the nine structural and accessory genes was ≥ % with only a small number of mutations among the four highly related viruses (table ) . mutations consisted of minor snp changes, and less commonly deletions that appeared to be randomly scattered among the genes that were sequenced; about one half of the mutations resulted in amino acid changes ( table ). among the structural and accessory genes of the four related cats, the highest genetic variability was in the c gene, followed by the s and m genes ( table ). the least variability was detected in the b, a, and a accessory genes. among all of the genes sequenced, only the c genes of the fipv isolates had snps that resulted in premature stop codons or deletions that caused frame shifts; both resulting in a variable truncation of the c protein ( figure ). the omentum viruses from red consisted of two distinct variants, as determined by sequences obtained from multiple overlapping pcr products (table ) . these variants were designated fipv-ucd a and -ucd b. there were only five snps scattered across the nine structural and accessory genes between the two variants. two variants were also sequenced from the omentum of toby, one with a non-functional c gene (fipv-ucd ) and one with a functional c gene (fecv-ucd ). these two variants were identical in sequence except for a single-base deletion in the c of one of the variants (fipv-ucd ). four additional unrelated cats ( , and ) from paradise, menlo park, and san jose, ca, respectively, and cat-t from mountlake terrace, wa were included in the study. the three cats seen at the vmth suffered from the non-effusive form of fip, while the washington state cat died of effusive fip. the e, m, n, a-c and a, b genes were amplified from the omentum or organ granulomas of all four animals. viruses were readily detected in the diseased tissues of cats , , cat-t, and and designated fipv-ucd to ucd , respectively (table ) . fipv-ucd a possessed a two-nucleotide deletion near the end of the c gene and a second deletion of nucleotide involving the terminus of b and beginning of c ( figure ). mutations of the c gene in fipv-ucd and -ucd involved premature stop codons. two variants with six scattered snps and an identical deletion in the c genes were identified in organ granulomas of cat and designated fipv-ucd a and -ucd b (table ). all of the structural and accessory genes that were sequenced for the eight different fip cats appeared to be intact, except for the c genes. the c genes from all eight isolates contained deletions or snps that either produced truncating frame shifts or premature stop codons ( figure ). the sequence relationship of the four unrelated fipv isolates to each other and to the fipv isolates from the four related scottish fold cats is shown in figure . the overall genetic similarity for the e, m, n, and a-c, a, b genes ranged from - % among the fipv isolates. the four fipvs from unrelated cats showed sequence identity of - % to each other and to the fipvs from the four related cats. feces or colonic scrapings from the four related cats and a fifth unrelated housemate contained feline coronaviruses ( table ). the amount of viral rna in feces in cats with fip was much lower than in diseased omentum and obtaining complete sequences of all genes was not always possible. therefore, the actual genes sequenced for each fecal coronavirus isolate are shown in table . coronaviruses isolated from the feces of two cats, tux and toby, were ≥ % identical and contained identical c gene mutations to the omental viruses from the same cats. the coronavirus isolated from lucy's feces (designated fecv-ucd ) had an intact (i.e., wild type or non-deliterious) c and its sequence was otherwise % identical to the sequence of fipv-ucd found in her diseased omentum. the sequence obtained from the fecal virus of simba, a housemate of lucy, also had an intact c gene and was designated fecv-ucd . fecv-ucd , was most closely related to the fipv isolated from lucy and was . % related to the consensus nucleotide sequences of coronaviruses obtained from the four related fip cats ( figure , table ). c there were snps between fipvs found in colonic scraping and the diseased omentum. d the biotypes of the virus isolated from the feces of these cats were not determined due to an inability to amplify the c gene. a similar finding was found for the cats that were unrelated to those described above and that were from disparate geographic regions. three of four fecal samples ( , and cat-t) contained amplifiable rna and complete c genes were sequenced in / of these cats ( and ). the c gene sequence of the fecal virus of cat was intact and ≥ % related to the fipv found in diseased tissue ( table ). this fecal isolate was designated fecv-ucd . the c gene of fecal virus contained a deliterious two-nucleotide deletion near the end of the c gene and was designated fipv-ucd b. this same deletion was also detected in the lesional fipv-ucd a. however, fipv-ucd a did not contain the -nucleotide deletion involving b and c of fipv-ucd b ( figure ). only the a, b genes were sequenced from the feces of cat-t and the sequence was % identical to the a, b sequence from the omental fipv-ucd . this study of lesional and/or fecal coronaviruses from nine cats both supported and modified the previous conclusions of vennema et al. [ ] . viruses from diseased tissues from all eight cats in this study had truncating mutations, either in the form of deletions leading to frame shifts or coding changes causing premature stop codons in the c gene. such damaging mutations were not present in other accessory and structural genes in this or in a previous study [ ] . as with the earlier study [ ] , all or almost all of the fecal isolates from diseased cats and a healthy contact control animal had intact c genes. taken as a whole, the present study supported a role for deleterious c gene mutations in the genesis of fipvs from fecvs. however, not all fipv isolates have deleterious c gene mutations. although / ( %) of lesional isolates in the present study had functional mutations in their c genes, only / ( %) of the fipvs reported by vennema et al. [ ] had deliterious c gene mutations. we have also recently observed what appeared to be intact c genes in / random breed cats that were adopted from a large shelter in northern california and died of fip. however, several of these isolates contained mutated c genes as minor variants, and without animal inoculation studies it is not possible to say whether or not these or the remaining isolates were capable of causing fip. the existence of helper/defective virus replication in the latter situation also needs to be considered. animal inoculation studies to determine the biotype of a given feline coronavirus are critical for determining the ultimate biotype of any isolate, regardless of its sequence regularities or irregularities. it was therefore important to demonstrate herein that an isolate from the four related cats reported herein was capable of causing fip. since some fipvs appear to have intact c genes, it may be premature to ascribe the fip biotype solely to deleterious mutations in the c gene. however, what are the alternatives? it can be argued that mutations in the conserved replicase/transcriptase genes may have a similar effect; that small mutations in other structural and accessory genes, collectively or singly, will have the same effect; that fipv and fecv are identical viruses; or that deleterious c gene mutations are an effect of the disease and not its cause. involvement of the replicase/transcriptase genes is unlikely, because the replicase/transcriptase region is highly conserved among feline coronaviruses and unlikely to be involved in cell tropism or evasion of the host's immune response. one study of a natural serotype i fipv isolate (c je) showed a high degree of sequence conservation within the replicase/transcriptase genes compared to other feline coronaviruses, while a premature stop codon limited the c gene product to the first amino acids [ ] . it is also unlikely that mutations in other accessory or structural genes are involved, even though such mutations have been frequently found in feline coronaviruses. firstly, c gene mutations in fipvs occur significantly out of proportion to mutations in other structural or accessory genes. secondly, there is little scientific evidence, especially based on animal inoculation, that other accessory genes are involved in fip. in the original report that proposed the internal mutation theory, / of the fipvs had c mutations, while / isolates had only b mutations [ ] . however, both of the latter cats were related and had been experimentally infected with an identical fecv (fecv-rm); a third sibling cat from this group had the same b mutation but with a unique functional c mutation. variants were not tested at the time and it is possible that c mutants would have been present if the two discordant isolates had been adequately sequenced. earlier studies have also demonstrated an absence of b mutations in almost all fecvs and other fipvs and indicate that such mutations are most likely tissue culture artifacts [ , ] . yet other studies suggest that a and b mutations occur in nature in both fip and enteric infections and are therefore not directly linked to pathogenicity [ , , ] . there is a general belief that host and environmental factors, and not virus mutation, are the basic determinants of whether a cat develops fip or just a mild enteritis following exposure to the common feline coronavirus [ ] [ ] [ ] [ ] [ ] . for such a theory to be correct, fecvs and fipvs would have to be identical in both genetic structure and virulence. the evidence that fecvs and fipvs cause very different diseases is strong [ ] [ ] [ ] [ ] . even though environmental and host factors are admittedly important in fip [ , ] , lesional viruses from the eight fip cats in this study, even though highly related to fecal isolates, were easily differentiated from each other based on deliterious c gene mutations alone. moreover, an infectious inoculum made from the diseased omentum of one of the fip cats induced fip in of cats that were experimentally infected (see section . .). confirmation of biotype by animal inoculation, such as described herein, is rarely done in published reports concerned with feline coronavirus infection [reviewed in ] . the possibility that deleterious c gene mutations are an effect of the disease and not a cause also has to be considered. however, there is little precedence for this and given the ability of a lesional isolate from the present study to produce fip, it is counterintuitive for a functional c gene mutant to be both a cause and effect of its own disease. this theory would also not explain why all non-tissue culture adapted fecv strains used for experimental inoculation studies have intact c genes, while all tissue culture adapted and non-adapted strains have mutated c genes [ ] . the existence of feline coronavirus variants was not a novel observation [ , , ] , but their frequency and fate has not been previously addressed. variant forms were found in both extraintestinal tissues and feces of the cats in this study, but only one variant became predominant upon experimental passage from one cat to another (see section . ). the infecting variant may have been merely the first virus into a macrophage, or its selection may have involved more complex host/virus interactions. we also found that subtle, and sometimes significant, genetic mutations (usually snps and deletions) occurred upon primary replication in a new host. therefore, genetic variation among feline coronaviruses occurs both within and between host cats. selective infection with a single variant can also rapidly lead to genetically distinct clades of coronavirus, especially when combined with a high intrinsic and extrinsic mutation rate. twelve laboratory cats were inoculated intraperitoneally with a cell-free inoculum prepared from the diseased omentum of red, which contained two variant forms of the virus (fipv-ucd a and -ucd b). three of these cats developed effusive fip within - weeks. viral rna was isolated from the omentum of each experimentally infected cat at the time of necropsy. the s (one cat) and e, m, n and a-c, a, b genes (all three cats) were sequenced. one of the cats was found to be infected with fipv-ucd a, while two of the cats were each infected with fipv-ucd b. each of these cats had a nearly identical variant of ucd- a or ucd- b in its diseased omentum ( table ). the premature stop codon of parental c gene was preserved in fipv isolates from all three cats. however, fipv-ucd b. isolated from one of the three cats had acquired two additional large deletions affecting both the b and c genes that were not in the infecting virus (table and figure ). table . name and biotype designation of coronavirus isolates from three cats dying of experimentally induced fip. the genes that were sequenced, their mutability, degree of relatedness to the consensus sequence of fipv-ucd a, b, and nature of the functional mutation in the c gene are given for each cat. it is important to determine by animal inoculation studies the true biotype of a feline coronavirus that is being reported, rather than always referring to a generic feline coronavirus [reviewed in ] . feline coronaviruses that possess the fip biotype, such as fipv-ucd a,b, will readily induce fip in from - % of infected individuals, depending on the strain being tested [reviewed in ]. however, bonifed (cat-to-cat passaged, non-tissue culture adapted) fecv strains will rarely induce fip in healthy cats [ , , , ]. the present study adds to our knowledge of genetic drift among feline coronaviruses that inhabit the same cat, multi-cat household, cattery, or geographically distant region. all of the fipvs and fecvs isolated from the five cats that had close contact with each other in sonoma, california were ≥ % related (tables and ; figure ). based on gene sequences and historical facts, it can be reasonably concluded that cat simba was infected with an fecv following contact with cat lucy. this supported another correlate of the internal mutation theory; fecvs are easily spread cat-to-cat, while fipvs are not. addie et al. [ ] also noted that the same strain of coronavirus tended to persist among any given group of cats. however, coronaviruses within a closely housed group of cats, and even within the same cat, undergo continuous genetic drift. we observed sequence differences of - % or less in cats from the same group, while genetic drift between cats from distant areas of the western us was on the order of - %. herewegh et al. [ ] also found that feline coronaviruses from individuals within the same environment had unique genetic fingerprints and fell within the same clade, while geographically distant isolates belonged to genetically unique clades. the notable mutational drift observed among feline coronaviruses across geographic regions, in the face of genetic conservation within stable groups of cats, is paradoxical. however, the evidence indicates that coronavirus infection in any group of cats originates from a single founder virus, that virtually every cat in a group is infected rapidly and efficiently, and that cats appear to resist superinfection with closely related strains [ ] . the single founder virus effect was confirmed in the present study (table ) . thus, marked genetic drift occurs when a single coronavirus strain is serially passed from one susceptible population to the next. this scenario was supported by our animal transmission studies; when cats were simultaneously infected with two closely related variants of fipv, each variant segregated into different cats. therefore, minor mutants may become predominate when passed cat-to-cat. the s sequences of fipv-ucd to were compared to that of previously reported fipvs and to a purported fecv (wsu- - ) (data not shown). the s protein shared % sequence identity among the four fipv isolates, and - % sequence identity to other published serotype i fipvs. however, when compared to the s protein of serotype ii feline coronaviruses wsu- - and wsu- - , there was only - % sequence identity (data not shown). based on the comparison of s proteins, fipv-ucd to were classified as serotype i feline coronaviruses. however, these studies demonstrate that serotype designation of feline coronaviruses can be more easily made from comparisons of the much smaller a rather than significantly larger s sequences ( vs amino acids in the respective gene products) (figure ). similar to the s protein comparison, the a sequence of all four fipvs from the related cats shared - % sequence identity to each other, and - % sequence identity to the four fipvs from unrelated cats and from published serotype i fipvs ( figure ). however, when compared to the a protein of serotype ii feline coronaviruses wsu- - and wsu- - or to a of canine coronavirus, there was only - % sequence identity. these results indicate all eight fipvs from this study clearly belonged to serotype i based on their a protein sequences, while known serotype ii viruses and the canine coronavirus formed a separate group (figure ). fipv is unique from most other viruses, because it is infrequently spread from animal-to-animal in a horizontal manner, yet it is highly infectious when extracts of diseased tissues or fluids are inoculated into naïve cats by a number of routes [reviewed in ] . the general belief is that enteric biotypes are compartmentalized to the gut, while fip biotypes are found only within internal organs [ ] . however, viruses with c mutations identical to fipvs from lesional tissues were present in the feces of some cats in this study (table ) , thus making horizontal transmission theoretically possible in certain circumstances. there is also evidence that fipv may have been shed in urine of fipv infected cats [ ] , and that coronavirus may be present in the blood, especially among younger cats [ ] . there are also several reports of fip outbreaks of sufficient magnitude and acuteness to suggest horizontal transmission [reviewed in ] . while this study did not answer the question as to the relative importance of vertical and horizontal transmission, it indicated the need to carefully study fecal and lesional virus isolates that are involved in explosive, large scale, epizootics of fip and not just the common enzootic form. amount of sequence homology among this protein, the similarities in their hydropathy profiles, both to each other and to the corresponding m proteins, as well as to the sars-cob a protein, are quite remarkable. nothing is known about these proteins, but it is clear that it will be interesting to learn more about their biological features." a great deal of research has been reported, and is being conducted, on the sars coronavirus a gene and protein and it is evident that this gene and its product play an important role in viral assembly, spread and pathogenesis, as well as to protective immunity [ ] [ ] [ ] . if the c protein of feline coronavirus truly has an analogous function to sars coronavirus a protein, sars coronavirus research might be applicable to feline coronaviruses and how they cause disease. the authors have used the original names of fecv to refer the enteric biotype of feline coronavirus, and fipv for the fip biotype. published non-tissue culture adapted coronavirus isolates from the feces of healthy cats always possess an intact or wildtype c, while strains from fip diseased tissues have mutated c genes [ ] . therefore, the designation of fecv or fipv in this study was applied to isolates with c genes that yielded either intact or truncated proteins, respectively. the generic term "coronavirus" or "feline coronavirus" was used herein when not referring to a specific biotype. four scottish fold kittens were born into the same cattery in sonoma, california; red, toby and lucy were from the same litter of three, while tux was born a week later in a litter of three to a sister queen and the same tom. simba, an year-old american curl, was born in an unrelated cattery and resided in another sonoma household as a pet. lucy was placed into this household with simba when she was weeks old, while red went to live in another home with two other older cats when at weeks of age. tux and toby remained in their home cattery with several other cats. lucy, tux, red and toby first showed signs of indicative fip at , , and , weeks of age, and were euthanatized with confirmed disease at , , and weeks of age, respectively. all other contact cats have remained healthy to this time. four additional cats were recruited from the western us. two of them were -and -month old burmese ( and ) from paradise and menlo park, ca, respectively. the third was a month old birman ( ) from san jose, ca, and the fourth was a -year old sphinx (cat-t) from mountlake terrace, wa (courtesy dr. tracy tomlinson). full necropsies on all cats, except cat-t were performed at the school of veterinary medicine teaching hospital (vmth), university of california, davis, ca. cat-t was necropsied at a private veterinary diagnostic laboratory (phoenix central laboratory, everett, wa). a definitive diagnosis of fip was confirmed on all eight cats by gross and microscopic examination of tissues and immunohistochemistry. the four related scottish folds and cat-t suffered from the effusive form of fip, while the two burmese and one birman cats suffered from non-effusive fip. samples of diseased omentum (effusive fip) or kidney granulomas (non-effusive fip), along with feces (or colonic mucus/mucosal scrapings from one cat) were collected at the time of necropsy and stored at - °c. feces from the healthy sentinel cat, simba, were also collected. a cell free inoculum was made from the diseased omentum of red, one of the four related cats. omentum was frozen in liquid nitrogen and ground to a powder. the frozen omental powder was reconstituted in . g/ml hbss (hanks buffered saline solution) and centrifuged twice at , x g for minutes. the supernatant was stored at - °c as viral stock. the viral stock was diluted : with hbss when used as inoculum for the fipv transmission study. adult specific pathogen free cats were obtained from the breeding colony of the feline health and pet care center, school of veterinary medicine, university of california, davis, ca. a total of twelve cats were inoculated intraperitoneally with ml of cell free viral inoculum. three cats developed fip within - weeks and complete necropsies established that all three cats had effusive fip. diseased tissues and feces were collected for isolation of feline coronavirus rna. viral rna was extracted from omentum, granulomas of kidney, and colonic mucus/mucosal scrapings using qiagen raeasy mini kit (qiagen, usa). about mg ground lesional tissues were lysed with µl lysis buffer containing b-mercaptoethanol. after thoroughly mixing, the lysate was homogenized with qiashredder (qiagen, usa) and an equal volume of % ethanol was added to the homogenized lysate. the lysate mixture was applied to rneasy spin column and the rna binding to the column was achieved by centrifugation. the rneasy spin column was then washed and the rna was eluted with µl rnase-free water and stored at - °c. feces from / cats were suspended with volumes of phosphate buffer saline (pbs) by vortexing. the suspension was centrifuged at , x g for min and the supernatant transferred to a new tube and centrifuged at , x g for min. the pellet containing the virus was suspended with ml pbs and centrifuged again at , x g for min. the pellet was suspended in µl pbs and the viral rna extracted using a qiaamp viral rna mini kit (qiagen, usa). briefly, µl lysis buffer containing carrier rna was mixed with the µl viral suspension and incubated at ambient temperature for min; µl % ethanol was added to the lysate. the lysate mixture was applied to qiaamp mini spin column and the rna binding to the column was achieved by centrifugation. the column was then washed and the rna was eluted with ml rnase-free water and stored at - °c. the published sequences of feline coronaviruses in genbank were used to design the primers for a reverse transcripase polymerase chain reaction (rt-pcr). three primer pairs were designed from highly conserved regions and used to amplify three overlapping fragments containing the nine structural and accessory genes of feline coronavirus (figure and table ). the rt-pcr was carried out with qiagen longrange step rt-pcr kit (qiagen, usa). the viral rna was first denatured by incubating at °c for min and then chilled on ice. the reverse transcription was carried out in l reaction mixture containing units of longrange reverse transcriptase, . unit of rnase inhibitor, mm dntp, mm oligo dt , and l of denatured viral rna in x reaction buffer. the mixture was incubated at °c for hr followed by °c for min. the reverse transcribed cdna was stored at - °c or used immediately in pcr amplification. the viral cdna was amplified in l reaction mixture containing l cdna, unit longrange pcr enzyme mix, . mm dntp, . mm forward primer, and . mm reverse primer in x pcr buffer. the mixture was then incubated at °c for min and amplified for cycles at °c for s, °c for s, and °c for min per kb of pcr product, followed by a final extension for min at °c. the reverse transcribed viral rna from feces was amplified for cycles under the same condition. the pcr products were electrophoresed in tae buffer on a . % agarose gel. the pcr product was purified using a qiagen gel purification kit (qiagen, usa). sixty primers were ultimately used for sequencing, with the s gene requiring the most primer development and modification (primer sequences not shown). regions containing mixed sequences due to the presence of a minor variant were also resolved with overlapping primers. the purified overlapping pcr products encoding the nine structural and accessory genes were sequenced with a bigdye terminator v . cycle sequencing kit (applied biosystems, usa) in µl reaction containing µl big dye terminator mix, µl reaction buffer ( x), ng sequencing primer, and µl (out of µl) gel purified pcr product. the sequencing reaction was incubated at °c for min and then amplified for cycles at °c for s, °c for s, and °c for min. unincorporated dye terminators and dntp were removed by gel filtration based performa dtr ultra -well plate kit (edgebio, usa) and the cycle amplified products were analyzed by capillary electrophoresis using an abi genetic analyser (applied biosystems, usa). vector nti advance software (invitrogen, usa) was used for alignment of sequence data. the percent sequence identity for pairwise alignment and the phylogenetic relationship among different fipv isolates was analyzed using clustalw ii (www.ebi.ac.uk/tools/clustalw /). tables and list the various fipv and fecv isolates that were studied and the genes that were sequenced for each. these sequences have been deposited in the database of genbank. the ubiquitous form of feline coronavirus is readily passed cat-to-cat by the fecal-oral route and is the cause of a mild or unapparent enteritis. like coronaviruses in general, feline enteric coronavirus (fecv) is undergoing constant mutation within its accessory and structural genes. feline infectious peritonitis (fip), which is a highly fatal systemic disease, is a sequel of fecv infection in a small proportion of cats. the virus isolated from the diseased tissues of cats with fip is highly related to the fecv identified in the feces. although snp and deletion mutations were common between isolates from the same cat, only the c gene was rendered non functional by such mutations. deleterious mutations in c tend to be found in diseased internal tissues, while viruses with intact c are found mainly in the feces. while deleterious mutations of the c gene were seen in all fip cats in this study, and in virtually all previously reported fipvs, they are by no means a universal finding. however, there is compelling evidence that when they do occur, they are the cause of fip and not an effect of the disease. deleterious c gene mutants will readily cause fip when inoculated into laboratory cats, whereas their largely non-pathogenic fecal counterparts with intact c genes are readily transmitted from one cat to another. these gene c mutants, when they occur, are unique to each cat with fip, indicating that they arise independently in each host and not by mutation in one cat with subsequent horizontal spread to others. several minor variants can co-exist in both tissues and feces and when two variants are inoculated together into the same cat, one or the other, but not both, will predominate. the high mutability of feline coronaviruses leads to minor genetic differences between cats in one closely contained geographic area, while significant genetic differences are seen between isolates from geographically disparate regions. more in depth studies on the function of the feline coronavirus c gene will be important for determining its precise role in fip. some important disorders of cats clinico-pathology conference epidemiology of feline infectious peritonitis among cats examined at veterinary medical teaching hospitals feline infectious peritonitis ultrastructural evidence for the viral etiology of feline infectious peritonitis morphogenesis of a virus in cats with experimental feline infectious peritonitis extraperitoneal lesions in feline infectious peritonitis antigenic relationship of the feline infections peritonitis virus to coronaviruses of other species pathogenic differences between various feline coronavirus isolates. coronaviruses; molecular biology and pathogenesis an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis a review of feline infectious peritonitis virus infection: - pathogenesis of feline enteric coronavirus infection virologic and immunologic aspects of feline infectious peritonitis virus infection perspectives on feline coronavirus evolution elimination of feline coronavirus infection from a large experimental specific pathogen-free cat breeding colony by serologic testing and isolation two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein genomic rna sequence of feline coronavirus strain fcov c je persistence and evolution of feline coronavirus in a closed catbreeding colony field strain feline coronaviruses with small deletions in orf b associated with both enteric infection and feline infectious peritonitis deletions in the a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis belák s. preliminary studies on feline coronavirus distribution in naturally and experimentally infected cats feline leucocyte antigen class ii polymorphism and susceptibility to feline infectious peritonitis natural fcov infection: cats with fip exhibit significantly higher viral loads than healthy infected cats natural feline coronavirus infection: differences in cytokine patterns in association with the outcome of infection high viral loads despite absence of clinical and pathological findings in cats experimentally infected with feline coronavirus (fcov) type i and in naturally fcov-infected cats association between faecal shedding of feline coronavirus and serum alpha -acid glycoprotein sialylation inheritance of susceptibility of feline infectious peritonitis in purebred catteries risk factors for feline infectious peritonitis among cats in multiple-cat environments with endemic feline enteric coronavirus quasispecies composition and pylogenetic analysis of feline coronaviruses (fcovs) in naturally infected cats. fems antigenic and plaque variations of serotype ii feline infectious peritonitis coronaviruses persistence and transmission of natural type i feline coronavirus infection the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf a/ b transcription unit of different biotypes feline infectious peritonitis: experimental studies the detection of feline coronaviruses in blood samples from cats by mrna rt-pcr glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins a and m severe acute respiratory syndrome coroanvirus a protein is a viral structural protein amino terminus of the sars coronavirus protein a elicits strong, potentially protective humoral responses in infected patients the severe acute respiratory syndrome (sars)-coronavirus a protein may function as a modulator of the trafficking properties of the spike protein this research was funded by the center for companion animal health, school of veterinary medicine, university of california, davis, ca . we are also grateful for anonymous and named donations from owners of cats dying from fip. the authors are grateful for the assistance of sue weitendorf for allowing access to a critical group of cats used in this study. positive proof that the c protein is responsible for the fip phenotype, in at least a proportion of cats dying of fip, will require knowledge of its exact function, of which we currently know very little. a genblank blast search shows a % genetic homology between feline coronavirus c and sars coronavirus a (data not shown). moreover, the c protein of feline coronavirus also has an identical hydrophillicity profile to its own m protein and to the m and a proteins of sars coronavirus [ ] . these similarities prompted oostra and colleagues [ ] to state -"(…) it appears that all group [corona] viruses expresss group-specific proteins predicted to be triple-spanning membrane proteins. examples are the feline orf c protein and the hcov-nl orf a protein (…) despite the small key: cord- - djwbivp authors: xiang, qi; wan, pin; yang, ge; huang, siyu; qin, mengying; yang, hua; luo, zhen; wu, kailang; wu, jianguo title: beclin binds to enterovirus d protein to promote the virus replication date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: djwbivp enterovirus (ev ) is the main pathogen causing hand-foot-mouth disease (hfmd) in infants and children, which can also lead to severe neurological diseases and even death. therefore, understanding the replication mechanism of ev is of great significance for the prevention and control of ev -induced diseases. beclin (becn , a mammalian homologue of atg in yeast) is an important core protein for the initiation and the normal process of autophagy in cells. in addition to its involvement in autophagy, beclin has also been reported to play an important role in cancer and innate immune signaling pathways. however, the role of beclin in ev replication remains elusive. here, we primarily found that beclin facilitates ev replication in human rhabdomyosarcoma (rd) cells and the autophagy was actually induced, but beclin was not significantly affected at either mrna level or protein level during early ev infection. further studies discovered that beclin could interacts with ev non-structural protein d mainly through its evolutionary conserved domain (ecd) and coiled-coiled domain (ccd), thus promoting the replication of ev in human rhabdomyosarcoma (rd) cells and human astroglioma (u ) cells. collectively, we reveal a novel regulatory mechanism associated with beclin to promote ev replication, thus providing a potential therapeutic target for the prevention and control of ev -associated diseases. enterovirus (ev ) is a single-stranded positive rna virus of the picornaviridae family with a genome of approximately . kb. the ev genome encodes a long polyprotein with a single open reading frame followed by a polya tract. the single polyprotein is flanked by untranslated regions at both the and ends and can be divided into three different genomic regions (p , p and p ). the p genomic region encodes the structural proteins vp -vp . the p and p genomic regions encode the nonstructural proteins a, b, c, a, b, c and d [ ] . ev causes hand, foot and mouth disease (hfmd) and can even cause fatal central nervous system (cns) infections in young children, including encephalitis, aseptic meningitis and acute flaccid paralysis [ ] . since it was first identified from a child with neurologic symptoms in california in [ ] , epidemic and sporadic outbreaks of neurovirulent ev infections have been reported worldwide especially in the asia-pacific region [ ] [ ] [ ] [ ] [ ] [ ] . it is highlighted that inactivated ev vaccine elicited ev -specific immune responses and protection rabbit anti-p polyclonal antibody( - -ap) were purchased from proteintech group (chicago, il, usa). rabbit anti-β-actin (ac ) and anti-ev - d polyclonal antibodies were produced by abclonal technology (wuhan, china). rabbit anti-ev -vp (pab -d p) polyclonal antibody was purchased from abnova (taipei city, taiwan). rabbit anti-beclin ( ) antibody was purchased from zenbio (chengdu, china). delbecco modified eagle medium (dmem), and fetal bovine serum (fbs) were purchased from gibco (grand island, ny, usa). lipofectamine , normal rabbit igg and normal mouse igg were purchased from invitrogen corporation (carlsbad, ca, usa). protein ladder ( ) was purchased from thermo scientific (rockford, il, usa). complete, edta-free protease inhibitor cocktail was purchased from roche (basel, switzerland). chloroquine diphosphate were purchased from selleck (houston, tx, usa). rapamycin ( - - ) was obtained from target mol (shanghai, china). the cdna regions of human beclin were generously provided by the han jiahuai laboratory of xiamen university and then cloned into pcdna . (+)- × flag and pcaggs-ha vectors. the beclin mutants flag-beclin -bd, flag-beclin -bd+ccd, flag-beclin -bd+ccd+ecd, flag-beclin -ccd+ecd, and flag-beclin -ecd, were all constructed into pcdna . (+)- × flag vector. to construct plasmids expressing ev nonstructural protein d, the coding sequence of d was cloned into pcaggs-ha and pegfp-c vectors. total rna was extracted from cells using trizol reagent (invitrogen life technologies; carlsbad, ca, usa) according to the manufacturer's instructions. rna was reversed by hiscript ii q rt supermix (vazyme biotech co, nanjing, china) to obtain complementary dna and the following quantitative real-time analysis was performed using chamq sybr qpcr master mix (vazyme biotech co, nanjing, china) on a roche lc (roche diagnostics; penzberg, germany). all testing primers were designed by primer premier . and their sequences were as follows: ev -vp forward: -aattgagt tccataggtg- , and ev -vp reverse: -ctgtgcgaattaaggacag- ; becn forward: -ccatgcaggtgagcttcgt- , and becn reverse: -gaatctgcgagagacaccatc- , gapdh forward: -ggagcgagatccctccaaaat- , and gapdh reverse: -ggctgttgtc atacttctcatgg- . rd cells were grown in -well plates to % confluence and inoculated with the supernatants from infected rd cells for h. cells were washed with phosphate buffer saline (pbs) and cultured with % fbs in dmem. at day three post-infection, the plates were examined for the lowest dilution at which % of the wells showed the cytopathic effect using the reed-muench method. the targeting sequences of short hairpin rna (shrna) for the human becn were: shbecn - : -cccgtggaatggaatgagatt- , shbecn - : -ctcaagttcatgctgacgaat- . after annealing with the complementary shrna, the double stranded shrna for becn was ligated into the plko. saint louis, mo, usa) for seven days continuously. knockdown efficiency of shrna-targeted becn was identified by rt-pcr and immunoblot analyses. for western blot, cells were washed twice in ice-cold pbs and then lysed in lysis buffer ( mm hepes, ph . , mm nacl, mm edta, mm egta and % triton-x ) in the presence of cocktail (roche) on ice. protein concentration was determined by bradford assay (bio-rad, hercules, ca, usa). after adding × sds loading buffer, cell lysates were electrophoresed by - % sds-page gel and then transferred onto a nitrocellulose membrane (millipore sigma-aldrich, st. louis, mo, usa). the membranes were blocked in % nonfat dried milk dissolved in pbs with . % tween- and incubated with specific primary antibodies overnight at • c. after incubating with the secondary antibodies at room temperature for h, protein bands were detected with the clarity western ecl substrate (bio-rad) and luminescent image analyzer (las- , fujifilm, tokyo, japan). for co-ip assays, cells were washed with cold pbs and lysed in ml ripa buffer ( mm tris-hcl, ph . , mm nacl, % np- , mm edta, and % glycerol) containing protease inhibitors. µl of the lysates were subjected for immunoblot analysis to detect the expression of target proteins. the rest of the lysates were incubated with a control igg or the indicated primary antibodies at • c overnight and were further incubated with protein a/g-agarose (ge healthcare, waukesha, wi, usa) for - h. the beads were washed five times by washing buffer ( mm tris-hcl, ph . , mm nacl, % np- , mm edta, and % glycerol) and resuspended in ml × sds loading buffer before western blot analysis. processed cells were washed with pbs containing . % bsa and fixed with % paraformaldehyde for min at room temperature, followed by permeabilization with . % triton-x for min, and blocked with % bsa for h. anti-flag and ha or anti- d antibodies were added and incubated at • c subsequently, and then fitc-conjugated anti-mouse and cy -conjugated anti-rabbit secondary antibodies were incubated with cells for min away from light. eventually, cells were washed three times with pbs containing . % bsa and stained with dapi for min at • c, and confocal imaging was performed using fluo view fv (olympus, tokyo, japan). all experiments were repeated at least three times with similar results. all data were expressed as the mean ± sd (standard deviation). statistical analysis was carried out using the student's t test for two groups and one-way anova for multiple groups (graphpad prism . , inc. la jolla, ca, usa). differences were considered statistically significant when p < . . when studying beclin as a core protein for the initiation and normal process of autophagy in cells, we firstly verified whether autophagy was induced during ev infection. a dual fluorescent tagged-plasmid gfp-mcherry-lc was used to indicate the course of autophagic flux by confocal assay as previously reported [ ] . it is well known that lc -attached autophagosomes are formed in the cytoplasm when autophagy is induced and then fuse with endosomes or lysosomes to form autolysosomes, which provide an acidic environment and digestive function to the interior of the autophagosome. as the mcherry is acid stable while gfp is acid labile, so, if autophagic flux is increased, both yellow and red punctate are increased; however, if autophagosome maturation into autolysosomes is blocked, only yellow punctate is increased without a concomitant increase in red punctate. we found ev infection induced autophagy at and h post-infection in rd cells figure a,b) . as a positive control, it is apparently that autophagy was activated by rapamycin (rap) and autolysosomes were blocked by chloroquine (cq) ( figure a ,b), which is similar as previously reported [ , ] . to further confirm the influence of ev on beclin during infection, we infected rd cells with ev in a time-dependent manner or dose-dependent manner. in ev -infected rd cells, the levels of ev vp rna ( figure d ,h) and d protein ( figure e ,i) were found to be significantly increased at each indicated time point or moi post-infection as expected. however, the becn rna level ( figure c ,g) and beclin protein level ( figure e ,i) were not significantly altered. previous studies have illustrated that ev infection led to the upregulation of usp [ ] and usp could stabilize beclin by decreasing the k -linked ubiquitin chains leading to the inhibition of beclin degradation [ ] , which may provide partial explanation of our results. besides, we measured the ratio of lc -i to lc -ii and the protein levels of a well characterized autophagic substrate, p /sqstm , which binds to lc and is specifically degraded as a result of complete autophagic flux; the conversion to lc -ii increased and the level of the p was reduced with the increase of infection time and infection concentration, indicating that autophagy was induced and promoted upon ev infection. in conclusion, these results suggested that ev infection induced autophagy and maintained the expression level of beclin . viruses , , x for peer review of previously reported [ , ] . to further confirm the influence of ev on beclin during infection, we infected rd cells with ev in a time-dependent manner or dose-dependent manner. in ev infected rd cells, the levels of ev vp rna ( figure d and h) and d protein ( figure e and i) were found to be significantly increased at each indicated time point or moi post-infection as expected. however, the becn rna level ( figure c and g) and beclin protein level ( figure e and i) were not significantly altered. previous studies have illustrated that ev infection led to the upregulation of usp [ ] and usp could stabilize beclin by decreasing the k -linked ubiquitin chains leading to the inhibition of beclin degradation [ ] , which may provide partial explanation of our results. besides, we measured the ratio of lc -i to lc -ii and the protein levels of a well characterized autophagic substrate, p /sqstm , which binds to lc and is specifically degraded as a result of complete autophagic flux; the conversion to lc -ii increased and the level of the p was reduced with the increase of infection time and infection concentration, indicating that autophagy was induced and promoted upon ev infection. in conclusion, these results suggested that ev infection induced autophagy and maintained the expression level of beclin . . graph expressed as mean ± sd, ns, not-significant; * p < . ; ** p < . ; *** p < . . to explore the role of beclin in ev infection, rd cells were transfected with flag-tagged beclin by an increased degree; at h post-transfection, cells were infected with ev at a moi = for another h. the capsid protein of ev -vp , which is usually used to identify the replicating efficiency of ev , was significantly enhanced in both mrna level ( figure a ) and protein level ( figure b ), which indicated that beclin was able to promote ev replication. furthermore, we also demonstrated that ev replication was promoted by overexpression of beclin in a time-dependent manner ( figure c,d) . collectively, the results of beclin overexpression in ev -infected rd cells proved that beclin could promote ev replication in host cells. viruses , , x for peer review of i ratio was measured (j). graph expressed as mean ± sd, ns, not-significant; *, p < . ; **, p < . ; ***, p < . . to explore the role of beclin in ev infection, rd cells were transfected with flag-tagged beclin by an increased degree; at h post-transfection, cells were infected with ev at a moi = for another h. the capsid protein of ev -vp , which is usually used to identify the replicating efficiency of ev , was significantly enhanced in both mrna level ( figure a ) and protein level ( figure b ), which indicated that beclin was able to promote ev replication. furthermore, we also demonstrated that ev replication was promoted by overexpression of beclin in a time-dependent manner ( figure c and d) . collectively, the results of beclin overexpression in ev -infected rd cells proved that beclin could promote ev replication in host cells. flag-vector or flag-beclin for h and then infected with ev (moi = ) at the indicated times. the rna and protein levels of ev -vp were detected by real-time pcr with gapdh as an internal control (c) and western blot (d), respectively. graph expressed as mean ± sd, ns, not-significant; *, p < . ; **, p < . . since the overexpression of beclin can strengthen ev replication and ev infection does not change the expression level of beclin , we next identified the effects of knockdown of becn on ev infection. firstly, we synthesized two different becn (beclin )-targeted shrnas and transfected into rd cells so as to identify the knockdown efficiency and its influence on ev replication. the results show that the mrna level and protein level of beclin were significantly reduced in rd cells; moreover, knockdown of becn inhibited ev replication ( figure a and b ). in addition, supernatants from infected rd cells were transfected with flag-vector or flag-beclin , plko. -shnc or plko. -shbeclin for h and then collected for tcid assay. viral tcid was obviously upregulated in beclin overexpressed cells compared with mock cells, while viral titer was significantly reduced in beclin -knockdown cells compared to beclin normally expressed cells, further indicating that beclin promoted ev replication ( figure c ). next, we utilized lentivirus to establish stable becn -knockdown rd cells and u (human astroglioma) cells, then we infected the rna and protein levels of ev -vp were detected by real-time pcr with gapdh as an internal control (c) and western blot (d), respectively. graph expressed as mean ± sd, ns, not-significant; * p < . ; ** p < . . since the overexpression of beclin can strengthen ev replication and ev infection does not change the expression level of beclin , we next identified the effects of knockdown of becn on ev infection. firstly, we synthesized two different becn (beclin )-targeted shrnas and transfected into rd cells so as to identify the knockdown efficiency and its influence on ev replication. the results show that the mrna level and protein level of beclin were significantly reduced in rd cells; moreover, knockdown of becn inhibited ev replication ( figure a,b) . in addition, supernatants from infected rd cells were transfected with flag-vector or flag-beclin , plko. -shnc or plko. -shbeclin for h and then collected for tcid assay. viral tcid was obviously upregulated in beclin overexpressed cells compared with mock cells, while viral titer was significantly reduced in beclin -knockdown cells compared to beclin normally expressed cells, further indicating that beclin promoted ev replication ( figure c ). next, we utilized lentivirus to establish stable becn -knockdown rd cells and u (human astroglioma) cells, then we infected stable rd and u cells with ev and checked replication efficiency at different time points in two different cells. we discovered that the mrna level and protein level of ev -vp were remarkably decreased compared with responding controls viruses , , of both in rd ( figure d ,e) and u ( figure g ,h) cells. interestingly, on account of beclin playing a pivotal role in autophagy [ ] [ ] [ ] , we have also tested lc (a marker of autophagy) change after knockdown of becn ( figure f,i) . it was shown that the lc -ii/lc -i ratio was attenuated when beclin interfered, which may indicate that autophagy flux was involved in beclin enhancing ev replication. some studies have reported the relationship between ev and autophagy [ , , ] ; however, the molecular mechanism is still indistinct. with all the above results, we firstly provided the evidence that beclin promoted ev infection in rd and u cells. viruses , , x for peer review of stable rd and u cells with ev and checked replication efficiency at different time points in two different cells. we discovered that the mrna level and protein level of ev -vp were remarkably decreased compared with responding controls both in rd ( figure d and e) and u ( figure g and h) cells. interestingly, on account of beclin playing a pivotal role in autophagy [ ] [ ] [ ] , we have also tested lc (a marker of autophagy) change after knockdown of becn ( figure f and i) . it was shown that the lc -ii/lc -i ratio was attenuated when beclin interfered, which may indicate that autophagy flux was involved in beclin enhancing ev replication. some studies have reported the relationship between ev and autophagy [ , , ] ; however, the molecular mechanism is still indistinct. with all the above results, we firstly provided the evidence that beclin promoted ev infection in rd and u cells. to further evaluate the relationship between ev and beclin , and the possible mechanism utilized by beclin , we screened the interactions among ev nonstructural proteins and beclin . coimmunoprecipitation (co-ip) assay was carried out as previously described [ ] . briefly, plasmid encoding flag-beclin and plasmids encoding each of the ev non-structure proteins, ha- a, ha- b, ha- c, ha- ab, ha- c, ha- d and flag-beclin , were co-transfected into human embryonic kidney (hek t) cells. co-ip results showed that ev d specifically interacted with beclin , whereas other ev viral proteins failed to interact with beclin ( figure a ). additionally, we . the transfected rd cells were infected with ev (moi = ), and the protein levels of ev -vp and beclin were detected by western blot with β-actin as an internal control (b). (c) rd cells were transfected with flag-vector or flag-beclin , plko. -shnc or plko. -shbeclin and infected with ev for h, and supernatants were collected for tcid assay. (d-f) the stable rd cells were infected with ev (moi = ) for different periods. the rna and protein levels of ev -vp were detected by real-time pcr with gapdh (d) and western blot with β-actin as controls (e), and the lc -ii/lc -i ratio was measured(f), respectively. (g-i) the stable u cells were infected with ev (moi = ) for different periods. the rna and protein levels of ev vp were detected by real-time pcr (g) and western blot (h), and the lc -ii/lc -i ratio was measured (i), respectively. graph expressed as mean ± sd, ns, not-significant; * p < . ; ** p < . ; *** p < . . to further evaluate the relationship between ev and beclin , and the possible mechanism utilized by beclin , we screened the interactions among ev nonstructural proteins and beclin . co-immunoprecipitation (co-ip) assay was carried out as previously described [ ] . briefly, plasmid encoding flag-beclin and plasmids encoding each of the ev non-structure proteins, ha- a, ha- b, ha- c, ha- ab, ha- c, ha- d and flag-beclin , were co-transfected into human embryonic kidney (hek t) cells. co-ip results showed that ev d specifically interacted with beclin , whereas other ev viral proteins failed to interact with beclin ( figure a ). additionally, we repeated the co-ip assay by co-transfecting d and beclin in hek t cells. further results confirmed the interaction between d and beclin ( figure b ). immunofluorescence analysis also showed that d and beclin viruses , , of could co-localize in the cytoplasm ( figure c ). moreover, rd cells were infected with ev at a moi = for h and cell lysates were immunoprecipitated with anti-beclin antibody. it is shown that there was an endogenous interaction between ev - d and beclin ( figure d ). altogether, these results consistently revealed the interaction between d and beclin . viruses , , x for peer review of repeated the co-ip assay by co-transfecting d and beclin in hek t cells. further results confirmed the interaction between d and beclin ( figure b ). immunofluorescence analysis also showed that d and beclin could co-localize in the cytoplasm ( figure c ). moreover, rd cells were infected with ev at a moi = for h and cell lysates were immunoprecipitated with anti-beclin antibody. it is shown that there was an endogenous interaction between ev - d and beclin ( figure d ). altogether, these results consistently revealed the interaction between d and beclin . to further evaluate the region of beclin responsible for interacting with d and facilitating ev replication, a series of mutants were constructed based on the functional domains of beclin , including beclin mutants beclin -bd, beclin -bd+ccd, beclin -bd+ccd+ecd, beclin -ccd+ecd, and beclin -ecd ( figure a ). to determine that the domain of beclin is responsible for interacting with d, plasmids expressing gfp- d and flag-beclin or various mutants were transfected into hek t cells. co-ip assay revealed that beclin coprecipitated with d, whereas this interaction was weakened when ccd and ecd domains were deleted ( figure b) , suggesting ccd and ecd domains were indispensable in the interaction between beclin and d. to further assess the functional domain of beclin for promoting ev replication, plasmids expressing flagvector, flag-beclin and various mutants were transfected into rd cells followed by ev infection. subsequently, real-time pcr and western blot results showed that ccd and ecd domains were essential for beclin to maintain its function in the promotion of ev replication ( figure c and d) . to further evaluate the region of beclin responsible for interacting with d and facilitating ev replication, a series of mutants were constructed based on the functional domains of beclin , including beclin mutants beclin -bd, beclin -bd+ccd, beclin -bd+ccd+ecd, beclin -ccd+ecd, and beclin -ecd ( figure a ). to determine that the domain of beclin is responsible for interacting with d, plasmids expressing gfp- d and flag-beclin or various mutants were transfected into hek t cells. co-ip assay revealed that beclin coprecipitated with d, whereas this interaction was weakened when ccd and ecd domains were deleted ( figure b ), suggesting ccd and ecd domains were indispensable in the interaction between beclin and d. to further assess the functional domain of beclin for promoting ev replication, plasmids expressing flag-vector, flag-beclin and various mutants were transfected into rd cells followed by ev infection. subsequently, real-time pcr and western blot results showed that ccd and ecd domains were essential for beclin to maintain its function in the promotion of ev replication ( figure c,d) . thus, beclin ccd and ecd domains were essential for the interaction between beclin and d in promoting ev replication. thus, beclin ccd and ecd domains were essential for the interaction between beclin and d in promoting ev replication. graph expressed as mean ± sd, ns, not-significant, **, p < . . enterovirus (ev ) is the major pathogen of hand-foot-mouth disease (hfmd), which affects mainly children of age five and below [ , ] . unlike coxsackieviruses, another member of picornaviridae family, also leading to hfmd [ , ] , ev infections are usually associated with severe neurological diseases such as poliomyelitis-like paralysis, aseptic meningitis and encephalitis [ ] , which accounts for % of severe hfmd cases and % of hfmd-related deaths [ ] . although many vaccines and antiviral drugs against ev have been developed [ ] [ ] [ ] , there are still challenges involving the worldwide epidemic of ev , including their applicability against various ev pandemic strains in other countries, international requirements on vaccine production and quality control, etc. therefore, it is highly important to discover new mechanisms underlying host regulating ev infection and find new potential therapeutic targets against ev . our group has previously reported several host factors regulating ev replication; for example, sirt can inhibit ev replication and rna translation by interfering with the viral polymerase and ′utr rna [ ] , and polyc-binding protein interacts with '-untranslated region of ev rna in membraneassociated complex to facilitate viral replication [ ] . all of these findings provide new insights into the development of anti-ev therapeutic strategies. graph expressed as mean ± sd, ns, not-significant, ** p < . . enterovirus (ev ) is the major pathogen of hand-foot-mouth disease (hfmd), which affects mainly children of age five and below [ , ] . unlike coxsackieviruses, another member of picornaviridae family, also leading to hfmd [ , ] , ev infections are usually associated with severe neurological diseases such as poliomyelitis-like paralysis, aseptic meningitis and encephalitis [ ] , which accounts for % of severe hfmd cases and % of hfmd-related deaths [ ] . although many vaccines and antiviral drugs against ev have been developed [ ] [ ] [ ] , there are still challenges involving the worldwide epidemic of ev , including their applicability against various ev pandemic strains in other countries, international requirements on vaccine production and quality control, etc. therefore, it is highly important to discover new mechanisms underlying host regulating ev infection and find new potential therapeutic targets against ev . our group has previously reported several host factors regulating ev replication; for example, sirt can inhibit ev replication and rna translation by interfering with the viral polymerase and utr rna [ ] , and polyc-binding protein interacts with -untranslated region of ev rna in membrane-associated complex to facilitate viral replication [ ] . all of these findings provide new insights into the development of anti-ev therapeutic strategies. beclin plays a central role in the autophagy [ , ] . in addition, it has also been reported that beclin plays an important role in viral replication. coronavirus membrane-associated papain-like proteases induce incomplete autophagy through interacting with beclin and negatively regulate antiviral innate immunity to promote viral replication [ ] . the nef protein of hiv functions in preventing destruction of hiv components in autolysosomes by interacting with beclin , thus shielding hiv from autophagy in its role of a cell autonomous antimicrobial defense [ ] . in fmdv (foot-and-mouth disease virus)-infected cells, the largest viral protein in the replication complex, c, would bind to beclin to prevent the fusion of lysosomes to autophagosomes, allowing for virus survival [ ] . beclin- has also been shown to interact with cgas or mavs to suppress cgamp synthesis or rig-i-mavs interaction, halting type i interferon (ifn) production upon recognition of external pathogens [ , ] . nevertheless, the mechanisms of beclin in ev replication is not clearly known and ev -mediated effects on beclin are not reported. in our study, we firstly identified a novel role of beclin in ev infection and identified beclin as an essential component of the autophagy pathway to ev . when ev infects host cells, beclin will bind with d protein and promote the replication of ev . furthermore, early ev infection has few influences on the protein and mrna level of beclin . the different domains of beclin , shown in figure a , mediate communications with multiple interaction partners that can alter its conformation and binding accessibility, thus making beclin an important molecular platform for the regulation of pi k activity and autophagy. beclin interacts with the pi k core component vps and lipid membranes via its evolutionary conserved domain (ecd) in c-terminal [ , ] and binds to atg l or uvrag in a mutually exclusive manner via its coiled-coiled domain (ccd) [ ] . the bh domain (bd) of beclin mediates its interaction with the antiapoptotic protein bcl and this interaction causes a steric block inhibiting pi k complex formation [ ] , and ambra is a positive regulator of autophagy and competes with bcl for binding to the beclin bh domain [ , ] . interestingly, we found that the ccd and ecd domains of beclin are most effective for interacting with d and promoting ev replication, but it is hard to clearly understand why these two domains play the most important role. ev d protein is an rna-dependent rna polymerase and is responsible for the genomic rna replication of ev [ , ] . a previous study has clarified that ev -activated sirt binds with the d protein and attenuates the acetylation and rdrp activity of d, resulting in the repression of viral genome replication [ ] . beclin could assist viral reproduction through impacting the rdrp activity of d. some studies have also reported other functions of d; one research study reported that ev mediated cell cycle arrest in s phase through d [ ] , and another claimed that d protein facilitates the release of il- β by binding with nlrp and enhancing the assembly of inflammasome complex [ ] . recently, a new finding pointed out that ev d inhibits mda -mediated ifn-β promoter activation via interacting with the card domain of mda protein [ ] . our study may provide further evidence for d inhibiting ifn responses and promoting viral replication. ev - d makes use of the interaction with beclin to suppress type i ifn signaling pathway due to beclin acting as a negative regulator of rig-i-mavs mediated ifn response [ ] . however, the precise molecular mechanism is still indistinct. in addition, there may be effects other than the interaction of beclin alone with the d protein involved, as shown in figure . when beclin interfered, we found that higher levels of lc -ii were accumulated in mock-cells than in beclin knockdown-cells upon ev infection, and higher levels of ev were replicated in mock-cells than in beclin knockdown-cells, which may indicate that higher ev replication is positively correlated to a higher level of autophagy, because knockdown of beclin is likely to alter functions of other host proteins of the autophagy pathways and weaken the dynamism of the autophagy [ ] . even though ev -induced autophagy and its pro-viral role have been documented [ ] , disagreement still exists [ ] and which components of the autophagy pathway are used still need to be determined. in fact, our research might have discovered one of the specific elements involved in ev infection, i.e., ev possibly propels d to interact with beclin in order to regulate the process of autophagy so as to achieve the purpose of massive viral proliferation. however, a finding published in demonstrated that the knockout of beclin has no effect on replication of poliovirus (pv), and pv belongs to the enteroviruses, like ev [ ] . in our opinion, each virus, even closely related viruses like pv and ev , have different structural features and life cycles, and cells used in experiments are also different. furthermore, this paper has also illustrated that even denv and zikv, both of which are members of flavivirus, will use an exclusive component of the autophagy pathway for their own replication. therefore, it is not incompatible that each virus uses slightly different components. a human genome-wide rnai screen carried out in has also identified that beclin is involved in ev replication in human rhabdomyosarcoma cells [ ] , which is consistent with our results. as mentioned above, autophagy was induced to offer a favorable micro-environment for ev 's propagation within an infected host, and treatment of cells with tamoxifen, rapamycin and serum starvation, which are agonists of autophagy, promoted viral yields, while inhibition of autophagy by -ma, bafilomycin a or saikosaponin d, suppressed viral production [ , , ] . however, it is unknown whether ev induces autophagic flux, since no reports have actually shown that the number of autolysosomes is increased in ev infected cells. interestingly, our results may provide the first evidence that ev induces the formation of autolysosomes, because red dots in the h ev infected group, which represented autolysosomes, increased significantly compared with the mock group and h infected group in figure a . the detailed mechanism of how ev activates autophagy and makes use of autophagy for its own benefit is not well understood, and the relationship between other enteroviruses, such as poliovirus and cvb , and autophagy is also different than ev 's relationship with autophagy. in cvb infected hek and hela cells, the formation of a double-membranous autophagosome-like vesicle was induced, but sqstm was accumulated and the fusion of autophagosome with lysosome or degradation in autolysosome was inhibited, suggesting autophagic flux is blocked in cvb infected cells. likewise, inhibition of the autophagy pathway by antagonists, e.g., -ma, reduced viral production of cvb . conversely, nutrient deprivation (hbss incubation) or mtor inhibitor (rapamycin) enhanced cvb propagation [ ] [ ] [ ] [ ] [ ] [ ] . similar to ev and cvb , poliovirus infection was found to recruit lc to the viral replication complex, which was also co-localized with lamp (autolysosome/lysosome marker). likewise, knockdown of autophagy-related proteins (lc and atg ), or treatment with -ma decreased poliovirus viral load, while activation of autophagy by tamoxifen or rapamycin promoted viral production [ ] . recently, two groups have reported completely different roles of ulk and fip in poliovirus replication, corona velazquez et al. found that it failed to affect poliovirus production, while abernathy et al. considered that with depletion of ulk , fip suppresses poliovirus production [ , ] . taken together, though the detailed mechanism of how enterovirus utilizes the autophagy network reported from different research teams is not be perfectly consistent, these works strongly show that enteroviruses are good at manipulating the autophagy network to achieve efficient propagation. our results further enrich our understanding of the relationship between ev and autophagy and possibly provide a new therapeutic target against ev infection. an overview of the evolution of enterovirus and its clinical and public health significance an apparently new enterovirus isolated from patients with disease of the central nervous system identification of enterovirus isolates from an outbreak of hand, foot and mouth disease (hfmd) with fatal cases of encephalomyelitis in malaysia clinical features and risk factors of pulmonary oedema after enterovirus- -related hand, foot, and mouth disease neurological manifestations of enterovirus infection in children during an outbreak of hand, foot, and mouth disease in western australia the largest outbreak of hand; foot and mouth disease in singapore in : the role of enterovirus and coxsackievirus a strains characterization of full-length enterovirus strains from severe and mild disease patients in northeastern china epidemiological and genetic characteristics of ev in hand, foot, and mouth disease in guangxi, southern china, from five-year immunity persistence following immunization with inactivated enterovirus type (ev ) vaccine in healthy children: a further observation cleavage of the adaptor protein trif by enterovirus c inhibits antiviral responses mediated by toll-like receptor cleavage of interferon regulatory factor by enterovirus c suppresses cellular responses downregulation of microrna mir- a by enterovirus inhibits rig-i-dependent innate immune response enterovirus protease apro targets mavs to inhibit anti-viral type i interferon responses enterovirus apro targets mda and mavs in infected cells enterovirus c protein inhibits nf-kappab activation by binding to rela(p ) enterovirus disrupts interferon signaling by reducing the level of interferon receptor usp suppresses cellular type i interferon signaling by targeting traf for deubiquitination inhibition of mir- a prevents enterovirus-induced death by restoring the production of type i interferon exosome-mediated mir- a transfer suppresses type i interferon response and facilitates ev infection induction of autophagy and inhibition of tumorigenesis by beclin protection against fatal sindbis virus encephalitis by beclin, a novel bcl- -interacting protein the beclin -vps complex-at the crossroads of autophagy and beyond beclin -binding uvrag targets the class c vps complex to coordinate autophagosome maturation and endocytic trafficking two beclin -binding proteins, atg l and rubicon, reciprocally regulate autophagy at different stages distinct regulation of autophagic activity by atg l and rubicon associated with beclin -phosphatidylinositol- -kinase complex autophagic and tumour suppressor activity of a novel beclin -binding protein uvrag beclin , an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor the autophagy-related protein beclin shows reduced expression in early alzheimer disease and regulates amyloid beta accumulation in mice regulation of intracellular accumulation of mutant huntingtin by beclin a becn mutation mediates hyperactive autophagic sequestration of amyloid oligomers and improved cognition in alzheimer's disease autophagy pathway intersects with hiv- biosynthesis and regulates viral yields in macrophages matrix protein of influenza a virus blocks autophagosome fusion with lysosomes a l, a new viral bcl homolog targeting beclin autophagy related protein foot-and-mouth disease virus nonstructural protein c interacts with beclin , modulating virus replication hsv- icp . confers neurovirulence by targeting the beclin autophagy protein. cell host microbe the human cytomegalovirus protein trs inhibits autophagy via its interaction with beclin early growth response- facilitates enterovirus replication by direct binding to the viral genome rna phosphoprotein of human parainfluenza virus type blocks autophagosome-lysosome fusion to increase virus production enterovirus -induced autophagy detected in vitro and in vivo promotes viral replication enterovirus -induced autophagy increases viral replication and pathogenesis in a suckling mouse model usp modulates autophagy and antiviral immune responses by deubiquitinating beclin- the evolutionarily conserved domain of beclin is required for vps binding, autophagy and tumor suppressor function two distinct vps phosphatidylinositol -kinase complexes function in autophagy and carboxypeptidase y sorting in saccharomyces cerevisiae the pre-autophagosomal structure organized by concerted functions of apg genes is essential for autophagosome formation antiviral protection against enterovirus mediated by autophagy induction following flice-inhibitory protein inactivation cullin binds and promotes nlrp ubiquitination to repress systematic inflammasome activation an epidemic of enterovirus infection in taiwan. taiwan enterovirus epidemic working group deaths of children during an outbreak of hand, foot, and mouth disease in sarawak, malaysia: clinical and pathological characteristics of the disease. outbreak study group group b coxsackievirus infections in infants younger than three months of age: a serious childhood illness outbreak of hand, foot and mouth disease/herpangina associated with coxsackievirus a and a infections in , france: a large citywide, prospective observational study neural pathogenesis of enterovirus infection. microbes infect comparative epidemiology and virology of fatal and nonfatal cases of hand, foot and mouth disease in mainland china from recent progress towards novel ev anti-therapeutics and vaccines. viruses progress on the research and development of inactivated ev whole-virus vaccines an inactivated enterovirus vaccine in healthy children sirt inhibits ev genome replication and rna translation by interfering with the viral polymerase and utr rna polyc-binding protein interacts with -untranslated region of enterovirus rna in membrane-associated complex to facilitate viral replication beclin-phosphatidylinositol -kinase complex functions at the trans-golgi network coronavirus membrane-associated papain-like proteases induce autophagy through interacting with beclin to negatively regulate antiviral innate immunity crosstalk between the cgas dna sensor and beclin- autophagy protein shapes innate antimicrobial immune responses the autophagy-related beclin protein requires the coiled-coil and bara domains to form a homodimer with submicromolar affinity beclin forms two distinct phosphatidylinositol -kinase complexes with mammalian atg and uvrag bcl- antiapoptotic proteins inhibit beclin -dependent autophagy mitochondrial bcl- inhibits ambra -induced autophagy the dynamic interaction of ambra with the dynein motor complex regulates mammalian autophagy structures of ev rna-dependent rna polymerase in complex with substrate and analogue provide a drug target against the hand-foot-and-mouth disease pandemic in china enterovirus vpg uridylation uses a two-molecular mechanism of d polymerase enterovirus mediates cell cycle arrest in s phase through non-structural protein d ev d protein binds with nlrp and enhances the assembly of inflammasome complex role of enteroviral rna-dependent rna polymerase in regulation of mda -mediated beta interferon activation ulk induces autophagy by phosphorylating beclin- and activating vps lipid kinase differential and convergent utilization of autophagy components by positive-strand rna viruses human genome-wide rnai screen reveals host factors required for enterovirus replication saikosaponin d suppresses enterovirus a infection by inhibiting autophagy the cytotoxicity of coxsackievirus b is associated with a blockage of autophagic flux mediated by reduced syntaxin expression enteroviral infection inhibits autophagic flux via disruption of the snare complex to enhance viral replication coxsackievirus infection induces autophagy-like vesicles and megaphagosomes in pancreatic acinar cells in vivo autophagosome supports coxsackievirus b replication in host cells the pi k/akt/mtor pathway is involved in cvb -induced autophagy of hela cells the role of autophagy during coxsackievirus infection of neural progenitor and stem cells subversion of cellular autophagosomal machinery by rna viruses poliovirus induces autophagic signaling independent of the ulk complex this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank jiahua han of xiamen university, china for kindly providing cdna regions of human beclin ; fengmei han of hubei university, china for providing the fluorescent tagged-plasmid, gfp-mcherry-lc . the authors declare no conflict of interest.viruses , , key: cord- -eeyqh nc authors: zhou, yusen; yang, yang; huang, jingwei; jiang, shibo; du, lanying title: advances in mers-cov vaccines and therapeutics based on the receptor-binding domain date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: eeyqh nc middle east respiratory syndrome (mers) coronavirus (mers-cov) is an infectious virus that was first reported in . the mers-cov genome encodes four major structural proteins, among which the spike (s) protein has a key role in viral infection and pathogenesis. the receptor-binding domain (rbd) of the s protein contains a critical neutralizing domain and is an important target for development of mers vaccines and therapeutics. in this review, we describe the relevant features of the mers-cov s-protein rbd, summarize recent advances in the development of mers-cov rbd-based vaccines and therapeutic antibodies, and illustrate potential challenges and strategies to further improve their efficacy. middle east respiratory syndrome (mers) coronavirus (cov) is an infectious virus that was first reported in june [ ] . mers-cov may infect people of any age, but older age, underlying comorbidity (such as diabetes mellitus, renal disease, respiratory disease, heart disease, and hypertension), and delayed confirmation or late diagnosis are all factors that affect mers disease outcomes and mortality [ ] [ ] [ ] [ ] [ ] [ ] . sex could be a factor in mers epidemiology, as more males seem to be affected than females [ ] [ ] [ ] . mers-cov infection of women during pregnancy has adverse outcomes, with fetal mortality of~ %; however, only a limited number of pediatric mers-cov infections occur [ ] [ ] [ ] [ ] . at the end of december , , laboratory-confirmed mers infections were reported globally (in countries), leading to deaths, and a mortality of . %. among these infections, , ( . %) were reported in saudi arabia, with mortality in individuals ( . %) (http://www.emro.who.int/health-topics/mers-cov/mers-outbreaks.html). the largest mers outbreak outside saudi arabia occurred in south korea in , with cases and deaths [ , , ] . the most recent mers cases were reported in in south korea, the united kingdom, and malaysia, in addition to saudi arabia, the united arab emirates, and oman (http://www.who.int/emergencies/mers-cov/en/). mers-cov is thought to have originated in bats [ ] [ ] [ ] [ ] . mers-like viruses have been isolated from bats that use (at lower efficiency) the same receptor for cell entry as the mers-cov isolated from humans [ ] [ ] [ ] . dromedary camels are potential intermediates for long-term evolution of mers-cov and seasonal zoonotic transfer of virus to humans [ ] [ ] [ ] [ ] . antibodies specific to host cellular proteases for its activity in viral entry, but although evidence initially indicated that cellular furin activates s protein, subsequent results have demonstrated no evidence for the involvement of furin during viral entry [ , ] . the dpp receptor varies among different host species, and mers-cov is thought to use multiple pathways to enable rapid adaptation to speciesspecific variations [ ] [ ] [ ] . in addition to dpp , mers-cov can bind to sialic acid via the s subunit of s protein, or utilize the membrane-associated kda glucose-regulated protein (grp ) to attach to target cells, suggesting that these proteins may also have roles in virion attachment [ , ] . the structures of mers-cov rbd alone and complexed with dpp have been determined ( figure ) [ , , ] . the rbd has a fold-rich tertiary structure, which consists of a core and a receptor-binding motif (rbm), with stabilization provided by four disulfide bonds and two glycans [ ] . a number of rbd residues are located at the dpp -binding interface, and they have a critical role in rbd-dpp binding [ , , ] . structural analysis of mers-cov trimeric s protein has identified specific features of the rbd and its complex with dpp . notably, in the prefusion conformation of the s trimer, individual rbds are either buried (lying state) or exposed (standing state), and this flexibility presumably facilitates recognition by dpp [ ] . other structural studies have revealed four s-trimer conformational states, in which each rbd is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation, suggesting fusion initiation through sequential rbd events [ ] . in configurations with one, two, or three rbds rotated out, rbd determinants are exposed at the apex of the rbd-dpp complex, and they are accessible for interaction with dpp ( figure ) [ ] . mers-cov s protein has an important role in viral pathogenesis, determining host tropism and entry into host cells [ , , ] . the s protein contains an s subunit at the n terminus and an s subunit at the c terminus. the s subunit is composed of the n-terminal domain (ntd) and rbd [ , , ] . the rbd has a key role in the mediation of binding of mers-cov to cells expressing dipeptidyl peptidase (dpp ) receptor, enabling the virus to enter into target cells by fusing with cell membranes through the formation of a fusion core ( figure c ) [ ] [ ] [ ] [ ] . the s protein requires host cellular proteases for its activity in viral entry, but although evidence initially indicated that cellular furin activates s protein, subsequent results have demonstrated no evidence for the involvement of furin during viral entry [ , ] . the dpp receptor varies among different host species, and mers-cov is thought to use multiple pathways to enable rapid adaptation to species-specific variations [ ] [ ] [ ] . in addition to dpp , mers-cov can bind to sialic acid via the s subunit of s protein, or utilize the membrane-associated kda glucose-regulated protein (grp ) to attach to target cells, suggesting that these proteins may also have roles in virion attachment [ , ] . the structures of mers-cov rbd alone and complexed with dpp have been determined ( figure ) [ , , ] . the rbd has a fold-rich tertiary structure, which consists of a core and a receptor-binding motif (rbm), with stabilization provided by four disulfide bonds and two glycans [ ] . a number of rbd residues are located at the dpp -binding interface, and they have a critical role in rbd-dpp binding [ , , ] . structural analysis of mers-cov trimeric s protein has identified specific features of the rbd and its complex with dpp . notably, in the prefusion conformation of the s trimer, individual rbds are either buried (lying state) or exposed (standing state), and this flexibility presumably facilitates recognition by dpp [ ] . other structural studies have revealed four s-trimer conformational states, in which each rbd is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation, suggesting fusion initiation through sequential rbd the mers-cov rbd core is colored in blue, the rbm is colored in red, and dpp is colored in green. the rbm residues directly involved in dpp binding are shown as sticks. dpp , dipeptidyl peptidase ; rbd, receptor-binding domain; rbm, receptor-binding motif; s, spike protein. the function and structure of the s-protein rbd demonstrate that it is an important target for development of vaccines and therapeutic agents against mers-cov. a number of mers vaccines have been developed based on viral rbd, including nanoparticles, virus-like particles (vlps), and recombinant proteins, and their protective efficacy has been evaluated in animal models, including mice with adenovirus (ad )-directed expression of human dpp (ad /hdpp ), hdpp -transgenic (hdpp -tg) mice, and non-human primates (nhps) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . features of these rbd-based vaccines, in terms of functionality, antigenicity, immunogenicity, and protective ability, are shown in table . the function and structure of the s-protein rbd demonstrate that it is an important target for development of vaccines and therapeutic agents against mers-cov. a number of mers vaccines have been developed based on viral rbd, including nanoparticles, virus-like particles (vlps), and recombinant proteins, and their protective efficacy has been evaluated in animal models, including mice with adenovirus (ad )-directed expression of human dpp (ad /hdpp ), hdpp -transgenic (hdpp -tg) mice, and non-human primates (nhps) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . features of these rbd-based vaccines, in terms of functionality, antigenicity, immunogenicity, and protective ability, are shown in table . a soluble nanoparticle vaccine formed in escherichia coli by the rna-mediated folding of a rbd-ferritin (fr) hybrid elicits robust rbd-specific antibody and cellular immune responses in mice, producing antisera that effectively block the binding of rbd to hdpp in vitro [ ] . the adjuvants alum and the squalene-based mf significantly augment the antibody titers and t-cell responses induced by rbd-fr nanoparticle vaccines engineered with or without a ssg linker [ ] . similarly, a chimeric, spherical vlp (svlp) vaccine expressing mers-cov rbd induces specific antibody and cellular immune responses in mice, preventing pseudotyped mers-cov entry into susceptible cells [ ] . the protective efficacy of these two types of mers vaccine does not yet seem to have been investigated in a viral-challenge animal model. recombinant vaccines involving rbd subunits have been extensively studied for protection against mers-cov infection in mers-cov-susceptible animal models [ , [ ] [ ] [ ] , ] . a recombinant rbd (rrbd) fragment (residues - ) expressed in insect cells elicits an antibody response and the production of neutralizing antibodies in mice and nhps [ , ] . it gives incomplete protection in mers-cov-challenged nhps, with the alleviation of pneumonia and clinical manifestations, as well as the reduction of viral load in lung, trachea, and oropharyngeal swabs [ ] . a mers-cov s-protein rbd fragment containing residues - has been identified as a critical neutralizing domain [ ] . a treatment regimen involving two doses of a fusion of this fragment and the fc region of human igg (s - -fc) four weeks apart is able to induce strong, long-term antibody responses (including production of neutralizing antibodies) in mice [ ] . these responses are significantly greater than those with a single dose or two doses at intervals of one, two, or three weeks [ ] . rrbds with single or multiple mutations corresponding to s-protein sequences of mers-cov strains isolated from humans or camels from to have also been studied [ ] . all these rrbds bind rbd-specific neutralizing monoclonal antibodies (mabs) and dpp , and are highly immunogenic, eliciting the production of s -specific antibodies in mice, which cross-neutralizes multiple mers pseudoviruses and live mers-cov [ ] . a trimeric rbd-fd protein formed by fusing a mers-cov rbd fragment (residues - ) to the foldon trimerization motif, binds strongly to dpp , and elicits robust and long-term responses with the production of mers-cov s -specific antibodies and neutralizing antibodies in mice, and protects hdpp -tg mice against mers-cov infection [ ] . the protection provided by existing subunit vaccines based on wild-type mers-cov rbd is not complete, with survival rates in hdpp -tg mice after a mers-cov challenge of~ % for s - -fc and % for rbd-fd [ , ] . however, a variant rbd (t n) vaccine produced by masking a non-neutralizing epitope at residue with a glycan probe has both functionality in binding dpp , and antigenicity in binding four potent mers-cov rbd-specific neutralizing mabs (hhs- , m , m , and m ) [ ] . the t n vaccine has significantly greater efficacy than the wild-type rbd vaccine, and it fully protects against a lethal mers-cov challenge in immunized hdpp -tg mice [ ] , demonstrating the possibility of developing rbd-based mers-cov vaccines with high efficacy. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these antibodies generally have greater neutralizing activity against mers-cov infection than non-rbd s -based or s -based antibodies [ , , , ] . the prophylactic and therapeutic efficacies of rbd-targeting antibodies have been tested in ad /hdpp mice, hdpp -tg mice, and nhps [ , , [ ] [ ] [ ] . in an earlier review, we described the antiviral mechanisms, in vivo protection, and crystal structures of previously reported mers-cov rbd-specific mabs, including mouse mabs mersmab , e , c , f , and d , and human mabs lca , mers- , mers- , regn , regn , e , f , a , b , b , b -n, c , m d , m , m , m , hms- , and c h [ ] . in this review, we focus on newly reported antibodies targeting mers-cov s-protein rbd, or on newly identified features of existing mabs that were not described previously (table ) [ , [ ] [ ] [ ] [ ] . rbd-targeting human mabs have been extensively reported. most of these mabs can neutralize pseudotyped or live mers-cov in vitro, and some have shown protection against mers-cov infection in animal models in vivo [ , [ ] [ ] [ ] [ ] . the structures of several of these mabs with their antigen-binding fragments (fabs) or single-chain variable fragments (scfvs) complexed with rbd are known ( figure ) [ , [ ] [ ] [ ] [ ] . binding of these mabs to rbd involves two major recognition modes, with binding to rbd residues contacted by or overlapping with dpp (as is the case for gd- , mca , and cdc -c ), or with binding to the rbd residues outside of the dpp -binding interface (as seen with mers- ) ( table ) . infection in animal models in vivo [ , [ ] [ ] [ ] [ ] . the structures of several of these mabs with their antigen-binding fragments (fabs) or single-chain variable fragments (scfvs) complexed with rbd are known ( figure ) [ , [ ] [ ] [ ] [ ] . binding of these mabs to rbd involves two major recognition modes, with binding to rbd residues contacted by or overlapping with dpp (as is the case for gd- , mca , and cdc -c ), or with binding to the rbd residues outside of the dpp -binding interface (as seen with mers- ) ( table ) . the human mabs mers-gd and mers-gd each recognize distinct regions of the rbd [ ] . these mabs have a synergistic effect in the neutralization of pseudotyped mers-cov in vitro, with a much lower half-maximal inhibitory concentration (ic ) for their use in combination than separately [ ] . an analysis of crystal structures has indicated that mers-gd binds rbd at the dpp -binding site, and that the neutralization and recognition epitopes almost completely overlap this site, as seen previously for mers-cov rbd-targeting neutralizing mabs, such as m [ , ] . the mers-gd mab protects hdpp -tg mice from mers-cov challenge, both preventively and therapeutically, with significantly lower lung virus titers and rna copy numbers at day postchallenge, and higher survival rates ( % for pre-challenge vaccination and % for post-challenge vaccination) relative to control mice treated with an irrelevant mab [ ] . the human mab mca was isolated from a mers survivor via the construction of a phagedisplay antibody library from peripheral b cells [ ] . crystal structure analysis indicates that mca binds mers-cov s-protein rbd at residues involved in receptor binding, thus interfering with rbd binding to hdpp ( figure a ) [ ] . this mab prophylactically and therapeutically inhibits mers-cov replication in common marmosets, resulting in significantly improved outcomes and reduced the human mabs mers-gd and mers-gd each recognize distinct regions of the rbd [ ] . these mabs have a synergistic effect in the neutralization of pseudotyped mers-cov in vitro, with a much lower half-maximal inhibitory concentration (ic ) for their use in combination than separately [ ] . an analysis of crystal structures has indicated that mers-gd binds rbd at the dpp -binding site, and that the neutralization and recognition epitopes almost completely overlap this site, as seen previously for mers-cov rbd-targeting neutralizing mabs, such as m [ , ] . the mers-gd mab protects hdpp -tg mice from mers-cov challenge, both preventively and therapeutically, with significantly lower lung virus titers and rna copy numbers at day post-challenge, and higher survival rates ( % for pre-challenge vaccination and % for post-challenge vaccination) relative to control mice treated with an irrelevant mab [ ] . the human mab mca was isolated from a mers survivor via the construction of a phage-display antibody library from peripheral b cells [ ] . crystal structure analysis indicates that mca binds mers-cov s-protein rbd at residues involved in receptor binding, thus interfering with rbd binding to hdpp ( figure a ) [ ] . this mab prophylactically and therapeutically inhibits mers-cov replication in common marmosets, resulting in significantly improved outcomes and reduced lung disease, compared with unvaccinated controls, and undetectable virus titers days post-challenge [ ] . a probe-based single-b-cell cloning strategy has been used for the isolation of cdc -c and cdc -c mabs from a patient convalescing from mers, as well as for the isolation of jc - and jc - mabs from nhps immunized with mers-cov full-length s dna and protein [ ] . all these antibodies have neutralizing activities against both pseudotyped and live mers-cov. among them, cdc -c is the most potent against pseudotyped mers-cov strains, with neutralization ic values ranging from . µg/ml to . µg/ml [ ] . crystal-structure analysis of the cdc -c and jc - fab-rbd complexes indicates that both mabs bind rbd in the "out" (exposed) position, with the cdc -c rbd binding overlapping with the dpp -contacting residues ( figure b ,c) [ ] . in addition, cdc -c prophylactically protects hdpp -tg mice from mers-cov infection, resulting in no detectable viral replication in the lungs three days post-challenge, and no fatalities over days of observation [ ] . the human mab mers- also neutralizes pseudotyped mers-cov and, notably, displays synergistic neutralization in combination with the mers-cov s-protein rbd-targeting mers- and m mabs [ , ] , as well as the s-protein ntd-targeting f mab, in each case with dramatic reduction of the ic compared with individual mabs [ ] . structural analysis of a mers- -fab-rbd complex revealed that mers- binds the rbd from outside the dpp -binding interface, rather than competing with dpp ( figure d ). unlike mers- , which binds rbd regardless of its conformational state within the s trimer, mers- binds rbd in the "standing" position where its epitopes are readily exposed and accessible [ ] . thus, mers- displays unique epitope specificity, and an unusual mechanism of action involving indirect interference with dpp binding through conformational changes, which may explain the observation of synergistic neutralization in combination with other mabs [ ] . single-domain antibody fragments (vhhs), or nanobodies, are the antigen-recognition regions of camelid heavy-chain-only antibodies (hcabs), which do not contain light chains. vhhs are easily expressed with high yield, and they have intrinsic stability, strong binding affinity, and specificity to target antigens, and they have therefore been developed as important therapeutic tools against viral infection, including that of mers-cov [ , , [ ] [ ] [ ] [ ] [ ] . four vhhs (vhh- , vhh- , vhh- , and vhh- ) have been identified from bone marrow cells of dromedary camels immunized with modified vaccinia virus (mva) expressing mers-cov s protein, and challenged with mers-cov [ ] . these vhhs bind mers-cov s protein with low k d values ( . - nm), recognize an epitope at residue d of rbd, and neutralize mers-cov (prnt , . - . µg/ml) [ ] . these four monomeric vhhs have each been fused with a c-terminal human igg tag to generate four hcabs (hcab- , hcab- , hcab- , and hcab- ), with a higher binding affinity and a longer half-life than the free vhhs [ ] . studies of protective efficacy show that hdpp -tg mice (k ) injected with monomeric vhh- ( or µg per mouse) lose weight, and die within seven days post-infection, possibly because of the short half-life of the vhh. however, when the mice are injected with hcab- ( µg per mouse), which has an extended half-life (~ . days), protection against mers-cov is complete, with no viral titers or pathological changes in the lungs of virally challenged mice [ ] . by immunizing llamas with a recombinant rbd fragment (residues - ) fused to a c-terminal human igg fc tag (s - -fc), we constructed a vhh library, and we used it to generate a monomeric vhh, nbms , and a human fc-fused vhh, nbms -fc [ ] . both vhhs can be expressed in a yeast expression system to high purity, and bind rbd with high affinity, recognizing a conformational epitope (residue ) at the rbd-dpp interface, and blocking the binding of rbd to dpp . these vhhs, particularly nbms -fc, potently cross-neutralize pseudotyped mers-cov strains isolated from different countries, hosts, and time periods [ ] . importantly, the fc-fused nbms -fc significantly improves the serum half-life of nbms , and a single-dose treatment of hdpp -tg mice with this agent completely protects them against lethal mers-cov challenge [ ] . these single-domain vhhs demonstrate the feasibility of developing cost-effective, potent, and broad-spectrum therapeutic antibodies against mers-cov infection. compared with vaccines based on mers-cov full-length s protein, which have the potential to attenuate neutralizing activity or enhance immune pathology, vaccines developed from mers-cov s-protein rbd are safer, and they do not cause immunological toxicity or eosinophilic immune enhancement [ , , , ] . moreover, rbd-based therapeutic antibodies are generally more potent than non-rbd s -based or s -based antibodies [ , , ] . hence, rbd-based vaccines and therapeutic antibodies have the potential for further development as effective tools to prevent and treat mers-cov infection. despite their acknowledged advantages, there are some issues associated with rbd-based interventions that need to be addressed. for example, rbd is under a high level of pressure of positive selection, and mutations occur in the rbd-dpp binding interface that might reduce the efficacy of these treatments [ , [ ] [ ] [ ] . one possible way to avoid this effect, and to delay the emergence of escape mutants is to combine rbd-targeting therapeutics with those targeting other regions of the s protein, or to combine antibodies recognizing distinct epitopes within the rbd [ , ] . such combinatorial strategies could also dramatically reduce antibody neutralization doses, providing feasible means to combat the continual threat of mers-cov. some recent advances have been made in the structure-guided design of anti-mers-cov interventions. structurally designed inhibitors of the cl protease have demonstrated potency against mers-cov [ ] . also, a structurally designed s-protein trimer in the optimal prefusion conformation is shown to elicit production of high titers of anti-mers-cov neutralizing antibodies [ ] . indeed, based on the previous studies on the structural design of mers-cov rbd, non-neutralizing epitopes in the rbd can be masked, to refocus the immunogenicity of the rbd on the neutralizing epitopes, and thus to enhance its ability to confer immune protection [ ] . results from these structure-based studies will help to inform the design of innovative rbd-based anti-mers-cov vaccines and therapeutics with improved efficacy. isolation of a novel coronavirus from a man with pneumonia in saudi arabia fatality risks for nosocomial outbreaks of middle east respiratory syndrome coronavirus in the middle east and south korea risks of death and severe disease in patients with middle east respiratory syndrome coronavirus impact of comorbidity on fatality rate of patients with middle east respiratory syndrome clinical determinants of the severity of middle east respiratory 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against pseudotyped mers-cov with or without mutations in rbd structure-guided design of potent and permeable inhibitors of mers coronavirus cl protease that utilize a piperidine moiety as a novel design element this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: this study was supported by the nsfc grant , and the nih grants r ai , r ai , and r ai . the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. the authors declare no competing interests. key: cord- -ujz nkk authors: pirnay, jean-paul; selhorst, philippe; cochez, christel; petrillo, mauro; claes, vincent; van der beken, yolien; verbeken, gilbert; degueldre, julie; t’sas, france; van den eede, guy; weuts, wouter; smets, cedric; mertens, jan; geeraerts, philippe; ariën, kevin k.; neirinckx, pierre; soentjens, patrick title: study of a sars-cov- outbreak in a belgian military education and training center in maradi, niger date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ujz nkk coronavirus disease (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ) compromises the ability of military forces to fulfill missions. at the beginning of may , out of belgian soldiers deployed to a military education and training center in maradi, niger, developed mild covid- compatible symptoms. immediately upon their return to belgium, and two weeks later, all seventy soldiers were tested for sars-cov- rna (rt-qpcr) and antibodies (two immunoassays). nine soldiers had at least one positive covid- diagnostic test result. five of them exhibited covid- symptoms (mainly anosmia, ageusia, and fever), while four were asymptomatic. in four soldiers, sars-cov- viral load was detected and the genomes were sequenced. conventional and genomic epidemiological data suggest that these genomes have an african most recent common ancestor and that the belgian military service men were infected through contact with locals. the medical military command implemented testing of all belgian soldiers for sars-cov- viral load and antibodies, two to three days before their departure on a mission abroad or on the high seas, and for specific missions immediately upon their return in belgium. some military operational settings (e.g., training camps in austere environments and ships) were also equipped with mobile infectious disease (covid- ) testing capacity. coronavirus disease (covid- ) is an infectious disease caused by the newly emerged severe acute respiratory syndrome coronavirus (sars-cov- ), a single stranded-stranded rna beta-coronavirus with a , nucleotides-long genome [ ] . it was first identified in january in wuhan, china, and spread rapidly causing an ongoing worldwide pandemic. the republic of the niger is the largest country in west africa, landlocked by libya, chad, nigeria, benin, mali, burkina faso, and algeria. it has a population of about million, living mostly in clusters in the far south and west of the country. niger faces serious development challenges due to its desert terrain and overpopulation, but also due to attacks by "al-qaeda in the islamic maghreb", its use as a transit country for migrants, and the spillover of nigeria's boko haram insurgency into southeastern niger. french, american, and belgian armed forces are currently assisting niger in countering jihadist groups present in the sahel [ ] . since the end of august , belgian military personnel have been deployed in the maradi region nearby the southern border with nigeria in the context of operation new nero (onn). the belgian military are based on the outskirts of the city of maradi, the second-or third-largest city in the country. in , it had about , inhabitants and it is one of niger's most economically prosperous cities and the major transport, trade, and agricultural hub of south-central niger. a group of belgian soldiers was sent in two waves, one on december and the other on february , to the maradi camp. they formed the "mobile education and training team" (mett), which brings together military trainers, members of the special forces group, around forty members of the rd battalion para and a "special operations surgical team". they trained a special intervention company made up of nigerien soldiers. the first case of covid- in belgium was confirmed on february [ ] . on march, the first case of covid- in niger was reported in the nigerien capital, niamey: a -year-old nigerien or nigerien national (depending on the report), a storekeeper in a land transport company, who had travelled along the lomé (togo)-accra (ghana)-abidjan (ivory coast)-ouagadougou (burkina faso) axis [ ] . after that, two other covid- cases were confirmed in niamey: a brazilian woman, who returned to niger on march , coming from switzerland, and a -year-old italian man, who returned to niger on february , from his home country of italy [ ] . on march, the first death from the new coronavirus in niger was recorded: a -year-old male, brazilian national, who entered niger from senegal on february [ ] . niger declared a national state of emergency due to the covid- pandemic on march. gatherings of more than people were banned, places of worship closed, and obligatory epidemiological safety measures were instituted, including the wear of facemasks and social distancing in public places and public transport. on april, the first two covid- cases outside niamey were confirmed in two residents of maradi. at the beginning of may , a handful of belgian military service men developed covid- suspicious symptoms. the mett returned to belgium in two waves, on may and may . immediately upon their arrival in brussels, nasopharyngeal swabs and blood samples were collected from all soldiers, symptomatic, and asymptomatic alike, for covid- diagnostic testing. the returning military personnel were placed in quarantine nearby the military airport, awaiting test results and medical consultation later in the day. sars-cov- viral loads of the nasopharyngeal samples were determined using quantitative rt-pcr (rt-qpcr), while two immunoassays were used to assess their antibody response to the virus. a rapid covid- antibody test was used for first line screening, and a more widely available and evaluated double antigen sandwich assay was performed later on frozen serum samples. two weeks later, all individuals were re-tested. the sars-cov- genomes present in rt-qpcr positive nasopharyngeal samples were sequenced. viral genomes were evaluated and a retrospective epidemiological study was conducted to identify possible origins and routes of spread and to discuss measures to prevent or control future covid- outbreaks, which pose a threat to the readiness of military forces and their ability to fulfill missions [ ] . nasopharyngeal sample collection kits (copan diagnostics, murrieta, ca, usa), consisting of a flexible minitip flocked swab in a tube filled with ml universal transport medium (utm), were used for covid- sample collection in all returning service members, immediately upon arrival in belgium (d ) and two weeks later (d ). blood samples were collected, using vacuette serum gel tubes (greiner bio-one, vilvoorde, belgium), for covid- antibody testing in all returning personnel on d and d . blood was allowed to clot for a minimum of min in a vertical position and then centrifuged at room temperature in a swinging bucket rotor for min at g. serum samples were tested immediately and stored at − • c ± • c for possible re-testing. rt-qpcr was performed on the nasopharyngeal swabs using the novel coronavirus ( -ncov) nucleic acid diagnostic kit and sample release reagent (both sansure biotech inc., changsha, china), according to the manufacturer's instructions, with some adaptations for the lightcycler . instrument (roche diagnostics belgium, machelen, belgium). the assay targets the orf ab and n genes, coding for a well-conserved replicase polyprotein and the nucleocapsid protein, respectively. each test also contains an rna internal control (rnase p genes), which detects rt-pcr failure and/or inhibition. swab samples were first inactivated at • c for min in a bead bath. two hundred microliters of swab medium were pipetted into a . ml tube, which was centrifuged at , g for min. the supernatant was discarded and µl of sterile saline were added. the tubes were vortexed and again centrifuged at , g for min, and after discarding the supernatant, about - µl of the liquid were retained. thirty microliters of sample release reagent (denatures proteins and frees sars-cov- rna) were added and mixed by pipetting up and down. the tubes were incubated at room temperature for min. eight microliters of the sample supernatants, of the negative control (physiological saline), and of the positive control (in vitro transcriptional rna of target genes orf ab and n) were subsequently pipetted into the corresponding capillaries, which were preloaded with µl pcr master mix. the program cycle parameters were as follows: a reverse transcription step at • c for min, followed by a cdna pre-denaturation at • c for min and then cycles of denaturation at • c for s, and annealing, extension and fluorescence collection at • c for s. there was no need to apply color compensation files. two sars-cov- immunoassays were performed on the serum samples. the multi-g covid- igg/igm rapid test (multi-g, antwerpen, belgium), a lateral flow chromatographic membrane-based immunoassay, was used for the in vitro qualitative detection of igg and igm antibodies to -ncov in serum specimens. in this assay, the samples first react with sars-cov- antigen-coated particles and then migrate to react with anti-human igg and/or igm antibodies. according to the package insert, the relative specificity of the test is . % for igg and . % for igm. the relative sensitivity is % for igg and . % for igm. the elecsys anti-sars-cov- immunoassay on the cobas e analyzer (roche diagnostics belgium, machelen, belgium) was used for the in vitro qualitative detection of antibodies (including igg) to sars-cov- in serum samples. this double antigen sandwich assay uses a mix of biotinylated and ruthenylated recombinant nucleocapsid (n) antigen of sars-cov- for determination of antibodies. a signal threshold ≥ was defined as positive. a specificity of . % and a sensitivity of % (≥ days post pcr confirmation) were reported in the package insert. all immunoassays were performed according to the manufacturers' instructions. d nasopharyngeal samples with positive rt-qpcr results were selected for sequencing on an oxford nanopore minion device using r . flow cells (oxford nanopore technologies, oxford, uk), based on a protocol from quick et al., [ ] . briefly, rna was extracted from µl utm transport medium and eluted in µl water using a maxwell rsc instrument. µl rna was then converted to cdna using random hexamers and the protoscript ii first strand cdna synthesis kit (new england biolabs, hitchin, uk). subsequently, a strain-specific multiplex pcr was performed in six reactions using an bp sars-cov- primer scheme (table s ) and q high-fidelity dna polymerase (new england biolabs). cycling conditions were s at • c, followed by - cycles at • c for s, • c for . min, • c for min, and a final elongation step at • c for min. the resulting bp pcr products were pooled and cleaned up using ampurexp magnetic beads (beckman coulter, high wycombe, uk) and quantified using a qubit dsdna high sensitivity assay on a qubit . instrument (thermo fisher scientific, waltham, ma, usa). samples were then barcoded using the ultra ii end repair/da-tailing module (new england biolabs) and the native barcoding kits nbd and nbd (oxford nanopore technologies), cleaned up with magnetic beads and pooled at equimolar ratios prior to ligation of the amii adapters with blunt/ta ligase master mix (new england biolabs). sequencing libraries were loaded onto the r . flow cell using the ligation sequencing kit lsk (oxford nanopore technologies) and sequencing data were collected overnight. sequence reads were base called in high accuracy mode and demultiplexed using the guppy algorithm v . (oxford nanopore technologies). consensus genome sequences were produced by comparing two distinct bioinformatics pipelines. first, an in-house customized pipeline, which aligns reads to a sars-cov- reference genome (genbank mn . ) using minimap , was applied. after removal of primer sequences using a custom python script, a majority rule consensus was produced for positions with ≥ x genome coverage, while regions with lower coverage, were masked with n characters [ ] . in parallel, the artic network's bioinformatics pipeline [ ] was run, which incorporates base quality data as well as the nanopolish algorithm [ ] to improve consensus sequence for a draft genome assembly. whole genome sequences were compared to the reference sars-cov- wuhan genome (nc_ . ) and to all sars-cov- genomes submitted to the gisaid (global initiative on sharing all influenza data) repository (https://www.gisaid.org) [ ] (july status) and to the national center for biotechnology information (ncbi). blosum substitution scores were calculated to assess amino acid changes and their potential to alter or change the functionality of the involved protein [ ] . negative scores are predicted to be "bad" in terms of substitution, i.e., they are not frequently observed in nature. after submission of the obtained sars-cov- genome sequences to gisaid (accession ids: epi_isl_ to epi_isl_ ) they were automatically included into nextstrain [ ] , an open-source project providing a continually updated phylogenetic analysis and visualization of the evolutionary relationships of publicly available sars-cov- genomes. this analysis includes subsampling, alignment, maximum likelihood phylogenetic inference, temporal dating of ancestral nodes, and discrete trait geographic reconstruction. nextstrain phylogeny is rooted relative to early samples from wuhan. full details on bioinformatic processing can be found at [ ] . demographics, covid- suspicious symptoms and signs (cough, fever, sore throat, myalgia, headache, fatigue, anosmia, ageusia, and/or diarrhea) and dates of symptom onset were extracted from the medical files of the belgian soldiers who took part in onn in maradi, niger. information on the duration of covid- compatible signs and symptoms was not systematically recorded. possible contacts between the belgian soldiers and externs were traced. background covid- related epidemiological data were retrieved from online local media and from the european centre for disease prevention and control (ecdc) (https://www.ecdc.europa.eu/en/geographical-distribution- -ncov-cases). clinical samples were routinely processed in view of diagnosis. results were analyzed in the context of retrospective epidemiological analysis and were not deemed prospective research. seventy belgian male soldiers returned from a mission in maradi in two waves, one on may and another on may . they had arrived in the maradi military education and training center on december and february , a few days before the first diagnosis of covid- in belgium, in a -year-old man, who was repatriated from wuhan city on february [ ] . mild symptoms indicative of covid- were recorded in ( . %, / ) of these soldiers in the week period ( april to may) preceding their return to belgium (tables and ). during that same period, approximately one sixth of the nigerien military trainees ran a fever. the belgian soldiers were immediately tested (rt-qpcr and two immunoassays), nearby the military airport, upon their return to belgium (d ) and again two weeks later (d ). nine belgian soldiers ( . %, / ) had at least one positive covid- diagnostic test result ( table ). five of them ( . %, / ) were trainers. the total number of trainers involved in the training mission was ( . %, / ). trainers were thus disproportionately represented in the covid- positive diagnosis population. sars-cov- viral load was detected in the d nasopharyngeal samples of four ( . %, / ) soldiers (m -m ), which persisted at d in three of them ( table ). the four rt-qpcr positive soldiers remained in isolation for two weeks. in these four d rt-qpcr positive soldiers, antibodies were not observed at d , but all had seroconverted on d (table ). this indicates that these soldiers were recently infected and that the maradi outbreak was still ongoing. the remaining five soldiers with negative rt-qpcr results, showed positive immunoassays at d , which highlights the importance of serological testing to achieve more accurate estimates of the extent of a covid- outbreak. however, for two individuals (m and m ), only the rapid antibody tests were positive, making it uncertain whether they actually had the disease ( table ) . seroconversion is usually, and depending on the applied method, observed after a median of approximately two weeks after symptom onset for both igm and igg [ ] and in contrast to igg, the positive rate of igm drops after weeks [ ] . although seroconversion as well as the loss of igm can clearly be observed in our antibody data, the timings do not always correspond. for example, in some patients igms disappeared more rapidly (e.g., m and m ) or were not detected at all (e.g., m ), but this was somewhat expected given person-related variations as well as the sensitivity of the different tests used ( table ) . of interest, four (m -m ) of the nine covid- positive soldiers (including soldiers m and m with doubtful diagnosis) were asymptomatic ( table ). in two of them, sars-cov- viral load was detected by rt-qpcr (table ). these infected asymptomatic soldiers (one of them a trainer) would have continued to work while shedding the virus. in addition, for two soldiers (m and m ), the onset of covid- symptoms occurred on may and , respectively, a few days before their return to belgium. hence, it is very likely that the timely return to belgium prevented this becoming worse. for comparison, a covid- outbreak on the uss theodore roosevelt was characterized by widespread transmission with mild symptoms and asymptomatic infection ( %) among a similar population of mostly young healthy adults living in a congregate environment [ ] . of note, just like with the belgian soldiers, anosmia and ageusia were the symptoms most strongly associated with infection, followed by fever [ ] . some scientists suggested that onset of anosmia should be considered sars-cov- related until proven otherwise and recommended immediate isolation and confirmatory testing [ ] . to analyze possible patterns of spread across local and global epidemiological scales, we further determined the whole-genome sequences of the sars-cov- isolates from the four d rt-qpcr positive belgian soldiers (sequencing statistic are given in table s ). the genome sequences were submitted to gisaid under accession ids epi_isl_ - , and were re-named c , c , c , and c in nextstrain (table ) . no other sars-cov- genome sequences from patients with documented history of exposure or residence in niger were present in the gisaid and ncbi databases. the four genome sequences were compared to the reference sars-cov- wuhan genome (nc_ . ) and with all sars-cov- genomes submitted to the gisaid and ncbi repositories. genomes c and c are identical and were found in soldiers m and m , both trainers (table ) . genomic sequences of samples collected from covid- cases within one infection cluster are often identical or highly similar, especially within household pairs [ ] . however, due to the relatively limited mutation rate of sars-cov- , it is impossible to know if both trainers were infected by the same source, by different sources, or passed it on to each other. genome c has one additional difference in comparison to genomes c and c , i.e., mutation g t corresponding to a glycine (g) to valine (v) amino acid change in protein orf ab. genome c has two differences in comparison to genomes c and c : mutations a c and c t, both in orf ab and both silent (table ) . table . nucleotide and amino acid comparison of the maradi genomes to the reference sars-cov- wuhan genome (nc_ . ). reference genome nucleotides and amino acids showing variations in any of the maradi genomes are presented in dark green. matches with the reference genome are indicated in light green, variations in red. the sarscov- genes harboring these variations are marked in orange, sky blue, air force blue, greige, purple and grey. orf ab with two genomes being indistinguishable and one differing by one single-nucleotide polymorphism (snp), and another by two snps, the genomic variations observed in our cluster correspond with those observed in "institutional outbreaks". in three reported outbreaks where , , and individuals had contracted the virus within one institution, up to two snps were detected [ ] . to date, five major clades of sars-cov- were defined (i.e., gr, g, gh, l, o, s, and v), based on characteristic mutational events observed in the genomes submitted to gisaid (https: //www.gisaid.org/epiflu-applications/next-hcov- -app/) (figure ) [ ] . phylogenetic examination using nextstrain (accessed august ) showed that all four maradi genomes belonged to cluster b of gisaid's clade s (figure ). the s clade was first observed among travelers from wuhan in the early days of the outbreak [ ] and was prevalent in north america and in europe [ ] [ ] [ ] , but has now declined worldwide in favor of the g clades ( figure b) . all four maradi genomes bear the two signature mutations of clade s: the silent mutation c t and mutation t c, which causes a leucine (l) to serine (s) (hence the clade's name) change in the orf protein [ ] . in addition, our cluster shared other mutations when compared to the reference nc_ . , four of which were previously reported and determine the tree topology (g a, g t, a g, and g a), as indicated by the arrows in figures and . mutation g a, which causes a glycine (g) to arginine (r) amino acid change in orf a, and the silent mutation c t were previously observed in other sars-cov- genomes. the c t mutation, however, is unique to the maradi genomes and results in an alanine (a) to valine (v) amino acid change in orf ab. other infrequent variations, which are present in less than reported genomes, are shown in table . the rare silent mutation a g, present in all four maradi genomes, was recently observed in benin, nigeria, and mali (table ) , three neighboring countries of niger, and places our genomes in a cluster of recent african descent ( % confidence), with its most recent common ancestor (mrca) estimated between february and march (figure ). this is compatible with the suspected first covid- case in niger (reported on march ), who had travelled to togo, ghana, ivory coast, and burkina faso [ ] . the dates on which the first confirmed covid- cases were reported in these countries, according to ecdc, were ( case), ( cases), ( case), and march ( cases), respectively. the rare silent mutation a g, present in all four maradi genomes, was recently observed in benin, nigeria, and mali (table ) , three neighboring countries of niger, and places our genomes in a cluster of recent african descent ( % confidence), with its most recent common ancestor (mrca) estimated between february and march (figure ). this is compatible with the suspected first covid- case in niger (reported on march ), who had travelled to togo, ghana, ivory coast, and burkina faso [ ] . the dates on which the first confirmed covid- cases were reported in these countries, according to ecdc, were ( case), ( cases), ( case), and march ( cases), respectively. three amino acid changes, resulting from the individual mutation g t, the shared mutation g a, and the s clade signature mutation t c are predicted to result in significant alterations of the functionality of the involved proteins (negative blosum scores) ( table ) [ ] . the mutation predicted to have the most significant impact (exhibiting a blosum score of − ), was g t, but has only been observed once so far, in march , in isolate ncov- /france/ bi/ (table ). it was suggested that sars-cov- protein sequence diversity could be associated with virus pathogenicity and/or transmission. in europe, several countries were associated with specific virus clades or mutations while the frequency in clinical symptoms of covid- patients also varied between these countries. it was therefore suggested that genome and patient symptom data should be combined to better understand sars-cov- pathogenicity and help develop adapted treatments [ ] . a military camp is a semi-enclosed environment with a high population density and contained spaces (e.g., dining halls and recreational spaces) in which many soldiers (e.g., trainers and trainees) and a number of civilians interact. communal living and training environments are renowned for person-to-person transmission of disease agents, a frequent problem for militaries worldwide [ ]. during their entire stay at the maradi training facility, the belgian military service men had sporadic contacts with nigerien civilians (in the camp and in the city of maradi), but especially the trainers had frequent contacts with nigerien military trainees (table ). other non-nigerien contacts occurred at least two weeks before the date on which the first covid- cases were presumed to have appeared in maradi ( april) and more than six weeks before the onset of covid- -like symptoms in the belgian military service men ( april- may) (tables and ). considering that the incubation period for covid- is currently estimated to be between one and days [ ] , it is unlikely that the belgian soldiers contracted covid- from these non-nigerien contacts. in fact, the possible window of covid- infection of the belgian soldiers occurred short after high daily growth increases of covid- cases in niger (more than doubling for some days) (figure ) , as derived from data reported by the ecdc (https://www.ecdc.europa.eu/en/ geographical-distribution- -ncov-cases). overall, the data produced by this combined conventional (i.e., contact tracing) and genomic (i.e., african mrca) epidemiological analysis, suggest that the belgian soldiers were infected via direct contacts with locals and subsequently among themselves. overall, the data produced by this combined conventional (i.e., contact tracing) and genomic (i.e., african mrca) epidemiological analysis, suggest that the belgian soldiers were infected via direct contacts with locals and subsequently among themselves. the sars-cov- outbreak in a belgian military education and training center in maradi, niger, was characterized by mild symptoms in five soldiers and asymptomatic infection in two soldiers (one trainer), both having a viral load, as diagnosed upon their timely return to belgium. the symptoms most strongly associated with covid- infection were anosmia and ageusia. trainers were disproportionately infected. conventional and genomic epidemiological data suggest that the the sars-cov- outbreak in a belgian military education and training center in maradi, niger, was characterized by mild symptoms in five soldiers and asymptomatic infection in two soldiers (one trainer), both having a viral load, as diagnosed upon their timely return to belgium. the symptoms most strongly associated with covid- infection were anosmia and ageusia. trainers were disproportionately infected. conventional and genomic epidemiological data suggest that the belgian military servicemen were infected by a locally transmitted virus with a recent african common ancestor. based, among others, on this epidemiological study, the medical military command decided to continue testing belgian soldiers for sars-cov- viral load and antibodies, and this two to three days before a mission abroad or on the high seas, and for specific missions immediately upon their return. soldiers in whom sars-cov- viral load is detected are isolated. some military operational settings (e.g., training camps in austere environments and ships) were also equipped with mobile infectious disease (covid- ) testing capacity. more specifically, the training center in maradi is now using a genexpert machine for the rapid detection of sars-cov- viral load in nasopharyngeal swabs. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : sars-cov- primer scheme, table s : sars-cov- sequencing statistics. a new coronavirus associated with human respiratory disease in china unhcr: attacks in nw nigeria send thousands fleeing to niger. news laboratory information system requirements to manage the covid- pandemic: a report from the belgian national reference testing center measures to limit covid- outbreak effects among military personnel: preliminary data multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples data, disease and diplomacy: gisaid's innovative contribution to global health amino acid substitution matrices from protein blocks nextstrain: real-time tracking of pathogen evolution antibody responses to sars-cov- in patients with covid- evaluation of nucleocapsid and spike protein-based enzyme-linked immunosorbent assays for detecting antibodies against sars-cov- sars-cov- infections and serologic responses from a sample of anosmia and covid- revealing covid- transmission in australia by sars-cov- genome sequencing and agent-based modeling geographic and genomic distribution of sars-cov- mutations. front. microbiol. , , variant analysis of sars-cov- genomes severe acute respiratory syndrome coronavirus : virus mutations in specific european populations european centre for disease prevention and control (ecdc). q & a on novel coronavirus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank all the authors behind the sars-cov- global genomic data as well as the nextstrain consortium (https://nextstrain.org/ncov/global). we thank petra andries, jacintha vandevelde, griet steurs, johann griselain, lilo tranchina, and jan moens for their technical support, and heidi verelst for contacting patients. the authors declare no conflict of interest. key: cord- -s x grh authors: payne, natalie; kraberger, simona; fontenele, rafaela s; schmidlin, kara; bergeman, melissa h; cassaigne, ivonne; culver, melanie; varsani, arvind; van doorslaer, koenraad title: novel circoviruses detected in feces of sonoran felids date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: s x grh sonoran felids are threatened by drought and habitat fragmentation. vector range expansion and anthropogenic factors such as habitat encroachment and climate change are altering viral evolutionary dynamics and exposure. however, little is known about the diversity of viruses present in these populations. small felid populations with lower genetic diversity are likely to be most threatened with extinction by emerging diseases, as with other selective pressures, due to having less adaptive potential. we used a metagenomic approach to identify novel circoviruses, which may have a negative impact on the population viability, from confirmed bobcat (lynx rufus) and puma (puma concolor) scats collected in sonora, mexico. given some circoviruses are known to cause disease in their hosts, such as porcine and avian circoviruses, we took a non-invasive approach using scat to identify circoviruses in free-roaming bobcats and puma. three circovirus genomes were determined, and, based on the current species demarcation, they represent two novel species. phylogenetic analyses reveal that one circovirus species is more closely related to rodent associated circoviruses and the other to bat associated circoviruses, sharing highest genome-wide pairwise identity of approximately % and %, respectively. at this time, it is unknown whether these scat-derived circoviruses infect felids, their prey, or another organism that might have had contact with the scat in the environment. further studies should be conducted to elucidate the host of these viruses and assess health impacts in felids. the sonoran desert is a unique ecosystem in which four species of felids are known to coexist: pumas (puma concolor), bobcats (lynx rufus), ocelots (leopardus pardalis), and jaguars (panthera onca) [ ] . these felids play a crucial role in maintaining a functional ecosystem. pumas mainly regulate populations of ungulates, including deer, bighorn sheep, and javelina [ ] [ ] [ ] , while bobcats and ocelots tend to prey upon small mammals, such as lagomorphs and rodents, and reptiles [ , [ ] [ ] [ ] . ocelots and jaguars are recognized as endangered in the region [ ] [ ] [ ] ; however, the status of all four felid species are likely threatened by shared environmental pressures, including drought [ ] , habitat fragmentation and encroachment (which can lead to human-wildlife conflict), and emerging diseases. while antibodies to canine distemper virus (cdv) have been detected in sonoran jaguars [ ] and antibodies to cdv, feline panleukopenia virus, feline calicivirus, and feline enteric coronavirus have been detected in pumas from southern arizona [ ] , other viruses circulating in populations of sonoran felids are largely unknown. cataloging the diversity of viruses present in these felids could reveal an abundance of both known and novel viruses; although most viruses are not pathogenic, some may cause disease and be relevant to conservation. high throughput sequencing technologies have allowed for unprecedented advances in identifying known and novel viruses and characterizing viral communities through viral metagenomics. taking advantage of metagenomic approaches to monitor viral communities associated with wildlife could be instrumental for conservation; however, this is not routinely performed. altered viral evolutionary dynamics (largely due to anthropogenic factors such as facilitating viral movement around the world, spillover from domestic animals, increasingly dense populations of wildlife due to habitat encroachment, and climate change) and altered exposure of wildlife to viruses through vector range expansion create conditions for accelerated emergence of viruses, some of which may cause new disease outbreaks in wildlife populations [ , ] . notable examples include the spillover of feline leukemia virus (felv) from domestic cats into the endangered florida panther [ ] and spillover of cdv from domestic dogs into wildlife populations within serengeti national park, tanzania, affecting spotted hyenas, african lions, and other species [ , ] . this may be especially problematic for already threatened populations, as small populations typically have lower genetic diversity (and possibly stress-induced immunosuppression) and, therefore, decreased adaptive potential to assist survival of a proportion of the population experiencing the effects of a novel viral disease [ , [ ] [ ] [ ] . genomes from several families of circular rep-encoding single-stranded dna viruses (cress-dna viruses) are part of the phylum cressdnaviricota [ ] and have been identified in fecal samples of other mammals, including domestic cats [ , ] , bobcats, african lions [ ] , capybaras [ ] , and tasmanian devils [ ] . circoviridae is one of the families in the cressdnaviricota phylum and is composed of the genera circovirus and cyclovirus. circoviruses have ambisense genomes of approximately . - . kb in length and encode two proteins, rep and the capsid protein (cp) [ ] . circoviruses have implications for wildlife management because they are associated with disease in some vertebrates, including life-threatening hemorrhagic gastroenteritis in dogs [ ] [ ] [ ] , psittacine beak and feather disease in parrots [ ] , and postweaning multisystemic wasting syndrome in pigs [ , ] . importantly, several studies suggest that these life-threatening diseases may be largely due to coinfection with porcine parvovirus or porcine reproductive and respiratory syndrome virus [ , ] , or canine coronavirus, canine parvovirus, or cdv [ ] [ ] [ ] , in pigs and dogs respectively. no circoviruses are known to infect felids, although a cyclovirus (feline associated cyclovirus ) has been identified in the feces of domestic cats [ ] . additionally, a feline stool-associated circular dna cress-dna virus has recently been identified from cats with diarrhea [ ] . endogenous fragments of circoviruses have also been detected in feline genomes, indicating the susceptibility of the ancestors of modern felids to circovirus infection [ , ] . here we used a metagenomic approach to identify novel circoviruses in the feces of two species of sonoran felids, the puma and bobcat; although not endangered, knowledge of viral threats facing these species could help prevent future population decline, as well as indicate potential threats to the endangered ocelot and jaguar. for the two novel circoviruses identified, we sought to determine relationships with known circoviruses and characterize their genomes. these novel feline feces associated circoviruses may represent the first known feline circoviruses. scat samples from bobcats and pumas were collected from sonora, mexico, between and . the samples were desiccated at room temperature prior to shipping and long-term storage at − • c. to determine the species, dna was extracted by swabbing the scat surface. the swab was deposited into lysis buffer and dna extracted using qiagen's dneasy blood and tissue kit as previously described by cassaigne et al. [ ] . this dna was used as template for pcr of the mitochondrial cytochrome b gene [ ] with confirmation by sanger sequencing of the amplicon (~ bp region) as previously described [ ] . we randomly selected fecal samples (bobcats (n = ) and pumas (n = )) for this study. of each of the fecal samples, g was homogenized in sm buffer and the homogenate was centrifuged at × g for min. the supernatant was sequentially filtered through . µm and . µm syringe filters and viral particles in the filtrate were precipitated with % (w/v) peg- with overnight incubation at • c followed by centrifugation at , ×g as described in fontenele et al. [ ] . the pellet was resuspended in µl of sm buffer and µl of this was used for viral dna extraction using the high pure viral nucleic acid kit (roche diagnostics, indianapolis, in, usa). circular viral dna was amplified by rolling circle amplification (rca) using the illustra templiphi amplification kit (ge healthcare, chicago, il, usa). sequencing libraries were prepared from the rca products using the nextera dna flex library prep kit (illumina, san diego, ca, usa) and sequenced on an illumina hiseq ( × bp). the paired-end raw reads were trimmed using default settings within trimmomatic v . [ ] and the trimmed reads were de novo assembled using k-mer values of , , and within metaspades v . . [ ] . contigs greater than nucleotides were analyzed by blastx [ ] against a local viral protein database constructed from available ncbi refseq viral protein sequences (https://ftp.ncbi.nlm.nih.gov/refseq/release/viral/). based on the de novo assembled contigs (> nts) that had blastx hits to circovirus sequences, two pairs of abutting primers were designed manually to recover and verify the full genomes of circoviruses: uoa _ f -ctatagaacagatatgcaaattatggccgg- and uoa _ r -atatctcaaaaagaggaaccgaaaccttgg- (complementarity to cp gene/ stem loop region) and uoa f -gaccgatacccattgaaagtggagactaag- and uoa r -catcactcgaagcaggtcatcatag- (complementary to the rep gene region). as a template, . µl rca product was used with kapa hifi hotstart dna polymerase (kapa biosystems, wilmington, ma, usa) and the specific abutting primers described above were used for each of the fecal samples to screen and recover the full genomes of the circoviruses using the manufacturer's recommended thermal cycling conditions. the pcr amplicons were resolved on a . % agarose gel, recovered with gel purification, cloned into the plasmid pjet . (thermofisher, waltham, ma, usa), and sanger-sequenced at macrogen inc. (seoul, south korea) by primer walking. the sanger sequence contigs were assembled using the "assembly module" in geneious prime v [ ] . open reading frames in the genomes were identified using orffinder (https://www.ncbi.nlm.nih. gov/orffinder/). the genomes and amino acid sequences of rep and cp of representative circoviruses and those identified in this study were aligned using muscle [ ] , and pairwise percent identities were obtained using sdt v . [ ] (file s ). the optimal substitution model based on akaike information criterion with correction for small sample size (aicc) for the genome alignment was identified as gtr+i+g using jmodeltest [ , ] , and prottest [ ] identified lg+i+g as the optimal model for the rep alignment and vt+i+g+f as the optimal model for the cp alignment. phylogenetic analyses for each alignment were performed with phyml . [ ] . for visualization purposes, all trees were rooted with sequences from the duck associated cyclovirus (genbank: ky ) and horse associated cyclovirus (genbank: kr ) (not shown in the tree). branches with sh-like alrt support less than . [ , ] were collapsed using ips [ ] and ape [ ] packages in r [ ] . the viral genomes described in this manuscript were submitted to genbank (accession numbers: mt -mt ). based on the metagenomic analysis, we assembled a partial viral genome in two of the samples. based on this partial sequence data, we designed abutting primers to screen all the available scat samples. of the samples screened with the two primer pairs, three circovirus genomes were identified and recovered ( figure a ) from three fecal samples of bobcats. two of the genomes (genbank: mt and mt ) share greater than % pairwise identity to each other (file s ) and are nucleotides in length, having a rep coding sequence (cds) of nucleotides ( amino acids) on the virion-sense strand and cp cds of nucleotides ( amino acids) on the complementary strand. based on the species-demarcation threshold for circoviruses which is % genome-wide identity [ ] , both of these belong to a new species which we refer to as sonfela (derived from sonoran felid associated) circovirus . the third genome (genbank: mt ) of nucleotides, referred to as sonfela circovirus , is more distantly related, sharing approximately % identity with the two sonfela circovirus genomes (file s ), and contains a rep cds of nucleotides ( amino acids) on the virion-sense strand and cp cds of nucleotides ( amino acids) on the complementary strand. the stem loop and nonanucleotide motif "tagtattac" were identified in the genomes and correspond to the origin of replication. conserved motifs within rep (rc endonuclease motifs i, ii, and iii and sf helicase domains walker a, walker b, motif c, and arg finger) [ ] were all detected. the genome ( figure a ) and protein ml phylogenetic trees ( figure b ,c) demonstrate that canine circovirus (genbank: kc ), rodent associated circoviruses (roacv , , , , and ) (genbank: ky , ky , ky , ky , and mf ), bat associated circovirus (genbank: kx ), and the sonfela circoviruses cluster in a separate clade with sh-like alrt support between . - . . sonfela circovirus is most closely related to a group of three rodent-derived viruses (roacv - ; genbank: ky , ky , and ky ), sharing a maximum of approximately % genome-wide identity, % rep identity, and % cp identity with roacv (genbank: ky ) (file s ). the phylogenetic trees reveal sonfela circovirus and bat associated circovirus (genbank: kx ) to be sister taxa, sharing approximately % genome-wide identity, % rep identity, and % cp identity according to sdt; however, pairwise percent identity calculations reveal maximum genome-wide identity with batacv (genbank: kj ) ( . %) and cp identity with roacv (genbank: ky ) ( %) (file s ). sharing less than % genome-wide identity with known circoviruses, both sonfela circoviruses and represent novel species (file s ). based on the circovirus species demarcation threshold of % identity [ ] , the circovirus genomes identified and recovered in this study represent two new species. these feline associated viral genomes have a typical circovirus length, contain both circovirus rep and cp cds (in appropriate orientation), and have a well-defined nonanucleotide sequence. the health implications of these circoviruses for these populations are currently unclear given the viruses' true hosts and pathogenicity are unknown. as the viral genomes were derived from scat samples, the circoviruses could have infected the bobcat prey species or the felids themselves or be environmentally derived. the phylogenetic clustering of sonfela circovirus and several rodent circoviruses suggests the virus may be rodent-derived; similarly, sonfela circovirus may be bat-derived. as with these novel feline associated viruses, many of the recently described viruses have not been associated with their mammalian hosts. the lack of formal host association limits our ability to directly interpret the biological relevance of these viruses. however, in the meantime, it is critical to continue to describe the viral diversity associated with unconventional hosts. to our knowledge, the circoviruses described here may represent the first known feline associated circoviruses. detection, or lack thereof, of the circoviruses in other tissues within felids could help discern the viruses' true hosts. screening for the viruses in sympatric populations of rodents, bats, and other prey species could also be utilized to rule out or confirm the sources of these viruses. if felids are the host for these viruses, affected individuals should be monitored for possible symptoms of disease; however, further investigations linking these viruses to their natural host are needed as well as investigations into the prevalence of the viruses within felid populations in the sonoran desert and across the americas. funding: np was supported by funds from the genetics graduate interdisciplinary program and the technology and research initiative fund at university of arizona. the high throughput sequencing work and viral molecular work was supported by a startup grant awarded to av by arizona state university. sample collection of bobcat and puma scat was supported by primero conservation, a nonprofit wildlife conservation organization. wildlife survey and monitoring in the sky island region with an emphasis on neotropical felids food habits of pumas in northwestern sonora abundance and food habits of cougars and bobcats in the sierra leopardus pardalis) food habits in a tropical deciduous forest of jalisco diets of sympatric bobcats and coyotes during years of varying rainfall in central arizona food habits of ocelots and potential for competition with bobcats in southern texas interior. endangered and threatened wildlife and plants; final rule to extend endangered status for the jaguar in the united states protección ambiental-especies nativas de méxico de flora y fauna silvestres-categorías de riesgo y especificaciones para su inclusión, exclusión o cambio-lista de especies en riesgo recovery plan 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alignment search tool geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data multiple sequence alignment with high accuracy and high throughput a virus classification tool based on pairwise sequence alignment and identity calculation fast, and accurate algorithm to estimate large phylogenies by maximum likelihood jmodeltest , more models, new heuristics and parallel computing prottest , fast selection of best-fit models of protein evolution new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml . approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative phyloch: r language tree plotting tools and interfaces to diverse phylogenetic software packages ape . , an environment for modern phylogenetics and evolutionary analyses in r r: a language and environment for statistical computing a field guide to eukaryotic circular single-stranded dna viruses: insights gained from metagenomics we would like to thank interns meagan bethel and anna kegan scott and work study student emma froelich for help with dna extraction of bobcat and puma scat samples. we acknowledge jana jandova for her assistance with extracting viral dna. we also thank alex erwin, eldridge wisely, karla vargas, conor handley, and hans-werner herrmann for feedback during the early stages of manuscript preparation and robert jackson for critical review of this manuscript. any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the u.s. government. the authors declare that there are no conflict of interest. key: cord- -no mbg d authors: vandegrift, kurt j.; wale, nina; epstein, jonathan h. title: an ecological and conservation perspective on advances in the applied virology of zoonoses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: no mbg d the aim of this manuscript is to describe how modern advances in our knowledge of viruses and viral evolution can be applied to the fields of disease ecology and conservation. we review recent progress in virology and provide examples of how it is informing both empirical research in field ecology and applied conservation. we include a discussion of needed breakthroughs and ways to bridge communication gaps between the field and the lab. in an effort to foster this interdisciplinary effort, we have also included a table that lists the definitions of key terms. the importance of understanding the dynamics of zoonotic pathogens in their reservoir hosts is emphasized as a tool to both assess risk factors for spillover and to test hypotheses related to treatment and/or intervention strategies. in conclusion, we highlight the need for smart surveillance, viral discovery efforts and predictive modeling. a shift towards a predictive approach is necessary in today’s globalized society because, as the h n pandemic demonstrated, identification post-emergence is often too late to prevent global spread. integrating molecular virology and ecological techniques will allow for earlier recognition of potentially dangerous pathogens, ideally before they jump from wildlife reservoirs into human or livestock populations and cause serious public health or conservation issues. generated concern for the environment and thrust ecologists into a new political field where preserving the integrity of our global ecosystems was the priority [ ] . even so, the society for conservation biology was not established until [ ] . as a part of this transition, ecology shifted from a descriptive science to one of prediction, reflecting the hope that ecologists might mitigate changes which can have negative impacts upon the ecosystem. ecologists have branched out into the study of parasites and disease as it has become increasingly apparent that parasites are inextricably linked to the ecology of their hosts and environments, to the point where they have been a driving force in the evolution of sexual reproduction and in the shaping of biodiversity [ , ] . over the past years, disease ecologists have developed the study of parasites and pathogens in the wild. this knowledge has been synthesized into mathematical models which describe the dynamic properties of ecosystems and predict how parasites and pathogens flow through them. [ , ] . these models are becoming more commonly integrated into epidemiological studies that seek to predict outbreaks or periods of time when cross-species spillover risk is highest. parallel to this progress, the field of virology, particularly the subfields of molecular virology and viral evolution, have also been burgeoning, largely due to advances in technology that have made molecular assays and genetic sequencing more accessible to a greater number of scientists. the development of high-throughput sequencing has greatly increased our ability to efficiently detect known viruses as well as to discover new types of viruses, thereby improving our understanding of viral diversity, pathology and evolution. this increased capacity has spawned the development of new fields of study. for example, phylodynamics allows researchers to determine the origin of circulating viruses in space and time. mutations among viral strains can be used to investigate interactions among host species as well as long-range host movement via corridors and flyways. phylodynamic analyses can also inform livestock management practices, as was the case with foot and mouth disease in the united kingdom [ ] . conducting viral surveillance in animal reservoirs and invertebrate vectors can help explain circulation within host species; observed patterns of zoonotic transmission; and even allow for the prediction of periods of increased risk of zoonotic transmission (e.g., rift valley fever and rainfall [ ] ; west nile virus (wnv) and american robin (turdus turdus) migration [ ] ; as well as hantavirus in mice [ , ] ). understanding viral ecology in wildlife reservoirs and identifying high-risk human-wildlife interfaces is especially critical in the context of ever increasing globalization, whereby transportation networks facilitate rapid spread of pathogens well beyond bounds where traditional epidemiological methods can be effective [ ] [ ] [ ] . the influenza a (h n ) pandemic spread from the presumptive point of emergence in la gloria mexico to new zealand in just under a month [ ] while sars radiated from guangdong, china to different countries within several months [ ] . the negative impacts of emerging infectious diseases are not limited to humans. indeed, wildlife conservationists have documented several mass mortality events in other animal species. western lowland gorillas (gorilla gorilla gorilla) have been decimated by ebola virus [ ] and an especially virulent calicivirus, rabbit hemorrhagic disease virus, spread through both domestic and wild rabbit populations, resulting in tens of millions of deaths [ ] . in some instances the viruses have attenuated, while in others the animal populations have been brought to the brink of extinction. importantly, the risk from disease to humans and animals should not be separated. the global transportation network facilitated the introduction of infected vectors (e.g., mosquitoes) into new york and wnv caused both avian and human mortality, and this virus has subsequently spread across the united states [ ] . table . definitions of key terms used in the text. the formation of a hybrid population through the mixing of two ancestral, or long-separated, populations. a method for estimating historical population dynamics from a sample of sequences without assuming a predefined demographic model. coalescent theory a mathematical framework which describes the distribution of gene trees in populations. it provides mathematical methods for connecting demographic or ecological models with a phylogenetic tree. demography is the statistical study of populations. in the field of ecology, demography encompasses the study of the size, structure and distribution of populations, and spatial and/or temporal changes in them in response to birth, migration, aging and death. however, here we use a more rigid definition of demography-as the pattern and rate of population growth. the number of breeding individuals in an idealized population that would show the same amount of dispersion of allele frequencies under random genetic drift, or the same amount of inbreeding, as the natural population under consideration. the analogue of ne for viruses, the ‗effective number of infections' is related to the number of infected host individuals and to the number of new transmission events [ ] . a discipline which uses next-generation sequencing technologies to characterize the entirety of genomic material found in environmental samples. the molecular clock is derived from the hypothesis that sequence evolution, while random, occurs at stable rate such that the time since the divergence of two or more sequences can be estimated. recent ‗relaxed molecular clock' analysis can account for variation in the rate of sequence evolution through time or between lineages. the relations among a set of sequences showing which shares a most recent common ancestor with other sequences. the study of the principles and processes governing the geographical distribution of genealogical lineages. in the field of population genetics, population structure is defined as the absence of random mating within a population. this is the definition used here. in ecology, population structure is defined by several key parameters including number of individuals in a population, age distribution of individuals, probabilities of survival (or mortality), and rates of fecundity. a process that occurs in segmented viruses by which one or more segments ‗swap' to create a new viral genome. this drives the process of antigenic shift in influenza a viruses. the process by which new genotypes are created by the combination of distinct lineages. in sexual organisms it occurs during meiotic division, by the exchange of dna between different chromosomes or ‗crossing-over'. viral recombination occurs during viral replication and is an important factor in viral evolution (for more details see worobey and holmes, [ ] ). mutation of a virus such that it changes ‗back' to its wild-type state. the timing of an organism's schedule of reproduction and death. species with long life histories, also known as ‗k' strategists, tend to have low reproductive rates, stable populations, long generation times and long lifespans. where the parasite population is not randomly distributed among hosts, such that the variance is greater than the mean. the macroparasites in a host population are often best described by the negative binomial distribution such that a minority of hosts possess the majority of the parasites. r (basic reproduction number) in the case of viruses and other microparasites, r is the average number of secondary infections which an infection produces. as such it is a measure of parasite fitness. a host species that can independently maintain a disease and act as a source of infection to other host species. infection in reservoirs is usually more persistent and less harmful than that of other hosts. zoonotic disease a disease transmissible from animals to humans or vice versa. globalization, host ecology, host-virus dynamics, climate change, and anthropogenic landscape changes all contribute to the complexity of zoonotic viral emergence and disease, and create significant conservation and public health challenges. comprehensive and collaborative scientific approaches that transcend disciplinary boundaries are necessary to address these challenges. it is the goal of this paper to review new methods for understanding viral dynamics and illustrate how and when these techniques can be used by not only public health officials, but also disease ecologists and conservation biologists. the phylodynamic paradigm, established in [ ] , exemplifies the power of a multidisciplinary approach. it unites the ecological and evolutionary study of viruses and builds upon advances in sequencing technologies and coalescent theory, by which gene genealogies are reconstructed backward in time [ ] . the analysis of phylogenetic trees enables researchers to address many of the primary questions posed by disease ecologists (figure ). in some cases this approach can provide an estimate of a virus's basic reproductive number (r ), which is a measure of parasite fitness [ ] . phylodynamics has far-reaching applications for the control of viruses in both human and animal populations, in addition to being vital to our understanding of the interconnectedness between them. phylodynamic studies can be used to identify reservoir species as well as defining the spatial and temporal origin of emerging infectious diseases [ ] . they can also help to elucidate how these viruses spread following their emergence [ ] . firstly, chains of viral transmission can be extrapolated from the branching topology of phylogenetic trees. one example of the utility of this approach is with rabies virus. rabies causes thousands of human deaths a year in africa and has been implicated in the decline and local extinction of several populations of african wild dog (lycaon pictus) [ , ] . lembo et al. analyzed sequences of rabies virus from the serengeti, revealing that domestic dogs were the reservoir of the virus and that they had transmitted it to other resident carnivore populations on repeated occasions [ ] . this work has applicability in that it can be used to design efficient and effective vaccination strategies, both to alleviate current distress and prevent future outbreaks [ ] . secondly, by mapping the geographical origin of each sequence onto the nodes of phylogenetic trees, the geographical origin of a virus might be identified. wallace et al. ( ) [ ] used this phylogeographic approach to identify guangdong province, china as the most parsimonious origin of highly pathogenic h n strain of avian influenza and to delineate the most likely pathways of viral spread [ ] . however, a recent bayesian analysis of this data did not support the conclusion that h n had dispersed from guangdong to indonesia [ ] . instead, the bayesian analysis suggested it had spread to indonesia from guangxi or hunan in china. this example demonstrates that different statistical techniques may yield different conclusions. as yet, neither the bayesian nor the frequentist method is universally considered to be superior and there is much room for improvement as statistical phylogeography develops as a field. phylogeographic tools have also been applied by walsh et al. ( ) [ ] to locate the putative origin of the zaire strain of ebola virus. in an attempt to resolve the controversy over the time of emergence and spreading trajectory of ebola in the congo basin, they then used spatial data and two different tests for the impact of selection on the virus genome [ ] . where a virus is expanding in range, as the zaire strain of ebola virus appears to be, using a ‗landscape genetics' approach may help identify geographical barriers to viral spread and help identify vulnerable human or wildlife populations lying in the path of infection [ , ] . these phylogenies may reflect transmission chains, however sampling must be sufficient for them to do so, while recombination may obscure ‗true' relationships between viral sequences. (b) simple molecular clock theory, predicated on the neutral theory of molecular evolution [ ] assumes that mutation occurs at a constant rate over time, thus the time that has elapsed since a pair of virus strains diverged from a common ancestor may be quantified. methods that account for differences in the evolutionary rates of different strains, and for variation in these rates through time, have been recently developed [ ] . here variants represented by thick lines evolve much faster than those represented by thin lines. (c) using a phylogeographic approach, the location at which a sequence was sampled may be mapped onto the viral phylogeny and the likely spreading trajectory of the virus inferred. while parsimony approaches have been popular, powerful bayesian methods that account for uncertainty of dispersal process and historical phylogeny have been developed to reconstruct viral dispersal events [ ] . crosses on the phylogeny represent such viral dispersal events, in this example. (d) coalescent theory provides the basis for many phylodynamic approaches. here, circles on the same row represent temporally simultaneous infections. working back from sampled infections (red circles), lineages can be traced back to the most recent common ancestor (black circle) via hypothetical, unsampled ancestors (grey circles). the time it takes for sampled lineages to coalesce is dependent on a variety of variables (i.e., viral effective population size, population structure, selection, stochastic infection die-out and recombination). a variety of methods are available to test for selection and recombination. phylodynamic analyses are not without limitations. dense and representative sampling at a scale equivalent to epidemiological surveys is required to fulfill the potential of phylodynamics for understanding epidemics of rapidly-evolving viruses [ ] . the construction of phylogenetic trees from these viral sequences may be complicated by recombination and, in the case of segmented viruses, reassortment of viral genomes [ ] . as a result of these processes, the genes on a single viral sequence may have very different origins (see the discussion of the different origins of the hemagglutinin and neuraminidase segments of h n avian influenza in lemey et al. [ ] ). therefore, concatenated analysis of multiple genes may be confounded. the ability to construct phylogenies is further limited by the total viral genetic information available. genbank is a vast public database that contains records of genetic sequences; however, its usefulness is dependent upon the willingness and/or ability of individuals and organizations to submit viral sequences. governments and industrial institutions may be reluctant to report sequences of economically important viruses (i.e., avian influenza) due to the potential negative economic impacts that may ensue. although phylodynamics is currently encumbered by the aforementioned factors, there is hope for progress. advancements in coalescent theory will help us to deal with the phylogeny construction problems caused by recombination and reassortment. they will also facilitate better utilization of genomic and spatial data, provided these advancements are also accompanied by a simultaneous increase in computing power, which is also currently limiting. the utility of phylodynamics is not limited to questions of interest to virologists and disease ecologists. this approach may also inform investigations of host population biology and, in so doing, aid in the development of conservation policy. host molecular markers (e.g., microsatellites, mitochondrial dna) are used by conservation biologists and ecologists to infer population structure, historical demography and other critical features of wildlife populations [ ] and have proved particularly powerful when analyzed in combination (i.e., [ , ] ). recently, it has been demonstrated that the pathogens of host populations might also be useful to this end. research using helminths and bacteria has revealed patterns of ancient human migration and dispersal [ , ] identified ancient refuges of rodent and bird taxa [ , ] and shown that there was past contact between contemporary non-sympatric bat species [ ] . however, there have been few attempts to utilize viruses to this end (save [ , ] ). it is surprising that viruses have not been used more for the inference of host population biology since some of their characteristics make them ideal for doing so. most viruses have large population sizes and short generation times, and many replicate using a highly error-prone rna-dependent rna polymerase, causing them to accumulate many more mutations (nucleotide changes) per unit time than the host genomes [ , ] . consequently, viruses may provide information about host demographics on a shorter timescale than molecular markers of the host. one of the signature tools of phylodynamics, the bayesian skyline plot, might also be utilized to infer changes in historical population size of the host. these plots incorporate the use of a molecular clock and coalescent theory to infer historical changes in virus population sizes without assuming a predefined demographic model [ ] . it is important, however, that the timescale over which the evolutionary dynamics of the virus population can be reliably reconstructed is appropriate for the parameters of interest in the host population. unfortunately, the very characteristics that make viruses useful for estimating host population structure and demography may also impede the analyses. multiple substitutions can occur quickly in the viral genome and this will obscure the host population's actual evolutionary history. meanwhile, variations in the transmission mechanisms of viruses (vertical vs. horizontal) can alter the ability to accurately infer a virus' relationship to a host population. cross-species transmission is also problematic in that it can cause pathogen phylogenies to inaccurately reflect the history of their hosts [ ] . therefore, before the genetic information contained within a virus population can be used to infer the population structure and demography of the host, it is critical to test for congruence in the evolutionary history of the host and virus populations. this is accomplished by statistically comparing the respective phylogenies within the relevant timescale. viruses with high host specificity have a greater likelihood of exhibiting such congruence. feline immunodeficiency virus (fiv) is known for high host specificity and is, thus far, the only virus to have been used to elucidate changes in host population structure and size. from a phylogenetic analysis of fivpco, the fiv type specific to the cougar (puma concolor), biek, drummond, and poss ( ) [ ] inferred that the north american population of cougars became subdivided during the last century but subsequently expanded in both size and range. subsequently, antunes et al. ( ) [ ] used the distribution of fiv ple subtypes in the serengeti to infer that recent admixture has occurred between the region's lion (panthera leo) populations. these recent changes in felid population size and structure could not have been inferred from host genetic data. there is great potential for the further use of this technique by conservation biologists and ecologists, and for it to complement existing methods which utilize host genetic data. host genetic markers are used to define management units for conservation purposes [ ] . many ‗flagship' endangered species have long life histories [ ] , a feature that correlates with both extinction risk in certain regions [ ] and difficulty in reconstructing recent demographic history from molecular markers. because of the latter, the use of viral genetics to define management units may be an important avenue of exploration. in addition to aiding the definition of management units, viral data could be used to analyze the consequences of management activities and other environmental changes on target species. where viral genetic diversity exists in a spatially heterogeneous distribution, viral movement patterns could be used to study the migratory behavior of animals (as macroparasites have been [ , ] ). as such, researchers could monitor the use of wildlife corridors and the efficacy of control measures aimed at limiting the range of a host species [ , ] . where viruses can be readily amplified from non-invasively collected samples (see [ ] ), the above objectives could be achieved in a cost effective manner with minimal disturbance of the study species. viruses with specific transmission routes may also serve as proxies for behaviors related to transmission (i.e., sexually transmitted diseases). similarly, where cross-species transmission occurs, viruses might be indicative of types of sustained, direct contact between different, sympatric taxa which facilitate such transmission, for example predator-prey interactions [ ] . at the broader ecosystem level, inferences about long-term evolutionary processes might also be made by examining the phylogeographic structure of numerous host and virus populations of a region (see [ ] ). a major hurdle for both virologists and ecologists is defining the biodiversity of life. at present, scientists do not know the actual number of mammal species, much less the diversity of viruses they harbor [ ] . indeed, the diversity of viruses known to infect the house mouse (mus musculus), a staple in biomedical research, is not yet completely known. recent advances in genetic sequencing, including high-throughput sequencing and other -next-generation sequencing‖ techniques, as well as masstag pcr and microarray multiplex assays [ , ] have made the study of microbial diversity feasible [ ] . these technologies have facilitated a movement from classical virology, where the focus was on disease etiology, toward a broader discipline that considers the rest of the viral diversity or -the virosphere‖. metagenomic studies have used next-generation sequencing to study biodiversity in substrates such as ocean water and soil [ , ] . metagenomics has also been used to screen human and animal clinical samples in order to determine etiologic agents of disease or to describe the microbial flora normally present in a vertebrate host-in many cases the result has been the discovery both of novel pathogens and novel associations between clinical disease and agent [ ] [ ] [ ] [ ] . as technology becomes more affordable, and thus accessible, there will be increasing opportunities to ask large-scale questions such as: how does the virosphere vary across space and time? how does it vary across species? can we use this to define risk of cross-species transmission or to inform conservation efforts? and how might co-infections with these undiscovered viruses influence the dynamics of the more well known viruses? the paucity of information about viral diversity within a host poses problems for research progress. it is difficult to understand viral pathogenesis and transmission without completely understanding the dynamics of co-infections. indeed, it is currently difficult to ascribe a host's symptoms to an individual virus with any certainty because a virus' actions, and even its ability to infect the host, could be a function of another (possibly undetected) co-habitant of the host. evidence of interactions between co-infecting species has been clearly demonstrated [ , ] and it will be critically important to elucidate the interactions that occur between multiple pathogens as well as the combined effects they may have on a host's immune system. broadening our understanding of the diversity of pathogens that exist in human and animal hosts through wildlife and domestic animal surveillance will significantly improve our ability to recognize novel zoonotic agents in the context of a disease outbreak. phylogenetic information obtained from comparative sequence analyses can improve our understanding of the impact of sequence mutation on virulence, as well as inform decisions about vaccine development. a final noteworthy benefit of viral discovery efforts is that these techniques should be important for identifying candidates for future vaccines as a virus's most worthy competitor is often another virus. from a health perspective, vaccination is arguably the most important technology that has arisen from the study of viruses. vaccination offers a direct means of intervening in a host-pathogen system and it has become routine in many parts of the world. efforts to this end have resulted in the eradication and/or control of smallpox, polio, mumps, measles, rubella and most recently, rinderpest. vaccines take several forms including live-attenuated viruses; inactivated whole viruses; inactivated toxins and viral protein subunits, and these are often delivered in combination. while live-attenuated vaccines have been predominant, a new generation of techniques including gene delivery and nano-technologies are being used to develop highly-efficacious and safer vaccines, that have less risk of reversion [ ] . new types of administration methods are also being developed with oral, aerosolized and nasal vaccines currently on the market. these less invasive administration techniques decrease labor costs associated with administration and offer increased capacity for mass-dispersal of vaccines to both humans and free-ranging wildlife [ ] . vaccination campaigns aimed at both protecting threatened species and decreasing public health risks via animal vaccination have taken place. swiss health officials were pioneers in this field, using oral vaccines to control rabies in wild red foxes (vulpes vulpes) [ , ] . these vaccines were inserted into chicken heads which were distributed in the wild beginning in [ ] . as can be observed from the supplemental movie (video s ), their initial barrier approach evolved into a large-scale treatment of infected areas and resulted in rabies being successfully pushed back to and then eliminated from the swiss alps [ , ] . following the success of these trials, campaigns were conducted in western europe [ ] and canada [ ] with similar results, though the situation in the united states has proven more challenging. other successful vaccination examples include canine distemper virus in black-footed ferrets (mustela nigripes; [ ] ) and ethiopian wolves (canis simensis; [ ] ), as well as rabies in florida panthers (felis concolor coryi; [ ] ) and african wild dogs [ , ] . a caveat to this success is that there is growing evidence that vaccination against a specific strain of pathogen can result in inadvertent selection for related co-infecting strains. thus vaccination can influence the dynamics of a pathogen [ ] . the possibility of inadvertent viral strain selection highlights the importance of understanding the long-term evolutionary and ecological consequences of vaccination. indeed, where threatened or endangered animals are concerned, mishaps may prove disastrous. attenuated canine distemper vaccines did not provide immunity to critically endangered black-footed ferrets, while the use of a live canine distemper virus vaccine resulted in clinical distemper arising in one of the few remaining populations [ ] . ideally, long-term clinical trials with suitable animal models might avert these problems. these trials should also be used to provide an a priori understanding of how vaccination might shape future evolutionary processes. in contrast to the ferret experience, efforts with the endangered ethiopian wolf serve as an example of a successful vaccination program. wolf populations were suffering severe mortality due to rabies and distemper acquired from the wild dogs that shared their home range [ ] . on the basis of a spatially explicit individual-based model, which indicated rabies could be controlled in dogs given just over % coverage [ ] , knobel et al. executed an intensive vaccination plan [ ] . both the extent and duration of outbreaks in the treated areas were limited and, although monitoring and continued vaccination are required, the situation appeared to be under control in [ ] . wildlife vaccination campaigns are also being investigated as tools to limit public health risks. tsao et al. vaccinated mice in an effort to break the cycle of lyme disease and reduce the risk of emergence in human populations, in which it causes tens of thousands of deaths per year in the us [ , ] . in the same vein, griffing et al. [ ] have tested the efficacy of vaccinating american robins to interrupt the wnv transmission cycle. this species can absorb up to % of the potentially infective mosquito bites in early spring and is thus a key host in the wnv system [ ] . targeted vaccination of this single species could potentially result in herd immunity and reduce the risk of human infection as well as decreasing wildlife mortality. these works exemplify how ecological knowledge can be used to identify and exploit some of the heterogeneities which so often dominate the dynamics of pathogens. while the lasting efficacy of wildlife vaccination efforts has yet to be demonstrated with either endangered species or in breaking the transmission cycle of human pathogens, an increasing number of researchers are drawing attention to systems where it seems feasible [ , ] ; demonstrating that intricate knowledge of host and virus ecology can greatly reduce the amount of vaccine coverage that is necessary to control these viruses. the problems entailed by the sheer number of viruses, viral resistance, the explosive potential for spread, and the economic burden, make it clear that currently available vaccination methods do not provide a sustainable solution for either human or animal disease. the unambiguous indication is that researchers need to work towards the goal of developing a predictive framework where risk can be defined for different scenarios and not only to rank pathogens, and species, but also, places and times of year that can be identified as more or less precarious for global health. pending questions include: which geographic areas will experience more disease and conservation problems? which areas pose the highest risk for pandemic spread of pathogens? what characteristics of hosts and viruses make them more or less likely to be involved in cross-species transmission events? and what are the relative roles of genetic relatedness and contact rate for transmission? some modeling work and reviews of historic data have been informative [ , ] , but novel uses of phylogenies of both viruses and hosts (as discussed above) provide promise for progress to this end, especially when coupled with high quality surveillance data. once we have this information, scientists will be able to design -smart surveillance‖ strategies whereby valuable vaccine resources can be efficiently targeted and efficiently distributed. ecological studies can effectively inform conservation as well as public health policy. gaining knowledge of reservoir host ecology can be critical for the development of eradication strategies. most viral disease systems are dominated by heterogeneities and identifying and understanding these can be crucially important when trying to interrupt the chain of events that leads to persistence. ecological studies of wnv have shown how forest fragmentation and decreased biodiversity can alter transmission among avian hosts as well as to humans [ ] . likewise, researchers have used satellite imagery to identify habitat characteristics that accurately predict the prevalence of sin nombre virus [ , ] , a hantavirus that uses the deer mouse (peromyscus maniculatus) as a reservoir host and it occasionally infects and kills humans [ ] . these studies epitomize the type of effort scientists will need to successfully fight viral pathogens in the future. however, piecing together emerging disease and conservation problems ex posto facto is only of limited value. increased pathogen surveillance and ecosystem process monitoring may provide the insight necessary to mitigate problems before they become serious human health or conservation concerns. this is especially the case for zoonotic viral pathogens where the reservoir hosts are known and a targeted approach is feasible. rodents rank as the number one reservoir of emerging and re-emerging zoonotic viruses [ ] . conveniently, these small mammals also present a manageable system for studying disease dynamics [ ] [ ] [ ] . individuals can be marked and sampled individually through time. their locations as well as their contacts with other individuals can be measured. as such, wild populations of rodents can be valuable as model disease systems to address relevant questions like: are there key hosts for transmission? how does prevalence vary seasonally or over time? what is the contact rate between the reservoir and humans? how do these pathogens flow through populations? the answers are of critical importance because they provide an indication of when and where there is increased risk of a zoonotic event whereby a human becomes infected, or when a species becomes at genuine risk of extinction. by monitoring and manipulating wild populations, one might also be able to identify factors that may increase a pathogens chance of emerging. for instance, what characteristics of hosts and viruses make them more or less likely to be involved in cross-species transmission? and what are the relative roles of genetic relatedness and contact rate for transmission? long-term monitoring and surveillance in reservoirs will also enlighten us to the kind of aggregations and other heterogeneities that exist through time and that and can be exploited with efficient vaccination campaigns. we are experiencing a global increase in the rate of emerging viral zoonoses, which are primarily driven by anthropogenic activities such as land-use change, agricultural intensification, and driven by global travel and trade [ ] . in order to adequately understand, predict and ultimately interrupt the processes by which zoonoses cross the species barrier from their natural reservoirs to humans, and then become established as human pathogens, comprehensive scientific studies that use the tools of ecology, virology, microbiology, and epidemiology are needed [ ] . the study of disease ecology has become an established discipline with advances in both the formulation of new theory as well as the integration of molecular virological techniques that provide important information about epidemiology, ecology and viral evolution, all of which has been applied to both health and conservation [ ] [ ] [ ] . because ecological systems are rife with heterogeneities and often have non-intuitive processes underlying their dynamics, it is critically important for scientists to use a comprehensive approach to understanding the population processes of an ecosystem before successful intervention strategies can be developed or implemented. admittedly this is a daunting task and it is often the case that scientists need to operate with less than complete information. where this is the case, a modeling approach is necessary to identify key processes that allow successful interventions. technological advances in molecular virology and genetics, as well as the expanded use of mathematical models in epidemiology and disease ecology have dramatically changed our ability to manage both conservation and health. finally, it is only with this type of interdisciplinary approach that considers free-ranging wildlife, domestic animals and humans as inextricable components of a single disease system, that progress can be made that will satisfy both conservation and public health needs. this is the essence of conservation medicine [ ] and indeed we believe a -one health‖ approach to infectious disease is necessarily the way forward. supplemental movie (video s ). are we in the midst of the sixth mass extinction? a view from the world of amphibians how many species? philos the future of biodiversity the impact of infection and disease on animal populations: implications for conservation biology disease and conservation emerging viruses emerging infectious diseases of wildlife-threats to biodiversity and human health risk factors for human disease emergence host range and emerging and reemerging pathogens rates of evolutionary change in viruses: patterns and determinants years of hiv the re-emergence 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endangered species an ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the lyme disease cycle mosquito landing rates on nesting american robins (turdus migratorius). vector borne zoonotic dis vaccination of wildlife to control zoonotic disease: west nile virus as a case study transmission dynamics and prospects for the elimination of canine rabies global trends in emerging infectious diseases ability to replicate in the cytoplasm predicts zoonotic transmission of livestock viruses land use and west nile virus seroprevalence in wild mammals satellite imagery characterizes local animal reservoir populations of sin nombre virus in the southwestern united states ecology of hantaviruses and their hosts in north america. vector borne zoonotic dis chain reactions linking acorns to gypsy moth outbreaks and lyme disease risk could parasites destabilize mouse populations? the potential role of pterygodermatites peromysci in the population dynamics of free-living mice, peromyscus leucopus predicting the emergence of human hantavirus disease using a combination of viral dynamics and rodent demographic patterns anthropogenic environmental change and the emergence of infectious diseases in wildlife collaborative research approaches to the role of wildlife in zoonotic disease emergence ecology of infectious diseases in natural populations the ecology of wildlife diseases conservation medicine and a new agenda for emerging diseases this work was supported in part by nih/nsf -ecology of infectious diseases‖ awards from the john e. fogarty international center (grant r -tw - s ) and an nih award (k ai ) from the national institute of allergy and infectious diseases. we thank c. j. wale, b. wale and h. ewing for their help in proofing the manuscript. key: cord- - qlk y h authors: friedrich, brian m.; trefry, john c.; biggins, julia e.; hensley, lisa e.; honko, anna n.; smith, darci r.; olinger, gene g. title: potential vaccines and post-exposure treatments for filovirus infections date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qlk y h viruses of the family filoviridae represent significant health risks as emerging infectious diseases as well as potentially engineered biothreats. while many research efforts have been published offering possibilities toward the mitigation of filoviral infection, there remain no sanctioned therapeutic or vaccine strategies. current progress in the development of filovirus therapeutics and vaccines is outlined herein with respect to their current level of testing, evaluation, and proximity toward human implementation, specifically with regard to human clinical trials, nonhuman primate studies, small animal studies, and in vitro development. contemporary methods of supportive care and previous treatment approaches for human patients are also discussed. the family filoviridae includes two accepted genera, ebolavirus and marburgvirus. the genus ebolavirus includes five species (each represented by a single virus): zaire ebolavirus (ebola virus, ebov), sudan ebolavirus (sudan virus, sudv), reston ebolavirus (reston virus, restv), taï forest ebolavirus (tai forest virus, tafv), and bundibugyo ebolavirus (bundibugyo virus, bdbv). the genus marburgvirus includes a single species, marburg marburgvirus, which has two members: marburg virus (marv) and ravn virus (ravv) [ , ] . in , the first cases of filoviral infection were documented in three simultaneous outbreaks in west germany and yugoslavia. the virus responsible for the outbreaks was named "marburg virus" after the german city of marburg in which it was first recognized [ , ] . from documented instances of infection, it seems that the members of the filovirus genera may exist in quite opposite climates of africa with marburgvirus infections occurring more frequently in the dry woodlands, while ebolavirus infections occur more frequently in rain forests [ ] . more than years of effort have focused on the search for the reservoir of these viruses in africa, and while the search is still ongoing, recent evidence indicates that bats may be reservoirs for both marburgviruses and ebolaviruses [ , [ ] [ ] [ ] [ ] [ ] [ ] . however, the recent outbreak of restv in domestic pigs in the philippines demonstrated the potential for animals other than primates and bats to be infected and potentially spread or amplify outbreaks [ ] . filoviruses are named for their long, filamentous shape which can been seen on the order of micrometers in length, while their width is more narrow (usually around nm) with little fluctuation [ ] . contained within this filamentous virus is a single, -kb negative-sense rna genome that encodes seven proteins [ , ] . the seven filoviral proteins are the glycoprotein (gp), the polymerase (l), the nucleoprotein (np), a secondary matrix protein (vp ), the transcriptional activator (vp ), the polymerase cofactor (vp ), and the matrix protein (vp ) [ , ] . homotrimers of the viral gp cover the surface of the virion, and this viral gp is believed to be the sole host attachment factor for filoviruses [ , ] . candidates for filoviral receptor and co-factors include transferrin, dc-sign, tim- , and npc [ , [ ] [ ] [ ] [ ] . after entry, filoviruses replicate their genomes and viral proteins in the cytoplasm using a rna-dependent rna-polymerase which is carried in with the virus. wild-type filovirus infection has been associated with severe case fatality rates in humans, as high as % [ ] . in humans, filovirus infection is characterized by an abrupt onset of flu-like illness, after an initial incubation period of - days. following this initial illness, signs and symptoms of disease include anorexia, nausea, vomiting, chest pain, cough, edema, postural hypotension, neurologic complications, petechiae, and mucosal hemorrhage. there have also been several observed wild-type filovirus outbreaks among great apes in africa that have demonstrated similarly high mortality rates [ ] . in an effort to create cost and time-effective models of filoviral disease for the development of vaccines and therapeutics, small animals, such as mice and guinea pigs, are often used. however, these animals usually demonstrate significant resistance to wild-type filovirus infection, and only demonstrate mortality rates similar to primates when the filovirus in question has been adapted to the model species [ ] . due to the difficulties in evaluating wild-type filovirus infection in small animals and the generally high level of immune protection correlates derived from non-human primate (nhp) models of infection, therapeutics and vaccines are ultimately evaluated in nhp species for efficacy against filovirus. of the nhp models available for filovirus study, rhesus and cynomolgus macaques have been the most highly characterized and utilized for therapeutic and vaccine development, respectively. however, the starting point of vaccine and therapeutic development remains small animal models due to the cost, ethical, and time-associated benefits [ ] . this review will highlight the current research into filovirus vaccines and therapeutics. the current clinical standard for filoviral infection is supportive care as there are currently no fda-approved treatment strategies. supportive care consists of oral fluid rehydration, oral medication, nutritional supplementation, and psychosocial support [ ] . nasogastric feeding tubes and i.v. administration of both fluids and medication are increasingly considered supportive care where possible during outbreak scenarios to prevent dehydration and facilitate support of blood pressure [ , ] . however, given the limited equipment and laboratory support during outbreaks, care must be taken to prevent overaggressive fluid administration [ ] . fluid replacement was evaluated briefly in rhesus macaques, and while there was no significant benefit to survival, a less severe renal compromise was observed [ ] . while supportive care may (or may not) reduce the overall case fatality rate in humans, the true impact of simple interventions such as fluid management has yet to be fully evaluated and the potential for benefit in combination with direct antiviral measures has yet to be assessed [ ] . treatment of filovirus infection with passive transfer of antibodies is an attractive therapy. while there have been conflicting results in vitro, in animals, and in humans, recent breakthroughs have solidified the potential for this strategy of intervention. in addition, there have been a number of immunotherapies developed for other agents, such as respiratory syncytial virus (rsv), which can provide potential roadmaps or precedence to facilitate the advancement of these products through the necessary regulatory hurdles [ , ] . transfer of immune serum for the treatment of filovirus infection in humans has previously been attempted. however, interpretation of these results has been cautious due to the study conditions as well as the uncertainty of the disease stage at which the individuals were treated [ ] . as a result, much attention has focused on animal studies evaluating candidate products. while many of the early studies in mice and guinea pigs were successful, these successes did not translate to nhp studies and tempered the enthusiasm for further evaluation of candidate products. [ , [ ] [ ] [ ] [ ] . when similar passive transfer strategies were attempted in nhps, viremia onset and outward signs of disease were reduced, but the treatment did not affect survivorship [ ] [ ] [ ] . in addition, the suggestion that antibodies could enhance filovirus infections in vitro caused further concern [ ] [ ] [ ] . however, recently a series of experiments have made researchers, developers, and funding agencies reconsider the potential of this category of products for filoviruses. both polyclonal and monoclonal passive therapies have been shown to be efficacious in rodents for filovirus infection [ ] [ ] [ ] . furthermore, evidence of enhancing antibodies exists in the antibody response to ebov [ ] . more recent studies have demonstrated protection in macaques with polyclonal and monoclonal passive therapy [ ] [ ] [ ] . these sources of monoclonal antibodies have ranged from murine monoclonal antibodies to recombinant-derived cloned human monoclonal antibodies from survivors of filovirus infection [ , ] . development of new antibodies to be used for post-exposure treatment is on-going. in one study, an antibody ( f ) targeting the ebov gp mucin-like domain was generated and subsequently shown to protect % of mice against a lethal ebov challenge when given days post-exposure [ ] . this antibody was then modified to generate h- f , a human recombinant antibody. this human recombinant antibody also significantly protected mice against a lethal challenge of ebov [ ] . in another method, a recombinant vsvΔg/ebovgp was used to generate a total of monoclonal antibodies which were subsequently characterized. all monoclonal antibodies improved survival rate of mice ( %- %) against a high-dose lethal challenge by mouse-adapted ebov [ ] . another antibody, kz , was isolated from the bone marrow of a human survivor of ebov infection and is specific for the complex of gp and gp [ ] . kz neutralized ebov in vitro and offered protection from lethal ebov challenge in a rodent model [ ] , but was non-protective in nhps [ ] . table . . . dna vaccines the first clinical trials involving filovirus vaccines were based off of plasmids expressing ebov np and gp as well as sudv gp [ ] . this strategy proved safe in subjects involved with phase i testing. however, the prime/boost dna vaccine strategy covering four separate plasmid doses administered three times each was ineffective at creating durable immunity as evidenced by the near non-existent antibody titer in these subjects after year [ ] . while clinical trials with this vaccine have halted, it may be possible that this strategy can supplement another vaccine technology in a prime/boost capacity. while no vaccines or therapeutics are currently licensed for use by the fda, phase i clinical trial safety tests have been performed on one particular platform for an ebov vaccine. this vaccine is based on a replication deficient, recombinant adenovirus serotype (rad ) vector genetically engineered to carry the genes for ebov glycoprotein (gp (z)) and sudv glycoprotein (gp (s/g)). as a common human pathogen, this vector vaccine utilized the broad-tropism of the adenovirus vector to infect cells and once inside the inserted ebolavirus glycoproteins are expressed. upon expression of these inserted ebolavirus genes, the host immune system will recognize them as foreign and mount a response against them. the advancement of this vaccine technology to phase i trials manifested from its ability to provide % protection among cynomolgus macaques vaccinated - days prior to challenge and its ability to generate potent humoral and cell-mediated immune responses [ , ] . in the clinical trial participants remained asymptomatic after a single vaccination with either × or × viral particles [ ] . furthermore, for both doses of the vaccine significant antibody titers were observed at weeks post-vaccination with % and % of participants receiving × viral particles being positive for gp (s/g) and gp (z), respectively. significant antibody titers were observed again at weeks post-vaccination and, while decreased from weeks, demonstrated the potential durability of this vaccine over time [ ] . t-cell activation was also examined for these individuals and found to directly correlate with the dose administered, but to a lesser extent than the previously mentioned antibody response. cd + activation was observed with greater frequency than cd + activation in those receiving the vaccine. importantly, these results were obtained in the context of significant pre-existing immunity to ad as pre-entry evaluation of antibody titers revealed that % of participants were positive against ad , showing that pre-existing immunity to ad still resulted in protection against ebov [ ] . while this study shows great promise, further development and additional studies in nhps and humans are needed. table . in addition to the adenovirus platform in clinical trials, additional variations of the rad vaccine are also in development and have been evaluated in nhp models. based on the adenovirus vector platform, the complex adenovirus (cadvax) technology substantially increased the genetic payload capacity of the vector, up to kb. additionally, this strategy involved the blending of four separate vectors expressing the glycoproteins of ebov, sudv, and marv along with the nucleoproteins of ebov and marv. when administered in a prime/boost strategy, this technology offered % protection against ebov, sudv, and marv [ ] . another variation of the cadvax system designed to express modified ebov glycoprotein and sudv glycoprotein was effective in protecting against both parenteral and aerosol challenge when administered in a prime/boost strategy [ ] . both implementations of the cadvax technology demonstrated significant antibody titers. further improvement upon the adenovirus-based ebov vaccine technology is ongoing. richardson et al. reformatted the genetic insert for the vector which included the addition of a cytomegalovirus (cmv)-chicken β-actin hybrid promoter, optimized codons and a consensus kozak sequence [ ] . these improvements led to three-to seven-fold increases in ebov glycoprotein expression. neutralizing antibody titers were found at doses as low as infectious forming units (ifu) with comparable titers requiring ifu of the unmodified vaccine in mice. these modifications demonstrated % protection of mice at doses two orders of magnitude lower than the unmodified vaccine. interestingly, at min post-challenge, the modified ad-cmvzgp/ad-cagoptzgp offered % protection compared to the % protection of mice offered from the original vaccine [ ] . this vector format has also recently showed promise when administered sublingually in mice, therefore eliminating the complexities of parenteral administration such as the necessity for sterile tools, aseptic chemicals, and the risks of potential blood-borne pathogen exposure [ ] . while this adenovirus vector vaccine technology is promising, demonstrations that pre-existing immunity to the ad vector depressed the desired immune response may impede its implementation. in efforts to circumvent issues of pre-existing immunity to ad , geisbert et al. sought out a less prevalent serovariation [ ] . in their study, a heterologous prime/boost strategy with recombinant adenovirus serotypes and carrying gp (z) and gp (s/g) demonstrated complete protection among nhps. each of these vectors was capable of stimulating humoral and cell-mediated immune responses in the context of nhps pre-vaccinated with rad as evidenced by antibody titers reaching an order of magnitude above those achieved in rad vaccinated subjects ( : , compared to : , ), and cd + intracellular cytokine staining was . -fold greater among heterologous prime/boosted subjects ( . % compared to . %) [ ] . rhabdoviruses have recently offered unique vaccine platforms to generate both genus/species specific immunity as well as potential for cross-protective immunity for filoviruses. for example, based on an attenuated recombinant vesicular stomatitis virus (rvsv), the replication-competent virus expresses the glycoprotein of the target filovirus in place of its wild-type membrane glycoprotein. as this virus is primarily an agricultural pathogen, pre-existing immunity interfering with the desired immune response and subsequent protection is unlikely [ ] . several studies have begun to address the safety of the filovirus vsv platforms. evaluation of this platform in immunocompromised nhps has suggested that this technology may be safe among similarly immunocompromised humans [ ] . further encouragement for the safety of this live-attenuated vaccine came recently from mire et al. who showed that ebov and marv rvsv showed no signs of neurovirulence associated with vsv [ ] . the utility of the vsv-based vaccine for protection against filoviral hemorrhagic fever was highlighted by geisbert et al. [ ] . using a blended vaccine consisting of equal amounts of three different vsv vectors each carrying the ebov, sudv or marv glycoprotein, they were able to generate % protection of nhps against challenges with ebov, sudv, tafv, and marv with no observed ill effects from this replication-competent vaccine. of all vaccinated nhps, only one showed signs of viremia as assayed by rt-pcr. each of the vaccinated nhps also demonstrated elevated antibody responses after vaccination, with titers ranging from : to : for all three glycoprotein components of the blended vaccine [ ] . in addition to providing such high levels of protection as a prophylactic vaccine strategy, the vsv-based technology has demonstrated post-exposure protection for both ebov and marv when administered via intramuscular (i.m.) injection [ ] . when marv-rvsv was administered i.m. - min post-challenge with marv, % of nhps survived. in this study viral rna was observed in the blood on day three post-challenge when assayed by rt-pcr, but active virus was unobservable by traditional plaque assay. clinical chemistry results demonstrated that these surviving nhps experienced significant rises in aspartate aminotransferase, gamma-glutamyl transferase, total bilirubin, and blood urea nitrogen indicating that, while protective, the post-exposure treatment did not completely prevent typical pathogenic events associated with marv infection. similar experiments demonstrated sudv-rvsv delivered - min after challenge offered % protection [ ] . post-exposure protection for ebov-rvsv was less effective at - min but still afforded % protection to eight nhps [ ] . as a post-exposure treatment, ebov glycoprotein rvsv was used recently h after a suspected human exposure via needlestick in the laboratory. while there is no direct evidence the laboratory worker was indeed exposed, that person survived the experience with no discernible sequelae from the treatment outside of a transient fever occurring h after an injection of × plaque forming units (pfu) [ ] . also of note for rhabdovirus-based filoviral vaccines was a recent report that generated dual immunity for both ebov and rabies virus infection. ebov gp was efficiently expressed from an attenuated vaccine used for wildlife against rabies virus in place of the wild-type rabies envelope glycoprotein, g [ ] . this vaccine vector was capable of inducing protective immunity to ebov infection as well as to rabies virus infection in both live-attenuated format and β-propriolactone inactivated vaccine. neurovirulence of the recombinant vector was unobserved in suckling mice when compared to the unaltered vaccine [ ] . this versatility offers increased storage options with an inactivated vaccine as well as the opportunity to vaccinate for each disease where they are both endemic. alphavirus particle-based vaccines also provide high levels of protection to nhps against lethal filovirus challenge. these vaccines for ebov, sudv, and marv are composed of venezuelan equine encephalitis virus (veev)-based replicon particles (vrps) that express the viral glycoprotein of interest [ ] . replicon particles are replication-defective, single-cycle vectors which cannot spread from cell-to-cell. the vrps are composed of an attenuated veev replicon that contains veev non-structural genes and the filovirus glycoprotein. vrps are generated when the replicon is co-transfected into cells with helper plasmids containing the veev structural genes. the resulting single-cycle propagation defective particles are then administered to the appropriate animal model for efficacy testing [ , ] . this technology offers several advantages, including high expression levels of heterologous genes, dendritic cell tropism, induction of robust cellular and antibody immune responses, rapid construction into single and multivalent vaccines, and relative resistance to anti-vector immunity [ , ] . in vivo studies with the marv vrp offered the first demonstration of a fully protective filovirus vaccine. nhps exhibited % survival after vaccination with focus forming units (ffu) of vrp in three consecutive doses spaced at day intervals prior to challenge with marv [ ] . this protection was offered when the vrps were constructed to express gp alone or gp + np; however, the nhps vaccinated with np alone all exhibited clinical symptoms of illness and only two out of three survived the challenge. substantial antibody titers were found in each of the vaccinated nhps. additionally, no conspicuous elevations in clinical chemistries were observed in nhps throughout the experiment. experiments performed on mice and guinea pigs supported the ability of vrps expressing gp to mediate complete protection from lethal marv and ebov challenge [ ] . in mice, adoptive transfer of cd + cells, but not cd + cells or passive antibody transfer, from vrp-np-immunized mice was protective, suggesting this vaccine may be most protective by stimulating the host cell-mediated immune response [ ] . additionally, adoptive transfer of cd + t-cells after activation via specific ebov peptides provided mice complete protection indicating a mechanism for vrp-based immunity [ ] . recent studies indicate that a vrp-based vaccine is fully protective in cynomolgus macaques against ebov, sudv, and marv parenteral and aerosol virus challenges (unpublished observations, olinger). paramyxovirus-based vectors for vaccination against filoviral threats have recently demonstrated the capacity to protect nhps from infection and stimulate strong immune responses. paramyxoviruses have a natural tropism for the respiratory tract and, as filoviruses are both emerging diseases and potential weaponized threats, the idea of targeting vaccines to this area is ideal. two candidates for this category of vaccines have been investigated to date: human parainfluenza virus (hpiv- ) and newcastle disease virus (ndv). of these two systems, the hpiv- system has been evaluated in nhp models of ebov infection. combinations of ebov gp alone, ebov gp + np, and ebov gp + human granulocyte macrophage colony stimulating factor (gm-csf) were inserted into the genome of hpiv- . each of these vaccine vectors was used to vaccinate nhps both intranasally (i.n.) and intratracheally (i.t.) as initial studies offered complete protection of guinea pigs vaccinated via the respiratory route [ ] . at least one nhp in all three vaccine groups receiving × tcid (median tissue culture infective dose) of their respective vector displayed clinical signs of illness after challenge during the study. each group held two nhps and out of the three groups only one vaccinated animal from the ebov gp + np succumbed to the disease. immune responses from these subjects, prior to challenge, revealed antibody titers ranging from : to : , [ ] . by manipulating dose and administration strategies, bukreyev et al. were able to achieve complete protection of nhps after two successive doses of × tcid given at day zero and again at day with challenge occurring on day [ ] . the two-dose strategy produced igg titers ranging between : , and : , , much higher than the single dose. each of these experiments highlights the potential of the hpiv- platform for ebov vaccination but the known prevalence of pre-existing immunity to hpiv- in humans could hinder the generation of targeted immunity [ ] . to address these concerns, bukreyev et al. compared the immunogenicity of ebov gp expressing hpiv- vector among naïve and pre-immune nhps [ ] . in these experiments ebov-specific igg levels were substantially decreased among hpiv- pre-immune nhps; however, this hindrance was overcome when the nhps were vaccinated with two doses of recombinant vector which was previously shown to offer complete protection against ebov challenge [ ] . in efforts to diversify the paramyxovirus-based vectors and avoid issues surrounding the pre-existing immunity found for hpiv- , a new vector design based on ndv was established. ndv is an avian paramyxovirus that infects the respiratory tract. this virus has been shown to be highly attenuated in nhps due to natural host restriction processes [ ] . additionally, this vector system has proven successful as a vaccine platform for severe acute respiratory syndrome-associated coronavirus (sars-cov) and influenza h n in nhps [ ] . although this system has yet to be evaluated in the context of nhp models of ebov infection and disease, it was recently shown to be immunogenic in nhps. single vaccination with ndv expressing ebov gp produced lower titers than the hpiv- platform, demonstrating this vector is less immunogenic; however, in a homologous prime/boost vaccination strategy ebov-specific mucosal iga levels reached those similar to the hpiv- homologous prime boost vaccination strategy [ ] . igg specific for ebov did not reach levels comparable to the previous hpiv- platform. these reports support the potential use of paramyxoviruses as vaccine candidates, but further examination of immunostimulatory effects and pre-existing immunity will require investigation. the vlp technology works by generating non-replicating virus particles that do not contain any filoviral genetic material. immune responses generated in response to exposure to vlps are derived from the filoviral protein shell that is the vlp itself. vlps are constructed through the matrix protein vp 's ability to drive the budding process. by simple transfection and expression of vp into target cells, filamentous structures can be generated [ ] . co-expression of additional filoviral proteins, such as gp and np, can dramatically enhance and stabilize the production of vlps from target cells [ ] . when traditional target/production cells were swapped out for insect cells and filoviral protein expression was driven from a baculovirus vector, vlps were generated and found to have filamentous structures resembling wild-type virus. vlps have demonstrated immunogenicity and the ability to protect nhps from lethal challenge. the first such study involving nhps utilized the baculovirusproduced vlps containing ebov gp, np, and vp [ ] . five cynomolgus macaques were vaccinated three times at -day intervals with µg of vlp with ribi adjuvant. after the full vaccination schedule, % survived a lethal ebov challenge, and there was a three-to ten-fold increase in ebov specific antibodies which possessed an % plaque reduction at titers between : and : [ ] . additionally, these antibodies were shown to have both compliment-mediated and antibody-dependent cell-mediated cytotoxic properties [ ] . using this same vlp technology, swenson et al. were able to show a similar effect for marv. three i.m. injections of mg of vlp in . ml of qs- adjuvant spaced days apart offered % protection against marv and ravv; however, one animal challenged with ravv did exhibit slight morbidity [ ] . all animals had no detectable viremia as determined by plaque assay at days , , , , , , and post-exposure. additionally, two injections of the vlps correlated with a peak in homologous antibody titer while three injections correlated to peak heterologous antibody titer [ ] . the ability of vlps to generate protective immunity against filoviral challenge has been demonstrated in guinea pigs as well [ ] . table . immunogenic virus-like particles (vlps) of ebov and marv can be easily generated in mammalian systems. ebov-like particles (vlps) can be efficiently generated in mammalian cells after expression of vp alone, but other filovirus proteins can be co-expressed as well [ ] [ ] [ ] . baculovirus-derived vlps have also been successfully generated in insect cells and stimulated both cellular and antibody immune responses against hepatitis e virus, human papilloma virus, rotavirus, and simian immunodeficiency virus [ ] [ ] [ ] [ ] . several groups have made and tested baculovirus-generated filovirus-vlps as vaccines in small animal models. warfield et al. generated ebola-vlps (evlp) and marburg-vlp (mvlp) containing vp , np, and gp. this vaccine had up to % survival following a lethal infection in mice (vaccine dose-dependent) [ ] . sun et al. produced an evlp containing vp and gp. this vaccine was also up to % effective following a lethal ebov infection in mice (vaccine dose-dependent) [ , ] . many reports document the ability of filoviral gp to act as a potent immunogen, and as such, viral vectors are a popular method of vaccinating through the expression of gp by the host and subsequent immune recognition. however, a recent report sought to utilize gp itself as the vaccination agent. in order to efficiently produce the protein, an expression vector was constructed such that the fc portion of a human igg was fused to ebov-gp [ ] [ ] [ ] . this gp-fc fusion protein induced both cell-mediated and humoral immune responses, and mice vaccinated with zebovgp-fc demonstrated % protection against a lethal ebov challenge. [ ] . while further studies are required, these results show that vaccination with ebovgp-fc alone is effective against a lethal ebov infection, and thus fusion proteins could potentially be used as an effective vaccine against filoviruses [ ] . the use of plants to produce vaccine antigens and antibodies has been demonstrated previously and is attractive for several reasons, including low manufacturing cost, efficient production, minimal risk of contamination with human pathogens or toxins, and the fact that plants have similar secretory pathways and endosomal systems as mammalian cells [ ] [ ] [ ] [ ] . recently, a bean yellow dwarf virus (beydv)-derived replicon system was developed and shown to efficiently produce antibodies against ebov in nicotiana benthamiana leaves [ ] . poolcharoen et al. used this system to develop and fuse ebov-gp to the c-terminus of an igg heavy chain [ ] . these ebov immune complexes (eic) were then used as a vaccine to immunize mice. serum antibody response tests showed that this eic was highly immunogenic in mice and produced antibody levels similar to mice protected from a lethal ebov challenge [ ] . while antibody titers alone do not fully correlated with protection from lethal challenge, there is potential for further development of this vaccine. a replicating vaccine that could spread through the target population after initial inoculation would be an attractive, alternative approach to filovirus development. this approach could provide high coverage with minimal initial vaccinations. a cmv-based vaccine would allow for this type of spread [ ] . cmv, a member of the family betaherpesvirinae, induces a high "effector" memory t-cell (t em ) response before establishing a low-level persistent infection [ ] [ ] [ ] . this high immunogenicity makes cmv a good potential vaccine vector, and the cmv vector has been previously shown to be effective as a vaccine for siv in rhesus macaques [ , ] . tsuda et al. constructed a mouse cmv vector expressing a cd + t cell epitope from the nucleoprotein (np) of ebov (mcmv/ebov-npctl). this vaccine induced high levels of ebov-specific cd + t cells, and subsequently, protected % of mice against a lethal ebov challenge. this shows a proof-ofconcept for cmv as a potential vaccine vector for ebov [ ] . as discussed previously, paramyxovirus-based vectors have demonstrated the capacity to induce strong immune responses and protect animals from infection. as such, a chimeric hpiv was developed where all the hpiv surface markers/receptors were deleted and replaced with ebov-gp [ ] . this chimeric virus can be amplified and recovered easily. additionally, this chimeric virus has -fold greater incorporation of ebov-gp into the virion due to the lack of competition with hpiv surface proteins [ ] . testing in guinea pigs showed that hpiv /Δf-hn/ebogp is highly attenuated, as compared to both hpiv and hpiv /ebogp, and immunogenic, as % developed neutralizing antibodies against ebov. finally, a × pfu i.n. inoculation of hpiv /Δf-hn/ebogp was able to protect % of guinea pigs against a lethal ebov challenge [ ] . table . . . rnapc severe coagulation disorders are one of the most prominent features of filoviral infection. in the event of a breach in vascular integrity a strict balance of pro-and anti-coagulant host factors must be maintained to successfully clot the breach and to prevent too much or too little clot formation. in the instance of filovirus infection, sustained microvascular injury in effected organs results in host coagulation inhibitor depletion which results in disseminated intravascular coagulation (dic). in dic, tissue factor (tf), a clotting factor normally present on cells not exposed to blood, complexes with circulating factor vii leading to clot formation and fibrin deposition through the extrinsic pathway [ , ] . as numerous studies have demonstrated a clear link with dic and resultant organ failure, geisbert et al, sought to eliminate potential tf pathogenesis during filovirus infection by using recombinant nematode anticoagulant protein c (rnapc ) [ ] . they demonstrated that rnapc , an inhibitor of the tf pathway, provided partial post-exposure protection to rhesus macaques during filovirus infection [ ] . previous studies with rnapc have already gone through phase ii trials in orthopedic surgery [ ] and coronary revascularization [ ] . geisbert et al. showed a % survival rate, in addition to a . -day increase in mean time-to-death, for ebov-infected rhesus macaques and a % survival rate, with a . -day increase in mean time-to-death, in marv-infected rhesus macaques, when treated with rnapc post-exposure [ , ] . in a normally % lethal model of filovirus infection, rnapc demonstrated a clear benefit to survival as an increase in the mean time-todeath was observed. rnapc has completed phase ii clinical trials with a good safety record. primates. comparison of current drug candidates at the highest level of development, either in human clinical trials or those that have shown promise in nhps. also listed are the afforded levels of protection in nhps, the type of drug used to induce immunity and the dosing paradigm used to achieve the listed results. phosphorodiamidate morpholino oligomers (pmos) exert their anti-translation effects through steric hindrance of the translation machinery. this steric hindrance is possible due to a morpholino group, similar to a ribose base in rna, and a methylene phosphorodiamidate linking moiety that physically bind to mrna and prevent the translation machinery from accessing the mrna [ ] . once the antisense pmos bind to their target mrna, they are highly stable and highly soluble which would allow high levels of translation inhibition and predictably low levels of potential cytotoxicity [ ] [ ] [ ] [ ] . pmos have previously demonstrated effective antiviral activity against coronaviruses and flaviviruses [ , ] . swenson et al. initially utilized pmos targeting ebov vp and ebov vp to highly protect mice and guinea pigs against a lethal challenge with ebov and marv [ , ] . subsequently, avi- (a combination of pmos against both ebov vp and vp ) and avi- (a combination of pmos against both marv vp and np) were developed and tested in a nhp post-exposure scenario. these pmos, delivered - min post-exposure, protected > % of rhesus macaques against lethal ebov infection and % of cynomolgus macaques against marv infection [ ] . both the ease of controlled manufacture and their efficacy in nhp models to combat filovirus infection, pmos represent a viable therapeutic strategy [ ] . currently, avi- and avi- are in phase i clinical trials. table . . . recombinant human activated protein c ebolavirus disease (evd) and severe sepsis (or septic shock) share many clinical features including fever, hypotension, increased production of tissue factor, elevated levels of nitric oxide, and elevated levels of d-dimers [ ] [ ] [ ] . in addition, the most prominent and consistent finding in severe sepsis is severe protein c deficiency [ , ] . it was shown that treatment of patients with severe sepsis with recombinant human activated protein c (rhapc) resulted in improved survival [ ] . later experiments in nhps demonstrated that ebov infection results in rapid reduction of circulating protein c levels [ , ] . therefore, it was tested if treatment with rhapc could protect against lethal ebov infection in rhesus macaques. fourteen rhesus macaques were infected with a lethal dose of ebov; eleven were then treated with iv rhapc - min after challenge, continuing for days. all control animals died on day post-exposure; however, of the rhapc-treated animals survived (~ % survival). additionally, the mean time-to-death for rhapc-treated animals was . days, which is significant compared to the . days observed in placebo and historical controls [ ] . this product was pulled as a single post-exposure treatment, but given that this intervention is not directly targeting the virus, there may be additional merit in assessing this product in conjunction with a direct antiviral. rna interference (rnai) represents a powerful, naturally occurring biological strategy for inhibiting gene expression. rnai interferes with the translation of mrna to protein products by either sterically blocking mrna or by triggering rnase h-mediated cleavage of the dna/rna duplex, resulting in inhibition of gene expression [ ] . for many years rnai as demonstrated clear efficacy in preventing viral replication in vitro against a number of viruses, including coxsackieviruses, hepatitis b virus (hbv), hepatitis c virus (hcv), herpesviruses, human immunodeficiency virus (hiv), human papillomavirus, rsv, influenza a virus, lymphocytic choriomeningitis virus, polioviruses, and sars-cov [ ] [ ] [ ] [ ] . fowler et al. demonstrated that small-interfering rna (sirna) downregulation of various marv mrna transcripts was able to significantly decrease viral protein production and subsequent viral release in cell culture [ ] . unfortunately, efficient delivery vehicles providing effective drug targeting and stability have hindered the application of rnai in the clinical setting. however, recent developments in the field of nanotechnology have made nanoparticles the solution to increasing pharmacokinetic profiles for rnai therapies [ ] . additionally, tekmira, inc. developed proprietary lipid encapsulation as a means of improving the pharmacology of sirna targeting the ebola rna polymerase l protein, as demonstrated by geisbert et al. [ , ] . to efficiently deliver the sirna to target cells, a mixture of lipids forming a bilayered liposome, or stable nucleic acid-lipid particles (snalp), was designed. the snalp ensures cell entry by preferential fusing with the endosomal membrane upon exposure to the decreasing ph of the endosome. the snalps were further modified by conjugation with polyethylene glycol (peg) ensuring stability and efficient delivery of the sirna payload by neutralizing surface charges and presenting a hydrophilic exterior. this encapsulation was initially demonstrated to significantly increase the stability, half-life, and effectiveness of sirna directed against hbv [ ] . the snalp-encapsulation of sirna targeting the ebov l protein was initially shown to completely protect guinea pigs when administered shortly after a lethal ebov challenge [ ] . this treatment was then assessed for efficacy in rhesus macaques. snalp-encapsulated sirnas targeting ebov l polymerase, vp , and vp were given to rhesus monkeys either four or seven times following a lethal challenge of ebov. two of the three monkeys given four doses survived lethal infection, while all four monkeys given seven doses survived infection [ ] . the enhanced survivorship among the snalp-treated group highlights the efficacy of this potential therapeutic. additionally, there was little to no evidence of side effects associated with the treatment group, aside from mildly altered liver enzyme levels (which could have been an artifact separate from the challenge course) [ ] . table . innate immunity is often the first line of defense against invading pathogens. one mechanism by which innate immunity functions relies on the identification of pathogen-associated molecular patterns (pamps). pamps consist of unique carbohydrate moieties on the external surfaces of foreign microbes, such as hexose and mannose, which are not expressed on the surfaces of the host cells. these pamps are then recognized by host proteins such as mannose-binding lectins (mbl), which recognize these high hexose and mannose contents [ ] . filoviruses have large amounts of mannose comprising their glycoproteins and thus are a target of mbl [ ] . upon exposure, host mbl targets filoviruses for compliment-dependent virus neutralization through the lectin pathway of the compliment cascade [ ] . when administered in supraphysiological doses before or after lethal challenge with ebov, recombinant mbl treatment protected % of mice [ ] . these studies showed that mbl may be a potential post-exposure prophylactic. table . post-exposure treatments effective in small animal models. comparison of current treatment candidates at the small animal model level of development, specifically mouse models. also listed are the afforded levels of protection, the type of drug used to induce immunity and the dosing paradigm used to achieve the listed results. high-throughput screening (hts) is a significant tool for novel drug discovery. hts involves screening libraries consisting of thousands to hundreds of thousands of unique molecules against specific targets. available libraries used in hts have included natural compounds [ , ] , peptides [ ] , drugs [ ] , and synthetic compounds [ , ] . recently, compound fgi- was identified during a screen with an ebov-gfp pseudotyped virus and has shown strong antiviral activity in vitro against high doses of ebov and marv. fgi- was subsequently shown to protect mice against lethal challenges of both ebov and marv [ ] . additionally, compound fgi- was initially identified in a similar manner and was shown to exhibit strong antiviral activity in vitro against ebov, rift valley fever virus (rvfv), all four types of dengue virus (denv), hcv, and hiv- . fgi- also protected mice against a lethal challenge of ebov when given postexposure [ ] . taken together, this suggests that fgi- probably acts on a conserved pathway common to these four viruses, and potentially makes for a very intriguing antiviral. a second screen was done using a collection of , small molecule compounds obtained from the nci and the ebov-gfp pseudotyped virus [ ] . as a result, nsc , a reactive oxygen species scavenger, was identified and shown to have high antiviral activity against ebov, marv, lassa virus, rvfv, and veev. in vivo studies demonstrated that this compound protected mice against a lethal challenge of ebov and marv when given either pre-or post-exposure [ ] . metal ion-based therapeutics are a new potential class of drugs because they differ from carbon-based compounds due to the charged central ion which determines the molecular geometry of the compound. through these unique molecular geometries, specific compounds can be isolated that inhibit biological processes, and are unlike traditional carbon-based compounds because of their unique geometry. this difference allows these compounds to form octahedral and square planar molecular geometries. hexamminecobalt (iii) chloride (cohex) is a complex of a cobalt (iii) ion surrounded by six ammonia ligands in a full octahedral coordination. cohex was initially reported to have antiviral activity against adenovirus and sindbis virus, and was subsequently thought to have potential broad-spectrum antiviral activities [ ] . cohex was shown to be well-tolerated in mice with no apparent toxicity. mice were treated with cohex daily and infected with a lethal dose of ebov. cohex-treated mice had a significant increase in mean time-to-death, and the highest concentration treatment group had a % survival rate [ ] . this suggests that cohex has the potential to be an effective treatment against ebov infection. niemann-pick c (npc ), a cholesterol transporter found in endosomes and lysosomes, was recently identified as being required for ebov replication during a gene trap screen in hap cells using a replication-competent vsv bearing the ebov glycoprotein (rvsv-gp-ebov). in these experiments, cells with non-functional npc demonstrated decreased infectivity by rvsv-gp-ebov; however, expression of a functional npc rescued the normal infectivity of these viruses [ ] . npc is known to affect calcium homeostasis as well as endosomal and lysosomal fission and fusion [ ] [ ] [ ] . it has also been shown to be involved in hiv- release [ ] . loss of npc causes a neurological disorder called niemann-pick disease, which is characterized by cholesterol accumulation in lysosomes [ ] . while heterozygous npc knockout mice (npc +/− ) do not show evidence of niemann-pick disease, most npc +/− knockout mice were protected against a lethal challenge of mouse adapted ebov ( % survival) and marv ( % survival) [ ] . additionally, small molecules, such as u a, have been identified that interfere with npc and cause a cellular phenotype similar to npc deficiency [ ] . as such, u a was subsequently shown to block infection of ebov in vitro [ ] . heat-shock protein (hsp ) is a molecular chaperone that aids in the folding, trafficking, and proteolytic processing of many proteins [ , ] . as a result of their many functionalities, hsp inhibitors have been designed to combat diseases such as cancer, and there are currently several drugs now in phase i and ii clinical trials [ , ] additionally, hsp has shown to be important for replication of negative-strand viruses, as well as hcv, hbv, and polio [ ] [ ] [ ] [ ] . the effects of several natural and synthetic hsp inhibitors on ebov replication were tested in vitro. results of this study demonstrated that three hsp inhibitors significantly inhibited the replication of ebov in vero cells and primary human monocytes, suggesting their use as a potential therapeutic [ ] . ebolaviruses express two secreted glycoproteins, soluble gp (sgp) and small soluble gp (ssgp) [ ] . sgp has been associated with stabilization of the endothelial barrier function and reduction of endothelial barrier permeability by opposing the effects of tnf-α. these effects are in direct opposition to the observed roles for gp, which has been associated with endothelial cell destruction [ ] . ssgp has yet to have a clear role during infection [ ] . each of the gp forms contains identical n-termini but differ in the structure of their c-termini. during the differentiation process of the c-termini, the homodimer sgp is cleaved by furin to yield the mature sgp and a Δ-peptide which is retained in cells longer as compared to sgp. when these Δ-peptides from ebov, sudv, and tafv were fused with fc tags and transfected into cells before infection, they were capable of inhibiting both ebov and marv infection in a dose dependent fashion [ ] . these observations indicated that Δ-peptides might play an important role in filovirus pathogenesis, and could be exploited as a novel anti-filoviral therapeutic [ ] . while many events in filovirus entry remain undiscovered, a fusion mechanism similar to hiv and sars-cov is thought to occur such that a conformational rearrangement of glycoproteins on the viral surface results in viral fusion with the cellular membrane. inhibitors of viral fusion have been developed for hiv- and sars-cov. these inhibitors prevent the c-terminal heptad repeat in the envelope protein from interacting with the cellular membrane proteins by directly competing and blocking its interaction with the n-terminal heptad repeat, which normally would result in the formation of the six-helix bundle required for membrane fusion. c-peptides, which are inhibitors of viral envelope fusion, have had limited success against filoviruses in the past, most likely due to the suggestive evidence that filovirus fusion occurs far along in the endosomal maturation process [ ] [ ] [ ] [ ] [ ] . however, when these c-peptides were conjugated with an argenine-rich segment of hiv- 's tat protein (a protein known for its endosomal localization) the conjugated c-peptide exhibited marked antiviral effects, up to % inhibition of ebov and marv in vitro [ ] [ ] [ ] . unfortunately, the concentrations used to generate this inhibition in vitro were not possible in the clinical setting, but this report indicates that future c-peptide research may result in filovirus entry prevention for future therapeutics. a recent report that screened , molecules demonstrated that chlorophyllide was able to decrease the section of hbv dna in a hbv antiviral assay. these results were obtained at compound concentrations which exhibited no cytotoxic effects. this molecule is an alkylated porphyrin containing copper and as such this compound is carries a charge at neutral phs [ ] . during these screens, the chlorin e compound, a metal-free chlorophyllide-like molecule, was found to be the most potent and was subsequently tested against other viruses, including marv. during testing, the chlorine e compound showed significant antiviral activity in vitro against marv. this compound also inhibited junin virus, denv, hcv, and hiv- [ ] . another recent study that examined a library of , compounds yielded candidates capable of generating ≥ % inhibition of a hiv- /ebov-gp pseudotyped virus, while not interfering with the hiv- /vsv-g control virus. from these candidates, compound , a benzodiazepine derivative, showed an ability to inhibit both ebov and marv to a similar degree in vitro [ ] . results from these experiments suggested that compound acts at an early stage of viral entry, preventing infection by an unknown mechanism [ ] . lj , an aryl methyldiene rhodanine derivative, was identified during a high-throughput screening of inhibitors for nipah virus entry in the context of a vsv-pseudotyped vector [ ] . this compound was subsequently shown to inhibit a variety of enveloped viruses, including marv, ebov, nipah, rvfv, hiv- , hcv, wnv, etc. [ ] . however, it did not inhibit nonenveloped viruses such as adenovirus and reovirus. further testing demonstrated that lj binds to the viral membrane and prevents virus-cell fusion. while initial testing in mice did not show antiviral efficacy, further development of this compound to improve pharmacokinetics and potency is distinctly possible [ ] . development of medical countermeasures for ebov and marv remain a high priority and substantial progress has been made over the past decade. we have moved from the inability to protect from infection in various animal models of disease to a realm of medical countermeasures that protect prophylactically and more recently successful treatments that can be employed following known exposure to the viruses. initial efforts, focused on preventing the disease with vaccination strategies, ranged from subunit vaccines to vlps, vectored systems, dna vaccines, and live-attenuated virus systems that express the ebov or marv glycoproteins. to that aim, vaccine efficacy has been achieved by multiple vaccines against parenteral and aerosol routes of exposure. with the success of these new vaccine platforms, the attention of the past years has focused on the ability to treat infected patients. in the animal models, success has been demonstrated with traditional small molecules and antibodies directed against the virus or critical host proteins or pathways associated with pathogenesis. the ability to utilize various rna silencing technologies has been a focus for therapeutics that could be beneficial for filovirus infection, other infectious diseases and cancer therapy. despite these successes, there is much work to do to adequately prepare for this infectious threat. the ability to provide a beneficial therapeutic impact at a point when patients experience clinical symptoms and seek relief from caregivers remains a hurdle for the medical countermeasure development. moreover, the quality of life of patients following infection and treatment may require additional development efforts or the combination of multiple therapeutic approaches. as seen in outbreaks, the clinical sequelae observed in patients that survive infection are severe and life changing. these observations emphasize the need for medical countermeasures that not only provide survival but also decrease morbidity and long-term pathological outcomes following infection. lastly, the funding resources fortitude and the ability to navigate regulatory pathways will be essential to reaching either emergency use authorization (eua) status or licensed drug status for therapeutics and vaccines. however, the field remains optimistic that medical countermeasure solutions for human use are possible. proposal for a revised taxonomy of the family filoviridae: classification, names of taxa and viruses, and virus abbreviations virus taxonomy-ninth report of the international committee on taxonomy of viruses a hitherto unknown infectious disease contracted from monkeys filoviridae: a taxonomic home for marburg and ebola viruses? a compendium of years of epidemiological, clinical, and laboratory studies drug targets in infections with ebola and marburg viruses ebolavirus and other 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molecular chaperone hsp is important for vaccinia virus growth in cells heat-shock protein is essential for stabilization of the hepatitis c virus nonstructural protein ns ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry effects of ebola virus glycoproteins on endothelial cell activation and barrier function heptad repeat-derived peptides block protease-mediated direct entry from the cell surface of severe acute respiratory syndrome coronavirus but not entry via the endosomal pathway peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type gp are potent inhibitors of virus infection evidence that a prominent cavity in the coiled coil of hiv type gp is an attractive drug target capture of an early fusion-active conformation of hiv- gp inhibition of ebola virus entry by a c-peptide targeted to endosomes tat transduction: the molecular mechanism and therapeutic prospects cellular uptake of unconjugated tat peptide involves clathrin-dependent endocytosis and heparan sulfate receptors alkylated porphyrins have broad antiviral activity against hepadnaviruses, flaviviruses, filoviruses, and arenaviruses identification of a small-molecule entry inhibitor for filoviruses a broad-spectrum antiviral targeting entry of enveloped viruses the authors declare no conflict of interest. key: cord- - rxradwz authors: kohl, claudia; kurth, andreas title: european bats as carriers of viruses with zoonotic potential date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: rxradwz bats are being increasingly recognized as reservoir hosts of highly pathogenic and zoonotic emerging viruses (marburg virus, nipah virus, hendra virus, rabies virus, and coronaviruses). while numerous studies have focused on the mentioned highly human-pathogenic bat viruses in tropical regions, little is known on similar human-pathogenic viruses that may be present in european bats. although novel viruses are being detected, their zoonotic potential remains unclear unless further studies are conducted. at present, it is assumed that the risk posed by bats to the general public is rather low. in this review, selected viruses detected and isolated in europe are discussed from our point of view in regard to their human-pathogenic potential. all european bat species and their roosts are legally protected and some european species are even endangered. nevertheless, the increasing public fear of bats and their viruses is an obstacle to their protection. educating the public regarding bat lyssaviruses might result in reduced threats to both the public and the bats. the european continent is inhabited by hibernation. many bat species migrate over vast distances while others are rather territorial. all bats in europe utilize echolocation to navigate. contrary to the worldwide efforts in protecting bats, they have been increasingly gaining attention as potential reservoir hosts of some of the most virulent viruses we know. various publications reviewed bats globally as carriers and potential reservoir hosts of human-pathogenic and zoonotic viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , while hardly anything is known about human-pathogenicity of european bat viruses apart from lyssaviruses. in this review, we discuss a selection of viruses as possible threats posed by european bats to the public from our point of view. a summary of viruses that have been detected in european bats is given in table at the end of the manuscript. a more comprehensive and up-to-date list of bat-associated viruses can be found online at the database of bat-associated viruses (dbatvir) [ ] . european bat lyssaviruses (family rhabdoviridae) are the most important zoonotic bat-borne viruses in europe and have been comprehensively reviewed by banyard et al. in this special issue on bat viruses (title: lyssavirus infections of bats: emergence and zoonotic threat) [ ] . therefore, we will provide a short overview. [ , ] nyctalus noctula rhinolophus ferrumequinum hungary pcr [ ] myotis myotis germany pcr [ ] astroviridae myotis myotis germany pcr [ ] mamastrovirus italy pcr [ , ] the postulates drafted by jacob henle and robert koch in the late th century constitute a framework regarding the principles of cause-and-effect in microbiology [ ] . back then, it was comparatively straightforward to limit cause-and-effect to four postulates, although viruses had not yet been discovered nor was molecular biology developed ( table ). all of the postulates are hard to fulfill for viruses, as they do not grow on nutrient media, but require living cells for replication. when looking for viruses on a molecular level, it is necessary to consider that only the first postulate can be accomplished. studies identifying a host-pathogen relationship solely at the molecular level do not take into consideration that detection does not equal etiology. even though polymerase chain reaction (pcr) screening and metagenomic studies are indispensable and valuable tools, virologists should stay close to the henle-koch postulates when assuming a possible virulence of viruses detected in bat hosts. a plethora of coronaviruses has been detected in bats, mostly belonging to the alphaand betacoronaviruses [ , ] . the genus alphacoronavirus hosts human-pathogenic strains (i.e., human cov e and nl ); however, in this review, we will focus on selected highly human-pathogenic betacoronaviruses and their european bat virus relatives [ ] . from november until july the world was confronted with the first pandemic of the new millennium, caused by a novel coronavirus (cov) inducing the severe acute respiratory syndrome in humans (sars) [ ] [ ] [ ] . the pandemic spread from its origin, a wet-market in the guangdong province in china, through countries on five continents resulted in more than infected humans of whom more than eventually died [ , ] . the search for the animal reservoir began, identifying masked palm civets and bats as possible sources. subsequently, a plethora of diverse coronaviruses of distinct groups have been detected in various bat species around the world via molecular-biological techniques. in , another human-pathogenic coronavirus, called middle east respiratory syndrome coronavirus (mers-cov), began spreading from the arabian peninsula, so far resulting in globally laboratory-confirmed cases of infection with mers-cov, including at least deaths [ ] . dromedaries and bats are suspected as reservoirs for mers-cov [ ] . recent findings support the plausibility of dromedaries as reservoir species [ ] . although numerous studies in european bats report the presence of sars-like-cov and mers-like-cov sequences [ , [ ] [ ] [ ] ] , no final conclusion can be drawn regarding their zoonotic potential. a related virus detected in bats cannot necessarily be considered as zoonotic. a few alterations in the sars-cov spike protein enabled its binding to the host receptor ace- , thus sars-cov became capable of infecting humans [ ] . so far, the sars-like cov detected in european bats lack these alterations and thus are not predicted to be capable of infecting humans. although virus strains might be similar or related on a nucleic acid level, the distinct function of proteins is crucial when determining the host range. therefore, mere similarity is not sufficient to examine the potential of viruses to infect humans or even predict their virulence. it took ten years from the emergence of sars-cov for the first bat cov to be isolated from rhinolophus bats in china, that displayed the human ace- receptor, which enabled the virus to infect human cells [ ] . these findings provide evidence for the reservoir theory. from the european perspective, nevertheless, no sars-like cov or mers-like cov has been isolated from any european bat, nor has any transmission of sars-like cov or mers-like cov to humans been reported. the case of mers-cov is slightly different, as a sequence of base pairs with % identity to mers-cov was detected in a bat (taphozous perforates-the species identification performed was not beyond doubt, as it was based on exclusion criteria (no cytochrome b sequence of taphozous perforates is available in genbank [ ] )) in saudi arabia [ ] . this finding initiated a controversy among leading cov experts, as the journal nature recently reported [ ] . they discussed that the complete genome sequence of mers-cov obtained from the bat should confirm that the virus was indeed identical and not coincidentally just a short conserved region of the virus genome. furthermore, a prevalence study might provide insights into the distribution of mers-cov in bat populations. although taphozous perforates are not abundant in europe, climate change and environmental factors may have an effect on the future distribution of this bat species (figure ) [ ] . the case of mers-cov emergence impressively demonstrates the necessity of virus discovery and prevalence studies. with the first sequence of mers-cov that became available, bats were suspected as reservoir hosts, not only because mers-cov is a sars-cov relative, but also because previous bat virus discovery studies had provided eligible sequences of bat cov to genbank, allowing for correlations with the novel mers-cov. recently, a quasi-species of mers covs was recovered from nasal swabs of dromedaries of the kingdom of saudi arabia [ ] . the mers cov consensus genome variants from dromedaries and humans are indistinguishable, supporting the plausibility of dromedaries in the role of transmission [ ] . in , the first reported outbreak of filovirus, named lloviu virus (llov), in a european bat population occurred in france, spain, and portugal [ ] . several colonies of schreiber's bats (miniopterus schreibersii) suddenly declined due to an unknown disease. llov was found in animals that showed signs of viral infection, but not in healthy bats co-roosting in the caves (myotis myotis). llov is distinctly related to filoviruses found in african bats and was classified in as type species of the novel genus cuevavirus [ ] . unfortunately, the lack of successful isolation of llov prohibits the experimental infection of schreiber's bats to clarify whether llov is the first filovirus capable of inducing disease in bats. this would challenge the hypothesis of bats as potential reservoir hosts for other filoviruses like ebola and marburg virus. schreiber's bats are distributed in distinct lineages throughout oceania, africa, southern europe, and south-east asia (figure ) [ ] . they are thought to transmit and maintain llov across different lineages throughout their habitats, although no studies are available to prove this hypothesis. consequently, the sole demonstration of a novel filovirus sequence does not provide evidence of a possible public health threat. following the henle-koch postulates, the virus should be isolated and further characterized to draw conclusions on the evolution of filoviruses in their respective bat host. as most filoviruses are described as highly pathogenic for humans, the occurrence of llov should be carefully monitored by prevalence studies in the highly abundant miniopterus schreibersii (figure ). in , three distinct paramyxoviruses were detected in german bats, two of which were related to the proposed genus jeilongvirus (myotis mystacinus, pipistrellus pipistrellus) and one was related to the genus rubulavirus (nyctalus noctula) [ ] . another study published in the same year described another different paramyxoviruses in bats from germany (myotis bechsteinii, m. daubentonii, m. myotis, and m. mystacinus) and bulgaria (myotis alcathoe and m. capaccinii), all of which belong to the genus morbillivirus [ ] . none of the novel bat paramyxoviruses are closely related to viruses of the highly pathogenic genus henipavirus or other human-pathogenic paramyxoviruses [ , ] . there is no evidence to suggest that any of these novel paramyxoviruses are capable of infecting humans. similar to the case of the llov filovirus, virus isolates and prevalence studies in both humans and bats could improve knowledge and clarify their zoonotic potential. few studies have documented the negative results from pcr testing of european bats for other human-pathogenic viruses. for instance following generic pcr screening for flavi-, hanta-and influenza-a viruses in european bats in [ ] , testing of another central european bats for influenza-a viruses [ ] and testing european bats for hepadnaviruses in did not lead to the detection of any viral nucleic acids [ ] . pcr screening of european bats for orthopoxviruses has not revealed any known or novel virus sequences [ ] . so far, the only virus isolates (beside lyssaviruses) obtained from european bats are one bunyavirus, one adenovirus and orthoreoviruses [ , , , ] . these represent the only isolates that would allow for further characterization and potential clarification of their zoonotic potential. nevertheless, recombinant viruses, constructed on sequence information, are also valuable tools to study prevalence and pathogenicity in vitro. toscana virus (tosv) was isolated from a bat's brain in , while simultaneously tosv was isolated from sandflies in the laboratory [ ] . as tosv has never been reported in bats afterwards and no hemagglutination-inhibiting antibodies has been initially found in the bat, there is a reasonable chance that this tosv isolation may have been a cross-contamination [ ] . bat adenovirus (bat adv- ) was isolated from a bat's intestine in [ ] , and the whole genome was obtained and circumstantially analyzed [ , ] . bat adv- displays a monophyletic relationship to the adenoviruses of canids (cadv). moreover, open trading frames (orf) in the bat adv- genome and the cadv are identical and not present in other members of the mastadenoviruses. the closely related canine adv contribute to the severe kennel cough syndrome in canids and show an unusually broad host range [ ] . this provides evidence suggesting an ancestral inter-species transmission of mastadenoviruses between bats and canids. like in the case of rabies virus, which is prevalent in both bats and terrestrial mammals (e.g., dogs, raccoons, skunks, and foxes) of the americas, a continuing exchange and transmission between bats and canids or other terrestrial animals might be possible [ ] . there is no evidence of a zoonotic potential of bat adv- . in , three novel orthoreoviruses were isolated from plecotus auritus and myotis mystacinus in germany [ ] . a subsequent pcr screening obtained identical viral sequences also in other bat species: pipistrellus pipistrellus, pipistrellus nathusii, pipistrellus kuhlii, and nyctalus noctula. at the same time, a group in italy detected further orthoreoviruses in myotis kuhlii, rhinolophus hyposideros, tadarida teniotis, and vespertilio murinus [ ] . summing up the data for the reovirus isolates from germany and italy, a close relationship was revealed to the genus mammalian orthoreovirus (mrv), in particular to an orthoreovirus obtained from a dog (strain t /d ) with hemorrhagic enteritis in italy [ , , ] . no ancestral relationship was assumed here, but rather an opportunistic -behavior‖ of the novel closely related mrvs, as they were detected in various different bat species. moreover, the newly isolated mrvs are phylogenetically related to viruses capable of inducing severe meningitis in humans [ ] . recently, a study published by steyer et al. described the detection of an mrv from a child hospitalized with acute gastroenteritis in slovenia [ ] . the causative agent was determined to be an mrv with the highest similarity of . %- . % in the respective segments to a bat mrv (t /bat/germany/ / ) [ ] . this might indicate a human-pathogenic potential of strain t /bat/germany/ / . as the case of sars-cov has shown that even small changes in the genome are important for determining the host range, this has to be determined for the bat mrvs in further studies. interestingly, no contact was reported between the infected child and bats, but contact to a domestic dog was assumed [ ] . the isolated viruses will allow for a seroprevalence study (cross-reactivity and cross-neutralization with other strains) in humans, which shall be initiated to examine the prevalence of specific antibodies to bat mrvs in germany and italy (where these viruses have been found) to clarify their zoonotic potential. this is especially interesting as asian bat orthoreoviruses of the genus pteropine orthoreovirus have already been linked to potentially zoonotic respiratory diseases in humans [ , ] . rhabdoviruses of the genus lyssavirus that have been detected in europe are considerably harmful and truly zoonotic agents, inevitably causing the death of unvaccinated humans if not treated in time before onset of the rabies disease [ ] . even though bat-transmitted lyssaviruses have a fatality rate of virtually % and are suspected to be transmissible by bat biting and scratching, the reported total number of human fatalities in europe is low (n = - since ) [ ] [ ] [ ] . all described hosts of european bat lyssaviruses (eblv- and eblv- ) are synanthropic, hence sharing their habitats with humans [ ] . eblv- has been predominantly detected in eptesicus serotinus and e. isabellinus in europe, both living in buildings, roofs, and attics usually in the southern regions of europe (e. serotinus until ° north, e. isabellinus in southern portugal-e. isabellinus is a north african population of e. serotinus that is controversially but not concludingly discussed as a novel species [ ]), and male bats are reported to co-roost with multiple bat species [ ] . eblv- was also detected in v. murinus, m. schreibersii, m. myotis, m. nattereri, r. ferrumequinum, and t. teniotis. whether these bat species constitute accidental hosts infected by spillover from co-roosting e. serotinus species, or whether they are additional reservoirs, has not yet been determined [ ] [ ] [ ] [ ] ] . two human cases described by johnson et al. were confirmed as infected with eblv- , which is prevalent in european m. daubentonii and m. dasycneme [ , ] . m. daubentonii is prevalent in north-eastern europe and is frequently found co-roosting with p. pipistrellus and m. nattereri, whereas m. dasycneme is found throughout europe and in the mediterranean, co-roosting with m. capaccinii. so far, none of the co-roosting bats have been reported to carry eblv- [ ] . however, spillover transmission to other animals (stone-marten, sheep, and cat) was described for eblv- [ ] [ ] [ ] . overall, lyssaviruses prevalent in european bats pose a risk to public health, and preventive measures have already been implemented by many european countries for decades (e.g., surveillance, vaccination plans, and post exposure prophylaxis) [ ] . especially the high-risk occupational groups (i.e., bat workers, bat carers in bat bat hospitals) are at increased risk. however, lyssavirus prevalence in european bats is very difficult to determine and results are very heterogenic [ ] . the lyssavirus prevalences are considerably low, but changes of behavior as a result of a lyssavirus infection may be more likely to bring bats into contact with humans. however, it is necessary to balance the risk with the total number of fatal human cases during the last years (five cases in million people living in greater europe) [ ] . accordingly, the risk is relatively low and would probably fall to zero if people were educated appropriately. direct contact (bites and scratches) with certain bat species might be risky and require post exposure prophylaxis. only few of the european bat species are known to be reservoirs of eblv- and eblv- , but all of the european species are endangered or close to extinction. relocation or culling of bat colonies, in spite of being an obvious solution from the viewpoint of the general public, increases the risk of lyssavirus exposure and transmission and should not be considered [ ] . only education can channel public fear to avoid further threats to the bats and the general public. alexander von humboldt discovered the latitudinal gradient in species diversity as early as [ ] : the richness of species is subject to a global diversity gradient, abating from the species-rich tropics toward the higher latitudes [ ] . bats influence this gradient significantly. more than bat species have been described worldwide. although they are abundant worldwide except for the polar regions, a steep diversity gradient is present from the tropics towards the poles [ ] [ ] [ ] [ ] . are fewer viruses prevalent in european bats because of the lower abundance of species in the more temperate europe? and is the zoonotic risk posed by bats decreased accordingly? only few studies on the biogeography of microorganisms are available. these studies indicate that the latitudinal diversity gradient has either no or a top-down effect on microbial diversity [ ] [ ] [ ] [ ] [ ] . two studies hypothesized that the local diversity and dispersal of viruses is very high, though overall, the viral diversity is limited on the global scale [ , ] . therefore, no assumptions can be made regarding the viral diversity in species abundant in temperate climates. as the total number of abundant species might not be essential, the change in biodiversity might play a role. the effect of decline in biodiversity on the emergence of diseases is subject of numerous publications [ ] [ ] [ ] [ ] [ ] [ ] [ ] . basically, there are arguments in favor of two controversial theories; reduced biodiversity could either increase (dilution effect) or decrease the risk of disease transmission. for almost half of the zoonotic diseases that have newly emerged by spillover since , a preceding change in land-use, agriculture, and wildlife hunting was reported [ ] . all of the above-mentioned effects contribute to changes in biodiversity and increased contact situations between human and animal hosts, also in europe. once spillover in novel hosts has occurred, a high density of the novel host population eventually facilitates the establishment in the novel niche. thus, human overpopulation and a decreased biodiversity might be mutual factors promoting the establishment of emerging infectious diseases. in conclusion, the baas becking hypothesis from might still be appropriate: -everything is everywhere, but the environment selects‖ [ ] . until now, lyssaviruses have been the only proven zoonotic viruses in european bats and may cause rabies in humans. however, only few bat species are known to transmit lyssaviruses in europe, and the number of human cases is rather low. nevertheless, education of the general public should be intensified to avoid easily preventable infections. although viruses with zoonotic potential have been detected in european bats, no clear assumption can be made without further studies. sero-prevalence studies should be conducted on the orthoreoviruses isolated from european bats, especially as a closely related virus was detected in a diseased child in slovenia [ ] . other bat viruses detected by using molecular techniques should be isolated (e.g., mers-like cov or bat bunyavirus) to allow for characterization and follow-up sero-prevalence studies. in general, bats are special reservoir hosts because of their biological features, long-time co-evolution and high diversity of viruses that can be found. furthermore, there is neither a clearly decreased risk in the emergence of zoonotic viruses in temperate climates compared to the tropics nor a decreased risk in regions of lower biodiversity. in conclusion, many drivers of emergence in the tropics also have validity in europe. however, european bats are endangered species, and some of them are threatened by extinction. although lyssaviruses are prevalent in european 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